U.S. patent application number 17/312442 was filed with the patent office on 2022-09-22 for compositions comprising streptococcus pneumoniae polysaccharide-protein conjugates and methods of use thereof.
This patent application is currently assigned to Merck Sharp & Dohme Corp.. The applicant listed for this patent is Chitrananda ABEYGUNAWARDANA, Yadong Adam CUI, Romulo FERRERO, Jian HE, Merck Sharp & Dohme Corp., Luwy MUSEY, Tanaz PETIGARA, Julie M. SKINNER. Invention is credited to Chitrananda Abeygunawardana, Yadong Adam Cui, Romulo Ferrero, Jian He, Luwy Musey, Tanaz Petigara, Julie M. Skinner.
Application Number | 20220296695 17/312442 |
Document ID | / |
Family ID | 1000006417171 |
Filed Date | 2022-09-22 |
United States Patent
Application |
20220296695 |
Kind Code |
A1 |
Abeygunawardana; Chitrananda ;
et al. |
September 22, 2022 |
COMPOSITIONS COMPRISING STREPTOCOCCUS PNEUMONIAE
POLYSACCHARIDE-PROTEIN CONJUGATES AND METHODS OF USE THEREOF
Abstract
The invention is related to multivalent immunogenic compositions
comprising more than one S. pneumoniae polysaccharide protein
conjugates, wherein each of the conjugates comprises a
polysaccharide from an S. pneumoniae serotype conjugated to a
carrier protein, wherein the serotypes of S. pneumoniae are as
defined herein. In some embodiments, at least one of the
polysaccharide protein conjugates is formed by a conjugation
reaction comprising an aprotic solvent. In further embodiments,
each of the polysaccharide protein conjugates is formed by a
conjugation reaction comprising an aprotic solvent. Also provided
are methods for inducing a protective immune response in a human
patient comprising administering the multivalent immunogenic
compositions of the invention to the patient. The multivalent
immunogenic compositions are useful for providing protection
against S. pneumoniae infection and/or pneumococcal diseases caused
by S. pneumoniae. The compositions of the invention are also useful
as part of treatment regimes that provide complementary protection
for patients that have been vaccinated with a multivalent vaccine
indicated for the prevention of pneumococcal disease.
Inventors: |
Abeygunawardana; Chitrananda;
(Ambler, PA) ; Cui; Yadong Adam; (Norristown,
PA) ; Ferrero; Romulo; (Westfield, NJ) ; He;
Jian; (Blue Bell, PA) ; Musey; Luwy; (Blue
Bell, PA) ; Petigara; Tanaz; (Philadelphia, PA)
; Skinner; Julie M.; (Phoenixville, PA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ABEYGUNAWARDANA; Chitrananda
CUI; Yadong Adam
FERRERO; Romulo
HE; Jian
MUSEY; Luwy
PETIGARA; Tanaz
SKINNER; Julie M.
Merck Sharp & Dohme Corp. |
North Wales
North Wales
North Wales
West Point
North Wales
North Wales
West Point
Rahway |
PA
PA
PA
PA
PA
PA
PA
NJ |
US
US
US
US
US
US
US
US |
|
|
Assignee: |
Merck Sharp & Dohme
Corp.
Rahway
NJ
|
Family ID: |
1000006417171 |
Appl. No.: |
17/312442 |
Filed: |
December 17, 2019 |
PCT Filed: |
December 17, 2019 |
PCT NO: |
PCT/US2019/066682 |
371 Date: |
June 10, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62853331 |
May 28, 2019 |
|
|
|
62781835 |
Dec 19, 2018 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 37/04 20180101;
A61K 2039/6068 20130101; A61P 31/04 20180101; A61K 2039/70
20130101; A61K 47/20 20130101; A61K 39/092 20130101; A61K
2039/55588 20130101; A61K 2039/6037 20130101 |
International
Class: |
A61K 39/09 20060101
A61K039/09; A61K 47/20 20060101 A61K047/20; A61P 31/04 20060101
A61P031/04; A61P 37/04 20060101 A61P037/04 |
Claims
1. A multivalent immunogenic composition comprising S. pneumoniae
polysaccharide protein conjugates, wherein each of the conjugates
comprises a polysaccharide from a S. pneumoniae serotype conjugated
to a carrier protein, and wherein the polysaccharide protein
conjugates include polysaccharides of a group of S. pneumoniae
serotypes selected from the group consisting of: a) 1, 3, 4, 5, 6A,
6B, 7F, 9V, 10A, 12F, 14, 15A, DeOAc15B, 18C, 19A, 19F, 22F, 23B,
23F, 24F, 33F and 35B; b) 1, 3, 4, 5, 6A, 6B, 7F, 9V, 10A, 12F, 14,
15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; c) 1, 3,
4, 5, 6A, 6B, 7F, 8, 9V, 10A, 12F, 14, 15A, DeOAc15B, 18C, 19A,
19F, 22F, 23B, 23F, 24F, 33F and 35B; d) 1, 3, 4, 5, 6A, 6B, 7F, 8,
9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F
and 35B; e) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 12F, 14, 15B, 18C,
19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; f) 1, 3, 4, 5, 6A, 6B,
7F, 8, 9V, 10A, 11A, 12F, 14, 15A, DeOAc15B, 18C, 19A, 19F, 22F,
23B, 23F, 24F, 33F and 35B; g) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A,
11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and
35B; h) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B,
18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; i) 1, 3, 4, 5, 6A,
6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23B,
23F, 24F, 33F and 35B; j) 1, 3, 4, 5, 6A, 7F, 9V, 10A, 12F, 14,
15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; k) 1, 3,
4, 5, 6B, 7F, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B,
23F, 24F, 33F and 35B; l) 1, 3, 4, 5, 6C, 7F, 9V, 10A, 12F, 14,
15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; m) 1, 3,
4, 5, 6A, 7F, 8, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F,
23B, 23F, 24F, 33F and 35B; n) 1, 3, 4, 5, 6B, 7F, 8, 9V, 10A, 12F,
14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; o) 1,
3, 4, 5, 6C, 7F, 8, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F,
23B, 23F, 24F, 33F and 35B; p) 1, 3, 4, 5, 6A, 7F, 8, 9V, 10A, 11A,
12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;
q) 1, 3, 4, 5, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15C, 18C,
19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; r) 1, 3, 4, 5, 6C, 7F,
8, 9V, 10A, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F,
24F, 33F and 35B; s) 1, 3, 4, 5, 6A, 7F, 9V, 10A, 12F, 14, 15A,
15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; t) 1, 3, 4, 5,
6B, 7F, 9V, 10A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F,
24F, 33F and 35B; u) 1, 3, 4, 5, 6C, 7F, 9V, 10A, 12F, 14, 15A,
15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; v) 1, 3, 4, 5,
6A, 7F, 8, 9V, 10A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B,
23F, 24F, 33F and 35B; w) 1, 3, 4, 5, 6B, 7F, 8, 9V, 10A, 12F, 14,
15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; x) 1, 3,
4, 5, 6C, 7F, 8, 9V, 10A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F,
23B, 23F, 24F, 33F and 35B; y) 1, 3, 4, 5, 6A, 7F, 8, 9V, 10A, 11A,
12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;
z) 1, 3, 4, 5, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C,
19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; aa) 1, 3, 4, 5, 6C, 7F,
8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F,
24F, 33F and 35B; bb) 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 15A,
DeOAc15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; cc)
1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F,
23B, 23F, 24F, 33F, 35B and 39; dd) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V,
12F, 14, 15A, DeOAc15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B
and 39; ee) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 12F, 14, 15A, 15C, 18C,
19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; ff) 1, 3, 4, 5, 6A,
6B, 7F, 8, 9V, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F,
33F, 35B and 39; gg) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 11A, 12F, 14,
15A, DeOAc15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39;
hh) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 11A, 12F, 14, 15A, 15C, 18C,
19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; ii) 1, 3, 4, 5, 6A,
6B, 7F, 8, 9V, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B,
23F, 24F, 33F, 35B and 39; jj) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 11A,
12F, 14, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39;
kk) 1, 3, 4, 5, 6A, 7F, 9V, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F,
23B, 23F, 24F, 33F, 35B and 39; ll) 1, 3, 4, 5, 6B, 7F, 9V, 12F,
14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39;
mm) 1, 3, 4, 5, 6C, 7F, 9V, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F,
23B, 23F, 24F, 33F, 35B and 39; nn) 1, 3, 4, 5, 6A, 7F, 8, 9V, 12F,
14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39;
oo) 1, 3, 4, 5, 6B, 7F, 8, 9V, 12F, 14, 15A, 15C, 18C, 19A, 19F,
22F, 23B, 23F, 24F, 33F, 35B and 39; pp) 1, 3, 4, 5, 6C, 7F, 8, 9V,
12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and
39; qq) 1, 3, 4, 5, 6A, 7F, 8, 9V, 11A, 12F, 14, 15A, 15C, 18C,
19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; rr) 1, 3, 4, 5, 6B,
7F, 8, 9V, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F,
24F, 33F, 35B and 39; ss) 1, 3, 4, 5, 6C, 7F, 8, 9V, 11A, 12F, 14,
15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; tt)
1, 3, 4, 5, 6A, 7F, 9V, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B,
23F, 24F, 33F, 35B and 39; uu) 1, 3, 4, 5, 6B, 7F, 9V, 12F, 14,
15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; vv)
1, 3, 4, 5, 6C, 7F, 9V, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B,
23F, 24F, 33F, 35B and 39; ww) 1, 3, 4, 5, 6A, 7F, 8, 9V, 12F, 14,
15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; xx)
1, 3, 4, 5, 6B, 7F, 8, 9V, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F,
23B, 23F, 24F, 33F, 35B and 39; yy) 1, 3, 4, 5, 6C, 7F, 8, 9V, 12F,
14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39;
zz) 1, 3, 4, 5, 6A, 7F, 8, 9V, 11A, 12F, 14, 15A, 15B, 18C, 19A,
19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; aaa) 1, 3, 4, 5, 6B, 7F,
8, 9V, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F,
33F, 35B and 39; and bbb) 1, 3, 4, 5, 6C, 7F, 8, 9V, 11A, 12F, 14,
15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39.
2. The multivalent immunogenic composition of claim 1, wherein the
immunogenic composition does not comprise polysaccharide protein
conjugates having polysaccharides from any further S. pneumoniae
serotypes.
3. The multivalent immunogenic composition of claim 1, wherein at
least one of the polysaccharide protein conjugates is formed by a
conjugation reaction comprising an aprotic solvent.
4. The multivalent immunogenic composition of claim 1, wherein each
of the polysaccharide protein conjugates is formed by a conjugation
reaction comprising an aprotic solvent.
5. The multivalent immunogenic composition of claims 3, wherein the
aprotic solvent is dimethylsulfoxide (DMSO).
6. The multivalent immunogenic composition of claim 1, wherein the
carrier protein is selected from the group consisting of Outer
Membrane Protein Complex (OMPC), tetanus toxoid, diphtheria toxoid,
protein D and CRM197.
7. The multivalent immunogenic composition of claim 1, wherein the
carrier protein is CRM197.
8. The multivalent immunogenic composition of claim 1, wherein the
composition further comprises an adjuvant.
9. The multivalent immunogenic composition of claim 1, wherein the
composition does not comprise an adjuvant.
10. A method for inducing an immune response in a human patient
comprising administering the multivalent immunogenic composition of
claim 1 to a patient.
11. A method for inducing a protective immune response in a human
patient comprising administering the multivalent immunogenic
composition of claim 1 to a patient.
12. A method for inducing a protective immune response against S.
pneumoniae in a human patient comprising administering the
multivalent immunogenic composition of claim 1 to a patient.
13. The method of claim 10, wherein the patient was previously
treated with a multivalent pneumococcal vaccine.
14. The method of claim 10, wherein the patient is 6 weeks through
17 years of age.
15. A method for the prevention of pneumococcal pneumonia and/or
invasive pneumococcal disease in patients 6 weeks to 17 years of
age comprising administering the multivalent immunogenic
composition of claim 1 to patients 6 weeks to 17 years of age.
16. A multivalent immunogenic composition comprising 23 distinct S.
pneumoniae polysaccharide protein conjugates, wherein each of the
conjugates comprises a capsular polysaccharide from a S. pneumoniae
serotype conjugated to a carrier protein, wherein each distinct
polysaccharide protein conjugate comprises a polysaccharide from S.
pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 12F, 14,
15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B,
respectively, and wherein the carrier protein is CRM197.
17. The multivalent immunogenic composition of claim 16, wherein
the immunogenic composition does not comprise polysaccharide
protein conjugates having polysaccharides from any further S.
pneumoniae serotypes.
18. The multivalent immunogenic composition of claim 16, wherein
each of the polysaccharide protein conjugates is formed by a
conjugation reaction comprising an aprotic solvent, wherein the
aprotic solvent is dimethylsulfoxide (DMSO).
19. The multivalent immunogenic composition of claim 16, wherein
the composition comprises an adjuvant.
20. A multivalent immunogenic composition comprising 24 distinct S.
pneumoniae polysaccharide protein conjugates, wherein each of the
conjugates comprises a capsular polysaccharide from a S. pneumoniae
serotype conjugated to a carrier protein, wherein each distinct
polysaccharide protein conjugate comprises a polysaccharide from S.
pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F,
14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B,
respectively, and wherein the carrier protein is CRM197.
21. The multivalent immunogenic composition of claim 20, wherein
the immunogenic composition does not comprise polysaccharide
protein conjugates having polysaccharides from any further S.
pneumoniae serotypes.
22. The multivalent immunogenic composition of claim 20, wherein
each of the polysaccharide protein conjugates is formed by a
conjugation reaction comprising an aprotic solvent, wherein the
aprotic solvent is dimethylsulfoxide (DMSO).
23. The multivalent immunogenic composition of claim 20, wherein
the composition comprises an adjuvant.
24. A multivalent immunogenic composition comprising 24 distinct S.
pneumoniae polysaccharide protein conjugates, wherein each of the
conjugates comprises a capsular polysaccharide from a S. pneumoniae
serotype conjugated to a carrier protein, wherein each distinct
polysaccharide protein conjugate comprises a polysaccharide from S.
pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F,
14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B,
respectively, and wherein the carrier protein is CRM197.
25. The multivalent immunogenic composition of claim 24, wherein
the immunogenic composition does not comprise polysaccharide
protein conjugates having polysaccharides from any further S.
pneumoniae serotypes.
26. The multivalent immunogenic composition of claim 24, wherein
each of the polysaccharide protein conjugates is formed by a
conjugation reaction comprising an aprotic solvent, wherein the
aprotic solvent is dimethylsulfoxide (DMSO).
27. The multivalent immunogenic composition of claim 24, wherein
the composition comprises an adjuvant.
28. A multivalent immunogenic composition comprising up to 30
distinct S. pneumoniae polysaccharide protein conjugates, wherein
each of the conjugates comprises a polysaccharide from a S.
pneumoniae serotype conjugated to a carrier protein, and wherein
the up to 30 polysaccharide protein conjugates include
polysaccharides of a group of S. pneumoniae serotypes selected from
the group consisting of: 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A,
12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and
35B.
29. The multivalent immunogenic composition of claim 28, wherein
the up to 30 polysaccharide protein conjugates further include one,
two, three, four, five or six additional S. pneumoniae serotypes
selected from 7C, 9N, 16F, 23A, 35F and 38.
30. A multivalent immunogenic composition comprising up to 30
distinct S. pneumoniae polysaccharide protein conjugates, wherein
each of the conjugates comprises a polysaccharide from a S.
pneumoniae serotype conjugated to a carrier protein, and wherein
the up to 30 polysaccharide protein conjugates include
polysaccharides of a group of S. pneumoniae serotypes selected from
the group consisting of: 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A,
12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and
35B.
31. The multivalent immunogenic composition of claim 30, wherein
the up to 30 polysaccharide protein conjugates further include one,
two, three, four, five or six additional S. pneumoniae serotypes
selected from 7C, 9N, 16F, 23A, 35F and 38.
32. The multivalent immunogenic composition of claim 28, wherein
the carrier protein is CRM197.
33. The multivalent immunogenic composition of claim 28, wherein
each of the polysaccharide protein conjugates is formed by a
conjugation reaction comprising an aprotic solvent, wherein the
aprotic solvent is dimethylsulfoxide (DMSO).
34. The multivalent immunogenic composition of claim 28, wherein
the composition comprises an adjuvant.
Description
FIELD OF THE INVENTION
[0001] The present invention provides multivalent immunogenic
compositions having distinct polysaccharide-protein conjugates.
Each conjugate consists of a capsular polysaccharide prepared from
a different serotype of Streptococcus pneumoniae conjugated to a
carrier protein, preferably CRM197. The immunogenic compositions
provide broad coverage against pneumococcal disease.
[0002] The instant application contains a Sequence Listing which
has been submitted electronically in ASCII format and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Dec. 3, 2019, is named 24683WOPCT-SEQTXT-03DEC2019 and is 6
kilobytes in size.
BACKGROUND OF THE INVENTION
[0003] Streptococcus pneumoniae is a Gram-positive bacterium and
the most common cause of invasive bacterial disease (such as
pneumonia, bacteraemia, meningitis and Otitis media) in infants and
young children. Pneumococcus is encapsulated with a chemically
linked polysaccharide which confers serotype specificity. There are
over 90 known serotypes of pneumococci, and the capsule is the
principle virulence determinant for pneumococci, as the capsule not
only protects the inner surface of the bacteria from complement,
but is itself poorly immunogenic. Polysaccharides are T-cell
independent antigens, and, in most cases, can not be processed or
presented on MHC molecules to interact with T-cells. They can
however, stimulate the immune system through an alternate mechanism
which involves cross-linking of surface receptors on B cells.
[0004] The multivalent pneumococcal polysaccharide vaccines that
have been licensed for many years have proved valuable in
preventing pneumococcal disease in adults, particularly, the
elderly and those at high-risk. However, infants and young children
respond poorly to unconjugated pneumococcal polysaccharides. The
pneumococcal conjugate vaccine, Prevnar.RTM., containing the 7 most
frequently isolated serotypes (4, 6B, 9V, 14, 18C, 19F and 23F)
causing invasive pneumococcal disease in young children and infants
at the time, was first licensed in the United States in February
2000. Following universal use of Prevnar.RTM. in the United States,
there has been a significant reduction in invasive pneumococcal
disease in children due to the serotypes present in Prevnar.RTM..
See Centers for Disease Control and Prevention, MMWR Morb Mortal
Wkly Rep 2005, 54(36):893-7. However, there are limitations in
serotype coverage with Prevnar.RTM. in certain regions of the world
and some evidence of certain emerging serotypes in the United
States (for example, 19A and others). See O'Brien et al., 2004, Am
J Epidemiol 159:634-44; Whitney et al., 2003, N Engl J Med
348:1737-46; Kyaw et al., 2006, N Engl J Med 354:1455-63; Hicks et
al., 2007, J Infect Dis 196:1346-54; Traore et al., 2009, Clin
Infect Dis 48:S181-S189.
[0005] U.S. Patent Application Publication No. US 2006/0228380
describes a 13-valent pneumococcal polysaccharide-protein conjugate
vaccine including serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C,
19A, 19F and 23F. Chinese Patent Application Publication No. CN
101590224 A describes a 14-valent pneumococcal
polysaccharide-protein conjugate vaccine including serotypes 1, 2,
4, 5, 6A, 6B, 7F, 9N, 9V, 14, 18C, 19A, 19F and 23F.
[0006] Other PCVs have covered 7, 10, 11, or 13 of the serotypes
contained in PCV-15 (U.S Pub. No. 2011/0195086), but immune
interference has been observed for some serotypes (e.g. lower
protection for serotype 3 in GSK's PCV-11) and lower response rates
to serotype 6B in Pfizer's PCV-13 (PREVNAR.RTM. 13). See Prymula et
al., 2006, Lancet 367:740-48 and Kieninger et al., Safety and
Immunologic Non-inferiority of 13-valent Pneumococcal Conjugate
Vaccine Compared to 7-valent Pneumococcal Conjugate Vaccine Given
as a 4-Dose Series in Healthy Infants and Toddlers, presented at
the 48t.sup.h Annual ICAAC/ISDA 46.sup.th Annual Meeting,
Washington DC, Oct. 25-28, 2008.
[0007] The current multivalent pneumococcal vaccines have been
effective in reducing the incidence of pneumococcal disease
associated with those serotypes present in the vaccines. However,
the prevalence of the pneumococci expressing serotypes not present
in the currently available vaccines has been increasing.
Accordingly, there is a need for additional pneumococcal vaccine
compositions which can provide protection against pneumococcal
serotypes not present in currently available vaccines.
SUMMARY OF THE INVENTION
[0008] The invention provides multivalent immunogenic compositions
comprising S. pneumoniae polysaccharide protein conjugates, wherein
each of the conjugates comprises a polysaccharide from a S.
pneumoniae serotype conjugated to a carrier protein, and wherein
the polysaccharide protein conjugates include polysaccharides of a
group of S. pneumoniae serotypes selected from the group consisting
of: [0009] a) 1, 3, 4, 5, 6A, 6B, 7F, 9V, 10A, 12F, 14, 15A,
DeOAc15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; [0010] b)
1, 3, 4, 5, 6A, 6B, 7F, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F,
22F, 23B, 23F, 24F, 33F and 35B; [0011] c) 1, 3, 4, 5, 6A, 6B, 7F,
8, 9V, 10A, 12F, 14, 15A, DeOAc15B, 18C, 19A, 19F, 22F, 23B, 23F,
24F, 33F and 35B; [0012] d) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A,
12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;
[0013] e) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 12F, 14, 15B, 18C,
19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; [0014] f) 1, 3, 4, 5,
6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, DeOAc15B, 18C, 19A, 19F,
22F, 23B, 23F, 24F, 33F and 35B; [0015] g) 1, 3, 4, 5, 6A, 6B, 7F,
8, 9V, 10A, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F,
24F, 33F and 35B; [0016] h) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A,
11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and
35B; [0017] i) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14,
15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; [0018] j) 1,
3, 4, 5, 6A, 7F, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F,
23B, 23F, 24F, 33F and 35B; [0019] k) 1, 3, 4, 5, 6B, 7F, 9V, 10A,
12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;
[0020] l) 1, 3, 4, 5, 6C, 7F, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A,
19F, 22F, 23B, 23F, 24F, 33F and 35B; [0021] m) 1, 3, 4, 5, 6A, 7F,
8, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F,
33F and 35B; [0022] n) 1, 3, 4, 5, 6B, 7F, 8, 9V, 10A, 12F, 14,
15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; [0023] o)
1, 3, 4, 5, 6C, 7F, 8, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F,
22F, 23B, 23F, 24F, 33F and 35B; [0024] p) 1, 3, 4, 5, 6A, 7F, 8,
9V, 10A, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F,
33F and 35B; [0025] q) 1, 3, 4, 5, 6B, 7F, 8, 9V, 10A, 11A, 12F,
14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;
[0026] r) 1, 3, 4, 5, 6C, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15C,
18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; [0027] s) 1, 3, 4,
5, 6A, 7F, 9V, 10A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B,
23F, 24F, 33F and 35B; [0028] t) 1, 3, 4, 5, 6B, 7F, 9V, 10A, 12F,
14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;
[0029] u) 1, 3, 4, 5, 6C, 7F, 9V, 10A, 12F, 14, 15A, 15B, 18C, 19A,
19F, 22F, 23B, 23F, 24F, 33F and 35B; [0030] v) 1, 3, 4, 5, 6A, 7F,
8, 9V, 10A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F,
33F and 35B; [0031] w) 1, 3, 4, 5, 6B, 7F, 8, 9V, 10A, 12F, 14,
15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; [0032] x)
1, 3, 4, 5, 6C, 7F, 8, 9V, 10A, 12F, 14, 15A, 15B, 18C, 19A, 19F,
22F, 23B, 23F, 24F, 33F and 35B; [0033] y) 1, 3, 4, 5, 6A, 7F, 8,
9V, 10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F,
33F and 35B; [0034] z) 1, 3, 4, 5, 6B, 7F, 8, 9V, 10A, 11A, 12F,
14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; and
[0035] aa) 1, 3, 4, 5, 6C, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B,
18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B.
[0036] The invention provides a multivalent immunogenic composition
comprising 22 distinct polysaccharide protein conjugates, wherein
each of the conjugates comprises a capsular polysaccharide from a
S. pneumoniae serotype conjugated to a carrier protein, wherein the
polysaccharide are prepared from S. pneumoniae serotypes 1, 3, 4,
5, 6A, 6B, 7F, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B,
23F, 24F, 33F and 35B.
[0037] The invention provides a multivalent immunogenic composition
comprising 22 distinct polysaccharide protein conjugates, wherein
each of the conjugates comprises a polysaccharide from a S.
pneumoniae serotype conjugated to a carrier protein, and wherein
the polysaccharide protein conjugates include polysaccharides of a
group of S. pneumoniae serotypes selected from the group consisting
of: 1, 3, 4, 5, 6A, 6B, 7F, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A,
19F, 22F, 23B, 23F, 24F, 33F and 35B.
[0038] The invention provides a multivalent immunogenic composition
comprising 23 distinct polysaccharide protein conjugates, wherein
each of the conjugates comprises a capsular polysaccharide from a
S. pneumoniae serotype conjugated to a carrier protein, wherein the
polysaccharide are prepared from S. pneumoniae serotypes 1, 3, 4,
5, 6A, 6B, 7F, 8, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F,
23B, 23F, 24F, 33F and 35B.
[0039] The invention provides a multivalent immunogenic composition
comprising 23 distinct S. pneumoniae polysaccharide protein
conjugates, wherein each of the conjugates comprises a capsular
polysaccharide from a S. pneumoniae serotype conjugated to a
carrier protein, wherein each distinct polysaccharide protein
conjugate comprises a polysaccharide from S. pneumoniae serotypes
1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A,
19F, 22F, 23B, 23F, 24F, 33F and 35B, respectively, and wherein the
carrier protein is CRM197.
[0040] The invention provides a multivalent immunogenic composition
comprising 24 distinct polysaccharide protein conjugates, wherein
each of the conjugates comprises a capsular polysaccharide from a
S. pneumoniae serotype conjugated to a carrier protein, wherein the
polysaccharide are prepared from S. pneumoniae serotypes 1, 3, 4,
5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F,
22F, 23B, 23F, 24F, 33F and 35B.
[0041] The invention provides a multivalent immunogenic composition
comprising 24 distinct S. pneumoniae polysaccharide protein
conjugates, wherein each of the conjugates comprises a capsular
polysaccharide from a S. pneumoniae serotype conjugated to a
carrier protein, wherein each distinct polysaccharide protein
conjugate comprises a polysaccharide from S. pneumoniae serotypes
1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15C, 18C,
19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B, respectively, and
wherein the carrier protein is CRM197.
[0042] The invention provides a multivalent immunogenic composition
comprising 24 distinct polysaccharide protein conjugates, wherein
each of the conjugates comprises a capsular polysaccharide from a
S. pneumoniae serotype conjugated to a carrier protein, wherein the
polysaccharide are prepared from S. pneumoniae serotypes 1, 3, 4,
5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F,
22F, 23B, 23F, 24F, 33F and 35B.
[0043] The invention provides a multivalent immunogenic composition
comprising 24 distinct S. pneumoniae polysaccharide protein
conjugates, wherein each of the conjugates comprises a capsular
polysaccharide from a S. pneumoniae serotype conjugated to a
carrier protein, wherein each distinct polysaccharide protein
conjugate comprises a polysaccharide from S. pneumoniae serotypes
1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C,
19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B, respectively, and
wherein the carrier protein is CRM197.
[0044] The invention provides a multivalent immunogenic composition
comprising up to 33 distinct polysaccharide protein conjugates,
wherein each of the conjugates comprises a polysaccharide from a S.
pneumoniae serotype conjugated to a carrier protein, and wherein
the polysaccharide protein conjugates include polysaccharides of a
group of S. pneumoniae serotypes selected from the group consisting
of: 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15C,
18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B, further including
one, two, three, four, five, six, seven, eight or nine additional
S. pneumoniae serotypes selected from 7C, 9N, 16F, 21, 23A, 31, 34,
35F and 38.
[0045] The invention provides a multivalent immunogenic composition
comprising up to 30 distinct polysaccharide protein conjugates,
wherein each of the conjugates comprises a polysaccharide from a S.
pneumoniae serotype conjugated to a carrier protein, and wherein
the polysaccharide protein conjugates include polysaccharides of a
group of S. pneumoniae serotypes selected from the group consisting
of: 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15C,
18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B, further including
one, two, three, four, five or six additional S. pneumoniae
serotypes selected from 7C, 9N, 16F, 23A, 35F and 38. The invention
provides a multivalent immunogenic composition comprising up to 33
distinct polysaccharide protein conjugates, wherein each of the
conjugates comprises a polysaccharide from a S. pneumoniae serotype
conjugated to a carrier protein, and wherein the polysaccharide
protein conjugates include polysaccharides of a group of S.
pneumoniae serotypes selected from the group consisting of: 1, 3,
4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C, 19A,
19F, 22F, 23B, 23F, 24F, 33F and 35B, further including one, two,
three, four, five, six, seven, eight or nine additional S.
pneumoniae serotypes selected from 7C, 9N, 16F, 21, 23A, 31, 34,
35F and 38.
[0046] The invention provides a multivalent immunogenic composition
comprising up to 30 distinct polysaccharide protein conjugates,
wherein each of the conjugates comprises a polysaccharide from a S.
pneumoniae serotype conjugated to a carrier protein, and wherein
the polysaccharide protein conjugates include polysaccharides of a
group of S. pneumoniae serotypes selected from the group consisting
of: 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B,
18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B, further including
one, two, three, four, five or six additional S. pneumoniae
serotypes selected from 7C, 9N, 16F, 23A, 35F and 38.
[0047] In some embodiments, at least one of the polysaccharide
protein conjugates is formed by a conjugation reaction comprising
an aprotic solvent, e.g. dimethylsulfoxide (DMSO). In specific
embodiments, each of the polysaccharide protein conjugates is
formed by a conjugation reaction comprising an aprotic solvent,
e.g. (DMSO).
[0048] Also provided are methods for inducing a protective immune
response in a human patient comprising administering the
multivalent immunogenic compositions of the invention to the
patient. In some embodiments of the methods of the invention, the
patient was previously treated with a multivalent pneumococcal
vaccine.
[0049] A multivalent immunogenic composition of the invention may
be used as part of a treatment regimen with a different,
complementary pneumococcal vaccine. Accordingly, the invention
provides a method of inducing a protective immune response in a
human patient comprising administering a multivalent immunogenic
composition of the invention to the patient, further comprising
administering a multivalent pneumococcal vaccine to the patient in
any order. In particular embodiments, the multivalent pneumococcal
vaccine is comprised of multiple S. pneumoniae polysaccharide
protein conjugates wherein each of the conjugates comprises
polysaccharide from an S. pneumoniae serotype conjugated to a
carrier protein. In other embodiments, the multivalent pneumococcal
vaccine is comprised of unconjugated capsular polysaccharides.
[0050] The invention also provides multivalent immunogenic
compositions comprising S. pneumoniae polysaccharide protein
conjugates wherein each of the conjugates comprises a
polysaccharide from a S. pneumoniae serotype conjugated to a
carrier protein, wherein select serotypes of S. pneumoniae provide
cross-reactivity to other select serotypes.
BRIEF DESCRIPTION OF THE DRAWINGS
[0051] FIG. 1. Pre-immune (Pre), post-dose 1 (PD1), 2 (PD2) and 3
(PD3) IgG antibody dilution titers as determined by ECL for mice
immunized with PCV22 unadjuvanted (PCV22 unadj) or formulated with
aluminum phosphate adjuvant (PCV22/APA). Reading from left to
right; Pre PCV22 unadj, Pre PCV22/APA, PD1 PCV22 unadj, PD1
PCV22/APA, PD2 PCV22 unadj, PD2 PCV22/APA, PD3 PCV22 unadj, and PD3
PCV22/APA.
[0052] FIG. 2. ECL dilution titer ratio of PCV22/APA compared to
PCV22 unadjuvanted (PCV22 unadj) at PD3.
[0053] FIG. 3. Serotype specific OPA dilution titers (pre-immune,
PD1, PD2, PD3) for mice immunized with PCV22 unadjuvanted (PCV22
unadj) or formulated with APA (PCV22/APA). Reading from left to
right; Pre PCV22 unadj, Pre PCV22/APA, PD1 PCV22 unadj, PD1
PCV22/APA, PD2 PCV22 unadj, PD2 PCV22/APA, PD3 PCV22 unadj, and PD3
PCV22/APA.
[0054] FIG. 4. OPA dilution titer ratio of PCV22/APA compared to
PCV22 unadjuvanted (PCV22 unadj) at PD3.
[0055] FIG. 5. PCV22 immunized mice are protected from S.
pneumoniae 24F intra tracheal challenge.
[0056] FIG. 6. Pre-immune (Pre), PD1 and PD2 IgG antibody dilution
titers as determined by ECL for rabbits immunized with PCV22
unadjuvanted or PCV22/APA. Error bars represent the 95% confidence
intervals (CI) of the geometric mean titer (GMT). Reading from left
to right; Pre PCV22 unadj, Pre PCV22/APA, PD1 PCV22 unadj, PD1
PCV22/APA, PD2 PCV22 unadj, and PD2 PCV22/APA.
[0057] FIG. 7. ECL GMT ratio of PCV22/APA compared to PCV22
unadjuvanted at PD2. Error bars represent the 95% confidence
intervals (CI).
[0058] FIG. 8. Serotype specific OPA dilution titers (pre-immune
"Pre" and PD2) for rabbits immunized with PCV22 unadjuvanted or
PCV22/APA. Error bars represent the variation in functional
antibody titers for five rabbits.
[0059] FIG. 9. Pre-immune (Pre), PD1, PD2, and PD3 IgG antibody
dilution titers as determined by ECL for IRMs immunized with A)
PCV23 unadjuvanted, B) PCV23 (DMSO)/APA, C) PCV23(DMSO+Aq)/APA, and
D) PCV15/APA+PCV8/APA. Error bars represent the 95% confidence
intervals (CI) of the geometric mean titer (GMT).
[0060] FIG. 10. Comparison of ECL antibody responses in IRMs (8-9
per group) following vaccination with PCV23 with or without APA.
Symbols indicate ratios at A) PD1, B) PD2, or C) PD3. GMT ratios
with error bars representing the 95% CIs.
[0061] FIG. 11. Comparison of ECL antibody responses in IRMs (9 per
group) following vaccination with PCV23 (DMSO+Aq)/APA or
co-administrated with PCV15/APA +PCV8/APA. Symbols indicate ratios
at A) PD1, B) PD2, and C) PD3. GMT ratios with error bars
representing the 95% CIs.
[0062] FIG. 12. Comparison of boosted ECL antibody responses in
IRMs (8-9 per group) following vaccination with PCV23 unadjuvanted,
PCV23(DMSO)/APA, PCV23(DMSO+Aq)/APA or PCV15/APA+PCV8/APA. A)
PD1/Pre, B) PD2/Pre, C) PD3/Pre, D) PD2/PD1 and E) PD3/PD2. Symbols
are GMT ratios with error bars representing the 95% CIs.
[0063] FIG. 13. (A) Pre-immune (Pre), post-dose 1 (PD1) and 2 (PD2)
IgG antibody dilution titers as determined by ECL for New Zealand
white rabbits immunized with PCV24 formulated with aluminum
phosphate adjuvant (PCV24/APA). Error bars represent the 95%
confidence intervals (CI) of the geometric mean titer (GMT). (B)
Serotype specific OPA dilution titers (pre-immune and PD2) for
NZWRs immunized with PCV24/APA. Error bars represent the variation
in functional antibody titers for eight NZWRs.
[0064] FIG. 14. (A) Pre-immune (Pre), post-dose 1 (PD1), 2 (PD2)
and 3 (PD3) IgG antibody dilution titers as determined by ECL for
infant Rhesus monkeys (IRMs) immunized with PCV24 formulated with
aluminum phosphate adjuvant (PCV24/APA). Error bars represent the
95% confidence intervals (CI) of the geometric mean titer (GMT).
(B) Serotype specific OPA dilution titers (pre-immune and PD3) for
IRMs immunized with PCV24/APA. Error bars represent the variation
in functional antibody titers for five IRMs.
DETAILED DESCRIPTION OF THE INVENTION
[0065] The present invention provides multivalent immunogenic
compositions comprising pneumococcal polysaccharide-protein
conjugates, wherein each of the conjugates comprises a
polysaccharide from an S. pneumoniae serotype conjugated to a
carrier protein, wherein the serotypes of S. pneumoniae are as
defined herein.
[0066] In some embodiments the invention provides multivalent
immunogenic compositions comprising S. pneumoniae polysaccharide
protein conjugates, wherein each of the conjugates comprises a
polysaccharide from a S. pneumoniae serotype conjugated to a
carrier protein, and wherein the polysaccharide protein conjugates
include polysaccharides of a group of S. pneumoniae serotypes
selected from the group consisting of: [0067] a) 1, 3, 4, 5, 6A,
6B, 7F, 9V, 10A, 12F, 14, 15A, DeOAc15B, 18C, 19A, 19F, 22F, 23B,
23F, 24F, 33F and 35B; [0068] b) 1, 3, 4, 5, 6A, 6B, 7F, 9V, 10A,
12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;
[0069] c) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 12F, 14, 15A,
DeOAc15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; [0070] d)
1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A,
19F, 22F, 23B, 23F, 24F, 33F and 35B; [0071] e) 1, 3, 4, 5, 6A, 6B,
7F, 8, 9V, 10A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F,
33F and 35B; [0072] f) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A,
12F, 14, 15A, DeOAc15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and
35B; [0073] g) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14,
15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; [0074] h)
1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C,
19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; [0075] i) 1, 3, 4, 5,
6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23B,
23F, 24F, 33F and 35B; [0076] j) 1, 3, 4, 5, 6A, 7F, 9V, 10A, 12F,
14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;
[0077] k) 1, 3, 4, 5, 6B, 7F, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A,
19F, 22F, 23B, 23F, 24F, 33F and 35B; [0078] l) 1, 3, 4, 5, 6C, 7F,
9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F
and 35B; [0079] m) 1, 3, 4, 5, 6A, 7F, 8, 9V, 10A, 12F, 14, 15A,
15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; [0080] n) 1,
3, 4, 5, 6B, 7F, 8, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F,
23B, 23F, 24F, 33F and 35B; [0081] o) 1, 3, 4, 5, 6C, 7F, 8, 9V,
10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and
35B; [0082] p) 1, 3, 4, 5, 6A, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A,
15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; [0083] q) 1,
3, 4, 5, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F,
22F, 23B, 23F, 24F, 33F and 35B; [0084] r) 1, 3, 4, 5, 6C, 7F, 8,
9V, 10A, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F,
33F and 35B; [0085] s) 1, 3, 4, 5, 6A, 7F, 9V, 10A, 12F, 14, 15A,
15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; [0086] t) 1,
3, 4, 5, 6B, 7F, 9V, 10A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F,
23B, 23F, 24F, 33F and 35B; [0087] u) 1, 3, 4, 5, 6C, 7F, 9V, 10A,
12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;
[0088] v) 1, 3, 4, 5, 6A, 7F, 8, 9V, 10A, 12F, 14, 15A, 15B, 18C,
19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; [0089] w) 1, 3, 4, 5,
6B, 7F, 8, 9V, 10A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B,
23F, 24F, 33F and 35B; [0090] x) 1, 3, 4, 5, 6C, 7F, 8, 9V, 10A,
12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;
[0091] y) 1, 3, 4, 5, 6A, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B,
18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; [0092] z) 1, 3, 4,
5, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F,
23B, 23F, 24F, 33F and 35B; [0093] aa) 1, 3, 4, 5, 6C, 7F, 8, 9V,
10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F
and 35B; [0094] bb) 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 15A,
DeOAc15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39;
[0095] cc) 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 15A, 15C, 18C, 19A,
19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; [0096] dd) 1, 3, 4, 5,
6A, 6B, 7F, 8, 9V, 12F, 14, 15A, DeOAc15B, 18C, 19A, 19F, 22F, 23B,
23F, 24F, 33F, 35B and 39; [0097] ee) 1, 3, 4, 5, 6A, 6B, 7F, 8,
9V, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B
and 39; [0098] ff) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 12F, 14, 15B,
18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; [0099] gg) 1,
3, 4, 5, 6A, 6B, 7F, 8, 9V, 11A, 12F, 14, 15A, DeOAc15B, 18C, 19A,
19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; [0100] hh) 1, 3, 4, 5,
6A, 6B, 7F, 8, 9V, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B,
23F, 24F, 33F, 35B and 39; [0101] ii) 1, 3, 4, 5, 6A, 6B, 7F, 8,
9V, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F,
35B and 39; [0102] jj) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 11A, 12F, 14,
15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; [0103] kk)
1, 3, 4, 5, 6A, 7F, 9V, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B,
23F, 24F, 33F, 35B and 39; [0104] ll) 1, 3, 4, 5, 6B, 7F, 9V, 12F,
14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39;
[0105] mm) 1, 3, 4, 5, 6C, 7F, 9V, 12F, 14, 15A, 15C, 18C, 19A,
19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; [0106] nn) 1, 3, 4, 5,
6A, 7F, 8, 9V, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F,
24F, 33F, 35B and 39; [0107] oo) 1, 3, 4, 5, 6B, 7F, 8, 9V, 12F,
14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39;
[0108] pp) 1, 3, 4, 5, 6C, 7F, 8, 9V, 12F, 14, 15A, 15C, 18C, 19A,
19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; [0109] qq) 1, 3, 4, 5,
6A, 7F, 8, 9V, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B,
23F, 24F, 33F, 35B and 39; [0110] rr) 1, 3, 4, 5, 6B, 7F, 8, 9V,
11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B
and 39; [0111] ss) 1, 3, 4, 5, 6C, 7F, 8, 9V, 11A, 12F, 14, 15A,
15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; [0112] tt)
1, 3, 4, 5, 6A, 7F, 9V, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B,
23F, 24F, 33F, 35B and 39; [0113] uu) 1, 3, 4, 5, 6B, 7F, 9V, 12F,
14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39;
[0114] vv) 1, 3, 4, 5, 6C, 7F, 9V, 12F, 14, 15A, 15B, 18C, 19A,
19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; [0115] ww) 1, 3, 4, 5,
6A, 7F, 8, 9V, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F,
24F, 33F, 35B and 39; [0116] xx) 1, 3, 4, 5, 6B, 7F, 8, 9V, 12F,
14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39;
[0117] yy) 1, 3, 4, 5, 6C, 7F, 8, 9V, 12F, 14, 15A, 15B, 18C, 19A,
19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; [0118] zz) 1, 3, 4, 5,
6A, 7F, 8, 9V, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B,
23F, 24F, 33F, 35B and 39; [0119] aaa) 1, 3, 4, 5, 6B, 7F, 8, 9V,
11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B
and 39; and [0120] bbb) 1, 3, 4, 5, 6C, 7F, 8, 9V, 11A, 12F, 14,
15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39.
[0121] In some embodiments, the multivalent immunogenic composition
comprises pneumococcal serotypes selected from the group consisting
of: i) 1, 3, 4, 5, 6A, 6B, 7F, 9V, 10A, 12F, 14, 15A, 15C, 18C,
19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; or ii) 1, 3, 4, 5, 6A,
6B, 7F, 8, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B,
23F, 24F, 33F and 35B; or iii) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A,
11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and
35B; or iv) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A,
15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B. In a
particular embodiment, a multivalent immunogenic composition of the
invention comprises multiple pneumococcal S. pneumoniae
polysaccharide protein conjugates wherein each of the conjugates
comprises a polysaccharide from an S. pneumoniae serotype
conjugated to a carrier protein, wherein the serotypes of S.
pneumoniae comprise serotypes: i) 1, 3, 4, 5, 6A, 6B, 7F, 9V, 10A,
12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;
or ii) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 12F, 14, 15A, 15C, 18C,
19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; or iii) 1, 3, 4, 5, 6A,
6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F,
23B, 23F, 24F, 33F and 35B. Said compositions were found to be
immunogenic in mice, rabbits and/or monkeys and generate functional
antibody which killed vaccine-type bacterial strains at all doses
tested.
[0122] The multivalent immunogenic compositions of the invention
are useful for immunizing a patient against vaccine-type S.
pneumoniae serotypes and/or as part of a treatment regimen with
different, complementary pneumococcal vaccine(s). Accordingly, the
invention provides a method of inducing a protective immune
response in a human patient comprising administering a multivalent
immunogenic composition of the invention to the patient, and
further comprising administering a multivalent pneumococcal vaccine
to the patient, in any order. In other embodiments, the multivalent
immunogenic compositions of the invention are administered to a
patient who had been previously immunized with a different
multivalent pneumococcal vaccine.
[0123] In embodiments of the invention, conjugates from at least
one pneumococcal serotype are prepared using reductive amination in
an aprotic solvent such as DMSO. In further embodiments, the
multivalent immunogenic composition comprises pneumococcal
conjugates that are each prepared using reductive amination in an
aprotic solvent. The use of DMSO solvent enhances the covalent
associations of polysaccharide to protein through direct
consumption of lysine residues on the surface of the carrier
protein. The increased covalent association has a direct benefit to
increasing the stability of the polysaccharide protein conjugate of
multivalent immunogenic compositions comprising polysaccharide
antigens conjugated in DMSO.
[0124] I. Definitions and Abbreviations
[0125] As used throughout the specification and appended claims,
the following abbreviations apply:
[0126] APA aluminum phosphate adjuvant
[0127] APC antigen presenting cell
[0128] CI confidence interval
[0129] DMSO dimethylsulfoxide
[0130] DS polysaccharide-protein Drug Substance
[0131] GMC geometric mean concentration
[0132] GMT geometric mean titer
[0133] HPSEC high performance size exclusion chromatography
[0134] IM intra-muscular or intra-muscularly
[0135] IRM infant rhesus macaque
[0136] LOS lipo-oligosaccharide
[0137] LPS lipopolysaccharide
[0138] MALS multi-angle light scattering
[0139] MBC monovalent bulk conjugate
[0140] Mn number averaged molecular weight
[0141] MOPA multiplexed opsonophagocytic assays
[0142] MW molecular weight
[0143] NMWCO nominal molecular weight cut off
[0144] NZWR New Zealand White rabbit
[0145] OPA opsonophagocytosis assay
[0146] PCV pneumococcal conjugate vaccine
[0147] PD1 post-dose 1
[0148] PD2 post-dose 2
[0149] PD3 post-dose 3
[0150] PnPs Pneumococcal Polysaccharide
[0151] Ps polysaccharide
[0152] PS-20 polysorbate-20
[0153] RI refractive index
[0154] UV ultraviolet
[0155] w/v weight per volume
[0156] So that the invention may be more readily understood,
certain technical and scientific terms are specifically defined
below. Unless specifically defined elsewhere in this document, all
other technical and scientific terms used herein have the meaning
commonly understood by one of ordinary skill in the art to which
this invention belongs.
[0157] As used throughout the specification and in the appended
claims, the singular forms "a," "an," and "the" include the plural
reference unless the context clearly dictates otherwise.
[0158] Reference to "or" indicates either or both possibilities
unless the context clearly dictates one of the indicated
possibilities. In some cases, "and/or" was employed to highlight
either or both possibilities.
[0159] The terms "aqueous solvent" or "aqueous conditions" when
used with conjugation, such as reductive amination, refers to use
of water as the solvent for the conjugation reaction. The water may
contain buffers and other components except that no organic solvent
is present.
[0160] The terms "aprotic solvent", "DMSO solvent" or "DMSO
conditions" when used with conjugation, such as reductive
amination, refers to use of an aprotic solvent, or a combination of
aprotic solvents, (or DMSO, as applicable) as the solvent for the
conjugation reaction. The aprotic solvent may have some water
present, for example, up to 1%, 2%, 5%, 10% or 20%.
[0161] The term "comprises" when used with the immunogenic
composition of the invention refers to the inclusion of any other
components, such as adjuvants and excipients, or the addition of
one or more polysaccharide-protein conjugates that are not
specifically enumerated. The term "consisting of" when used with
the multivalent polysaccharide-protein conjugate mixture refers to
a mixture having those particular S. pneumoniae polysaccharide
protein conjugates and no other S. pneumoniae polysaccharide
protein conjugates from a different serotype. "Consists essentially
of" and variations such as "consist essentially of" or "consisting
essentially of," indicate the inclusion of any recited elements or
group of elements, and the optional inclusion of other elements, of
similar or different nature than the recited elements, which do not
materially change the basic or novel properties of the specified
dosage regimen, method, or composition.
[0162] "Effective amount" of a composition of the invention refers
to a dose required to elicit antibodies that significantly reduce
the likelihood or severity of infectivity of a microbe, e.g., S.
pneumoniae, during a subsequent challenge.
[0163] As used herein, the phrase "indicated for the prevention of
pneumococcal disease" means that a vaccine or immunogenic
composition is approved by one or more regulatory authorities, such
as the US Food and Drug Administration, for the prophylaxis of one
or more diseases caused by any serotype of S. pneumoniae,
including, but not limited to:
[0164] pneumococcal disease generally, pneumococcal pneumonia,
pneumococcal meningitis, pneumococcal bacteremia, invasive disease
caused by S. pneumoniae, and otitis media caused by S.
pneumoniae.
[0165] A "multivalent pneumococcal vaccine" is a pharmaceutical
preparation comprising more than one active agent (e.g.,
pneumococcal capsular polysaccharide or pneumococcal polysaccharide
protein conjugate) that provides active immunity to disease or
pathological condition caused by more than one serotype of S.
pneumoniae.
[0166] The term "polysaccharide" is meant to include any antigenic
saccharide element (or antigenic unit) commonly used in the
immunologic and bacterial vaccine arts, including, but not limited
to, a "saccharide", an "oligosaccharide", a "polysaccharide", a
"liposaccharide", a "lipo-oligosaccharide (LOS)", a
"lipopolysaccharide (LPS)", a "glycosylate", a "glycoconjugate" and
the like.
[0167] The term "unadjuvanted", in the context of a vaccine or
immunogenic composition of the instant invention, means a
pneumococcal polysaccharide composition, including but not limited
to PCV8, PCV15, PCV22, PCV23 and PCV24, wherein the composition
contains no adjuvant.
[0168] "PCV8" refers to an immunogenic composition containing S.
pneumoniae polysaccharide (PnPs) serotypes -8, -10A, -12F, -15A,
-15C, -23B, -24F and -35B.
[0169] "PCV15" refers to an immunogenic composition containing S.
pneumoniae polysaccharide (PnPs) serotypes -1, -3, -4, -5, -6A,
-6B, -7F, -9V, -14, -18C, -19A, -19F, -22F, -23F, and -33F.
[0170] "PCV22" refers to an immunogenic composition containing S.
pneumoniae polysaccharide (PnPs) serotypes -1, -3, -4, -5, -6A,
-6B, -7F, -9V, -10A, -12F,-14, -15A, -15C, -18C, -19A, -19F, -22F,
-23B, -23F, -24F, -33F, and -35B.
[0171] "PCV23" refers to an immunogenic composition containing S.
pneumoniae polysaccharide (PnPs) serotypes -1, -3, -4, -5, -6A,
-6B, -7F, -8, -9V, -10A, -12F,-14, -15A, -15C, -18C, -19A, -19F,
-22F, -23B, -23F, -24F, -33F, and -35B.
[0172] "PCV24" refers to an immunogenic composition containing S.
pneumoniae polysaccharide (PnPs) serotypes -1, -3, -4, -5, -6A,
-6B, -7F, -8, -9V, -10A, -11A, -12F,-14, -15A, -15C, -18C, -19A,
-19F, -22F, -23B, -23F, -24F, -33F, and -35B.
[0173] "CpG-containing nucleotide," "CpG-containing
oligonucleotide," "CpG oligonucleotide," and similar terms refer to
a nucleotide molecule of 6-50 nucleotides in length that contains
an unmethylated CpG moiety. See, e.g., Wang et al., 2003, Vaccine
21:4297. CpG-containing oligonucleotides include modified
oligonucleotides using any synthetic internucleoside linkages,
modified base and/or modified sugar.
[0174] An "adjuvant," as defined herein, is a substance that serves
to enhance the immunogenicity of an immunogenic composition of the
invention. An immune adjuvant may enhance an immune response to an
antigen that is weakly immunogenic when administered alone, e.g.,
inducing no or weak antibody titers or cell-mediated immune
response, increase antibody titers to the antigen, and/or lowers
the dose of the antigen effective to achieve an immune response in
the individual. Thus, adjuvants are often given to boost the immune
response and are well known to the skilled artisan.
[0175] A "patient" (alternatively referred to herein as a
"subject") refers to a mammal capable of being infected with a S.
pneumoniae. In preferred embodiments, the patient is a human. A
patient can be treated prophylactically or therapeutically.
Prophylactic treatment provides sufficient protective immunity to
reduce the likelihood or severity of a pneumococcal infection or
the effects thereof, e.g., pneumococcal pneumonia. Therapeutic
treatment can be performed to reduce the severity or prevent
recurrence of a S. pneumoniae infection or the clinical effects
thereof. Prophylactic treatment can be performed using a
multivalent immunogenic composition of the invention, as described
herein. The composition of the invention can be administered to the
general population or to those persons at an increased risk of
pneumococcal infection, e.g. the elderly, or those who live with or
care for the elderly.
[0176] Those "in need of treatment" include those previously
exposed to or infected with S. pneumoniae, those who were
previously vaccinated against S. pneumoniae, as well as those prone
to have an infection or any person in which a reduction in the
likelihood of infection is desired, e.g., the immunocompromised,
the elderly, children, adults, or healthy individuals.
[0177] A "stable" multivalent immunogenic composition is a
composition which has no significant changes observed at a
refrigerated temperature (e.g., 2-8.degree. C. or 4.degree. C.) for
at least 1 month, 2 months, 3 months, 6 months, 12 months and/or 24
months. Additionally, a "stable" composition includes one that
exhibits desired features at temperatures including at 25.degree.
C. and 37.degree. C. for periods including 1 month, 3 months, 6
months, 12 months, and/or 24 months. Typical acceptable criteria
for stability are as follows: no more than about 5%, about 10%,
about 15%, or about 20% variability in one or more of the
following: (a) the number average molecular weight (Mn) of the S.
pneumoniae polysaccharide protein conjugates in the composition,
(b) weight average molecular weight (Mw) of the S. pneumoniae
polysaccharide protein conjugates in the composition, (c) total
polysaccharide concentration in the composition, (d) emission
maximum of the composition measured using intrinsic protein
fluorescence spectroscopy at a particular excitation wavelength,
e.g. 280 nanometers, and (e) the fluorescence intensity of the
composition measured using intrinsic protein fluorescence
spectroscopy at a particular excitation wavelength.
[0178] The term "stable" may also be used to refer to a particular
pneumococcal conjugate within a multivalent immunogenic
composition. In such use, the term refers to a conjugate that
exhibits the desired properties over time, at a particular
temperature, and such properties vary no more that about 5%, about
10%, about 15%, or about 20% over the time and temperature
noted.
[0179] IL Multivalent Immunogenic Compositions
[0180] The invention provides multivalent immunogenic compositions
comprising multiple S. pneumoniae polysaccharide protein conjugates
wherein each of the conjugates comprises a polysaccharide from an
S. pneumoniae serotype conjugated to a carrier protein. Different
aspects and embodiments of the multivalent immunogenic compositions
of the invention are described, infra.
[0181] In one embodiment (Embodiment E1), the invention provides a
multivalent immunogenic composition comprising multiple S.
pneumoniae polysaccharide protein conjugates, each comprising
capsular polysaccharide from an S. pneumoniae serotype conjugated
to a carrier protein, wherein the serotypes of S. pneumoniae
comprise, consist, or consist essentially of: i) 1, 3, 4, 5, 6A,
6B, 7F, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F,
24F, 33F and 35B or ii) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 12F,
14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B or
iii) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15C,
18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B. In sub-embodiments
of Embodiment E1, the immunogenic composition does not comprise any
further S. pneumoniae polysaccharide protein conjugates.
[0182] As used herein, de-O-acetylated serotype 15B (DeOAcl5B)
pneumococcal polysaccharide is substantially equivalent to serotype
15C pneumococcal polysaccharide and has a substantially identical
NMR spectra (data not shown). As used herein, de-O-acetylated
serotype 15B pneumococcal polysaccharide and serotype 15C
pneumococcal polysaccharide may each have an 0-Acetyl content per
repeating unit in the range of 0-5%, or in the range of 0-4%, or in
the range of 0-3%, or in the range of 0-2%, or in the range of
0-1%, or in the range of 0-0.5%, or in the range of 0-0.1%, or no
O-acetyl content. In a report by Spencer B. L., et al.,
pneumococcal polysaccharide 15C may be slightly o-acetylated
(Spencer, B.L. et al., Clin. Vac. Immuno. (2017) 24(8): 1-13).
Thus, in any of the embodiments of the multivalent immunogenic
compositions herein, de-O-acetylated serotype 15B (DeOAcl5B) can be
used in place of serotype 15C. Processes for de-O-acetylation are
known in the art, for example as described in Rajam et al.,
Clinical and Vaccine Immunology, 2007, 14(9):1223-1227.
[0183] In certain embodiments of any of the multivalent immunogenic
compositions of the invention, including Embodiment E1 and any
sub-embodiment thereof, the composition further comprises a
pharmaceutically acceptable carrier.
Cross-Reactivity
[0184] In an embodiment the invention provides multivalent
immunogenic compositions comprising S. pneumoniae polysaccharide
protein conjugates wherein each of the conjugates comprises a
polysaccharide from a S. pneumoniae serotype, including serotype
6C, conjugated to a carrier protein, wherein serotype 6C of S.
pneumoniae provides cross-reactivity against serotypes 6A and 6B of
S. pneumoniae.
[0185] In an embodiment the invention provides multivalent
immunogenic compositions comprising S. pneumoniae polysaccharide
protein conjugates wherein each of the conjugates comprises a
polysaccharide from an S. pneumoniae serotype, including serotype
6A, conjugated to a carrier protein, wherein serotype 6A of S.
pneumoniae provides cross-protection against serotypes 6B and/or 6C
of S. pneumoniae.
[0186] In an embodiment the invention provides multivalent
immunogenic compositions comprising S. pneumoniae polysaccharide
protein conjugates wherein each of the conjugates comprises a
polysaccharide from an S. pneumoniae serotype, including serotype
6B, conjugated to a carrier protein, wherein serotype 6B of S.
pneumoniae provides cross-protection against serotypes 6A and/or 6C
of S. pneumoniae.
[0187] In an embodiment the invention provides multivalent
immunogenic compositions comprising S. pneumoniae polysaccharide
protein conjugates wherein each of the conjugates comprises a
polysaccharide from an S. pneumoniae serotype, including serotype
15C, conjugated to a carrier protein, wherein serotype 15C of S.
pneumoniae provides cross-protection against serotype 15B of S.
pneumoniae.
[0188] In an embodiment the invention provides multivalent
immunogenic compositions comprising S. pneumoniae polysaccharide
protein conjugates wherein each of the conjugates comprises a
polysaccharide from an S. pneumoniae serotype, including serotype
15B, conjugated to a carrier protein, wherein serotype 15B of S.
pneumoniae provides cross-protection against serotype 15C of S.
pneumoniae.
Carrier Protein
[0189] In particular embodiments of the present invention, CRM197
is used as the carrier protein. CRM197 is a non-toxic variant
(i.e., toxoid) of diphtheria toxin having the following sequence of
amino acids:
TABLE-US-00001 (SEQ ID NO: 1) GADDVVDSSK SFVMENFSSY HGTKPGYVDS
IQKGIQKPKS GTQGNYDDDW KEFYSTDNKY DAAGYSVDNE NPLSGKAGGV VKVTYPGLTK
VLALKVDNAE TIKKELGLSL TEPLMEQVGT EEFIKRFGDG ASRVVLSLPF AEGSSSVEYI
NNWEQAKALS VELEINFETR GKRGQDAMYE YMAQACAGNR VRRSVGSSLS CINLDWDVIR
DKIKTKIESL KEHGPIKNKM SESPNKTVSE EKAKQYLEEF HQTALEHPEL SELKTVTGTN
PVFAGANYAA WAVNVAQVID SETADNLEKT TAALSILPGI GSVMGIADGA VHHNTEEIVA
QSIALSSLMV AQAIPLVGEL VDIGFAAYNF VESIINLFQV VHNSYNRPAY SPGHKTQPFL
HDGYAVSWNT VEDSIIRTGF QGESGHDIKI TAENTPLPIA GVLLPTIPGK LDVNKSKTHI
SVNGRKIRMR CRAIDGDVTF CRPKSPVYVG NGVHANLHVA FHRSSSEKIH SNEISSDSIG
VLGYQKTVDH TKVNSKLSLF FEIKS
[0190] In one embodiment, CRM197 is isolated from cultures of
Corynebacterium diphtheria strain C7 (p197) grown in casamino acids
and yeast extract-based medium. In another embodiment, CRM197 is
prepared recombinantly in accordance with the methods described in
U.S. Pat. No. 5,614,382. Typically, CRM197 is purified through a
combination of ultra-filtration, ammonium sulfate precipitation,
and ion-exchange chromatography. In some embodiments, CRM197 is
prepared in Pseudomonas fluorescens using Pfenex Expression
Technology.TM. (Pfenex Inc., San Diego, Calif.).
[0191] Other suitable carrier proteins include additional
inactivated bacterial toxins such as DT (Diphtheria toxoid) or
fragment B of DT (DTFB), TT (tetanus toxid) or fragment C of TT,
pertussis toxoid, cholera toxoid (e.g., as described in WO
2004/083251), E. coli LT, E. coli ST, and exotoxin A from
Pseudomonas aeruginosa. Bacterial outer membrane proteins such as
outer membrane protein complex (OMPC), porins, transferrin binding
proteins, pneumococcal surface protein A (PspA; See WO 02/091998),
pneumococcal adhesin protein (PsaA), C5a peptidase from Group A or
Group B streptococcus, or Haemophilus influenzae protein D,
pneumococcal pneumolysin (Kuo et al., 1995, Infect Immun 63;
2706-13) including ply detoxified in some fashion for example
dPLY-GMBS (See WO 04/081515) or dPLY-formol, PhtX, including PhtA,
PhtB, PhtD, PhtE and fusions of Pht proteins for example PhtDE
fusions, PhtBE fusions (See WO 01/98334 and WO 03/54007), can also
be used. Other proteins, such as ovalbumin, keyhole limpet
hemocyanin (KLH), bovine serum albumin (BSA) or purified protein
derivative of tuberculin (PPD), PorB (from N meningitidis), PD
(Haemophilus influenzae protein D; see, e.g., EP 0 594 610 B), or
immunologically functional equivalents thereof, synthetic peptides
(See EP0378881 and EP0427347), heat shock proteins (See WO 93/17712
and WO 94/03208), pertussis proteins (See WO 98/58668 and
EP0471177), cytokines, lymphokines, growth factors or hormones (See
WO 91/01146), artificial proteins comprising multiple human CD4+ T
cell epitopes from various pathogen derived antigens (See Falugi et
al., 2001, Eur J Immunol 31:3816-3824) such as N19 protein (See
Baraldoi et al., 2004, Infect Immun 72:4884-7), iron uptake
proteins (See WO 01/72337), toxin A or B of C. difficile (See WO
00/61761), and flagellin (See Ben-Yedidia et al., 1998, Immunol
Lett 64:9) can also be used as carrier proteins.
[0192] Other DT mutants can be used as the carrier protein, such as
CRM176, CRM228, CRM45 (Uchida et al., 1973, J Biol Chem
218:3838-3844); CRM9, CRM45, CRM102, CRM103 and CRM107 and other
mutations described by Nicholls and Youle in Genetically Engineered
Toxins, Ed: Frankel, Maecel Dekker Inc, 1992; deletion or mutation
of Glu-148 to Asp, Gln or Ser and/or Ala 158 to Gly and other
mutations disclosed in U.S. 4,709,017 or U.S. Pat. No. 4,950,740;
mutation of at least one or more residues Lys 516, Lys 526, Phe 530
and/or Lys 534 and other mutations disclosed in U.S. Pat. Nos.
5,917,017 or 6,455,673; or fragment disclosed in U.S. Pat. No.
5,843,711. Such DT mutants can also be used to make DTFB variants
where the variants comprise the B fragment contain the epitope
regions.
[0193] In certain embodiments, the carrier protein is selected from
the group consisting of: Outer Membrane Protein Complex (OMPC),
tetanus toxoid, diphtheria toxoid, protein D and CRM197.
[0194] In some embodiments of the invention, a second carrier can
be used for one or more of the polysaccharide protein conjugates in
the multivalent immunogenic composition.
[0195] The second carrier protein is preferably a protein that is
non-toxic and non-reactogenic and obtainable in sufficient amount
and purity. The second carrier protein is also conjugated or joined
with the S. pneumoniae polysaccharide to enhance immunogenicity of
the antigen. Carrier proteins should be amenable to standard
conjugation procedures. In one embodiment, each capsular
polysaccharide not conjugated to the first carrier protein is
conjugated to the same second carrier protein (e.g., each capsular
polysaccharide molecule being conjugated to a single carrier
protein). In another embodiment, the capsular polysaccharides not
conjugated to the first carrier protein are conjugated to two or
more carrier proteins (each capsular polysaccharide molecule being
conjugated to a single carrier protein). In such embodiments, each
capsular polysaccharide of the same serotype is typically
conjugated to the same carrier protein.
[0196] In embodiments of the invention, including Embodiment E1 and
any sub-embodiment thereof, one or more (including 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 26, 27, 28, 29, 30 or more, where applicable) of the
polysaccharide serotypes is conjugated to CRM197. In further
embodiments of the invention, including Embodiment E1 and any
sub-embodiment thereof, each of the polysaccharide serotypes is
conjugated to CRM197.
[0197] Formulation of the polysaccharide-protein conjugates of the
present invention can be accomplished using art-recognized methods.
For instance, individual pneumococcal conjugates can be formulated
with a physiologically acceptable vehicle to prepare the
composition. Examples of such vehicles include, but are not limited
to, water, buffered saline, polyols (e.g., glycerol, propylene
glycol, liquid polyethylene glycol) and dextrose solutions.
[0198] In a preferred embodiment, the vaccine composition is
formulated in L-histidine buffer with sodium chloride.
[0199] In some embodiments of the invention, the multivalent
immunogenic composition comprises multiple S. pneumoniae
polysaccharide protein conjugates comprising capsular
polysaccharide from an S. pneumoniae serotype conjugated to a
carrier protein and an adjuvant, wherein the S. pneumoniae
serotypes are as described herein. Suitable adjuvants to enhance
effectiveness of the composition include, but are not limited
to:
[0200] (1) aluminum salts (alum), such as aluminum hydroxide,
aluminum phosphate, aluminum sulfate, etc.;
[0201] (2) oil-in-water emulsion formulations (with or without
other specific immunostimulating agents such as muramyl peptides
(defined below) or bacterial cell wall components), such as, for
example, (a) MF59 (International Patent Application Publication No.
WO 90/14837), containing 5% Squalene, 0.5% Tween 80, and 0.5% Span
85 (optionally containing various amounts of MTP-PE) formulated
into submicron particles using a microfluidizer such as Model 110Y
microfluidizer (Microfluidics, Newton, Mass.), (b) SAF, containing
10% Squalene, 0.4% Tween 80, 5% pluronic-blocked polymer L121, and
thr-MDP either microfluidized into a submicron emulsion or vortexed
to generate a larger particle size emulsion, (c) Ribi.TM. adjuvant
system (RAS), (Corixa, Hamilton, Mont.) containing 2% Squalene,
0.2% Tween 80, and one or more bacterial cell wall components from
the group consisting of 3-O-deacylated monophosphorylipid A
(MPL.TM.) described in U.S. Pat. No. 4,912,094, trehalose
dimycolate (TDM), and cell wall skeleton (CWS), preferably MPL+CWS
(Detox.TM.); and (d) a Montanide ISA;
[0202] (3) saponin adjuvants, such as Quil A or STIMULON.TM. QS-21
(Antigenics, Framingham, Mass.) (see, e.g., U.S. Pat. No.
5,057,540) may be used or particles generated therefrom such as
ISCOM (immunostimulating complexes formed by the combination of
cholesterol, saponin, phospholipid, and amphipathic proteins) and
Iscomatrix.RTM. (having essentially the same structure as an ISCOM
but without the protein);
[0203] (4) bacterial lipopolysaccharides, synthetic lipid A analogs
such as aminoalkyl glucosamine phosphate compounds (AGP), or
derivatives or analogs thereof, which are available from Corixa,
and which are described in U.S. Pat. No. 6,113,918; one such AGP is
2-[(R)-3-tetradecanoyloxytetradecanoylamino]ethyl
2-Deoxy-4-O-phosphono-3-O-[(R)-3-tetradecanoyloxytetradecanoyl]-2-[(R)-3--
tetradecanoyloxytetradecanoylamino]-.beta.-D-glucopyranoside, which
is also known as 529 (formerly known as RC529), which is formulated
as an aqueous form or as a stable emulsion
[0204] (5) synthetic polynucleotides such as oligonucleotides
containing CpG motif(s) (U.S. Pat. No. 6,207,646);
[0205] (6) cytokines, such as interleukins (e.g., IL-1, IL-2, IL-4,
IL-5, IL-6, IL-7, IL-12, IL-15, IL-18, etc.), interferons (e.g.,
gamma interferon), granulocyte macrophage colony stimulating factor
(GM-CSF), macrophage colony stimulating factor (M-CSF), tumor
necrosis factor (TNF), costimulatory molecules B7-1 and B7-2, etc;
and
[0206] (7) complement, such as a trimer of complement component
C3d.
[0207] In another embodiment, the adjuvant is a mixture of 2, 3, or
more of the above adjuvants, e.g., SBAS2 (an oil-in-water emulsion
also containing 3-deacylated monophosphoryl lipid A and QS21).
[0208] Muramyl peptides include, but are not limited to,
N-acetylmuramyl-L-threonyl-D-isoglutamine (thr-MDP),
N-acetyl-normuramyl-L-alanine-2-(1', 2'
dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (MTP-PE),
etc.
[0209] In certain embodiments, the adjuvant is an aluminum salt.
The aluminum salt adjuvant may be an alum-precipitated vaccine or
an alum-adsorbed vaccine. Aluminum-salt adjuvants are well known in
the art and are described, for example, in Harlow, E. and D. Lane
(1988; Antibodies: A Laboratory Manual Cold Spring Harbor
Laboratory) and Nicklas, W. (1992; Aluminum salts. Research in
Immunology 143:489-493). The aluminum salt includes, but is not
limited to, hydrated alumina, alumina hydrate, alumina trihydrate
(ATH), aluminum hydrate, aluminum trihydrate, alhydrogel, Superfos,
Amphogel, aluminum (III) hydroxide, aluminum hydroxyphosphate
sulfate, Aluminum Phosphate Adjuvant (APA), amorphous alumina,
trihydrated alumina, or trihydroxyaluminum.
[0210] APA is an aqueous suspension of aluminum hydroxyphosphate.
APA is manufactured by blending aluminum chloride and sodium
phosphate in a 1:1 volumetric ratio to precipitate aluminum
hydroxyphosphate. After the blending process, the material is
size-reduced with a high-shear mixer to achieve a monodisperse
particle size distribution. The product is then diafiltered against
physiological saline and steam sterilized. In one embodiment, the
dose of the aluminum salt is 10, 15, 20, 25, 30, 50, 70, 100, 125,
150, 200, 300, 500, or 700 .mu.g, or 1, 1.2, 1.5, 2, 3, 5 mg or
more. In yet another embodiment, the dose of alum salt described
above is per .mu.g of recombinant protein.
[0211] In certain embodiments, a commercially available
Al(OH).sub.3 (e.g. Alhydrogel or Superfos of Denmark/Accurate
Chemical and Scientific Co., Westbury, NY) is used to adsorb
proteins in a ratio of 50-200 .mu.g protein/mg aluminum hydroxide.
Adsorption of protein is dependent, in another embodiment, on the
pI (Isoelectric pH) of the protein and the pH of the medium. A
protein with a lower pI adsorbs to the positively charged aluminum
ion more strongly than a protein with a higher pI. Aluminum salts
may establish a depot of antigen that is released slowly over a
period of 2-3 weeks, be involved in nonspecific activation of
macrophages and complement activation, and/or stimulate innate
immune mechanism (possibly through stimulation of uric acid). See,
e.g., Lambrecht et al., 2009, Curr Opin Immunol 21:23.
[0212] Monovalent bulk aqueous conjugates are typically blended
together and diluted to target 4 .mu.g/mL for all serotypes except
6B, which may be diluted to target 8 .mu.g/mL. Once diluted, the
batch will be filter sterilized, and an equal volume of aluminum
phosphate adjuvant added aseptically to target a final aluminum
concentration of 250 .mu.g/mL. The adjuvanted, formulated batch
will be filled into single-use, 0.5 mL/dose vials.
[0213] In certain embodiments, the adjuvant is a CpG-containing
nucleotide sequence, for example, a CpG-containing oligonucleotide,
in particular, a CpG-containing oligodeoxynucleotide (CpG ODN). In
another embodiment, the adjuvant is ODN 1826, which may be acquired
from Coley Pharmaceutical Group.
[0214] Methods for use of CpG oligonucleotides are well known in
the art and are described, for example, in Sur et al., 1999, J
Immunol. 162:6284-93; Verthelyi, 2006, Methods Mol Med. 127:139-58;
and Yasuda et al., 2006, Crit Rev Ther Drug Carrier Syst.
23:89-110.
[0215] In alternative embodiments, the immunogenic composition
comprises multiple S. pneumoniae polysaccharide protein conjugates
as described herein, for example in Embodiment E1 or any
sub-embodiment thereof, and does not comprise an adjuvant.
Formulations
[0216] The multivalent immunogenic compositions of the invention
can be formulated as single dose vials, multi-dose vials or as
pre-filled glass or plastic syringes.
[0217] In another embodiment, the multivalent immunogenic
compositions of the present invention are administered orally, and
are thus formulated in a form suitable for oral administration,
i.e., as a solid or a liquid preparation. Solid oral formulations
include tablets, capsules, pills, granules, pellets and the like.
Liquid oral formulations include solutions, suspensions,
dispersions, emulsions, oils and the like.
[0218] Pharmaceutically acceptable carriers for liquid formulations
are aqueous or non-aqueous solutions, suspensions, emulsions or
oils. Examples of nonaqueous solvents are propylene glycol,
polyethylene glycol, and injectable organic esters such as ethyl
oleate.
[0219] Aqueous carriers include water, alcoholic/aqueous solutions,
emulsions or suspensions, including saline and buffered media.
Examples of oils are those of animal, vegetable, or synthetic
origin, for example, peanut oil, soybean oil, olive oil, sunflower
oil, fish-liver oil, another marine oil, or a lipid from milk or
eggs.
[0220] The multivalent immunogenic compositions of the instant
invention may be isotonic, hypotonic or hypertonic. However, it is
often preferred that a composition for infusion or injection be
essentially isotonic, when administrated. Hence, for storage, a
composition may preferably be isotonic or hypertonic. If the
composition is hypertonic for storage, it may be diluted to become
an isotonic solution prior to administration.
[0221] The isotonic agent may be an ionic isotonic agent such as a
salt or a non-ionic isotonic agent such as a carbohydrate. Examples
of ionic isotonic agents include but are not limited to NaCl,
CaCl.sub.2, KCl and MgCl.sub.2. Examples of non-ionic isotonic
agents include but are not limited to mannitol, sorbitol and
glycerol.
[0222] It is also preferred that at least one pharmaceutically
acceptable additive is a buffer. For some purposes, for example,
when the pharmaceutical composition is meant for infusion or
injection, it is often desirable that the composition comprises a
buffer, which is capable of buffering a solution to a pH in the
range of 4 to 10, such as 5 to 9, for example 6 to 8.
[0223] The buffer may, for example, be selected from the group
consisting of TRIS, acetate, glutamate, lactate, maleate, tartrate,
phosphate, citrate, carbonate, glycinate, histidine, glycine,
succinate and triethanolamine buffer.
[0224] The buffer may be selected from USP compatible buffers for
parenteral use, in particular, when the pharmaceutical formulation
is for parenteral use. For example the buffer may be selected from
the group consisting of monobasic acids such as acetic, benzoic,
gluconic, glyceric and lactic; dibasic acids such as aconitic,
adipic, ascorbic, carbonic, glutamic, malic, succinic and tartaric,
polybasic acids such as citric and phosphoric; and bases such as
ammonia, diethanolamine, glycine, triethanolamine, and TRIS.
[0225] Parenteral vehicles (for subcutaneous, intravenous,
intraarterial, or intramuscular injection) include sodium chloride
solution, Ringer's dextrose, dextrose and sodium chloride, lactated
Ringer's and fixed oils. Intravenous vehicles include fluid and
nutrient replenishers, electrolyte replenishers such as those based
on Ringer's dextrose, and the like. Examples are sterile liquids
such as water and oils, with or without the addition of a
surfactant and other pharmaceutically acceptable adjuvants. In
general, water, saline, aqueous dextrose and related sugar
solutions, glycols such as propylene glycols or polyethylene
glycol, Polysorbate 80 (PS-80), Polysorbate 20 (PS-20), and
Poloxamer 188 (P188) are preferred liquid carriers, particularly
for injectable solutions. Examples of oils are those of animal,
vegetable, or synthetic origin, for example, peanut oil, soybean
oil, olive oil, sunflower oil, fish-liver oil, another marine oil,
or a lipid from milk or eggs.
[0226] The formulations of the invention may also contain a
surfactant. Preferred surfactants include, but are not limited to:
Poloxamer-188 (P188; Pluoronic; F68 NF), the polyoxyethylene
sorbitan esters surfactants (commonly referred to as the Tweens),
especially PS-20 and PS-80; copolymers of ethylene oxide (EO),
propylene oxide (PO), and/or butylene oxide (BO), sold under the
DOWFAX.TM. tradename, such as linear EO/PO block copolymers;
octoxynols, which can vary in the number of repeating ethoxy
(oxy-1,2-ethanediyl) groups, with octoxynol-9 (Triton X-100, or
t-octylphenoxypolyethoxyethanol) being of particular interest;
(octylphenoxy)polyethoxyethanol (IGEPAL CA-630/NP-40);
phospholipids such as phosphatidylcholine (lecithin); nonylphenol
ethoxylates, such as the Tergitol.TM. NP series; polyoxyethylene
fatty ethers derived from lauryl, cetyl, stearyl and oleyl alcohols
(known as Brij surfactants), such as triethyleneglycol monolauryl
ether (Brij 30); and sorbitan esters (commonly known as the SPANs),
such as sorbitan trioleate (Span 85) and sorbitan monolaurate. A
preferred surfactant for including in the emulsion is PS-80.
[0227] Mixtures of surfactants can be used, e.g. PS-80/Span 85
mixtures. A combination of a polyoxyethylene sorbitan ester such as
polyoxyethylene sorbitan monooleate (PS-80) and an octoxynol such
as t-octylphenoxypolyethoxyethanol (Triton X-100) is also suitable.
Another useful combination comprises laureth 9 plus a
polyoxyethylene sorbitan ester and/or an octoxynol.
[0228] Preferred amounts of surfactants (% by weight) are:
polyoxyethylene sorbitan esters (such as PS-80) of from 0.01 to 1%,
in particular about 0.1%; octyl- or nonylphenoxy polyoxyethanols
(such as Triton X-100, or other detergents in the Triton series) of
from 0.001 to 0.1%, in particular 0.005 to 0.02%; polyoxyethylene
ethers (such as laureth 9) of from 0.1 to 20%, preferably 0.1 to
10% and in particular 0.1 to 1% or about 0.5%.
[0229] In certain embodiments, the composition consists essentially
of histidine (20 mM), saline (150 mM) and 0.2% PS-20 at a pH of 5.8
with 250 .mu.g/mL of APA (Aluminum Phosphate Adjuvant). PS-20 can
range from 0.005% to 0.3% (w/v). In another embodiment, PS-20 can
range from 0.025% to 0.8% (w/v). In another embodiment, PS-20 can
range from 0.05% to 0.8% (w/v). In another embodiment, PS-20 can
range from 0.05% to 0.2% (w/v). The process consists of combining a
blend of up to 24 serotypes in histidine, saline, and PS-20, then
combining this blended material with APA and saline with or without
antimicrobial preservatives.
[0230] In particular embodiments, the multivalent immunogenic
composition comprises S. pneumoniae polysaccharide protein
conjugates wherein each of the conjugates comprises a
polysaccharide from an S. pneumoniae serotype conjugated to a
carrier protein, wherein the serotypes of S. pneumoniae in the
polysaccharide protein conjugates comprise any of the sets of
serotypes set forth herein, and further comprises 20-80 mM
histidine pH 5.8 and 150 mM NaCl.
[0231] In some embodiments, the multivalent immunogenic composition
further comprises from 0.2% to 0.8% w/v polysorbate 20.
[0232] The multivalent immunogenic composition PCV24 is prepared by
individually conjugating the CRM197 protein to S. pneumoniae
polysaccharide (PnPs) serotypes -1, -3, -4, -5, -6A, -6B, -7F, -8,
-9V, -10A, -11A, -12F, -14, -15A, -15C, -18C, -19A, -19F, -22F,
-23B, -23F, -24F, -33F, and -35B using reductive amination in an
aprotic solvent (also referred to as DMSO chemistry) and formulated
in 20 mM L-Histidine pH 5.8, 150 mM NaCl and 0.1% w/v
Polysorbate-20 (PS-20) at 4 .mu.g/mL or 8 .mu.g/mL of each
polysacchardide serotype for a total polysaccharide concentration
of 96 .mu.g/mL or 192 .mu.g/mL, respectively, and referred to as
"PCV24 unadj". In another specific embodiment, the multivalent
immunogenic composition PCV24 is prepared in 20 mM L-Histidine pH
5.8, 150 mM NaCl and 0.2% w/v Polysorbate-20 (PS-20) at 4 .mu.g/mL
of each polysaccharide serotype for a total polysaccharide
concentration of 96 .mu.g/mL further comprising 250 ng [Al]/mL in
the form of Aluminum Phosphate Adjuvant. This is referred to as
"PCV24/APA".
[0233] The choice of surfactant may need to be optimized for
different drug products and drug substances. For multivalent
vaccines having 15 or more serotypes, PS-20 and P188 are preferred.
The choice of chemistry used to make conjugates can also play an
important role in the stabilization of the formulation. In
particular, when the conjugation reactions used to prepare
different polysaccharide protein conjugates in a multivalent
composition include both aqueous solvent and DMSO solvent,
particular surfactant systems provide significant differences in
stability. Improved stability of polysacharide protein conjugates
was seen with polysorbate 20 alone or with poloxamer 188 in
combination with a polyol.
[0234] The exact mechanism of how a specific detergent protects a
biotherapeutic is poorly understood and cannot be predicted a
priori. Possible stabilization mechanisms include preferential
hydration, preferential exclusion, air/liquid interface competition
between biotherapeutic and surface, surface tension, and/or direct
association of the detergent with the biotherapeutic to mask
hydrophobic patches which serve as seeds for aggregation.
[0235] Poloxamer may also be used in the compositions of the
invention. A poloxamer is a nonionic triblock copolymer composed of
a central hydrophobic chain of polyoxypropylene (poly(propylene
oxide)) flanked by two hydrophilic chains of polyoxyethylene
(poly(ethylene oxide)). Poloxamers are also known by the tradename
Pluronic.RTM.. Because the lengths of the polymer blocks can be
customized, many different poloxamers exist that have slightly
different properties. For the generic term "poloxamer", these
copolymers are commonly named with the letter "P" (for poloxamer)
followed by three digits, the first two digits x 100 give the
approximate molecular mass of the polyoxypropylene core, and the
last digit x 10 gives the percentage polyoxyethylene content (e.g.,
P407=Poloxamer with a polyoxypropylene molecular mass of 4,000
g/mol and a 70% polyoxyethylene content). For the Pluronic.RTM.
tradename, coding of these copolymers starts with a letter to
define its physical form at room temperature (L=liquid, P=paste,
F=flake (solid)) followed by two or three digits. The first digit
(two digits in a three-digit number) in the numerical designation,
multiplied by 300, indicates the approximate molecular weight of
the hydrophobe; and the last digit x 10 gives the percentage
polyoxyethylene content (e.g., L61=Pluronic.RTM. with a
polyoxypropylene molecular mass of 1,800 g/mol and a 10%
polyoxyethylene content). See U.S. Pat. No. 3,740,421.
[0236] Examples of poloxamers have the general formula:
HO(C.sub.2H.sub.4O).sub.a(C.sub.3H.sub.6O).sub.b(C.sub.2H.sub.4O).sub.aH,
wherein a and b blocks have the following values:
TABLE-US-00002 Pluronic .RTM. Poloxamer A B Molecular Weight L31 2
16 1100 (average) L35 1900 (average) L44NF 124 12 20 2090 to 2360
L64 2900 (average) L81 2800 (average) L121 4400 (average) P123 20
70 5750 (average) F68NF 188 80 27 7680 to 9510 F87NF 237 64 37 6840
to 8830 F108NF 338 141 44 12700 to 17400 F127NF 407 101 56 9840 to
14600
[0237] Molecular weight units, as used herein, are in Dalton (Da)
or g/mol.
[0238] Preferably, the poloxamer generally has a molecular weight
in the range from 1,100 to 17,400 Da, from 7,500 to 15,000 Da, or
from 7,500 to 10,000 Da. The poloxamer can be selected from
poloxamer 188 or poloxamer 407. The final concentration of the
poloxamer in the formulations is from 0.001% to 5% weight/volume,
or 0.025% to 1% weight/volume. In certain aspects, the polyol is
propylene glycol and is at final concentration from 1% to 20%
weight/volume. In certain aspects, the polyol is polyethylene
glycol 400 and is at final concentration from 1% to 20%
weight/volume.
[0239] Suitable polyols for the formulations of the invention are
polymeric polyols, particularly polyether diols including, but are
not limited to, propylene glycol and polyethylene glycol,
Polyethylene glycol monomethyl ethers. Propylene glycol is
available in a range of molecular weights of the monomer from
.about.425 to .about.2,700. Polyethylene glycol and Polyethylene
glycol monomethyl ether is also available in a range of molecular
weights ranging from --200 to 35,000 including but not limited to
PEG200, PEG300, PEG400, PEG1000, PEG MME 550, PEG MME 600, PEG MME
2000, PEG MME 3350 and PEG MME 4000. A preferred polyethylene
glycol is polyethylene glycol 400. The final concentration of the
polyol in the formulations of the invention may be 1% to 20%
weight/volume or 6% to 20% weight/volume.
[0240] The formulation also contains a pH-buffered saline solution.
The buffer may, for example, be selected from the group consisting
of TRIS, acetate, glutamate, lactate, maleate, tartrate, phosphate,
citrate, carbonate, glycinate, histidine, glycine, succinate, HEPES
(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), MOPS
(3-(N-morpholino)propanesulfonic acid), MES
(2-(N-morpholino)ethanesulfonic acid) and triethanolamine buffer.
The buffer is capable of buffering a solution to a pH in the range
of 4 to 10, 5.2 to 7.5, or 5.8 to 7.0. In certain aspect of the
invention, the buffer is selected from the group consisting of
phosphate, succinate, histidine, MES, MOPS, HEPES, acetate or
citrate. The buffer may furthermore, for example, be selected from
USP compatible buffers for parenteral use, in particular, when the
pharmaceutical formulation is for parenteral use. In one
embodiment, the concentration of buffer will range from 1 mM to 100
mM. In another embodiment, the concentration of buffer will range
from 10 mM to 80 mM. In another embodiment, the concentration of
buffer will range from 1 mM to 50 mM, or 5 mM to 50 mM. In certain
aspects, the buffer is histidine at a final concentration of 5 mM
to 50 mM, or succinate at a final concentration of 1 mM to 10 mM.
In certain aspects, the histidine buffer is at a final
concentration of 20 mM.+-.2 mM.
[0241] While the saline solution (e.g., a solution containing NaCl)
is preferred, other salts suitable for formulation include but are
not limited to, CaCl.sub.2, KCl and MgCl.sub.2 and combinations
thereof. Non-ionic isotonic agents including but not limited to
sucrose, trehalose, mannitol, sorbitol and glycerol may be used in
lieu of a salt. Suitable salt ranges include, but are not limited
to 20 mM to 500 mM or 40 mM to 170 mM. In one aspect, the saline is
NaCl, optionally present at a concentration from 25 mM to 170
mM.
[0242] In a preferred embodiment, the formulations comprise a
L-histidine buffer with sodium chloride.
[0243] In another embodiment, the pharmaceutical composition is
delivered in a controlled release system. For example, the agent
can be administered using intravenous infusion, a transdermal
patch, liposomes, or other modes of administration. In another
embodiment, polymeric materials are used; e.g. in microspheres in
or an implant.
[0244] The amount of conjugate in each dose of the composition is
selected as an amount that induces an immunoprotective response
without significant, adverse effects. Such amount can vary
depending upon the pneumococcal serotype. Generally, for
polysaccharide-based conjugates, each dose will comprise 0.08 to
100 .mu.g of each polysaccharide. In some embodiments of the
invention, the dose of each polysaccharide conjugate is from 0.08
to 10 .mu.g. In further embodiments, the dose of each conjugate is
from 1 to 5 .mu.g, from 0.4 to 4 .mu.g, from 0.4 to 3 .mu.g, from
0.4 to 2 .mu.g, or from 0.4 to 1 .mu.g. In some embodiments, the
dose of one or more polysaccharide conjugates is 100, 150, 200,
250, 300, 400, 500, or 750 ng or 0.4, 0.5, 0.6, 0.7, 0.75, 0.8,
0.9, 1, 1.5, 2, 3, 4, 5, 6, 7, 7.5, 8, 9, 10, 11, 12, 13, 14, 15,
16, 18, 20, 22, 25, 30, 40, 50, 60, 70, 80, 90, or 100 .mu.g.
[0245] In some embodiments of the compositions of the invention,
all of the polysaccharide conjugates are present in the composition
in the same amount. In further embodiments, the polysaccharide
conjugates are present in the composition in different amounts
(i.e., at least one polysaccharide conjugate is present in an
amount that is different than one or more of the other
polysaccharide conjugates of the composition).
[0246] Optimal amounts of components for a particular immunogenic
composition can be ascertained by standard studies involving
observation of appropriate immune responses in subjects. For
example, in another embodiment, the dosage for human vaccination is
determined by extrapolation from animal studies to human data. In
another embodiment, the dosage is determined empirically.
[0247] The compositions of this invention may also include one or
more proteins from S. pneumoniae. Examples of S. pneumoniae
proteins suitable for inclusion include those identified in
International Patent Application Publication Nos. WO 02/083855 and
WO 02/053761.
[0248] In certain embodiments, the compositions of the invention
are administered to a subject by one or more methods known to a
person skilled in the art, such as parenterally, transmucosally,
transdermally, intramuscularly, intravenously, intra-dermally,
intra-nasally, subcutaneously, intra-peritonealy, and formulated
accordingly. In one embodiment, compositions of the present
invention are administered via epidermal injection, intramuscular
injection, intravenous, intra-arterial, subcutaneous injection, or
intra-respiratory mucosal injection of a liquid preparation. Liquid
formulations for injection include solutions and the like.
[0249] III. Methods of Making
[0250] Capsular polysaccharides from Streptococcus pneumoniae can
be prepared by standard techniques known to those skilled in the
art. For example, polysaccharides can be isolated from bacteria and
may be sized to some degree by known methods (see, e.g., European
Patent Nos. EP497524 and EP497525); and preferably by
microfluidisation accomplished using a homogenizer or by chemical
hydrolysis. In one embodiment, each pneumococcal polysaccharide
serotype is grown in a soy-based medium. The individual
polysaccharides are then purified through standard steps including
centrifugation, precipitation, and ultra-filtration. See, e.g.,
U.S. Patent Application Publication No. 2008/0286838 and U.S. Pat.
No. 5,847,112. Polysaccharides can be sized in order to reduce
viscosity in polysaccharide samples and/or to improve filterability
for conjugated products using techniques such as mechanical or
chemical sizing. Chemical hydrolysis may be conducted using acetic
acid. Mechanical sizing may be conducted using High Pressure
Homogenization Shearing. The purified polysaccharides can be
chemically activated to make the saccharides capable of reacting
with the carrier protein. The purified polysaccharides can be
connected to a linker. Once activated or connected to a linker,
each capsular polysaccharide is separately conjugated to a carrier
protein to form a glycoconjugate. The polysaccharide conjugates may
be prepared by known coupling techniques. The polysaccharide can be
coupled to a linker to form a polysaccharide-linker intermediate in
which the free terminus of the linker is an ester group. The linker
is therefore one in which at least one terminus is an ester group.
The other terminus is selected so that it can react with the
polysaccharide to form the polysaccharide-linker intermediate.
[0251] The polysaccharide can be coupled to a linker using a
primary amine group in the polysaccharide. In this case, the linker
typically has an ester group at both termini. This allows the
coupling to take place by reacting one of the ester groups with the
primary amine group in the polysaccharide by nucleophilic acyl
substitution. The reaction results in a polysaccharide-linker
intermediate in which the polysaccharide is coupled to the linker
via an amide linkage. The linker is therefore a bifunctional linker
that provides a first ester group for reacting with the primary
amine group in the polysaccharide and a second ester group for
reacting with the primary amine group in the carrier molecule. A
typical linker is adipic acid N-hydroxysuccinimide diester
(SIDEA).
[0252] The coupling can also take place indirectly, i.e. with an
additional linker that is used to derivatise the polysaccharide
prior to coupling to the linker. The polysaccharide is coupled to
the additional linker using a carbonyl group at the reducing
terminus of the polysaccharide. This coupling comprises two steps:
(a1) reacting the carbonyl group with the additional linker; and
(a2) reacting the free terminus of the additional linker with the
linker. In these embodiments, the additional linker typically has a
primary amine group at both termini, thereby allowing step (a1) to
take place by reacting one of the primary amine groups with the
carbonyl group in the polysaccharide by reductive amination. A
primary amine group is used that is reactive with the carbonyl
group in the polysaccharide. Hydrazide or hydroxylamino groups are
suitable. The same primary amine group is typically present at both
termini of the additional linker. The reaction results in a
polysaccharide-additional linker intermediate in which the
polysaccharide is coupled to the additional linker via a C--N
linkage.
[0253] The polysaccharide can be coupled to the additional linker
using a different group in the polysaccharide, particularly a
carboxyl group. This coupling comprises two steps: (a1) reacting
the group with the additional linker; and (a2) reacting the free
terminus of the additional linker with the linker. In this case,
the additional linker typically has a primary amine group at both
termini, thereby allowing step (a1) to take place by reacting one
of the primary amine groups with the carboxyl group in the
polysaccharide by EDAC activation. A primary amine group is used
that is reactive with the EDAC-activated carboxyl group in the
polysaccharide. A hydrazide group is suitable. The same primary
amine group is typically present at both termini of the additional
linker. The reaction results in a polysaccharide-additional linker
intermediate in which the polysaccharide is coupled to the
additional linker via an amide linkage.
[0254] In one embodiment, the chemical activation of the
polysaccharides and subsequent conjugation to the carrier protein
by reductive amination can be achieved by means described in U.S.
Pat. Nos. 4,365,170, 4,673,574 and 4,902,506, U.S. Patent
Application Publication Nos. 2006/0228380, 2007/184072,
2007/0231340 and 2007/0184071, and WO2006/110381, WO2008/079653,
and WO2008/143709. The chemistry may include the activation of
pneumococcal polysaccharide by reaction with any oxidizing agent
which oxidizes a terminal hydroxyl group to an aldehyde, such as
periodate (including sodium periodate, potassium periodate, or
periodic acid). The reaction leads to a random oxidative cleavage
of vicinal hydroxyl groups of the carbohydrates with the formation
of reactive aldehyde groups.
[0255] Coupling to the carrier protein is by reductive amination
via direct amination to the lysyl groups of the protein. For
example, conjugation can be carried out by reacting a mixture of
the activated polysaccharide and carrier protein with a reducing
agent such as sodium cyanoborohydride. The conjugation reaction may
take place under aqueous solution or in the presence of DMSO. See,
e.g., US2015/0231270, US2011/0195086 and EP 0471 177 B1. Unreacted
aldehydes are then capped with the addition of a strong reducing
agent, such as sodium borohydride.
[0256] Reductive amination involves two steps, (1) oxidation of the
polysaccharide to form reactive aldehydes, (2) reduction of the
imine (Schiff base) formed between activated polysaccharide and a
carrier protein to form a stable amine conjugate bond. Before
oxidation, the polysaccharide is optionally size reduced.
Mechanical methods (e.g. homogenization) or chemical hydrolysis may
be employed. Chemical hydrolysis may be conducted using acetic
acid. The oxidation step may involve reaction with periodate. For
the purpose of the present invention, the term "periodate" includes
both periodate and periodic acid; the term also includes both
metaperiodate (IO.sub.4.sup.-) and orthoperiodate (IO.sub.6.sup.-)
and includes the various salts of periodate (e.g. , sodium
periodate and potassium periodate). In an embodiment the capsular
polysaccharide is oxidized in the presence of metaperiodate,
preferably in the presence of sodium periodate (NaIO.sub.4). In
another embodiment the capsular polysaccharide is oxidized in the
presence of orthoperiodate, preferably in the presence of periodic
acid. In one embodiment, the oxidizing agent is a stable nitroxyl
or nitroxide radical compound, such as piperidine-N-oxy or
pyrrolidine-N-oxy compounds, in the presence of an oxidant to
selectively oxidize primary hydroxyls (as described in WO
2014/097099). In said reaction, the actual oxidant is the
N-oxoammonium salt, in a catalytic cycle. In an aspect, said stable
nitroxyl or nitroxide radical compound are piperidine-N-oxy or
pyrrolidine-N-oxy compounds. In an aspect, said stable nitroxyl or
nitroxide radical compound bears a TEMPO
(2,2,6,6-tetramethyl-1-piperidinyloxy) or a PROXYL
(2,2,5,5-tetramethyl-1-pyrrolidinyloxy) moiety. In an aspect, said
stable nitroxyl radical compound is TEMPO or a derivative thereof.
In an aspect, said oxidant is a molecule bearing a N-halo moiety.
In an aspect, said oxidant is selected from the group consisting of
N-Chlorosuccinimide, N-Bromosuccinimide, N-lodosuccinimide,
Dichloroisocyanuric acid,
1,3,5-trichloro-1,3,5-triazinane-2,4,6-trione,
[0257] Dibromoisocyanuric acid,
1,3,5-tribromo-1,3,5-triazinane-2,4,6-trione, Diiodoisocyanuric
acid and 1,3,5-triiodo-1,3,5-triazinane-2,4,6-trione. Preferably
said oxidant is N- Chlorosuccinimide.
[0258] In certain aspects, the oxidizing agent is
2,2,6,6-Tetramethyl-1-piperidinyloxy (TEMPO) free radical and
N-Chlorosuccinimide (NCS) as the cooxidant (as described in WO
2014/097099). Therefore in one aspect, the glycoconjugates from S.
pneumoniae are obtainable by a method comprising the steps of: a)
reacting a saccharide with 2,2,6,6-tetramethyl-1-piperidinyloxy
(TEMPO) and N-chlorosuccinimide (NCS) in an aqueous solvent to
produce an activated saccharide; and b) reacting the activated
saccharide with a carrier protein comprising one or more amine
groups (said method is designated "TEMPO/NCS-reductive amination"
thereafter).
[0259] Optionally the oxidation reaction is quenched by addition of
a quenching agent. The quenching agent maybe selected from vicinal
diols, 1,2-aminoalcohols, amino acids, glutathione, sulfite,
bisulfate, dithionite, metabisulfite, thiosulfate, phosphites,
hypophosphites or phosphorous acid (such as glycerol, ethylene
glycol, propan-1,2-diol, butan-1 ,2-diol or butan-2,3-diol,
ascorbic acid).
[0260] In certain embodiments, the instant invention provides a
method for preparing a serotype 8 Streptococcus pneumoniae
polysaccharide-protein conjugate utilizing a conjugation reaction
in an aprotic solvent, wherein the conjugation reaction does not
use cyanoborohydride. In further embodiments, the conjugation
reaction is a Schiff base reduction or reductive amination. In
further embodiments, the protein is tetanus toxoid, diphtheria
toxoid, or CRM197.
[0261] In still further embodiments the protein is CRM197. In
further embodiments, the conjugation reaction is reductive
amination. In further embodiments, the reductive amination is
performed in dimethylsulfoxide (DMSO).
[0262] In some embodiments, the oxidized polysaccharides before
conjugation have a molecular weight of between 30 kDa and 1,000
kDa. Molecular weight can be calculated by size exclusion
chromatography (SEC) combined with multiangle light scattering
detector (MALS) and refractive index detector (RI). In some
embodiments, the polysaccharide has a molecular weight of between
50 kDa and 300 kDa. In some embodiments, the polysaccharide has a
molecular weight of between 50 kDa and 1,000 kDa. In additional
embodiments, the polysaccharide has a molecular weight of between
70 kDa and 900 kDa. In other embodiments, the polysaccharide has a
molecular weight of between 100 kDa and 800 kDa. In other
embodiments, the polysaccharide has a molecular weight of between
200 kDa and 600 kDa. In further embodiments, the polysaccharide has
a molecular weight of 100 kDa to 1,000 kDa; 100 kDa to 900 kDa; 100
kDa to 800 kDa; 100 kDa to 700 kDa; 100 kDa to 600 kDa; 100 kDa to
500 kDa; 100 kDa to 400 kDa; 100 kDa to 300 kDa; 150 kDa to 1,000
kDa; 150 kDa to 900 kDa; 150 kDa to 800 kDa; 150 kDa to 700 kDa;
150 kDa to 600 kDa; 150 kDa to 500 kDa; 150 kDa to 400 kDa; 150 kDa
to 300 kDa; 200 kDa to 1,000 kDa; 200 kDa to 900 kDa; 200 kDa to
800 kDa; 200 kDa to 700 kDa; 200 kDa to 600 kDa; 200 kDa to 500
kDa; 200 kDa to 400 kDa; 200 kDa to 300; 250 kDa to 1,000 kDa; 250
kDa to 900 kDa; 250 kDa to 800 kDa; 250 kDa to 700 kDa; 250 kDa to
600 kDa; 250 kDa to 500 kDa; 250 kDa to 400 kDa; 250 kDa to 350
kDa; 300 kDa to 1 ,000 kDa; 300 kDa to 900 kDa; 300 kDa to 800 kDa;
300 kDa to 700 kDa; 300 kDa to 600 kDa; 300 kDa to 500 kDa; 300 kDa
to 400 kDa; 400 kDa to 1,000 kDa; 400 kDa to 900 kDa; 400 kDa to
800 kDa; 400 kDa to 700 kDa; 400 kDa to 600 kDa; or 500 kDa to 600
kDa.
[0263] The second step of the conjugation process is the reduction
of the imine (Schiff base) bond between activated polysaccharide
and a carrier protein to form a stable conjugate bond (so-called
reductive amination), using a reducing agent. Reducing agents which
are suitable include the cyanoborohydrides (such as sodium
cyanoborohydride or sodium borohydride). In one embodiment the
reducing agent is sodium cyanoborohydride.
[0264] In certain embodiments, the reductive amination reaction is
carried out in aprotic solvent (or a mixture of aprotic solvents).
In one embodiment, the reduction reaction is carried out in DMSO or
in DMF (dimethylformamide) solvent. The DMSO or DMF solvent may be
used to reconstitute the activated polysaccharide and carrier
protein, if lyophilized. In one embodiment, the aprotic solvent is
DMSO.
[0265] At the end of the reduction reaction, there may be unreacted
aldehyde groups remaining in the conjugates, which may be capped
using a suitable capping agent. In one embodiment this capping
agent is sodium borohydride (NaBH4). Suitable alternatives include
sodium triacetoxyborohydride or sodium or zinc borohydride in the
presence of Bronsted or Lewis acids), amine boranes such as
pyridine borane, 2-Picoline Borane, 2,6-diborane-methanol,
dimethylamine-borane, t-BuMe'PrN-BH3, benzylamine-BH3 or
5-ethyl-2-methylpyridine borane (PEMB) or borohydride exchange
resin. Following the conjugation (the reduction reaction and
optionally the capping), the glycoconjugates may be purified
(enriched with respect to the amount of polysaccharide-protein
conjugate) by a variety of techniques known to the skilled person.
These techniques include dialysis, concentration/diafiltration
operations, tangential flow filtration,
precipitation/elution,column chromatography (ion exchange
chromatography, multimodal ion exchange chromatography, DEAE, or
hydrophobic interaction chromatography), and depth filtration. In
an embodiment, the glycoconjugates are purified by diafilitration
or ion exchange chromatography or size exclusion
chromatography.
[0266] Glycoconjugates prepared using reductive amination in an
aprotic solvent are generally used in multivalent pneumococcal
conjugate vaccines. Thus, in certain embodiments for multivalent
compositions where not all the serotypes are prepared in an aprotic
solvent, the reduction reaction for the remaining seroytpes is
carried out in aqueous solvent (e.g., selected from PBS (phosphate
buffered saline), MES (2-(N-morpholino)ethanesulfonic acid), HEPES
(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), Bis-tris, ADA
(N-(2-Acetamido)iminodiacetic acid), PIPES
(piperazine-N,N'-bis(2-ethanesulfonic acid)), MOPSO
(3-Morpholino-2-hydroxypropanesulfonic acid), BES
(N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid), MOPS
(3-(N-morpholino)propanesulfonic acid), DIPSO
(3-Bis(2-hydroxyethyl) amino-2-hydroxypropane-1-sulfonic acid),
MOBS (4-(N-morpholino)butanesulfonic acid), HEPPSO
(N-(2-Hydroxyethyl)piperazine-N'-(2-hydroxypropanesulfonic acid)),
POPSO (Piperazine-1,4-bis(2-hydroxy-3-propanesulfonic acid)), TEA
(triethanolamine), EPPS
(4-(2-Hydroxyethyl)piperazine-1-propanesulfonic acid), or Bicine at
a pH between 6.0 and 8.5, 7.0 and 8.0, or 7.0 and 7.5).
[0267] S. pneumoniae capsular polysaccharide-protein conjugates
that can be prepared using reductive amination in an aprotic
solvent, include, but are not limited to, S. pneumoniae serotypes:
1, 3, 4, 5, 6A, 6B, 6C, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B,
15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B. The
polysaccharides may be used in the form of oligosaccharides. These
are conveniently formed by fragmentation of purified polysaccharide
(e.g. by hydrolysis), which will usually be followed by
purification of the fragments of the desired size.
[0268] In certain embodiments, pneumococcal polysaccharide-protein
conjugates of one or more of the S. pneumoniae serotypes 1, 3, 4,
5, 6A, 6B, 6C, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 18C,
19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B are prepared using
reductive amination in an aprotic solvent. In certain embodiments,
each of the conjugates in the multivalent immunogenic composition
is prepared using reductive amination in an aprotic solvent. In
certain embodiments, polysaccharides of one or more serotypes in a
multivalent composition of the invention are conjugated to a
carrier protein using reductive amination in an aprotic solvent and
polysaccharides of one or more serotypes are conjugated using
reductive amination in an aqueous solvent. In certain embodiments,
polysaccharides of two or more serotypes in a multivalent
composition of the invention are conjugated to a carrier protein
using reductive amination in an aprotic solvent. In other
embodiments, polysaccharides of three or more, four or more, five
or more, six or more, seven or more, eight or more, nine or more,
ten or more, eleven or more, twelve or more, thirteen or more,
fourteen or more, fifteen or more, sixteen or more, seventeen or
more, eighteen or more, nineteen or more, twenty or more,
twenty-one or more, twenty-two or more, twenty-three or more, or
twenty-four or more serotypes in a multivalent composition of the
invention are conjugated to a carrier protein using reductive
amination in an aprotic solvent. In certain embodiments,
polysaccharides from one or more serotypes in a multivalent
composition of the invention are conjugated to a carrier protein
using other chemistries which may be in an aprotic solvent or in an
aqueous solvent.
[0269] Thus, the invention relates to a multivalent immunogenic
composition comprising multiple S. pneumoniae polysaccharide
protein conjugates, each comprising capsular polysaccharide from an
S. pneumoniae serotype conjugated to a carrier protein, wherein the
serotypes of S. pneumoniae are as described herein (i.e. in Section
II, "Multivalent Immunogenic Compositions"), wherein the
conjugation reaction whereby the S. pneumonia polysaccharide of one
or more of the polysaccharide protein conjugates is conjugated to
the carrier protein is in an aprotic solvent. In certain
embodiments, at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,
75%, 80%, 85%, 90%, 95%, or 100% of the pneumococcal serotypes in a
multivalent immunogenic composition are conjugated in an aprotic
solvent. The remainder of the serotypes are conjugated using an
alternative chemistry and/or in an aqueous solvent.
[0270] It was determined that the use of DMSO as a solvent during
reductive amination of polysaccharide-protein conjugates results in
the unexpectedly superior stability and enhanced immunogenicity for
those serotypes relative to the same conjugates prepared under
aqueous conditions (See U.S. application Ser. Nos. 62/463,216 and
62/555,444).
[0271] In certain embodiments of the invention the total
polysaccharide concentration in the composition is from about 0.02
to about 0.288 mg/mL. In certain embodiments of the invention the
total polysaccharide concentration in the composition is from about
0.03 to about 0.192 mg/mL. In certain embodiments of the invention
the total polysaccharide concentration in the composition is from
about 0.04 to about 0.192 mg/mL. In other embodiments, the total
polysaccharide concentration in the composition is from about 0.065
to about 0.096 mg/mL, about 0.070 to about 0.080 mg/mL, about 0.065
to about 0.080 mg/mL, about 0.070 to about 0.085 mg/mL, about 0.110
to about 0.128 mg/mL, about 0.110 to about 0.175 mg/mL, about 0.10
to about 0.175 mg/mL, about 0.110 to about 0.170 mg/mL, about 0.115
to about 0.15 mg/mL, about 0.110 to about 0.15 mg/mL, about 0.110
to about 0.125 mg/mL, about 0.150 to about 0.170 mg/mL, about 0.150
to about 0.165 mg/mL, about 0.140 to about 0.170 mg/mL, about 0.130
to about 0.170 mg/mL, about 0.150 to about 0.175 mg/mL, about 0.070
to about 0.170 mg/mL, about 0.065 to about 0.175 mg/mL, or about
0.065 to about 0.180 mg/mL.
[0272] In embodiments of the invention wherein one or more, or all,
of the polysaccharide-protein conjugates in the multivalent
immunogenic compositions are prepared in an aprotic solvent, the
total polysaccharide concentration in the composition is stable for
4 weeks or more at 37.degree. C., 4 weeks or more at 25.degree. C.,
or 12 weeks or more at 4.degree. C.
[0273] In certain embodiments of the invention wherein one or more,
or all, of the polysaccharide-protein conjugates in the multivalent
immunogenic compositions are prepared in an aprotic solvent, the
average molecular weight (Mw) of all of the S. pneumoniae
polysaccharide protein conjugates in the composition (average of
all conjugates in the composition) is from about 2,000 to about
6,500 kDa, from about 2,500 to about 6,000 kDa, from about 3,000 to
about 5,500 kDa, from about 3,500 to about 5,000 kDa, from about
3,500 to about 4,500 kDa, from about 3,500 to about 4,700 kDa, from
about 3,500 to about 4,600 kDa, from about 3,500 to about 4,500
kDa, from about 3,500 to about 4,400 kDa, from about 3,500 to about
4,300 kDa, from about 3,500 to about 4,200 kDa, from about 3,600 to
about 4,700 kDa, from about 3,600 to about 4,600 kDa, from about
3,600 to about 4,500 kDa, from about 3,600 to about 4,400 kDa, from
about 3,600 to about 4,300 kDa, from about 3,600 to about 4,200
kDa, from about 3,700 to about 4,700 kDa, from about 3,700 to about
4,600 kDa, from about 3,700 to about 4,500 kDa, from about 3,700 to
about 4,400 kDa, from about 3,700 to about 4,300 kDa, from about
3,700 to about 4,200 kDa, from about 3,800 to about 4,700 kDa, from
about 3,800 to about 4,600 kDa, from about 3,800 to about 4,500
kDa, from about 3,800 to about 4,400 kDa, from about 3,800 to about
4,300 kDa, from about 3,800 to about 4,200 kDa, from about 3,900 to
about 4,700 kDa, from about 3,900 to about 4,600 kDa, from about
3,900 to about 4,500 kDa, from about 3,900 to about 4,400 kDa, from
about 3,900 to about 4,300 kDa, or from about 3,900 to about 4,200
kDa.
[0274] In certain embodiments of the invention wherein the
polysaccharide-protein conjugates in the multivalent immunogenic
compositions are prepared in an aprotic solvent, the Mw of each of
the S. pneumoniae polysaccharide protein conjugates in the
composition (for a single serotype) is from about 1,000 to about
10,000 kDa, from about 1,500 to about 5,500 kDa, from about 1,500
to about 5,600 kDa, from about 1,500 to about 5,700 kDa, from about
1,500 to about 5,800 kDa, from about 1,500 to about 5,900 kDa, from
about 1,500 to about 6,000 kDa, from about 1,000 to about 5,500
kDa, from about 1,000 to about 5,000 kDa, from about 1,000 to about
4,000 kDa, from about 1,000 to about 4,500 kDa, from about 1,000 to
about 4,000 kDa, or from about 1,000 to about 3,500 kDa. In other
embodiments, the Mw of a conjugate from a single serotype within
the composition is about 1,000 kDa, about 1,100 kDa, about 1,200
kDa, about 1,300 kDa, about 1,400 kDa, about 1,500 kDa, about 1,600
kDa, about 1,700 kDa, about 1,800 kDa, about 1,900 kDa, about 2,000
kDa, about 2,100 kDa, about 2,200 kDa, about 2,300 kDa, about 2,400
kDa, about 2,500 kDa, about 2,600 kDa, about 2,700 kDa, about 2,800
kDa, about 2,900 kDa, about 3,000 kDa, about 3,100 kDa, about 3,200
kDa, about 3,300 kDa, about 3,400 kDa, about 3,500 kDa, about 3,600
kDa, about 3,700 kDa, about 3,800 kDa, about 3,900 kDa, about 4,000
kDa, about 4,100 kDa, about 4,200 kDa, about 4,300 kDa, about 4,400
kDa, about 4,500 kDa, about 4,600 kDa, about 4,700 kDa, about 4,800
kDa, about 4,900 kDa, about 5,000 kDa, about 5,100 kDa, about 5,200
kDa, about 5,300 kDa, about 5,400 kDa, or about 5,500 kDa.
[0275] In certain embodiments of the invention the
polysaccharide-protein conjugates in the multivalent immunogenic
compositions are prepared in an aprotic solvent. In certain
embodiments, the percentage (as calculated by the number of
polysaccharide serotypes prepared in an aprotic solvent divided by
the total number of polysaccharide serotypes, where total number
includes those prepared in an aprotic solvent or a protic solvent)
of S. pneumoniae serotype specific conjugates prepared in an
aprotic solvent may be greater than 50%, or greater than 60%, or
greater than 70%, or greater than 80%, or greater than 90% or are
100%.
[0276] In certain embodiments of the invention, the serotype 3
polysaccharide-protein conjugate in the composition is prepared in
an aprotic solvent and the Mw of said conjugate is from about 1,000
to about 5,000 kDa, or from about 1,000 to about 4,000 kDa, or from
about 1,000 to about 3,000 kDa, or from about 1,000 to about 2,500
kDa, or from about 1,000 to about 2,000 kDa.
[0277] In certain embodiments of the invention wherein one or more,
or all, of the polysaccharide-protein conjugates in the multivalent
immunogenic compositions are prepared in an aprotic solvent, the
number average molecular weight (Mn) of the S. pneumoniae
polysaccharide protein conjugates in the composition (average of
all conjugates in the composition) is from about 900 to about 3,000
kDa, from about 1,000 to about 3,000 kDa, from about 1,000 to about
2,500 kDa, from about 1,500 to about 2,500 kDa, from about 1,800 to
about 2,500 kDa, from about 1,900 to about 2,500 kDa, or from about
2,000 to about 2,500 kDa.
[0278] In certain embodiments of the invention wherein one or more,
or all, of the polysaccharide-protein conjugates in the multivalent
immunogenic compositions are prepared in an aprotic solvent, the Mn
of each of the S. pneumoniae polysaccharide protein conjugates in
the composition (for a single serotype) is from about 700 to about
7,000 kDa, from about 1,000 to about 6,000 kDa, from about 1,000 to
about 5,000 kDa, from about 1,000 to about 4,000 kDa, from about
1,000 to about 3,000 kDa, from about 900 to about 5,500 kDa, from
about 900 to about 5,000 kDa, from about 900 to about 4,500 kDa,
from about 900 to about 4,000 kDa, from about 900 to about 3,500
kDa, or from about 900 to about 3,000 kDa.
[0279] In embodiments of the invention, the Mw and/or Mn of the S.
pneumoniae polysaccharide protein conjugates in the composition is
stable for 4 weeks or more at 37.degree. C., 4 weeks or more at
25.degree. C., and/or 12 weeks or more at 4.degree. C.
[0280] In embodiments of the invention, the polysaccharide
concentration, Mw, and/or Mn are determined using HPSEC
UV/MALS/RI.
[0281] In some embodiment of the invention, wherein one or more, or
all, of the polysaccharide-protein conjugates in the multivalent
immunogenic compositions are prepared in an aprotic solvent, the
emission maximum of the composition measured using intrinsic
protein fluorescence spectroscopy with an excitation wavelength at
280 nanometers (nm) is from about 335 nm to about 342 nm. In some
embodiments, the emission maximum remains from about 335 nm to
about 342 nm and the fluorescence intensity is stable for at least
1 week at 37.degree. C. In some embodiments, the emission maximum
remains from about 335 nm to about 342 nm and the fluorescence
intensity is stable for 1 week at 37.degree. C.
[0282] In some embodiments, all of the pneumococcal polysaccharide
conjugates in the multivalent composition are prepared using
reductive amination in DMSO. In certain sub-embodiments, the
multivalent composition comprising polysaccharide conjugates which
were all prepared using DMSO does not comprise an adjuvant.
[0283] Without being bound by any particular theory, one possible
mechanism for the enhanced immunogenicity observed with
glycoconjugates prepared in DMSO include an increased number of
linkages between the carbohydrate (capsular polysaccharide) and
lysine residues on the surface of the carrier protein which would
result in additional attachment points between the protein and
polysaccharide to impart stability and counter chemical
depolymerization or breakdown of the peptide carbohydrate bond.
See, e.g., Hsieh, Characterization of Saccharide-CRM197 Conjugate
Vaccines in Brown F, Corbel M, Griffiths E (eds): Physico-Chemical
Procedures for the Characterization of Vaccines. Dev. Biol. Basel,
Karger, 2000, vol 103, pp. 93-104. An additional benefit of the
increased polysaccharide-protein linkages that are created during
conjugation in the DMSO solvent could be additional opportunities
for successful presentation of peptide-carbohydrate to T-cells. A
possible mechanism of enhanced immunogenicity observed by
conjugation in the DMSO solvent could be due to the denaturation of
CRM197 in organic solvent, which exposes additional lysines for
polysaccharide linkages giving increased chances for glycopeptide
presentation at the surface of an APC for T-cell dependent response
to different peptide epitopes. See Avci et al., 2011, Nature
Medicine 17: 1602-1610.
[0284] Yet another benefit of conjugation in an organic solvent
generating denatured CRM197 in the conjugates could be reduced
immunological interference of antibodies against native CRM197
epitopes. A further benefit of the increased polysaccharide-protein
linkages that are created during conjugation in the DMSO solvent
could be the formation of larger sized polysaccharide protein
conjugates resulting in enhanced immunogenicity. The compositions
of the invention are believed to provide significant advantages in
eliciting a human response.
[0285] In certain embodiments, the conjugation reaction is
performed by reductive amination wherein nickel is used for greater
conjugation reaction efficiency and to aid in free cyanide removal.
Transition metals are known to form stable complexes with cyanide
and are known to improve reductive methylation of protein amino
groups and formaldehyde with sodium cyanoborohydride (S Gidley et
al., Biochem 1 1982, 203: 331-334; Jentoft et al. Anal Biochem.
1980, 106: 186-190). By complexing residual, inhibitory cyanide,
the addition of nickel increases the consumption of protein during
the conjugation and leads to formation of larger, potentially more
immunogenic conjugates.
[0286] Differences in starting cyanide levels in sodium
cyanoborohydride reagent lots also lead to inconsistent conjugation
performance, resulting in variable product attributes, such as
conjugate size and conjugate Ps-to-CRM197 ratio. The addition of
nickel reduced conjugation inconsistency by complexing cyanide,
eliminating differences in sodium cyanoborohydride lots.
[0287] Suitable alternative chemistries include the activation of
the saccharide with 1-cyano-4-dimethylamino pyridinium
tetrafluoroborate (CDAP) to form a cyanate ester. The activated
saccharide may thus be coupled directly or via a spacer (linker)
group to an amino group on the carrier protein. For example, the
spacer could be cystamine or cysteamine to give a thiolated
polysaccharide which could be coupled to the carrier via a
thioether linkage obtained after reaction with a
maleimide-activated carrier protein (for example using GMBS) or a
haloacetylated carrier protein (for example using iodoacetimide
[e.g. ethyl iodoacetimide HCl] or N-succinimidyl bromoacetate or
SIAB, or SIA, or SBAP). Preferably, the cyanate ester (optionally
made by CDAP chemistry) is coupled with hexane diamine or adipic
acid dihydrazide (ADH) and the amino-derivatised saccharide is
conjugated to the carrier protein using carbodiimide (e.g. EDAC or
EDC) chemistry via a carboxyl group on the protein carrier. Such
conjugates are described in International Patent Application
Publication Nos. WO 93/15760, WO 95/08348 and WO 96/29094; and Chu
et al., 1983, Infect. Immunity 40:245-256.
[0288] Other suitable conjugation techniques use carbodiimides,
hydrazides, active esters, norborane, p-nitrobenzoic acid,
N-hydroxysuccinimide, S--NHS, EDC, TSTU. Many are described in
International Patent Application Publication No. WO 98/42721.
Conjugation may involve a carbonyl linker which may be formed by
reaction of a free hydroxyl group of the saccharide with CDI (See
Bethell et al., 1979, J. Biol. Chem. 254:2572-4; Hearn et al.,
1981, J. Chromatogr. 218:509-18) followed by reaction with a
protein to form a carbamate linkage. This may involve reduction of
the anomeric terminus to a primary hydroxyl group, optional
protection/deprotection of the primary hydroxyl group, reaction of
the primary hydroxyl group with CDI to form a CDI carbamate
intermediate and coupling the CDI carbamate intermediate with an
amino group on a protein.
[0289] After conjugation of the capsular polysaccharide to the
carrier protein, the polysaccharide-protein conjugates are purified
(enriched with respect to the amount of polysaccharide-protein
conjugate) by one or more of a variety of techniques. Examples of
these techniques are well known to the skilled artisan and include
concentration/diafiltration operations, ultrafiltration,
precipitation/elution, column chromatography, and depth filtration.
See, e.g., U.S. Pat. No. 6,146,902.
[0290] After the individual glycoconjugates are purified, they are
compounded to formulate the immunogenic composition of the present
invention. These pneumococcal conjugates may be prepared by
separate processes and bulk formulated into a single dosage
formulation.
[0291] An alternative method for characterizing the glycoconjugates
of the invention is by the number of lysine residues in the carrier
protein (e.g., CRM197) that become conjugated to the saccharide
which can be characterized as a range of conjugated lysines (degree
of conjugation). The evidence for lysine modification of the
carrier protein, due to covalent linkages to the polysaccharides,
can be obtained by amino acid analysis using routine methods known
to those of skill in the art. Conjugation results in a reduction in
the number of lysine residues recovered, compared to the carrier
protein starting material used to generate the conjugate materials.
In a preferred embodiment, the extent of conjugation, as measured
by lysine consumption of the glycoconjugate of the invention is
between 2 and 15, between 2 and 13, between 2 and 10, between 2 and
8, between 2 and 6, between 2 and 5, between 2 and 4, between 3 and
15, between 3 and 13, between 3 and 10, between 3 and 8, between 3
and 6, between 3 and 5, between 3 and 4, between 5 and 15, between
5 and 10, between 8 and 15, between 8 and 12, between 10 and 15 or
between 10 and 12. In an embodiment, the degree of conjugation of
the glycoconjugate of the invention is about 2, about 3, about 4,
about 5, about 6, about 7, about 8, about 9, about 10, about 11 ,
about 12, about 13, about 14 or about 15. In another embodiment,
the degree of conjugation of the glycoconjugate of the invention is
between 4 and 7. In some such embodiments, the carrier protein is
CRM197.
[0292] The glycoconjugates of the compositions of the invention may
also be characterized by the ratio (weight/weight) of
polysaccharide to carrier protein (Ps:Pr). In some embodiments, the
ratio of polysaccharide to carrier protein of the glycoconjugates
(w/w) in the composition is between 0.5 and 3.0 (e.g., about 0.5,
about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about 1.1 ,
about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7,
about 1.8, about 1.9, about 2.0, about 2.1 , about 2.2, about 2.3,
about 2.4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9,
or about 3.0). In other embodiments, the polysaccharide to carrier
protein ratio (w/w) is between 0.5 and 2.5, between 0.5 and 1.5,
between 0.8 and 2.5, between 0.5 and 1.0, between 1.0 and 1.5,
between 1.0 and 2.0, between 0.8 and 2.4, between 0.8 and 2.3,
between 0.8 and 2.2, between 0.8 and 2.1, between 0.8 and 2.0,
between 0.8 and 1.9, between 0.8 and 1.8, between 0.8 and 1.7,
between 0.8 and 1.6, between 0.8 and 1.5, between 0.8 and 1.4,
between 0.8 and 1.3, between 0.9 and 2.4, between 0.9 and 2.3,
between 0.9 and 2.2, between 0.9 and 2.1, between 0.9 and 2.0,
between 0.9 and 1.9, between 0.9 and 1.8, between 0.9 and 1.7,
between 0.9 and 1.6, between 0.9 and 1.5, between 0.9 and 1.4,
between 0.9 and 1.3, between 0.9 and 1.2, between 1.0 and 2.4,
between 1.0 and 2.3, between 1.0 and 2.2, between 1.0 and 2.1,
between 1.0 and 2.0, between 1.0 and 1.9, between 1.0 and 1.8,
between 1.0 and 1.7, between 1.0 and 1.6, between 1.0 and 1.5,
between 1.0 and 1.4, between 1.0 and 1.3 or between 1.0 and 1.2. In
further embodiments, the saccharide to carrier protein ratio (w/w)
is between 0.8 and 1.2. In some such embodiments, the carrier
protein is CRM197. The glycoconjugates and immunogenic compositions
of the invention may contain free saccharide that is not covalently
conjugated to the carrier protein, but is nevertheless present in
the glycoconjugate composition. The free saccharide may be
non-covalently associated with (e.g., non-covalently bound to,
adsorbed to, or entrapped in or with) the glycoconjugate.
[0293] In specific embodiments, the saccharide to carrier protein
ratio (w/w) for the serotype 15A conjugate is from about 1.0 to
about 2.0, from about 1.25 to about 1.75, or from about 1.3 to
about 1.7. In other embodiments, the saccharide to carrier protein
ratio (w/w) for serotype 15A is about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6,
1.7, or 1.8.
[0294] In specific embodiments, the saccharide to carrier protein
ratio (w/w) for the serotype 15C conjugate is from about 1.0 to
about 2.0, from about 1.25 to about 1.75, or from about 1.3 to
about 1.7. In other embodiments, the saccharide to carrier protein
ratio (w/w) for serotype 15C is about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6,
1.7, or 1.8.
[0295] In specific embodiments, the saccharide to carrier protein
ratio (w/w) for the serotype 33F conjugate is from about 1.0 to
about 2.0, from about 1.25 to about 1.75, or from about 1.3 to
about 1.7. In other embodiments, the saccharide to carrier protein
ratio (w/w) for serotype 33F is about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6,
1.7, or 1.8.
[0296] In specific embodiments, the saccharide to carrier protein
ratio (w/w) for the serotype 35B conjugate is from about 1.25 to
about 2.25, from about 1.25 to about 2.0, or from about 1.3 to
about 1.8. In other embodiments, the saccharide to carrier protein
ratio (w/w) for serotype 35B is about 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,
1.8, 1.9, or 2.0.
[0297] In specific embodiments, the saccharide to carrier protein
ratio (w/w) for the serotype 24F conjugate is from about 0.5 to
about 1.5, from about 0.75 to about 1.25, or from about 0.8 to
about 1.0. In other embodiments, the saccharide to carrier protein
ratio (w/w) for serotype 24F is about 0.5, 0.6, 0.7, 0.8, 0.9, or
1.0.
[0298] In a preferred embodiment, the glycoconjugate composition
comprises less than about 50%, 45%, 40%, 35%, 30%, 25%, 20% or 15%
of free polysaccharide compared to the total amount of
polysaccharide. In a preferred embodiment the glycoconjugate
composition comprises less than about 25% of free polysaccharide
compared to the total amount of polysaccharide. In a preferred
embodiment the glycoconjugate composition comprises less than about
20% of free polysaccharide compared to the total amount of
polysaccharide. In a preferred embodiment the glycoconjugate
composition comprises less than about 15% of free polysaccharide
compared to the total amount of polysaccharide.
[0299] IV. Methods of Use
[0300] Embodiments of the invention also include one or more of the
multivalent immunogenic compositions described herein (i) for use
in, (ii) for use as a medicament or composition for, or (iii) for
use in the preparation of a medicament for: (a) therapy (e.g., of
the human body); (b) medicine; (c) inhibition of infection with
Streptococcus pneumoniae; (d) induction of an immune response or a
protective immune response against S. pneumoniae; (e) prophylaxis
of infection by S. pneumoniae; (0 prevention of recurrence of S.
pneumoniae infection; (g) reduction of the progression, onset or
severity of pathological symptoms associated with S. pneumoniae
infection including the prevention of associated complications such
as brain damage, hearing loss, and seizures, (h) reduction of the
likelihood of a S. pneumoniae infection or, (i) treatment,
prophylaxis of, or delay in the onset, severity, or progression of
pneumococcal disease(s), including, but not limited to:
pneumococcal pneumonia, pneumococcal bacteremia, pneumococcal
meningitis, otits media and sinusitis. In these uses, the
multivalent pneumococcal polysaccharide-conjugate compositions of
the invention can optionally be employed in combination with one or
more adjuvants, or without an adjuvant.
[0301] Accordingly, the invention provides methods for the
prophylactic treatment of (i.e. protection against) S. pneumoniae
infection or pneumococcal disease comprising administering one or
more of the multivalent immunogenic pneumococcal
polysaccharide-protein conjugate compositions of the invention to a
patient in need of treatment.
[0302] The compositions and formulations of the present invention
can be used to protect or treat a human susceptible to infection,
e.g., a pneumococcal infection, by means of administering such
composition or formulation via a systemic or mucosal route.
[0303] In one embodiment, the invention provides a method of
inducing an immune response to S. pneumoniae, comprising
administering to a patient an immunologically effective amount of a
multivalent immunogenic composition of the invention. In another
embodiment, the invention provides a method of vaccinating a human
against a pneumococcal infection, comprising the step of
administering to the human an immunogically effective amount of a
multivalent immunogenic composition of the invention.
[0304] Thus, in one aspect, the invention provides a method for (1)
inducing an immune response in a human patient, (2) inducing a
protective immune response in a human patient, (3) vaccinating a
human patient against an infection with S. pneumoniae, or (4)
reducing the likelihood of a S. pneumoniae infection in a human
patient, the method comprising administering a multivalent
immunogenic composition of the invention to the patient (i.e. any
multivalent immunogenic composition described herein, such as the
multivalent immunogenic compositions described in Section II,
entitled "Multivalent Immunogenic Compositions," supra).
[0305] In one embodiment, the invention provides a method for the
prevention of pneumococcal pneumonia and/or invasive pneumococcal
disease in an infant (less than 1 year of age), toddler
(approximately 12 to 24 months), or young child (approximately 2 to
5 years).
[0306] In another embodiment, the invention provides a method for
the prevention of pneumococcal pneumonia and/or invasive
pneumococcal disease in a 6 week through 17 year old patient.
[0307] In another embodiment, the invention provides a method for
the prevention of pneumococcal pneumonia and/or invasive
pneumococcal disease in a 6 month through 17 year old patient.
[0308] In another embodiment, the invention provides a method for
the prevention of pneumococcal pneumonia and/or invasive
pneumococcal disease in adults 18 years of age and older.
[0309] In another embodiment, the invention provides a method for
the prevention of pneumococcal pneumonia and/or invasive
pneumococcal disease in adults 50 years of age and older.
[0310] In another embodiment, the invention provides a method for
the prevention of pneumococcal pneumonia and/or invasive
pneumococcal disease in adults 65 years of age and older.
[0311] In another embodiment, the invention provides a method for
the prevention of pneumococcal pneumonia and/or invasive
pneumococcal disease caused by one or more of the following
Streptococcus pneumoniae strains: 1, 3, 4, 5, 6A, 6B, 6C, 7F, 8,
9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 18C, 19A, 19F, 22F, 23B, 23F,
24F, 33F and 35B.
[0312] In one embodiment of the methods above, the composition
comprises multiple S. pneumoniae polysaccharide protein conjugates
wherein each of the conjugates comprises a polysaccharide from an
S. pneumoniae serotype conjugated to a carrier protein, wherein the
serotypes of S. pneumoniae comprise serotypes: 1, 3, 4, 5, 6A, 6B,
6C, 7F, 8, 9V, 10A, 12F, 14, 15A, 15B, 15C, 18C, 19A, 19F, 22F,
23B, 23F, 24F, 33F and 35B or serotypes: 1, 3, 4, 5, 6A, 6B, 6C,
7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 18C, 19A, 19F, 22F,
23B, 23F, 24F, 33F and 35B. In another embodiment of the methods
above, the composition comprises multiple S. pneumoniae
polysaccharide protein conjugates wherein each of the conjugates
comprises a polysaccharide from an S. pneumoniae serotype
conjugated to a carrier protein, wherein the serotypes of S.
pneumoniae consist of serotypes: 1, 3, 4, 5, 6A, 6B, 6C, 7F, 8, 9V,
10A, 12F, 14, 15A, 15B, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F
and 35B or serotypes: 1, 3, 4, 5, 6A, 6B, 6C, 7F, 8, 9V, 10A, 11A,
12F, 14, 15A, 15B, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and
35B. In one embodiment of the methods above, the composition
comprises multiple S. pneumoniae polysaccharide protein conjugates
wherein each of the conjugates comprises a polysaccharide from an
S. pneumoniae serotype conjugated to a carrier protein, wherein the
serotypes of S. pneumoniae comprise serotypes: 1, 3, 4, 5, 6A, 6B,
7F, 8, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F,
24F, 33F and 35B or serotypes: 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A,
11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and
35B. In another embodiment of the methods above, the composition
comprises multiple S. pneumoniae polysaccharide protein conjugates
wherein each of the conjugates comprises a polysaccharide from an
S. pneumoniae serotype conjugated to a carrier protein, wherein the
serotypes of S. pneumoniae consist of serotypes: 1, 3, 4, 5, 6A,
6B, 7F, 8, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B,
23F, 24F, 33F and 35B or serotypes: 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V,
10A, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F
and 35B. In one embodiment of the methods above, the composition
comprises multiple S. pneumoniae polysaccharide protein conjugates
wherein each of the conjugates comprises a polysaccharide from an
S. pneumoniae serotype conjugated to a carrier protein, wherein the
serotypes of S. pneumoniae comprise serotypes: 1, 3, 4, 5, 6A, 6B,
7F, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F,
33F and 35B. In another embodiment of the methods above, the
composition comprises multiple S. pneumoniae polysaccharide protein
conjugates wherein each of the conjugates comprises a
polysaccharide from an S. pneumoniae serotype conjugated to a
carrier protein, wherein the serotypes of S. pneumoniae consist of
serotypes: 1, 3, 4, 5, 6A, 6B, 7F, 9V, 10A, 12F, 14, 15A, 15C, 18C,
19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B or serotypes: .
[0313] It has been shown that a pneumococcal conjugate vaccine
comprising serotype 6A polysaccharide may provide some
cross-protection against serotype 6C (Cooper et al., Vaccine 29
(2011) 7207-7211). Therefore, in some embodiments of the methods
above, the invention also provides use of multivalent immunogenic
compositions that do not comprise serotype 6C polysaccharide
conjugate, but instead comprise serotype 6A polysaccharide
conjugate or serotypes 6A and 6B polysaccharide conjugates. In
other embodiments, the immunogenic composition comprises
pneumococcal polysaccharide conjugates of serotypes 6A, 6B, and
6C.
[0314] In particular embodiments of the methods above, the
multivalent immunogenic compositions comprise pneumococcal
conjugates that include polysaccharides of a group of S. pneumoniae
serotypes selected from the group consisting of: [0315] a) 1, 3, 4,
5, 6A, 6B, 7F, 9V, 10A, 12F, 14, 15A, DeOAc15B, 18C, 19A, 19F, 22F,
23B, 23F, 24F, 33F and 35B; [0316] b) 1, 3, 4, 5, 6A, 6B, 7F, 9V,
10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and
35B; [0317] c) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 12F, 14, 15A,
DeOAcl5B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; [0318] d)
1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A,
19F, 22F, 23B, 23F, 24F, 33F and 35B; [0319] e) 1, 3, 4, 5, 6A, 6B,
7F, 8, 9V, 10A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F,
33F and 35B; [0320] f) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A,
12F, 14, 15A, DeOAc15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and
35B; [0321] g) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14,
15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; [0322] h)
1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C,
19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; [0323] i) 1, 3, 4, 5,
6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23B,
23F, 24F, 33F and 35B; [0324] j) 1, 3, 4, 5, 6A, 7F, 9V, 10A, 12F,
14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;
[0325] k) 1, 3, 4, 5, 6B, 7F, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A,
19F, 22F, 23B, 23F, 24F, 33F and 35B; [0326] l) 1, 3, 4, 5, 6C, 7F,
9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F
and 35B; [0327] m) 1, 3, 4, 5, 6A, 7F, 8, 9V, 10A, 12F, 14, 15A,
15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; [0328] n) 1,
3, 4, 5, 6B, 7F, 8, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F,
23B, 23F, 24F, 33F and 35B; [0329] o) 1, 3, 4, 5, 6C, 7F, 8, 9V,
10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and
35B; [0330] p) 1, 3, 4, 5, 6A, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A,
15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; [0331] q) 1,
3, 4, 5, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F,
22F, 23B, 23F, 24F, 33F and 35B; [0332] r) 1, 3, 4, 5, 6C, 7F, 8,
9V, 10A, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F,
33F and 35B; [0333] s) 1, 3, 4, 5, 6A, 7F, 9V, 10A, 12F, 14, 15A,
15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; [0334] t) 1,
3, 4, 5, 6B, 7F, 9V, 10A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F,
23B, 23F, 24F, 33F and 35B; [0335] u) 1, 3, 4, 5, 6C, 7F, 9V, 10A,
12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;
[0336] v) 1, 3, 4, 5, 6A, 7F, 8, 9V, 10A, 12F, 14, 15A, 15B, 18C,
19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; [0337] w) 1, 3, 4, 5,
6B, 7F, 8, 9V, 10A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B,
23F, 24F, 33F and 35B; [0338] x) 1, 3, 4, 5, 6C, 7F, 8, 9V, 10A,
12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;
[0339] y) 1, 3, 4, 5, 6A, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B,
18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; [0340] z) 1, 3, 4,
5, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F,
23B, 23F, 24F, 33F and 35B; and [0341] aa) 1, 3, 4, 5, 6C, 7F, 8,
9V, 10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F,
33F and 35B.
[0342] In further embodiments of the methods above, the composition
comprises multiple S. pneumoniae polysaccharide protein conjugates
wherein each of the conjugates comprises a polysaccharide from an
S. pneumoniae serotype conjugated to a carrier protein, wherein the
serotypes of S. pneumoniae comprise serotypes: i) 1, 3, 4, 5, 6A,
6B, 7F, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F,
24F, 33F and 35B; or ii) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 12F,
14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; or
iii) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15C,
18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; or iv) 1, 3, 4, 5,
6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F,
23B, 23F, 24F, 33F and 35B.
[0343] It has also been shown that a pneumococcal conjugate vaccine
comprising serotype 10A polysaccharide may provide some
cross-protection against serotype 39 (see WO 2017/ 085586).
Therefore, in some embodiments of the methods above, the invention
also provides use of multivalent immunogenic compositions that do
not comprise serotype 10A polysaccharide conjugate, but instead
comprise serotype 39 polysaccharide conjugate. In other
embodiments, the immunogenic composition comprises pneumococcal
polysaccharide conjugates of serotypes 10A and 39. In particular
embodiments of the methods above, the serotypes of S. pneumoniae
comprise a group of serotypes selected from the group consisting
of: [0344] bb) 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 15A, DeOAc15B,
18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; [0345] cc) 1,
3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F,
23B, 23F, 24F, 33F, 35B and 39; [0346] dd) 1, 3, 4, 5, 6A, 6B, 7F,
8, 9V, 12F, 14, 15A, DeOAciSB, 18C, 19A, 19F, 22F, 23B, 23F, 24F,
33F, 35B and 39; [0347] ee) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 12F, 14,
15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39;
[0348] ff) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 12F, 14, 15B, 18C, 19A,
19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; [0349] gg) 1, 3, 4, 5,
6A, 6B, 7F, 8, 9V, 11A, 12F, 14, 15A, DeOAc15B, 18C, 19A, 19F, 22F,
23B, 23F, 24F, 33F, 35B and 39; [0350] hh) 1, 3, 4, 5, 6A, 6B, 7F,
8, 9V, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F,
33F, 35B and 39; [0351] ii) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 11A,
12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and
39; [0352] jj) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 11A, 12F, 14, 15B,
18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; [0353] kk) 1,
3, 4, 5, 6A, 7F, 9V, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B,
23F, 24F, 33F, 35B and 39; [0354] ll) 1, 3, 4, 5, 6B, 7F, 9V, 12F,
14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39;
[0355] mm) 1, 3, 4, 5, 6C, 7F, 9V, 12F, 14, 15A, 15C, 18C, 19A,
19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; [0356] nn) 1, 3, 4, 5,
6A, 7F, 8, 9V, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F,
24F, 33F, 35B and 39; [0357] oo) 1, 3, 4, 5, 6B, 7F, 8, 9V, 12F,
14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39;
[0358] pp) 1, 3, 4, 5, 6C, 7F, 8, 9V, 12F, 14, 15A, 15C, 18C, 19A,
19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; [0359] qq) 1, 3, 4, 5,
6A, 7F, 8, 9V, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B,
23F, 24F, 33F, 35B and 39; [0360] rr) 1, 3, 4, 5, 6B, 7F, 8, 9V,
11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B
and 39; [0361] ss) 1, 3, 4, 5, 6C, 7F, 8, 9V, 11A, 12F, 14, 15A,
15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; [0362] tt)
1, 3, 4, 5, 6A, 7F, 9V, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B,
23F, 24F, 33F, 35B and 39; [0363] uu) 1, 3, 4, 5, 6B, 7F, 9V, 12F,
14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39;
[0364] vv) 1, 3, 4, 5, 6C, 7F, 9V, 12F, 14, 15A, 15B, 18C, 19A,
19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; [0365] ww) 1, 3, 4, 5,
6A, 7F, 8, 9V, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F,
24F, 33F, 35B and 39; [0366] xx) 1, 3, 4, 5, 6B, 7F, 8, 9V, 12F,
14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39;
[0367] yy) 1, 3, 4, 5, 6C, 7F, 8, 9V, 12F, 14, 15A, 15B, 18C, 19A,
19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; [0368] zz) 1, 3, 4, 5,
6A, 7F, 8, 9V, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B,
23F, 24F, 33F, 35B and 39; [0369] aaa) 1, 3, 4, 5, 6B, 7F, 8, 9V,
11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B
and 39; and [0370] bbb) 1, 3, 4, 5, 6C, 7F, 8, 9V, 11A, 12F, 14,
15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39.
[0371] It has also been shown that immunogenic conjugates
comprising S. pneumoniae serotype 15B capsular polysaccharide
covalently linked to a carrier protein may provide some
cross-protection against serotype 15C and/or serotype 15A (see WO
2015/110942). Therefore, in some embodiments of the methods above,
the invention also provides use of multivalent immunogenic
compositions that do not comprise serotype 15C (or de-O-acetylated
15B) polysaccharide conjugate, but instead comprise serotype 15B
polysaccharide conjugate (i.e. the serotype 15B polysaccharide is
not substantially de-O-acetylated). In other embodiments, the
immunogenic composition comprises pneumococcal polysaccharide
conjugates of serotypes 15B and 15C (or de-O-acetylated 15B).
[0372] The compositions of the invention are useful in methods for
providing complementary protection against S. pneumoniae in
patients who had previously received a multivalent pneumococcal
vaccine. In this use, the compositions of the invention can provide
protection against particular S. pneumoniae serotypes that a
patient had not been previously vaccinated against, can provide
additional protection against S. pneumoniae serotypes that a
patient had been previously vaccinated against, or can provide
protection against both S. pneumoniae serotypes that a patient had
not been previously vaccinated against and S. pneumoniae serotypes
that a patient had been previously vaccinated against.
[0373] Thus, the invention provides a method of inducing an immune
response, vaccinating, or inducing a protective immune response
against S. pneumoniae in a patient, comprising administering a
multivalent immunogenic composition to the patient, the composition
comprising multiple S. pneumoniae polysaccharide protein
conjugates, wherein the polysaccharide protein conjugates comprise
capsular polysaccharide from a S. pneumoniae serotype conjugated to
a carrier protein, wherein the patient had previously been
vaccinated against S. pneumoniae. In embodiments of this aspect of
the invention, the multivalent immunogenic composition can be any
multivalent immunogenic composition described herein.
[0374] In particular embodiments of the methods of the invention,
the multivalent immunogenic composition is administered to a
patient who was previously treated with a multivalent pneumococcal
vaccine. The multivalent immunogenic vaccine may be any vaccine
that is indicated for the prevention of pneumococcal disease caused
by more than one serotype of S. pneumoniae.
[0375] In specific embodiments of the method above, the patient was
previously treated with a multivalent pneumococcal vaccine that is
indicated for the prevention of pneumococcal disease caused by one
or more S. pneumoniae serotypes within a group of serotypes
selected from the group consisting of: [0376] i. 4, 6B, 9V, 14,
18C, 19F and 23F; [0377] ii. 4, 6B, 9V, 14, 18C, 19F, 23F, 1, 3, 5,
6A, 7F, and 19A; [0378] iii. 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and
23F; [0379] iv. 4, 6B, 9V, 14, 18C, 19F, 23F, 1, 3, 5, 6A, 7F, 19A,
22F, and 33F; [0380] v. 4, 6B, 9V, 14, 18C, 19F, 23F, 1, 3, 5, 7F,
19A, 22F, 33F, 2, 8, 9N, 10A, 11A, 12F, 15B, 17F, and 20; and
[0381] vi. 4, 6B, 9V, 14, 18C, 19F, 23F, 1, 3, 5, 6A, 7F, 19A, 22F,
33F, 8, 10A, 11A, 12F and 15B.
[0382] In specific embodiments of the method above, the multivalent
pneumococcal vaccine comprises multiple polysaccharide protein
conjugates, wherein the polysaccharide protein conjugates comprise
polysaccharide from a S. pneumoniae serotype conjugated to a
carrier protein. In other embodiments, the multivalent pneumococcal
vaccine comprises multiple S. pneumoniae capsular polysaccharides
that are not conjugated to a carrier protein.
[0383] In additional embodiments of the method above, the patient
was previously treated with PREVNAR.RTM. 13 (Pneumococcal 13-valent
Conjugate Vaccine [Diphtheria CRM197 Protein], Pfizer, Inc.,
Philadelphia, Pa., USA).
[0384] In further embodiments of the method above, the patient was
previously treated with PNEUMOVAX.RTM. 23 (Pneumococcol Vaccine
Polyvalent, Merck & Co., Inc., Kenilworth, NJ, USA).
[0385] In still further embodiments of the method above, the
patient was previously treated with SYNFLORIX.TM. (Pneumococcal
polysaccharide conjugate vaccine (adsorbed), GlaxoSmithKline
Biologicals s.a., Rixensart, Belgium).
[0386] In embodiments of the method above, the multivalent
immunogenic composition of the invention is administered to a
patient at any time after the patient has received a multivalent
pneumococcal vaccine, according to the treatment regimen provided
by the medical professional, e.g. a physician. In particular
embodiments, the multivalent immunogenic composition of the
invention is administered to a patient from about 1 month to about
5 years after the patient has received the multivalent pneumococcal
vaccine, alternatively, from 1 month to 1 year, from 1 month to 2
years, from 1 month to 3 years, from 1 month to 4 years, from 1
month to 6 months, from 2 months to 6 months, from 2 months to 1
year, from 1 year to 5 years, from 6 months to 5 years, from 6
months to 4 years, from 6 months to 3 years, from 6 months to 2
years, from 6 months to 1 year, from 1 year to 4 years, from 1 year
to 3 years, or from 1 year to 2 years, after the patient has
received the multivalent pneumococcal vaccine. In further
embodiments, the multivalent immunogenic composition is
administered to the patient about 1 month, about 2 months, about 3
months, about 4 months, about 5 months, about 6 months, about 7
months, about 8 months, about 9 months, about 10 months, about 11
months, about 1 year, about 1.25 years, about 1.5 years, about 1.75
years, about 2 years, about 2.25 years, about 2.5 years, about 2.75
years, about 3 years, about 3.25 years, about 3.5 years, about 3.75
years, about 4 years, about 4.25 years, about 4.5 years, about 4.75
years, or about 5 years after the patient has received the
multivalent pneumococcal vaccine.
[0387] In further embodiments, the invention provides a method for
(1) inducing an immune response in a human patient, (2) inducing a
protective immune response in a human patient, (3) vaccinating a
human patient against an infection with S. pneumoniae, or (4)
reducing the likelihood of a S. pneumoniae infection in a human
patient, the method comprising administering a multivalent
immunogenic composition of the invention and administering a
multivalent pneumococcal vaccine to the patient, in any order. For
example, the patient is administered a multivalent pneumococcal
vaccine first, and the patient is administered a multivalent
immunogenic composition of the invention second. Alternatively, the
patient is administered a multivalent immunogenic composition of
the invention first and is administered a multivalent pneumococcal
vaccine second. The multivalent pneumococcal vaccine may be any
vaccine indicated for the prevention of pneumococcal disease caused
by more than one serotype of S. pneumoniae.
[0388] In specific embodiments of the method above, the patient is
treated with a multivalent immunogenic composition of the invention
and a multivalent pneumococcal vaccine that is indicated for the
prevention of pneumococcal disease caused by one or more S.
pneumoniae serotypes of a group of serotypes selected from the
group consisting of:
[0389] i. 4, 6B, 9V, 14, 18C, 19F and 23F;
[0390] ii. 4, 6B, 9V, 14, 18C, 19F, 23F, 1, 3, 5, 6A, 7F, and
19A;
[0391] iii. 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F;
[0392] iv. 4, 6B, 9V, 14, 18C, 19F, 23F, 1, 3, 5, 6A, 7F, 19A, 22F,
and 33F;
[0393] v. 4, 6B, 9V, 14, 18C, 19F, 23F, 1, 3, 5, 7F, 19A, 22F, 33F,
2, 8, 9N, 10A, 11A, 12F, 15B, 17F, and 20; and
[0394] vi. 4, 6B, 9V, 14, 18C, 19F, 23F, 1, 3, 5, 6A, 7F, 19A, 22F,
33F, 8, 10A, 11A, 12F and 15B.
[0395] In specific embodiments of the method above, the multivalent
pneumococcal vaccine comprises capsular polysaccharides of S.
pneumoniae serotypes 4, 6B, 9V, 14, 18C, 19F, 23F, 1, 3, 5, 7F,
19A, 22F, 33F, 2, 8, 9N, 10A, 11A, 12F, 15B, 17F, and 20A.
[0396] In specific embodiments of the method above, the multivalent
pneumococcal vaccine comprises multiple polysaccharide protein
conjugates, wherein the polysaccharide protein conjugates comprise
polysaccharide from a S. pneumoniae serotype conjugated to a
carrier protein. In other embodiments, the multivalent pneumococcal
vaccine comprises multiple S. pneumoniae capsular polysaccharides
that are not conjugated to a carrier protein.
[0397] In additional embodiments of the method above, the patient
is treated with a multivalent immunogenic composition of the
invention and is treated with PREVNAR.RTM. 13 (Pneumococcal
13-valent Conjugate Vaccine [Diphtheria CRM197 Protein], Pfizer,
Inc., Philadelphia, Pa., USA), in any order. In one embodiment, the
patient is administered PREVNAR.RTM. 13 first, and the patient is
administered a multivalent immunogenic composition of the invention
second. In alternative embodiments, the patient is administered a
multivalent immunogenic composition of the invention first and is
administered PREVNAR.RTM. 13 second.
[0398] In further embodiments of the method above, the patient is
treated with a multivalent immunogenic composition of the invention
and is treated with PNEUMOVAX.RTM. 23 (pneumococcal vaccine
polyvalent, Merck & Co., Inc., Kenilworth, NJ, USA), in any
order. In one embodiment, the patient is administered PNEUMOVAX
.RTM. 23 first, and the patient is administered a multivalent
immunogenic composition of the invention second. In alternative
embodiments, the patient is administered a multivalent immunogenic
composition of the invention first and is administered PNEUMOVAX
.RTM. 23 second. In still further embodiments of the method above,
the patient is treated with a multivalent immunogenic composition
of the invention and is treated with SYNFLORIX.TM. (Pneumococcal
polysaccharide conjugate vaccine (adsorbed), GlaxoSmithKline
Biologicals s.a., Rixensart, Belgium), in any order. In one
embodiment, the patient is administered SYNFLORIX.TM. first, and
the patient is administered a multivalent immunogenic composition
of the invention second. In an alternative embodiment, the patient
is administered a multivalent immunogenic composition of the
invention first and is administered SYNFLORIX.TM. second.
[0399] In some embodiments of the method above, the multivalent
immunogenic composition and the multivalent pneumococcal vaccine
are administered concurrently. As used herein, "concurrent
administration" is not limited to dosing of two compositions at the
same time, but includes administration one right after the other in
any order. In some embodiments, the multivalent immunogenic
composition and the multivalent pneumococcal vaccine are
administered via intramuscular or subcutaneous administration into
separate anatomical sites, e.g. two different arms.
[0400] In some embodiments of the method above, the amount of time
between administration of the multivalent immunogenic composition
of the invention and the multivalent pneumococcal vaccine is from
about 4 weeks to about 1 year. In alternative embodiments, the
amount of time is from about 1 month to about 5 years.
[0401] In one embodiment, the patient is administered the
multivalent pneumococcal vaccine first and the multivalent
immunogenic composition of the invention second. In alternative
embodiments, the patient is administered a multivalent immunogenic
composition of the invention first and is administered the
multivalent pneumococcal vaccine second.
[0402] Also provided is a method of inducing an immune response,
vaccinating or inducing a protective immune response against S.
pneumoniae in a patient, comprising:
[0403] (1) administering a multivalent immunogenic composition of
the invention to the patient,
[0404] (2) waiting for a pre-determined amount of time to pass,
and
[0405] (3) administering a multivalent pneumococcal vaccine to the
patient.
[0406] In this method, the multivalent immunogenic composition can
comprise any combination of S. pneumoniae polysaccharide protein
conjugates set forth herein and the multivalent pneumococcal
vaccine can be any vaccine indicated for the prevention of disease
caused by more than one serotype of S. pneumoniae.
[0407] Also provided by the invention is a method of inducing an
immune response, vaccinating or inducing a protective immune
response against S. pneumoniae in a patient, comprising:
[0408] (1) administering a multivalent pneumococcal vaccine to the
patient,
[0409] (2) waiting for a pre-determined amount of time to pass,
and
[0410] (3) administering a multivalent immunogenic composition of
the invention to the patient.
[0411] In this method, the multivalent immunogenic composition can
comprise any combination of S. pneumoniae polysaccharide protein
conjugates set forth herein and the multivalent pneumococcal
vaccine can be any vaccine indicated for the prevention of disease
caused by more than one serotype of S. pneumoniae.
[0412] In some embodiments of the methods above, the multivalent
pneumococcal vaccine comprises multiple S. pneumoniae
polysaccharide protein conjugates, wherein the polysaccharide
protein conjugates comprise capsular polysaccharide from a S.
pneumoniae serotype conjugated to a carrier protein. In alternative
embodiments, the multivalent pneumococcal vaccine comprises S.
pneumoniae capsular polysaccharides that are not conjugated to a
carrier protein.
[0413] In any embodiments of the methods of the invention (i.e. any
of the methods described herein), the method may further comprise
administering one or more additional doses of a multivalent
immunogenic composition of the invention to the patient. In such
methods, the patient may have already received a multivalent
pneumococcal vaccine prior to receiving a first dose of a
multivalent immunogenic composition of the invention, supra, or may
not have been vaccinated against S. pneumoniae prior to receiving a
multivalent immunogenic composition of the invention. Thus, in one
embodiment, a patient who had received a multivalent pneumococcal
vaccine indicated for the prevention of pneumococcal disease caused
by S. pneumoniae is administered two or more doses of a multivalent
immunogenic composition of the invention. In alternative
embodiments, a patient who had not been previously treated with any
vaccine indicated for the prevention of pneumococcal disease, is
administered two or more doses of a multivalent immunogenic
composition of the invention.
[0414] In embodiments of the method above, the two or more doses
are of the same multivalent immunogenic composition of the
invention. In alternative embodiments, the two or more doses are of
different multivalent immunogenic compositions of the invention. In
specific embodiments of any of these methods, the patient is
administered two, three, or four doses of a multivalent immunogenic
composition of the invention. In particular embodiments, the
patient is immunocompromised (e.g., on an immunosuppressive regimen
following a stem cell transplant).
[0415] In some embodiments, the amount of time between
administration of each dose of multivalent immunogenic composition
of the invention is from about 4 weeks to about 1 year. In
alternative embodiment, the amount of time between administration
of each dose of multivalent immunogenic composition of the
invention is from about 1 month to about 5 years.
[0416] In embodiments of any of the methods of the invention, the
patient to be treated with the composition(s) of the invention is a
human. In certain embodiments, the human patient is an infant
(approximately 6 weeks to 12 months). In certain embodiments, the
human patient is a toddler (approximately 12 to 24 months), or
young child (approximately 2 to 5 years). The compositions of this
invention are also suitable for use with older children,
adolescents and adults (e.g., aged 18 to 45 years, aged 18 to 50
years, aged 18 to 55 years, aged 18 to 60 years or 18 to 65 years).
In other embodiments of any of the methods of the invention, the
patient is from about 2 to about 18 years of age. In further
embodiments of any of the methods of the invention, the patient is
18 years of age or older.
[0417] In further embodiments of the methods of the invention, the
patient is an infant and the infant is administered 1, 2, or 3
doses of a multivalent immunogenic composition of the instant
invention. The amount of time between administration of each dose
can vary, but an example of a dosing schedule includes
administration of a dose at 2 months of age, then another
administration of a dose at 4 months of age, and finally a final
administration of a dose at 6 months of age. Another example of an
administration schedule in infants is administration of a dose at 2
months of age and then another administration of a dose at 3 months
of age. Another example of an administration schedule in infants is
administration of a dose at 2 months of age and then another
administration of a dose at 3 months of age, and finally a final
administration of a dose at 6 months of age. In further
embodiments, an infant patient can receive an additional "booster"
dose of a multivalent immunogenic composition of the instant
invention when the infant becomes a toddler. For example, an infant
is dosed at 2 months of age, then another administration of a dose
at 4 months of age, and finally a final administration of a dose at
6 months of age, then, when the infant reaches the age of a
toddler, an additional "booster" dose of a multivalent immunogenic
composition of the instant invention is administered between 11 to
15 months of age.
[0418] In an embodiment, an infant is administered 2 doses of a
multivalent immunogenic composition of the instant invention.
[0419] In an embodiment, an infant is administered 3 doses of a
multivalent immunogenic composition of the instant invention.
[0420] In an embodiment, a patient is administered 3 doses of a
multivalent immunogenic composition of the instant invention,
wherein the first and second doses are administered between 2 and
10 months of age and the third dose is administered between 11 to15
months of age.
[0421] In an embodiment, a patient is administered 4 doses of a
multivalent immunogenic composition of the instant invention,
wherein the first dose is administered at 2 months of age, the
second dose is administered at 4 months of age, the third dose is
administered at 6 months of age and the fourth dose is administered
between 11 to15 months of age.
[0422] In further embodiments of the methods of the invention, the
human patient is elderly. In some embodiments of any of the methods
of the invention, the patient is 50 years of age or older. In some
embodiments of any of the methods of the invention, the patient is
55 years of age or older. In some embodiments of any of the methods
of the invention, the patient is 60 years of age or older. In still
further embodiments of any of the methods of the invention, the
patient is 65 years of age or older. In additional embodiments of
any of the methods of the invention, the patient is 70 years of age
or older.
[0423] In some embodiments of any of the methods of the invention,
the patient to be treated with an immunogenic composition of the
invention is immunocompromised.
[0424] In some embodiments of any of the methods of the invention,
the multivalent immunogenic composition of the invention is
administered concomitantly with a vaccine against influenza. In
certain embodiments, the influenza vaccine is a "senior flu
vaccine," a high dose flu vaccine indicated for the elderly, e.g.
persons aged 65 and older.
[0425] The invention provides a method for inducing a protective
immune response in a patient against a pneumococcal infection
comprising the step of administering to the patient an
immunologically effective amount of any of the multivalent
immunogenic pneumococcal polysaccharide-protein conjugate
compositions described herein. Optimal amounts of components for a
particular vaccine (e.g. a multivalent immunogenic composition of
the invention) can be ascertained by standard studies involving
observation of appropriate immune responses in subjects. For
example, in another embodiment, the dosage for human vaccination is
determined by extrapolation from animal studies to human data. In
another embodiment, the dosage is determined empirically.
[0426] The methods of the invention can be used for the prevention
and/or reduction of primary clinical syndromes caused by microbes,
e.g., S. pneumoniae, including both invasive infections
(meningitis, pneumonia, and bacteremia), and noninvasive infections
(acute otitis media, and sinusitis).
[0427] Administration of the compositions of the invention can
include one or more of: injection via the intramuscular,
intraperitoneal, intradermal or subcutaneous routes; or via mucosal
administration to the oral/alimentary, respiratory or genitourinary
tracts. In one embodiment, intranasal administration is used for
the treatment of pneumonia or otitis media (as nasopharyngeal
carriage of pneumococci can be more effectively prevented, thus
attenuating infection at its earliest stage). In specific
embodiments, the compositions of the invention are administered to
the patient via intramuscular or subcutaneous administration.
[0428] All publications mentioned herein are incorporated by
reference for the purpose of describing and disclosing
methodologies and materials that might be used in connection with
the present invention.
[0429] Having described different embodiments of the invention
herein with reference to the accompanying drawings, it is to be
understood that the invention is not limited to those precise
embodiments, and that various changes and modifications may be
effected therein by one skilled in the art without departing from
the scope or spirit of the invention as defined in the appended
claims.
[0430] The following examples illustrate, but do not limit the
invention.
EXAMPLE 1
[0431] Preparation of S. pneumoniae Capsular Polysaccharides
[0432] Methods of culturing pneumococci are well known in the art.
See, e.g., Chase, 1967, Methods of Immunology and Immunochemistry
1:52. Methods of preparing pneumococcal capsular polysaccharides
are also well known in the art. See, e.g., European Patent No. EP 0
497 524 B1. The process described below generally follows the
method described in European Patent No. EP 0 497 524 B1 and is
generally applicable to all pneumococcal serotypes.
[0433] Isolates of pneumococcal strains for serotypes 1, 3, 4, 5,
6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F,
23F, 33F, and 35B were obtained from Merck Culture Collection.
Strains for serotypes 23B were obtained from Centers of Disease
Control and Prevention and University of Alabama Birmingham.
Strains for serotype 24F were obtained from Merck Culture
Collection and University of Alabama Birmingham. Where needed,
subtypes were differentiated on the basis of Quellung reaction
using specific antisera. See, e.g.,
[0434] U.S. Pat. No. 5,847,112. The obtained isolates were further
clonally isolated by plating serially in two stages on agar plates
consisting of an animal-component free medium containing soy
peptone, yeast extract, and glucose without hemin. For serotype 7F,
the agar plates used also contained hemin. Clonal isolates for each
serotype were further expanded in liquid culture using
animal-component free media containing soy peptone, yeast extract,
HEPES, sodium chloride, sodium bicarbonate, potassium phosphate,
glucose, and glycerol to prepare the pre-master cell banks.
[0435] The production of each serotype of pneumococcal
polysaccharide consisted of a cell expansion and batch production
fermentation followed by chemical inactivation prior to downstream
purification. A thawed cell bank vial from each serotype was
expanded using a shake flask or culture bottle containing a
pre-sterilized animal-component free growth media containing soy
peptone or soy peptone ultrafiltrate, yeast extract or yeast
extract ultrafiltrate, HEPES, sodium chloride, sodium bicarbonate,
potassium phosphate, and glucose. The cell expansion culture was
grown in a sealed shake flask or bottle to minimize gas exchange
with temperature and agitation control. During the cell expansion
of these serotypes, temperature, pH, pressure, and agitation were
controlled. Airflow overlay was also controlled as sparging was not
used. After achieving a specified culture density, as measured by
optical density at 600 nm, a portion of the cell expansion culture
was transferred to a production fermentor containing pre-sterilized
animal-component free growth media containing soy peptone or soy
peptone ultrafiltrate, yeast extract or yeast extract
ultrafiltrate, sodium chloride, potassium phosphate, and glucose.
Temperature, pH, pressure, and agitation were controlled. Airflow
overlay was also controlled as sparging was not used.
[0436] The batch fermentation was terminated via the addition of a
chemical inactivating agent, phenol, when glucose was nearly
exhausted. Pure phenol was added to a final concentration of
0.8-1.2% to inactivate the cells and liberate the capsular
polysaccharide from the cell wall. Primary inactivation occurs for
a specified time within the fermentor where temperature and
agitation continue to be controlled. After primary inactivation,
the batch was transferred to another vessel where it was held for
an additional specified time at controlled temperature and
agitation for complete inactivation. This was confirmed by either
microbial plating techniques or by verification of the phenol
concentration and specified time. The inactivated broth was then
purified.
EXAMPLE 2
Purification of Pneumococcal Polysaccharides
[0437] The purification process for the pneumococcal
polysaccharides consisted of several centrifugation, depth
filtration, concentration/diafiltration operations, and
precipitation steps. All procedures were performed at room
temperature unless otherwise specified.
[0438] Inactivated broth from the fermentor cultures of S.
pneumoniae were flocculated with a cationic polymer (such as
BPA-1000, TRETOLITE.RTM. (Baker Hughes Inc., Houston, Tex.),
Spectrum 8160, poly(ethyleneimine), and Millipore pDADMAC). The
cationic polymers binded to the impurity proteins, nucleic acids
and cell debris. Following the flocculation step and an aging
period, flocculated solids were removed via centrifugation and
multiple depth filtration steps. Clarified broth was concentrated
and diafiltered using a 100 kDa to 500 kDa MWCO (molecular weight
cutoff) filter. Diafiltration was accomplished using Tris,
MgCl.sub.2 buffer and sodium phosphate buffer. Diafiltration
removed residual nucleic acid and protein.
[0439] Removal of further impurities was accomplished by
reprecipitation of the polysaccharide in sodium acetate and phenol
with denatured alcohol and/or isopropanol. During the phenol
precipitation step, sodium acetate in sodium phosphate saline
buffer and phenol (liquefied phenols or solid phenols) were charged
to the diafiltered retentate. Alcohol fractionation of the
polysaccharide was then conducted in two stages. In the first stage
a low percent alcohol was added to the preparation to precipitate
cellular debris and other unwanted impurities, while the crude
polysaccharide remained in solution. The impurities were removed
via centrifugation followed by a depth filtration step. The
polysaccharide was then recovered from the solution by adding
additional isopropanol or denatured alcohol to the batch. The
precipitated polysaccharide pellet was recovered by centrifugation,
triturated and dried as a powder and stored frozen at -70.degree.
C.
EXAMPLE 3
Preparation of Serotype 1 Conjugate for PCV23 (DMSO) Polyvalent
Study Using DMSO Conjugation
[0440] Polysaccharide was dissolved, sized to a target molecular
mass, chemically activated and buffer-exchanged by ultrafiltration.
Activated polysaccharide and purified CRM197 were individually
lyophilized and redissolved in dimethylsulfoxide (DMSO).
Redissolved polysaccharide and CRM197 solutions were then combined
and conjugated as described below. The resulting conjugate was
purified by dialysis prior to a final 0.2-micron filtration.
Several process parameters within each step, such as pH,
temperature, concentration, and time were controlled to yield
conjugates with desired attributes.
Polysaccharide Size Reduction and Oxidation
[0441] Purified pneumococcal capsular Ps powder was dissolved in
water and 0.45-micron filtered. Dissolved polysaccharide was
homogenized to reduce the molecular mass of the Ps. Homogenization
pressure and number of passes through the homogenizer were
controlled to 250 bar/5 passes.
[0442] Size-reduced polysaccharide was concentrated and diafiltered
against water using a 10 kDa NMWCO tangential flow ultrafiltration
membrane.
[0443] The polysaccharide solution was then adjusted to 22.degree.
C. and pH 5 with a sodium acetate buffer to minimize polysaccharide
size reduction due to activation. Polysaccharide activation was
initiated with the addition of a 100 mM sodium metaperiodate
solution. The oxidation reaction proceeded for 15 hours at
22.degree. C.
[0444] The activated product was diafiltered against 10 mM
potassium phosphate, pH 6.4 using a 10 kDa NMWCO tangential flow
ultrafiltration membrane followed by diafiltration against water
using a 5 kDa NMWCO tangential flow ultrafiltration membrane.
Ultrafiltration was conducted at 2-8.degree. C.
Polysaccharide Conjugation to CRM197
[0445] Purified CRM197, obtained through expression in Pseudomonas
fluorescens as previously described (WO 2012/173876 A1), was
diafiltered against 2 mM phosphate, pH 7.2 buffer using a 5 kDa
NMWCO tangential flow ultrafiltration membrane and 0.2-micron
filtered.
[0446] Activated polysaccharide was formulated for lyophilization
at 2.5 mg Ps/mL with sucrose concentration of 10% w/v. CRM197 was
formulated for lyophilization at 6 mg Pr/mL with sucrose
concentration of 1% w/v.
[0447] Formulated Ps and CRM197 solutions were individually
lyophilized. Lyophilized Ps and CRM197 materials were redissolved
individually in equal volumes of DMSO. The polysaccharide and
CRM197 solutions were blended to achieve a polysaccharide
concentration of 1.0 g Ps/L and a polysaccharide to CRM197 mass
ratio of 1.5. The mass ratio was selected to control the
polysaccharide to CRM197 ratio in the resulting conjugate. Sodium
cyanoborohydride (1 mole per mole of polysaccharide repeating unit)
was added, and conjugation proceeded at 22.degree. C.
Reduction with Sodium Borohydride
[0448] Sodium borohydride (2 moles per mole of polysaccharide
repeating unit) was added following the conjugation reaction and
incubated for 1 hour at 22.degree. C. The batch was diluted into
150 mM sodium chloride, with approximately 0.025% (w/v) polysorbate
20, at approximately 4.degree. C. Potassium phosphate buffer was
then added to neutralize the pH. The batch was dialyzed at
approximately 4.degree. C. for 3 days against 150 mM sodium
chloride, 0.05% (w/v) polysorbate 20, using a 300 kDa NMWCO
dialysis casette.
Final Filtration and Product Storage
[0449] The batch was 0.2 micron filtered (with 0.5 micron
prefilter) , dispensed into aliquots and frozen at
.ltoreq.-60.degree. C.
EXAMPLE 4
Preparation of Serotype 1 Conjugate for PCV23 (DMSO+Aq) Polyvalent
Study Using Aqueous Conjugation
[0450] Polysaccharide was dissolved, size reduced, chemically
activated and buffer-exchanged by ultrafiltration. Purified CRM197
was then conjugated to the activated polysaccharide utilizing
nickel chloride in the reaction mixture, and the resulting
conjugate was purified by ultrafiltration prior to a final
0.2-micron filtration. Several process parameters within each step,
such as pH, temperature, concentration, and time were controlled to
yield conjugates with desired attributes.
Polysaccharide Size Reduction and Oxidation
[0451] Purified pneumococcal capsular polysaccharide powder was
dissolved in water, and 0.45-micron filtered. Dissolved
polysaccharide was homogenized to reduce the molecular mass.
Homogenization pressure and number of passes through the
homogenizer were controlled to 250 bar/5 passes to size-reduce to a
target molecular mass. Size-reduced polysaccharide was then
concentrated and diafiltered against water using a 10 kDa NMWCO
tangential flow ultrafiltration membrane.
[0452] The polysaccharide solution was then adjusted to 22.degree.
C. and pH 5 with a sodium acetate buffer to minimize polysaccharide
size reduction due to activation. Polysaccharide activation was
initiated with the addition of a 100 mM sodium metaperiodate
solution. The oxidation reaction proceeded for 15 hours at
22.degree. C.
[0453] The activated product was diafiltered against 10 mM
potassium phosphate, pH 6.4 using a 10 kDa NMWCO tangential flow
ultrafiltration membrane. Ultrafiltration was conducted at
2-8.degree. C.
Polysaccharide Conjugation to CRM197
[0454] Oxidized polysaccharide solution was mixed with water and
1.5 M potassium phosphate pH 7.0. The buffer pH selected was to
improve the stability of activated polysaccharide during the
conjugation reaction. Purified CRM197, obtained through expression
in Pseudomonas fluorescens as previously described (WO 2012/173876
A1), was combined with the buffered polysaccharide solution at a
polysaccharide to CRM197 mass ratio of 0.5. The mass ratio was
selected to control the polysaccharide to CRM197 ratio in the
resulting conjugate. The polysaccharide and phosphate
concentrations were 6.9 g/L and 100 mM respectively. The
polysaccharide concentration was selected to control the size of
the resulting conjugate. Nickel chloride was added to approximately
2 mM using a 100 mM nickel chloride solution. Sodium
cyanoborohydride (2 moles per mole of polysaccharide repeating
unit) was added. Conjugation proceeded for 120 hours to maximize
consumption of polysaccharide and protein.
Reduction with Sodium Borohydride
[0455] Following the conjugation reaction, the batch was diluted to
a polysaccharide concentration of approximately 3.5 g/L, cooled to
2-8.degree. C., and 1.2-micron filtered. The batch was diafiltered
against 100 mM potassium phosphate, pH 7.0 at 2-8.degree. C. using
a 100 kDa NMWCO tangential flow ultrafiltration membrane. The
batch, recovered in the retentate, was then diluted to
approximately 2.0 g polysaccharide/L and pH-adjusted with the
addition of 1.2 M sodium bicarbonate, pH 9.4. Sodium borohydride (1
mole per mole of polysaccharide repeating unit) was added. 1.5 M
potassium phosphate, pH 6.0 was later added.
Final Filtration and Product Storage
[0456] The batch was then concentrated and diaftiltered against 10
mM L-histidine in 150 mM sodium chloride, pH 7.0 at 4.degree. C.
using a 300 kDa NMWCO tangential flow ultrafiltration membrane.
Polysorbate 20 was added to the retentate batch to a concentration
of 0.05% (w/v) then the batch was 0.2 micron filtered.
[0457] The batch was adjusted to a polysaccharide concentration of
1.0 g/L with additional 10 mM L-histidine in 150 mM sodium
chloride, pH 7.0 buffer with 0.015% (w/v) polysorbate 20. The batch
was dispensed into aliquots and frozen at .ltoreq.-60.degree.
C.
EXAMPLE 5
Preparation of Serotype 1 Conjugate for PCV22 Polyvalent Study
Using Aqueous Conjugation
[0458] Polysaccharide was dissolved, size reduced, chemically
activated and buffer-exchanged by ultrafiltration. Purified CRM197
was then conjugated to the activated polysaccharide utilizing
nickel chloride in the reaction mixture, and the resulting
conjugate was purified by ultrafiltration prior to a final
0.2-micron filtration. Several process parameters within each step,
such as pH, temperature, concentration, and time were controlled to
yield conjugates with desired attributes.
Polysaccharide Size Reduction and Oxidation
[0459] Purified pneumococcal capsular polysaccharide powder was
dissolved in water, and 0.45-micron filtered. Dissolved
polysaccharide was homogenized to reduce the molecular mass.
Homogenization pressure and number of passes through the
homogenizer were controlled to 250 bar/5 passes to size-reduce to a
target molecular mass. Size-reduced polysaccharide was then
concentrated and diafiltered against water using a 10 kDa NMWCO
tangential flow ultrafiltration membrane.
[0460] The polysaccharide solution was then adjusted to 22.degree.
C. and pH 5 with a sodium acetate buffer to minimize polysaccharide
size reduction due to activation. Polysaccharide activation was
initiated with the addition of a 100 mM sodium metaperiodate
solution. The oxidation reaction proceeded for 15 hours at
22.degree. C.
[0461] The activated product was diafiltered against 10 mM
potassium phosphate, pH 6.4 using a 10 kDa NMWCO tangential flow
ultrafiltration membrane. Ultrafiltration was conducted at
2-8.degree. C.
Polysaccharide Conjugation to CRM197
[0462] Oxidized polysaccharide solution was mixed with water and
1.5 M potassium phosphate pH 7.0. The buffer pH selected was to
improve the stability of activated polysaccharide during the
conjugation reaction. Purified CRM197, obtained through expression
in Pseudomonas fluorescens as previously described (WO 2012/173876
A1), was combined with the buffered polysaccharide solution at a
polysaccharide to CRM197 mass ratio of 0.5. The mass ratio was
selected to control the polysaccharide to CRM197 ratio in the
resulting conjugate. The polysaccharide and phosphate
concentrations were 6.9 g/L and 100 mM respectively. The
polysaccharide concentration was selected to control the size of
the resulting conjugate. The solution was then 0.2-micron filtered.
Nickel chloride was added to approximately 2 mM using a 100 mM
nickel chloride solution. Sodium cyanoborohydride (2 moles per mole
of polysaccharide repeating unit) was added. Conjugation proceeded
for 120 hours to maximize consumption of polysaccharide and
protein.
Reduction with Sodium Borohydride
[0463] Following the conjugation reaction, the batch was diluted to
a polysaccharide concentration of approximately 3.5 g/L, cooled to
2-8.degree. C., and 1.2-micron filtered. The batch was diafiltered
against 100 mM potassium phosphate, pH 7.0 at 2-8.degree. C. using
a 100 kDa NMWCO tangential flow ultrafiltration membrane. The
batch, recovered in the retentate, was then diluted to
approximately 2.0 g polysaccharide/L and pH-adjusted with the
addition of 1.2 M sodium bicarbonate, pH 9.4. Sodium borohydride (1
mole per mole of polysaccharide repeating unit) was added. 1.5 M
potassium phosphate, pH 6.0 was later added.
Final Filtration and Product Storage
[0464] The batch was then concentrated and diaftiltered against 10
mM L-histidine in 150 mM sodium chloride, pH 7.0 at 4.degree. C.
using a 300 kDa NMWCO tangential flow ultrafiltration membrane.
Polysorbate 20 was added to the retentate batch to a concentration
of 0.05% (w/v) then the batch was 0.2 micron filtered.
[0465] The batch was adjusted to a polysaccharide concentration of
1.0 g/L with additional 10 mM L-histidine in 150 mM sodium
chloride, pH 7.0 buffer with 0.015% (w/v) polysorbate 20. The batch
was dispensed into aliquots and frozen at .ltoreq.-60.degree.
C.
EXAMPLE 6
Preparation of Serotype 3 Conjugate for PCV 23 (DMSO) Polyvalent
Study Using DMSO Conjugation
[0466] Polysaccharide was dissolved, sized to a target molecular
mass, chemically activated and buffer-exchanged by ultrafiltration.
Activated polysaccharide and purified CRM197 were individually
lyophilized and redissolved in dimethylsulfoxide (DMSO).
Redissolved polysaccharide and CRM197 solutions were then combined
and conjugated as described below.
[0467] The resulting conjugate was purified by ultrafiltration
prior to a final 0.2-micron filtration. Several process parameters
within each step, such as pH, temperature, concentration, and time
were controlled to yield conjugates with desired attributes.
Polysaccharide Size Reduction and Oxidation
[0468] Purified pneumococcal capsular Ps powder was dissolved in
water and 0.45-micron filtered. Dissolved polysaccharide was
homogenized to reduce the molecular mass of the Ps. Homogenization
pressure and number of passes through the homogenizer were
controlled to 810 bar/6 passes followed by 900 bar/3 passes.
[0469] Size-reduced polysaccharide was concentrated and diafiltered
against water using a 5 kDa NMWCO tangential flow ultrafiltration
membrane.
[0470] The polysaccharide solution was then adjusted to 22.degree.
C. and pH 5 with a sodium acetate buffer to minimize polysaccharide
size reduction due to activation. Polysaccharide activation was
initiated with the addition of a 100 mM sodium metaperiodate
solution. The oxidation reaction proceeded for 12 hours at
22.degree. C.
[0471] The activated product was diafiltered against 10 mM
potassium phosphate, pH 6.4 followed by diafiltration against water
using a 5 kDa NMWCO tangential flow ultrafiltration membrane.
Ultrafiltration was conducted at 2-8.degree. C.
Polysaccharide Conjugation to CRM197
[0472] Purified CRM197, obtained through expression in Pseudomonas
fluorescens as previously described (WO 2012/173876 A1), was
diafiltered against 2 mM phosphate, pH 7.2 buffer using a 5 kDa
NMWCO tangential flow ultrafiltration membrane and 0.2-micron
filtered.
[0473] Activated polysaccharide was formulated for lyophilization
at 2.5 mg Ps/mL with sucrose concentration of 10% w/v. CRM197 was
formulated for lyophilization at 6 mg Pr/mL with sucrose
concentration of 1% w/v.
[0474] Formulated Ps and CRM197 solutions were individually
lyophilized. Lyophilized Ps and CRM197 materials were redissolved
individually in equal volumes of DMSO. The polysaccharide and
CRM197 solutions were blended to achieve a polysaccharide
concentration of 2.25 g Ps/L and a polysaccharide to CRM197 mass
ratio of 1.35. The mass ratio was selected to control the
polysaccharide to CRM197 ratio in the resulting conjugate. Sodium
cyanoborohydride (1 mole per mole of polysaccharide repeating unit)
was added, and conjugation proceeded at 22.degree. C.
Reduction with Sodium Borohydride
[0475] Sodium borohydride (2 moles per mole of polysaccharide
repeating unit) was added following the conjugation reaction and
incubated for 1 hour at 22.degree. C. The batch was diluted into
150 mM sodium chloride, with approximately 0.025% (w/v) polysorbate
20, at approximately 4.degree. C. Potassium phosphate buffer was
then added to neutralize the pH.
Final Filtration and Product Storage
[0476] The batch was then concentrated and diafiltered against 10
mM histidine in 150 mM sodium chloride, pH 7.0, with 0.015% (w/v)
polysorbate 20, at 4.degree. C. using a 300 kDa NMWCO tangential
flow ultrafiltration membrane.
[0477] The retentate batch was 0.2 micron filtered (with 0.5 micron
prefilter) then diluted with additional 10 mM histidine in 150 mM
sodium chloride, pH 7.0 with 0.015% (w/v) polysorbate 20, dispensed
into aliquots and frozen at .ltoreq.-60.degree. C.
EXAMPLE 7
Preparation of Serotype 3 Conjugate for PCV22 and PCV23 (DMSO+Aq)
Polyvalent Studies Using Aqueous Conjugation
[0478] Polysaccharide was dissolved, size reduced, chemically
activated and buffer-exchanged by ultrafiltration. Purified CRM197
was then conjugated to the activated polysaccharide utilizing
nickel chloride in the reaction mixture, and the resulting
conjugate was purified by ultrafiltration prior to a final
0.2-micron filtration. Several process parameters within each step,
such as pH, temperature, concentration, and time were controlled to
yield conjugates with desired attributes.
Polysaccharide Size Reduction and Oxidation
[0479] Purified pneumococcal capsular polysaccharide powder was
dissolved in water, and 0.45-micron filtered. Dissolved
polysaccharide was homogenized to reduce the molecular mass.
Homogenization pressure and number of passes through the
homogenizer were controlled to 380 bar/5 passes to size-reduce to a
target molecular mass. Size-reduced polysaccharide was then
concentrated and diafiltered against water using a 10 kDa NMWCO
tangential flow ultrafiltration membrane.
[0480] The polysaccharide solution was then adjusted to 22.degree.
C. and pH 5 with a sodium acetate buffer to minimize polysaccharide
size reduction due to activation. Polysaccharide activation was
initiated with the addition of a 100 mM sodium metaperiodate
solution. The oxidation reaction proceeded for 12 hours at
22.degree. C.
[0481] The activated product was diafiltered against 10 mM
potassium phosphate, pH 6.4 using a 10 kDa NMWCO tangential flow
ultrafiltration membrane. Ultrafiltration was conducted at
2-8.degree. C.
Polysaccharide Conjugation to CRM197
[0482] Oxidized polysaccharide solution was mixed with water and
1.5 M potassium phosphate pH 6.0. The buffer pH selected was to
improve the stability of activated polysaccharide during the
conjugation reaction. Purified CRM197, obtained through expression
in Pseudomonas fluorescens as previously described (WO 2012/173876
A1), was 0.2-micron filtered and combined with the buffered
polysaccharide solution at a polysaccharide to CRM197 mass ratio of
0.6. The mass ratio was selected to control the polysaccharide to
CRM197 ratio in the resulting conjugate. The polysaccharide and
phosphate concentrations were 4.1 g/L and 150 mM respectively. The
polysaccharide concentration was selected to control the size of
the resulting conjugate. The solution was then 0.2-micron filtered.
Nickel chloride was added to approximately 2 mM using a 100 mM
nickel chloride solution. Sodium cyanoborohydride (2 moles per mole
of polysaccharide repeating unit) was added. Conjugation proceeded
for 120 hours at 10.degree. C. to maximize consumption of
polysaccharide and protein.
Reduction with Sodium Borohydride
[0483] Following the conjugation reaction, the batch was diluted to
a polysaccharide concentration of approximately 3.5 g/L, cooled to
2-8.degree. C., and 1.2-micron filtered. The batch was diafiltered
against 100 mM potassium phosphate, pH 7.0 at 2-8.degree. C. using
a 100 kDa NMWCO tangential flow ultrafiltration membrane. The
batch, recovered in the retentate, was then diluted to
approximately 2.0 g polysaccharide/L and pH-adjusted with the
addition of 1.2 M sodium bicarbonate, pH 9.4. Sodium borohydride (1
mole per mole of polysaccharide repeating unit) was added. 1.5 M
potassium phosphate, pH 6.0 was later added.
[0484] Final filtration and product storage
[0485] The batch was then concentrated and diaftiltered against 10
mM L-histidine in 150 mM sodium chloride, pH 7.0 at 4.degree. C.
using a 300 kDa NMWCO tangential flow ultrafiltration membrane. The
batch was 0.2 micron filtered.
[0486] The batch was adjusted to a polysaccharide concentration of
1.0 g/L with additional 10 mM L-histidine in 150 mM sodium
chloride, pH 7.0 buffer. The batch was dispensed into aliquots and
frozen at .ltoreq.-60.degree. C.
EXAMPLE 8
Preparation of Serotype 4 Conjugate for PCV23 (DMSO) Polyvalent
Study Using DMSO Conjugation
[0487] Polysaccharide was dissolved, sized to a target molecular
mass, chemically activated and buffer-exchanged by ultrafiltration.
Activated polysaccharide and purified CRM197 were individually
lyophilized and redissolved in DMSO. Redissolved polysaccharide and
CRM197 solutions were then combined and conjugated as described
below. The resulting conjugate was purified by dialysis prior to a
final 0.2-micron filtration. Several process parameters within each
step, such as pH, temperature, concentration, and time were
controlled to yield conjugates with desired attributes.
Polysaccharide Size Reduction and Oxidation
[0488] Purified pneumococcal capsular Ps powder was dissolved in
water and 0.45-micron filtered. Dissolved polysaccharide was
homogenized to reduce the molecular mass of the Ps. Homogenization
pressure and number of passes through the homogenizer were
controlled to 300 bar/5 passes.
[0489] Size-reduced polysaccharide was concentrated and diafiltered
against water using a 10 kDa NMWCO tangential flow ultrafiltration
membrane.
[0490] The polysaccharide solution was then adjusted to 50.degree.
C. and pH 4.1 with a sodium acetate buffer to partially deketalize
the polysaccharide. The polysaccharide solution was then cooled to
22.degree. C. prior to activation. Polysaccharide activation was
initiated with the addition of a 100 mM sodium metaperiodate
solution. The oxidation reaction proceeded for 4 hours at
22.degree. C.
[0491] The activated product was diafiltered against 10 mM
potassium phosphate, pH 6.4 using a 10 kDa NMWCO tangential flow
ultrafiltration membrane followed by diafiltration against water
using a 5 kDa NMWCO tangential flow ultrafiltration membrane.
Ultrafiltration was conducted at 2-8.degree. C.
[0492] Polysaccharide Conjugation to CRM197
[0493] Purified CRM197, obtained through expression in Pseudomonas
fluorescens as previously described (WO 2012/173876 A1), was
diafiltered against 2 mM phosphate, pH 7.2 buffer using a 5 kDa
NMWCO tangential flow ultrafiltration membrane and 0.2-micron
filtered.
[0494] Activated polysaccharide was formulated for lyophilization
at 6 mg Ps/mL with sucrose concentration of 5% w/v. CRM197 was
formulated for lyophilization at 6 mg Pr/mL with sucrose
concentration of 1% w/v.
[0495] Formulated Ps and CRM197 solutions were individually
lyophilized. Lyophilized Ps and CRM197 materials were redissolved
individually in equal volumes of DMSO. The polysaccharide and
CRM197 solutions were blended to achieve a polysaccharide
concentration of 5.0 g Ps/L and a polysaccharide to CRM197 mass
ratio of 2.0. The mass ratio was selected to control the
polysaccharide to CRM197 ratio in the resulting conjugate. Sodium
cyanoborohydride (1 mole per mole of polysaccharide repeating unit)
was added, and conjugation proceeded at 22.degree. C.
Reduction with Sodium Borohydride
[0496] Sodium borohydride (2 moles per mole of polysaccharide
repeating unit) was added following the conjugation reaction and
incubated for 1 hour at 22.degree. C. The batch was diluted into
150 mM sodium chloride, with approximately 0.025% (w/v) polysorbate
20, at approximately 4.degree. C. Potassium phosphate buffer was
then added to neutralize the pH. The batch was dialyzed at
approximately 4.degree. C. for 3 days against 150 mM sodium
chloride, 0.05% (w/v) polysorbate 20, using a 300 kDa NMWCO
dialysis cassette.
Final Filtration and Product Storage
[0497] The batch was 0.2 micron filtered (with 0.5 micron
prefilter), dispensed into aliquots and frozen at
.ltoreq.-60.degree. C.
EXAMPLE 9
Preparation of Serotype 4 Conjugate for PCV23 (DMSO+Aq) and PCV22
Polyvalent Studies Using Aqueous Conjugation
[0498] Polysaccharide was dissolved, size reduced, chemically
activated and buffer-exchanged by ultrafiltration. Purified CRM197
was then conjugated to the activated polysaccharide utilizing
nickel chloride in the reaction mixture, and the resulting
conjugate was purified by ultrafiltration prior to a final
0.2-micron filtration. Several process parameters within each step,
such as pH, temperature, concentration, and time were controlled to
yield conjugates with desired attributes.
Polysaccharide Size Reduction and Oxidation
[0499] Purified pneumococcal capsular polysaccharide powder was
dissolved in water, and 0.45-micron filtered. Dissolved
polysaccharide was homogenized to reduce the molecular mass.
Homogenization pressure and number of passes through the
homogenizer were controlled to 300 bar/5 passes to size-reduce to a
target molecular mass. Size-reduced polysaccharide was then
concentrated and diafiltered against water using a 10 kDa NMWCO
tangential flow ultrafiltration membrane.
[0500] The polysaccharide solution was then adjusted to 50.degree.
C. and pH 4.1 with a sodium acetate buffer to partially deketalize
the polysaccharide. The polysaccharide solution was then cooled to
22.degree. C. prior to activation. Polysaccharide activation was
initiated with the addition of a 100 mM sodium metaperiodate
solution. The oxidation reaction proceeded for 4 hours at
22.degree. C. The activated product was diafiltered against 10 mM
potassium phosphate, pH 6.4 using a 10 kDa NMWCO tangential flow
ultrafiltration membrane. Ultrafiltration was conducted at
2-8.degree. C.
[0501] Polysaccharide Conjugation to CRM197
[0502] Oxidized polysaccharide solution was mixed with water and
1.5 M potassium phosphate pH 7.0. The buffer pH selected was to
improve the stability of activated polysaccharide during the
conjugation reaction. Purified CRM197, obtained through expression
in Pseudomonas fluorescens as previously described (WO 2012/173876
A1), was 0.2-micron filtered and combined with the buffered
polysaccharide solution at a polysaccharide to CRM197 mass ratio of
0.5. The mass ratio was selected to control the polysaccharide to
CRM197 ratio in the resulting conjugate. The polysaccharide and
phosphate concentrations were 8.3 g/L and 100 mM respectively. The
polysaccharide concentration was selected to control the size of
the resulting conjugate. The solution was then 0.2-micron filtered.
Nickel chloride was added to approximately 2 mM using a 100 mM
nickel chloride solution. Sodium cyanoborohydride (2 moles per mole
of polysaccharide repeating unit) was added. Conjugation proceeded
for 120 hours to maximize consumption of polysaccharide and
protein.
[0503] Reduction with Sodium Borohydride
[0504] Following the conjugation reaction, the batch was diluted to
a polysaccharide concentration of approximately 3.5 g/L, cooled to
2-8.degree. C., and 1.2-micron filtered. The batch was diafiltered
against 100 mM potassium phosphate, pH 7.0 at 2-8.degree. C. using
a 100 kDa NMWCO tangential flow ultrafiltration membrane. The
batch, recovered in the retentate, was then diluted to
approximately 2.0 g polysaccharide/L and pH-adjusted with the
addition of 1.2 M sodium bicarbonate, pH 9.4. Sodium borohydride (1
mole per mole of polysaccharide repeating unit) was added. 1.5 M
potassium phosphate, pH 6.0 was later added.
Final Filtration and Product Storage
[0505] The batch was then concentrated and diaftiltered against 10
mM L-histidine in 150 mM sodium chloride, pH 7.0 at 4.degree. C.
using a 300 kDa NMWCO tangential flow ultrafiltration membrane.
Then the batch was 0.2 micron filtered.
[0506] The batch was adjusted to a polysaccharide concentration of
1.0 g/L with additional 10 mM L-histidine in 150 mM sodium
chloride, pH 7.0 buffer. The batch was dispensed into aliquots and
frozen at .ltoreq.-60.degree. C.
EXAMPLE 10
Preparation of Serotype 5 Conjugate for PCV23 (DMSO) Polyvalent
Study Using DMSO Conjugation
[0507] Polysaccharide was dissolved, sized to a target molecular
mass, chemically activated and buffer-exchanged by ultrafiltration.
Activated polysaccharide and purified CRM197 were individually
lyophilized and redissolved in DMSO. Redissolved polysaccharide and
CRM197 solutions were then combined and conjugated as described
below. The resulting conjugate was purified by dialysis prior to a
final 0.2-micron filtration. Several process parameters within each
step, such as pH, temperature, concentration, and time were
controlled to yield conjugates with desired attributes.
Polysaccharide Size Reduction and Oxidation
[0508] Purified pneumococcal capsular Ps powder was dissolved in
water and 0.45-micron filtered. Dissolved polysaccharide was
homogenized to reduce the molecular mass of the Ps. Homogenization
pressure and number of passes through the homogenizer were
controlled to 600 bar/5 passes.
[0509] Size-reduced polysaccharide was concentrated and diafiltered
against water using a 10 kDa NMWCO tangential flow ultrafiltration
membrane.
[0510] The polysaccharide solution was then adjusted to 4.degree.
C. and pH 4.1 with a sodium acetate buffer to minimize
polysaccharide size reduction due to activation. Polysaccharide
activation was initiated with the addition of a 100 mM sodium
metaperiodate solution. The oxidation reaction proceeded for 4
hours at 4.degree. C. The activated product was diafiltered against
10mM Sodium Acetate, pH 4.1 using a 10 kDa NMWCO tangential flow
ultrafiltration membrane followed by diafiltration against water
using a 5 kDa NMWCO tangential flow ultrafiltration membrane.
Ultrafiltration was conducted at 2-8.degree. C.
Polysaccharide Conjugation to CRM197
[0511] Purified CRM197, obtained through expression in Pseudomonas
fluorescens as previously described (WO 2012/173876 A1), was
diafiltered against 2 mM phosphate, pH 7.2 buffer using a 5 kDa
NMWCO tangential flow ultrafiltration membrane and 0.2-micron
filtered.
[0512] Activated polysaccharide was formulated for lyophilization
at 6 mg Ps/mL with sucrose concentration of 5% w/v. CRM197 was
formulated for lyophilization at 6 mg Pr/mL with sucrose
concentration of 1% w/v.
[0513] Formulated Ps and CRM197 solutions were individually
lyophilized. Lyophilized Ps and CRM197 materials were redissolved
individually in equal volumes of DMSO. The polysaccharide solution
was spiked with sodium chloride to a concentration of 20 mM. The
polysaccharide and CRM197 solutions were blended to achieve a
polysaccharide concentration of 2.0 g Ps/L and a polysaccharide to
CRM197 mass ratio of 1.5. The mass ratio was selected to control
the polysaccharide to CRM197 ratio in the resulting conjugate.
Sodium cyanoborohydride (1 mole per mole of polysaccharide
repeating unit) was added, and conjugation proceeded at 22.degree.
C.
Dilution and Neutralization
[0514] The batch was diluted into 150 mM sodium chloride, with
approximately 0.025% (w/v) polysorbate 20, at approximately
4.degree. C. Potassium phosphate buffer was then added to
neutralize the pH. The batch was dialyzed at approximately
4.degree. C. for 3 days against 150 mM sodium chloride, 0.05% (w/v)
polysorbate 20, using a 300 kDa NMWCO dialysis cassette.
Final Filtration and Product Storage
[0515] The retentate batch was 0.2 micron filtered (with 0.5 micron
prefilter), dispensed into aliquots and frozen at
.ltoreq.-60.degree. C.
EXAMPLE 11
[0516] Preparation of Serotype 5 Conjugate for PCV22 and PCV23
(DMSO+Aq) Polyvalent Studies Using Aqueous Conjugation
[0517] Polysaccharide was dissolved, size reduced, chemically
activated and buffer-exchanged by ultrafiltration. Purified CRM197
was then conjugated to the activated polysaccharide utilizing
nickel chloride in the reaction mixture, and the resulting
conjugate was purified by ultrafiltration prior to a final
0.2-micron filtration. Several process parameters within each step,
such as pH, temperature, concentration, and time were controlled to
yield conjugates with desired attributes.
Polysaccharide Size Reduction and Oxidation
[0518] Purified pneumococcal capsular polysaccharide powder was
dissolved in water, and 0.45-micron filtered. Dissolved
polysaccharide was homogenized to reduce the molecular mass.
Homogenization pressure and number of passes through the
homogenizer were controlled to 600 bar/5 passes to size-reduce to a
target molecular mass. Size-reduced polysaccharide was then
concentrated and diafiltered against water using a 10 kDa NMWCO
tangential flow ultrafiltration membrane.
[0519] The polysaccharide solution was then adjusted to 4.degree.
C. and pH 4.1 with a sodium acetate buffer to minimize
polysaccharide size reduction due to activation. Polysaccharide
activation was initiated with the addition of a 100 mM sodium
metaperiodate solution. The oxidation reaction proceeded for 4
hours at 4.degree. C.
[0520] The activated product was diafiltered against 10 mM sodium
acetate, pH 4.1 using a 10 kDa NMWCO tangential flow
ultrafiltration membrane. Ultrafiltration was conducted at
2-8.degree. C.
Polysaccharide Conjugation to CRM197
[0521] Oxidized polysaccharide solution was mixed with water and
1.5 M potassium phosphate pH 6.0. The buffer pH selected was to
improve the stability of activated polysaccharide during the
conjugation reaction. Purified CRM197, obtained through expression
in Pseudomonas fluorescens as previously described (WO 2012/173876
A1), was combined with the buffered polysaccharide solution at a
polysaccharide to CRM197 mass ratio of 0.4. The mass ratio was
selected to control the polysaccharide to CRM197 ratio in the
resulting conjugate. The polysaccharide and phosphate
concentrations were 3.8 g/L and 150 mM respectively. The
polysaccharide concentration was selected to control the size of
the resulting conjugate. The solution was then 0.2-micron filtered.
Nickel chloride was added to approximately 2 mM using a 100 mM
nickel chloride solution. Sodium cyanoborohydride (2 moles per mole
of polysaccharide repeating unit) was added. Conjugation proceeded
for 96 hours to maximize consumption of polysaccharide and
protein.
Purification and Neutralization
[0522] Following the conjugation reaction, the batch was diluted to
a polysaccharide concentration of approximately 3.5 g/L, cooled to
2-8.degree. C., and 1.2-micron filtered. The batch was diafiltered
against 300 mM sodium bicarbonate pH 9.3 at 2-8.degree. C. using a
100 kDa NMWCO tangential flow ultrafiltration membrane. The batch,
recovered in the retentate, was then neutralized with 1.5 M
potassium phosphate, pH 6.0.
Final Filtration and Product Storage
[0523] The batch was then concentrated and diaftiltered against 10
mM L-histidine in 150 mM sodium chloride, pH 7.0 at 4.degree. C.
using a 300 kDa NMWCO tangential flow ultrafiltration membrane.
Polysorbate 20 was added to the retentate batch to a concentration
of 0.05% (w/v) and then the batch was 0.2 micron filtered.
[0524] The batch was adjusted to a polysaccharide concentration of
1.0 g/L with additional 10 mM L-histidine in 150 mM sodium
chloride, pH 7.0 buffer with 0.015% (w/v) polysorbate 20. The batch
was dispensed into aliquots and frozen at .ltoreq.-60.degree.
C.
EXAMPLE 12
[0525] Preparation of Serotype 6A Conjugate for PCV23 (DMSO) and
PCV23 (DMSO+Aq) and PCV 22 Polyvalent Studies Using DMSO
Conjugation
[0526] Polysaccharide was dissolved, chemically activated and
buffer-exchanged by ultrafiltration. Activated polysaccharide and
purified CRM197 were individually lyophilized and redissolved in
DMSO. Redissolved polysaccharide and CRM197 solutions were then
combined and conjugated as described below. The resulting conjugate
was purified by ultrafiltration prior to a final 0.2-micron
filtration. Several process parameters within each step, such as
pH, temperature, concentration, and time were controlled to yield
conjugates with desired attributes.
Polysaccharide Size Reduction and Oxidation
[0527] Purified pneumococcal capsular Ps powder was dissolved in
water and 0.45-micron filtered. Dissolved polysaccharide was
homogenized to reduce the molecular mass of the Ps. Homogenization
pressure and number of passes through the homogenizer were
controlled to 200 bar/5 passes. Size-reduced polysaccharide was
concentrated and diafiltered against water using a 10 kDa NMWCO
tangential flow ultrafiltration membrane.
[0528] The polysaccharide solution was then adjusted to 22.degree.
C. and pH 5 with a sodium acetate buffer to minimize polysaccharide
size reduction due to activation. Polysaccharide activation was
initiated with the addition of a 100 mM sodium metaperiodate
solution. The oxidation reaction proceeded for 2 hours at
22.degree. C.
[0529] The activated product was diafiltered against 10 mM
potassium phosphate, pH 6.4 followed by diafiltration against water
using a 10 kDa NMWCO tangential flow ultrafiltration membrane.
Ultrafiltration was conducted at 2-8.degree. C.
Polysaccharide Conjugation to CRM197
[0530] Purified CRM197, obtained through expression in Pseudomonas
fluorescens as previously described (WO 2012/173876 Al), was
diafiltered against 2 mM phosphate, pH 7.2 buffer using a 5 kDa
NMWCO tangential flow ultrafiltration membrane and 0.2-micron
filtered.
[0531] Activated polysaccharide was formulated for lyophilization
at 6 mg Ps/mL with sucrose concentration of 5% w/v. CRM197 was
formulated for lyophilization at 6 mg Pr/mL with sucrose
concentration of 1% w/v.
[0532] Formulated Ps and CRM197 solutions were individually
lyophilized. Lyophilized Ps and CRM197 materials were redissolved
individually in equal volumes of DMSO. The polysaccharide and
CRM197 solutions were blended to achieve a polysaccharide
concentration of 1.5 g Ps/L and a polysaccharide to CRM197 mass
ratio of 1.4. The mass ratio was selected to control the
polysaccharide to CRM197 ratio in the resulting conjugate. Sodium
cyanoborohydride (1 mole per mole of polysaccharide repeating unit)
was added, and conjugation proceeded at 22.degree. C.
[0533] Reduction with sodium b or ohydride
[0534] Sodium borohydride (2 moles per mole of polysaccharide
repeating unit) was added following the conjugation reaction and
incubated for 3 hours at 22.degree. C. The batch was diluted into
150 mM sodium chloride, with approximately 0.025% (w/v) polysorbate
20, at approximately 4.degree. C. Potassium phosphate buffer was
then added to neutralize the pH. The batch was concentrated and
diafiltered at approximately 4.degree. C. against 150 mM sodium
chloride, 25 mM potassium phosphate pH 7, using a 30 kDa NMWCO
tangential flow ultrafiltration membrane.
Final Filtration and Product Storage
[0535] The batch was then concentrated and diafiltered against 10
mM histidine in 150 mM sodium chloride, pH 7.0, with 0.015% (w/v)
polysorbate 20, at 4.degree. C. using a 300 kDa NMWCO tangential
flow ultrafiltration membrane.
[0536] The retentate batch was 0.2 micron filtered then diluted
with additional 10 mM histidine in 150 mM sodium chloride, pH 7.0
with 0.015% (w/v) polysorbate 20, dispensed into aliquots and
frozen at .ltoreq.-60.degree. C.
EXAMPLE 13
[0537] Preparation of Serotype 6B Conjugate for PCV 23 (DMSO) and
PCV 23 (DMSO/Aq) Polyvalent Studies Using DMSO Conjugation
[0538] Polysaccharide was dissolved, sized to a target molecular
mass, chemically activated and buffer-exchanged by ultrafiltration.
Activated polysaccharide and purified CRM197 were individually
lyophilized and redissolved in DMSO. Redissolved polysaccharide and
CRM197 solutions were then combined and conjugated as described
below. The resulting conjugate was purified by ultrafiltration
prior to a final 0.2-micron filtration. Several process parameters
within each step, such as pH, temperature, concentration, and time
were controlled to yield conjugates with desired attributes.
Polysaccharide Size Reduction and Oxidation
[0539] Purified pneumococcal capsular Ps powder was dissolved in
water and 0.45-micron filtered. Dissolved polysaccharide was
homogenized to reduce the molecular mass of the Ps. Homogenization
pressure and number of passes through the homogenizer were
controlled to 200 bar/5 passes.
[0540] Size-reduced polysaccharide was concentrated and diafiltered
against water using a 10 kDa NMWCO tangential flow ultrafiltration
membrane.
[0541] The polysaccharide solution was then adjusted to 22.degree.
C. and pH 5 with a sodium acetate buffer to minimize polysaccharide
size reduction due to activation. Polysaccharide activation was
initiated with the addition of a 100 mM sodium metaperiodate
solution. The oxidation reaction proceeded for 2 hours at
22.degree. C.
[0542] The activated product was diafiltered against 10 mM
potassium phosphate, pH 6.4 followed by diafiltration against water
using a 10 kDa NMWCO tangential flow ultrafiltration membrane.
Ultrafiltration was conducted at 2-8.degree. C.
[0543] Polysaccharide Conjugation to CRM197
[0544] Purified CRM197, obtained through expression in Pseudomonas
fluorescens as previously described (WO 2012/173876 A1), was
diafiltered against 2 mM phosphate, pH 7.2 buffer using a 5 kDa
NMWCO tangential flow ultrafiltration membrane and 0.2-micron
filtered.
[0545] Activated polysaccharide was formulated for lyophilization
at 6 mg Ps/mL with sucrose concentration of 5% w/v. CRM197 was
formulated for lyophilization at 6 mg Pr/mL with sucrose
concentration of 1% w/v.
[0546] Formulated Ps and CRM197 solutions were individually
lyophilized. Lyophilized Ps and CRM197 materials were redissolved
individually in equal volumes of DMSO. The polysaccharide and
CRM197 solutions were blended to achieve a polysaccharide
concentration of 1.85 g Ps/L and a polysaccharide to CRM197 mass
ratio of 1.35. The mass ratio was selected to control the
polysaccharide to CRM197 ratio in the resulting conjugate. Sodium
cyanoborohydride (1 mole per mole of polysaccharide repeating unit)
was added, and conjugation proceeded at 22.degree. C.
Reduction with Sodium Borohydride
[0547] Sodium borohydride (2 moles per mole of polysaccharide
repeating unit) was added following the conjugation reaction and
incubated for 3 hours at 22.degree. C. The batch was diluted into
150 mM sodium chloride, with approximately 0.025% (w/v) polysorbate
20, at approximately 4.degree. C. Potassium phosphate buffer was
then added to neutralize the pH. The batch was concentrated and
diafiltered at approximately 4.degree. C. against 150 mM sodium
chloride, 25 mM potassium phosphate pH 7, using a 30 kDa NMWCO
tangential flow ultrafiltration membrane.
Final Filtration and Product Storage
[0548] The batch was then concentrated and diafiltered against 10
mM histidine in 150 mM sodium chloride, pH 7.0, with 0.015% (w/v)
polysorbate 20, at 4.degree. C. using a 300 kDa NMWCO tangential
flow ultrafiltration membrane.
[0549] The retentate batch was 0.2 micron filtered then diluted
with additional 10 mM histidine in 150 mM sodium chloride, pH 7.0
with 0.015% (w/v) polysorbate 20, dispensed into aliquots and
frozen at .ltoreq.-60.degree. C.
EXAMPLE 14
Preparation of Serotype 6B Conjugate for PCV22 Polyvalent Study
Using DMSO Conjugation
[0550] Polysaccharide was dissolved, sized to a target molecular
mass, chemically activated and buffer-exchanged by ultrafiltration.
Activated polysaccharide and purified CRM197 were individually
lyophilized and redissolved in DMSO. Redissolved polysaccharide and
CRM197 solutions were then combined and conjugated as described
below. The resulting conjugate was purified by ultrafiltration
prior to a final 0.2-micron filtration. Several process parameters
within each step, such as pH, temperature, concentration, and time
were controlled to yield conjugates with desired attributes.
Polysaccharide Size Reduction and Oxidation
[0551] Purified pneumococcal capsular Ps powder was dissolved in
water and 0.45-micron filtered. Dissolved polysaccharide was
homogenized to reduce the molecular mass of the Ps. Homogenization
pressure and number of passes through the homogenizer were
controlled to 200 bar/5 passes. Size-reduced polysaccharide was
concentrated and diafiltered against water using a 10 kDa NMWCO
tangential flow ultrafiltration membrane.
[0552] The polysaccharide solution was then adjusted to 22.degree.
C. and pH 5 with a sodium acetate buffer to minimize polysaccharide
size reduction due to activation. Polysaccharide activation was
initiated with the addition of a 100 mM sodium metaperiodate
solution. The oxidation reaction proceeded for 2 hours at
22.degree. C.
[0553] The activated product was diafiltered against 10 mM
potassium phosphate, pH 6.4 followed by diafiltration against water
using a 10 kDa NMWCO tangential flow ultrafiltration membrane.
Ultrafiltration was conducted at 2-8.degree. C.
Polysaccharide Conjugation to CRM197
[0554] Purified CRM197, obtained through expression in Pseudomonas
fluorescens as previously described (WO 2012/173876 A1), was
diafiltered against 2 mM phosphate, pH 7.2 buffer using a 5 kDa
NMWCO tangential flow ultrafiltration membrane and 0.2-micron
filtered.
[0555] Activated polysaccharide was formulated for lyophilization
at 6 mg Ps/mL with sucrose concentration of 5% w/v. CRM197 was
formulated for lyophilization at 6 mg Pr/mL with sucrose
concentration of 1% w/v.
[0556] Formulated Ps and CRM197 solutions were individually
lyophilized. Lyophilized Ps and CRM197 materials were redissolved
individually in equal volumes of DMSO. The polysaccharide and
CRM197 solutions were blended to achieve a polysaccharide
concentration of 1.75 g Ps/L and a polysaccharide to CRM197 mass
ratio of 1.35. The mass ratio was selected to control the
polysaccharide to CRM197 ratio in the resulting conjugate. Sodium
cyanoborohydride (1 mole per mole of polysaccharide repeating unit)
was added, and conjugation proceeded at 22.degree. C.
Reduction with Sodium Borohydride
[0557] Sodium borohydride (2 moles per mole of polysaccharide
repeating unit) was added following the conjugation reaction and
incubated for 3 hours at 22.degree. C. The batch was diluted into
150 mM sodium chloride, with approximately 0.025% (w/v) polysorbate
20, at approximately 4.degree. C. Potassium phosphate buffer was
then added to neutralize the pH. The batch was concentrated and
diafiltered at approximately 4.degree. C. against 150 mM sodium
chloride, 25 mM potassium phosphate pH 7, using a 30 kDa NMWCO
tangential flow ultrafiltration membrane.
Final Filtration and Product Storage
[0558] The batch was then concentrated and diafiltered against 10
mM histidine in 150 mM sodium chloride, pH 7.0, with 0.015% (w/v)
polysorbate 20, at 4.degree. C. using a 300 kDa NMWCO tangential
flow ultrafiltration membrane.
[0559] The retentate batch was 0.2 micron filtered then diluted
with additional 10 mM histidine in 150 mM sodium chloride, pH 7.0
with 0.015% (w/v) polysorbate 20, dispensed into aliquots and
frozen at .ltoreq.-60.degree. C.
EXAMPLE 15
Preparation of Serotype 7F Conjugate for PCV23 (DMSO) and PCV23
(DMSO+Aq) Polyvalent Studies Using DMSO Conjugation
[0560] Polysaccharide was dissolved, sized to a target molecular
mass, chemically activated and buffer-exchanged by ultrafiltration.
Activated polysaccharide and purified CRM197 were individually
lyophilized and redissolved in DMSO. Redissolved polysaccharide and
CRM197 solutions were then combined and conjugated as described
below. The resulting conjugate was purified by ultrafiltration
prior to a final 0.2-micron filtration. Several process parameters
within each step, such as pH, temperature, concentration, and time
were controlled to yield conjugates with desired attributes.
Polysaccharide Size Reduction and Oxidation
[0561] Purified pneumococcal capsular Ps powder was dissolved in
water and 0.45-micron filtered. Dissolved polysaccharide was
homogenized to reduce the molecular mass of the Ps. Homogenization
pressure and number of passes through the homogenizer were
controlled to 150 bar/7 passes. Size-reduced polysaccharide was
concentrated and diafiltered against water using a 10 kDa NMWCO
tangential flow ultrafiltration membrane.
[0562] The polysaccharide solution was then adjusted to 4.degree.
C. and pH 5 with a sodium acetate buffer to minimize polysaccharide
size reduction due to activation. Polysaccharide activation was
initiated with the addition of a 100 mM sodium metaperiodate
solution. The oxidation reaction proceeded for 4 hours at 4.degree.
C.
[0563] The activated product was diafiltered against 10 mM
potassium phosphate, pH 6.4 followed by diafiltration against water
using a 10 kDa NMWCO tangential flow ultrafiltration membrane.
Ultrafiltration was conducted at 2-8.degree. C.
Polysaccharide Conjugation to CRM197
[0564] Purified CRM197, obtained through expression in Pseudomonas
fluorescens as previously described (WO 2012/173876 A1), was
diafiltered against 2 mM phosphate, pH 7.2 buffer using a 5 kDa
NMWCO tangential flow ultrafiltration membrane and 0.2-micron
filtered.
[0565] Activated polysaccharide was formulated for lyophilization
at 6 mg Ps/mL with sucrose concentration of 5% w/v. CRM197 was
formulated for lyophilization at 6 mg Pr/mL with sucrose
concentration of 1% w/v.
[0566] Formulated Ps and CRM197 solutions were individually
lyophilized. Lyophilized Ps and CRM197 materials were redissolved
individually in equal volumes of DMSO. The polysaccharide and
CRM197 solutions were blended to achieve a polysaccharide
concentration of 2.6 g Ps/L and a polysaccharide to CRM197 mass
ratio of 1.5. The mass ratio was selected to control the
polysaccharide to CRM197 ratio in the resulting conjugate. Sodium
cyanoborohydride (1 mole per mole of polysaccharide repeating unit)
was added, and conjugation proceeded at 22.degree. C.
Reduction with Sodium Borohydride
[0567] Sodium borohydride (2 moles per mole of polysaccharide
repeating unit) was added following the conjugation reaction and
incubated for 3 hours at 22.degree. C. The batch was diluted into
150 mM sodium chloride, with approximately 0.025% (w/v) polysorbate
20, at approximately 4.degree. C. Potassium phosphate buffer was
then added to neutralize the pH. The batch was concentrated and
diafiltered at approximately 4.degree. C. against 150 mM sodium
chloride, 25 mM potassium phosphate pH 7, using a 30 kDa NMWCO
tangential flow ultrafiltration membrane.
Final Filtration and Product Storage
[0568] The batch was then concentrated and diafiltered against 10
mM histidine in 150 mM sodium chloride, pH 7.0, with 0.015% (w/v)
polysorbate 20, at 4.degree. C. using a 300 kDa NMWCO tangential
flow ultrafiltration membrane.
[0569] The retentate batch was 0.2 micron filtered then diluted
with additional 10 mM histidine in 150 mM sodium chloride, pH 7.0
with 0.015% (w/v) polysorbate 20, dispensed into aliquots and
frozen at .ltoreq.-60.degree. C.
EXAMPLE 16
Preparation of Serotype 7F Conjugate for PCV22 Polyvalent Study
Using DMSO Conjugation
[0570] Polysaccharide was dissolved, sized to a target molecular
mass, chemically activated and buffer-exchanged by ultrafiltration.
Activated polysaccharide and purified CRM197 were individually
lyophilized and redissolved in DMSO. Redissolved polysaccharide and
CRM197 solutions were then combined and conjugated as described
below. The resulting conjugate was purified by ultrafiltration
prior to a final 0.2-micron filtration. Several process parameters
within each step, such as pH, temperature, concentration, and time
were controlled to yield conjugates with desired attributes.
Polysaccharide Size Reduction and Oxidation
[0571] Purified pneumococcal capsular Ps powder was dissolved in
water and 0.45-micron filtered. Dissolved polysaccharide was
homogenized to reduce the molecular mass of the Ps. Homogenization
pressure and number of passes through the homogenizer were
controlled to 150 bar/7 passes. Size-reduced polysaccharide was
concentrated and diafiltered against water using a 10 kDa NMWCO
tangential flow ultrafiltration membrane. The polysaccharide
solution was then adjusted to 4.degree. C. and pH 5 with a sodium
acetate buffer to minimize polysaccharide size reduction due to
activation. Polysaccharide activation was initiated with the
addition of a 100 mM sodium metaperiodate solution. The oxidation
reaction proceeded for 4 hours at 4.degree. C.
[0572] The activated product was diafiltered against 10 mM
potassium phosphate, pH 6.4 followed by diafiltration against water
using a 10 kDa NMWCO tangential flow ultrafiltration membrane.
Ultrafiltration was conducted at 2-8.degree. C.
Polysaccharide Conjugation to CRM197
[0573] Purified CRM197, obtained through expression in Pseudomonas
fluorescens as previously described (WO 2012/173876 Al), was
diafiltered against 2 mM phosphate, pH 7.2 buffer using a 5 kDa
NMWCO tangential flow ultrafiltration membrane and 0.2-micron
filtered.
[0574] Activated polysaccharide was formulated for lyophilization
at 6 mg Ps/mL with sucrose concentration of 5% w/v. CRM197 was
formulated for lyophilization at 6 mg Pr/mL with sucrose
concentration of 1% w/v.
[0575] Formulated Ps and CRM197 solutions were individually
lyophilized. Lyophilized Ps and CRM197 materials were redissolved
individually in equal volumes of DMSO. The polysaccharide and
CRM197 solutions were blended to achieve a polysaccharide
concentration of 2.04 g Ps/L and a polysaccharide to CRM197 mass
ratio of 1.5. The mass ratio was selected to control the
polysaccharide to CRM197 ratio in the resulting conjugate. Sodium
cyanoborohydride (1 mole per mole of polysaccharide repeating unit)
was added, and conjugation proceeded at 22.degree. C.
Reduction with Sodium Borohydride
[0576] Sodium borohydride (2 moles per mole of polysaccharide
repeating unit) was added following the conjugation reaction and
incubated for 3 hours at 22.degree. C. The batch was diluted into
150 mM sodium chloride, with approximately 0.025% (w/v) polysorbate
20, at approximately 4.degree. C. Potassium phosphate buffer was
then added to neutralize the pH. The batch was concentrated and
diafiltered at approximately 4.degree. C. against 150 mM sodium
chloride, 25 mM potassium phosphate pH 7, using a 30 kDa NMWCO
tangential flow ultrafiltration membrane.
Final Filtration and Product Storage
[0577] The batch was then concentrated and diafiltered against 10
mM histidine in 150 mM sodium chloride, pH 7.0, with 0.015% (w/v)
polysorbate 20, at 4.degree. C. using a 300 kDa NMWCO tangential
flow ultrafiltration membrane.
[0578] The retentate batch was 0.2 micron filtered then diluted
with additional 10 mM histidine in 150 mM sodium chloride, pH 7.0
with 0.015% (w/v) polysorbate 20, dispensed into aliquots and
frozen at .ltoreq.-60.degree. C.
EXAMPLE 17
Preparation of Serotype 8 Conjugate for PCV23 (DMSO) and PCV23
(DMSO+Aq) Polyvalent Studies Using DMSO Conjugation
[0579] Polysaccharide was dissolved, sized to a target molecular
mass, chemically activated and buffer-exchanged by ultrafiltration.
Activated polysaccharide and purified CRM197 were individually
lyophilized and redissolved in DMSO. Redissolved polysaccharide and
CRM197 solutions were then combined and conjugated as described
below. The resulting conjugate was purified by ultrafiltration
prior to a final 0.2-micron filtration. Several process parameters
within each step, such as pH, temperature, concentration, and time
were controlled to yield conjugates with desired attributes.
Polysaccharide Size Reduction and Oxidation
[0580] Purified pneumococcal capsular Ps powder was dissolved in
water and 0.45-micron filtered. Dissolved polysaccharide was
homogenized to reduce the molecular mass of the Ps. Homogenization
pressure and number of passes through the homogenizer were
controlled to 600 bar/6 passes. Size-reduced polysaccharide was
concentrated and diafiltered against water using a 5 kDa NMWCO
tangential flow ultrafiltration membrane.
[0581] The polysaccharide solution was then adjusted to 22.degree.
C. and pH 5 with a sodium acetate buffer to minimize polysaccharide
size reduction due to activation. Polysaccharide activation was
initiated with the addition of a 100 mM sodium metaperiodate
solution. The oxidation reaction proceeded for 4 hours at
22.degree. C.
[0582] The activated product was diafiltered against 10 mM
potassium phosphate, pH 6.4 followed by diafiltration against water
using a 5 kDa NMWCO tangential flow ultrafiltration membrane.
Ultrafiltration was conducted at 2-8.degree. C.
Polysaccharide Conjugation to CRM197
[0583] Purified CRM197, obtained through expression in Pseudomonas
fluorescens as previously described (WO 2012/173876 A1), was
diafiltered against 2 mM phosphate, pH 7.2 buffer using a 5 kDa
NMWCO tangential flow ultrafiltration membrane and 0.2-micron
filtered.
[0584] Activated polysaccharide was formulated for lyophilization
at 6 mg Ps/mL with sucrose concentration of 5% w/v. CRM197 was
formulated for lyophilization at 6 mg Pr/mL with sucrose
concentration of 1% w/v.
[0585] Formulated Ps and CRM197 solutions were individually
lyophilized. Lyophilized Ps and CRM197 materials were redissolved
individually in equal volumes of DMSO. The polysaccharide and
CRM197 solutions were blended to achieve a polysaccharide
concentration of 4.5 g Ps/L and a polysaccharide to CRM197 mass
ratio of 1.5. The mass ratio was selected to control the
polysaccharide to CRM197 ratio in the resulting conjugate. After
the blend, the conjugation reaction proceeded at 22.degree. C.
Reduction with Sodium Borohydride
[0586] Sodium borohydride (2 moles per mole of polysaccharide
repeating unit) was added following the conjugation reaction and
incubated for 1 hour at 22.degree. C. The batch was diluted into
150 mM sodium chloride, with approximately 0.025% (w/v) polysorbate
20, at approximately 4.degree. C. Potassium phosphate buffer was
then added to neutralize the pH.
Final Filtration and Product Storage
[0587] The batch was then concentrated and diafiltered against 10
mM histidine in 150 mM sodium chloride, pH 7.0, with 0.015% (w/v)
polysorbate 20, at 4.degree. C. using a 300 kDa NMWCO tangential
flow ultrafiltration membrane.
[0588] The retentate batch was 0.2 micron filtered (with 0.5 micron
prefilter) then diluted with additional 10 mM histidine in 150 mM
sodium chloride, pH 7.0 with 0.015% (w/v) polysorbate 20, dispensed
into aliquots and frozen at .ltoreq.-60.degree. C.
EXAMPLE 18
[0589] Preparation of Serotype 9V Conjugate for PCV23 (DMSO)
Polyvalent Study Using DMSO Conjugation
[0590] Polysaccharide was dissolved, sized to a target molecular
mass, chemically activated and buffer-exchanged by ultrafiltration.
Activated polysaccharide and purified CRM197 were individually
lyophilized and redissolved in DMSO. Redissolved polysaccharide and
CRM197 solutions were then combined and conjugated as described
below. The resulting conjugate was purified by ultrafiltration
prior to a final 0.2-micron filtration. Several process parameters
within each step, such as pH, temperature, concentration, and time
were controlled to yield conjugates with desired attributes.
Polysaccharide Size Reduction and Oxidation
[0591] Purified pneumococcal capsular Ps powder was dissolved in
water and 0.45-micron filtered. Dissolved polysaccharide was
homogenized to reduce the molecular mass of the Ps. Homogenization
pressure and number of passes through the homogenizer were
controlled to 230 bar/5.5 passes. Size-reduced polysaccharide was
concentrated and diafiltered against water using a 10 kDa NMWCO
tangential flow ultrafiltration membrane.
[0592] The polysaccharide solution was then adjusted to 22.degree.
C. and pH 5 with a sodium acetate buffer to minimize polysaccharide
size reduction due to activation. Polysaccharide activation was
initiated with the addition of a 100 mM sodium metaperiodate
solution. The oxidation reaction proceeded for 6 hours at
22.degree. C.
[0593] The activated product was diafiltered against 10 mM
potassium phosphate, pH 6.4 followed by diafiltration against water
using a 10 kDa NMWCO tangential flow ultrafiltration membrane.
Ultrafiltration was conducted at 2-8.degree. C.
[0594] Polysaccharide Conjugation to CRM197
[0595] Purified CRM197, obtained through expression in Pseudomonas
fluorescens as previously described (WO 2012/173876 A1), was
diafiltered against 2 mM phosphate, pH 7.2 buffer using a 5 kDa
NMWCO tangential flow ultrafiltration membrane and 0.2-micron
filtered. Activated polysaccharide was formulated for
lyophilization at 6 mg Ps/mL with sucrose concentration of 5% w/v.
CRM197 was formulated for lyophilization at 6 mg Pr/mL with sucrose
concentration of 1% w/v.
[0596] Formulated Ps and CRM197 solutions were individually
lyophilized. Lyophilized Ps and CRM197 materials were redissolved
individually in equal volumes of DMSO. The polysaccharide and
CRM197 solutions were blended to achieve a polysaccharide
concentration of 3.0g Ps/L and a polysaccharide to CRM197 mass
ratio of 1.3. The mass ratio was selected to control the
polysaccharide to CRM197 ratio in the resulting conjugate. Sodium
cyanoborohydride (1 mole per mole of polysaccharide repeating unit)
was added, and conjugation proceeded at 22.degree. C.
Reduction with Sodium Borohydride
[0597] Sodium borohydride (2 moles per mole of polysaccharide
repeating unit) was added following the conjugation reaction and
incubated for 3 hours at 22.degree. C. The batch was diluted into
150 mM sodium chloride, with approximately 0.025% (w/v) polysorbate
20, at approximately 4.degree. C. Potassium phosphate buffer was
then added to neutralize the pH. The batch was concentrated and
diafiltered at approximately 4.degree. C. against 150 mM sodium
chloride, 25 mM potassium phosphate pH 7, using a 30 kDa NMWCO
tangential flow ultrafiltration membrane.
Final Filtration and Product Storage
[0598] The batch was then concentrated and diafiltered against 10
mM histidine in 150 mM sodium chloride, pH 7.0, with 0.015% (w/v)
polysorbate 20, at 4.degree. C. using a 300 kDa NMWCO tangential
flow ultrafiltration membrane.
[0599] The retentate batch was 0.2 micron filtered (with a 0.5
micron prefilter) then diluted with additional 10 mM histidine in
150 mM sodium chloride, pH 7.0 with 0.015% (w/v) polysorbate 20,
dispensed into aliquots and frozen at .ltoreq.-60.degree. C.
EXAMPLE 19
Preparation of Serotype 9V Conjugate for PCV22 Polyvalent Study
Using Aqueous Conjugation
[0600] Polysaccharide was dissolved, size reduced, chemically
activated and buffer-exchanged by ultrafiltration. Purified CRM197
was then conjugated to the activated polysaccharide utilizing
nickel chloride in the reaction mixture, and the resulting
conjugate was purified by ultrafiltration prior to a final
0.2-micron filtration. Several process parameters within each step,
such as pH, temperature, concentration, and time were controlled to
yield conjugates with desired attributes.
Polysaccharide Size Reduction and Oxidation
[0601] Purified pneumococcal capsular polysaccharide powder was
dissolved in water, and 0.45-micron filtered. Dissolved
polysaccharide was homogenized to reduce the molecular mass.
Homogenization pressure and number of passes through the
homogenizer were controlled to 100 bar/5 passes to size-reduce to a
target molecular mass. Size-reduced polysaccharide was then
concentrated and diafiltered against water using a 10 kDa NMWCO
tangential flow ultrafiltration membrane.
[0602] The polysaccharide solution was then adjusted to 22.degree.
C. and pH 5 with a sodium acetate buffer to minimize polysaccharide
size reduction due to activation. Polysaccharide activation was
initiated with the addition of a 100 mM sodium metaperiodate
solution. The oxidation reaction proceeded for 6 hours at
22.degree. C.
[0603] The activated product was diafiltered against 10 mM
potassium phosphate, pH 6.4 using a 10 kDa NMWCO tangential flow
ultrafiltration membrane. Ultrafiltration was conducted at
2-8.degree. C.
Polysaccharide Conjugation to CRM197
[0604] Oxidized polysaccharide solution was mixed with water and
1.5 M potassium phosphate pH 7.0. The buffer pH selected was to
improve the stability of activated polysaccharide during the
conjugation reaction. Purified CRM197, obtained through expression
in Pseudomonas fluorescens as previously described (WO 2012/173876
A1), was 0.2-micron filtered and combined with the buffered
polysaccharide solution at a polysaccharide to CRM197 mass ratio of
0.7. The mass ratio was selected to control the polysaccharide to
CRM197 ratio in the resulting conjugate. The polysaccharide and
phosphate concentrations were 10.0 g/L and 100 mM respectively. The
polysaccharide concentration was selected to control the size of
the resulting conjugate. The solution was then 0.2-micron filtered.
Nickel chloride was added to approximately 2 mM using a 100 mM
nickel chloride solution. Sodium cyanoborohydride (2 moles per mole
of polysaccharide repeating unit) was added. Conjugation proceeded
for 120 hours to maximize consumption of polysaccharide and
protein.
Reduction with Sodium Borohydride
[0605] Following the conjugation reaction, the batch was diluted to
a polysaccharide concentration of approximately 3.5 g/L, cooled to
2-8.degree. C., and 1.2-micron filtered. The batch was diafiltered
against 100 mM potassium phosphate, pH 7.0 at 2-8.degree. C. using
a 100 kDa NMWCO tangential flow ultrafiltration membrane. The
batch, recovered in the retentate, was then diluted to
approximately 2.0 g polysaccharide/L and pH-adjusted with the
addition of 1.2 M sodium bicarbonate, pH 9.4. Sodium borohydride (1
mole per mole of polysaccharide repeating unit) was added. 1.5 M
potassium phosphate, pH 6.0 was later added.
Final Filtration and Product Storage
[0606] The batch was then concentrated and diaftiltered against 10
mM L-histidine in 150 mM sodium chloride, pH 7.0 at 4.degree. C.
using a 300 kDa NMWCO tangential flow ultrafiltration membrane.
Polysorbate 20 was added to the retentate batch to a concentration
of 0.05% (w/v) then the batch was 0.2 micron filtered.
[0607] The batch was adjusted to a polysaccharide concentration of
1.0 g/L with additional 10 mM L-histidine in 150 mM sodium
chloride, pH 7.0 buffer with 0.015% (w/v) polysorbate 20. The batch
was dispensed into aliquots and frozen at .ltoreq.-60.degree.
C.
EXAMPLE 20
Preparation of Serotype 9V Conjugate for PCV23 (DMSO+Aq) Polyvalent
Study Using Aqueous Conjugation
[0608] Polysaccharide was dissolved, size reduced, chemically
activated and buffer-exchanged by ultrafiltration. Purified CRM197
was then conjugated to the activated polysaccharide utilizing
nickel chloride in the reaction mixture, and the resulting
conjugate was purified by ultrafiltration prior to a final
0.2-micron filtration. Several process parameters within each step,
such as pH, temperature, concentration, and time were controlled to
yield conjugates with desired attributes.
Polysaccharide Size Reduction and Oxidation
[0609] Purified pneumococcal capsular polysaccharide powder was
dissolved in water, and 0.45-micron filtered. Dissolved
polysaccharide was homogenized to reduce the molecular mass.
Homogenization pressure and number of passes through the
homogenizer were controlled to 100 bar/5 passes to size-reduce to a
target molecular mass. Size-reduced polysaccharide was then
concentrated and diafiltered against water using a 10 kDa NMWCO
tangential flow ultrafiltration membrane.
[0610] The polysaccharide solution was then adjusted to 22.degree.
C. and pH 5 with a sodium acetate buffer to minimize polysaccharide
size reduction due to activation. Polysaccharide activation was
initiated with the addition of a 100 mM sodium metaperiodate
solution. The oxidation reaction proceeded for 6 hours at
22.degree. C.
[0611] The activated product was diafiltered against 10 mM
potassium phosphate, pH 6.4 using a 10 kDa NMWCO tangential flow
ultrafiltration membrane. Ultrafiltration was conducted at
2-8.degree. C.
Polysaccharide Conjugation to CRM197
[0612] Oxidized polysaccharide solution was mixed with water and
1.5 M potassium phosphate pH 7.0. The buffer pH selected was to
improve the stability of activated polysaccharide during the
conjugation reaction. Purified CRM197, obtained through expression
in Pseudomonas fluorescens as previously described (WO 2012/173876
A1), was 0.2-micron filtered and combined with the buffered
polysaccharide solution at a polysaccharide to CRM197 mass ratio of
0.7. The mass ratio was selected to control the polysaccharide to
CRM197 ratio in the resulting conjugate. The polysaccharide and
phosphate concentrations were 10.0 g/L and 100 mM respectively. The
polysaccharide concentration was selected to control the size of
the resulting conjugate. The solution was then 0.2-micron filtered.
Nickel chloride was added to approximately 2 mM using a 100 mM
nickel chloride solution. Sodium cyanoborohydride (2 moles per mole
of polysaccharide repeating unit) was added. Conjugation proceeded
for 120 hours to maximize consumption of polysaccharide and
protein.
Reduction with Sodium Borohydride
[0613] Following the conjugation reaction, the batch was diluted to
a polysaccharide concentration of approximately 3.5 g/L, cooled to
2-8.degree. C., and 1.2-micron filtered. The batch was diafiltered
against 100 mM potassium phosphate, pH 7.0 at 2-8.degree. C. using
a 100 kDa NMWCO tangential flow ultrafiltration membrane. The
batch, recovered in the retentate, was then diluted to
approximately 2.0 g polysaccharide/L and pH-adjusted with the
addition of 1.2 M sodium bicarbonate, pH 9.4. Sodium borohydride (1
mole per mole of polysaccharide repeating unit) was added. 1.5 M
potassium phosphate, pH 6.0 was later added.
Final Filtration and Product Storage
[0614] The batch was then concentrated and diaftiltered against 10
mM L-histidine in 150 mM sodium chloride, pH 7.0 at 4.degree. C.
using a 300 kDa NMWCO tangential flow ultrafiltration membrane.
Polysorbate 20 was added to the retentate batch to a concentration
of 0.05% (w/v) then the batch was 0.2 micron filtered.
[0615] The batch was adjusted to a polysaccharide concentration of
1.0 g/L with additional 10 mM L-histidine in 150 mM sodium
chloride, pH 7.0 buffer with 0.015% (w/v) polysorbate 20. The batch
was dispensed into aliquots and frozen at .ltoreq.-60.degree.
C.
EXAMPLE 21
Preparation of Serotype 10A Conjugate for PCV23 Polyvalent Study
Using DMSO Conjugation
[0616] Polysaccharide was dissolved, sized to a target molecular
mass, chemically activated and buffer-exchanged by ultrafiltration.
Activated polysaccharide and purified CRM197 were individually
lyophilized and redissolved in DMSO. Redissolved polysaccharide and
CRM197 solutions were then combined and conjugated as described
below. The resulting conjugate was purified by ultrafiltration
prior to a final 0.2-micron filtration. Several process parameters
within each step, such as pH, temperature, concentration, and time
were controlled to yield conjugates with desired attributes.
Polysaccharide Size Reduction and Oxidation
[0617] Purified pneumococcal capsular Ps powder was dissolved in
water and 0.45-micron filtered. Dissolved polysaccharide was
homogenized to reduce the molecular mass of the Ps. Homogenization
pressure and number of passes through the homogenizer were
controlled to 615 bar/5 passes.
[0618] Size-reduced polysaccharide was concentrated and diafiltered
against water using a 10 kDa NMWCO tangential flow ultrafiltration
membrane.
[0619] The polysaccharide solution was then adjusted to 22.degree.
C. and pH 5 with a sodium acetate buffer to minimize polysaccharide
size reduction due to activation. Polysaccharide activation was
initiated with the addition of a 100 mM sodium metaperiodate
solution. The oxidation reaction proceeded for 2 hours at
22.degree. C.
[0620] The activated product was diafiltered against 10 mM
potassium phosphate, pH 6.4 followed by diafiltration against water
using a 10 kDa NMWCO tangential flow ultrafiltration membrane.
Ultrafiltration was conducted at 2-8.degree. C.
Polysaccharide Conjugation to CRM197
[0621] Purified CRM197, obtained through expression in Pseudomonas
fluorescens as previously described (WO 2012/173876 A1), was
diafiltered against 2 mM phosphate, pH 7.2 buffer using a 5 kDa
NMWCO tangential flow ultrafiltration membrane and 0.2-micron
filtered.
[0622] Activated polysaccharide was formulated for lyophilization
at 6 mg Ps/mL with sucrose concentration of 5% w/v. CRM197 was
formulated for lyophilization at 6 mg Pr/mL with sucrose
concentration of 1% w/v.
[0623] Formulated Ps and CRM197 solutions were individually
lyophilized. Lyophilized Ps and CRM197 materials were redissolved
individually in equal volumes of DMSO. The polysaccharide and
CRM197 solutions were blended to achieve a polysaccharide
concentration of 3.5 g Ps/L and a polysaccharide to CRM197 mass
ratio of 1.6. The mass ratio was selected to control the
polysaccharide to CRM197 ratio in the resulting conjugate. Sodium
cyanoborohydride (1 mole per mole of polysaccharide repeating unit)
was added, and conjugation proceeded at 22.degree. C.
Reduction with Sodium Borohydride
[0624] Sodium borohydride (2 moles per mole of polysaccharide
repeating unit) was added following the conjugation reaction and
incubated for 1 hour at 22.degree. C. The batch was diluted into
150 mM sodium chloride, with approximately 0.025% (w/v) polysorbate
20, at approximately 4.degree. C. Potassium phosphate buffer was
then added to neutralize the pH.
Final Filtration and Product Storage
[0625] The batch was concentrated and diafiltered against 10 mM
histidine in 150 mM sodium chloride, pH 7.0, with 0.015% (w/v)
polysorbate 20, at 4.degree. C. using a 300 kDa NMWCO tangential
flow ultrafiltration membrane.
[0626] The retentate batch was 0.2 micron filtered (with 0.5 micron
prefilter) then diluted with additional 10 mM histidine in 150 mM
sodium chloride, pH 7.0 with 0.015% (w/v) polysorbate 20, dispensed
into aliquots and frozen at .ltoreq.-60.degree. C.
[0627] EXAMPLE 22
Preparation of Serotype 10A Conjugate for PCV23 (DMSO+Aq)
Polyvalent Study Using DMSO Conjugation
[0628] Polysaccharide was dissolved, sized to a target molecular
mass, chemically activated and buffer-exchanged by ultrafiltration.
Activated polysaccharide and purified
[0629] CRM197 were individually lyophilized and redissolved in
DMSO. Redissolved polysaccharide and CRM197 solutions were then
combined and conjugated as described below. The resulting conjugate
was purified by ultrafiltration prior to a final 0.2-micron
filtration. Several process parameters within each step, such as
pH, temperature, concentration, and time were controlled to yield
conjugates with desired attributes.
Polysaccharide Size Reduction and Oxidation
[0630] Purified pneumococcal capsular Ps powder was dissolved in
water and 0.45-micron filtered. Dissolved polysaccharide was
homogenized to reduce the molecular mass of the Ps. Homogenization
pressure and number of passes through the homogenizer were
controlled to 600 bar/5 passes. Size-reduced polysaccharide was
concentrated and diafiltered against water using a 10 kDa NMWCO
tangential flow ultrafiltration membrane.
[0631] The polysaccharide solution was then adjusted to 22.degree.
C. and pH 5 with a sodium acetate buffer to minimize polysaccharide
size reduction due to activation. Polysaccharide activation was
initiated with the addition of a 100 mM sodium metaperiodate
solution. The oxidation reaction proceeded for 2 hours at
22.degree. C.
[0632] The activated product was diafiltered against 10 mM
potassium phosphate, pH 6.4 followed by diafiltration against water
using a 10 kDa NMWCO tangential flow ultrafiltration membrane.
Ultrafiltration was conducted at 2-8.degree. C.
Polysaccharide Conjugation to CRM197
[0633] Purified CRM197, obtained through expression in Pseudomonas
fluorescens as previously described (WO 2012/173876 A1), was
diafiltered against 2 mM phosphate, pH 7.2 buffer using a 5 kDa
NMWCO tangential flow ultrafiltration membrane and 0.2-micron
filtered.
[0634] Activated polysaccharide was formulated for lyophilization
at 6 mg Ps/mL with sucrose concentration of 5% w/v. CRM197 was
formulated for lyophilization at 6 mg Pr/mL with sucrose
concentration of 1% w/v.
[0635] Formulated Ps and CRM197 solutions were individually
lyophilized. Lyophilized Ps and CRM197 materials were redissolved
individually in equal volumes of DMSO. The polysaccharide and
CRM197 solutions were blended to achieve a polysaccharide
concentration of 3.4 g Ps/L and a polysaccharide to CRM197 mass
ratio of 1.6. The mass ratio was selected to control the
polysaccharide to CRM197 ratio in the resulting conjugate. Sodium
cyanoborohydride (1 mole per mole of polysaccharide repeating unit)
was added, and conjugation proceeded at 22.degree. C.
Reduction with Sodium Borohydride
[0636] Sodium borohydride (2 moles per mole of polysaccharide
repeating unit) was added following the conjugation reaction and
incubated for 1 hour at 22.degree. C. The batch was diluted into
150 mM sodium chloride, with approximately 0.025% (w/v) polysorbate
20, at approximately 4.degree. C. Potassium phosphate buffer was
then added to neutralize the pH.
[0637] Final filtration and product storage
[0638] The batch was then concentrated and diafiltered against 150
mM sodium chloride, 25 mM potassium phosphate, pH 7 followed by
diafiltration against 10 mM histidine in 150 mM sodium chloride, pH
7.0, with 0.015% (w/v) polysorbate 20, at 4.degree. C. using a 300
kDa NMWCO tangential flow ultrafiltration membrane.
[0639] The retentate batch was 0.2 micron filtered (with 0.5 micron
prefilter) then diluted with additional 10 mM histidine in 150 mM
sodium chloride, pH 7.0 with 0.015% (w/v) polysorbate 20, dispensed
into aliquots and frozen at .ltoreq.-60.degree. C.
EXAMPLE 23
[0640] Preparation of Serotype 10A Conjugate for PCV22 Polyvalent
Study Using DMSO Conjugation
[0641] Polysaccharide was dissolved, sized to a target molecular
mass, chemically activated and buffer-exchanged by ultrafiltration.
Activated polysaccharide and purified CRM197 were individually
lyophilized and redissolved in DMSO. Redissolved polysaccharide and
CRM197 solutions were then combined and conjugated as described
below. The resulting conjugate was purified by ultrafiltration
prior to a final 0.2-micron filtration. Several process parameters
within each step, such as pH, temperature, concentration, and time
were controlled to yield conjugates with desired attributes.
Polysaccharide Size Reduction and Oxidation
[0642] Purified pneumococcal capsular Ps powder was dissolved in
water and 0.45-micron filtered. Dissolved polysaccharide was
homogenized to reduce the molecular mass of the Ps. Homogenization
pressure and number of passes through the homogenizer were
controlled to 600 bar/5 passes. Size-reduced polysaccharide was
concentrated and diafiltered against water using a 10 kDa NMWCO
tangential flow ultrafiltration membrane.
[0643] The polysaccharide solution was then adjusted to 22.degree.
C. and pH 5 with a sodium acetate buffer to minimize polysaccharide
size reduction due to activation. Polysaccharide activation was
initiated with the addition of a 100 mM sodium metaperiodate
solution. The oxidation reaction proceeded for 2 hours at
22.degree. C.
[0644] The activated product was diafiltered against 10 mM
potassium phosphate, pH 6.4 followed by diafiltration against water
using a 10 kDa NMWCO tangential flow ultrafiltration membrane.
Ultrafiltration was conducted at 2-8.degree. C.
Polysaccharide Conjugation to CRM197
[0645] Purified CRM197, obtained through expression in Pseudomonas
fluorescens as previously described (WO 2012/173876 A1), was
diafiltered against 2 mM phosphate, pH 7.2 buffer using a 5 kDa
NMWCO tangential flow ultrafiltration membrane and 0.2-micron
filtered.
[0646] Activated polysaccharide was formulated for lyophilization
at 6 mg Ps/mL with sucrose concentration of 5% w/v. CRM197 was
formulated for lyophilization at 6 mg Pr/mL with sucrose
concentration of 1% w/v.
[0647] Formulated Ps and CRM197 solutions were individually
lyophilized. Lyophilized Ps and CRM197 materials were redissolved
individually in equal volumes of DMSO. The polysaccharide and
CRM197 solutions were blended to achieve a polysaccharide
concentration of 3.8 g Ps/L and a polysaccharide to CRM197 mass
ratio of 1.75. The mass ratio was selected to control the
polysaccharide to CRM197 ratio in the resulting conjugate. Sodium
cyanoborohydride (1 mole per mole of polysaccharide repeating unit)
was added, and conjugation proceeded at 22.degree. C.
Reduction with Sodium Borohydride
[0648] Sodium borohydride (2 moles per mole of polysaccharide
repeating unit) was added following the conjugation reaction and
incubated for 1 hour at 22.degree. C. The batch was diluted into
150 mM sodium chloride, with approximately 0.025% (w/v) polysorbate
20, at approximately 4.degree. C. Potassium phosphate buffer was
then added to neutralize the pH. The batch was concentrated and
diafiltered at approximately 4.degree. C. against 150 mM sodium
chloride, 25 mM potassium phosphate pH 7, using a 30 kDa NMWCO
tangential flow ultrafiltration membrane.
[0649] Final Filtration and Product Storage
[0650] The batch was then concentrated and diafiltered against 10
mM histidine in 150 mM sodium chloride, pH 7.0, with 0.015% (w/v)
polysorbate 20, at 4.degree. C. using a 300 kDa NMWCO tangential
flow ultrafiltration membrane.
[0651] The retentate batch was 0.2 micron filtered (with 0.5 micron
prefilter) then diluted with additional 10 mM histidine in 150 mM
sodium chloride, pH 7.0 with 0.015% (w/v) polysorbate 20, dispensed
into aliquots and frozen at .ltoreq.-60.degree. C.
EXAMPLE X
Preparation of Serotype 11A Conjugate for PCV24 Polyvalent Study
Using DMSO Conjugation
[0652] Polysaccharide was dissolved, sized to a target molecular
mass, chemically activated and buffer-exchanged by ultrafiltration.
Activated polysaccharide and purified CRM197 were individually
lyophilized and redissolved in DMSO. Redissolved polysaccharide and
CRM197 solutions were then combined and conjugated as described
below. The resulting conjugate was purified by ultrafiltration
prior to a final 0.2-micron filtration. Several process parameters
within each step, such as pH, temperature, concentration, and time
were controlled to yield conjugates with desired attributes.
Polysaccharide Size Reduction and Oxidation
[0653] Purified pneumococcal capsular Ps powder was dissolved in
water and 0.45-micron filtered. Dissolved polysaccharide was
homogenized to reduce the molecular mass of the Ps. Homogenization
pressure and number of passes through the homogenizer were
controlled to 800 bar/8 passes. Size-reduced polysaccharide was
concentrated and diafiltered against water using a 5 kDa NMWCO
tangential flow ultrafiltration membrane.
[0654] The polysaccharide solution was then adjusted to 22.degree.
C. and pH 5 with a sodium acetate buffer to minimize polysaccharide
size reduction due to activation. Polysaccharide activation was
initiated with the addition of a 100 mM sodium metaperiodate
solution. The oxidation reaction proceeded for 2 hours at
22.degree. C.
[0655] The activated product was diafiltered against 10 mM
potassium phosphate, pH 6.4 followed by diafiltration against water
using a 5 kDa NMWCO tangential flow ultrafiltration membrane.
Ultrafiltration was conducted at 2-8.degree. C.
Polysaccharide Conjugation to CRM197
[0656] Purified CRM197, obtained through expression in Pseudomonas
fluorescens as previously described (WO 2012/173876 A1), was
diafiltered against 2 mM phosphate, pH 7.2 buffer using a 5 kDa
NMWCO tangential flow ultrafiltration membrane and 0.2-micron
filtered.
[0657] Activated polysaccharide was formulated for lyophilization
at 6 mg Ps/mL with sucrose concentration of 5% w/v. CRM197 was
formulated for lyophilization at 6 mg Pr/mL with sucrose
concentration of 1% w/v.
[0658] Formulated Ps and CRM197 solutions were individually
lyophilized. Lyophilized Ps and CRM197 materials were redissolved
individually in equal volumes of DMSO. The polysaccharide and
CRM197 solutions were blended to achieve a polysaccharide
concentration of 2.3 g Ps/L and a polysaccharide to CRM197 mass
ratio of 1.5. The mass ratio was selected to control the
polysaccharide to CRM197 ratio in the resulting conjugate. Sodium
cyanoborohydride (1 mole per mole of polysaccharide repeating unit)
was added, and conjugation proceeded at 22.degree. C.
Reduction with Sodium Borohydride
[0659] Sodium borohydride (2 moles per mole of polysaccharide
repeating unit) was added following the conjugation reaction and
incubated for 1 hour at 22.degree. C. The batch was diluted into
150 mM sodium chloride, with approximately 0.025% (w/v) polysorbate
20, at approximately 4.degree. C. Potassium phosphate buffer was
then added to neutralize the pH.
Final Filtration and Product Storage
[0660] The batch was then concentrated and diafiltered against 10
mM histidine in 150 mM sodium chloride, pH 7.0, with 0.015% (w/v)
polysorbate 20, at 4.degree. C. using a 300 kDa NMWCO tangential
flow ultrafiltration membrane.
[0661] The retentate batch was 0.2 micron filtered (with 0.5 micron
prefilter) then diluted with additional 10 mM histidine in 150 mM
sodium chloride, pH 7.0 with 0.015% (w/v) polysorbate 20, dispensed
into aliquots and frozen at .ltoreq.-60.degree. C.
EXAMPLE 24
Preparation of Serotype 12F Conjugate for PCV23 (DMSO) and PCV23
(DMSO+Aq) Polyvalent Studies Using DMSO Conjugation
[0662] Polysaccharide was dissolved, sized to a target molecular
mass, chemically activated and buffer-exchanged by ultrafiltration.
Activated polysaccharide and purified CRM197 were individually
lyophilized and redissolved in DMSO. Redissolved polysaccharide and
CRM197 solutions were then combined and conjugated as described
below. The resulting conjugate was purified by ultrafiltration
prior to a final 0.2-micron filtration. Several process parameters
within each step, such as pH, temperature, concentration, and time
were controlled to yield conjugates with desired attributes.
[0663] Polysaccharide Size Reduction and Oxidation
[0664] Purified pneumococcal capsular Ps powder was dissolved in
water and 0.45-micron filtered. Dissolved polysaccharide was
size-reduced by acid hydrolysis by adding acetic acid to 200 mM,
incubating at 80.degree. C. for 155 minutes, then neutralizing by
adding cold potassium phosphate pH 7 buffer to 400 mM.
[0665] Size-reduced polysaccharide was concentrated and diafiltered
against water using a 5 kDa NMWCO tangential flow ultrafiltration
membrane.
[0666] The polysaccharide solution was then adjusted to 22.degree.
C. and pH 5 with a sodium acetate buffer to minimize polysaccharide
size reduction due to activation. Polysaccharide activation was
initiated with the addition of a 100 mM sodium metaperiodate
solution. The oxidation reaction proceeded for 2 hours at
22.degree. C.
[0667] The activated product was diafiltered against 10 mM
potassium phosphate, pH 6.4 followed by diafiltration against water
using a 5 kDa NMWCO tangential flow ultrafiltration membrane.
Ultrafiltration was conducted at 2-8.degree. C.
Polysaccharide Conjugation to CRM197
[0668] Purified CRM197, obtained through expression in Pseudomonas
fluorescens as previously described (WO 2012/173876 A1), was
diafiltered against 2 mM phosphate, pH 7.2 buffer using a 5 kDa
NMWCO tangential flow ultrafiltration membrane and 0.2-micron
filtered.
[0669] Activated polysaccharide was formulated for lyophilization
at 6 mg Ps/mL with sucrose concentration of 5% w/v. CRM197 was
formulated for lyophilization at 6 mg Pr/mL with sucrose
concentration of 1% w/v.
[0670] Formulated Ps and CRM197 solutions were individually
lyophilized. Lyophilized Ps and CRM197 materials were redissolved
individually in equal volumes of DMSO. The polysaccharide and
CRM197 solutions were blended to achieve a polysaccharide
concentration of 2.7 g Ps/L and a polysaccharide to CRM197 mass
ratio of 1.8. The mass ratio was selected to control the
polysaccharide to CRM197 ratio in the resulting conjugate. Sodium
cyanoborohydride (1 mole per mole of polysaccharide repeating unit)
was added, and conjugation proceeded at 22.degree. C.
Reduction with Sodium Borohydride
[0671] Sodium borohydride (2 moles per mole of polysaccharide
repeating unit) was added following the conjugation reaction and
incubated for 1 hour at 22.degree. C. The batch was diluted into
150 mM sodium chloride, with approximately 0.025% (w/v) polysorbate
20, at approximately 4.degree. C. Potassium phosphate buffer was
then added to neutralize the pH.
Final Filtration and Product Storage
[0672] The batch was then concentrated and diafiltered against 10
mM histidine in 150 mM sodium chloride, pH 7.0, with 0.015% (w/v)
polysorbate 20, at 4.degree. C. using a 300 kDa NMWCO tangential
flow ultrafiltration membrane.
[0673] The retentate batch was 0.2 micron filtered (with 0.5 micron
prefilter) then diluted with additional 10 mM histidine in 150 mM
sodium chloride, pH 7.0 with 0.015% (w/v) polysorbate 20, dispensed
into aliquots and frozen at .ltoreq.-60.degree. C.
EXAMPLE 25
Preparation of Serotype 12F Conjugate for PCV23 (DMSO) and PCV23
(DMSO+Aq) Polyvalent Studies Using DMSO Conjugation
[0674] Polysaccharide was dissolved, sized to a target molecular
mass, chemically activated and buffer-exchanged by ultrafiltration.
Activated polysaccharide and purified CRM197 were individually
lyophilized and redissolved in DMSO. Redissolved polysaccharide and
CRM197 solutions were then combined and conjugated as described
below. The resulting conjugate was purified by ultrafiltration
prior to a final 0.2-micron filtration. Several process parameters
within each step, such as pH, temperature, concentration, and time
were controlled to yield conjugates with desired attributes.
Polysaccharide Size Reduction and Oxidation
[0675] Purified pneumococcal capsular Ps powder was dissolved in
water and 0.45-micron filtered. Dissolved polysaccharide was
size-reduced by acid hydrolysis by adding acetic acid to 200 mM,
incubating at 90.degree. C. for 60 minutes, then neutralizing by
adding cold potassium phosphate pH 7 buffer to 400 mM.
[0676] Size-reduced polysaccharide was concentrated and diafiltered
against water using a 5 kDa NMWCO tangential flow ultrafiltration
membrane.
[0677] The polysaccharide solution was then adjusted to 22.degree.
C. and pH 5 with a sodium acetate buffer to minimize polysaccharide
size reduction due to activation. Polysaccharide activation was
initiated with the addition of a 100 mM sodium metaperiodate
solution. The oxidation reaction proceeded for 2 hours at
22.degree. C.
[0678] The activated product was diafiltered against 10 mM
potassium phosphate, pH 6.4 followed by diafiltration against water
using a 5 kDa NMWCO tangential flow ultrafiltration membrane.
Ultrafiltration was conducted at 2-8.degree. C.
Polysaccharide Conjugation to CRM197
[0679] Purified CRM197, obtained through expression in Pseudomonas
fluorescens as previously described (WO 2012/173876 A1), was
diafiltered against 2 mM phosphate, pH 7.2 buffer using a 5 kDa
NMWCO tangential flow ultrafiltration membrane and 0.2-micron
filtered.
[0680] Activated polysaccharide was formulated for lyophilization
at 6 mg Ps/mL with sucrose concentration of 5% w/v. CRM197 was
formulated for lyophilization at 6 mg Pr/mL with sucrose
concentration of 1% w/v.
[0681] Formulated Ps and CRM197 solutions were individually
lyophilized. Lyophilized Ps and CRM197 materials were redissolved
individually in equal volumes of DMSO. The polysaccharide and
CRM197 solutions were blended to achieve a polysaccharide
concentration of 3.0 g Ps/L and a polysaccharide to CRM197 mass
ratio of 1.5. The mass ratio was selected to control the
polysaccharide to CRM197 ratio in the resulting conjugate. Sodium
cyanoborohydride (1 mole per mole of polysaccharide repeating unit)
was added, and conjugation proceeded at 22.degree. C.
Reduction with Sodium Borohydride
[0682] Sodium borohydride (2 moles per mole of polysaccharide
repeating unit) was added following the conjugation reaction and
incubated for 1 hour at 22.degree. C. The batch was diluted into
150 mM sodium chloride, with approximately 0.025% (w/v) polysorbate
20, at approximately 4.degree. C. Potassium phosphate buffer was
then added to neutralize the pH. The batch was concentrated and
diafiltered at approximately 4.degree. C. against 150 mM sodium
chloride, 25 mM potassium phosphate pH 7.0, using a 30 kDa NMWCO
tangential flow ultrafiltration membrane.
Final Filtration and Product Storage
[0683] The batch was then concentrated and diafiltered against 10
mM histidine in 150 mM sodium chloride, pH 7.0, with 0.015% (w/v)
polysorbate 20, at 4.degree. C. using a 300 kDa NMWCO tangential
flow ultrafiltration membrane.
[0684] The retentate batch was 0.2 micron filtered (with 0.5 micron
prefilter) then diluted with additional 10 mM histidine in 150 mM
sodium chloride, pH 7.0 with 0.015% (w/v) polysorbate 20, dispensed
into aliquots and frozen at .ltoreq.-60.degree. C.
EXAMPLE 26
Preparation of Serotype 14 Conjugate for PCV23 (DMSO) Polyvalent
Study Using DMSO Conjugation
[0685] Polysaccharide was dissolved, sized to a target molecular
mass, chemically activated and buffer-exchanged by ultrafiltration.
Activated polysaccharide and purified CRM197 were individually
lyophilized and redissolved in DMSO. Redissolved polysaccharide and
CRM197 solutions were then combined and conjugated as described
below. The resulting conjugate was purified by dialysis prior to a
final 0.2-micron filtration. Several process parameters within each
step, such as pH, temperature, concentration, and time were
controlled to yield conjugates with desired attributes.
Polysaccharide Size Reduction and Oxidation
[0686] Purified pneumococcal capsular Ps powder was dissolved in
water and 0.45-micron filtered. Dissolved polysaccharide was
homogenized to reduce the molecular mass of the Ps. Homogenization
pressure and number of passes through the homogenizer were
controlled to 200 bar/6 passes.
[0687] Size-reduced polysaccharide was concentrated and diafiltered
against water using a 10 kDa NMWCO tangential flow ultrafiltration
membrane.
[0688] The polysaccharide solution was then adjusted to 22.degree.
C. and pH 5 with a sodium acetate buffer to minimize polysaccharide
size reduction due to activation. Polysaccharide activation was
initiated with the addition of a 100 mM sodium metaperiodate
solution. The oxidation reaction proceeded for 4 hours at
22.degree. C.
[0689] The activated product was diafiltered against 10 mM
potassium phosphate, pH 6.4 followed by diafiltration against water
using a 10 kDa NMWCO tangential flow ultrafiltration membrane.
Ultrafiltration was conducted at 2-8.degree. C.
Polysaccharide Conjugation to CRM197
[0690] Purified CRM197, obtained through expression in Pseudomonas
fluorescens as previously described (WO 2012/173876 A1), was
diafiltered against 2 mM phosphate, pH 7.2 buffer using a 5 kDa
NMWCO tangential flow ultrafiltration membrane and 0.2-micron
filtered.
[0691] Activated polysaccharide was formulated for lyophilization
at 6 mg Ps/mL with sucrose concentration of 5% w/v. CRM197 was
formulated for lyophilization at 6 mg Pr/mL with sucrose
concentration of 1% w/v.
[0692] Formulated Ps and CRM197 solutions were individually
lyophilized. Lyophilized Ps and CRM197 materials were redissolved
individually in equal volumes of DMSO. The polysaccharide and
CRM197 solutions were blended to achieve a polysaccharide
concentration of 1.8 g Ps/L and a polysaccharide to CRM197 mass
ratio of 1.5. The mass ratio was selected to control the
polysaccharide to CRM197 ratio in the resulting conjugate. Sodium
cyanoborohydride (1 mole per mole of polysaccharide repeating unit)
was added, and conjugation proceeded at 22.degree. C.
Reduction with Sodium Borohydride
[0693] Sodium borohydride (2 moles per mole of polysaccharide
repeating unit) was added following the conjugation reaction and
incubated for 1 hour at 22.degree. C. The batch was diluted into
150 mM sodium chloride, with approximately 0.025% (w/v) polysorbate
20, at approximately 4.degree. C. Potassium phosphate buffer was
then added to neutralize the pH. The batch was dialyzed at
approximately 4.degree. C. for 22.5 hours against 150 mM sodium
chloride, 0.05% polysorbate 20, using a 300 kDa MWCO dialysis
cassette.
Final Filtration and Product Storage
[0694] The retentate batch was 0.2 micron filtered (with 0.5 micron
prefilter), dispensed into aliquots and frozen at
.ltoreq.-60.degree. C.
EXAMPLE 27
[0695] Preparation of Serotype 14 Conjugate for PCV22 and PCV23
(DMSO+Aq) Polyvalent Study Using Aqueous Conjugation
[0696] Polysaccharide was dissolved, size reduced, chemically
activated and buffer-exchanged by ultrafiltration. Purified CRM197
was then conjugated to the activated polysaccharide utilizing
nickel chloride in the reaction mixture, and the resulting
conjugate was purified by ultrafiltration prior to a final
0.2-micron filtration. Several process parameters within each step,
such as pH, temperature, concentration, and time were controlled to
yield conjugates with desired attributes.
Polysaccharide Size Reduction and Oxidation
[0697] Purified pneumococcal capsular polysaccharide powder was
dissolved in water, and 0.45-micron filtered. Dissolved
polysaccharide was homogenized to reduce the molecular mass.
Homogenization pressure and number of passes through the
homogenizer were controlled to 200 bar/6 passes to size-reduce to a
target molecular mass. Size-reduced polysaccharide was then
concentrated and diafiltered against water using a 10 kDa NMWCO
tangential flow ultrafiltration membrane.
[0698] The polysaccharide solution was then adjusted to 22.degree.
C. and pH 5 with a sodium acetate buffer to minimize polysaccharide
size reduction due to activation. Polysaccharide activation was
initiated with the addition of a 100 mM sodium metaperiodate
solution. The oxidation reaction proceeded for 4 hours at
22.degree. C.
[0699] The activated product was diafiltered against 10 mM
potassium phosphate, pH 6.4 using a 10 kDa NMWCO tangential flow
ultrafiltration membrane. Ultrafiltration was conducted at
2-8.degree. C.
Polysaccharide Conjugation to CRM197
[0700] Oxidized polysaccharide solution was mixed with water and
1.5 M potassium phosphate pH 7.0. The buffer pH selected was to
improve the stability of activated polysaccharide during the
conjugation reaction. Purified CRM197, obtained through expression
in Pseudomonas fluorescens as previously described (WO 2012/173876
A1), was 0.2-micron filtered and combined with the buffered
polysaccharide solution at a polysaccharide to CRM197 mass ratio of
1.0. The mass ratio was selected to control the polysaccharide to
CRM197 ratio in the resulting conjugate. The polysaccharide and
phosphate concentrations were 3.8 g/L and 100 mM respectively. The
polysaccharide concentration was selected to control the size of
the resulting conjugate. The solution was then 0.2-micron filtered.
Nickel chloride was added to approximately 2 mM using a 100 mM
nickel chloride solution. Sodium cyanoborohydride (2 moles per mole
of polysaccharide repeating unit) was added. Conjugation proceeded
for 72 hours to maximize consumption of polysaccharide and
protein.
Reduction with Sodium Borohydride
[0701] Following the conjugation reaction, the batch was diluted to
a polysaccharide concentration of approximately 3.5 g/L, cooled to
2-8.degree. C., and 1.2-micron filtered. The batch was diafiltered
against 100 mM potassium phosphate, pH 7.0 at 2-8.degree. C. using
a 100 kDa NMWCO tangential flow ultrafiltration membrane. The
batch, recovered in the retentate, was then diluted to
approximately 2.0 g polysaccharide/L and pH-adjusted with the
addition of 1.2 M sodium bicarbonate, pH 9.4. Sodium borohydride (1
mole per mole of polysaccharide repeating unit) was added. 1.5 M
potassium phosphate, pH 6.0 was later added.
Final Filtration and Product Storage
[0702] The batch was then concentrated and diaftiltered against 10
mM L-histidine in 150 mM sodium chloride, pH 7.0 at 4.degree. C.
using a 300 kDa NMWCO tangential flow ultrafiltration membrane. The
batch was then 0.2 micron filtered.
[0703] The batch was adjusted to a polysaccharide concentration of
1.0 g/L with additional 10 mM L-histidine in 150 mM sodium
chloride, pH 7.0 buffer. The batch was dispensed into aliquots and
frozen at .ltoreq.-60.degree. C.
EXAMPLE 28
Preparation of Serotype 15A Conjugate for PCV23 (DMSO) and PCV23
(DMSO+Aq) Polyvalent Studies Using DMSO Conjugation
[0704] Polysaccharide was dissolved, sized to a target molecular
mass, chemically activated and buffer-exchanged by ultrafiltration.
Activated polysaccharide and purified CRM197 were individually
lyophilized and redissolved in DMSO. Redissolved polysaccharide and
CRM197 solutions were then combined and conjugated as described
below. The resulting conjugate was purified by ultrafiltration
prior to a final 0.2-micron filtration. Several process parameters
within each step, such as pH, temperature, concentration, and time
were controlled to yield conjugates with desired attributes.
Polysaccharide Size Reduction and Oxidation
[0705] Purified pneumococcal capsular Ps powder was dissolved in
water and 0.45-micron filtered. Dissolved polysaccharide was
homogenized to reduce the molecular mass of the Ps. Homogenization
pressure and number of passes through the homogenizer were
controlled to 210 bar/5 passes.
[0706] Size-reduced polysaccharide was concentrated and diafiltered
against water using a 10 kDa NMWCO tangential flow ultrafiltration
membrane.
[0707] The polysaccharide solution was then adjusted to 22.degree.
C. and pH 5 with a sodium acetate buffer to minimize polysaccharide
size reduction due to activation. Polysaccharide activation was
initiated with the addition of a 100 mM sodium metaperiodate
solution. The oxidation reaction proceeded for 20 hours at
22.degree. C.
[0708] The activated product was diafiltered against 10 mM
potassium phosphate, pH 6.4 followed by diafiltration against water
using a 10 kDa NMWCO tangential flow ultrafiltration membrane.
Ultrafiltration was conducted at 2-8.degree. C.
Polysaccharide Conjugation to CRM197
[0709] Purified CRM197, obtained through expression in Pseudomonas
fluorescens as previously described (WO 2012/173876 A1), was
diafiltered against 2 mM phosphate, pH 7.2 buffer using a 5 kDa
NMWCO tangential flow ultrafiltration membrane and 0.2-micron
filtered.
[0710] Activated polysaccharide was formulated for lyophilization
at 6 mg Ps/mL with sucrose concentration of 5% w/v. CRM197 was
formulated for lyophilization at 6 mg Pr/mL with sucrose
concentration of 1% w/v.
[0711] Formulated Ps and CRM197 solutions were individually
lyophilized. Lyophilized Ps and CRM197 materials were redissolved
individually in equal volumes of DMSO which was pre-heated to
34.degree. C. The polysaccharide solution was spiked with sodium
chloride to a concentration of 25 mM. The polysaccharide and CRM197
solutions were blended to achieve a polysaccharide concentration of
5.0 g Ps/L and a polysaccharide to CRM197 mass ratio of 2.0. The
mass ratio was selected to control the polysaccharide to CRM197
ratio in the resulting conjugate. Sodium cyanoborohydride (1 mole
per mole of polysaccharide repeating unit) was added, and
conjugation proceeded at 34.degree. C.
Reduction with Sodium Borohydride
[0712] Sodium borohydride (2 moles per mole of polysaccharide
repeating unit) was added following the conjugation reaction and
incubated for 1 hour at 34.degree. C. The batch was diluted into
150 mM sodium chloride, with approximately 0.025% (w/v) polysorbate
20, at approximately 4.degree. C. Potassium phosphate buffer was
then added to neutralize the pH.
Final Filtration and Product Storage
[0713] The batch was then concentrated and diafiltered against 10
mM histidine in 150 mM sodium chloride, pH 7.0, with 0.015% (w/v)
polysorbate 20, at 4.degree. C. using a 300 kDa NMWCO tangential
flow ultrafiltration membrane.
[0714] The retentate batch was 0.2 micron filtered (with 0.5 micron
prefilter) then diluted with additional 10 mM histidine in 150 mM
sodium chloride, pH 7.0 with 0.015% (w/v) polysorbate 20, dispensed
into aliquots and frozen at .ltoreq.-60.degree. C.
EXAMPLE 29
Preparation of Serotype 15A Conjugate for PCV22 Polyvalent Study
Using DMSO Conjugation
[0715] Polysaccharide was dissolved, sized to a target molecular
mass, chemically activated and buffer-exchanged by ultrafiltration.
Activated polysaccharide and purified CRM197 were individually
lyophilized and redissolved in DMSO. Redissolved polysaccharide and
CRM197 solutions were then combined and conjugated as described
below. The resulting conjugate was purified by ultrafiltration
prior to a final 0.2-micron filtration. Several process parameters
within each step, such as pH, temperature, concentration, and time
were controlled to yield conjugates with desired attributes.
Polysaccharide Size Reduction and Oxidation
[0716] Purified pneumococcal capsular Ps powder was dissolved in
water and 0.45-micron filtered. Dissolved polysaccharide was
homogenized to reduce the molecular mass of the Ps. Homogenization
pressure and number of passes through the homogenizer were
controlled to 200 bar/5 passes.
[0717] Size-reduced polysaccharide was concentrated and diafiltered
against water using a 10 kDa NMWCO tangential flow ultrafiltration
membrane.
[0718] The polysaccharide solution was then adjusted to 22.degree.
C. and pH 5 with a sodium acetate buffer to minimize polysaccharide
size reduction due to activation. Polysaccharide activation was
initiated with the addition of a 100 mM sodium metaperiodate
solution. The oxidation reaction proceeded for 20 hours at
22.degree. C.
[0719] The activated product was diafiltered against 10 mM
potassium phosphate, pH 6.4 followed by diafiltration against water
using a 10 kDa NMWCO tangential flow ultrafiltration membrane.
Ultrafiltration was conducted at 2-8.degree. C.
Polysaccharide Conjugation to CRM197
[0720] Purified CRM197, obtained through expression in Pseudomonas
fluorescens as previously described (WO 2012/173876 A1), was
diafiltered against 2 mM phosphate, pH 7.2 buffer using a 5 kDa
NMWCO tangential flow ultrafiltration membrane and 0.2-micron
filtered.
[0721] Activated polysaccharides were formulated for lyophilization
at 6 mg Ps/mL with sucrose concentration of 5% w/v. CRM197 was
formulated for lyophilization at 6 mg Pr/mL with sucrose
concentration of 1% w/v.
[0722] Formulated Ps and CRM197 solutions were individually
lyophilized. Lyophilized Ps and CRM197 materials were redissolved
individually in equal volumes of DMSO which was pre-heated to
34.degree. C. The polysaccharide solution was spiked with sodium
chloride to a concentration of 25 mM. The polysaccharide and CRM197
solutions were blended to achieve a polysaccharide concentration of
5.0 g Ps/L and a polysaccharide to CRM197 mass ratio of 2.0. The
mass ratio was selected to control the polysaccharide to CRM197
ratio in the resulting conjugate. Sodium cyanoborohydride (1 mole
per mole of polysaccharide repeating unit) was added, and
conjugation proceeded at 34.degree. C.
Reduction with Sodium Borohydride
[0723] Sodium borohydride (2 moles per mole of polysaccharide
repeating unit) was added following the conjugation reaction and
incubated for 1 hour at 22.degree. C. The batch was diluted into
150 mM sodium chloride, with approximately 0.025% (w/v) polysorbate
20, at approximately 4.degree. C. Potassium phosphate buffer was
then added to neutralize the pH. The batch was concentrated and
diafiltered at approximately 4.degree. C. against 150 mM sodium
chloride, 25 mM potassium phosphate pH 7, using a 30 kDa NMWCO
tangential flow ultrafiltration membrane.
Final Filtration and Product Storage
[0724] The batch was then concentrated and diafiltered against 10
mM histidine in 150 mM sodium chloride, pH 7.0, with 0.015% (w/v)
polysorbate 20, at 4.degree. C. using a 300 kDa NMWCO tangential
flow ultrafiltration membrane.
[0725] The retentate batch was 0.2 micron filtered (with 0.5 micron
prefilter) then diluted with additional 10 mM histidine in 150 mM
sodium chloride, pH 7.0 with 0.015% (w/v) polysorbate 20, dispensed
into aliquots and frozen at .ltoreq.-60.degree. C.
EXAMPLE 30
Preparation of Serotype 15C Conjugate for PCV23 (DMSO) and PCV23
(DMSO+Aq) Polyvalent Studies Using DMSO Conjugation
[0726] Polysaccharide derived from Streptococcus pneumoniae
serotype 15B was dissolved, sized to a target molecular mass,
subjected to mild base hydrolysis to release 0-acetyl groups,
chemically activated and buffer-exchanged by ultrafiltration.
Activated polysaccharide and purified CRM197 were individually
lyophilized and redissolved in DMSO. Redissolved polysaccharide and
CRM197 solutions were then combined and conjugated as described
below. The resulting conjugate was purified by ultrafiltration
prior to a final 0.2-micron filtration. Several process parameters
within each step, such as pH, temperature, concentration, and time
were controlled to yield conjugates with desired attributes.
Polysaccharide Size Reduction, Base Hydrolysis and Oxidation
[0727] Purified serotype 15B pneumococcal capsular Ps powder was
dissolved in water and 0.45-micron filtered. Dissolved
polysaccharide was homogenized to reduce the molecular mass of the
Ps. Homogenization pressure and number of passes through the
homogenizer were controlled to 300 bar/5 passes.
[0728] Size-reduced polysaccharide was concentrated and diafiltered
against water using a 10 kDa NMWCO tangential flow ultrafiltration
membrane.
[0729] The polysaccharide solution was heated to 60.degree. C. and
sodium bicarbonate pH 9 buffer was added to a final concentration
of 50 mM. The batch was incubated with mixing for 13 hours at
60.degree. C. to release 0-acetyl groups. Potassium phosphate pH 6
buffer was added to a final concentration of 136 mM to neutralize
pH and the solution was cooled to ambient temperature. The solution
was then concentrated and diafiltered against water using a 10 kDa
NMWCO tangential flow ultrafiltration membrane.
[0730] The polysaccharide solution was adjusted to 22.degree. C.
and pH 5 with a sodium acetate buffer to minimize polysaccharide
size reduction due to activation. Polysaccharide activation was
initiated with the addition of a 100 mM sodium metaperiodate
solution. The oxidation reaction proceeded for 2 hours at
22.degree. C.
[0731] The activated product was diafiltered against 10 mM
potassium phosphate, pH 6.4 followed by diafiltration against water
using a 10 kDa NMWCO tangential flow ultrafiltration membrane.
Ultrafiltration was conducted at 2-8.degree. C.
Polysaccharide Conjugation to CRM197
[0732] Purified CRM197, obtained through expression in Pseudomonas
fluorescens as previously described (WO 2012/173876 A1), was
diafiltered against 2 mM phosphate, pH 7.2 buffer using a 5 kDa
NMWCO tangential flow ultrafiltration membrane and 0.2-micron
filtered.
[0733] Activated polysaccharide was formulated for lyophilization
at 6 mg Ps/mL with sucrose concentration of 5% w/v. CRM197 was
formulated for lyophilization at 6 mg Pr/mL with sucrose
concentration of 1% w/v.
[0734] Formulated Ps and CRM197 solutions were individually
lyophilized. Lyophilized Ps and CRM197 materials were redissolved
individually in equal volumes of DMSO. The polysaccharide and
CRM197 solutions were blended to achieve a polysaccharide
concentration of 3.0 g Ps/L and a polysaccharide to CRM197 mass
ratio of 1.75. The mass ratio was selected to control the
polysaccharide to CRM197 ratio in the resulting conjugate. Sodium
cyanoborohydride (1 mole per mole of polysaccharide repeating unit)
was added, and conjugation proceeded at 22.degree. C.
Reduction with Sodium Borohydride
[0735] Sodium borohydride (2 moles per mole of polysaccharide
repeating unit) was added following the conjugation reaction and
incubated for 1 hour at 22.degree. C. The batch was diluted into
150 mM sodium chloride, with approximately 0.025% (w/v) polysorbate
20, at approximately 4.degree. C. Potassium phosphate buffer was
then added to neutralize the pH. The batch was concentrated and
diafiltered at approximately 4.degree. C. against 150 mM sodium
chloride, 25 mM potassium phosphate pH 7, using a 30 kDa NMWCO
tangential flow ultrafiltration membrane.
Final Filtration and Product Storage
[0736] The batch was then concentrated and diafiltered against 10
mM histidine in 150 mM sodium chloride, pH 7.0, with 0.015% (w/v)
polysorbate 20, at 4.degree. C. using a 300 kDa NMWCO tangential
flow ultrafiltration membrane.
[0737] The retentate batch was 0.2 micron filtered (with 0.5 micron
prefilter) then diluted with additional 10 mM histidine in 150 mM
sodium chloride, pH 7.0 with 0.015% (w/v) polysorbate 20, dispensed
into aliquots and frozen at .ltoreq.-60.degree. C.
EXAMPLE 31
Preparation of Serotype 15C Conjugate for PCV22 Polyvalent Study
Using DMSO Conjugation
[0738] Polysaccharide derived from Streptococcus pneumoniae
serotype 15B was dissolved, sized to a target molecular mass,
subjected to mild base hydrolysis to release O-acetyl groups,
chemically activated and buffer-exchanged by ultrafiltration.
Activated polysaccharide and purified CRM197 were individually
lyophilized and redissolved in DMSO. Redissolved polysaccharide and
CRM197 solutions were then combined and conjugated as described
below. The resulting conjugate was purified by ultrafiltration
prior to a final 0.2-micron filtration. Several process parameters
within each step, such as pH, temperature, concentration, and time
were controlled to yield conjugates with desired attributes.
Polysaccharide Size Reduction, Base Hydrolysis and Oxidation
[0739] Purified serotype 15B pneumococcal capsular Ps powder was
dissolved in water and 0.45-micron filtered. Dissolved
polysaccharide was homogenized to reduce the molecular mass of the
Ps. Homogenization pressure and number of passes through the
homogenizer were controlled to 300 bar/5 passes.The size-reduced
polysaccharide solution was heated to 60.degree. C. and sodium
bicarbonate pH 9.4 buffer was added to a final concentration of 50
mM. The batch was incubated with mixing for 12 hours at 60.degree.
C. to release 0-acetyl groups. Potassium phosphate pH 6 buffer was
added to a final concentration of 150 mM to neutralize pH and the
solution was cooled to ambient temperature. The solution was then
concentrated and diafiltered against water using a 10 kDa NMWCO
tangential flow ultrafiltration membrane.
[0740] The polysaccharide solution was adjusted to 22.degree. C.
and pH 5 with a sodium acetate buffer to minimize polysaccharide
size reduction due to activation. Polysaccharide activation was
initiated with the addition of a 100 mM sodium metaperiodate
solution. The oxidation reaction proceeded for 2 hours at
22.degree. C.
[0741] The activated product was diafiltered against 10 mM
potassium phosphate, pH 6.4 followed by diafiltration against water
using a 10 kDa NMWCO tangential flow ultrafiltration membrane.
Ultrafiltration was conducted at 2-8.degree. C.
Polysaccharide Conjugation to CRM197
[0742] Purified CRM197, obtained through expression in Pseudomonas
fluorescens as previously described (WO 2012/173876 A1), was
diafiltered against 2 mM phosphate, pH 7.2 buffer using a 5 kDa
NMWCO tangential flow ultrafiltration membrane and 0.2-micron
filtered.
[0743] Activated polysaccharide was formulated for lyophilization
at 6 mg Ps/mL with sucrose concentration of 5% w/v. CRM197 was
formulated for lyophilization at 6 mg Pr/mL with sucrose
concentration of 1% w/v.
[0744] Formulated Ps and CRM197 solutions were individually
lyophilized. Lyophilized Ps and CRM197 materials were redissolved
individually in equal volumes of DMSO. The polysaccharide and
CRM197 solutions were blended to achieve a polysaccharide
concentration of 3.2 g Ps/L and a polysaccharide to CRM197 mass
ratio of 1.75. The mass ratio was selected to control the
polysaccharide to CRM197 ratio in the resulting conjugate. Sodium
cyanoborohydride (1 mole per mole of polysaccharide repeating unit)
was added, and conjugation proceeded at 22.degree. C.
Reduction with Sodium Borohydride
[0745] Sodium borohydride (2 moles per mole of polysaccharide
repeating unit) was added following the conjugation reaction and
incubated for 1 hour at 22.degree. C. The batch was diluted into
150 mM sodium chloride, with approximately 0.025% (w/v) polysorbate
20, at approximately 4.degree. C. Potassium phosphate buffer was
then added to neutralize the pH. The batch was concentrated and
diafiltered at approximately 4.degree. C. against 150 mM sodium
chloride, 25 mM potassium phosphate pH 7, using a 30 kDa NMWCO
tangential flow ultrafiltration membrane.
Final Filtration and Product Storage
[0746] The batch was then concentrated and diafiltered against 10
mM histidine in 150 mM sodium chloride, pH 7.0, with 0.015% (w/v)
polysorbate 20, at 4.degree. C. using a 300 kDa NMWCO tangential
flow ultrafiltration membrane.
[0747] The retentate batch was 0.2 micron filtered (with 0.5 micron
prefilter) then diluted with additional 10 mM histidine in 150 mM
sodium chloride, pH 7.0 with 0.015% (w/v) polysorbate 20, dispensed
into aliquots and frozen at .ltoreq.-60.degree. C.
EXAMPLE 32
Preparation of Serotype 18C Conjugate for PCV23 (DMSO) and PCV23
(DMSO+Aq) Polyvalent Studies Using DMSO Conjugation
[0748] Polysaccharide was dissolved, sized to a target molecular
mass, chemically activated and buffer-exchanged by ultrafiltration.
Activated polysaccharide and purified CRM197 were individually
lyophilized and redissolved in DMSO. Redissolved polysaccharide and
CRM197 solutions were then combined and conjugated as described
below. The resulting conjugate was purified by ultrafiltration
prior to a final 0.2-micron filtration. Several process parameters
within each step, such as pH, temperature, concentration, and time
were controlled to yield conjugates with desired attributes.
Polysaccharide Size Reduction and Oxidation
[0749] Purified pneumococcal capsular Ps powder was dissolved in
water and 0.45-micron filtered. Dissolved polysaccharide was
size-reduced by acid hydrolysis by adding acetic acid to 200 mM,
incubating at 90.degree. C. for 160 minutes, then neutralizing by
adding cold potassium phosphate pH 7 buffer to 400 mM.
[0750] Size-reduced polysaccharide was concentrated and diafiltered
against water using a 5 kDa NMWCO tangential flow ultrafiltration
membrane.
[0751] The polysaccharide solution was then adjusted to 22.degree.
C. and pH 5 with a sodium acetate buffer to minimize polysaccharide
size reduction due to activation. Polysaccharide activation was
initiated with the addition of a 100 mM sodium metaperiodate
solution. The oxidation reaction proceeded for 2 hours at
22.degree. C.
[0752] The activated product was diafiltered against 10 mM
potassium phosphate, pH 6.4 followed by diafiltration against water
using a 10 kDa NMWCO tangential flow ultrafiltration membrane.
Ultrafiltration was conducted at 2-8.degree. C.
Polysaccharide Conjugation to CRM197
[0753] Purified CRM197, obtained through expression in Pseudomonas
fluorescens as previously described (WO 2012/173876 A1), was
diafiltered against 2 mM phosphate, pH 7.2 buffer using a 5 kDa
NMWCO tangential flow ultrafiltration membrane and 0.2-micron
filtered.
[0754] Activated polysaccharide was formulated for lyophilization
at 6 mg Ps/mL with sucrose concentration of 5% w/v. CRM197 was
formulated for lyophilization at 6 mg Pr/mL with sucrose
concentration of 1% w/v.
[0755] Formulated Ps and CRM197 solutions were individually
lyophilized. Lyophilized Ps and CRM197 materials were redissolved
individually in equal volumes of DMSO. The polysaccharide and
CRM197 solutions were blended to achieve a polysaccharide
concentration of 3.49 g Ps/L and a polysaccharide to CRM197 mass
ratio of 1.5. The mass ratio was selected to control the
polysaccharide to CRM197 ratio in the resulting conjugate. Sodium
cyanoborohydride (1 mole per mole of polysaccharide repeating unit)
was added, and conjugation proceeded at 22.degree. C.
Reduction with Sodium Borohydride
[0756] Sodium borohydride (2 moles per mole of polysaccharide
repeating unit) was added following the conjugation reaction and
incubated for 3 hours at 22.degree. C. The batch was diluted into
150 mM sodium chloride, with approximately 0.025% (w/v) polysorbate
20, at approximately 4.degree. C. Potassium phosphate buffer was
then added to neutralize the pH. The batch was concentrated and
diafiltered at approximately 4.degree. C. against 150 mM sodium
chloride, 25 mM potassium phosphate pH 7, using a 30 kDa NMWCO
tangential flow ultrafiltration membrane.
[0757] Final Filtration and Product Storage
[0758] The batch was then concentrated and diafiltered against 10
mM histidine in 150 mM sodium chloride, pH 7.0, with 0.015% (w/v)
polysorbate 20, at 4.degree. C. using a 300 kDa NMWCO tangential
flow ultrafiltration membrane.
[0759] The retentate batch was 0.2 micron filtered (with 0.5 micron
prefilter) then diluted with additional 10 mM histidine in 150 mM
sodium chloride, pH 7.0 with 0.015% (w/v) polysorbate 20, dispensed
into aliquots and frozen at .ltoreq.-60.degree. C.
EXAMPLE 33
Preparation of Serotype 18C Conjugate for PCV22 Polyvalent Study
Using DMSO Conjugation
[0760] Polysaccharide was dissolved, sized to a target molecular
mass, chemically activated and buffer-exchanged by ultrafiltration.
Activated polysaccharide and purified CRM197 were individually
lyophilized and redissolved in DMSO. Redissolved polysaccharide and
CRM197 solutions were then combined and conjugated as described
below. The resulting conjugate was purified by ultrafiltration
prior to a final 0.2-micron filtration. Several process parameters
within each step, such as pH, temperature, concentration, and time
were controlled to yield conjugates with desired attributes.
Polysaccharide Size Reduction and Oxidation
[0761] Purified pneumococcal capsular Ps powder was dissolved in
water and 0.45-micron filtered. Dissolved polysaccharide was
size-reduced by acid hydrolysis by adding acetic acid to 200 mM,
incubating at 90.degree. C. for 160 minutes, then neutralizing by
adding cold potassium phosphate pH 7 buffer to 400 mM.
[0762] Size-reduced polysaccharide was concentrated and diafiltered
against water using a 5 kDa NMWCO tangential flow ultrafiltration
membrane.
[0763] The polysaccharide solution was then adjusted to 22.degree.
C. and pH 5 with a sodium acetate buffer to minimize polysaccharide
size reduction due to activation. Polysaccharide activation was
initiated with the addition of a 100 mM sodium metaperiodate
solution. The oxidation reaction proceeded for 2 hours at
22.degree. C.
[0764] The activated product was diafiltered against 10 mM
potassium phosphate, pH 6.4 followed by diafiltration against water
using a 5 kDa NMWCO tangential flow ultrafiltration membrane.
Ultrafiltration was conducted at 2-8.degree. C.
Polysaccharide Conjugation to CRM197
[0765] Purified CRM197, obtained through expression in Pseudomonas
fluorescens as previously described (WO 2012/173876 A1), was
diafiltered against 2 mM phosphate, pH 7.2 buffer using a 5 kDa
NMWCO tangential flow ultrafiltration membrane and 0.2-micron
filtered.
[0766] Activated polysaccharide was formulated for lyophilization
at 6 mg Ps/mL with sucrose concentration of 5% w/v. CRM197 was
formulated for lyophilization at 6 mg Pr/mL with sucrose
concentration of 1% w/v.
[0767] Formulated Ps and CRM197 solutions were individually
lyophilized. Lyophilized Ps and CRM197 materials were redissolved
individually in equal volumes of DMSO. The polysaccharide and
CRM197 solutions were blended to achieve a polysaccharide
concentration of 2.49 g Ps/L and a polysaccharide to CRM197 mass
ratio of 1.5. The mass ratio was selected to control the
polysaccharide to CRM197 ratio in the resulting conjugate. Sodium
cyanoborohydride (1 mole per mole of polysaccharide repeating unit)
was added, and conjugation proceeded at 22.degree. C.
Reduction with Sodium Borohydride
[0768] Sodium borohydride (2 moles per mole of polysaccharide
repeating unit) was added following the conjugation reaction and
incubated for 3 hours at 22.degree. C. The batch was diluted into
150 mM sodium chloride, with approximately 0.025% (w/v) polysorbate
20, at approximately 4.degree. C. Potassium phosphate buffer was
then added to neutralize the pH. The batch was concentrated and
diafiltered at approximately 4.degree. C. against 150 mM sodium
chloride, 25 mM potassium phosphate pH 7, using a 30 kDa NMWCO
tangential flow ultrafiltration membrane.
Final Filtration and Product Storage
[0769] The batch was then concentrated and diafiltered against 10
mM histidine in 150 mM sodium chloride, pH 7.0, with 0.015% (w/v)
polysorbate 20, at 4.degree. C. using a 300 kDa NMWCO tangential
flow ultrafiltration membrane.
[0770] The retentate batch was 0.2 micron filtered (with 0.5 micron
prefilter) then diluted with additional 10 mM histidine in 150 mM
sodium chloride, pH 7.0 with 0.015% (w/v) polysorbate 20, dispensed
into aliquots and frozen at .ltoreq.-60.degree. C.
EXAMPLE 34
Preparation of Serotype 19A Conjugate for PCV23 (DMSO) and PCV23
(DMSO+Aq) Polyvalent Studies Using DMSO Conjugation
[0771] Polysaccharide was dissolved, chemically activated and
buffer-exchanged by ultrafiltration. Activated polysaccharide and
purified CRM197 were individually lyophilized and redissolved in
DMSO. Redissolved polysaccharide and CRM197 solutions were then
combined and conjugated as described below. The resulting conjugate
was purified by ultrafiltration prior to a final 0.2-micron
filtration. Several process parameters within each step, such as
pH, temperature, concentration, and time were controlled to yield
conjugates with desired attributes.
Polysaccharide Oxidation
[0772] Purified pneumococcal capsular Ps powder was dissolved in
water and 0.22-micron filtered. The polysaccharide was concentrated
and diafiltered against water using a 10 kDa NMWCO tangential flow
ultrafiltration membrane.
[0773] The polysaccharide solution was then adjusted to 22.degree.
C. and pH 5 with a sodium acetate buffer to minimize polysaccharide
size reduction due to activation. Polysaccharide activation was
initiated with the addition of a 100 mM sodium metaperiodate
solution. The oxidation reaction proceeded for 20 hours at
22.degree. C. The activated product was diafiltered against 10 mM
potassium phosphate, pH 6.4 followed by diafiltration against water
using a 10 kDa NMWCO tangential flow ultrafiltration membrane.
Ultrafiltration was conducted at 2-8.degree. C.
Polysaccharide Conjugation to CRM197
[0774] Purified CRM197, obtained through expression in Pseudomonas
fluorescens as previously described (WO 2012/173876 Al), was
diafiltered against 2 mM phosphate, pH 7.2 buffer using a 5 kDa
NMWCO tangential flow ultrafiltration membrane and 0.2-micron
filtered.
[0775] Activated polysaccharide was formulated for lyophilization
at 6 mg Ps/mL with sucrose concentration of 5% w/v. CRM197 was
formulated for lyophilization at 6 mg Pr/mL with sucrose
concentration of 1% w/v.
[0776] Formulated Ps and CRM197 solutions were individually
lyophilized. Lyophilized Ps and CRM197 materials were redissolved
individually in equal volumes of DMSO. The polysaccharide and
CRM197 solutions were blended to achieve a polysaccharide
concentration of 3.8 g Ps/L and a polysaccharide to CRM197 mass
ratio of 1.33. The mass ratio was selected to control the
polysaccharide to CRM197 ratio in the resulting conjugate. Sodium
cyanoborohydride (1 mole per mole of polysaccharide repeating unit)
was added, and conjugation proceeded at 22.degree. C.
Reduction with Sodium Borohydride
[0777] Sodium borohydride (2 moles per mole of polysaccharide
repeating unit) was added following the conjugation reaction and
incubated for 3 hours at 22.degree. C. The batch was diluted into
150 mM sodium chloride, with approximately 0.025% (w/v) polysorbate
20, at approximately 4.degree. C. Potassium phosphate buffer was
then added to neutralize the pH. The batch was concentrated and
diafiltered at approximately 4.degree. C. against 150 mM sodium
chloride, 25 mM potassium phosphate pH 7, using a 30 kDa NMWCO
tangential flow ultrafiltration membrane.
Final Filtration and Product Storage
[0778] The batch was then concentrated and diafiltered against 10
mM histidine in 150 mM sodium chloride, pH 7.0, with 0.015% (w/v)
polysorbate 20, at 4.degree. C. using a 300 kDa NMWCO tangential
flow ultrafiltration membrane.
[0779] The retentate batch was 0.2 micron filtered then diluted
with additional 10 mM histidine in 150 mM sodium chloride, pH 7.0
with 0.015% (w/v) polysorbate 20, dispensed into aliquots and
frozen at .ltoreq.-60.degree. C.
EXAMPLE 35
Preparation of Serotype 19A Conjugate for PCV22 Polyvalent Study
Using DMSO Conjugation
[0780] Polysaccharide was dissolved, chemically activated and
buffer-exchanged by ultrafiltration. Activated polysaccharide and
purified CRM197 were individually lyophilized and redissolved in
DMSO. Redissolved polysaccharide and CRM197 solutions were then
combined and conjugated as described below. The resulting conjugate
was purified by ultrafiltration prior to a final 0.2-micron
filtration. Several process parameters within each step, such as
pH, temperature, concentration, and time were controlled to yield
conjugates with desired attributes.
Polysaccharide Oxidation
[0781] Purified pneumococcal capsular Ps powder was dissolved in
water and 0.22-micron filtered. The polysaccharide was concentrated
and diafiltered against water using a 10 kDa NMWCO tangential flow
ultrafiltration membrane.
[0782] The polysaccharide solution was then adjusted to 22.degree.
C. and pH 5 with a sodium acetate buffer to minimize polysaccharide
size reduction due to activation. Polysaccharide activation was
initiated with the addition of a 100 mM sodium metaperiodate
solution. The oxidation reaction proceeded for 20 hours at
22.degree. C.
[0783] The activated product was diafiltered against 10 mM
potassium phosphate, pH 6.4 followed by diafiltration against water
using a 10 kDa NMWCO tangential flow ultrafiltration membrane.
Ultrafiltration was conducted at 2-8.degree. C.
Polysaccharide Conjugation to CRM197
[0784] Purified CRM197, obtained through expression in Pseudomonas
fluorescens as previously described (WO 2012/173876 A1), was
diafiltered against 2 mM phosphate, pH 7.2 buffer using a 5 kDa
NMWCO tangential flow ultrafiltration membrane and 0.2-micron
filtered.
[0785] Activated polysaccharide was formulated for lyophilization
at 6 mg Ps/mL with sucrose concentration of 5% w/v. CRM197 was
formulated for lyophilization at 6 mg Pr/mL with sucrose
concentration of 1% w/v.
[0786] Formulated Ps and CRM197 solutions were individually
lyophilized. Lyophilized Ps and CRM197 materials were redissolved
individually in equal volumes of DMSO. The polysaccharide and
CRM197 solutions were blended to achieve a polysaccharide
concentration of 3.8 g Ps/L and a polysaccharide to CRM197 mass
ratio of 1.33. The mass ratio was selected to control the
polysaccharide to CRM197 ratio in the resulting conjugate. Sodium
cyanoborohydride (1 mole per mole of polysaccharide repeating unit)
was added, and conjugation proceeded at 22.degree. C.
Reduction with Sodium Borohydride
[0787] Sodium borohydride (2 moles per mole of polysaccharide
repeating unit) was added following the conjugation reaction and
incubated for 3 hours at 22.degree. C. The batch was diluted into
150 mM sodium chloride, with approximately 0.025% (w/v) polysorbate
20, at approximately 4.degree. C. Potassium phosphate buffer was
then added to neutralize the pH. The batch was concentrated and
diafiltered at approximately 4.degree. C. against 150 mM sodium
chloride, 25 mM potassium phosphate pH 7, using a 30 kDa NMWCO
tangential flow ultrafiltration membrane.
Final Filtration and Product Storage
[0788] The batch was then concentrated and diafiltered against 10
mM histidine in 150 mM sodium chloride, pH 7.0, with 0.015% (w/v)
polysorbate 20, at 4.degree. C. using a 300 kDa NMWCO tangential
flow ultrafiltration membrane.
[0789] The retentate batch was 0.2 micron filtered then diluted
with additional 10 mM histidine in 150 mM sodium chloride, pH 7.0
with 0.015% (w/v) polysorbate 20, dispensed into aliquots and
frozen at .ltoreq.-60.degree. C.
[0790] EXAMPLE 36
Preparation of Serotype 19F Conjugate for PCV23 (DMSO), PCV23
(DMSO+Aq) and PCV22 Polyvalent Studies Using DMSO Conjugation
[0791] Polysaccharide was dissolved, sized to a target molecular
mass, chemically activated and buffer-exchanged by ultrafiltration.
Activated polysaccharide and purified CRM197 were individually
lyophilized and redissolved in DMSO. Redissolved polysaccharide and
CRM197 solutions were then combined and conjugated as described
below. The resulting conjugate was purified by ultrafiltration
prior to a final 0.2-micron filtration. Several process parameters
within each step, such as pH, temperature, concentration, and time
were controlled to yield conjugates with desired attributes.
Polysaccharide Size Reduction and Oxidation
[0792] Purified pneumococcal capsular Ps powder was dissolved in
water and 0.45-micron filtered. Dissolved polysaccharide was
homogenized to reduce the molecular mass of the Ps. Homogenization
pressure and number of passes through the homogenizer were
controlled to 150 bar/5 passes.
[0793] Size-reduced polysaccharide was concentrated and diafiltered
against water using a 10 kDa NMWCO tangential flow ultrafiltration
membrane.
[0794] The polysaccharide solution was then adjusted to 4.degree.
C. and pH 5 with a sodium acetate buffer to minimize polysaccharide
size reduction due to activation. Polysaccharide activation was
initiated with the addition of a 100 mM sodium metaperiodate
solution. The oxidation reaction proceeded for 4 hours at 4.degree.
C.
[0795] The activated product was diafiltered against 10 mM
potassium phosphate, pH 6.4 followed by diafiltration against water
using a 10 kDa NMWCO tangential flow ultrafiltration membrane.
Ultrafiltration was conducted at 2-8.degree. C.
Polysaccharide Conjugation to CRM197
[0796] Purified CRM197, obtained through expression in Pseudomonas
fluorescens as previously described (WO 2012/173876 A1), was
diafiltered against 2 mM phosphate, pH 7.2 buffer using a 5 kDa
NMWCO tangential flow ultrafiltration membrane and 0.2-micron
filtered.
[0797] Activated polysaccharide was formulated for lyophilization
at 6 mg Ps/mL with sucrose concentration of 5% w/v. CRM197 was
formulated for lyophilization at 6 mg Pr/mL with sucrose
concentration of 1% w/v.
[0798] Formulated Ps and CRM197 solutions were individually
lyophilized. Lyophilized Ps and CRM197 materials were redissolved
individually in equal volumes of DMSO. The polysaccharide and
CRM197 solutions were blended to achieve a polysaccharide
concentration of 2.0 g Ps/L and a polysaccharide to CRM197 mass
ratio of 1.2. The mass ratio was selected to control the
polysaccharide to CRM197 ratio in the resulting conjugate. Sodium
cyanoborohydride (1 mole per mole of polysaccharide repeating unit)
was added, and conjugation proceeded at 22.degree. C.
Reduction with Sodium Borohydride
[0799] Sodium borohydride (2 moles per mole of polysaccharide
repeating unit) was added following the conjugation reaction and
incubated for 3 hours at 22.degree. C. The batch was diluted into
150 mM sodium chloride, with approximately 0.025% (w/v) polysorbate
20, at approximately 4.degree. C. Potassium phosphate buffer was
then added to neutralize the pH. The batch was concentrated and
diafiltered at approximately 4.degree. C. against 150 mM sodium
chloride, 25 mM potassium phosphate pH 7, using a 30 kDa NMWCO
tangential flow ultrafiltration membrane. The retentate batch was
0.2 micron filtered then incubated at 22.degree. C. for 4.5
days.
Final Filtration and Product Storage
[0800] The batch was then concentrated and diafiltered against 10
mM histidine in 150 mM sodium chloride, pH 7.0 at 4.degree. C.
using a 300 kDa NMWCO tangential flow ultrafiltration membrane.
[0801] The retentate batch was 0.2 micron filtered then diluted
with additional 10 mM histidine in 150 mM sodium chloride, pH 7.0
with 0.015% (w/v) polysorbate 20, dispensed into aliquots and
frozen at .ltoreq.-60.degree. C.
EXAMPLE 37
Preparation of Serotype 22F Conjugate for PCV23 (DMSO) Polyvalent
Study Using DMSO Conjugation
[0802] Polysaccharide was dissolved, sized to a target molecular
mass, chemically activated and buffer-exchanged by ultrafiltration.
Activated polysaccharide and purified CRM197 were individually
lyophilized and redissolved in DMSO. Redissolved polysaccharide and
CRM197 solutions were then combined and conjugated as described
below. The resulting conjugate was purified by ultrafiltration
prior to a final 0.2-micron filtration. Several process parameters
within each step, such as pH, temperature, concentration, and time
were controlled to yield conjugates with desired attributes.
Polysaccharide Size Reduction and Oxidation
[0803] Purified pneumococcal capsular Ps powder was dissolved in
water and 0.45-micron filtered. Dissolved polysaccharide was
homogenized to reduce the molecular mass of the Ps. Homogenization
pressure and number of passes through the homogenizer were
controlled to 810 bar/5 passes.
[0804] Size-reduced polysaccharide was concentrated and diafiltered
against water using a 5 kDa NMWCO tangential flow ultrafiltration
membrane.
[0805] The polysaccharide solution was then adjusted to 22.degree.
C. and pH 5 with a sodium acetate buffer to minimize polysaccharide
size reduction due to activation. Polysaccharide activation was
initiated with the addition of a 100 mM sodium metaperiodate
solution. The oxidation reaction proceeded for 2 hours at
22.degree. C.
[0806] The activated product was diafiltered against 10 mM
potassium phosphate, pH 6.4 followed by diafiltration against water
using a 5 kDa NMWCO tangential flow ultrafiltration membrane.
Ultrafiltration was conducted at 2-8.degree. C.
Polysaccharide Conjugation to CRM197
[0807] Purified CRM197, obtained through expression in Pseudomonas
fluorescens as previously described (WO 2012/173876 A1), was
diafiltered against 2 mM phosphate, pH 7.2 buffer using a 5 kDa
NMWCO tangential flow ultrafiltration membrane and 0.2-micron
filtered.
[0808] Activated polysaccharide was formulated for lyophilization
at 6 mg Ps/mL with sucrose concentration of 5% w/v. CRM197 was
formulated for lyophilization at 6 mg Pr/mL with sucrose
concentration of 1% w/v.
[0809] Formulated Ps and CRM197 solutions were individually
lyophilized. Lyophilized Ps and CRM197 materials were redissolved
individually in equal volumes of DMSO. The polysaccharide and
CRM197 solutions were blended to achieve a polysaccharide
concentration of 2.3 g Ps/L and a polysaccharide to CRM197 mass
ratio of 1.5. The mass ratio was selected to control the
polysaccharide to CRM197 ratio in the resulting conjugate. Sodium
cyanoborohydride (1 mole per mole of polysaccharide repeating unit)
was added, and conjugation proceeded at 22.degree. C.
Reduction with Sodium Borohydride
[0810] Sodium borohydride (2 moles per mole of polysaccharide
repeating unit) was added following the conjugation reaction and
incubated for 1 hour at 22.degree. C. The batch was diluted into
150 mM sodium chloride, with approximately 0.025% (w/v) polysorbate
20, at approximately 4.degree. C. Potassium phosphate buffer was
then added to neutralize the pH.
Final Filtration and Product Storage
[0811] The batch was then concentrated and diafiltered against 10
mM histidine in 150 mM sodium chloride, pH 7.0, with 0.015% (w/v)
polysorbate 20, at 4.degree. C. using a 300 kDa NMWCO tangential
flow ultrafiltration membrane.
[0812] The retentate batch was 0.2 micron filtered with 0.5 micron
prefilter then diluted with additional 10 mM histidine in 150 mM
sodium chloride, pH 7.0 with 0.015% (w/v) polysorbate 20, dispensed
into aliquots and frozen at .ltoreq.-60.degree. C.
EXAMPLE 38
Preparation of Serotype 22F Conjugate for PCV22 and PCV23 (DMSO+Aq)
Polyvalent Studies Using Aqueous Conjugation
[0813] Polysaccharide was dissolved, size reduced, chemically
activated and buffer-exchanged by ultrafiltration. Purified CRM197
was then conjugated to the activated polysaccharide utilizing
nickel chloride in the reaction mixture, and the resulting
conjugate was purified by ultrafiltration prior to a final
0.2-micron filtration. Several process parameters within each step,
such as pH, temperature, concentration, and time were controlled to
yield conjugates with desired attributes.
Polysaccharide Size Reduction and Oxidation
[0814] Purified pneumococcal capsular polysaccharide powder was
dissolved in water, and 0.45-micron filtered. Dissolved
polysaccharide was homogenized to reduce the molecular mass.
Homogenization pressure and number of passes through the
homogenizer were controlled to 350 bar/5 passes to size-reduce to a
target molecular mass. Size-reduced polysaccharide was then
concentrated and diafiltered against water using a 10 kDa NMWCO
tangential flow ultrafiltration membrane.
[0815] The polysaccharide solution was then adjusted to 22.degree.
C. and pH 5 with a sodium acetate buffer to minimize polysaccharide
size reduction due to activation. Polysaccharide activation was
initiated with the addition of a 100 mM sodium metaperiodate
solution. The oxidation reaction proceeded for 2 hours at
22.degree. C.
[0816] The activated product was diafiltered against 10 mM
potassium phosphate, pH 6.4 using a 10 kDa NMWCO tangential flow
ultrafiltration membrane. Ultrafiltration was conducted at
2-8.degree. C.
Polysaccharide Conjugation to CRM197
[0817] Oxidized polysaccharide solution was mixed with water and
1.5 M potassium phosphate pH 7.0. The buffer pH selected was to
improve the stability of activated polysaccharide during the
conjugation reaction. Purified CRM197, obtained through expression
in Pseudomonas fluorescens as previously described (WO 2012/173876
A1), was 0.2-micron filtered and combined with the buffered
polysaccharide solution at a polysaccharide to CRM197 mass ratio of
0.6. The mass ratio was selected to control the polysaccharide to
CRM197 ratio in the resulting conjugate. The polysaccharide and
phosphate concentrations were 7.5 g/L and 100 mM respectively. The
polysaccharide concentration was selected to control the size of
the resulting conjugate. The solution was then 0.2-micron filtered.
Nickel chloride was added to approximately 2 mM using a 100 mM
nickel chloride solution. Sodium cyanoborohydride (2 moles per mole
of polysaccharide repeating unit) was added. Conjugation proceeded
for 120 hours to maximize consumption of polysaccharide and
protein.
Reduction with Sodium Borohydride
[0818] Following the conjugation reaction, the batch was diluted to
a polysaccharide concentration of approximately 3.5 g/L, cooled to
2-8.degree. C., and 1.2-micron filtered. The batch was diafiltered
against 100 mM potassium phosphate, pH 7.0 at 2-8.degree. C. using
a 100 kDa NMWCO tangential flow ultrafiltration membrane. The
batch, recovered in the retentate, was then diluted to
approximately 2.0 g polysaccharide/L and pH-adjusted with the
addition of 1.2 M sodium bicarbonate, pH 9.4. Sodium borohydride (1
mole per mole of polysaccharide repeating unit) was added. 1.5 M
potassium phosphate, pH 6.0 was later added.
Final Filtration and Product Storage
[0819] The batch was then concentrated and diaftiltered against 10
mM L-histidine in 150 mM sodium chloride, pH 7.0 at 4.degree. C.
using a 300 kDa NMWCO tangential flow ultrafiltration membrane.
[0820] The batch was 0.2 micron filtered and was adjusted to a
polysaccharide concentration of 1.0 g/L with additional 10 mM
L-histidine in 150 mM sodium chloride, pH 7.0 buffer. The batch was
dispensed into aliquots and frozen at .ltoreq.-60.degree. C.
[0821] EXAMPLE 39
[0822] Preparation of Serotype 23B Conjugate for PCV23 (DMSO),
PCV23 (DMSO+Aq) and PCV22 Polyvalent Studies Using DMSO
Conjugation
[0823] Polysaccharide was dissolved, sized to a target molecular
mass, chemically activated and buffer-exchanged by ultrafiltration.
Activated polysaccharide and purified CRM197 were individually
lyophilized and redissolved in DMSO. Redissolved polysaccharide and
CRM197 solutions were then combined and conjugated as described
below. The resulting conjugate was purified by ultrafiltration
prior to a final 0.2-micron filtration. Several process parameters
within each step, such as pH, temperature, concentration, and time
were controlled to yield conjugates with desired attributes.
Polysaccharide Size Reduction and Oxidation
[0824] Purified pneumococcal capsular Ps powder was dissolved in
water and 0.45-micron filtered. Dissolved polysaccharide was
homogenized to reduce the molecular mass of the Ps. Homogenization
pressure and number of passes through the homogenizer were
controlled to 400 bar/5 passes.
[0825] Size-reduced polysaccharide was concentrated and diafiltered
against water using a 10 kDa NMWCO tangential flow ultrafiltration
membrane.
[0826] The polysaccharide solution was then adjusted to 22.degree.
C. and pH 5 with a sodium acetate buffer to minimize polysaccharide
size reduction due to activation. Polysaccharide activation was
initiated with the addition of a 100 mM sodium metaperiodate
solution. The oxidation reaction proceeded for 2 hours at
22.degree. C.
[0827] The activated product was diafiltered against 10 mM
potassium phosphate, pH 6.4 followed by diafiltration against water
using a 10 kDa NMWCO tangential flow ultrafiltration membrane.
Ultrafiltration was conducted at 2-8.degree. C.
Polysaccharide Conjugation to CRM197
[0828] Purified CRM197, obtained through expression in Pseudomonas
fluorescens as previously described (WO 2012/173876 A1), was
diafiltered against 2 mM phosphate, pH 7.2 buffer using a 5 kDa
NMWCO tangential flow ultrafiltration membrane and 0.2-micron
filtered.
[0829] Activated polysaccharide was formulated for lyophilization
at 6 mg Ps/mL with sucrose concentration of 5% w/v. CRM197 was
formulated for lyophilization at 6 mg Pr/mL with sucrose
concentration of 1% w/v.
[0830] Formulated Ps and CRM197 solutions were individually
lyophilized. Lyophilized Ps and CRM197 materials were redissolved
individually in equal volumes of DMSO. The polysaccharide and
CRM197 solutions were blended to achieve a polysaccharide
concentration of 5.0 g Ps/L and a polysaccharide to CRM197 mass
ratio of 1.5. The mass ratio was selected to control the
polysaccharide to CRM197 ratio in the resulting conjugate. Sodium
cyanoborohydride (1 mole per mole of polysaccharide repeating unit)
was added, and conjugation proceeded at 22.degree. C.
Reduction with Sodium Borohydride
[0831] Sodium borohydride (2 moles per mole of polysaccharide
repeating unit) was added following the conjugation reaction and
incubated for 1 hour at 22.degree. C. The batch was diluted into
150 mM sodium chloride, with approximately 0.025% (w/v) polysorbate
20, at approximately 4.degree. C. Potassium phosphate buffer was
then added to neutralize the pH. The batch was concentrated and
diafiltered at approximately 4.degree. C. against 150 mM sodium
chloride, 25 mM potassium phosphate pH 7, using a 30 kD NMWCO
tangential flow ultrafiltration membrane.
Final Filtration and Product Storage
[0832] The batch was then concentrated and diafiltered against 10
mM histidine in 150 mM sodium chloride, pH 7.0, with 0.015% (w/v)
polysorbate 20, at 4.degree. C. using a 300 kDa NMWCO tangential
flow ultrafiltration membrane.
[0833] The retentate batch was 0.2 micron filtered (with 0.5 micron
prefilter) then diluted with additional 10 mM histidine in 150 mM
sodium chloride, pH 7.0 with 0.015% (w/v) polysorbate 20, dispensed
into aliquots and frozen at .ltoreq.-60.degree. C.
EXAMPLE 40
Preparation of Serotype 23F Conjugate for PCV23 (DMSO) and PCV23
(DMSO+Aq) Polyvalent Studies Using DMSO Conjugation
[0834] Polysaccharide was dissolved, sized to a target molecular
mass, chemically activated and buffer-exchanged by ultrafiltration.
Activated polysaccharide and purified CRM197 were individually
lyophilized and redissolved in DMSO. Redissolved polysaccharide and
CRM197 solutions were then combined and conjugated as described
below. The resulting conjugate was purified by ultrafiltration
prior to a final 0.2-micron filtration. Several process parameters
within each step, such as pH, temperature, concentration, and time
were controlled to yield conjugates with desired attributes.
[0835] Polysaccharide size reduction and oxidation
[0836] Purified pneumococcal capsular Ps powder was dissolved in
water and 0.45-micron filtered. Dissolved polysaccharide was
homogenized to reduce the molecular mass of the Ps. Homogenization
pressure and number of passes through the homogenizer were
controlled to 400 bar/5 passes.
[0837] Size-reduced polysaccharide was concentrated and diafiltered
against water using a 10 kDa NMWCO tangential flow ultrafiltration
membrane.
[0838] The polysaccharide solution was then adjusted to 22.degree.
C. and pH 5 with a sodium acetate buffer to minimize polysaccharide
size reduction due to activation. Polysaccharide activation was
initiated with the addition of a 100 mM sodium metaperiodate
solution. The oxidation reaction proceeded for 5 hours at
22.degree. C.
[0839] The activated product was diafiltered against 10 mM
potassium phosphate, pH 6.4 followed by diafiltration against water
using a 10 kDa NMWCO tangential flow ultrafiltration membrane.
Ultrafiltration was conducted at 2-8.degree. C.
Polysaccharide Conjugation to CRM197
[0840] Purified CRM197, obtained through expression in Pseudomonas
fluorescens as previously described (WO 2012/173876 A1), was
diafiltered against 2 mM phosphate, pH 7.2 buffer using a 5 kDa
NMWCO tangential flow ultrafiltration membrane and 0.2-micron
filtered.
[0841] Activated polysaccharide was formulated for lyophilization
at 6 mg Ps/mL with sucrose concentration of 5% w/v. CRM197 was
formulated for lyophilization at 6 mg Pr/mL with sucrose
concentration of 1% w/v.
[0842] Formulated Ps and CRM197 solutions were individually
lyophilized. Lyophilized Ps and CRM197 materials were redissolved
individually in equal volumes of DMSO. The polysaccharide and
CRM197 solutions were blended to achieve a polysaccharide
concentration of 2.1 g Ps/L and a polysaccharide to CRM197 mass
ratio of 1.25. The mass ratio was selected to control the
polysaccharide to CRM197 ratio in the resulting conjugate. Sodium
cyanoborohydride (1 mole per mole of polysaccharide repeating unit)
was added, and conjugation proceeded at 22.degree. C.
Reduction with Sodium Borohydride
[0843] Sodium borohydride (2 moles per mole of polysaccharide
repeating unit) was added following the conjugation reaction and
incubated for 3 hours at 22.degree. C. The batch was diluted into
150 mM sodium chloride, with approximately 0.025% (w/v) polysorbate
20, at approximately 4.degree. C. Potassium phosphate buffer was
then added to neutralize the pH. The batch was concentrated and
diafiltered at approximately 4.degree. C. against 150 mM sodium
chloride, 25 mM potassium phosphate pH 7, using a 30 kDa NMWCO
tangential flow ultrafiltration membrane.
Final Filtration and Product Storage
[0844] The batch was then concentrated and diafiltered against 10
mM histidine in 150 mM sodium chloride, pH 7.0, with 0.015% (w/v)
polysorbate 20, at 4.degree. C. using a 300 kDa NMWCO tangential
flow ultrafiltration membrane.
[0845] The retentate batch was 0.2 micron filtered then diluted
with additional 10 mM histidine in 150 mM sodium chloride, pH 7.0
with 0.015% (w/v) polysorbate 20, dispensed into aliquots and
frozen at .ltoreq.-60.degree. C.
EXAMPLE 41
Preparation of Serotype 23F Conjugate for PCV22 Polyvalent Study
Using DMSO Conjugation
[0846] Polysaccharide was dissolved, sized to a target molecular
mass, chemically activated and buffer-exchanged by ultrafiltration.
Activated polysaccharide and purified CRM197 were individually
lyophilized and redissolved in DMSO. Redissolved polysaccharide and
CRM197 solutions were then combined and conjugated as described
below. The resulting conjugate was purified by ultrafiltration
prior to a final 0.2-micron filtration. Several process parameters
within each step, such as pH, temperature, concentration, and time
were controlled to yield conjugates with desired attributes.
Polysaccharide Size Reduction and Oxidation
[0847] Purified pneumococcal capsular Ps powder was dissolved in
water and 0.45-micron filtered. Dissolved polysaccharide was
homogenized to reduce the molecular mass of the Ps. Homogenization
pressure and number of passes through the homogenizer were
controlled to 400 bar/5 passes.
[0848] Size-reduced polysaccharide was concentrated and diafiltered
against water using a 10 kDa NMWCO tangential flow ultrafiltration
membrane.
[0849] The polysaccharide solution was then adjusted to 22.degree.
C. and pH 5 with a sodium acetate buffer to minimize polysaccharide
size reduction due to activation. Polysaccharide activation was
initiated with the addition of a 100 mM sodium metaperiodate
solution. The oxidation reaction proceeded for 4 hours at
22.degree. C.
[0850] The activated product was diafiltered against 10 mM
potassium phosphate, pH 6.4 followed by diafiltration against water
using a 10 kDa NMWCO tangential flow ultrafiltration membrane.
Ultrafiltration was conducted at 2-8.degree. C.
Polysaccharide Conjugation to CRM197
[0851] Purified CRM197, obtained through expression in Pseudomonas
fluorescens as previously described (WO 2012/173876 Al), was
diafiltered against 2 mM phosphate, pH 7.2 buffer using a 5 kDa
NMWCO tangential flow ultrafiltration membrane and 0.2-micron
filtered.
[0852] Activated polysaccharide was formulated for lyophilization
at 6 mg Ps/mL with sucrose concentration of 5% w/v. CRM197 was
formulated for lyophilization at 6 mg Pr/mL with sucrose
concentration of 1% w/v.
[0853] Formulated Ps and CRM197 solutions were individually
lyophilized. Lyophilized Ps and CRM197 materials were redissolved
individually in equal volumes of DMSO. The polysaccharide and
CRM197 solutions were blended to achieve a polysaccharide
concentration of 2.1 g Ps/L and a polysaccharide to CRM197 mass
ratio of 1.25. The mass ratio was selected to control the
polysaccharide to CRM197 ratio in the resulting conjugate. Sodium
cyanoborohydride (1 mole per mole of polysaccharide repeating unit)
was added, and conjugation proceeded at 22.degree. C.
Reduction with Sodium Borohydride
[0854] Sodium borohydride (2 moles per mole of polysaccharide
repeating unit) was added following the conjugation reaction and
incubated for 3 hours at 22.degree. C. The batch was diluted into
150 mM sodium chloride, with approximately 0.025% (w/v) polysorbate
20, at approximately 4.degree. C. Potassium phosphate buffer was
then added to neutralize the pH. The batch was concentrated and
diafiltered at approximately 4.degree. C. against 150 mM sodium
chloride, 25 mM potassium phosphate pH 7, using a 30 kDa NMWCO
tangential flow ultrafiltration membrane.
Final Filtration and Product Storage
[0855] The batch was then concentrated and diafiltered against 10
mM histidine in 150 mM sodium chloride, pH 7.0, with 0.015% (w/v)
polysorbate 20, at 4.degree. C. using a 300 kDa NMWCO tangential
flow ultrafiltration membrane.
[0856] The retentate batch was 0.2 micron filtered then diluted
with additional 10 mM histidine in 150 mM sodium chloride, pH 7.0
with 0.015% (w/v) polysorbate 20, dispensed into aliquots and
frozen at .ltoreq.-60.degree. C.
EXAMPLE 42
Preparation of Serotype 24F Conjugate for PCV23 (DMSO) and
PCV23(DMSO+Aq) Polyvalent Studies Using DMSO Conjugation
[0857] Polysaccharide was dissolved, sized to a target molecular
mass, chemically activated and buffer-exchanged by ultrafiltration.
Activated polysaccharide and purified CRM197 were individually
lyophilized and redissolved in DMSO. Redissolved polysaccharide and
CRM197 solutions were then combined and conjugated as described
below. The resulting conjugate was purified by ultrafiltration
prior to a final 0.2-micron filtration. Several process parameters
within each step, such as pH, temperature, concentration, and time
were controlled to yield conjugates with desired attributes.
Polysaccharide Size Reduction and Oxidation
[0858] Purified pneumococcal capsular Ps powder was dissolved in
water and 0.45-micron filtered. Dissolved polysaccharide was
size-reduced by acid hydrolysis by adding acetic acid to 200 mM,
incubating at 80.degree. C. for 150 minutes, then neutralizing by
adding cold potassium phosphate pH 7 buffer to 400 mM.
[0859] Size-reduced polysaccharide was concentrated and diafiltered
against water using a 5 kDa NMWCO tangential flow ultrafiltration
membrane.
[0860] The polysaccharide solution was then adjusted to 22.degree.
C. and pH 5 with a sodium acetate buffer to minimize polysaccharide
size reduction due to activation. Polysaccharide activation was
initiated with the addition of a 100 mM sodium metaperiodate
solution. The oxidation reaction proceeded for 2 hours at
22.degree. C.
[0861] The activated product was diafiltered against 10 mM
potassium phosphate, pH 6.4 followed by diafiltration against water
using a 5 kDa NMWCO tangential flow ultrafiltration membrane.
Ultrafiltration was conducted at 2-8.degree. C.
Polysaccharide Conjugation to CRM197
[0862] Purified CRM197, obtained through expression in Pseudomonas
fluorescens as previously described (WO 2012/173876 A1), was
diafiltered against 2 mM phosphate, pH 7.2 buffer using a 5 kDa
NMWCO tangential flow ultrafiltration membrane and 0.2-micron
filtered.
[0863] Activated polysaccharide was formulated for lyophilization
at 2 mg Ps/mL with sucrose concentration of 10% w/v. CRM197 was
formulated for lyophilization at 6 mg Pr/mL with sucrose
concentration of 1% w/v.
[0864] Formulated Ps and CRM197 solutions were individually
lyophilized. Lyophilized Ps and CRM197 materials were redissolved
individually in equal volumes of DMSO. The polysaccharide solution
was spiked with sodium chloride to a final concentration of 10 mM.
The polysaccharide and CRM197 solutions were blended to achieve a
polysaccharide concentration of 1.4 g Ps/L and a polysaccharide to
CRM197 mass ratio of 1.5. The mass ratio was selected to control
the polysaccharide to CRM197 ratio in the resulting conjugate.
Sodium cyanoborohydride (1 mole per mole of polysaccharide
repeating unit) was added, and conjugation proceeded at 22.degree.
C.
Reduction with Sodium Borohydride
[0865] Sodium borohydride (2 moles per mole of polysaccharide
repeating unit) was added following the conjugation reaction and
incubated for 1 hour at 22.degree. C. The batch was diluted into
150 mM sodium chloride, with approximately 0.025% (w/v) polysorbate
20, at approximately 4.degree. C. Potassium phosphate buffer was
then added to neutralize the pH. The batch was concentrated and
diafiltered at approximately 4.degree. C. against 150 mM sodium
chloride, 25 mM potassium phosphate pH 7, using a 30 kD NMWCO
tangential flow ultrafiltration membrane.
Final Filtration and Product Storage
[0866] The batch was then concentrated and diafiltered against 10
mM histidine in 150 mM sodium chloride, pH 7.0, with 0.015% (w/v)
polysorbate 20, at 4.degree. C. using a 300 kDa NMWCO tangential
flow ultrafiltration membrane.
[0867] The retentate batch was 0.2 micron filtered (with 0.5 micron
prefilter) then diluted with additional 10 mM histidine in 150 mM
sodium chloride, pH 7.0 with 0.015% (w/v) polysorbate 20, dispensed
into aliquots and frozen at .ltoreq.-60.degree. C.
EXAMPLE 43
Preparation of Serotype 24F Conjugate for PCV22 Polyvalent Study
Using DMSO Conjugation
[0868] Polysaccharide was dissolved, sized to a target molecular
mass, chemically activated and buffer-exchanged by ultrafiltration.
Activated polysaccharide and purified CRM197 were individually
lyophilized and redissolved in DMSO. Redissolved polysaccharide and
CRM197 solutions were then combined and conjugated as described
below. The resulting conjugate was purified by ultrafiltration
prior to a final 0.2-micron filtration. Several process parameters
within each step, such as pH, temperature, concentration, and time
were controlled to yield conjugates with desired attributes.
Polysaccharide Size Reduction and Oxidation
[0869] Purified pneumococcal capsular Ps powder was dissolved in
water and 0.45-micron filtered. Dissolved polysaccharide was
size-reduced by acid hydrolysis by adding acetic acid to 200 mM,
incubating at 80.degree. C. for 150 minutes, then neutralizing by
adding cold potassium phosphate pH 7 buffer to 400 mM.
[0870] Size-reduced polysaccharide was concentrated and diafiltered
against water using a 5 kDa NMWCO tangential flow ultrafiltration
membrane.
[0871] The polysaccharide solution was then adjusted to 22.degree.
C. and pH 5 with a sodium acetate buffer to minimize polysaccharide
size reduction due to activation. Polysaccharide activation was
initiated with the addition of a 100 mM sodium metaperiodate
solution. The oxidation reaction proceeded for 2 hours at
22.degree. C.
[0872] The activated product was diafiltered against 10 mM
potassium phosphate, pH 6.4 followed by diafiltration against water
using a 5 kDa NMWCO tangential flow ultrafiltration membrane.
Ultrafiltration was conducted at 2-8.degree. C.
Polysaccharide Conjugation to CRM197
[0873] Purified CRM197, obtained through expression in Pseudomonas
fluorescens as previously described (WO 2012/173876 Al), was
diafiltered against 2 mM phosphate, pH 7.2 buffer using a 5 kDa
NMWCO tangential flow ultrafiltration membrane and 0.2-micron
filtered.
[0874] Activated polysaccharide was formulated for lyophilization
at 2 mg Ps/mL with sucrose concentration of 10% w/v. CRM197 was
formulated for lyophilization at 6 mg Pr/mL with sucrose
concentration of 1% w/v.
[0875] Formulated Ps and CRM197 solutions were individually
lyophilized. Lyophilized Ps and CRM197 materials were redissolved
individually in equal volumes of DMSO. The polysaccharide solution
was spiked with sodium chloride to a final concentration of 25 mM.
The polysaccharide and CRM197 solutions were blended to achieve a
polysaccharide concentration of 1.4 g Ps/L and a polysaccharide to
CRM197 mass ratio of 1.5. The mass ratio was selected to control
the polysaccharide to CRM197 ratio in the resulting conjugate.
Sodium cyanoborohydride (1 mole per mole of polysaccharide
repeating unit) was added, and conjugation proceeded at 22.degree.
C.
Reduction with Sodium Borohydride
[0876] Sodium borohydride (2 moles per mole of polysaccharide
repeating unit) was added following the conjugation reaction and
incubated for 1 hour at 22.degree. C. The batch was diluted into
150 mM sodium chloride, with approximately 0.025% (w/v) polysorbate
20, at approximately 4.degree. C. Potassium phosphate buffer was
then added to neutralize the pH. The batch was concentrated and
diafiltered at approximately 4.degree. C. against 150 mM sodium
chloride, 25 mM potassium phosphate pH 7, using a 30 kD NMWCO
tangential flow ultrafiltration membrane.
Final Filtration and Product Storage
[0877] The batch was then concentrated and diafiltered against 10
mM histidine in 150 mM sodium chloride, pH 7.0, with 0.015% (w/v)
polysorbate 20, at 4.degree. C. using a 300 kDa NMWCO tangential
flow ultrafiltration membrane.
[0878] The retentate batch was 0.2 micron filtered (with 0.5 micron
prefilter) then diluted with additional 10 mM histidine in 150 mM
sodium chloride, pH 7.0 with 0.015% (w/v) polysorbate 20, dispensed
into aliquots and frozen at .ltoreq.-60.degree. C.
EXAMPLE 44
Preparation of Serotype 33F Conjugate for PCV23 (DMSO) Polyvalent
Study Using DMSO Conjugation
[0879] Polysaccharide was dissolved, sized to a target molecular
mass, chemically activated and buffer-exchanged by ultrafiltration.
Activated polysaccharide and purified CRM197 were individually
lyophilized and redissolved in DMSO. Redissolved polysaccharide and
CRM197 solutions were then combined and conjugated as described
below. The resulting conjugate was purified by ultrafiltration
prior to a final 0.2-micron filtration. Several process parameters
within each step, such as pH, temperature, concentration, and time
were controlled to yield conjugates with desired attributes.
Polysaccharide Size Reduction and Oxidation
[0880] Purified pneumococcal capsular Ps powder was dissolved in
water and 0.45-micron filtered. Dissolved polysaccharide was
homogenized to reduce the molecular mass of the Ps. Homogenization
pressure and number of passes through the homogenizer were
controlled to 510 bar/5 passes.
[0881] Size-reduced polysaccharide was concentrated and diafiltered
against water using a 10 kDa NMWCO tangential flow ultrafiltration
membrane.
[0882] The polysaccharide solution was then adjusted to 22.degree.
C. and pH 5 with a sodium acetate buffer to minimize polysaccharide
size reduction due to activation. Polysaccharide activation was
initiated with the addition of a 100 mM sodium metaperiodate
solution. The oxidation reaction proceeded for 2 hours at
22.degree. C.
[0883] The activated product was diafiltered against 10 mM
potassium phosphate, pH 6.4 followed by diafiltration against water
using a 10 kDa NMWCO tangential flow ultrafiltration membrane.
Ultrafiltration was conducted at 2-8.degree. C.
Polysaccharide Conjugation to CRM197
[0884] Purified CRM197, obtained through expression in Pseudomonas
fluorescens as previously described (WO 2012/173876 A1), was
diafiltered against 2 mM phosphate, pH 7.2 buffer using a 5 kDa
NMWCO tangential flow ultrafiltration membrane and 0.2-micron
filtered.
[0885] Activated polysaccharide was formulated for lyophilization
at 6 mg Ps/mL with sucrose concentration of 5% w/v. CRM197 was
formulated for lyophilization at 6 mg Pr/mL with sucrose
concentration of 1% w/v.
[0886] Formulated Ps and CRM197 solutions were individually
lyophilized. Lyophilized Ps and CRM197 materials were redissolved
individually in equal volumes of DMSO. The polysaccharide and
CRM197 solutions were blended to achieve a polysaccharide
concentration of 2.5 g Ps/L and a polysaccharide to CRM197 mass
ratio of 1.75. The mass ratio was selected to control the
polysaccharide to CRM197 ratio in the resulting conjugate. Sodium
cyanoborohydride (1 mole per mole of polysaccharide repeating unit)
was added, and conjugation proceeded at 22.degree. C.
Reduction with Sodium Borohydride
[0887] Sodium borohydride (2 moles per mole of polysaccharide
repeating unit) was added following the conjugation reaction and
incubated for 1 hour at 22.degree. C. The batch was diluted into
150 mM sodium chloride, with approximately 0.025% (w/v) polysorbate
20, at approximately 4.degree. C. Potassium phosphate buffer was
then added to neutralize the pH.
Final Filtration and Product Storage
[0888] The batch was then concentrated and diafiltered against 10
mM histidine in 150 mM sodium chloride, pH 7.0, with 0.015% (w/v)
polysorbate 20, at 4.degree. C. using a 300 kDa NMWCO tangential
flow ultrafiltration membrane.
[0889] The retentate batch was 0.2 micron filtered (with 0.5 micron
prefilter) then diluted with additional 10 mM histidine in 150 mM
sodium chloride, pH 7.0 with 0.015% (w/v) polysorbate 20, dispensed
into aliquots and frozen at .ltoreq.-60.degree. C.
EXAMPLE 45
Preparation of Serotype 33F Conjugate for PCV22 and PCV23 (DMSO+Aq)
Polyvalent Studies Using Aqueous Conjugation
[0890] Polysaccharide was dissolved, size reduced, chemically
activated and buffer-exchanged by ultrafiltration. Purified CRM197
was then conjugated to the activated polysaccharide utilizing
nickel chloride in the reaction mixture, and the resulting
conjugate was purified by ultrafiltration prior to a final
0.2-micron filtration. Several process parameters within each step,
such as pH, temperature, concentration, and time were controlled to
yield conjugates with desired attributes.
Polysaccharide Size Reduction and Oxidation
[0891] Purified pneumococcal capsular polysaccharide powder was
dissolved in water, and 0.45-micron filtered. Dissolved
polysaccharide was homogenized to reduce the molecular mass.
Homogenization pressure and number of passes through the
homogenizer were controlled to 350 bar/4 passes to size-reduce to a
target molecular mass. Size-reduced polysaccharide was then
concentrated and diafiltered against water using a 10 kDa NMWCO
tangential flow ultrafiltration membrane.
[0892] The polysaccharide solution was then adjusted to 22.degree.
C. and pH 5 with a sodium acetate buffer to minimize polysaccharide
size reduction due to activation. Polysaccharide activation was
initiated with the addition of a 100 mM sodium metaperiodate
solution. The oxidation reaction proceeded for 2 hours at
22.degree. C.
[0893] The activated product was diafiltered against 10 mM
potassium phosphate, pH 6.4 using a 10 kDa NMWCO tangential flow
ultrafiltration membrane. Ultrafiltration was conducted at
2-8.degree. C.
Polysaccharide Conjugation to CRM197
[0894] Oxidized polysaccharide solution was mixed with water and
1.5 M potassium phosphate pH 7.0. The buffer pH selected was to
improve the stability of activated polysaccharide during the
conjugation reaction. Purified CRM197, obtained through expression
in Pseudomonas fluorescens as previously described (WO 2012/173876
A1), was 0.2-micron filtered and combined with the buffered
polysaccharide solution at a polysaccharide to CRM197 mass ratio of
0.7. The mass ratio was selected to control the polysaccharide to
CRM197 ratio in the resulting conjugate. The polysaccharide and
phosphate concentrations were 6.5 g/L and 100 mM respectively. The
polysaccharide concentration was selected to control the size of
the resulting conjugate. The solution was then 0.2-micron filtered.
Nickel chloride was added to approximately 2 mM using a 100 mM
nickel chloride solution. Sodium cyanoborohydride (2 moles per mole
of polysaccharide repeating unit) was added. Conjugation proceeded
for 96 hours to maximize consumption of polysaccharide and
protein.
Reduction with Sodium Borohydride
[0895] Following the conjugation reaction, the batch was diluted to
a polysaccharide concentration of approximately 3.5 g/L, cooled to
2-8.degree. C., and 1.2-micron filtered. The batch was diafiltered
against 100 mM potassium phosphate, pH 7.0 at 2-8.degree. C. using
a 100 kDa NMWCO tangential flow ultrafiltration membrane. The
batch, recovered in the retentate, was then diluted to
approximately 2.0 g polysaccharide/L and pH-adjusted with the
addition of 1.2 M sodium bicarbonate, pH 9.4. Sodium borohydride (1
mole per mole of polysaccharide repeating unit) was added. 1.5 M
potassium phosphate, pH 6.0 was later added.
Final Filtration and Product Storage
[0896] The batch was then concentrated and diaftiltered against 10
mM L-histidine in 150 mM sodium chloride, pH 7.0 at 4.degree. C.
using a 300 kDa NMWCO tangential flow ultrafiltration membrane. The
batch was 0.2 micron filtered and was adjusted to a polysaccharide
concentration of 1.0 g/L with additional 10 mM L-histidine in 150
mM sodium chloride, pH 7.0 buffer. The batch was dispensed into
aliquots and frozen at .ltoreq.-60.degree. C.
EXAMPLE 46
Preparation of Serotype 35B Conjugate for PCV23 (DMSO) and PCV23
(DMSO+Aq) Polyvalent Studies Using DMSO Conjugation
[0897] Polysaccharide was dissolved, chemically activated and
buffer-exchanged by ultrafiltration. Activated polysaccharide and
purified CRM197 were individually lyophilized and redissolved in
DMSO. Redissolved polysaccharide and CRM197 solutions were then
combined and conjugated as described below. The resulting conjugate
was purified by ultrafiltration prior to a final 0.2-micron
filtration. Several process parameters within each step, such as
pH, temperature, concentration, and time were controlled to yield
conjugates with desired attributes.
Polysaccharide Oxidation
[0898] Purified pneumococcal capsular Ps powder was dissolved in
water and 0.45-micron filtered. Dissolved polysaccharide was
concentrated and diafiltered against water using a 10 kDa NMWCO
tangential flow ultrafiltration membrane.
[0899] The polysaccharide solution was then adjusted to 22.degree.
C. and pH 5 with a sodium acetate buffer to minimize polysaccharide
size reduction due to activation. Polysaccharide activation was
initiated with the addition of a 100 mM sodium metaperiodate
solution. The oxidation reaction proceeded for 2 hours at
22.degree. C.
[0900] The activated product was diafiltered against 10 mM
potassium phosphate, pH 6.4 followed by diafiltration against water
using a 5 kDa NMWCO tangential flow ultrafiltration membrane.
Ultrafiltration was conducted at 2-8.degree. C.
Polysaccharide Conjugation to CRM197
[0901] Purified CRM197, obtained through expression in Pseudomonas
fluorescens as previously described (WO 2012/173876 Al), was
diafiltered against 2 mM phosphate, pH 7.2 buffer using a 5 kDa
NMWCO tangential flow ultrafiltration membrane and 0.2-micron
filtered.
[0902] Activated polysaccharide was formulated for lyophilization
at 6 mg Ps/mL with sucrose concentration of 5% w/v. CRM197 was
formulated for lyophilization at 6 mg Pr/mL with sucrose
concentration of 1% w/v.
[0903] Formulated Ps and CRM197 solutions were individually
lyophilized. Lyophilized Ps and CRM197 materials were redissolved
individually in equal volumes of DMSO. The polysaccharide solution
was spiked with sodium chloride to a final concentration of 20 mM.
The polysaccharide and CRM197 solutions were blended to achieve a
polysaccharide concentration of 6.0 g Ps/L and a polysaccharide to
CRM197 mass ratio of 3.0. The mass ratio was selected to control
the polysaccharide to CRM197 ratio in the resulting conjugate.
Conjugation proceeded at 34.degree. C.
Reduction with Sodium Borohydride
[0904] Sodium borohydride (2 moles per mole of polysaccharide
repeating unit) was added following the conjugation reaction and
incubated for 1 hour at 34.degree. C. The batch was diluted into
150 mM sodium chloride, with approximately 0.025% (w/v) polysorbate
20, at approximately 4.degree. C. Potassium phosphate buffer was
then added to neutralize the pH. The batch was concentrated and
diafiltered at approximately 4.degree. C. against 150 mM sodium
chloride, 25 mM potassium phosphate pH 7, using a 30 kD NMWCO
tangential flow ultrafiltration membrane.
Final Filtration and Product Storage
[0905] The batch was then concentrated and diafiltered against 10
mM histidine in 150 mM sodium chloride, pH 7.0, with 0.015% (w/v)
polysorbate 20, at 4.degree. C. using a 300 kDa NMWCO tangential
flow ultrafiltration membrane.
[0906] The retentate batch was 0.2 micron filtered (with 0.5 micron
prefilter) then diluted with additional 10 mM histidine in 150 mM
sodium chloride, pH 7.0 with 0.015% (w/v) polysorbate 20, dispensed
into aliquots and frozen at .ltoreq.-60.degree. C.
EXAMPLE 47
Preparation of Serotype 35B Conjugate for PCV22 Polyvalent Study
Using DMSO Conjugation
[0907] Polysaccharide was dissolved, chemically activated and
buffer-exchanged by ultrafiltration. Activated polysaccharide and
purified CRM197 were individually lyophilized and redissolved in
DMSO. Redissolved polysaccharide and CRM197 solutions were then
combined and conjugated as described below. The resulting conjugate
was purified by ultrafiltration prior to a final 0.2-micron
filtration. Several process parameters within each step, such as
pH, temperature, concentration, and time were controlled to yield
conjugates with desired attributes.
Polysaccharide Oxidation
[0908] Purified pneumococcal capsular Ps powder was dissolved in
water and 0.45-micron filtered. Dissolved polysaccharide was
concentrated and diafiltered against water using a 10 kDa NMWCO
tangential flow ultrafiltration membrane.
[0909] The polysaccharide solution was then adjusted to 22.degree.
C. and pH 5 with a sodium acetate buffer to minimize polysaccharide
size reduction due to activation. Polysaccharide activation was
initiated with the addition of a 100 mM sodium metaperiodate
solution. The oxidation reaction proceeded for 2 hours at
22.degree. C.
[0910] The activated product was diafiltered against 10 mM
potassium phosphate, pH 6.4 followed by diafiltration against water
using a 5 kDa NMWCO tangential flow ultrafiltration membrane.
Ultrafiltration was conducted at 2-8.degree. C.
Polysaccharide Conjugation to CRM197
[0911] Purified CRM197, obtained through expression in Pseudomonas
fluorescens as previously described (WO 2012/173876 A1), was
diafiltered against 2 mM phosphate, pH 7.2 buffer using a 5 kDa
NMWCO tangential flow ultrafiltration membrane and 0.2-micron
filtered.
[0912] Activated polysaccharide was formulated for lyophilization
at 6 mg Ps/mL with sucrose concentration of 5% w/v. CRM197 was
formulated for lyophilization at 6 mg Pr/mL with sucrose
concentration of 1% w/v.
[0913] Formulated Ps and CRM197 solutions were individually
lyophilized. Lyophilized Ps and CRM197 materials were redissolved
individually in equal volumes of DMSO. The polysaccharide solution
was spiked with sodium chloride to a final concentration of 20 mM.
The polysaccharide and CRM197 solutions were blended to achieve a
polysaccharide concentration of 6.0 g Ps/L and a polysaccharide to
CRM197 mass ratio of 3.0. The mass ratio was selected to control
the polysaccharide to CRM197 ratio in the resulting conjugate.
Conjugation proceeded at 34.degree. C.
Reduction with Sodium Borohydride
[0914] Sodium borohydride (2 moles per mole of polysaccharide
repeating unit) was added following the conjugation reaction and
incubated for 1 hour at 34.degree. C. The batch was diluted into
150 mM sodium chloride, with approximately 0.025% (w/v) polysorbate
20, at approximately 4.degree. C. Potassium phosphate buffer was
then added to neutralize the pH. The batch was concentrated and
diafiltered at approximately 4.degree. C. against 150 mM sodium
chloride, 25 mM potassium phosphate pH 7, using a 30 kD NMWCO
tangential flow ultrafiltration membrane.
Final Filtration and Product Storage
[0915] The batch was then concentrated and diafiltered against 10
mM histidine in 150 mM sodium chloride, pH 7.0, with 0.015% (w/v)
polysorbate 20, at 4.degree. C. using a 300 kDa NMWCO tangential
flow ultrafiltration membrane.
[0916] The retentate batch was 0.2 micron filtered (with 0.5 micron
prefilter) then diluted with additional 10 mM histidine in 150 mM
sodium chloride, pH 7.0 with 0.015% (w/v) polysorbate 20, dispensed
into aliquots and frozen at .ltoreq.-60.degree. C.
EXAMPLE Y
Preparation of Serotypes for PCV24 Polyvalent Study Using DMSO
Conjugation
[0917] Conjugates were prepared for the PCV24 study using methods
similar to those described in prior Examples. For each serotype,
polysaccharide was dissolved, chemically activated and
buffer-exchanged by ultrafiltration. Activated polysaccharide and
purified CRM197 were individually lyophilized and redissolved in
DMSO. Redissolved polysaccharide and CRM197 solutions were then
combined and conjugated as described below. The resulting conjugate
was purified by ultrafiltration prior to a final 0.2-micron
filtration.
[0918] Several process parameters within each step, such as pH,
temperature, concentration, and time were controlled to yield
conjugates with desired attributes. Differences from prior Examples
may include: homogenization pressure and number of passes,
oxidation time, polysaccharide and sucrose concentration for
lyophilization, polysaccharide concentration during conjugation,
polysaccharide to CRM197 mass ratio and salt concentration in the
conjugation reaction.
EXAMPLE 48
Formulation of Pneumococcal Conjugate Vaccines
[0919] Individual pneumococcal polysaccharide-protein conjugates
prepared utilizing different chemistries as described in the
Examples, supra, were used for the formulation of an 8-, 15-, 22-,
23-& 24-valent pneumococcal conjugate vaccines.
[0920] The PCV8/APA vaccine drug product is prepared by
individually conjugating the CRM197 protein to Pneumococcal
polysaccharide (PnPs) Types (-8, -10A, -12F, -15A, -15C, -23B, -24F
and -35B) using reductive amination in an aprotic solvent (also
referred to as DMSO chemistry) and formulated in 20 mM L-Histidine
pH 5.8 and 150 mM NaCl 0.1% w/v Polysorbate-20 (PS-20) and 250
.mu.g [Al]/mL in the form of Aluminum Phosphate Adjuvant as the
adjuvant at 4 .mu.g/mL each serotype for a total polysaccharide
concentration of 32 .mu.g/mL.
[0921] The PCV15/APA vaccine drug product is prepared by
individually conjugating the CRM197 protein to Pneumococcal
polysaccharide (PnPs) Types (-6A, -6B, -7F, -9V, -18C, -19A, -19F,
-23F) using reductive amination in an aprotic solvent (also
referred to as DMSO chemistry) or for Types -1, -3,-4, -5, -14,
-22F and -33F using reductive amination in a protic solvent (also
referred to as aqueous chemistry) and formulated in 20 mM
L-Histidine pH 5.8 and 150 mM NaCl 0.2% w/v Polysorbate-20 (PS-20)
and 250 .mu.g [Al]/mL in the form of Aluminum Phosphate as the
adjuvant at 4 .mu.g/mL each serotype (except 6B at 8 .mu.g/mL) for
a total polysaccharide concentration of 64 .mu.g/mL.
[0922] The PCV22 vaccine drug product used to immunize mice and
rabbits was prepared by individually conjugating the CRM197 protein
to Pneumococcal polysaccharide (PnPs) Types (-1, -3, -4, -5, -6A,
-6B, -7F, -9V, -10A, -12F,-14, -15A, -15C, -18C, -19A, -19F, -22F,
-23B, -23F, -24F, -33F, and -35B) using reductive amination in a
protic and aprotic solutions (DMSO/Aqueous "Aq") and formulated in
20 mM L-Histidine pH 5.8 and 150 mM
[0923] NaCl and 0.2% w/v Polysorbate-20 (PS-20) at 0.8 .mu.g/mL
each serotype (except 6B at 1.6 .mu.g/mL) for a total
polysaccharide concentration of 18.4 .mu.g/mL referred to as PCV22
unadjuvanted or unadj. In another specific embodiment, the
formulation is prepared with 50 .mu.g [Al]/mL in the form of
Aluminum Phosphate as the adjuvant referred to as PCV22/APA.
[0924] The PCV23 vaccine drug product is prepared by individually
conjugating the CRM197 protein to Pneumococcal polysaccharide
(PnPs) Types (-1, -3, -4, -5, -6A, -6B, -7F, -8, -9V, -10A, -12F,
-14, -15A, -15C, -18C, -19A, -19F, -22F, -23B, -23F, -24F, -33F,
and -35B) using reductive amination in an aprotic solvent (also
referred to as DMSO chemistry) and formulated in 20 mM L-Histidine
pH 5.8 and 150 mM NaCl and 0.2% w/v Polysorbate-20 (PS-20) at 4
.mu.g/mL each -serotype for a total polysaccharide concentration of
92 .mu.g/mL referred to as PCV23 unadjuv. In another specific
embodiment, the formulation is prepared with 250 .mu.g [Al]/mL in
the form of Aluminum Phosphate Adjuvant as the adjuvant referred to
as PCV23/APA. In another embodiment, the PCV23 vaccine drug product
is prepared by individually conjugating the CRM197 protein to
Pneumococcal polysaccharide (PnPs) Types (-1, -3, -4, -5, -9V, -14,
-22F, and -33F) using reductive amination in an protic solvent
(also referred to as aqueous chemistry) and conjugating the CRM197
protein to Pneumococcal polysaccharide (PnPs) Types (-6A, -6B, -7F,
-8, -10A, -12F, -15A, -15C, -18C, -19A, -19F, -23B, -23F, -24F, and
-35B) using reductive amination in an aprotic solvent (also
referred to as DMSO chemistry). The vaccine drug product is
formulated in 20 mM L-Histidine pH 5.8 and 150 mM NaCl and 0.1% w/v
Polysorbate-20 (PS-20) with 0.01% CarboxyMethyl Cellulose (CMC) at
4 .mu.g/mL each serotype (except 6B at 8 .mu.g/mL) for a total
polysaccharide concentration of 96 .mu.g/mL and prepared with 250
.mu.g [Al]/mL in the form of Aluminum Phosphate Adjuvant as the
adjuvant and referred to as PCV23 (DMSO+Aq)/APA.
[0925] The multivalent immunogenic composition PCV24 is prepared by
individually conjugating the CRM197 protein to S. pneumoniae
polysaccharide (PnPs) serotypes -1, -3, -4, -5, -6A, -6B, -7F, -8,
-9V, -10A, -11A, -12F, -14, -15A, -15C, -18C, -19A, -19F, -22F,
-23B, -23F, -24F, -33F, and -35B using reductive amination in an
aprotic solvent (also referred to as DMSO chemistry) and formulated
in 20 mM L-Histidine pH 5.8, 150 mM NaCl and 0.1% w/v
Polysorbate-20 (PS-20) at 4 .mu.g/mL or 8 .mu.g/mL of each
polysacchardide serotype for a total polysaccharide concentration
of 96 .mu.g/mL or 192 .mu.g/mL, respectively, and referred to as
"PCV24 unadj". In another specific embodiment, the multivalent
immunogenic composition PCV24 is prepared in 20 mM L-Histidine pH
5.8, 150 mM NaCl and 0.2% w/v Polysorbate-20 (PS-20) at 4 .mu.g/mL
of each polysaccharide serotype for a total polysaccharide
concentration of 96 .mu.g/mL further comprising 250 .mu.g [Al]/mL
in the form of Aluminum Phosphate Adjuvant. This is referred to as
"PCV24/APA".
[0926] The required volume of bulk conjugates needed to obtain the
target concentration of individual serotypes were calculated based
on batch volume and concentration of individual bulk polysaccharide
concentrations. The individual conjugates were added to a solution
of histidine, sodium chloride and Polysorbate-20 (PS-20) to produce
a 2.times.-4.times. conjugate blend. The formulation vessel
containing the conjugate blend is mixed using a magnetic stir bar,
sterile filtered into another vessel. The sterile filtered
2.times.-4.times. blend is either added to another vessel
containing Aluminum Phosphate Adjuvant or diluted with saline to
achieve the desired target total polysaccharide, excipient and APA
adjuvant (if required) concentrations. The formulations are then
filled into glass vials or syringes and stored at 2-8.degree.
C.
EXAMPLE 49
PCV22 Immunogenicity and Functional Antibody in Mice
[0927] Young female Balb/c mice (6-8 weeks old, n=10/group) were
immunized with 0.1mL of a 22-valent pneumococcal conjugate vaccine
(PCV22/APA or PCV22 unadjuvanted) on day 0, day 14 and day 28.
PCV22 was dosed at 0.08 .mu.g of each pneumococcal polysaccharide
(1, 3, 4, 5, 6A, 7F, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F,
22F, 23B, 23F, 24F, 33F, 35B) and 6B at 0.16 .mu.g and all
conjugated to CRM197 unadjuvanted or with 5 .mu.g aluminum
phosphate adjuvant (APA) per immunization. Mice were observed at
least daily by trained animal care staff for any signs of illness
or distress. The vaccine formulations in mice were deemed to be
safe and well tolerated, as no vaccine-related adverse events were
noted. On day 52 the mice were intratracheally challenged with
Streptococcus pneumoniae serotype 24F. Exponential phase cultures
of S. pneumoniae were centrifuged, washed, and suspended in sterile
PBS. Mice were anesthetized with isoflurane prior to challenge.
10.sup.5 cfu of S. pneumoniae in 0.1mL of PBS was placed in the
throat of mice hung upright by their incisors. Aspiration of the
bacteria was induced by gently pulling the tongue outward and
covering the nostrils. Mice were weighed daily and euthanized if
weight loss exceeded 20% of starting weight. Blood was collected at
24 hours, 48 hours and 72 hours to assess for bacteremia. Mice were
observed at least twice daily by trained animal care staff for any
signs of illness or distress. All animal experiments were performed
in strict accordance with the recommendations in the Guide for Care
and Use of Laboratory Animals of the National Institutes of Health.
The mouse experimental protocol was approved by the Institutional
Animal Care and Use Committee at Merck & Co., Inc.
[0928] Mouse sera were evaluated for IgG immunogenicity using a
multiplexed electrochemiluminescence (ECL) assay. This assay was
developed for use with mouse serum based on the human assay
described by Marchese et al. .sup.[3] using technology developed by
MesoScale Discovery (a division of MesoScale Diagnostics, LLC,
Gaithersburg, MD) which utilizes a SULFO-TAGTM label that emits
light upon electrochemical stimulation. SULFO-TAG.TM.-labeled
anti-mouse IgG was used as the secondary antibody for testing mouse
serum samples. Functional antibody was determined through
multiplexed opsonophagocytic assays (MOPA) based on previously
described protocols at www.vaccine.uab.edu and Opsotiter.RTM. 3
software owned by and licensed from University of Alabama (UAB)
Research Foundation .sup.[1,2].
[0929] Mouse sera were pooled for each group and tested in
multiplexed electrochemiluminescent assays to determine antibody
titers. PCV22 generated antibody titers in mice for all serotypes
following 1, 2 and 3 immunizations with the vaccine (FIG. 1). PCV22
showed cross-reactivity to serotype 15B, as evidenced by IgG titers
(FIG. 1). PCV22 formulated with APA trended toward higher
immunogenicity compared to unadjuvanted PCV22 in mice at PD2 (data
not shown) and PD3 (FIG. 2).
[0930] Mouse sera were pooled for each group and tested in
multiplexed opsonophagocytic assays (MOPA) to determine functional
antibody titers. PCV22 generated functional antibody titers in mice
which killed vaccine-type bacterial serotypes following 3
immunizations with the vaccine (FIG. 3). PCV22 formulated with APA
trended toward higher functional antibody titers compared to PCV22
unadjuvanted at PD2 (data not shown) and PD3 (FIG. 4), similar to
IgG titers (FIG. 2).
[0931] PCV22 immunized mice were protected from intratracheal
challenge with S. pneumoniae 24F (FIG. 5). Mantel Cox log-rank test
indicated that both the PCV22 unadjuvanted (PCV22 unadj) and
PCV22/APA groups were significantly protected from challenge when
compared to the naive group (P<0.0001). Likewise, both PCV22
immunized mouse groups had little to no bacteremia, which was
significantly less when compared to the naive group (data not
shown).
EXAMPLE 50
PCV22 Immunogenicity and Functional Antibody in Rabbits
[0932] Adult New Zealand white rabbits (NZWR, n=5/group) were
intramuscularly (IM) immunized with 0.1 mL of a 22-valent
pneumococcal conjugate vaccine (PCV22/APA or PCV22 unadjuvanted) on
day 0 and day 14 (alternating sides). PCV22 was dosed at 0.08 .mu.g
of each pneumococcal polysaccharide (1, 3, 4, 5, 6A, 7F, 9V, 10A,
12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B) and
6B at 0.16 .mu.g and all conjugated to CRM197 and either
unadjuvanted or formulated with 5.mu.g APA per immunization. Sera
were collected prior to study start (pre-immune) and on days 14
(PD1) and 28 (PD2). NZWRs were observed at least daily by trained
animal care staff for any signs of illness or distress. The vaccine
formulations in NZWRs were deemed to be safe and well tolerated, as
no vaccine-related adverse events were noted. All animal
experiments were performed in strict accordance with the
recommendations in the Guide for Care and Use of Laboratory Animals
of the National Institutes of Health. The NZWR experimental
protocol was approved by the Institutional Animal Care and Use
Committees at both Merck & Co., Inc and Covance (Denver,
Pa.).
[0933] Rabbit sera were evaluated for IgG immunogenicity using a
multiplexed electrochemiluminescence (ECL) assay. This assay was
developed for use with rabbit serum based on the human assay
described by Marchese et al. .sup.[3]using technology developed by
MesoScale Discovery (a division of MesoScale Diagnostics, LLC,
Gaithersburg, MD) which utilizes a SULFO-TAG.TM. label that emits
light upon electrochemical stimulation. SULFO-TAG.TM.-labeled
anti-rabbit IgG was used as the secondary antibody for testing NZWR
serum samples. Functional antibody was determined through
multiplexed opsonophagocytic assays (MOPA) based on previously
described protocols at www.vaccine.uab.edu and Opsotiter.RTM. 3
software owned by and licensed from University of Alabama (UAB)
Research Foundation .sup.[1,21].
[0934] Rabbit sera were tested individually in multiplexed
electrochemiluminescent assays to determine antibody titers. PCV22
generated antibody titers in rabbits for all serotypes following
immunizations with the vaccine (FIG. 6). Immunization of rabbits
with PCV22 also generates antibodies that bind to serotype 15B
polysaccharide (FIG. 6). There was no benefit to including APA with
PCV22 in rabbits, as the immunogenicity was comparable to or lower
than (serotype 1) PCV22 unadjuvanted at PD2 (FIG. 7).
[0935] Rabbit sera were tested individually in multiplexed
opsonophagocytic assays (MOPA) to determine functional antibody
titers. PCV22 generated functional antibody titers in rabbits which
killed vaccine-type bacterial serotypes following 2 immunizations
with the vaccine
[0936] (FIG. 8). PCV22 unadjuvanted had higher functional antibody
titers at PD1 for serotype 4 (data not shown) and at PD2 for
serotypes 1, 3 and 4 compared to PCV22 formulated with APA. PCV22
formulated with APA did not have higher functional antibody titers
at PD1 (data not shown) and PD2 for most of the serotypes compared
to PCV22 unadjuvanted (FIG. 8).
EXAMPLE 51
PCV23 Immunogenicity in Infant Rhesus Macaques
[0937] Infant Rhesus macaques (IRM, 2-3 months old, n=8-9/group)
were intramuscularly immunized with 0.1mL of a 23-valent
pneumococcal conjugate vaccine (PCV23) on days 0, 28 and 56. PCV23
was dosed at 9.6.mu.g of total pneumococcal polysaccharide (1, 3,
4, 5, 6A, 7F, 8, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F,
23B, 23F, 24F, 33F and 35B at 0.4 .mu.g, 6B at 0.8 .mu.g and all
conjugated to CRM197) unadjuvanted or formulated with 25 .mu.g
aluminum phosphate adjuvant (APA) per immunization. An additional
group of IRMs were intramuscularly immunized with a 0.1mL of PCV15.
PCV15 was dosed at 6.4 .mu.g of total pneumococcal polysaccharide
(1, 3, 4, 5, 6A, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F at 0.4
.mu.g, 6B at 0.8 .mu.g and all conjugated to CRM197 with 25 .mu.g
APA per immunization) in one quadricep and 0.1 mL of PCV8 (8, 10A,
12F, 15A, 15C, 23B, 24F and 35B at 0.4 .mu.g and all conjugated to
CRM197 with 25 .mu.g APA per immunization) in a separate quadricep
following the same schedule as described above. Sera were collected
prior to study start (pre-immune, day 0) and on days 14 (PD1), 28,
42 (PD2), 56, and 70 (PD3). IRMs were observed at least daily by
trained animal care staff for any signs of illness or distress. All
animal experiments were performed in strict accordance with the
recommendations in the Guide for Care and Use of Laboratory Animals
of the National Institutes of Health. The experimental protocol was
approved by the Institutional Animal Care and Use Committee at
Merck & Co., Inc and New Iberia Research Center.
[0938] Rhesus sera were evaluated for IgG immunogenicity using a
multiplexed electrochemiluminescence (ECL) assay. This assay was
developed for use with Rhesus serum based on the human assay
described by Marchese et al. and Skinner et al.sup.[3,4] using
technology developed by MesoScale Discovery (a division of
MesoScale Diagnostics, LLC, Gaithersburg, MD) which utilizes a
SULFO-TAG.TM. label that emits light upon electrochemical
stimulation. SULFO-TAG.TM.-labeled anti-human IgG was used as the
secondary antibody for testing Rhesus serum samples.
[0939] IRM immunization with PCV23 generated antibody titers for
all serotypes for all of the PCV23 vaccine formulations evaluated
(FIGS. 9A-9D). It is also of note that PCV23, which contains
polysaccharide conjugates 15A-CRM197 and 15C-CRM197, also provides
cross-reactivity to 15B, as evidenced in ECL (FIGS. 9A-9D). PCV23
was immunogenic with one dose of vaccine in the IRMs (FIGS.
9A-9D).
[0940] The PCV23 (DMSO)/APA formulation had higher immunogenicity
for serotype 22F compared to PCV23 unadjuvanted at PD1 while PCV23
(DMSO+Aq)/APA had higher immunogenicity for serotype 1 and serotype
15C compared to PCV23 unadjuvanted at PD1 (FIG. 10A). The PCV23
(DMSO)/APA formulation had higher immunogenicity for serotype 18C
compared to PCV23 unadjuvanted at PD2 (FIG. 10B). The PCV23
(DMSO+Aq)/APA had higher immunogenicity for the majority of
serotypes (1, 3, 4, 7F, 8, 9V, 12F, 14, 15A, 15B, 15C, 18C, 19F,
22F and 23F) compared to PCV23 unadjuvanted at PD2 (FIG. 10B). The
PCV23 (DMSO+Aq)/APA had higher immunogenicity for serotypes 1, 4
and 9V compared to PCV23 unadjuvanted at PD3 (FIG. 10C).
[0941] IRMs vaccinated with PCV23 (DMSO+Aq)/APA or with a
co-administration of PCV15/APA+PCV8/APA in separate limbs did not
show many immunogenicity differences at PD1, with the exception of
serotype 10A which had higher immunogenicity in the
co-administration group (FIG. 11A). However, IRMs vaccinated with
PCV23 (DMSO+Aq)/APA had higher PD2 immunogenicity for the majority
of serotypes 1, 3, 4, 5, 7F, 8, 9V, 14, 15A, 15B, 15C, 18C, 19F,
22F, 23B and 23F compared to co-administration of
PCV15/APA+PCV8/APA (FIG. 11B). There were no differences in
immunogenicity between the two vaccines at PD3 (FIG. 11C).
[0942] When evaluating the antibody boosting effects, all of the
PCVs generated primary immune responses at PD1 compared to
pre-immune sera for all serotypes with the exception of serotype
23B in IRMs immunized with PCV23 (DMSO+Aq)/APA (FIG. 12A). All PCVs
evaluated generated significantly higher antibody titers at PD2 and
PD3 when compared to pre-immune sera for all serotypes (FIGS. 12B
and 12C). A second dose of PCV results in increased antibody titers
from PD2 compared to PD1 for all serotypes except for serotype 3
and serotype 1 in IRMs immunized with PCV23 unadjuvanted, PCV23
(DMSO)/APA and PCV15/APA+PCV8/APA (FIG. 12D). A third dose of PCV
results a boost in antibody titers for the majority of serotypes,
with the exception of IRMs immunized with PCV23 (DMSO+Aq)/APA where
there is a decrease at PD3 compared to PD2, suggesting that PCV23
(DMSO+Aq)/APA immunized IRMs reached the maximum antibody response
at PD2 (FIG. 12E).
EXAMPLE Z
PCV24 Immunogenicity in New Zealand White Rabbits and Infant Rhesus
Macaques
[0943] New Zealand white rabbits (NZWR, n=8/group) were
intramuscularly immunized with 0.1mL of a 24-valent pneumococcal
conjugate vaccine (PCV24) on days 0 and 14. PCV24 was dosed at 9.6
.mu.g of total pneumococcal polysaccharide (1, 3, 4, 5, 6A, 6B, 7F,
8, 9V, 10A, 11A, 12F, 14, 15A, deOAc15B, 18C, 19A, 19F, 22F, 23B,
23F, 24F, 33F and 35B at 0.4 .mu.g and all conjugated to CRM197)
and formulated with aluminum phosphate adjuvant (APA, 25 .mu.g) per
immunization. Sera were collected prior to study start (pre-immune,
day 0) and on days 14 (PD1) and 28 (PD2). NZWRs were observed at
least daily by trained animal care staff for any signs of illness
or distress.
[0944] Infant Rhesus macaques (IRM, 2-3 months old, n=5/group) were
intramuscularly immunized with 0.1mL of a 24-valent pneumococcal
conjugate vaccine (PCV24) on days 0, 28 and 56. PCV24 was dosed at
9.6 .mu.g of total pneumococcal polysaccharide (1, 3, 4, 5, 6A, 6B,
7F, 8, 9V, 10A, 11A, 12F, 14, 15A, deOAc15B, 18C, 19A, 19F, 22F,
23B, 23F, 24F, 33F and 35B at 0.4 .mu.g and all conjugated to
CRM197) and formulated with aluminum phosphate adjuvant (APA, 25
.mu.g) per immunization. Sera were collected prior to study start
(pre-immune, day 0) and on days 14 (PD1), 28, 42 (PD2), 56, and 70
(PD3). IRMs were observed at least daily by trained animal care
staff for any signs of illness or distress. All animal experiments
were performed in strict accordance with the recommendations in the
Guide for Care and Use of Laboratory Animals of the National
Institutes of Health. The experimental protocol was approved by the
Institutional Animal Care and Use Committee at Merck & Co., Inc
and New Iberia Research Center.
[0945] Rhesus sera were evaluated for IgG immunogenicity using a
multiplexed electrochemiluminescence (ECL) assay. This assay was
developed for use with Rhesus serum based on the human assay
described by Marchese et al. and Skinner et al [3, 4] using
technology developed by MesoScale Discovery (a division of
MesoScale Diagnostics, LLC, Gaithersburg, MD) which utilizes a
SULFO-TAGTM label that emits light upon electrochemical
stimulation. SULFO-TAG.TM.-labeled anti-human IgG was used as the
secondary antibody for testing Rhesus serum samples and a
SULFO-TAG.TM.-labeled anti-rabbit IgG for the New Zealand white
rabbit samples.
[0946] Functional antibody was determined through multiplexed
opsonophagocytic assays (MOPA) based on previously described
protocols at www.vaccine.uab.edu and Opsotiter.RTM. 3 software
owned by and licensed from University of Alabama (UAB) Research
Foundation [1, 2].
[0947] NZWR immunization with PCV24 generated antibody titers for
all serotypes in the vaccine (FIG. 13A). It is also of note that
PCV24, which contains polysaccharide conjugates 15A-CRM197,
deOAc15B-CRM197, 6A-CRM197, 6B-CRM197 also provides
cross-reactivity to 15B and 6C, as evidenced in ECL (FIG. 13A).
[0948] NZWR sera were tested individually in multiplexed
opsonophagocytic assays (MOPA) to determine functional antibody
titers. PCV24 generated functional antibody titers in NZWRs which
killed all vaccine-type bacterial serotypes (FIG. 13B).
[0949] IRM immunization with PCV24 generated antibody titers for
all serotypes in the vaccine (FIG. 14A). It is also of note that
PCV24, which contains polysaccharide conjugates 15A-CRM197,
deOAc15B-CRM197, 6A-CRM197, 6B-CRM197 also provides
cross-reactivity to 15B and 6C, as evidenced in ECL (FIG. 14A).
[0950] IRM sera were tested individually in multiplexed
opsonophagocytic assays (MOPA) to determine functional antibody
titers. PCV24 generated functional antibody titers in IRMs which
killed vaccine-type bacterial serotypes (FIG. 14B), with the
exception of 33F, which also had lower PD3/Pre binding antibody
titers in ECL. However, when PCV24/APA was evaluated in New Zealand
white rabbits, PD2 33F OPA titers were over 58-fold higher than
pre-immune titers (FIG. 13B).
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of immunoglobulin G serotype-specific antipneumococcal antibodies
in human serum. Clin Vaccine Immunol. 2009 Mar.; 16(3): 387-96.
[0954] 4. Skinner, J. M., et al., Pre-clinical evaluation of a
15-valent pneumococcal conjugate vaccine (PCV15-CRM197) in an
infant-rhesus monkey immunogenicity model. Vaccine, 2011. 29(48):
p. 8870-8876.
EXAMPLE 52
Materials and Methods
[0955] Free polysaccharide Testing
[0956] Free polysaccharide (polysaccharide that is not conjugated
with CRM197) in conjugate sample is measured by first precipitating
free protein and conjugates with deoxycholate (DOC) and
hydrochloric acid. Precipitates are then filtered out and the
filtrates are analyzed for free polysaccharide concentration by
HPSEC/UV/MALS/RI. Free polysaccharide is calculated as a percentage
of total polysaccharide measured by HPSEC/UV/MALS/RI.
Free Protein Testing
[0957] Free polysaccharide, polysaccharide-CRM197 conjugate, and
free CRM197 in conjugate samples are separated by capillary
electrophoresis in micellar electrokinetic chromatography (MEKC)
mode. Briefly, samples are mixed with MEKC running buffer
containing 25 mM borate, 100 mM SDS, pH 9.3, and are separated in a
preconditioned bare-fused silica capillary. Separation is monitored
at 200 nm and free CRM197 is quantified with a CRM197 standard
curve. Free protein results are reported as a percentage of total
protein content determined by the HPSEC/UV/MALS/RI procedure.
Molecular Weight and Concentration Analysis of Conjugates using HP
SEC/UV/MALS/RI Assay
[0958] Conjugate samples were injected and separated by high
performance size-exclusion chromatography (HPSEC). Detection was
accomplished with ultraviolet (UV), multi-angle light scattering
(MALS) and refractive index (RI) detectors in series. Protein
concentration was calculated from UV280 using an extinction
coefficient. Polysaccharide concentration was deconvoluted from the
RI signal (contributed by both protein and polysaccharide) using
the do/dc factors which are the change in a solution's refractive
index with a change in the solute concentration reported in mL/g.
Average molecular weight of the samples were calculated by Astra
software (Wyatt Technology Corporation, Santa Barbara, CA) using
the measured concentration and light scattering information across
the entire sample peak. There are multiple form of average values
of molecular weight for polydispersed molecules. For example
number-average molecular weight Mn, weight-average molecular weight
Mw, and z-average molecular weight Mz (Molecules, 2015, 20,
10313-10341). Unless specified, the molecular weights are
weight-average molecular weight.
Determination of Lysine Consumption in Conjugated Protein as a
Measure of the Number of Covalent Attachments Between
Polysaccharide and Carrier Protein
[0959] The Waters AccQ-Tag amino acid analysis (AAA) was used to
measure the extent of conjugation in conjugate samples. Samples
were hydrolyzed using vapor phase acid hydrolysis in the Eldex
workstation, to break the carrier proteins down into their
component amino acids. The free amino acids were derivatized using
6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC). The
derivatized samples were then analyzed using UPLC with UV detection
on a C18 column. The average protein concentration was obtained
using representative amino acids other than lysine. Lysine
consumption during conjugation (i.e., lysine loss) was determined
by the difference between the average measured amount of lysine in
the conjugate and the expected amount of lysine in the starting
protein.
Sequence CWU 1
1
11535PRTArtificial SequenceCRM197 variant of diphtheria toxin 1Gly
Ala Asp Asp Val Val Asp Ser Ser Lys Ser Phe Val Met Glu Asn1 5 10
15Phe Ser Ser Tyr His Gly Thr Lys Pro Gly Tyr Val Asp Ser Ile Gln
20 25 30Lys Gly Ile Gln Lys Pro Lys Ser Gly Thr Gln Gly Asn Tyr Asp
Asp 35 40 45Asp Trp Lys Glu Phe Tyr Ser Thr Asp Asn Lys Tyr Asp Ala
Ala Gly 50 55 60Tyr Ser Val Asp Asn Glu Asn Pro Leu Ser Gly Lys Ala
Gly Gly Val65 70 75 80Val Lys Val Thr Tyr Pro Gly Leu Thr Lys Val
Leu Ala Leu Lys Val 85 90 95Asp Asn Ala Glu Thr Ile Lys Lys Glu Leu
Gly Leu Ser Leu Thr Glu 100 105 110Pro Leu Met Glu Gln Val Gly Thr
Glu Glu Phe Ile Lys Arg Phe Gly 115 120 125Asp Gly Ala Ser Arg Val
Val Leu Ser Leu Pro Phe Ala Glu Gly Ser 130 135 140Ser Ser Val Glu
Tyr Ile Asn Asn Trp Glu Gln Ala Lys Ala Leu Ser145 150 155 160Val
Glu Leu Glu Ile Asn Phe Glu Thr Arg Gly Lys Arg Gly Gln Asp 165 170
175Ala Met Tyr Glu Tyr Met Ala Gln Ala Cys Ala Gly Asn Arg Val Arg
180 185 190Arg Ser Val Gly Ser Ser Leu Ser Cys Ile Asn Leu Asp Trp
Asp Val 195 200 205Ile Arg Asp Lys Thr Lys Thr Lys Ile Glu Ser Leu
Lys Glu His Gly 210 215 220Pro Ile Lys Asn Lys Met Ser Glu Ser Pro
Asn Lys Thr Val Ser Glu225 230 235 240Glu Lys Ala Lys Gln Tyr Leu
Glu Glu Phe His Gln Thr Ala Leu Glu 245 250 255His Pro Glu Leu Ser
Glu Leu Lys Thr Val Thr Gly Thr Asn Pro Val 260 265 270Phe Ala Gly
Ala Asn Tyr Ala Ala Trp Ala Val Asn Val Ala Gln Val 275 280 285Ile
Asp Ser Glu Thr Ala Asp Asn Leu Glu Lys Thr Thr Ala Ala Leu 290 295
300Ser Ile Leu Pro Gly Ile Gly Ser Val Met Gly Ile Ala Asp Gly
Ala305 310 315 320Val His His Asn Thr Glu Glu Ile Val Ala Gln Ser
Ile Ala Leu Ser 325 330 335Ser Leu Met Val Ala Gln Ala Ile Pro Leu
Val Gly Glu Leu Val Asp 340 345 350Ile Gly Phe Ala Ala Tyr Asn Phe
Val Glu Ser Ile Ile Asn Leu Phe 355 360 365Gln Val Val His Asn Ser
Tyr Asn Arg Pro Ala Tyr Ser Pro Gly His 370 375 380Lys Thr Gln Pro
Phe Leu His Asp Gly Tyr Ala Val Ser Trp Asn Thr385 390 395 400Val
Glu Asp Ser Ile Ile Arg Thr Gly Phe Gln Gly Glu Ser Gly His 405 410
415Asp Ile Lys Ile Thr Ala Glu Asn Thr Pro Leu Pro Ile Ala Gly Val
420 425 430Leu Leu Pro Thr Ile Pro Gly Lys Leu Asp Val Asn Lys Ser
Lys Thr 435 440 445His Ile Ser Val Asn Gly Arg Lys Ile Arg Met Arg
Cys Arg Ala Ile 450 455 460Asp Gly Asp Val Thr Phe Cys Arg Pro Lys
Ser Pro Val Tyr Val Gly465 470 475 480Asn Gly Val His Ala Asn Leu
His Val Ala Phe His Arg Ser Ser Ser 485 490 495Glu Lys Ile His Ser
Asn Glu Ile Ser Ser Asp Ser Ile Gly Val Leu 500 505 510Gly Tyr Gln
Lys Thr Val Asp His Thr Lys Val Asn Ser Lys Leu Ser 515 520 525Leu
Phe Phe Glu Ile Lys Ser 530 535
* * * * *
References