U.S. patent application number 13/850849 was filed with the patent office on 2014-09-25 for stable igg4 based binding agent formulations.
This patent application is currently assigned to Sanofi. The applicant listed for this patent is Sanofi. Invention is credited to Annika Hagendorf, Martina KIRSCH, Sabrina RUGGEBERG, Julia SCHNIEDERS, Dirk USENER, Ahmed YOUSSEF.
Application Number | 20140286933 13/850849 |
Document ID | / |
Family ID | 48048304 |
Filed Date | 2014-09-25 |
United States Patent
Application |
20140286933 |
Kind Code |
A9 |
SCHNIEDERS; Julia ; et
al. |
September 25, 2014 |
STABLE IGG4 BASED BINDING AGENT FORMULATIONS
Abstract
The present invention provides stable pharmaceutical antibody
formulations, including liquid drug product formulations and
lyophilized drug product formulations, comprising an IgG4 binding
agent and a citrate buffer, wherein the pH of the formulation is at
or below both pH 6 and the pI of the binding agent. The
formulations can be used in the treatment of chronic bowel diseases
or rheumatoid arthritis.
Inventors: |
SCHNIEDERS; Julia;
(Frankfurt, DE) ; USENER; Dirk; (Frankfurt,
DE) ; RUGGEBERG; Sabrina; (Frankfurt, DE) ;
YOUSSEF; Ahmed; (Frankfurt, DE) ; KIRSCH;
Martina; (Frankfurt, DE) ; Hagendorf; Annika;
(Hattersheim, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Sanofi; |
|
|
US |
|
|
Assignee: |
Sanofi
Paris
FR
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20140004106 A1 |
January 2, 2014 |
|
|
Family ID: |
48048304 |
Appl. No.: |
13/850849 |
Filed: |
March 26, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61615539 |
Mar 26, 2012 |
|
|
|
Current U.S.
Class: |
424/133.1 ;
424/158.1; 424/173.1 |
Current CPC
Class: |
C07K 2317/52 20130101;
A61K 39/3955 20130101; C07K 16/2866 20130101; A61K 39/39591
20130101; C07K 16/2875 20130101; C07K 2317/21 20130101; A61K 47/12
20130101; A61K 47/26 20130101; C07K 2317/565 20130101; A61K 47/22
20130101; A61K 9/19 20130101; C07K 2317/515 20130101; A61K 39/39558
20130101; C07K 2317/51 20130101 |
Class at
Publication: |
424/133.1 ;
424/158.1; 424/173.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 6, 2013 |
FR |
1351013 |
Claims
1. A stable formulation comprising: a binding agent comprising at
least a portion of a Fc region of an IgG4 antibody; and about 5 to
about 50 mM citrate as a buffering agent; wherein the pH of the
formulation is at or below both about pH 6 and the pI of the
binding agent.
2. The formulation of claim 1, wherein the binding agent is an
antibody.
3. The formulation of claim 1, wherein the binding agent binds to
lymphotoxin-like, exhibits inducible expression and competes with
herpes virus glycoprotein D for herpes virus entry mediator, a
receptor expressed on lymphocytes (LIGHT).
4. The formulation of claim 3, wherein the anti-LIGHT binding agent
comprises a heavy chain variable region and a light chain variable
region, the heavy chain variable region comprising complementary
determining regions (CDRs) comprising the amino acid sequences of
SEQ ID NOS: 1, 2, and 3, and the light chain variable region
comprising CDRs comprising the amino acid sequences of SEQ ID NOS:
4, 5, and 6.
5. The formulation of claim 3, wherein the binding agent is a fully
human IgG4 anti-LIGHT antibody comprising a heavy chain comprising
the amino acid sequence of SEQ ID NO: 7 and a light chain
comprising the amino acid sequence of SEQ ID NO: 8.
6. The formulation of claim 2, wherein the binding agent or
antibody binds to C-X-C chemokine receptor type 5 (CXCR5).
7. The formulation of claim 6, wherein the anti-CXCR5 binding agent
or antibody comprises a heavy chain variable region and a light
chain variable region, the heavy chain variable region comprising
complementary determining regions (CDRs) comprising the amino acid
sequences of SEQ ID NOS: 15, 16, and 17, and the light chain
variable region comprising CDRs comprising the amino acid sequences
of SEQ ID NOS: 18, 19, and 20.
8. The formulation of claim 6, wherein the binding agent or
antibody is a humanized IgG4 anti-CXCR5 antibody comprising a heavy
chain comprising the amino acid sequence of SEQ ID NO: 25 and a
light chain comprising the amino acid sequence of SEQ ID NO:
26.
9. The formulation of claim 2, wherein the antibody concentration
is from about 5 to about 280 mg/mL.
10. (canceled)
11. (canceled)
12. (canceled)
13. (canceled)
14. The formulation of claim 1, wherein the citrate concentration
is from about 5 to about 15 mM.
15. (canceled)
16. The formulation of claim 1, wherein the citrate buffer is
sodium citrate dihydrate.
17. The formulation of claim 1, wherein the pH of the formulation
is from about pH 5 and about pH 6.
18. The formulation of claim 17, wherein the pH of the formulation
is selected from the group consisting of about pH 5.0, about pH
5.5, and about pH 6.0.
19. The formulation of claim 5, wherein the pI of the binding agent
or antibody is from about 6.8 and about 7.2.
20. The formulation of claim 8, wherein the pI of the binding agent
or antibody is from about 7.6 and about 8.4.
21. The formulation of claim 1, wherein the formulation further
comprises a surfactant.
22. The formulation of claim 21, wherein the concentration of
surfactant is between about 0.001% and about 0.1% w/v.
23. The formulation of claim 21, wherein the surfactant is a
polysorbate.
24. The formulation of claim 23, wherein the polysorbate is
polysorbate 20.
25. (canceled)
26. (canceled)
27. (canceled)
28. The formulation of claim 1, wherein the formulation further
comprises a tonicity agent.
29. The formulation of claim 28, wherein the concentration of
tonicity agent is between about 0.1% and about 10% w/v.
30. The formulation of claim 28, wherein the tonicity agent is a
saccharide.
31. The formulation of claim 30, wherein the saccharide is
mannitol.
32. (canceled)
33. (canceled)
34. The formulation of claim 30, wherein the saccharide is
sucrose.
35. (canceled)
36. (canceled)
37. (canceled)
38. (canceled)
39. The formulation of claim 28, wherein the tonicity agent is
sodium chloride.
40. The formulation of claim 39, wherein the concentration of
sodium chloride is between about 0.01% and about 1%.
41. (canceled)
42. The formulation of claim 28, wherein the tonicity agent is a
combination of sucrose and sodium chloride.
43. The formulation of claim 42, wherein the concentration of
sucrose is between about 1% and about 10% w/v.
44. The formulation of claim 42, wherein the concentration of
sodium chloride is between about 0.01% and about 1%.
45. (canceled)
46. (canceled)
47. The formulation of claim 1, wherein the formulation further
comprises an amino acid.
48. The formulation of claim 47, wherein the amino acid
concentration is between about 0.1% and about 5% w/v.
49. The formulation of claim 47, wherein the amino acid is proline
or arginine.
50. (canceled)
51. (canceled)
52. (canceled)
53. The formulation of claim 1, wherein the formulation is a liquid
formulation.
54. The formulation of claim 1, wherein the formulation is a
lyophilized formulation.
55. (canceled)
56. (canceled)
57. The formulation of claim 1, wherein the formulation exhibits a
reduced amount of at least one byproduct selected from the group
consisting of aggregates, half-molecules, degradation products, low
molecular weight proteins, high molecular weight proteins,
rearrangements of acidic/basic/neutral isoforms of the antibody as
compared to either a reference anti-LIGHT formulation comprising an
anti-LIGHT antibody in phosphate buffered saline at pH 7.3 or a
reference anti-CXCR5 formulation comprising an anti-LIGHT antibody
in phosphate buffered saline at pH 7.3.
58. A stable liquid antibody formulation suitable for subcutaneous
administration, the formulation comprising: a) about 150 mg/mL of a
fully human IgG4 anti-LIGHT (lymphotoxin-like, exhibits inducible
expression and competes with HSV glycoprotein D for HVEM, a
receptor expressed by T lymphocytes) antibody comprising a heavy
chain comprising the amino acid sequence of SEQ ID NO: 7 and a
light chain comprising the amino acid sequence of SEQ ID NO: 8; b)
about 10 mM citrate buffer; c) about 0.005% polysorbate 20; and d)
about 4% mannitol; wherein the pH of the formulation is about pH
5.5.
59. A stable liquid antibody formulation suitable for intravenous
administration, the formulation comprising: a) about 50 mg/mL of a
fully human IgG4 anti-LIGHT (lymphotoxin-like, exhibits inducible
expression and competes with HSV glycoprotein D for HVEM, a
receptor expressed by T lymphocytes) antibody comprising a heavy
chain comprising the amino acid sequence of SEQ ID NO: 7 and a
light chain comprising the amino acid sequence of SEQ ID NO: 8; b)
about 10 mM citrate buffer; and c) about 0.01% polysorbate 20;
wherein the pH of the formulation is about pH 5.5.
60. A stable lyophilized antibody formulation suitable for
intravenous administration, the formulation comprising: a) about 50
mg/mL of a fully human IgG4 anti-LIGHT (lymphotoxin-like, exhibits
inducible expression and competes with HSV glycoprotein D for HVEM,
a receptor expressed by T lymphocytes) antibody comprising a heavy
chain comprising the amino acid sequence of SEQ ID NO: 7 and a
light chain comprising the amino acid sequence of SEQ ID NO: 8; b)
about 10 mM citrate buffer; c) about 0.01% polysorbate 20; d) about
5% sucrose; and e) about 1.5% proline; wherein the pH of the
formulation is about pH 5.5.
61. A stable antibody formulation comprising: a) about 20 mg/mL of
a humanized IgG4 anti-CXCR5 (C-X-C chemokine receptor type 5)
antibody comprising a heavy chain comprising the amino acid
sequence of SEQ ID NO: 25 and a light chain comprising the amino
acid sequence of SEQ ID NO: 26; b) about 10 mM citrate buffer; c)
about 0.02% polysorbate 20; d) about 6% sucrose; and e) about 0.2%
sodium chloride; wherein the pH of the formulation is about pH
6.0.
62. A stable antibody formulation comprising: a) about 100 mg/mL of
a humanized IgG4 anti-CXCR5 (C-X-C chemokine receptor type 5)
antibody comprising a heavy chain comprising the amino acid
sequence of SEQ ID NO: 25 and a light chain comprising the amino
acid sequence of SEQ ID NO: 26; b) about 10 mM citrate buffer; c)
about 0.01% polysorbate 20; d) about 4.5% sucrose; e) about 0.2%
sodium chloride; and f) about 1% arginine; wherein the pH of the
formulation is about pH 6.0.
63. A kit comprising a container comprising: 1) the formulation of
claim 1, and 2) a label or instructions for the administration and
use of the formulation.
64. The kit of claim 63, wherein the label comprises one or more of
the following: instructions for the administration of the
formulation, instructions for use of the formulation, instructions
concerning the storage conditions of the formulation, information
concerning lot and batch number of the formulation and/or kit,
information concerning the composition of the formulation, safety
information, information concerning possible adverse reactions,
secondary effects, and/or side effects in connection with the
administration of the formulation, or information concerning
possible indications and/or contra-indications of the
formulation.
65. A pre-filled device or pre-filled container comprising the
formulation of claim 1.
66. The kit of claim 63 comprising the pre-filled device of claim
65.
67. A method for treating an inflammatory bowel disease comprising
administering to a subject in need thereof the formulation of claim
5.
68. A method for treating rheumatoid arthritis comprising
administering to a subject in need thereof the formulation of claim
8.
69. (canceled)
70. (canceled)
71. (canceled)
72. (canceled)
73. A method for preparing a stable antibody formulation
comprising: a) providing an anti-LIGHT binding agent; b)
resuspending the anti-LIGHT binding agent in about 5 to about 50 mM
citrate buffer; and c) adjusting the pH of the formulation to about
pH 5.0 to about pH 6.0.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional
Application Ser. No. 61/615,539, filed Mar. 26, 2012, which is
incorporated herein by reference in its entirety.
BACKGROUND OF THE INVENTION
[0002] The human LIGHT antigen is one potential cytokine target
that has been implicated in the processes of chronic inflammatory
autoimmune disease. As a member of the TNF superfamily (TNFSF) of
ligands, LIGHT is also known as TNFSF14 or CD258. LIGHT is
expressed on the surface of T cells upon activation in a tightly
regulated manner. However, LIGHT is also present at detectable
levels constitutively on the surface of immature dendritic cells
and on T cells and natural killer (NK) cells of the gut. LIGHT
mediates its biologic effects by binding three TNF superfamily
receptors, including the lymphotoxin .beta. receptor (LT.beta.R),
the herpes virus entry mediator (HVEM), and decoy receptor 3
(DcR3). LIGHT-expressing lymphocytes can induce IBD-like symptoms
in humans, and increases of LIGHT expression have been observed in
patients with active Crohn's disease and other inflammatory
disorders such as Graft-vs.-Host Disease.
[0003] CXCR5, also known as Burkitt lymphoma receptor (BLR1),
CD185, MDR15, and MGC117347, is a G protein-coupled receptor that
is a member of the CXC chemokine receptor family. The unprocessed
CXCR5 precursor is 372 amino acids in length with a molecular
weight of 42 K.sub.D. CXCR5 has a role in B cell migration and
localization within particular anatomic compartments. Knockout mice
lack peripheral lymph nodes, have fewer Peyer's patches and have
decreased B cell levels. CXCL13, also known as BLC, is a ligand for
CXCR5. CXCL13 is a B cell chemoattractant.
[0004] Anti-LIGHT binding agents and anti-CXCR5 binding agents are
each therapeutically relevant, and a need exists to formulate each
of these binding agents into drug products that may be administered
to subjects, particularly human subjects, for the treatment of
inflammatory diseases.
[0005] In order to develop a pharmaceutical formulation containing
an anti-LIGHT binding agent or an anti-CXCR5 binding agent suitable
for intravenous or subcutaneous administration, the binding agent
must be concentrated to about 20 mg/mL or greater, usually about
100-150 mg/mL, and even up to 250 mg/mL. Many complications can
arise at such high concentrations, including an increase in
viscosity, a shift of pH, a change of the color of the solution,
and the formation of visible and sub-visible particles.
[0006] The formulation of these binding agents is further
complicated by the fact that these agents are highly prone to
aggregation at such high concentrations.
[0007] The formulation of IgG4 antibodies is even further
complicated by the fact that IgG4 antibodies tend to form
half-molecules at high concentrations in solution. However, IgG4
antibodies are of therapeutic interest because they have reduced
effector function.
SUMMARY OF THE INVENTION
[0008] To meet these and other needs, provided herein are highly
stable IgG4 binding agent formulations. Highly stable IgG4 binding
agent formulations have surprisingly been found in the form of
liquids and lyophilized powders that comprise an IgG4 binding agent
and a citrate buffer, wherein the pH of the formulation is at or
below both about pH 6 and the isoelectric point (pI) of the binding
agent. These formulations improve upon conventional formulations,
which often lead to dimerization of the binding agent, such as an
antibody, upon increasing the concentration of the binding agent,
such as an antibody, in the formulation. In particular, the
formulations of the invention reduce the amount of unwanted
byproducts, including aggregates, half-molecules, degradation
products, low molecular weight proteins (LMWPs), high molecular
weight proteins (HMWPs), and rearrangements of acid, basic, and
neutral isoforms of the binding agent, such as an antibody,
component in the formulation.
[0009] In certain aspects, the invention provides a stable
formulation comprising: a binding agent comprising at least a
portion of a Fc region of an IgG4 antibody; and about 5 to about 50
mM citrate as a buffering agent; wherein the pH of the formulation
is at or below both about pH 6 and the pI of the binding agent. In
certain embodiments of the invention, the binding agent is an
antibody.
[0010] In certain embodiments of the invention, the binding agent
or antibody binds to lymphotoxin-like, exhibits inducible
expression and competes with herpes virus glycoprotein D for herpes
virus entry mediator, a receptor expressed on lymphocytes (LIGHT).
In specific embodiments of the invention, the anti-LIGHT binding
agent or antibody comprises a heavy chain variable region and a
light chain variable region, the heavy chain variable region
comprising complementary determining regions (CDRs) comprising the
amino acid sequences of SEQ ID NOS: 1, 2, and 3, and the light
chain variable region comprising CDRs comprising the amino acid
sequences of SEQ ID NOS: 4, 5, and 6. In other specific embodiments
of the invention, the antibody is a fully human IgG4 anti-LIGHT
antibody comprising a heavy chain comprising the amino acid
sequence of SEQ ID NO: 7 and a light chain comprising the amino
acid sequence of SEQ ID NO: 8.
[0011] In certain embodiments of the invention, the binding agent
or antibody binds to C--X--C chemokine receptor type 5 (CXCR5). In
specific embodiments of the invention, the anti-CXCR5 binding agent
or antibody comprises a heavy chain variable region and a light
chain variable region, the heavy chain variable region comprising
complementary determining regions (CDRs) comprising the amino acid
sequences of SEQ ID NOS: 15, 16, and 17, and the light chain
variable region comprising CDRs comprising the amino acid sequences
of SEQ ID NOS: 18, 19, and 20. In other specific embodiments of the
invention, the antibody is a humanized IgG4 anti-CXCR5 antibody
comprising a heavy chain comprising the amino acid sequence of SEQ
ID NO: 25 and a light chain comprising the amino acid sequence of
SEQ ID NO: 26.
[0012] In certain embodiments of the invention, the antibody
concentration is from about 5 to about 280 mg/mL. In certain
specific embodiments of the invention, the antibody concentration
is about 150 mg/mL. In other specific embodiments of the invention,
the antibody concentration is about 50 mg/mL. In further specific
embodiments of the invention, the antibody concentration is about
20 mg/mL. In yet further specific embodiments of the invention, the
antibody concentration is about 100 mg/mL.
[0013] In certain embodiments of the invention, the citrate
concentration is from about 5 to about 15 mM. In some embodiments
of the invention, the citrate concentration is about 10 mM. In some
embodiments of the invention, the citrate buffer is sodium citrate
dihydrate.
[0014] In certain embodiments of the invention, the pH of the
formulation is from about pH 5 and about pH 6. In specific
embodiments of the invention, the pH of the formulation is selected
from the group consisting of about pH 5.0, about pH 5.5, and about
pH 6.0.
[0015] In certain specific embodiment of the invention, the pI of
the binding agent or antibody is from about 6.8 and about 7.2. In
alternative specific embodiments of the invention, the pI of the
binding agent or antibody is from about 7.6 and about 8.4.
[0016] In certain specific embodiments of the invention, the
formulation further comprises a surfactant. In certain specific
embodiments of the invention, the concentration of surfactant is
between about 0.001% and about 0.1% w/v. In certain embodiments of
the invention, the surfactant is a polysorbate. In certain specific
embodiments of the invention, the polysorbate is polysorbate 20. In
some specific embodiments of the invention, the concentration of
polysorbate 20 is about 0.005% w/v. In alternative specific
embodiments of the invention, the concentration of polysorbate 20
is about 0.01% w/v. In further alternative specific embodiments of
the invention, the concentration of polysorbate 20 is about 0.02%
w/v.
[0017] In certain embodiments of the invention, the formulation
further comprises a tonicity agent. In certain specific embodiments
of the invention, the concentration of tonicity agent is between
about 0.1% and about 10% w/v. In certain specific embodiments of
the invention, the tonicity agent is a saccharide. In some specific
embodiments of the invention, the saccharide is mannitol. In other
specific embodiments of the invention, the concentration of
mannitol is between about 1% and about 10% w/v. In yet other
specific embodiments of the invention, the concentration of
mannitol is about 4%. In alternative specific embodiments of the
invention, the saccharide is sucrose. In some specific embodiments
of the invention, the concentration of sucrose is between about 1%
and about 10% w/v. In some specific embodiments of the invention,
the concentration of sucrose is about 5% w/v. In alternative
specific embodiments of the invention, the concentration of sucrose
is about 6% w/v. In yet other specific embodiments of the
invention, the concentration of sucrose is about 4.5% w/v. In
further specific alternative embodiments of the invention, the
tonicity agent is sodium chloride. In some specific embodiments of
the invention, the concentration of sodium chloride is between
about 0.01% and about 1%. In some specific embodiments of the
invention, the concentration of sodium chloride is about 0.2%. In
other specific embodiments of the invention, the tonicity agent is
a combination of sucrose and sodium chloride. In specific
embodiments of the invention, the concentration of sucrose is
between about 1% and about 10% w/v. In other specific embodiments
of the invention, the concentration of sodium chloride is between
about 0.01% and about 1%. In alternative specific embodiments of
the invention, the concentration of sucrose is about 6% w/v and the
concentration of sodium chloride is about 0.2%. In yet further
alternative specific embodiments of the invention, the
concentration of sucrose is about 4.5% w/v and the concentration of
sodium chloride is about 0.2%.
[0018] In certain embodiments of the invention, the formulation
further comprises an amino acid. In certain specific embodiments of
the invention, the amino acid concentration is between about 0.1%
and about 5% w/v. In certain specific embodiments of the invention,
the amino acid is proline or arginine. In specific embodiments of
the invention, the proline or arginine concentration is between
about 1% and about 2% w/v. In other specific embodiments of the
invention, the proline concentration is about 1.5% w/v. In
alternative specific embodiments of the invention, the arginine
concentration is about 1% w/v.
[0019] In certain embodiments of the invention, the formulation is
a liquid formulation. In other specific embodiments of the
invention, the formulation is a lyophilized formulation.
[0020] In certain embodiments of the invention, the formulation is
stable for at least 6 months at +5.degree. C. In alterntative
embodiments of the invention, the formulation is stable for at
least 9 months at +5.degree. C.
[0021] In certain embodiments of the invention, the formulation
exhibits a reduced amount of at least one byproduct selected from
the group consisting of aggregates, half-molecules, degradation
products, low molecular weight proteins, high molecular weight
proteins, and rearrangements of acidic/basic/neutral isoforms of
the antibody as compared to either a reference anti-LIGHT
formulation comprising an anti-LIGHT antibody in phosphate buffered
saline at pH 7.3 or a reference anti-CXCR5 formulation comprising
an anti-LIGHT antibody in phosphate buffered saline at pH 7.3.
[0022] In certain specific embodiments of the invention, the
invention provides a stable liquid antibody formulation suitable
for subcutaneous administration, the formulation comprising:
[0023] a) about 150 mg/mL of a fully human IgG4 anti-LIGHT
(lymphotoxin-like, exhibits inducible expression and competes with
HSV glycoprotein D for HVEM, a receptor expressed by T lymphocytes)
antibody comprising a heavy chain comprising the amino acid
sequence of SEQ ID NO: 7 and a light chain comprising the amino
acid sequence of SEQ ID NO: 8;
[0024] b) about 10 mM citrate buffer;
[0025] c) about 0.005% polysorbate 20; and
[0026] d) about 4% mannitol;
wherein the pH of the formulation is about pH 5.5.
[0027] In other specific embodiments of the invention, the
invention provides a stable liquid antibody formulation suitable
for intravenous administration, the formulation comprising:
[0028] a) about 50 mg/mL of a fully human IgG4 anti-LIGHT
(lymphotoxin-like, exhibits inducible expression and competes with
HSV glycoprotein D for HVEM, a receptor expressed by T lymphocytes)
antibody comprising a heavy chain comprising the amino acid
sequence of SEQ ID NO: 7 and a light chain comprising the amino
acid sequence of SEQ ID NO: 8;
[0029] b) about 10 mM citrate buffer; and
[0030] c) about 0.01% polysorbate 20;
wherein the pH of the formulation is about pH 5.5.
[0031] In yet other specific embodiments of the invention, the
invention provides a stable lyophilized antibody formulation
suitable for intravenous administration, the formulation
comprising:
[0032] a) about 50 mg/mL of a fully human IgG4 anti-LIGHT
(lymphotoxin-like, exhibits inducible expression and competes with
HSV glycoprotein D for HVEM, a receptor expressed by T lymphocytes)
antibody comprising a heavy chain comprising the amino acid
sequence of SEQ ID NO: 7 and a light chain comprising the amino
acid sequence of SEQ ID NO: 8;
[0033] b) about 10 mM citrate buffer;
[0034] c) about 0.01% polysorbate 20;
[0035] d) about 5% sucrose; and
[0036] e) about 1.5% proline;
wherein the pH of the formulation is about pH 5.5.
[0037] In alternative specific embodiments of the invention, the
invention provides a stable antibody formulation comprising:
[0038] a) about 20 mg/mL of a humanized IgG4 anti-CXCR5 (C--X--C
chemokine receptor type 5) antibody comprising a heavy chain
comprising the amino acid sequence of SEQ ID NO: 25 and a light
chain comprising the amino acid sequence of SEQ ID NO: 26;
[0039] b) about 10 mM citrate buffer;
[0040] c) about 0.02% polysorbate 20;
[0041] d) about 6% sucrose; and
[0042] e) about 0.2% sodium chloride;
wherein the pH of the formulation is about pH 6.0.
[0043] In further alternative specific embodiments of the
invention, the invention provides a stable antibody formulation
comprising:
[0044] a) about 100 mg/mL of a humanized IgG4 anti-CXCR5 (C-X-C
chemokine receptor type 5) antibody comprising a heavy chain
comprising the amino acid sequence of SEQ ID NO: 25 and a light
chain comprising the amino acid sequence of SEQ ID NO: 26;
[0045] b) about 10 mM citrate buffer;
[0046] c) about 0.01% polysorbate 20;
[0047] d) about 4.5% sucrose;
[0048] e) about 0.2% sodium chloride; and
[0049] f) about 1% arginine;
wherein the pH of the formulation is about pH 6.0.
[0050] In certain embodiments of the invention, the invention
provides a kit comprising a container comprising: 1) the
formulation of any one of the previous claims, and 2) a lable or
instructions for the administration and use of the formulation. In
certain embodiments of the invention, the label comprises one or
more of the following: instructions for the administration of the
formulation, instructions for use of the formulation, instructions
concerning the storage conditions of the formulation, information
concerning lot and batch number of the formulation and/or kit,
information concerning the composition of the formulation, safety
information, information concerning possible adverse reactions,
secondary effects, and/or side effects in connection with the
administration of the formulation, or information concerning
possible indications and/or contra-indications of the
formulation.
[0051] In certain embodiments of the invention, the invention
provides a pre-filled device or pre-filled container, such as a
syringe, cartridge, vial, ampoule, or autoinjector comprising the
formulation of the invention. In certain other embodiments, the
invention provides a kit comprising such pre-filled syringe,
cartridge, vial, ampoule, or autoinjector.
[0052] In certain embodiments, the invention provides a method for
treating an inflammatory bowel disease comprising administering to
a subject in need thereof a formulation of the invention.
[0053] In other certain embodiments, the invention provides a
method for treating rheumatoid arthritis comprising administering
to a subject in need thereof a formulation of the invention.
[0054] In certain embodiments, the invention provides a formulation
for use in a method of diagnosis or treatment of the human or
animal body. In specific embodiments, the formulation is used in
the treatment of inflammatory bowel disease. In alternative
embodiments, the formulation is used in the treatment of rheumatoid
arthritis.
[0055] In certain embodiments of the invention, the invention
provides a method for preparing a formulation of the invention
comprising mixing the components of the formulation and adjusting
the pH, wherein the preparation is performed under sterile
conditions or the formulation is sterilized after the mixing of the
components and the pH adjustment or both.
[0056] In certain specific embodiments of the invention, the
invention provides a method for preparing a stable antibody
formulation comprising: a) providing an anti-LIGHT binding agent;
b) resuspending the anti-LIGHT binding agent in about 5 to about 50
mM citrate buffer; and c) adjusting the pH of the formulation to
about pH 5.0 to about pH 6.0.
BRIEF DESCRIPTION OF THE FIGURES
[0057] FIG. 1 is a picture of a gel showing the results of
denatured isoelectric focusing experiments that were used to
determine the isoelectric point (pI) of the fully human IgG4
anti-LIGHT antibody comprising a heavy chain comprising the amino
acid sequence of SEQ ID NO: 7 and a light chain comprising the
amino acid sequence of SEQ ID NO: 8 formulated in phosphate
buffered saline at pH 7.3 at a concentration of 5.5 mg/mL (the
"Original Formulation", "PBS Formulation", or "Reference Lot").
Lanes 1 & 5: IEF Calibration Kit High Range pI 5-10.5; lanes 2
& 4: a first batch of Reference Lot; lanes 3 & 4: a second
batch of Reference Lot. The pI values are indicated by numbers.
[0058] FIG. 2 is a picture of an SDS-PAGE gel that compared
different Reference Lot batches under reducing and non-reducing
conditions. Lanes 1 & 10: Biorad Precision Plus Protein
Standard; lane 5: empty; lane 2: a first batch of Reference Lot
under non-reduced conditions; lanes 3 & 4: a second batch of
Reference Lot under non-reduced conditions; lane 6: a first batch
of Reference Lot under reduced conditions; lanes 7 & 8: a
second batch of Reference Lot under reduced conditions; and lane 9:
system control. The sizes are indicated by numbers within the
rows.
[0059] FIG. 3 shows an ELISA graph that was used to determine the
antigen binding activity of the first and second batches of
Reference Lot.
[0060] FIG. 4 shows a size exclusion chromatography (SEC)
chromatogram of the first batch of Reference Lot. As shown in FIG.
4, SEC detected high molecular weight proteins (HMWP), e.g.,
di-/oligomers (RRT0.8) or aggregates, and low molecular weight
proteins (LMWPs) or degradation products. The first batch of
Reference Lot batch had a purity of 97% monomer content.
[0061] FIG. 5 shows a weak cation exchange chromatogram for the
first batch of Reference Lot. As shown in FIG. 5, rearrangements of
acidic, neutral, and basic isoforms occurred during stability
studies. The first batch of Reference Lot had a distribution of
acidic/neutral/basic isoforms of 42.3/55.6/1.9%.
[0062] FIG. 6 shows a differencial scanning calorimetry thermogram
of the first batch of Reference Lot. As shown in FIG. 6, the three
domains of the antibody unfold at 68.degree. C., 75.degree. C., and
78.degree. C.
[0063] FIG. 7 shows a dynamic light scattering pattern of the first
batch of Reference Lot, which was unfiltered. DLS was used to
determine the hydrodynamic diameter of the first batch of Reference
Lot antibody monomer and potential soluble aggregates.
[0064] FIG. 8 shows a dynamic light scattering pattern of the first
batch of Reference Lot, which was filetered. DLS was used to
determine the hydrodynamic diameter of the first batch of Reference
Lot antibody monomer and potential soluble aggregates.
[0065] FIG. 9 is a flow diagram of the drug product manufacturing
process for the high antibody concentration formulation.
[0066] FIG. 10 shows a dynamic light scattering pattern of
Formulation 14. DLS was used to determine the hydrodynamic diameter
of the antibody monomer and potential soluble aggregates.
[0067] FIG. 11 is a picture of a gel showing the results of
isoelectric focusing to determine the pI (isoelectric point) of the
Lead CXCR5 Antibody. Lanes 1,6: IEF Calibration High Range pI Kit;
Lanes 2,4: Reference Standard Lead Antibody LP08031; and Lanes 3,5:
Lead Antibody Drug Substance, RSN0151.
[0068] FIG. 12 is a picture of an SDS-PAGE gel that compared
different drug substance batches under reducing and non-reducing
conditions. The gel was also used to determine the molecular weight
of the Lead CXCR5 Antibody, and the presence of any aggregates.
[0069] FIG. 13 is an ELISA graph that was used to determine antigen
binding activity of the Lead CXCR5 Antibody to a 28mer peptide of
the CXCR5 antigen.
[0070] FIG. 14 is a SEC chromatogram of stressed Lead CXCR5
Antibody. SEC could detect high molecular weight proteins (HMWP),
e.g., di-/oligomers or aggregatres and low molecular weight
proteins (LMWP) or degradation products. The Lead CXCR5 Antibody
had a purity of 99% monomer content.
[0071] FIG. 15 is a WCX chromatogram that was used to determine
acidic, neutral, and basic isoforms of the Lead CXCR5 Antibody. The
Lead CXCR5 Antibody had a distribution of acidic/neutral/basic
isoforms of 14/85/1%.
[0072] FIG. 16 is a DLS measurement that was used to determine the
hydrodynamic diameter of the antibody monomer and potential soluble
aggregates.
[0073] FIG. 17 is a picture of the Lead CXCR5 Antibody in acetate
buffer pH 5.0 (left) and pH 5.5 (right); each v. WFI (water for
injection) and after thermal stress. This figure shows that acetate
is a suitable buffer system.
[0074] FIG. 18 is a picture of the Lead CXCR5 Antibody in histidine
buffer pH 6.0 (left), pH 5.5 (middle), and pH 5.0 (right); each v.
WFI (water for injection) and after thermal stress. This figure
shows that histidine is a suitable buffer.
[0075] FIG. 19 is a picture of the Lead CXCR5 Antibody in TRIS
buffer pH 7.5 after UF/DF (left) and after filtration (right); each
v. WFI (water for injection) and after thermal stress. This figure
shows that TRIS is an incompatible buffer system.
[0076] FIG. 20 is a picture of the Lead CXCR5 Antibody in citrate
buffer pH 6.0 after UF/DF and filtration.
[0077] FIG. 21 is a picture of the Lead CXCR5 Antibody in acetate
buffer pH 5.5 after UF/DF and filtration.
[0078] FIG. 22 is a picture of the Lead CXCR5 Antibody in succinate
buffer pH 5.0 after UF/DF and filtration.
[0079] FIG. 23 is a picture of the Lead CXCR5 Antibody in histidine
buffer pH 5.0 after UF/DF and filtration.
[0080] FIG. 24 is a picture of the Lead CXCR5 Antibody in arginine
buffer pH 6.0 after UF/DF and filtration.
[0081] FIG. 25 is a picture of the appearance of Lead CXCR5
Antibody LA.sub.--09.sub.--016 solutions with different surfactants
(without surfactant, polysorbate 20, polysorbate 80, Lutrol F68,
Cremophor RH40, Solutol HS15, and SDS) after mechanical stress (350
rpm, 2.5 h, RT).
[0082] FIG. 26 is a graph that shows an increase of dimers under
accelerated conditions, as analyzed by SEC. An increase of dimer
formation up to 10% after three months of storage in all four
histidine formulation can be seen. Acetate formulations showed an
increase of dimer content up to 6%. In all four citrate
formulations, the dimer concentration was below 2%, even after
three months at +40.degree. C.
[0083] FIG. 27 is a graph showing an increase of basic isoforms
under accelerated conditions, as analyzed by WCX. Histidine is
worse for Lead CXCR5 Antibody stability under accelerated
conditions. A slight increase of basic isoforms can be noticed for
all four acetate formultions. Interestingly, it was not possible to
discriminate between the four citrate formulations.
[0084] FIG. 28 is a graph showing a decrease of neutral isoforms
under accelerated conditions, as analyzed by WCX. This figure shows
a strong decrease in neutral isoforms for the histidine
formulations. A slight decrease was seen in acetate. Citrate was
affected the least.
[0085] FIG. 29 shows the delta pH of all four formulations (A-D) in
citrate buffer at accelerated conditions. The most pH stabilizing
formulations are the citrate buffered, and especially formulation B
and D.
[0086] FIG. 30 shows the delta pH of all four formulations (A-D) in
acetate buffer at accelerated conditions. In acetate buffered
solutions of the Lead CXCR5 Antibody, the pH was shifted towards
higher value.
[0087] FIG. 31 shows the delta pH of all four formulations (A-D) in
histidine buffer at accelerated conditions. In histidine buffered
solutions of the Lead CXCR5 Antibody, the pH was slightly
decreasing.
[0088] FIG. 32 is a graph showing the hydrodynamic diameter of
CXCR5 LA.sub.--09.sub.--027 A-D after 3 months storage at
40.degree. C. Citrate buffered formulations showed only slight
aggregates after three weeks in formulation C, and after six weeks
of storage in formulation A. Some aggregates could be detected
after three months in formulation B as well. But, compared to
acetate buffered formulations, the amount was very little.
[0089] FIG. 33 is a graph showing the hydrodynamic diameter of
CXCR5 LA.sub.--09.sub.--028 A-D after 3 months storage at
40.degree. C. The acetate buffered formulation C showed some
aggregates <200 nm after three weeks. Formulation A showed some
aggregates after three months.
[0090] FIG. 34 is a chart showing the effect of increasing Lead
CXCR5 Antibody concentration on the Z-average. The Lead CXCR5
Antibody showed a significant increase in the hydrodynamic diameter
(Z-Average) by increasing the concentration of the antibody.
[0091] FIG. 35 is a chart showing the effect of different
stabilizers (excipients) on the Z-Average at 100 mg/mL of Lead
CXCR5 Antibody after thermal stress. Z-Average was measured before
and after thermal stress. The stabilizing effect was simiar to all
tested excipients, but the increase in Z-average was generally
reduced by using amino acids as stabilizers (argining, lysing, or
glycine). Lysine was excluded due to a higher content of aggregates
after stress. Arginine showed a better effect than glycine.
[0092] FIG. 36 is a chart showing the effect of different
stabilizers on the Z-Average at 100 mg/mL Lead CXCR5 Antibody after
mechanical stress. Z-Average was measured before and after
mechanical stress. The same reduction in Z-average was noticed in
the presence of amino acids. Sucrose had a better protective effect
than trehalose against mechanical stress. Arginine and glycine
performed better in combination with NaCl.
[0093] FIG. 37 is a set of graphs showing particle size
distribution, as measured by DLS, of Lead CXCR5 Antibody formulated
in 10 mM citrate buffer at pH 6 before mechanical stress (A) and
after mechanical stress (B). A higher molecular weight species was
measured by DLS after mechanical stress of DS.
[0094] FIG. 38 is a set of graphs showing particle size
distribution, as measured by DLS, of Lead CXCR5 Antibody drug
product prototype formulations (A-D; Table 111) before (A) and
after (B) mechanical stress.
DETAILED DESCRIPTION
A. Definitions
[0095] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as is commonly understood by one
of ordinary skill in the art.
[0096] It is noted here that as used in this specification and the
appended claims, the singular forms "a", "an", and "the" also
include plural reference, unless the context clearly dictates
otherwise.
[0097] The term "about" or "approximately" means within 10%, such
as within 5% (or 1% or less) of a given value or range.
[0098] The terms "administer" or "administration" refers to the act
of injecting or otherwise physically delivering a substance as it
exists outside the body (e.g., a formulation of the invention) into
a patient, such as by mucosal, intradermal, intravenous,
subcutaneous, intramuscular delivery and/or any other method of
physical delivery described herein or known in the art. When a
disease, or a symptom thereof, is being treated, administration of
the substance typically occurs after the onset of the disease or
symptoms thereof. When a disease or its symptoms are being
prevented, administration of the substance typically occurs before
the onset of the disease or symptoms thereof.
[0099] In the context of a polypeptide, the term "analog" refers to
a polypeptide that possesses a similar or identical function as a
LIGHT or CXCR5 polypeptide, a fragment of a LIGHT or CXCR5
polypeptide, a LIGHT or CXCR5 epitope, or an anti-LIGHT or
anti-CXCR5 antibody, but does not necessarily comprise a similar or
identical amino acid sequence of a LIGHT or CXCR5 polypeptide, a
fragment of a LIGHT or CXCR5 polypeptide, a LIGHT or CXCR5 epitope,
or an anti-LIGHT or anti-CXCR5 antibody, or possess a similar or
identical structure of a LIGHT or CXCR5 polypeptide, a fragment of
a LIGHT or CXCR5 polypeptide, a LIGHT or CXCR5 epitope, or an
anti-LIGHT or anti-CXCR5 antibody. A polypeptide that has a similar
amino acid sequence refers to a polypeptide that satisfies at least
one of the following: (a) a polypeptide having an amino acid
sequence that is at least 30%, at least 35%, at least 40%, at least
45%, at least 50%, at least 55%, at least 60%, at least 65%, at
least 70%, at least 75%, at least 80%, at least 85%, at least 90%,
at least 95%, or at least 99% identical to the amino acid sequence
of a LIGHT or CXCR5 polypeptide (e.g., SEQ ID NO: 9 or SEQ ID NO:
14, respectively), a fragment of a LIGHT or CXCR5 polypeptide, a
LIGHT or CXCR5 epitope, or an anti-LIGHT or anti-CXCR5 antibody
described herein; (b) a polypeptide encoded by a nucleotide
sequence that hybridizes under stringent conditions to a nucleotide
sequence encoding a LIGHT or CXCR5 polypeptide, a fragment of a
LIGHT or CXCR5 polypeptide, a LIGHT or CXCR5 epitope, or an
anti-LIGHT or anti-CXCR5 antibody (or VH or VL region thereof)
described herein of at least 5 amino acid residues, at least 10
amino acid residues, at least 15 amino acid residues, at least 20
amino acid residues, at least 25 amino acid residues, at least 40
amino acid residues, at least 50 amino acid residues, at least 60
amino residues, at least 70 amino acid residues, at least 80 amino
acid residues, at least 90 amino acid residues, at least 100 amino
acid residues, at least 125 amino acid residues, or at least 150
amino acid residues (see, e.g., Sambrook et al. (2001) Molecular
Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y.; Maniatis et al. (1982) Molecular Cloning:
A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor,
N.Y.); and (c) a polypeptide encoded by a nucleotide sequence that
is at least 30%, at least 35%, at least 40%, at least 45%, at least
50%, at least 55%, at least 60%, at least 65%, at least 70%, at
least 75%, at least 80%, at least 85%, at least 90%, at least 95%,
or at least 99% identical to the nucleotide sequence encoding a
LIGHT or CXCR5 polypeptide, a fragment of a LIGHT or CXCR5
polypeptide, a LIGHT or CXCR5 epitope, or an anti-LIGHT or
anti-CXCR5 antibody (or VH or VL region thereof) described herein.
A polypeptide with similar structure to a LIGHT or CXCR5
polypeptide, a fragment of a LIGHT or CXCR5 polypeptide, a LIGHT or
CXCR5 epitope, or an anti-LIGHT or anti-CXCR5 antibody refers to a
polypeptide that has a similar secondary, tertiary or quaternary
structure of a LIGHT or CXCR5 polypeptide, a fragment of a LIGHT or
CXCR5 polypeptide, a LIGHT or CXCR5 epitope, or a LIGHT or CXCR5
antibody. The structure of a polypeptide can determined by methods
known to those skilled in the art, including but not limited to,
X-ray crystallography, nuclear magnetic resonance, and
crystallographic electron microscopy.
[0100] To determine the percent identity of two amino acid
sequences or of two nucleic acid sequences, the sequences are
aligned for optimal comparison purposes (e.g., gaps can be
introduced in the sequence of a first amino acid or nucleic acid
sequence for optimal alignment with a second amino acid or nucleic
acid sequence). The amino acid residues or nucleotides at
corresponding amino acid positions or nucleotide positions are then
compared. When a position in the first sequence is occupied by the
same amino acid residue or nucleotide as the corresponding position
in the second sequence, then the molecules are identical at that
position. The percent identity between the two sequences is a
function of the number of identical positions shared by the
sequences (i.e., % identity=number of identical overlapping
positions/total number of positions X 100%). In one embodiment, the
two sequences are the same length.
[0101] The determination of percent identity between two sequences
(e.g., amino acid sequences or nucleic acid sequences) can also be
accomplished using a mathematical algorithm. A non-limiting example
of a mathematical algorithm utilized for the comparison of two
sequences is the algorithm of Karlin and Altschul, 1990, Proc.
Natl. Acad. Sci. U.S.A. 87:2264 2268, modified as in Karlin and
Altschul, 1993, Proc. Natl. Acad. Sci. U.S.A. 90:5873 5877. Such an
algorithm is incorporated into the NBLAST and XBLAST programs of
Altschul et al., 1990, J. Mol. Biol. 215:403. BLAST nucleotide
searches can be performed with the NBLAST nucleotide program
parameters set, e.g., for score=100, wordlength=12 to obtain
nucleotide sequences homologous to nucleic acid molecules of
interest. BLAST protein searches can be performed with the XBLAST
program parameters set, e.g., to score 50, wordlength=3 to obtain
amino acid sequences homologous to a protein molecule of interest.
To obtain gapped alignments for comparison purposes, Gapped BLAST
can be utilized as described in Altschul et al., 1997, Nucleic
Acids Res. 25:3389 3402. Alternatively, PSI BLAST can be used to
perform an iterated search which detects distant relationships
between molecules (Id.). When utilizing BLAST, Gapped BLAST, and
PSI Blast programs, the default parameters of the respective
programs (e.g., of XBLAST and NBLAST) can be used (see, e.g.,
National Center for Biotechnology Information (NCBI) on the
worldwide web at ncbi dot nlm dot nih dot gov). Another non
limiting example of a mathematical algorithm utilized for the
comparison of sequences is the algorithm of Myers and Miller, 1988,
CABIOS 4:11 17. Such an algorithm is incorporated in the ALIGN
program (version 2.0), which is part of the GCG sequence alignment
software package. When utilizing the ALIGN program for comparing
amino acid sequences, a PAM120 weight residue table, a gap length
penalty of 12, and a gap penalty of 4 can be used.
[0102] The percent identity between two sequences can be determined
using techniques similar to those described above, with or without
allowing gaps. In calculating percent identity, typically only
exact matches are counted.
[0103] An "antagonist" or "inhibitor" refers to a molecule capable
of inhibiting one or more biological activities of a target
molecule. Antagonists may interfere with the binding of a receptor
to a ligand and vice versa, by incapacitating or killing cells
activated by a ligand, and/or by interfering with receptor or
ligand activation (e.g., tyrosine kinase activation) or signal
transduction after ligand binding to a receptor. The antagonist may
completely block receptor-ligand interactions or may substantially
reduce such interactions. All such points of intervention by an
antagonist shall be considered equivalent for purposes of the
instant invention.
[0104] For example, an "antagonist" or "inhibitor" of LIGHT refers
to a molecule that is capable of inhibiting or otherwise decreasing
one or more of the biological activities of LIGHT, such as in a
cell expressing LIGHT or in a cell expressing a LIGHT ligand, such
as a LIGHT receptor. For example, in certain embodiments,
antibodies of the invention are antagonist antibodies that inhibit
or otherwise decrease secretion of CCL20, IL-8, and/or RANTES from
a cell having a cell surface-expressed LIGHT receptor (e.g., HVEM,
LT.beta.R and/or DcR3) when said antibody is contacted with said
cell. In some embodiments, an antagonist of LIGHT (e.g., an
antagonistic antibody of the invention) may, for example, act by
inhibiting or otherwise decreasing the activation and/or cell
signaling pathways of the cell expressing a LIGHT receptor, thereby
inhibiting a LIGHT-mediated biological activity of the cell
relative to the LIGHT-mediated biological activity in the absence
of antagonist. In certain embodiments of the invention, the
anti-LIGHT antibodies are fully human, antagonistic anti-LIGHT
antibodies, such as fully human, monoclonal, antagonistic
anti-LIGHT antibodies.
[0105] For example, an "antagonist" or "inhibitor" of CXCR5 refers
to a molecule capable of inhibiting one or more biological
activities, such as signaling, by CXCR5. Thus, included within the
scope of the invention are antagonists (e.g., neutralizing
antibodies) that bind to CXCR5, CXCL13 or other ligands of CXCR5,
or a complex of CXCR5 and a ligand thereof, such as CXCL13; amino
acid sequence variants or derivatives of CXCR5 or CXCL13 which
antagonize the interaction between CXCR5 and a ligand, such as
CXCL13; soluble CXCR5, optionally fused to a heterologous molecule
such as an immunoglobulin region (e.g., an immunoadhesin); a
complex comprising CXCR5 in association with another receptor or
biological molecule; synthetic or native sequence peptides which
bind to CXCR5; and so on.
[0106] The terms "antibody", "immunoglobulin", or "Ig" may be used
interchangeably herein. The term antibody includes, but is not
limited to, synthetic antibodies, monoclonal antibodies,
recombinantly produced antibodies, multispecific antibodies
(including bi-specific antibodies), human antibodies, humanized
antibodies, chimeric antibodies, intrabodies, single-chain Fvs
(scFv) (e.g., including monospecific, bispecific, etc.), camelized
antibodies, Fab fragments, F(ab') fragments, disulfide-linked Fvs
(sdFv), anti-idiotypic (anti-Id) antibodies, and epitope-binding
fragments of any of the above. In particular, antibodies include
immunoglobulin molecules and immunologically active portions of
immunoglobulin molecules, i.e., antigen binding domains or
molecules that contain an antigen-binding site that specifically
binds to a LIGHT antigen (e.g., one or more complementarity
determining regions (CDRs) of an anti-LIGHT antibody) or CXCR5
antigen (e.g., one or more complementarity determining regions
(CDRs) of an anti-CXCR5 antibody). The anti-LIGHT or anti-CXCR5
antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and
IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2), or
any subclass (e.g., IgG2a and IgG2b) of immunoglobulin molecule. In
some embodiments, the anti-LIGHT antibodies are fully human, such
as fully human monoclonal anti-LIGHT antibodies. In certain
embodiments, the anti-LIGHT antibodies are IgG antibodies, human
IgG4 antibodies. Alternatively, in some embodiments, the anti-CXCR5
antibodies are humanized, such as humanized monoclonal anti-CXCR5
antibodies. In certain embodiments, the anti-CXCR5 antibodies are
IgG antibodies, humanized IgG4 antibodies.
[0107] As used herein, the term "anti-LIGHT antibody" means an
antibody or polypeptide derived therefrom (a derivative) that binds
specifically to human LIGHT as defined herein, including, but not
limited to, molecules that inhibit or substantially reduce the
binding of LIGHT to its ligands or inhibit LIGHT activity.
[0108] As used herein, the term "anti-CXCR5 antibody" means an
antibody or polypeptide derived therefrom (a derivative) that binds
specifically to human CXCR5 as defined herein, including, but not
limited to, molecules that inhibit or substantially reduce the
binding of CXCR5 to its ligands or inhibit CXCR5 activity.
[0109] The term "B cell activity" means higher than normal B cell
levels, which can be local, or evidence of a biological
manifestation or function of a B cell, such as antibody expression,
Bruton's tyrosine kinase presence or activity, expression or
presence of CD19, expression or presence of B cell activating
factor and so on.
[0110] The term "binding agent" means any molecule, such as an
antibody, a siRNA, a nucleic acid, an aptamer, a protein, or a
small molecule organic compound, that binds or specifically binds
to LIGHT or CXCR5, or a variant or a fragment thereof.
[0111] The term "by-product" includes undesired products, which
detract or diminish the proportion of therapeutic/prophylactic
binding agent, such as an antibody, in a given formulation. For
example, typical by-products include aggregates of the antibody,
fragments of the antibody, e.g. produced by degradation of the
antibody by deamidation or hydrolysis, or mixtures thereof.
Typically, aggregates are complexes that have a molecular weight
greater than the monomer antibody. Antibody degradation products
may include, for example, fragments of the antibody, for example,
brought about by deamidation or hydrolysis. Typically, degradation
products are complexes that have a molecular weight less than the
monomer antibody. In the case of an IgG antibody, such degradation
products are less than about 150 kD.
[0112] The terms "composition" and "formulation" are intended to
encompass a product containing the specified ingredients (e.g., an
anti-LIGHT antibody or an anti-CXCR5 antibody) in, optionally, the
specified amounts, as well as any product that results, directly or
indirectly, from the combination of the specified ingredients in,
optionally, the specified amounts.
[0113] The terms "constant region" or "constant domain" refer to a
carboxy terminal portion of the light and heavy chain which is not
directly involved in binding of the antibody to antigen but
exhibits various effector functions, such as interaction with the
Fc receptor. The terms refer to the portion of an immunoglobulin
molecule having a more conserved amino acid sequence relative to
the other portion of the immunoglobulin, the variable domain, which
contains the antigen binding site. The constant domain contains the
CH1, CH2 and CH3 domains of the heavy chain and the CHL domain of
the light chain.
[0114] The term "CXCR5" relates to the naturally occurring, known
molecule found on lymphocytes, particularly B cells, and
particularly naive B cells; to such a molecule isolated from such
cells; to such a molecule manufactured recombinantly using known
materials and means, and using a nucleic acid encoding a CXCR5; as
well as to portions of CXCR5, such as the extracellular (EC)
domain, that retain the characteristics and properties relevant to
the practice of the instant invention, such as CXCL13 binding. A
soluble CXCR5 molecule can consist essentially of the EC domain of
CXCR5, which includes, generally, about the first sixty amino acids
of the molecule, that is, the amino terminal portion of CXCR5.
[0115] CXCR5 is a non-promiscuous receptor. CXCL13 is a ligand of
CXCR5 and is expressed constitutively on stromal cells, such as
follicular dendritic cells, and in lymphoid tissues. CXCL13
specifically attracts B cells and a small subset of T cells called
B helper follicular T cells, TFH. This may not be unexpected given
the many interactions between T cell and B cell populations in the
immune system. Moreover, activated T cells induce or upregulate
CXCR5 expression. Infiltration of lymphocytes into tertiary,
ectopic germinal centers (GCs) has been found to correlate well
with increased disease severity and tolerance breakdown in certain
disorders that present with such atypical lymph node-like
structures. Using in vivo murine models, such as CXCR5-/- and
CXCL13-/- mice, the absence of either the receptor or the ligand
results in an altered GC fine architecture due to altered T and B
cell localization, and possibly interaction. These mice are also
protected against developing severe collagen-induced arthritis
(CIA). As CXCR5 is selectively expressed on mature B cells, which
are linked to the pathogenesis of RA, blocking this receptor will
modulate the arthritogenic response in affected individuals.
Rheumatoid arthritis treatment with biologics (i.e.,
anti-TNF.alpha. and anti-CD20 antibodies, Rituximab) has shown to
be clinically effective; in particular, patients on B cell-directed
therapy have shown long-lasting improvements in clinical signs and
symptoms. Selective targeting of CXCR5, which is only expressed on
mature B cells and B helper T cells, will not affect B cell
development or immunocompromise the patient. Unlike Rituximab, the
instant anti-CXCR5 antibody is a neutralizing antibody that does
not mediate cell cytotoxicity.
[0116] A "CXCR5 disease" is a malady, disorder, disease, condition,
abnormality and so on, that is characterized by or caused by
overexpression or increased levels of CXCL13 or other CXCR5 ligand,
increased levels of B cells, increased levels of B cell activity,
increased levels of CXCR5, or improper metabolism and activity of
CXCR5.
[0117] The term "epitope" refers to a localized region on the
surface of an antigen, such as a LIGHT or CXCR5 polypeptide, or
LIGHT or CXCR5 polypeptide fragment, that is capable of being bound
to one or more antigen binding regions of a binding agent, such as
an antibody, and that has antigenic or immunogenic activity in an
animal, such a mammal, such as in a human, that is capable of
eliciting an immune response. An epitope having immunogenic
activity is a portion of a polypeptide that elicits an antibody
response in an animal. An epitope having antigenic activity is a
portion of a polypeptide to which an antibody specifically binds,
as determined by any method well known in the art, for example,
such as an immunoassay. Antigenic epitopes need not necessarily be
immunogenic. Epitopes usually consist of chemically active surface
groupings of molecules, such as amino acids or sugar side chains,
and have specific three dimensional structural characteristics, as
well as specific charge characteristics. A region of a polypeptide
contributing to an epitope may be contiguous amino acids of the
polypeptide or the epitope may come together from two or more
non-contiguous regions of the polypeptide. The epitope may or may
not be a three-dimensional surface feature of the antigen. In
certain embodiments, a LIGHT or CXCR5 epitope is a
three-dimensional surface feature of a LIGHT or CXCR5 polypeptide
(e.g., in a trimeric form of a LIGHT polypeptide). In other
embodiments, a LIGHT epitope is a linear feature of a LIGHT or
CXCR5 polypeptide (e.g., in a trimeric form or monomeric form of
the LIGHT polypeptide). Anti-LIGHT or anti-CXCR5 antibodies may
specifically bind to an epitope of the monomeric (denatured) form
of LIGHT or CXCR5, an epitope of the trimeric (native) form of
LIGHT or CXCR5, or both the monomeric (denatured) form and the
trimeric (native) form of LIGHT or CXCR5. In specific embodiments,
the anti-LIGHT antibodies specifically bind to an epitope of the
trimeric form of LIGHT but do not specifically bind the monomeric
form of LIGHT.
[0118] The term "excipients" refers to inert substances that are
commonly used as a diluent, vehicle, preservative, binder,
stabilizing agent, etc. for drugs and includes, but is not limited
to, proteins (e.g., serum albumin, etc.), amino acids (e.g.,
aspartic acid, glutamic acid, lysine, arginine, glycine, histidine,
etc.), fatty acids and phospholipids (e.g., alkyl sulfonates,
caprylate, etc.), surfactants (e.g., SDS, polysorbate, nonionic
surfactant, etc.), saccharides (e.g., sucrose, maltose, trehalose,
etc.) and polyols (e.g., mannitol, sorbitol, etc.). See, also,
Remington's Pharmaceutical Sciences (1990) Mack Publishing Co.,
Easton, Pa., which is hereby incorporated by reference in its
entirety.
[0119] In the context of a peptide or polypeptide, the term
"fragment" refers to a peptide or polypeptide that comprises less
than the full length amino acid sequence. Such a fragment may
arise, for example, from a truncation at the amino terminus, a
truncation at the carboxy terminus, and/or an internal deletion of
a residue(s) from the amino acid sequence. Fragments may, for
example, result from alternative RNA splicing or from in vivo
protease activity. In certain embodiments, hLIGHT or hCXCR5
fragments include polypeptides comprising an amino acid sequence of
at least 5 contiguous amino acid residues, at least 10 contiguous
amino acid residues, at least 15 contiguous amino acid residues, at
least 20 contiguous amino acid residues, at least 25 contiguous
amino acid residues, at least 40 contiguous amino acid residues, at
least 50 contiguous amino acid residues, at least 60 contiguous
amino residues, at least 70 contiguous amino acid residues, at
least 80 contiguous amino acid residues, at least 90 contiguous
amino acid residues, at least contiguous 100 amino acid residues,
at least 125 contiguous amino acid residues, at least 150
contiguous amino acid residues, at least 175 contiguous amino acid
residues, at least 200 contiguous amino acid residues, or at least
250 contiguous amino acid residues of the amino acid sequence of a
LIGHT or CXCR5 polypeptide or an antibody that specifically binds
to a LIGHT or CXCR5 polypeptide. In a specific embodiment, a
fragment of a LIGHT or CXCR5 polypeptide or an antibody that
specifically binds to a LIGHT or CXCR5 antigen retains at least 1,
at least 2, or at least 3 functions of the polypeptide or
antibody.
[0120] The terms "fully human antibody" or "human antibody" are
used interchangeably herein and refer to an antibody that comprises
a human variable region and, possibly a human constant region. In
specific embodiments, the terms refer to an antibody that comprises
a variable region and constant region of human origin. "Fully
human" anti-LIGHT antibodies, in certain embodiments, can also
encompass antibodies that bind LIGHT polypeptides and are encoded
by nucleic acid sequences that are naturally occurring somatic
variants of a human germline immunoglobulin nucleic acid sequence.
In a specific embodiment, the anti-LIGHT antibodies are fully human
antibodies. The term "fully human antibody" includes antibodies
having variable and constant regions corresponding to human
germline immunoglobulin sequences as described by Kabat et al. (See
Kabat et al. (1991) Sequences of Proteins of Immunological
Interest, Fifth Edition, U.S. Department of Health and Human
Services, NIH Publication No. 91-3242). Methods of producing fully
human antibodies are known in the art.
[0121] The phrase "recombinant human antibody" includes human
antibodies that are prepared, expressed, created, or isolated by
recombinant means, such as antibodies expressed using a recombinant
expression vector transfected into a host cell, antibodies isolated
from a recombinant, combinatorial human antibody library,
antibodies isolated from an animal (e.g., a mouse or cow) that is
transgenic and/or transchromosomal for human immunoglobulin genes
(see, e.g., Taylor, L. D. et al. (1992) Nucl. Acids Res.
20:6287-6295) or antibodies prepared, expressed, created, or
isolated by any other means that involves splicing of human
immunoglobulin gene sequences to other DNA sequences. Such
recombinant human antibodies can have variable and constant regions
derived from human germline immunoglobulin sequences (See Kabat, E.
A. et al. (1991) Sequences of Proteins of Immunological Interest,
Fifth Edition, U.S. Department of Health and Human Services, NIH
Publication No. 91-3242). In certain embodiments, however, such
recombinant human antibodies are subjected to in vitro mutagenesis
(or, when an animal transgenic for human Ig sequences is used, in
vivo somatic mutagenesis) and thus the amino acid sequences of the
VH and VL regions of the recombinant antibodies are sequences that,
while derived from and related to human germline VH and VL
sequences, may not naturally exist within the human antibody
germline repertoire in vivo.
[0122] An "IgG4 binding agent" or a "binding agent comprising at
least a portion of an IgG4 Fc region" both refer to binding agents
described herein that include at least a fragment of IgG4 Fc. In
certain embodiments, the fragment comprises 10, 20, 30, 40, 50,
100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210 or 220
amino acids of the IgG4 Fc region. In other embodiments, the
fragment includes 10-50, 50-100, 100-150, or 150-200 amino acids of
the IgG4 Fc region. In other embodiments, the portion of the IgG4
Fc region can have a certain homology to the IgG4 Fc region. For
example, the IgG4 binding agent may include a portion of a protein
with greater than 50, 60, 70, 80, 90, 93, 95, 96, 97, 98, 99, or
100% homology to the IgG4 Fc region. Exemplary Fc regions of IgG4
are described throughout the specification.
[0123] The term "heavy chain", when used in reference to an
antibody, refers to five distinct types, called alpha (.alpha.),
delta (.DELTA.), epsilon (.epsilon.), gamma (.gamma.), and mu
(.mu.), based on the amino acid sequence of the heavy chain
constant domain. These distinct types of heavy chains are well
known in the art and give rise to five classes of antibodies, IgA,
IgD, IgE, IgG, and IgM, respectively, including four subclasses of
IgG, namely IgG1, IgG1, IgG3, and IgG4. In some embodiments, the
heavy chain is a human heavy chain.
[0124] "Humanized" forms of non-human (e.g., murine) antibodies are
chimeric immunoglobulins, immunoglobulin chains or fragments
thereof (such as F.sub.v, F.sub.ab, F.sub.ab', F.sub.(ab')2 or
other target-binding subsequences of antibodies) that contain
sequences derived from non-human immunoglobulin, as compared to a
human antibody. In general, the humanized antibody will comprise
substantially all of one, and typically two, variable domains, in
which all or substantially all of the CDR regions correspond to
those of a non-human immunoglobulin and all or substantially all of
the FR regions are those of a human immunoglobulin template
sequence. The humanized antibody may also comprise at least a
portion of an immunoglobulin constant region (F.sub.c), typically
that of the human immunoglobulin template chosen. In general, the
goal is to have an antibody molecule that is minimally immunogenic
in a human. Thus, it is possible that one or more amino acids in
one or more CDRs also can be changed to one that is less
immunogenic to a human host, without substantially minimizing the
specific binding function of the one or more CDRs to CXCR5 or to
CXCL13. Alternatively, the FR can be non-human but those amino
acids most immunogenic are replaced with ones less immunogenic.
Nevertheless, CDR grafting, as discussed above, is not the only way
to obtain a humanized antibody. For example, modifying just the CDR
regions may be insufficient as it is not uncommon for framework
residues to have a role in determining the three-dimensional
structure of the CDR loops and the overall affinity of the antibody
for its ligand. Hence, any means can be practiced so that the
non-human parent antibody molecule is modified to be one that is
less immunogenic to a human, and global sequence identity with a
human antibody is not always a necessity. So, humanization also can
be achieved, for example, by the mere substitution of just a few
residues, particularly those which are exposed on the antibody
molecule and not buried within the molecule, and hence, not readily
accessible to the host immune system. Such a method is taught
herein with respect to substituting "mobile" or "flexible" residues
on the antibody molecule, the goal being to reduce or dampen the
immunogenicity of the resultant molecule without comprising the
specificity of the antibody for its epitope or determinant. See,
for example, Studnicka et al., Prot Eng 7(6)805-814, 1994; Mol 1 mm
44:1986-1988, 2007; Sims et al., J Immunol 151:2296 (1993); Chothia
et al., J Mol Biol 196:901 (1987); Carter et al., Proc Natl Acad
Sci USA 89:4285 (1992); Presta et al., J Immunol 151:2623 (1993),
WO 2006/042333 and U.S. Pat. No. 5,869,619.
[0125] An "isolated" or "purified" binding agent, such as an
antibody, is substantially free of cellular material or other
contaminating proteins from the cell or tissue source from which
the binding agent is derived, or substantially free of chemical
precursors or other chemicals when chemically synthesized. For
example, the language "substantially free of cellular material"
includes preparations of an antibody in which the antibody is
separated from cellular components of the cells from which it is
isolated or recombinantly produced. Thus, an antibody that is
substantially free of cellular material includes preparations of
antibody having less than about 30%, 20%, 10%, or 5% (by dry
weight) of heterologous protein (also referred to herein as a
"contaminating protein"). When the antibody is recombinantly
produced, it is also desirable to be substantially free of culture
medium, i.e., culture medium represents less than about 20%, 10%,
or 5% of the volume of the protein preparation. When the antibody
is produced by chemical synthesis, in some embodiments it is
substantially free of chemical precursors or other chemicals, i.e.,
it is separated from chemical precursors or other chemicals that
are involved in the synthesis of the protein. Accordingly, such
preparations of the antibody have less than about 30%, 20%, 10%, 5%
(by dry weight) of chemical precursors or compounds other than the
antibody of interest. In some embodiments, anti-LIGHT or anti-CXCR5
antibodies are isolated or purified.
[0126] The term "human LIGHT," "hLIGHT" or "hLIGHT polypeptide" and
similar terms refer to the polypeptides ("polypeptides," "peptides"
and "proteins" are used interchangeably herein) comprising the
amino acid sequence of SEQ ID NO: 9 and related polypeptides,
including SNP variants thereof. Related polypeptides include
allelic variants (e.g., SNP variants); splice variants; fragments;
derivatives; substitution, deletion, and insertion variants; fusion
polypeptides; and interspecies homologs, in some embodiments, which
retain LIGHT activity and/or are sufficient to generate an
anti-LIGHT immune response. Also encompassed are soluble forms of
LIGHT that are sufficient to generate an anti-LIGHT immunological
response. As those skilled in the art will appreciate, an
anti-LIGHT binding agent, such as an antibody, can bind to a LIGHT
polypeptide, polypeptide fragment, antigen, and/or epitope, as an
epitope is part of the larger antigen, which is part of the larger
polypeptide fragment, which, in turn, is part of the larger
polypeptide. hLIGHT can exist in a trimeric (native) or monomeric
(denatured) form.
[0127] The term "human CXCR5," "hCXCR5" or "hCXCR5 polypeptide" and
similar terms refer to the polypeptides ("polypeptides," "peptides"
and "proteins" are used interchangeably herein) comprising the
amino acid sequence of SEQ ID NO: 14 and related polypeptides,
including SNP variants thereof. Related polypeptides include
allelic variants (e.g., SNP variants); splice variants; fragments;
derivatives; substitution, deletion, and insertion variants; fusion
polypeptides; and interspecies homologs, in some embodiments, which
retain CXCR5 activity and/or are sufficient to generate an
anti-CXCR5 immune response. Also encompassed are soluble forms of
CXCR5 that are sufficient to generate an anti-CXCR5 immunological
response. As those skilled in the art will appreciate, an
anti-CXCR5 binding agent, such as an antibody, can bind to a CXCR5
polypeptide, polypeptide fragment, antigen, and/or epitope, as an
epitope is part of the larger antigen, which is part of the larger
polypeptide fragment, which, in turn, is part of the larger
polypeptide.
[0128] The term "Kabat numbering," and like terms are recognized in
the art and refer to a system of numbering amino acid residues that
are more variable (i.e. hypervariable) than other amino acid
residues in the heavy and light chain variable regions of an
antibody, or an antigen binding portion thereof (Kabat et al.
(1971) Ann. NY Acad. Sci. 190:382-391 and, Kabat et al. (1991)
Sequences of Proteins of Immunological Interest, Fifth Edition,
U.S. Department of Health and Human Services, NIH Publication No.
91-3242). For the heavy chain variable region, the hypervariable
region typically ranges from amino acid positions 31 to 35 for
CDR1, amino acid positions 50 to 65 for CDR2, and amino acid
positions 95 to 102 for CDR3. For the light chain variable region,
the hypervariable region typically ranges from amino acid positions
24 to 34 for CDR1, amino acid positions 50 to 56 for CDR2, and
amino acid positions 89 to 97 for CDR3.
[0129] The term "light chain" when used in reference to an antibody
refers to two distinct types, called kappa (.kappa.) of lambda
(.lamda.) based on the amino acid sequence of the constant domains.
Light chain amino acid sequences are well known in the art. In some
embodiments, the light chain is a human light chain.
[0130] The terms "manage", "managing", and "management" refer to
the beneficial effects that a subject derives from a therapy (e.g.,
a prophylactic or therapeutic agent), which does not result in a
cure of the infection. In certain embodiments, a subject is
administered one or more therapies (e.g., prophylactic or
therapeutic agents, such as a formulation of the invention) to
"manage" a LIGHT-mediated disease (e.g., chronic bowel disease,
IBD, Crohn's disease, ulcerative colitis, or GVHD) or
CXCR5-mediated disease (e.g., rheumatoid arthritis), one or more
symptoms thereof, so as to prevent the progression or worsening of
the disease.
[0131] The term "monoclonal antibody" refers to an antibody
obtained from a population of homogenous or substantially
homogeneous antibodies, and each monoclonal antibody will typically
recognize a single epitope on the antigen. In some embodiments, a
"monoclonal antibody" is an antibody produced by a single hybridoma
or other cell. The term "monoclonal" is not limited to any
particular method for making the antibody. For example, monoclonal
antibodies may be made by the hybridoma method as described in
Kohler et al.; Nature, 256:495 (1975) or may be isolated from phage
libraries. Other methods for the preparation of clonal cell lines
and of monoclonal antibodies expressed thereby are well known in
the art (see, for example, Chapter 11 in: Short Protocols in
Molecular Biology, (2002) 5th Ed.; Ausubel et al., eds., John Wiley
and Sons, New York).
[0132] The term "pharmaceutically acceptable" means being approved
by a regulatory agency of the Federal or a state government, or
listed in the U.S. Pharmacopeia, European Pharmacopeia or other
generally recognized Pharmacopeia for use in animals, and more
particularly in humans.
[0133] By "pharmaceutically acceptable excipient" is meant any
inert substance that is combined with an active molecule, such as a
monoclonal antibody, for preparing an agreeable or convenient
dosage form. The "pharmaceutically acceptable excipient" is an
excipient that is non-toxic to recipients at the dosages and
concentrations employed, and is compatible with other ingredients
of the formulation comprising the monoclonal antibody.
[0134] The terms "prevent", "preventing", and "prevention" refer to
the total or partial inhibition of the development, recurrence,
onset or spread of a LIGHT-mediated or CXCR5-mediated disease
and/or symptom related thereto, resulting from the administration
of a therapy or combination of therapies provided herein (e.g., a
combination of prophylactic or therapeutic agents, such as a
formulation of the invention).
[0135] The term "prophylactic agent" refers to any agent that can
totally or partially inhibit the development, recurrence, onset or
spread of a LIGHT-mediated or CXCR5-mediated disease and/or symptom
related thereto in a subject. In certain embodiments, the term
"prophylactic agent" refers to a formulation of the invention. In
certain other embodiments, the term "prophylactic agent" refers to
an agent other than a formulation of the invention. In some
embodiments, a prophylactic agent is an agent that is known to be
useful to or has been or is currently being used to prevent a
LIGHT-mediated or CXCR5-mediated disease and/or a symptom related
thereto, or impede the onset, development, progression and/or
severity of a LIGHT-mediated or CXCR5-mediated disease and/or a
symptom related thereto. In specific embodiments, the prophylactic
agent is a fully human anti-LIGHT antibody, such as a fully human
anti-LIGHT monoclonal antibody, or a humanized anti-CXCR5 antibody,
such as a humanized anti-CXCR5 monoclonal antibody.
[0136] The term "LIGHT antigen" refers to that portion of a LIGHT
polypeptide to which a binding agent, such as an antibody,
specifically binds. A LIGHT antigen also refers to an analog or
derivative of a LIGHT polypeptide or fragment thereof to which a
binding agent, such as an antibody, specifically binds. In some
embodiments, a LIGHT antigen is a monomeric LIGHT antigen or a
trimeric LIGHT antigen. A region of a LIGHT polypeptide
contributing to an epitope may be contiguous amino acids of the
polypeptide, or the epitope may come together from two or more
non-contiguous regions of the polypeptide. The epitope may or may
not be a three-dimensional surface feature of the antigen. A
localized region on the surface of a LIGHT antigen that is capable
of eliciting an immune response is a LIGHT epitope. The epitope may
or may not be a three-dimensional surface feature of the
antigen.
[0137] The term "CXCR5 antigen" refers to that portion of a CXCR5
polypeptide to which a binding agent, such as an antibody,
specifically binds. A CXCR5 antigen also refers to an analog or
derivative of a CXCR5 polypeptide or fragment thereof to which a
binding agent, such as an antibody, specifically binds. A region of
a CXCR5 polypeptide contributing to an epitope may be contiguous
amino acids of the polypeptide, or the epitope may come together
from two or more non-contiguous regions of the polypeptide. The
epitope may or may not be a three-dimensional surface feature of
the antigen. A localized region on the surface of a CXCR5 antigen
that is capable of eliciting an immune response is a CXCR5 epitope.
The epitope may or may not be a three-dimensional surface feature
of the antigen.
[0138] The terms "LIGHT-mediated disease" and "LIGHT-mediated
disorder" are used interchangeably and refer to any disease that is
completely or partially caused by or is the result of LIGHT. In
certain embodiments, LIGHT is aberrantly (e.g., highly) expressed
on the surface of a cell. In some embodiments, LIGHT may be
aberrantly upregulated on a particular cell type. In other
embodiments, normal, aberrant, or excessive cell signaling is
caused by binding of LIGHT to a LIGHT ligand. In certain
embodiments, the LIGHT ligand is a LIGHT receptor (e.g., HVEM,
LT.beta.R, or DCR3), for example, that is expressed on the surface
of a cell, such as a colonic epithelial cell. In certain
embodiments, the LIGHT-mediated disease is a chronic bowel disease,
an inflammatory bowel disease (IBD), such as Crohn's disease (CD)
or ulcerative colitis (UC). In other embodiments, the
LIGHT-mediated disease is graft-versus-host disease (GVHD).
[0139] The terms "CXCR5-mediated disease" and "CXCR5-mediated
disorder" are used interchangeably and refer to any disease that is
completely or partially caused by or is the result of CXCR5. In
certain embodiments, CXCR5 is aberrantly (e.g., highly) expressed
on the surface of a cell. In some embodiments, CXCR5 may be
aberrantly upregulated on a particular cell type. In other
embodiments, normal, aberrant, or excessive cell signaling is
caused by binding of CXCR5 to a CXCR5 ligand. In certain
embodiments, the CXCR5 ligand is CXCL13. In certain embodiments,
the CXCR5-mediated disease is rheumatoid arthritis (RA).
[0140] The term "saccharide" refers to a class of molecules that
are derivatives of polyhydric alcohols. Saccharides are commonly
referred to as carbohydrates and may contain different amounts of
sugar (saccharide) units, e.g., monosaccharides, disaccharides, and
polysaccharides.
[0141] The terms "specifically binds" or "specifically binding"
mean specifically binding to an antigen or a fragment thereof and
not specifically binding to other antigens. For example, an
antibody that specifically binds to an antigen may bind to other
peptides or polypeptides with lower affinity, as determined by,
e.g., radioimmunoassays (RIA), enzyme-linked immunosorbent assays
(ELISA), BIACORE, or other assays known in the art. Antibodies or
variants or fragments thereof that specifically bind to an antigen
may be cross-reactive with related antigens. In some embodiments,
antibodies or variants or fragments thereof that specifically bind
to an antigen do not cross-react with other antigens. An antibody
or a variant or a fragment thereof that specifically binds to a
LIGHT or CXCR5 antigen can be identified, for example, by
immunoassays, BIAcore, or other techniques known to those of skill
in the art. Typically a specific or selective reaction will be at
least twice background signal or noise, and more typically more
than 10 times background. See, e.g., Paul, ed., 1989, Fundamental
Immunology Second Edition, Raven Press, New York at pages 332-336
for a discussion regarding antibody specificity.
[0142] A "stable" or "stabilized" formulation is one in which the
binding agent, such as an antibody, therein essentially retains its
physical stability, identity, integrity, and/or chemical stability,
identity, integrity, and/or biological activity upon storage.
Various analytical techniques for measuring protein stability are
available in the art and are reviewed in Peptide and Protein Drug
Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York,
N.Y., Pubs. (1991) and Jones, A. Adv. Drug Delivery Rev. 10:29-90
(1993), for example. Stability can be measured at a selected
temperature and other storage conditions for a selected time
period. The stability may be determined by at least one of the
methods selected from the group consisting of visual inspection,
SDS-PAGE, IEF, HPSEC, RFFIT, and kappa/lambda ELISA. For example,
an antibody "retains its physical stability" in a pharmaceutical
formulation, if it shows no signs of aggregation, precipitation,
and/or denaturation upon visual examination of color and/or
clarity, or as measured by UV light scattering, SDS-PAGE, or by
(high pressure) size exclusion chromatography (HPSEC). In some
embodiments, when using the formulations of the invention, 5% or
less, typically 4% or less, typically 3% or less, more typically 2%
or less, and particularly 1% or less of the antibodies forms
aggregates, as measured by HPSEC or any other suitable method for
measuring aggregation formation. For example, an antibody is
considered stable in a particular formulation if the antibody
monomer has a purity of about 90%, typically about 95%, in
particular about 98% after a certain predetermined period of time
under certain storage conditions in a particular formulation.
Chemical stability can be assessed by detecting and quantifying
chemically altered forms of the protein. Chemical alteration may
involve size modification (e.g., clipping), which can be evaluated
using (HP)SEC, SDS-PAGE, and/or matrix-assisted laser desorption
ionization/time-of-flight mass spectrometry (MALDI/TOF MS), for
example. Other types of chemical alteration include charge
alteration (e.g., occurring as a result of deamidation), which can
be evaluated by ion-exchange chromatography, for example. An
antibody "retains its biological activity" in a pharmaceutical
formulation at a given time, if the biological activity of the
antibody at a given time is at least about 90% (within the errors
of the assay) of the biological activity exhibited at the time the
pharmaceutical formulation was prepared, as determined in an
antigen binding assay or virus neutralizing assay, for example.
[0143] The terms "subject" and "patient" are used interchangeably.
As used herein, a subject is typically a mammal, such as a
non-primate (e.g., cows, pigs, horses, cats, dogs, rats, etc.) or a
primate (e.g., monkey and human), and in some embodiments a human.
In one embodiment, the subject is a mammal, such as a human, having
a LIGHT-mediated or CXCR5-mediated disease. In another embodiment,
the subject is a mammal, such as a human, at risk of developing a
LIGHT-mediated or CXCR5-mediated disease.
[0144] The term "therapeutically effective amount" refers to the
amount of a therapy (e.g., a formulation of the invention) that is
sufficient to reduce and/or ameliorate the severity and/or duration
of a given disease and/or a symptom related thereto. This term also
encompasses an amount necessary for the reduction or amelioration
of the advancement or progression of a given disease, reduction or
amelioration of the recurrence, development or onset of a given
disease, and/or to improve or enhance the prophylactic or
therapeutic effect(s) of another therapy (e.g., a therapy other
than a formulation of the invention). In some embodiments, the
therapeutically effective amount of an antibody of the invention is
from about 0.1 mg/kg (mg of antibody per kg weight of the subject)
to about 100 mg/kg. In certain embodiments, a therapeutically
effective amount of an antibody provided therein is about 0.1
mg/kg, about 0.5 mg/kg, about 1 mg/kg, 3 mg/kg, 5 mg/kg, about 10
mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30
mg/kg, about 35 mg/kg, about 40 mg/kg, about 45 mg/kg, about 50
mg/kg, about 60 mg/kg, about 70 mg/kg, about 80 mg/kg about 90
mg/kg or about 100 mg/kg (or a range therein). In some embodiments,
"therapeutically effective amount" as used herein also refers to
the amount of an antibody of the invention to achieve a specified
result (e.g., inhibition of a LIGHT biological activity of a cell,
such as inhibition of secretion of CCL20, IL-8, or RANTES from the
cell; or inhibition of a CXCR5 biological activity of a cell, such
as binding to CXCL13).
[0145] The term "therapeutic agent" refers to any agent that can be
used in the treatment, management or amelioration of a
LIGHT-mediated or CXCR5-mediated disease and/or a symptom related
thereto. In certain embodiments, the term "therapeutic agent"
refers to a formulation of the invention. In certain other
embodiments, the term "therapeutic agent" refers to an agent other
than a formulation of the invention. In some embodiments, a
therapeutic agent is an agent that is known to be useful for, or
has been or is currently being used for the treatment, management
or amelioration of a LIGHT-mediated or CXCR5-mediated disease or
one or more symptoms related thereto.
[0146] The term "therapy" refers to any protocol, method, and/or
agent that can be used in the prevention, management, treatment,
and/or amelioration of a LIGHT-mediated disease (e.g., IBD or GVHD)
or CXCR5-mediated disease (e.g., rheumatoid arthritis). In certain
embodiments, the terms "therapies" and "therapy" refer to a
biological therapy, supportive therapy, and/or other therapies
useful in the prevention, management, treatment, and/or
amelioration of a LIGHT-mediated or CXCR5-mediated disease known to
one of skill in the art, such as medical personnel.
[0147] The terms "treat", "treatment", and "treating" refer to the
reduction or amelioration of the progression, severity, and/or
duration of a LIGHT-mediated disease (e.g., chronic bowel disease,
IBD, or GVHD) or CXCR5-mediated disease (e.g., rheumatoid
arthritis) resulting from the administration of one or more
therapies (including, but not limited to, the administration of one
or more prophylactic or therapeutic agents, such as a formulation
of the invention). In specific embodiments for LIGHT, such terms
refer to the reduction or inhibition of the binding of LIGHT to
HVEM, the reduction or inhibition of the binding of LIGHT to
LT.beta.R, the reduction or inhibition of the binding of LIGHT to
DcR3, the reduction or inhibition of the production or secretion of
CCL20 from a cell expressing a LIGHT receptor of a subject, the
reduction or inhibition of the production or secretion of IL-8 from
a cell expressing a LIGHT receptor of a subject, the reduction or
inhibition of the production or secretion of RANTES from a cell
expressing a LIGHT receptor of a subject, and/or the inhibition or
reduction of one or more symptoms associated with a LIGHT-mediated
disease, such as a chronic bowel disease, IBD, or GVHD. In specific
embodiments for CXCR5, such terms refer to the reduction or
inhibition of the binding of CXCR5 to CXCL13, and/or the inhibition
or reduction of one or more symptoms associated with a
CXCR5-mediated disease, such as rheumatoid arthritis.
[0148] The terms "variable region" or "variable domain" refer to a
portion of the light and heavy chains, typically about the
amino-terminal 120 to 130 amino acids in the heavy chain and about
100 to 110 amino acids in the light chain, which differ extensively
in sequence among antibodies and are used in the binding and
specificity of each particular antibody for its particular antigen.
The variability in sequence is concentrated in those regions called
complementarity determining regions (CDRs), while the more highly
conserved regions in the variable domain are called framework
regions (FR). The CDRs of the light and heavy chains are primarily
responsible for the interaction of the antibody with antigen.
Numbering of amino acid positions is according to the EU Index, as
in Kabat et al. (1991) Sequences of proteins of immunological
interest. (U.S. Department of Health and Human Services,
Washington, D.C.) 5.sup.th ed. ("Kabat et al."). In some
embodiments, the variable region is a human variable region.
B. Formulations and Formulation Components
[0149] As stated previously, the formulations of the invention have
surprisingly been found in the form of liquids and lyophilized
powders that comprise an IgG4 binding agent and a citrate buffer,
wherein the pH of the formulation is at or below both about pH 6
and the isoelectric point (pI) of the binding agent. The
formulations of the invention provide significant improvements over
conventional IgG4 binding agent formulations (e.g., phosphate
buffered saline (PBS) formulations), which form unwanted byproducts
upon increasing the concentration of the binding agent in the
formulation. In particular, the formulations of the invention have
a reduced amount of aggregates, half-molecules, degradation
products, low molecular weight proteins (LMWPs), high molecular
weight proteins (HMWPs), and rearrangements of acid, basic, and
neutral isoforms of the binding agent in the formulations.
i. Anti-LIGHT Binding Agents, and Variants and Fragments
Thereof.
[0150] In certain embodiments, the formulations of the invention
include an anti-LIGHT binding agent. The binding agents may be any
molecule, such as an antibody, a siRNA, a nucleic acid, an aptamer,
a protein, or a small molecule organic compound, that binds or
specifically binds to LIGHT, or a variant or a fragment thereof. In
some embodiments, the binding agent is an anti-LIGHT antibody, or a
variant thereof, or an antigen binding fragment thereof. Anti-LIGHT
antibodies specifically bind to a LIGHT (lymphotoxin-like, exhibits
inducible expression and competes with HSV glycoprotein D for HVEM,
a receptor expressed by T lymphocytes) protein, polypeptide
fragment, or epitope. The LIGHT molecule may be from any species.
In some embodiments, the LIGHT molecule is from a human, known
herein as "hLIGHT". hLIGHT has the following amino acid sequence,
which is identified as SEQ ID NO: 9:
TABLE-US-00001 (SEQ ID NO: 9) 1 MEESVVRPSV FVVDGQTDIP FTRLGRSHRR
QSCSVARVGL GLLLLLMGAG 51 LAVQGWFLLQ LHWRLGEMVT RLPDGPAGSW
EQLTQERRSH EVNPAAHLTG 101 ANSSLTGSGG PLLWETQLGL AFLRGLSYHD
GALVVTKAGY YYIYSKVQLG 150 GVGCPLGLAS TITHGLYKRT PRYPEELELL
VSQQSPCGRA TSSSRVWWDS 200 SFLGGVVHLE AGEEVVVRVL DERLVRLRDG
TRSYFGAFMV
[0151] In certain exemplary embodiments, the anti-LIGHT antibody is
a humanized antibody, a fully human antibody, or a variant thereof
or an antigen-binding fragment thereof. In some embodiments,
anti-LIGHT antibodies prevent binding of LIGHT with its receptors
and inhibit LIGHT biological activity (e.g., the LIGHT-mediated
production or secretion of CCL20, IL-8, or RANTES from cells
expressing a LIGHT ligand, such as a LIGHT receptor (e.g., HVEM,
LT.beta.R, and/or DcR3).
[0152] In certain specific embodiments, the anti-LIGHT antibody
comprises a heavy chain variable region (VH) comprising the amino
acid sequence of any one or more of the following complementary
determining regions (CDRs):
TABLE-US-00002 HCDR1 - GYNWH; (SEQ ID NO: 1) HCDR2 -
EITHSGSTNYNPSLKS; (SEQ ID NO: 2) or HCDR3 - EIAVAGTGYYGMDV. (SEQ ID
NO: 3)
[0153] In other specific embodiments, the anti-LIGHT antibody
comprises a light chain variable region (VL) comprising the amino
acid sequence of any one or more of the following complementary
determining regions (CDRs):
TABLE-US-00003 LCDR1 - RASQGINSAFA; (SEQ ID NO: 4) LCDR2 - DASSLES;
(SEQ ID NO: 5) or LCDR3 - QQFNSYPLT. (SEQ ID NO: 6)
[0154] In one specific embodiment, the anti-LIGHT antibody
comprises a heavy chain variable region (VH) comprising the amino
acid sequences of SEQ ID NOs: 1, 2, and 3.
[0155] In another specific embodiment, the anti-LIGHT antibody
comprises a light chain variable region (VL) comprising the amino
acid sequences of SEQ ID NOs: 4, 5, and 6.
[0156] In more specific embodiments, the anti-LIGHT antibody
comprises a heavy chain variable region comprising the amino acid
sequences of SEQ ID NOs: 1, 2, and 3; and a light chain variable
region comprising the amino acid sequences of SEQ ID NOs: 4, 5, and
6.
[0157] In specific embodiments, the anti-LIGHT antibody comprises a
heavy chain comprising the amino acid sequence of SEQ ID NO: 7:
TABLE-US-00004 1 QVQLQQWGAG LLKPSETLSL TCAVYGGSFS GYNWHWIRQP
PGKGLEWIGE (SEQ ID NO: 7) 51 ITHSGSTNYN PSLKSRVTIS VDTSKNQFSL
KLSSVTAADT AVYYCVREIA 101 VAGTGYYGMD VWGQGTTVTV SSASTKGPSV
FPLAPCSRST SESTAALGCL 151 VKDYFPEPVT VSWNSGALTS GVHTFPAVLQ
SSGLYSLSSV VTVPSSSLGT 201 KTYTCNVDHK PSNTKVDKRV ESKYGPPCPP
CPAPEFEGGP SVFLFPPKPK 251 DTLMISRTPE VTCVVVDVSQ EDPEVQFNWY
VDGVEVHNAK TKPREEQFNS 301 TYRVVSVLTV LHQDWLNGKE YKCKVSNKGL
PSSIEKTISK AKGQPREPQV 351 YTLPPSQEEM TKNQVSLTCL VKGFYPSDIA
VEWESNGQPE NNYKTTPPVL 401 DSDGSFFLYS RLTVDKSRWQ EGNVFSCSVM
HEALHNHYTQ KSLSLSLG
[0158] Positions 1-122: variable region of the heavy chain (VH).
The CDRs (complementary determining regions, according to Kabat
definition) are underlined. [0159] Positions 123-448: constant
region of human IgG4 (SwissProt IGHG4_HUMAN with the two mutations
S241P and L248E, according to Kabat numbering).
[0160] In other specific embodiments, the anti-LIGHT antibody
comprises a light chain comprising the amino acid sequence of SEQ
ID NO: 8:
TABLE-US-00005 1 AIQLTQSPSS LSASVGDRVT ITCRASQGIN SAFAWYQQKP
GKAPKLLIYD (SEQ ID NO: 8) 51 ASSLESGVPS RFSGSGSGTD FTLTISSLQP
EDFATYYCQQ FNSYPLTFGG 101 GTKVEIKRTV AAPSVFIFPP SDEQLKSGTA
SVVCLLNNFY PREAKVQWKV 151 DNALQSGNSQ ESVTEQDSKD STYSLSSTLT
LSKADYEKHK VYACEVTHQG 201 LSSPVTKSFN RGEC
[0161] Positions 1-107: variable region of the light chain (VL).
The CDRs (complementary determining regions, according to Kabat
definition) are underlined. [0162] Positions 108-214: constant
region of human C.kappa..
[0163] In further embodiments, the anti-LIGHT antibody comprises a
heavy chain comprising the amino acid sequence of SEQ ID NO: 7, and
a light chain comprising the amino acid sequence of SEQ ID NO:
8.
[0164] In certain embodiments, the anti-LIGHT antibody comprises a
heavy chain derived from the amino acid sequence of SEQ ID NO: 10,
which may be encoded by the nucleic acid sequence of SEQ ID NO:
11.
TABLE-US-00006 M K H L W F F L L L V A A P R W V L S Q V Q L Q Q W
G .cndot. 1
ATGAAGCACCTGTGGTTCTTTCTGCTGCTGGTGGCCGCTCCTAGATGGGTGCTGTCCCAGGTGCAGCTG-
CAGCAGTGGGG .cndot. A G L L K P S E T L S L T C A V Y G G S F S G Y
N W H .cndot. 81
CGCTGGCCTGCTGAAGCCTTCCGAGACACTGTCCCTGACCTGCGCCGTGTACGGCGGCTCCTTCTCCGG-
CTACAACTGGC .cndot. W I R Q P P G K G L E W I G E I T H S G S T N Y
N P 161
ACTGGATCAGGCAGCCTCCCGGCAAGGGCCTGGAATGGATCGGCGAGATCACCCACTCCGGCTCCACCA-
ACTACAACCCT S L K S R V T I S V D T S K N Q F S L K L S S V T A A
.cndot. 241
AGCCTGAAGTCCAGAGTGACCATCTCCGTGGACACCTCCAAGAACCAGTTCTCCCTGAAGCTGTCCTCT-
GTGACCGCCGC .cndot. D T A V Y Y C V R E I A V A G T G Y Y G M D V W
G Q G .cndot. 321
TGACACCGCCGTGTACTACTGTGTGCGGGAGATCGCCGTGGCTGGCACCGGCTACTACGGCATGGATGT-
GTGGGGCCAGG .cndot. T T V T V S S A S T K G P S V F P L A P C S R S
T S 401
GCACCACCGTGACCGTGTCCAGCGCTTCTACCAAGGGCCCTTCCGTGTTCCCTCTGGCCCCTTGCTCCC-
GGTCCACCTCC E S T A A L G C L V K D Y F P E P V T V S W N S G A L
.cndot. 481
GAGTCCACCGCCGCTCTGGGCTGCCTGGTGAAGGACTACTTCCCTGAGCCTGTGACCGTGTCCTGGAAC-
TCTGGCGCCCT .cndot. T S G V H T F P A V L Q S S G L Y S L S S V V T
V P S .cndot. 561
GACCTCCGGCGTGCACACCTTCCCTGCCGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTGGT-
GACCGTGCCTT .cndot. S S L G T K T Y T C N V D H K P S N T K V D K R
V E 641
CCTCCTCCCTGGGCACCAAGACCTACACCTGTAACGTGGACCACAAGCCTTCCAACACCAAGGTGGACA-
AGCGGGTGGAG S K Y G P P C P P C P A P E F E G G P S V F L F P P K
.cndot. 721
TCCAAGTACGGCCCTCCTTGCCCTCCCTGCCCTGCCCCTGAGTTCGAGGGCGGACCTAGCGTGTTCCTG-
TTCCCTCCTAA .cndot. P K D T L M I S R T P E V T C V V V D V S Q E D
P E V .cndot. 801
GCCTAAGGACACCCTGATGATCTCCCGGACCCCTGAGGTGACCTGTGTGGTGGTGGACGTGTCCCAGGA-
GGACCCTGAGG .cndot. Q F N W Y V D G V E V H N A K T K P R E E Q F N
S T 881
TCCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCTCGGGAGGAGCAGT-
TCAATTCCACC Y R V V S V L T V L H Q D W L N G K E Y K C K V S N K
.cndot. 961
TACCGGGTGGTGTCTGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAAGAATACAAGTGTAAG-
GTCTCCAACAA .cndot. G L P S S I E K T I S K A K G Q P R E P Q V Y T
L P P .cndot. 1041
GGGCCTGCCCTCCTCCATCGAGAAAACCATCTCCAAGGCCAAGGGCCAGCCTAGGGAGCCTCAGGTGTA-
CACCCTGCCTC .cndot. S Q E E M T K N Q V S L T C L V K G F Y P S D I
A V 1121
CTAGCCAGGAAGAGATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGAAGGGCTTCTACCCTTCCG-
ACATCGCCGTG E W E S N G Q P E N N Y K T T P P V L D S D G S F F L
.cndot. 1201
GAGTGGGAGTCCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCTCCTGTGCTGGACTCCGACGGC-
TCCTTCTTCCT .cndot. Y S R L T V D K S R W Q E G N V F S C S V M H E
A L H .cndot. 1281
GTACTCCAGGCTGACCGTGGACAAGTCCCGGTGGCAGGAGGGCAACGTCTTTTCCTGCTCCGTGATGCA-
CGAGGCCCTGC .cndot. N H Y T Q K S L S L S L G * (SEQ ID NO: 10)
1361 ACAACCACTACACCCAGAAGTCCCTGTCCCTGTCTCTGGGCTGA (SEQ ID NO:
11)
Amino acids 1-19: signal peptide Amino acids 20-141: 124F19k2
variable region (VH) Amino acids 142-end: hIgG4 PE constant region
Nucleic acids 1-57: nucleic acids encoding the signal peptide
Nucleic acids 58-423: nucleic acids encoding the 124F19k2 variable
region (VH) Nucleic acids 424-end: nucleic acids encoding the hIgG4
PE constant region
[0165] In alternative specific embodiments, the anti-LIGHT antibody
comprises a light chain derived from the amino acid sequence of SEQ
ID NO: 12, which may be encoded by the nucleic acid sequence of SEQ
ID NO: 13.
TABLE-US-00007 M D M R V P A Q L L G L L L L W L P G A R C A I Q L
T .cndot. 1
ATGGACATGAGAGTGCCTGCTCAGCTGCTGGGACTGCTGCTGCTGTGGCTGCCTGGCGCTAGATGCGCCA-
TCCAGCTGAC .cndot. Q S P S S L S A S V G D R V T I T C R A S Q G I
N S A .cndot. 81
CCAGTCCCCCTCCTCTCTGTCCGCCTCCGTGGGCGACAGAGTGACCATCACCTGTCGGGCCTCCCAGGGC-
ATCAACTCCG .cndot. F A W Y Q Q K P G K A P K L L I Y D A S S L E S
G V 161
CCTTCGCCTGGTATCAGCAGAAGCCTGGCAAGGCCCCTAAGCTGCTGATCTACGACGCCTCCTCCCTGGA-
ATCCGGCGTG P S R F S G S G S G T D F T L T I S S L Q P E D F A T
.cndot. 241
CCCTCCAGATTTTCCGGCTCCGGCTCTGGCACCGACTTCACCCTGACCATCTCCAGCCTGCAGCCTGAGG-
ACTTCGCCAC .cndot. Y Y C Q Q F N S Y P L T F G G G T K V E I K R T
V A A .cndot. 321
CTACTACTGCCAGCAGTTCAACTCCTACCCTCTGACCTTCGGCGGAGGCACCAAGGTGGAGATCAAGCGT-
ACGGTGGCTG .cndot. P S V F I F P P S D E Q L K S G T A S V V C L L
N N 401
CACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCT-
GCTGAATAAC F Y P R E A K V Q W K V D N A L Q S G N S Q E S V T E
.cndot. 481
TTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGA-
GTGTCACAGA .cndot. Q D S K D S T Y S L S S T L T L S K A D Y E K H
K V Y .cndot. 561
GCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAA-
CACAAAGTCT .cndot. A C E V T H Q G L S S P V T K S F N R G E C *
(SEQ ID NO: 12) 641
ACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTA-
G (SEQ ID NO: 13)
Amino acids 1-22: signal peptide Amino acids 23-129: 124F19k2
variable region (VL) Amino acids 130-end: hKappa constant region
Nucleic acids 1-66: nucleic acids encoding the signal peptide
Nucleic acids 67-387: nucleic acids encoding the 124F19k2 variable
region (VL) Nucleic acids 388-end: nucleic acids encoding the
hKappa constant region
[0166] In an embodiment of the invention, the anti-LIGHT antibody
is a fully human antibody. Examples of fully human antibody
isotypes include IgA, IgD, IgE, IgG, and IgM. In some embodiments,
the anti-LIGHT antibody is an IgG antibody. There are four forms of
IgG. In some embodiments, the anti-LIGHT antibody is an IgG4
antibody. In some embodiments of the invention, the anti-LIGHT
antibody is a fully human IgG4 antibody.
[0167] In some embodiments, the anti-LIGHT antibody further
comprises a constant region, e.g., a human IgG constant region. In
some embodiments, the constant region is a human IgG4 constant
region. In additional embodiments, the constant region is a
modified human IgG4 constant region. In other embodiments, the
constant region is a human C.kappa. constant region.
[0168] In some embodiments of the invention, the anti-LIGHT
antibody is a fully human IgG4 anti-LIGHT antibody comprising a
heavy chain comprising the amino acid sequence of SEQ ID NO: 7 and
a light chain comprising the amino acid sequence of SEQ ID NO: 8
(the "Lead LIGHT Antibody"). In alternative embodiments of the
invention, the anti-LIGHT antibody is a fully human IgG4 anti-LIGHT
antibody comprising a heavy chain variable region and a light chain
variable region, the heavy chain variable region comprising 3
complementary determining regions (CDRs) comprising the amino acid
sequences of SEQ ID NOs: 1, 2, and 3, and the light chain variable
region comprising 3 CDRs comprising the amino acid sequences of SEQ
ID NOs: 4, 5, and 6. Identification, isolation, preparation, and
characterization of anti-LIGHT antibodies, including the anti-LIGHT
antibody comprising a heavy chain amino acid sequence comprising
SEQ ID NO: 7 and a light chain amino acid sequence comprising SEQ
ID NO: 8, have been described in detail in U.S. Pat. No. 8,058,402,
corresponding to PCT Publication WO 2008/027338, which are
incorporated herein by reference.
[0169] Certain embodiments of formulations of the invention also
include variants of anti-LIGHT binding agents, such as antibodies.
Specifically, the formulations of the invention may include
variants of the anti-LIGHT antibody that is a fully human IgG4
anti-LIGHT antibody comprising a heavy chain comprising the amino
acid sequence of SEQ ID NO: 7 and a light chain comprising the
amino acid sequence of SEQ ID NO: 8. Variants of anti-LIGHT
antibodies may have similar physicochemical properties based on
their high similarity, and therefore are also included within the
scope of the invention. Variants are defined as antibodies with an
amino acid sequence that is at least 95%, at least 97%, for
instance at least 98% or 99% homologous to an anti-LIGHT antibody,
and capable of competing for binding to a LIGHT polypeptide, a
LIGHT polypeptide fragment, or a LIGHT epitope. In some
embodiments, the variants will ameliorate, neutralize, or otherwise
inhibit LIGHT biological activity (e.g., the LIGHT-mediated
production or secretion of CCL20, IL-8, or RANTES from cells
expressing a LIGHT ligand, such as a LIGHT receptor (e.g., HVEM,
LT.beta.R, and/or DcR3). Determining competition for binding to the
target can be done by routine methods known to the skilled person
in the art. In some embodiments, the variants are human antibodies,
and, in some embodiments, are IgG4 molecules. In some embodiments,
a variant is at least 95%, 96%, 97%, 98%, or 99% identical in amino
acid sequence with the Lead Antibody. The term "variant" refers to
an antibody that comprises an amino acid sequence that is altered
by one or more amino acids compared to the amino acid sequences of
the anti-LIGHT antibody. The variant may have conservative sequence
modifications, including amino acid substitutions, modifications,
additions, and deletions.
[0170] Examples of modifications include, but are not limited to,
glycosylation, acetylation, pegylation, phosphorylation, amidation,
derivatization by known protecting/blocking groups, proteolytic
cleavage, and linkage to a cellular ligand or other protein. Amino
acid modifications can be introduced by standard techniques known
in the art, such as site-directed mutagenesis, molecular cloning,
oligonucleotide-directed mutagenesis, and random PCR-mediated
mutagenesis in the nucleic acid encoding the antibodies.
Conservative amino acid substitutions include the ones in which the
amino acid residue is replaced with an amino acid residue having
similar structural or chemical properties. Families of amino acid
residues having similar side chains have been defined in the art.
These families include amino acids with basic side chains (e.g.,
lysine, arginine, histidine), acidic side chains (e.g., aspartic
acid, glutamic acid), uncharged polar side chains (e.g.,
asparagine, glutamine, serine, threonine, tyrosine, cysteine,
tryptophan), nonpolar side chains (e.g., glycine, alanine, valine,
leucine, isoleucine, proline, phenylalanine, methionine),
beta-branched side chains (e.g., threonine, valine, isoleucine),
and aromatic side chains (e.g., tyrosine, phenylalanine,
tryptophan). It will be clear to the skilled artisan that
classifications of amino acid residue families other than the one
used above can also be employed. Furthermore, a variant may have
non-conservative amino acid substitutions, e.g., replacement of an
amino acid with an amino acid residue having different structural
or chemical properties. Similar minor variations may also include
amino acid deletions or insertions, or both. Guidance in
determining which amino acid residues may be substituted, modified,
inserted, or deleted without abolishing immunological activity may
be found using computer programs well known in the art. Computer
algorithms, such as, inter alia, Gap or Bestfit, which are known to
a person skilled in the art, can be used to optimally align amino
acid sequences to be compared and to define similar or identical
amino acid residues. Variants may have the same or different,
either higher or lower, binding affinities compared to an
anti-LIGHT antibody, but are still capable of specifically binding
to LIGHT, and may have the same, higher or lower, biological
activity as the anti-LIGHT antibody.
[0171] Embodiments of the invention also include antigen binding
fragments of the anti-LIGHT binding agents, such as antibodies. The
term "antigen binding domain," "antigen binding region," "antigen
binding fragment," and similar terms refer to that portion of an
antibody which comprises the amino acid residues that interact with
an antigen and confer on the binding agent its specificity and
affinity for the antigen (e.g., the complementary determining
regions (CDR)). The antigen binding region can be derived from any
animal species, such as rodents (e.g., rabbit, rat or hamster) and
humans. In some embodiments, the antigen binding region will be of
human origin. Non-limiting examples of antigen binding fragments
include: Fab fragments, F(ab')2 fragments, Fd fragments, Fv
fragments, single chain Fv (scFv) molecules, dAb fragments, and
minimal recognition units consisting of the amino acid residues
that mimic the hypervariable region of the antibody.
[0172] In some embodiments of the invention, the anti-LIGHT binding
agents (or a variant thereof or an antigen binding fragment
thereof) will ameliorate, neutralize, or otherwise inhibit LIGHT
biological activity in vivo (e.g., the LIGHT-mediated production or
secretion of CCL20, IL-8, or RANTES from a cell expressing a LIGHT
receptor, e.g., HVEM, LT.beta.R, and/or DcR3).
[0173] In some embodiments of the invention, the anti-LIGHT binding
agents (or a variant thereof or an antigen binding fragment
thereof) are antagonist binding agents that ameliorate, neutralize,
or otherwise inhibit LIGHT biological activity in vivo (e.g., the
LIGHT-mediated production or secretion of CCL20, IL-8, or RANTES
from cells expressing a LIGHT ligand, such as a LIGHT receptor,
(e.g., HVEM, LT.beta.R, and/or DcR3).
[0174] In some embodiments, the anti-LIGHT binding agent (or a
variant thereof or an antigen binding fragment thereof) is present
in the formulations in an amount from about 5 mg/mL to about 280
mg/mL, e.g., about 5 mg/mL to about 200 mg/mL, about 50 mg/mL to
about 250 mg/mL, about 100 mg/mL to about 250 mg/mL, about 50 mg/mL
to about 200 mg/mL, and about 100 mg/mL to about 200 mg/mL. For
example, the anti-LIGHT binding agent may be present in the
formulation in an amount of about 5 mg/mL, about 10 mg/mL, about 15
mg/mL, about 20 mg/mL, about 25 mg/mL, about 30 mg/mL, about 35
mg/mL, about 40 mg/mL, about 45 mg/mL, about 50 mg/mL, about 55
mg/mL, about 60 mg/mL, about 65 mg/mL, about 70 mg/mL, about 75
mg/mL, about 80 mg/mL, about 85 mg/mL, about 90 mg/mL, about 95
mg/mL, about 100 mg/mL, about 105 mg/mL, about 110 mg/mL, about 115
mg/mL, about 120 mg/mL, about 125 mg/mL, about 130 mg/mL, about 135
mg/mL, about 140 mg/mL, about 145 mg/mL, about 150 mg/mL, about 155
mg/mL, about 160 mg/mL, about 165 mg/mL, about 170 mg/mL, about 175
mg/mL, about 180 mg/mL, about 185 mg/mL, about 190 mg/mL, about 195
mg/mL, about 200 mg/mL, about 205 mg/mL, about 210 mg/mL, about 215
mg/mL, about 220 mg/mL, about 225 mg/mL, about 230 mg/mL, about 235
mg/mL, about 240 mg/mL, about 245 mg/mL, about 250 mg/mL, about 255
mg/mL, about 260 mg/mL, about 265 mg/mL, about 270 mg/mL, about 275
mg/mL, or about 280 mg/mL.
[0175] In alternative embodiments, the anti-LIGHT binding agent may
be present in the formulation in an amount from about 5 to about 25
mg/mL, from about 26 to about 50 mg/mL, from about 51 to about 75
mg/mL, from about 76 to about 100 mg/mL, from about 101 to about
125 mg/mL, from about 126 to about 150 mg/mL, from about 151 to
about 175 mg/mL, from about 176 to about 200 mg/mL, from about 201
mg/mL to about 225 mg/mL, from about 226 mg/mL to about 250 mg/mL,
from about 251 to about 280 mg/mL, from about 5 to about 10 mg/mL,
from about 40 to about 60 mg/mL, from about 75 to about 85 mg/mL,
or from about 140 to about 160 mg/mL.
[0176] In certain exemplary embodiments, the anti-LIGHT binding
agent is present in the formulation in an amount from about 50
mg/mL to about 170 mg, about 100 mg/mL to about 160 mg/mL, for
example about 150 mg/mL. Alternatively, the anti-LIGHT binding
agent is present in an amount of about 50 mg/mL. In another
exemplary embodiment, a fully human IgG4 anti-LIGHT antibody
comprising a heavy chain comprising the amino acid sequence of SEQ
ID NO: 7 and a light chain comprising the amino acid sequence of
SEQ ID NO: 8 is present in the formulation in an amount of about
150 mg/mL.
ii. Anti-CXCR5 Binding Agents, and Variants and Fragments
Thereof.
[0177] In certain embodiments, the formulations of the invention
include an anti-CXCR5 binding agent. The binding agents may be any
molecule, such as an antibody, a siRNA, a nucleic acid, an aptamer,
a protein, or a small molecule organic compound, that binds or
specifically binds to CXCR5, or a variant or a fragment thereof. In
some embodiments, the binding agent is an anti-CXCR5 antibody, or a
variant thereof, or an antigen binding fragment thereof. Anti-CXCR5
antibodies specifically bind to a CXCL13 (also known as BLC)
protein, polypeptide fragment, or epitope. The CXCR5 molecule may
be from any species. In some embodiments, the CXCR5 molecule is
from a human, known herein as "hCXCR5". hCXCR5 has the following
amino acid sequence, which is identified as SEQ ID NO: 14:
TABLE-US-00008 (SEQ ID NO: 14) MNYPLTLEMD LENLEDLFWE LDRLDNYNDT
SLVENHLCPA TEGPLMASFK AVFVPVAYSL IFLLGVIGNV LVLVILERHR QTRSSTETFL
FHLAVADLLL VFILPFAVAE GSVGWVLGTF LCKTVIALHK VNFYCSSLLL ACIAVDRYLA
IVHAVHAYRH RRLLSIHITC GTIWLVGFLL ALPEILFAKV SQGHHNNSLP RCTFSQENQA
ETHAWFTSRF LYHVAGFLLP MLVMGWCYVG VVHRLRQAQR RPQRQKAVRV AILVTSIFFL
CWSPYHIVIF LDTLARLKAV DNTCKLNGSL PVAITMCEFL GLAHCCLNPM LYTFAGVKFR
SDLSRLLTKL GCTGPASLCQ LFPSWRRSSL SESENATSLT TF.
[0178] In certain exemplary embodiments, the anti-CXCR5 antibody is
a humanized antibody, a fully human antibody, or a variant thereof
or an antigen-binding fragment thereof. In some embodiments,
anti-CXCR5 antibodies prevent binding of CXCR5 with its ligands and
inhibit CXCR5 biological activity (e.g., the binding of CXCR5 to
CXCL13).
[0179] In certain specific embodiments, the anti-CXCR5 antibody
comprises a heavy chain variable region (VH) comprising the amino
acid sequence of any one or more of the following complementary
determining regions (CDRs):
TABLE-US-00009 HCDR1 - GFSLIDYGVN; (SEQ ID NO: 15) HCDR2 -
VIWGDGTTY; (SEQ ID NO: 16) or HCDR3 - IVY. (SEQ ID NO: 17)
[0180] In other specific embodiments, the anti-CXCR5 antibody
comprises a light chain variable region (VL) comprising the amino
acid sequence of any one or more of the following complementary
determining regions (CDRs):
TABLE-US-00010 LCDR1 - RSSKSLLHSSGKTYLY; (SEQ ID NO: 18) LCDR2 -
RLSSLA; (SEQ ID NO: 19) or LCDR3 - MQHLEYPYT. (SEQ ID NO: 20)
[0181] In one specific embodiment, the anti-CXCR5 antibody
comprises a heavy chain variable region (VH) comprising the amino
acid sequences of SEQ ID NOs: 15, 16, and 17.
[0182] In another specific embodiment, the anti-CXCR5 antibody
comprises a light chain variable region (VL) comprising the amino
acid sequences of SEQ ID NOs: 18, 19, and 20.
[0183] In more specific embodiments, the anti-CXCR5 antibody
comprises a heavy chain variable region comprising the amino acid
sequences of SEQ ID NOs: 15, 16, and 17; and a light chain variable
region comprising the amino acid sequences of SEQ ID NOs: 18, 19,
and 20.
[0184] In a specific embodiment, the anti-CXCR5 antibody comprises
a heavy chain variable region comprising the amino acid sequence of
SEQ ID NO: 21:
TABLE-US-00011 (SEQ ID NO: 21) QVQLKESGPG LVAPSESLSI TCTVSGFSLI
DYGVNWIRQP PGKGLEWLGV IWGDGTTYYN PSLKSRLSIS KDNSKSQVFL KVTSLTTDDT
AMYYCARIVY WGQGTLVTVS A.
[0185] In another specific embodiment, the anti-CXCR5 antibody
comprises a light chain variable region comprising the amino acid
sequence of SEQ ID NO: 22:
TABLE-US-00012 (SEQ ID NO: 22) DIVMTQAAPS VAVTPGASVS ISCRSSKSLL
HSSGKTYLYW FLQRPGQSPQ LLIYRLSSLA SGVPDRFSGS GSGTAFTLRI SRVEAEDVGV
YYCMQHLEYP YTFGGGTKLE IK.
[0186] In more specific embodiments, the anti-CXCR5 antibody
comprises a heavy chain variable region comprising the amino acid
sequence of SEQ ID NO: 21; and a light chain variable region
comprising the amino acid sequence of SEQ ID NO: 22.
[0187] In some embodiments, the anti-CXCR5 antibody further
comprises a constant region, e.g., a human IgG constant region. In
some embodiments, the constant region is a human IgG4 constant
region. In additional embodiments, the constant region is a
modified human IgG4 constant region. In some embodiments, the human
IgG4 constant region has the following modifications: S241P (shown
below in SEQ ID NO: 23 in bold), L248E (shown below in SEQ ID NO:
23 in bold), and the lack of a terminal lysine in order to avoid
heterogeneity. In some embodiments, the IgG4 constant region
comprises the amino acid sequence of SEQ ID NO: 23:
TABLE-US-00013 (SEQ ID NO: 23) ASTKGPSVFP LAPCSRSTSE STAALGCLVK
DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTKT YTCNVDHKPS
NTKVDKRVES KYGPPCPPCP APEFEGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSQED
PEVQFNWYVD GVEVHNAKTK PREEQFNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKGLPS
SIEKTISKAK GQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN
YKTTPPVLDS DGSFFLYSRL TVDKSRWQEG NVFSCSVMHE ALHNHYTQKS LSLSLG.
[0188] In other embodiments, the constant region is a human
C.kappa. constant region. In some embodiments, the C.kappa.
constant region comprises the amino acid sequence of SEQ ID NO:
24:
TABLE-US-00014 (SEQ ID NO: 24) RTVAAPSVFI FPPSDEQLKS GTASVVCLLN
NFYPREAKVQ WKVDNALQSG NSQESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVT
HQGLSSPVTK SFNRGEC.
[0189] In specific embodiments, the anti-CXCR5 antibody comprises a
heavy chain comprising the amino acid sequence of SEQ ID NO:
25:
TABLE-US-00015 (SEQ ID NO: 25) QVQLKESGPG LVAPSESLSI TCTVSGFSLI
DYGVNWIRQP PGKGLEWLGV IWGDGTTYYN PSLKSRLSIS KDNSKSQVFL KVTSLTTDDT
AMYYCARIVY WGQGTLVTVS AASTKGPSVF PLAPCSRSTS ESTAALGCLV KDYFPEPVTV
SWNSGALTSG VHTFPAVLQS SGLYSLSSVV TVPSSSLGTK TYTCNVDHKP SNTKVDKRVE
SKYGPPCPPC PAPEFEGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSQE DPEVQFNWYV
DGVEVHNAKT KPREEQFNST YRVVSVLTVL HQDWLNGKEY KCKVSNKGLP SSIEKTISKA
KGQPREPQVY TLPPSQEEMT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD
SDGSFFLYSR LTVDKSRWQE GNVFSCSVMH EALHNHYTQK SLSLSLG.
[0190] Positions 1-111: variable region of the heavy chain (VH).
The CDRs (complementarity determining regions, according to Kabat
definition) are underlined. [0191] Positions 112-432: constant
region of human IgG4 (SwissProt IGHG4_HUMAN, including the
following modifications: S241P, L248E, and the lack of a terminal
lysine in order to avoid heterogeneity).
[0192] In other specific embodiments, the anti-CXCR5 antibody
comprises a light chain comprising the amino acid sequence of SEQ
ID NO: 26:
TABLE-US-00016 (SEQ ID NO: 26) DIVMTQAAPS VAVTPGASVS ISCRSSKSLL
HSSGKTYLYW FLQRPGQSPQ LLIYRLSSLA SGVPDRFSGS GSGTAFTLRI SRVEAEDVGV
YYCMQHLEYP YTFGGGTKLE IKRTVAAPSV FIFPPSDEQL KSGTASVVCL LNNFYPREAK
VQWKVDNALQ SGNSQESVTE QDSKDSTYSL SSTLTLSKAD YEKHKVYACE VTHQGLSSPV
TKSFNRGEC.
[0193] Positions 1-112: variable region of the light chain (VL).
The CDRs (complementarity determining regions, according to Kabat
definition) are underlined. [0194] Positions 113-182: constant
region of human C.kappa..
[0195] In further embodiments, the anti-CXCR5 antibody comprises a
heavy chain comprising the amino acid sequence of SEQ ID NO: 25,
and a light chain comprising the amino acid sequence of SEQ ID NO:
26.
[0196] In some embodiments, the anti-CXCR5 antibody further
comprises a leader sequence. The leader sequence, in some
embodiments, comprises an amino acid sequence from 1-30 amino acids
in length, such as 25-25 amino acids, and typically 19 amino acids.
The heavy chain, light chain, or both the heavy and light chain may
comprise a leader sequence. In some embodiments, the leader
sequence comprises the amino acid sequence of SEQ ID NO: 16:
MGWSCIILFL VATATGVHS (SEQ ID NO: 27).
[0197] In specific embodiments, the anti-CXCR5 antibody comprises a
heavy chain derived from the amino acid sequence of SEQ ID NO:
28:
TABLE-US-00017 (SEQ ID NO: 28) MGWSCIILFL VATATGVHSQ VQLKESGPGL
VAPSESLSIT CTVSGFSLID YGVNWIRQPP GKGLEWLGVI WGDGTTYYNP SLKSRLSISK
DNSKSQVFLK VTSLTTDDTA MYYCARIVYW GQGTLVTVSA ASTKGPSVFP LAPCSRSTSE
STAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTKT
YTCNVDHKPS NTKVDKRVES KYGPPCPPCP APEFEGGPSV FLFPPKPKDT LMISRTPEVT
CVVVDVSQED PEVQFNWYVD GVEVHNAKTK PREEQFNSTY RVVSVLTVLH QDWLNGKEYK
CKVSNKGLPS SIEKTISKAK GQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE
WESNGQPENN YKTTPPVLDS DGSFFLYSRL TVDKSRWQEG NVFSCSVMHE ALHNHYTQKS
LSLSLG.
[0198] Positions 1-19: leader sequence [0199] Positions 20-130:
variable region of the heavy chain (VH). The CDRs (complementarity
determining regions, according to Kabat definition) are underlined.
[0200] Positions 131-456: constant region of human IgG4 (SwissProt
IGHG4_HUMAN, including the following modifications: S241P, L248E,
and the lack of a terminal lysine in order to avoid
heterogeneity).
[0201] In other specific embodiments, the anti-CXCR5 antibody
comprises a light chain derived from the amino acid sequence of SEQ
ID NO: 29:
TABLE-US-00018 (SEQ ID NO: 29) MGWSCIILFL VATATGVHSD IVMTQAAPSV
AVTPGASVSI SCRSSKSLLH SSGKTYLYWF LQRPGQSPQL LIYRLSSLAS GVPDRFSGSG
SGTAFTLRIS RVEAEDVGVY YCMQHLEYPY TFGGGTKLEI KRTVAAPSVF IFPPSDEQLK
SGTASVVCLL NNFYPREAKV QWKVDNALQS GNSQESVTEQ DSKDSTYSLS STLTLSKADY
EKHKVYACEV THQGLSSPVT KSFNRGEC.
[0202] Positions 1-19: leader sequence [0203] Positions 20-131:
variable region of the light chain (VL). The CDRs (complementarity
determining regions, according to Kabat definition) are underlined.
[0204] Positions 132-238: constant region of human C.kappa..
[0205] In further embodiments, the anti-CXCR5 antibody comprises a
heavy chain comprising the amino acid sequence of SEQ ID NO: 28,
and a light chain comprising the amino acid sequence of SEQ ID NO:
29.
[0206] In some embodiments of the invention, the anti-CXCR5
antibody is a humanized or a fully human antibody. Examples of
humanized and fully human antibody isotypes include IgA, IgD, IgE,
IgG, and IgM. In some embodiments, the anti-CXCR5 antibody is an
IgG antibody. There are four forms of IgG. In some embodiments, the
anti-CXCR5 antibody is an IgG4 antibody. In some embodiments of the
invention, the anti-CXCR5 antibody is a humanized IgG4
antibody.
[0207] In some embodiments of the invention, the anti-CXCR5
antibody is a humanized IgG4 anti-CXCR5 antibody comprising a heavy
chain comprising the amino acid sequence of SEQ ID NO: 25 and a
light chain comprising the amino acid sequence of SEQ ID NO: 26
(the "Lead CXCR5 Antibody"). In alternative embodiments of the
invention, the anti-CXCR5 antibody is a humanized IgG4 anti-CXCR5
antibody comprising a heavy chain variable region and a light chain
variable region, the heavy chain variable region comprising 3
complementary determining regions (CDRs) comprising the amino acid
sequences of SEQ ID NOs: 15, 16, and 17, and the light chain
variable region comprising 3 CDRs comprising the amino acid
sequences of SEQ ID NOs: 18, 19, and 20. Identification, isolation,
preparation, and characterization of anti-CXCR5 antibodies,
including the anti-CXCR5 antibody comprising a heavy chain amino
acid sequence comprising SEQ ID NO: 25 and a light chain amino acid
sequence comprising SEQ ID NO: 26, have been described in detail in
PCT Publication WO 2009/032661, which are incorporated herein by
reference.
[0208] Certain embodiments of formulations of the invention also
include variants of anti-CXCR5 binding agents, such as antibodies.
Specifically, the formulations of the invention may include
variants of the anti-CXCR5 antibody that is a humanized IgG4
anti-CXCR5 antibody comprising a heavy chain comprising the amino
acid sequence of SEQ ID NO: 25 and a light chain comprising the
amino acid sequence of SEQ ID NO: 26. Variants of anti-CXCR5
antibodies may have similar physicochemical properties based on
their high similarity, and therefore are also included within the
scope of the invention. Variants are defined as antibodies with an
amino acid sequence that is at least 95%, at least 97%, for
instance at least 98% or 99% homologous to an anti-CXCR5 antibody,
and capable of competing for binding to a CXCR5 polypeptide, a
CXCR5 polypeptide fragment, or a CXCR5 epitope. In some
embodiments, the variants will ameliorate, neutralize, or otherwise
inhibit CXCR5 biological activity (e.g., the binding of CXCL13 to
CXCR5). Determining competition for binding to the target can be
done by routine methods known to the skilled person in the art. In
some embodiments, the variants are human antibodies, and, in some
embodiments, are IgG4 molecules. In some embodiments, a variant is
at least 95%, 96%, 97%, 98%, or 99% identical in amino acid
sequence with the Lead Antibody. The term "variant" refers to an
antibody that comprises an amino acid sequence that is altered by
one or more amino acids compared to the amino acid sequences of the
anti-CXCR5 antibody. The variant may have conservative sequence
modifications, including amino acid substitutions, modifications,
additions, and deletions.
[0209] Examples of modifications include, but are not limited to,
glycosylation, acetylation, pegylation, phosphorylation, amidation,
derivatization by known protecting/blocking groups, proteolytic
cleavage, and linkage to a cellular ligand or other protein. Amino
acid modifications can be introduced by standard techniques known
in the art, such as site-directed mutagenesis, molecular cloning,
oligonucleotide-directed mutagenesis, and random PCR-mediated
mutagenesis in the nucleic acid encoding the antibodies.
Conservative amino acid substitutions include the ones in which the
amino acid residue is replaced with an amino acid residue having
similar structural or chemical properties. Families of amino acid
residues having similar side chains have been defined in the art.
These families include amino acids with basic side chains (e.g.,
lysine, arginine, histidine), acidic side chains (e.g., aspartic
acid, glutamic acid), uncharged polar side chains (e.g.,
asparagine, glutamine, serine, threonine, tyrosine, cysteine,
tryptophan), nonpolar side chains (e.g., glycine, alanine, valine,
leucine, isoleucine, proline, phenylalanine, methionine),
beta-branched side chains (e.g., threonine, valine, isoleucine),
and aromatic side chains (e.g., tyrosine, phenylalanine,
tryptophan). It will be clear to the skilled artisan that
classifications of amino acid residue families other than the one
used above can also be employed. Furthermore, a variant may have
non-conservative amino acid substitutions, e.g., replacement of an
amino acid with an amino acid residue having different structural
or chemical properties. Similar minor variations may also include
amino acid deletions or insertions, or both. Guidance in
determining which amino acid residues may be substituted, modified,
inserted, or deleted without abolishing immunological activity may
be found using computer programs well known in the art. Computer
algorithms, such as, inter alia, Gap or Bestfit, which are known to
a person skilled in the art, can be used to optimally align amino
acid sequences to be compared and to define similar or identical
amino acid residues. Variants may have the same or different,
either higher or lower, binding affinities compared to an
anti-CXCR5 antibody, but are still capable of specifically binding
to CXCR5, and may have the same, higher or lower, biological
activity as the anti-CXCR5 antibody.
[0210] Embodiments of the invention also include antigen binding
fragments of the anti-CXCR5 binding agents, such as antibodies. The
term "antigen binding domain," "antigen binding region," "antigen
binding fragment," and similar terms refer to that portion of an
antibody which comprises the amino acid residues that interact with
an antigen and confer on the binding agent its specificity and
affinity for the antigen (e.g., the complementary determining
regions (CDR)). The antigen binding region can be derived from any
animal species, such as rodents (e.g., rabbit, rat or hamster) and
humans. In some embodiments, the antigen binding region will be of
human origin. Non-limiting examples of antigen binding fragments
include: Fab fragments, F(ab')2 fragments, Fd fragments, Fv
fragments, single chain Fv (scFv) molecules, dAb fragments, and
minimal recognition units consisting of the amino acid residues
that mimic the hypervariable region of the antibody.
[0211] In some embodiments of the invention, the anti-CXCR5 binding
agents (or a variant thereof or an antigen binding fragment
thereof) will ameliorate, neutralize, or otherwise inhibit CXCR5
biological activity in vivo (e.g., the binding of CXCL13 to
CXCR5).
[0212] In some embodiments of the invention, the anti-CXCR5 binding
agents (or a variant thereof or an antigen binding fragment
thereof) are antagonist binding agents that ameliorate, neutralize,
or otherwise inhibit CXCR5 biological activity in vivo (e.g., the
binding of CXCL13 to CXCR5).
[0213] In some embodiments, the anti-CXCR5 binding agent (or a
variant thereof or an antigen binding fragment thereof) is present
in the formulations in an amount from about 5 mg/mL to about 280
mg/mL, e.g., about 5 mg/mL to about 200 mg/mL, about 5 mg/mL to
about 125 mg/mL, about 5 mg/mL to about 75 mg/mL, about 5 mg/mL to
about 50 mg/mL, and about 5 mg/mL to about 25 mg/mL. For example,
the anti-CXCR5 binding agent may be present in the formulation in
an amount of about 5 mg/mL, about 10 mg/mL, about 15 mg/mL, about
20 mg/mL, about 25 mg/mL, about 30 mg/mL, about 35 mg/mL, about 40
mg/mL, about 45 mg/mL, about 50 mg/mL, about 55 mg/mL, about 60
mg/mL, about 65 mg/mL, about 70 mg/mL, about 75 mg/mL, about 80
mg/mL, about 85 mg/mL, about 90 mg/mL, about 95 mg/mL, about 100
mg/mL, about 105 mg/mL, about 110 mg/mL, about 115 mg/mL, about 120
mg/mL, about 125 mg/mL, about 130 mg/mL, about 135 mg/mL, about 140
mg/mL, about 145 mg/mL, about 150 mg/mL, about 155 mg/mL, about 160
mg/mL, about 165 mg/mL, about 170 mg/mL, about 175 mg/mL, about 180
mg/mL, about 185 mg/mL, about 190 mg/mL, about 195 mg/mL, about 200
mg/mL, about 205 mg/mL, about 210 mg/mL, about 215 mg/mL, about 220
mg/mL, about 225 mg/mL, about 230 mg/mL, about 235 mg/mL, about 240
mg/mL, about 245 mg/mL, about 250 mg/mL, about 255 mg/mL, about 260
mg/mL, about 265 mg/mL, about 270 mg/mL, about 275 mg/mL, or about
280 mg/mL.
[0214] In alternative embodiments, the anti-CXCR5 binding agent may
be present in the formulation in an amount from about 5 to about 25
mg/mL, from about 26 to about 50 mg/mL, from about 51 to about 75
mg/mL, from about 76 to about 100 mg/mL, from about 101 to about
125 mg/mL, from about 126 to about 150 mg/mL, from about 151 to
about 175 mg/mL, from about 176 to about 200 mg/mL, from about 201
mg/mL to about 225 mg/mL, from about 226 mg/mL to about 250 mg/mL,
from about 251 to about 280 mg/mL, from about 5 to about 25 mg/mL,
from about 40 to about 60 mg/mL, from about 75 to about 85 mg/mL,
or from about 90 to about 110 mg/mL.
[0215] In certain exemplary embodiments, the anti-CXCR5 binding
agent is present in the formulation in an amount of about 20 mg/mL.
Alternatively, the anti-CXCR5 binding agent is present in an amount
of about 100 mg/mL. In another exemplary embodiment, a humanized
IgG4 anti-CXCR5 antibody comprising a heavy chain comprising the
amino acid sequence of SEQ ID NO: 25 and a light chain comprising
the amino acid sequence of SEQ ID NO: 26 is present in the
formulation in an amount of about 20 mg/mL or 100 mg/mL.
iii. Buffering Agents
[0216] The formulations of the invention comprise a citrate buffer
as a buffering agent. A buffering agent maintains a physiologically
suitable pH. In addition, a buffering agent enhances isotonicity
and chemical stability of the formulation. In some embodiments, the
citrate buffer is present in the formulations at a concentration
from about 0.5 mM to about 50 mM, e.g., about 5 mM to about 15 mM.
For example, the citrate buffer may be present in the formulation
at a concentration about 5 mM, about 6 mM, about 7 mM, about 8 mM,
about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM,
about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM,
about 19 mM, about 20 mM, about 21 mM, about 22 mM, about 23 mM,
about 24 mM, about 25 mM, about 26 mM, about 27 mM, about 28 mM,
about 29 mM, about 30 mM, about 31 mM, about 32 mM, about 33 mM,
about 34 mM, about 35 mM, about 36 mM, about 37 mM, about 38 mM,
about 39 mM, about 40 mM, about 41 mM, about 42 mM, about 43 mM,
about 44 mM, about 45 mM, about 46 mM, about 47 mM, about 48 mM,
about 49 mM, and about 50 mM. In some embodiments, the citrate
buffer is present in the formulation at a concentration from about
7 mM to about 13 mM, such as from about 9 mM to about 11 mM. In
some embodiments, the citrate buffer is present at a concentration
of about 10 mM.
[0217] In certain embodiments, the formulations of the invention
have a pH at or below pH 6. In some embodiments, the pH of the
formulations ranges from about 5.0 to about 6.0. For example, the
pH of the formulations may be about 5.0, about 5.1, about 5.2,
about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8,
about 5.9, and about 6.0. In some embodiments, the pH of the
formulations may range from about 5.5 to about 6.0. In some
embodiments, the pH is either about 5.5 or about 6.0. The pH of the
formulation may be measured by any means known to those of skill in
the art. A means for measuring pH is using a pH meter with a
micro-electrode. The pH of the formulation may be adjusted using
any means known in the art. Exemplary chemicals for altering the pH
of the formulations are hydrochloric acid (HCl) and sodium
hydroxide (NaOH).
[0218] In certain embodiments, the formulations of the invention
have a pH at or below the isoelectric point (pI) of the binding
agent, such as an antibody. The isoelectric point is the pH at
which a particular molecule or surface carries no net electrical
charge. The pI of an anti-LIGHT or an anti-CXCR5 binding agent may
be determined by any means known to those of skill in the art. In
some embodiments, the pI of an anti-LIGHT or anti-CXCR5 antibody is
determined by denaturated isoelectric focusing. As shown in FIG. 1,
the pI of a fully human IgG4 anti-LIGHT antibody comprising a heavy
chain comprising the amino acid sequence of SEQ ID NO: 7 and a
light chain comprising the amino acid sequence of SEQ ID NO: 8 is
6.8-7.2. As shown in FIG. 11, the pI of a humanized IgG4 anti-CXCR5
antibody comprising a heavy chain comprising the amino acid
sequence of SEQ ID NO: 25 and a light chain comprising the amino
acid sequence of SEQ ID NO: 26 is 7.6-8.4.
iv. Surfactants
[0219] The formulations of the invention may, optionally, further
comprise a surfactant, which is also known as a stabilizing agent.
Surfactants/stabilizing agents are chemical compounds that interact
and stabilize biological molecules and/or general pharmaceutical
excipients in a formulation. In certain embodiments, surfactants
may be used in conjunction with lower temperature storage.
Surfactants generally protect the binding agent from air/solution
interface induced stresses and solution/surface induced stresses,
which may otherwise result in protein aggregation. Surfactants may
include, but are not limited to, polysorbates, glycerin,
dicarboxylic acids, oxalic acid, succinic acid, fumaric acids,
phthalic acids, and combinations thereof. Those skilled in the art
are aware that other surfactants, e.g. non-ionic or ionic
detergents, can be used as long as they are pharmaceutically
acceptable, i.e. suitable for administration to subjects. The
surfactant is, in some embodiments, a polysorbate. Examples of
polysorbates include polysorbate 20, polysorbate 40, polysorbate
60, polysorbate 65, and polysorbate 80.
[0220] In exemplary embodiments, the surfactant is present in the
formulations in an amount from about 0.001% to about 0.1% (w/v).
For example, the surfactant may be present in the formulations in
an amount of about 0.001% (w/v), about 0.002% (w/v), about 0.003%
(w/v), about 0.004% (w/v), about 0.005% (w/v), about 0.006% (w/v),
about 0.007% (w/v), about 0.008% (w/v), about 0.009% (w/v), about
0.01% (w/v), about 0.02% (w/v), about 0.03% (w/v), about 0.04%
(w/v), about 0.05% (w/v), about 0.06% (w/v), about 0.07% (w/v),
about 0.08% (w/v), about 0.09% (w/v), and about 0.1% (w/v). In
particular embodiments, the surfactant is present in the
formulations from about 0.003% to about 0.05% (w/v), about 0.004%
to about 0.025% (w/v), or about 0.005% to about 0.02% (w/v), e.g.
about 0.005% (w/v). For example, polysorbate 20 may be present in
an amount from about 0.001% to about 0.1% (w/v), about 0.002% to
about 0.01% (w/v), about 0.003% to about 0.008% (w/v), and about
0.004% to about 0.006% (w/v), e.g., about 0.005% (w/v). In
alternative embodiments, polysorbate 20 is present in an amount
from about 0.001% to about 0.1% (w/v), about 0.005% to about 0.05%
(w/v), and about 0.0075% to about 0.025% (w/v), e.g., about 0.01%
(w/v). In further alternative embodiments, polysorbate 20 is
present in an amount from about 0.001% to about 0.1% (w/v), about
0.005% to about 0.05% (w/v), and about 0.01% to about 0.03% (w/v),
e.g., about 0.02% (w/v).
v. Tonicity Agents
[0221] The formulations of the invention may, optionally, further
comprise a tonicity agent. Typically, tonicity agents are used to
adjust or maintain the osmolality of the formulations in order to
bring it closer to the osmotic pressure of body fluids, such as
blood or plasma. Tonicity agents may also maintain the binding
agent levels in a formulation. In part, the tonicity agent
contributes to preserving the level, ratio, or proportion of the
therapeutically active binding agent present in the formulation. As
used herein, the term "tonicity" refers to the behavior of biologic
components in a fluid enviornment or solution. Isotonic solutions
possess the same osmotic pressure as blood plasma, and can be
intravenously infused into a subject without changing the osmotic
pressure of the subject's blood plasma. Indeed, in certain
embodiments of the invention, the tonicity agent is present in an
amount sufficient to render the formulation suitable for
intravenous infusion. Often, the tonicity agent serves as a bulking
agent or a stabilizing agent as well. As such, the tonicity agent
may allow the binding agent to overcome various stresses, such as
freezing and shear. Tonicity agents may include, but are not
limited to, saccharides, sugars, glycerol, sorbitol, mannitol,
sodium chloride, potassium chloride, magnesium chloride, and other
inorganic salts. Those skilled in the art are aware that other
tonicity agents can be used as long as they are pharmaceutically
acceptable, i.e. suitable for administration to subjects.
[0222] In certain embodiments, the tonicity agent is present in the
formulations in an amount from about 0.1% to 10% (w/v). For
example, the tonicity agent may be present in the formulation in an
amount of about 0.1% (w/v), about 0.2% (w/v), about 0.3% (w/v),
about 0.4% (w/v), about 0.5% (w/v), about 0.6% (w/v), about 0.7%
(w/v), about 0.8% (w/v), about 0.9% (w/v), about 1% (w/v), about 2%
(w/v), about 3% (w/v), about 4% (w/v), about 4.5% (w/v), about 5%
(w/v), about 5.5% (w/v), about 6% (w/v), about 7% (w/v), about 8%
(w/v), about 9% (w/v), and about 10% (w/v). Alternatively, the
tonicity agent may be present in the formulation in an amount from
about 2% to about 8% (w/v), from about 3% to about 7% (w/v), and
from about 4% to about 6% (w/v). In further alternative
embodiments, the tonicity agent may be present in the formulation
in an amount from about 0.1% to about 1%, from about 0.1% to about
0.5%, from about 0.1 to about 0.3%, and about 0.2%.
[0223] In certain exemplary embodiments, the tonicity agent is a
saccharide. Examples of saccharides include glucose, sucrose (which
is also known as saccharose), maltose, trehalose, dextrose,
xylitol, fructose and mannitol. For example, mannitol may be
present in an amount of about 1% to about 10% (w/v), about 2% to
about 8% (w/v), or about 3% to about 5% (w/v), e.g., about 4%
(w/v). Alternatively, sucrose (which is also known as saccharose)
may be present in an amount of about 1% to about 10% (w/v), about
3% to about 8% (w/v), or about 4% to about 6% (w/v), e.g., about
4.5, 5, 5.5, or 6% (w/v).
[0224] In certain other exemplary embodiments, the tonicity agent
is sodium chloride. For example, sodium chloride may be present in
an amount of about 0.1% (w/v), about 0.2% (w/v), about 0.3% (w/v),
about 0.4% (w/v), about 0.5% (w/v), about 0.6% (w/v), about 0.7%
(w/v), about 0.8% (w/v), about 0.9% (w/v), and about 1% (w/v).
Alternatively, sodium chloride may be present in the formulation in
an amount from about 0.1% to about 1%, from about 0.1% to about
0.5%, from about 0.1 to about 0.3%, and about 0.2%.
[0225] In further exemplary embodiments, the formulations may
comprise one or more tonicity agents. For example, the formulations
may comprise one or more of the above tonicity agents in the above
concentrations. In certain specific embodiments, the formulations
may comprise sucrose and sodium chloride, wherein each of the
sucrose and sodium chloride concentrations is between about 0.1% to
about 10% (w/v). In some embodiments, the sucrose concentration is
about 6% and the sodium chloride concentration is about 0.2%.
Alternatively, the sucrose concentration is about 4.5% and the
sodium chloride concentration is about 0.2%.
[0226] In certain embodiments of the invention, the osmolality of
the formulations range from about 200 mOsm/kg to about 350 mOsm/kg,
about 270 mOsm/kg to about 330 mOsm/kg, about 280 mOsm/kg to about
320 mOsm/kg, or about 290 mOsm/kg to about 310 mOsm/kg, e.g., about
300 mOsm/kg. In other words, the formulations of the invention are,
in some embodiments, substantially isotonic, i.e. having
substantially the same osmotic pressure as human blood. Osmolality
can be measured by any means known to those of skill in the art,
such as using vapor pressure or ice-freezing type osmometers. The
osmolality of the formulations of the invention can, for instance,
be regulated by the one or more tonicity agents described
herein.
vi. Amino Acids
[0227] The formulations of the invention may, optionally, further
comprise an amino acid. Examples of amino acids include, but are
not limited to, glycine, alanine, aspartic acid, lysine, serine,
tyrosine, cysteine, glutamine, methionine, arginine, and proline.
In exemplary embodiments, the amino acid is present in the
formulations in an amount from about 0.1% to 5% (w/v). For example,
the amino acid may be present in the formulation in an amount of
about 0.1% (w/v), about 0.2% (w/v), about 0.3% (w/v), about 0.4%
(w/v), about 0.5% (w/v), about 0.6% (w/v), about 0.7% (w/v), about
0.8% (w/v), about 0.9% (w/v), about 1.0% (w/v), about 1.1% (w/v),
about 1.2% (w/v), about 1.3% (w/v), about 1.4% (w/v), about 1.5%
(w/v), about 1.6% (w/v), about 1.7% (w/v), about 1.8% (w/v), about
1.9% (w/v), about 2.0% (w/v), about 3% (w/v), about 4% (w/v), and
about 5% (w/v). Alternatively, the amino acid is present in the
formulation in an amount from about 1.3% to about 1.8% (w/v), or
about 1.4% to about 1.6% (w/v), e.g., about 1.5% (w/v). In further
alternative embodiments, the amino acid is present in the
formulation in an amount from about 0.5% to about 1.5% (w/v), or
about 0.8% to about 1.2% (w/v), e.g., about 1.0% (w/v). An
exemplary amino acid is proline or arginine. For example, proline
may be present in the formulation in an amount from about 1% to
about 2%, (w/v) about 1.3% to about 1.8% (w/v), about 1.4% to about
1.6% (w/v), e.g., about 1.5% (w/v). Alternatively, arginine may be
present in the formulation in an amount from about 0.5% to about
1.5% (w/v), or about 0.8% to about 1.2% (w/v), e.g., about 1.0%
(w/v).
vii. Other Excipients
[0228] Furthermore, the formulations of the invention may comprise
other excipients including, but not limited to, water for
injection, diluents, solubilizing agents, soothing agents,
additional buffers, inorganic or organic salts, antioxidants, or
the like. In some embodiments, however, the formulations of the
invention comprise no other excipients, except those described
above. Other pharmaceutically acceptable carriers, excipients, or
stabilizers, such as those described in Remington's Pharmaceutical
Sciences 16.sup.th edition, Osol, A. Ed. (1980) may be included in
the formulation provided that they do not adversely affect the
desired characteristics of the formulation. In a particular
embodiment, the formulation is substantially free of preservatives,
although, in alternative embodiments, preservatives may be added as
necessary. For example, cryoprotectants or lyoprotectants may be
included in lyophilized formulations.
viii. Liquid or Lyophilized Formulations
[0229] The formulations of the invention may either be liquid
formulations or lyophilized formulations. In some embodiments, the
formulations are liquid formulations. In some embodiments, the
liquid formulations are ready for injection. Alternatively, the
formulations may be lyophilized powders. In some embodiments, the
lyophilized powders are ready to be combined with a solvent just
prior to administration.
ix. Exemplary Formulations
[0230] In one exemplary embodiment of the invention, the invention
provides a stable liquid antibody formulation suitable for
subcutaneous administration, the formulation comprising:
[0231] a) greater than about 80 mg/ml, e.g., about 150 mg/ml, of a
fully human IgG4 anti-LIGHT (lymphotoxin-like, exhibits inducible
expression and competes with HSV glycoprotein D for HVEM, a
receptor expressed by T lymphocytes) antibody comprising a heavy
chain comprising the amino acid sequence of SEQ ID NO: 7 and a
light chain comprising the amino acid sequence of SEQ ID NO: 8;
[0232] b) about 10 mM citrate buffer;
[0233] c) about 0.005% (w/v) polysorbate 20; and
[0234] d) about 4% (w/v) mannitol;
wherein the pH of the formulation is about pH 5.5
[0235] In certain exemplary embodiments, this formulation may be
manufactured by:
[0236] a) dissolving about 10 mM sodium citrate dihydrate in water
for injection and adjusting the pH of the buffered solution to
about pH 5.5, e.g., using either hydrochloric acid or sodium
hydroxide;
[0237] b) adding greater than about 80 mg/ml, e.g., about 150
mg/ml, of a fully human IgG4 anti-LIGHT (lymphotoxin-like, exhibits
inducible expression and competes with HSV glycoprotein D for HVEM,
a receptor expressed by T lymphocytes) antibody comprising a heavy
chain comprising the amino acid sequence of SEQ ID NO: 7 and a
light chain comprising the amino acid sequence of SEQ ID NO: 8,
about 4% (w/v) mannitol, and 0.005% (w/v) polysorbate 20 to the
buffered solution from step a) while stirring in a vessel made of
inert material until completely dissolved, and then adjusting the
pH of the resulting formulation to about pH 5.5 using either
hydrochloric acid or sodium hydroxide, and then adding buffered
solution from step a) to adjust the final weight of the resulting
formulation;
[0238] c) filtering the formulation from step b) under aseptic
conditions using a sterilized, compatible membrane filter having a
nominal pore size of 0.2 .mu.M, and then sterilizing the
formulation by filtration under aseptic conditions into sterilized
containers made out of inert material using a sterilized,
compatible membrane filter having a nominal pore size of 0.2
.mu.M;
[0239] d) filling the formulation from step c) under aseptic
conditions into sterilized vials that are closed with stoppers and
flip-off caps with a flange; and, optionally,
[0240] e) inspecting the containers from step d) for coarse
contaminants, intact sealing, and visible particles.
[0241] In another exemplary embodiment of the invention, the
invention provides a stable liquid antibody formulation suitable
for intravenous administration, the formulation comprising:
[0242] a) about 5 to about 80 mg/mL, e.g., about 50 mg/mL, of a
fully human IgG4 anti-LIGHT (lymphotoxin-like, exhibits inducible
expression and competes with HSV glycoprotein D for HVEM, a
receptor expressed by T lymphocytes) antibody comprising a heavy
chain comprising the amino acid sequence of SEQ ID NO: 7 and a
light chain comprising the amino acid sequence of SEQ ID NO: 8;
[0243] b) about 10 mM citrate buffer; and
[0244] c) about 0.01% (w/v) polysorbate 20;
wherein the pH of the formulation is about pH 5.5.
[0245] In an alternative exemplary embodiment of the invention, the
invention provides a stable lyophilized antibody formulation
suitable for intravenous administration, the formulation
comprising:
[0246] a) about 5 to about 80 mg/mL, e.g., about 50 mg/mL, of a
fully human IgG4 anti-LIGHT (lymphotoxin-like, exhibits inducible
expression and competes with HSV glycoprotein D for HVEM, a
receptor expressed by T lymphocytes) antibody comprising a heavy
chain comprising the amino acid sequence of SEQ ID NO: 7 and a
light chain comprising the amino acid sequence of SEQ ID NO: 8;
[0247] b) about 10 mM citrate buffer;
[0248] c) about 0.01% (w/v) polysorbate 20;
[0249] d) about 5% (w/v) sucrose; and
[0250] e) about 1.5% (w/v) proline;
wherein the pH of the formulation is about pH 5.5.
[0251] In an exemplary embodiment of the invention, the invention
provides a stable antibody formulation comprising:
[0252] a) about 20 mg/mL of a humanized IgG4 anti-CXCR5 (C-X-C
chemokine receptor type 5) antibody comprising a heavy chain
comprising the amino acid sequence of SEQ ID NO: 25 and a light
chain comprising the amino acid sequence of SEQ ID NO: 26;
[0253] b) about 10 mM citrate buffer;
[0254] c) about 0.02% polysorbate 20;
[0255] d) about 6% sucrose; and
[0256] e) about 0.2% sodium chloride;
wherein the pH of the formulation is about pH 6.0.
[0257] In an alternative exemplary embodiment of the invention, the
invention provides a stable antibody formulation comprising:
[0258] a) about 100 mg/mL of a humanized IgG4 anti-CXCR5 (C-X-C
chemokine receptor type 5) antibody comprising a heavy chain
comprising the amino acid sequence of SEQ ID NO: 25 and a light
chain comprising the amino acid sequence of SEQ ID NO: 26;
[0259] b) about 10 mM citrate buffer;
[0260] c) about 0.01% polysorbate 20;
[0261] d) about 4.5% sucrose;
[0262] e) about 0.2% sodium chloride; and
[0263] f) about 1% arginine;
wherein the pH of the formulation is about pH 6.0. x. Stability
[0264] The formulations of the invention are stable at 5.degree. C.
for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months
or more, and typically at least about 12, 18 or 24 months or more.
In exemplary embodiments, they are stable at 5.degree. C. for at
least about 6 months or more. In other exemplary embodiments, they
are stable at 5.degree. C. for at least about 9 months. In further
exemplary embodiments, they are stable at 5.degree. C. for at least
about 1 year or more, and typically about 2 years.
C. Modes of Administration
[0265] In certain embodiments of the invention, the formulations
are suitable for administration parenterally, intravenously,
intramuscularly, intradermally, subcutaneously, or a combination
thereof. The formulations of the invention are suitable for
delivery by a variety of techniques.
[0266] In some embodiments of the invention, the formulation is
administered intravenously. For example, it is desirable that
formulations containing 80 mg/mL of IgG4 binding agent, such as an
antibody, or less are administered intravenously. Therefore, the
formulations are typically sterile. Methods for making formulations
sterile are well known in the art and include, for example,
filtration through sterile filtration membranes or autoclaving the
ingredients of the formulation, with the exception of the
antibodies, at about 120.degree. C. for about 30 minutes. For
example, the invention provides a stable liquid antibody
formulation suitable for intravenous administration, the
formulation comprising: a) about 5 to about 80 mg/mL, e.g., about
50 mg/mL, of a fully human IgG4 anti-LIGHT (lymphotoxin-like,
exhibits inducible expression and competes with HSV glycoprotein D
for HVEM, a receptor expressed by T lymphocytes) antibody
comprising a heavy chain comprising the amino acid sequence of SEQ
ID NO: 7 and a light chain comprising the amino acid sequence of
SEQ ID NO: 8; b) about 10 mM citrate buffer; and c) about 0.01%
(w/v) polysorbate 20; wherein the pH of the formulation is about pH
5.5. Alternatively, the invention provides a stable antibody
formulation comprising: a) about 20 mg/mL of a humanized IgG4
anti-CXCR5 (C-X-C chemokine receptor type 5) antibody comprising a
heavy chain comprising the amino acid sequence of SEQ ID NO: 25 and
a light chain comprising the amino acid sequence of SEQ ID NO: 26;
b) about 10 mM citrate buffer; c) about 0.02% polysorbate 20; d)
about 6% sucrose; and e) about 0.2% sodium chloride; wherein the pH
of the formulation is about pH 6.0.
[0267] In some embodiments of the invention, the formulation is
administered subcutaneously. For example, it is desirable that
formulations containing more than 80 mg/mL of IgG4 binding agent,
such as an antibody, are administered subcutaneously. In a specific
embodiment, it is desirable to administer subcutaneously to
subjects a stable liquid antibody formulation comprising: a) about
150 mg/mL of a fully human IgG4 anti-LIGHT (lymphotoxin-like,
exhibits inducible expression and competes with HSV glycoprotein D
for HVEM, a receptor expressed by T lymphocytes) antibody
comprising a heavy chain comprising the amino acid sequence of SEQ
ID NO: 7 and a light chain comprising the amino acid sequence of
SEQ ID NO: 8; b) about 10 mM citrate buffer; c) about 0.005% (w/v)
polysorbate 20; d) about 4% (w/v) mannitol; and wherein the pH of
the formulation is about pH 5.5. Alternatively, the invention
provides a stable antibody formulation comprising: a) about 100
mg/mL of a humanized IgG4 anti-CXCR5 (C-X-C chemokine receptor type
5) antibody comprising a heavy chain comprising the amino acid
sequence of SEQ ID NO: 25 and a light chain comprising the amino
acid sequence of SEQ ID NO: 26; b) about 10 mM citrate buffer; c)
about 0.01% polysorbate 20; d) about 4.5% sucrose; e) about 0.2%
sodium chloride; and f) about 1% arginine; wherein the pH of the
formulation is about pH 6.0.
D. Dosages and Dosage Forms
[0268] Effective doses of the formulations of the invention vary
depending upon many different factors, including means of
administration, target site, physiological state of the subject,
whether the subject is human or an animal, other medications
administered, and whether treatment is prophylactic or therapeutic.
Usually, the subject is a human, but non-human mammals including
transgenic mammals can also be treated. Treatment dosages may need
to be titrated to optimize safety and efficacy.
[0269] The formulations of the invention may be administered on
multiple occasions. Intervals between single dosages can be daily,
weekly, biweekly, monthly or yearly. Intervals can also be
irregular. In some methods, the dosage is adjusted to achieve a
certain plasma binding agent, such as an antibody, concentration.
Dosage and frequency will vary depending on the half-life of the
anti-LIGHT or anti-CXCR5 binding agent, such as an antibody, in the
subject. In general, human antibodies show the longest half-life,
followed by humanized antibodies, chimeric antibodies, and nonhuman
antibodies.
[0270] In further embodiments, the invention provides a
pharmaceutical unit dosage form comprising a therapeutically
effective amount of a formulation of the invention for the
treatment of one or more diseases in a subject through
administration of the dosage form to the subject. In some
embodiments, the subject is a human. The human may be an adult or
may be an infant. The term "pharmaceutical unit dosage form" refers
to a physically discrete unit suitable as unitary dosages for the
subjects to be treated, each unit containing a predetermined
quantity of active compound calculated to produce the desired
therapeutic/prophylactic effect in association with the required
citrate buffer and pH.
[0271] The unit dosage form may be a container comprising the
formulation. Suitable containers include, but are not limited to,
sealed ampoules, vials, bottles, syringes, and test tubes. The
containers may be formed from a variety of materials, such as glass
or plastic, and may have a sterile access port (for example, the
container may be a vial having a stopper pierceable by a hypodermic
injection needle). In some embodiments, the container is a vial.
Generally, the container should maintain the sterility and
stability of the formulation.
[0272] In specific embodiments, the formulations are packaged in 2
mL vials that are made of clear, colorless type I glass, and closed
with a stopper (fluoropolymer-coated bromobutyl) sealed with
flip-of caps with flange (polypropylene). The vials are, in some
embodiments, filled with 1.2 mL of the formulations so that the
vial has an overfill volume of about 0.2 mL per vial, and an
extractable volume of 1.0 mL. For example, this means that the
dosage strength of anti-LIGHT antibody (e.g., 150 mg/mL) will be
contained within 1 mL of solution.
[0273] In specific embodiment, the formulations are secondarily
packaged in a container, such as a cardboard box, that protects the
vials from light.
E. Methods of Treatment
[0274] Further provided herein are methods for treating a
LIGHT-mediated disease or disorder, the methods comprising
administering a formulation of the invention to a subject. The
invention further relates to a formulation of the invention for use
in a herein-described method for treating a LIGHT-mediated disease
or disorder. In certain embodiments, the LIGHT-mediated disease is
a chronic bowel disease, or an inflammatory bowel disease (IBD),
such as Crohn's disease (CD) or ulcerative colitis (UC). In other
embodiments, the LIGHT mediated disease is graft-versus-host
disease (GVHD).
[0275] Also provided herein are methods for treating a
CXCR5-mediated disease or disorder, the methods comprising
administering a formulation of the invention to a subject. The
invention further relates to a formulation of the invention for use
in a herein-described method for treating a CXCR-5 mediated disease
or disorder. In certain embodiments, the anti-CXCR5 binding agent
is used for reduction of signs and symptoms, inhibition of
progression of structural damage, induction of a major clinical
response, and prevention of disability in adult patients with
moderately to severely active Rheumatoid Arthritis (RA) who have
had inadequate response to one or more Disease-Modifying
Anti-Rheumatic Drugs (DMARDs), such as methotrexate (MTX), or
TNF.alpha. antagonists. The anti-CXCR5 binding agent may be used in
combination with DMARDs or anti-TNF.alpha. agonists.
[0276] In certain embodiments, the formulations of the invention
can be administered in combination with one or more therapies
(e.g., therapies that are not the formulations of the invention
that are currently administered to prevent, treat, manage, and/or
ameliorate a LIGHT-mediated disease or a CXCR5-mediated disease.
The use of the term "in combination" does not restrict the order in
which therapies are administered to a subject. A first therapy can
be administered before (e.g., 1 minute, 45 minutes, 30 minutes, 45
minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48
hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5
weeks, 6 weeks, 8 weeks, or 12 weeks), concurrently, or after
(e.g., 1 minute, 45 minutes, 30 minutes, 45 minutes, 1 hour, 2
hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96
hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8
weeks, or 12 weeks) the administration of a second therapy to a
subject that had, has, or is susceptible to a LIGHT-mediated
disease or a CXCR5-mediated disease. Any additional therapy can be
administered in any order with the other additional therapies.
Non-limiting examples of therapies that can be administered in
combination with an antibody of the invention include approved
anti-inflammatory agents listed in the U.S. Pharmacopoeia and/or
Physician's Desk Reference.
F. Kits
[0277] Certain embodiments of the invention include a kit
comprising a formulation of the invention. The kit may further
comprise one or more containers comprising pharmaceutically
acceptable excipients, and include other materials desirable from a
commercial and user standpoint, including filters, needles and
syringes. Associated with the kits can be instructions customarily
included in commercial packages of therapeutic, prophylactic or
diagnostic products, that contain information about, for example,
the indications, usage, dosage, manufacture, administration,
contra-indications, and/or warnings concerning the use of such
therapeutic, prophylactic or diagnostic products. The kit can also
be associated with a label that can be any kind of data carrier
(e.g., a leaflet, sticker, chip, print or bar code) comprising
information. In certain embodiments, the instructions etc. as
listed above can be comprised in or on the label. The kit can
further comprise a device for administration of the formulation,
and particularly a device that contains the formulation, i.e., a
pre-filled device such as, but not limited to, a pre-filled syringe
or a pre-filled autoinjector. The kit can also comprise a container
comprising the formulation, i.e., a pre-filled container, such as a
pre-filled vial, cartouche, sachet, or ampoule.
G. Combination of Different Embodiments
[0278] In the context of the present invention, any of the herein
described embodiments can be combined with one or more of the other
herein described embodiments unless explicitly stated to the
contrary. Particularly, any of the herein described binding agents
and antibodies and the herein described formulations thereof can be
used in combination with any of the kits, pre-filled devices or
pre-filled containers, or can be used in the methods of treatment
or medical uses as described herein in connection with the
respective antibody (e.g., the stable formulations comprising the
anti-LIGHT antibodies or anti-CXCR5 antibodies can be combined with
any of the herein described kits, containers or devices). Any of
the herein described binding agents specifically binding an antigen
(e.g., a binding agent specifically binding LIGHT or a binding
agent specifically binding CXCR5) can also be used in any of the
methods of treatment that are described herein in connection with
the respective antibodies (i.e., anti-LIGHT or anti-CXCR5) and vice
versa.
EXAMPLES
[0279] To help illustrate the invention, the following examples are
provided. The examples are not intended to limit the scope of the
invention in any way. In general, the practice of the present
invention employs, unless otherwise indicated, conventional
techniques of pharmaceutical formulation, chemistry, molecular
biology, recombinant DNA technology, immunology such as antibody
technology, and standard techniques of polypeptide preparation as
described, for example, in Sambrook, Fritsch and Maniatis,
Molecular Cloning: Cold Spring Harbor Laboratory Press (1989);
Antibody Engineering Protocols (Methods in Molecular Biology),
volume 51, Ed.: Paul S., Humana Press (1996); Antibody Engineering:
A Practical Approach (Practical Approach Series, 169), Eds.:
McCafferty J. et al., Humana Press (1996); Antibodies: A Laboratory
Manual, Harlow and Lane, Cold Spring Harbor Laboratory Press
(1999); and Current Protocols in Molecular Biology, Eds. Ausubel et
al., John Wiley & Sons (1992).
Anti-LIGHT
[0280] A fully human IgG4 anti-LIGHT antibody comprising a heavy
chain comprising the amino acid sequence of SEQ ID NO: 7 and a
light chain comprising the amino acid sequence of SEQ ID NO: 8 (the
"Lead LIGHT Antibody") was used in Examples 1-9 in order to
determine optimal formulation conditions.
Materials
Drug Substance Batch
[0281] The Lead Antibody, formulated in phosphate buffered saline
(PBS) at a concentration of 5.5 mg/mL and at a pH of 7.3 (the
"Original Formulation", "PBS Formulation", or "Reference Lot"), was
used in the following examples.
Excipients
[0282] Table 1 shows the excipients that were used in the following
examples, which were chosen according to their
acceptability/suitability for use in parenteral products.
TABLE-US-00019 TABLE 1 Excipients used in this study Excipients
Art. No./Charge Supplier Arginine 1.01587 Merck Citric acid 100241
Merck HCl 114027 Merck Sodium acetate 1.06265 Merck Sodium chloride
10158 Riedel de Haen Sodium hydroxide 114076 Merck Sodium citrate
114196 Boehringer Ingelheim KG Polysorbate 20 139850 Fluka
Trehalose-dihydrat T9531 Sigma-Aldrich
Methods
[0283] The following methods were used to manufacture the
experimental formulations and the formulations of the invention
containing the Lead LIGHT Antibody.
Manufacturing & Composition of Buffers
[0284] All buffers were manufactured under stirring to dissolve the
respective excipients. pH was adjusted using 0.1 M HCl or 0.1 M
NaOH. The general concentration of all buffers was 10 mM.
Manufacturing & Composition of Excipient Stock Solutions
[0285] All stock solutions were manufactured under stirring to
dissolve the excipients. Concentration was given as weight/weight
(w/w).
Sterile Filtration of Samples
[0286] All samples, solutions, buffers, etc. were sterile filtered
(0.22 .mu.m) using a Sartopore-2 membrane. The samples were
filtered into sterilized bottles or vials and closed under aseptic
conditions inside a clean-bench to prevent microbiological
contamination.
Mechanical Stress Test
[0287] Mechanical stress with an agitation speed of 350/minute for
2.5 hours at room temperature was performed using a horizontal
laboratory shaker with a 26 mm distance (shaker & incubation
hood from Buhler Company). 2R vials were filled with 1 mL solution
with a head space of about 2.5 mL. The mechanical stress test was
planned and performed during the first pre-formulation studies and
during relevant studies for surfactant selection.
Thermal Stress Test
[0288] Thermal stress was used as a stress test during all steps of
the pre-formulation program. The samples were stored at +40.degree.
C. for either 24 hours or 7 days, depending on the study.
Analytical Methods in Formulation Fill and Finish
[0289] The following analytical methods were used in the
formulation fill and finish in the following examples.
Appearance
[0290] Appearance of the antibody solutions were checked visually,
and additionally documented by taking a picture with a digital
camera.
pH
[0291] All pH measurements were performed using a pH-meter with a
micro-electrode.
Concentration Using UV
[0292] The protein concentrations of all antibody solutions were
measured against buffer using a NanoDrop ND 1000. Proteins
concentrations near or below 5 mg/mL were diluted 1:3, while higher
protein concentrations near 20 mg/mL were diluted 1:20, and the
absorption was measured at 215 nm and 280 nm.
Dynamic Light Scattering (DLS)
[0293] The hydrodynamic diameter of the molecule was measured using
laser light scattering. The samples were sterile filtered prior to
the analytics if turbidity was observed, thus only soluble
aggregates could be detected.
Differencial Scanning Calorimetry (DSC)
[0294] Aliquots of most pre-formulation samples were examined by
DSC using a VPCapillary DSC from Microcal and scanned in the
autosampling instrument at 90.degree. C./hour with a filter time of
2 seconds. 400 .mu.l samples were placed into 96-well plates and
analyzed for the unfolding temperature Tm.
Osmolarity
[0295] Osmolarity was measured using an automated Knaur
Osmometer.
Density
[0296] Density of the formulations was measured using a falling
sphere viscosimeter DMA4500 Anton Paar.
Analytical methods in Bioanalytics FF
[0297] The following analytical methods were used in the
bioanalytics fill and finish in the following examples.
Size Exclusion Chromatography (SEC)
[0298] Aggregates, as well as degradation products of the Lead
Antibody, were quantified using size exclusion GL chromatography.
The test was carried out by isocratic HPLC with a SUPERDEX 200
10/300 column.
SDS-PAGE, Reducing and Non-Reducing
[0299] Sodium dodecyl sulfate polyacrylamide gel electrophoresis
(SDS-PAGE) was used to analyze the molecular integrity (e.g., half
molecules) and the purity. This electrophoretic analysis was
performed with 4-12% gradient gels under reducing and non-reducing
conditions. The proteins were visualized with Coomassie staining
after electrophoretic separation.
Weak Cation Exchange (WCX)
[0300] Weak cation exchange chromatography was used to monitor the
charge heterogeneity of the antibody. The percentages of basic,
neutral, and acidic isoforms were reported. The test was carried
out by discontinuous high performance liquid chromatography (HPLC)
with a ProPac WCX10 column.
Antigen-Enzyme Linked Immunosorbent Assay (Antigen-ELISA)
[0301] Antigen-ELISA was performed to determine the functionality
of the antibody. The binding properties to native LIGHT protein
were monitored in comparison to the current standard of the
antibody. This potency was reported as the relative EC.sub.50.
Isoelectric Focusing (IEF)
[0302] IEF was performed. The isoelectric pattern was specific for
the Lead Antibody and served as an identification test. Degradation
could be seen by a different charge pattern.
Storage
[0303] All buffer solutions, excipient solutions, and samples were
stored at 5.degree. C. (.+-.3.degree. C.), if not otherwise
mentioned.
Summary of all Formulations Prepared & Analyzed
[0304] Table 2 below shows a summary of all of the formulations
that were prepared and analyzed in the following examples. Each of
the formulations contained the Lead LIGHT Antibody at the
concentration listed.
TABLE-US-00020 TABLE 2 Summary of all formulations prepared and
analyzed Com- Sample number Buffer pH Concentration ment
Formulation 1.1 Citrate 10 mM 5.0 5.5 mg/mL Formulation 1.2 Citrate
10 mM 5.5 5.5 mg/mL Formulation 1.3 Citrate 10 mM 6.0 5.5 mg/mL
Formulation 2 PBS 7.3 <80 5 mg/mLmg/mL Very Citrate 10 mM 5.0
turbid Formulation 3.1 PS20 0.01% 5 mg/mL Citrate 10 mM 5.5
Formulation 3.2 PS20 0.01% 5 mg/mL Citrate 10 mM 5.5 Formulation 4
PS20 0.01% 80 mg/mL clear Citrate 10 mM 5.0 Formulation 5 PS20
0.01% 5 mg/mL Citrate 10 mM 5.5 PS20 0.01% Proline 1.5% Formulation
6.1 Sucrose 5% 50 mg/mL Lyo Citrate 10 mM 5.5 PS20 0.01%
Formulation 6.2 Sucrose 5% 50 mg/mL Lyo Formulation 7 Histidine 5.5
50 mg/mL 10 mM Histidine 5.5 10 mM Formulation 8 PS20 0.01% 50
mg/mL Formulation 9 Citrate 10 mM 5.5 50 mg/mL Citrate 10 mM 5.5
Formulation 10 PS20 0.01% 50 mg/mL Citrate 10 mM 5.5 Formulation 11
Sucrose 5% 50 mg/mL lyo Formulation 12 Citrate 10 mM 7.0 5 mg/mL
.mu.DSC Formulation 13 PBS 5.0 5 mg/mL .mu.DSC
Example 1
Characterization of a Phosphate Buffered Saline (PBS) Formulation
and Disadvantages Associated Therewith
[0305] In this example, the Reference Lot was characterized. As
stated in the Materials section above, the Reference Lot contains
the Lead LIGHT Antibody formulated in phosphate buffered saline
(PBS) at a concentration of 5.5 mg/mL and at a pH of 7.3, and
produced in research solutions Vitry (BioSCP).
[0306] Isoelectric focusing (IEF) was used to determine the
isoelectric point (pI) of the Lead Antibody. The pI of the Lead
LIGHT Antibody was theoretically calculated as 6.28, and then
measured by denaturated isoelectric focusing standard methods known
in the art. As shown in FIG. 1, the main bands show that the pI of
the Lead LIGHT Antibody was 6.8-7.2.
[0307] SDS-PAGE was used to identify the molecular weight of the
antibody monomer, potential aggregates, or the presence of
half-molecules. FIG. 2 shows an SDS-PAGE gel that compared
different Reference Lot batches under reducing and non-reducing
conditions. An ELISA was used to determine the antigen binding
activity of the Lead LIGHT Antibody. FIG. 3 shows an ELISA graph
that was used to determine the antigen binding activity of the
first and second batches of Reference Lot.
[0308] SEC was used to determine the presence of aggregates, as
well as degradation products of the first batch of Reference Lot.
As shown in FIG. 4, size exclusion chromatography detected high
molecular weight proteins (HMWP), e.g., di-/oligomers (RRT0.8) or
aggregates, and low molecular weight proteins (LMWPs) or
degradation products. The first batch of Reference Lot had a purity
of 97% monomer content.
[0309] WCX was used to monitor the charge heterogeneity of the
first batch of Reference Lot. As shown in FIG. 5, rearrangements of
acidic, neutral, and basic isoforms occurred during stability
studies. The first batch of Reference Lot had a distribution of
acidic/neutral/basic isoforms of 42.3/55.6/1.9%.
[0310] DSC was used to analyze the unfolding temperature Tm of the
first batch of Reference Lot. As shown in FIG. 6, the three domains
of the antibody unfold at 68.degree. C., 75.degree. C., and
78.degree. C.
[0311] DLS was used to determine the hydrodynamic diameter of the
antibody monomer and potential soluble aggregates. As shown in
FIGS. 7 & 8, a hydrodynamic diameter of about 10 nm was
detected, but aggregates were seen in PBS. However, aggregates were
not seen in citrate buffer (FIG. 10).
Example 2
Development of Citrate-Buffered Formulations, and Advantages
Associated Therewith
[0312] The original buffer, phosphate buffered saline (PBS) at a pH
of 7.3, was, in terms of pH, very close to the isoelectric point
(pI) of the Lead Antibody (see Example 1). In addition, the
Original Formulation exhibited aggregates; half-molecules;
degradation products; low molecular weight proteins (LMWPs); high
molecular weight proteins (HMWPs); and rearrangements of acidic,
basic, and neutral antibody isoforms (see Example 1). Thus, there
was a need for an improved formulation that does not suffer from
these disadvantages.
[0313] Formulations of the Lead LIGHT Antibody (a fully human IgG4
anti-LIGHT antibody comprising a heavy chain comprising the amino
acid sequence of SEQ ID NO: 7 and a light chain comprising the
amino acid sequence of SEQ ID NO: 8) containing 10 mM citrate
buffer at a pH of 5, 5.5, and 6, with and without polysorbate 20
were tested. Table 3 shows the analytical results of the first
batch of Reference Lot, and the various experimental formulations
of the Lead LIGHT Antibody formulated into citrate, at a pH of 5.0
and 5.5 and 6.0, with and without polysorbate 20. Aggregates were
found in dynamic light scattering (DLS) measurements for the
Reference Lot, but not in all other tested formulations. Tm, as
measured by differencial scanning calorimetry (.mu.DSC), indicated
that the higher the pH, the higher the thermodynamic stability
could be assumed. But for high antibody concentrated formulations,
the pH had to be chosen below the pI of the antibody.
[0314] As shown in Table 4, size exclusion chromatography (SEC)
data showed a significantly reduced amount of high molecular weight
proteins (HMWPs) for the Lead LIGHT Antibody in citrate buffer as
compared to the Reference Lot (phosphate buffer at pH 7.3). In
contrast, no differences could be detected with SDS-PAGE (Table
5).
TABLE-US-00021 TABLE 3 Analytical Results of Formulations Concen-
Sample Tm1 Tm2 Tm3 ZAve tration number [.degree. C.] [.degree. C.]
[.degree. C.] pH [nm] [mg/mL] Buffer Reference 67.94 75.00 77.37
7.3 179.85 5.5 PBS Lot Formulation 58.39 69.98 75.75 5.0 10.97 5.0
Citrate 1.1 10 mM Formulation 62.02 72.26 76.59 5.5 10.71 5.0
Citrate 1.2 10 mM Formulation 65.46 73.74 77.02 6.0 10.81 5.0
Citrate 1.3 10 mM Formulation 58.33 69.93 75.74 5.0 13.14 5.0
Citrate 3.1 10 mM PS20 0.01% Formulation 61.42 71.97 76.45 5.5
12.79 5.0 Citrate 3.2 10 mM PS20 0.01%
TABLE-US-00022 TABLE 4 SEC data of Formulations ANTIBODY RRT0.8
LMWP HMWPs Rel. Rel. Rel. Rel. Monomer Area Area Area Area Area
Area Area Area Content Sample Name mAU*min % mAU*min % mAU*min %
mAU*min % [mg/mL] Ref. Lot 255.61 98.00 3.98 1.52 1.50 0.57 0.59
0.23 Formulation 223.23 98.07 3.22 1.42 1.01 0.44 0.16 0.07 45.49
3.1 Formulation 257.09 98.24 3.74 1.43 0.79 0.30 0.09 0.03 48.92
3.2
TABLE-US-00023 TABLE 5 SDS-PAGE data of Formulations size Rel. QTY
size Rel. QTY size Rel. QTY size Rel. QTY Sample Name kDa % kDa %
kDa % kDa % comment Ref. Lot 172.5 98.4 150.1 1.4 68.4 0.2
Additional bands <0.5% Formulation 166.1 97.7 147.8 2 71.5 0.3
Identical pattern to Ref. Lot 3.1 Formulation 166.2 96.2 147.2 3.4
71.4 0.4 Identical pattern to Ref. Lot 3.2
Example 3
Development of High-Concentration Antibody Formulations
[0315] In view of the improvements provided by the Citrate-Buffered
Antibody Formulation of Example 2, the citrate buffer components
were optimized for increased concentrations of Lead LIGHT Antibody.
Table 6 shows the analytical results of the first batch of high
concentration (about 40 mg/ml) antibody formulations: high
phosphate buffered saline (PBS) at a pH of 7.3 (Formulation 2) or
citrate at a pH of 5.5 with polysorbate 20 (Formulation 4).
TABLE-US-00024 TABLE 6 Analytical results of Formulations 2 & 4
Sample Tm1 Tm2 Tm3 ZAve Concentration number [.degree. C.]
[.degree. C.] [.degree. C.] pH [nm] [mg/mL] Buffer Reference 67.94
75.00 77.37 7.3 10.05 5.5 PBS Lot Formulation 67.87 74.87 77.28 7.3
12.89 42.1 PBS 2 Formulation 61.55 72.00 76.48 5.5 16.71 39.97
Citrate 4 10 mM PS20 0.01%
[0316] Slightly reduced monomer content was observed after
concentrating the protein solution in citrate buffer. Moreover,
dimer concentration was reduced and high molecular weight proteins
(HMWPs) could be significantly reduced as well (see Table 7). In
contrast, these impurities and byproducts were increased by
increasing the concentration in phosphate buffer. No differences
could be detected with SDS-PAGE analysis (Table 8).
TABLE-US-00025 TABLE 7 SEC data of Formulations 2 & 4 SEC
Analysis ANTIBODY RRT0.8 LMWP HMWPs Rel. Rel. Rel. Rel. Monomer
Area Area Area Area Area Area Area Area Gehalt Sample Name mAU*min
% mAU*min % mAU*min % mAU*min % [mg/mL] Ref. Lot 255.61 98.00 3.98
1.52 1.50 0.57 0.59 0.23 Formulation 2 121.42 97.39 2.13 1.71 0.98
0.79 0.15 0.12 44.08 Formulation 4 141.90 97.65 2.17 1.49 1.16 0.80
0.09 0.06 45.83
TABLE-US-00026 TABLE 8 SDS-PAGE data of Formulations 2 & 4
SDS-PAGE Analysis Antibody Main 2. band Half molecules Additional
bands size Rel. QTY size Rel. QTY size Rel. QTY size Rel. QTY
Sample Name kDa % kDa % kDa % kDa % comment Ref. Lot 172.5 98.4
150.1 1.4 68.4 0.2 Additional bands <0.5% Formulation 2 170.6
97.9 147.6 1.9 72.2 0.2 Identical pattern to Ref. Lot Formulation 4
171 97.2 149 2.5 70.5 0.3 Identical pattern to Ref. Lot
Example 4
Development of Lyophilized Antibody Formulations
[0317] To test the feasibility of lyophilization, different
lyophilized experimental formulations were manufactured and
subjected to stability analysis. The concentration of the Lead
LIGHT Antibody was increased to 50 mg/mL.
[0318] Table 9 shows the freeze drying program that was used in
this example.
TABLE-US-00027 TABLE 9 Freeze drying program Lyo program (vacuum)
N.degree. 8 Chamber loading 5 min/RT/100% Freezing 2 h/-45.degree.
C./100% Main drying I 30 min/-45.degree. C./30% Main drying II 5
h/-20.degree. C./30% Main drying III 8 h/+20.degree. C./30% Final
drying 2 h/+20.degree. C./3%
[0319] Table 10 shows the analytical results of the first batch of
Reference Lot, and the various experimental lyophilized
formulations of the Lead LIGHT Antibody formulated into various
combinations of citrate buffer, sucrose, polysorbate 20, and
proline.
[0320] As shown in Table 11, high molecular weight proteins (HMWPs)
could clearly be reduced by using citrate buffer. No differences in
dimer content were seen over the time of storage at 40.degree. C.
An increase of low molecular weight proteins (LMWPs) after freeze
drying was observed. As before, these differences could not be
detected with SDS-PAGE analysis (Table 12).
TABLE-US-00028 TABLE 10 Analytical data of Formulations 6-6.2 &
11 Time/ ZAve Sample number Tm1 Tm2 Tm3 Temp. pH [nm] Concentration
Buffer Reference 67.94 75.00 77.37 7.3 10.05 5.5 mg/mL PBS Lot
Formulation Nd Nd Nd N/A 5.7 17.46 57.32 Citrate 6 10 mM PS20 0.01%
Formulation 64.30 72.61 77.02 T0 5.7 59.66 Nd Citrate 6.1
T1/5.degree. C. 5.7 18.85 Nd 10 mM T1/40.degree. C. 5.7 19.12 Nd
PS20 T2/5.degree. C. 5.7 Nd Nd 0.01% T2/40.degree. C. 5.7 Nd Nd
Prolin 1.5% Sucrose 5% Formulation 65.45 75.08 79.37 T0 5.7 19.58
Nd Citrate 6.2 T1/5.degree. C. 5.7 31.34 Nd 10 mM T1/40.degree. C.
5.7 18.1 Nd PS20 T2/5.degree. C. 5.7 Nd Nd 0.01% T2/40.degree. C.
5.7 Nd Nd Sucrose 5% Formulation 68.84 75.61 77.91 T0 7.0 98.60
56.49 PBS 11 T1/5.degree. C. 5.7 20.22 Nd Sucrose T1/40.degree. C.
5.7 22.68 Nd 5% T2/5.degree. C. 5.7 Nd Nd T2/40.degree. C. 5.7 Nd
Nd
TABLE-US-00029 TABLE 11 SEC data of Formulations 6.1-6.2 & 11
SEC Analysis ANTIBODY RRT0.8 LMWP HMWPs Area Rel. Area Area Rel.
Area Area Rel. Area Area Rel. Area Sample Name Time [mAU*min] [%]
[mAU*min] [%] [mAU*min] [%] [mAU*min] [%] Ref. Lot 255.61 98.00
3.98 1.52 1.50 0.57 0.59 0.23 Formulation 6.1 T0 222.94 97.43 3.89
1.70 1.91 0.83 0.09 0.04 Formulation 6.1 T1 5.degree. C. 369.72
97.57 6.31 1.66 2.75 0.73 0.18 0.05 Formulation 6.1 T1 40.degree.
C. 405.49 97.35 7.33 1.76 3.60 0.86 0.12 0.03 Formulation 6.1 T2
5.degree. C. 422.46 97.59 7.01 1.62 2.74 0.63 0.68 0.16 Formulation
6.1 T2 40.degree. C. 289.65 97.28 5.50 1.85 2.13 0.72 0.48 0.16
Formulation 6.2 T0 230.06 97.61 3.93 1.67 1.64 0.70 0.07 0.03
Formulation 6.2 T1 5.degree. C. 407.17 97.56 6.81 1.63 3.23 0.77
0.17 0.04 Formulation 6.2 T1 40.degree. C. 468.74 97.36 8.79 1.83
3.78 0.78 0.16 0.03 Formulation 6.2 T2 5.degree. C. 552.31 97.64
9.80 1.73 2.96 0.52 0.61 0.11 Formulation 6.2 T2 40.degree. C.
249.95 96.78 5.39 2.09 2.39 0.93 0.52 0.20 Formulation 11 T0 211.45
97.49 3.64 1.68 1.47 0.68 0.35 0.16 Formulation 11 T1 5.degree. C.
339.08 97.71 5.45 1.57 2.28 0.66 0.23 0.07 Formulation 11 T1
40.degree. C. 700.91 97.30 12.69 1.76 5.19 0.72 1.60 0.22
Formulation 11 T2 5.degree. C. 325.80 97.17 5.80 1.73 2.17 0.65
1.52 0.45 Formulation 11 T2 40.degree. C. 229.29 96.96 4.33 1.83
1.78 0.75 1.09 0.46
TABLE-US-00030 TABLE 12 SDS-PAGE data of Formulations 6.1-6.2 &
11 SDS-PAGE Analysis ANTIBODY Main 2.Band HM Additional bands Rel.
Rel. Rel. Rel. Sample Name Sample ID size QTY size QTY size QTY
size QTY comment Ref. Lot 182.9 95.6 161.2 2.3 73.8 0.5 Formulation
6.1 T0 175.6 94.7 156.1 2.7 73.5 0.5 Formulation 6.1 T1 5.degree.
C. 180.2 86.9 159.9 11.4 75.9 0.1 Formulation 6.1 T1 40.degree. C.
179.2 90.4 158.5 7.5 76.1 0.4 Formulation 6.1 T2 5.degree. C. 177.3
95.6 157.9 2.1 74.9 0.3 Formulation 6.1 T2 40.degree. C. 179.8 94.7
159.8 2.9 75.4 0.3 Formulation 6.2 T0 176.6 94.9 156.3 2.6 73.6 0.5
Formulation 6.2 T1 5.degree. C. 180.2 89.8 159.3 7.9 76.3 0.4
Formulation 6.2 T1 40.degree. C. 182.1 88.7 160.9 9.4 76.3 0.1
Formulation 6.2 T2 5.degree. C. 177.5 95.5 160.2 2.9 75.4 0.2
Formulation 6.2 T2 40.degree. C. 180.9 95.5 161.5 2.4 75.7 0.3
Formulation 11 T0 178.7 95.1 156.5 2.3 73.7 0.4 Formulation 11 T1
5.degree. C. 181.0 70.0 154.7 25.7 74.5 0.3 Formulation 11 T1
40.degree. C. 181.3 66.2 154.2 28.9 74.5 0.3 Formulation 11 T2
5.degree. C. 177.7 87.5 155.9 10.9 75.2 0.3 Formulation 11 T2
40.degree. C. 176.8 86.2 155.2 12.0 74.5 0.3
Example 5
Accelerated Stability Study
[0321] An accelerated stability study was performed with citrate
and histidine buffers. Table 13 shows the analytical results of the
first batch of Reference Lot, and the various xperimental
formulations of the Lead LIGHT Antibody formulated into various
combinations of citrate buffer or histidine buffer. Notably, the
citrate formulation of the invention appeared in all experiments to
perform better than histidine. In particular, citrate formulations
had a higher monomer content compared to the both the Reference Lot
batch and the histidine (Table 13) and the content or low molecular
weight proteins (LMWPs) and high molecular weight proteins (HMWPs)
were also significantly lower (Table 14). As before, these
differences could not be detected with SDS-PAGE analysis (Table
15).
TABLE-US-00031 TABLE 13 Analytical data of Formulations 7, 8, 9
& 10 Sample Tm1 Tm2 Tm3 ZAve Concentration number [.degree. C.]
[.degree. C.] [.degree. C.] pH [nm] [mg/mL] Buffer Ref. Lot 67.94
75.00 77.37 7.3 10.05 5.5 PBS Formulation 7 58.95 68.51 76.20 5.5
12.97 53.65 Histidine 10 mM Formulation 8 58.69 68.23 76.12 5.4
13.29 58.72 Histidine 10 mM PS20 0.01% Formulation 9 61.67 72.01
76.53 5.6 59.26 55.01 Citrate 10 mM Formulation 10 62.24 72.32
76.61 5.6 17.3 55.8 Citrate 10 mM PS20 0.01%
TABLE-US-00032 TABLE 14 SEC Analysis of Formulations 7 & 8
& 9 & 10 ANTIBODY RRT0.8 LMWP HMWPs Monomer Area Rel. Area
Area Rel. Area Area Rel. Area Area Rel. Area Content Sample Name
Time [mAU*min] [%] [mAU*min] [%] [mAU*min] [%] [mAU*min] [%]
[mg/mL] Ref. Lot 282.42 97.40 4.55 1.57 2.15 0.74 0.85 0.29
Formulation 7 T0 184.4 95.1 163.8 1.6 72.8 1 Formulation 7 T1
5.degree. C. 390.84 97.85 5.72 1.43 2.80 0.70 0.06 0.01 Formulation
7 T1 40.degree. C. 379.81 96.74 7.04 1.79 5.77 1.47 0 0 Formulation
7 T2 5.degree. C. 863.01 97.75 14.19 1.61 4.14 0.47 1.54 0.18
164.88 Formulation 7 T2 40.degree. C. 1085.91 95.22 29.59 2.60
23.25 2.04 1.69 0.15 207.47 Formulation 8 T0 184.6 94.9 165.9 2.1
72.7 0.8 Formulation 8 T1 5.degree. C. 507.64 97.74 7.52 1.45 4.19
0.81 0.05 0.01 Formulation 8 T1 40.degree. C. 461.44 96.98 8.05
1.69 6.19 1.30 0 0 Formulation 8 T2 5.degree. C. 416.54 97.46 6.59
1.54 3.49 0.82 0.79 0.18 79.58 Formulation 8 T2 40.degree. C.
422.21 93.23 11.17 2.47 18.40 4.06 1.11 0.25 80.66 Formulation 9 T0
229.01 97.63 3.75 1.60 1.63 0.70 0.19 0.08 45.28 Formulation 9 T1
5.degree. C. 307.94 97.96 4.2 1.34 2.20 0.7 0 0 Formulation 9 T1
40.degree. C. 319.10 97.54 5.24 1.60 2.59 0.79 0.23 0.07
Formulation 9 T2 5.degree. C. 337.15 97.48 5.41 1.56 2.84 0.82 0.49
0.14 64.41 Formulation 9 T2 40.degree. C. 325.54 96.26 7.78 2.30
3.66 1.08 1.20 0.36 62.20 Formulation 10 T0 233.11 97.54 3.84 1.61
1.97 0.82 0.08 0.03 46.09 Formulation 10 T1 5.degree. C. 343.38
97.77 5.21 1.48 2.58 0.73 0.04 0.01 Formulation 10 T1 40.degree. C.
329.56 97.21 5.06 1.49 4.29 1.26 0.13 0.04 Formulation 10 T2
5.degree. C. 343.33 97.43 5.47 1.55 3.06 0.87 0.53 0.15 65.59
Formulation 10 T2 40.degree. C. 257.20 94.59 5.59 2.06 8.98 3.30
0.15 0.05 49.14
TABLE-US-00033 TABLE 15 SDS-PAGE data of Formulations 7 & 8
& 9 & 10 SDS-PAGE Analysis ANTIBODY Main 2.band Half
molecules Additional bands size Rel. QTY size Rel. QTY size Rel.
QTY size Rel. QTY Sample Name Time/Temp [kDa] [%] [kDa] [%] [kDa]
[%] [kDa] [%] comment Ref. Lot 173.6 96.3 155.8 2.2 74 0.4
Formulation 7 T0 184.4 95.1 163.8 1.6 72.8 1 Formulation 7 T1
5.degree. C. 183.0 91.1 159.9 7.2 76.1 0.4 Formulation 7 T1
40.degree. C. 182.2 83.1 158.4 13.8 74.0 0.4 Formulation 7 T2
5.degree. C. 181.5 95.7 160.3 2.7 75.6 0.3 Formulation 7 T2
40.degree. C. 173.0 84.6 151.1 10.3 73.9 0.7 12.1 0.9 more LMWPs
Formulation 8 T0 184.6 94.9 165.9 2.1 72.7 0.8 Formulation 8 T1
5.degree. C. 180.1 86.2 158.3 11.4 73.9 0.4 Formulation 8 T1
40.degree. C. 180.9 79.4 158.2 16.9 74.0 0.3 Formulation 8 T2
5.degree. C. 175.1 95.2 154.9 3.1 74.4 0.3 Formulation 8 T2
40.degree. C. 174.8 84.7 150.5 9.2 74.0 0.9 12.1 1.5 more LMWPs
Formulation 9 T0 187.7 95.5 163.1 1.1 72.9 0.9 Formulation 9 T1
5.degree. C. 178.9 65.8 160.4 29.6 73.7 0.9 Formulation 9 T1
40.degree. C. 184.7 82.9 160.3 14.8 74.4 0.3 Formulation 9 T2
5.degree. C. 176.2 95.6 155.6 2.6 73.6 0.3 Formulation 9 T2
40.degree. C. 174.3 91.5 153.9 3.1 73.1 0.3 12.1 0.2 more LMWPs
Formulation 10 T0 182.5 95.2 161.3 1.6 72.3 0.8 Formulation 10 T1
5.degree. C. 184.5 68.4 156.4 26.6 75.1 0.3 Formulation 10 T1
40.degree. C. 180.8 65.4 153.8 28.8 74.8 0.3 Formulation 10 T2
5.degree. C. 188.7 88.6 165.0 9.6 73.5 0.2 Formulation 10 T2
40.degree. C. 181.7 78.9 158.2 15.8 75.7 0.8 12.6 1.3 more
LMWPs
Example 6
Development of High Antibody Concentration Formulation for
Subcutaneous Administration
[0322] Based on the successful results of the citrate-buffered
formulations of Examples 2-5, a high-concentration (150 mg/ml)
antibody formulation suitable for subcutaneous administration was
developed. Formulation development was performed on the Lead LIGHT
Antibody with the goal of developing a liquid dosage form with an
acceptable shelf life when stored at +2 to +8.degree. C.
Preliminary stress studies showed the formation of subvisible and
visible particles, high molecular weight species and more basic
species. Therefore, these parameters were monitored during the
screening of formulation candidates using visual assessment,
dynamic light scattering, light obscuration, size exclusion
chromatography, sodium dodocyl sulphate polyacrylamide gel
electrophoresis, and weak cationic exchange chromatography.
Different liquid formulations were used in the pre-formulation and
formulation trials prior to selection of the clinical formulation.
According to the findings, a formulation in 10 mM citrate buffer
adjusted to pH 5.5 (Formulation 14) was selected for further
development. The pH of the formulation is in the region of optimal
physical and chemical stability of the drug substance and
acceptable physiological tolerability (e.g., osmolarity).
[0323] As shown in Table 16, Formulation 14 is a solution for
injection and is an aqueous, sterile, and clear solution containing
the Lead LIGHT Antibody, sodium citrate dihydrate (buffering
agent), polysorbate 20 (stabilizing agent), and mannitol (tonicity
agent). Sodium hydroxide solution and hydrochloric acid were used
to adjust the pH to 5.5.
[0324] All excipients were soluble and well tolerated
pharmacopoeial standard excipients for parenterals and listed in
Ph. Eur. and USP.
TABLE-US-00034 TABLE 16 Composition Composition per vial per mL
(1.2 mL) Reference to Components.sup.a (mg) (mg) Function
standards.sup.b Lead Antibody 150.00 180.00 Drug substance In-house
Sodium citrate 2.94 3.53 Buffering agent Ph. Eur., dehydrate USP
Mannitol 40.00 48.00 Tonicity agent Ph. Eur., USP Polysorbate 20
0.05 0.06 Stabilizing Ph. Eur., agent NF, JP Hydrochloric q.s pH
5.5 q.s. pH 5.5 Acidifying Ph. Eur., acid, agent NF concentrated
[Hydrochloric acid] Sodium q.s.pH 5.5 q.s. pH 5.5 Alkalizing Ph.
Eur., hydroxide agent NF Water for q.s. 1 mL q.s. 1.2 mL Solvent
Ph. Eur., injection USP Nitrogen Process aid for filtration Ph.
Eur., NF .sup.aComponents are listed according to their
pharmacopoeial names. If more than one monograph exists, other
names are given in brackets, along with the compendial origin.
.sup.bReference is made to the current edition of the
Pharmacopoeia.
Example 7
Manufacturing Process for Subcutaneous Antibody Formulation
[0325] A GMP-compliant manufacturing process was developed for the
subcutaneous, high-concentration antibody formulation (Formulation
14) of Example 6. The manufacturing procedure consisted of
dissolving, pH adjustment, sterile filtration, filling, and
packaging steps.
[0326] Drug substance (the Lead LIGHT Antibody) is provided in a
liquid form in the formulation buffer (10 mM citrate buffer at pH
5.5). The excipients were all water-soluble and dissolved in the
initial aqueous portion of the formulation buffer during
manufacture. The bulk drug substance solution was further diluted
with the same formulation buffer to reach the concentration of 150
mg/mL of Lead LIGHT Antibody. The bulk solution was well mixed to
facilitate the dissolution process and to ensure homogeneity.
[0327] Sterilization by filtration was carried out (according to
Ph. Eur. and USP) using bacteria retentive filters having a nominal
pore size of 0.2 .mu.m. An additional filtration procedure before
"sterilization by filtration" was performed to ensure a low
bioburden. Bioburden sampling was done before the pre-filtration
step as well as the sterile filtration step.
[0328] Preparation and filling of the sterilized solution into the
suitable containers was performed under aseptic conditions. This
was in accordance with pharmacopoeial monographs and related
guidelines, which required sterilization by filtration and
subsequent aseptic processing. The equipment, which comes into
contact with the product after the step "sterilization by
filtration", was sterilized by heat or steam using a validated
method (according to Ph. Eur./USP).
[0329] Vials were filled to about 1.2 mL to ensure an extractable
volume of 1.0 mL. The 2 mL vials were made of clear, colorless type
I glass, and closed with a stopper (fluoropolymer-coated
bromobutyl) sealed with flip-off caps with a flange
(polypropylene). The primary packaging materials met the
requirements of the Ph. Eur. and USP. The suitability of the
primary packaging materials was substantiated by the results of the
stability tests. Incompatibilities with the primary packaging
material used were not observed. Secondary packaging which provides
protection of the product from light.
[0330] Compatibility with application syringes was assessed using 3
different syringe sizes and needles diameters (between 25 and 28
gauges) on the drug product solution. No differences in terms of
product quality were obtained. Compatibility was proven for a time
period of 8 hours.
[0331] Formulation 14 was made in 5 L batches, the composition of
which is shown in Table 17. However, the batch size may be adjusted
according to clinical requirements.
TABLE-US-00035 TABLE 17 Batch formula Batch size 5 Liter.sup.a
Components [g] Lead Antibody .sup.b 750.00 Mannitol 200.00
Polysorbate 20 0.25 Sodium citrate dihydrate 14.71 Hydrochloric
acid, concentrated .sup.c q.s. pH 5.5 Sodium hydroxide .sup.c q.s.
pH 5.5 Water for injection Ad 5285.25 .sup.d Nitrogen Process aid
for filtration .sup.aThe vials were filled with a volume of 1.2 mL
to ensure an extractable volume of 1.0 mL. A 6.0 L batch size
therefore results in a theoretical yield of 5000 vials. .sup.b For
pH adjustment. .sup.c This was calculated according to the density
of the final drug product solution (1.05705 mg/mL)
[0332] The manufacturing process and process controls for
Formulation 14 are shown in the flow diagram in FIG. 9. Batch
manufacturing included the following steps: [0333] I. Sodium
citrate dihydrate was dissolved in water for injection while
stirring in a vessel made of inert material (e.g., stainless steel
or glass), until completely dissolved. The pH value was adjusted to
5.5 using hydrochloric acid, diluted (e.g., 0.1 M hydrochloric
acid) and/or sodium hydroxide solution (e.g., 0.1 M sodium
hydroxide), if necessary. [0334] II. Lead Antibody, mannitol, and
polysorbate 20 were diluted in the buffer solution from step 1
while stirring in a vessel made of inert material (e.g., stainless
steel or glass) until completely dissolved. If necessary, the pH
value was adjusted to 5.5 using hydrochloric acid, diluted (e.g., 1
M hydrochloric acid) or sodium hydroxide solution (e.g., 1 M sodium
hydroxide). Buffer solution from step 1 (remainder) was added to
adjust the final weight. [0335] III. a) Pre-filtration: [0336]
Solution from step II was filtered under aseptic conditions using a
sterilized, compatible membrane filter (e.g., polyether sulfone or
polyvinylidene difluoride) having a nominal pore size of 0.2 .mu.m.
[0337] b) Sterilization by filtration: [0338] Solution from step
III.a was sterilized by filtration under aseptic conditions into
sterilized containers made out of inert material (e.g., stainless
steel or glass) using a sterilized, compatible membrane filter
(e.g., polyether sulfone or polyvinylidene difluoride) having a
nominal pore size of 0.2 .mu.m. [0339] IV. Solution from step III.b
was filled under aseptic conditions into sterilized vials, which
were closed with stoppers and flip-off caps with a flange. [0340]
V. The containers from step IV were inspected for coarse
contaminants, intact sealing, and visible particles. [0341] VI. The
inspected containers from step V were additionally packaged in
suitable containers (e.g., cardboard boxes).
[0342] In addition, DLS was used to determine the hydrodynamic
diameter of the antibody monomer and potential soluble aggregates.
As shown in FIG. 10, aggregates were not seen in citrate buffer.
However, as shown in FIGS. 7 & 8, aggregates were seen in PBS.
Due to the higher concentration of antibody, an increase in ZAve to
28 nm was observed, compared to the sample in PBS.
Example 8
Stability Profile for Subcutaneous Antibody Formulation
[0343] The stability profile of the clinical batch (batch 2) of
Example 7 was assessed for storage under long term and accelerated
testing conditions according to ICH guidelines. Samples were packed
and stored in 2 mL clear and colorless vials (glass type I) closed
with stoppers (fluoropolymer-coated bromobutyl) and flip-off caps
with a flange (polypropylene).
[0344] The following tests were performed during stability:
appearance (clarity, color), assay (antigen ELISA, UV), purity
(SEC, SDS-PAGE under reducing and non-reducing conditions),
molecular integrity (SDS-PAGE under non-reducing conditions),
charge heterogeneity (weak cation exchange chromatography,
isoelectric focusing), pH, sterility, bacterial endotoxins,
particulate matter (visible and subvisible particles), and closure
integrity.
[0345] The samples were stored in inverted and upright positions.
The results of the inverted storage were presented as the more
stringent condition. Stability data at -20.degree. C., +5.degree.
C. and +25.degree. C. are presented in Tables 18-20, respectively.
The investigations on physical, chemical, and biological properties
of storage under long term testing conditions confirmed a good
stability of the drug product at 5.degree. C. (see Table 19). Under
accelerated testing conditions (+25.degree. C.), only a slight
decrease in the purity was detected by size exclusion
chromatography (see Table 20). Therefore, it was concluded that the
drug product should be stored at +2.degree. C. to +8.degree. C.
protected from light.
TABLE-US-00036 TABLE 18 Long term stability at -20.degree. C. for
drug product Drug product: Lead LIGHT Antibody Batch no.: 11_021
solution for injection Dosage strength: 150 mg/mL Formulation no.:
14 Container/closure: 2 mL glass vials Storage condition:
-20.degree. C. .+-. 5.degree. C. Storage orientation: Upright Time
Initial 1 3 6 12 18 24 Test item results month months months months
months months Appearance of solution Clarity <I III II >IV IV
>IV Color BY7 BY7 BY6 BY6 BY6 BY7 Identification IEF Isoelectric
pattern Conforms Conforms Conforms Conforms Conforms Conforms Assay
Antigen-ELISA EC50 value (in 76% 110% 76% 103% 96% 105% comparison
to reference Total protein content 153 mg/mL 148 mg/mL 151 mg/mL
151 mg/mL 149 mg/mL 156 mg/mL (UV) Purity HPLC (SEC) Monomer (%
area) 98.2%.sup. 97.5% 96.2%.sup. 94.5% 94.3%.sup. 94.1% HMWPs (%
area) 1.3% 2.3% 3.7% 5.4% 5.5% 5.5% LMWPs (% area) 0.4% 0.2% 0.1%
0.0% 0.1% 0.5% SDS-PAGE under non reducing conditions Half
molecules <1.0%.sup. <1.0% <1.0%.sup. 2.7% <1.0%.sup.
<5.0% SDS-PAGE under reducing conditions Relative purity 98%
100% 100% 100% 96% 100% Molecular integrity SDS-PAGE under non-
reducing conditions Gel pattern Conforms Does not Conforms Conforms
Conforms Conforms conform Charge heterogeneity HPLC (WCX) acidic
40% 36% 44% 42% 43% 41% neutral 55% 60% 54% 47% 51% 56% basic
isoforms (% area) 5% 4% 2% .sup. 2% 6% 3% pH (potentiometry) 5.5
5.5 5.5 5.5 5.5 5.5 Particulate matter Complies Complies Complies
Complies Complies Complies (visible particles) Particulate matter
Not Not Not Not (subvisible particles) tested tested tested tested
Number of particles per 313 33 vial .gtoreq.10 .mu.m Number of
particles per 10 5 vial .gtoreq.25 .mu.m Closure integrity Complies
Not Not Not Complies Not tested tested tested tested Microbial
contamination TAMC <1 CFU/2 mL Not Not Not Not Not tested tested
tested tested tested TAnMC <1 CFU/2 mL Not Not Not Not Not
tested tested tested tested tested
TABLE-US-00037 TABLE 19 Long term stability at +5.degree. C. for
drug product Drug product: Lead LIGHT Antibody Batch no.: 11_021
solution for injection Dosage strength: 150 mg/mL Formulation no.:
14 Container/closure: 2 mL glass vials Storage condition:
+5.degree. C. .+-. 3.degree. C. Storage orientation: Upright Time
Initial 1 3 6 12 18 24 Test item results month months months months
months months Appearance of solution Clarity <I <I <I
<I <IError! >IV Reference source not found. Color BY7 BY7
BY7 BY7 BY6 BY7 Identification IEF Isoelectric pattern Conforms
Conforms Conforms Conforms Conforms Conforms Assay Antigen-ELISA
EC50 value (in 76% 119% 83% 107% 96% 115% comparison to reference
Total protein content 153 mg/mL 150 mg/mL 150 mg/mL 151 mg/mL 148
mg/mL 155 mg/mL (UV) Purity HPLC (SEC) Monomer (% area) 98.2%.sup.
98.5% 98.5%.sup. 98.3% 98.0%.sup. 97.4% HMWPs (% area) 1.3% 1.4%
1.5% 1.7% 1.9% 2.0% LMWPs (% area) 0.4% 0.0% 0.0% 0.0% 0.1% 0.6%
SDS-PAGE under non reducing conditions Half molecules <1.0%.sup.
<1.0% <1.0%.sup. 1.8% <1.0%.sup. <1.0% SDS-PAGE under
reducing conditions Relative purity 98% 100% 100% 100% 96% 100%
Molecular integrity SDS-PAGE under non- reducing conditions Gel
pattern Conforms Does not Conforms Conforms Conforms Conforms
conform Charge heterogeneity HPLC (WCX) acidic 40% 36% 44% 42% 43%
39% neutral 55% 60% 54% 57% 52% 57% basic isoforms (% area) 5% 4%
2% .sup. 2% 5% 4% pH (potentiometry) 5.5 5.5 5.5 5.5 5.5 5.5
Particulate matter Complies Complies Complies Complies Complies
Complies (visible particles) Particulate matter Not Not Not Not
(subvisible particles) tested tested tested tested Number of
particles per 313 35 vial .gtoreq.10 .mu.m Number of particles per
10 5 vial .gtoreq.25 .mu.m Closure integrity complies Not Not Not
Complies Complies tested tested tested Microbial contamination TAMC
<1 CFU/2 mL Not Not Not Not Not tested tested tested tested
tested TAnMC <1 CFU/2 mL Not Not Not Not Not tested tested
tested tested tested
TABLE-US-00038 TABLE 20 Accelerated stability at +25.degree. C. for
drug product Drug product: Lead LIGHT Antibody Batch no.: 11_021
solution for injection Dosage strength: 150 mg/mL Formulation no.:
14 Container/closure: 2 mL glass vials Storage condition:
+25.degree. C. .+-. 2.degree. C./60% .+-. 5% RH Storage
orientation: Upright Time Test Item Initial results 1 month 3
months 6 months Appearance of solution Clarity <I <I <I
<I Color BY7 BY7 BY7 BY7 Identification IEF Conforms Conforms
Conforms Conforms Isoelectric pattern Assay Antigen-ELISA 76% 108%
96% 111% EC50 value (in comparison to reference Total protein
content (UV) 153 mg/mL 149 mg/mL 150 mg/mL 151 mg/mL Purity HPLC
(SEC) Monomer (% area) 98.2%.sup. 98.2% 97.7%.sup. 96.8% HMWPs (%
area) 1.3% 1.7% 2.2% 3.1% LMWPs (% area) 0.4% 0.1% 0.1% 0.1%
SDS-PAGE under non <1.0%.sup. <1.0% <1.0%.sup. <1.0%
reducing conditions Half molecules 98% 100% 100% 100% SDS-PAGE
under reducing conditions Relative purity Molecular integrity
SDS-PAGE under non- Conforms Does not Conforms Conforms reducing
conditions conform Gel pattern Charge heterogeneity HPLC (WCX)
acidic 40% 36% 44% 41% neutral 55% 59% 53% 56% basic isoforms (%
area) 5% 5% 2% 3% pH (potentiometry) 5.5 5.5 5.5 5.5 Particulate
matter (visible Complies Complies Complies Complies particles)
Particulate matter (subvisible particles) Number of particles per
313 Not tested Not tested 22 vial .gtoreq.10 .mu.m Number of
particles per 10 1 vial .gtoreq.25 .mu.m Closure integrity complies
Not tested Not tested Complies Microbial contamination TAMC <1
CFU/2 mL Not tested Not tested <1 CFU/2 mL TAnMC <1 CFU/2 mL
Not tested Not tested <1 CFU/2 mL
Example 9
Development of Ultra-High Antibody Concentration Formulation for
Subcutaneous Administration
[0346] Based upon the successful results of the citrate-buffered
formulations for antibody concentrations up to 150 mg/mL in Example
7, higher concentrated (up to 250 mg/ml) antibody formulations
suitable for subcutaneous administration were developed.
[0347] Preliminary data showed that the formulation of antibody
concentrations above 150 mg/mL may lead to higher viscosities
affecting usage of the formulation.
TABLE-US-00039 TABLE 21 Ultra high concentrations with formulation
14 DLS Size exclusion Concentration Density Viscosity z-average
chromatography Sample [mg/mL] [kg/m-3] [mPa] [nm] HMWPs Monomer
LMWPs Lead LIGHT 237 1.066 42.29 30 1.3 98.6 0.0 Antibody_11_30A
Lead LIGHT 212 1.059 22.58 39 1.3 98.7 0.0 Antibody_11_30B Lead
LIGHT 181 1.052 13.57 28 1.3 98.7 0.1 Antibody_11_30C Lead LIGHT
173 1.046 8.8 27 1.2 98.8 0.0 Antibody_11_30D Lead LIGHT 143 1.039
6.16 25 1.1 98.8 0.1 Antibody_11_30E
[0348] As can be seen in Table 21, the viscosity decreases with
lower antibody concentrations, yet still being in an acceptable
range at the higher concentration formulated with formulation 14.
All other parameters seemed to be unaffected or just slightly
affected by the ultra-high concentrations.
[0349] As shown in Table 22, the antibody concentrations did not
affect the stability of the formulations, which was indicated by
identical 1 month stability data at long term and stress
conditions.
TABLE-US-00040 TABLE 22 1 month stability data of ultra high
concentrated Lead Antibody formulations Concen- tration Mono-
[mg/mL] HMWPs mer LMWPs Lead LIGHT 237 4.7 95.2 0.2 Antibody_11_30A
40.degree. C. Lead LIGHT 212 4.4 95.4 0.2 Antibody_11_30B
40.degree. C. Lead LIGHT 181 5.8 91.7 2.6 Antibody_11_30C
40.degree. C. Lead LIGHT 173 3.9 96.0 0.2 Antibody_11_30D
40.degree. C. Lead LIGHT 143 4.2 94.7 1.1 Antibody_11_30E
40.degree. C. Lead LIGHT 237 1.4 98.6 0.0 Antibody_11_30A 5.degree.
C. Lead LIGHT 212 1.3 98.7 0.0 Antibody_11_30B 5.degree. C. Lead
LIGHT 181 1.3 98.7 0.0 Antibody_11_30C 5.degree. C. Lead LIGHT 173
1.2 98.8 0.0 Antibody_11_30D 5.degree. C. Lead LIGHT 143 1.1 98.9
0.0 Antibody_11_30E 5.degree. C.
Anti-CXCR5 (20 mg/mL) Pre-Formulation Studies
[0350] A humanized IgG4 anti-CXCR5 antibody comprising a heavy
chain comprising the amino acid sequence of SEQ ID NO: 25 and a
light chain comprising the amino acid sequence of SEQ ID NO: 26
(the "Lead CXCR5 Antibody") was used in Examples 10-12 in order to
determine optimal formulation conditions for a 20 mg/mL
formulation.
[0351] The Lead Antibody is a humanized monoclonal antibody (mAB)
specific to human CXCR5, with an engineered IgG4 framework
containing 2 amino acid substitutions aimed at reducing
half-molecules (S241P) and effector functions (L248E). The Lead
CXCR5 Antibody protein structure is comprised of two kappa light
chains, each with a molecular weight of approximately 24 kDa, and
two IgG4 heavy chains, each with a molecular weight of
approximately 48 kDa linked through disulfide bridges. Each light
chain consists of 219 amino acid residues, and each heavy chain
consists of 437 amino acid residues.
[0352] The data in Examples 10-12 were collected during
preformulation activities for the Lead CXCR5 Antibody and its drug
product for intravenous and subcutaneous administration. The
objective of the preformulation studies was to provide good
stability of buffered Lead CXCR5 Antibody solutions with a target
concentration of 20 mg/mL, with special emphasis on the aggregation
behavior of the Lead CXCR5 Antibody and its tendency to form
half-molecules, as the Lead Antibody is an IgG4 subclass antibody,
which is prone to aggregation and the formation of particles.
Materials
Drug Substance (DS)
[0353] The Lead CXCR5 Antibody batch RSN0151 was formulated in PBS
pH 7.2 with a concentration of 5.13 mg/mL.
Excipients
[0354] Table 23 shows excipients that were used during the
preformulation studies.
TABLE-US-00041 TABLE 23 Excipients used during preformulation Art.
No./ Excipients Charge Supplier Acetic acid A002630 MTP/VWR
International SAS Arginine-HCl A1700 AppliChem Arginine 1.01587
Merck Benzyl alcohol 113594 Industrial Affairs/Harrmann &
Reimer Citric acid 100241 Merck Dextran 40 CL-AO19A Meito Sangyo
Glycine 113560 Industrial Affairs.sup.1/Tessenderlo Chemie. HCl
114027 Industrial Affairs.sup.1/Merck Histidine 1.04352 Merck
Potassium dihydrogen 1.04871 Merck phosphate Lysine 62840 Fluka
Magnesium chloride 814733 Merck Maltose 105911 Merck Mannitol
A000780 MTP/Roquette Freres Sodium acetate 1.06265 Merck Sodium
chloride 10158 Industrial Affairs.sup.1/Riedel de Haen Sodium
hydroxide 114076 Industrial Affairs.sup.1/Merck Sodium citrate
114196 Industrial Affairs.sup.1/Boehringer Ingelheim KG
Di-sodiumhydogen- 1.06586 Merck phosphate anhydrous Polysorbate 20
139850 Industrial Affairs.sup.1/Fluka Succinat/Succinic acid 14079
Fluka Sucrose S3929 Sigma-Aldrich Trehalose-dihydrat T9531
Sigma-Aldrich Trometamol 114011 Industrial Affairs/Merck
Methods
[0355] The following methods were used to manufacture the
experimental formulations and the formulations of the invention
containing the Lead CXCR5 Antibody.
Manufacturing & Composition of Buffers
[0356] All buffers were manufactured by stirring constantly to
dissolve the respective excipients. pH was adjusted using 0.1 M HCl
or 0.1 M NaOH. The general concentration of all buffers was 10
mM.
Manufacturing & Composition of Excipient Stock Solutions
[0357] All stock solutions were manufactured by stirring constantly
to dissolve the excipients. Concentrations were given as
weight/weight (w/w).
UF/DF--Small Scale
[0358] UF/DF experiments were performed using Vivaspin units
(Sartorius Stedim, Gottingen, Germany) with a Hydrosart membrane
and a 30 kDa cut-off for removing phosphate buffer and replacing it
with the appropriate buffers and to increase the concentration.
These units were placed in a common laboratory centrifuge
(Multifuge 3S, Haereus, Germany) and centrifuged at 2000 rpm (860
G, rotor radius 192 mm) at +5.degree. C.
UF/DF--Larger Scale
[0359] UF/DF experiments were performed using Vivaflow units
(Sartorius Stedim, Gottingen, Germany) with a Hydrosart membrane
and a 30 kDa cut-off for removing phosphate buffer and replacing it
with the appropriate buffers and to increase the concentration. The
equipment was placed inside a clean-bench under aseptic conditions
and the process was performed at room temperature.
Sterile Filtration of Samples
[0360] All samples, solutions, buffers, etc. were sterile filtered
(0.22 .mu.m) using a Sartopore-2 membrane. The samples were
filtered into sterilized bottles or vials and closed under aseptic
conditions inside a clean-bench to prevent microbiological
contamination.
Mechanical Stress Test
[0361] Mechanical stress with an agitation speed of 350 rpm/min for
2.5 hours at room temperature was performed using a horizontal
laboratory shaker with a 26 mm distance (shaker & incubation
hood from Baler Company). 2R vials were filled with 1 mL solution
with a head space of about 2.5 cm.sup.3.
[0362] A mechanical stress test was planned and performed during
the first preformulation studies. As the analytical results did not
show any additional information compared to the thermal stress
tests, during buffer selection or pH selection, this test was only
used during surfactant selection.
Thermal Stress Test
[0363] Thermal stress was used as an accelerated stress test during
all steps of the preformulation program. The samples were stored at
+40.degree. C. either for 24 h, 7 days, or 3 months, depending on
the study.
Analytical Methods in Formulation Fill and Finish
[0364] The following analytical methods were used in the following
examples.
Appearance
[0365] Appearance of the antibody solutions was checked visually
and additionally documented by taking a picture with a camera.
pH
[0366] All pH measurements were performed using a pH-meter with a
micro-electrode.
Concentration Using UV
[0367] The protein concentration of all antibody solutions was
measured against buffer using a Nanodrop ND1000. Protein
concentrations near or below 5 mg/mL were diluted 1:3, and higher
protein concentrations near 20 mg/mL were diluted 1:20, before
measuring the absorption at 215 nm and 280 nm.
Dynamic Light Scattering (DLS)
[0368] The hydrodynamic diameter of the molecule was measured using
laser light scattering. The samples were sterile filtered prior to
the analytics if turbidity was observed, thus only soluble
aggregates could be detected.
Thioflavine-Ttest
[0369] Fluorescence measurements of some preformulation samples
were carried out using a Tecan GENios Plus, XFLOUR4. The samples
were stressed mechanically (4 h at +37.degree. C., agitation speed
300 units/min and 26 mm distance in a shaker & incubator hood
from Buhler company). Thioflavin-T fluorescence spectra were
measured at room temperature. 10 .mu.l Thioflavin-T solution (10.1
mM in ultrapure water) was added to 1 ml of the formulations and
gently mixed. The mixture was then dispensed into a black Eppendorf
V-shaped cup, and then into a 96-well plate (100 .mu.L per
well).
[0370] The thioflavin-T test was used in the beginning of
preformulation activities to detect insoluble aggregates. But, as
these aggregates can be seen visually as a turbidity of the
solution, this method was not used for the whole preformulation
program.
Differential Scanning Calorimetry (DSC)
[0371] Aliquots of the preformulation samples were examined by DSC
using a VPCapillary DSC from Microcal and scanned in the auto
sampling instrument at 90.degree. C./h with a filter time of 2 sec.
400 .mu.l samples were placed into 96-well plates and analyzed for
the unfolding temperature Tm.
Osmolarity
[0372] Osmolarity was measured using an automated Knaur
Osmometer.
Density
[0373] Density of the formulations was measured using a falling
sphere viscosimeter DMA4500 Anton Paar.
Analytical Methods in Bioanalytics FF
Size Exclusion Chromatography (SEC)
[0374] Oligomers/dimers of the antibodies were quantified by using
size exclusion chromatography. The test was carried out by
isocratic HPLC with a SUPERDEX 200 10/300 column
SDS-PAGE, Reducing and Non-Reducing
[0375] Sodium dodecyl sulfate polyacrylamide gel electrophoresis
(SDS-PAGE) was used to analyze the molecular integrity (e.g., half
molecules) and appearance of degradation products. This
electrophoresis analysis was performed with 15% homogenous gels
under reducing and non-reducing conditions. The proteins were
visualized with silver staining after electrophoresis
separation.
WCX
[0376] Weak cationic exchange chromatography (WCX) was used to
monitor the charge heterogeneity of the antibody. The percentage of
basic, neutral, and acidic isoforms was reported. The test was
carried out by discontinuous HPLC with a ProPac WCX10 column
Antigen-ELISA
[0377] Antigen-ELISA was performed to determine the functionality
of the antibody. The binding property to a 28mer peptide of the
CXCR5 antigen was monitored in comparison to the current standard
of the antibody. This potency was reported as the relative
EC50.
Isoelectric Focusing (IEF)
[0378] IEF was performed.
Storage
[0379] All buffer solutions, excipient solutions, and samples were
stored at +5.degree. C., if not otherwise mentioned.
Summary of all Formulations Prepared & Analyzed
[0380] Table 24 below shows a summary of all of the formulations
that were prepared and analyzed in Examples 10-12. Each of the
formulations contained the Lead CXCR5 Antibody. PBS stands for
phosphate buffered saline. PB stands for phosphate buffer. PS
stands for polysorbate. LA stands for the Lead CXCR5 Antibody.
TABLE-US-00042 TABLE 24 Summary of all formulations prepared and
analyzed Sample number Buffer pH LA_09_05-1 PBS 155 mM 7.5
LA_09_05-2 PBS 155 mM 7.0 LA_09_05-3 PBS 155 mM 6.5 LA_09_06-1 PB 5
mM 7.5 LA_09_06-2 PB 5 mM 7.0 LA_09_06-3 PB 5 mM 6.5 LA_09_07-1 PB
10 mM 7.5 LA_09_07-2 PB 10 mM 7.0 LA_09_07-3 PB 10 mM 6.5
LA_09_08-1 Citrate 10 mM 7.0 LA_09_08-2 Citrate 10 mM 6.5
LA_09_08-3 Citrate 10 mM 6.0 LA_09_08-4 Citrate 10 mM 5.5
LA_09_08-5 Citrate 10 mM 5.0 LA_09_09-1 Saline 150 mM 6.0
LA_09_10-1 Acetate 10 mM 5.5 LA_09_10-2 Acetate 10 mM 5.0
LA_09_11-1 Succinate 10 mM 6.0 LA_09_11-2 Succinate 10 mM 5.5
LA_09_11-3 Succinate 10 mM 5.0 LA_09_12-1 Histidine 10 mM 6.5
LA_09_12-2 Histidine 10 mM 6.0 LA_09_12-3 Histidine 10 mM 5.5
LA_09_13-1 Glycine 10 mM 8.0 LA_09_13-2 Glycine 10 mM 7.0
LA_09_14-1 Arginine 10 mM 8.0 LA_09_14-2 Arginine 10 mM 6.0
LA_09_15-1 TRIS 10 mM 8.5 LA_09_15-2 TRIS 10 mM 7.5 LA_09_16
Citrate 10 mM 6.0 LA_09_16_1 Citrate 10 mM/PS20 6.0 LA_09_16_2
Citrate 10 mM/PS80 6.0 LA_09_16_3 Citrate 10 mM/LutrolF68 6.0
LA_09_16_4 Citrate 10 mM/Cremophor 6.0 LA_09_16_5 Citrate 10
mM/SolutolHS15 6.0 LA_09_16_6 Citrate 10 mM/SDS 6.0 LA_09_17
Acetate 10 mM 5.5 LA_09_17_1 Acetate 10 mM + PS20 5.5 LA_09_17_2
Acetate 10 mM + PS80 5.5 LA_09_17_3 Acetate 10 mM + Lutrol F68 5.5
LA_09_17_4 Acetate 10 mM + Cremophor R40 5.5 LA_09_17_5 Acetate 10
mM + Solutol HS15 5.5 LA_09_17_6 Acetate 10 mM + SDS 5.5 LA_09_18
Succinate 10 mM 5.0 LA_09_18_1 Succinate 10 mM + PS20 5.0
LA_09_18_2 Succinate 10 mM + PS80 5.0 LA_09_18_3 Succinate 10 mM +
Lutrol F68 5.0 LA_09_18_4 Succinate 10 mM + Cremophor 5.0
LA_09_18_5 Succinate 10 mM + Solutol HS15 5.0 LA_09_19 Histidine 10
mM 5.5 LA_09_19_1 Histidine 10 mM + PS20 5.5 LA_09_19_2 Histidine
10 mM + PS80 5.5 LA_09_19_3 Histidine 10 mM + Lutrol F68 5.5
LA_09_19_4 Histidine 10 mM + Cremophor 5.5 LA_09_19_5 Histidine 10
mM + Solutol HS15 5.5 LA_09_20 Arginine 10 mM 6.0 LA_09_20_1
Arginine 10 mM + PS20 6.0 LA_09_20_2 Arginine 10 mM + PS80 6.0
LA_09_20_3 Arginine 10 mM + Lutrol F68 6.0 LA_09_20_4 Arginine 10
mM + Cremophor 6.0 LA_09_20_5 Arginine 10 mM + Solutol HS15 6.0
LA_09_21 Histidine 10 mM + PS20 5.5 LA_09_22 PBS 155 mM 7.2
LA_09_22_1 PBS 155 mM 7.2 LA_09_22_2 PBS 155 mM + NaCl 7.2
LA_09_22_3 PBS 155 mM + MgCl.sub.2 7.2 LA_09_22_4 PBS 155 mM +
CaCl.sub.2 7.2 LA_09_22_5 PBS 155 mM + Mannitol 7.2 LA_09_22_6 PBS
155 mM + Maltose 7.2 LA_09_22_7 PBS 155 mM + Trehalose 7.2
LA_09_22_8 PBS 155 mM + Sucrose 7.2 LA_09_22_9 PBS 155 mM +
Dextran40 7.2 LA_09_22_10 PBS 155 mM + Benzyl alcohol 7.2
LA_09_22_11 PBS 155 mM + Arginine 7.2 LA_09_22_12 PBS 155 mM +
Lysine 7.2 LA_09_23 Citrate 10 mM (=LA_09_16) 6.0 LA_09_23_1
Citrate 10 mM 6.0 LA_09_23_2 Citrate 10 mM + NaCl 6.0 LA_09_23_3
Citrate 10 mM + MgCl.sub.2 6.0 LA_09_23_4 Citrate 10 mM + Mannitol
6.0 LA_09_23_5 Citrate 10 mM + Maltose 6.0 LA_09_23_6 Citrate 10 mM
+ Trehalose 6.0 LA_09_23_7 Citrate 10 mM + Sucrose 6.0 LA_09_23_8
Citrate 10 mM + Benzyl alcohol 6.0 LA_09_23_9 Citrate 10 mM +
Arginine 6.0 LA_09_23_10 Citrate 10 mM + Lysine 6.0 LA_09_24
Acetate 10 mM (=LA_09_17) 5.5 LA_09_24_1 Acetate 10 mM 5.5
LA_09_24_2 Acetate 10 mM + NaCl 5.5 LA_09_24_3 Acetate 10 mM +
MgCl.sub.2 5.5 LA_09_24_4 Acetate 10 mM + Mannitol 5.5 LA_09_24_5
Acetate 10 mM + Maltose 5.5 LA_09_24_6 Acetate 10 mM + Trehalose
5.5 LA_09_24_7 Acetate 10 mM + Sucrose 5.5 LA_09_24_8 Acetate 10 mM
+ Benzyl alcohol 5.5 LA_09_24_9 Acetate 10 mM + Arginine 5.5
LA_09_24_10 Acetate 10 mM + Lysine 5.5 LA_09_25 Histidine 10 mM
(=LA_09_19) 5.5 LA_09_25_1 Histidine 10 mM + NaCl 50 mM 5.5
LA_09_25_2 Histidine 10 mM + MgCl.sub.2 50 mM 5.5 LA_09_25_3
Histidine 10 mM + Mannitol 5% 5.5 LA_09_25_4 Histidine 10 mM +
Maltose 10% 5.5 LA_09_26_1 Histidine 10 mM + PS20 (=LA_09_21) 5.5
LA_09_26_2 Histidine 10 mM + PS20 + NaCl 50 mM 5.5 LA_09_26_3
Histidine 10 mM + PS20 + MgC.sub.l2 50 mM 5.5 LA_09_26_4 Histidine
+ PS20 + 5% Mannitol 5.5 LA_09_26_5 Histidine + PS20 + 10% Maltose
5.5 LA_09_26_6 Histidine + PS20 + 6% Trehalose 5.5 LA_09_26_7
Histidine + PS20 + 5% Sucrose 5.5 LA_09_26_8 Histidine + PS20 + 9
mg Benzyl alcohol 5.5 LA_09_26_9 Histidine + PS20 + 20 mM
Arginine-HCl 5.5 LA_09_26_10 Histidine + PS20 + 20 mM Lysine 5.5
LA_09_27 Citrate 10 mM + PS20 6.0 LA_09_27_A Citrate 10 mM + PS20
Prototype formulation 6.0 LA_09_27_B Citrate 10 mM + PS20 Prototype
formulation 6.0 LA_09_27_C Citrate 10 mM + PS20 Prototype
formulation 6.0 LA_09_27_D Citrate 10 mM + PS20 Prototype
formulation 6.0 LA_09_28 Acetate 10 mM + PS20 5.5 LA_09_28_A
Acetate 10 mM + PS20 Prototype formulation 5.5 LA_09_28_B Acetate
10 mM + PS20 Prototype formulation 5.5 LA_09_28_C Acetate 10 mM +
PS20 Prototype formulation 5.5 LA_09_28_D Acetate 10 mM + PS20
Prototype formulation 5.5 LA_09_29 Histidine 10 mM + PS20 5.0
LA_09_29_A Histidine 10 mM + PS20 Prototype formulation 5.0
LA_09_29_B Histidine 10 mM + PS20 Prototype formulation 5.0
LA_09_29_C Histidine 10 mM + PS20 Prototype formulation 5.0
LA_09_29_D Histidine 10 mM + PS20 Prototype formulation 5.0
Example 10
Phosphate Buffer Formulation
[0381] The following will give an overview on results of the
characterization of the Lead CXCR5 Antibody drug substance in
phosphate buffer.
IEF
[0382] The pI (isoelectric point) of the Lead CXCR5 Antibody was
theoretically calculated as 7.6, and confirmed by denaturized
isoelectric focusing (pI of 7.6-8.4). See FIG. 11.
SDS-PAGE
[0383] SDS-PAGE was used to determine the molecular weight of the
antibody monomer, potential aggregates, or the presence of
half-molecules. FIG. 12 showed an example of an SDS-PAGE gel to
compare different drug substance batches under reducing and
non-reducing conditions.
ELISA
[0384] FIG. 13 shows an example of an ELISA graph to determine
antigen binding activity of the Lead Antibody.
SEC
[0385] As shown in FIG. 14, size exclusion chromatography detected
high molecular weight proteins (HMWP), e.g., di-/oligomers or
aggregates and low molecular weight proteins (LMWPs) or degradation
products. The Lead CXCR5 Antibody batch had a purity of 99% monomer
content.
WCX
[0386] Weak cationic exchange chromatography for the Lead Antibody
shows in FIG. 15, display charge heterogeneity. During stability
studies, the arrangement of the acidic peaks changed and the
percentage of basic isoforms increased. The Lead CXCR5 Antibody had
a distribution of acidic/neutral/basic isoforms of 14/85/1%.
Dynamic Light Scattering
[0387] As shown in FIG. 16, DLS was used to determine the
hydrodynamic diameter of the antibody monomer and potential soluble
aggregates.
[0388] In conclusion, the Lead Antibody might be stable in PBS, but
aggregate formation is easy to generate by shear forces or light
stress.
[0389] In addition, the pH of PBS is close to the pI of the Lead
CXCR5 Antibody. Therefore, the formulation should be formulated at
least one pH step below the pI.
[0390] Table 25 shows 3 the results of a three month stability
study for the Lead CXCR5 Antibody. The Lead Antibody was stored at
different temperatures and analyzed after one and three months.
TABLE-US-00043 TABLE 25 Analytical results of a 3-month stability
study of DS Temperature 5.degree. C. -20.degree. C. 25.degree. C.
Test Spec for 1 3 1 3 1 3 Method item release Release month months
month months month months Appearance Color Monitoring >B9,
>B9, >B9, >B9, >B9, >B9, >B9, >BY7 >BY7
>BY7 >BY7 >BY7 >BY7 >BY7 Clarity Monitoring <I
<I <I <I <I <I <I Identity IEF Conform 8.30-7.50*
8.31-7.60 8.31-7.51 8.30-7.63 8.34-7.57 8.30-7.58 8.31-7.61 Potency
UV mg/mL Monitoring 5.13 5.26 5.18 5.14 5.16 5.20 5.11 SEC Monomer
Monitoring 5.34 5.04 5.13 5.02 5.11 5.00 5.08 (mg/mL) Ag-ELISA
EC50% 50-200 100 83 112 132 93 106 108 Purity SDS-PAGE kD values
Monitoring 46.8/26.1 47.4/25.5 47.4/25.0 46.8/25.2 47.9/25.2
46.5/25.7 47.2/25.2 reduced gel Monitoring does not No No No No No
No pattern conform changes changes changes changes changes changes
SDS-PAGE kD values Monitoring 134.6 128.0 147.5 128.4 145.9 131.3
147.1 non-reduced gel Monitoring conforms No No No No No No pattern
changes changes changes changes changes changes Half- <5% <5%
<5% <5% <5% <5% <5% molecules Western gel does not
No No No No No Additional Blot pattern conform changes changes
changes changes changes bands reduced (155.0 kD, 134.4 kD) Western
gel does not No No No No No Additional Blot pattern conform changes
changes changes changes changes bands non-reduced (117.2 kD, 33.0
kD) SDS-PAGE gel does not No No No No No No reduced pattern conform
changes changes changes changes changes changes silver SDS-PAGE gel
conforms No No No No No Additional non-reduced pattern changes
changes changes changes changes band silver (122.5 kD) SEC %
Monomer .gtoreq.90 99.8 99.8 99.7 99.7 99.7 99.5 99.4 Charge WCX
(acidic/ 13.9/ 13.2/ 13.1/ 13.1/ 13.3/ 13.0/ 12.6/ hetero- neutral/
84.9/1.2 86.2/0.6 86.1/0.8 86.1/0.9 85.5/1.2 85.8/1.3 85.2/2.2
geneity basic) (%) pH pH 6.5-8.0 7.2 7.2 7.2 7.2 7.2 7.2 7.2
*Initially reported pH 8.61-7.66
[0391] The 3-month stability data with the Lead CXCR5 Antibody
buffered in PBS indicated no relevant changes at +5.degree. C. and
-20.degree. C. storage. After 3 months at accelerated conditions
(+25.degree. C.), significant changes could be observed. Additional
bands, as analyzed by SDS-PAGE and Western-Blot analysis, showed an
increase of basic- and decrease of acidic-isoforms, suggesting
degradation products.
Example 11
Buffer and pH Optimization
[0392] PBS pH 7.2 showed aggregation and degradation after
freeze/thaw cycles and after freezing storage. Thus, it was
necessary to find another buffer and a better pH range. In
addition, PBS is not suitable for freezing of the solutions, as a
pH shift occurs.
[0393] 30 different buffers with various pH and buffer systems were
used to select the best pH range. These experiments were run in a
very small scale, and analyzed intensively.
Best Buffer & pH Selection Screening--Small Scale (Yield 5
mL)
[0394] The analytical results are summarized in Table 40, Table 41,
Table 42, and Table 43,
[0395] In FIG. 17 and FIG. 18, the appearance of two suitable
buffer systems (acetate & histidine) after thermal stress are
shown. pH 5.5 in acetate and pH 5.0 in histidine were chosen for
further evaluation. By way of contrast, in FIG. 19, the appearance
of an incompatible buffer system (TRIS buffer) is shown.
[0396] The following buffers were selected to test in larger UF/DF
scale:
[0397] Citrate 10 mM, pH 16
[0398] Acetate 10 mM, pH 5.5
[0399] Succinate 10 mM, pH 5
[0400] Histidine 10 mM, pH 5
[0401] Arginine 10 mM, pH 6
Best Buffer & pH Selection Screening--Large Scale (Yield
.about.20 g)
[0402] After the best buffers and pH could be selected, a larger
quantity of Lead CXCR5
[0403] Antibody in each buffer system was prepared by using the
Sartorius Vivaflow system. Each batch was analytically tested and
the results are described below.
Citrate Buffer 10 mM, pH 6 (LA.sub.--09.sub.--016)
[0404] The UF/DF step worked well and only a slightly turbid
solution was obtained; no difficulties during sterile filtration
were encountered. No increase of hydrodynamic diameter, as analyzed
by DLS, was seen.
[0405] The analytical results indicated no increase in dimers, and
no changes in basic or acidic isoforms compared to the Lead CXCR5
Antibody batch material. See Table 26 and FIG. 20.
TABLE-US-00044 TABLE 26 Analytical results of Lead Antibody in
citrate buffer pH 6 Conc. Sample pH Appearance UV DLS Yield Tm
LA_09_016 6.0 Slightly 18.2 12.73 20.8 79.4.degree. turbid after
UF/DF, mg/mL nm g C. Clear after filtration
Acetate Buffer 10 mM, pH 5.5 (LA.sub.--09.sub.--017)
[0406] The UF/DF step worked well, but a turbid solution was
obtained; filter blockage during sterile filtration.
[0407] The analytical results indicated no increase in dimers, and
no changes in basic or acidic isoforms compared to the Lead CXCR5
Antibody batch material. See Table 27 and FIG. 21.
TABLE-US-00045 TABLE 27 Analytical results of Lead Antibody in
acetate buffer pH 5.5 Conc. Sample pH Appearance UV DLS Yield Tm
LA_09_017 5.5 Slightly 17.8 12.22 20.4 77.7.degree. turbid after
UF/DF, mg/mL nm g C. Clear after filtration
Succinate Buffer 10 mM, pH 5 (LA.sub.--09.sub.--018)
[0408] The sterile filtration after UF/DF was difficult to perform
because of filter blockage. The yield of 12 g was very low.
[0409] The analytical results indicated a slight decrease in
dimers, and no changes in basic or acidic isoforms compared to the
Lead CXCR5 Antibody batch material. After mechanical stress, the
dimer concentration increased slightly, and the acidic isoforms
peak in WCX decreased as the basic isoforms increased. See Table 28
and FIG. 22.
TABLE-US-00046 TABLE 28 Analytical results of Lead Antibody in
succinate buffer pH 5 Conc. Sample pH Appearance UV Yield DLS Tm
LA_09_018 4.9 Slightly 22.4 12 12.82 73.3.degree. turbid after
UF/DF, mg/mL g nm C. Clear after filtration
Histidine Buffer 10 mM, pH 5 (LA.sub.--09.sub.--019)
[0410] The sterile filtration after UF/DF was very difficult to
perform because of filter blockage. The yield of 10.5 g was very
low.
[0411] The analytical results indicated a slight decrease in
dimers, and no changes in basic or acidic isoforms compared to the
Lead CXCR5 Antibody batch material. After mechanical stress, the
dimer concentration increased slightly and the acidic isoforms peak
in WCX decreased as the basic isoforms increased. See Table 29 and
FIG. 23.
TABLE-US-00047 TABLE 29 Analytical results of Lead Antibody in
histidine buffer pH 5 Conc. Sample pH Appearance UV Yield DLS Tm
LA_09_019 5.4 Slightly 23.4 10.5 11.32 nd turbid after UF/DF, mg/mL
g nm Clear after filtration
Arginine Buffer 10 mM, pH 6 (LA.sub.--09.sub.--020)
[0412] The sterile filtration after UF/DF was very difficult to
perform. DLS showed a brought peak with a hydrodynamic diameter of
21.08 nm, which might indicate dimer formation.
[0413] The analytical results indicated a slight increase in dimers
from 0.29% in the Lead CXCR5 Antibody batch to 0.49% in arginine
After mechanical stress, 0.61% dimers were found and an increase in
basic isoforms in WCX was detected. See Table 30 and FIG. 24.
TABLE-US-00048 TABLE 30 Analytical results of Lead Antibody in
arginine buffer pH 5 Conc. Sample pH Appearance UV Yield DLS Tm
LA_09_020 6.2 Slightly 22.5 15.3 21.08 nd turbid after UF/DF, mg/mL
g nm Clear after filtration
[0414] In conclusion, three of the five batches are compatible with
Lead CXCR5 Antibody in 20 mg/ml concentration:
[0415] Citrate pH 6.0
[0416] Acetate pH 5.5
[0417] Histidine pH 5.0
These batches were characterized in terms compatibility and
stability more in detail.
Example 12
Compatibility with Excipients
[0418] All the above mentioned batches were used for compatibility
studies with surfactants. Compatibility studies were performed with
Lead CXCR5 Antibody and four selected buffers. Succinate pH 5.0 and
arginine pH 6.0 were not tested with excipients anymore, as these
buffers were not compatible with the Lead CXCR5 Antibody.
Excipients were classified as follows:
[0419] Surfactants
[0420] Sugars
[0421] Salts
[0422] Others (amino acids, preservative)
Mechanical stress (agitator speed 350/min, 2.5 h, room temperature)
was applied to test the effect of surfactants, and thermal stress
(+40.degree. C., one week) was used to test all other
excipients.
Surfactants
[0423] Orientating studies on selection of type of surfactants
(LA.sub.--08.sub.--001) and surfactant concentration
(LA.sub.--09.sub.--003; 0.01%, 0.05%, and 0.1%) indicated that a
concentration of 0.01% was sufficient to prevent visible
aggregates. The following surfactants were not suitable for the
Lead CXCR5 Antibody: PVP K12 and K17, as both showed turbidity
before mechanical stress was applied. Additionally, it was shown
that ionic surfactants such as sodium dodecyl sulfate were not
compatible with Lead CXCR5 Antibody protein solutions.
[0424] As an example, FIG. 25 shows the appearance of different
citrate buffered solution with various surfactants after mechanical
stress, and in comparison to a solution without any surfactant.
Analytical results are collected in Table 44 and Table 45.
Other Excipients
[0425] After thermal stress of +40.degree. C. for one week and
analytical determination, a selection of compatible excipients with
Lead CXCR5 Antibody in different buffer systems could be given.
[0426] Some excipients could not be tested in all four buffer
systems, as there was only little sample volume available.
[0427] After reviewing all analytical data, the excipients in Table
31 were identified to be compatible with the Lead CXCR5 Antibody.
These excipients did not show a significant increase in dimers,
HMWPs or basic isoforms analyzed by SEC and WCX.
[0428] All hydrodynamic diameter measurements were indicating a
sharp monomer peak and the Tm of the suitable excipients was not
decreasing compared to Lead CXCR5 Antibody in the respective buffer
system. All analytical data were summarized in Table 46, Table 47,
Table 48 and Table 49.
TABLE-US-00049 TABLE 31 Compatibility of all tested excipients in
the different buffer systems PBS Citrate Acetate Histidine pH 7.2
pH 6.0 pH 5.5 pH 5.0 NaCl X X X MgCl.sub.2 X X X X CaCl.sub.2 X Nd
Nd Nd Mannitol X X X Maltose Trehalose X X Nd Sucrose X X X Nd
Dextran Nd Nd Nd Benzyl alcohol Nd Arginine-HCl X X X Nd Lysine X
Nd
[0429] In conclusion, compatibility studies with surfactants show
clearly that polysorbate 20 is suited for all selected buffers in
combination with the Lead CXCR5 Antibody at 20 mg/mL. The
surfactant prevents particle formation during mechanical stress.
Nearly all other surfactants led to an increase of HMWPs.
[0430] The following excipients were selected to formulate the
different prototype formulations: NaCl, Trehalose (sucrose is more
or less comparable to trehalose in terms of stability), and
Arginine-HCl.
3-Months Prototype Stability Study
[0431] To support the formulation development of the Lead CXCR5
Antibody, twelve different prototype formulations were manufactured
and put on stability at different conditions (-20.degree. C.,
+5.degree. C. and +40.degree. C.) for three months.
[0432] Three different buffer systems were selected based on the
before described buffer, pH and excipients screening.
[0433] Citrate pH 6.0 (formulation number LA.sub.--09.sub.--027),
acetate pH 5.5 (LA.sub.--09.sub.--028), and histidine pH 5.0
(LA.sub.--09.sub.--029) were used as 10 mM buffer solutions with 20
mg/mL Lead Antibody and four different excipient combinations
(Table 32).
[0434] These four excipients showed promising results after the
excipient screening. NaCl was selected to adjust the osmolarity,
trehalose was chosen for tonicity adjustment and to have a sugar
for a lyophilization option, if needed. Additionally trehalose can
stabilize the antibody, and arginine-HCl was selected as a
stabilizer as well. Polysorbate 20 was found to be helpful to
prevent aggregation during mechanical stress.
[0435] The following paragraphs show selected data that were
compiled during the stability study to select the best buffer
system and the best excipients for formulation development. In
Table 33 all storage conditions, time points and analytical methods
were collected.
TABLE-US-00050 TABLE 32 Compositions of four different formulation
options Formulation NaCl .alpha.,.alpha.-Trehalose* H.sub.2O
Arginine Polysorbate 20 A 3 mg 25 mg 20 mM 10 mg B 50 mg 10 mg C 6
mg 20 mM 10 mg D 50 mg 20 mM 10 mg
TABLE-US-00051 TABLE 33 Storage conditions and time points for the
analytical testing Storage conditions T0 T 21 days T 6 weeks T 3
months -80.degree. C. SEC, WCX, SDS- SEC, WCX, SDS- PAGE, ELISA,
Tm, PAGE*, Tm, pH, DLS, UV pH, DLS, UV, Appearance, HIAC,
Osmolarity -20.degree. C. SEC, WCX, SDS- SEC, WCX, SDS- SEC, WCX,
SDS- PAGE*, Tm, pH, DLS, UV PAGE*, Tm, pH, DLS, UV PAGE*, Tm, pH,
DLS UV, Appearance +5.degree. C. SEC, WCX, SDS- SEC, WCX SDS- SEC,
WCX, SDS- PAGE*, Tm, pH, DLS, PAGE, Tm, pH, DLS, UV PAGE*, Tm, pH,
DLS, UV, HIAC UV, Appearance +40.degree. C. SEC, WCX, SDS- SEC,
WCX, SDS- SEC, WCX, SDS- PAGE*, Tm, pH, DLS, UV PAGE*, Tm, pH, DLS,
UV PAGE*, Tm, pH, DLS, UV *if reasonable
Unfortunately, the 3 month stability data of the Lead CXCR5
Antibody in PBS buffer was not comparable to the prototype
stability due to batch differences and due to different accelerated
conditions.
Size Exclusion Chromatography (SEC)
[0436] In FIG. 26, an increase of dimer formation up to 10% after
three months of storage in all four histidine formulations can be
clearly seen. Acetate formulations showed an increase of dimer
content up to 6%. In all four citrate formulations, the dimer
concentration was below 2%, even after three months at +40.degree.
C.
Weak Cationic Exchange Chromatography (WCX)
[0437] As the determination of neutral, basic, and acidic isoforms
is a good indicator for the stability of different formulations,
theses methods were used to amend the SEC data.
[0438] In FIG. 27 it can be seen again that histidine is worse for
the Lead CXCR5 Antibody stability under accelerated conditions. A
slight increase of basic isoforms can be noticed for all four
acetate formulations, but interestingly for citrate formulations,
discrimination between the four formulations is not possible here.
In addition, FIG. 28 shows a strong decrease in neutral isoforms
for the histidine formulations, and a slight decrease in acetate.
Again, the Lead CXCR5 Antibody in citrate is affected the
least.
SDS-PAGE
[0439] The results of SDS-PAGE measurements can be found in the
result tables in the appendix. See Tables 37-61.
Unfolding Temperature (Tm)
[0440] The unfolding temperature can be used to predict the
stability of different formulations and was measured here with the
Microcal equipment. The higher the Tm, the more promising the
formulations were. Precision of the Tm measurements were
+/-0.4.degree. C.
[0441] Between citrate and acetate formulations, nearly no
differences between Tm at T0 were noticed. In addition,
formulations A, B, and D did have a slightly higher Tm, compared to
C. The formulations A, B, and D all contain trehalose.
[0442] Histidine formulations did have a significantly lower Tm in
all cases.
TABLE-US-00052 TABLE 34 Unfolding temperatures at T.sub.0
Formulation LA_09_027 LA_09_028 LA_09_29 A 81.4.degree. C.
81.1.degree. C. 79.4.degree. C. B 81.5.degree. C. 81.6.degree. C.
81.0.degree. C. C 80.7.degree. C. 80.5.degree. C. 78.9.degree. C. D
81.6.degree. C. 81.7.degree. C. 80.7.degree. C.
pH
[0443] As the pH is of major interest and importance for the
stability of an antibody solution, the pH was monitored. The
following figures show the delta pH between T0, T1, T2, and T3 at
accelerated storage conditions.
[0444] The most pH stabilizing formulations are the citrate
buffered, and especially formulations B and D (FIG. 29). In acetate
buffered solutions of Lead CXCR5 Antibody, the pH was shifted
towards higher values (FIG. 30). In histidine buffered solutions,
the pH was slightly decreasing (FIG. 31).
DLS
[0445] The hydrodynamic diameter of the monomer and potential
soluble aggregates were measured using dynamic light
scattering.
[0446] Only after storage under accelerated conditions (+40.degree.
C.), soluble aggregates <200 nm could be seen. These aggregates
mainly occurred in histidine buffered formulation
LA.sub.--09.sub.--029 A, C, D after 3 weeks of storage.
[0447] Citrate buffered formulations showed only slightly
aggregates (FIG. 32) after three weeks in formulation C, and after
six weeks of storage in formulation A. Some aggregates could be
detected after three months in formulation B as well. But, compared
to acetate buffered formulations, the amount was very little.
[0448] Acetate buffered formulation LA.sub.--09.sub.--028 C showed
some aggregates <200 nm after three weeks, and after three
months as well in formulation A. See FIG. 33.
UV
[0449] By monitoring the protein concentration by UV measurements,
no significant differences between all time points, samples, and
formulations were noticeable. As the sample volume was very little,
the concentration was measured with a Nanodrop equipment. The
results did vary +/-5%. For detailed information, see Tables
50-61.
Appearance
[0450] After the three month storage period, all samples remained
clear and colorless without any turbidity, even in histidine. This
observation indicates as well, that all measured aggregates in DLS
were soluble. Insoluble and sub visible aggregates could be
detected by light blockage measurement by HIAC.
HIAC
[0451] Sub visible particles were detected at T0 and after three
weeks of storage at +5.degree. C. It can be clearly seen in Table
36 that the formation of particles were mainly observed in acetate
buffer. Interestingly, histidine showed good results for all four
different formulations. In citrate formulations A, B, and C are
good as well. As the level of particles >10 .mu.m and >25
.mu.m and the values in all formulations are far below the limits
defined in Ph. Eur. and USP, particle formation is of no
concern.
Osmolarity
[0452] The quantification of the excipients to adjust the
osmolarity were done prior the manufacturing of the samples by
calculation, as no samples volume was available for orientating
experiments. Therefore, the osmolarity was lower then it should be
(ideally between 280 and 320 mOsmol/kg) Table 35. Further studies
for better adjustment will be done during formulation optimization
studies.
TABLE-US-00053 TABLE 35 Osmolarity at T0 for all prototype
formulations. Formulation LA_09_027 LA_09_028 LA_09_29 A 241 221
220 B 181 160 165 C 238 220 220 D 214 192 197
Conclusion
[0453] In conclusion, citrate buffer, acetate buffer, and histidine
buffer showed no changes after storage at +5.degree. C. and
-20.degree. C., and only a minor increase in degradation products
was seen with acetate-buffer after 3 months.
[0454] The storage of Lead CXCR5 Antibody under accelerated
conditions led to significant changes of the DS. While minor
changes in citrate buffer were observed, acetate buffer showed a
significant increase of degradation- and aggregation products and a
decrease of neutral isoforms in acidic- or basic isoforms.
[0455] A tremendous effect on the Lead CXCR5 Antibody was observed
under accelerated conditions (up to 29.6% high molecular weight
proteins and up to 8.2% di-/oligomer and up to 1.3% low molecular
weight proteins). Also, cationic exchange chromatography revealed a
decrease of the neutral isoforms to 50%.
[0456] The target concentration of 20 mg/mL could be achieved with
all tested buffers, e.g. citrate, acetate, and histidine.
[0457] The pH range of a stable DP could be defined as pH
5-6.5.
[0458] Two scale-up steps (4 mL/E 100 mL-1000 mL UF/DF) with three
selected buffers were successfully performed.
[0459] The reduction of aggregate formation with 0.01% polysorbate
20 in all selected buffers after mechanical stress (agitator speed
350/min, 2.5 h, RT) was evaluated and analytically confirmed.
[0460] The absence or decrease of HMWPs could be observed, thus
increasing filterability (0.22 .mu.m) by adding 0.01% polysorbate
20 could be achieved.
[0461] The amount of dimers/oligomers was highly dependent on
buffer and pH and was analyzed by using SEC, SDS-PAGE and DLS.
Characterization of Drug Substance
[0462] The Lead CXCR5 Antibody molecule is very stable in terms of
degradation or half molecules formation, but it turned out during
preformulation activities, that Lead CXCR5 Antibody dissolved in
PBS at pH 7.2 does have an aggregation tendency. Therefore, this
buffer is not suitable for long term stability. The formation of
visible and sub-visible particles during storage or freeze/thaw
cycles should be monitored carefully during formulation development
and stability studies.
Best Buffer & pH Selection
[0463] After the best buffer and pH selection, citrate buffer 10 mM
at pH 6.0 was identified to be suitable for 20 mg/mL Lead CXCR5
Antibody solutions. 10 mM histidine buffer pH 5 or 10 mM acetate
buffer pH 5.5 could serve as backup options.
Compatibility Study with Excipients
[0464] The following excipients are recommended for prototype
formulations: [0465] Polysorbate 20 [0466] Trehalose/sucrose [0467]
NaCl [0468] Arginine-HCl
[0469] The following excipients are not recommended for
development: [0470] Benzyl alcohol [0471] Maltose [0472] Mannitol
[0473] Dextran [0474] Lysin-HCl
Prototype Formulation 3-Months Stability Study
[0475] Excellent stability of 20 mg/mL Lead CXCR5 Antibody in
citrate buffer pH 6.0, acetate buffer pH 5.5, and histidine buffer
pH 5.0 was seen at +5.degree. C. and -20.degree. C. after three
months of storage. A slight degradation at +40.degree. C. (<5%
reduction of monomer content) was observed with citrate buffer,
while acetate buffer showed low, but significant--and histidine
buffer strong artefact increases.
[0476] All tested formulations showed significant reduction of
particle formation during storage compared to the generic discovery
formulation in PBS pH 7.2.
[0477] Thus, the recommendation of this preformulation study is to
use 10 mM citrate buffer pH 6 for DS and DP of Lead CXCR5 Antibody.
A sterile filtered buffered solution with 20 mg/mL Lead CXCR5
Antibody, and stability increasing excipients should be feasible
with a storage recommendation at +5.degree. C. in vials.
[0478] For tonicity adjustment trehalose and NaCl could be used and
polysorbate 20 should be used to prevent the formation of
aggregates.
[0479] The feasibility of UF/DF experiments to either change the
buffer system and/or to increase the mAB concentration from 5 mg/mL
to 20 mg/mL could be shown in different scales.
TABLE-US-00054 TABLE 37 Explanation of data assessment Lead CXCR5
Antibody Preformulation Data Assessment T0 Process Ranking
Assessment Processability Duration Apperance personal in principle
small scale small scale clearity particle pH assessment DLS UV good
good good clear no ok 0 no aggregates no subvisible if pH
aggregates easy to <10 h @ no no visible differes <0.3 not
observed in handle, +5.degree. C. turbidity particles from basic
acceptable DLS viscosity for 4 ml observed value in for further
measurements ok row C studies medium clear- 0.5 basic turbid not
totally value no clear clear, if measured assessment acceptable at
T0 applicable or not bad bad bad turbid yes not ok 1 aggregates
subvisible particles highly >10 h @ strong visible if pH buffer
can be observed in viscous, +5.degree. C. turbidity particles
differes >0.31 recommended DSL (in difficult for 4 ml observed
observed from basic for further 0.22 .mu.m to handle value in
studies filtered row C sample) after 1 week at +40.degree. C.
Ranking Assessment Apperance personal in principle clearity
particle pH assessment DLS UV good clear no ok 0 no aggregates no
ok subvisible if pH aggregates no no visible differes <0.3 not
observed in if value after turbidity particles from basic
acceptable DLS thermal stress observed value in for further
measurements differs from row C studies basic value in row K
<10% medium clear- 0.5 turbid not totally no clear clear, if
assessment acceptable applicable or not bad turbid yes not ok 1
aggregates not ok subvisible particles strong visible if pH buffer
can be observed in if value after turbidity particles differes
>0.31 recommended DSL (in thermal stress observed observed from
basic for further 0.22 .mu.m differs from value in studies filtered
basic value row C sample) in row K <10%
TABLE-US-00055 TABLE 38 Results of preliminary packaging material
testing (data assessment) ASD PSD SEC Elisa Formula- Packaging DLS
UV Monomer Dimer/ EC Buffer tionnumber material Stress pH [nm]
[mg/mL] [%] Oligomer [%] 50% EC50 slope PBS LA_09_004_1 Clear glas
24 7.1 11.4 4.8 99.793 0.207 131.1 5.18E-13 0.98 1.12 155 Standard:
RSN0151 type I h +40.degree. C. 99.780 0.22 100 3.95E-13 1.18 1.12
mM LA_09_004_2 Amber glass 24 7.1 10.5 4.6 99.793 0.207 90.1
3.56E-13 1.16 1.12 Standard: RSN0151 type II h +40.degree. C.
99.780 0.22 100 3.95E-13 1.08 1.12 LA_09_004_3 Polyethylen- 24 7.1
11.1 5.3 99.767 0.233 157 6.20E-13 1.06 Standard: RSN0151 high
density h +40.degree. C. 99.780 0.22 100 3.95E-13 1 LA_09_004_4
Polyethylen- 24 7.1 11.0 4.4 99.722 0.278 104.3 4.12E-13 Standard:
RSN0151 Low density h +40.degree. C. 99.780 0.22 100 3.95E-13
LA_09_004_5 Polypropylen 24 7.1 11.2 5.1 99.781 0.219 106.9
5.11E-13 h +40.degree. C. 99.780 0.22 100 4.78E-13 indicates data
missing or illegible when filed
TABLE-US-00056 TABLE 39 Results of preliminary various stress tests
(data assessment) ASD PSD SEC UV Dimer/ Elisa Formula- Temper-
Appear- DLS [mg/ Monomer Oligomer EC Buffer tionnumber Stress ature
ance pH [nm] mL] [%] [%] 50% EC50 slope PBS LA_09_004_6 Clear glas
type I + 24 h Clear 7.1 11.9 4.9 99.551 0.391 80.8 3.86E-13 1.15
155 Standard: RSN0151 V2A piece 99.780 0.22 100 4.78E-13 1 mM
LA_09_004_7 Clear glas type I + 24 h Turbid 7.1 11.2 4.6 99.727
0.273 89.3 4.27E-13 1.04 Standard: RSN0151 1 h purged with N2
99.780 0.22 100 4.78E-13 1 LA_09_004_8 Clear glas type I + 24 h
Turbid 7.0 11.0 4.4 99.791 0.265 84.3 4.03E-13 1.05 Standard:
RSN0151 1 h purged with air 99.780 0.22 100 4.78E-13 1 LA 09_004_9
Clear glas type I + 24 h Turbid 7.1 10.1 4.7 99.662 0.338 72.3
3.55E-13 1.06 Standard: RSN0151 light stress 99.780 0.22 100
4.91E-13 1.15 LA_09_004_10 Clear glass type I 24 h Clear 7.1 11.1
4.3 99.773 0.227 62.3 3.06E-13 1.19 Standard: RSN0151 as T0 99.780
0.22 100 4.91E-13 1.15
TABLE-US-00057 TABLE 40 Results of small scale buffer selection
(data assessment) Lead CXCR5 Antibody Preformulation Data
Assessment Process T0 Formulation Processability Duration smal
Apperance number Buffer pH small scale scale clearity particle pH
Ranking LA_09_05-1 PBS 155 mM 7.50 good good clear-turbid yes 7.5 0
LA_09_05-2 PBS 155 mM 7.00 good good clear-turbid yes 7 0.5
LA_09_05-3 PBS 155 mM 6.50 good good clear-turbid yes 6.6 1
LA_09_06-1 PB 5 mM 7.50 bad bad turbid yes 7.4 0 LA_09_06-2 PB 5 mM
7.00 bad bad turbid yes 7 0 LA_09_06-3 PB 5 mM 6.50 bad bad turbid
yes 6.6 0 LA_09_07-1 PB 10 mM 7.50 bad bad turbid yes 7.5 0
LA_09_07-2 PB 10 mM 7.00 bad bad turbid yes 7 0 LA_09_07-3 PB 10 mM
6.50 bad bad turbid yes 6.6 0 LA_09_08-1 Citrate 10 mM 7.00 good
good clear no 7 1 LA_09_08-2 Citrate 10 mM 6.50 good good clear no
8.5 1 LA_09_08-3 Citrate 10 mM 6.00 good good clear no 6 1
LA_09_08-4 Citrate 10 mM 5.50 good good clear no 5.5 1 LA_09_08-5
Citrate 10 mM 5.00 good good clear no 5 1 LA_09_09-1 Saline 150 mM
6.00 good good clear-turbid yes 7 0 LA_09_10-1 Acetate 10 mM 5.50
good good clear yes 5.8 0.5 LA_09_10-2 Acetate 10 mM 5.00 good good
clear-turbid yes 5.2 0 LA_09_11-1 Succinate 10 mM 6.00 bad good
clear-turbid yes 6.1 1 LA_09_11-2 Succinate 10 mM 5.50 good good
clear-turbid yes 5.7 0.5 LA_09_11-3 Succinate 10 mM 5.00 good good
clear-turbid yes 6.1 0 LA_09_12-1 Histidine 10 mM 6.5 good good
clear-turbid yes 6.6 0.5 LA_09_12-2 Histidine 10 mM 6 good good
clear-turbid no 6.1 1 LA_09_12-3 Histidine 10 mM 5.5 good good
clear-turbid yes 5.1 0 LA_09_13-1 Glycine 10 mM 8 bad bad turbid
yes 0 0 LA_09_13-2 Glycine 10 mM 7 good good clear-turbid yes 6.91
0 LA_09_14-1 Arginine 10 mM 8 bad bad turbid yes 7.7 0 LA_09_14-2
Arginine 10 mM 6 good good clear yes 6.4 0.5 LA_09_15-1 TRIS 10 mM
8.5 bad bad turbid yes 3.4 0 LA_09_15-2 TRIS 10 mM 7.5 bad bad
turbid yes 7.5 0 after 1 week at +40.degree. C. Formulation T0
Apperance number DLS UV clearity particle pH Ranking DLS UV
LA_09_05-1 no aggregate 23.39 clear no 7.39 1 no aggregate 18
LA_09_05-2 no aggregate 22.63 clear yes 6.99 0 no aggregate 21.71
LA_09_05-3 no aggregate 22.73 clear yes 6.64 0.5 no aggregate 23.77
LA_09_06-1 no aggregate 13.44 turbid yes 7.45 0 no aggregate 12.72
LA_09_06-2 no aggregate 18.34 turbid no 7 0.5 no aggregate 16.94
LA_09_06-3 no aggregate 20.75 turbid no 6.5 0.5 no aggregate 20.4
LA_09_07-1 no aggregate 18.12 clear no 7.43 1 no aggregate
LA_09_07-2 no aggregate 16.97 clear-turbid no 7 0 no aggregate
17.42 LA_09_07-3 no aggregate 20.73 clear-turbid yes 6.5 0 no
aggregate 18.88 LA_09_08-1 no aggregate 19.69 clear no 7.4 1 no
aggregate 23 LA_09_08-2 no aggregate 23.77 clear no 6.8 1 no
aggregate 24.55 LA_09_08-3 no aggregate 21.93 clear no 6.1 1 no
aggregate 22.58 LA_09_08-4 no aggregate 23.67 clear no 5.6 1 no
aggregate 24.6 LA_09_08-5 no aggregate 22.97 clear no 5.1 1
aggregate 23.42 LA_09_09-1 no aggregate 24.13 clear no 6.8 1 no
aggregate 22.48 LA_09_10-1 no aggregate 24.86 clear no 5.8 1 no
aggregate 24.32 LA_09_10-2 no aggregate 24.99 clear yes 5.2 0.5 no
aggregate LA_09_11-1 no aggregate 23.99 clear no 6.2 0.5 no
aggregate 26.13 LA_09_11-2 no aggregate 24.54 clear no 5.6 0.5 no
aggregate 23.33 LA_09_11-3 no aggregate 24.22 clear no 5.2 1 no
aggregate 26.1 LA_09_12-1 no aggregate 19.75 clear-turbid yes 6.57
1 no aggregate 19.38 LA_09_12-2 no aggregate 20.95 clear-turbid no
6.09 0.5 no aggregate 20.74 LA_09_12-3 no aggregate 21.37
clear-turbid no 5.56 0 no aggregate 21.98 LA_09_13-1 aggregate 0
turbid yes 0 0 aggregate 0 LA_09_13-2 aggregate 21.19 clear-turbid
yes 7.09 0 aggregate 20.4 LA_09_14-1 no aggregate 10.06 turbid yes
7.6 0 no aggregate 9.6 LA_09_14-2 no aggregate 21.17 turbid yes 6.4
0.5 no aggregate 21.5 LA_09_15-1 aggregate 6.37 clear no 6.5 0.5 no
aggregate 6.12 LA_09_15-2 aggregate 14.28 turbid yes 7.5 0 no
aggregate 13.26 indicates data missing or illegible when filed
TABLE-US-00058 TABLE 41 Results of small scale buffer selection, T0
(data assessment) Lead CXCR5 Antibody Preformulation Data
Assessment T0 SDS- SDS-PAGE PAGE SEC WCX red. non-red. Formulation
Monomer Dimer/ Elisa % % % HC LC main number Buffer pH [%] Oligomer
[%] EC50 % EC50 slope acic neutral basic [kDa] [kDa] band
LA_09_05-1 PBS 155 mM 7.50 99.543 0.457 81 4.00E-13 0.96 13.4 85.6
1.1 49.81 25.79 141.88 LA_09_05-2 PBS 155 mM 7.00 99.546 0.452 74
3.82E-13 1.01 13.6 85.4 1 50.81 26.37 136.83 LA_09_05-3 PBS 155 mM
6.50 99.582 0.416 139 1.43E-12 0.72 13.3 85.6 1.1 50.91 26.87
136.69 LA_09_06-1 PB 5 mM 7.50 99.437 0.563 176 1.81E-12 0.78 12
86.5 1.5 47.62 25.94 161.36 LA_09_06-2 PB 5 mM 7.00 99.194 0.606 68
9.48E-13 0.76 14 84.6 1.4 48.06 25.19 168.27 LA_09_06-3 PB 5 mM
6.50 99.266 0.714 109.63 8.54E-13 1.12 14.2 84.4 1.3 49.79 26.56
157.05 LA_09_07-1 PB 10 mM 7.50 98.676 1.124 117 1.44E-12 0.81 14.2
83.7 2.1 52.1 25.31 148.91 LA_09_07-2 PB 10 mM 7.00 98.670 1.130
144.66 1.49E-12 0.87 13.9 84.2 1.9 50.16 25.81 140.47 LA_09_07-3 PB
10 mM 6.50 99.342 0.658 105 1.29E-12 0.73 13.9 85.1 1 50.34 25.61
131.66 LA_09_08-1 Citrate 10 mM 7.00 99.394 0.606 59.85 7.90E-13
0.90 12.6 86.0 1.1 49.5 26.1 157.3 LA_09_08-2 Citrate 10 mM 6.50
99.596 0.404 59 1.26E-12 0.53 12.7 85.6 1.4 49.7 26.4 147.0
LA_09_08-3 Citrate 10 mM 6.00 99.681 0.339 52 1.10E-12 0.50 12.7
86.0 1.2 50.9 26.8 144.5 LA_09_08-4 Citrate 10 mM 5.50 99.766 0.234
112 8.83E-13 1.01 12.8 88.1 1.1 49.1 26.0 147.9 LA_09_08-5 Citrate
10 mM 5.00 99.830 0.17 79 1.46E-12 0.66 12.7 86.1 1.2 49.77 26.18
143.43 LA_09_09-1 Saline 150 mM 6.00 99.232 0.768 96.18 1.51E-12
0.71 13.4 84.9 1.6 50 26.26 128.56 LA_09_10-1 Acetate 10 mM 5.50
99.507 0.493 180.25 2.83E-12 0.55 13.5 85.2 1.3 49.46 25.31 126.83
LA_09_10-2 Acetate 10 mM 5.00 99.622 0.378 146.86 9.81E-13 0.77
13.4 85.2 1.4 50.35 26.61 127.54 LA_09_11-1 Succinate 10 mM 6.00
99.447 0.553 112.43 7.51E-13 0.81 13.0 85.7 1.3 49.45 25.17 165.19
LA_09_11-2 Succinate 10 mM 5.50 99.582 0.418 13.0 85.8 1.3 50.3
25.44 155.06 LA_09_11-3 Succinate 10 mM 5.00 99.703 0.297 124
1.81E-12 0.72 12.9 85.8 1.2 50.15 25.51 150.47 LA_09_12-1 Histidine
10 mM 6.5 99.547 0.453 110 1.60E-12 0.60 13.3 86 0.7 47.7 24.5
157.44 LA_09_12-2 Histidine 10 mM 6 99.630 0.37 184 2.68E-12 0.55
13.1 86.2 0.6 47.77 24.7 157.86 LA_09_12-3 Histidine 10 mM 5.5
99.683 0.317 57 1.71E-12 0.71 13.2 86.1 0.7 47.41 25 156.38
LA_09_13-1 Glycine 10 mM 8 n/a LA_09_13-2 Glycine 10 mM 7 99.176
0.324 127 3.81E-12 0.57 13.6 85.5 0.8 48.74 25.04 163.07 LA_09_14-1
Arginine 10 mM 8 99.705 0.295 121 2.10E-12 0.71 15.9 83.6 0.5 49
25.22 157.49 LA_09_14-2 Arginine 10 mM 6 99.582 0.438 114 1.97E-12
0.66 13.1 88.3 0.6 49.43 25.96 147.5 LA_09_15-1 TRIS 10 mM 8.5
99.226 0.774 118 2.05E-12 0.71 25.5 74.1 0.4 50.06 25.8 147.5
LA_09_15-2 TRIS 10 mM 7.5 99.294 0.706 104 1.64E-i2 0.71 13 88.1
0.8 48.75 25.41 159.79 Buffers selected for larger scale
testing
TABLE-US-00059 TABLE 42 Results of small scale buffer selection, T
one week +40.degree. C. (ASD data assessment) Lead CXCR5 Antibody
Preformulation Data Assessment after 1 week at +40.degree. C. SEC
Formulation Monomer Dimer/ HMW Elisa number Buffer pH [%] Oligomer
[%] [%] EC50 % EC50 slope LA_09_05-1 PBS 155 mM 7.50 52 2.67E-13
1.11 LA_09_05-2 PBS 155 mM 7.00 99.279 0.721 97 9.96E-13 0.86
LA_09_05-3 PBS 155 mM 6.50 99.233 115 1.18E-12 0.84 LA_09_06-1 PB 5
mM 7.50 141 1.96E-12 0.88 LA_09_06-2 PB 5 mM 7.00 68 9.49E-13 0.81
LA_09_06-3 PB 5 mM 6.50 98.644 77 1.07E-12 0.75 LA_09_07-1 PB 10 mM
7.50 0.014 68 8.40E-13 0.81 LA_09_07-2 PB 10 mM 7.00 109 1.34E-12
0.68 LA_09_07-3 PB 10 mM 6.50 78.79 1.04E-12 0.78 LA_09_08-1
Citrate 10 mM 7.00 99.214 111 1.36E-12 0.72 LA_09_08-2 Citrate 10
mM 6.50 99.588 0.404 46 9.79E-13 0.60 LA_09_08-3 Citrate 10 mM 6.00
99.661 0.338 98 7.77E-13 1.01 LA_09_08-4 Citrate 10 mM 5.50 99.766
0.234 0.84 LA_09_08-5 Citrate 10 mM 5.00 99.772 0.228 75 1.38E-12
0.82 LA_09_09-1 Saline 150 mM 6.00 80.89 1.27E-12 0.59 LA_09_10-1
Acetate 10 mM 5.50 99.106 0.834 94.9 1.49E-12 0.55 LA_09_10-2
Acetate 10 mM 5.00 99.463 0.537 101.5 6.78E-13 0.79 LA_09_11-1
Succinate 10 mM 6.00 99.145 125.90 8.41E-13 0.74 LA_09_11-2
Succinate 10 mM 5.50 99.410 0.890 LA_09_11-3 Succinate 10 mM 5.00
99.521 0.479 157 2.29E-12 0.64 LA_09_12-1 Histidine 10 mM 6.5
99.300 0.700 175 2.55E-12 0.57 LA_09_12-2 Histidine 10 mM 6 99.508
0.492 77 2.32E-12 0.65 LA_09_12-3 Histidine 10 mM 5.5 99.567 0.333
81 2.45E-12 0.64 LA_09_13-1 Glycine 10 mM 8 LA_09_13-2 Glycine 10
mM 7 98.446 1.554 65 1.95E-12 0.70 LA_09_14-1 Arginine 10 mM 8
99.247 0.783 88 1.53E-12 0.80 LA_09_14-2 Arginine 10 mM 6 99.277
0.723 99 1.72E-12 0.76 LA_09_15-1 TRIS 10 mM 8.5 99.456 1.644 237
3.72E-12 0.84 LA_09_15-2 TRIS 10 mM 7.5 1.002 184 2.89E-12 0.69
after 1 week at +40.degree. C. SDS-PAGE SDS-PAGE WCX red. non-red.
Formulation % % % HC LC main number acic neutral basic [kDa] [kDa]
band comment LA_09_05-1 14.8 83.5 1.7 50.25 26.07 142.27 LA_09_05-2
13.4 85.2 1.4 51.14 26.27 135.83 LA_09_05-3 13.3 85.3 1.4 49.79
25.9 141 LA_09_06-1 82.6 2 46.92 26.73 166.69 LA_09_06-2 11.7 86.1
2.2 49 26.11 159.55 LA_09_06-3 13.9 84.1 2 49.35 26.15 164.49
LA_09_07-1 82.2 2.6 50.18 25.41 137.71 LA_09_07-2 14.8 88.2 2 49.89
25.52 131.79 LA_09_07-3 14.1 84 1.9 51.63 26.2 139.92 LA_09_08-1
10.500 88.000 1.5 50.060 26.830 153.790 LA_09_08-2 12.700 85.800
1.4 49.690 26.390 146.970 LA_09_08-3 12.700 86.000 1.2 50.880
26.780 144.500 LA_09_08-4 12.5 86.1 1.4 49.23 25.9 144.79
LA_09_08-5 12.7 86 1.3 49.05 25.84 144.336 LA_09_09-1 13 85.1 1.3
49.33 25.23 127.67 LA_09_10-1 13.5 84.7 1.9 50.17 26.14 126.06
LA_09_10-2 13.4 84.8 1.8 49.86 26.18 128.96 LA_09_11-1 12.7 85.6
1.7 49.95 49.4 163.2 LA_09_11-2 12.6 85.7 1.6 49.84 25.23 161.74
LA_09_11-3 12.8 49.72 25.01 LA_09_12-1 13.4 85.7 0.9 47.73 24.7
158.71 LA_09_12-2 12.6 86.9 0.5 48.03 24.9 159.02 LA_09_12-3 13.1
88.2 0.8 47.67 24.69 153.93 LA_09_13-1 LA_09_13-2 13.4 85.1 1.3
49.02 25.27 160.97 LA_09_14-1 14.1 84.6 1.3 49.13 25.52 154.37
LA_09_14-2 12.9 88.3 0.8 49.57 25.56 149.34 LA_09_15-1 73.9 2.3
49.37 25.05 150.98 LA_09_15-2 13.7 85 1.3 49.25 25.14 158.5 Buffers
selected for larger scale testing indicates data missing or
illegible when filed
TABLE-US-00060 TABLE 43 Results of small scale buffer selection,
after mechanical stress (data assessment) Lead CXCR5 Antibody
Preformulation Data Assessment after mechanical stress 350 rpm, 2.5
h SEC WCX SDS-PAGE SDS-PAGE Mono- mer/ % red. non-red. Formulation
mer Oligo- HMW Elisa % neu- % HC LC main com- number Buffer pH [%]
mer [%] EC50 % EC50 slope acic tral basic [kDa] [kDa] band ment
LA_09_05-1 PBS 155 mM 7.50 99.263 0.658 0.080 71 3.64E-13 0.9 10.2
88.6 1.2 51.01 26.64 144.8 LA_09_05-2 PBS 155 mM 7.00 0.650 50
2.58E-13 1.02 10.1 88.6 1.3 51.38 26.94 138.72 LA_09_05-3 PBS 155
mM 6.50 0.527 79.3 9.12E-13 0.77 11.6 87.4 1.2 50.4 26.17 135.68
LA_09_06-1 PB 5 mM 7.50 99.677 0.423 1.54E-12 0.79 17.1 82 0.9
47.57 25.79 163.25 LA_09_06-2 PB 5 mM 7.00 99.084 110.14 8.58E-13
0.89 11.2 87.6 1.2 47.36 24.75 166.93 LA_09_06-3 PB 5 mM 6.50
99.202 150 1.23E-12 0.94 13.6 86.2 1.3 50.68 26.88 168.84
LA_09_07-1 PB 10 mM 7.50 60.85 4.99E-13 0.87 12.9 86.3 1.8 50.47
26.26 147.83 LA_09_07-2 PB 10 mM 7.00 99.053 61.36 6.32E-13 0.92
14.8 83.6 1.6 52.27 26.31 136.88 LA_09_07-3 PB 10 mM 6.50 99.272
89.39 1.18E-12 0.94 13.6 86.4 1 50.05 25.62 131.53 LA_09_08-1
Citrate 10 mM 7.00 99.138 0.024 100.76 1.33E-12 0.74 10.5 88.0 1.5
50.06 26.83 153.79 LA_09_08-2 Citrate 10 mM 6.50 99.517 0.468 0.015
41 8.61E-13 0.51 10.6 88.2 1.2 50.82 26.79 143.82 LA_09_08-3
Citrate 10 mM 6.00 99.608 0.334 0.088 136 1.07E-12 0.85 10.3 88.4
1.3 49.57 25.38 145.79 LA_09_08-4 Citrate 10 mM 5.50 98.317 0.328
79 1.46E-12 0.73 10.1 88.6 1.4 49.29 25.88 144.93 LA_09_08-5
Citrate 10 mM 5.00 99.299 0.269 99 1.84E-12 0.71 10.1 83.4 1.5
49.31 26.02 143.32 LA_09_09-1 Saline 150 mM 6.00 N/A LA_09_10-1
Acetate 10 mM 5.50 LA_09_10-2 Acetate 10 mM 5.00 LA_09_11-1
Succinate 6.00 10 mM LA_09_11-2 Succinate 5.50 10 mM LA_09_11-3
Succinate 5.00 10 mM LA_09_12-1 Histidine 6.5 10 mM LA_09_12-2
Histidine 6 10 mM LA_09_12-3 Histidine 5.5 10 mM LA_09_13-1 Glycine
10 mM 8 LA_09_13-2 Glycine 10 mM 7 LA_09_14-1 Arginine 8 10 mM
LA_09_14-2 Arginine 6 10 mM LA_09_15-1 TRIS 10 mM 8.5 LA_09_15-2
TRIS 10 mM 7.5 Buffers selected for larger scale testing indicates
data missing or illegible when filed
As there were seen no major differences after mechanical stress,
samples LA.sub.--09.sub.--09 to 15 were not stressed
mechanically
TABLE-US-00061 TABLE 44 Results - Surfactant selection data
assessment Lead CXCR5 Antibody Assessment - Surfactant data PSD
Formulation Mechanical Stress Tm DLS UV number 350 rpm 2.5 h
Surfactant pH [.degree. C.] pH [nm] [mg/mL] Appearance Citrate
LA_09_16 no non 79.4 6.0 12.7 18.2 Clear 10 mM LA_09_16 yes non nd
6.1 N/A 18.1 Turbid LA_09_16-1 yes Polysorbate 20 79.1 6.1 12.2
17.1 Clear LA_09_16-2 yes Polysorbate 80 78.6 6.1 12.5 18.6 Clear
LA_09_16-3 yes Lutrol F68 6.0 78.6 6.1 12.7 18.5 Clear LA_09_16-4
yes Cremophor RH40 78.6 6.1 13.0 17.7 Clear LA_09_16-5 yes Solutol
HS15 78.4 6.1 12.8 19.2 Clear LA_09_16_6 N/A SDS nd 6.1 N/A N/A
Turbid Acetate LA_09_17 no non 77.7 5.5 12.2 17.8 Clear 10 mM
LA_09_17 yes non nd 5.6 13.4 13.2 Turbid LA_09_17-1 yes Polysorbate
20 77.4 5.5 12.5 17.7 Clear LA_09_17-2 yes Polysorbate 80 76.4 5.6
12.8 17.8 Clear 5.5 LA_09_17-3 yes Lutrol F68 76.7 5.5 12.5 18.0
Clear LA_09_17-4 yes Cremophor RH40 76.7 5.5 13.3 17.7 Clear
LA_09_17-5 yes Solutol HS15 76.4 5.5 12.9 17.7 Clear LA_09_17_6 yes
SDS nd 5.6 N/A N/A Turbid Succinate LA_09_18 no non 73.3 4.9 12.8
20.1 Clear 10 mM LA_09_18 yes non nd 5.0 13.0 20.8 Clear LA_09_18-1
yes Polysorbate 20 72.9 5.0 12.6 20.4 Clear LA_09_18-2 yes
Polysorbate 80 5.0 72.6 5.0 12.7 20.1 Clear LA_09_18-3 yes Lutrol
F68 72.6 5.0 12.7 21.0 Clear LA_09_18-4 yes Cremophor RH40 72.4 5.0
12.9 21.1 Clear LA_09_18-5 yes Solutol HS15 nd 5.0 12.6 20.4 Clear
ASD SEC Dimer/ WCX SDS-PAGE Formulation Monomer Oligomer HMW % % %
non- number [%] [% [%] acic neutral basic Comment main band comment
Citrate LA_09_16 99.74 0.260 12.79 86.98 1.28 153.79 10 mM 99.74
0.260 13.43 85.41 1.16 146.19 LA_09_16 N/A LA_09_16-1 99.72 0.280
12.28 85.92 1.28 151.33 99.74 0.260 13.43 85.41 1.16 146.19
LA_09_16-2 99.71 0.290 12.71 86.04 1.26 149.35 99.74 0.260 13.43
85.41 1.16 146.19 LA_09_16-3 99.73 0.270 13.13 85.6 1.27 147.77
99.74 0.260 13.43 85.41 1.16 146.19 LA_09_16-4 0.440 11.37 86.86
1.19 P 149.68 99.74 0.260 13.43 85.41 1.16 146.19 LA_09_16-5 99.1
0.560 12.92 88.52 1.28 P 151.82 99.74 0.260 13.43 85.41 1.16 146.19
LA_09_16_6 N/A Acetate LA_09_17 99.74 0.260 12.92 89.98 1.11 173.63
10 mM 99.75 0.250 13.42 85.43 1.14 169.47 LA_09_17 N/A LA_09_17-1
99.74 0.280 12.88 86.02 1.12 175.92 99.75 0.250 13.42 85.43 1.14
169.47 LA_09_17-2 99.32 0.369 12.76 89.03 1.21 178.34 99.75 0.250
13.42 85.43 1.14 169.47 LA_09_17-3 98.79 0.240 12 85.86 1.14 176.33
99.75 0.250 13.42 85.43 1.14 169.47 LA_09_17-4 11.51 87.18 1.31
174.25 99.75 0.250 13.42 85.43 1.14 169.47 LA_09_17-5 0.450 12.28
89.67 1.1 169.78 99.75 0.250 13.42 85.43 1.14 169.47 LA_09_17_6 N/A
Succinate LA_09_18 98.76 0.240 13.318 85.425 1.258 159.8 10 mM
99.71 0.290 13.8 85.06 1.13 160.51 LA_09_18 99.7 0.300 12.189 1.423
155.65 99.71 0.290 13.8 85.06 1.13 160.51 LA_09_18-1 N/A LA_09_18-2
LA_09_18-3 LA_09_18-4 LA_09_18-5 indicates data missing or
illegible when filed
TABLE-US-00062 TABLE 45 Results - Surfactant selection data
assessment Histidin LA_09_19 non 99.76 10 mM no nd nd N/A 11.3 23.1
Clear Turbid LA_09_19 non nd nd 5.4 99.7 yes 21.9 23.4 Clear Clear
97.98 LA_09_19-1 Polysorbate 20 nd 5.3 99.7 yes 21.0 21.1 Turbid
LA_09_19-2 Polysorbate 80 99.3 yes 5.0 5.3 21.0 21.0 99.7
LA_09_19-3 Lutrol F68 5.3 21.0 21.3 99.53 99.7 LA_09_19-4 yes
Cremophor RH40 nd 5.3 N/A 20.1 Clear 99.7 LA_09_19-5 yes Solutol
HS15 nd 5.3 21.5 21.2 Clear 99.14 99.7 Arginine LA_09_20 no non nd
20.2 Clear 99.61 10 mM 99.71 LA_09_20-1 yes non nd 6.2 N/A 22.5
Turbid 99.29 99.71 LA_09_20-2 yes Polysorbate 20 71.8 6.2 11.9
22.64 Clear N/A LA_09_20-3 yes Polysorbate 80 6.0 nd 6.2 12.22
22.16 Clear LA_09_20-4 yes Lutrol F68 nd 6.2 12.34 22.79 Turbid
LA_09_20-5 yes Cremophor RH40 nd 6.2 13.2 22.36 Clear LA_09_20-1
yes Solutol HS15 nd 6.2 N/A 22.04 Turbid Histidin LA_09_19 0.240
13.967 84.657 1.457 157.26 10 mM 13.662 85.263 1.173 160.51
LA_09_19 0.300 1.6 12.237 86.306 1.257 rer Peak k 162.08 Zusatzbade
0.420 13.662 85.263 1.173 160.51 LA_09_19-1 0.300 12.756 84.86
1.384 159.2 13.662 85.263 1.173 160.51 LA_09_19-2 0.320 0.13 12.516
84.949 1.535 161.06 0.300 13.662 85.263 1.173 160.51 Zusatzbade
LA_09_19-3 0.350 1.23 12.182 86.561 1.257 rer Peak k 163.05 0.300
13.662 85.263 1.173 160.51 LA_09_19-4 10.437 87.667 1.906 rer Peak
k 168.73 0.300 13.662 85.263 1.173 160.51 LA_09_19-5 0.330 12.561
85.891 1.548 rer Peak k 160.22 0.300 13.662 85.263 1.173 160.51
Arginine LA_09_20 0.490 85.224 1.267 157.89 10 mM 0.290 13.8 85.06
1.13 155.98 LA_09_20-1 0.610 3.1 12.32 88.015 1.636 152.77 0.290
13.8 85.06 1.13 155.98 LA_09_20-2 N/A LA_09_20-3 LA_09_20-4
LA_09_20-5 LA_09_20-1 N/A, samples were not tested analytically, as
too little sample volume was available. indicates data missing or
illegible when filed
Excipients and Lead CXCR5 Antibody (LA.sub.--09 022)
TABLE-US-00063 [0480] TABLE 46 Results - compatibility of Lead
Antibody with excipients Lead CXCR5 Antibody Assessment -
Compatibility PSD ASD UV SEC Thermal Tm DLS [mg/ Monomer Dimer/ HMW
pH [.degree. C.] pH [nm] m Appearance [%] Oligimer [% [%] RSN0151
LA_09_22 no non 80.3 6.0 12.7 18.2 Clear 99.689 0.314 155 mM PBS
Standard: RSN0151 99.707 0.293 LA_09_22_1 yes non 6.1 N/A 18.1
Turbid 99.388 0.542 Standard: RSN0151 99.707 0.293 LA_09_22_2 yes
NaCl 80.3 6.1 12.2 17.1 Clear 99.538 0.4 Standard: RSN0151 99.707
0.293 LA_09_22_3 yes MgCl2 nd 6.1 12.5 18.6 Clear 99.697 0.303
Standard: RSN0151 99.707 0.293 LA_09_22_4 yes CaCl2 78.2 6.1 12.7
18.5 Clear 99.656 0.132 Standard: RSN0151 99.707 0.293 LA_09_22_5
yes Mannitol nd 6.1 13.0 17.7 Clear 89.488 0.489 0.013 Standard:
RSN0151 99.707 0.293 LA_09_22_6 yes Maltose nd 6.1 12.8 19.2 Clear
Standard: RSN0151 99.707 0.293 LA_09_22_7 yes Trehalose 80.8 6.1
N/A N/A Turbid 98.507 0.389 standard: RSN0151 99.366 0.343
LA_09_22_8 yes Sucrose 80.5 5.5 12.2 17.8 Clear 99.307 0.535
Standard: RSN0151 99.366 0.343 LA_09_22_9 yes Dextran 40 79.3 5.6
13.4 13.2 Turbid 99.821 Standard: RSN0151 99.366 0.343 LA_09_22_10
yes Benzylalkohol 75.8 5.5 12.5 17.7 Clear Standard: RSN0151 99.366
0.343 LA_09_22_11 yes Arginine-HCl 80.0 5.6 12.8 17.8 Clear 99.622
0.331 Standard: RSN0151 99.366 0.343 LA_09_22_12 yes Lysin 80.9 5.5
12.5 18.0 Clear 99.047 0.701 Standard: RSN0151 99.366 0.343 ASD
SDS-PAGE WCX non-red. Thermal % % % main acic neutral basic Comment
band comment RSN0151 LA_09_22 14.323 84.306 1.373 155.76 155 mM PBS
Standard: RSN0151 13.867 85.032 1.102 155.98 LA_09_22_1 13.878
84.289 1.833 142.95 Standard: RSN0151 13.867 85.032 1.102 154.13
LA_09_22_2 13.606 84.404 1.790 147.97 Standard: RSN0151 13.867
85.032 1.102 154.13 LA_09_22_3 13.353 81.235 1.412 149.38 Standard:
RSN0151 13.867 85.032 1.102 154.13 LA_09_22_4 14.887 83.908 1.296
148.81 Standard: RSN0151 13.867 85.032 1.102 154.13 LA_09_22_5
82.849 1.824 150.42 Standard: RSN0151 13.867 85.032 1.102 154.13
LA_09_22_6 82.913 152.49 Standard: RSN0151 13.867 85.032 1.102
154.13 LA_09_22_7 81.167 1.552 154.74 standard: RSN0151 13.867
84.575 1.407 154.13 LA_09_22_8 83.506 1.508 158.76 Standard:
RSN0151 14.019 84.575 1.407 154.13 LA_09_22_9 13.181 77.996 1.596
166.61 Standard: RSN0151 14.019 84.575 1.407 154.13 LA_09_22_10
83.508 2.17 168.13 Standard: RSN0151 14.019 84.575 1.407 154.13
LA_09_22_11 18.282 83.116 1.621 169.77 Standard: RSN0151 14.019
84.575 1.407 154.13 LA_09_22_12 83.104 169.9 ad Standard: RSN0151
14.019 84.575 1.407 154.13 indicates data missing or illegible when
filed
Excipients and Citrate Buffered (LA.sub.--09.sub.--023)
TABLE-US-00064 [0481] TABLE 47 Results - compatibility of Lead
Antibody in citrate buffer with excipients Lead CXCR5 Antibody
Assessment - Compatibility in citrate buffer ASD SEC Dimer/ DLS UV
Monomer Oligomer Excipient pH [.degree. C.] [nm] [mg/mL] [%] [%
RSN0151 LA_09_23 no non 81.5 12.6 18.6 99.596 0.404 155 Standard:
RSN0151 99.686 0.314 mMPBS LA_09_23_1 yes non 12.4 18.7 99.493
0.309 Standard: RSN0151 99.686 0.314 LA_09_23_2 yes NaCl 81.7 12.2
21.8 99.498 0.302 Standard: RSN0151 99.686 0.314 LA_09_23_3 yes
MgCl2 77.2 12.3 14.7 99.538 0.462 Standard: RSN0151 99.686 0.314
LA_09_23_4 yes Mannitol 82.1 13.5 23.3 99.533 0.467 standard:
RSN0151 99.686 0.314 LA_09_23_5 0.677 Standard: RSN0151 99.686
0.314 LA_09_23_6 yes Trehalose 82.1 13.9 17.6 99.656 0.344
standard: RSN0151 99.686 0.314 LA_09_23_7 yes Sucrose 81.9 13.5
17.5 99.594 0.406 Standard: RSNU151 99.686 0.314 LA_09_23_8 yes
Benzylalkohol 77.1 13.9 20.5 0.507 Standard: RSN0151 99.686 0.314
LA_09_23_9 yes Arginine-HCl 80.7 14.7 17.6 99.327 0.412 Standard:
RSN0151 99.686 0.314 LA_09_23_10 yes Lysin nd 12.0 16.7 Standard:
RSN0151 99.686 0.314 ASD SEC WCX SDS-PAGE non-red. HMW % % % main
[%] acic neutral basic Comment band comment RSN0151 LA_09_23 13.307
85.310 1.383 155.59 155 Standard: RSN0151 14.058 84.609 1.333
146.11 mMPBS LA_09_23_1 12.807 85.597 1.396 151.85 Standard:
RSN0151 14.058 84.609 1.333 146.11 LA_09_23_2 12.803 85.641 1.354
145.12 Standard: RSN0151 14.058 84.609 1.333 146.11 LA_09_23_3
12.799 85.692 1.509 151.67 Standard: RSN0151 14.058 84.609 1.333
146.11 LA_09_23_4 12.703 85.302 1.495 147.96 standard: RSN0151
14.058 84.609 1.333 146.11 LA_09_23_5 82.106 1.631 155.02 Standard:
RSN0151 14.058 84.609 1.333 146.11 LA_09_23_6 12.693 85.848 1.465
151.43 standard: RSN0151 14.058 84.609 1.333 146.11 LA_09_23_7
12.758 85.754 1.487 152.67 Standard: RSNU151 14.058 84.609 1.333
146.11 LA_09_23_8 11.591 87.216 1.192 145.93 Standard: RSN0151
14.058 84.609 1.333 146.11 LA_09_23_9 13.047 85.453 1.500 146.01
Standard: RSN0151 14.058 84.609 1.333 146.11 LA_09_23_10 13.642
80.787 144.05 Standard: RSN0151 14.058 84.609 1.333 146.11
indicates data missing or illegible when filed
Excipients and Acetate Buffered (LA.sub.--09.sub.--024)
TABLE-US-00065 [0482] TABLE 48 Results - compatibility with
excipients and Lead Antibody in acetate buffer Lead CXCR5 Antibody
Assessment - Compatibility in acetate buffer - data ASD SEC Dimer/
DLS UV Monomer Oligomer HMW Excipient pH [.degree. C.] [nm] [mg/mL]
[%] [% [%] RSN0151 LA_09_24 no non 81.8 12.2 17.8 99.603 0.397 155
mM Standard: RSN0151 99.628 0.309 PBS LA_09_24_1 yes non nd 12.5
16.5 99.405 0.595 Standard: RSN0151 99.628 0.309 LA_09_24_2 yes
NaCl 81.3 12.3 17.4 99.479 0.521 Standard: RSN0151 99.628 0.309
LA_09_24_3 yes MgCl2 81.0 12.3 16.8 99.517 0.483 Standard: RSN0151
99.628 0.309 LA_09_24_4 yes Mannitol 82.5 14.2 15.3 99.546 0.431
0.023 Standard: RSN0151 99.628 0.309 0.539 0.029 Standard: RSN0151
99.628 0.309 LA_09_24_6 yes Trehalose 82.5 14.0 15.4 99.522 0.478
Standard: RSN0151 99.628 0.309 LA_09_24_7 yes Sucrose 82.3 14.0
19.1 99.542 0.458 Standard: RSN0151 99.628 0.309 LA_09_24_8 yes
Benzylalkohol 78.0 13.2 16.4 0.67 Standard: RSN0151 99.628 0.309
LA_09_24_9 yes Arginine-HCl 81.5 12.2 16.9 99.226 0.439 Standard:
RSN0151 99.628 0.309 LA_09_24_10 yes Lysin 81.2 12.3 15.3 99.134
0.692 Standard: RSN0151 99.628 0.309 ASD SEC WCX SDS-PAGE non-red.
HMW % % % main [%] acic neutral basic Comment band comment RSN0151
LA_09_24 13.408 85.225 1.368 173.63 155 mM Standard: RSN0151 14.230
84.407 1.363 169.47 PBS LA_09_24_1 13.368 85.052 1.580 149.18
Standard: RSN0151 14.230 84.407 1.363 151.63 LA_09_24_2 13.507
84.895 1.598 156.31 Standard: RSN0151 14.230 84.407 1.363 151.63
LA_09_24_3 13.470 84.915 1.615 142.23 Standard: RSN0151 14.230
84.407 1.363 151.63 LA_09_24_4 0.023 13.567 84.965 1.478 155.69
Standard: RSN0151 14.230 84.407 1.363 151.63 0.029 82.016 1.680
156.15 Standard: RSN0151 14.230 84.407 1.363 151.63 LA_09_24_6
13.558 84.905 1.537 146.61 yes Trehalose Standard: RSN0151 14.230
84.407 1.363 151.63 LA_09_24_7 13.587 84.923 1.490 149.12 Standard:
RSN0151 14.230 84.407 1.363 151.63 LA_09_24_8 12.739 85.960 1.301
152.48 Standard: RSN0151 14.230 84.407 1.363 151.63 LA_09_24_9
13.412 84.978 1.611 152.88 Standard: RSN0151 14.230 84.407 1.363
151.63 LA_09_24_10 13.546 84.814 1.639 153.32 Standard: RSN0151
14.230 84.407 1.363 151.63 indicates data missing or illegible when
filed
Excipients and Histidine Buffered (LA.sub.--09.sub.--025)
TABLE-US-00066 [0483] TABLE 49 Results - compatibility with
excipients and Lead Antibody in histidine buffer Lead CXCR5
Antibody Assessment - Compatibility in Histidine buffer - data ASD
SEC Dimer/ Formulation Excip- DLS UV Monomer Oligomer number ient
pH [.degree. C.] [nm] [mg/mL] [%] [%] no non 28.1 Standard: RSN0151
yes NaCl 78.8 25.3 Standard: RSN0151 yes MgCl2 nd 12.5 Standard:
RSN0151 yes Mannitol 22.1 Standard: RSN0151 yes 12.5 21.5 Standard:
RSN0151 ASD SEC WCX SDS-PAGE non-red Formulation HMW % % % main
number [%] acid neutral basic Comment band comment Standard:
RSN0151 Standard: RSN0151 Standard: RSN0151 Standard: RSN0151
Standard: RSN0151 indicates data missing or illegible when
filed
TABLE-US-00067 TABLE 50 Results - Prototype formulation LA_09_27A
ASD SEC PSD Dimer/ Formulation Storage Tm DLS UV Monomer Oligomer
HMW NP Buffer number Time point condition .degree. C. [.degree. C.]
pH [nm] [mg/mL] [%] [%] [%] [%] Citrate 10 mM. LA_09_27A T0 N/A
81.4 N/A 12.2 20.0 99.712 0.217 3 mg/mL NaCl. Standard: RSN0151
99.691 0.309 25 mg/mL LA_09_27A T: 3 weeks {grave over (
)}+5.degree. C. 81.6 5.8 12.2 19.7 99.670 0.309 0.021 Trehalose, 20
mM Standard: RSN0151 99.463 0.563 Arginine-HCl. LA_09_27A T: 3
weeks {grave over ( )}-20.degree. C. 81.6 5.8 12.2 20.1 99.649
0.313 0.033 0.01% Standard: RSN0151 99.463 0.563 Polysorbate 20
LA_09_27A T: 3 weeks {grave over ( )}+40.degree. C. 81.6 5.8 13.4
19.3 0.807 0.547 Standard: RSN0151 99.463 0.563 LA_09_27A T: 6
weeks {grave over ( )}+5.degree. C. 81.6 5.9 12.3 24.8 99.673 0.327
Standard: RSN0151 99.460 0.54 LA_09_27A T: 6 weeks {grave over (
)}-20.degree. C. 6.0 81.4 5.9 12.2 24.9 99.670 0.33 Standard:
RSN0151 99.460 0.54 LA_09_27A T: 6 weeks {grave over (
)}+40.degree. C. 81.4 5.9 25.9 0.731 Standard: RSN0151 99.460 0.54
LA_09_27A T: 6 weeks {grave over ( )}-80.degree. C. N/A 5.9 12.2
24.7 99.667 0.333 Standard: RSN0151 99.460 0.54 LA_09_27A T: 3
months {grave over ( )}+5.degree. C. 81.7 5.8 12.2 19.9 99.582
0.418 Standard: RSN0151 99.427 0.573 LA_09_27A T: 3 months {grave
over ( )}-20.degree. C. 81.7 5.8 12.4 20.4 99.684 0.316 Standard:
RSN0151 99.427 0.573 LA_09_27A T: 3 months {grave over (
)}+40.degree. C. 81.6 5.8 20.3 99.427 0.573 ASD WCX SDS-PAGE
non-red. Formulation % % % main Elisa Buffer number acic neutral
basic Comment band comment EC50 % EC50 slope Citrate 10 mM.
LA_09_27A 13.988 84.86 1.151 118.94 112 2.09E+12 0.53 3 mg/mL NaCl.
Standard: RSN0151 13.759 85.041 1.2 122.88 100 1.86E+12 0.56 25
mg/mL LA_09_27A 14.143 84.561 1.292 N/A Trehalose, 20 mM Standard:
RSN0151 14.000 84.450 1.551 N/A 112 2.09E+12 0.53 Arginine-HCl.
LA_09_27A 14.382 84.423 1.195 100 1.86E+12 0.56 0.01% Standard:
RSN0151 14.000 84.450 1.551 Polysorbate 20 LA_09_27A 12.133 85.819
2.048 Standard: RSN0151 14.000 84.450 1.551 LA_09_27A 13.798 85.176
1.026 Standard: RSN0151 14.054 84.285 1.662 LA_09_27A 13.756 86.192
1.052 Standard: RSN0151 14.054 84.285 1.662 N/A LA_09_27A 11.826
85.289 2.886 Standard: RSN0151 14.054 84.285 1.662 LA_09_27A 13.298
86.669 1.033 Standard: RSN0151 14.054 84.285 1.662 LA_09_27A 14.528
83.928 1.543 Standard: RSN0151 14.413 84.08 1.507 LA_09_27A 14.936
83.655 1.409 Standard: RSN0151 14.413 84.08 1.507 LA_09_27A 14.671
80.287 4.843 14.413 84.08 1.507 indicates data missing or illegible
when filed
TABLE-US-00068 TABLE 51 Results - Prototype formulation LA_09_27B
ASD SEC PSD Dimer/ Formulation Storage Tm DLS UV Monomer Oligomer
HMW NP Buffer number Time point condition pH [.degree. C.] pH [nm]
[mg/mL] [%] [%] [%] [%] Citrate 10 mM, LA_09_27B T0 N/A 81.5 N/A
13.6 20.9 99.688 0.312 50 mg Trehalose, Standard: RSN0151 99.691
0.309 0.01% LA_09_27B T: 3 weeks {grave over ( )}+5.degree. C. 6.1
13.6 20.5 99.321 0.384 0.295 Polysorbate 20 Standard: RSN0151
99.463 0.563 LA_09_27B T: 3 weeks {grave over ( )}-20.degree. C.
6.0 13.6 19.7 99.626 0.374 Standard: RSN0151 99.463 0.563 LA_09_27B
T; 3 weeks {grave over ( )}+40.degree. C. 6.0 14.4 19.9 0.934
Standard: RSN0151 99.463 0.563 LA_09_27B T: 6 weeks {grave over (
)}+5.degree. C. 6.1 13.6 25.6 99.612 0.388 Standard: RSN0151 6.0
N/A 99.460 0.54 LA_09_27B T: 6 weeks {grave over ( )}-20.degree. C.
6.1 13.6 25.0 0.372 Standard: RSN0151 99.460 0.54 LA_09_27B T: 6
weeks {grave over ( )}+40.degree. C. 6.2 14.9 24.9 Standard:
RSN0151 99.460 0.54 LA_09_27B T: 6 weeks {grave over (
)}-80.degree. C. 6.1 13.7 25.0 Standard: RSN0151 99.460 0.54
LA_09_27B T: 3 months {grave over ( )}+5.degree. C. 6.1 13.7 20.8
99.636 0.349 Standard: RSN0151 99.427 0.573 LA_09_27B T: 3 months
{grave over ( )}-20.degree. C. 6.1 13.6 19.9 99.621 0.379 Standard:
RSN0151 99.427 0.573 LA_09_27B T: 3 months {grave over (
)}+40.degree. C. 6.0 19.4 Standard: RSN0151 99.427 0.573 ASD WCX
SDS-PAGE non-red. Formulation % % % main Elisa Buffer number acic
neutral basic Comment band comment EC50 % EC50 slope Citrate 10 mM,
LA_09_27B 13.723 85.084 1.194 117.40 104 1.93E+12 0.62 50 mg
Trehalose, Standard: RSN0151 13.759 85.041 1.2 122.88 100 1.86E+12
0.56 0.01% LA_09_27B 13.911 84.772 1.317 Polysorbate 20 Standard:
RSN0151 14.000 84.450 1.551 N/A LA_09_27B 14.14 84.541 1.318
Standard: RSN0151 14.000 84.450 1.551 LA_09_27B 12.455 85.392 2.153
Standard: RSN0151 14.000 84.450 1.551 LA_09_27B 13.747 84.98 1.273
Standard: RSN0151 14.054 84.285 1.662 LA_09_27B 84.901 1.264
Standard: RSN0151 14.054 84.285 1.662 LA_09_27B 218.32 90. 28. 14
kD N/A Standard: RSN0151 14.054 84.285 1.662 189.54 LA_09_27B
84.783 1.336 Standard: RSN0151 14.054 84.285 1.662 LA_09_27B 14.63
83.798 1.571 Standard: RSN0151 14.413 84.08 1.507 N/A LA_09_27B
13.128 83.406 1.466 Standard: RSN0151 14.413 84.08 1.507 LA_09_27B
15.373 5.25 Standard: RSN0151 14.413 84.08 1.507 indicates data
missing or illegible when filed
TABLE-US-00069 TABLE 52 Results - Prototype formulation LA_09_027C
ASD SEC PSD Dimer/ Formulation Storage Tm DLS UV Monomer Oligomer
HMW NP Buffer number Time point condition pH [.degree. C.] pH [nm]
[mg/mL] [%] [%] [%] [%] Citrate 10 mM, LA_09_27C T0 N/A 80.7 N/A
11.6 99.688 0.312 6 mg/mL NaCl, Standard: RSN0151 19.9 21.1 99.691
0.309 0.01% LA_09_27C T: 3 weeks {grave over ( )}+5.degree. C. 80.9
5.8 11.6 99.321 0.384 0.295 Polysorbate 20 Standard: RSN0151 20.6
20.4 99.463 0.563 LA_09_27C T: 3 weeks {grave over ( )}-20.degree.
C. 80.7 5.8 11.6 99.626 0.374 Standard: RSN0151 25.4 25.4 99.463
0.563 LA_09_27C T: 3 weeks {grave over ( )}+40.degree. C. 80.4 5.8
0.934 0.515 Standard: RSN0151 5.3 25.7 99.463 0.563 LA_09_27C T: 6
weeks {grave over ( )}+5.degree. C. 80.7 5.9 11.6 99.612 0.388
Standard: RSN0151 21.4 20.9 99.460 0.54 LA_09_27C T: 6 weeks {grave
over ( )}-20.degree. C. 80.9 5.9 11.5 99.623 0.372 Standard:
RSN0151 21.4 99.460 0.54 LA_09_27C T: 6 weeks {grave over (
)}+40.degree. C. 80.7 5.9 0.729 Standard: RSN0151 99.460 0.54
LA_09_27C T: 6 weeks {grave over ( )}-80.degree. C. 6.0 N/A 5.8
18.7 Standard: RSN0151 99.460 0.54 LA_09_27C T: 3 months {grave
over ( )}+5.degree. C. 80.9 5.8 11.6 99.636 0.349 Standard: RSN0151
99.427 0.573 LA_09_27C T: 3 months {grave over ( )}-20.degree. C.
81.2 5.8 11.7 99.621 0.379 Standard: RSN0151 99.427 0.573 LA_09_27C
T: 3 months {grave over ( )}+40.degree. C. 80.9 5.8 99.427 0.573
ASD WCX SDS-PAGE non-red. Formulation % % % main Elisa Buffer
number acic neutral basic Comment band comment EC50 % EC50 slope
Citrate 10 mM, LA_09_27C 13.723 85.084 1.194 117.40 104 1.93E+12
0.62 6 mg/mL NaCl, Standard: RSN0151 13.759 85.041 1.2 122.88 100
1.86E+12 0.56 0.01% LA_09_27C 13.911 84.772 1.317 Polysorbate 20
Standard: RSN0151 14.000 84.450 1.551 LA_09_27C 14.14 84.541 1.318
Standard: RSN0151 14.000 84.450 1.551 LA_09_27C 12.455 85.392 2.153
Standard: RSN0151 14.000 84.450 1.551 N/A LA_09_27C 13.747 84.98
1.273 Standard: RSN0151 14.054 84.285 1.662 LA_09_27C 13.335 84.901
1.264 Standard: RSN0151 14.054 84.285 1.662 LA_09_27C 12.685 83.332
3.483 218.32 90. 28. 14 kD N/A Standard: RSN0151 14.054 84.285
1.662 189.54 LA_09_27C 13.382 84.789 1.336 Standard: RSN0151 14.054
84.285 1.662 LA_09_27C 14.63 83.798 1.571 Standard: RSN0151 14.413
84.08 1.507 N/A LA_09_27C 15.128 83.406 1.466 Standard: RSN0151
14.413 84.08 1.507 LA_09_27C 16.373 79.378 5.25 14.413 84.08 1.507
indicates data missing or illegible when filed
TABLE-US-00070 TABLE 53 Results - Prototype formulation LA_09_027D
ASD SEC PSD Dimer/ Formulation Storage Tm DLS UV Monomer Oligomer
HMW NP Buffer number Time point condition pH [.degree. C.] pH [nm]
[mg/mL] [%] [%] [%] [%] Citrate 10 mM, LA_09_27D T0 N/A 81.6 N/A
13.3 19.8 99.579 0.283 50 mg/mL Standard: RSN0151 99.691 0.309
Trehalose, LA_09_27D T: 3 weeks {grave over ( )}+5.degree. C. 81.4
6.0 13.1 19.5 99.676 0.313 0.011 20 mM Arginine- Standard: RSN0151
99.463 0.563 HCl, 0.01% LA_09_27D T: 3 weeks {grave over (
)}-20.degree. C. 81.4 5.8 13.2 20.4 99.633 0.338 0.024 Polysorbate
20 Standard: RSN0151 99.463 0.563 LA_09_27D T: 3 weeks {grave over
( )}+40.degree. C. 81.4 6.0 13.9 19.6 0.809 0.592 Standard: RSN0151
99.463 0.563 LA_09_27D T: 6 weeks {grave over ( )}+5.degree. C.
80.9 6.0 13.1 24.4 99.660 0.34 Standard: RSN0151 99.460 0.54
LA_09_27D T: 6 weeks {grave over ( )}-20.degree. C. 6.0 80.6 6.0
13.2 24.2 99.643 0.352 Standard: RSN0151 99.460 0.54 LA_09_27D T: 6
weeks {grave over ( )}+40.degree. C. 80.9 6.0 14.5 24.8 0.763
Standard: RSN0151 99.460 0.54 LA_09_27D T: 6 weeks {grave over (
)}-80.degree. C. N/A 6.0 13.2 24.5 99.664 0.336 Standard: RSN0151
99.460 0.54 LA_09_27D T: 3 months {grave over ( )}+5.degree. C.
82.1 5.9 13.1 20.4 99.693 0.341 Standard: RSN0151 99.427 0.573
LA_09_27D T: 3 months {grave over ( )}-20.degree. C. 82.1 5.9 13.2
19.8 99.655 0.394 Standard: RSN0151 99.427 0.573 LA_09_27D T: 3
months {grave over ( )}+40.degree. C. 81.9 5.9 15.2 20.4 1.12
99.427 0.573 ASD WCX SDS-PAGE non-red. Formulation % % % main Elisa
Buffer number acic neutral basic Comment band comment EC50 % EC50
slope Citrate 10 mM, LA_09_27D 13.974 84.912 1.114 117.29 94
1.75E+12 0.61 50 mg/mL Standard: RSN0151 13.759 85.041 1.2 122.88
100 1.86E+12 0.56 Trehalose, LA_09_27D 13.825 84.918 1.256 20 mM
Arginine- Standard: RSN0151 14.000 84.450 1.551 HCl, 0.01%
LA_09_27D 14.028 84.745 1.227 Polysorbate 20 Standard: RSN0151
14.000 84.450 1.551 LA_09_27D 12.315 85.553 2.131 Standard: RSN0151
14.000 84.450 1.551 N/A LA_09_27D 13.475 85.337 1.188 Standard:
RSN0151 14.054 84.285 1.662 LA_09_27D 13.445 85.502 1.053 Standard:
RSN0151 14.054 84.285 1.662 LA_09_27D 11.916 85.099 2.985 192.29
90. 14 kD N/A Standard: RSN0151 14.054 84.285 1.662 189.54
LA_09_27D 13.485 83.357 1.159 Standard: RSN0151 14.054 84.285 1.662
LA_09_27D 14.09 84.528 1.38 Standard: RSN0151 14.413 84.08 1.507
N/A LA_09_27D 14.521 84.08 1.398 Standard: RSN0151 14.413 84.08
1.507 LA_09_27D 14.173 81.003 4.823 14.413 84.08 1.507 indicates
data missing or illegible when filed
TABLE-US-00071 TABLE 54 Results - Prototype formulation LA_09_028A
ASD SEC PSD Dimer/ Formulation Storage Tm DLS UV Monomer Oligomer
HMW NP Buffer number Time point condition pH [.degree. C.] pH [nm]
[mg/mL] [%] [%] [%] [%] Acetate 10 mM, LA_09_28A T0 N/A 81.1 N/A
12.9 17.4 99.503 0.26 3 mg/mL NaCl, Standard: RSN0151 99.691 0.309
25 mg/mL LA_09_28A T: 3 weeks {grave over ( )}+5.degree. C. 81.1
5.6 12.6 16.1 99.650 0.33 Trehalose, Standard: RSN0151 99.481 0.519
20 mM Arginine- LA_09_28A T: 3 weeks {grave over ( )}-20.degree. C.
81.1 5.6 12.5 15.7 99.639 0.315 0.046 HCl, = .01% Standard: RSN0151
99.481 0.519 Polysorbate 20 LA_09_28A T: 3 weeks {grave over (
)}+40.degree. C. 81.1 5.6 13.9 15.6 0.478 Standard: RSN0151 99.481
0.519 LA_09_28A T: 6 weeks {grave over ( )}+5.degree. C. 81.1 5.6
12.6 16.4 99.634 0.366 Standard: RSN0151 99.460 0.545 LA_09_28A T:
6 weeks {grave over ( )}-20.degree. C. 5.5 81.1 5.6 12.7 16.6
99.650 0.35 Standard: RSN0151 99.460 0.545 LA_09_28A T: 6 weeks
{grave over ( )}+40.degree. C. N/A 5.7 14.7 16.5 1.121 Standard:
RSN0151 99.460 0.545 LA_09_28A T: 6 weeks {grave over (
)}-80.degree. C. N/A 5.6 12.8 16.6 99.623 0.372 Standard: RSN0151
99.460 0.545 LA_09_28A T: 3 months {grave over ( )}+5.degree. C.
81.1 5.7 12.5 16.6 99.564 0.415 Standard: RSN0151 99.421 0.579
LA_09_28A T: 3 months {grave over ( )}-20.degree. C. 81.3 5.7 12.7
16.4 99.594 0.388 Standard: RSN0151 99.421 0.579 LA_09_28A T: 3
months {grave over ( )}+40.degree. C. 80.7 5.7 16.5 1.872 99.421
0.579 ASD WCX SDS-PAGE non-red. Formulation % % % main Elisa Buffer
number acic neutral basic Comment band comment EC50 % EC50 slope
Acetate 10 mM, LA_09_28A 14.153 84.625 1.221 118.94 112 2.09E+12
0.53 3 mg/mL NaCl, Standard: RSN0151 13.772 85.024 1.204 122.88 100
1.86E+12 0.56 25 mg/mL LA_09_28A 14.036 84.717 1.247 Trehalose,
Standard: RSN0151 14.278 84.346 1.377 N/A 20 mM Arginine- LA_09_28A
13.99 84.778 1.231 N/A 112 2.09E+12 0.53 HCl, = .01% Standard:
RSN0151 14.278 84.346 1.377 100 1.86E+12 0.56 Polysorbate 20
LA_09_28A 13.418 83.352 2.730 Standard: RSN0151 14.278 84.346 1.377
LA_09_28A 13.603 84.981 1.216 N/A Standard: RSN0151 13.864 85.048
1.088 LA_09_28A 13.844 84.952 1.205 Standard: RSN0151 13.864 85.048
1.088 LA_09_28A 13.593 82.452 3.952 195.57 66. 28. 14 kDa N/A
Standard: RSN0151 13.864 85.048 1.088 189.54 LA_09_28A Standard:
RSN0151 13.864 85.048 1.088 LA_09_28A 14.363 83.628 2.009 Standard:
RSN0151 14.618 83.409 1.973 N/A LA_09_28A 14.314 83.738 1.948
Standard: RSN0151 83.409 1.973 LA_09_28A 74.736 7.147 83.409 1.973
indicates data missing or illegible when filed
TABLE-US-00072 TABLE 55 Results - Prototype formulation LA_09_028B
ASD SEC PSD Dimer/ Formulation Storage Tm DLS UV Monomer Oligomer
HMW NP Buffer number Time point condition pH [.degree. C.] pH [nm]
[mg/mL] [%] [%] [%] [%] Acetate 10 mM, LA_09_28B T0 N/A 81.6 N/A
14.4 17.1 99.590 0.41 50 mg Trehalose, Standard: RSN0151 99.691
0.309 0.01% LA_09_28B T: 3 weeks {grave over ( )}+5.degree. C. 81.6
5.6 14.2 16.3 99.476 0.476 0.048 Polysorbate 20 Standard: RSN0151
99.481 0.519 LA_09_28B T: 3 weeks {grave over ( )}-20.degree. C.
81.6 5.7 14.2 16.2 99.411 0.497 0.092 Standard: RSN0151 99.481
0.519 LA_09_28B T: 3 weeks {grave over ( )}+40.degree. C. 81.6 5.6
14.8 16.2 0.639 Standard: RSN0151 99.481 0.519 LA_09_28B T: 6 weeks
{grave over ( )}+5.degree. C. 81.6 5.7 14.3 16.7 99.510 0.49
Standard: RSN0151 99.460 0.545 LA_09_28B T: 6 weeks {grave over (
)}-20.degree. C. 5.5 81.6 5.7 14.6 17.4 99.590 0.47 Standard:
RSN0151 99.460 0.545 LA_09_28B T: 6 weeks {grave over (
)}+40.degree. C. 81.6 5.7 15.4 16.7 1.043 Standard: RSN0151 99.460
0.545 LA_09_28B T: 6 weeks {grave over ( )}-80.degree. C. N/A 5.7
14.5 17.2 99.536 0.464 Standard: RSN0151 99.460 0.545 LA_09_28B T:
3 months {grave over ( )}+5.degree. C. 82.1 5.7 14.04 16.9 99.446
0.554 Standard: RSN0151 99.421 0.579 LA_09_28B T: 3 months {grave
over ( )}-20.degree. C. 81.9 5.7 14.1 16.8 99.548 0.452 Standard:
RSN0151 99.421 0.579 LA_09_28B T: 3 months {grave over (
)}+40.degree. C. 81.9 5.7 16.3 16.8 1.383 99.421 0.579 ASD WCX
SDS-PAGE non-red. Formulation % % % main Elisa Buffer number acic
neutral basic Comment band comment EC50 % EC50 slope Acetate 10 mM,
LA_09_28B 14.257 84.511 1.232 118.94 112 2.09E+12 0.53 50 mg
Trehalose, Standard: RSN0151 13.772 85.024 1.204 122.88 100
1.86E+12 0.56 0.01% LA_09_28B 14.56 84.112 1.328 Polysorbate 20
Standard: RSN0151 14.278 84.346 1.377 N/A LA_09_28B 14.174 84.403
1.423 N/A 112 2.09E+12 0.53 Standard: RSN0151 14.278 84.346 1.377
100 1.86E+12 0.56 LA_09_28B 14.243 82.977 2.78 Standard: RSN0151
14.278 84.346 1.377 LA_09_28B 13.844 84.829 1.327 N/A Standard:
RSN0151 13.864 85.048 1.088 LA_09_28B 13.755 84.949 1.302 Standard:
RSN0151 13.864 85.048 1.088 LA_09_28B 14.485 80.651 4.864 203.14
66. 28. 14 kDa N/A Standard: RSN0151 13.864 85.048 1.088 189.54
LA_09_28B 13.98 84.747 1.273 Standard: RSN0151 13.864 85.048 1.088
LA_09_28B 14.511 83.408 2.081 Standard: RSN0151 14.618 83.409 1.973
N/A LA_09_28B 15.255 82.643 2.103 Standard: RSN0151 83.409 1.973
LA_09_28B 71.010 9.177 83.409 1.973 indicates data missing or
illegible when filed
TABLE-US-00073 TABLE 56 Results - Prototype formulation LA_09_028C
ASD SEC PSD Dimer/ Formulation Storage Tm DLS UV Monomer Oligomer
HMW NP Buffer number Time point condition pH [.degree. C.] pH [nm]
[mg/mL] [%] [%] [%] [%] Acetate 10 mM, LA_09_28C T0 N/A 80.5 N/A
11.9 18.0 99.561 0.439 6 mg NaCl, Standard: RSN0151 99.691 0.309
0.01% LA_09_28C T: 3 weeks {grave over ( )}+5.degree. C. 80.5 5.6
11.9 17.1 99.560 0.44 Polysorbate 20 Standard: RSN0151 99.481 0.519
LA_09_28C T: 3 weeks {grave over ( )}-20.degree. C. 80.7 5.6 11.8
16.3 99.611 0.389 Standard: RSN0151 99.481 0.519 LA_09_28C T: 3
weeks {grave over ( )}+40.degree. C. 80.7 5.5 16.4 0.831 Standard:
RSN0151 99.481 0.519 LA_09_28C T: 6 weeks {grave over (
)}+5.degree. C. 80.5 5.6 12.1 17.7 99.586 0.414 Standard: RSN0151
99.460 0.545 LA_09_28C T: 6 weeks {grave over ( )}-20.degree. C.
5.5 80.7 5.6 12.1 17.6 99.584 0.416 Standard: RSN0151 99.460 0.545
LA_09_28C T: 6 weeks {grave over ( )}+40.degree. C. 80.5 5.7 17.2
0.834 Standard: RSN0151 99.460 0.545 LA_09_28C T: 6 weeks {grave
over ( )}-80.degree. C. N/A 5.6 12.1 16.1 99.524 0.476 Standard:
RSN0151 99.460 0.545 LA_09_28C T: 3 months {grave over (
)}+5.degree. C. 80.5 5.660 11.64 17.2 99.522 0.478 Standard:
RSN0151 99.421 0.579 LA_09_28C T: 3 months {grave over (
)}-20.degree. C. 80.5 5.6 11.7 17.2 99.565 0.435 Standard: RSN0151
99.421 0.579 LA_09_28C T: 3 months {grave over ( )}+40.degree. C.
80.5 5.7 17.2 1.278 99.421 0.579 ASD WCX SDS-PAGE non-red.
Formulation % % % main Elisa Buffer number acic neutral basic
Comment band comment EC50 % EC50 slope Acetate 10 mM, LA_09_28C
14.329 84.427 1.244 130.69 142 1.52E+12 0.87 6 mg NaCl, Standard:
RSN0151 13.772 85.024 1.204 122.88 100 1.07E+12 0.81 0.01%
LA_09_28C 14.581 84.001 1.418 Polysorbate 20 Standard: RSN0151
14.278 84.346 1.377 LA_09_28C 14.478 84.192 1.330 Standard: RSN0151
14.278 84.346 1.377 LA_09_28C 14.989 83.971 3.04 N/A Standard:
RSN0151 14.278 84.346 1.377 LA_09_28C 13.913 84.88 1.207 Standard:
RSN0151 13.864 85.048 1.088 LA_09_28C 13.975 84.683 1.332 Standard:
RSN0151 13.864 85.048 1.088 LA_09_28C 14.449 82.466 4.085 203.41
66. 28. 14 kDa Standard: RSN0151 13.864 85.048 1.088 189.54 N/A
LA_09_28C 13.949 84.815 1.235 Standard: RSN0151 13.864 85.048 1.088
LA_09_28C 14.849 83.298 1.853 Standard: RSN0151 14.618 83.409 1.973
N/A LA_09_28C 14.827 2.277 Standard: RSN0151 83.409 1.973 LA_09_28C
74.293 7.488 83.409 1.973 indicates data missing or illegible when
filed
TABLE-US-00074 TABLE 57 Results - Prototype formulation LA_09_028D
ASD SEC PSD Dimer/ Formulation Storage Tm DLS UV Monomer Oligomer
HMW NP Buffer number Time point condition pH [.degree. C.] pH [nm]
[mg/mL] [%] [%] [%] [%] Acetate 10 mM, LA_09_28D T0 N/A 81.7 N/A
14.3 16.2 99.231 0.336 50 mg Trehalose, Standard: RSN0151 99.691
0.309 20 mM Arginine- LA_09_28D T: 3 weeks {grave over (
)}+5.degree. C. 81.7 5.6 14.1 15.9 99.603 0.363 0.039 HCl, 0.01%
Standard: RSN0151 99.481 0.519 Polysorbate 20 LA_09_28D T: 3 weeks
{grave over ( )}-20.degree. C. 81.5 5.6 14.1 15.5 99.558 0.395
0.047 Standard: RSN0151 99.481 0.519 LA_09_28D T: 3 weeks {grave
over ( )}+40.degree. C. 81.7 5.6 14.8 15.7 0.736 Standard: RSN0151
99.481 0.519 LA_09_28D T: 6 weeks {grave over ( )}+5.degree. C.
81.5 5.6 14.3 16.5 99.603 0.4 Standard: RSN0151 99.460 0.545
LA_09_28D T: 6 weeks {grave over ( )}-20.degree. C. 5.5 81.5 5.6
14.4 16.5 99.633 0.367 Standard: RSN0151 99.460 0.545 LA_09_28D T:
6 weeks {grave over ( )}+40.degree. C. 81.8 5.7 16.0 16.4 1.095
Standard: RSN0151 99.460 0.545 LA_09_28D T: 6 weeks {grave over (
)}-80.degree. C. N/A 5.6 14.4 15.5 99.616 0.384 Standard: RSN0151
99.460 0.545 LA_09_28D T: 3 months {grave over ( )}+5.degree. C.
81.8 5.7 13.8 16.3 99.446 0.416 Standard: RSN0151 99.421 0.579
LA_09_28D T: 3 months {grave over ( )}-20.degree. C. 81.7 5.7 14.1
15.8 99.548 0.380 Standard: RSN0151 99.421 0.579 LA_09_28D T: 3
months {grave over ( )}+40.degree. C. 81.5 5.7 16.9 16.4 1.894
99.421 0.579 ASD WCX SDS-PAGE non-red. Formulation % % % main Elisa
Buffer number acic neutral basic Comment band comment EC50 % EC50
slope Acetate 10 mM, LA_09_28D 14.336 84.478 1.186 131.16 91
9.75E+12 0.60 50 mg Trehalose, Standard: RSN0151 13.772 85.024
1.204 122.88 100 1.07E+12 0.81 20 mM Arginine- LA_09_28D 14.309
84.351 1.34 HCl, 0.01% Standard: RSN0151 14.278 84.346 1.377
Polysorbate 20 LA_09_28D 14.048 84.700 1.252 Standard: RSN0151
14.278 84.346 1.377 LA_09_28D 14.872 83.307 2.821 N/A Standard:
RSN0151 14.278 84.346 1.377 LA_09_28D 13.991 84.894 1.175 Standard:
RSN0151 13.864 85.048 1.088 LA_09_28D 13.683 84.139 1.179 Standard:
RSN0151 13.864 85.048 1.088 LA_09_28D 14.794 82.051 4.155 203.72
66. 28. 14 kDa N/A Standard: RSN0151 13.864 85.048 1.088 189.54
LA_09_28D 13.795 84.888 1.323 Standard: RSN0151 13.864 85.048 1.088
LA_09_28D 14.089 83.957 1.954 Standard: RSN0151 14.618 83.409 1.973
N/A LA_09_28D 14.488 83.570 1.942 Standard: RSN0151 83.409 1.973
LA_09_28D 74.345 8.697 83.409 1.973 indicates data missing or
illegible when filed
TABLE-US-00075 TABLE 58 Results - Prototype formulation LA_09_029A
ASD SEC PSD Dimer/ Formulation Storage Tm DLS UV Monomer Oligomer
HMW NP Buffer number Time point condition pH [.degree. C.] pH [nm]
[mg/mL] [%] [%] [%] [%] Histidine 10 mM, LA_09_29A T0 N/A 79.4 N/A
12.5 20.3 99.748 0.252 3 mg/mL NaCl, Standard: RSN0151 99.691 0.309
25 mg/mL LA_09_29A T: 3 weeks {grave over ( )}+5.degree. C. 79.7
5.1 12.5 18.1 99.690 0.31 Trehalose, Standard: RSN0151 99.504 0.496
20 mM Arginine- LA_09_29A T: 3 weeks {grave over ( )}-20.degree. C.
79.3 5.2 12.5 17.9 99.625 0.338 0.037 HCl, 0.01% Standard: RSN0151
99.504 0.496 Polysorbate 20 LA_09_29A T: 3 weeks {grave over (
)}+40.degree. C. 78.4 5.0 18.6 0.569 Standard: RSN0151 99.504 0.496
LA_09_29A T: 6 weeks {grave over ( )}+5.degree. C. 78.3 5.1 12.3
18.9 99.664 0.336 Standard: RSN0151 99.461 0.539 LA_))9_29A T: 6
weeks {grave over ( )}-20.degree. C. 5.5 78.9 5.1 12.6 18.7 99.683
0.317 Standard: RSN0151 99.461 0.539 LA_09_29A T: 6 weeks {grave
over ( )}+40.degree. C. 75.8 4.9 17.9 1.237 Standard: RSN0151
99.461 0.539 LA_09_29A T: 6 weeks {grave over ( )}-80.degree. C.
N/A 5.2 12.5 19.3 99.666 0.334 Standard: RSN0151 99.461 0.539
LA_09_29A T: 3 months {grave over ( )}+5.degree. C. 79.2 99.642
0.358 Standard: RSN0151 99.424 0.576 LA_09_29A T: 3 months {grave
over ( )}-20.degree. C. 79.2 N/A 99.684 0.313 Standard: RSN0151
99.424 0.576 LA_09_29A T: 3 months {grave over ( )}+40.degree. C.
77.3 3.104 99.424 0.576 ASD WCX SDS-PAGE non-red. Formulation % % %
main Elisa Buffer number acic neutral basic Comment band comment
EC50 % EC50 slope Histidine 10 mM, LA_09_29A 13.946 84.881 1.174
123.08 174 1.71E+12 0.77 3 mg/mL NaCl, Standard: RSN0151 13.755
85.045 1.2 122.88 100 9.84E+13 0.64 25 mg/mL LA_09_29A 1.077
Trehalose, Standard: RSN0151 20 mM Arginine- LA_09_29A 1.217 HCl,
0.01% Standard: RSN0151 Polysorbate 20 LA_09_29A 7.197 N/A
Standard: RSN0151 LA_09_29A 13.962 84.777 1.261 Standard: RSN0151
13.982 84.916 1.102 LA_))9_29A 16.596 85.942 1.061 Standard:
RSN0151 13.982 84.916 1.102 LA_09_29A 12.029 89.19 18.78 205.8 90.
14 kDa N/A Standard: RSN0151 13.982 84.916 1.102 189.54 LA_09_29A
13.621 85.201 1.177 Standard: RSN0151 13.982 94.916 1.102 LA_09_29A
14.401 83.691 1.908 Standard: RSN0151 14.922 83.041 2.037 N/A
LA_09_29A 14.189 83.583 2.228 Standard: RSN0151 83.041 2.037
LA_09_29A 41.194 32.04 83.041 2.037 indicates data missing or
illegible when filed
TABLE-US-00076 TABLE 59 Results - Prototype formulation LA_09_029B
ASD SEC PSD Dimer/ Formulation Storage Tm DLS UV Monomer Oligomer
HMW NP Buffer number Time point condition pH [.degree. C.] pH [nm]
[mg/mL] [%] [%] [%] [%] Histidine 10 LA_09_29B T0 N/A 81.0 N/A 10.7
20.2 99.679 0.321 mM, 50 mg Standard: RSN0151 99.691 0.309
Trehalose, LA_09_29B T: 3 weeks {grave over ( )}+5.degree. C. 81.3
5.0 10.5 19.3 99.622 0.351 0.01% Standard: RSN0151 99.504 0.496
Polysorbate LA_09_29B T: 3 weeks {grave over ( )}-20.degree. C. N/A
5.1 10.4 17.6 99.623 0.342 20 Standard: RSN0151 99.504 0.496
LA_09_29B T: 3 weeks {grave over ( )}+40.degree. C. N/A 4.9 11.2
18.2 0.368 Standard: RSN0151 99.504 0.496 LA_09_29B T: 6 weeks
{grave over ( )}+5.degree. C. 81.0 5.1 10.4 19.9 99.613 0.387
Standard: RSN0151 99.461 0.539 LA_09_29B T: 6 weeks {grave over (
)}-20.degree. C. 5.5 5.1 10.4 19.2 99.653 0.347 Standard: RSN0151
99.461 0.539 LA_09_29B T: 6 weeks {grave over ( )}+40.degree. C.
4.9 11.98 + A 19.4 0.761 Standard: RSN0151 99.461 0.539 LA_09_29B
T: 6 weeks {grave over ( )}-80.degree. C. 5.1 10.3 19.0 99.654
0.346 Standard: RSN0151 99.461 0.539 LA_09_29B T: 3 months {grave
over ( )}+5.degree. C. N/A 99.603 0.397 Standard: RSN0151 N/A
99.424 0.576 LA_09_29B T: 3 months {grave over ( )}-20.degree. C.
99.650 0.350 Standard: RSN0151 99.424 0.576 LA_09_29B T: 3 months
{grave over ( )}+40.degree. C. 3.009 Standard: RSN0151 99.424 0.576
ASD WCX SDS-PAGE non-red. Formulation % % % main Elisa Buffer
number acic neutral basic Comment band comment EC50 % EC50 slope
Histidine 10 LA_09_29B 13.891 84.907 1.202 122.26 118 1.16E+12 0.73
mM, 50 mg Standard: RSN0151 13.755 85.045 1.2 122.88 100 9.84E+13
0.64 Trehalose, LA_09_29B 14.145 84.674 1.182 0.01% Standard:
RSN0151 14.208 84.672 1.22 Polysorbate LA_09_29B 14.369 84.470
1.162 20 Standard: RSN0151 14.208 84.672 1.22 LA_09_29B 59.191
5.247 N/A Standard: RSN0151 14.208 84.672 1.22 LA_09_29B 13.622
85.381 0.988 Standard: RSN0151 13.982 84.916 1.102 LA_09_29B 13.758
86.184 1.058 Standard: RSN0151 13.982 84.916 1.102 LA_09_29B 42.961
11.071 200.33 90. 14 kDa N/A Standard: RSN0151 13.982 84.916 1.102
189.54 LA_09_29B 13.454 83.382 1.164 Standard: RSN0151 13.982
84.916 1.102 LA_09_29B 14.238 83.852 1.910 Standard: RSN0151 14.922
83.041 2.037 N/A LA_09_29B 14.108 83.764 2.128 Standard: RSN0151
14.922 83.041 2.037 LA_09_29B 42.311 16.113 14.922 83.041 2.037
indicates data missing or illegible when filed
TABLE-US-00077 TABLE 60 Results - Prototype formulation LA_09_029C
ASD SEC PSD Dimer/ Formulation Storage Tm DLS UV Monomer Oligomer
HMW NP Buffer number Time point condition pH [.degree. C.] pH [nm]
[mg/mL] [%] [%] [%] [%] Histidine 10 mM, LA_09_29C T0 N/A 78.9 N/A
11.9 19.4 99.741 0.259 6 mg/mL NaCl, Standard: RSN0151 99.691 0.309
0.01% LA_09_29C T: 3 weeks {grave over ( )}+5.degree. C. 79.0 5.1
11.9 19.0 99.643 0.357 Polysorbate 20 Standard: RSN0151 99.504
0.496 LA_09_29C T: 3 weeks {grave over ( )}-20.degree. C. 78.4 5.2
12.0 18.6 99.674 0.326 Standard: RSN0151 99.504 0.496 LA_09_29C T:
3 weeks {grave over ( )}+40.degree. C. 77.4 5.0 18.7 0.562
Standard: RSN0151 99.504 0.496 LA_09_29C T: 6 weeks {grave over (
)}+5.degree. C. 78.1 5.2 11.8 19.7 99.627 0.373 Standard: RSN0151
99.461 0.539 LA_09_29C T: 6 weeks {grave over ( )}-20.degree. C.
5.0 77.8 5.2 12.0 20.8 99.661 0.339 Standard: RSN0151 99.461 0.539
LA_09_29C T: 6 weeks {grave over ( )}+40.degree. C. 76.6 4.9 19.2
1.359 Standard: RSN0151 99.461 0.539 LA_09_29C T: 6 weeks {grave
over ( )}-80.degree. C. N/A 5.2 11.9 19.9 99.663 0.337 Standard:
RSN0151 99.461 0.539 LA_09_29C T: 3 months {grave over (
)}+5.degree. C. 78.4 99.594 0.406 Standard: RSN0151 99.424 0.576
LA_09_29C T: 3 months {grave over ( )}-20.degree. C. 78.5 N/A
99.680 0.32 Standard: RSN0151 99.424 0.576 LA_09_29C T: 3 months
{grave over ( )}+40.degree. C. 76.6 3.54 99.424 0.576 ASD WCX
SDS-PAGE non-red. Formulation % % % main Elisa Buffer number acic
neutral basic Comment band comment EC50 % EC50 slope Histidine 10
mM, LA_09_29C 14.119 84.681 1.2 122.43 6 mg/mL NaCl, Standard:
RSN0151 13.755 85.045 1.2 122.88 0.01% LA_09_29C 14.046 84.796
1.158 Polysorbate 20 Standard: RSN0151 14.208 84.672 1.22 LA_09_29C
14.171 84.763 1.076 Standard: RSN0151 14.208 84.672 1.22 LA_09_29C
14.068 47.301 11.631 N/A Standard: RSN0151 14.208 84.672 1.22
LA_09_29C 13.871 84.916 1.213 Standard: RSN0151 13.982 84.916 1.102
LA_09_29C 13.764 85.153 1.083 Standard: RSN0151 84.916 1.102
LA_09_29C 85.038 21.636 199.58 90. 14 kDa N/A Standard: RSN0151
84.916 1.102 189.54 LA_09_29C 13.521 85.426 1.053 Standard: RSN0151
13.982 85.916 1.102 LA_09_29C 14.214 83.772 1.984 Standard: RSN0151
14.922 83.041 2.037 N/A LA_09_29C 14.199 83.324 1.977 Standard:
RSN0151 83.041 2.037 LA_09_29C 37.193 37.198 83.041 2.037 indicates
data missing or illegible when filed
TABLE-US-00078 TABLE 61 Results - Prototype formulation LA_09_029D
ASD SEC PSD Dimer/ Formulation Storage Tm DLS UV Monomer Oligomer
HMW NP Buffer number Time point condition pH [.degree. C.] pH [nm]
[mg/mL] [%] [%] [%] [%] Histidine 10 mM, LA_09_29D T0 N/A 80.7 N/A
13.1 19.4 99.789 0.211 50 mg Trehalose, Standard: RSN0151 99.691
0.309 20 mM Arginine- LA_09_29D T: 3 weeks {grave over (
)}+5.degree. C. 80.3 5.1 12.6 17.8 99.670 0.33 HCl, 0.01% Standard:
RSN0151 99.504 0.496 Polysorbate 20 LA_09_29D T: 3 weeks {grave
over ( )}-20.degree. C. 75.5 5.1 12.8 17.1 99.663 0.337 Standard:
RSN0151 99.504 0.496 LA_09_29D T: 3 weeks {grave over (
)}+40.degree. C. 78.6 4.9 17.2 0.553 Standard: RSN0151 99.504 0.496
LA_09_29D T: 6 weeks {grave over ( )}+5.degree. C. 80.0 5.1 12.6
18.7 99.655 0.345 Standard: RSN0151 99.461 0.539 LA_09_29D T: 6
weeks {grave over ( )}-20.degree. C. 5.0 72.1 5.1 12.6 18.9 99.687
0.313 Standard: RSN0151 99.461 0.539 LA_09_29D T: 6 weeks {grave
over ( )}+40.degree. C. 72.7 4.9 17.9 1.054 Standard: RSN0151
99.461 0.539 LA_09_29D T: 6 weeks {grave over ( )}-80.degree. C.
N/A 5.1 12.5 18.5 99.680 0.32 Standard: RSN0151 99.461 0.539
LA_09_29D T: 3 months {grave over ( )}+5.degree. C. 80.0 99.632
0.368 Standard: RSN0151 99.424 0.576 LA_09_29D T: 3 months {grave
over ( )}-20.degree. C. 80.0 N/A 99.677 0.323 Standard: RSN0151
99.424 0.576 LA_09_29D T: 3 months {grave over ( )}+40.degree. C.
78.0 2.8 99.424 0.576 ASD WCX SDS-PAGE non-red. Formulation % % %
main Elisa Buffer number acic neutral basic Comment band comment
EC50 % EC50 slope Histidine 10 mM, LA_09_29D 14.446 84.366 1.189
124.60 169 1.66E+12 0.66 50 mg Trehalose, Standard: RSN0151 13.755
85.045 1.2 122.88 100 9.84E+13 0.64 20 mM Arginine- LA_09_29D
14.168 84.691 1.141 HCl, 0.01% Standard: RSN0151 14.208 84.672 1.22
Polysorbate 20 LA_09_29D 14.366 84.473 1.161 Standard: RSN0151
14.208 84.672 1.22 LA_09_29D 14.697 83.391 10.712 N/A Standard:
RSN0151 14.208 84.672 1.22 LA_09_29D 13.796 84.86 1.344 Standard:
RSN0151 13.982 84.916 1.102 LA_09_29D 13.806 84.995 1.2 Standard:
RSN0151 13.982 84.916 1.102 LA_09_29D 14.302 41.575 14.124 198.81
90. 14 kDa N/A Standard: RSN0151 13.982 84.916 1.102 189.54
LA_09_29D 13.995 84.985 1.020 Standard: RSN0151 13.982 84.916 1.102
LA_09_29D 14.623 83.446 1.931 Standard: RSN0151 14.922 83.041 2.037
N/A LA_09_29D 14.150 83.575 2.276 Standard: RSN0151 83.041 2.037
LA_09_29D 41.914 27.279 83.041 2.037 indicates data missing or
illegible when filed
Anti-CXCR5 (20 mg/mL) Formulation Studies
[0484] The data in Examples 13-16 were collected during formulation
studies for the Lead CXCR5 Antibody and its drug product for
intravenous and subcutaneous administration. The objective of the
formulation studies was to provide a stable, clear or slightly
opalescent, and colorless or slightly yellow, visual particle-free
Lead CXCR5 Antibody solution for injection for phase I.
Materials
Drug Substance (DS)
[0485] Two drug substance batches were used for these formulation
studies. One was formulated in phosphate buffered saline (PBS) and
the other was formulated in citrate buffer. See Table 62.
TABLE-US-00079 TABLE 62 Available drug substance batches Lead Ab
pH-value Batch no. Amount concentration Buffer [--] RSN0151 10 g
5.0 mg/mL 155 mM PBS 7.2 SCB0001 20 g 20.30 mg/mL 10 mM Citrate
6.0
Excipients
[0486] Table 63 shows excipients that were used during the
formulation studies.
TABLE-US-00080 TABLE 63 Excipients Excipient Material no. Supplier
Arginine 1.01587 Merck Citric acid 100241 Merck Histidine 1.04352
Merck Hydrochloric acid 114027 H600 Saccharose S3929 Sigma-Aldrich
Sodium acetate 1.06265 Merck Sodium chloride 10158 H600 Sodium
citrate 114196 H600 Sodium hydroxide 114076 H600 Polysorbate 20
139850 H600 .alpha.,.alpha.-Trehalose T9531 Sigma-Aldrich
Methods
Sample Preparation
[0487] Ultrafiltration/Diafiltration was performed on a small scale
using VivaSpin devices with a Hydrosart membrane and a 30 kDa
cut-off. RSN material was concentrated from 5 mg/mL to 20 mg/mL,
and phosphate (PBS) buffer was exchanged to either 10 mM citrate
buffer pH 6.0, acetate buffer pH 5.5, or histidine buffer pH 5.0.
The VivaSpin units were placed at room temperature (RT) in a common
laboratory centrifuge and centrifuged with 2000 rpm. The solution
was filtered over a 0.2 .mu.m Minisart before analytical testing.
All samples were stored between +2.degree. and +8.degree. C.,
tightly closed, and protected from light, until analytical testing
at T0 and after one week thermal stress at +40.degree. C. or after
mechanical stress for 2.5 hours, 300 rpm at RT (only for evaluation
of polysorbate 20 concentration).
Analytical Methods
[0488] The following techniques were used for sample analysis:
TABLE-US-00081 TABLE 64 Analytical techniques used Technique
(Company) Parameter to investigate Organoleptic (--) Appearance
Nephelometer (Hach Lange) Turbidity pH-meter (WTW) pH-value UV
(Perkin Elmer) Concentration of mAB Densimeter (Paar) Density
Osmometer (Knauer) Osmolality Viscosimeter (Paar) Viscosity Dynamic
Light Scattering Hydrodynamic diameter (Malvern) SEC (N/A)
Mono-/Di-/Oligomer and High Molecular Weight Protein (HMWP)/ Low
Molecular Weight (LMW) content WCX(N/A) Isoforms
(acid/basic/neutral) ELISA*(N/A) Potency (Binding) SDS-Page
(red.)** (N/A) HC/LC, mAB-fragments SDS-Page (non-red.)** (N/A)
Aggregation and degradation products HIAC*(N/A) Particulate matter
*Some samples will be analyzed. **SDS-Page will be performed in
case SEC shows unusual results.
Example 13
Additional pH Optimization
[0489] Preformulation studies identified 10 mM citrate buffer at pH
6.0 as the best buffer with less Lead CXCR5 Antibody aggregation
tendency. To obtain a pH-profile in citrate buffer, stepwise
pH-dependent stability from pH 5.0 to 7.0 was evaluated. Due to
limited drug substance availability, in-depth pH-screening was
performed only with 10 mM citrate buffer. Samples were taken at T0
and after one week thermal stress at +40.degree. C. See Tables
65-69.
TABLE-US-00082 TABLE 65 Overview of samples Target pH-value
Measured pH-value Batch no. (--) (--) LA_09_030 5.0 5.1 LA_09_031
5.3 5.4 LA_09_032 5.5 5.6 LA_09_033 5.7 5.8 LA_09_034 6.0 6.1
LA_09_035 6.3 6.4 LA_09_036 6.5 6.6 LA_09_037 6.7 6.8 LA_09_038 7.0
7.1
TABLE-US-00083 TABLE 66 results T0 Appear- Measured pH- mAB conc.
Hydrodynamic Batch no. ance value (--) (mg/mL) diameter (nm)
LA_09_030 Clear 5.1 23.35 15.42 + aggr. LA_09_031 Clear 5.4 21.95
12.85 LA_09_032 Clear 5.6 23.00 12.98 LA_09_033 Clear 5.8 21.21
12.99 LA_09_034 Clear 6.1 22.77 12.83 LA_09_035 Clear 6.4 23.87
13.22 LA_09_036 Clear 6.6 23.74 13.04 LA_09_037 Clear 6.8 22.85
13.00 LA_09_038 Clear 7.1 21.96 13.32
TABLE-US-00084 TABLE 67 results T1 week +40.degree. C. Appear-
Measured pH mAB conc. Hydrodynamic Batch no. ance value (--)
[mg/mL] diameter (nm) LA_09_030 Clear 5.2 15.62* 15.27 + aggr.
LA_09_031 Clear 5.5 21.91 16.74 + aggr. LA_09_032 Clear 5.6 24.32
13.59 LA_09_033 Clear 5.8 24.74 13.83 LA_09_034 Clear 6.1 24.18
13.25 LA_09_035 Clear 6.5 N/A 13.41 LA_09_036 Clear 6.6 23.03 13.37
LA_09_037 Clear 6.9 22.68 13.24 LA_09_038 Clear 7.2 23.33 14.40
*Unusual result due to dilution mistake
TABLE-US-00085 TABLE 68 ASD results T0 % Mono- % Di-/ % % Batch no.
mer Oligomer HMWP % % % basic LA_09_030 99.63 0.37 -- -- 13.60
85.18 1.22 LA_09_031 99.57 0.43 -- -- 13.52 85.24 1.24 LA_09_032
99.48 0.52 -- -- 13.71 85.04 1.25 LA_09_033 99.51 0.49 -- -- 13.99
84.61 1.40 LA_09_034 99.41 0.59 -- -- 13.62 85.17 1.21 LA_09_035
99.24 0.76 -- -- 13.72 84.64 1.64 LA_09_036 98.72 1.28 -- -- 13.72
84.45 1.83 LA_09_037 98.95 1.05 -- -- 13.60 84.73 1.67 LA_09_038
98.58 1.42 -- -- 13.84 84.13 2.03
TABLE-US-00086 TABLE 69 results T1 week +40.degree. C. % % % % %
Batch no. % Monomer % Di-/Oligomer HMWP LMW acidic neutral basic
LA_09_030 95.19 0.97 3.29 0.55 11.90 83.92 4.18 LA_09_031 96.47
0.89 2.14 0.50 12.19 84.70 3.11 LA_09_032 96.82 0.92 1.69 0.57
12.14 85.48 2.38 LA_09_033 97.13 0.94 1.48 0.45 12.41 85.04 2.55
LA_09_034 97.73 0.97 0.82 0.48 12.35 85.69 1.96 LA_09_035 97.58
1.12 0.89 0.41 11.93 85.74 2.33 LA_09_036 97.47 1.32 0.86 0.35
12.01 85.46 2.53 LA_09_037 97.35 1.41 0.87 0.37 12.08 85.28 2.64
LA_09_038 96.97 1.62 0.97 0.44 11.65 85.10 3.25
[0490] In conclusion, the data confirm the results already
generated during preformulation studies: increasing the pH causes
the monomer content to decrease and dimer rate to increase. Samples
at +40.degree. C. showed with lower pH-value decrease in HMWs up to
pH 6.0 and then increase up to pH 5.0.
Example 14
Additional Buffer Optimization
[0491] Next, citrate, acetate, and histidine (as back-up buffer)
buffers were screened at 5/10/25/50 mM at the selected pH-values.
See Tables 70-84.
TABLE-US-00087 TABLE 70 Overview on samples - Citrate buffer pH 6.0
Batch no. Citrate buffer conc. [mM] LA_09_040 5 LA_09_034 10
LA_09_041 25 LA_09_042 50
TABLE-US-00088 TABLE 71 results after T0 Appear- Measured pH mAB
conc. Hydrodynamic Batch no. ance value (--) [mg/mL] diameter (nm)
LA_09_040 Clear 6.1 20.05 13.47 LA_09_034 Clear 6.1 22.77 12.83
LA_09_041 Clear 6.2 20.48 11.91 LA_09_042 Clear 6.1 22.19 11.87
TABLE-US-00089 TABLE 72 results after T1 week +40.degree. C.
Appear- Measured pH mAB conc. Hydrodynamic Batch no. ance value
(--) [mg/mL] diameter (nm) LA_09_040 Clear 6.3 21.62 13.78
LA_09_034 Clear 6.1 24.18 13.25 LA_09_041 Clear 6.2 18.37 12.50
LA_09_042 Clear 6.2 20.59 12.07
TABLE-US-00090 TABLE 73 results T0 % Di-/ % % Oligomer HMWP Mono-
(RRT (RRT % Batch no. mer 0.84) 0.68) % % % basic LA_09_040 99.49
0.51 -- -- 13.19 85.81 1.00 LA_09_034 99.41 0.59 -- -- 13.62 85.17
1.21 LA_09_041 99.55 0.42 0.03 -- 13.24 85.67 1.09 LA_09_042 99.60
0.39 0.01 -- 13.41 85.48 1.11
TABLE-US-00091 TABLE 74 results after thermal stress 1
week/+40.degree. C. % Di-/ % % Oligomer HMWP Mono- (RRT (RRT %
Batch no. mer 0.84) 0.68) % % % basic LA_09_040 98.58 0.86 0.17
0.39 12.52 85.95 1.53 LA_09_034 97.73 0.97 0.82 0.48 12.35 85.69
1.96 LA_09_041 98.81 0.65 0.21 0.33 12.54 86.07 1.38 LA_09_042
98.87 0.59 0.14 0.40 12.45 86.10 1.45
TABLE-US-00092 TABLE 75 Overview on samples - Histidine buffer pH
5.0 Batch no. Histidine buffer conc. [mM] LA_09_043 5 LA_09_044 10
LA_09_045 25 LA_09_046 50
TABLE-US-00093 TABLE 76 results after T0 Appearance mAB Hydro-
Measured pH conc. diameter Batch no. dynamic value (--) [mg/mL]
(nm) LA_09_043 Clear 5.5 21.89 8.40 + aggr. LA_09_044 Clear N/A
6.95* 11.34 LA_09_045 Clear 5.2 21.78 11.86 + aggr. LA_09_046 Clear
5.1 20.04 11.86 *Low data due to sample dilution mistake
TABLE-US-00094 TABLE 77 results after T1 week +40.degree. C.
Appear- Measured pH mAB conc. Hydrodynamic Batch no. ance value
(--) [mg/mL] diameter LA_09_043 Clear 5.5 21.34 8.81 LA_09_044
Clear 5.5 24.18 13.25 LA_09_045 Clear 5.2 23.62 11.88 LA_09_046
Clear 5.1 21.41 12.50 + aggr.
TABLE-US-00095 TABLE 78 ASD results T0 % % % Batch no. % % Di- HMWP
% acidic % basic LA_09_043 99.55 0.45 -- -- 13.69 85.16 1.15
LA_09_044* N/A N/A N/A N/A N/A N/A N/A LA_09_045 99.68 0.32 -- --
13.73 85.00 1.27 LA_09_046 99.70 0.30 -- -- 13.43 85.49 1.08 *not
analyzed due to dilution mistake
TABLE-US-00096 TABLE 79 results after thermal stress 1
week/+40.degree. C. % % % Batch no. % % Di- HMWP % acidic % basic
LA_09_043 98.72 0.82 -- 0.46 13.63 84.60 1.75 LA_09_044* N/A N/A
N/A N/A N/A N/A N/A LA_09_045 98.30 0.80 0.44 0.56 12.79 85.19 2.02
LA_09_046 97.79 0.68 1.07 0.46 12.61 84.75 2.64 *not analyzed due
to dilution mistake
TABLE-US-00097 TABLE 80 Overview of samples - Acetate buffer pH 5.5
Batch no. Acetate buffer conc. [mM] LA113244_09_053 5
LA113244_09_054 10 LA113244_09_055 25 LA113244_09_056 50
TABLE-US-00098 TABLE 81 results after T0 Appear- Measured pH mAB
conc. Hydrodynamic Batch no. ance value (--) [mg/mL] diameter
LA_09_053 Clear 5.88 25.41 10.44 LA_09_054 Clear 5.68 21.91 13.21 +
aggr. LA_09_055 Clear 5.56 21.53 14.06 LA_09_056 Clear 5.56 22.08
13.54
TABLE-US-00099 TABLE 82 results after T0 Appear- Measured pH mAB
conc. Hydrodynamic Batch no. ance value (--) [mg/mL] diameter
LA_09_053 Clear 5.88 26.43 11.48 LA_09_054 Clear 5.69 23.40 13.44
LA_09_055 Clear 5.61 21.53 14.46 LA_09_056 Clear 5.56 21.68
13.71
TABLE-US-00100 TABLE 83 results T0 % % acid- % Batch no. % % Di-
HMWP % ic % basic LA_09_053 98.89 0.99 0.02 0.10 14.44 83.67 1.89
LA_09_054 98.84 1.09 0.07 -- 11.30 86.80 1.90 LA_09_055 98.91 0.99
0.07 0.03 11.30 86.77 1.93 LA_09_056 98.97 0.87 0.10 0.06 11.27
86.90 1.83
TABLE-US-00101 TABLE 84 results after thermal stress 1
week/+40.degree. C. % Di-/ % % Oligomer HMWP % Mono- (RRT (RRT
acid- % Batch no. mer 0.84) 0.68) % ic % basic LA_09_053 97.68 1.96
0.04 0.32 14.53 81.50 3.91 LA_09_054 97.83 1.99 0.09 0.09 11.13
85.79 3.08 LA_09_055 97.88 2.00 0.09 0.09 11.09 85.76 3.15
LA_09_056 98.10 1.22 0.59 0.09 10.92 86.24 2.74
[0492] In conclusion, the data confirm the results generated during
the preformulation studies. Using citrate as the buffer agent, the
monomer content is slightly higher than with acetate buffer and
histidine buffer. With histidine, high aggregation behavior is
observable, even at T0, leading to difficulties in analytical
sample preparation. A significant difference between the tested
buffer concentrations cannot be measured, so all three buffers
citrate, histidine, and acetate will be used with a concentration
of 10 mM.
Example 15
Additional Surfactant Optimization
[0493] Based on preformulation trials, the addition of non-ionic
surfactant polysorbate 20 (0.01%) showed beneficial effects on
stability, so further evaluation of its concentration was performed
by adding the following polysorbate 20 concentrations to the
respective buffers: 0.0025%/0.005%/0.01%/0.02%. See Tables
85-94.
TABLE-US-00102 TABLE 85 Overview of samples in acetate buffer
Polysorbate 20 concentration Batch no. in [mg/mL] as percent[%]
LA_09_058 0.2 0.02 LA_09_059 0.1 0.01 LA_09_060 0.05 0.005
LA_09_061 0.025 0.0025
TABLE-US-00103 TABLE 86 results after T0 Appear- Measured pH mAB
conc. Hydrodynamic Batch no. ance value (--) [mg/mL] diameter (nm)
LA_09_058 Clear 5.61 23.71 12.50 LA_09_059 Clear 5.64 22.76 12.94
LA_09_060 Clear 5.63 23.89 12.83 LA_09_061 Clear 5.64 25.79
12.88
TABLE-US-00104 TABLE 87 results after mechanical stress 300 rpm/150
min Appear- Measured pH mAB conc. Hydrodynamic Batch no. ance value
(--) [mg/mL] diameter (nm) LA_09_058 Clear 5.61 22.82 12.48
LA_09_059 Clear 5.67 22.47 12.73 LA_09_060 Clear 5.55 22.90 12.59
LA_09_061 Clear 5.65 25.19 12.76
TABLE-US-00105 TABLE 88 results T0 % % % Batch no. % % Di- HMWP %
acidic % basic LA_09_058 99.17 0.80 0.03 -- 11.25 86.94 1.81
LA_09_059 99.16 0.81 0.03 -- 11.27 86.94 1.79 LA_09_060 99.17 0.81
0.03 -- 11.44 86.80 1.76 LA_09_061 99.13 0.84 0.03 -- 11.31 86.91
1.78
TABLE-US-00106 TABLE 89 results after mechanical stress 300 rpm/150
min % Di-/ % % Oligomer HMWP Mono- (RRT (RRT % % Batch no. mer
0.84) 0.68) % acidic % basic LA_09_058 99.16 0.81 0.03 -- 11.27
86.95 1.78 LA_09_059 99.16 0.82 0.02 -- 11.24 86.91 1.85 LA_09_060
99.17 0.81 0.03 -- 11.46 86.77 1.77 LA_09_061 99.16 0.81 0.02 11.22
86.97 1.79
TABLE-US-00107 TABLE 90 Overview of samples in citrate buffer
Polysorbate 20 concentration Batch no. in (mg/mL) as percent (%)
LA_09_062 0.2 0.02 LA_09_063 0.1 0.01 LA_09_064 0.05 0.005
LA_09_065 0.025 0.0025
TABLE-US-00108 TABLE 91 results after T0 Appear- Measured pH mAB
conc. Hydrodynamic Batch no. ance value (--) (mg/mL) diameter (nm)
LA_09_062 Clear 6.05 23.72 12.67 LA_09_063 Clear 6.03 25.18 12.73
LA_09_064 Clear 6.04 23.85 12.47 LA_09_065 Clear 6.04 22.65
12.46
TABLE-US-00109 TABLE 92 results after T1 week +40.degree. C.
Appear- Measured pH mAB conc. Hydrodynamic Batch no. ance value
(--) [mg/mL] diameter (nm) LA_09_062 Clear 6.07 23.44 12.99
LA_09_063 Clear 6.03 24.39 12.59 LA_09_064 Clear 6.04 23.93 12.39
LA_09_065 Clear 6.04 22.27 12.37
TABLE-US-00110 TABLE 93 results T0 % Mono- % Di-/ % % % Batch no.
mer Oligomer HMWP % acidic % basic LA_09_062 99.25 0.70 0.05 --
11.33 86.24 2.43 LA_09_063 99.28 0.68 0.04 -- 11.00 86.36 2.64
LA_09_064 99.23 0.74 0.03 -- 10.93 86.45 2.62 LA_09_065 99.28 0.69
0.03 -- 10.97 86.25 2.77
TABLE-US-00111 TABLE 94 results after mechanical stress 300 rpm/150
min % Mono- % Di-/ % % % Batch no. mer Oligomer HMWP % acidic %
basic LA_09_062 99.27 0.69 0.04 -- 11.25 86.27 2.48 LA_09_063 99.32
0.65 0.03 -- 10.87 86.59 2.54 LA_09_064 99.19 0.78 0.03 -- 10.91
86.56 2.53 LA_09_065 99.16 0.80 0.04 -- 10.79 86.51 2.70
[0494] In conclusion, no significant differences in samples
containing acetate or citrate buffer with various polysorbate
concentration were measurable. To ensure mAb prevention against
mechanical stress over a longer period of time than tested for 150
min, the polysorbate concentration was set to 0.2 mg/mL. This
amount was also proposed based on preformulation studies.
Example 16
Additional Isotonicity Optimization
[0495] During preformulation studies, NaCl, Trehalose, and
Arginine-HCl were identified as additives for isotonicity and
stability purposes. Arginine-HCl was then dropped due to less mAb
stability effects. Depending on buffer concentration and pH-value,
isotonant/stabilizer amount is adapted to achieve osmolality of at
least 240 mOsmol/kg according to Ph. Eur.
[0496] The use of trehalose was challenged as it is not a
compendial excipient and is high priced. During preformulation
studies, sucrose (saccharose) caused slightly more aggregation, but
was not followed-up and verified in further studies. Therefore, a
new short-term stability study over four weeks was designed,
including trehalose as well as saccharose in both 10 mM citrate and
acetate buffer with storage temperatures at +5.degree.,
+25.degree., and +40.degree. C. See Tables 95-103.
[0497] Fine-tuning of osmolality of at least 240 mOsmol/kg was
performed with NaCl.
TABLE-US-00112 TABLE 95 Overview of samples Target pH- Stabi- value
Poly- lizing Batch no. Buffer [--] sorbate 20 NaCl agent LA_09_051A
10 mM 6.0 0.2 mg/mL 2 mg/mL Sucrose Citrate 60 mg/mL LA_09_051B 10
mM 6.0 0.2 mg/mL 2 mg/mL Trehalose Citrate 60 mg/mL LA_09_052A 10
mM 5.5 0.2 mg/mL 2 mg/mL Sucrose Citrate 60 mg/mL LA_09_052B 10 mM
5.5 0.2 mg/mL 2 mg/mL Trehalose Citrate 60 mg/mL
TABLE-US-00113 TABLE 96 results T0 Appear- Measured pH mAB-conc.
Osmolality Batch no. ance value (--) (mg/mL) (mOsmol/kg) LA_09_051A
Clear 5.89 21.46 289 LA_09_051B Clear 5.94 21.46 268 LA_09_052A
Clear 5.82 22.07 273 LA_09_052B Clear 5.80 22.07 256
TABLE-US-00114 TABLE 97 results T 4 weeks, +5.degree. C. Measured
pH- mAB-conc. Hydrodynamic Batch no. value (--) (mg/mL) diameter
(nm) LA_09_051A 6.02 21.81 13.58 LA_09_051B 5.95 22.10 13.35
LA_09_052A 5.86 21.70 15.12 LA_09_052B N/A N/A N/A
TABLE-US-00115 TABLE 98 results T 4 weeks, +25.degree. C. Measured
pH- mAB-conc. Hydrodynamic Batch no. value (mg/mL) diameter (nm)
LA_09_051A 6.06 22.30 13.57 LA_09_051B 6.02 22.09 13.41 LA_09_052A
5.91 21.98 15.14 LA_09_052B N/A N/A N/A
TABLE-US-00116 TABLE 99 results T 4 weeks, +40.degree. C. Measured
pH- mAB-conc. Hydrodynamic Batch no. value (mg/mL) diameter (nm)
LA_09_051A 6.04 22.21 14.73 LA_09_051B 5.95 21.86 14.11 LA_09_052A
5.91 22.23 16.37 LA_09_052B 5.89 22.84 16.02
TABLE-US-00117 TABLE 100 results T0 mAB- conc. Batch no. % % Di- %
% % (mg/mL) LA_09_051A 99.53 0.47 13.78 83.85 2.37 23.45 LA_09_051B
99.54 0.46 13.73 84.83 1.94 22.91 LA_09_052A 99.44 0.56 13.83 83.99
2.18 22.54 LA_09_052B 99.44 0.56 14.39 83.38 2.23 23.19
TABLE-US-00118 TABLE 101 esults after thermal stress 4
weeks/+5.degree. C. % Di- % HMWP mAB-conc. Batch no. % (RRT 0.84)
(RRT 0.68) % % % % (mg/mL) LA_09_05 1A 99.21 0.38 -- 0.41 11.34
87.11 1.55 24.24 LA_09_05 1B 98.97 0.46 -- 0.57 11.26 87.17 1.58
23.71 LA_09_05 2A 98.81 0.54 -- 0.65 11.47 86.86 1.67 22.63
LA_09_05 2B 99.00 0.55 -- 0.45 11.46 86.90 1.64 23.17
TABLE-US-00119 TABLE 102 results after thermal stress 4
weeks/+25.degree. C. mAB-conc. Batch no. % % Di- % % LMW % % %
(mg/mL) LA_09_051A 98.87 0.46 -- 0.67 11.01 87.31 1.69 26.16
LA_09_051B 98.72 0.53 -- 0.75 11.02 87.29 1.70 23.63 LA_09_052A
98.25 0.83 -- 0.92 11.69 86.35 1.96 24.27 LA_09_052B 98.52 0.74 --
0.74 11.46 86.62 1.92 23.52
TABLE-US-00120 TABLE 103 results after thermal stress 4
weeks/+40.degree. C. mAB-conc. Batch no. % % Di- % % LMW % % %
(mg/mL) LA_09_051A 96.84 0.95 1.06 1.15 10.16 87.05 2.79 25.04
LA_09_051B 96.96 0.97 1.01 1.06 10.01 87.05 2.83 23.60 LA_09_052A
96.00 1.63 1.17 1.20 11.32 84.89 3.79 24.11 LA_09_052B 96.23 1.49
1.21 1.07 11.01 85.30 3.69 24.29
[0498] In conclusion, no significant differences between citrate
and acetate buffer were measured, and no difference at accelerated
conditions between trehalose and saccharose was visible. Citrate
buffer with saccharose was selected for further studies.
Determination of DP Manufacturing Process Parameters
[0499] DS batch in citrate buffer was used to determine
manufacturing process parameters. Preformulation studies indicated
that the DS was not that susceptible to oxidation, and that light
protection or nitrogen overlay or purging during manufacturing was
required. Standard glass equipment as well as silicone tubings
(SaniTech65) were used.
Adding Order
[0500] Experiments evaluating the adding order of the excipients
were limited due to the small dilution volume of DS.
[0501] The DS was weighed in a glass bottle, polysorbate 20 as
first excipient, saccharose as the second excipient, and NaCl as
third excipient were added and rinsed with citrate buffer 10 mM pH
6.0 to dilute the content of DS to 20 mg/mL.
Stirring Speed and Time
[0502] Stirring speed was set at 100 rpm to reduce mechanical
stress for the DS. Due to the fact that all excipients were well
water-soluble, stirring time was set to 5 minutes.
Monitoring Parameters and IPCs
[0503] Monitoring parameters such as appearance, turbidity,
density, and viscosity, and IPCs such as pH-value and osmolality
were routinely checked during sample manufacturing according to the
following Table 104:
TABLE-US-00121 TABLE 104 Before Filtration After Filtration After
Filling Appearance colorless to colorless to colorless to slightly
yellow slightly yellow slightly yellow Density 1.006 mg/mL Not
measured Not measured Turbidity Clear Clear Clear Viscosity Not
measured Not measured <5 mPa s pH-value 6.0 .+-. 0.2 6.0 .+-.
0.2 Not measured (20-25.degree. C.) (20-25.degree. C.) Osmolality
290 .+-. 40 290 .+-. 40 Not measured mOsmol/kg mOsmol/kg
No issues were observed during manufacturing. The limits for
osmolality were set-up based on measured data.
Filtration Process
[0504] According to preformulation studies, polyethersulfone was a
suitable membrane for sterile filtration (Sartorius, 0.22 .mu.m).
No potential pH-shifts after filtration could be observed, as
filtration rate and time showed standard values for filtration of
an aqueous solution. Filter integrity testing was routinely
performed without any issues.
Filling Process
[0505] Standard dosing equipment made of stainless steel, such as
the filling pump and filling needle were investigated. Also,
duration and filling speed was monitored. Extractable volume of
filled DP was determined. An overfilling of 0.2 mL was required to
ensure an extractable volume of 1.5 mL.
Material Compatibility
[0506] All preformulation and formulation studies were performed in
glass as standard manufacturing equipment, which is also the
recommendation for equipment to be used for GMP manufacturing.
Cleaning Agents
[0507] Cleaning of manufacturing equipment was performed according
to the respective SOPs using the dishwasher with standard cleaning
agent Neodisher.RTM.. A manual pre-cleaning with water for
injection was routinely done before. No harmful effects of cleaning
agents were observed.
Summary of Additional Formulation Studies for Lead CXCR5 Antibody
(20 mg/mL)
[0508] For selection of phase I Lead CXCR5 Antibody DP formulation,
citrate 10 mM at pH 6.0 was selected as the buffer over histidine
and acetate. The pH-value of the solution was set at 6.0, as
increasing or decreasing the pH-value means a reduction in monomer
content. The buffer concentration was set at a medium concentration
of 10 mM, although there was no significant difference between
concentrations of 5-50 mM.
[0509] Polysorbate 20 was chosen as the surfactant with 0.2 mg/mL
(0.02%), sufficient to stabilize the DS against mechanical
stress.
[0510] Sucrose (saccharose) was selected as the stabilizer against
thermal stress in favour of trehalose. The concentration of
saccharose was set at 60 mg/mL (6%).
[0511] NaCl will be used as the isotonant agent in a concentration
of 2.0 mg/mL (0.2%) in order to achieve an osmolality of DP of
about 300 mOsmol/kg.
Anti-CXCR5 (100 mg/mL) Formulation Studies
[0512] The data in Examples 17-21 were collected during formulation
studies for the Lead CXCR5 Antibody and its drug product for
intravenous and subcutaneous administration. The objective of the
formulation studies was to provide a stable, clear or slightly
opalescent, and colorless or slightly yellow, visual particle-free
Lead CXCR5 Antibody solution for injection for phase I.
Methods
Sample Preparation
[0513] UF/DF was performed on a small scale using VivaSpin devices
with a Hydrosart membrane and a 30 kDa cut-off. RSN material was
concentrated from ca. 20 mg/mL to 100 mg/mL. All solutions were
already in the final formulation buffer (10 mM citrate buffer at pH
6.0).
[0514] The VivaSpin units were placed at RT in a common laboratory
centrifuge and centrifuged at 2000 rpm. Solution was filtered over
0.2 .mu.m Minisart before analytical testing.
[0515] All samples were stored between +2.degree. and +8.degree.
C., tightly closed and protected from light, until analytical
testing at T0 and after one week thermal stress at +40.degree. C.
or after mechanical stress.
Analytical Methods
[0516] The following techniques were used for sample analysis:
TABLE-US-00122 TABLE 105 Analytical techniques used Technique
Parameter to investigate Organoleptic Appearance Nephelometer
Turbidity pH-meter pH-value UV mAB-concentration Densimeter Density
Osmometer Osmolality Viscosimeter Viscosity DLS Hydrodynamic
diameter DSC* Unfolding temperature SEC Mono-/Di-/Oligomer/HMW
content WCX Isoforms (acid/basic/neutral) ELISA* Potency (Binding)
SDS-Page (red.)** HC/LC, mAB-fragments SDS-Page (non-red.)**
Aggregation and degradation products HIAC* Subvisible particles
*Some samples will be analyzed. **SDS-Page will be performed in
case SEC shows unusual results.
Example 17
Excipient Screening
[0517] Preformulation studies identified 10 mM citrate buffer at pH
6.0 as the best buffer with less Lead CXCR5 Antibody aggregation
tendency. In previous studies at 20 mg/mL, a formulation containing
10 mM citrate buffer, 60 mg/mL (6%) sucrose, 2 mg/mL (0.2%) NaCl,
and 0.2 mg/mL (0.02%) Polysorbate 20 was selected. Those excipients
plus some alternatives were tested to confirm the suitability of
the selected formulation at a higher concentration (100 mg/mL).
[0518] Different formulations were stressed thermally at 40.degree.
C. for 7 days and mechanically at 100 rpm for 5 hrs. Additionally,
the unfolding temperature for the different formulations were
screened at 100 mg/mL using DSC (Differential scanning
calorimetry).
[0519] The following excipients were tested:
Sucrose.fwdarw.60 mg/mL Trehalose.fwdarw.60 mg/mL
Arginine.fwdarw.30 mg/mL Lysine.fwdarw.30 mg/mL Glycine.fwdarw.30
mg/mL
[0520] NaCl or Mannitol was added as an isotonant. No salts were
needed for viscosity reduction (around 2.1 cP).
[0521] The results of T0 and T7 days are shown in Table 106.
TABLE-US-00123 TABLE 106 Excipients screening SEC Isoforms by WCX
Activity SDS-Page Formulation HMW Mono. acidic neutral basic rel.
potency non-reducing T composition (mg/mL) Nr. [%] [%] [%] [%] [%]
[%] conditions T zero Sucrose (60) + NaCl (2) 72_A1 2.7 97.0 11.7
84.2 4.0 165 comparable to reference Sucrose (60) + Mannitol (15)
72_A2 2.8 97.1 11.9 84.4 3.7 205 comparable to reference Trehalose
(60) + NaCl (2) 72_B1 2.8 97.0 11.7 84.8 3.6 110 comparable to
reference Trehalose (60) + Mannitol (15) 72_B2 2.8 97.0 11.8 84.2
4.0 156 comparable to reference Arginine (30) 72_C1 2.5 97.2 11.9
84.3 3.8 160 comparable to reference Arginine (20) + NaCl (2) 72_C2
2.6 97.2 11.8 84.6 3.6 144 comparable to reference Arginine (20) +
Mannitol (15) 72_C3 2.6 97.2 12.2 83.8 3.9 117 comparable to
reference Lysine (30) + NaCl (2) 72_D1 2.6 97.1 12.7 82.1 5.3 130
comparable to reference Lysine (30) + Mannitol (15) 72_D21 2.6 97.1
12.6 82.2 5.3 88 comparable to reference Glycine (20) 72_E1 2.7
97.1 12.4 83.5 4.1 170 comparable to reference Glycine (20) + NaCl
(2) 72_E2 2.7 97.1 12.4 83.5 4.1 174 comparable to reference
Glycine (20) + Mannitol (15) 72_E3 2.7 97.0 12.7 83.3 4.1 111
comparable to reference T 7 days Sucrose (60) + NaCl (2) 72_A1 3.5
96.3 11.4 84.4 4.2 188 comparable to reference at 40.degree. C.
Sucrose (60) + Mannitol (15) 72_A2 3.5 96.3 11.3 84.6 4.2 243
comparable to reference Trehalose (60) + NaCl (2) 72_B1 3.4 96.4
11.5 84.4 4.1 191 comparable to reference Trehalose (60) + Mannitol
(15) 72_B2 3.5 96.3 11.4 84.3 4.3 266 comparable to reference
Arginine (30) 72_C1 3.6 96.1 11.2 84.9 4.0 143 comparable to
reference Arginine (20) + NaCl (2) 72_C2 3.4 96.4 11.6 84.5 3.9 164
comparable to reference Arginine (20) + Mannitol (15) 72_C3 3.3
96.4 11.3 85.3 3.4 not tested comparable to reference Lysine (30) +
NaCl (2) 72_D1 6.5 93.1 30.9 48.0 21.1 264 comparable to reference
Lysine (30) + Mannitol (15) 72_D21 5.9 93.7 31.2 47.9 20.9 297
comparable to reference Glycine (20) 72_E1 3.2 96.6 11.3 84.4 4.3
180 comparable to reference Glycine (20) + NaCl (2) 72_E2 3.3 96.4
11.6 84.4 4.0 not tested comparable to reference Glycine (20) +
Mannitol (15) 72_E3 3.2 96.6 11.2 84.6 4.2 not tested comparable to
reference
Thermal Stress
[0522] None of the samples showed turbidity before or after
stress.
[0523] Lysine showed: a pH shift to 9.8, a very high tendency to
aggregate, a very high increase in acidic and basic isoforms, and
high molecular weight bands in SDS-PAGE. As a result, it was
excluded from further consideration.
[0524] Formulations with mannitol showed bad binding in an ELISA
assay after stress. As a result, NaCl is the favored isotonant.
[0525] Sucrose showed slightly better chemical stability than
trehalose, but additional bands were seen in SDS-PAGE after stress
(for both).
[0526] Arginine (especially in the presence of NaCl) and glycine
had a similar SEC profile, but no additional bands were seen in
SDS-PAGE after stress.
Protein Associated Formation Measured by Dynamic Light Scattering
(DLS)
[0527] Lead CXCR5 Antibody showed a significant increase in the
hydrodynamic diameter (Z-Average) by increasing the concentration
(FIG. 34). This behavior was fully reversible upon dilution. For
further investigation of this effect, the different Lead CXCR5
Antibody concentrations were measured by analytical ultra
centrifugation (AUC) and aggregation was excluded. The conclusion
of the AUC study was that this behavior was due to the formation of
protein associates.
[0528] The effect of the above listed excipient on this behavior
was studied and the results are shown in FIG. 35. The Z-Average was
measure before and after thermal stress. The stabilizing effect was
similar to all tested ecipients, but the increase in Z-average was
generally reduced by using amino acids as stabilizers (Arginine,
Lysine or Glycine). Lysine was excluded due to higher content of
aggregates after stress. Arginine showed a better effect than
Glycine. Both amino acids were considered for the final design of
experiment in order to choose the best excipient combination.
Mechanical Stress
[0529] Lysine formulations were excluded as well as all
formulations containing mannitol. SEC data showed no effect of the
stress on the tested samples. See Table 107.
TABLE-US-00124 TABLE 107 Mechanical stress SEC before SEC after
Formulation mechanical stress mechanical stress composition
Formula- HMW monomer HMW monomer (mg/mL) tion No. [%] [%] [%] [%]
Sucrose (60) + 080_A 2.6 97.3 2.7 97.2 NaCl (2) Trehalose (60) +
080_B 2.7 97.2 2.6 97.3 NaCl (2) Arginine (30) 080_C1 2.5 97.5 2.3
97.6 Arginine (20) + 080_C2 2.5 97.4 2.5 97.4 NaCl (2) Glycine (20)
080_D1 2.5 97.5 2.4 97.5 Glycine (20) + 080_D2 2.5 97.5 2.4 97.5
NaCl (2)
The same reduction in Z-average was noticed in the presence of
amino acids. Sucrose had a better protective effect than trehalose
against mechanical stress. Arginine and glycine performed better in
combination with NaCl. See FIG. 36.
Differential Scanning Calorimetry (DSC) Screening
[0530] A screening study to determine the unfolding temperature of
Lead CXCR5 Antibody was performed using Differential scanning
calorimetry (DSC). Sucrose, trehalo se, argenine, and glycine were
screened.
[0531] The Tm results are listed in Table 108.
TABLE-US-00125 TABLE 108 Effect of different excipients on the Tm
values of Lead CXCR5 Antibody. All formulations were in 10 mM
citrate buffer at pH 6 Excipient screened Tm1 Tm2 Tm3 Sucrose +
NaCl 65.3 73.6 83.8 Trehalose + NaCl 65.5 73.9 83.9 Arginine 63.8
72.2 82.6 Arginine + NaCl 64.3 72.8 82.6 Glycine 64.8 74.1 84.2
Glycine + NaCl 64.9 73.6 83.8 Based on Tm1, sucrose and trehalose
showed the highest values. Arginine performed better in combination
with NaCl.
[0532] In conclusion, the data collected suggests that the final
Lead CXCR5 Antibody 100 mg/ml formulation would contain a
combination of a sugar (in some embodiments, sucrose) and an amino
acid (in some embodiments, arginine or glycine) in the presence of
NaCl as the isotonant.
Example 18
Surfactant Screening
[0533] Polysorbate as a stabilizer was evaluated for protection of
Lead CXCR5 Antibody against both thermal and mechanical
stresses.
[0534] Polysorbate 20 and 80 were tested in two different
concentrations: 0.1 and 0.2 mg/ml.
Thermal Stress
[0535] DLS showed no effect by the addition of Polysorbate after
thermal stress. The formation of HMWs and fragments after 7 days
storage at 40.degree. C. was noticed in all samples, as detected by
SEC. No additional bands in SDS-PAGE were detected. Slight changes
were seen after thermal stress, but no differences between PS20 and
PS80, as well as between the 2 concentrations, were seen (data not
shown).
Mechanical Stress
[0536] DLS showed no changes after mechanical stress. Polysorbate
20 showed no aggregations after mechanical stress. Polysorbate 80
showed aggregates formation after mechanical stress. No additional
bands in SDS-PAGE (data not shown) were seen.
[0537] In conclusion, Polysorbate 20 was the desired surfactant due
to superiority in mechanical stabilization of the Lead CXCR5
Antibody.
Example 19
Prototype Formulation Pre-Selection Using DSC
[0538] Based on the excipient screening and the surfactant
screening studies, 12 different excipient combinations were
suggested (see Tables 109 and 110)
[0539] The unfolding temperature for all formulations was
determined using DSC and the resulting Tms, as well as the
osmolality for each formulation, are listed in Tables 109 and
110.
TABLE-US-00126 TABLE 109 Excipient combinations for prototype
formulations (Arginine) pre-selection study using DSC. Tm values
and osmolality are listed as well Composition mg/mL DSC Osmo.
Formulation Sucrose Arginine NaCl PS 20 Tm1 Tm2 Tm3 (mosmol/kg)
LA_10_087_A 60 20 2 0.1 65.2 73.3 83.2 495 LA_10_087_C 60 20 2 0.2
65.1 73.0 83.2 486 LA_10_087_E 30 10 2 0.1 64.5 72.8 83.0 304
LA_10_087_G 30 10 2 0.2 64.4 72.7 83.0 304 LA_10_087_L 45 10 2 0.1
64.7 73 83.2 349 LA_10_087_M 45 10 2 0.2 64.6 72.8 83.1 357
TABLE-US-00127 TABLE 110 Excipient combinations for prototype
formulations (glycine) pre-selection study using DSC. Tm values and
osmolality are listed as well Composition mg/mL DSC Osmo.
Formulation Sucrose Glycine NaCl PS 20 Tm1 Tm2 Tm3 (mosmol/kg)
LA_10_087_B 60 15 2 0.1 66.2 74.1 84.3 539 LA_10_087_D 60 15 2 0.2
65.8 74.1 84.3 533 LA_10_087_F 30 7.5 2 0.1 65.0 73.3 83.5 330
LA_10_087_H 30 7.5 2 0.2 64.8 73.1 83.4 320 LA_10_090_A 45 7.5 2
0.1 65.3 73.7 83.6 408 LA_10_090_B 45 7.5 2 0.2 65.3 73.7 83.9
391
The formulations didn't show great differences in Tm, but the
osmolality varied a lot. The pre-selection of the prototype
formulations were made based on Tm and omolality. Accordingly, in
each excipient group (arginine and glycine), the highest Tm was
selected (regardless of the osmolality). In addition, the highest
Tm in the isotonic region was also selected.
Example 20
Prototype Expolatory Stability Study
[0540] The above prototype selection resulted in 4 prototype
formulations, which are listed in Table 111. Those formulations
were tested for mechanical stress (100 rpm for 5 hours), 5
freeze/thaw cycles and isothermal stress at 5, 20, and 40.degree.
C.
TABLE-US-00128 TABLE 111 Prototype formulations for the 100 mg/mL
Lead CXCR5 Antibody formulation Composition Su- Argi- Gly- PS Osmo.
Formulation crose nine cine NaCl 20 (mosmol/kg) LA_10_102_A 60 20 2
0.1 518 LA_10_102_B 45 10 2 0.1 374 LA_10_102_C 60 15 2 0.1 550
LA_10_102_D 30 7.5 2 0.1 325
Mechanical Stability
[0541] Lead CXCR5 Antibody in 10 mM citrate buffer at pH 6, without
addition of any excipients (DS formulation), was also stressed in
parallel with the prototype formulations. A higher molecular weight
species was measured by DLS after mechanical stress of DS (FIG.
37), stress where no changes have been seen in all tested
formulations after mechanical stress. The formation of aggregates
after mechanical stress was measured using size exclusion
chromatography (SEC) and the results are shown in Table 112. In
general, the 4 formulations were equally stable to mechanical
stress except formulation A, where more HMWs were found by SEC
after mechanical stress. See FIG. 38.
TABLE-US-00129 TABLE 112 Size exclusion chromatography (SEC)
results of the prototype formulations before and after mechanical
stress SEC For_A For_B For_C For_D % Mon. % Mon. % Mon. % Mon.
-20.degree. C. Before 99.6 99.5 99.5 99.5 After 98.1 99.5 99.3
99.4
Freeze/Thaw Stability
[0542] No significant differences were detected, either on DS or
DP, after 5 freeze/thawing cycles. Therefore, there should be no
instability issues by freezing and thawing (data are not
shown).
Exploratory Prototype Stability Study
[0543] The prototype formulations were stored at -20, 5, 20, and
40.degree. C. They were analyzed at the start of the study, after 1
month, after 3 months, and after 6 months. The formulations were
selected based on the 3 months results (Tables 113-115). The
results showed that formulation B performed the best with regard to
SEC, WCX, and sub-visible particles, especially at 40.degree.
C.
TABLE-US-00130 TABLE 113 Size exclusion chromatography (SEC)
results of the prototype formulations after 3 months SEC For_A
For_B For_C For_D % Mon. % Mon. % Mon. % Mon. -20.degree. C. T0
99.6 99.5 99.5 99.5 T1 month N/A N/A N/A N/A T3 months 99.1 99.1 99
99.5 5.degree. C. T0 99.6 99.5 99.5 99.5 T1 month 99.3 99.4 99.5
99.5 T3 months 99 99.4 98.8 99.4 20.degree. C. T0 99.6 99.5 99.5
99.5 T1 month 99.5 99.5 99.4 99.4 T3 months 99 98.9 98.6 99.1
40.degree. C. T0 99.6 99.5 99.5 99.5 T1 month 96.9 96.7 96.5 96.3
T3 months 91.5 91.6 89.5 90.2
TABLE-US-00131 TABLE 114 Weak Cationic exchange chromatography
(WCX) results of the prototype formulations after 3 months WCX
For_A For_B For_C For_D % Basic % Basic % Basic % Basic -20.degree.
C. T0 2 2.2 2.1 2.2 T1 month N/A N/A N/A N/A T3 months 1.5 1.6 1.6
1.6 5.degree. C. T0 2 2.2 2.1 2.2 T1 month 1.1 1.2 1.2 1.3 T3
months 1.5 1.6 1.7 1.6 20.degree. C. T0 2 2.2 2.1 2.2 T1 month 1.3
1.3 1.4 1.3 T3 months 1.7 1.9 2 2 40.degree. C. T0 2 2.2 2.1 2.2 T1
month 2.2 2.2 2.7 2.5 T3 months 6.5 5.1 8.6 8.2
TABLE-US-00132 TABLE 115 Sub-visible particles measured by Light
blockage at T zero and after 3 months (5.degree. C.) For_A For_B
For_C For_D >10 >25 >10 >25 >10 >25 >10 >25
.mu.m .mu.m .mu.m .mu.m .mu.m .mu.m .mu.m .mu.m T0 4 3 4 4 5 4 4 3
T2 8 1 5 1 34 14 6 2
[0544] In conclusion, the studies showed better results for the
formulation LA.sub.--10.sub.--102_B. This formulated had a
concentration of 100 mg/mL Lead CXCR5 Antibody in 10 mM citrate
buffer at pH 6 and contained the following excipients:
TABLE-US-00133 Sucrose 45 mg/mL (4.5%); Arginine 10 mg/mL (1%);
NaCl 2 mg/mL (0.2%); and Polysorbate 20 0.1 mg/mL (0.01%).
Example 21
Supporting Stability Data for the 100 mg/mL Formulation
[0545] Additional stability studies were done on the 100 mg/mL Lead
CXCR5 Antibody formulation identified in Example 20. The additional
studies were performed at -20, 5, and 25.degree. C. The results are
shown in Tables 116-118.
TABLE-US-00134 TABLE 116 Stability Data for 100 mg/mL Lead CXCR5
Antibody formulation at -20.degree. C. Drug product: Lead CXCR5
Antibody- Batch no.: 11_106/LST0008 solution for injection Dosage
100 mg/mL Manufacturer 11_106 strength: batch no.: Storage
-20.degree. C. .+-. 5.degree. C. condition: Storage Inverted
orientation: Time Initial 1 3 6 9 12 18 24 Test item results month
months months months months months months Appearance of solution
Clarity I <I <I <I II <IV <IV (20 NTU) (19 NTU)
Color Y7 Y7 Y7 Y7 Y7 Y6 Y6 Assay Potency (Antigen 75% 107% 84% 96%
101% 96% 127% ELISA) EC.sub.50 value (in comparison to reference)
Total protein 103 101 101 101 102 101 102 content (UV) mg/mL mg/mL
mg/mL mg/mL mg/mL mg/mL mg/mL Molecular integrity SDS-PAGE under
Conforms Conforms Conforms Conforms Conforms Conforms Conforms
non-reducing to to to to to to to conditions (Band reference
reference reference reference reference reference reference
pattern) Purity HPLC (SEC) Monomer (% area) 99.1%.sup. 99.0%
99.0%.sup. 98.8%.sup. 98.9% 98.8%.sup. 98.9% High molecular 0.8%
0.8% 0.8% 0.8% 0.8% 0.8% 0.8% weight proteins (% area) SDS-PAGE
under <1.0%.sup. <1.0% <1.0%.sup. <1.0%.sup. <1.0%
<1.0%.sup. <1.0% non-reducing conditions Half molecules (%)
SDS-PAGE under 99% 97% 99% 97% 99% 98% 97% reducing conditions
Relative purity (%) Charge heterogeneity HPLC (weak cation
4%/94%/2% 4%/94%/2% 4%/94%/2% 4%/94%/2% 4%/94%/2% 4%/94%/2%
4%/95%/2% exchange) Isoforms (acidic/neutral/basic) (% area) IEF
Conforms Conforms Conforms Conforms Conforms Conforms Conforms to
to to to to to to reference reference reference reference reference
reference reference pH 5.9 5.8 6.0 5.9 5.9 5.9 5.9 (potentiometry)
Particulate matter Practically Complies Complies Complies Complies
Complies Complies (visible particles) free from particles
Particulate matter (subvisible particles) Number of 2 7 particles
per vial .gtoreq.10 .mu.m Number of 0 1 particles per vial
.gtoreq.25 .mu.m Microbial <1 cfu/ contamination 2 mL Closure
integrity No trace No trace of of coloration coloration visible
visible Dynamic light z- z- z- z- z- z- z- scattering average:
average: average: average: average: average: average: 8.1 r nm 8.0
r nm 8.0 r nm 8.1 r nm 8.1 r nm 8.1 r nm 8.1 r nm Pdl: 0.05 Pdl:
0.05 Pdl: 0.05 Pdl: 0.05 Pdl: 0.05 Pdl: 0.07 Pdl: 0.05
TABLE-US-00135 TABLE 117 Stability Data for 100 mg/mL Lead CXCR5
Antibody formulation at 5.degree. C. Drug product: Lead CXCR5
Antibody - Batch no.: 11_106/ solution for injection LST0008 Dosage
100 mg/mL Manufacturer 11/106 strength: batch no.: Storage
+5.degree. C. .+-. 3.degree. C. condition: Storage Inverted
orientation: Time Initial 1 3 6 9 12 15 18 24 Test item results
month months months months months months months months Appearance
of solution Clarity I <I <I <I I <IV <IV <IV (22
NTU) (22 NTU) (21 NTU) Color Y7 Y7 Y7 Y7 Y7 Y6 Y6 Y6 Assay Potency
(Antigen ELISA) EC.sub.50 value (in comparison 75% 105% 95% 97% 92%
93% 114% 119% to reference) Total protein content (UV) 103 101 102
101 102 102 101 102 mg/mL mg/mL mg/mL mg/mL mg/mL mg/mL mg/mL mg/mL
Molecular integrity SDS-PAGE under non- Conforms Conforms Conforms
Conforms Conforms Conforms Conforms Conforms reducing conditions
(Band to to to to to to to to pattern) reference reference
reference reference reference reference reference reference Purity
HPLC (SEC) Monomer (% area) 99.1%.sup. 99.0% 99.1%.sup. 98.8%.sup.
98.8%.sup. 98.7%.sup. 98.9% 98.8% High molecular weight 0.8% 0.7%
0.7% 0.7% 0.8% 0.8% 0.9% 0.9% proteins (% area) SDS-PAGE under non-
<1.0%.sup. <1.0% <1.0%.sup. <1.0%.sup. <1.0%.sup.
<1.0%.sup. <1.0% <1.0% reducing conditions Half molecules
(%) SDS-PAGE under reducing 99% 98% 99% 95% 99% 97% 99% 98%
conditions Relative purity (%) Charge heterogeneity HPLC (weak
cation 4%/94%/ 4%/94%/ 3%/94%/ 4%/94%/ 4%/94%/ 4%/94%/ 4%/94%/
4%/94%/ exchange) 2% 2% 2% 2% 2% 2% 2% 2% Isoforms
(acidic/neutral/basic) (% area) IEF Conforms Conforms Conforms
Conforms Conforms Conforms Conforms Conforms to to to to to to to
to reference reference reference reference reference reference
reference reference pH (potentiometry) 5.9 5.9 6.0 5.9 5.9 5.9 5.9
5.9 Particulate matter (visible Complies Complies Complies Complies
Complies Complies Complies Complies particles) Particulate matter
(subvisible particles) Number of particles per 2 14 2 16 vial
.gtoreq.10 .mu.m Number of particles per 0 2 0 0 vial .gtoreq.25
.mu.m Microbial contamination <1 cfu/ 2 mL Closure integrity No
trace No trace of of coloration coloration visible visible Dynamic
light scattering z- z- z- z- z- z- z- z- average: average: average:
average: average: average: average: average: 8.1 8.0 7.9 8.0 8.1
8.1 8.0 8.1 r nm r nm r nm r nm r nm r nm r nm r nm Pdl: Pdl: Pdl:
Pdl: Pdl: Pdl: Pdl: Pdl: 0.05 0.05 0.04 0.04 0.06 0.06 0.05
0.05
TABLE-US-00136 TABLE 118 Stability Data for 100 mg/mL Lead CXCR5
Antibody formulation at 25.degree. C. Drug product: Lead CXCR5
Antibody- Batch no.: 11_106/ solution for injection LST0008 Dosage
strength: 100 mg/mL Manufacturer 11_106 batch no.: Storage
condition: +25.degree. C. .+-. 2.degree. C./60% .+-. 5% RH Storage
orientation: Inverted Time Test item Initial results 1 month 3
months 6 months Appearance of solution Clarity I <I <I <I
Color Y7 Y7 Y7 Y7 Assay Potency (Antigen ELISA) EC.sub.50 value (in
comparison 75% 121% 96% 104% to reference) Total protein content
(UV) 103 mg/mL 101 mg/mL 102 mg/mL 102 mg/mL Molecular integrity
SDS-PAGE under non- Conforms to Conforms to Conforms to Conforms to
reducing conditions (Band reference reference reference reference
pattern) Purity HPLC (SEC) Monomer (% area) 99.1%.sup. 98.9%
98.8%.sup. 98.2% High molecular weight 0.8% 0.8% 1.0% 1.2% proteins
(% area) SDS-PAGE under non- <1.0%.sup. <1.0% <1.0%.sup.
<1.0% reducing conditions Half molecules (%) SDS-PAGE under
reducing 99% 96% 99% 96% conditions Relative purity (%) Charge
heterogeneity HPLC (weak cation 4%/94%/2% 4%/94%/2% 4%/94%/3%
4%/93%/3% exchange) Isoforms (acidic/neutral/basic) (% area) IEF
Conforms to Conforms to Conforms to Conforms to reference reference
reference reference pH (potentiometry) 5.9 5.9 6.0 6.1 Particulate
matter (visible Complies Complies Complies Complies particles)
Particulate matter (subvisible particles) Number of particles per 2
17 vial .gtoreq.10 .mu.m Number of particles per 0 1 vial
.gtoreq.25 .mu.m Microbial contamination <1 cfu/2 mL <1 cfu/2
mL Closure integrity No trace of No trace of coloration visible
coloration visible Dynamic light scattering z-average: 8.1
z-average: 8.0 z-average: 8.1 z-average: 8.1 r nm; r nm r nm r nm
Pdl: 0.05 Pdl: 0.05 Pdl: 0.05 Pdl: 0.06
Sequence CWU 1
1
2915PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 1Gly Tyr Asn Trp His 1 5 216PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 2Glu
Ile Thr His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys Ser 1 5 10
15 314PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 3Glu Ile Ala Val Ala Gly Thr Gly Tyr Tyr Gly Met
Asp Val 1 5 10 411PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 4Arg Ala Ser Gln Gly Ile Asn Ser Ala Phe
Ala 1 5 10 57PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 5Asp Ala Ser Ser Leu Glu Ser 1 5
69PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 6Gln Gln Phe Asn Ser Tyr Pro Leu Thr 1 5
7448PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 7Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu
Leu Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Tyr
Gly Gly Ser Phe Ser Gly Tyr 20 25 30 Asn Trp His Trp Ile Arg Gln
Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Thr His
Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val
Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys
Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Val 85 90
95 Arg Glu Ile Ala Val Ala Gly Thr Gly Tyr Tyr Gly Met Asp Val Trp
100 105 110 Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys
Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr
Ser Glu Ser Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala
Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser
Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp 195 200 205 His
Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr 210 215
220 Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro
225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
Val Ser Gln Glu Asp 260 265 270 Pro Glu Val Gln Phe Asn Trp Tyr Val
Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu
Glu Gln Phe Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys
Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys 325 330 335
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340
345 350 Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu
Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Arg Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Glu Gly
Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly 435 440 445
8214PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 8Ala Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Gly Ile Asn Ser Ala 20 25 30 Phe Ala Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Ser
Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Asn Ser Tyr Pro Leu 85 90
95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys
Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu
Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp
Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu
Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu
Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe
Asn Arg Gly Glu Cys 210 9240PRTHomo sapiens 9Met Glu Glu Ser Val
Val Arg Pro Ser Val Phe Val Val Asp Gly Gln 1 5 10 15 Thr Asp Ile
Pro Phe Thr Arg Leu Gly Arg Ser His Arg Arg Gln Ser 20 25 30 Cys
Ser Val Ala Arg Val Gly Leu Gly Leu Leu Leu Leu Leu Met Gly 35 40
45 Ala Gly Leu Ala Val Gln Gly Trp Phe Leu Leu Gln Leu His Trp Arg
50 55 60 Leu Gly Glu Met Val Thr Arg Leu Pro Asp Gly Pro Ala Gly
Ser Trp 65 70 75 80 Glu Gln Leu Thr Gln Glu Arg Arg Ser His Glu Val
Asn Pro Ala Ala 85 90 95 His Leu Thr Gly Ala Asn Ser Ser Leu Thr
Gly Ser Gly Gly Pro Leu 100 105 110 Leu Trp Glu Thr Gln Leu Gly Leu
Ala Phe Leu Arg Gly Leu Ser Tyr 115 120 125 His Asp Gly Ala Leu Val
Val Thr Lys Ala Gly Tyr Tyr Tyr Ile Tyr 130 135 140 Ser Lys Val Gln
Leu Gly Gly Val Gly Cys Pro Leu Gly Leu Ala Ser 145 150 155 160 Thr
Ile Thr His Gly Leu Tyr Lys Arg Thr Pro Arg Tyr Pro Glu Glu 165 170
175 Leu Glu Leu Leu Val Ser Gln Gln Ser Pro Cys Gly Arg Ala Thr Ser
180 185 190 Ser Ser Arg Val Trp Trp Asp Ser Ser Phe Leu Gly Gly Val
Val His 195 200 205 Leu Glu Ala Gly Glu Glu Val Val Val Arg Val Leu
Asp Glu Arg Leu 210 215 220 Val Arg Leu Arg Asp Gly Thr Arg Ser Tyr
Phe Gly Ala Phe Met Val 225 230 235 240 10467PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
10Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp 1
5 10 15 Val Leu Ser Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu
Lys 20 25 30 Pro Ser Glu Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly
Gly Ser Phe 35 40 45 Ser Gly Tyr Asn Trp His Trp Ile Arg Gln Pro
Pro Gly Lys Gly Leu 50 55 60 Glu Trp Ile Gly Glu Ile Thr His Ser
Gly Ser Thr Asn Tyr Asn Pro 65 70 75 80 Ser Leu Lys Ser Arg Val Thr
Ile Ser Val Asp Thr Ser Lys Asn Gln 85 90 95 Phe Ser Leu Lys Leu
Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr 100 105 110 Tyr Cys Val
Arg Glu Ile Ala Val Ala Gly Thr Gly Tyr Tyr Gly Met 115 120 125 Asp
Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr 130 135
140 Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser
145 150 155 160 Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
Phe Pro Glu 165 170 175 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu
Thr Ser Gly Val His 180 185 190 Thr Phe Pro Ala Val Leu Gln Ser Ser
Gly Leu Tyr Ser Leu Ser Ser 195 200 205 Val Val Thr Val Pro Ser Ser
Ser Leu Gly Thr Lys Thr Tyr Thr Cys 210 215 220 Asn Val Asp His Lys
Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu 225 230 235 240 Ser Lys
Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu 245 250 255
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 260
265 270 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser 275 280 285 Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp
Gly Val Glu 290 295 300 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Phe Asn Ser Thr 305 310 315 320 Tyr Arg Val Val Ser Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn 325 330 335 Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys Gly Leu Pro Ser Ser 340 345 350 Ile Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 355 360 365 Val Tyr
Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val 370 375 380
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 385
390 395 400 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
Thr Pro 405 410 415 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Arg Leu Thr 420 425 430 Val Asp Lys Ser Arg Trp Gln Glu Gly Asn
Val Phe Ser Cys Ser Val 435 440 445 Met His Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu 450 455 460 Ser Leu Gly 465
111404DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 11atg aag cac ctg tgg ttc ttt ctg ctg ctg
gtg gcc gct cct aga tgg 48Met Lys His Leu Trp Phe Phe Leu Leu Leu
Val Ala Ala Pro Arg Trp 1 5 10 15 gtg ctg tcc cag gtg cag ctg cag
cag tgg ggc gct ggc ctg ctg aag 96Val Leu Ser Gln Val Gln Leu Gln
Gln Trp Gly Ala Gly Leu Leu Lys 20 25 30 cct tcc gag aca ctg tcc
ctg acc tgc gcc gtg tac ggc ggc tcc ttc 144Pro Ser Glu Thr Leu Ser
Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe 35 40 45 tcc ggc tac aac
tgg cac tgg atc agg cag cct ccc ggc aag ggc ctg 192Ser Gly Tyr Asn
Trp His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu 50 55 60 gaa tgg
atc ggc gag atc acc cac tcc ggc tcc acc aac tac aac cct 240Glu Trp
Ile Gly Glu Ile Thr His Ser Gly Ser Thr Asn Tyr Asn Pro 65 70 75 80
agc ctg aag tcc aga gtg acc atc tcc gtg gac acc tcc aag aac cag
288Ser Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln
85 90 95 ttc tcc ctg aag ctg tcc tct gtg acc gcc gct gac acc gcc
gtg tac 336Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala
Val Tyr 100 105 110 tac tgt gtg cgg gag atc gcc gtg gct ggc acc ggc
tac tac ggc atg 384Tyr Cys Val Arg Glu Ile Ala Val Ala Gly Thr Gly
Tyr Tyr Gly Met 115 120 125 gat gtg tgg ggc cag ggc acc acc gtg acc
gtg tcc agc gct tct acc 432Asp Val Trp Gly Gln Gly Thr Thr Val Thr
Val Ser Ser Ala Ser Thr 130 135 140 aag ggc cct tcc gtg ttc cct ctg
gcc cct tgc tcc cgg tcc acc tcc 480Lys Gly Pro Ser Val Phe Pro Leu
Ala Pro Cys Ser Arg Ser Thr Ser 145 150 155 160 gag tcc acc gcc gct
ctg ggc tgc ctg gtg aag gac tac ttc cct gag 528Glu Ser Thr Ala Ala
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 165 170 175 cct gtg acc
gtg tcc tgg aac tct ggc gcc ctg acc tcc ggc gtg cac 576Pro Val Thr
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 180 185 190 acc
ttc cct gcc gtg ctg cag tcc tcc ggc ctg tac tcc ctg tcc tcc 624Thr
Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser 195 200
205 gtg gtg acc gtg cct tcc tcc tcc ctg ggc acc aag acc tac acc tgt
672Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys
210 215 220 aac gtg gac cac aag cct tcc aac acc aag gtg gac aag cgg
gtg gag 720Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg
Val Glu 225 230 235 240 tcc aag tac ggc cct cct tgc cct ccc tgc cct
gcc cct gag ttc gag 768Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro
Ala Pro Glu Phe Glu 245 250 255 ggc gga cct agc gtg ttc ctg ttc cct
cct aag cct aag gac acc ctg 816Gly Gly Pro Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu 260 265 270 atg atc tcc cgg acc cct gag
gtg acc tgt gtg gtg gtg gac gtg tcc 864Met Ile Ser Arg Thr Pro Glu
Val Thr Cys Val Val Val Asp Val Ser 275 280 285 cag gag gac cct gag
gtc cag ttc aac tgg tac gtg gac ggc gtg gag 912Gln Glu Asp Pro Glu
Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu 290 295 300 gtg cac aac
gcc aag acc aag cct cgg gag gag cag ttc aat tcc acc 960Val His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr 305 310 315 320
tac cgg gtg gtg tct gtg ctg acc gtg ctg cac cag gac tgg ctg aac
1008Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
325 330 335 ggc aaa gaa tac aag tgt aag gtc tcc aac aag ggc ctg ccc
tcc tcc 1056Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro
Ser Ser 340 345 350 atc gag aaa acc atc tcc aag gcc aag ggc cag cct
agg gag cct cag 1104Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln 355 360 365 gtg tac acc ctg cct cct agc cag gaa gag
atg acc aag aac cag gtg 1152Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu
Met Thr Lys Asn Gln Val 370 375 380 tcc ctg acc tgt ctg gtg aag ggc
ttc tac cct tcc gac atc gcc gtg 1200Ser Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val 385 390 395 400 gag tgg gag tcc aac
ggc cag cct gag aac aac tac aag acc acc cct 1248Glu Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 405 410 415 cct gtg ctg
gac tcc gac ggc tcc ttc ttc ctg tac tcc agg ctg acc 1296Pro Val Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr 420 425 430 gtg
gac aag tcc cgg tgg cag gag ggc aac gtc ttt tcc tgc tcc gtg 1344Val
Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val
435 440 445 atg cac gag gcc ctg cac aac cac tac acc cag aag tcc ctg
tcc ctg 1392Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
Ser Leu 450 455 460 tct ctg ggc tga 1404Ser Leu Gly 465
12236PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 12Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly
Leu Leu Leu Leu Trp 1 5 10 15 Leu Pro Gly Ala Arg Cys Ala Ile Gln
Leu Thr Gln Ser Pro Ser Ser 20 25 30 Leu Ser Ala Ser Val Gly Asp
Arg Val Thr Ile Thr Cys Arg Ala Ser 35 40 45 Gln Gly Ile Asn Ser
Ala Phe Ala Trp Tyr Gln Gln Lys Pro Gly Lys 50 55 60 Ala Pro Lys
Leu Leu Ile Tyr Asp Ala Ser Ser Leu Glu Ser Gly Val 65 70 75 80 Pro
Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 85 90
95 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
100 105 110 Phe Asn Ser Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys Val
Glu Ile 115 120 125 Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe
Pro Pro Ser Asp 130 135 140 Glu Gln Leu Lys Ser Gly Thr Ala Ser Val
Val Cys Leu Leu Asn Asn 145 150 155 160 Phe Tyr Pro Arg Glu Ala Lys
Val Gln Trp Lys Val Asp Asn Ala Leu 165 170 175 Gln Ser Gly Asn Ser
Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 180 185 190 Ser Thr Tyr
Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr 195 200 205 Glu
Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser 210 215
220 Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235
13711DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 13atg gac atg aga gtg cct gct cag ctg ctg
gga ctg ctg ctg ctg tgg 48Met Asp Met Arg Val Pro Ala Gln Leu Leu
Gly Leu Leu Leu Leu Trp 1 5 10 15 ctg cct ggc gct aga tgc gcc atc
cag ctg acc cag tcc ccc tcc tct 96Leu Pro Gly Ala Arg Cys Ala Ile
Gln Leu Thr Gln Ser Pro Ser Ser 20 25 30 ctg tcc gcc tcc gtg ggc
gac aga gtg acc atc acc tgt cgg gcc tcc 144Leu Ser Ala Ser Val Gly
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser 35 40 45 cag ggc atc aac
tcc gcc ttc gcc tgg tat cag cag aag cct ggc aag 192Gln Gly Ile Asn
Ser Ala Phe Ala Trp Tyr Gln Gln Lys Pro Gly Lys 50 55 60 gcc cct
aag ctg ctg atc tac gac gcc tcc tcc ctg gaa tcc ggc gtg 240Ala Pro
Lys Leu Leu Ile Tyr Asp Ala Ser Ser Leu Glu Ser Gly Val 65 70 75 80
ccc tcc aga ttt tcc ggc tcc ggc tct ggc acc gac ttc acc ctg acc
288Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
85 90 95 atc tcc agc ctg cag cct gag gac ttc gcc acc tac tac tgc
cag cag 336Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys
Gln Gln 100 105 110 ttc aac tcc tac cct ctg acc ttc ggc gga ggc acc
aag gtg gag atc 384Phe Asn Ser Tyr Pro Leu Thr Phe Gly Gly Gly Thr
Lys Val Glu Ile 115 120 125 aag cgt acg gtg gct gca cca tct gtc ttc
atc ttc ccg cca tct gat 432Lys Arg Thr Val Ala Ala Pro Ser Val Phe
Ile Phe Pro Pro Ser Asp 130 135 140 gag cag ttg aaa tct gga act gcc
tct gtt gtg tgc ctg ctg aat aac 480Glu Gln Leu Lys Ser Gly Thr Ala
Ser Val Val Cys Leu Leu Asn Asn 145 150 155 160 ttc tat ccc aga gag
gcc aaa gta cag tgg aag gtg gat aac gcc ctc 528Phe Tyr Pro Arg Glu
Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu 165 170 175 caa tcg ggt
aac tcc cag gag agt gtc aca gag cag gac agc aag gac 576Gln Ser Gly
Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 180 185 190 agc
acc tac agc ctc agc agc acc ctg acg ctg agc aaa gca gac tac 624Ser
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr 195 200
205 gag aaa cac aaa gtc tac gcc tgc gaa gtc acc cat cag ggc ctg agc
672Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser
210 215 220 tcg ccc gtc aca aag agc ttc aac agg gga gag tgt tag
711Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235
14372PRTHomo sapiens 14Met Asn Tyr Pro Leu Thr Leu Glu Met Asp Leu
Glu Asn Leu Glu Asp 1 5 10 15 Leu Phe Trp Glu Leu Asp Arg Leu Asp
Asn Tyr Asn Asp Thr Ser Leu 20 25 30 Val Glu Asn His Leu Cys Pro
Ala Thr Glu Gly Pro Leu Met Ala Ser 35 40 45 Phe Lys Ala Val Phe
Val Pro Val Ala Tyr Ser Leu Ile Phe Leu Leu 50 55 60 Gly Val Ile
Gly Asn Val Leu Val Leu Val Ile Leu Glu Arg His Arg 65 70 75 80 Gln
Thr Arg Ser Ser Thr Glu Thr Phe Leu Phe His Leu Ala Val Ala 85 90
95 Asp Leu Leu Leu Val Phe Ile Leu Pro Phe Ala Val Ala Glu Gly Ser
100 105 110 Val Gly Trp Val Leu Gly Thr Phe Leu Cys Lys Thr Val Ile
Ala Leu 115 120 125 His Lys Val Asn Phe Tyr Cys Ser Ser Leu Leu Leu
Ala Cys Ile Ala 130 135 140 Val Asp Arg Tyr Leu Ala Ile Val His Ala
Val His Ala Tyr Arg His 145 150 155 160 Arg Arg Leu Leu Ser Ile His
Ile Thr Cys Gly Thr Ile Trp Leu Val 165 170 175 Gly Phe Leu Leu Ala
Leu Pro Glu Ile Leu Phe Ala Lys Val Ser Gln 180 185 190 Gly His His
Asn Asn Ser Leu Pro Arg Cys Thr Phe Ser Gln Glu Asn 195 200 205 Gln
Ala Glu Thr His Ala Trp Phe Thr Ser Arg Phe Leu Tyr His Val 210 215
220 Ala Gly Phe Leu Leu Pro Met Leu Val Met Gly Trp Cys Tyr Val Gly
225 230 235 240 Val Val His Arg Leu Arg Gln Ala Gln Arg Arg Pro Gln
Arg Gln Lys 245 250 255 Ala Val Arg Val Ala Ile Leu Val Thr Ser Ile
Phe Phe Leu Cys Trp 260 265 270 Ser Pro Tyr His Ile Val Ile Phe Leu
Asp Thr Leu Ala Arg Leu Lys 275 280 285 Ala Val Asp Asn Thr Cys Lys
Leu Asn Gly Ser Leu Pro Val Ala Ile 290 295 300 Thr Met Cys Glu Phe
Leu Gly Leu Ala His Cys Cys Leu Asn Pro Met 305 310 315 320 Leu Tyr
Thr Phe Ala Gly Val Lys Phe Arg Ser Asp Leu Ser Arg Leu 325 330 335
Leu Thr Lys Leu Gly Cys Thr Gly Pro Ala Ser Leu Cys Gln Leu Phe 340
345 350 Pro Ser Trp Arg Arg Ser Ser Leu Ser Glu Ser Glu Asn Ala Thr
Ser 355 360 365 Leu Thr Thr Phe 370 1510PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 15Gly
Phe Ser Leu Ile Asp Tyr Gly Val Asn 1 5 10 169PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 16Val
Ile Trp Gly Asp Gly Thr Thr Tyr 1 5 173PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 17Ile
Val Tyr 1 1816PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 18Arg Ser Ser Lys Ser Leu Leu His Ser
Ser Gly Lys Thr Tyr Leu Tyr 1 5 10 15 196PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 19Arg
Leu Ser Ser Leu Ala 1 5 209PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 20Met Gln His Leu Glu Tyr Pro
Tyr Thr 1 5 21111PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 21Gln Val Gln Leu Lys Glu Ser Gly
Pro Gly Leu Val Ala Pro Ser Glu 1 5 10 15 Ser Leu Ser Ile Thr Cys
Thr Val Ser Gly Phe Ser Leu Ile Asp Tyr 20 25 30 Gly Val Asn Trp
Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Val
Ile Trp Gly Asp Gly Thr Thr Tyr Tyr Asn Pro Ser Leu Lys 50 55 60
Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu 65
70 75 80 Lys Val Thr Ser Leu Thr Thr Asp Asp Thr Ala Met Tyr Tyr
Cys Ala 85 90 95 Arg Ile Val Tyr Trp Gly Gln Gly Thr Leu Val Thr
Val Ser Ala 100 105 110 22112PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 22Asp Ile Val Met Thr Gln
Ala Ala Pro Ser Val Ala Val Thr Pro Gly 1 5 10 15 Ala Ser Val Ser
Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser 20 25 30 Ser Gly
Lys Thr Tyr Leu Tyr Trp Phe Leu Gln Arg Pro Gly Gln Ser 35 40 45
Pro Gln Leu Leu Ile Tyr Arg Leu Ser Ser Leu Ala Ser Gly Val Pro 50
55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Ala Phe Thr Leu Arg
Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys
Met Gln His 85 90 95 Leu Glu Tyr Pro Tyr Thr Phe Gly Gly Gly Thr
Lys Leu Glu Ile Lys 100 105 110 23326PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
23Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1
5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala
Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser
Ser Ser Leu Gly Thr Lys Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His
Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Ser Lys
Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro 100 105 110 Glu Phe Glu
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120 125 Asp
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 130 135
140 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp
145 150 155 160 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
Glu Gln Phe 165 170 175 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
Val Leu His Gln Asp 180 185 190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys Gly Leu 195 200 205 Pro Ser Ser Ile Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg 210 215 220 Glu Pro Gln Val Tyr
Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 225 230 235 240 Asn Gln
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260
265 270 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser 275 280 285 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn
Val Phe Ser 290 295 300 Cys Ser Val Met His Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser 305 310 315 320 Leu Ser Leu Ser Leu Gly 325
24107PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 24Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe
Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val
Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val
Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser
Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90
95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105
25437PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 25Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu
Val Ala Pro Ser Glu 1 5 10 15 Ser Leu Ser Ile Thr Cys Thr Val Ser
Gly Phe Ser Leu Ile Asp Tyr 20 25 30 Gly Val Asn Trp Ile Arg Gln
Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Val Ile Trp Gly
Asp Gly Thr Thr Tyr Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Leu
Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu 65 70 75 80 Lys
Val Thr Ser Leu Thr Thr Asp Asp Thr Ala Met Tyr Tyr Cys Ala 85 90
95 Arg Ile Val Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala Ala
100 105 110 Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser
Arg Ser 115 120 125 Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val
Lys Asp Tyr Phe 130 135 140 Pro Glu Pro Val Thr Val Ser Trp Asn Ser
Gly Ala Leu Thr Ser Gly 145 150 155 160 Val His Thr Phe Pro Ala Val
Leu Gln Ser Ser Gly Leu Tyr Ser Leu 165 170 175 Ser Ser Val Val Thr
Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr 180 185 190 Thr Cys Asn
Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg 195 200 205 Val
Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu 210 215
220 Phe Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
225 230 235 240 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
Val Val Asp 245 250 255 Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn
Trp Tyr Val Asp Gly 260 265 270 Val Glu Val His Asn Ala Lys Thr Lys
Pro Arg Glu Glu Gln Phe Asn 275 280 285 Ser Thr Tyr Arg Val Val Ser
Val Leu Thr Val Leu His Gln Asp Trp 290 295 300 Leu Asn Gly Lys Glu
Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro 305 310 315 320 Ser Ser
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 325 330 335
Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn 340
345 350 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile 355 360 365 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr 370 375 380 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Arg 385 390 395 400 Leu Thr Val Asp Lys Ser Arg
Trp Gln Glu Gly Asn Val Phe Ser Cys 405 410 415 Ser Val Met His Glu
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 420 425 430 Ser Leu Ser
Leu Gly 435 26219PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 26Asp Ile Val Met Thr Gln Ala Ala
Pro Ser Val Ala Val Thr Pro Gly 1 5 10 15 Ala Ser Val Ser Ile Ser
Cys Arg Ser Ser Lys Ser Leu Leu His Ser 20 25 30 Ser Gly Lys Thr
Tyr Leu Tyr Trp Phe Leu Gln Arg Pro Gly Gln Ser 35 40 45 Pro Gln
Leu Leu Ile Tyr Arg Leu Ser Ser Leu Ala Ser Gly Val Pro 50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Ala Phe Thr Leu Arg Ile 65
70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met
Gln His 85 90 95 Leu Glu Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys
Leu Glu Ile Lys 100 105 110 Arg Thr Val Ala Ala Pro Ser Val Phe Ile
Phe Pro Pro Ser Asp Glu 115 120 125 Gln Leu Lys Ser Gly Thr Ala Ser
Val Val Cys Leu Leu Asn Asn Phe 130 135 140 Tyr Pro Arg Glu Ala Lys
Val Gln Trp Lys Val Asp Asn Ala Leu Gln 145 150 155 160 Ser Gly Asn
Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 165 170 175 Thr
Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 180 185
190 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
195 200 205 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215
2719PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 27Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala
Thr Ala Thr Gly 1 5 10 15 Val His Ser 28456PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
28Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1
5 10 15 Val His Ser Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val
Ala 20 25 30 Pro Ser Glu Ser Leu Ser Ile Thr Cys Thr Val Ser Gly
Phe Ser Leu 35 40 45 Ile Asp Tyr Gly Val Asn Trp Ile Arg Gln Pro
Pro Gly Lys Gly Leu 50 55 60 Glu Trp Leu Gly Val Ile Trp Gly Asp
Gly Thr Thr Tyr Tyr Asn Pro 65 70 75 80 Ser Leu Lys Ser Arg Leu Ser
Ile Ser Lys Asp Asn Ser Lys Ser Gln 85 90 95 Val Phe Leu Lys Val
Thr Ser Leu Thr Thr Asp Asp Thr Ala Met Tyr 100 105 110 Tyr Cys Ala
Arg Ile Val Tyr Trp Gly Gln Gly Thr Leu Val Thr Val 115 120 125 Ser
Ala Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys 130 135
140 Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys
145 150 155 160 Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser
Gly Ala Leu 165 170 175 Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
Gln Ser Ser Gly Leu 180 185 190 Tyr Ser Leu Ser Ser Val Val Thr Val
Pro Ser Ser Ser Leu Gly Thr 195 200 205 Lys Thr Tyr Thr Cys Asn Val
Asp His Lys Pro Ser Asn Thr Lys Val 210 215 220 Asp Lys Arg Val Glu
Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro 225 230 235 240 Ala Pro
Glu Phe Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 245 250 255
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 260
265 270 Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp
Tyr 275 280 285 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
Arg Glu Glu 290 295 300 Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val
Leu Thr Val Leu His 305 310 315 320 Gln Asp Trp Leu Asn Gly Lys Glu
Tyr Lys Cys Lys Val Ser Asn Lys 325 330 335 Gly Leu Pro Ser Ser Ile
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 340 345 350 Pro Arg Glu Pro
Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met 355 360 365 Thr Lys
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 370 375 380
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 385
390 395 400 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
Phe Leu 405 410 415 Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln
Glu Gly Asn Val 420 425 430 Phe Ser Cys Ser Val Met His Glu Ala Leu
His Asn His Tyr Thr Gln 435 440 445 Lys Ser Leu Ser Leu Ser Leu Gly
450 455 29238PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 29Met Gly Trp Ser Cys Ile Ile Leu
Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser Asp Ile Val
Met Thr Gln Ala Ala Pro Ser Val Ala Val 20 25 30 Thr Pro Gly Ala
Ser Val Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu 35 40 45 Leu His
Ser Ser Gly Lys Thr Tyr Leu Tyr Trp Phe Leu Gln Arg Pro 50 55 60
Gly Gln Ser Pro Gln Leu Leu Ile Tyr Arg Leu Ser Ser Leu Ala Ser 65
70 75 80 Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Ala
Phe Thr 85 90 95 Leu Arg Ile Ser Arg Val Glu Ala Glu Asp Val Gly
Val Tyr Tyr Cys 100 105 110 Met Gln His Leu Glu Tyr Pro Tyr Thr Phe
Gly Gly Gly Thr Lys Leu 115 120 125 Glu Ile Lys Arg Thr Val Ala Ala
Pro Ser Val Phe Ile Phe Pro Pro 130 135 140 Ser Asp Glu Gln Leu Lys
Ser Gly Thr Ala Ser Val Val Cys Leu Leu 145 150 155 160 Asn Asn Phe
Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn 165 170 175 Ala
Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser 180 185
190 Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala
195 200 205 Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His
Gln Gly 210 215 220 Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly
Glu Cys 225 230 235
* * * * *