U.S. patent application number 15/326341 was filed with the patent office on 2017-07-13 for crystalline antibody formulations.
This patent application is currently assigned to AMGEN INC.. The applicant listed for this patent is AMGEN INC.. Invention is credited to Nazer KHALAF, Saraswathi MANDAPATI, Reena J. PATEL.
Application Number | 20170198059 15/326341 |
Document ID | / |
Family ID | 53836192 |
Filed Date | 2017-07-13 |
United States Patent
Application |
20170198059 |
Kind Code |
A1 |
KHALAF; Nazer ; et
al. |
July 13, 2017 |
CRYSTALLINE ANTIBODY FORMULATIONS
Abstract
Described herein are anti-PCSK9 antibody crystals, methods of
making such antibody crystals and formulations comprising the
antibody crystals.
Inventors: |
KHALAF; Nazer; (Millbury,
MA) ; MANDAPATI; Saraswathi; (Brookline, MA) ;
PATEL; Reena J.; (Woburn, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
AMGEN INC. |
Thousand Oaks |
CA |
US |
|
|
Assignee: |
AMGEN INC.
Thousand Oaks
CA
|
Family ID: |
53836192 |
Appl. No.: |
15/326341 |
Filed: |
July 13, 2015 |
PCT Filed: |
July 13, 2015 |
PCT NO: |
PCT/US2015/040217 |
371 Date: |
January 13, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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62024393 |
Jul 14, 2014 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 16/40 20130101;
C07K 2317/14 20130101; A61P 3/06 20180101; C07K 2317/565 20130101;
A61K 39/39591 20130101 |
International
Class: |
C07K 16/40 20060101
C07K016/40; A61K 39/395 20060101 A61K039/395 |
Claims
1. A crystal of an anti-PCSK9 IgG antibody comprising a light chain
complementarity determining region (CDR) of the CDRL1 sequence in
SEQ ID NO:9, a CDRL2 of the CDRL2 sequence in SEQ ID NO:9, and a
CDRL3 of the CDRL3 sequence in SEQ ID NO:9, and a heavy chain
complementarity determining region (CDR) of the CDRH1 sequence in
SEQ ID NO:5, a CDRH2 of the CDRH2 sequence in SEQ ID NO:5, and a
CDRH3 of the CDRH3 sequence in SEQ ID NO:5.
2. The crystal of claim 1, wherein the anti-PCSK9 IgG antibody
comprises a light chain variable region that is at least 90%
identical to that of SEQ ID NO:9 or SEQ ID NO:11 and a heavy chain
variable region that is at least 90% identical to that of SEQ ID
NO:5 or SEQ ID NO:7.
3. The crystal of claim 2, wherein the anti-PCSK9 IgG antibody
comprises a light chain variable region having the amino acid
sequence set forth in SEQ ID NO:9 or SEQ ID NO:11 and a heavy chain
variable region comprising the amino acid sequence set forth in SEQ
ID NO:5 or SEQ ID NO:7.
4. The crystal of claim 1, wherein the crystal has a length of
about 5 .mu.M to about 50 .mu.M.
5. The crystal of claim 1 wherein the crystal has a rod or needle
shape.
6. The crystal of any of claim 1, wherein the crystal comprises a
salt selected from the group consisting of sodium di-hydrogen
phosphate, di-potassium hydrogen phosphate, sodium chloride,
ammonium sulfate, potassium sodium tartrate tetrahydrate, sodium
citrate dihydrate, sodium acetate trihydrate, di-ammonium hydrogen
phosphate, potassium sodium tartrate, calcium acetate, cacodylate,
CHES, CAPS, Tris, lithium sulfate, sodium phosphate, potassium
phosphate, sodium sulfate.
7. A method of making a crystal of an anti-PCSK-9 antibody
comprising combining a solution of the antibody with a
crystallization reagent comprising a salt selected from the group
consisting of sodium di-hydrogen phosphate, di-potassium hydrogen
phosphate, sodium chloride, ammonium sulfate, potassium sodium
tartrate tetrahydrate, sodium citrate dihydrate, sodium acetate
trihydrate, di-ammonium hydrogen phosphate, potassium sodium
tartrate, calcium acetate, cacodylate, CHES, CAPS, Tris, lithium
sulfate, sodium phosphate, potassium phosphate, and sodium sulfate
in a crystallization buffer, wherein the anti-PCSK-9 antibody is
selected from the group consisting of an antibody comprising: (a) a
light chain variable region having the amino acid sequence set
forth in SEQ ID NO:9 or SEQ ID NO:11 and a heavy chain variable
region comprising the amino acid sequence set forth in SEQ ID NO:5
or SEQ ID NO:7; (b) a light chain variable region that is at least
90% identical to that of SEQ ID NO:9 or SEQ ID NO:11 and a heavy
chain variable region that is at least 90% identical to that of SEQ
ID NO:5 or SEQ ID NO:7; (c) a light chain complementarity
determining region (CDR) of the CDRL1 sequence in SEQ ID NO:9, a
CDRL2 of the CDRL2 sequence in SEQ ID NO:9, and a CDRL3 of the
CDRL3 sequence in SEQ ID NO:9, and a heavy chain complementarity
determining region (CDR) of the CDRH1 sequence in SEQ ID NO:5, a
CDRH2 of the CDRH2 sequence in SEQ ID NO:5, and a CDRH3 of the
CDRH3 sequence in SEQ ID NO:5; and (d) a light chain
complementarity determining region of the CDRL1 sequence of SEQ ID
NO:24, the CDRL2 sequence of SEQ ID NO:25, the CDRL3 sequence of
SEQ ID NO:26, and a heavy chain complementarity determining region
(CDR) of the CDRH1 sequence of SEQ ID NO:20 or SEQ ID NO:21, the
CDRH2 sequence of SEQ ID NO:22, and the CDRH3 sequence of SEQ ID
NO:23.
8. The method of claim 7, wherein the concentration of salt in the
crystallization buffer is from about 0.1M to about 10M.
9. The method of claim 7, further comprising removing at least a
portion of the crystallization buffer after crystals have
formed.
10. The method of claim 9, wherein the portion of crystallization
buffer is removed by centrifugation.
11. The method of claim 10, wherein the crystals are placed in a
solution containing an organic additive.
12. The method of claim 11, further comprising the addition of an
excipient to the solution.
13. The method of claim 7, further comprising drying crystals that
have formed.
14. The method of claim 13, wherein the crystals are dried by
exposure to air, or by exposure to a vacuum, or by exposure to
nitrogen gas.
15. An antibody crystal produced by the method of claim 7.
16. A crystalline formulation of the antibody of claim 1.
17. A method of lowering serum LDL cholesterol or treating a
disorder associated with increased levels of serum LDL cholesterol
in a mammalian subject in need thereof comprising administering the
crystal of claim 1 or the crystalline formulation of claim 16 in an
amount effective to lower serum LDL cholesterol levels in the
subject as compared to a predose serum LDL cholesterol level.
18. The method of claim 7, wherein the crystallization buffer
further comprises polyethylene glycol (PEG).
19. The method of claim 18, wherein the PEG has a molecular weight
of about 400 kDa to about 20,000 kDa.
20. The method of claim 19, wherein the PEG is present at a
concentration of about 0.1% to about 50%.
21. The crystalline formulation of claim 16, wherein the crystal
comprises a salt selected from the group consisting of sodium
dihydrogen phosphate, di-potassium hydrogen phosphate, sodium
chloride, ammonium sulfate, potassium sodium tartrate tetrahydrate,
tacsimate, sodium citrate dihydrate, sodium acetate trihydrate,
di-ammonium tartrate, sodium malonate, acetate, calcium acetate,
cacodylate, CHES, lithium sulfate, magnesium chloride, zinc
acetate, cesium chloride, ammonium phosphate, sodium phosphate,
potassium phosphate, sodium fluoride, potassium iodide, sodium
idodide, ammonium iodide, sodium thiocyanate, potassium
thiocyanate, sodium formate, potassium formate, and ammonium
formate.
22. The crystalline formulation of claim 16, wherein the crystals
have a length of about 5 .mu.m to about 50 .mu.m and a morphology
selected from the group consisting of rod shapes and needle shapes,
or a mixture thereof.
23. The crystalline formulation of claim 16, comprising a crystal
of any one of claims 1-6.
24. The crystalline formulation of claim 16, comprising at least
20% PEG3350 precipitant.
25. The crystalline formulation of claim 16, wherein the osmolality
of the formulation ranges from about 180 to about 420 mOsm/kg.
26. A container comprising at least 100 to about 450 mg or more of
the crystals of claim 6 for reconstitution in a volume of 0.5-2
mL.
27. A container comprising the crystalline formulation of claim 16
at a concentration of at least 100 mg/ml.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application No. 62/024,393 filed Jul. 14, 2014, which is
incorporated in its entirety by reference herein.
INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY
[0002] This application contains, as a separate part of disclosure,
a Sequence Listing in computer-readable form (filename: Seq
List10-07-13_ST25.txt, created Jun. 26, 2014 which is 42 KB in
size), which is incorporated by reference in its entirety.
BACKGROUND
[0003] Monoclonal antibodies are extensively used as
biotherapeutics with an increasing demand to meet high
concentrations of over a 100 mg/m1 for delivery. This presents a
challenge for solubility limited proteins via a subcutaneous route,
since the preferred subcutaneous administration limit is 1.2 ml
(Yang, M. X., Shenoy, B., Disttler, M., Patel, R., McGrath, M.,
Pechenov, S., Margolin, A. L. (2003) Crystalline monoclonal
antibodies for subcutaneous delivery, PNAS 100, 6934-6939).
Development of high concentration formulation poses a lot of
challenges from a formulation, analytical, stability, manufacturing
and drug delivery point of view (Shire, S. J., Zahra, S., Liu, J.
(2004) Challenges in the development of high concentration
formulations, J. Pharm. Sci. 93, 1390-1402). So far, high
concentration formulation demands have been met by addition of
excipients like amino acids, sugars and salts that increase
stability, reduce aggregation and viscosity (Shire, supra and
Jenkins, T. W. (1998) Three solutions of the protein solubility
problem, Protein Science 7: 376-382).
[0004] Protein crystals are often viewed as only the intermediates
to a protein structure but they also have an important role from a
formulation perspective. Protein molecules in the crystalline form
have the lowest entropy thus making them 3-6 kcal/ml more stable
than in the liquid state (Dreuth, J., Haas, C. (1992) Protein
crystals and their stability, J. Crystal Growth 122, 107-109). The
main advantages of crystalline formulation include high protein
concentration, lower viscosity, stability, elimination of frequent
dosage due to high concentration and controlled release properties
(Yang, supra, and Basu, S. K., Govardhan, C. P., Jung, C. W.,
Margolin, A. L. (2004) Protein crystals for the delivery of
biopharmaceuticals, Expert Opin. Biol. Thera. 4, 301-317).
[0005] Crystallization conditions can be manipulated to achieve
different morphologies for desired controlled release properties
(Pechenov, S., Shenoy, B., Yang, M. X., Basu, S., Margolin, A. L.
(2004) Injectable controlled release formulations incorporating
protein crystals, Journal of Controlled Release 96, 149-158).
Insulin crystalline formulations were first reported in 1920's and
today, it is not only the first recombinant protein therapeutic
approved by the FDA, it is also the first approved crystalline
protein therapeutic (Hagedorn H. C.; Jensen, B. N.; Krarup, N. B.;
Wodstrup, I. Protamine insulinate, (1936) J. Am. Med. Assn. 106,
177-180; Johnson, I. S. (2003) The trials and tribulations of
producing the first genetically engineered drug. Nat. Rev. Drug.
Discovery 2, 747-751; and Basu, S. K., Govardhan, C. P., Jung, C.
W., Margolin, A. L. (2004) Protein crystals for the delivery of
biopharmaceuticals, Expert Opin. Biol. Thera. 4, 301-317).
Macromolecules are challenging to crystallize due to their inherent
flexibility, but, once crystallized, often pose challenges from a
formulation and regulatory perspective (Basu, supra, and Jen, A.,
Merkle, H. P. (2001) Diamonds in the rough: Protein crystals from a
formulation perspective, Pharm. Res. 18, 1483-1488.).
SUMMARY OF THE INVENTION
[0006] The invention relates to crystals of anti-PCSK9
immunoglobulin type G (IgG) antibodies (more specifically, antibody
21B12) that are suitable for use in crystalline formulations for
parenteral administration; solutions, salts and methods for
producing such crystals; methods of using such crystals to prepare
crystalline formulations for use as medicaments, and methods of
using such crystalline formulations for treating mammals,
specifically humans.
[0007] In the crystals or formulations described herein, the
anti-PCSK9 IgG can comprise the heavy and light chain complementary
determining regions (CDRs) of antibody, 21B12. Thus, in some
embodiments, the antibody is an IgG comprising a light chain
complementarity region (CDR) of the CDRL1 sequence in SEQ ID NO:9,
a CDRL2 of the CDRL2 sequence in SEQ ID NO:9, and a CDRL3 of the
CDRL3 sequence in SEQ ID NO:9, and a heavy chain complementarity
determining region (CDR) of the CDRH1 sequence in SEQ ID NO:5, a
CDRH2 of the CDRH2 sequence in SEQ ID NO:5, and a CDRH3 of the
CDRH3 sequence in SEQ ID NO:5. In some other embodiments, the
antibody is an IgG comprising a light chain complementarity region
(CDR) of the CDRL1 sequence in SEQ ID NO:11, a CDRL2 of the CDRL2
sequence in SEQ ID NO:11, and a CDRL3 of the CDRL3 sequence in SEQ
ID NO:11, and a heavy chain complementarity determining region
(CDR) of the CDRH1 sequence in SEQ ID NO:7, a CDRH2 of the CDRH2
sequence in SEQ ID NO:7, and a CDRH3 of the CDRH3 sequence in SEQ
ID NO:7. In some embodiments, the antibody is an IgG comprising the
amino acid sequences of: SEQ ID NO:20 or SEQ ID NO:21 (21B12
CDRH1), and SEQ ID NO:22 (21B12 CDRH2), and SEQ ID NO:23 (21B12
CDRH3), and SEQ ID NO:24 (21B12 CDRL1), and SEQ ID NO:25 (21B12
CDRL2), and SEQ ID NO:26 (21B12 CDRL3).
[0008] In the crystals or formulations described herein, the
anti-PCSK9 IgG antibody can comprise the heavy and light chain
variable regions of an antibody having at least 70%, at least 80%,
at least 90%, at least 95%, at least 98%, at least 99% sequence
identity to antibody, 21B12. Thus, in some embodiments, the
antibody is an IgG comprising a light chain variable region
comprising an amino acid sequence that is at least 70% identical to
that of SEQ ID NO:9 or SEQ ID NO:11 and a heavy chain variable
region that comprises an amino acid sequence that is at least 70%
identical to that of SEQ ID NO:5 or SEQ ID NO:7.
[0009] In some embodiments, the antibody is an IgG comprising a
light chain variable region comprising an amino acid sequence that
is at least 80% identical to that of SEQ ID NO:9 or SEQ ID NO:11
and a heavy chain variable region that comprises an amino acid
sequence that is at least 80% identical to that of SEQ ID NO:5 or
SEQ ID NO:7. In some embodiments, the antibody is an IgG comprising
a light chain variable region comprising an amino acid sequence
that is at least 90% identical to that of SEQ ID NO:9 or SEQ ID
NO:11 and a heavy chain variable region that comprises an amino
acid sequence that is at least 90% identical to that of SEQ ID NO:5
or SEQ ID NO:7. In some embodiments, the antibody is an IgG
comprising a light chain variable region comprising an amino acid
sequence that is at least 95% identical to that of SEQ ID NO:9 or
SEQ ID NO:11 and a heavy chain variable region that comprises an
amino acid sequence that is at least 95% identical to that of SEQ
ID NO:5 or SEQ ID NO:7. In some embodiments, the antibody is an IgG
comprising a light chain variable region comprising an amino acid
sequence that is at least 98% identical to that of SEQ ID NO:9 or
SEQ ID NO:11 and a heavy chain variable region that comprises an
amino acid sequence that is at least 98% identical to that of SEQ
ID NO:5 or SEQ ID NO:7. In some embodiments, the antibody is an IgG
comprising a light chain variable region comprising an amino acid
sequence that is at least 99% identical to that of SEQ ID NO:9 or
SEQ ID NO:11 and a heavy chain variable region that comprises an
amino acid sequence that is at least 99% identical to that of SEQ
ID NO:5 or SEQ ID NO:7. In some embodiments, the antibody is an IgG
comprising a light chain variable region comprising the amino acid
sequence of SEQ ID NO:9 or SEQ ID NO:11 and a heavy chain variable
region that comprises an amino acid sequence of SEQ ID NO:5 or SEQ
ID NO:7. In some embodiments, the antibody comprises a light chain
variable region comprising the amino acid sequence of SEQ ID NO:9
and a heavy chain variable region that comprises an amino acid
sequence of SEQ ID NO:5. In some embodiments, the antibody
comprises a light chain variable region comprising the amino acid
sequence of SEQ ID NO:11 and a heavy chain variable region that
comprises an amino acid sequence of SEQ ID NO:7.
[0010] In the crystals or formulations described herein, the
anti-PCSK9 IgG antibody can comprise the heavy and light chain
variable regions described above that are each fused to a suitable
constant region. In some embodiments, the antibody comprises the
mature heavy and light chains of antibody 21B12, (SEQ ID NOS:16 or
17, 21B12 mature light chain and SEQ ID NOS:18 or 19, 21B12 mature
heavy chain). In some embodiments, the antibody comprises SEQ ID
NO:16 and SEQ ID NO:18. In some embodiments, the antibody comprises
SEQ ID NO:17 and SEQ ID NO:19. In some embodiments, the antibody
comprises amino acid sequences obtainable by expressing in
mammalian host cells the cDNA encoding the heavy and/or light
chain, or alternatively the heavy and/or light chain variable
regions, each fused to a suitable constant region, of antibody,
21B12, as described herein. In some embodiments, the antibody binds
to PCSK9 of SEQ ID NO: 1 with a KD binding affinity of 10.sup.-7 or
less (lower numbers meaning higher binding affinity.
[0011] The antibody crystals described herein can be characterized,
for example, by size, shape, morphology, salt content, crystal
packing, and other properties. In some embodiments, the crystal
length ranges from about 5 .mu.M to about 50 .mu.M, optionally with
a morphology that is needle shaped, hexagonal rod shaped,
plate-shaped, football shaped (almond shaped), or mixtures thereof.
Optionally, the crystals are in clusters. The crystals are also
characterized by x-ray diffraction. For example, antibody 21B12
crystals may exhibit a needle shape, hexagonal rod shape,
plate-shape, football shape (almond shape), or a mixture thereof,
or other shapes. In some embodiments, antibody 21B12 crystals
exhibited hexagonal rod shapes.
[0012] In some or any embodiments, the antibody crystals described
herein are characterized by the type of salt. Suitable salts for
the production of antibody 21B12 crystals include, but are not
limited to, one or more of the following: sodium di-hydrogen
phosphate, di-potassium hydrogen phosphate, sodium chloride,
ammonium sulfate, potassium sodium tartrate tetrahydrate, sodium
citrate dihydrate, sodium acetate trihydrate, di-ammonium hydrogen
phosphate, potassium sodium tartrate, calcium acetate, cacodylate,
CHES, CAPS, Tris, lithium sulfate, sodium phosphate, potassium
phosphate, sodium sulfate . For example, other salts (including
hydrates) for the production of antibody 21B12 crystals can include
other dihydrogen phosphate salts, hydrogen phosphate salts,
phosphate salts, chloride salts, sulfate salts, tartrate salts,
citrate salts, acetate salts, cacodylate salts, and; with, for
example, monovalent (e.g. sodium, potassium, ammonium) or divalent
cations (e.g. including but not limited to zinc, magnesium,
calcium). In some or any embodiments, antibody 21B12 crystals are
produced with dihydrogen phosphate salts, hydrogen phosphate salts
and/or tartrate.
[0013] In some or any embodiments, the antibody crystals are
characterized by crystallization additives, which can influence the
crystal growth and/or shape. Suitable crystallization additives
include, but are not limited to, precipitants such as PEG having a
molecular weight of about 400 kD to about 20,000 kD, or about 1000
kD to about 5000 kD (e.g., PEG3350) In some or any embodiments, the
crystals are also characterized by the process by which they are
produced, including remaining impurities. In some embodiments, the
additives (e.g., PEG, glycerol) are at 0.1% to about 75% w/v or
v/v, or about 0.1-50%, or about 0.1-10%, or about 10% to about 50%,
or about 20%-50%, or at least 10%, or at least 20%. Another aspect
of the invention provides methods of making the crystals described
herein. In some embodiments, the method comprises combining a
solution of antibody 21B12 with a crystallization reagent
comprising an appropriate salt, including any of the previously
described salts, and/or a crystallization additive, including any
of the previously described additives. In any of the embodiments
described herein, the salt in the crystallization reagent is
present at a concentration of about 0.1M to about 30M, optionally
0.1M to about 10M, or about 0.1 to about 2M, or about 1M to about
10M. In any of the embodiments described herein, the additives
(e.g., PEG, glycerol) are present at a concentration of about 0.1%
to about 75% w/v or v/v, or about 0.1% to about 50%, or about 0.1%
to about 10%, or about 10% to about 50%, or about 20%-50%, or at
least 10%, or at least 20%.
[0014] Methods of making antibody crystals optionally further
comprise removing at least a portion of the crystallization buffer
(e.g., by centrifugation) after the crystals are formed.
[0015] The methods of making the antibody crystals optionally
further comprise the step of drying the crystals that have formed
(e.g., by air drying the crystals or exposing the crystals to a
vacuum or nitrogen gas).
[0016] Exemplary methods for producing the antibody crystals
described herein include vapor diffusion and batch crystallization,
which are known in the art.
[0017] Another aspect described herein are crystalline formulations
(e.g., powder crystalline and liquid crystalline formulations) and
methods of using antibody crystals described herein to prepare
medicaments, such as crystalline formulations, for therapy of
mammals including humans. Therapy of any of the conditions
described herein is contemplated, optionally using any of the
dosing and timing regimens described herein. The crystalline
formulations comprise antibody crystals, e.g. antibody, 21B12
having one or more of the properties described herein (e.g. size,
length, shape, salt content, additive content, crystal packing or
other properties).
[0018] The crystalline formulations are suitable for parenteral
administration, e.g. are sterile, have endotoxin levels acceptable
for parenteral administration, e.g. <0.25 EU/mL or 0.008 EU/mg,
and comprise pharmaceutically acceptable excipients. The
crystalline formulations are also preferably of high protein
concentrations, e.g. at least 100 mg/ml, 120 mg/ml 140 mg/mL, 150
mg/mL, 160 mg/mL, 170 mg/mL, 180 mg/mL, 190 mg/mL, 200 mg/mL, 210
mg/mL, 220 mg/mL, 230 mg/mL, 240 mg/mL, 250 mg/mL, 260 mg/mL, 270
mg/mL, 280 mg/mL, 290 mg/mL, 300 mg/mL, 310 mg/mL, 320 mg/mL, 330
mg/mL, 340 mg/mL, 350 mg/mL, 360 mg/mL, 370 mg/mL, 380 mg/mL, 390
mg/mL, 400 mg/mL, 410 mg/mL, 420 mg/mL, 430 mg/mL, 440 mg/mL, 450
mg/mL, 460 mg/mL, 480 mg/mL, 500 mg/mL or higher.
[0019] In some or any embodiments, the crystal formulation
comprises excipients including, but not limited to amino acids,
sucrose, trehalose and sorbitol, or other sugars or polyols.
[0020] In some or any embodiments, the crystalline formulations
have a pH ranging from about 2 to about 12, or about 6 to about 9,
or about 6 to 8.5, or about 7 to about 7.5 and an osmolality
ranging from about 180 to about 420 mOsm/kg, or about 200 to about
400 mOsm/kg, or about 250 to about 350 mOsm/kg. While isotonic
(250-350 mOsm/kg) and physiologic pH (about 7-7.5) is preferred,
formulations may be prepared outside of these ranges as long as the
crystals are formulated in physiological relevant conditions.
[0021] Optionally, the crystalline formulation suitable for
parenteral administration (e.g., subcutaneous or intramuscular) is
presented in a container, such as a single dose vial, multidose
vial, syringe, pre-filled syringe or injection device. In some or
any embodiments, the container comprises a single dose of an
anti-PCSK9 antibody (e.g., about 100 mg to about 500 mg of
anti-PCSK9 antibody). In one exemplary embodiment, a container may
contain about 100 mg or 110 mg or 120 mg 130 mg or 140 mg or 150 mg
160 mg or 170 mg or 180 mg or 190 mg or 200 mg or 210 mg or 220 mg
230 mg or 240 mg or 250 mg 260 mg or 270 mg or 280 mg or 290 mg or
300 mg of the crystalline formulation of anti-PCSK9 antibody and
would be suitable for administering a single dose of about 2, 3, 4,
5 or 6 up to about 16 mg/kg body weight. In other embodiments, a
container may contain about 150 mg, or about 160 mg, or about 170
mg, or about 180 mg, or about 190 mg, or about 200 mg, or about 210
mg or about 220 mg or about 230 mg; or about 240 mg, or at about
250 mg; or about 250-450 mg; or about 280 mg, or about 290 mg or
about 300 mg, or about 350 mg or about 360 mg; or about 420 mg or
about 430 mg or about 440 mg or about 450 mg; or about 500 mg to
about 1200 mg; or about 550 mg, or about 600 mg, or about 700 mg,
or about 800 mg, or about 900 mg, or about 1000 mg, or about 1100
mg, or about 1200 mg of the crystalline formulation of anti-PCSK9
antibody. In any of such embodiments, the container may be suitable
for administering a single dose of about 2, 3, 4, 5 or 6 up to
about 16 mg/kg body weight. In any of these embodiments, the
container may comprise the antibody at a high protein concentration
such as those described herein. In any of these embodiments, the
container may comprise a powdered formulation and be for
reconstitution in a volume of about 0.5-2 mL.
[0022] Also disclosed are methods of reconstituting any of the
foregoing powdered formulations comprising adding a sterile diluent
to achieve a high protein constitution such as those described
herein.
[0023] Also disclosed herein is a kit comprising such a container
and a label comprising instructions to use the appropriate volume
or amount of the crystalline formulation necessary to achieve a
dose of from about 100 mg to about 1200 mg of anti-PCSK9 antibody,
or from about 2-16 mg/kg of patient body weight.
[0024] Also disclosed herein are crystalline formulations (e.g.,
powder crystalline and/or liquid crystalline formulations) that are
stable at room temperature for at least 1 month, 3 months, 6
months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year,
18 months, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8
years, 9 years, 10 years or longer. In some embodiments, the
crystalline formulation comprises antibody 21B12 crystals and the
formulation is stable at room temperature for at least 1 month, 3
months, 6 months, 7 months, 8 months, 9 months, 10 months, 11
months, 1 year, 18 months, 2 years, 3 years, 4 years, 5 years, 6
years, 7 years, 8 years, 9 years, 10 years or more.
[0025] Also described herein are methods of using the formulations
described herein to treat and/or prevent cholesterol related
disorders. In some embodiments, a "cholesterol related disorder"
(which includes "serum cholesterol related disorders") includes any
one or more of the following: familial hypercholesterolemia,
non-familial hypercholesterolemia, hyperlipidemia, heart disease,
metabolic syndrome, diabetes, coronary heart disease, stroke,
cardiovascular diseases, Alzheimer's disease and generally
dyslipidemias, which can be manifested, for example, by an elevated
total serum cholesterol, elevated LDL, elevated triglycerides,
elevated VLDL, and/or low HDL. Some non-limiting examples of
primary and secondary dyslipidemias that can be treated using the
formulations describe herein, either alone, or in combination with
one or more other agents include the metabolic syndrome, diabetes
mellitus, familial combined hyperlipidemia, familial
hypertriglyceridemia, familial hypercholesterolemia, including
heterozygous hypercholesterolemia, homozygous hypercholesterolemia,
familial defective apoplipoprotein B-100; polygenic
hypercholesterolemia; remnant removal disease, hepatic lipase
deficiency; dyslipidemia secondary to any of the following: dietary
indiscretion, hypothyroidism, drugs including estrogen and
progestin therapy, beta-blockers, and thiazide diuretics; nephrotic
syndrome, chronic renal failure, Cushing's syndrome, primary
biliary cirrhosis, glycogen storage diseases, hepatoma,
cholestasis, acromegaly, insulinoma, isolated growth hormone
deficiency, and alcohol-induced hypertriglyceridemia. The
formulations described herein can also be useful in preventing or
treating atherosclerotic diseases, such as, for example,
cardiovascular death, non-cardiovascular or all-cause death,
coronary heart disease, coronary artery disease, peripheral
arterial disease, stroke (ischaemic and hemorrhagic), angina
pectoris, or cerebrovascular disease and acute coronary syndrome,
myocardial infarction and unstable angina. In some embodiments, the
formulations described here are useful in reducing the risk of:
fatal and nonfatal heart attacks, fatal and non-fatal strokes,
certain types of heart surgery, hospitalization for heart failure,
chest pain in patients with heart disease, and/or cardiovascular
events because of established heart disease such as prior heart
attack, prior heart surgery, and/or chest pain with evidence of
clogged arteries and/or transplant-related vascular disease. In
some embodiments, the formulations described herein are useful in
preventing or reducing the cardiovascular risk due to elevated CRP
or hsCRP. In some embodiments, the formulations described herein
can be used to reduce the risk of recurrent cardiovascular events.
Exemplary doses of anti-PCSK9 antibody to treat or prevent
cholesterol related disorders range from about 100 mg to about 1200
mg, or about 220 mg to about 450 mg, or about 280 mg to about 450
mg of anti-PCSK9 antibody or 1 mg/kg to about 16 mg/kg, or about 3
mg/kg to 10 mg/k, or about 5-7 mg/kg body weight of anti-PCSK9
antibody.
[0026] As will be appreciated by one of skill in the art, diseases
or disorders that are generally addressable (either treatable or
preventable) through the use of statins can also benefit from the
application of the formulations described herein. In addition, in
some embodiments, disorders or disease that can benefit from the
prevention of cholesterol synthesis or increased LDLR expression
can also be treated by the formulations described herein. In
addition, as will be appreciated by one of skill in the art, the
use of the formulations described herein can be especially useful
in the treatment of diabetes. Not only is diabetes a risk factor
for coronary heart disease, but insulin increases the expression of
PCSK9. That is, people with diabetes have elevated plasma lipid
levels (which can be related to high PCSK9 levels) and can benefit
from lowering those levels. This is generally discussed in more
detail in Costet et al. ("Hepatic PCSK9 Expression is Regulated by
Nutritional Status via Insulin and Sterol Regulatory
Element-binding Protein 1C", J. Biol. Chem., 281: 6211-6218, 2006),
the entirety of which is incorporated herein by reference.
[0027] In another aspect, described herein are methods of lowering
the serum LDL cholesterol level in a mammalian subject comprising
administering a crystalline formulation described herein to the
mammalian subject in an amount effective to lower serum LDL
cholesterol level, as compared to a predose serum LDL cholesterol
level. In some embodiments, the serum LDL cholesterol level in the
mammalian subject is reduced by at least about 15%, 20%, 25%, 30%,
35%. 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or more,
as compared to a predose serum LDL cholesterol level . In some
embodiments the serum LDL cholesterol level is reduced and the
reduction is sustained for a period of at least about 7 days, 2
weeks, 3 weeks, 4 weeks, 1 month, 5 weeks, 6 weeks, 7 weeks, 8
weeks, 2 months, 3 months or longer.
[0028] In another aspect, described herein are methods of lowering
the PCSK9 values in a mammalian subject comprising administering a
crystalline formulation described herein to the mammalian subject
in an amount effective to lower PCSK9 values, as compared to a
predose PCSK9 value. In some embodiments, the PCSK9 value in the
mammalian subject is reduced by at least about 60%, 65%, 70%, 75%,
80%, 85%, 90% or more, as compared to a predose PCSK9 value . In
some embodiments the PCSK9 value is reduced and the reduction is
sustained for a period of at least about 7 days, 2 weeks, 3 weeks,
4 weeks, 1 month, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 2 months, 3
months or longer.
[0029] In another aspect, described herein are methods of lowering
the total cholesterol level in a mammalian subject comprising
administering a crystalline formulation described herein to the
mammalian subject in an amount effective to lower total cholesterol
level, as compared to a predose total cholesterol level. In some
embodiments, the total cholesterol level in the mammalian subject
is reduced by at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%,
55%, 60% or more, as compared to a predose total cholesterol level
. In some embodiments the total cholesterol level is reduced and
the reduction is sustained for a period of at least about 7 days, 2
weeks, 3 weeks, 4 weeks, 1 month, 5 weeks, 6 weeks, 7 weeks, 8
weeks, 2 months, 3 months or longer.
[0030] In another aspect, described herein are methods of lowering
the non-HDL cholesterol level in a mammalian subject comprising
administering a crystalline formulation described herein to the
mammalian subject in an amount effective to lower non-HDL
cholesterol level, as compared to a predose non-HDL cholesterol
level. In some embodiments, the total cholesterol level in the
mammalian subject is reduced by at least about 30%, 35%, 40%, 45%,
50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or more, as compared to a
predose non-HDL cholesterol level. In some embodiments the non-HDL
cholesterol level is reduced and the reduction is sustained for a
period of at least about 7 days, 2 weeks, 3 weeks, 4 weeks, 1
month, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 2 months, 3 months or
longer.
[0031] In another aspect, described herein are methods of lowering
the ApoB level in a mammalian subject comprising administering a
crystalline formulation described herein to the mammalian subject
in an amount effective to lower the ApoB level, as compared to a
predose ApoB level. In some embodiments, the ApoB level in the
mammalian subject is reduced by at least about 20%, 25%, 30%, 35%,
40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more, as compared to a
predose ApoB level . In some embodiments the ApoB level is reduced
and the reduction is sustained for a period of at least about 7
days, 2 weeks, 3 weeks, 4 weeks, 1 month, 5 weeks, 6 weeks, 7
weeks, 8 weeks, 2 months, 3 months or longer.
[0032] In another aspect, described herein are methods of lowering
the Lipoprotein A ("Lp(a)" level in a mammalian subject comprising
administering a crystalline formulation described herein to the
mammalian subject in an amount effective to lower the Lp(a) level,
as compared to a predose Lp(a) level. In some embodiments, the
Lp(a) level in the mammalian subject is reduced by at least about
20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or more, as
compared to a predose Lp(a) level . In some embodiments the Lp(a)
level is reduced and the reduction is sustained for a period of at
least about 7 days, 2 weeks, 3 weeks, 4 weeks, 1 month, 5 weeks, 6
weeks, 7 weeks, 8 weeks, 2 months, 3 months or longer.
[0033] It should be understood that while various embodiments in
the specification are presented using "comprising" language, under
various circumstances, a related embodiment may also be described
using "consisting of" or "consisting essentially" of language. It
is to be noted that the term "a" or "an", refers to one or more,
for example, "an immunoglobulin molecule," is understood to
represent one or more immunoglobulin molecules. As such, the terms
"a" (or "an"), "one or more," and "at least one" can be used
interchangeably herein.
[0034] It should also be understood that when describing a range of
values, the characteristic being described could be an individual
value found within the range. For example, "a pH from about pH 4 to
about pH 6," could be, but is not limited to, pH 4, 4.2, 4.6, 5.1,
5.5, etc. and any value in between such values. Additionally, "a pH
from about pH 4 to about pH 6," should not be construed to mean
that the pH of a formulation in question varies 2 pH units in the
range from pH 4 to pH 6 during storage, but rather a value may be
picked in that range for the pH of the solution, and the pH remains
buffered at about that pH. In some embodiments, when the term
"about" is used, it means the recited number plus or minus 5%, 10%,
15% or more of that recited number. The actual variation intended
is determinable from the context.
[0035] In any of the ranges described herein, the endpoints of the
range are included in the range. However, the description also
contemplates the same ranges in which the lower and/or the higher
endpoint is excluded. Additional features and variations of the
invention will be apparent to those skilled in the art from the
entirety of this application, including the drawing and detailed
description, and all such features are intended as aspects of the
invention. Likewise, features of the invention described herein can
be re-combined into additional embodiments that also are intended
as aspects of the invention, irrespective of whether the
combination of features is specifically mentioned above as an
aspect or embodiment of the invention. Also, only such limitations
which are described herein as critical to the invention should be
viewed as such; variations of the invention lacking limitations
which have not been described herein as critical are intended as
aspects of the invention.
BRIEF DESCRIPTION OF THE FIGURES
[0036] FIG. 1A depicts an amino acid sequence of the mature form of
the PCSK9 with the pro-domain underlined.
[0037] FIGS. 1B.sub.1-1B.sub.4 depict amino acid and nucleic acid
sequences of PCSK9 with the pro-domain underlined and the signal
sequence in bold.
[0038] FIGS. 2A and 2B depict the amino acid and nucleic acid
sequences for the variable domains of antibody, 21B12, CDRs are
underlined and/or boxed.
[0039] FIG. 3 depicts the amino acid sequences for various constant
domains.
[0040] FIGS. 4A and 4B depict the amino acid sequences for mature
heavy chains and mature light chains of antibody, 21B12.
DETAILED DESCRIPTION OF THE INVENTION
[0041] Described herein are crystals of anti-PCSK9 immunoglobulin
type G (IgG) antibodies. In some embodiments, the crystals of
anti-PCSK9 immunoglobulin type G (IgG) antibodies are suitable for
use in crystalline formulations for parenteral administration. In
some embodiments, the crystals of anti-PCSK9 immunoglobulin type G
(IgG) antibodies are suitable for purification and drug substance
storage. Also described herein are methods of using such crystals
of anti-PCSK9 immunoglobulin type G (IgG) antibodies to prepare
crystalline formulations for use as medicaments; formulations
comprising high concentrations of a crystalline anti-PCSK9
antibodies, methods of using these formulations for treatment,
methods of administering these formulations, e.g. subcutaneously or
intramuscularly, and containers or kits comprising these
formulations.
I. Antibodies in the Formulation
[0042] In some embodiments, the anti-PCSK9 antibody in the
formulation is present at a concentration (a "high protein
concentration") of at least about 100 mg/ml, about 101 mg/ml, about
102 mg/ml, about 103 mg/ml, about 104 mg/ml, about 105 mg/ml, about
106 mg/ml, about 107 mg/ml, about 108 mg/ml, about 109 mg/ml, about
110 mg/ml, about 111 mg/ml, about 112 mg/ml, about 113 mg/ml, about
114 mg/ml, about 115 mg/ml, about 116 mg/ml, about 117 mg/ml, about
118 mg/ml, about 119 mg/ml, about 120 mg/ml, about 121 mg/ml, about
122 mg/ml, about 123 mg/ml, about 124 mg/ml, about 125 mg/ml, about
126 mg/ml, about 127 mg/ml, about 128 mg/ml, about 129 mg/ml, about
130 mg/ml, about 131 mg/ml, about 132 mg/ml, about 132 mg/ml, about
133 mg/ml, about 134 mg/ml, about 135 mg/ml, about 136 mg/ml, about
137 mg/ml, about 138 mg/ml, about 139 mg/ml, about 140 mg/ml, about
141 mg/ml, about 142 mg/ml, about 143 mg/ml, about 144 mg/ml, about
145 mg/ml, about 146 mg/ml, about 147 mg/ml, about 148 mg/ml, about
149 mg/ml, about 150 mg/ml, about 151 mg/ml, about 152 mg/ml, about
153 mg/ml, about 154 mg/ml, about 155 mg/ml, about 156 mg/ml, about
157 mg/ml, about 158 mg/ml, about 159 mg/ml, about 160 mg/ml, about
161 mg/ml, about 162 mg/ml, about 163 mg/ml, about 164 mg/ml, about
165 mg/ml, about 166 mg/ml, about 167 mg/ml, about 168 mg/ml, about
169 mg/ml, about 170 mg/ml, about 171 mg/ml, about 172 mg/ml, about
173 mg/ml, about 174 mg/ml, about 175 mg/ml, about 176 mg/ml, about
177 mg/ml, about 178 mg/ml, about 179 mg/ml, about 180 mg/ml, about
181 mg/ml, about 182 mg/ml, about 183 mg/ml, about 184 mg/ml, about
185 mg/ml, about 186 mg/ml, about 187 mg/ml, about 188 mg/ml, about
189 mg/ml, about 190 mg/ml, about 191 mg/ml, about 192 mg/ml, about
193 mg/ml, about 194 mg/ml, about 195 mg/ml, about 196 mg/ml, about
197 mg/ml, about 198 mg/ml, about 199 mg/ml, about 200 mg/ml, about
201 mg/ml, about 202 mg/ml, about 203 mg/ml, about 204 mg/ml, about
205 mg/ml, about 206 mg/ml, about 207 mg/ml, about 208 mg/ml, about
209 mg/ml, about 210 mg/ml, about 211 mg/ml, about 212 mg/ml, about
213 mg/ml, about 214 mg/ml, about 215 mg/ml, about 216 mg/ml, about
217 mg/ml, about 218 mg/ml, about 219 mg/ml, about 220 mg/ml, about
221 mg/ml, about 222 mg/ml, about 223 mg/ml, about 224 mg/ml, about
225 mg/ml, about 226 mg/ml, about 227 mg/ml, about 228 mg/ml, about
229 mg/ml, about 230 mg/ml, about 231 mg/ml, about 232 mg/ml, about
232 mg/ml, about 233 mg/ml, about 234 mg/ml, about 235 mg/ml, about
236 mg/ml, about 237 mg/ml, about 238 mg/ml, about 239 mg/ml, about
240 mg/ml, about 241 mg/ml, about 242 mg/ml, about 243 mg/ml, about
244 mg/ml, about 245 mg/ml, about 246 mg/ml, about 247 mg/ml, about
248 mg/ml, about 249 mg/ml, about 250 mg/ml, and may range up to
e.g., about 450 mg/ml, about 440 mg/ml, 430 mg/ml, 420 mg/ml, 410
mg/ml, 400 mg/ml, about 390 mg/ml, about 380 mg/ml, about 370
mg/ml, about 360 mg/ml, about 350 mg/ml, about 340 mg/ml, about 330
mg/ml, about 320 mg/ml, about 310 mg/ml, about 300 mg/ml, about 290
mg/ml, about 280 mg/ml, about 270 mg/ml, or about 260 mg/ml. Any
range featuring a combination of the foregoing endpoints is
contemplated, including but not limited to: about 70 mg/ml to about
250 mg/ml, about 100 mg/ml to about 250 mg/ml, about 150 mg/ml to
about 250 mg/ml, about 150 mg/ml to about 300 mg/ml, about 150
mg/ml to about 320 mg/ml or about 150 mg/ml to about 350 mg/ml.
[0043] In some embodiments, the anti-PCSK9 antibody is antibody
21B12. Antibody 21B12 was previously described in U.S. Pat. No.
8,030,457, the disclosure of which including sequence listing is
incorporated herein by reference in its entirety.
[0044] The anti-PCSK9 antibody described herein binds to PCSK9 of
SEQ ID NO: 1 with a KD of 10.sup.-6 or less, or 10.sup.-7 or less,
or 10.sup.-8 or less, or 10.sup.-9 or less (lower numbers meaning
higher binding affinity). Affinity can be determined by any means
known in the art, including via Biacore technology.
[0045] The term "21B12 antibody" as used herein refers to an IgG
immunoglobulin composed of two light chains and two heavy chains,
wherein the light chain comprises a light chain complementarity
region (CDR) of the CDRL1 sequence in SQ ID NO:9, a CDRL2 of the
CDRL2 sequence in SQ ID NO:9, and a CDRL3 of the CDRL3 sequence in
SQ ID NO:9, and the heavy chain comprises a heavy chain
complementarity determining region (CDR) of the CDRH1 sequence in
SEQ ID NO:5, a CDRH2 of the CDRH2 sequence in SEQ ID NO:5, and a
CDRH3 of the CDRH3 sequence in SEQ ID NO:5. In some other
embodiments, the antibody is an IgG comprising a light chain
complementarity region (CDR) of the CDRL1 sequence in SEQ ID NO:11,
a CDRL2 of the CDRL2 sequence in SEQ ID NO:11, and a CDRL3 of the
CDRL3 sequence in SEQ ID NO:11, and a heavy chain complementarity
determining region (CDR) of the CDRH1 sequence in SEQ ID NO:7, a
CDRH2 of the CDRH2 sequence in SEQ ID NO:7, and a CDRH3 of the
CDRH3 sequence in SEQ ID NO:7. In some embodiments, the 21B12
antibody comprises the amino acid sequences of: SEQ ID NO:24 (21B12
CDRL1), and SEQ ID NO:25 (21B12 CDRL2), and SEQ ID NO:26 (21B12
CDRL3) and SEQ ID NO:20 or SEQ ID NO:21 (21B12 CDRH1), and SEQ ID
NO:22 (21B12 CDRH2), and SEQ ID NO:23 (21B12 CDRH3).
[0046] In some embodiments, the anti-PCSK9 IgG antibody comprises
the heavy and light chain variable regions of an antibody having at
least 70%, at least 80%, at least 90%, at least 95%, at least 98%,
at least 99% sequence identity to antibody, 21B12. Thus, in some
embodiments, the antibody is an IgG comprising a light chain
variable region comprising an amino acid sequence that is at least
70% identical to that of SEQ ID NO:9 or SEQ ID NO:11 and a heavy
chain variable region that comprises an amino acid sequence that is
at least 70% identical to that of SEQ ID NO:5 or SEQ ID NO:7. In
some embodiments, the antibody is an IgG comprising a light chain
variable region comprising an amino acid sequence that is at least
80% identical to that of SEQ ID NO:9 or SEQ ID NO:11 and a heavy
chain variable region that comprises an amino acid sequence that is
at least 80% identical to that of SEQ ID NO:5 or SEQ ID NO:7. In
some embodiments, the antibody is an IgG comprising a light chain
variable region comprising an amino acid sequence that is at least
90% identical to that of SEQ ID NO:9 or SEQ ID NO:11 and a heavy
chain variable region that comprises an amino acid sequence that is
at least 90% identical to that of SEQ ID NO:5 or SEQ ID NO:7. In
some embodiments, the antibody is an IgG comprising a light chain
variable region comprising an amino acid sequence that is at least
95% identical to that of SEQ ID NO:9 or SEQ ID NO:11 and a heavy
chain variable region that comprises an amino acid sequence that is
at least 95% identical to that of SEQ ID NO:5 or SEQ ID NO:7. In
some embodiments, the antibody is an IgG comprising a light chain
variable region comprising an amino acid sequence that is at least
98% identical to that of SEQ ID NO:9 or SEQ ID NO:11 and a heavy
chain variable region that comprises an amino acid sequence that is
at least 98% identical to that of SEQ ID NO:5 or SEQ ID NO:7. In
some embodiments, the antibody is an IgG comprising a light chain
variable region comprising an amino acid sequence that is at least
99% identical to that of SEQ ID NO:9 or SEQ ID NO:11 and a heavy
chain variable region that comprises an amino acid sequence that is
at least 99% identical to that of SEQ ID NO:5 or SEQ ID NO:7.
[0047] In some embodiments, the light chain of the 21B12 antibody
comprises the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:11
(21B12 light chain variable region) and the heavy chain of the
21B12 antibody comprises the amino acid sequence of SEQ ID NO:5 or
SEQ ID NO:7 (21B12 heavy chain variable domain). In some
embodiments, the light chain of the 21B12 antibody comprises the
amino acid sequence of SEQ ID NO:9 (21B12 light chain variable
region) and heavy chain of the 21B12 antibody comprises the amino
acid sequence of SEQ ID NO:5 (21B12 heavy chain variable region).
In some embodiments, the light chain of the 21B12 antibody
comprises the amino acid sequence of SEQ ID NO:11 (21B12 light
chain variable region) and heavy chain of the 21B12 antibody
comprises the amino acid sequence of SEQ ID NO:7 (21B12 heavy chain
variable region). In some embodiments, the light chain variable
region is fused to a light chain constant region, and the heavy
chain variable region is fused to an IgG constant region. In some
embodiments, the 21B12 antibody comprises the heavy and/or light
chain variable region of antibody 21B12, SEQ ID NO:5 (21B12 heavy
chain variable region) fused to a human heavy chain constant region
of isotype IgG1, 2, 3 or 4 (e.g., native, consensus or modified,
and a number of modifications that are known not to affect binding
are known in the art), and/or SEQ ID NO:9 (21B12 light chain
variable region) fused to a human light chain constant region
(e.g., native, consensus or modified and a number of modifications
that are known not to affect binding are known in the art), or SEQ
ID NO:7 (21B12 heavy chain variable region) fused to a human heavy
chain constant region of isotype IgG1, 2, 3 or 4, and/or SEQ ID
NO:11 (21B12 light chain variable region) fused to a human light
chain constant region. In some embodiments, the antibody comprises
the mature heavy and light chains of antibody 21B12, (SEQ ID NO:16
or 17, 21B12 mature light chain and SEQ ID NO:18 or 19, 21B12
mature heavy chain). In some embodiments, the antibody comprises
SEQ ID NO:16 and SEQ ID NO:18. In some embodiments, the antibody
comprises SEQ ID NO:17 and SEQ ID NO:19.
[0048] In some embodiments, the antibody comprises amino acid
sequences obtainable by expressing in mammalian host cells the cDNA
encoding the heavy and/or light chain, or alternatively the heavy
and/or light chain variable region, of antibody 21B12. The term
"antibody" refers to an intact immunoglobulin, e.g. in the case of
IgG a tetrameric immunoglobulin composed of two heavy chains and
two light chains. (e.g., chimeric, humanized, or human versions
preferably having full length heavy and/or light chains, optionally
with mutations within the framework or constant regions that retain
the anti-PCSK9 binding properties).
[0049] An "isolated" antibody refers to an antibody, as that term
is defined herein, that has been identified and separated from a
component of its natural environment. Contaminant components of its
natural environment are materials that would interfere with
diagnostic or therapeutic uses for the antibody, and may include
enzymes, hormones, and other proteinaceous or nonproteinaceous
solutes. In certain embodiments, the antibody will be purified (1)
to greater than 95% by weight of antibody, and most preferably more
than 99% by weight, (2) to a degree sufficient to obtain at least
15 residues of N-terminal or internal amino acid sequence, or (3)
to homogeneity by SDS-PAGE under reducing or nonreducing conditions
using Coomassie blue or, preferably, silver stain. Isolated
naturally occurring antibody includes the antibody in situ within
recombinant cells since at least one component of the antibody's
natural environment will not be present. Ordinarily, however,
isolated antibody will be prepared by at least one purification
step.
[0050] A "monoclonal" antibody refers to an antibody obtained from
a population of substantially homogeneous antibodies, i.e., the
individual antibodies comprising the population are identical
except for possible naturally occurring mutations that may be
present in minor amounts, compared to a "polyclonal" antibody which
refers to a mixed population of antibodies of diverse sequence that
bind diverse epitopes. The phrase "humanized antibody" refers to an
antibody derived from a sequence of a non-human antibody, typically
a rodent monoclonal antibody, which comprises modifications that
render the sequence more human-like. Alternatively, a humanized
antibody may be derived from a chimeric antibody. The phrase
"human" antibody refers to an antibody derived from human
sequences, e.g. through screening libraries of human antibody genes
through known techniques such as phage display, or produced using
transgenic animals that have no endogenous immunoglobulin
production and are engineered to contain human immunoglobulin
loci.
[0051] An "immunoglobulin G" or "native IgG antibody" is a
tetrameric glycoprotein. In a naturally-occurring immunoglobulin,
each tetramer is composed of two identical pairs of polypeptide
chains, each pair having one "light" (about 25 kDa) and one "heavy"
chain (about 50-70 kDa). The amino-terminal portion of each chain
includes a "variable" ("V") region of about 100 to 110 or more
amino acids primarily responsible for antigen recognition. The
carboxy-terminal portion of each chain defines a constant region
primarily responsible for effector function. Immunoglobulins can be
assigned to different classes depending on the amino acid sequence
of the constant domain of their heavy chains. Heavy chains are
classified as mu (.mu.), delta (.DELTA.), gamma (.gamma.), alpha
(.alpha.), and epsilon (.epsilon.), and define the antibody's
isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Several of
these may be further divided into subclasses or isotypes, e.g.
IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. Different isotypes have
different effector functions; for example, IgG1 and IgG3 isotypes
have antibody-dependent cellular cytotoxicity (ADCC) activity.
Human light chains are classified as kappa (.kappa.) and lambda
(.lamda.) light chains. Within light and heavy chains, the variable
and constant regions are joined by a "J" region of about 12 or more
amino acids, with the heavy chain also including a "D" region of
about 10 more amino acids. See generally, Fundamental Immunology,
Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)).
[0052] The term "hypervariable" region refers to amino acid
residues from a complementarity determining region or CDR (i.e.,
residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the light chain
variable domain and 31-35 (H1), 50-65 (H2) and 95-102 (H3) in the
heavy chain variable domain as described by Kabat et al., Sequences
of Proteins of Immunological Interest, 5th Ed. Public Health
Service, National Institutes of Health, Bethesda, Md. (1991)).
"Framework" or FR residues are those variable region residues other
than the hypervariable region residues.
[0053] The term "variant" when used in connection with antibodies
refers to a polypeptide sequence of an antibody that contains at
least one amino acid substitution, deletion, or insertion in the
variable region or the portion equivalent to the variable region,
provided that the variant retains the desired binding affinity or
biological activity. In addition, the antibodies as described
herein may have amino acid modifications in the constant region to
modify effector function of the antibody, including half-life or
clearance, ADCC and/or CDC activity. Such modifications can enhance
pharmacokinetics or enhance the effectiveness of the antibody in
treating cancer, for example. See Shields et al., J. Biol. Chem.,
276(9):6591-6604 (2001), incorporated by reference herein in its
entirety. In the case of IgG1, modifications to the constant
region, particularly the hinge or CH2 region, may increase or
decrease effector function, including ADCC and/or CDC activity. In
other embodiments, an IgG2 constant region is modified to decrease
antibody-antigen aggregate formation. In the case of IgG4,
modifications to the constant region, particularly the hinge
region, may reduce the formation of half-antibodies.
[0054] The term "modification" when used in connection with
antibodies or polypeptides described herein, includes but is not
limited to, one or more amino acid change (including substitutions,
insertions or deletions); chemical modifications that do not
interfere with PCSK9-binding activity; covalent modification by
conjugation to therapeutic or diagnostic agents; labeling (e.g.,
with radionuclides or various enzymes); covalent polymer attachment
such as pegylation (derivatization with polyethylene glycol) and
insertion or substitution by chemical synthesis of non-natural
amino acids. In some embodiments, modified polypeptides (including
antibodies) of the invention will retain the binding properties of
unmodified molecules of the invention.
[0055] The term "derivative" when used in connection with
antibodies or polypeptides of the invention refers to antibodies or
polypeptides that are covalently modified by conjugation to
therapeutic or diagnostic agents, labeling (e.g., with
radionuclides or various enzymes), covalent polymer attachment such
as pegylation (derivatization with polyethylene glycol) and
insertion or substitution by chemical synthesis of non-natural
amino acids. In some embodiments, derivatives of the invention will
retain the binding properties of underivatized molecules of the
invention.
[0056] Proteins and non-protein agents may be conjugated to the
antibodies by methods that are known in the art. Conjugation
methods include direct linkage, linkage via covalently attached
linkers, and specific binding pair members (e.g., avidin-biotin).
Such methods include, for example, that described by Greenfield et
al., Cancer Research 50, 6600-6607 (1990) for the conjugation of
doxorubicin and those described by Arnon et al., Adv. Exp. Med.
Biol. 303, 79-90 (1991) and by Kiseleva et al., Mol. Biol. (USSR)
25, 508-514 (1991) for the conjugation of platinum compounds.
II. Production of Crystals, Crystal Formulations and
Compositions
[0057] Polypeptide crystals are grown by controlled crystallization
of polypeptides from aqueous solutions or from aqueous solutions
containing organic solvents or additives. Solution conditions that
may be controlled include, for example, the rate of evaporation of
solvent, organic solvents or additives, the presence of appropriate
co-solutes and buffers, pH, and temperature. A comprehensive review
of the various factors affecting the crystallization of proteins
has been published by McPherson (1985, Methods Enzymol 114:
112-120). In addition, McPherson and Gilliland (1988, J Crystal
Growth, 90: 51-59) have compiled comprehensive lists of
polypeptides that have been crystallized, as well as the conditions
under which they were crystallized. A compendium of crystals and
crystallization recipes, as well as a repository of coordinates of
solved protein structures, is maintained by the Protein Data Bank
at the Brookhaven National Laboratory (www.rcsb.org/pdb/; Bernstein
et al., 1977, J Mol Biol 112: 535-542). It should be noted,
however, that the conditions reported in most of the above-cited
references have been optimized to yield, in most instances, a few
large, diffraction quality crystals. Accordingly, it will be
appreciated by those of skill in the art that these conditions vary
from protein to protein, and do not provide a high yielding process
for the large scale production of crystals of any given
polypeptide.
[0058] In general, crystals are produced by combining the
polypeptide (i.e., antibody) to be crystallized with an appropriate
aqueous solvent or aqueous solvent containing appropriate
crystallization agents, such as salts or organic solvents or
additives (collectively the "crystallization reagent"). The solvent
is combined with the polypeptide and may be subjected to agitation
at a temperature determined experimentally to be appropriate for
the induction of crystallization and acceptable for the maintenance
of polypeptide activity and stability. Laboratory-scale methods for
crystallization include hanging drop vapor diffusion, sitting drop
vapor diffusion, microdialysis, microbatch, under oil, in gel and
sandwich drop methods. The solvent can optionally include
co-crystallization additives, such as precipitants, fatty acids,
reducing agents, glycerol, sulfobetaine, surfactants, polyols,
divalent cations, co-factors, or chaotropes, and amino acids as
well as buffer species to control pH.
[0059] "Co-crystallization additives" include compounds that
facilitate crystallization of a polypeptide and/or compounds that
stabilize the protein and protect against denaturation. Examples of
co-solutes include ammonium acetate, ammonium chloride, ammonium
fluoride, ammonium formate, ammonium nitrate, ammonium phosphate,
ammonium sulfate, cadmium chloride, cadmium sulfate, calcium
acetate, calcium chloride, cesium chloride, cobaltous chloride,
CH.sub.3(CH.sub.2).sub.15N(CH.sub.3).sub.3.sup.+Br..sup.- (CTAB),
di-ammonium citrate, di-ammonium hydrogen phosphate, di-ammonium
phosphate, di-ammonium tartrate, di-potassium phosphate, di-sodium
phosphate, di-sodium tartrate, DL-malic acid, ferric chloride,
L-proline, lithium acetate, lithium chloride, lithium nitrate,
lithium sulfate, magnesium acetate, magnesium chloride, magnesium
formate, magnesium nitrate, magnesium sulfate, nickel chloride,
potassium acetate, potassium bromide, potassium chloride, potassium
citrate, potassium fluoride, potassium formate, potassium nitrate,
potassium phosphate, potassium sodium tartrate, potassium sulfate,
potassium thiocyanate, sodium acetate, sodium bromide, sodium
chloride, sodium citrate, sodium fluoride, sodium formate, sodium
malonate, sodium nitrate, sodium phosphate, sodium sulfate, sodium
thiocyanate, succinic acid, tacsimate, tri-ammonium citrate,
tri-lithium citrate, trimethylamine N-oxide, tri-potassium citrate,
tri-sodium citrate, zinc acetate, zinc sulfate, and other compounds
that function to supply co-solutes. "Crystallization" include
compounds that maintain the pH of a solution in a desired range to
facilitate crystallization of a polypeptide. Examples include ACES
(N-(2-acetamido)-2-aminoethanesulfonic acid), BES
(N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid), Bicine
(N,N-Bis(2-hydroxyethyl)glycine), BIS-TRIS
(2,2-bis-(hydroxymethyl)-2,2',2''-nitrilotriethanol), boric acid,
CAPS (3-[cyclohexylamino]-1-propanesulfonic acid), citric acid,
EPPS (HEPPS, 4-(2-Hydroxyethyl)piperazine-l-propanesulfonic acid),
Gly-Gly (NH.sub.2CH.sub.2CONHCH.sub.2COOH, glycyl-glycine), HEPES
(4-(2-hydroxyethyl)piperazine-l-ethanesulfonic acid), imidazole,
MES (2-morpholinoethanesulfonic acid), MOPS
(3-(N-morpholino)-propanesulfonic acid), PIPES
(piperazine-1,4-bis(2-ethanesulfonic acid)), potassium chloride,
sodium acetate, sodium bicarbonate, sodium phosphate monobasic
(sodium dihydrogen phosphate), sodium phosphate dibasic,
TAPS(N[tris-(hydroxymethyl)methyl]-3-aminopropanesulfonic acid),
TAPSO(N-[tris(hydroxymethyl)methyl]-3-amino-2-hydroxypropanesulfonic
acid), TES (N-[tris(hydroxymethyl)methyl]-2-aminoethanesulfonic
acid), Tricine (N-[tris(hydroxymethyl)methyl]glycine), Tris-HCl,
TRIZMA (2-amino-2-(hydroxymethyl)-1,3-propanediol), and other
compounds that function to maintain a solution at or near a
specified pH.
[0060] The selection of precipitants are one factor affecting
crystallization. For example, PEG products, e.g. of molecular
weight 200 to 20,000 kD, can be used. PEG3350 is a long polymer
precipitant or dehydrant which works by volume exclusion effect.
Lyotropic salts, such as ammonium sulfate, promote precipitation
processes, as do short-chain fatty acids, such as caprylic acid.
Polyionic species also are useful precipitants.
[0061] Antibodies for use in formulations for subcutaneous
injection, for example, preferably are precipitated at a
physiologic pH range and in a crystallization reagent that provides
isotonic osmolality.
[0062] The need for additives, co-solutes, buffers, etc. and their
concentrations are determined experimentally to facilitate
crystallization. Some examples of suitable crystallization
conditions for a polypeptide are described in the Examples
below.
[0063] Antibody 21B12 is crystallized under a variety of
conditions. Various morphologies of Antibody 21B12 crystals can be
grown under scale-up conditions whereby the antibody in a liquid
formulation is added to a volume of known crystallization reagent
and stored in a sealed container. Antibody 21B12 crystals can be
grown under these conditions in less than 24 hours, at room
temperature and have been shown to produce between about 30% to
about 99%% yield.
[0064] In an industrial-scale process, the controlled precipitation
leading to crystallization can best be carried out by the simple
combination of polypeptide, precipitant, co-solutes and,
optionally, buffers in a batch process. As another option,
polypeptides may be crystallized by using polypeptide precipitates
as the starting material ("seeding"). In this case, polypeptide
precipitates are added to a crystallization solution and incubated
until crystals form. Alternative laboratory crystallization
methods, such as dialysis or vapor diffusion, can also be adopted.
McPherson, supra and Gilliland, supra, include a comprehensive list
of suitable conditions in their reviews of the crystallization
literature. Occasionally, in cases in which the crystallized
polypeptide is to be crosslinked, incompatibility between an
intended crosslinking agent and the crystallization medium might
require exchanging the crystals into a more suitable solvent
system.
[0065] According to some embodiments, polypeptide crystals, crystal
formulations and compositions are prepared by the following
process: first, the polypeptide is crystallized. Next, excipients
or ingredients as described herein are added directly to the mother
liquor. Alternatively, the crystals are suspended in a solution of
excipient or other formulary ingredients, after the mother liquor
is removed, for a minimum of 1 hour to a maximum of 24 hours. The
excipient concentration is typically between about 0.01 to 30% w/w,
which corresponds to a polypeptide crystal concentration of 99.99
to 70% w/w, respectively. In one embodiment, the excipient
concentration is between about 0.1 to 10%, which corresponds to a
crystal concentration of 99.9 to 90% w/w, respectively. The mother
liquor can be removed from the crystal slurry either by filtration,
buffer exchange, or by centrifugation. Subsequently, the crystals
are washed with any isotonic injectable vehicle as long as the
these vehicles do not dissolve the crystals, optionally with
solutions of 50 to 100% of one or more organic solvents or
additives such as, for example, ethanol, methanol, isopropanol or
ethyl acetate, or polyethelene glycol (PEG), either at room
temperature or at temperatures between -20.degree. C. to 25.degree.
C. In addition, water can be used to wash the crystals. The
crystals are the dried either by passing a stream of nitrogen, air,
or inert gas over the crystals. Finally, micronizing of the
crystals can be performed if necessary. The drying of polypeptide
crystals is the removal of water, organic solvent or additive, or
liquid polymer by means including drying with N.sub.2, air, or
inert gases; vacuum oven drying; lyophilization; washing with a
volatile organic solvent or additive followed by evaporation of the
solvent; or evaporation in a fume hood. Typically, drying is
achieved when the crystals become a free-flowing powder. Drying may
be carried out by passing a stream of gas over wet crystals. The
gas may be selected from the group consisting of: nitrogen, argon,
helium, carbon dioxide, air or combinations thereof The diameter of
the particles achieved can be in the range of 0.1 to 100
micrometers, or in the range of 0.2 to 10 micrometers, or in the
range of 10 to 50 micrometers, or in the range of 0.5 to 2
micrometers. For formulations to be administered by inhalation, in
one embodiment the particles formed from the polypeptide crystals
are in the range of 0.5 to 1 micrometers.
[0066] According to some embodiments, when preparing protein
crystals, protein crystal formulations or compositions, enhancers,
such as surfactants are not added during crystallization. According
to some other embodiments, when preparing protein crystals, protein
crystal formulations or compositions, enhancers, such as
surfactants are added during crystallization. Excipients or
ingredients are added to the mother liquor after crystallization,
at a concentration of between about 1-10% w/w, alternatively at a
concentration of between about 0.1-25% w/w, alternatively at a
concentration of between about 0.1-50% w/w. These concentrations
correspond to crystal concentrations of 99-90% w/w, 99.9-75% w/w
and 99.9-50% w/w, respectively. The excipient or ingredient is
incubated with the crystals in the mother liquor for about 0.1-3
hrs, alternatively the incubation is carried out for 0.1-12 hrs,
alternatively the incubation is carried out for 0.1-24 hrs.
[0067] In some or any embodiments, the ingredient or excipient is
dissolved in a solution other than the mother liquor, and the
protein crystals are removed from the mother liquor and suspended
in the excipient or ingredient solution. In some embodiments, the
excipient or ingredient solution (or resuspension vehicle) is a
mixture of excipients or ingredients or surfactants that is
isotonic and injectable. In some embodiments, the excipient or
ingredient solution (or resuspension vehicle) is not a mixture of
excipients or ingredients or surfactants that is isotonic and
injectable. The ingredient or excipient concentrations and the
incubation times are the same as those described above.
Polypeptide Crystals
[0068] As used herein, "crystal" or "crystalline" refers to one
form of the solid state of matter, which is distinct from a second
form--the amorphous solid state. Crystals display characteristic
features including a lattice structure, characteristic shapes, and
optical properties such as refractive index and birefringence. A
crystal consists of atoms arranged in a pattern that repeats
periodically in three dimensions (C. S. Barrett, Structure of
Metals, 2nd ed., McGraw-Hill, New York, 1952, p. 1). In contrast,
amorphous material is a non-crystalline solid form of matter,
sometimes referred to as an amorphous precipitate. Such
precipitates have no molecular lattice structure characteristic of
the crystalline solid state and do not display birefringence or
other spectroscopic characteristics typical of the crystalline
forms of matter.
[0069] Polypeptide crystals are polypeptide molecules arranged in a
crystal lattice. Polypeptide crystals contain a pattern of specific
polypeptide-polypeptide interactions that are repeated periodically
in three dimensions. The polypeptide crystals of this invention are
to be distinguished from amorphous solid forms or precipitates of
polypeptides, such as those obtained by lyophilizing a polypeptide
solution.
[0070] In polypeptide crystals, the polypeptide molecules form
asymmetric units which are arranged together to form symmetric
units. The geometric structure of the symmetric units of
polypeptide crystals can be, for example, cubic, hexagonal,
monoclinic, orthorhombic, tetragonal, triclinic, or trigonal. The
overall structure of the crystals in their entirety can be, for
example, in the form of bipyramids, cubes, needles, plates, prisms,
rhomboids, rods, or spheres, or combinations thereof. Other
observed forms include block-shaped, UFO shaped, football shaped,
leaf shaped, wheat shaped, singlet shaped, feather-shaped,
straw-shaped, chrysanthemum-shaped, spherical or mixtures thereof.
In some embodiments, the crystals are observed in clusters.
Crystals that are of the "cubic" structural class can more
specifically have octadecahedral or dodecahedral crystal forms. The
diameter of the crystals is defined as the Martin's diameter. It is
measured as the length of the line, parallel to the ocular scale,
that divides the randomly oriented crystals into two equal
projected areas. Crystals in forms such as needles or rods will
also have a maximal dimension that is referred to herein as the
length of the crystal. The crystals are also characterized by x-ray
diffraction.
Testing Properties of Crystalline Polypeptides
[0071] After polypeptide crystals are formed, they can be subjected
to various analyses to confirm their polypeptide content and to
further examine their physical structure. For example, if necessary
individual crystals can be removed from the crystallization
solution and washed with aqueous or organic solvents or additives,
then dried (for example, by air drying, by passing a stream of
inert gas over the crystal, by lyophilization, or by vacuum).
Crystals can be isolated, removed from the crystal growth drop, and
then mounted for X-ray diffraction.
[0072] Crystals can also be characterized by a variety of means
described in the art. See, e.g., Basu et al., Expert Opin. Biol.
Thera. 4, 301-317 (2004), incorporated herein by reference in its
entirety for its disclosure of protein crystal production and
formulation procedures, and analytical tools for characterizing
crystals and their component protein. While powder X-ray
diffraction is commonly used to identify crystalline material, it
requires very large and perfect protein crystals and is not
commonly applied to the protein microcrystals typically used in
crystalline formulations. Electron diffraction and solid state
nuclear magnetic resonance (ssNMR) can be applied to characterize
crystals. Crystal size, shape and morphology (e.g. surface
morphology) can be inspected, for example, by light microscopy,
transmission electron microscopy, scanning electron microscopy,
atomic force microscopy, and/or light scattering (e.g. photon
correlation spectroscopy or DLS, low angle laser light scattering
or LAALS). Total surface area and porosity of crystals can also be
characterized. Mass spectrometry, micro-attenuated total
reflectance Fourier transform infrared spectroscopy (FTIR) and/or
differential scanning calorimetry (DSC) can provide information
about protein primary and secondary structure.
[0073] As another example, polypeptide crystals can be removed from
crystallization solution and washed or rinsed, or the majority of
crystallization solution can be removed from the crystals and
replaced with a different solution. In this way, the particular
salt that was using in the crystallization procedure can be
replaced in the crystal lattice with a different salt. In one
embodiment of the invention, crystallized Antibody 21B12 is
separated from the crystallization buffer and placed in a solution
containing a salt of sodium, potassium, or magnesium (for example,
sodium acetate, sodium chloride, sodium citrate, sodium phosphate,
sodium sulfate, potassium chloride, potassium citrate, or magnesium
sulfate). For X-ray diffraction, the replacement solution can
contain heavy atoms useful in determining the atomic coordinates of
the crystallized polypeptide.
[0074] In a further example, polypeptide crystals can be removed
from crystallization solution and solubilized in an appropriate
buffer for further testing, such as an SDS-containing buffer for
analysis of the polypeptide that had been crystallized by gel
electrophoresis. Methods for analysis of proteins by gel
electrophoresis are well known and include staining a gel with
silver or Coomassie blue dye, and comparing the electrophoretic
migration of the polypeptide that had been crystallized with the
migration of polypeptide markers of known molecular weight. In
another method, the polypeptide is visualized in the gel by use of
a labeled antibody that specifically binds to the polypeptide.
Polypeptides that have been crystallized can also be solubilized in
buffers appropriate for amino acid sequencing by Edman degradation,
for mass spectrometry, for other spectrographic scattering,
refraction, diffraction, or absorption studies, or for labeling of
the polypeptide by attachment of a label molecule to the
polypeptide.
III. Formulations for Therapeutic Administration
[0075] As used herein, the term "composition" as used herein means
a mixture comprising at least two components. In particular,
described herein are compositions comprising a crystalline
anti-PCSK9 antibody, or prepared using a crystalline anti-PCSK9
antibody. In some embodiments, the composition or formulation
comprising or prepared using a crystalline anti-PCSK9 antibody is
prepared such that it is suitable for injection and/or
administration to a patient in need thereof. Compositions to be
administered for pharmaceutical purposes to patients are
substantially sterile and do not contain any agents that are unduly
toxic or infectious to the recipient.
[0076] In some embodiments, crystalline anti-PCSK9 antibodies, such
as crystalline antibody 21B12, are administered in the form of a
physiologically acceptable composition (also referred to herein as
a pharmaceutical composition or as a pharmaceutical formulation)
comprising a crystalline anti-PCSK9 antibody that is formulated
with one or more of the following: physiologically acceptable
carriers, excipients, or diluents. Such carriers, excipients, or
diluents are nontoxic to recipients at the dosages and
concentrations employed. Ordinarily, the preparation of such
compositions entails combining the crystalline anti-PCSK9 antibody
with one or more of the following: buffers, antioxidants such as
ascorbic acid, low molecular weight polypeptides (such as those
having fewer than 10 amino acids), proteins, amino acids such as
Leucine, Proline, Alanine, Valine, Glycine, Serine, Asparagine,
Glutamine, Aspartic acid, Glutamic acid, Methionine, Tryptophan,
Phenylalanine, Isoleucine, Threonine, Cysteine, Tyrosine,
Histidine, Lysine and Arginine, carbohydrates such as glucose,
sucrose or dextrins, chelating agents such as EDTA, glutathione and
other stabilizers and excipients. In liquid formulations, neutral
buffered saline or saline mixed with nonspecific serum albumin are
exemplary appropriate diluents. In accordance with appropriate
industry standards, preservatives may also be added, such as benzyl
alcohol. Further examples of components that may be employed in
pharmaceutical formulations are presented in Remington's
Pharmaceutical Sciences, 16.sup.th Ed., Mack Publishing Company,
Easton, Pa., 1980, and in the Handbook of Pharmaceutical
Excipients, published jointly by the American Pharmaceutical
Association and the Pharmaceutical Society of Great Britain.
[0077] In one embodiment, it is contemplated that the formulation
described herein is prepared in a bulk formulation and as such, the
components of the pharmaceutical composition are adjusted so that
they are higher than would be required for administration, and are
diluted appropriately prior to administration.
[0078] The antibody crystals described herein can be formulated as
a solid crystalline or powder formulation in forms suitable for
storage and handling, and in forms suitable for inhalation or
pulmonary administration, for example in the form of powders for
the preparation of aerosol formulations. In an further embodiment,
the antibody crystals can be formulated in a liquid solution of
such crystals, or in a slurry of such crystals. In another
embodiment, the antibody crystals are used to prepare a liquid
formulation, such as an aqueous formulation, for therapeutic
administration.
A. Solid Crystalline Formulations
[0079] Solid formulations of antibody crystals include crystals
that have been substantially isolated from liquid solution or
dried, and are present as free crystals or as particles in for
example powder form. In the present context the expression "powder"
refers to a collection of essentially dry particles, i.e. the
moisture content being below about 10% by weight, or below 6% by
weight, or below 4% by weight. Polypeptide crystals or powders can
be optionally combined with carriers or surfactants. Suitable
carrier agents include 1) carbohydrates, e.g. monosaccharides such
as fructose, galactose, glucose, sorbose, and the like; 2)
disaccharides, such as lactose, trehalose and the like; 3)
polysaccharides, such as raffmose, maltodextrins, dextrans, and the
like; 4) alditols, such as mannitol, xylitol, and the like; 5)
inorganic salts, such as sodium chloride, and the like; and 6)
organic salts, such as sodium citrate, sodium ascorbate and the
like. In certain embodiments, the carrier is selected from the
group consisting of trehalose, raffinose, mannitol, sorbitol,
xylitol, inositol, sucrose, sodium chloride, and sodium citrate.
Surfactants can be selected from the group consisting of salts of
fatty acids, bile salts, phospholipids or polysorbates. Fatty acids
salts include salts of C.sub.10-14 fatty acids, such as sodium
caprate, sodium laurate, and sodium myristate. Bile salts include
salts of ursodeoxycholate, taurocholate, glycocholate, and
taurodihydrofusidate. Polysorbates include polysorbate 20 and
polysorbate 80. In one embodiment, the surfactant is a salt of
taurocholate such as sodium taurocholate. Phospholipids that can be
used as surfactants include lysophosphatidylcholine. In one
embodiment, the surfactant is polysorbate 20, and in another
embodiment, the surfactant is polysorbate 80.
B. Crystals in Solution or Slurries
[0080] Also described herein is a method for rendering polypeptide
crystals suitable for storage in suspensions comprising replacing
the crystallization buffer (the mother liquor) with a non-aqueous
solvent. In yet another embodiment, the crystalline slurry can be
rendered solid by spinning out the first solvent and washing the
remaining crystalline solid using a second organic solvent or
additive to remove water, followed by evaporation of the
non-aqueous solvent. Non-aqueous slurries of crystalline
therapeutic proteins are especially useful for subcutaneous
delivery.
[0081] In one such embodiment, the polypeptide crystals described
herein are combined with liquid organic additives with the object
of stabilizing the polypeptide crystals. Such a mixture can be
characterized as an aqueous-organic mixture that comprises n%
organic additive, where n is between 1 and 99 and m% aqueous
solution, where m is 100-n. Examples of organic additives include
phenolic compounds, such as m-cresol or phenol or a mixture
thereof, and acetone, methyl alcohol, methyl isobutyl ketone,
chloroform, 1-propanol, isopropanol, 2-propanol, acetonitrile,
1-butanol, 2-butanol, ethyl alcohol, cyclohexane, dioxane, ethyl
acetate, dimethylformamide, dichloroethane, hexane, isooctane,
methylene chloride, tert-butyl alchohol, toluene, carbon
tetrachloride, or combinations thereof
C. Liquid Formulations
[0082] Another embodiment provided herein is an aqueous formulation
that allows for stable long-term storage of a pharmaceutical
composition wherein a crystalline anti-PCSK9 antibody is the active
ingredient used in the preparation of the pharmaceutical
composition. This formulation is useful, in part, because it is
more convenient to use for the patient, as this formulation does
not require any extra steps such as rehydrating. As used herein, a
"solution" or "liquid formulation" is meant to mean a liquid
preparation that contains one or more chemical substances dissolved
in a suitable solvent or mixture of mutually miscible solvents.
Reconstitution is the dissolution of polypeptide crystals or
crystal formulations or compositions in an appropriate buffer or
pharmaceutical formulation.
D. Components of Pharmaceutical Formulations
[0083] The present pharmaceutical composition is prepared by
combining, in addition to a crystalline anti-PCSK9 antibody as
described above, one or more of the following types of ingredients
or excipients listed in the paragraphs below, many or all of which
are available from commercial suppliers. It will be understood by
one of ordinary skill in the art that the combining of the various
components to be included in the composition can be done in any
appropriate order, namely, the buffer can be added first, middle or
last and the tonicity modifier can also be added first, middle or
last. It is also to be understood by one of ordinary skill in the
art that some of these chemicals can be incompatible in certain
combinations, and accordingly, are easily substituted with
different chemicals that have similar properties but are compatible
in the relevant mixture. There is knowledge in the art regarding
the suitability of various combinations of excipients and other
ingredients or materials present in, for example, the containers
used for storage of the pharmaceutical composition and/or the
devices used for therapeutic administration (see, for example,
Akers, 2002, J Pharm Sci 91: 2283-2300).
[0084] Non-limiting examples of additional agents that can be
included in the formulations described herein include acidifying
agents (including, but not limited to, acetic acid, glacial acetic
acid, citric acid, fumaric acid, hydrochloric acid, diluted
hydrochloric acid, malic acid, nitric acid, phosphoric acid,
diluted phosphoric acid, sulfuric acid, tartaric acid, and other
suitable acids); active ingredients (including, but not limited to,
additional active ingredients to reduce injection site discomfort,
and non-steroidal anti-inflammatory drugs such as, for example,
tromethamine, in an appropriate dosage); aerosol propellants
(including, but not limited to, butane, dichlorodifluoromethane,
dichlorotetrafluoroethane, isobutane, propane and
trichloromonofluoromethane); alcohol denaturants (including, but
not limited to, denatonium benzoate, methyl isobutyl ketone,
sucrose octacetate); alkalizing agents (including, but not limited
to, strong ammonia solution, ammonium carbonate, diethanolamine,
diisopropanolamine, potassium hydroxide, sodium bicarbonate, sodium
borate, sodium carbonate, sodium hydroxide, trolamine); anticaking
agents (including, but not limited to, calcium silicate, magnesium
silicate, colloidal silicon dioxide and talc); antifoaming agents
(including, but not limited to, dimethicone and simethicone);
chelating agents (also called sequestering agents) (including, but
not limited to, edetate disodium, ethylenediaminetetraacetic acid
and salts and edetic acid); coating agents (including, but not
limited to, sodium carboxymethylcellulose, cellulose acetate,
cellulose acetate phthalate, ethylcellulose, gelatin,
pharmaceutical glaze, hydroxypropyl cellulose, hydroxypropyl
methylcellulose, hydroxypropyl methylcellulose phthalate,
methacrylic acid copolymer, methylcellulose, polyethylene glycol,
polyvinyl acetate phthalate, shellac, sucrose, titanium dioxide,
carnauba wax, microcystalline wax and zein); colors (including, but
not limited to, caramel, erythrosine (FD&C Red No. 3); FD&C
Red No. 40; FD&C Yellow No. 5; FD&C Yellow No. 6; FD&C
Blue No. 1; red, yellow, black, blue or blends and ferric oxide);
complexing agents (including, but not limited to,
ethylenediaminetetraacetic acid (EDTA)and salts thereof, edetic
acid, gentisic acid ethanolmaide and oxyquinoline sulfate);
desiccants (including, but not limited to, calcium chloride,
calcium sulfate and silicon dioxide); filtering aids (including,
but not limited to, powdered cellulose and purified siliceous
earth); flavors and perfumes (including, but not limited to,
anethole, anise oil, benzaldehyde, cinnamon oil, cocoa, ethyl
vanillin, menthol, methyl salicylate, monosodium glutamate, orange
flower oil, orange oil, peppermint, peppermint oil, peppermint
spirit, rose oil, stronger rose water, thymol, tolu balsam
tincture, vanilla, vanilla tincture and vanillin); humectants
(including, but not limited to, glycerin, hexylene glycol,
propylene glycol and sorbitol); ointment bases (including, but not
limited to, lanolin, anhydrous lanolin, hydrophilic ointment, white
ointment, yellow ointment, polyethylene glycol ointment,
petrolatum, hydrophilic petrolatum, white petrolatum, rose water
ointment and squalane); plasticizers (including, but not limited
to, castor oil, diacetylated monoglycerides, diethyl phthalate,
glycerin, mono- and di-acetylated monoglycerides, polyethylene
glycol, propylene glycol, triacetin and triethyl citrate); polymer
membranes (including, but not limited to, cellulose acetate);
solvents (including, but not limited to, acetone, alcohol, diluted
alcohol, amylene hydrate, benzyl benzoate, butyl alcohol, carbon
tetrachloride, chloroform, corn oil, cottonseed oil, ethyl acetate,
glycerin, hexylene glycol, isopropyl alcohol, methyl alcohol,
methylene chloride, methyl isobutyl ketone, mineral oil, peanut
oil, polyethylene glycol, propylene carbonate, propylene glycol,
sesame oil, water for injection, sterile water for injection,
sterile water for irrigation and purified water); sorbents
(including, but not limited to powdered cellulose, charcoal,
purified siliceous earth; and carbon dioxide sorbents: barium
hydroxide lime and soda lime); stiffening agents (including, but
not limited to, hydrogenated castor oil, cetostearyl alcohol, cetyl
alcohol, cetyl esters wax, hard fat, paraffin, polyethylene
excipient, stearyl alcohol, emulsifying wax, white wax and yellow
wax); suppository bases (including, but not limited to, cocoa
butter, hard fat and polyethylene glycol);Suspending and/or
viscosity-increasing agents (including, but not limited to, acacia,
agar, alginic acid, aluminum monostearate, bentonite, purified
bentonite, magma bentonite, carbomer 934p, carboxymethylcellulose
calcium, carboxymethylcellulose sodium, carboxymethycellulose
sodium 12, carrageenan, microcrystalline and carboxymethylcellulose
sodium cellulose, dextrin, gelatin, guar gum, hydroxyethyl
cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose,
magnesium aluminum silicate, methylcellulose, pectin, polyethylene
oxide, polyvinyl alcohol, povidone, propylene glycol alginate,
silicon dioxide, colloidal silicon dioxide, sodium alginate,
tragacanth and xanthan gum); sweetening agents (including, but not
limited to, aspartame, dextrates, dextrose, excipient dextrose,
fructose, mannitol, saccharin, calcium saccharin, sodium saccharin,
sorbitol, solution sorbitol, sucrose, compressible sugar,
confectioner's sugar and syrup); tablet binders (including, but not
limited to, acacia, alginic acid, sodium carboxymethylcellulose,
microcrystalline cellulose, dextrin, ethylcellulose, gelatin,
liquid glucose, guar gum, hydroxypropyl methylcellulose,
methycellulose, polyethylene oxide, povidone, pregelatinized starch
and syrup); tablet and/or capsule diluents (including, but not
limited to, calcium carbonate, dibasic calcium phosphate, tribasic
calcium phosphate, calcium sulfate, microcrystalline cellulose,
powdered cellulose, dextrates, dextrin, dextrose excipient,
fructose, kaolin, lactose, mannitol, sorbitol, starch,
pregelatinized starch, sucrose, compressible sugar and
confectioner's sugar); tablet disintegrants (including, but not
limited to, alginic acid, microcrystalline cellulose,
croscarmellose sodium, corspovidone, polacrilin potassium, sodium
starch glycolate, starch and pregelatinized starch); tablet and/or
capsule lubricants (including, but not limited to, calcium
stearate, glyceryl behenate, magnesium stearate, light mineral oil,
polyethylene glycol, sodium stearyl fumarate, stearic acid,
purified stearic acid, talc, hydrogenated vegetable oil and zinc
stearate); vehicles (include, but are not limited to flavored
and/or sweetened (aromatic elixir, compound benzaldehyde elixir,
iso-alcoholic elixir, peppermint water, sorbitol solution, syrup,
tolu balsam syrup); oleaginous (almond oil, corn oil, cottonseed
oil, ethyl oleate, isopropyl myristate, isopropyl palmitate,
mineral oil, light mineral oil, myristyl alcohol, octyldodecanol,
olive oil, peanut oil, persic oil, sesame oil, soybean oil,
squalane); solid carriers such as sugar spheres; and sterile
vehicles (bacteriostatic water for injection, bacteriostatic sodium
chloride injection); and water-repelling agents (including, but not
limited to, cyclomethicone, dimethicone and simethicone);
[0085] Aggregation inhibitors, reduce a polypeptide's tendency to
associate in inappropriate or unwanted ternary or quaternary
complexes, can also be included in the formulations described
herein. Suitable aggregation inhibitors include the amino acids
L-arginine and/or, L-cysteine, which can act to reduce aggregation
of polypeptides containing an Fc domain over long periods, e.g.,
two years or more. The concentration of the aggregation inhibitor
in the formulation can be between about 1 mM to 1M, or about 10 mM
to about 200 mM, or about 10 mM to about 100 mM, or about 15 MM to
about 75 mM, or about 150 mM to about 250 mM, or about 25 mM.
[0086] Antioxidants may also be included in the formulations
described herein. Antioxidants contemplated for use in the
preparation of the formulations include amino acids such as glycine
and lysine, chelating agents such as EDTA and DTPA, and
free-radical scavengers such as sorbitol and mannitol. Additional
antioxidants include ascorbic acid, ascorbyl palmitate, butylated
hydroxyanisole, butylated hydroxytoluene, hypophosphorous acid,
monothioglycerol, propyl gallate, sodium bisulfite, sodium
formaldehyde sulfoxylate, sodium metabisulfite, sodium thiosulfate,
sulfur dioxide, tocopherol, and tocopherols excipient. Also
contemplated for use in inhibiting oxidation is nitrogen or carbon
dioxide overlay. Nitrogen or carbon dioxide overlay can be
introduced to the headspace of a vial or prefilled syringe during
the filling process.
[0087] Buffering agents, which maintain the pH of the
pharmaceutical formulation in a desired range, can also be included
in the formulations described herein. When the pH of the
pharmaceutical composition is set at or near physiological levels,
comfort of the patient upon administration is maximized. In
particular, in certain embodiments the pH of a pharmaceutical
composition is within a pH range of about 4.0 to 8.4, or a pH range
of about 5.0 to 8.0, or a pH range of about 5.8 to 7.4, or about
6.2 to 7.0. It is to be understood that the pH can be adjusted as
necessary to maximize stability and solubility of the polypeptide
in a particular formulation and as such, a pH outside of
physiological ranges, yet tolerable to the patient, is within the
scope of the invention. Various buffers suitable for use in the
pharmaceutical composition of the invention include histidine,
alkali salts (sodium or potassium phosphate or their hydrogen or
dihydrogen salts), sodium citrate/citric acid, sodium
acetate/acetic acid, potassium citrate, maleic acid, ammonium
acetate, tris-(hydroxymethyl)-aminomethane (tris), various forms of
acetate and diethanolamine, ammonium carbonate, ammonium phosphate,
boric acid, lactic acid, phosphoric acid, potassium metaphosphate,
potassium phosphate monobasic, sodium lactate solution, and any
other pharmaceutically acceptable pH buffering agent known in the
art. pH-adjusting agents such as hydrochloric acid, sodium
hydroxide, or a salt thereof, may also be included in order to
obtain the desired pH. One suitable buffer is sodium phosphate for
maintaining pharmaceutical compositions at or near pH 6.2. In
another example, acetate is a more efficient buffer at pH 5 than pH
6 so less acetate may be used in a solution at pH 5 than at pH 6.
The concentration of the buffer in the formulation can be between
about 1 mM to about 1M, or about 10 mM to about 300 mM.
[0088] Polymeric carriers can also be included in the formulations
described herein. Polymeric carriers are polymers used for
encapsulation of polypeptide crystals for delivery of polypeptide,
including biological delivery. Such polymers include biocompatible
and biodegradable polymers. The polymeric carrier may be a single
polymer type or it may be composed of a mixture of polymer types.
Polymers useful as the polymeric carrier, include for example,
poly(acrylic acid), poly(cyanoacrylates), poly(amino acids),
poly(anhydrides), poly(depsipeptide), poly(esters) such as
poly(lactic acid) or PLA, poly(lactic-co-glycolic acid) or PLGA,
poly(B-hydroxybutryate), poly(caprolactone) and poly(dioxanone);
poly(ethylene glycol), poly((hydroxypropyl)methacrylamide, poly
[(organo)phosphazene], poly(ortho esters), poly(vinyl alcohol),
poly(vinylpyrrolidone), maleic anhydride-alkyl vinyl ether
copolymers, pluronic polyols, albumin, natural and synthetic
polypeptides, alginate, cellulose and cellulose derivatives,
collagen, fibrin, gelatin, hyaluronic acid, oligosaccharides,
glycaminoglycans, sulfated polysaccharides, or any conventional
material that will encapsulate polypeptide crystals.
[0089] Preservatives, such as antimicrobial preservatives, are also
contemplated for use in the formulations described herein. Suitable
preservatives include, but are not limited to, benzalkonium
chloride, benzalkonium chloride solution, benzelthonium chloride,
benzoic acid, benzyl alcohol, butylparaben, cetylpyridinium
chloride, chlorobutanol, chlorocresol, cresol, dehydroacetic acid,
ethylparaben, methylparaben, methylparaben sodium, phenol,
phenylethyl alcohol, phenylmercuric acetate, phenylmercuric
nitrate, potassium benzoate, potassium sorbate, propylparaben,
propylparaben sodium, sodium benzoate, sodium dehydroacetate,
sodium propionate, sorbic acid, thimerosal, and thymol. The amount
of preservative included will be in the range of 0% to 2% (w/v) or
about 1% (w/v).
[0090] Solubilizing agents and stabilizers (also referred to as
emulsifying agents, co-solutes, or co-solvents) that increase the
solubility of the polypeptide and/or stabilize the polypeptide
while in solution (or in dried or frozen forms) can also be added
to a pharmaceutical composition. Examples of solubilizing and
stabilizing agents include but are not limited to sugars/polyols
such as: sucrose, lactose, glycerol, xylitol, sorbitol, mannitol,
maltose, inositol, trehalose, glucose; polymers such as: serum
albumin (bovine serum albumin (BSA), human SA (HSA), or recombinant
HA), dextran, PVA, hydroxypropyl methylcellulose (HPMC),
polyethyleneimine, gelatin, polyvinylpyrrolidone (PVP),
hydroxyethylcellulose (HEC); non-aqueous solvents such as:
polyhydric alcohols (e.g., PEG, ethylene glycol and glycerol),
dimethysulfoxide (DMSO), and dimethylformamide (DMF); amino acids
such as: proline, L-methionine, L-serine, sodium glutamic acid,
alanine, glycine, lysine hydrochloride, sarcosine, and
gamma-aminobutyric acid; surfactants such as: Tween-80, Tween-20,
SDS, polysorbate, polyoxyethylene copolymer; and miscellaneous
stabilizing excipients such as: potassium phosphate, sodium
acetate, ammonium sulfate, magnesium sulfate, sodium sulfate,
trimethylamine N-oxide, betaine, metal ions (e.g., zinc, copper,
calcium, manganese, and magnesium), CHAPS, monolaurate,
2-0-beta-mannoglycerate; or any of the following: acacia,
cholesterol, diethanolamine (adjunct), glyceryl monostearate,
lanolin alcohols, lecithin, mono- and di-glycerides,
monoethanolamine (adjunct), oleic acid (adjunct), oleyl alcohol
(stabilizer), poloxamer, polyoxyethylene 50 stearate, polyoxyl 35
caster oil, polyoxyl 40 hydrogenated castor oil, polyoxyl 10 oleyl
ether, polyoxyl 20 cetostearyl ether, polyoxyl 40 stearate,
polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80,
polysorbate 100, Triton X-100, propylene glycol diacetate,
propylene glycol monostearate, sodium lauryl sulfate, sodium
stearate, sorbitan monolaurate, sorbitan monooleate, sorbitan
monopalmitate, sorbitan monostearate, stearic acid, trolamine,
emulsifying wax; wetting and/or solubilizing agents such as
benzalkonium chloride, benzethonium chloride, cetylpyridinium
chloride, docusate sodium, nonoxynol 9, nonoxynol 10, octoxynol 9,
polyoxyl 50 stearate, tyloxapol; or any combination of the above.
The concentration of solubilizers/stabilizers in the formulation
can be between about 0.001 to 5 weight percent, or about 0.1 to 2
weight percent. In one embodiment, the stabilizer is selected from
sorbitan mono-9-octadecenoate poly(oxy-1,2-ethanediyl) derivatives,
including but not limited to, polysorbate 80 or polysorbate 20. The
amount of polysorbate 20 or 80 to be used in this embodiment is in
the range of 0.001% to 1.0% (w/v), such as 0.005% (w/v), in single
use or in multi-dose formulations. In another embodiment, free
L-methionine in the range of 0.05 mM to 50 mM is included in the
formulation: the amount of free L-methionine is 0.05 mM to 5 mM for
single use formulations, and 1 mM to 10 mM for multi-dose
formulations.
[0091] Tonicity modifiers can also be included in the formulations
described herein. Tonicity modifiers are understood to be molecules
that contribute to the osmolality of a solution. The osmolality of
a pharmaceutical composition is preferably regulated in order to
maximize the active ingredient's stability and also to minimize
discomfort to the patient upon administration. Serum is
approximately 300+/-50 milliosmolals per kilogram. It is generally
preferred that a pharmaceutical composition be isotonic with serum,
i.e., having the same or similar osmolality, which is achieved by
addition of a tonicity modifier, thus it is contemplated that the
osmolality will be from about 180 to about 420 milliosmolals,
however, it is to be understood that the osmolality can be either
higher or lower as specific conditions require. Examples of
tonicity modifiers suitable for modifying osmolality include, but
are not limited to amino acids (e.g., arginine, cysteine, histidine
and glycine), salts (e.g., sodium chloride, potassium chloride and
sodium citrate) and/or saccharides (e.g., sucrose, glucose,
dextrose, glycerin, and mannitol). The concentration of the
tonicity modifier in the formulation can be between about 1 mM to
1M, or about 10 mM to about 200 mM. In one embodiment, the tonicity
modifier is sodium chloride within a concentration range of 0 mM to
200 mM. In another embodiment, the tonicity modifier is sorbitol or
trehalose and no sodium chloride is present.
[0092] In certain embodiments, the formulation comprises a compound
selected from the following, or any combination thereof: salts of
1) amino acids such as glycine, arginine, aspartic acid, glutamic
acid, lysine, asparagine, glutamine, proline; 2) carbohydrates,
e.g. monosaccharides such as glucose, fructose, galactose, mannose,
arabinose, xylose, ribose; 3) disaccharides, such as lactose,
trehalose, maltose, sucrose; 4) polysaccharides, such as
maltodextrins, dextrans, starch, glycogen; 5) alditols, such as
mannitol, xylitol, lactitol, sorbitol; 6) glucuronic acid,
galacturonic acid; 7) cyclodextrins, such as methyl cyclodextrin,
hydroxypropyl-.beta.-cyclodextrin and alike; 8) inorganic salts,
such as sodium chloride, potassium chloride, magnesium chloride,
phosphates of sodium and potassium, boric acid ammonium carbonate
and ammonium phosphate; 9) organic salts, such as acetates,
citrate, ascorbate, lactate; 10) emulsifying or solubilizing agents
like acacia, diethanolamine, glyceryl monostearate, lecithin,
monoethanolamine, oleic acid, oleyl alcohol, poloxamer,
polysorbates, sodium lauryl sulfate, stearic acid, sorbitan
monolaurate, sorbitan monostearate, and other sorbitan derivatives,
polyoxyl derivatives, wax, polyoxyethylene derivatives, sorbitan
derivatives; 11) viscosity increasing reagents like, agar, alginic
acid and its salts, guar gum, pectin, polyvinyl alcohol,
polyethylene oxide, cellulose and its derivatives propylene
carbonate, polyethylene glycol, hexylene glycol, tyloxapol; and 12)
particular ingredients such as sucrose, trehalose, lactose,
sorbitol, lactitol, inositol, salts of sodium and potassium such as
acetate, phosphates, citrates, borate, glycine, arginine,
polyethylene oxide, polyvinyl alcohol, polyethylene glycol,
hexylene glycol, methoxy polyethylene glycol, gelatin,
hydroxypropyl-P-cyclodextrin.
E. Sustained-Release Forms
[0093] In some embodiments, sustained-release forms (also called
"controlled-release" forms) of crystalline anti-PCSK9 antibodies
are used, including sustained-release forms of crystalline antibody
21B12; sustained- or controlled-release forms comprising
crystalline antibody 21B12, and a substance for extending the
physical release or biological availability of the crystalline
antibody 21B12 over a desired period of time.
[0094] Sustained-release forms suitable for use in the disclosed
methods include, but are not limited to, crystalline antibody 21B12
that is encapsulated in a sustained-release means such as a
slowly-dissolving biocompatible polymer (for example, the polymeric
carriers described herein, the alginate microparticles described in
U.S. Pat. No. 6,036,978, or the polyethylene-vinyl acetate and
poly(lactic-glucolic acid) compositions described in U.S. Pat. No.
6,083,534), admixed with such a polymer (including topically
applied hydrogels), and or encased in a biocompatible
semi-permeable implant. Further embodiments of the invention
include additional sustained-release forms such as polymeric
microparticles, wherein a mixture of the active ingredient and
sustained-release means such as polymers (for example, PLGA) are
dispersed within a continuous phase, and the resulting dispersion
is directly lyophilized to remove water and organic solvents or
additives and form said microparticles (U.S. Pat. No. 6,020,004,
incorporated herein by reference in its entirety); injectable gel
compositions comprising a biodegradable anionic polysaccharide such
as an alginate ester, a polypeptide, and at least one bound
polyvalent metal ion (U.S. Pat. No. 6,432,449, incorporated herein
by reference in its entirety); injectable biodegradable polymeric
matrices having reverse thermal gelation properties and optionally
pH-responsive gelation/de-gelation properties (U.S. Pat. Nos.
6,541,033 and 6,451,346, incorporated herein by reference in their
entireties); biocompatible polyol:oil suspensions, such as those
wherein the suspension comprises polyol in the range of from about
15% to about 30% by weight (U.S. Pat. No. 6,245,740, incorporated
by reference in its entirety). Such sustained release forms are
suitable for continuous delivery of polypeptides through
administration in the form of a depot, wherein the depot can be an
implant, or can be in the form of injectable microspheres,
nanospheres, or gels. The above listed U.S. patents (U.S. Pat. Nos.
6,036,978; 6,083,534; 6,020,004; 6,432,449; 6,541,033; 6,451,346,
and 6,245,740) are incorporated in their entirety by reference
herein. In addition, sustained- or controlled-release forms of
crystalline polypeptides of the invention comprise types of
sustained release means such as those described in Kim, C., 2000,
"Controlled Release Dosage Form Design", Techonomic Publishing Co.,
Lancaster Pa., which include the following: natural polymers
(gelatin, sodium alginic acid, xanthan gum, arabic gum, or
chitosan), semi-synthetic polymers or cellulose derivatives
(methylcellulose, ethylcellulose, hydroxyethylcellulose,
hydroxypropylcellulose, hydroxyethylmethylcellulose,
hydroxypropylmethylcellulose, sodium carboxymethylcellulose,
cellulose acetate, cellulose acetate butyrate, cellulose acetate
proprionate, cellulose acetatephthalate, or
hydroxypropylmethylcellulose phthalate), and synthetic polymers
(ion exchange resins (methacrylic acid, sulfonated
polystyrene/divinylbenzene), polyacrylic acid (Carbopol),
poly(MMA/MAA), poly(MMA/DEAMA), poly(MMA/EA), poly(vinylacetate
phthalate), poly(vinyl alcohol), poly(vinyl pyrrolidone),
poly(lactic acid), poly(glycolic acid), poly(lactic/glycolic acid),
polyethylene glycol, polyethylene oxide, poly(dimethyl silicone),
poly(hydroxyethyl methacrylate), poly(ethylene/vinyl acetate),
poly(ethylene/vinyl alcohol), polybutadiene, poly(anhydride),
poly(orthoester), and poly(glutamic acid)).
[0095] Further embodiments disclosed herein include antibody 21B12
crystals encapsulated in at least one polymeric carrier to form
microspheres by virtue of encapsulation within the matrix of the
polymeric carrier to preserve their native and biologically active
tertiary structure, as described in U.S. Pat. No. 6,541,606, which
is incorporated in its entirety by reference herein. Antibody 21B12
crystals or formulations thereof to be encapsulated are suspended
in a polymeric carrier such as PLGA which is dissolved in an
organic solvent or additive. Such encapsulated Antibody 21B12
crystals maintain the biological activity of antibody 21B12 for a
longer period of time than antibody 21B12 in solution when stored
under comparable conditions.
IV. Kits
[0096] As an additional aspect, described herein are kits which
comprise one or more formulations described herein packaged in a
manner which facilitates their use for administration to subjects.
In one embodiment, such a kit includes a formulation described
herein (e.g., a composition comprising any of the antibodies
described therein), packaged in a container such as a sealed
bottle, vessel, single-use or multi-use vial, prefilled syringe, or
prefilled injection device, optionally with a label affixed to the
container or included in the package that describes use of the
compound or composition in practicing the method. In one aspect,
the compound or composition is packaged in a unit dosage form. The
kit may further include a device suitable for administering the
composition according to a specific route of administration.
Preferably, the kit contains a label that describes use of an
antibody described herein or formulation described herein.
V. Dosages
[0097] The dosage regimen involved in a method for treating a
condition described herein will be determined by the attending
physician, considering various factors which modify the action of
drugs, e.g. the age, condition, body weight, sex and diet of the
patient, the severity of any infection, time of administration and
other clinical factors. In various aspects, the dosage is in the
range of 0.1-50 mg of a preparation of antibody per kilogram of
body weight (calculating the mass of the protein alone, without
chemical modification). In some embodiments, the dosage is about
0.5 mg/kg to 20 mg/kg, or about 0.5-10 mg/kg. In some embodiments,
the dosage is about 120 mg to about 1200 mg, or about 280 to about
450 mg.
[0098] In various aspects, the dosage of an anti-PCSK9 antibody,
e.g., antibody 21B12, can range from at least about 100 mg to at
about 1400 mg; or about 120 mg to about 1200 mg; or about 120 mg to
about 1000 mg; or about 120 mg to about 800 mg; or about 120 mg to
about 700 mg; or about 120 mg to about 480 mg; or about 120 mg up
to about 480 mg; or about 100 mg up to about 480 mg; or about 1200
mg to about 480 mg; or about 140 mg to about 480 mg; or about 145
mg to about 480 mg; or about 150 mg to about 480 mg; or about 160
mg to about 480 mg; or about 170 mg to about 480 mg; or about 180
mg to about 480 mg or about 190 mg to about 480 mg or about 200 mg
to about 480 mg; or about 210 mg to about 480 mg; or about 220 mg
to about 480 mg; or about 230 mg to about 480 mg; or about 240 mg
to about 480 mg; or about 250 mg to about 480 mg; or about 260 mg
to about 480 mg; or about 270 mg to about 480 mg; or about 280 mg
to about 480 mg; or about 290 mg to about 480 mg; or about 300 mg
to about 480 mg; or about 310 mg to about 480 mg; or about 320 mg
to about 480 mg; or about 330 mg to about 480 mg; or about 340 mg
to about 480 mg; or about 350 mg to about 480 mg; or about 360 mg
to about 480 mg; or about 370 mg to about 480 mg; or about 380 mg
to about 480 mg; or about 390 mg to about 480 mg; or about 400 mg
to about 480 mg; or about 410 mg to about 480 mg; or about 420 mg
to about 480 mg; or about 430 mg to about 480 mg; or about 440 mg
to about 480 mg; or about 450 mg to about 480 mg; or about 460 mg
to about 480 mg; or about 470 mg to about 480 mg of an anti-PCSK9
antibody, e.g., antibody 21B12.
[0099] In certain embodiments, the frequency of dosing will take
into account the pharmacokinetic parameters of an anti-PCSK
antibody and/or any additional therapeutic agents in the
formulation used. In certain embodiments, a clinician will
administer the formulation until a dosage is reached that achieves
the desired effect. In certain embodiments, the formulation can
therefore be administered as a single dose, or as two, three, four
or more doses (which may or may not contain the same amount of the
desired molecule) over time, or as a continuous infusion via an
implantation device or catheter. The formulation can also be
delivered subcutaneously or intravenously with a standard needle
and syringe. In addition, with respect to subcutaneous delivery,
pen delivery devices, as well as autoinjector delivery devices,
have applications in delivering a pharmaceutical formulation of the
present invention. Further refinement of the appropriate dosage is
routinely made by those of ordinary skill in the art and is within
the ambit of tasks routinely performed by them. In certain
embodiments, appropriate dosages can be ascertained through use of
appropriate dose-response data. In some embodiments, the amount and
frequency of administration can take into account the desired
cholesterol level (serum and/or total) to be obtained and the
subject's present cholesterol level, LDL level, and/or LDLR levels,
all of which can be obtained by methods that are well known to
those of skill in the art.
[0100] In some embodiments, a dosage of at least about 100 mg; or
up to about 110 mg; or up to about 115 mg, or up to about 120 mg;
or up to about 140 mg; or up to about 160 mg; or up to about 200
mg; or up to about 250 mg; or up to 280 mg; or up to 300 mg; or up
to 350 mg; or up to 400 mg; or up to 420 mg of an anti-PCSK9
antibody, e.g., antibody 21B12, is administered once every other
week, (or every two weeks)(Q2W), to a patient in need thereof.
[0101] In certain other embodiments, a dosage of at least about 250
mg; or up to about 280 mg; or up to about 300 mg; or up to about
350 mg; or up to about 400 mg; or up to about 420 mg; or up to
about 450 mg; or up to 480 mg of a an anti-PCSK9 antibody, e.g.,
antibody 21B12, is administered once every four weeks, (or once a
month), to a patient in need thereof
[0102] The formulations are generally administered parenterally,
e.g. intravenously, subcutaneously, intramuscularly, via aerosol
(intrapulmonary or inhalational administration), or via depot for
long-term release. In some embodiments, the formulation is
administered intravenously by an initial bolus followed by a
continuous infusion to maintain therapeutic circulating levels of
drug product. In other embodiments, the formulation is administered
as a one-time dose. Those of ordinary skill in the art will readily
optimize effective dosages and administration regimens as
determined by good medical practice and the clinical condition of
the individual patient. The frequency of dosing will depend on the
pharmacokinetic parameters of the agents and the route of
administration. The optimal pharmaceutical formulation will be
determined by one skilled in the art depending upon the route of
administration and desired dosage. See for example, Remington's
Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co.,
Easton, Pa. 18042) pages 1435-1712, the disclosure of which is
hereby incorporated by reference. Such formulations may influence
the physical state, stability, rate of in vivo release, and rate of
in vivo clearance of the administered agents. Depending on the
route of administration, a suitable dose may be calculated
according to body weight, body surface area or organ size. Further
refinement of the calculations necessary to determine the
appropriate dosage for treatment involving each of the above
mentioned formulations is routinely made by those of ordinary skill
in the art without undue experimentation, especially in light of
the dosage information and assays disclosed herein, as well as the
pharmacokinetic data observed in the human clinical trials
discussed above. Appropriate dosages may be ascertained through use
of established assays for determining blood level dosages in
conjunction with appropriate dose-response data. The final dosage
regimen will be determined by the attending physician, considering
various factors which modify the action of drugs, e.g. the drug's
specific activity, the severity of the damage and the
responsiveness of the patient, the age, condition, body weight, sex
and diet of the patient, the severity of any infection, time of
administration and other clinical factors. As studies are
conducted, further information will emerge regarding the
appropriate dosage levels and duration of treatment for various
diseases and conditions.
VI. Therapeutic Uses of the Formulation
[0103] As will be appreciated by one of skill in the art, disorders
that relate to, involve, or can be influenced by varied
cholesterol, LDL, LDLR, PCSK9, VLDL-C, apoprotein B ("ApoB"),
lipoprotein A ("Lp(a)"), triglycerides, HDL-C, non-HDL-C, and total
cholesterol levels can be addressed by the pharmaceutical
formulations of the present invention. In one aspect, the anti-PCS9
antibody formulations can be used in methods to treat and/or
prevent and/or reduce the risk of disorders that relate to elevated
serum cholesterol levels or in which elevated serum cholesterol
levels are relevant. In one aspect, the anti-PCS9 antibody
formulations can be used in methods to treat and/or prevent and/or
reduce the risk of disorders that relate to elevated PCSK9 values
or in which elevated PCSK9 values are relevant. In one aspect, the
anti-PCS9 antibody formulations can be used in methods to treat
and/or prevent and/or reduce the risk of disorders that relate to
elevated total cholesterol levels or in which elevated total
cholesterol levels are relevant. In one aspect, the anti-PCS9
antibody formulations can be used in methods to treat and/or
prevent and/or reduce the risk of disorders that relate to elevated
non-HDL cholesterol levels or in which elevated non-HDL cholesterol
levels are relevant. In one aspect, the anti-PCS9 antibody
formulations can be used in methods to treat and/or prevent and/or
reduce the risk of disorders that relate to elevated ApoB levels or
in which elevated ApoB levels are relevant. In one aspect, the
anti-PCS9 antibody formulations can be used in methods to treat
and/or prevent and/or reduce the risk of disorders that relate to
elevated Lp(a) levels or in which elevated Lp(a) levels are
relevant. In one aspect, the anti-PCS9 antibody formulations can be
used in methods to treat and/or prevent and/or reduce the risk of
disorders that relate to elevated triglyceride levels or in which
elevated triglyceride levels are relevant. In one aspect, the
anti-PCS9 antibody formulations can be used in methods to treat
and/or prevent and/or reduce the risk of disorders that relate to
elevated VLDL-C levels or in which elevated VLDL-C levels are
relevant.
[0104] As will be appreciated by one of skill in the art, the
anti-PCS9 antibody formulations of the present invention can be
therapeutically useful in treating and/or preventing cholesterol
related disorders. Exemplary, non-limiting diseases and disorders
that can be treated or prevented by the administration of the
pharmaceutical formulations of the present invention include
familial hypercholesterolemia, non-familial hypercholesterolemia,
hyperlipidemia, heart disease, metabolic syndrome, diabetes,
coronary heart disease, stroke, cardiovascular diseases,
Alzheimer's disease and generally dyslipidemias, which can be
manifested, for example, by an elevated total serum cholesterol,
elevated LDL, elevated triglycerides, elevated VLDL, and/or low
HDL. Some non-limiting examples of primary and secondary
dyslipidemias that can be treated using the formulations described
herein, either alone, or in combination with one or more other
agents include the metabolic syndrome, diabetes mellitus, familial
combined hyperlipidemia, familial hypertriglyceridemia, familial
hypercholesterolemia, including heterozygous hypercholesterolemia,
homozygous hypercholesterolemia, familial defective apoplipoprotein
B-100; polygenic hypercholesterolemia; remnant removal disease,
hepatic lipase deficiency; dyslipidemia secondary to any of the
following: dietary indiscretion, hypothyroidism, drugs including
estrogen and progestin therapy, beta-blockers, and thiazide
diuretics; nephrotic syndrome, chronic renal failure, Cushing's
syndrome, primary biliary cirrhosis, glycogen storage diseases,
hepatoma, cholestasis, acromegaly, insulinoma, isolated growth
hormone deficiency, and alcohol-induced hypertriglyceridemia. The
formulations of the present invention can also be useful in
preventing or treating atherosclerotic diseases, such as, for
example, cardiovascular death, non-cardiovascular or all-cause
death, coronary heart disease, coronary artery disease, peripheral
arterial disease, stroke (ischaemic and hemorrhagic), angina
pectoris, or cerebrovascular disease and acute coronary syndrome,
myocardial infarction and unstable angina. In some embodiments, the
formulations are useful in reducing the risk of: fatal and nonfatal
heart attacks, fatal and non-fatal strokes, certain types of heart
surgery, hospitalization for heart failure, chest pain in patients
with heart disease, and/or cardiovascular events because of
established heart disease such as prior heart attack, prior heart
surgery, and/or chest pain with evidence of clogged arteries and/or
transplant-related vascular disease. In some embodiments, the
formulations are useful in preventing or reducing the
cardiovascular risk due to elevated CRP or hsCRP. In some
embodiments, the formulations and methods can be used to reduce the
risk of recurrent cardiovascular events.
[0105] As will be appreciated by one of skill in the art, diseases
or disorders that are generally addressable (either treatable or
preventable) through the use of statins can also benefit from the
application of formulations of this invention. In addition, as will
be appreciated by one of skill in the art, the use of formulations
of this invention can be especially useful in the treatment of
diabetes.
[0106] In some embodiments, the formulations of the present
invention are administered to those who have diabetes mellitus,
abdominal aortic aneurysm, atherosclerosis and/or peripheral
vascular disease in order to decrease their serum cholesterol
levels to a safer range. In some embodiments, the formulations of
this invention are administered to patients at risk of developing
any of the herein described disorders. In some embodiments, the
formulations of this invention are administered to subjects that
smoke, or used to smoke (i.e., former smokers), have hypertension
or a familial history of early heart attacks.
[0107] The formulation need not cure the subject of the disorder.
The formulation may be used therapeutically to ameliorate, in whole
or in part, a cholesterol-related disorder or symptom thereof, or
to protect, in whole or in part, against further progression of a
cholesterol-related disorder or symptom thereof Indeed, the
materials and methods of the invention are particularly useful for
lowering serum LDL cholesterol and maintaining the reduction in
serum LDL cholesterol over a period of time.
[0108] One or more administrations of a formulation described
herein may be carried out over a therapeutic period of, for
example, about 2 weeks to about 12 months (e.g., about 1 month,
about 2 months, about 3 months, about 4 months, about 5 months,
about 6 months, about 7 months, about 8 months, about 9 months,
about 10 months, or about 11 months). In some embodiments, a
subject is administered one or more doses of the formulation to
lower serum LDL cholesterol. The term "maintain reduction of serum
LDL cholesterol" as used herein means the reduction of serum LDL
cholesterol resulting the initial dose of the formulation does not
fall more than about 1% to about 5% over the course of about 2
weeks, about 1 month, about 2 months, about 3 months, about 6
months, about 9 months about 1 year, about 18 months, about 2
years, or over the course of the patient's life).
[0109] In addition, it may be advantageous to administer multiple
doses of the formulation or space out the administration of doses,
depending on the therapeutic regimen selected for a particular
subject. The formulation can be administered periodically over a
time period of one year or less (e.g., 9 months or less, 6 months
or less, or 3 months or less). In this regard, the formulation can
be administered to the human once every about 7 days, or 2 weeks,
or 3 weeks, or 1 month, or 5 weeks, or 6 weeks, or 7 weeks, or 2
months, or 9 weeks, or 10 weeks, or 11 weeks, or 3 months, or 13
weeks, or 14 weeks, or 15 weeks, or 4 months, or 17 weeks, or 18
weeks, or 19 weeks, or 5 months, or 21 weeks, or 22 weeks, or 23
weeks, or 6 months, or 12 months.
VII. Combination therapy
[0110] Treatment of a pathology by combining two or more agents
that target the same pathogen or biochemical pathway sometimes
results in greater efficacy and diminished side effects relative to
the use of the therapeutically relevant dose of each agent alone.
In some cases, the efficacy of the drug combination is additive
(the efficacy of the combination is approximately equal to the sum
of the effects of each drug alone), but in other cases the effect
can be synergistic (the efficacy of the combination is greater than
the sum of the effects of each drug given alone). As used herein,
the term "combination therapy" means the two compounds can be
delivered in a simultaneous manner, e.g. concurrently, or wherein
one of the compounds is administered first, followed by the second
agent, e.g., sequentially. The desired result can be either a
subjective relief of one or more symptoms or an objectively
identifiable improvement in the recipient of the dosage.
[0111] In some embodiments, the formulation is administered prior
to, concurrent with, or subsequent to, a standard of care
therapeutic for the treatment of decreased bone mineral density. As
used herein, the term "standard of care" refers to a treatment that
is generally accepted by clinicians for a certain type of patient
diagnosed with a type of illness. In some embodiments, the standard
of care therapeutic is at least one other cholesterol-lowering
(serum and/or total body cholesterol) agent. Exemplary agents
include, but are not limited to, statins (atorvastatin,
cerivastatin, fluvastatin, lovastatin, mevastatin, pitavastatin,
pravastatin, rosuvastatin, simvastatin), Nicotinic acid (Niacin)
(NIACOR, NIASPAN (slow release niacin), SLO-NIACIN (slow release
niacin), CORDAPTIVE (laropiprant)), Fibric acid (LOPID
(Gemfibrozil), TRICOR (fenofibrate), Bile acid sequestrants
(QUESTRAN (cholestyramine), colesevelam (WELCHOL), COLESTID
(colestipol)), Cholesterol absorption inhibitors (ZETIA
(ezetimibe)), Combining nicotinic acid with statin (ADVICOR
(LOVASTATIN and NIASPAN), Combining a statin with an absorption
inhibitor (VYTORIN (ZOCOR and ZETIA) and/or lipid modifying agents.
In some embodiments, the formulation is combined with PPAR gamma
agonists, PPAR alpha/gamma agonists, squalene synthase inhibitors,
CETP inhibitors, anti-hypertensives, anti-diabetic agents (such as
sulphonyl ureas, insulin, GLP-1 analogs, DDPIV inhibitors, e.g.,
metaformin), ApoB modulators, such as mipomersan, MTP inhibitors
and /or arteriosclerosis obliterans treatments. In some
embodiments, the formulation is combined with an agent that
increases the level of LDLR protein in a subject, such as statins,
certain cytokines like oncostatin M, estrogen, and/or certain
herbal ingredients such as berberine. In some embodiments, the
formulation is combined with an agent that increases serum
cholesterol levels in a subject (such as certain anti-psychotic
agents, certain HIV protease inhibitors, dietary factors such as
high fructose, sucrose, cholesterol or certain fatty acids and
certain nuclear receptor agonists and antagonists for RXR, RAR,
LXR, FXR). In some embodiments, the formulation is combined with an
agent that increases the level of PCSK9 in a subject, such as
statins and/or insulin. The combination of the two can allow for
the undesirable side-effects of other agents to be mitigated by the
formulation.
[0112] In some embodiments, the formulation is administered to a
subject when treatment of a standard of care therapeutic described
herein is contraindicated.
EXAMPLES
Example 1
Crystallization of Antibody, 21B12
[0113] Antibody 21B12 (SEQ ID NOs:17 and 19, FIGS. 4A and 4B) (120
mg/ml) in 20 mM sodium acetate, 220 mM proline, 0.010% polysorbate
80, pH5.0 was desalted in 20 mM acetate buffer pH 5.0 using a
desalting column from Bio-Rad. Antibody 21B12 was crystallized
under a variety of conditions.
[0114] Crystallization of antibody 21B12 was achieved using 3
different crystallization screens (Emerald BioSystems), which
employ a method for crystallization of macromolecules known as
`hanging drop` vapor diffusion. A drop composed of a mixture of the
protein and the crystallization reagent (the "crystallization
buffer" or the "mother liquor" or "crystal growth solution" or the
"reservoir solution") is deposited on the underside of a
siliconized coverslip, and then the drop on the coverslip is placed
over typically a 24 well VDX tray (Hampton Research, Aliso Viejo,
Calif. (HR3-140) after applying oil to well lips for sealing
causing a vapor equilibrium with a liquid reservoir of reagent. To
achieve equilibrium, water vapor exchanges between the drop and a
500-600 .mu.L reservoir solution in the well of the tray. As water
leaves the drop, the protein undergoes an increase in relative
concentration which may eventually lead to supersaturation. It is
the super-saturation of protein that is required for
crystallization to take place. Typically the drop contains a lower
concentration of reagent than the reservoir, and typically, the
drop contained half the concentration of reagent in the reservoir,
because equal volumes of sample and reagent were mixed to form the
drop.
[0115] In these experiments, the initial protein concentration in
the drop was approximately 10 mg/ml. The crystallization screens
were set up in 24-well VDX trays with sealant. Each position in the
VDX tray contained 500-600 .mu.L of reagent reservoir, with the
reagent reservoir in each well differing in composition from that
in the other wells, to establish an array of differing
crystallization buffer conditions. 1-10 .mu.L of protein was added
to 1-10 .mu.l of reservoir solution to form the drops. Trays were
incubated at ambient room temperature.
[0116] Crystallization screens: Antibody 21B12 was screened in a
total of approximately 144 conditions using 3 different
crystallization screens which resulted in 21 crystal hits.
[0117] Antibody 21B12 was screened in Wizard I (Emerald Biosystems
EBS-WIZ-1), Wizard II (Emerald Biosystems EBS-WIZ-2) and Cryo I
(Emerald biosystems EBS-CRYO-1). The primary screen variables are
buffers, salts covering a broad range of crystallization space at
pH4.5 to pH 10.5. A total of 144 conditions were screened and
antibody 21B12 crystallized in the following conditions: Wizard I
#5 (30% PEG400, 0.1M CAPS pH10.5) after 14 days with crystal
morphology of needles; Wizard I #9 (1.0M (NH4)2HPO4, 0.1M Acetate
pH4.5) after 1 day with crystal morphology of hexagonal rods;
Wizard I #10 (20% PEG2K MME, 0.1M Tris pH7) after 1 day with
crystal morphology of tiny needles; Wizard I #13 (1.26 M (NH4)2SO4,
0.1M cacodylate pH6.5) after 14 days with crystal morphology of
hexagonal rods; Wizard I #15 (10% PEG3000, 0.1M imidazole pH8.0,
0.2M Li2SO4) after 14 days with crystal morphology of pointed ovals
(like almond shaped ovals); Wizard I #20 (0.4M/1.6M NaH2/K2HPO4,
0.1M imidazole pH8.0, 0.2M NaCl) after 1 day with crystal
morphology of hexagonal rods; Wizard I #27 (1.2M/0.8M NaH2/K2HPO4,
0.1M CAPS pH10.5, 0.2M Li2SO4) after 14 days with crystal
morphology of long rods; Wizard I #34 (1.0M (NH4)2HPO4, 0.1
imidazole pH 8.0) after 1 day with crystal morphology of hexagonal
rods; Wizard I #46 (10% PEG8K, 0.1M imidazole pH8.0, 0.2M Ca
acetate) after 14 days with crystal morphology of thread like
needles; Wizard I #47 (1.26M (NH4)2SO4, 0.1 Tris pH8.5, 0.2M
Li2SO4) after 1 day with crystal morphology of hexagonal rods;
Wizard II #6 (10% IPA, 0.1M Phosphate citrate pH4.2, 0.2M Li2SO4)
after 22 days with crystal morphology of hexagonal rods; Wizard II
#10 (1.0M (NH4)2HPO4, 0.1M Tris pH8.5) after 1 day with crystal
morphology of hexagonal rods; Wizard II #19 (1.6M/0.4M
NaH2PO4/K2HPO4, 0.1M Phosphate citrate pH4.2) after 1 day with
crystal morphology of hexagonal rods; Wizard II #26 (30% PEG400,
0.1M CHES pH9.5) after 22 days with crystal morphology of thin
needles; Wizard II #31 (1.0M Na citrate, 0.1M Tris pH7.0, 0.2M
NaCl) after 22 days with crystal morphology of hexagonal rods;
Wizard II #33 (1.0M (NH4)2HPO4, 0.1M Citrate pH5.5, 0.2 NaCl) after
1 day with crystal morphology of hexagonal rods; Wizard II #34 (10%
PEG8000, 0.1 imidazole pH8.0) after 22 days with crystal morphology
of tiny needles; Wizard II #37 (1.0M Na/K Tartrate, 0.1M Tris
pH7.0, 0.2M Li2SO4) after 1 day with crystal morphology of
hexagonal rods; Wizard II #39 (20% PEG8000, 0.1M CAPS pH10.5, 0.2M
NaCl) after 22 days with crystal morphology of needles; Wizard II
#46 (1.0M (NH4)2HPO4, 0.1M imidazole pH8.0, 0.2 NaCl) after 1 day
with crystal morphology of hexagonal rods; and Wizard II #48 (1.0M
Na/K Tartrate, 0.1M MES pH6.0) after 1 day with crystal morphology
of hexagonal rods.
[0118] Various morphologies of antibody 21B12 crystals can be grown
under scale-up conditions whereby the antibody in a liquid
formulation is added to a volume of known crystallization reagent
and stored in a sealed container. Antibody 21B12 crystals can be
grown under these conditions in less than 24 hours.
[0119] This Example demonstrates that antibody 21B12 was
crystallizable under a variety of crystallization conditions, but
crystals did not form under every condition tested. Approximately
144 crystallization conditions were tested in a number of different
commercially-available (i.e., Hampton Research, Emerald BioSystems)
and proprietary screens.
Example 2
Antibody 21B12 Crystal Hits Micro Batch Optimization
[0120] Certain conditions that proved successful in generating
Antibody 21B12 crystals as described in Example 1 were selected for
micro batch optimization as follows. Unless noted otherwise, all
conditions used Antibody 21B12 (120 mg/ml) in 10 mM sodium acetate,
220 mM proline, 0.010% polysorbate 80, pH5.0 that was desalted in
20 mM acetate buffer pH5.0 using a desalting column from Bio-Rad.
Antibody 21B12 consisted of two mature heavy chains (SEQ ID NO:19)
and two mature light chains (SEQ ID NO:17) recombinantly produced
by DNA encoding each of these chains was crystallized under a
variety of conditions.
Wizard II #10 (1.0M (NH4)2HPO4, 0.1M Tris pH8.5) at 7-10 mg/ml:
[0121] Antibody, 21B12, was screened in Wizard II #10 (1.0M
(NH4)2HPO4, 0.1M Tris pH8.5) at 7-10 mg/ml and optimized using the
conditions described in Table 2.1 below.
TABLE-US-00001 TABLE 2.1 Antibody (NH.sub.4).sub.2HPO.sub.4 21B12
Batch # [Conc.] Buffer [Conc.] mg/ml Results 838-21-16 0.65M 0.05M
Tris pH8.5 6.8 Some long rods 838-21-17 0.70M 0.05M Tris pH8.5 6.8
More long rods 838-21-18 0.75M 0.05M Tris pH8.5 6.8 Smaller rods
with precipitates 838-21-19 0.80M 0.05M Tris pH8.5 6.8 Smaller rods
with precipitates 838-21-20 0.70M 0.05M Acetate pH4.0 7.1 Few long
rods 838-21-21 0.70M 0.05M Acetate pH5.0 7.1 More long rods
838-21-22 0.70M 0.05M Acetate pH6.0 7.1 Long rods 838-21-23 0.70M
0.05M Tris pH7 7.1 Few long rods 838-21-24 0.70M 0.05M Tris pH8 7.1
Few long rods 838-21-25 0.70M 0.05M Tris pH9 7.1 Few long rods
838-37-1 0.65M 0.05M Tris pH8.5 10.0 One or two rods 838-37-2 0.70M
0.05M Tris pH8.5 10.0 Few rods 838-37-3 0.75M 0.05M Tris pH8.5 10.0
More fat short rods 838-37-4 0.80M 0.05M Tris pH8.5 10.0 More fat
short tiny rods 838-37-5 0.65M 0.05M Acetate pH5.0 10.0 Few flat
big and tiny rods 838-37-6 0.65M 0.05M Acetate pH5.5 10.0 Few flat
big and tiny rods 838-37-7 0.65M 0.05M Acetate pH6.0 10.0 More flat
big and tiny rods 838-37-8 0.65M 0.05M Citrate pH5.0 10.0 More rods
838-37-9 0.65M 0.05M Citrate pH5.5 10.0 More rods nice 838-37-10
0.65M 0.05M Citrate pH6.0 10.0 More rods very nice
[0122] Antibody, 21B12, crystals were observed after 1 day at pH
4-9 with salt concentrations between 0.65M and 0.8M . The crystal
morphology was rods (short and long based on conditions).
Wizard II #33 (1.0M (NH.sub.4).sub.2HPO.sub.4, 0.1M Citrate pH 5.5,
0.2M NaCl):
[0123] For this condition, different (NH.sub.4).sub.2HPO.sub.4,
different NaCl, and different Antibody 21B12 concentrations were
tested. The best condition was 0.6M (NH.sub.4).sub.2HPO.sub.4,
0.05M citrate pH5.5, 0.1M NaCl. Crystals were heterogeneous in size
and (NH.sub.4).sub.2HPO.sub.4 salt crystallized antibody 21B12
without NaCl at lower pH. Optimization conditions for Wizard II #33
are described in Tables 2.2 and 2.3 below
TABLE-US-00002 TABLE 2.2 Antibody (NH.sub.4).sub.2HPO.sub.4 NaCl
21B12 Batch # [Conc.] Buffer [Conc.] [Conc.] mg/ml Results 838-14-5
0.6M 0.05M Citrate pH 5.5 0.1M 17.1 Big crystals 838-14-6 0.8M
0.05M Citrate pH 5.5 0.1M 17.1 Small rods with lots of phase
838-14-7 1.0M 0.05M Citrate pH 5.5 0.1M 17.1 Very few rods,
denatured protein 838-14-8 1.2M 0.05M Citrate pH 5.5 0.1M 17.1 Very
few rods, denatured protein 838-36-22 0.6M 0.05M Citrate pH 5.5
0.1M 13.0 Few crystals not well formed 838-36-23 0.65M 0.05M
Citrate pH 5.5 0.1M 13.0 Few crystals not well formed 838-36-24
0.70M 0.05M Citrate pH 5.5 0.1M 13.0 More crystals, not well formed
838-36-25 0.65M 0.05M Citrate pH 5.5 0.0M 13.0 Few crystals not
well formed 838-36-26 0.65M 0.05M Citrate pH 5.5 0.05M 13.0 Few
crystals not well formed 838-36-27 0.65M 0.05M Citrate pH 5.5 0.15M
13.0 Few crystals not well formed 838-36-28 0.65M 0.05M Citrate pH
5.0 0.1M 13.0 Few crystals not well formed 838-36-29 0.65M 0.05M
Citrate pH 6.0 0.1M 13.0 More crystals not well formed
TABLE-US-00003 TABLE 2.3 Antibody (NH.sub.4).sub.2HPO.sub.4 21B12
Batch # [Conc.] Buffer [Conc.] mg/ml Results 838-50-1 0.65M 0.05M
Citrate pH5.5 10.0 Short hexagonal rods 838-50-2 0.70M 0.05M
Citrate pH5.5 10.0 Short rods with precipitates 838-50-3 0.75M
0.05M Citrate pH5.5 10.0 Fewer rods, more precipitates 838-50-4
0.80M 0.05M Citrate pH5.5 10.0 Fewer rods, more precipitates
838-50-5 0.65M 0.05M Citrate pH6.0 10.0 Short hexagonal rods
838-50-6 0.70M 0.05M Citrate pH6.0 10.0 Fewer rods, more
precipitates 838-50-7 0.75M 0.05M Citrate pH6.0 10.0 Fewer rods,
more precipitates 838-50-8 0.80M 0.05M Citrate pH6.0 10.0
Precipitates 838-50-9 0.65M 0.05M Citrate pH6.0 05.1 Long rods
838-50-10 0.65M 0.05M Citrate pH6.0 07.4 Short rods 838-50-11 0.65M
0.05M Citrate pH6.0 12.5 Short rods with precipitates 838-50-12
0.65M 0.05M Citrate pH6.0 15.1 Short rods with precipitates
Wizard II #48 (1.0M Na/K Tartrate, 0.1M MES pH 6.0):
[0124] For this condition, different Na/K tartrate and different
Antibody 21B12 concentrations were tested. Also different buffers
were tested to replace MES buffer. The best condition was 0.7M Na/K
tartrate, 0.05M acetate, pH6.0. Optimization conditions are
described in table 2.4 below
TABLE-US-00004 TABLE 2.4 Na/K Antibody tartrate 21B12 Batch #
[Conc.] Buffer [Conc.] mg/ml Results 838-21-26 0.65M 0.05M MES pH
6.0 12.8 Rods 838-21-27 0.70M 0.05M MES pH 6.0 12.8 Nice rods
838-21-28 0.75M 0.05M MES pH 6.0 12.8 Short rods with precipitates
838-21-29 0.80M 0.05M MES pH 6.0 12.8 Precipitates 838-37-15 0.70M
0.05M Acetate pH 6.0 12.8 Nice small rods 838-37-16 0.70M 0.05M
Citrate pH 6.0 12.8 Precipitates with little spikes 838-37-17 0.70M
0.05M MES pH 6.0 15.7 Nice rods, longer, thicker 838-37-18 0.70M
0.05M MES pH 6.0 18.5 Nice rods, longer, thicker 838-50-13 0.70M
0.05M Acetate pH 6.0 13.1 Short rods with precipitates 838-50-14
0.73M 0.05M Acetate pH 6.0 13.1 Fewer short rods, more precipitates
838-50-15 0.75M 0.05M Acetate pH 6.0 13.1 Fewer short rods, more
precipitates 838-50-16 0.70M 0.05M Acetate pH 6.0 07.4 Short rods
with precipitates 838-50-17 0.70M 0.05M Acetate pH 6.0 10.0 Short
rods with precipitates 838-50-18 0.70M 0.05M Acetate pH 6.0 15.1
Precipitates 838-50-19 0.70M 0.05M Acetate 5.0 13.1 Rods 838-50-20
0.70M 0.05M Acetate 5.5 13.1 Rods 838-50-21 0.70M 0.05M Acetate 6.5
13.1 Precipitates 838-50-22 0.70M 0.05M Tris 7.0 13.1 Rods with
precipitates
Wizard II #19 (1.6M/0.4M NaH.sub.2PO.sub.4/K.sub.2HPO.sub.4, 0.1M
Phosphate Citrate pH4.2) For this condition different
NaH.sub.2PO.sub.4/K.sub.2HPO.sub.4 buffer concentrations (phosphate
concentrations), different pH and different antibody 21B12
concentrations were tested. The best condition for desalted
antibody 21B12 was 1.4 M phosphate, pH5.3, 13 mg/ml antibody 21B12
resulted in a crystallization yield of greater than 95%. The best
condition for antibody 21B12 in 10 mM Acetate, 220 mM Proline,
0.01% Polysorbate 80, pH 5.0 was 1.3M Phosphate pH 5.3, 64 mg/mL
Antibody 21B12 with yield of 99%. Optimization conditions are
described in Tables 2.5 and 2.6
TABLE-US-00005 TABLE 2.5 Antibody 21B12 Batch # Buffer [Conc.]
mg/ml Results 838-37-11 0.50M Phosphate pH 5.3 10.0 Few
precipitates 838-37-12 0.60M Phosphate pH 5.3 10.0 Few precipitates
838-37-13 0.70M Phosphate pH 5.3 10.0 More shiny precipitates
838-37-14 0.80M Phosphate pH 5.3 10.0 Few long rods 838-50-23 0.9M
Phosphate pH 5.3 13.1 Rods 838-50-24 1.0M Phosphate pH 5.3 13.1
Rods with precipitates 838-50-25 1.1M Phosphate pH 5.3 13.1 Few
rods with more precipitates 838-50-26 1.2M Phosphate pH 5.3 13.1
Precipitates 838-50-27 1.3M Phosphate pH 5.3 13.1 Shiny centered
precipitates 838-50-28 1.4M Phosphate pH 5.3 13.1 Rods 838-61-11
1.35M Phosphate pH 5.3 13.1 Small thick short rods 838-61-12 1.40M
Phosphate pH 5.3 13.1 Small thick short rods with phase 838-61-13
1.45M Phosphate pH 5.3 13.1 Shiny phase separation 838-67-1 1.35M
Phosphate pH 5.3 13.1 Small rods 838-67-2 1.20M Phosphate pH 5.3
20.0 Precipitates 838-67-3 1.30M Phosphate pH 5.3 20.0 Nice clean
small rods 838-67-4 1.40M Phosphate pH 5.3 20.0 Little bigger rods
838-67-5 1.20M Phosphate pH 5.3 25.1 Precipitates 838-67-6 1.30M
Phosphate pH 5.3 25.1 Small rods, clean 838-67-7 1.40M Phosphate pH
5.3 25.1 Little bigger rods 838-67-8 1.20M Phosphate pH 5.3 29.9
Precipitates 838-67-9 1.30M Phosphate pH 5.3 29.9 Small rods
838-67-10 1.40M Phosphate pH 5.3 29.9 Rods with precipitates, black
film 838-67-11 1.20M Phosphate pH 5.3 35.1 Precipitates 838-67-12
1.30M Phosphate pH 5.3 35.1 Very tiny rods 838-67-13 1.40M
Phosphate pH 5.3 35.1 Rods 838-67-14 1.3M Phosphate pH 5.3 13.1
Clean tiny rods 838-67-15 1.4M Phosphate pH 5.3 13.1 Bigger rods
838-67-16 1.3M Phosphate pH 4.5 13.1 Precipitates 838-67-17 1.4M
Phosphate pH 4.5 13.1 Very tiny rods or precipitates 838-67-18 1.3M
Phosphate pH 5.0 13.1 Very tiny rods or precipitates 838-67-19 1.4M
Phosphate pH 5.0 13.1 Small rods clean 838-67-20 1.3M Phosphate pH
5.5 13.1 Nice clean small rods 838-67-21 1.4M Phosphate pH 5.5 13.1
Rods with precipitates, black film 838-67-26 0.4M Phosphate pH 5.3
51.3 Clear 838-67-27 0.8M Phosphate pH 5.3 45.6 Big rods with
precipitates, film 838-67-28 1.2M Phosphate pH 5.3 39.9
Precipitates 838-73-1 0.8M Phosphate pH 5.3 39.9 Gel with few big
flat rods 838-73-2 0.9M Phosphate pH 5.3 39.9 Nice hexagonal rods
838-73-3 1.0M Phosphate pH 5.3 39.9 Gel and precipitates 838-73-4
1.1M Phosphate pH 5.3 39.9 Precipitates
TABLE-US-00006 TABLE 2.6 Antibody 21B12 Batch # Buffer [Conc.]
mg/ml Results 838-67-22 1.35M Phosphate pH 5.3 13.7 Rods with
precipitates, black film 838-67-23 1.40M Phosphate pH 5.3 13.7
Fewer rods with precipitates, black 838-67-24 1.45M Phosphate pH
5.3 13.7 Fewer rods with precipitates, black 838-67-25 1.35M
Phosphate pH 5.3 27.4 Fewer rods with precipitates, black 838-73-5
0.6M Phosphate pH 5.3 30.1 Clear 838-73-6 0.7M Phosphate pH 5.3
30.1 Few black threads 838-73-7 0.8M Phosphate pH 5.3 30.1 Phase
and precipitates 838-73-8 0.9M Phosphate pH 5.3 30.1 Big crystals
with gel and precipitates 838-73-9 1.0M Phosphate pH 5.3 30.1
Crystals, not well formed 838-73-10 0.6M Phosphate pH 5.3 41.0
Clear 838-73-11 0.7M Phosphate pH 5.3 41.0 Phase and precipitates
838-73-12 0.8M Phosphate pH 5.3 41.0 Crystals big and small rods
838-73-13 0.9M Phosphate pH 5.3 41.0 More crystals, big and small
rods 838-73-14 1.0M Phosphate pH 5.3 41.0 Crystals, not well formed
838-73-15 0.8M Phosphate pH 5.3 20.5 Very few big crystals
838-73-16 0.8M Phosphate pH 5.3 27.4 Phase and precipitates
838-73-17 0.8M Phosphate pH 5.3 34.2 More phase and precipitates
838-73-18 0.8M Phosphate pH 5.3 47.9 Shiny precipitates or phase
838-73-19 0.8M Phosphate pH 5.3 54.7 Big crystals with
precipitates, gel 838-73-20 0.8M Phosphate pH 5.3 61.6 Big crystals
with more gel 838-73-21 0.8M Phosphate pH 5.3 91.2 Crystals with
phase separation 838-73-22 0.8M Phosphate pH 5.3 68.4 Flat big rods
with phase 838-73-23 0.8M Phosphate pH 5.3 75.2 Flat big rods with
phase 838-73-24 0.8M Phosphate pH 5.3 82.1 Phase and precipitates
838-73-25 0.8M Phosphate pH 5.3 88.9 Big and small flat crystals,
not good 838-73-26 0.8M Phosphate pH 5.3 95.8 Big and small flat
crystals, not good 838-73-21A 1.0M Phosphate pH 5.3 85.5 Crystals
with phase separation 838-73-21B 1.0M Phosphate pH 5.3 85.5
Crystals with phase separation 838-95-1 1.24M Phosphate pH 5.3 54.7
Crystals with lots of phase 838-95-2 1.28M Phosphate pH 5.3 54.7
More crystals with some phase 838-95-3 1.32M Phosphate pH 5.3 54.7
Crystals with some gel 838-95-4 1.36M Phosphate pH 5.3 54.7
Crystals bigger with some gel 838-95-5 1.30M Phosphate pH 5.3 48.3
Crystals small rods 838-95-6 1.30M Phosphate pH 5.3 61.1 Crystals
rods 838-95-7 1.30M Phosphate pH 5.3 67.6 Crystals bigger rods
838-95-8 1.30M Phosphate pH 5.3 74.0 Crystals 838-95-9 1.3M
Phosphate pH 5.0 54.7 Tiny rods 838-95-10 1.3M Phosphate pH 5.5
54.7 Nice clean rods
Wizard II #34 (10% PEG8000, 0.1M Imidazole pH8.0):
[0125] For this condition different PEG and Antibody 21B12
concentrations were tested with Tris buffer pH8.0. Fewer hair-like,
thin needles were observed in batches. Optimization conditions are
described in Tables 2.7 and 2.8 below
TABLE-US-00007 TABLE 2.7 Antibody 21B12 Batch # PEG [Conc.] Buffer
[Conc.] mg/ml Results 838-37-19 5.5% PEG 8000 0.05M Tris pH 8.0
18.5 Phase, gel, black hair like needles 838-37-20 6.0% PEG 8000
0.05M Tris pH 8.0 18.5 Phase, gel, more hair like needles 838-37-21
6.5% PEG 8000 0.05M Tris pH 8.0 18.5 Phase, gel, more hair like
needles 838-51-1 7.0% PEG 8000 0.05M Tris pH 8.0 18.5 Thin needles
838-51-2 8.0% PEG 8000 0.05M Tris pH 8.0 18.5 Phase separation and
gel 838-51-3 9.0% PEG 8000 0.05M Tris pH 8.0 18.5 Phase, gel, few
hair like needles 838-51-4 7.0% PEG 1000 0.05M Tris pH 8.0 18.5
Little phase 838-51-5 8.0% PEG 1000 0.05M Tris pH 8.0 18.5 Little
phase 838-51-6 9.0% PEG 1000 0.05M Tris pH 8.0 18.5 Little phase
841-38-1 10.0% PEG 8000 0.05M Acetate pH 5.5 71.0 Phase separation,
some crystals 841-38-2 10.0% PEG 8000 0.05M Acetate pH 5.5 58.1
Phase separation, more crystals 841-38-3 10.0% PEG 8000 0.05M
Acetate pH 5.5 45.2 Phase separation, bigger crystals
TABLE-US-00008 TABLE 2.8 Antibody 21B12 Batch # PEG 8000 Acetate pH
5.5 mg/ml Results 841-43-1 10.0% PEG 0.05M Acetate pH 5.5 58.1
phase chunks with some crystals 841-43-2 10.0% PEG 0.05M Acetate pH
5.5 64.5 solid phase 841-43-3 10.0% PEG 0.00M Acetate pH 5.5 58.1
thin black ppts 841-43-4 10.0% PEG 0.02M Acetate pH 5.5 58.1 phase
big broken crystals irregular 841-43-5 10.0% PEG 0.04M Acetate pH
5.5 58.1 phase chunks with more crystals 841-43-6 10.0% PEG 0.06M
Acetate pH 5.5 58.1 phase chunks with bigger crystals 841-43-7
10.0% PEG 0.08M Acetate pH 5.5 58.1 phase chunks, fewer bigger
crystals 841-43-8 10.0% PEG 0.10M Acetate pH 5.5 58.1 phase chunks,
fewer smaller crystals 841-43-9 05.0% PEG 0.05M Acetate pH 5.5 58.1
phase chunks, bigger crystals 841-43-10 07.5% PEG 0.05M Acetate pH
5.5 58.1 Solid phase 841-43-11 10.0% PEG 0.05M Acetate pH 5.5 58.1
Solid phase with film 841-43-12 05.0% PEG 0.05M Acetate pH 6.0 58.1
phase with big broken crystals, gel 841-43-13 07.5% PEG 0.05M
Acetate pH 6.0 58.1 phase chunks, big and small crystals 841-43-14
10.0% PEG 0.05M Acetate pH 6.0 58.1 phase with film 841-43-15 05.0%
PEG 0.05M Tris pH 7.0 58.1 phase and gel with broken crystals
841-43-16 07.5% PEG 0.05M Tris pH 7.0 58.1 phase and gel with big
crystals 841-43-17 10.0% PEG 0.05M Tris pH 7.0 58.1 Solid phase
with film 841-43-18 05.0% PEG 0.05M Tris pH 8.0 58.1 phase and gel,
few thread like crystals 841-43-19 07.5% PEG 0.05M Tris pH 8.0 58.1
Gel, more needle like crystals 841-43-20 10.0% PEG 0.05M Tris pH
8.0 58.1 phase with film
Example 3
1 ml Batch Crystallization and Phase Diagram
[0126] Antibody 21B12 (SEQ ID NOs:17 and 19, FIGS. 4A and 4B), in
20 mM Acetate, 220 mM Proline, 0.01% polysorbate 80, pH 5.0 was
batch crystallized in 1.5 ml centrifuge tubes with a final volume
of 1.0 ml. Crystallization condition for Antibody 21B12 was 1.3M
Phosphate pH 5.3, 64 mg/mL Antibody 21B12 with yield of 99%.
Antibody 21B12 that was desalted in 20 mM Acetate pH 5.0 was batch
crystallized in 1.5 ml centrifuge tube with a final volume of lml
with final concentration of 1.4M Phosphate pH 5.3, with 13 mg/mL
antibody 21B12 with yield of 97%
[0127] Batches were made by adding phosphate and antibody 21B12 in
1.5 mL micro centrifuge tubes and quick vortexing to mix (Few
seconds until mixture looked homogenously mixed) at setting 3000 on
fisher digital mixer. Higher concentration batches took few more
seconds to mix then lower antibody 21B12 concentration batches
(lower concentration took about 5 seconds for higher concentration
about 8 seconds). After this initial mixing, batches were incubated
at room temperature in static condition overnight or for two
days.
[0128] Phase diagram was determined for this crystallization
condition at lml scale to see effect of change in crystallization
parameters such as phosphate pH, phosphate concentration, antibody
21B12 concentration and temperature.
Phosphate pH Screen:
[0129] Crystals formation was faster at pH 4.2 with sharps edges
compared other pHs. Therefore this pH was chosen to test the
phosphate concentration. Phosphate buffer stock solutions were made
at 3.5M concentration in the pH range from 3.7 to 4.7 in increments
of 0.2 pH units. Batches were made to provide a final antibody
21B12 concentration of 82.8 mg/mL with a final phosphate
concentration of 1.3M phosphate at different test pH's. Phosphate
was added in three parts with 30 minute incubation between
additions. The first 0.9M was added all at once and then remaining
volume to make total phosphate concentration to 1.3M was added in
two parts of 0.2 M. Batches were made in 2 mL centrifuge tubes,
mixed on vortex mixer at every addition of phosphate for
.about.10-15 seconds at 3000 rpm and incubated at room temperature
in static condition. Yield and crystal size were measured between
3.0-3.5 hours. A summary of the experiments are shown in Table 3.1
below.
TABLE-US-00009 TABLE 3.1 Phosphate concentration Final mAb YIELD
CRYSTAL SIZE BATCH # [M] (mg/mL) pH TEMPERATURE (%) (.mu.M)
RP1120-01 1.3 82.8 3.7 Room 97.9 No crystals, gel, temperature
phase RP1120-02 1.3 82.8 3.9 Room 95.8 Very few crystals,
temperature gel, phase RP1120-03 1.3 82.8 4.1 Room 96.3 ~10
temperature RP1120-04 1.3 82.8 4.3 Room 97.8 ~5 temperature
RP1120-05 1.3 82.8 4.5 Room 98.4 ~5 temperature RP1120-06 1.3 82.8
4.7 Room 98.8 ~7 temperature RP1120-07 1.3 82.8 4.2 Room 97.9 ~7
temperature
Phosphate Concentration Screen:
[0130] Phosphate buffer stock solution was made at 3.5M
concentration with pH 4.2. Phosphate concentrations from 0.5M to
1.5M were tested with 0.2M increments. Batches were made in 2 mL
centrifuge tubes to provide a final Antibody 21B12 concentration of
75 mg/mL with a final phosphate concentration from 0.5 to 1.5M at
pH 4.2. With increase in phosphate volume to reach to final
concentration of phosphate to 1.5M the antibody 21B12 gets diluted
to 75 mg/mL. Phosphate was added to the antibody 21B12 all at once
for 0.5M to 1.5M batches and vortex mixed for .about.10 seconds at
highest speed setting and kept static at room temperature. Yield
and crystal sizes were measured between 3.0-3.5 hours. For final
phosphate of 1.3 and 1.5M concentrations in addition to at once
addition, 2 stepwise additions for 1.3M (0.9M+0.4M) and 2stepwise
additions (0.9M+0.6M) and 3 stepwise additions (0.9+0.3M+0.3M) for
1.5M phosphate were performed with 30 min intervals for each
addition to check the tolerability of phosphate at each addition. A
summary of the experiments are shown in Table 3.2. Phosphate
addition at once to from 1.1 to 1.5M was not tolerated and resulted
in gel formation. Therefore, the following experiments stepwise
addition of phosphate was chosen for crystallization yield
improving.
TABLE-US-00010 TABLE 3.2 Phosphate concentration Final mAb YIELD
CRYSTAL SIZE BATCH # [M] (mg/mL) pH TEMPERATURE (%) (.mu.M)
SM1114-4 0.5 75 4.2 Room 0 No crystals Clear temperature SM1114-5
0.7 75 4.2 Room 40 >20 .mu.m temperature SM1114-6 0.9 75 4.2
Room 78 ~10 .mu.m temperature SM1114-7 1.1 75 4.2 Room 92 <5
.mu.m with some temperature gel SM1114-8 1.3 75 4.2 Room 98 <5
.mu.m with some temperature gel SM1114-9 1.5 75 4.2 Room 99 No
crystals, gel temperature SM1114-10 1.5 75 4.2 Room 99 ~10 .mu.m
temperature SM1114-11 1.5 75 4.2 Room 99 ~10 .mu.m temperature
SM1114-12 1.3 75 4.2 Room 96 ~10 .mu.m
Antibody 21B12 Concentration Screen:
[0131] Crystallization batches were made at 1 mL scale at final
antibody 21B12 concentrations of 60, 70 and 80 mg/mL. Batches were
made with 1.3M phosphate concentration at a pH 4.2 in 2M1
centrifuge tubes. Phosphate was added in three parts with 30 minute
incubation between additions. The first 0.9M was added all at once
and then remaining volume to make total phosphate concentration to
1.3M was added in two parts of 0.2 M. Batches were mixed on vortex
mixer at every addition of phosphate for 10-15 seconds at highest
speed setting and incubated at room temperature. Yield and crystal
size was tested around 3.5 hours. A summary of the experiments are
shown in Table 3.3. A protein concentration of 80 mg/m1 gave good
quality crystals and good yield as 60 mg/m1 protein batch, hence,
approximately 80 mg/m1 protein was selected in the further
optimization experiments.
TABLE-US-00011 TABLE 3.3 Phosphate concentration Final mAb YIELD
CRYSTAL SIZE BATCH # [M] (mg/mL) pH TEMPERATURE (%) (.mu.M)
SM1114-1 1.3 60 4.2 Room 97 ~5-10 temperature SM1114-2 1.3 70 4.2
Room 98 ~5-10 temperature SM1114-3 1.3 80 4.2 Room 98 ~10
temperature
Temperature Screen:
[0132] Crystallization batches were performed at 1 mL scale at
different temperatures ranging from 15.degree. C. to 25.degree. C.
in 2.degree. C. increments. Batches were made with 1.3M phosphate
concentration at a pH 4.2 to provide a final antibody 21B12
concentration of 82.8 mg/mL. Batches were made in 2 mL centrifuge
tubes with antibody 21B12 and reagents equilibrated to the testing
temperature. Phosphate was added in three parts with 30 minute
incubation between additions. The first 0.9M was added all at once
and then remaining volume to make total phosphate concentration to
1.3M was added in two parts of 0.2 M. Batches were mixed on vortex
mixer at every phosphate addition for approximately 10-15 seconds
at highest speed setting and incubated in temperature controlled
water bath. Yield and crystal size were tested at approximately 3.5
hours. A summary of the experiments are shown in Table 3.4.
Crystallization at higher from 19-25.degree. C. produced high
quality crystals with increase in temperature.
TABLE-US-00012 TABLE 3.4 Phosphate concentration Final mAb
TEMPERATURE YIELD CRYSTAL SIZE BATCH # [M] (mg/mL) pH (.degree. C.)
(%) (.mu.M) SM1120-2 1.3 82.8 4.2 15 97 <5 SM1121-1 1.3 82.8 4.2
17 98 <5 SM1122-1 1.3 82.8 4.2 19 98 ~5 SM1122-3 1.3 82.8 4.2 21
98 ~5 SM1125-1 1.3 82.8 4.2 23 98 ~5 SM1125-3 1.3 82.8 4.2 25 98
~5
Example 4
Batch Optimization at 10 ml Scale
[0133] Batches were made at 10 mL scale for further confirmation of
phase diagram that was generated at 1 mL scale as in Example 3. In
the 10 mL experiments the data was generated with continuous mixing
as this parameter could not be investigated at the 1 mL scale.
[0134] Crystallization at 10 mL scale was performed in round bottom
glass tubes and overhead continuous mixing using pitched blade
propeller size A511 and digital stirrer. Mixing speed was set to
500 rpm for 10 mL batches. Phosphate was pumped in to the Antibody
21B12 solution using peristaltic pump at controlled flow rate using
a two steps addition process. First, 2.18 mL of 3.5M phosphate (to
bring the total phosphate concentration to 0.9M) was added at 0.4
mL/min flow rate and after 1 hour incubation remaining phosphate
(1.56 mL) was added at 0.2 mL/min flow rate (to bring the total
phosphate concentration to 1.3M). Crystallization was carried out
for total of 3 hours and then crystal size and yield were measured.
Under the continuous mixing conditions parameters like reagent pH,
reagent concentration and crystallization temperature were
reevaluated at 10 ml scale.
Phosphate pH:
[0135] Phosphate solutions at pH from 4.1 to 4.7 in increments of
0.2 pH units were screened. A summary of the experiments are shown
in Table 4.1 below. Batches were made to provide a final antibody
21B12 concentration of approximately 80 mg/mL and 1.3M phosphate at
room temperature. Results were summarized in Table 4.1. Reagent pH
4.4 was selected to be in the middle of the range of
crystallization pH for next parameter testing.
TABLE-US-00013 TABLE 4.1 Final antibody Phosphate 21B12 BATCH #
concentration mg/mL PH TEMPERATURE YIELD CRYSTAL SIZE 1205-4.1 1.3M
80.8 mg/mL 4.1 22.4.degree. C. 96.86% ~10 .mu.m 23.6.degree. C.
1205-4.3 1.3M 80.8 mg/mL 4.3 22.2.degree. C. 97.20% ~5 .mu.m
23.5.degree. C. 1205-4.5 1.3M 80.8 mg/mL 4.5 23.5.degree. C. 97.30%
~5 .mu.m 23.8.degree. C. 1205-4.7 1.3M 80.8 mg/mL 4.7 23.6.degree.
C. 97.30% ~10 .mu.m 23.8.degree. C.
Phosphate Concentrations:
[0136] Phosphate concentrations at 1.0M, 1.2M, 1.4M and 1.5M were
tested at a pH of 4.4. A summary of the experiments are shown in
Table 4.2 below. Batches were made to provide a final antibody
21B12 concentration of approximately 80 mg/mL at room temperature
with 1.0M, 1.2M and 1.3M phosphate. For 1.4M and 1.5M phosphate,
antibody 21B12 concentrations were reduced to approximately 77
mg/mL and approximately 73 mg/mL respectively as more stock
solution was needed to achieve the desired phosphate
concentrations. Resulted were summarized in Table 4.2. Phosphate
concentration of 1.5M had relatively easy flow of crystal
suspension for a naked eye observation compared to lower phosphate
concentrations. Therefore, phosphate 1.5M was selected with pH 4.4
to optimize the next parameter.
TABLE-US-00014 TABLE 4.2 Final antibody Phosphate 21B12 BATCH #
concentration mg/mL PH TEMPERATURE YIELD CRYSTAL SIZE 1206-1.0M
1.0M 80.8 mg/mL 4.4 23.2.degree. C. 87.70% ~5 .mu.m 24.7.degree. C.
1206-1.2M 1.2M 80.8 mg/mL 4.4 23.2.degree. C. 95.80% ~5 .mu.m
24.4.degree. C. 1206-1.4M 1.4M 77.4 mg/mL 4.4 24.6.degree. C.
97.90% ~10 .mu.m 25.3.degree. C. 1206-1.5M 1.5M 73.8 mg/mL 4.4
24.3.degree. C. 99.20% ~10 .mu.m 25.3.degree. C.
Temperature: Temperatures at 21, 23, 25, 27, 30 and 35.degree. C.
were tested at 1.5M phosphate concentration at a pH of 4.4. A
summary of the experiments including results are shown in Table 4.3
below. Batches were made to provide a final antibody 21B12
concentration approximately 73 mg/mL.
TABLE-US-00015 TABLE 4.3 Final antibody Phosphate 21B12 BATCH #
concentration mg/mL PH TEMPERATURE YIELD CRYSTAL SIZE
1211-21.degree. C. 1.5M 73.8 mg/mL 4.4 21.degree. C. 99.38% ~2-5
.mu.m 1211-23.degree. C. 1.5M 73.8 mg/mL 4.4 23.degree. C. 99.40%
~2-5 .mu.m 1211-25.degree. C. 1.5M 73.8 mg/mL 4.4 25.degree. C.
99.10% ~10 .mu.m 1212-27.degree. C. 1.5M 73.8 mg/mL 4.4 27.degree.
C. 99.60% ~10 .mu.m 1212-30.degree. C. 1.5M 73.8 mg/mL 4.4
30.degree. C. 99.40% ~10 .mu.m 1213-35.degree. C. 1.5M 73.8 mg/mL
4.4 35.degree. C. 99.70% ~13 .mu.m
Example 5
20 ml Batch Crystallization and % Yield
[0137] Antibody 21B12 (SEQ ID NOs:17 and 19, FIGS. 4A and 4B), was
crystallized using a phosphate buffer having a final phosphate
concentration of 1.5 M at pH 4.4 and a final 21B12 Antibody
concentration of 73.8 mg/mL.
[0138] Effect of mixing speed was studied at 20 mL scale. Batches
were made with 500 rpm mixing and 800 rpm mixing side by side
keeping the same flow rate of phosphate addition for both mixing
speeds. In the first set of experiments, phosphate addition for the
first step was tested at 1.0, 2.0 and 3.0 mL/min and the rate of
phosphate addition in second step was fixed at 0.2 mL/min. In
second set of experiments, rate of first step phosphate addition
was fixed at 0.8 mL/min and rate of second step phosphate addition
was varied at 0.5, 1.0 and 2.0 mL/min. Experiments were set up
using conditions similar to the 10 mL scale batches described in
Example 4. Round bottom glass tubes with pitched blade propeller
size A511 and with overhead stirring were setup at room
temperature. The crystallization resulted in a final Antibody 21B12
concentration of approximately 73 mg/mL and 1.5M phosphate at pH of
4.4. Crystal size and yield were measured at 3 hours. The crystal
morphology was short hexagonal rods. Yields and crystal sizes were
approximately 5-10 nm and were measured 3.0 hours after start of
experiment. Crystal sizes were measured microscopically using image
pro software. A summary of the experiments, including results, are
shown in Tables 5.1 and 5.2 below.
TABLE-US-00016 TABLE 5.1 FIRST SECOND ADDITION ADDITION BATCH #
RATE RATE MIXING SPEED YIELD CRYSTAL SIZE 1206-1.0 mL-500 1.0
mL/min 0.2 mL/min 500 rpm 99.49% ~5 .mu.m 1206-1.0 mL-800 1.0
mL/min 0.2 mL/min 800 rpm 99.28% ~10 .mu.m 1209-2.0 mL-500 2.0
mL/min 0.2 mL/min 500 rpm 99.34% ~10 .mu.m 1209-2.0 mL-800 2.0
mL/min 0.2 mL/min 800 rpm 99.49% ~10 .mu.m 1209-3.0 mL-500 3.0
mL/min 0.2 mL/min 500 rpm 98.20% ~10 .mu.m 1209-3.0 mL-800 3.0
mL/min 0.2 mL/min 800 rpm 99.25% ~10 .mu.m
TABLE-US-00017 TABLE 5.2 FIRST SECOND ADDITION ADDITION BATCH #
RATE RATE MIXING SPEED YIELD CRYSTAL SIZE 1210-0.5 mL-500 0.8
mL/min 0.5 mL/min 500 rpm 99.20% ~5 .mu.m 1210-0.5 mL-800 0.8
mL/min 0.5 mL/min 800 rpm 99.35% ~5 .mu.m 1210-1.0 mL-500 0.8
mL/min 1.0 mL/min 500 rpm 98.96% ~5 .mu.m 1210-1.0 mL-800 0.8
mL/min 1.0 mL/min 800 rpm 99.00% ~5 .mu.m 1210-2.0 mL-500 0.8
mL/min 2.0 mL/min 500 rpm 99.30% ~5 .mu.m 1210-2.0 mL-800 0.8
mL/min 2.0 mL/min 800 rpm 99.32% ~5-10 .mu.m
Example 6
50 ml Batch Crystallization and % Yield
[0139] Certain conditions that proved successful in generating
Antibody 21B12 crystals as described in Example 5 were selected for
optimization as follows:
[0140] Crystallization was set up in a round bottom polycarbonate
tube (Nalgene centrifuge bottle with top cut off, diameter 5.8 cm)
and pitched blade propeller size A521. Two batches were made, one
with 500 rpm mixing and another with 700 rpm mixing. Phosphate
addition rate was 3 mL/min for the first step addition of phosphate
to reach to 0.9M of phosphate in the total crystallization volume.
After 1 hour of incubation, second addition of phosphate was done
at 1.0 mL/min to obtain a final phosphate concentration of 1.5M.
The final concentration of antibody 21B12 was approximately 73
mg/mL at pH of 4.4. Crystal size was approximately 5 .mu.m and
shape was hexagonal short rods. A summary of the experiments are
shown in Table 6.1 below. Crystallization with 500 rpm mixing speed
was not mixing uniformly after the first addition of reagent around
lhr from start of the experiment; but, the experiment with 700 rpm
mixing the suspension uniformly though out the crystallization.
After second addition both crystallization experiments at 500 rpm
and 700 rpm were mixing uniformly and resulted in similar type of
crystals quality.
TABLE-US-00018 TABLE 6.1 FIRST SECOND ADDITION ADDITION BATCH #
RATE RATE MIXING SPEED YIELD CRYSTAL SIZE 1211-50 mL-1 3.0 mL/min
1.0 mL/min 500 rpm 99.40% ~5 .mu.m 1211-50 mL-2 3.0 mL/min 1.0
mL/min 700 rpm 99.60% ~5 .mu.m
Example 7
100 ml Batch Crystallization and % Yield
[0141] Certain conditions that proved successful in generating
Antibody 21B12 crystals as described in Example 6 were selected for
optimization as follows:
[0142] Crystallization of 100 mL batches were conducted at
temperatures close to room temperatures 21 and 25.degree. C.
Protein solution was taken into a round bottom polycarbonate tube
(Nalgene centrifuge bottle with top cut off, diameter 5.8 cm) and
mixed with a propeller size A521 at 800 rpm. Phosphate addition
rate was 3 mL/min in first step of addition to reach to 0.9M
phosphate in the total crystallization volume. After lhour of
incubation, second addition of phosphate was done at 1.0 mL/min to
obtain a final phosphate concentration of 1.5M. The final
concentration of antibody 21B12 was approximately 73 mg/mL at pH of
4.4. The crystals size and yield were monitored at 15, 30, 60, 90
and 180 min from the start of phosphate addition. Crystals sizes
varied from approximately 2-5 .mu.m. Initially smaller crystals
were more and grew bigger to uniform size with time. Crystals shape
was hexagonal short rods. Yields were improved with time as well
from 74% (15 min) to 99.5% (90 min) and stayed same at 180 min.
[0143] For 100 mL batch, crystallization was carried out at
35.degree. C. by following the phosphate addition and mixing speeds
as above. The experiment did not produce crystals and resulted in
the formation of big chunks of gel formation. A summary of the
experiments, including the results, are shown in Table 7.1
below.
TABLE-US-00019 TABLE 7.1 FIRST SECOND ADDITION ADDITION BATCH #
RATE RATE MIXING SPEED YIELD CRYSTAL SIZE 1212-100 mL-1 3.0 mL/min
1.0 mL/min 800 rpm 99.55% ~2-5 .mu.m 1213-100 mL-1 3.0 mL/min 1.0
mL/min 800 rpm 99.50% ~2-5 .mu.m
Sequence CWU 1
1
261662PRTHomo sapiens 1Gln Glu Asp Glu Asp Gly Asp Tyr Glu Glu Leu
Val Leu Ala Leu Arg 1 5 10 15 Ser Glu Glu Asp Gly Leu Ala Glu Ala
Pro Glu His Gly Thr Thr Ala 20 25 30 Thr Phe His Arg Cys Ala Lys
Asp Pro Trp Arg Leu Pro Gly Thr Tyr 35 40 45 Val Val Val Leu Lys
Glu Glu Thr His Leu Ser Gln Ser Glu Arg Thr 50 55 60 Ala Arg Arg
Leu Gln Ala Gln Ala Ala Arg Arg Gly Tyr Leu Thr Lys 65 70 75 80 Ile
Leu His Val Phe His Gly Leu Leu Pro Gly Phe Leu Val Lys Met 85 90
95 Ser Gly Asp Leu Leu Glu Leu Ala Leu Lys Leu Pro His Val Asp Tyr
100 105 110 Ile Glu Glu Asp Ser Ser Val Phe Ala Gln Ser Ile Pro Trp
Asn Leu 115 120 125 Glu Arg Ile Thr Pro Pro Arg Tyr Arg Ala Asp Glu
Tyr Gln Pro Pro 130 135 140 Asp Gly Gly Ser Leu Val Glu Val Tyr Leu
Leu Asp Thr Ser Ile Gln 145 150 155 160 Ser Asp His Arg Glu Ile Glu
Gly Arg Val Met Val Thr Asp Phe Glu 165 170 175 Asn Val Pro Glu Glu
Asp Gly Thr Arg Phe His Arg Gln Ala Ser Lys 180 185 190 Cys Asp Ser
His Gly Thr His Leu Ala Gly Val Val Ser Gly Arg Asp 195 200 205 Ala
Gly Val Ala Lys Gly Ala Ser Met Arg Ser Leu Arg Val Leu Asn 210 215
220 Cys Gln Gly Lys Gly Thr Val Ser Gly Thr Leu Ile Gly Leu Glu Phe
225 230 235 240 Ile Arg Lys Ser Gln Leu Val Gln Pro Val Gly Pro Leu
Val Val Leu 245 250 255 Leu Pro Leu Ala Gly Gly Tyr Ser Arg Val Leu
Asn Ala Ala Cys Gln 260 265 270 Arg Leu Ala Arg Ala Gly Val Val Leu
Val Thr Ala Ala Gly Asn Phe 275 280 285 Arg Asp Asp Ala Cys Leu Tyr
Ser Pro Ala Ser Ala Pro Glu Val Ile 290 295 300 Thr Val Gly Ala Thr
Asn Ala Gln Asp Gln Pro Val Thr Leu Gly Thr 305 310 315 320 Leu Gly
Thr Asn Phe Gly Arg Cys Val Asp Leu Phe Ala Pro Gly Glu 325 330 335
Asp Ile Ile Gly Ala Ser Ser Asp Cys Ser Thr Cys Phe Val Ser Gln 340
345 350 Ser Gly Thr Ser Gln Ala Ala Ala His Val Ala Gly Ile Ala Ala
Met 355 360 365 Met Leu Ser Ala Glu Pro Glu Leu Thr Leu Ala Glu Leu
Arg Gln Arg 370 375 380 Leu Ile His Phe Ser Ala Lys Asp Val Ile Asn
Glu Ala Trp Phe Pro 385 390 395 400 Glu Asp Gln Arg Val Leu Thr Pro
Asn Leu Val Ala Ala Leu Pro Pro 405 410 415 Ser Thr His Gly Ala Gly
Trp Gln Leu Phe Cys Arg Thr Val Trp Ser 420 425 430 Ala His Ser Gly
Pro Thr Arg Met Ala Thr Ala Ile Ala Arg Cys Ala 435 440 445 Pro Asp
Glu Glu Leu Leu Ser Cys Ser Ser Phe Ser Arg Ser Gly Lys 450 455 460
Arg Arg Gly Glu Arg Met Glu Ala Gln Gly Gly Lys Leu Val Cys Arg 465
470 475 480 Ala His Asn Ala Phe Gly Gly Glu Gly Val Tyr Ala Ile Ala
Arg Cys 485 490 495 Cys Leu Leu Pro Gln Ala Asn Cys Ser Val His Thr
Ala Pro Pro Ala 500 505 510 Glu Ala Ser Met Gly Thr Arg Val His Cys
His Gln Gln Gly His Val 515 520 525 Leu Thr Gly Cys Ser Ser His Trp
Glu Val Glu Asp Leu Gly Thr His 530 535 540 Lys Pro Pro Val Leu Arg
Pro Arg Gly Gln Pro Asn Gln Cys Val Gly 545 550 555 560 His Arg Glu
Ala Ser Ile His Ala Ser Cys Cys His Ala Pro Gly Leu 565 570 575 Glu
Cys Lys Val Lys Glu His Gly Ile Pro Ala Pro Gln Gly Gln Val 580 585
590 Thr Val Ala Cys Glu Glu Gly Trp Thr Leu Thr Gly Cys Ser Ala Leu
595 600 605 Pro Gly Thr Ser His Val Leu Gly Ala Tyr Ala Val Asp Asn
Thr Cys 610 615 620 Val Val Arg Ser Arg Asp Val Ser Thr Thr Gly Ser
Thr Ser Glu Glu 625 630 635 640 Ala Val Thr Ala Val Ala Ile Cys Cys
Arg Ser Arg His Leu Ala Gln 645 650 655 Ala Ser Gln Glu Leu Gln 660
2 2076DNAHomo sapiens 2atgggcaccg tcagctccag gcggtcctgg tggccgctgc
cactgctgct gctgctgctg 60ctgctcctgg gtcccgcggg cgcccgtgcg caggaggacg
aggacggcga ctacgaggag 120ctggtgctag ccttgcgctc cgaggaggac
ggcctggccg aagcacccga gcacggaacc 180acagccacct tccaccgctg
cgccaaggat ccgtggaggt tgcctggcac ctacgtggtg 240gtgctgaagg
aggagaccca cctctcgcag tcagagcgca ctgcccgccg cctgcaggcc
300caggctgccc gccggggata cctcaccaag atcctgcatg tcttccatgg
ccttcttcct 360ggcttcctgg tgaagatgag tggcgacctg ctggagctgg
ccttgaagtt gccccatgtc 420gactacatcg aggaggactc ctctgtcttt
gcccagagca tcccgtggaa cctggagcgg 480attacccctc cgcggtaccg
ggcggatgaa taccagcccc ccgacggagg cagcctggtg 540gaggtgtatc
tcctagacac cagcatacag agtgaccacc gggaaatcga gggcagggtc
600atggtcaccg acttcgagaa tgtgcccgag gaggacggga cccgcttcca
cagacaggcc 660agcaagtgtg acagtcatgg cacccacctg gcaggggtgg
tcagcggccg ggatgccggc 720gtggccaagg gtgccagcat gcgcagcctg
cgcgtgctca actgccaagg gaagggcacg 780gttagcggca ccctcatagg
cctggagttt attcggaaaa gccagctggt ccagcctgtg 840gggccactgg
tggtgctgct gcccctggcg ggtgggtaca gccgcgtcct caacgccgcc
900tgccagcgcc tggcgagggc tggggtcgtg ctggtcaccg ctgccggcaa
cttccgggac 960gatgcctgcc tctactcccc agcctcagct cccgaggtca
tcacagttgg ggccaccaat 1020gcccaggacc agccggtgac cctggggact
ttggggacca actttggccg ctgtgtggac 1080ctctttgccc caggggagga
catcattggt gcctccagcg actgcagcac ctgctttgtg 1140tcacagagtg
ggacatcaca ggctgctgcc cacgtggctg gcattgcagc catgatgctg
1200tctgccgagc cggagctcac cctggccgag ttgaggcaga gactgatcca
cttctctgcc 1260aaagatgtca tcaatgaggc ctggttccct gaggaccagc
gggtactgac ccccaacctg 1320gtggccgccc tgccccccag cacccatggg
gcaggttggc agctgttttg caggactgtg 1380tggtcagcac actcggggcc
tacacggatg gccacagcca tcgcccgctg cgccccagat 1440gaggagctgc
tgagctgctc cagtttctcc aggagtggga agcggcgggg cgagcgcatg
1500gaggcccaag ggggcaagct ggtctgccgg gcccacaacg cttttggggg
tgagggtgtc 1560tacgccattg ccaggtgctg cctgctaccc caggccaact
gcagcgtcca cacagctcca 1620ccagctgagg ccagcatggg gacccgtgtc
cactgccacc aacagggcca cgtcctcaca 1680ggctgcagct cccactggga
ggtggaggac cttggcaccc acaagccgcc tgtgctgagg 1740ccacgaggtc
agcccaacca gtgcgtgggc cacagggagg ccagcatcca cgcttcctgc
1800tgccatgccc caggtctgga atgcaaagtc aaggagcatg gaatcccggc
ccctcagggg 1860caggtgaccg tggcctgcga ggagggctgg accctgactg
gctgcagcgc cctccctggg 1920acctcccacg tcctgggggc ctacgccgta
gacaacacgt gtgtagtcag gagccgggac 1980gtcagcacta caggcagcac
cagcgaagag gccgtgacag ccgttgccat ctgctgccgg 2040agccggcacc
tggcgcaggc ctcccaggag ctccag 20763692PRTHomo sapiens 3Met Gly Thr
Val Ser Ser Arg Arg Ser Trp Trp Pro Leu Pro Leu Leu 1 5 10 15 Leu
Leu Leu Leu Leu Leu Leu Gly Pro Ala Gly Ala Arg Ala Gln Glu 20 25
30 Asp Glu Asp Gly Asp Tyr Glu Glu Leu Val Leu Ala Leu Arg Ser Glu
35 40 45 Glu Asp Gly Leu Ala Glu Ala Pro Glu His Gly Thr Thr Ala
Thr Phe 50 55 60 His Arg Cys Ala Lys Asp Pro Trp Arg Leu Pro Gly
Thr Tyr Val Val 65 70 75 80 Val Leu Lys Glu Glu Thr His Leu Ser Gln
Ser Glu Arg Thr Ala Arg 85 90 95 Arg Leu Gln Ala Gln Ala Ala Arg
Arg Gly Tyr Leu Thr Lys Ile Leu 100 105 110 His Val Phe His Gly Leu
Leu Pro Gly Phe Leu Val Lys Met Ser Gly 115 120 125 Asp Leu Leu Glu
Leu Ala Leu Lys Leu Pro His Val Asp Tyr Ile Glu 130 135 140 Glu Asp
Ser Ser Val Phe Ala Gln Ser Ile Pro Trp Asn Leu Glu Arg 145 150 155
160 Ile Thr Pro Pro Arg Tyr Arg Ala Asp Glu Tyr Gln Pro Pro Asp Gly
165 170 175 Gly Ser Leu Val Glu Val Tyr Leu Leu Asp Thr Ser Ile Gln
Ser Asp 180 185 190 His Arg Glu Ile Glu Gly Arg Val Met Val Thr Asp
Phe Glu Asn Val 195 200 205 Pro Glu Glu Asp Gly Thr Arg Phe His Arg
Gln Ala Ser Lys Cys Asp 210 215 220 Ser His Gly Thr His Leu Ala Gly
Val Val Ser Gly Arg Asp Ala Gly 225 230 235 240 Val Ala Lys Gly Ala
Ser Met Arg Ser Leu Arg Val Leu Asn Cys Gln 245 250 255 Gly Lys Gly
Thr Val Ser Gly Thr Leu Ile Gly Leu Glu Phe Ile Arg 260 265 270 Lys
Ser Gln Leu Val Gln Pro Val Gly Pro Leu Val Val Leu Leu Pro 275 280
285 Leu Ala Gly Gly Tyr Ser Arg Val Leu Asn Ala Ala Cys Gln Arg Leu
290 295 300 Ala Arg Ala Gly Val Val Leu Val Thr Ala Ala Gly Asn Phe
Arg Asp 305 310 315 320 Asp Ala Cys Leu Tyr Ser Pro Ala Ser Ala Pro
Glu Val Ile Thr Val 325 330 335 Gly Ala Thr Asn Ala Gln Asp Gln Pro
Val Thr Leu Gly Thr Leu Gly 340 345 350 Thr Asn Phe Gly Arg Cys Val
Asp Leu Phe Ala Pro Gly Glu Asp Ile 355 360 365 Ile Gly Ala Ser Ser
Asp Cys Ser Thr Cys Phe Val Ser Gln Ser Gly 370 375 380 Thr Ser Gln
Ala Ala Ala His Val Ala Gly Ile Ala Ala Met Met Leu 385 390 395 400
Ser Ala Glu Pro Glu Leu Thr Leu Ala Glu Leu Arg Gln Arg Leu Ile 405
410 415 His Phe Ser Ala Lys Asp Val Ile Asn Glu Ala Trp Phe Pro Glu
Asp 420 425 430 Gln Arg Val Leu Thr Pro Asn Leu Val Ala Ala Leu Pro
Pro Ser Thr 435 440 445 His Gly Ala Gly Trp Gln Leu Phe Cys Arg Thr
Val Trp Ser Ala His 450 455 460 Ser Gly Pro Thr Arg Met Ala Thr Ala
Ile Ala Arg Cys Ala Pro Asp 465 470 475 480 Glu Glu Leu Leu Ser Cys
Ser Ser Phe Ser Arg Ser Gly Lys Arg Arg 485 490 495 Gly Glu Arg Met
Glu Ala Gln Gly Gly Lys Leu Val Cys Arg Ala His 500 505 510 Asn Ala
Phe Gly Gly Glu Gly Val Tyr Ala Ile Ala Arg Cys Cys Leu 515 520 525
Leu Pro Gln Ala Asn Cys Ser Val His Thr Ala Pro Pro Ala Glu Ala 530
535 540 Ser Met Gly Thr Arg Val His Cys His Gln Gln Gly His Val Leu
Thr 545 550 555 560 Gly Cys Ser Ser His Trp Glu Val Glu Asp Leu Gly
Thr His Lys Pro 565 570 575 Pro Val Leu Arg Pro Arg Gly Gln Pro Asn
Gln Cys Val Gly His Arg 580 585 590 Glu Ala Ser Ile His Ala Ser Cys
Cys His Ala Pro Gly Leu Glu Cys 595 600 605 Lys Val Lys Glu His Gly
Ile Pro Ala Pro Gln Gly Gln Val Thr Val 610 615 620 Ala Cys Glu Glu
Gly Trp Thr Leu Thr Gly Cys Ser Ala Leu Pro Gly 625 630 635 640 Thr
Ser His Val Leu Gly Ala Tyr Ala Val Asp Asn Thr Cys Val Val 645 650
655 Arg Ser Arg Asp Val Ser Thr Thr Gly Ser Thr Ser Glu Glu Ala Val
660 665 670 Thr Ala Val Ala Ile Cys Cys Arg Ser Arg His Leu Ala Gln
Ala Ser 675 680 685 Gln Glu Leu Gln 690 4345DNAHomo
sapiensmisc_featureNucleotide sequence of heavy chain variable
region 4caggttcagc tggtgcagtc tggagctgag gtgaagaagc ctggggcctc
agtgaaggtc 60tcctgcaagg cttctggtta caccttaacc agctatggta tcagctgggt
gcgacaggcc 120cctggacaag ggcttgagtg gatgggatgg gtcagttttt
ataatggtaa cacaaactat 180gcacagaagc tccagggcag aggcaccatg
accacagacc catccacgag cacagcctac 240atggagctga ggagcctgag
atctgacgac acggccgtgt attactgtgc gagaggctac 300ggtatggacg
tctggggcca agggaccacg gtcaccgtct cctct 3455115PRTHomo
sapiensMISC_FEATUREAmino acid sequence of heavy chain variable
region 5Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly
Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Leu
Thr Ser Tyr 20 25 30 Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln
Gly Leu Glu Trp Met 35 40 45 Gly Trp Val Ser Phe Tyr Asn Gly Asn
Thr Asn Tyr Ala Gln Lys Leu 50 55 60 Gln Gly Arg Gly Thr Met Thr
Thr Asp Pro Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser
Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly
Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr 100 105 110 Val
Ser Ser 115 6345DNAHomo sapiensmisc_featureAlternative Nucleotide
sequence of heavy chain variable region 6gaggttcagc tggtgcagtc
tggagctgag gtgaagaagc ctggggcctc agtgaaggtc 60tcctgcaagg cttctggtta
caccttaacc agctatggta tcagctgggt gcgacaggcc 120cctggacaag
ggcttgagtg gatgggatgg gtcagttttt ataatggtaa cacaaactat
180gcacagaagc tccagggcag aggcaccatg accacagacc catccacgag
cacagcctac 240atggagctga ggagcctgag atctgacgac acggccgtgt
attactgtgc gagaggctac 300ggtatggacg tctggggcca agggaccacg
gtcaccgtct cctct 3457115PRTHomo sapiensMISC_FEATUREAlternative
Amino acid sequence of heavy chain variable region 7Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Leu Thr Ser Tyr 20 25 30
Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35
40 45 Gly Trp Val Ser Phe Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys
Leu 50 55 60 Gln Gly Arg Gly Thr Met Thr Thr Asp Pro Ser Thr Ser
Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Tyr Gly Met Asp Val Trp
Gly Gln Gly Thr Thr Val Thr 100 105 110 Val Ser Ser 115 8327DNAHomo
sapiensmisc_featureNucleotide sequence of light chain variable
region 8cagtctgccc tgactcagcc tgcctccgtg tctgggtctc ctggacagtc
gatcaccatc 60tcctgcactg gaaccagcag tgacgttggt ggttataact ctgtctcctg
gtaccaacag 120cacccaggca aagcccccaa actcatgatt tatgaggtca
gtaatcggcc ctcaggggtt 180tctaatcgct tctctggctc caagtctggc
aacacggcct ccctgaccat ctctgggctc 240caggctgagg acgaggctga
ttattactgc aattcatata caagcaccag catggtattc 300ggcggaggga
ccaagctgac cgtccta 3279109PRTHomo sapiensMISC_FEATUREAmino acid
sequence of light chain variable region 9Gln Ser Ala Leu Thr Gln
Pro Ala Ser Val Ser Gly Ser Pro Gly Gln 1 5 10 15 Ser Ile Thr Ile
Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr 20 25 30 Asn Ser
Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu 35 40 45
Met Ile Tyr Glu Val Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe 50
55 60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly
Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Tyr
Thr Ser Thr 85 90 95 Ser Met Val Phe Gly Gly Gly Thr Lys Leu Thr
Val Leu 100 105 10327DNAHomo sapiensmisc_featureAlternative
Nucleotide sequence of light chain variable region 10gagtctgccc
tgactcagcc tgcctccgtg tctgggtctc ctggacagtc gatcaccatc 60tcctgcactg
gaaccagcag tgacgttggt ggttataact ctgtctcctg gtaccaacag
120cacccaggca aagcccccaa actcatgatt tatgaggtca gtaatcggcc
ctcaggggtt 180tctaatcgct tctctggctc
caagtctggc aacacggcct ccctgaccat ctctgggctc 240caggctgagg
acgaggctga ttattactgc aattcatata caagcaccag catggtattc
300ggcggaggga ccaagctgac cgtccta 32711109PRTHomo
sapiensMISC_FEATUREAlternative Amino acid sequence of light chain
variable region 11Glu Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly
Ser Pro Gly Gln 1 5 10 15 Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser
Ser Asp Val Gly Gly Tyr 20 25 30 Asn Ser Val Ser Trp Tyr Gln Gln
His Pro Gly Lys Ala Pro Lys Leu 35 40 45 Met Ile Tyr Glu Val Ser
Asn Arg Pro Ser Gly Val Ser Asn Arg Phe 50 55 60 Ser Gly Ser Lys
Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu 65 70 75 80 Gln Ala
Glu Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Tyr Thr Ser Thr 85 90 95
Ser Met Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105
12326PRTHomo sapiensMISC_FEATUREHuman IgG2 12Ala 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 Asn Phe Gly Thr
Gln Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr
Lys Val Asp Lys 85 90 95 Thr Val Glu Arg Lys Cys Cys Val Glu Cys
Pro Pro Cys Pro Ala Pro 100 105 110 Pro Val Ala Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp 115 120 125 Thr Leu Met Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val Asp 130 135 140 Val Ser His Glu
Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly 145 150 155 160 Val
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn 165 170
175 Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp
180 185 190 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly
Leu Pro 195 200 205 Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly
Gln Pro Arg Glu 210 215 220 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
Glu Glu Met Thr Lys Asn 225 230 235 240 Gln Val Ser Leu Thr Cys Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile 245 250 255 Ala Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 260 265 270 Thr Pro Pro
Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 275 280 285 Leu
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 290 295
300 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
305 310 315 320 Ser Leu Ser Pro Gly Lys 325 13327PRTHomo
sapiensMISC_FEATUREHuman IgG4 13Ala 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 Ser Cys
Pro Ala Pro 100 105 110 Glu Phe Leu 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 Lys 325 14105PRTHomo
sapiensMISC_FEATUREHuman lambda 14Gln Pro Lys Ala Ala Pro Ser Val
Thr Leu Phe Pro Pro Ser Ser Glu 1 5 10 15 Glu Leu Gln Ala Asn Lys
Ala Thr Leu Val Cys Leu Ile Ser Asp Phe 20 25 30 Tyr Pro Gly Ala
Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val 35 40 45 Lys Ala
Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys 50 55 60
Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser 65
70 75 80 His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr
Val Glu 85 90 95 Lys Thr Val Ala Pro Thr Glu Cys Ser 100 105
15106PRTHomo sapiensMISC_FEATUREHuman kappa 15Thr Val Ala Ala Pro
Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 1 5 10 15 Leu Lys Ser
Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 20 25 30 Pro
Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 35 40
45 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
50 55 60 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr
Glu Lys 65 70 75 80 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly
Leu Ser Ser Pro 85 90 95 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
100 105 16215PRTHomo sapiensMISC_FEATURE21B12 mature light chain
16Glu Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln 1
5 10 15 Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly
Tyr 20 25 30 Asn Ser Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala
Pro Lys Leu 35 40 45 Met Ile Tyr Glu Val Ser Asn Arg Pro Ser Gly
Val Ser Asn Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Asn Thr Ala
Ser Leu Thr Ile Ser Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp
Tyr Tyr Cys Asn Ser Tyr Thr Ser Thr 85 90 95 Ser Met Val Phe Gly
Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro 100 105 110 Lys Ala Ala
Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu 115 120 125 Gln
Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro 130 135
140 Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala
145 150 155 160 Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn
Lys Tyr Ala 165 170 175 Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln
Trp Lys Ser His Arg 180 185 190 Ser Tyr Ser Cys Gln Val Thr His Glu
Gly Ser Thr Val Glu Lys Thr 195 200 205 Val Ala Pro Thr Glu Cys Ser
210 215 17215PRTHomo sapiensMISC_FEATUREAlternative 21B12 mature
light chain 17Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser
Pro Gly Gln 1 5 10 15 Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser
Asp Val Gly Gly Tyr 20 25 30 Asn Ser Val Ser Trp Tyr Gln Gln His
Pro Gly Lys Ala Pro Lys Leu 35 40 45 Met Ile Tyr Glu Val Ser Asn
Arg Pro Ser Gly Val Ser Asn Arg Phe 50 55 60 Ser Gly Ser Lys Ser
Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu 65 70 75 80 Gln Ala Glu
Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Tyr Thr Ser Thr 85 90 95 Ser
Met Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro 100 105
110 Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu
115 120 125 Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe
Tyr Pro 130 135 140 Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser
Pro Val Lys Ala 145 150 155 160 Gly Val Glu Thr Thr Thr Pro Ser Lys
Gln Ser Asn Asn Lys Tyr Ala 165 170 175 Ala Ser Ser Tyr Leu Ser Leu
Thr Pro Glu Gln Trp Lys Ser His Arg 180 185 190 Ser Tyr Ser Cys Gln
Val Thr His Glu Gly Ser Thr Val Glu Lys Thr 195 200 205 Val Ala Pro
Thr Glu Cys Ser 210 215 18441PRTHomo sapiensMISC_FEATURE21B12
mature heavy chain 18Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Tyr Thr Leu Thr Ser Tyr 20 25 30 Gly Ile Ser Trp Val Arg Gln
Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Val Ser Phe
Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys Leu 50 55 60 Gln Gly Arg
Gly Thr Met Thr Thr Asp Pro Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met
Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Arg Gly Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr
100 105 110 Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
Ala Pro 115 120 125 Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu
Gly Cys Leu Val 130 135 140 Lys Asp Tyr Phe Pro Glu Pro Val Thr Val
Ser Trp Asn Ser Gly Ala 145 150 155 160 Leu Thr Ser Gly Val His Thr
Phe Pro Ala Val Leu Gln Ser Ser Gly 165 170 175 Leu Tyr Ser Leu Ser
Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly 180 185 190 Thr Gln Thr
Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys 195 200 205 Val
Asp Lys Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys 210 215
220 Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
225 230 235 240 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
Thr Cys Val 245 250 255 Val Val Asp Val Ser His Glu Asp Pro Glu Val
Gln Phe Asn Trp Tyr 260 265 270 Val Asp Gly Val Glu Val His Asn Ala
Lys Thr Lys Pro Arg Glu Glu 275 280 285 Gln Phe Asn Ser Thr Phe Arg
Val Val Ser Val Leu Thr Val Val His 290 295 300 Gln Asp Trp Leu Asn
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 305 310 315 320 Gly Leu
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln 325 330 335
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met 340
345 350 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro 355 360 365 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn 370 375 380 Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp
Gly Ser Phe Phe Leu 385 390 395 400 Tyr Ser Lys Leu Thr Val Asp Lys
Ser Arg Trp Gln Gln Gly Asn Val 405 410 415 Phe Ser Cys Ser Val Met
His Glu Ala Leu His Asn His Tyr Thr Gln 420 425 430 Lys Ser Leu Ser
Leu Ser Pro Gly Lys 435 440 19441PRTHomo
sapiensMISC_FEATUREAlternative 21B12 mature heavy chain 19Gln Val
Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Leu Thr Ser Tyr 20
25 30 Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp
Met 35 40 45 Gly Trp Val Ser Phe Tyr Asn Gly Asn Thr Asn Tyr Ala
Gln Lys Leu 50 55 60 Gln Gly Arg Gly Thr Met Thr Thr Asp Pro Ser
Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp
Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Tyr Gly Met Asp
Val Trp Gly Gln Gly Thr Thr Val Thr 100 105 110 Val Ser Ser Ala Ser
Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro 115 120 125 Cys Ser Arg
Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val 130 135 140 Lys
Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala 145 150
155 160 Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
Gly 165 170 175 Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
Asn Phe Gly 180 185 190 Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys
Pro Ser Asn Thr Lys 195 200 205 Val Asp Lys Thr Val Glu Arg Lys Cys
Cys Val Glu Cys Pro Pro Cys 210 215 220 Pro Ala Pro Pro Val Ala Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys 225 230 235 240 Pro Lys Asp Thr
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 245 250 255 Val Val
Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr 260 265 270
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 275
280 285 Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val
His 290 295 300 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn Lys 305 310 315 320 Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile
Ser Lys Thr Lys Gly Gln 325 330 335 Pro Arg Glu Pro Gln Val Tyr Thr
Leu Pro Pro Ser Arg Glu Glu Met 340 345 350 Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
355 360 365 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
Asn Asn 370 375 380 Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly
Ser Phe Phe Leu 385 390 395 400 Tyr Ser Lys Leu Thr Val Asp Lys Ser
Arg Trp Gln Gln Gly Asn Val 405 410 415 Phe Ser Cys Ser Val Met His
Glu Ala Leu His Asn His Tyr Thr Gln 420 425 430 Lys Ser Leu Ser Leu
Ser Pro Gly Lys 435 440 205PRTHomo sapiensMISC_FEATURECDRH1 20Ser
Tyr Gly Ile Ser 1 5 2110PRTHomo sapiensMISC_FEATURECDRH1 21Gly Tyr
Thr Leu Thr Ser Tyr Gly Ile Ser 1 5 10 2217PRTHomo
sapiensMISC_FEATURECDRH2 22Trp Val Ser Phe Tyr Asn Gly Asn Thr Asn
Tyr Ala Gln Lys Leu Gln 1 5 10 15 Gly 236PRTHomo
sapiensMISC_FEATURECDRH3 23Gly Tyr Gly Met Asp Val 1 5 2414PRTHomo
sapiensMISC_FEATURECDRL1 24Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr
Asn Ser Val Ser 1 5 10 257PRTHomo sapiensMISC_FEATURECDRL2 25Glu
Val Ser Asn Arg Pro Ser 1 5 269PRTHomo sapiensMISC_FEATURECDRL3
26Asn Ser Tyr Thr Ser Thr Ser Met Val 1 5
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