U.S. patent application number 15/558932 was filed with the patent office on 2018-03-15 for virus filtration.
This patent application is currently assigned to ALEXION PHARMACEUTICALS, INC.. The applicant listed for this patent is ALEXION PHARMACEUTICALS, INC.. Invention is credited to Bianca Olson, Saravanamoorthy Rajendran, Ryan Tedstone.
Application Number | 20180072769 15/558932 |
Document ID | / |
Family ID | 55752716 |
Filed Date | 2018-03-15 |
United States Patent
Application |
20180072769 |
Kind Code |
A1 |
Olson; Bianca ; et
al. |
March 15, 2018 |
VIRUS FILTRATION
Abstract
Provided herein are methods of performing viral filtration on a
fluid including a recombinant antibody, and the use of these
methods in methods of manufacturing or producing the recombinant
antibody.
Inventors: |
Olson; Bianca; (Watertown,
CT) ; Rajendran; Saravanamoorthy; (Middletown,
CT) ; Tedstone; Ryan; (New Haven, CT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ALEXION PHARMACEUTICALS, INC. |
New Haven |
CT |
US |
|
|
Assignee: |
ALEXION PHARMACEUTICALS,
INC.
New Haven
CT
|
Family ID: |
55752716 |
Appl. No.: |
15/558932 |
Filed: |
March 23, 2016 |
PCT Filed: |
March 23, 2016 |
PCT NO: |
PCT/US16/23746 |
371 Date: |
September 15, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62137187 |
Mar 23, 2015 |
|
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|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
B01D 71/10 20130101;
B01D 71/34 20130101; B01D 2311/04 20130101; C07K 2317/24 20130101;
B01D 61/58 20130101; C07K 16/18 20130101; B01D 69/08 20130101; B01D
2311/18 20130101; B01D 71/56 20130101; C07K 16/065 20130101; C07K
1/34 20130101; B01D 71/68 20130101 |
International
Class: |
C07K 1/34 20060101
C07K001/34; C07K 16/18 20060101 C07K016/18; B01D 71/56 20060101
B01D071/56; B01D 69/08 20060101 B01D069/08; B01D 71/10 20060101
B01D071/10; B01D 71/34 20060101 B01D071/34; B01D 71/68 20060101
B01D071/68; B01D 61/58 20060101 B01D061/58 |
Claims
1. A method of performing viral filtration, the method comprising:
(a) adjusting the pH of a fluid comprising a recombinant antibody
to between about 5.0 and about 6.7; and (b) flowing the fluid
through a virus filter to produce a filtrate comprising the
recombinant antibody.
2. The method of claim 1, wherein (a) comprises adjusting the pH of
the fluid to between about 5.0 and about 6.5.
3. The method of claim 2, wherein (a) comprises adjusting the pH of
the fluid to between about 5.0 and about 6.0.
4. The method of claim 3, wherein (a) comprises adjusting the pH of
the fluid to between about 5.5 and about 6.0.
5. The method of any one of claims 1-4, further comprising, prior
to (b): adding a stabilizing agent to the fluid in an amount
sufficient to yield a final concentration of between about 0.1 mM
and about 25 mM stabilizing agent in the fluid.
6. The method of claim 5, wherein adding the stabilizing agent to
the fluid yields a final concentration of between about 0.1 mM and
about 24 mM stabilizing agent in the fluid.
7. The method of claim 6, wherein adding the stabilizing agent to
the fluid yields a final concentration of between about 0.1 mM and
about 22 mM stabilizing agent in the fluid.
8. The method of claim 7, wherein adding the stabilizing agent to
the fluid yields a final concentration of between about 0.1 mM and
about 20 mM stabilizing agent in the fluid.
9. The method of claim 8, wherein adding the stabilizing agent to
the fluid yields a final concentration of between about 0.1 mM and
about 10 mM stabilizing agent in the fluid.
10. The method of claim 9, wherein adding the stabilizing agent to
the fluid yields a final concentration of between about 0.1 mM and
about 5 mM stabilizing agent in the fluid.
11. The method of any one of claims 1-10, further comprising,
immediately prior to (b): flowing the fluid through a
pre-filter.
12. The method of claim 11, wherein the pre-filter comprises a
polyamide membrane.
13. The method of claim 11, wherein the pre-filter is a depth
filter.
14. The method of claim 13, wherein the depth filter comprises a
porous filtration medium that is anionic and/or hydrophobic.
15. The method of any one of claims 11-14, wherein the fluid
further comprises between about 5 mM and about 300 mM sodium
chloride.
16. The method of claim 15, wherein the fluid comprises between
about 50 mM and about 300 mM sodium chloride.
17. The method of claim 16, wherein fluid comprises between about
100 mM and about 300 mM sodium chloride.
18. The method of claim 17, wherein the fluid comprises between
about 100 mM and about 250 mM sodium chloride.
19. A method of performing viral filtration, the method comprising:
(a) adding a stabilizing agent to a fluid comprising a recombinant
antibody in an amount sufficient to yield a final concentration of
between about 10 mM and about 100 mM stabilizing agent in the
fluid, wherein prior to adding, the fluid has a pH of between about
6.7 and about 8.5; and (b) flowing the fluid through a virus filter
to produce a filtrate comprising the recombinant antibody.
20. The method of claim 19, wherein prior to adding, the fluid has
a pH of between about 7.0 and about 7.8.
21. The method of claim 20, wherein prior to adding, the fluid has
a pH of between about 7.4 and about 7.8.
22. The method of claim 21, wherein prior to adding, the fluid has
a pH of about 7.6.
23. The method of any one of claims 19-22, wherein adding the
stabilizing agent to the fluid yields a final concentration of
between about 10 mM and about 90 mM stabilizing agent in the
fluid.
24. The method of claim 23, wherein adding the stabilizing agent to
the fluid yields a final concentration of between about 10 mM and
about 80 mM stabilizing agent in the fluid.
25. The method of claim 24, wherein adding the stabilizing agent to
the fluid yields a final concentration of between about 10 mM and
about 70 mM stabilizing agent in the fluid.
26. The method of claim 25, wherein adding the stabilizing agent to
the fluid yields a final concentration of between about 10 mM and
about 60 mM stabilizing agent in the fluid.
27. The method of claim 26, wherein adding the stabilizing agent to
the fluid yields a final concentration of between about 10 mM and
about 50 mM stabilizing agent in the fluid.
28. The method of claim 27, wherein adding the stabilizing agent to
the fluid yields a final concentration of between about 15 mM and
about 50 mM stabilizing agent in the fluid.
29. The method of claim 28, wherein adding the stabilizing agent to
the fluid yields a final concentration of between about 20 mM and
about 50 mM stabilizing agent in the fluid.
30. The method of any one of claims 19-29, further comprising:
further comprising, immediately prior to (b): flowing the fluid
through a pre-filter.
31. The method of claim 30, wherein the pre-filter comprises a
polyamide membrane.
32. The method of claim 30, wherein the pre-filter is a depth
filter.
33. The method of claim 32, wherein the pre-filter comprises a
porous filtration medium that is anionic and/or hydrophobic.
34. The method of any one of claims 19-33, wherein the fluid
comprises between about 1 mM and about 100 mM sodium chloride.
35. The method of claim 34, wherein the fluid comprises between
about 1 mM and about 80 mM sodium chloride.
36. The method of any one of claims 5-35, wherein the stabilizing
agent is selected from the group consisting of arginine, alanine,
aspartic acid, glutamic acid, leucine, lysine, histidine, glycine,
sucrose, trehalose, mannitol, and sorbitol.
37. The method of claim 36, wherein the stabilizing agent is
arginine.
38. The method of claim 37, wherein the arginine is L-arginine HCl
or L-arginine.
39. The method of any one of claims 1-38, wherein the virus filter
comprises a polyethersulfone membrane.
40. The method of any one of claims 1-38, wherein the virus filter
comprises a polyvinylidene fluoride (PVDF) membrane.
41. The method of claim 40, wherein the PVDF membrane is a hollow
fiber membrane.
42. The method of any one of claims 1-38, wherein the virus filter
comprises a cuprammonium-regenerated cellulose membrane.
43. The method of claim 42, wherein the cuprammonium-regenerated
cellulose membrane is a hollow fiber membrane.
44. The method of any one of claims 1-43, wherein prior to (a), the
fluid comprises between about 0.1 mg/mL and about 25 mg/mL
recombinant antibody.
45. The method of claim 44, wherein prior to (a), the fluid
comprises between about 0.1 mg/mL and about 15 mg/mL recombinant
antibody.
46. The method of claim 45, wherein prior to (a), the fluid
comprises between about 1 mg/mL and about 15 mg/mL recombinant
antibody.
47. The method of claim 46, wherein prior to (a), the fluid
comprises between about 5 mg/mL and about 15 mg/mL recombinant
antibody.
48. The method of any one of claims 1-18, wherein prior to (a), the
pH of the fluid is between about 7.4 and about 7.8.
49. The method of claim 48, wherein, prior to (a), the pH of the
fluid is between about 7.5 and about 7.7.
50. The method of claim 49, wherein, prior to (a), the pH of the
fluid is about 7.6.
51. The method of any one of claims 19-35, wherein the fluid has a
pH of between about 7.4 and about 7.8.
52. The method of claim 51, wherein the fluid has a pH of between
about 7.5 and about 7.7.
53. The method of claim 52, wherein the fluid has a pH of about
7.6.
54. The method of any one of claims 1-14 and 19-33, wherein the
fluid comprises between about 55 mM and about 90 mM sodium
chloride.
55. The method of claim 54, wherein the fluid comprises about 65 mM
sodium chloride.
56. The method of any one of claims 1-55, wherein the recombinant
antibody comprises one or both of: a heavy chain variable domain
that comprises a total of between one and six histidines in the set
of CDR1, CDR2, and CDR3; and a light chain variable domain that
comprises a total of between one and six histidines in the set of
CDR1, CDR2, and CDR3.
57. The method of claim 56, wherein the recombinant antibody
comprises a heavy chain variable domain that comprises a total of
between one and five histidines in the set of CDR1, CDR2, and
CDR3.
58. The method of claim 57, wherein the recombinant antibody
comprises a heavy chain variable domain that comprises a total of
between one and three histidines in the set of CDR1, CDR2, and
CDR3.
59. The method of claim 58, wherein the recombinant antibody
comprises a heavy chain variable domain that comprises a total of
two histidines in the set of CDR1, CDR2, and CDR3.
60. The method of claim 59, wherein the CDR1 comprises one
histidine residue and the CDR2 comprises one histidine residue.
61. The method of any one of claims 56-60, wherein the CDR1
comprises a sequence of SEQ ID NO: 1.
62. The method of any one of claims 56-61, wherein the CDR2
comprises a sequence of SEQ ID NO: 2.
63. The method of any one of claims 56-62, wherein the CDR3
comprises a sequence of SEQ ID NO: 3.
64. The method of any one of claims 56-62, wherein the heavy chain
variable domain comprises a sequence of SEQ ID NO: 4.
65. The method of any one of claims 56-64, wherein the recombinant
antibody comprises a heavy chain comprising a sequence of SEQ ID
NO: 5.
66. The method of any one of claims 56-65, wherein the recombinant
antibody comprises a light chain variable region comprising a CDR1
comprising a sequence of SEQ ID NO: 6, a CDR2 comprising a sequence
of SEQ ID NO: 7, and a CDR3 comprising a sequence of SEQ ID NO:
8.
67. The method of any one of claims 56-66, wherein the recombinant
antibody comprises a light chain variable region comprising a
sequence of SEQ ID NO: 9.
68. The method of any one of claims 56-67, wherein the recombinant
antibody comprises a light chain comprising a sequence of SEQ ID
NO: 10.
69. The method of any one of claims 1-55, wherein the recombinant
antibody comprises a heavy chain variable domain comprising a CDR1
comprising a sequence of SEQ ID NO: 11, a CDR2 comprising a
sequence of SEQ ID NO: 12, and a CDR3 comprising a sequence of SEQ
ID NO: 13.
70. The method of claim 69, wherein the heavy chain variable domain
comprises a sequence of SEQ ID NO: 14.
71. The method of claim 69 or 70, wherein the recombinant antibody
comprises a heavy chain comprising a sequence of SEQ ID NO: 15.
72. The method of any one of claims 69-71, wherein the recombinant
antibody comprises a light chain variable domain comprising a CDR1
comprising a sequence of SEQ ID NO: 16, a CDR2 comprising a
sequence of SEQ ID NO: 17, and a CDR3 comprising a sequence of SEQ
ID NO: 18.
73. The method of any one of claims 69-72, wherein the light chain
variable domain comprises a sequence of SEQ ID NO: 19.
74. The method of any one of claims 69-73, wherein the recombinant
antibody comprises a light chain comprising a sequence of SEQ ID
NO: 20.
75. The method of any one of claims 1-74, wherein the recombinant
antibody specifically binds to human complement protein C5.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. provisional patent
application 62/137,187, filed Mar. 23, 2015, the entire contents of
which are incorporated herein by reference.
TECHNICAL FIELD
[0002] This invention relates generally to methods of purifying
recombinant proteins and methods of manufacturing recombinant
protein products.
BACKGROUND
[0003] Recombinant proteins, such as monoclonal antibodies (mAb),
are an important and valuable class of therapeutic products for the
treatment of diseases, such as paroxysmal nocturnal hemoglobinuria
(PNH) and atypical hemolytic uremic syndrome (aHUS). Mammalian
cells including a nucleic acid that encodes a recombinant protein
are often used to produce the recombinant protein. The recombinant
protein is then purified from the mammalian cell culture using a
process that can include the passage of a fluid including the
recombinant protein through a virus filter. These purification
processes often experience slow flow rates and/or become fouled due
to the plugging of a virus filter in the process. The slow flow
rate and/or fouling of a virus filter in a purification process can
result in recombinant protein loss, can negatively impact the
safety of the resulting recombinant protein product, and/or reduce
the efficiency of the purification process.
SUMMARY
[0004] The present disclosure is based, at least in part, on the
discovery that a method of performing viral filtration that
includes one or more steps of (e.g., two, three, or four)
pre-filtering a fluid including a recombinant antibody (e.g., using
a Sartorius Virosart.RTM. Max pre-filter), adjusting the pH of a
fluid including a recombinant antibody, adding a stabilizing agent
to a fluid including a recombinant antibody, and adjusting the
sodium chloride concentration in a fluid including a recombinant
antibody, prior to flowing the fluid through a virus filter, has a
substantially improved throughput compared to methods of performing
viral filtration that do not include these one or more steps, prior
to flowing the fluid through a virus filter. In view of this
discovery, provided herein are methods of performing viral
filtration that include one or more (e.g., two, three, or four)
steps of pre-filtering a fluid including a recombinant antibody,
adjusting the pH of a fluid including a recombinant antibody,
adding a stabilizing agent to a fluid including a recombinant
antibody, and adjusting the sodium chloride concentration in a
fluid including a recombinant antibody, prior to flowing the fluid
through a virus filter, and methods of manufacturing or purifying a
recombinant antibody that include any of the viral filtration
methods described herein. Any of the viral filtration methods can
be performed as a large scale process or as part of a large scale
process.
[0005] Provided herein are methods of performing viral filtration
that include: (a) adjusting (e.g., increasing or decreasing) the pH
of a fluid including a recombinant antibody to between about 5.0
and about 6.7 (e.g., between about 5.0 and about 6.5, between about
5.0 and about 6.0, or between about 5.5 and about 6.0); and (b)
flowing the fluid through a virus filter to produce a filtrate
including the recombinant antibody. Some embodiments of any of the
methods described herein further include, prior to (b): adding a
stabilizing agent to the fluid in an amount sufficient to yield a
final concentration of between about 0.1 mM and about 25 mM (e.g.,
between about 0.1 mM and about 24 mM, between about 0.1 mM and
about 24 mM, between about 0.1 mM and about 22 mM, between about
0.1 mM and about 20 mM, between about 0.1 mM and about 10 mM, or
between about 0.1 mM and about 5 mM) stabilizing agent in the
fluid. Some embodiments of any of the methods described herein
further include, immediately prior to (b): flowing the fluid
through a pre-filter (e.g., a filter including a polyamide membrane
or a depth filter (e.g., a depth filter including a porous
filtration medium that is anionic and/or hydrophobic)). In some
embodiments of any of the methods described herein, the fluid
further includes between about 5 mM and about 300 mM (e.g., between
about 50 mM and about 300 mM, about 100 mM and about 300 mM, or
about 100 mM and about 250 mM) sodium chloride. In some embodiments
of any of the methods described herein, prior to (a), the pH of the
fluid is between about 7.4 and about 7.8 (e.g., between about 7.5
and about 7.7, or about 7.6).
[0006] Also provided are methods of performing viral filtration
that include: (a) adding a stabilizing agent to a fluid including a
recombinant antibody in an amount sufficient to yield a final
concentration of between about 10 mM and about 100 mM (e.g.,
between about 10 mM and about 90 mM, between about 10 mM and about
80 mM, between about 10 mM and about 70 mM, between about 10 mM and
about 60 mM, between about 10 mM and about 50 mM, between about 15
mM and about 50 mM, between about 20 mM and about 50 mM)
stabilizing agent in the fluid, wherein prior to adding, the fluid
has a pH of between about 6.7 and about 8.5 (e.g., between about
7.0 and about 7.8, between about 7.4 and about 7.8, or about 7.6);
and (b) flowing the fluid through a virus filter to produce a
filtrate including the recombinant antibody. Some embodiments of
any of the methods described herein further include, immediately
prior to (b): flowing the fluid through a pre-filter (e.g., a
pre-filter including a polyamide membrane or a depth filter (e.g.,
a depth filter including a porous filtration medium that is anionic
and/or hydrophobic). In some embodiments of any of the methods
described herein the fluid includes between about 1 mM and about
100 mM (e.g., between about 1 mM and about 80 mM) sodium chloride.
In some embodiments of any of the methods described herein, the
stabilizing agent is selected from the group of: arginine (e.g.,
L-arginine or L-arginine HCl), alanine, aspartic acid, glutamic
acid, leucine, lysine, histidine, glycine, sucrose, trehalose,
mannitol, sorbitol, and Polysorbate 80.
[0007] In some embodiments of any of the methods described herein,
the virus filter includes a polyethersulfone (PES) membrane, a
polyvinylidene fluoride (PVDF) membrane (e.g., a PVDF membrane that
is a hollow fiber membrane), or cuprammonium-regenerated cellulose
membrane (e.g., a cuprammonium-regenerated cellulose membrane that
is a hollow fiber membrane).
[0008] In some embodiments of any of the methods described herein,
prior to (a), the fluid includes between about 0.1 mg/mL and about
25 mg/mL (e.g., between about 0.1 mg/mL and about 15 mg/mL, between
about 1 mg/mL and about 15 mg/mL, or between about 5 mg/mL and
about 15 mg/mL) recombinant antibody. In some embodiments of any of
the methods described herein, the fluid has a pH of between about
7.4 and about 7.8 (e.g., between about 7.5 and about 7.7, or about
7.6). In some embodiments of any of the methods described herein,
the fluid includes between about 55 mM and about 90 mM (e.g., about
65 mM) sodium chloride.
[0009] In some embodiments of any of the methods described herein,
the recombinant antibody includes one or both of: a heavy chain
variable domain that includes a total of between one and six
histidines in the set of CDR1, CDR2, and CDR3; and a light chain
variable domain that includes a total of between one and six
histidines in the set of CDR1, CDR2, and CDR3. In some embodiments
of any one of the methods described herein, the recombinant
antibody includes a heavy chain variable domain that includes a
total of between one and five (e.g., between one and three, or two)
histidines in the set of CDR1, CDR2, and CDR3. In some embodiments
of any of the methods described herein, the CDR1 includes one
histidine residue and the CDR2 includes one histidine residue. In
some embodiments of any of the methods described herein, the CDR1
includes a sequence of SEQ ID NO: 1. In some embodiments of any of
the methods described herein, the CDR2 includes a sequence of SEQ
ID NO: 2. In some embodiments of any of the methods described
herein, the CDR3 includes a sequence of SEQ ID NO: 3. In some
embodiments of any of the methods described herein, the heavy chain
variable domain includes a sequence of SEQ ID NO: 4. In some
embodiments of any of the methods described herein, the recombinant
antibody includes a heavy chain including a sequence of SEQ ID NO:
5. In some embodiments of any of the methods described herein, the
recombinant antibody includes a light chain variable region
including a CDR1 including a sequence of SEQ ID NO: 6, a CDR2
including a sequence of SEQ ID NO: 7, and a CDR3 including a
sequence of SEQ ID NO: 8. In some embodiments of any of the methods
described herein, the recombinant antibody includes a light chain
variable region including a sequence of SEQ ID NO: 9. In some
embodiments of any of the methods described herein, recombinant
antibody includes a light chain including a sequence of SEQ ID NO:
10.
[0010] In any of the methods described herein, the recombinant
antibody includes a heavy chain variable domain including a CDR1
including a sequence of SEQ ID NO: 11, a CDR2 including a sequence
of SEQ ID NO: 12, and a CDR3 including a sequence of SEQ ID NO: 13.
In any of the methods described herein, the heavy chain variable
domain includes a sequence of SEQ ID NO: 14. In some embodiments of
any of the methods described herein, the recombinant antibody
includes a heavy chain including a sequence of SEQ ID NO: 15. In
some embodiments of any of the methods described herein, the
recombinant antibody includes a light chain variable domain
including a CDR1 including a sequence of SEQ ID NO: 16, a CDR2
including a sequence of SEQ ID NO: 17, and a CDR3 including a
sequence of SEQ ID NO: 18. In some embodiments of any of the
methods described herein, the light chain variable domain includes
a sequence of SEQ ID NO: 19. In some embodiments of any of the
methods described herein, the recombinant antibody includes a light
chain including a sequence of SEQ ID NO: 20.
[0011] In some embodiments of any of the methods described herein,
the recombinant antibody specifically binds to human complement
protein C5.
[0012] As used herein, the word "a" before a noun represents one or
more of the particular noun. For example, the phrase "a stabilizing
agent" represents "one or more stabilizing agents."
[0013] The term "mammalian cell" means any cell from or derived
from any mammal (e.g., a human, a hamster, a mouse, a green monkey,
a rat, a pig, a cow, or a rabbit). For example, a mammalian cell
can be an immortalized cell. The mammalian cell can be a
differentiated or undifferentiated cell. Non-limiting examples of
mammalian cells are described herein. Additional examples of
mammalian cells are known in the art.
[0014] The term "substantially free" means a composition (e.g., a
filtrate) that is at least or about 90% free, such as at least or
about 95%, 96%, 97%, 98%, or at least or about 99% free, or about
100% free of a specified substance, such as soluble antibody
aggregates or host cell proteins.
[0015] The term "culturing" or "cell culturing" means maintenance
or proliferation of a mammalian cell under a controlled set of
physical conditions.
[0016] The term "culture of mammalian cells" means a culture medium
(such as a liquid culture medium) including a plurality of
mammalian cells that is maintained or proliferated under a
controlled set of physical conditions.
[0017] The term "liquid culture medium" means a fluid that includes
sufficient nutrients to allow a cell (such as a mammalian cell) to
grow or proliferate in vitro. A liquid culture medium can include,
for example, one or more of: amino acids (such as 20 amino acids),
a purine (such as hypoxanthine), a pyrimidine (such as thymidine),
choline, inositol, thiamine, folic acid, biotin, calcium,
niacinamide, pyridoxine, riboflavin, thymidine, cyanocobalamin,
pyruvate, lipoic acid, magnesium, glucose, sodium, potassium, iron,
copper, zinc, and sodium bicarbonate. In some embodiments, a liquid
culture medium can include serum from a mammal. In some
embodiments, a liquid culture medium does not include serum or
another extract from a mammal (a defined liquid culture medium). A
liquid culture medium can also include trace metals, a mammalian
growth hormone, and/or a mammalian growth factor. An example of
liquid culture medium is minimal medium (such as a medium including
only inorganic salts, a carbon source, and water). Non-limiting
examples of liquid culture medium are described herein. Additional
examples of liquid culture medium are known in the art and are
commercially available. A liquid culture medium can include any
density of mammalian cells. For example, as used herein, a volume
of liquid culture medium removed from a vessel (such as a
bioreactor) can be substantially free of mammalian cells.
[0018] The term "antibody" means a polypeptide including an amino
acid sequence of at least 10 amino acids (such as at least 15, 20,
30, 40, 50, 60, 70, 80, 90, or 100 amino acids) of an
immunoglobulin protein (such as a variable domain sequence, a
framework sequence, or a constant domain sequence of a heavy or
light chain immunoglobulin). An antibody can be, e.g., an IgG, IgE,
IgD, IgA, or IgM. The antibody may be any subclass of IgG, such as
IgG1, IgG2, IgG3, or IgG4, or the chimeric IgG2/4 as found in
eculizumab. The antibody may be an antigen-binding antibody
fragment, such as a Fab fragment, a F(ab').sub.2 fragment, or an
scFv fragment. The antibody may be a bi-specific antibody or a
tri-specific antibody, or a dimer, trimer, or multimer antibody, or
a diabody, an AFFIBODY.RTM., or a NANOBODY.RTM.. The antibody can
be an engineered protein including at least one immunoglobulin
domain (such as a fusion protein including a Fc domain). The
antibody can be an engineered protein having four antibody binding
domains such as DVD-Ig and CODV-Ig. See, e.g., US2007/0071675 and
WO2012/135345. Non-limiting examples of antibodies are described
herein and additional examples of antibodies are known in the
art.
[0019] The term "capturing" means a step performed to partially
purify or isolate (such as at least or about 10%, 15%, 20%, 25%,
30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%,
or at least or about 99% pure by weight), concentrate, and/or
stabilize a recombinant antibody from one or more other components
present in a fluid including the recombinant antibody. Other
components may include buffers, salts, DNA, RNA, host cell
proteins, and aggregates of the desired recombinant antibody
present in or secreted from a mammalian cell. Capturing can be
performed using a chromatography resin that binds a recombinant
antibody through the use of a specific recognition and binding
interaction, such as with protein A chromatography or using antigen
chromatography. Non-limiting methods for capturing a recombinant
antibody from a fluid including the recombinant antibody or a
clarified liquid culture medium are described herein and others are
known in the art. A recombinant antibody can be captured from a
liquid culture medium using at least one chromatography column
(such as any of the chromatography columns described herein, such
as a chromatography column packed with an affinity chromatography
resin, an anionic exchange chromatography resin, a cationic
exchange chromatography resin, a mixed-mode chromatography resin, a
molecular sieve chromatography resin, or a hydrophobic interaction
chromatography resin). Capturing can be performed using a
chromatography resin that utilizes a protein A-binding capture
mechanism, an antibody- or antibody fragment-binding capture
mechanism, or an antigen-binding capture mechanism.
[0020] The term "purifying" means a method or step performed to
isolate a recombinant antibody from one or more other impurities or
components present in a fluid including a recombinant antibody. The
components being separated include liquid culture medium proteins,
host cell proteins, aggregates of the desired recombinant antibody,
DNA, RNA, other proteins, endotoxins, and viruses present in or
secreted from a mammalian cell. For example, a purifying step can
be performed before or after an initial capturing step and/or
before or after a step of flowing a recombinant antibody through a
depth filter or a pre-filter and/or a virus filter. A purifying
step can be performed using a resin, membrane, or any other solid
support that binds either a recombinant antibody or contaminants
(such as through the use of affinity chromatography, hydrophobic
interaction chromatography, anion or cation exchange
chromatography, mixed-mode chromatography resin, or molecular sieve
chromatography). A recombinant antibody can be purified from a
fluid including the recombinant antibody using at least one
chromatography column and/or chromatographic membrane (such as any
of the chromatography columns described herein).
[0021] The term "polishing" is a term of art and means a step
performed to remove remaining trace or small amounts of
contaminants or impurities from a fluid including a recombinant
antibody that is close to a final desired purity. For example,
polishing can be performed by passing a fluid including the
recombinant antibody through a chromatographic column(s) or
membrane absorber(s) that selectively binds to either the
recombinant antibody or small amounts of remaining contaminants or
impurities present in the fluid including the recombinant antibody.
In such an example, the eluate/filtrate of the chromatographic
column(s) or membrane absorber(s) includes the recombinant
antibody. As described herein, one or more unit operations of
polishing can be performed prior to flowing a fluid including the
recombinant antibody through a virus filter.
[0022] The term "filtrate" is a term of art and means a fluid that
is emitted from a filter (e.g., a depth filter, a pre-filter, or a
virus filter) that includes a detectable amount of a recombinant
antibody.
[0023] The term "filtering" means the removal of at least part of
(such as at least 90%, 95%, 96%, 97%, 98%, or 99%) undesired
biological contaminants (such as a mammalian cell, bacteria, yeast
cells, viruses, mycobacteria, or mycoplasma), impurities (such as
soluble antibody aggregates, host cell proteins, host cell DNA, and
other chemicals used in a method for purifying a recombinant
antibody or a method of manufacturing a recombinant antibody),
and/or particulate matter (such as precipitated antibodies) from a
fluid (such as a liquid culture medium or fluid present in any of
the processes described herein).
[0024] The term "viral filtration" means the removal of at least
part of (such as at least 90%, 95%, 96%, 97%, 98%, or 99%) viruses
from a fluid (e.g., such as a liquid culture medium or fluid
present in any of the processes described herein) that includes a
recombinant antibody. Methods for performing viral filtration are
described herein.
[0025] The term "virus filter" means a filter capable of removing
at least part of (such as at least 90%, 95%, 96%, 97%, 98%, or 99%,
or 100%) viruses from a fluid (e.g., such as a liquid culture
medium or fluid present in any of the processes described herein)
including a recombinant antibody when the fluid is flowed through
the filter. Non-limiting examples of virus filters are described
herein. Additional examples of virus filters are known in the
art.
[0026] The term "pre-filter" is a filter capable of removing at
least part of (such as at least 50%, 55%, 60%, 65%, 70%, 75%, 80%,
85%, 90%, 95%, 96%, 97%, 98%, or 99%, or 100%) soluble protein
aggregates and/or particles from a fluid (e.g., such as a liquid
culture medium or fluid present in any of the processes described
herein) when the fluid is flowed through the pre-filter. A fluid
can be, e.g., flowed through a pre-filter prior to flowing the
fluid through a virus filter. Non-limiting examples of pre-filters
are described herein. Additional examples of pre-filters are known
in the art.
[0027] The term "secreted antibody" or "secreted recombinant
antibody" means an antibody (such as a recombinant antibody) that
originally included at least one secretion signal sequence when it
is translated within a mammalian cell, and through, at least in
part, enzymatic cleavage of the secretion signal sequence in the
mammalian cell, is secreted at least partially into the
extracellular space (such as a liquid culture medium). Skilled
practitioners will appreciate that a "secreted" antibody need not
dissociate entirely from the cell to be considered a secreted
antibody.
[0028] The term "clarified liquid culture medium" means a liquid
culture medium obtained from a mammalian, bacterial, or yeast cell
culture that is substantially free (such as at least 90%, 92%, 94%,
96%, 98%, or 99% free) of mammalian, bacterial, or yeast cells. A
clarified liquid culture medium can be prepared, for example, by
filtering a cell culture (such as alternating tangential filtration
or tangential flow filtration), by centrifuging a cell culture and
collecting the supernatant, or by allowing the cells in the cell
culture settle and obtaining a fluid that is substantially free of
cells. The cells can also be separated from the medium by the use
of a cell separation device, such as the ATF system from Refine
Technology.
[0029] Purification or manufacture of recombinant antibodies
usually requires in series performance of multiple independent
purification operations or steps. The term "unit operation" is a
term of art and means a discreet step or mini-process performed in
a larger general process for purifying a recombinant antibody or a
method of manufacturing a recombinant antibody (such as a method of
manufacturing a recombinant antibody from a clarified liquid
culture medium). For example, a unit of operation can be a step of
capturing the recombinant antibody, ultrafiltration/diafiltration
to concentrate the recombinant antibody in a fluid, ion exchange
chromatography, hydrophobic interaction chromatography, polishing
the recombinant antibody, viral inactivation, viral filtration,
pre-filtration, adjustment of pH, adjustment of ionic strength, and
adjustment of both pH and ionic strength of a fluid including the
recombinant antibody.
[0030] The term "depth filter" is a term of art and means a filter
that includes a porous filtration medium that captures contaminants
and/or impurities (such as any of the contaminants and/or
impurities described herein) within its 3-dimensional structure and
not merely on the surface. Depth filters are characterized in that
they retain the contaminants or impurities within the filter and
can retain a relatively large quantity before becoming clogged.
Depth filter construction may include multiple layers, multiple
membranes, a single layer, or a resin material. Non-limiting
examples of depth filters include CUNO.RTM. Zeta PLUS.RTM. Delipid
filters (3M, St. Paul, Minn.), CUNO.RTM. Emphaze AEX filters (3M,
St. Paul, Minn.), CUNO.RTM. 90ZA08A filters (3M, St. Paul, Minn.),
CUNO.RTM. 90ZB08A filters (3M, St. Paul, Minn.), CUNO.RTM. DELI08A
Delipid filters (3M, St. Paul, Minn.), CUNO.RTM. DELIP08A Delipid
plus filters (3M, St. Paul, Minn.), Millipore X0HC filters (EMD
Millipore, Billerica, Mass.), MILLISTAK.RTM. pads (EMD Millipore,
Billerica, Mass.).
[0031] The term "soluble protein aggregates" is a term of art and
means complexes of two or more proteins (such as recombinant
antibodies) that are soluble in a fluid. Such complexes can form
through hydrophobic and/or ionic interactions between individual
recombinant protein molecules or fragments thereof.
[0032] The term "stabilizing agent" is an agent that reduces the
hydrodynamic radius of a recombinant antibody in a fluid and/or
minimizes the level of soluble and/or insoluble protein aggregates
(e.g., soluble and/or insoluble recombinant antibody aggregates
and/or soluble and/or insoluble host cell protein aggregates) in a
fluid including a recombinant protein. Non-limiting examples of
stabilizing agents are described herein. Additional examples of
stabilizing agents are known in the art.
[0033] Unless otherwise defined, all technical terms used herein
have the same meaning as commonly understood by one of ordinary
skill in the art to which this invention belongs. Methods and
materials are described herein for use in the present invention;
other, suitable methods and materials known in the art can also be
used. The materials, methods, and examples are illustrative only
and not intended to be limiting. All publications, patent
applications, patents, and other references mentioned herein are
incorporated by reference in their entirety. In case of conflict,
the present specification, including definitions, will control.
[0034] Other features and advantages of the invention will be
apparent from the following detailed description and figures, and
from the claims.
DESCRIPTION OF DRAWINGS
[0035] FIG. 1 is graph showing the flux decay as compared to the
throughput of a Virosart.RTM. CPV virus filter when a fluid
containing between 2.91 mg/mL to 3.54 mg/mL of BNJ441 human
monoclonal antibody, between 65 mM to 300 mM sodium chloride, and 0
or 50 mM L-arginine, and having a pH of between 5.5 and 7.6 is
passed through the filter. Each fluid passed through the
Virosart.RTM. CPV virus filter had first been pre-filtered using a
0.1 .mu.m filter or a Sartorius Virosart.RTM. Max pre-filter.
[0036] FIG. 2 is a table showing the throughput of a Virosart.RTM.
CPV virus filter at the time point at which 50% flow decay was
observed when a fluid including between 2.91 mg/mL to 3.54 mg/mL of
BNJ441 human monoclonal antibody, between 65 mM to 300 mM sodium
chloride, and 0 or 50 mM L-arginine, and having a pH of between 5.5
and 7.6 is passed through the filter. Each fluid passed through the
Virosart.RTM. CPV virus filter had been pre-filtered using a 0.1
.mu.m filter or a Sartorius Virosart.RTM. Max pre-filter.
[0037] FIG. 3 is a graph showing the relationship of the actual
Virosart.RTM. CPV virus filter throughput at the time point at
which 50% flow decay observed when fluids including between 2.91
mg/mL to 3.54 mg/mL BNJ441 human monoclonal antibody, between 65 mM
to 300 mM sodium chloride, and 0 or 50 mM L-arginine, and having a
pH of between 5.5 and 7.6, that have been pre-filtered using a 0.1
.mu.m or a Sartorius Virosart.RTM. Max pre-filter, are passed
through the Virosart.RTM. CPV virus filter, to the predicted
throughput at 50% flow decay for a Virosart.RTM. CPV virus filter
using statistical analysis.
[0038] FIG. 4 is a graph showing the significance of different
parameters on the Virosart.RTM. CPV virus filter throughput at the
time point at which 50% flow decay is observed (derived from data
gathered from flowing fluids including between 2.91 mg/mL to 3.54
mg/mL BNJ441 human monoclonal antibody, between 65 mM to 300 mM
sodium chloride, and 0 or 50 mM L-arginine, and having a pH of
between 5.5 and 7.6, that have been pre-filtered using a 0.1 .mu.m
or a Sartorius Virosart.RTM. Max pre-filter, through a
Virosart.RTM. CPV virus filter).
[0039] FIG. 5 is a graph showing the throughput at 50% flow decay
for a Virosart.RTM. CPV virus filter for fluids including between
2.91 mg/mL to 3.54 mg/mL BNJ441 human monoclonal antibody, between
65 mM to 300 mM sodium chloride, and 0 or 50 mM L-arginine, and
having a pH of between 5.5 and 7.6, that were either pre-filtered
using a 0.1 .mu.m filter (left bar) or a Sartorius Virosart.RTM.
Max pre-filter (right bar).
[0040] FIG. 6 is a graph showing the relationship of the throughput
at 50% flow decay for a Virosart.RTM. CPV virus filter for fluids
including between 2.91 mg/mL to 3.54 mg/mL BNJ441 human monoclonal
antibody, between 65 mM to 300 mM sodium chloride, and 0 or 50 mM
L-arginine, and having a pH of between 5.5 and 7.6, that were
either pre-filtered using a 0.1 .mu.m filter or a Sartorius
Virosart.RTM. Max pre-filter, to the hydrodynamic radius of BNJ441
human monoclonal antibody measured in each fluid.
[0041] FIG. 7 is a graph showing the mean throughput at 50% flow
decay for a Virosart.RTM. CPV virus filter for fluids having a pH
of 5.5, 6.5 or 7.5, and including between 2.91 mg/mL to 3.54 mg/mL
BNJ441 human monoclonal antibody, between 65 mM to 300 mM sodium
chloride, and 0 or 50 mM L-arginine, and the fluids having been
either pre-filtered using a 0.1 .mu.m filter or a Sartorius
Virosart.RTM. Max pre-filter.
[0042] FIG. 8 is a graph showing the relationship of the throughput
at 50% flow decay for a Virosart.RTM. CPV virus filter for fluids
including between 2.91 mg/mL to 3.54 mg/mL BNJ441 human monoclonal
antibody, between 65 mM to 300 mM sodium chloride, and 0 or 50 mM
L-arginine, and having a pH of between 5.5 and 7.6, that were
either pre-filtered using a 0.1 .mu.m filter or a Sartorius
Virosart.RTM. Max pre-filter, to the amount of L-arginine present
in each fluid.
[0043] FIG. 9 is a graph showing the relationship of the throughput
at 50% flow decay for a Virosart.RTM. CPV virus filter for fluids
including between 2.91 mg/mL to 3.54 mg/mL BNJ441 human monoclonal
antibody, between 65 mM to 300 mM sodium chloride, and 0 or 50 mM
L-arginine, and having a pH of between 5.5 and 7.6, that were
either pre-filtered using a 0.1 .mu.m filter or a Sartorius
Virosart.RTM. Max pre-filter, to the concentration of sodium
chloride present in each fluid.
[0044] FIG. 10 is a graph showing the relationship of the
throughput at 50% flow decay for a Virosart.RTM. CPV virus filter
for fluids including between 2.91 mg/mL to 3.54 mg/mL BNJ441 human
monoclonal antibody, between 65 mM to 300 mM sodium chloride, and 0
or 50 mM L-arginine, and having a pH of between 5.5 and 7.6, that
were either pre-filtered using 0.1 .mu.m filter or a Sartorius
Virosart.RTM. Max pre-filter, to the percentage of soluble protein
aggregates present in each fluid.
[0045] FIG. 11 is a graph showing the relationship of the
throughput at 50% flow decay for a Virosart.RTM. CPV virus filter
for fluids including between 2.91 mg/mL to 3.54 mg/mL BNJ441 human
monoclonal antibody, between 65 mM to 300 mM sodium chloride, and 0
or 50 mM L-arginine, and having a pH of between 5.5 and 7.6, that
were either pre-filtered using 0.1 .mu.m filter or a Sartorius
Virosart.RTM. Max pre-filter, to the average particle concentration
(number per mL of fluid).
[0046] FIG. 12 is a graph showing the relationship between
throughput at 50% flow decay for a Virosart.RTM. CPV virus filter
for fluids including between 2.91 mg/mL to 3.54 mg/mL BNJ441 human
monoclonal antibody, between 65 mM to 300 mM sodium chloride, and 0
or 50 mM L-arginine, and having a pH of between 5.5 and 7.6, that
were either pre-filtered using 0.1 .mu.m filter or a Sartorius
Virosart.RTM. Max pre-filter; the concentration of sodium chloride
in each fluid; and the pH of each fluid.
[0047] FIG. 13 is a graph showing the relationship between the
percentage of soluble protein aggregates present in fluids
including between 2.91 mg/mL to 3.54 mg/mL BNJ441 human monoclonal
antibody, between 65 mM to 300 mM sodium chloride, and 0 or 50 mM
L-arginine, and having a pH of between 5.5 and 7.6, that were
either pre-filtered using 0.1 .mu.m filter or a Virosart
Sartorius.RTM. Max pre-filter; the concentration of sodium chloride
in each fluid; and the pH of each fluid.
[0048] FIG. 14 is a graph showing the relationship between the
throughput at 50% flow decay for a Virosart.RTM. CPV virus filter
for fluids that had been pre-filtered using a 0.1 .mu.m filter or a
Sartorius Virosart.RTM. Max pre-filter, and including between 2.91
mg/mL to 3.54 mg/mL BNJ441 human monoclonal antibody, between 65 mM
to 300 mM sodium chloride, and 0 or 50 mM L-arginine; the pH of
each fluid; and the concentration of sodium chloride in each
fluid.
[0049] FIG. 15 is a graph showing the relationship between the
hydrodynamic radius of BNJ441 human monoclonal antibody in fluids
including between 2.91 mg/mL to 3.54 mg/mL BNJ441 human monoclonal
antibody, between 65 mM to 300 mM sodium chloride, and 0 or 50 mM
L-arginine, and having a pH of between 5.5 and 7.6, that were
either previously pre-filtered using 0.1 .mu.m filter or a
Sartorius Virosart.RTM. Max pre-filter; the pH of each fluid; and
the sodium chloride concentration of each fluid.
[0050] FIG. 16 is a graph showing the relationship between the
hydrodynamic radius of BNJ441 human monoclonal antibody in fluids
including between 2.91 mg/mL to 3.54 mg/mL BNJ441 human monoclonal
antibody, between 65 mM to 300 mM sodium chloride, and 0 or 50 mM
L-arginine, and having a pH of between 5.5 and 7.6, that were
either previously pre-filtered using 0.1 .mu.m filter or a
Sartorius Virosart.RTM. Max pre-filter; the pH of each fluid; and
the throughput at 50% flow decay when each fluid is flowed through
a Virosart.RTM. CPV virus filter.
[0051] FIG. 17 is a graph showing the relationship between the
percentage of aggregates of BNJ441 human monoclonal antibody in
fluids including between 2.91 mg/mL to 3.54 mg/mL BNJ441 human
monoclonal antibody, between 65 mM to 300 mM sodium chloride, and 0
or 50 mM L-arginine, and having a pH of between 5.5 and 7.6, that
were either previously pre-filtered using 0.1 .mu.m filter or a
Sartorius Virosart.RTM. Max pre-filter; the concentration of sodium
chloride in each fluid; and the throughput at 50% flow decay when
each fluid is flowed through a Virosart.RTM. CPV virus filter.
[0052] FIG. 18 is a graph showing the minimum concentration of
stabilizing agent necessary to achieve a Virosart.RTM. CPV virus
filter throughput of 750 g/m.sup.2 or higher for a fluid having a
pH of between 5.5 and 7.6, and including between 2.91 mg/mL to 3.54
mg/mL BNJ441 human monoclonal antibody.
[0053] FIG. 19 is a graph showing the flux as compared to
throughput when a fluid including 4 mg/mL BNJ441 human monoclonal
antibody and 65 mM sodium chloride, and having a pH of 7.0 was
flowed through an Asahi Planova.RTM. BioEx virus filter.
[0054] FIG. 20 is a graph showing the flux as compared to
throughput when a fluid including 4 mg/mL BNJ441 human monoclonal
antibody and 65 mM sodium chloride, and having a pH of 7.75 was
flowed through an Asahi Planova.RTM. BioEx virus filter.
[0055] FIG. 21 is a graph showing the flux as compared to
throughput when a fluid including 4 mg/mL BNJ441 human monoclonal
antibody and 65 mM sodium chloride, and having a pH of 8.5 was
flowed through an Asahi Planova.RTM. BioEx virus filter.
[0056] FIG. 22 is a graph showing the flux decay as compared to
throughput when a fluid including 4 mg/mL BNJ441 human monoclonal
antibody and 65 mM sodium chloride, and having a pH of 7.75 was
flowed through an Asahi Planova.RTM. 20N virus filter.
[0057] FIG. 23 is a graph showing the flux decay as compared to
throughput when a variety of fluids including between 5 mg/mL to 15
mg/mL samalizumab and between 75 mM to 300 mM sodium chloride, and
having a pH of 5.0, 5.5, or 6.0 were flowed through a Virosart.RTM.
CPV virus filter.
[0058] FIG. 24 is a set of three graphs showing the relationship
between throughput of a variety of fluids including between 5 mg/mL
to 15 mg/mL samalizumab and between 75 mM to 300 mM sodium
chloride, and having a pH of 5.0, 5.5, or 6.0 were flowed through a
Virosart.RTM. CPV virus filter and the pH of each fluid (left
graph), the concentration of sodium chloride in each fluid (center
graph), and the concentration of samalizumab present in each fluid
(right graph). The relationships were determined using statistical
analysis.
[0059] FIG. 25 is graph showing the flux decay as compared to
throughput when a variety of fluids including between 9.3 mg/mL to
10 mg/mL BNJ383 monoclonal antibody and between 80 mM to 300 mM
sodium chloride, and having a pH of between 7.0 and 8.5 were flowed
through a Virosart.RTM. CPV virus filter or a Virosart.RTM. HF
virus filter.
[0060] FIG. 26 is a set of two graphs showing the relationship
between throughput of a variety of fluids including between 9.3
mg/mL to 10 mg/mL BNJ383 monoclonal antibody and between 80 mM to
300 mM sodium chloride, and having a pH of between 7.0 and 8.5 were
flowed through a Virosart.RTM. CPV virus filter or a Virosart.RTM.
HF filter and the pH of each fluid (left graph) and the
concentration of sodium chloride in each fluid (right graph). The
relationships were determined using statistical analysis.
[0061] FIG. 27 is a graph showing the flux decay as compared to the
throughput of fluid including 7.1 mg/mL eculizumab, 20 mM sodium
phosphate, and 80 mM sodium chloride, and having a pH of 6.5, that
had been previously pre-filtered using a Millipore 0.5/0.2 .mu.m
and 0.5/0.1 .mu.m pre-filter, a Sartorius Virosart.RTM. Max
pre-filter, a Sartopore.RTM. 2 pre-filter, Sartobind STIC.RTM.
pre-filter, Sartobind.RTM. Q pre-filter, Sartobind.RTM. HIC Phenyl
pre-filter, or a Sartobind.RTM. S pre-filter, that is passed
through a Virosart.RTM. CPV virus filter.
[0062] FIG. 28 is a graph showing the percentage of protein
aggregates present in fluids including 7.1 mg/mL eculizumab, 20 mM
sodium phosphate, and 80 mM sodium chloride, and having a pH of
6.5, after the fluid had been pre-filtered using a Millipore
0.5/0.2 .mu.m and 0.5/0.1 .mu.m pre-filter, a Sartorius
Virosart.RTM. Max pre-filter, a Sartopore.RTM. 2 pre-filter,
Sartobind STIC.RTM. pre-filter, Sartobind.RTM. Q pre-filter,
Sartobind.RTM. HIC Phenyl pre-filter, or a Sartobind.RTM. S
pre-filter, as compared to the throughput of a Virosart.RTM. CPV
virus filter when each fluid is passed through the Virosart.RTM.
CPV virus filter.
[0063] FIG. 29 is a graph showing the number of particles present
in fluids including 7.1 mg/mL eculizumab, 20 mM sodium phosphate,
and 80 mM sodium chloride, and having a pH of 6.5, after the fluid
had been pre-filtered using Millipore 0.5/0.2 .mu.m and 0.5/0.1
.mu.m pre-filter, a Sartorius Virosart.RTM. Max pre-filter, a
Sartopore 2 pre-filter, Sartobind STIC pre-filter, Sartobind Q
pre-filter, Sartobind HIC Phenyl pre-filter, or a Sartobind S
pre-filter, and prior to each fluid being subsequently flowed
through a Virosart.RTM. CPV virus filter, as compared to the
throughput of the Virosart.RTM. CPV virus filter when each fluid is
passed through the Virosart.RTM. CPV virus filter.
[0064] FIG. 30 is a schematic showing the experimental protocols
used to perform pre-filtration and filtration with a Virosart.RTM.
CPV virus filter in Example 6.
[0065] FIG. 31 is a graph of the percentage flux decay as a
function of the loading capacity of a Virosart.RTM. CPV virus
filter from three different lots (Lots A-C). The material loaded
onto each Virosart.RTM. CPV virus filter was pre-filtered using a
3.5 cm.sup.2 Millipore 0.5/0.2 .mu.m pre-filter (SHC), and a 0.5
.mu.m/0.1 .mu.m pre-filter (SHR). A Virosart.RTM. CPV virus filter
from each lot was run in duplicate.
[0066] FIG. 32 is a graph of the flux over time of a Virosart.RTM.
CPV virus filter from three different lots (Lots A-C). The material
loaded onto each Virosart.RTM. CPV virus filter was pre-filtered
using a 3.5 cm.sup.2 Millipore 0.5/0.2 .mu.m pre-filter (SHC), and
a 0.5 .mu.m/0.1 .mu.m pre-filter (SHR). A Virosart.RTM. CPV virus
filter from each lot was run in duplicate.
[0067] FIG. 33 is a graph of the percentage flux decay as a
function of the loading capacity of a Virosart.RTM. CPV virus
filter loaded with eluate produced using a 3.5 cm.sup.2 Millipore
0.5/0.2 .mu.m pre-filter (SHC), and a 3.5 cm.sup.2 0.5 .mu.m/0.1
.mu.m pre-filter (SHR) run under one of two different conditions:
622 L/m.sup.2 (218 mL flowed through the 3.5 cm.sup.2 SHC and 3.5
cm.sup.2 SHR) or 311 L/m.sup.2 (109 mL through the 3.5 cm.sup.2 SHC
and 3.5 cm.sup.2 SHR).
[0068] FIG. 34 is a graph of the flux over time of a Virosart.RTM.
CPV virus filter loaded with eluate produced using a 3.5 cm.sup.2
Millipore 0.5/0.2 .mu.m pre-filter (SHC), and a 3.5 cm.sup.2 0.5
.mu.m/0.1 .mu.m pre-filter (SHR) run under one of two different
conditions: 622 L/m.sup.2 (218 mL flowed through the 3.5 cm.sup.2
SHC and 3.5 cm.sup.2 SEM) or 311 L/m.sup.2 (109 mL through the 3.5
cm.sup.2 SHC and 3.5 cm.sup.2 SHR).
[0069] FIG. 35 is a graph of the percentage flux decay as a
function of the loading capacity of a Virosart.RTM. CPV virus
filter runs, with a feed pressure of either 15 psi or 30 psi. The
material loaded onto the Virosart.RTM. CPV virus filter in-line
with a 3.5 cm.sup.2 Millipore 0.5/0.2 .mu.m pre-filter (SHC),
followed by a 3.5 cm.sup.2 0.5 .mu.m/0.1 .mu.m pre-filter (SHR), or
in-line with a Sartorius Virosart.RTM. Max pre-filter (5
cm.sup.2).
[0070] FIG. 36 is a graph of the flux over time of a Virosart.RTM.
CPV virus filter runs with a feed pressure of either 15 psi or 30
psi. The material was loaded onto the Virosart.RTM. CPV virus
filter in-line with a 3.5 cm.sup.2 Millipore 0.5/0.2 .mu.m
pre-filter (SHC), followed by a 3.5 cm.sup.2 0.5 .mu.m/0.1 .mu.m
pre-filter (SHR), or in-line with a Sartorius Virosart.RTM. Max
pre-filter (5 cm.sup.2).
[0071] FIG. 37 is a graph of the percentage flux decay as a
function of loading capacity of a Virosart.RTM. CPV virus filter
run with a feed pressure of 27-33 psi, and a buffer chase of 12.5
mL. The material loaded onto the Virosart.RTM. CPV virus filter was
generated by loading a liquid including 4 mg/mL or 8 mg/mL
eculizumab at 623 L/m.sup.2 onto a 3.5 cm.sup.2 Millipore 0.5/0.2
.mu.m pre-filter (SHC), and a 3.5 cm.sup.2 0.5 .mu.m/0.1 .mu.m
pre-filter (SHR), or a Sartorius Virosart.RTM. Max pre-filter (5
cm.sup.2).
[0072] FIG. 38 is a graph of the flux over time of a Virosart.RTM.
CPV virus filter run with a feed pressure of 27-33 psi, and a
buffer chase of 12.5 mL. The material loaded onto the Virosart.RTM.
CPV virus filter was generated by loading a liquid including 4
mg/mL or 8 mg/mL eculizumab at 623 L/m.sup.2 onto a 3.5 cm.sup.2
Millipore 0.5/0.2 .mu.m pre-filter (SHC), and a 3.5 cm.sup.2 0.5
.mu.m/0.1 .mu.m pre-filter (SHR), or a Sartorius Virosart.RTM. Max
pre-filter (5 cm.sup.2).
[0073] FIG. 39 is a graph of the percentage of flux decay as a
function of loading capacity Virosart.RTM. CPV virus filter run at
200 L/m.sup.2, with a feed pressure of 30 psi, and a buffer chase
of 12.5 mL. The material loaded onto the Virosart.RTM. CPV virus
filter was generated by loading a 3.5 cm.sup.2 Millipore 0.5/0.2
.mu.m pre-filter (SHC), and a 3.5 cm.sup.2 0.5 .mu.m/0.1 .mu.m
pre-filter (SHR), or a Sartorius Virosart.RTM. Max pre-filter (5
cm.sup.2). During filtration with the Virosart.RTM. CPV virus
filter, if an 80% flux decay was reached, the filtration was paused
for 60 minutes and the pre-filter eluate loaded onto the
Virosart.RTM. CPV virus filter was diluted 4-fold in buffer.
[0074] FIG. 40 is a graph of the flux over time of a Virosart.RTM.
CPV virus filter run at 200 L/m.sup.2, with a feed pressure of 30
psi, and a buffer chase of 12.5 mL. The material loaded onto the
Virosart.RTM. CPV virus filter was generated by loading a 3.5
cm.sup.2 Millipore 0.5/0.2 .mu.m pre-filter (SHC), and a 3.5
cm.sup.2 0.5 .mu.m/0.1 .mu.m pre-filter (SHR), or a Sartorius
Virosart.RTM. Max pre-filter (5 cm.sup.2). During filtration with
the Virosart.RTM. CPV virus filter, if an 80% flux decay was
reached, the filtration was paused for 60 minutes and the
pre-filter eluate loaded onto the Virosart.RTM. CPV virus filter
was diluted 4-fold in buffer.
[0075] FIG. 41 is a schematic showing the different recombinant
fusion protein purification processes tested in Example 10.
[0076] FIG. 42 is a graph of the percentage of flux decay as a
function of throughput of a Virosart.RTM. CPV virus filter in each
of the different recombinant fusion protein purification processes
tested in Example 10.
[0077] FIG. 43 is a graph showing the percentage of soluble protein
aggregates and the level of insoluble protein particles in
recombinant fusion protein purified at the end of each of the
different purification processes tested in Example 10.
[0078] FIG. 44A is a graph showing the ability of each of three
different tested depth filters to reduce the level of host cell
protein in a fluid including Alexion 1210 and host cell
protein.
[0079] FIG. 44B is a graph showing the level of host cell proteins
and soluble protein aggregates in Alexion 1210 purified using a
purification process that does not include the use of a depth
filter having anionic and hydrophobic properties prior to the viral
filtration step (as described in Example 10) and an optimized
purification process that includes the use of a wash buffer in the
protein A capture step and the use of a depth filter having anionic
and hydrophobic properties prior to the viral filtration step (as
described in Example 10).
DETAILED DESCRIPTION
[0080] During the manufacturing of a recombinant antibody, a fluid
including a recombinant antibody is typically flowed through a
virus filter in order to improve the safety of the manufactured
recombinant antibody. This step of viral filtration can be a rate
limiting step in the manufacture of a recombinant antibody if the
virus filter used to perform the viral filtration has a low
throughput (e.g., a throughput of less than, e.g., 500 g/m.sup.2).
Provided herein are methods of viral filtration that can result in
an throughput of a virus filter of greater than 550 g/m.sup.2
(e.g., between about 550 g/m.sup.2 and about 15,000 g/m.sup.2,
between about 550 g/m.sup.2 and about 14,000 g/m.sup.2, between
about 550 g/m.sup.2 and about 13,000 g/m.sup.2, between about 550
g/m.sup.2 and about 12,000 g/m.sup.2, between about 550 g/m.sup.2
and about 11,000 g/m.sup.2, between about 550 g/m.sup.2 and about
10,000 g/m.sup.2, between about 550 g/m.sup.2 and about 9,000
g/m.sup.2, between about 550 g/m.sup.2 and about 8,000 g/m.sup.2,
between about 550 g/m.sup.2 and about 7,000 g/m.sup.2, between
about 550 g/m.sup.2 and about 6,000 g/m.sup.2, between about 550
g/m.sup.2 and about 5,000 g/m.sup.2, between about 550 g/m.sup.2
and about 4,000 g/m.sup.2, between about 550 g/m.sup.2 and about
3,000 g/m.sup.2, between about 550 g/m.sup.2 and about 2,500
g/m.sup.2, between about 550 g/m.sup.2 and about 2,000 g/m.sup.2,
between about 550 g/m.sup.2 and about 1,500 g/m.sup.2, between
about 550 g/m.sup.2 and about 1,000 g/m.sup.2, between about 600
g/m.sup.2 and about 15,000 g/m.sup.2, between about 600 g/m.sup.2
and about 14,000 g/m.sup.2, between about 600 g/m.sup.2 and about
13,000 g/m.sup.2, between about 600 g/m.sup.2 and about 12,000
g/m.sup.2, between about 600 g/m.sup.2 and about 11,000 g/m.sup.2,
between about 600 g/m.sup.2 and about 10,000 g/m.sup.2, between
about 600 g/m.sup.2 and about 9,000 g/m.sup.2, between about 600
g/m.sup.2 and about 8,000 g/m.sup.2, between about 600 g/m.sup.2
and about 7,000 g/m.sup.2, between about 600 g/m.sup.2 and about
6,000 g/m.sup.2, between about 600 g/m.sup.2 and about 5,000
g/m.sup.2, between about 600 g/m.sup.2 and about 4,000 g/m.sup.2,
between about 600 g/m.sup.2 and about 3,000 g/m.sup.2, between
about 600 g/m.sup.2 and about 2,500 g/m.sup.2, between about 600
g/m.sup.2 and about 2,000 g/m.sup.2, between about 600 g/m.sup.2
and about 1,500 g/m.sup.2, between about 600 g/m.sup.2 and about
1,000 g/m.sup.2, between about 700 g/m.sup.2 and about 15,000
g/m.sup.2, between about 700 g/m.sup.2 and about 14,000 g/m.sup.2,
between about 700 g/m.sup.2 and about 13,000 g/m.sup.2, between
about 700 g/m.sup.2 and about 12,000 g/m.sup.2, between about 700
g/m.sup.2 and about 11,000 g/m.sup.2, between about 700 g/m.sup.2
and about 10,000 g/m.sup.2, between about 700 g/m.sup.2 and about
9,000 g/m.sup.2, between about 700 g/m.sup.2 and about 8,000
g/m.sup.2, between about 700 g/m.sup.2 and about 7,000 g/m.sup.2,
between about 700 g/m.sup.2 and about 6,000 g/m.sup.2, between
about 700 g/m.sup.2 and about 5,000 g/m.sup.2, between about 700
g/m.sup.2 and about 4,000 g/m.sup.2, between about 700 g/m.sup.2
and about 3,000 g/m.sup.2, between about 700 g/m.sup.2 and about
2,500 g/m.sup.2, between about 700 g/m.sup.2 and about 2,000
g/m.sup.2, between about 700 g/m.sup.2 and about 1,500 g/m.sup.2,
between about 700 g/m.sup.2 and about 1,000 g/m.sup.2, between
about 800 g/m.sup.2 and about 15,000 g/m.sup.2, between about 800
g/m.sup.2 and about 14,000 g/m.sup.2, between about 800 g/m.sup.2
and about 13,000 g/m.sup.2, between about 800 g/m.sup.2 and about
12,000 g/m.sup.2, between about 800 g/m.sup.2 and about 11,000
g/m.sup.2, between about 800 g/m.sup.2 and about 10,000 g/m.sup.2,
between about 800 g/m.sup.2 and about 9,000 g/m.sup.2, between
about 800 g/m.sup.2 and about 8,000 g/m.sup.2, between about 800
g/m.sup.2 and about 7,000 g/m.sup.2, between about 800 g/m.sup.2
and about 6,000 g/m.sup.2, between about 800 g/m.sup.2 and about
5,000 g/m.sup.2, between about 800 g/m.sup.2 and about 4,000
g/m.sup.2, between about 800 g/m.sup.2 and about 3,000 g/m.sup.2,
between about 800 g/m.sup.2 and about 2,500 g/m.sup.2, between
about 800 g/m.sup.2 and about 2,000 g/m.sup.2, between about 800
g/m.sup.2 and about 1,500 g/m.sup.2, between about 800 g/m.sup.2
and about 1,000 g/m.sup.2, between about 900 g/m.sup.2 and about
15,000 g/m.sup.2, between about 900 g/m.sup.2 and about 14,000
g/m.sup.2, between about 900 g/m.sup.2 and about 13,000 g/m.sup.2,
between about 900 g/m.sup.2 and about 12,000 g/m.sup.2, between
about 900 g/m.sup.2 and about 11,000 g/m.sup.2, between about 900
g/m.sup.2 and about 10,000 g/m.sup.2, between about 900 g/m.sup.2
and about 9,000 g/m.sup.2, between about 900 g/m.sup.2 and about
8,000 g/m.sup.2, between about 900 g/m.sup.2 and about 7,000
g/m.sup.2, between about 900 g/m.sup.2 and about 6,000 g/m.sup.2,
between about 900 g/m.sup.2 and about 5,000 g/m.sup.2, between
about 900 g/m.sup.2 and about 4,000 g/m.sup.2, between about 900
g/m.sup.2 and about 3,000 g/m.sup.2, between about 900 g/m.sup.2
and about 2,500 g/m.sup.2, between about 900 g/m.sup.2 and about
2,000 g/m.sup.2, between about 900 g/m.sup.2 and about 1,500
g/m.sup.2, between about 900 g/m.sup.2 and about 1,000 g/m.sup.2,
between about 1,000 g/m.sup.2 and about 15,000 g/m.sup.2, between
about 1,000 g/m.sup.2 and about 14,000 g/m.sup.2, between about
1,000 g/m.sup.2 and about 13,000 g/m.sup.2, between about 1,000
g/m.sup.2 and about 12,000 g/m.sup.2, between about 1,000 g/m.sup.2
and about 11,000 g/m.sup.2, between about 1,000 g/m.sup.2 and about
10,000 g/m.sup.2, between about 1,000 g/m.sup.2 and about 9,000
g/m.sup.2, between about 1,000 g/m.sup.2 and about 8,000 g/m.sup.2,
between about 1,000 g/m.sup.2 and about 7,000 g/m.sup.2, between
about 1,000 g/m.sup.2 and about 6,000 g/m.sup.2, between about
1,000 g/m.sup.2 and about 5,000 g/m.sup.2, between about 1,000
g/m.sup.2 and about 4,000 g/m.sup.2, between about 1,000 g/m.sup.2
and about 3,000 g/m.sup.2, between about 1,000 g/m.sup.2 and about
2,500 g/m.sup.2, between about 1,000 g/m.sup.2 and about 2,000
g/m.sup.2, between about 1,000 g/m.sup.2 and about 1,500 g/m.sup.2,
between about 1,100 g/m.sup.2 and about 15,000 g/m.sup.2, between
about 1,100 g/m.sup.2 and about 14,000 g/m.sup.2, between about
1,100 g/m.sup.2 and about 13,000 g/m.sup.2, between about 1,100
g/m.sup.2 and about 12,000 g/m.sup.2, between about 1,100 g/m.sup.2
and about 11,000 g/m.sup.2, between about 1,100 g/m.sup.2 and about
10,000 g/m.sup.2, between about 1,100 g/m.sup.2 and about 9,000
g/m.sup.2, between about 1,100 g/m.sup.2 and about 8,000 g/m.sup.2,
between about 1,100 g/m.sup.2 and about 7,000 g/m.sup.2, between
about 1,100 g/m.sup.2 and about 6,000 g/m.sup.2, between about
1,100 g/m.sup.2 and about 5,000 g/m.sup.2, between about 1,100
g/m.sup.2 and about 4,000 g/m.sup.2, between about 1,100 g/m.sup.2
and about 3,000 g/m.sup.2, between about 1,100 g/m.sup.2 and about
2,500 g/m.sup.2, between about 1,100 g/m.sup.2 and about 2,000
g/m.sup.2, between about 1,100 g/m.sup.2 and about 1,500 g/m.sup.2,
between about 1,200 g/m.sup.2 and about 15,000 g/m.sup.2, between
about 1,200 g/m.sup.2 and about 14,000 g/m.sup.2, between about
1,200 g/m.sup.2 and about 13,000 g/m.sup.2, between about 1,200
g/m.sup.2 and about 12,000 g/m.sup.2, between about 1,200 g/m.sup.2
and about 11,000 g/m.sup.2, between about 1,200 g/m.sup.2 and about
10,000 g/m.sup.2, between about 1,200 g/m.sup.2 and about 9,000
g/m.sup.2, between about 1,200 g/m.sup.2 and about 8,000 g/m.sup.2,
between about 1,200 g/m.sup.2 and about 7,000 g/m.sup.2, between
about 1,200 g/m.sup.2 and about 6,000 g/m.sup.2, between about
1,200 g/m.sup.2 and about 5,000 g/m.sup.2, between about 1,200
g/m.sup.2 and about 4,000 g/m.sup.2, between about 1,200 g/m.sup.2
and about 3,000 g/m.sup.2, between about 1,200 g/m.sup.2 and about
2,500 g/m.sup.2, between about 1,200 g/m.sup.2 and about 2,000
g/m.sup.2, between about 1,200 g/m.sup.2 and about 1,500 g/m.sup.2,
between about 1,300 g/m.sup.2 and about 15,000 g/m.sup.2, between
about 1,300 g/m.sup.2 and about 14,000 g/m.sup.2, between about
1,300 g/m.sup.2 and about 13,000 g/m.sup.2, between about 1,300
g/m.sup.2 and about 12,000 g/m.sup.2, between about 1,300 g/m.sup.2
and about 11,000 g/m.sup.2, between about 1,300 g/m.sup.2 and about
10,000 g/m.sup.2, between about 1,300 g/m.sup.2 and about 9,000
g/m.sup.2, between about 1,300 g/m.sup.2 and about 8,000 g/m.sup.2,
between about 1,300 g/m.sup.2 and about 7,000 g/m.sup.2, between
about 1,300 g/m.sup.2 and about 6,000 g/m.sup.2, between about
1,300 g/m.sup.2 and about 5,000 g/m.sup.2, between about 1,300
g/m.sup.2 and about 4,000 g/m.sup.2, between about 1,300 g/m.sup.2
and about 3,000 g/m.sup.2, between about 1,300 g/m.sup.2 and about
2,500 g/m.sup.2, between about 1,300 g/m.sup.2 and about 2,000
g/m.sup.2, between about 1,400 g/m.sup.2 and about 15,000
g/m.sup.2, between about 1,400 g/m.sup.2 and about 14,000
g/m.sup.2, between about 1,400 g/m.sup.2 and about 13,000
g/m.sup.2, between about 1,400 g/m.sup.2 and about 12,000
g/m.sup.2, between about 1,400 g/m.sup.2 and about 11,000
g/m.sup.2, between about 1,400 g/m.sup.2 and about 10,000
g/m.sup.2, between about 1,400 g/m.sup.2 and about 9,000 g/m.sup.2,
between about 1,400 g/m.sup.2 and about 8,000 g/m.sup.2, between
about 1,400 g/m.sup.2 and about 7,000 g/m.sup.2, between about
1,400 g/m.sup.2 and about 6,000 g/m.sup.2, between about 1,400
g/m.sup.2 and about 5,000 g/m.sup.2, between about 1,400 g/m.sup.2
and about 4,000 g/m.sup.2, between about 1,400 g/m.sup.2 and about
3,000 g/m.sup.2, between about 1,400 g/m.sup.2 and about 2,500
g/m.sup.2, between about 1,400 g/m.sup.2 and about 2,000 g/m.sup.2,
between about 1,500 g/m.sup.2 and about 15,000 g/m.sup.2, between
about 1,500 g/m.sup.2 and about 14,000 g/m.sup.2, between about
1,500 g/m.sup.2 and about 13,000 g/m.sup.2, between about 1,500
g/m.sup.2 and about 12,000 g/m.sup.2, between about 1,500 g/m.sup.2
and about 11,000 g/m.sup.2, between about 1,500 g/m.sup.2 and about
10,000 g/m.sup.2, between about 1,500 g/m.sup.2 and about 9,000
g/m.sup.2, between about 1,500 g/m.sup.2 and about 8,000 g/m.sup.2,
between about 1,500 g/m.sup.2 and about 7,000 g/m.sup.2, between
about 1,500 g/m.sup.2 and about 6,000 g/m.sup.2, between about
1,500 g/m.sup.2 and about 5,000 g/m.sup.2, between about 1,500
g/m.sup.2 and about 4,000 g/m.sup.2, between about 1,500 g/m.sup.2
and about 3,000 g/m.sup.2, between about 1,500 g/m.sup.2 and about
2,500 g/m.sup.2, between about 1,500 g/m.sup.2 and about 2,000
g/m.sup.2, between about 1,600 g/m.sup.2 and about 15,000
g/m.sup.2, between about 1,600 g/m.sup.2 and about 14,000
g/m.sup.2, between about 1,600 g/m.sup.2 and about 13,000
g/m.sup.2, between about 1,600 g/m.sup.2 and about 12,000
g/m.sup.2, between about 1,600 g/m.sup.2 and about 11,000
g/m.sup.2, between about 1,600 g/m.sup.2 and about 10,000
g/m.sup.2, between about 1,600 g/m.sup.2 and about 9,000 g/m.sup.2,
between about 1,600 g/m.sup.2 and about 8,000 g/m.sup.2, between
about 1,600 g/m.sup.2 and about 7,000 g/m.sup.2, between about
1,600 g/m.sup.2 and about 6,000 g/m.sup.2, between about 1,600
g/m.sup.2 and about 5,000 g/m.sup.2, between about 1,600 g/m.sup.2
and about 4,000 g/m.sup.2, between about 1,600 g/m.sup.2 and about
3,000 g/m.sup.2, between about 1,600 g/m.sup.2 and about 2,500
g/m.sup.2, between about 1,600 g/m.sup.2 and about 2,000 g/m.sup.2,
between about 1,700 g/m.sup.2 and about 15,000 g/m.sup.2, between
about 1,700 g/m.sup.2 and about 14,000 g/m.sup.2, between about
1,700 g/m.sup.2 and about 13,000 g/m.sup.2, between about 1,700
g/m.sup.2 and about 12,000 g/m.sup.2, between about 1,700 g/m.sup.2
and about 11,000 g/m.sup.2, between about 1,700 g/m.sup.2 and about
10,000 g/m.sup.2, between about 1,700 g/m.sup.2 and about 9,000
g/m.sup.2, between about 1,700 g/m.sup.2 and about 8,000 g/m.sup.2,
between about 1,700 g/m.sup.2 and about 7,000 g/m.sup.2, between
about 1,700 g/m.sup.2 and about 6,000 g/m.sup.2, between about
1,700 g/m.sup.2 and about 5,000 g/m.sup.2, between about 1,700
g/m.sup.2 and about 4,000 g/m.sup.2, between about 1,700 g/m.sup.2
and about 3,000 g/m.sup.2, between about 1,700 g/m.sup.2 and about
2,500 g/m.sup.2, between about 1,800 g/m.sup.2 and about 15,000
g/m.sup.2, between about 1,800 g/m.sup.2 and about 14,000
g/m.sup.2, between about 1,800 g/m.sup.2 and about 13,000
g/m.sup.2, between about 1,800 g/m.sup.2 and about 12,000
g/m.sup.2, between about 1,800 g/m.sup.2 and about 11,000
g/m.sup.2, between about 1,800 g/m.sup.2 and about 10,000
g/m.sup.2, between about 1,800 g/m.sup.2 and about 9,000 g/m.sup.2,
between about 1,800 g/m.sup.2 and about 8,000 g/m.sup.2, between
about 1,800 g/m.sup.2 and about 7,000 g/m.sup.2, between about
1,800 g/m.sup.2 and about 6,000 g/m.sup.2, between about 1,800
g/m.sup.2 and about 5,000 g/m.sup.2, between about 1,800 g/m.sup.2
and about 4,000 g/m.sup.2, between about 1,800 g/m.sup.2 and about
3,000 g/m.sup.2, between about 1,800 g/m.sup.2 and about 2,500
g/m.sup.2, between about 1,800 g/m.sup.2 and about 2,000 g/m.sup.2,
between about 2,000 g/m.sup.2 and about 15,000 g/m.sup.2, between
about 2,000 g/m.sup.2 and about 14,000 g/m.sup.2, between about
2,000 g/m.sup.2 and about 13,000 g/m.sup.2, between about 2,000
g/m.sup.2 and about 12,000 g/m.sup.2, between about 2,000 g/m.sup.2
and about 11,000 g/m.sup.2, between about 2,000 g/m.sup.2 and about
10,000 g/m.sup.2, between about 2,000 g/m.sup.2 and about 9,000
g/m.sup.2, between about 2,000 g/m.sup.2 and about 8,000 g/m.sup.2,
between about 2,000 g/m.sup.2 and about 7,000 g/m.sup.2, between
about 2,000 g/m.sup.2 and about 6,000 g/m.sup.2, between about
2,000 g/m.sup.2 and about 5,000 g/m.sup.2, between about 2,000
g/m.sup.2 and about 4,000 g/m.sup.2, between about 2,000 g/m.sup.2
and about 3,000 g/m.sup.2, between about 2,500 g/m.sup.2 and about
15,000 g/m.sup.2, between about 2,500 g/m.sup.2 and about 14,000
g/m.sup.2, between about 2,500 g/m.sup.2 and about 13,000
g/m.sup.2, between about 2,500 g/m.sup.2 and about 12,000
g/m.sup.2, between about 2,500 g/m.sup.2 and about 11,000
g/m.sup.2, between about 2,500 g/m.sup.2 and about 10,000
g/m.sup.2, between about 2,500 g/m.sup.2 and about 9,000 g/m.sup.2,
between about 2,500 g/m.sup.2 and about 8,000 g/m.sup.2, between
about 2,500 g/m.sup.2 and about 7,000 g/m.sup.2, between about
2,500 g/m.sup.2 and about 6,000 g/m.sup.2, between about 2,500
g/m.sup.2 and about 5,000 g/m.sup.2, between about 2,500 g/m.sup.2
and about 4,000 g/m.sup.2, between about 2,500 g/m.sup.2 and about
3,000 g/m.sup.2, between about 3,000 g/m.sup.2 and about 15,000
g/m.sup.2, between about 3,000 g/m.sup.2 and about 14,000
g/m.sup.2, between about 3,000 g/m.sup.2 and about 13,000
g/m.sup.2, between about 3,000 g/m.sup.2 and about 12,000
g/m.sup.2, between about 3,000 g/m.sup.2 and about 11,000
g/m.sup.2, between about 3,000 g/m.sup.2 and about 10,000
g/m.sup.2, between about 3,000 g/m
.sup.2 and about 9,000 g/m.sup.2, between about 3,000 g/m.sup.2 and
about 8,000 g/m.sup.2, between about 3,000 g/m.sup.2 and about
7,000 g/m.sup.2, between about 3,000 g/m.sup.2 and about 6,000
g/m.sup.2, between about 3,000 g/m.sup.2 and about 5,000 g/m.sup.2,
between about 3,000 g/m.sup.2 and about 4,000 g/m.sup.2, between
about 4,000 g/m.sup.2 and about 15,000 g/m.sup.2, between about
4,000 g/m.sup.2 and about 14,000 g/m.sup.2, between about 4,000
g/m.sup.2 and about 13,000 g/m.sup.2, between about 4,000 g/m.sup.2
and about 12,000 g/m.sup.2, between about 4,000 g/m.sup.2 and about
11,000 g/m.sup.2, between about 4,000 g/m.sup.2 and about 10,000
g/m.sup.2, between about 4,000 g/m.sup.2 and about 9,000 g/m.sup.2,
between about 4,000 g/m.sup.2 and about 8,000 g/m.sup.2, between
about 4,000 g/m.sup.2 and about 7,000 g/m.sup.2, between about
4,000 g/m.sup.2 and about 6,000 g/m.sup.2, between about 4,000
g/m.sup.2 and about 5,000 g/m.sup.2, between about 5,000 g/m.sup.2
and about 15,000 g/m.sup.2, between about 5,000 g/m.sup.2 and about
14,000 g/m.sup.2, between about 5,000 g/m.sup.2 and about 13,000
g/m.sup.2, between about 5,000 g/m.sup.2 and about 12,000
g/m.sup.2, between about 5,000 g/m.sup.2 and about 11,000
g/m.sup.2, between about 5,000 g/m.sup.2 and about 10,000
g/m.sup.2, between about 5,000 g/m.sup.2 and about 9,000 g/m.sup.2,
between about 5,000 g/m.sup.2 and about 8,000 g/m.sup.2, between
about 5,000 g/m.sup.2 and about 7,000 g/m.sup.2, between about
5,000 g/m.sup.2 and about 6,000 g/m.sup.2, between about 6,000
g/m.sup.2 and about 15,000 g/m.sup.2, between about 6,000 g/m.sup.2
and about 14,000 g/m.sup.2, between about 6,000 g/m.sup.2 and about
13,000 g/m.sup.2, between about 6,000 g/m.sup.2 and about 12,000
g/m.sup.2, between about 6,000 g/m.sup.2 and about 11,000
g/m.sup.2, between about 6,000 g/m.sup.2 and about 10,000
g/m.sup.2, between about 6,000 g/m.sup.2 and about 9,000 g/m.sup.2,
between about 6,000 g/m.sup.2 and about 8,000 g/m.sup.2, between
about 6,000 g/m.sup.2 and about 7,000 g/m.sup.2, between about
7,000 g/m.sup.2 and about 15,000 g/m.sup.2, between about 7,000
g/m.sup.2 and about 14,000 g/m.sup.2, between about 7,000 g/m.sup.2
and about 13,000 g/m.sup.2, between about 7,000 g/m.sup.2 and about
12,000 g/m.sup.2, between about 7,000 g/m.sup.2 and about 11,000
g/m.sup.2, between about 7,000 g/m.sup.2 and about 10,000
g/m.sup.2, between about 7,000 g/m.sup.2 and about 9,000 g/m.sup.2,
between about 7,000 g/m.sup.2 and about 8,000 g/m.sup.2, between
about 8,000 g/m.sup.2 and about 15,000 g/m.sup.2, between about
8,000 g/m.sup.2 and about 14,000 g/m.sup.2, between about 8,000
g/m.sup.2 and about 13,000 g/m.sup.2, between about 8,000 g/m.sup.2
and about 12,000 g/m.sup.2, between about 8,000 g/m.sup.2 and about
11,000 g/m.sup.2, between about 8,000 g/m.sup.2 and about 10,000
g/m.sup.2, between about 8,000 g/m.sup.2 and about 9,000 g/m.sup.2,
between about 9,000 g/m.sup.2 and about 15,000 g/m.sup.2, between
about 9,000 g/m.sup.2 and about 14,000 g/m.sup.2, between about
9,000 g/m.sup.2 and about 13,000 g/m.sup.2, between about 9,000
g/m.sup.2 and about 12,000 g/m.sup.2, between about 9,000 g/m.sup.2
and about 11,000 g/m.sup.2, between about 9,000 g/m.sup.2 and about
10,000 g/m.sup.2, between about 10,000 g/m.sup.2 and about 15,000
g/m.sup.2, between about 10,000 g/m.sup.2 and about 14,000
g/m.sup.2, between about 10,000 g/m.sup.2 and about 13,000
g/m.sup.2, between about 10,000 g/m.sup.2 and about 12,000
g/m.sup.2, or between about 10,000 g/m.sup.2 and about 11,000
g/m.sup.2) when a fluid including a recombinant antibody is flowed
through the virus filter.
[0081] Also provided herein are methods of manufacturing a
recombinant antibody or methods of purifying a recombinant antibody
that include performing a unit operation of viral filtration (e.g.,
using any of the methods of performing viral filtration described
herein). Non-limiting aspects of the methods of performing viral
filtration and methods of manufacturing or purifying a recombinant
antibody are described herein.
Recombinant Antibodies
[0082] Exemplary recombinant antibodies include an IgG, IgE, IgD,
IgA, or IgM. A recombinant antibody can be any subclass of IgG,
such as, e.g., IgG1, IgG2, IgG3, or IgG4, or a chimeric antibody
(e.g., a IgG2/4 chimeric antibody, such as eculizumab). A
recombinant antibody can be an antigen-binding antibody fragment,
such as a Fab fragment, a F(a').sub.2 fragment, or an scFv
fragment. The recombinant antibody may be a bi-specific antibody or
a tri-specific antibody, or a dimer, trimer, or multimer antibody,
or a diabody, an AFFIBODY.RTM., or a NANOBODY.RTM.. The recombinant
antibody can be an engineered protein having four antibody binding
domains such as DVD-Ig and CODV-Ig. See, e.g., US2007/0071675 and
WO2012/135345. Non-limiting examples of recombinant antibodies are
human or humanized antibodies.
[0083] Examples of recombinant antibodies can include one or both
of: a heavy chain variable domain that includes a total of between
one and six (e.g., one, two, three, four, five, or six) histidines
in the set of CDR1, CDR2, and CDR3; and a light chain variable
domain that includes a total of between one and six (e.g., one,
two, three, four, five, or six) histidines in the set of CDR1,
CDR2, and CDR3. In some examples, the recombinant antibody includes
a heavy chain variable domain that includes a total of between one
and six (e.g., one, two, three, four, five, or six) histidines in
the set of CDR1, CDR2, and CDR3. In some examples, the heavy chain
variable domain includes a CDR1 including one histidine residue and
a CDR2 including one histidine residue. In some examples, the
recombinant antibody includes one or more (e.g., one, two, or
three) of a CDR1 including a sequence of SEQ ID NO: 1, a CDR2
including a sequence of SEQ ID NO: 2, and a CDR3 including a
sequence of SEQ ID NO: 3. In some examples of any of the
recombinant antibodies described herein, the heavy chain variable
region includes a sequence of SEQ ID NO: 4. In some examples of any
of the recombinant antibodies described herein, the heavy chain
includes a sequence of SEQ ID NO: 5 (e.g., BNJ441 heavy chain). In
some examples of any of the recombinant antibodies described
herein, the light chain variable region includes one or more (e.g.,
one, two, or three) of a CDR1 including a sequence of SEQ ID NO: 6,
a CDR2 including a sequence of SEQ ID NO: 7, and a CDR3 including a
sequence of SEQ ID NO: 8. In some examples of any of the
recombinant antibodies described herein, the light chain variable
domain includes a sequence of SEQ ID NO: 9. In some examples of any
of the recombinant antibodies described herein, the light chain
includes a sequence of SEQ ID NO: 10 (e.g., BNJ441 light
chain).
[0084] Examples of recombinant antibodies can include a heavy chain
variable domain that includes one or more (e.g., one, two, or
three) of a CDR1 including a sequence of SEQ ID NO: 11, a CDR2
including a sequence of SEQ ID NO: 12, and a CDR3 including a
sequence of SEQ ID NO: 13. In some examples of any of the
recombinant antibodies described herein, the recombinant antibody
can include a heavy chain variable region including a sequence of
SEQ ID NO: 14. In some examples of any of the recombinant
antibodies described herein, the recombinant antibody can include a
heavy chain including a sequence of SEQ ID NO: 15 (e.g., eculizumab
heavy chain). In some examples of any of the recombinant antibodies
described herein, the light chain variable region includes one or
more (e.g., one, two, or three) of a CDR1 including a sequence of
SEQ ID NO: 16, a CDR2 including a sequence of SEQ ID NO: 17, and a
CDR3 including a sequence of SEQ ID NO: 18. In some examples of any
of the recombinant antibodies described herein, the recombinant
antibody can include a light chain variable region including a
sequence of SEQ ID NO: 19. In some examples of any of the
recombinant antibodies described herein, the recombinant antibody
can include a light chain including a sequence of SEQ ID NO: 20
(e.g., eculizumab light chain).
[0085] Examples of recombinant antibodies can include a heavy chain
variable domain that includes one or more (e.g., one, two, or
three) of a CDR1 including a sequence of SEQ ID NO: 21, a CDR2
including a sequence of SEQ ID NO: 22, and a CDR3 including a
sequence of SEQ ID NO: 23. In some examples of any of the
recombinant antibodies described herein, the recombinant antibody
can include a heavy chain variable region including a sequence of
SEQ ID NO: 24. In some examples of any of the recombinant
antibodies described herein, the recombinant antibody can include a
heavy chain including a sequence of SEQ ID NO: 25 (e.g., BNJ383
heavy chain). In some examples of any of the recombinant antibodies
described herein, the light chain variable region includes one or
more (e.g., one, two, or three) of a CDR1 including a sequence of
SEQ ID NO: 26, a CDR2 including a sequence of SEQ ID NO: 27, and a
CDR3 including a sequence of SEQ ID NO: 28. In some examples of any
of the recombinant antibodies described herein, the recombinant
antibody can include a light chain variable region including a
sequence of SEQ ID NO: 29. In some examples of any of the
recombinant antibodies described herein, the recombinant antibody
can include a light chain including a sequence of SEQ ID NO: 30
(e.g., BNJ383 light chain).
[0086] Examples of recombinant antibodies can include a heavy chain
variable domain that includes one or more (e.g., one, two, or
three) of a CDR1 including a sequence of SEQ ID NO: 31, a CDR2
including a sequence of SEQ ID NO: 32, and a CDR3 including a
sequence of SEQ ID NO: 33. In some examples of any of the
recombinant antibodies described herein, the recombinant antibody
can include a heavy chain variable region including a sequence of
SEQ ID NO: 34. In some examples of any of the recombinant
antibodies described herein, the recombinant antibody can include a
heavy chain including a sequence of SEQ ID NO: 35 (e.g.,
samalizumab heavy chain). In some examples of any of the
recombinant antibodies described herein, the light chain variable
region includes one or more (e.g., one, two, or three) of a CDR1
including a sequence of SEQ ID NO: 36, a CDR2 including a sequence
of SEQ ID NO: 37, and a CDR3 including a sequence of SEQ ID NO: 38.
In some examples of any of the recombinant antibodies described
herein, the recombinant antibody can include a light chain variable
region including a sequence of SEQ ID NO: 39. In some examples of
any of the recombinant antibodies described herein, the recombinant
antibody can include a light chain including a sequence of SEQ ID
NO: 40 (e.g., samalizumab light chain).
[0087] Additional examples of recombinant antibodies include
panitumumab, omalizumab, abagovomab, abciximab, actoxumab,
adalimumab, adecatumumab, afelimomab, afutuzumab, alacizumab,
alacizumab, alemtuzumab, alirocumab, altumomab, amatuximab,
amatuximab, anatumomab, anrukinzumab, apolizumab, arcitumomab,
atinumab, tocilizumab, basilizimab, bectumomab, belimumab,
bevacizumab, besilesomab, bezlotoxumab, biciromab, canakinumab,
certolizumab, cetuximab, cixutumumab, daclizumab, denosumab,
densumab, edrecolomab, efalizumab, efungumab, epratuzumab,
ertumaxomab, etaracizumab, figitumumab, golimumab, ibritumomab
tiuxetan, igovomab, imgatuzumab, infliximab, inolimomab,
inotuzumab, labetuzumab, lebrikizumab, moxetumomab, natalizumab,
obinutuzumab, oregovomab, palivizumab, panitumumab, pertuzumab,
ranibizumab, rituximab, tocilizumab, tositumomab, tralokinumab,
tucotuzumab, trastuzumab, veltuzumab, zalutumumab, and zatuximab.
Additional examples of recombinant antibodies are known in the
art.
Virus Filters
[0088] Virus filters can be a normal flow filter (NFF) or a
tangential flow filtration (TFF) filter such as is described in,
e.g., U.S. Pat. No. 6,365,395. In either TFF mode or NFF mode,
filtration is conducted under conditions to retain the virus, e.g.,
a virus having a 20 to 100 nanometer (nm) diameter, on the membrane
surface while permitting passage of the recombinant antibody
through the membrane.
[0089] Exemplary virus filters include those formed from
regenerated cellulose (e.g., cuprammonium-regenerated cellulose),
polyethersulfone, polyarylsulphones, polysulfone, polyimide,
polyamide, polyvinylidenedifluoride (PVDF) or the like.
Non-limiting examples of virus filters include VIRESOLVE.RTM.
membranes and RETROPORE.TM. membranes available from EMD Millipore,
Billerica, Mass. These can be supplied in either a cartridge (NFF)
form, such as VIRESOLVE.RTM. NFP viral filters, or as cassettes
(for TFF), such as PELLICON.RTM. cassettes, available from EMD
Millipore, Billerica, Mass.
[0090] Additional exemplary virus filters that can be used in any
of the methods described herein include Sartorius Virosart.RTM.
CPV, which includes a polyethersulfone membrane. Other examples of
a virus filter than can be used in any of the methods described
herein is Asahi BioEx and Millipore Viresolve.RTM. Pro filter,
which include a polyvinylidene fluoride (PVDF) membrane. In some
examples, a virus filter can include a hollow fiber PVDF membrane.
In some examples, the virus filter is an Asahi 20N filter, which
includes a cuprammonium-regenerated cellulose membrane (e.g., a
hollow fiber cuprammonium-regenerated cellulose membrane).
Additional virus filters are known in the art.
Pre-Filters
[0091] Some embodiments of any of the methods described herein
include flowing a fluid including a recombinant antibody through a
pre-filter, e.g., before the fluid is flowed through a virus
filter. Non-limiting examples of pre-filters include a Sartorius
Virosart.RTM. Max pre-filter, a Millipore pre-filter, a
Sartopore.RTM. 2 pre-filter, a Sartobind STIC.RTM. pre-filter, a
Sartobind.RTM. Q pre-filter, a Sartobind.RTM. HIC Phenyl
pre-filter, a Sartobind.RTM. S pre-filter, Millipore Viresolve.RTM.
Pro Shield pre-filter, CUNO delipid pre-filter, and Millipore X0HC
pre-filter. In some embodiments, a virus filter includes a
polyamide membrane (e.g., a Sartorius Virosart.RTM. Max
pre-filter), a cation exchange-based membrane, an anion
exchange-based membrane, or a hydrophobic interaction
chromatography (HIC)-based membrane. Additional examples of
pre-filters are known in the art.
Stabilizing Agents
[0092] A stabilizing agent is an agent that reduces (e.g., a
significant or detectable reduction) the hydrodynamic radius of a
recombinant antibody in a fluid and/or minimizes the level of
soluble and/or insoluble protein aggregates (e.g., soluble and/or
insoluble recombinant antibody aggregates and/or soluble and/or
insoluble host cell protein aggregates) in a fluid including a
recombinant antibody. The hydrodynamic radius of a recombinant
antibody in a fluid can be determined using methods well known in
the art, e.g., dynamic light scattering. Methods for detecting the
level or amount of soluble and insoluble protein aggregates are
known in the art. For example, size exclusion chromatography,
native (non-denaturing) gel chromatography, analytical
ultracentrifugation (AUC), field-flow fractionation (FFF), and
dynamic light scattering can be used to detect the amount of
soluble or insoluble protein aggregates present in a fluid.
[0093] Non-limiting examples of stabilizing agents include arginine
(e.g., L-arginine or L-arginine HCl), alanine (e.g., L-alanine),
aspartic acid (e.g., L-aspartic acid), glutamic acid (e.g.,
L-glutamic acid), leucine (e.g., L-leucine), lysine (e.g.,
L-lysine), histidine (e.g., L-histidine), glycine (e.g.,
L-glycine), sucrose, trehalose, mannitol, sorbitol, and Polysorbate
80. When Polysorbate 80 is used as a stabilizing agent, it may be
present in a fluid at a concentration of between about 0.005% v/v
and about 0.05% v/v (e.g., between about 0.005% v/v and about
0.04%, between about 0.005% v/v and about 0.03% v/v, between about
0.005% v/v and about 0.02% v/v, between about 0.005% v/v and about
0.01% v/v, between about 0.01% v/v and about 0.05% v/v, between
about 0.01% v/v and about 0.04% v/v, between about 0.01% v/v and
about 0.03% v/v, between about 0.01% v/v and about 0.02% v/v,
between about 0.02% v/v and about 0.05% v/v, between about 0.02%
v/v and about 0.04% v/v, between about 0.02% v/v and about 0.03%
v/v, between about 0.03% v/v and about 0.05% v/v, between about
0.03% v/v and about 0.04% v/v, or between about 0.04% v/v and about
0.05% v/v). Additional examples of stabilizing agents are known in
the art.
Methods of Performing Viral Filtration (Part A)
[0094] Provided herein are methods of performing viral filtration
that include: (a) adjusting (e.g., increasing or decreasing) the pH
of a fluid including a recombinant antibody (e.g., any of the
recombinant antibodies described herein) to between about 5.0 and
about 6.7 (e.g., between about 5.0 and about 6.6, between about 5.0
and about 6.5, between about 5.0 and about 6.4, between about 5.0
and about 6.3, between about 5.0 and about 6.2, between about 5.0
and about 6.1, between about 5.0 and about 6.0, between about 5.0
and about 5.9, between about 5.0 and about 5.8, between about 5.0
and about 5.7, between about 5.0 and about 5.6, between about 5.0
and about 5.5, between about 5.0 and about 5.4, between about 5.0
and about 5.3, between about 5.0 and about 5.2, between about 5.1
and about 6.7, between about 5.1 and about 6.6, between about 5.1
and about 6.5, between about 5.1 and about 6.4, between about 5.1
and about 6.3, between about 5.1 and about 6.2, between about 5.1
and about 6.1, between about 5.1 and about 6.0, between about 5.1
and about 5.9, between about 5.1 and about 5.8, between about 5.1
and about 5.7, between about 5.1 and about 5.6, between about 5.1
and about 5.5, between about 5.1 and about 5.4, between about 5.1
and about 5.3, between about 5.2 and about 6.7, between about 5.2
and about 6.6, between about 5.2 and about 6.5, between about 5.2
and about 6.4, between about 5.2 and about 6.3, between about 5.2
and about 6.2, between about 5.2 and about 6.1, between about 5.2
and about 6.0, between about 5.2 and about 5.9, between about 5.2
and about 5.8, between about 5.2 and about 5.7, between about 5.2
and about 5.6, between about 5.2 and about 5.5, between about 5.2
and about 5.4, between about 5.3 and about 6.7, between about 5.3
and about 6.6, between about 5.3 and about 6.5, between about 5.3
and about 6.4, between about 5.3 and about 6.3, between about 5.3
and about 6.2, between about 5.3 and about 6.1, between about 5.3
and about 6.0, between about 5.3 and about 5.9, between about 5.3
and about 5.8, between about 5.3 and about 5.7, between about 5.3
and about 5.6, between about 5.3 and about 5.5, between about 5.4
and about 6.7, between about 5.4 and about 6.6, between about 5.4
and about 6.5, between about 5.4 and about 6.4, between about 5.4
and about 6.3, between about 5.4 and about 6.2, between about 5.4
and about 6.1, between about 5.4 and about 6.0, between about 5.4
and about 5.9, between about 5.4 and about 5.8, between about 5.4
and about 5.7, between about 5.4 and about 5.6, between about 5.5
and about 6.7, between about 5.5 and about 6.6, between about 5.5
and about 6.5, between about 5.5 and about 6.4, between about 5.5
and about 6.3, between about 5.5 and about 6.2, between about 5.5
and about 6.1, between about 5.5 and about 6.0, between about 5.5
and about 5.9, between about 5.5 and about 5.8, between about 5.5
and about 5.7, between about 5.6 and about 6.7, between about 5.6
and about 6.6, between about 5.6 and about 6.5, between about 5.6
and about 6.4, between about 5.6 and about 6.3, between about 5.6
and about 6.2, between about 5.6 and about 6.1, between about 5.6
and about 6.0, between about 5.6 and about 5.9, between about 5.6
and about 5.8, between about 5.7 and about 6.7, between about 5.7
and about 6.6, between about 5.7 and about 6.5, between about 5.7
and about 6.4, between about 5.7 and about 6.3, between about 5.7
and about 6.2, between about 5.7 and about 6.1, between about 5.7
and about 6.0, between about 5.7 and about 5.9, between about 5.8
and about 6.7, between about 5.8 and about 6.6, between about 5.8
and about 6.5, between about 5.8 and about 6.4, between about 5.8
and about 6.3, between about 5.8 and about 6.2, between about 5.8
and about 6.1, between about 5.8 and about 6.0, between about 5.9
and about 6.7, between about 5.9 and about 6.6, between about 5.9
and about 6.5, between about 5.9 and about 6.4, between about 5.9
and about 6.3, between about 5.9 and about 6.2, between about 5.9
and about 6.1, between about 6.0 and about 6.7, between about 6.0
and about 6.6, between about 6.0 and about 6.5, between about 6.0
and about 6.4, between about 6.0 and about 6.3, between about 6.0
and about 6.2, between about 6.1 and about 6.7, between about 6.1
and about 6.6, between about 6.1 and about 6.5, between about 6.1
and about 6.4, between about 6.1 and about 6.3, between about 6.2
and about 6.7, between about 6.2 and about 6.6, between about 6.2
and about 6.5, between about 6.2 and about 6.4, between about 6.3
and about 6.7, between about 6.3 and about 6.6, between about 6.3
and about 6.5, between about 6.4 and about 6.7, between about 6.4
and about 6.6, or between about 6.5 and about 6.7); and (b) flowing
the fluid through a virus filter to produce a filtrate including
the recombinant antibody. Some embodiments of these methods further
include, prior to (b): adding a stabilizing agent (e.g., any of the
stabilizing agents described herein) to the fluid in an amount
sufficient to yield a final concentration of between about 0.1 mM
and about 25 mM (e.g., between about 0.1 mM and about 24 mM,
between about 0.1 mM and about 23 mM, between about 0.1 mM and
about 22 mM, between about 0.1 mM and about 21 mM, between about
0.1 mM and about 20 mM, between about 0.1 mM and about 19 mM,
between about 0.1 mM and about 18 mM, between about 0.1 mM and
about 17 mM, between about 0.1 mM and about 16 mM, between about
0.1 mM and about 15 mM, between about 0.1 mM and about 14 mM,
between about 0.1 mM and about 13 mM, between about 0.1 mM and
about 12 mM, between about 0.1 mM and about 11 mM, between about
0.1 mM and about 10 mM, between about 0.1 mM and about 9 mM,
between about 0.1 mM and about 8 mM, between about 0.1 mM and about
7 mM, between about 0.1 mM and about 6 mM, between about 0.1 mM and
about 5 mM, between about 0.1 mM and about 4 mM, between about 0.1
mM and about 3 mM, between about 0.1 mM and about 2 mM, between
about 0.5 mM and about 25 mM, between about 0.5 mM and about 24 mM,
between about 0.5 mM and about 23 mM, between about 0.5 mM and
about 22 mM, between about 0.5 mM and about 21 mM, between about
0.5 mM and about 20 mM, between about 0.5 mM and about 19 mM,
between about 0.5 mM and about 18 mM, between about 0.5 mM and
about 17 mM, between about 0.5 mM and about 16 mM, between about
0.5 mM and about 15 mM, between about 0.5 mM and about 14 mM,
between about 0.5 mM and about 13 mM, between about 0.5 mM and
about 12 mM, between about 0.5 mM and about 11 mM, between about
0.5 mM and about 10 mM, between about 0.5 mM and about 9 mM,
between about 0.5 mM and about 8 mM, between about 0.5 mM and about
7 mM, between about 0.5 mM and about 6 mM, between about 0.5 mM and
about 5 mM, between about 0.5 mM and about 4 mM, between about 0.5
mM and about 3 mM, between about 0.5 mM and about 2 mM, between
about 1 mM and about 25 mM, between about 1 mM and about 24 mM,
between about 1 mM and about 23 mM, between about 1 mM and about 22
mM, between about 1 mM and about 21 mM, between about 1 mM and
about 20 mM, between about 1 mM and about 19 mM, between about 1 mM
and about 18 mM, between about 1 mM and about 17 mM, between about
1 mM and about 16 mM, between about 1 mM and about 15 mM, between
about 1 mM and about 14 mM, between about 1 mM and about 13 mM,
between about 1 mM and about 12 mM, between about 1 mM and about 11
mM, between about 1 mM and about 10 mM, between about 1 mM and
about 9 mM, between about 1 mM and about 8 mM, between about 1 mM
and about 7 mM, between about 1 mM and about 6 mM, between about 1
mM and about 5 mM, between about 1 mM and about 4 mM, between about
1 mM and about 3 mM, between about 2.5 mM and about 25 mM, between
about 2.5 mM and about 24 mM, between about 2.5 mM and about 23 mM,
between about 2.5 mM and about 22 mM, between about 2.5 mM and
about 21 mM, between about 2.5 mM and about 20 mM, between about
2.5 mM and about 19 mM, between about 2.5 mM and about 18 mM,
between about 2.5 mM and about 17 mM, between about 2.5 mM and
about 16 mM, between about 2.5 mM and about 15 mM, between about
2.5 mM and about 14 mM, between about 2.5 mM and about 13 mM,
between about 2.5 mM and about 12 mM, between about 2.5 mM and
about 11 mM, between about 2.5 mM and about 10 mM, between about
2.5 mM and about 9 mM, between about 2.5 mM and about 8 mM, between
about 2.5 mM and about 7 mM, between about 2.5 mM and about 6 mM,
between about 2.5 mM and about 5 mM, between about 5 mM and about
25 mM, between about 5 mM and about 24 mM, between about 5 mM and
about 23 mM, between about 5 mM and about 22 mM, between about 5 mM
and about 21 mM, between about 5 mM and about 20 mM, between about
5 mM and about 19 mM, between about 5 mM and about 18 mM, between
about 5 mM and about 17 mM, between about 5 mM and about 16 mM,
between about 5 mM and about 15 mM, between about 5 mM and about 14
mM, between about 5 mM and about 13 mM, between about 5 mM and
about 12 mM, between about 5 mM and about 11 mM, between about 5 mM
and about 10 mM, between about 5 mM and about 9 mM, between about 5
mM and about 8 mM, between about 5 mM and about 7 mM, between about
10 mM and about 25 mM, between about 10 mM and about 24 mM, between
about 10 mM and about 23 mM, between about 10 mM and about 22 mM,
between about 10 mM and about 21 mM, between about 10 mM and about
20 mM, between about 10 mM and about 19 mM, between about 10 mM and
about 18 mM, between about 10 mM and about 17 mM, between about 10
mM and about 16 mM, between about 10 mM and about 15 mM, between
about 10 mM and about 14 mM, between about 10 mM and about 13 mM,
between about 10 mM and about 12 mM, between about 12.5 mM and
about 25 mM, between about 12.5 mM and about 24 mM, between about
12.5 mM and about 23 mM, between about 12.5 mM and about 22 mM,
between about 12.5 mM and about 21 mM, between about 12.5 mM and
about 20 mM, between about 12.5 mM and about 19 mM, between about
12.5 mM and about 18 mM, between about 12.5 mM and about 17 mM,
between about 12.5 mM and about 16 mM, between about 12.5 mM and
about 15 mM, between about 15 mM and about 25 mM, between about 15
mM and about 24 mM, between about 15 mM and about 23 mM, between
about 15 mM and about 22 mM, between about 15 mM and about 21 mM,
between about 15 mM and about 20 mM, between about 15 mM and about
19 mM, between about 15 mM and about 18 mM, between about 15 mM and
about 17 mM, between about 16 mM and about 25 mM, between about 16
mM and about 24 mM, between about 16 mM and about 23 mM, between
about 16 mM and about 22 mM, between about 16 mM and about 21 mM,
between about 16 mM and about 20 mM, between about 16 mM and about
19 mM, between about 16 mM and about 18 mM, between about 17 mM and
about 25 mM, between about 17 mM and about 24 mM, between about 17
mM and about 23 mM, between about 17 mM and about 22 mM, between
about 17 mM and about 21 mM, between about 17 mM and about 20 mM,
between about 17 mM and about 19 mM, between about 18 mM and about
25 mM, between about 18 mM and about 24 mM, between about 18 mM and
about 23 mM, between about 18 mM and about 22 mM, between about 18
mM and about 21 mM, between about 18 mM and about 20 mM, between
about 19 mM and about 25 mM, between about 19 mM and about 24 mM,
between about 19 mM and about 23 mM, between about 19 mM and about
22 mM, between about 19 mM and about 21 mM, between about 20 mM and
about 25 mM, between about 20 mM and about 24 mM, between about 20
mM and about 23 mM, between about 20 mM and about 22 mM, between
about 21 mM and about 25 mM, between about 21 mM and about 24 mM,
between about 21 mM and about 23 mM, between about 22 mM and about
25 mM, between about 22 mM and about 24 mM, or between about 23 mM
and about 25 mM) stabilizing agent in the fluid.
[0095] Some embodiments of these examples, further include,
immediately prior to (b): flowing the fluid through a pre-filter
(e.g., a pre-filter including a polyamide membrane, such as a
Sartorius Virosart.RTM. Max pre-filter). In some embodiments of
these examples, the fluid further includes between about 5 mM and
about 300 mM sodium chloride (e.g., between about 5 mM and about
280 mM, between about 5 mM and about 260 mM, between about 5 mM and
about 240 mM, between about 5 mM and about 220 mM, between about 5
mM and about 200 mM, between about 5 mM and about 180 mM, between
about 5 mM and about 160 mM, between about 5 mM and about 140 mM,
between about 5 mM and about 120 mM, between about 5 mM and about
100 mM, between about 5 mM and about 90 mM, between about 5 mM and
about 80 mM, between about 5 mM and about 70 mM, between about 5 mM
and about 60 mM, between about 5 mM and about 50 mM, between about
5 mM and about 40 mM, between about 5 mM and about 30 mM, between
about 10 mM and about 300 mM, between about 10 mM and about 280 mM,
between about 10 mM and about 260 mM, between about 10 mM and about
240 mM, between about 10 mM and about 220 mM, between about 10 mM
and about 200 mM, between about 10 mM and about 180 mM, between
about 10 mM and about 160 mM, between about 10 mM and about 140 mM,
between about 10 mM and about 120 mM, between about 10 mM and about
100 mM, between about 10 mM and about 90 mM, between about 10 mM
and about 80 mM, between about 10 mM and about 70 mM, between about
10 mM and about 60 mM, between about 10 mM and about 50 mM, between
about 10 mM and about 40 mM, between about 20 mM and about 300 mM,
between about 20 mM and about 280 mM, between about 20 mM and about
260 mM, between about 20 mM and about 240 mM, between about 20 mM
and about 220 mM, between about 20 mM and about 200 mM, between
about 20 mM and about 180 mM, between about 20 mM and about 160 mM,
between about 20 mM and about 140 mM, between about 20 mM and about
120 mM, between about 20 mM and about 100 mM, between about 20 mM
and about 90 mM, between about 20 mM and about 80 mM, between about
20 mM and about 70 mM, between about 20 mM and about 60 mM, between
about 20 mM and about 50 mM, between about 30 mM and about 300 mM,
between about 30 mM and about 280 mM, between about 30 mM and about
260 mM, between about 30 mM and about 240 mM, between about 30 mM
and about 220 mM, between about 30 mM and about 200 mM, between
about 30 mM and about 180 mM, between about 30 mM and about 160 mM,
between about 30 mM and about 140 mM, between about 30 mM and about
120 mM, between about 30 mM and about 100 mM, between about 30 mM
and about 90 mM, between about 30 mM and about 80 mM, between about
30 mM and about 70 mM, between about 30 mM and about 60 mM, between
about 40 mM and about 300 mM, between about 40 mM and about 280 mM,
between about 40 mM and about 260 mM, between about 40 mM and about
240 mM, between about 40 mM and about 220 mM, between about 40 mM
and about 200 mM, between about 40 mM and about 180 mM, between
about 40 mM and about 160 mM, between about 40 mM and about 140 mM,
between about 40 mM and about 120 mM, between about 40 mM and about
100 mM, between about 40 mM and about 90 mM, between about 40 mM
and about 80 mM, between about 40 mM and about 70 mM, between about
50 mM and about 300 mM, between about 50 mM and about 280 mM,
between about 50 mM and about 260 mM, between about 50 mM and about
240 mM, between about 50 mM and about 220 mM, between about 50 mM
and about 200 mM, between about 50 mM and about 180 mM, between
about 50 mM and about 160 mM, between about 50 mM and about 140 mM,
between about 50 mM and about 120 mM, between about 50 mM and about
100 mM, between about 50 mM and about 90 mM, between about 50 mM
and about 80 mM, between about 75 mM and about 300 mM, between
about 75 mM and about 280 mM, between about 75 mM and about 260 mM,
between about 75 mM and about 240 mM, between about 75 mM and about
220 mM, between about 75 mM and about 200 mM, between about 75 mM
and about 180 mM, between about 75 mM and about 160 mM, between
about 75 mM and about 140 mM, between about 75 mM and about 120 mM,
between about 75 mM and about 100 mM, between about 100 mM and
about 300 mM, between about 100 mM and about 280 mM, between about
100 mM and about 260 mM, between about 100 mM and about 240 mM,
between about 100 mM and about 220 mM, between about 100 mM and
about 200 mM, between about 100 mM and about 180 mM, between about
100 mM and about 160 mM, between about 100 mM and about 140 mM,
between about 100 mM and about 120 mM, between about 125 mM and
about 300 mM, between about 125 mM and about 280 mM, between about
125 mM and about 260 mM, between about 125 mM and about 240 mM,
between about 125 mM and about 220 mM, between about 125 mM and
about 200 mM, between about 125 mM and about 180 mM, between about
125 mM and about 160 mM, between about 150 mM and about 300 mM,
between about 150 mM and about 280 mM, between about 150 mM and
about 260 mM, between about 150 mM and about 240 mM, between about
150 mM and about 220 mM, between about 150 mM and about 200 mM,
between about 150 mM and about 180 mM, between about 175 mM and
about 300 mM, between about 175 mM and about 280 mM, between about
175 mM and about 260 mM, between about 175 mM and about 240 mM,
between about 175 mM and about 220 mM, between about 175 mM and
about 200 mM, between about 200 mM and about 300 mM, between about
200 mM and about 280 mM, between about 200 mM and about 260 mM,
between about 200 mM and about 240 mM, between about 200 mM and
about 220 mM, between about 225 mM and about 300 mM, between about
225 mM and about 280 mM, between about 225 mM and about 260 mM,
between about 250 mM and about 300 mM, between about 250 mM and
about 280 mM, or between about 275 mM and about 300 mM sodium
chloride). In some embodiments of any of these methods, the fluid
includes between about 10 mM and about 50 mM (e.g., between about
10 mM and about 45 mM, between about 10 mM and about 40 mM, between
about 10 mM and about 35 mM, between about 10 mM and about 30 mM,
between about 10 mM and about 25 mM, between about 10 mM and about
20 mM, between about 15 mM and about 50 mM, between about 15 mM and
about 45 mM, between about 15 mM and about 40 mM, between about 15
mM and about 35 mM, between about 15 mM and about 30 mM, between
about 15 mM and about 25 mM, between about 20 mM and about 50 mM,
between about 20 mM and about 45 mM, between about 20 mM and about
40 mM, between about 20 mM and about 35 mM, between about 20 mM and
about 30 mM, between about 25 mM and about 50 mM, between about 25
mM and about 45 mM, between about 25 mM and about 40 mM, between
about 25 mM and about 35 mM, between about 30 mM and about 50 mM,
between about 30 mM and about 45 mM, between about 30 mM and about
40 mM, between about 35 mM and about 50 mM, between about 35 mM and
about 45 mM, or between about 40 mM and about 50 mM) sodium
phosphate.
[0096] In some embodiments of these methods, prior to step (a), the
pH of the fluid is between about 7.4 and about 7.8 (e.g., between
about 7.4 and about 7.7, between about 7.4 and about 7.6, between
about 7.5 and about 7.8, between about 7.5 and about 7.7, between
about 7.6 and about 7.8, or about 7.6).
[0097] Also provided are methods of performing viral filtration
that include: (a) adding a stabilizing agent (e.g., any of the
stabilizing agent described herein) to a fluid including a
recombinant antibody in an amount sufficient to yield a final
concentration of between about 10 mM and about 100 mM (e.g.,
between about 10 mM and about 95 mM, between about 10 mM and about
90 mM, between about 10 mM and about 85 mM, between about 10 mM and
about 80 mM, between about 10 mM and about 75 mM, between about 10
mM and about 70 mM, between about 10 mM and about 65 mM, between
about 10 mM and about 60 mM, between about 10 mM and about 55 mM,
between about 10 mM and about 50 mM, between about 10 mM and about
45 mM, between about 10 mM and about 40 mM, between about 10 mM and
about 35 mM, between about 10 mM and about 30 mM, between about 15
mM and about 100 mM, between about 15 mM and about 95 mM, between
about 15 mM and about 90 mM, between about 15 mM and about 85 mM,
between about 15 mM and about 80 mM, between about 15 mM and about
75 mM, between about 15 mM and about 70 mM, between about 15 mM and
about 65 mM, between about 15 mM and about 60 mM, between about 15
mM and about 55 mM, between about 15 mM and about 50 mM, between
about 15 mM and about 45 mM, between about 15 mM and about 40 mM,
between about 15 mM and about 35 mM, between about 20 mM and about
100 mM, between about 20 mM and about 95 mM, between about 20 mM
and about 90 mM, between about 20 mM and about 85 mM, between about
20 mM and about 80 mM, between about 20 mM and about 75 mM, between
about 20 mM and about 70 mM, between about 20 mM and about 65 mM,
between about 20 mM and about 60 mM, between about 25 mM and about
55 mM, between about 25 mM and about 50 mM, between about 25 mM and
about 45 mM, between about 30 mM and about 100 mM, between about 30
mM and about 95 mM, between about 30 mM and about 90 mM, between
about 30 mM and about 85 mM, between about 30 mM and about 80 mM,
between about 30 mM and about 75 mM, between about 30 mM and about
70 mM, between about 30 mM and about 65 mM, between about 30 mM and
about 60 mM, between about 30 mM and about 55 mM, between about 30
mM and about 50 mM, between about 35 mM and about 100 mM, between
about 35 mM and about 95 mM, between about 35 mM and about 90 mM,
between about 35 mM and about 85 mM, between about 35 mM and about
80 mM, between about 35 mM and about 75 mM, between about 35 mM and
about 70 mM, between about 35 mM and about 65 mM, between about 35
mM and about 60 mM, between about 35 mM and about 55 mM, between
about 40 mM and about 100 mM, between about 40 mM and about 95 mM,
between about 40 mM and about 90 mM, between about 40 mM and about
85 mM, between about 40 mM and about 80 mM, between about 40 mM and
about 75 mM, between about 40 mM and about 70 mM, between about 40
mM and about 65 mM, between about 40 mM and about 60 mM, between
about 45 mM and about 100 mM, between about 45 mM and about 95 mM,
between about 45 mM and about 90 mM, between about 45 mM and about
85 mM, between about 45 mM and about 80 mM, between about 45 mM and
about 75 mM, between about 45 mM and about 70 mM, between about 45
mM and about 65 mM, between about 50 mM and about 100 mM, between
about 50 mM and about 95 mM, between about 50 mM and about 90 mM,
between about 50 mM and about 85 mM, between about 50 mM and about
80 mM, between about 50 mM and about 75 mM, between about 50 mM and
about 70 mM, between about 55 mM and about 100 mM, between about 55
mM and about 95 mM, between about 55 mM and about 90 mM, between
about 55 mM and about 85 mM, between about 55 mM and about 80 mM,
between about 55 mM and about 75 mM, between about 60 mM and about
100 mM, between about 60 mM and about 95 mM, between about 60 mM
and about 90 mM, between about 60 mM and about 85 mM, between about
60 mM and about 80 mM, between about 65 mM and about 100 mM,
between about 65 mM and about 95 mM, between about 65 mM and about
90 mM, between about 65 mM and about 85 mM, between about 70 mM and
about 100 mM, between about 70 mM and about 95 mM, between about 70
mM and about 90 mM, between about 75 mM and about 100 mM, between
about 75 mM and about 95 mM, or between about 80 mM and about 100
mM) stabilizing agent in the fluid, wherein prior to adding, the
fluid has a pH of between about 6.7 and about 8.5 (between about
6.7 and about 8.4, between about 6.7 and about 8.3, between about
6.7 and about 8.2, between about 6.7 and about 8.1, between about
6.7 and about 8.0, between about 6.7 and about 7.9, between about
6.7 and about 7.8, between about 6.7 and about 7.7, between about
6.7 and about 7.6, between about 6.7 and about 7.5, between about
6.7 and about 7.4, between about 6.7 and about 7.3, between about
6.7 and about 7.2, between about 6.7 and about 7.1, between about
6.7 and about 7.0, between about 6.7 and about 6.9, between about
6.8 and about 8.5, between about 6.8 and about 8.4, between about
6.8 and about 8.3, between about 6.8 and about 8.2, between about
6.8 and about 8.1, between about 6.8 and about 8.0, between about
6.8 and about 7.9, between about 6.8 and about 7.8, between about
6.8 and about 7.7, between about 6.8 and about 7.6, between about
6.8 and about 7.5, between about 6.8 and about 7.4, between about
6.8 and about 7.3, between about 6.8 and about 7.2, between about
6.8 and about 7.1, between about 6.8 and about 7.0, between about
6.9 and about 8.5, between about 6.9 and about 8.4, between about
6.9 and about 8.3, between about 6.9 and about 8.2, between about
6.9 and about 8.1, between about 6.9 and about 8.0, between about
6.9 and about 7.9, between about 6.9 and about 7.8, between about
6.9 and about 7.7, between about 6.9 and about 7.6, between about
6.9 and about 7.5, between about 6.9 and about 7.4, between about
6.9 and about 7.3, between about 6.9 and about 7.2, between about
6.9 and about 7.1, between about 7.0 and about 8.5, between about
7.0 and about 8.4, between about 7.0 and about 8.3, between about
7.0 and about 8.2, between about 7.0 and about 8.1, between about
7.0 and about 8.0, between about 7.0 and about 7.9, between about
7.0 and about 7.8, between about 7.0 and about 7.7, between about
7.0 and about 7.6, between about 7.0 and about 7.5, between about
7.0 and about 7.4, between about 7.0 and about 7.3, between about
7.0 and about 7.2, between about 7.1 and about 8.5, between about
7.1 and about 8.4, between about 7.1 and about 8.3, between about
7.1 and about 8.2, between about 7.1 and about 8.1, between about
7.1 and about 8.0, between about 7.1 and about 7.9, between about
7.1 and about 7.8, between about 7.1 and about 7.7, between about
7.1 and about 7.6, between about 7.1 and about 7.5, between about
7.1 and about 7.4, between about 7.1 and about 7.3, between about
7.2 and about 8.5, between about 7.2 and about 8.4, between about
7.2 and about 8.3, between about 7.2 and about 8.2, between about
7.2 and about 8.1, between about 7.2 and about 8.0, between about
7.2 and about 7.9, between about 7.2 and about 7.8, between about
7.2 and about 7.7, between about 7.2 and about 7.6, between about
7.2 and about 7.5, between about 7.2 and about 7.4, between about
7.3 and about 8.5, between about 7.3 and about 8.4, between about
7.3 and about 8.3, between about 7.3 and about 8.2, between about
7.3 and about 8.1, between about 7.3 and about 8.0, between about
7.3 and about 7.9, between about 7.3 and about 7.8, between about
7.3 and about 7.7, between about 7.3 and about 7.6, between about
7.3 and about 7.5, between about 7.4 and about 8.5, between about
7.4 and about 8.4, between about 7.4 and about 8.3, between about
7.4 and about 8.2, between about 7.4 and about 8.1, between about
7.4 and about 8.0, between about 7.4 and about 7.9, between about
7.4 and about 7.8, between about 7.4 and about 7.7, between about
7.4 and about 7.6, between about 7.5 and about 8.5, between about
7.5 and about 8.4, between about 7.5 and about 8.3, between about
7.5 and about 8.2, between about 7.5 and about 8.1, between about
7.5 and about 8.0, between about 7.5 and about 7.9, between about
7.5 and about 7.8, between about 7.5 and about 7.7, between about
7.6 and about 8.5, between about 7.6 and about 8.4, between about
7.6 and about 8.3, between about 7.6 and about 8.2, between about
7.6 and about 8.1, between about 7.6 and about 8.0, between about
7.6 and about 7.9, between about 7.6 and about 7.8, between about
7.7 and about 8.5, between about 7.7 and about 8.4, between about
7.7 and about 8.3, between about 7.7 and about 8.2, between about
7.7 and about 8.1, between about 7.7 and about 8.0, between about
7.7 and about 7.9, between about 7.8 and about 8.5, between about
7.8 and about 8.4, between about 7.8 and about 8.3, between about
7.8 and about 8.2, between about 7.8 and about 8.1, between about
7.8 and about 8.0, between about 7.9 and about 8.5, between about
7.9 and about 8.4, between about 7.9 and about 8.3, between about
7.9 and about 8.2, between about 7.9 and about 8.1, between about
8.0 and about 8.5, between about 8.0 and about 8.4, between about
8.0 and about 8.3, between about 8.0 and about 8.2, between about
8.1 and about 8.5, between about 8.1 and about 8.4, between about
8.1 and about 8.3, between about 8.2 and about 8.5, between about
8.2 and about 8.4, or between about 8.3 and about 8.5); and (b)
flowing the fluid through a virus filter to produce a filtrate
including the recombinant antibody. Some embodiments of these
methods further include, immediately prior to step (b), flowing the
fluid through a pre-filter (e.g., any of the pre-filters described
herein, such as a pre-filter including a polyamide membrane, such
as a Sartorius Virosart.RTM. Max pre-filter). In some embodiments
of these methods, the fluid includes between about 1 mM and about
100 mM sodium chloride (e.g., between about 1 mM and about 90 mM,
between about 1 mM and about 80 mM, between about 1 mM and about 70
mM, between about 1 mM and about 60 mM, between about 1 mM and
about 50 mM, between about 1 mM and about 40 mM, between about 1 mM
and about 30 mM, between about 1 mM and about 20 mM, between about
10 mM and about 100 mM, between about 10 mM and about 90 mM,
between about 10 mM and about 80 mM, between about 10 mM and about
70 mM, between about 10 mM and about 60 mM, between about 10 mM and
about 50 mM, between about 10 mM and about 40 mM, between about 10
mM and about 30 mM, between about 20 mM and about 100 mM, between
about 20 mM and about 90 mM, between about 20 mM and about 80 mM,
between about 20 mM and about 70 mM, between about 20 mM and about
60 mM, between about 20 mM and about 50 mM, between about 20 mM and
about 40 mM, between about 30 mM and about 100 mM, between about 30
mM and about 90 mM, between about 30 mM and about 80 mM, between
about 30 mM and about 70 mM, between about 30 mM and about 60 mM,
between about 30 mM and about 50 mM, between about 40 mM and about
100 mM, between about 40 mM and about 90 mM, between about 40 mM
and about 80 mM, between about 40 mM and about 70 mM, between about
40 mM and about 60 mM, between about 50 mM and about 100 mM,
between about 50 mM and about 90 mM, between about 50 mM and about
80 mM, between about 50 mM and about 70 mM, between about 60 mM and
about 100 mM, between about 60 mM and about 90 mM, between about 60
mM and about 80 mM, between about 70 mM and about 100 mM, between
about 70 mM and about 90 mM, or between about 80 mM and about 100
mM sodium chloride).
[0098] In any of the methods of performing viral filtration
described herein, wherein prior to (a), the fluid includes between
about 0.1 mg/mL and about 25 mg/mL (e.g., between about 0.1 mg/mL
and about 20 mg/mL, between about 0.1 mg/mL and about 24 mg/mL,
between about 0.1 mg/mL and about 22 mg/mL, between about 0.1 mg/mL
and about 20 mg/mL, between about 0.1 mg/mL and about 18 mg/mL,
between about 0.1 mg/mL and about 16 mg/mL, between about 0.1 mg/mL
and about 14 mg/mL, between about 0.1 mg/mL and about 12 mg/mL,
between about 0.1 mg/mL and about 10 mg/mL, between about 0.1 mg/mL
and about 8 mg/mL, between about 0.1 mg/mL and about 6 mg/mL,
between about 0.1 mg/mL and about 4 mg/mL, between about 0.1 mg/mL
and about 2 mg/mL, between about 0.5 mg/mL and about 25 mg/mL,
between about 0.5 mg/mL and about 24 mg/mL, between about 0.5 mg/mL
and about 22 mg/mL, between about 0.5 mg/mL and about 20 mg/mL,
between about 0.5 mg/mL and about 18 mg/mL, between about 0.5 mg/mL
and about 16 mg/mL, between about 0.5 mg/mL and about 14 mg/mL,
between about 0.5 mg/mL and about 12 mg/mL, between about 0.5 mg/mL
and about 10 mg/mL, between about 0.5 mg/mL and about 8 mg/mL,
between about 0.5 mg/mL and about 6 mg/mL, between about 0.5 mg/mL
and about 4 mg/mL, between about 0.5 mg/mL and about 2 mg/mL,
between about 1 mg/mL and about 25 mg/mL, between about 1 mg/mL and
about 24 mg/mL, between about 1 mg/mL and about 22 mg/mL, between
about 1 mg/mL and about 20 mg/mL, between about 1 mg/mL and about
18 mg/mL, between about 1 mg/mL and about 16 mg/mL, between about 1
mg/mL and about 14 mg/mL, between about 1 mg/mL and about 12 mg/mL,
between about 1 mg/mL and about 10 mg/mL, between about 1 mg/mL and
about 8 mg/mL, between about 1 mg/mL and about 6 mg/mL, between
about 1 mg/mL and about 4 mg/mL, between about 2 mg/mL and about 25
mg/mL, between about 2 mg/mL and about 24 mg/mL, between about 2
mg/mL and about 22 mg/mL, between about 2 mg/mL and about 20 mg/mL,
between about 2 mg/mL and about 18 mg/mL, between about 2 mg/mL and
about 16 mg/mL, between about 2 mg/mL and about 14 mg/mL, between
about 2 mg/mL and about 12 mg/mL, between about 2 mg/mL and about
10 mg/mL, between about 2 mg/mL and about 8 mg/mL, between about 2
mg/mL and about 6 mg/mL, between about 2 mg/mL and about 4 mg/mL,
between about 4 mg/mL and about 25 mg/mL, between about 4 mg/mL and
about 24 mg/mL, between about 4 mg/mL and about 22 mg/mL, between
about 4 mg/mL and about 20 mg/mL, between about 4 mg/mL and about
18 mg/mL, between about 4 mg/mL and about 16 mg/mL, between about 4
mg/mL and about 14 mg/mL, between about 4 mg/mL and about 12 mg/mL,
between about 4 mg/mL and about 10 mg/mL, between about 4 mg/mL and
about 8 mg/mL, between about 4 mg/mL and about 6 mg/mL, between
about 6 mg/mL and about 25 mg/mL, between about 6 mg/mL and about
24 mg/mL, between about 6 mg/mL and about 22 mg/mL, between about 6
mg/mL and about 20 mg/mL, between about 6 mg/mL and about 18 mg/mL,
between about 6 mg/mL and about 16 mg/mL, between about 6 mg/mL and
about 14 mg/mL, between about 6 mg/mL and about 12 mg/mL, between
about 6 mg/mL and about 10 mg/mL, between about 6 mg/mL and about 8
mg/mL, between about 8 mg/mL and about 25 mg/mL, between about 8
mg/mL and about 24 mg/mL, between about 8 mg/mL and about 22 mg/mL,
between about 8 mg/mL and about 20 mg/mL, between about 8 mg/mL and
about 18 mg/mL, between about 8 mg/mL and about 16 mg/mL, between
about 8 mg/mL and about 14 mg/mL, between about 8 mg/mL and about
12 mg/mL, between about 8 mg/mL and about 10 mg/mL, between about
10 mg/mL and about 25 mg/mL, between about 10 mg/mL and about 24
mg/mL, between about 10 mg/mL and about 22 mg/mL, between about 10
mg/mL and about 20 mg/mL, between about 10 mg/mL and about 18
mg/mL, between about 10 mg/mL and about 16 mg/mL, between about 10
mg/mL and about 14 mg/mL, between about 10 mg/mL and about 12
mg/mL, between about 12 mg/mL and about 25 mg/mL, between about 12
mg/mL and about 24 mg/mL, between about 12 mg/mL and about 22
mg/mL, between about 12 mg/mL and about 20 mg/mL, between about 12
mg/mL and about 18 mg/mL, between about 12 mg/mL and about 16
mg/mL, between about 12 mg/mL and about 14 mg/mL, between about 14
mg/mL and about 25 mg/mL, between about 14 mg/mL and about 24
mg/mL, between about 14 mg/mL and about 22 mg/mL, between about 14
mg/mL and about 20 mg/mL, between about 14 mg/mL and about 18
mg/mL, between about 14 mg/mL and about 16 mg/mL, between about 16
mg/mL and about 25 mg/mL, between about 16 mg/mL and about 24
mg/mL, between about 16 mg/mL and about 22 mg/mL, between about 16
mg/mL and about 20 mg/mL, between about 16 mg/mL and about 18
mg/mL, between about 18 mg/mL and about 25 mg/mL, between about 18
mg/mL and about 24 mg/mL, between about 18 mg/mL and about 22
mg/mL, between about 18 mg/mL and about 20 mg/mL, between about 20
mg/mL and about 25 mg/mL, between about 20 mg/mL and about 24
mg/mL, between about 20 mg/mL and about 22 mg/mL, between about 22
mg/mL and about 25 mg/mL, between about 22 mg/mL and about 24
mg/mL, or between about 23 mg/mL and about 25 mg/mL) recombinant
antibody. In some embodiments of these methods, the fluid has a pH
of between about 7.4 and about 7.8 (e.g., between about 7.4 and
about 7.7, between about 7.4 and about 7.6, between about 7.5 and
about 7.8, between about 7.5 and about 7.7, between about 7.6 and
about 7.8, or about 7.6).
[0099] In some embodiments of any of these methods, the fluid
includes between about 50 mM and about 90 mM sodium chloride (e.g.,
between about 50 mM and about 80 mM, between 50 mM and about 75 mM,
between about 50 mM and about 70 mM, between about 50 mM and about
65 mM, between about 50 mM and about 60 mM, between about 50 mM and
about 55 mM, between about 55 mM and about 90 mM, between about 55
mM and about 85 mM, between about 55 mM and about 80 mM, between
about 55 mM and about 75 mM, between about 55 mM and about 70 mM,
between about 55 mM and about 65 mM, between about 55 mM and about
60 mM, between about 60 mM and about 90 mM, between about 60 mM and
about 85 mM, between about 60 mM and about 80 mM, between about 60
mM and about 75 mM, between about 60 mM and about 70 mM, between
about 60 mM and about 65 mM, between about 65 mM and about 90 mM,
between about 65 mM and about 85 mM, between about 65 mM and about
80 mM, between about 65 mM and about 75 mM, between about 65 mM and
about 70 mM, between about 70 mM and about 90 mM, between about 70
mM and about 85 mM, between about 70 mM and about 80 mM, between
about 70 mM and about 75 mM, between about 75 mM and about 90 mM,
between about 75 mM and about 85 mM, between about 75 mM and about
80 mM, between about 80 mM and about 90 mM, between about 80 mM and
about 85 mM, or between about 85 mM and about 90 mM sodium
chloride).
[0100] In some embodiments of the methods described in this
section, the recombinant antibodies can include one or both of: a
heavy chain variable domain that includes a total of between one
and six (e.g., one, two, three, four, five, or six) histidines in
the set of CDR1, CDR2, and CDR3; and a light chain variable domain
that includes a total of between one and six (e.g., one, two,
three, four, five, or six) histidines in the set of CDR1, CDR2, and
CDR3. In some examples of the methods described in this section,
the recombinant antibody includes a heavy chain variable domain
that includes a total of between one and six (e.g., one, two,
three, four, five, or six) histidines in the set of CDR1, CDR2, and
CDR3. In some examples of the methods described in this section,
the heavy chain variable domain includes a CDR1 including one
histidine residue and a CDR2 including one histidine residue. In
some examples of the methods described in this section, the
recombinant antibody includes one or more (e.g., one, two, or
three) of a CDR1 including a sequence of SEQ ID NO: 1, a CDR2
including a sequence of SEQ ID NO: 2, and a CDR3 including a
sequence of SEQ ID NO: 3. In some examples of any the methods
described in this section, the heavy chain variable region includes
a sequence of SEQ ID NO: 4. In some examples of the methods
described in this section, the heavy chain includes a sequence of
SEQ ID NO: 5 (BNJ441 heavy chain). In some examples of any of the
methods described in this section, the light chain variable region
includes one or more (e.g., one, two, or three) of a CDR1 including
a sequence of SEQ ID NO: 6, a CDR2 including a sequence of SEQ ID
NO: 7, and a CDR3 including a sequence of SEQ ID NO: 8. In some
examples of the methods described in this section, the light chain
variable domain includes a sequence of SEQ ID NO: 9. In some
examples of the methods described in this section, the light chain
includes a sequence of SEQ ID NO: 10 (BNJ441 light chain).
[0101] In some examples of the methods described in this section,
the recombinant antibody can include a heavy chain variable domain
that includes one or more (e.g., one, two, or three) of a CDR1
including a sequence of SEQ ID NO: 11, a CDR2 including a sequence
of SEQ ID NO: 12, and a CDR3 including a sequence of SEQ ID NO: 13.
In some examples of the methods described in this section, the
recombinant antibody can include a heavy chain variable region
including a sequence of SEQ ID NO: 14. In some examples of any of
the methods described in this section, the recombinant antibody can
include a heavy chain including a sequence of SEQ ID NO: 15
(eculizumab heavy chain). In some examples of any of the methods
described in this section, the light chain variable region includes
one or more (e.g., one, two, or three) of a CDR1 including a
sequence of SEQ ID NO: 16, a CDR2 including a sequence of SEQ ID
NO: 17, and a CDR3 including a sequence of SEQ ID NO: 18. In some
examples of any of the methods described herein, the recombinant
antibody can include a light chain variable region including a
sequence of SEQ ID NO: 19. In some examples of any of the methods
described herein, the recombinant antibody can include a light
chain including a sequence of SEQ ID NO: 20 (eculizumab light
chain).
Methods of Performing Viral Filtration (Part B)
[0102] Also provided are methods of performing viral filtration
that include: (a) flowing a fluid including a recombinant antibody
(e.g., any of the recombinant antibodies described herein) through
a pre-filter (e.g., any of the pre-filters described herein) to
provide a first filtrate including the recombinant antibody; and
(b) flowing the first filtrate through a virus filter (e.g., any of
the virus filters described herein) to produce a second filtrate
including the recombinant antibody. Some embodiments further
include, prior to step (a): adding a stabilizing agent (e.g., any
of the stabilizing agent described herein) to a fluid including the
recombinant antibody in an amount sufficient to yield a final
concentration of between about 1 mM and about 100 mM (e.g., between
about 1 mM and about 95 mM, between about 1 mM and about 90 mM,
between about 1 mM and about 85 mM, between about 1 mM and about 80
mM, between about 1 mM and about 75 mM, between about 1 mM and
about 70 mM, between about 1 mM and about 65 mM, between about 1 mM
and about 60 mM, between about 1 mM and about 55 mM, between about
1 mM and about 50 mM, between about 1 mM and about 45 mM, between
about 1 mM and about 40 mM, between about 1 mM and about 35 mM,
between about 1 mM and about 30 mM, between about 1 mM and about 25
mM, between about 1 mM and about 20 mM, between about 1 mM and
about 15 mM, between about 1 mM and about 10 mM, between about 10
mM and about 100 mM, between about 10 mM and about 95 mM, between
about 10 mM and about 90 mM, between about 10 mM and about 85 mM,
between about 10 mM and about 80 mM, between about 10 mM and about
75 mM, between about 10 mM and about 70 mM, between about 10 mM and
about 65 mM, between about 10 mM and about 60 mM, between about 10
mM and about 55 mM, between about 10 mM and about 50 mM, between
about 10 mM and about 45 mM, between about 10 mM and about 40 mM,
between about 10 mM and about 35 mM, between about 10 mM and about
30 mM, between about 15 mM and about 100 mM, between about 15 mM
and about 95 mM, between about 15 mM and about 90 mM, between about
15 mM and about 85 mM, between about 15 mM and about 80 mM, between
about 15 mM and about 75 mM, between about 15 mM and about 70 mM,
between about 15 mM and about 65 mM, between about 15 mM and about
60 mM, between about 15 mM and about 55 mM, between about 15 mM and
about 50 mM, between about 15 mM and about 45 mM, between about 15
mM and about 40 mM, between about 15 mM and about 35 mM, between
about 20 mM and about 100 mM, between about 20 mM and about 95 mM,
between about 20 mM and about 90 mM, between about 20 mM and about
85 mM, between about 20 mM and about 80 mM, between about 20 mM and
about 75 mM, between about 20 mM and about 70 mM, between about 20
mM and about 65 mM, between about 20 mM and about 60 mM, between
about 25 mM and about 55 mM, between about 25 mM and about 50 mM,
between about 25 mM and about 45 mM, between about 30 mM and about
100 mM, between about 30 mM and about 95 mM, between about 30 mM
and about 90 mM, between about 30 mM and about 85 mM, between about
30 mM and about 80 mM, between about 30 mM and about 75 mM, between
about 30 mM and about 70 mM, between about 30 mM and about 65 mM,
between about 30 mM and about 60 mM, between about 30 mM and about
55 mM, between about 30 mM and about 50 mM, between about 35 mM and
about 100 mM, between about 35 mM and about 95 mM, between about 35
mM and about 90 mM, between about 35 mM and about 85 mM, between
about 35 mM and about 80 mM, between about 35 mM and about 75 mM,
between about 35 mM and about 70 mM, between about 35 mM and about
65 mM, between about 35 mM and about 60 mM, between about 35 mM and
about 55 mM, between about 40 mM and about 100 mM, between about 40
mM and about 95 mM, between about 40 mM and about 90 mM, between
about 40 mM and about 85 mM, between about 40 mM and about 80 mM,
between about 40 mM and about 75 mM, between about 40 mM and about
70 mM, between about 40 mM and about 65 mM, between about 40 mM and
about 60 mM, between about 45 mM and about 100 mM, between about 45
mM and about 95 mM, between about 45 mM and about 90 mM, between
about 45 mM and about 85 mM, between about 45 mM and about 80 mM,
between about 45 mM and about 75 mM, between about 45 mM and about
70 mM, between about 45 mM and about 65 mM, between about 50 mM and
about 100 mM, between about 50 mM and about 95 mM, between about 50
mM and about 90 mM, between about 50 mM and about 85 mM, between
about 50 mM and about 80 mM, between about 50 mM and about 75 mM,
between about 50 mM and about 70 mM, between about 55 mM and about
100 mM, between about 55 mM and about 95 mM, between about 55 mM
and about 90 mM, between about 55 mM and about 85 mM, between about
55 mM and about 80 mM, between about 55 mM and about 75 mM, between
about 60 mM and about 100 mM, between about 60 mM and about 95 mM,
between about 60 mM and about 90 mM, between about 60 mM and about
85 mM, between about 60 mM and about 80 mM, between about 65 mM and
about 100 mM, between about 65 mM and about 95 mM, between about 65
mM and about 90 mM, between about 65 mM and about 85 mM, between
about 70 mM and about 100 mM, between about 70 mM and about 95 mM,
between about 70 mM and about 90 mM, between about 75 mM and about
100 mM, between about 75 mM and about 95 mM, or between about 80 mM
and about 100 mM) stabilizing agent in the fluid.
[0103] In some embodiments of these methods, prior to step (a), the
pH of the fluid is between about 7.4 and about 7.8 (e.g., between
about 7.4 and about 7.7, between about 7.4 and about 7.6, between
about 7.5 and about 7.8, between about 7.5 and about 7.7, between
about 7.6 and about 7.8, or about 7.6). In some embodiments of any
of these methods, the fluid includes between about 10 mM and about
50 mM (e.g., between about 10 mM and about 45 mM, between about 10
mM and about 40 mM, between about 10 mM and about 35 mM, between
about 10 mM and about 30 mM, between about 10 mM and about 25 mM,
between about 10 mM and about 20 mM, between about 15 mM and about
50 mM, between about 15 mM and about 45 mM, between about 15 mM and
about 40 mM, between about 15 mM and about 35 mM, between about 15
mM and about 30 mM, between about 15 mM and about 25 mM, between
about 20 mM and about 50 mM, between about 20 mM and about 45 mM,
between about 20 mM and about 40 mM, between about 20 mM and about
35 mM, between about 20 mM and about 30 mM, between about 25 mM and
about 50 mM, between about 25 mM and about 45 mM, between about 25
mM and about 40 mM, between about 25 mM and about 35 mM, between
about 30 mM and about 50 mM, between about 30 mM and about 45 mM,
between about 30 mM and about 40 mM, between about 35 mM and about
50 mM, between about 35 mM and about 45 mM, or between about 40 mM
and about 50 mM) sodium phosphate.
[0104] In some embodiments of these methods, the fluid includes
between about 50 mM and about 90 mM sodium chloride (e.g., between
about 50 mM and about 80 mM, between 50 mM and about 75 mM, between
about 50 mM and about 70 mM, between about 50 mM and about 65 mM,
between about 50 mM and about 60 mM, between about 50 mM and about
55 mM, between about 55 mM and about 90 mM, between about 55 mM and
about 85 mM, between about 55 mM and about 80 mM, between about 55
mM and about 75 mM, between about 55 mM and about 70 mM, between
about 55 mM and about 65 mM, between about 55 mM and about 60 mM,
between about 60 mM and about 90 mM, between about 60 mM and about
85 mM, between about 60 mM and about 80 mM, between about 60 mM and
about 75 mM, between about 60 mM and about 70 mM, between about 60
mM and about 65 mM, between about 65 mM and about 90 mM, between
about 65 mM and about 85 mM, between about 65 mM and about 80 mM,
between about 65 mM and about 75 mM, between about 65 mM and about
70 mM, between about 70 mM and about 90 mM, between about 70 mM and
about 85 mM, between about 70 mM and about 80 mM, between about 70
mM and about 75 mM, between about 75 mM and about 90 mM, between
about 75 mM and about 85 mM, between about 75 mM and about 80 mM,
between about 80 mM and about 90 mM, between about 80 mM and about
85 mM, or between about 85 mM and about 90 mM sodium chloride).
[0105] The recombinant antibody in any of the methods described in
this section can be any of the recombinant antibodies described
herein. For example, the recombinant antibodies can include one or
both of: a heavy chain variable domain that includes a total of
between one and six (e.g., one, two, three, four, five, or six)
histidines in the set of CDR1, CDR2, and CDR3; and a light chain
variable domain that includes a total of between one and six (e.g.,
one, two, three, four, five, or six) histidines in the set of CDR1,
CDR2, and CDR3. The heavy chain variable domain e.g., can include a
total of between one and six (e.g., one, two, three, four, five, or
six) histidines in the set of CDR1, CDR2, and CDR3. In some
examples of the methods described in this section, the heavy chain
variable domain can include a CDR1 including one histidine residue
and a CDR2 including one histidine residue. In some examples of the
methods described in this section, the recombinant antibody
includes one or more (e.g., one, two, or three) of a CDR1 including
a sequence of SEQ ID NO: 1, a CDR2 including a sequence of SEQ ID
NO: 2, and a CDR3 including a sequence of SEQ ID NO: 3. In some
examples of any the methods described in this section, the heavy
chain variable region includes a sequence of SEQ ID NO: 4. In some
examples of the methods described in this section, the heavy chain
includes a sequence of SEQ ID NO: 5 (BNJ441 heavy chain). In some
examples of any of the methods described in this section, the light
chain variable region includes one or more (e.g., one, two, or
three) of a CDR1 including a sequence of SEQ ID NO: 6, a CDR2
including a sequence of SEQ ID NO: 7, and a CDR3 including a
sequence of SEQ ID NO: 8. In some examples of the methods described
in this section, the light chain variable domain includes a
sequence of SEQ ID NO: 9. In some examples of the methods described
in this section, the light chain includes a sequence of SEQ ID NO:
10 (BNJ441 light chain).
[0106] In some examples of the methods described in this section,
the recombinant antibody can include a heavy chain variable domain
that includes one or more (e.g., one, two, or three) of a CDR1
including a sequence of SEQ ID NO: 11, a CDR2 including a sequence
of SEQ ID NO: 12, and a CDR3 including a sequence of SEQ ID NO: 13.
In some examples of the methods described in this section, the
recombinant antibody can include a heavy chain variable region
including a sequence of SEQ ID NO: 14. In some examples of any of
the methods described in this section, the recombinant antibody can
include a heavy chain including a sequence of SEQ ID NO: 15
(eculizumab heavy chain). In some examples of any of the methods
described in this section, the light chain variable region includes
one or more (e.g., one, two, or three) of a CDR1 including a
sequence of SEQ ID NO: 16, a CDR2 including a sequence of SEQ ID
NO: 17, and a CDR3 including a sequence of SEQ ID NO: 18. In some
examples of any of the methods described herein, the recombinant
antibody can include a light chain variable region including a
sequence of SEQ ID NO: 19. In some examples of any of the methods
described herein, the recombinant antibody can include a light
chain including a sequence of SEQ ID NO: 20 (eculizumab light
chain).
Methods of Performing Viral Filtration (Part C)
[0107] Also provided are methods of performing viral filtration
that include: (a) adjusting (e.g., increasing or decreasing) the pH
of a fluid including a recombinant antibody (e.g., any of the
recombinant antibodies described herein) to between about 7.0 and
about 7.85 (e.g., between about 7.0 and about 7.8, between about
7.0 and about 7.75, between about 7.0 and about 7.7, between about
7.0 and about 7.65, between about 7.0 and about 7.6, between about
7.0 and about 7.55, between about 7.0 and about 7.5, between about
7.0 and about 7.45, between about 7.0 and about 7.4, between about
7.0 and about 7.35, between about 7.0 and about 7.3, between about
7.0 and about 7.25, between about 7.0 and about 7.2, between about
7.0 and about 7.15, between about 7.0 and about 7.1, between about
7.05 and about 7.85, between about 7.05 and about 7.80, between
about 7.05 and about 7.75, between about 7.05 and about 7.7,
between about 7.05 and about 7.65, between about 7.05 and about
7.6, between about 7.05 and about 7.55, between about 7.05 and
about 7.5, between about 7.05 and about 7.45, between about 7.05
and about 7.4, between about 7.05 and about 7.35, between about
7.05 and about 7.3, between about 7.05 and about 7.25, between
about 7.05 and about 7.2, between about 7.05 and about 7.15,
between about 7.1 and about 7.85, between about 7.1 and about 7.8,
between about 7.1 and about 7.75, between about 7.1 and about 7.7,
between about 7.1 and about 7.65, between about 7.1 and about 7.6,
between about 7.1 and about 7.55, between about 7.1 and about 7.5,
between about 7.1 and about 7.45, between about 7.1 and about 7.4,
between about 7.1 and about 7.35, between about 7.1 and about 7.3,
between about 7.1 and about 7.25, between about 7.1 and about 7.2,
between about 7.15 and about 7.85, between about 7.15 and about
7.8, between about 7.15 and about 7.75, between about 7.15 and
about 7.7, between about 7.15 and about 7.65, between about 7.15
and about 7.6, between about 7.15 and about 7.55, between about
7.15 and about 7.5, between about 7.15 and about 7.45, between
about 7.15 and about 7.4, between about 7.15 and about 7.35,
between about 7.15 and about 7.3, between about 7.15 and about
7.25, between about 7.2 and about 7.85, between about 7.2 and about
7.8, between about 7.2 and about 7.75, between about 7.2 and about
7.7, between about 7.2 and about 7.65, between about 7.2 and about
7.6, between about 7.2 and about 7.55, between about 7.2 and about
7.5, between about 7.2 and about 7.45, between about 7.2 and about
7.4, between about 7.2 and about 7.35, between about 7.2 and about
7.3, between about 7.25 and about 7.85, between about 7.25 and
about 7.8, between about 7.25 and about 7.75, between about 7.25
and about 7.7, between about 7.25 and about 7.65, between about
7.25 and about 7.6, between about 7.25 and about 7.55, between
about 7.25 and about 7.5, between about 7.25 and about 7.45,
between about 7.25 and about 7.4, between about 7.25 and about
7.35, between about 7.3 and about 7.85, between about 7.3 and about
7.8, between about 7.3 and about 7.75, between about 7.3 and about
7.7, between about 7.3 and about 7.65, between about 7.3 and about
7.6, between about 7.3 and about 7.55, between about 7.3 and about
7.5, between about 7.3 and about 7.45, between about 7.3 and about
7.4, between about 7.35 and about 7.85, between about 7.35 and
about 7.8, between about 7.35 and about 7.75, between about 7.35
and about 7.7, between about 7.35 and about 7.65, between about
7.35 and about 7.6, between about 7.35 and about 7.55, between
about 7.35 and about 7.5, between about 7.35 and about 7.45,
between about 7.4 and about 7.85, between about 7.4 and about 7.8,
between about 7.4 and about 7.75, between about 7.4 and about 7.7,
between about 7.4 and about 7.65, between about 7.4 and about 7.6,
between about 7.45 and about 7.85, between about 7.45 and about
7.8, between about 7.45 and about 7.75, between about 7.45 and
about 7.7, between about 7.45 and about 7.65, between about 7.45
and about 7.6, between about 7.45 and about 7.55, between about 7.5
and about 7.85, between about 7.5 and about 7.8, between about 7.5
and about 7.75, between about 7.5 and about 7.7, between about 7.5
and about 7.65, between about 7.5 and about 7.6, between about 7.55
and about 7.85, between about 7.55 and about 7.8, between about
7.55 and about 7.75, between about 7.55 and about 7.7, between
about 7.55 and about 7.65, between about 7.6 and about 7.85,
between about 7.6 and about 7.8, between about 7.6 and about 7.75,
between about 7.6 and about 7.7, between about 7.65 and about 7.85,
between about 7.65 and about 7.8, between about 7.65 and about
7.75, between about 7.7 and about 7.85, between about 7.7 and about
7.8, or between about 7.75 and about 7.85) and adjusting (e.g.,
increasing or decreasing) the sodium chloride concentration of the
fluid to between about 30 mM and about 200 mM (e.g., 30 mM and
about 190 mM, between about 30 mM and about 180 mM, between about
30 mM and about 170 mM, between about 30 mM and about 160 mM,
between about 30 mM and about 150 mM, between about 30 mM and about
140 mM, between about 30 mM and about 130 mM, between about 30 mM
and about 120 mM, between about 30 mM and about 110 mM, between
about 30 mM and about 100 mM, between about 30 mM and about 90 mM,
between about 30 mM and about 80 mM, between about 30 mM and about
70 mM, between about 30 mM and about 60 mM, between about 30 mM and
about 50 mM, between about 40 mM and about 200 mM, between about 40
mM and about 190 mM, between about 40 mM and about 180 mM, between
about 40 mM and about 170 mM, between about 40 mM and about 160 mM,
between about 40 mM and about 150 mM, between about 40 mM and about
140 mM, between about 40 mM and about 130 mM, between about 40 mM
and about 120 mM, between about 40 mM and about 110 mM, between
about 40 mM and about 100 mM, between about 40 mM and about 90 mM,
between about 40 mM and about 80 mM, between about 40 mM and about
70 mM, between about 40 mM and about 60 mM, between about 50 mM and
about 200 mM, between about 50 mM and about 190 mM, between about
50 mM and about 180 mM, between about 50 mM and about 170 mM,
between about 50 mM and about 160 mM, between about 50 mM and about
150 mM, between about 50 mM and about 140 mM, between about 50 mM
and about 130 mM, between about 50 mM and about 120 mM, between
about 50 mM and about 110 mM, between about 50 mM and about 100 mM,
between about 50 mM and about 90 mM, between about 50 mM and about
80 mM, between about 50 mM and about 70 mM, between about 60 mM and
about 200 mM, between about 60 mM and about 190 mM, between about
60 mM and about 180 mM, between about 60 mM and about 170 mM,
between about 60 mM and about 160 mM, between about 60 mM and about
150 mM, between about 60 mM and about 140 mM, between about 60 mM
and about 130 mM, between about 60 mM and about 120 mM, between
about 60 mM and about 110 mM, between about 60 mM and about 100 mM,
between about 60 mM and about 90 mM, between about 60 mM and about
80 mM, between about 70 mM and about 200 mM, between about 70 mM
and about 190 mM, between about 70 mM and about 180 mM, between
about 70 mM and about 170 mM, between about 70 mM and about 160 mM,
between about 70 mM and about 150 mM, between about 70 mM and about
140 mM, between about 70 mM and about 130 mM, between about 70 mM
and about 120 mM, between about 70 mM and about 110 mM, between
about 70 mM and about 100 mM, between about 70 mM and about 90 mM,
between about 80 mM and about 200 mM, between about 80 mM and about
190 mM, between about 80 mM and about 180 mM, between about 80 mM
and about 170 mM, between about 80 mM and about 160 mM, between
about 80 mM and about 150 mM, between about 80 mM and about 140 mM,
between about 80 mM and about 130 mM, between about 80 mM and about
120 mM, between about 80 mM and about 110 mM, between about 80 mM
and about 100 mM, between about 90 mM and about 200 mM, between
about 90 mM and about 190 mM, between about 90 mM and about 180 mM,
between about 90 mM and about 170 mM, between about 90 mM and about
160 mM, between about 90 mM and about 150 mM, between about 90 mM
and about 140 mM, between about 90 mM and about 130 mM, between
about 90 mM and about 120 mM, between about 90 mM and about 110 mM,
between about 100 mM and about 200 mM, between about 100 mM and
about 190 mM, between about 100 mM and about 180 mM, between about
100 mM and about 170 mM, between about 100 mM and about 160 mM,
between about 100 mM and about 150 mM, between about 100 mM and
about 140 mM, between about 100 mM and about 130 mM, between about
100 mM and about 120 mM, between about 110 mM and about 200 mM,
between about 110 mM and about 190 mM, between about 110 mM and
about 180 mM, between about 110 mM and about 170 mM, between about
110 mM and about 160 mM, between about 110 mM and about 150 mM,
between about 110 mM and about 140 mM, between about 110 mM and
about 130 mM, between about 120 mM and about 200 mM, between about
120 mM and about 190 mM, between about 120 mM and about 180 mM,
between about 120 mM and about 170 mM, between about 120 mM and
about 160 mM, between about 120 mM and about 150 mM, between about
120 mM and about 140 mM, between about 130 mM and about 200 mM,
between about 130 mM and about 190 mM, between about 130 mM and
about 180 mM, between about 130 mM and about 170 mM, between about
130 mM and about 160 mM, between about 130 mM and about 150 mM,
between about 140 mM and about 200 mM, between about 140 mM and
about 190 mM, between about 140 mM and about 180 mM, between about
140 mM and about 170 mM, between about 140 mM and about 160 mM,
between about 150 mM and about 200 mM, between about 140 mM and
about 190 mM, between about 150 mM and about 180 mM, between about
150 mM and about 170 mM, between about 160 mM and about 200 mM,
between about 160 mM and about 190 mM, between about 160 mM and
about 180 mM, between about 170 mM and about 200 mM, between about
170 mM and about 190 mM, or between about 180 mM and about 200 mM);
and (b) flowing the fluid through a virus filter (e.g., any of the
virus filters described herein) to produce a filtrate including the
recombinant antibody. Some embodiments of these methods further
include, immediately prior to step (b), flowing the fluid through a
pre-filter (e.g., any of the pre-filters described herein, such as
a pre-filter including a polyamide membrane, such as a Sartorius
Virosart.RTM. Max pre-filter).
[0108] In some embodiments of any of these methods, the fluid
includes between about 10 mM and about 50 mM (e.g., between about
10 mM and about 45 mM, between about 10 mM and about 40 mM, between
about 10 mM and about 35 mM, between about 10 mM and about 30 mM,
between about 10 mM and about 25 mM, between about 10 mM and about
20 mM, between about 15 mM and about 50 mM, between about 15 mM and
about 45 mM, between about 15 mM and about 40 mM, between about 15
mM and about 35 mM, between about 15 mM and about 30 mM, between
about 15 mM and about 25 mM, between about 20 mM and about 50 mM,
between about 20 mM and about 45 mM, between about 20 mM and about
40 mM, between about 20 mM and about 35 mM, between about 20 mM and
about 30 mM, between about 25 mM and about 50 mM, between about 25
mM and about 45 mM, between about 25 mM and about 40 mM, between
about 25 mM and about 35 mM, between about 30 mM and about 50 mM,
between about 30 mM and about 45 mM, between about 30 mM and about
40 mM, between about 35 mM and about 50 mM, between about 35 mM and
about 45 mM, or between about 40 mM and about 50 mM) sodium
phosphate.
[0109] In some examples of any of the methods in this section, the
recombinant antibody can include a heavy chain variable domain that
includes one or more (e.g., one, two, or three) of a CDR1 including
a sequence of SEQ ID NO: 21, a CDR2 including a sequence of SEQ ID
NO: 22, and a CDR3 including a sequence of SEQ ID NO: 23. In some
examples of any of the methods described in this section, the
recombinant antibody can include a heavy chain variable region
including a sequence of SEQ ID NO: 24. In some examples of any of
the methods described in this section, the recombinant antibody can
include a heavy chain including a sequence of SEQ ID NO: 25 (BNJ383
heavy chain). In some examples of any of the methods described in
this section, the light chain variable region includes one or more
(e.g., one, two, or three) of a CDR1 including a sequence of SEQ ID
NO: 26, a CDR2 including a sequence of SEQ ID NO: 27, and a CDR3
including a sequence of SEQ ID NO: 28. In some examples of any of
the methods described in this section, the recombinant antibody can
include a light chain variable region including a sequence of SEQ
ID NO: 29. In some examples of any of the methods described in this
section, the recombinant antibody can include a light chain
including a sequence of SEQ ID NO: 30 (BNJ383 light chain).
Methods of Performing Viral Filtration (Part D)
[0110] Also provided are methods of performing viral filtration
that include: (a) adjusting (e.g., increasing or decreasing) the pH
of a fluid including a recombinant antibody (e.g., any of the
recombinant antibodies described herein) to between about 5.0 and
about 7.0 (e.g., between about 5.0 and about 6.9, between about 5.0
and about 6.8, between about 5.0 and about 6.7, between about 5.0
and about 6.6, between about 5.0 and about 6.5, between about 5.0
and about 6.4, between about 5.0 and about 6.3, between about 5.0
and about 6.2, between about 5.0 and about 6.1, between about 5.0
and about 6.0, between about 5.0 and about 5.9, between about 5.0
and about 5.8, between about 5.0 and about 5.7, between about 5.0
and about 5.6, between about 5.0 and about 5.5, between about 5.0
and about 5.4, between about 5.0 and about 5.3, between about 5.0
and about 5.2, between about 5.1 and about 7.0, between about 5.1
and about 6.9, between about 5.1 and about 6.8, between about 5.1
and about 6.7, between about 5.1 and about 6.6, between about 5.1
and about 6.5, between about 5.1 and about 6.4, between about 5.1
and about 6.3, between about 5.1 and about 6.2, between about 5.1
and about 6.1, between about 5.1 and about 6.0, between about 5.1
and about 5.9, between about 5.1 and about 5.8, between about 5.1
and about 5.7, between about 5.1 and about 5.6, between about 5.1
and about 5.5, between about 5.1 and about 5.4, between about 5.1
and about 5.3, between about 5.2 and about 7.0, between about 5.2
and about 6.9, between about 5.2 and about 6.8, between about 5.2
and about 6.7, between about 5.2 and about 6.6, between about 5.2
and about 6.5, between about 5.2 and about 6.4, between about 5.2
and about 6.3, between about 5.2 and about 6.2, between about 5.2
and about 6.1, between about 5.2 and about 6.0, between about 5.2
and about 5.9, between about 5.2 and about 5.8, between about 5.2
and about 5.7, between about 5.2 and about 5.6, between about 5.2
and about 5.5, between about 5.2 and about 5.4, between about 5.3
and about 7.0, between about 5.3 and about 6.9, between about 5.3
and about 6.8, between about 5.3 and about 6.7, between about 5.3
and about 6.6, between about 5.3 and about 6.5, between about 5.3
and about 6.4, between about 5.3 and about 6.3, between about 5.3
and about 6.2, between about 5.3 and about 6.1, between about 5.3
and about 6.0, between about 5.3 and about 5.9, between about 5.3
and about 5.8, between about 5.3 and about 5.7, between about 5.3
and about 5.6, between about 5.3 and about 5.5, between about 5.4
and about 7.0, between about 5.4 and about 6.9, between about 5.4
and about 6.8, between about 5.4 and about 6.7, between about 5.4
and about 6.6, between about 5.4 and about 6.5, between about 5.4
and about 6.4, between about 5.4 and about 6.3, between about 5.4
and about 6.2, between about 5.4 and about 6.1, between about 5.4
and about 6.0, between about 5.4 and about 5.9, between about 5.4
and about 5.8, between about 5.4 and about 5.7, between about 5.4
and about 5.6, between about 5.5 and about 7.0, between about 5.5
and about 6.9, between about 5.5 and about 6.8, between about 5.5
and about 6.7, between about 5.5 and about 6.6, between about 5.5
and about 6.5, between about 5.5 and about 6.4, between about 5.5
and about 6.3, between about 5.5 and about 6.2, between about 5.5
and about 6.1, between about 5.5 and about 6.0, between about 5.5
and about 5.9, between about 5.5 and about 5.8, between about 5.5
and about 5.7, between about 5.6 and about 7.0, between about 5.6
and about 6.9, between about 5.6 and about 6.8, between about 5.6
and about 6.7, between about 5.6 and about 6.6, between about 5.6
and about 6.5, between about 5.6 and about 6.4, between about 5.6
and about 6.3, between about 5.6 and about 6.2, between about 5.6
and about 6.1, between about 5.6 and about 6.0, between about 5.6
and about 5.9, between about 5.6 and about 5.8, between about 5.7
and about 7.0, between about 5.7 and about 6.9, between about 5.7
and about 6.8, between about 5.7 and about 6.7, between about 5.7
and about 6.6, between about 5.7 and about 6.5, between about 5.7
and about 6.4, between about 5.7 and about 6.3, between about 5.7
and about 6.2, between about 5.7 and about 6.1, between about 5.7
and about 6.0, between about 5.7 and about 5.9, between about 5.8
and about 7.0, between about 5.8 and about 6.9, between about 5.8
and about 6.8, between about 5.8 and about 6.7, between about 5.8
and about 6.6, between about 5.8 and about 6.5, between about 5.8
and about 6.4, between about 5.8 and about 6.3, between about 5.8
and about 6.2, between about 5.8 and about 6.1, between about 5.8
and about 6.0, between about 5.9 and about 7.0, between about 5.9
and about 6.9, between about 5.9 and about 6.8, between about 5.9
and about 6.7, between about 5.9 and about 6.6, between about 5.9
and about 6.5, between about 5.9 and about 6.4, between about 5.9
and about 6.3, between about 5.9 and about 6.2, between about 5.9
and about 6.1, between about 6.0 and about 7.0, between about 6.0
and about 6.9, between about 6.0 and about 6.8, between about 6.0
and about 6.7, between about 6.0 and about 6.6, between about 6.0
and about 6.5, between about 6.0 and about 6.4, between about 6.0
and about 6.3, between about 6.0 and about 6.2, between about 6.1
and about 7.0, between about 6.1 and about 6.9, between about 6.1
and about 6.8, between about 6.1 and about 6.7, between about 6.1
and about 6.6, between about 6.1 and about 6.5, between about 6.1
and about 6.4, between about 6.1 and about 6.3, between about 6.2
and about 7.0, between about 6.2 and about 6.9, between about 6.2
and about 6.8, between about 6.2 and about 6.7, between about 6.2
and about 6.6, between about 6.2 and about 6.5, between about 6.2
and about 6.4, between about 6.3 and about 7.0, between about 6.3
and about 6.9, between about 6.3 and about 6.8, between about 6.3
and about 6.7, between about 6.3 and about 6.6, between about 6.3
and about 6.5, between about 6.4 and about 7.0, between about 6.4
and about 6.9, between about 6.4 and about 6.8, between about 6.4
and about 6.7, between about 6.4 and about 6.6, between about 6.5
and about 7.0, between about 6.5 and about 6.9, between about 6.5
and about 6.8, between about 6.5 and about 6.7, between about 6.6
and about 7.0, between about 6.6 and about 6.9, between about 6.6
and about 6.8, between about 6.7 and about 7.0, between about 6.7
and about 6.9, or between about 6.8 and about 7.0); and (b) flowing
the fluid through a virus filter (e.g., any of the virus filters
described herein) to produce a filtrate including the recombinant
antibody.
[0111] Some embodiments of these methods further include, prior to
step (b) adjusting (e.g., increasing or decreasing) the sodium
chloride concentration of the fluid to between about 60 mM and
about 300 mM (e.g., between about 60 mM and about 280 mM, between
about 60 mM and about 260 mM, between about 60 mM and about 240 mM,
between about 60 mM and about 220 mM, between about 60 mM and about
200 mM, between about 60 mM and about 180 mM, between about 60 mM
and about 160 mM, between about 60 mM and about 140 mM, between
about 60 mM and about 120 mM, between about 60 mM and about 100 mM,
between about 60 mM and about 80 mM, between about 80 mM and about
300 mM, between about 80 mM and about 280 mM, 80 mM and about 260
mM, between about 80 mM and about 240 mM, between about 80 mM and
about 220 mM, between about 80 mM and about 200 mM, between about
80 mM and about 180 mM, between about 80 mM and about 160 mM,
between about 80 mM and about 140 mM, between about 80 mM and about
120 mM, between about 80 mM and about 100 mM, between about 100 mM
and about 300 mM, between about 100 mM and about 280 mM, between
about 100 mM and about 260 mM, between about 100 mM and about 240
mM, between about 100 mM and about 220 mM, between about 100 mM and
about 200 mM, between about 100 mM and about 180 mM, between about
100 mM and about 160 mM, between about 100 mM and about 140 mM,
between about 100 mM and about 120 mM, between about 120 mM and
about 300 mM, between about 120 mM and about 280 mM, between about
120 mM and about 260 mM, between about 120 mM and about 240 mM,
between about 120 mM and about 220 mM, between about 120 mM and
about 200 mM, between about 120 mM and about 180 mM, between about
120 mM and about 160 mM, between about 120 mM and about 140 mM,
between about 140 mM and about 300 mM, between about 140 mM and
about 280 mM, between about 140 mM and about 260 mM, between about
140 mM and about 240 mM, between about 140 mM and about 220 mM,
between about 140 mM and about 200 mM, between about 140 mM and
about 180 mM, between about 140 mM and about 160 mM, between about
160 mM and about 300 mM, between about 160 mM and about 280 mM,
between about 160 mM and about 260 mM, between about 160 mM and
about 240 mM, between about 160 mM and about 220 mM, between about
160 mM and about 200 mM, between about 160 mM and about 180 mM,
between about 180 mM and about 300 mM, between about 180 mM and
about 280 mM, between about 180 mM and about 260 mM, between about
180 mM and about 240 mM, between about 180 mM and about 220 mM,
between about 180 mM and about 200 mM, between about 200 mM and
about 300 mM, between about 200 mM and about 280 mM, between about
200 mM and about 260 mM, between about 200 mM and about 240 mM,
between about 200 mM and about 220 mM, between about 220 mM and
about 300 mM, between about 220 mM and about 280 mM, between about
220 mM and about 260 mM, between about 220 mM and about 240 mM,
between about 240 mM and about 300 mM, between about 240 mM and
about 280 mM, between about 240 mM and about 260 mM, between about
260 mM and about 300 mM, between about 260 mM and about 280 mM, or
between about 280 mM and about 300 mM).
[0112] In some embodiments of any of these methods, the fluid
includes between about 10 mM and about 50 mM (e.g., between about
10 mM and about 45 mM, between about 10 mM and about 40 mM, between
about 10 mM and about 35 mM, between about 10 mM and about 30 mM,
between about 10 mM and about 25 mM, between about 10 mM and about
20 mM, between about 15 mM and about 50 mM, between about 15 mM and
about 45 mM, between about 15 mM and about 40 mM, between about 15
mM and about 35 mM, between about 15 mM and about 30 mM, between
about 15 mM and about 25 mM, between about 20 mM and about 50 mM,
between about 20 mM and about 45 mM, between about 20 mM and about
40 mM, between about 20 mM and about 35 mM, between about 20 mM and
about 30 mM, between about 25 mM and about 50 mM, between about 25
mM and about 45 mM, between about 25 mM and about 40 mM, between
about 25 mM and about 35 mM, between about 30 mM and about 50 mM,
between about 30 mM and about 45 mM, between about 30 mM and about
40 mM, between about 35 mM and about 50 mM, between about 35 mM and
about 45 mM, or between about 40 mM and about 50 mM) sodium
phosphate.
[0113] Some embodiments of these methods further include,
immediately prior to step (b), flowing the fluid through a
pre-filter (e.g., any of the pre-filters described herein, such as
a pre-filter including a polyamide membrane, such as a Sartorius
Virosart.RTM. Max pre-filter).
[0114] Examples of recombinant antibodies can include a heavy chain
variable domain that includes one or more (e.g., one, two, or
three) of a CDR1 including a sequence of SEQ ID NO: 31, a CDR2
including a sequence of SEQ ID NO: 32, and a CDR3 including a
sequence of SEQ ID NO: 33. In some examples of any of the
recombinant antibodies described herein, the recombinant antibody
can include a heavy chain variable region including a sequence of
SEQ ID NO: 34. In some examples of any of the recombinant
antibodies described herein, the recombinant antibody can include a
heavy chain including a sequence of SEQ ID NO: 35 (e.g.,
samalizumab heavy chain). In some examples of any of the
recombinant antibodies described herein, the light chain variable
region includes one or more (e.g., one, two, or three) of a CDR1
including a sequence of SEQ ID NO: 36, a CDR2 including a sequence
of SEQ ID NO: 37, and a CDR3 including a sequence of SEQ ID NO: 38.
In some examples of any of the recombinant antibodies described
herein, the recombinant antibody can include a light chain variable
region including a sequence of SEQ ID NO: 39. In some examples of
any of the recombinant antibodies described herein, the recombinant
antibody can include a light chain including a sequence of SEQ ID
NO: 40 (e.g., samalizumab light chain).
Methods of Purifying or Manufacturing a Recombinant Antibody
[0115] Provided herein are methods of purifying a recombinant
antibody and methods of manufacturing a recombinant antibody. The
methods can include, for example, (a) capturing a recombinant
antibody from a fluid including the recombinant antibody, e.g., a
clarified liquid culture medium or a buffered fluid including the
recombinant antibody; (b) following capturing, performing one or
more unit operations on the recombinant antibody; and following
steps (a) and (b), performing viral filtration on the recombinant
antibody to provide a filtrate that includes purified recombinant
antibody and, e.g., optionally is substantially free of soluble
protein aggregates (e.g., using any of the methods of performing
viral filtration described herein); and optionally, further (d)
performing one or more unit operations on the purified recombinant
antibody. Some embodiments further include, for example, performing
at least one (such as two, three, or four) unit operation before
the capturing step (e.g., selected from the group of clarifying a
culture medium, ultrafiltration/diafiltration to concentrate the
recombinant antibody in a fluid, viral inactivation, and adjusting
(e.g., increasing or decreasing) one or both of the pH and ionic
concentration of a fluid including the recombinant antibody). In
some embodiments, step (b) includes performing one or more (such as
two, three, or four) unit operations on the recombinant antibody,
e.g., selected from the group of ultrafiltration/diafiltration to
concentrate the recombinant antibody in a fluid, purifying the
recombinant antibody, polishing the recombinant antibody,
inactivating viruses, and adjusting (e.g., increasing or
decreasing) one or both of the pH and ionic concentration of a
fluid including the recombinant antibody. In some embodiments, step
(b) includes, immediately prior to performing viral filtration, a
step of performing the unit operation of pre-filtration by passing
a fluid including the recombinant antibody through a pre-filter. In
some embodiments, step (b) includes performing the following unit
operations of: purifying the recombinant antibody, inactivating
viruses, ultrafiltration/diafiltration to concentrate the
recombinant antibody in a fluid, depth filtration (e.g., using any
of the exemplary depth filters described herein), adjusting (e.g.,
increasing or decreasing) the pH and ionic concentration of a fluid
including the recombinant antibody, and pre-filtration (e.g., using
any of the exemplary pre-filters described herein).
[0116] Some embodiments further include performing one or more
(two, three, or four) unit operations after the step of performing
viral filtration (e.g., one or more unit operations selected from
the group of purifying the recombinant antibody, polishing the
recombinant antibody, adjusting (e.g., increasing or decreasing)
one or both of the pH and ionic concentration of a fluid including
the purified recombinant antibody, or passing the fluid through an
additional virus filter). Some embodiments further include
performing a step of formulating the purified recombinant antibody
after performing viral filtration.
[0117] The methods provided herein can result in a purified
recombinant antibody that is, e.g., at least or about 95%, 96%,
97%, 98%, 98.2%, 98.4%, 98.6%, 98.8%, 99.0%, 99.1%, 99.2%, 99.3%,
99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% free of soluble protein
aggregates or includes no detectable soluble protein aggregates.
The methods provided herein can also provide, e.g., for a
reduction, such as up to a 5% reduction, up to a 10% reduction, up
to a 15% reduction, up to a 20% reduction, up to a 25% reduction,
up to a 30% reduction, up to a 35% reduction, up to a 40%
reduction, up to a 45% reduction, up to a 50% reduction, up to a
55% reduction, up to a 60% reduction, up to a 65% reduction, up to
a 70% reduction, up to a 75% reduction, up to a 80% reduction, up
to an 85% reduction, up to a 90% reduction, up to a 95% reduction,
or up to a 99% reduction in host cell protein present in a purified
recombinant antibody (e.g., as compared to a purified recombinant
antibody produced by a method that does not include a viral
filtration step performed using any of the methods described herein
(and optionally also does not include a pre-filtration step prior
to the viral filtration step), after a step of capturing the
recombinant protein (and optionally further after one or more
additional unit operations)). Methods for determining the level of
host cell protein are well known in the art. For example, kits for
detecting the level of host cell protein are commercially available
from Cygnus Technologies (Southport, N.C.), ArrayBridge (St. Louis,
Mo.), Cisbio (Bedford, Mass.), and Lonza (Basel, Switzerland).
[0118] In some embodiments of any of the methods described herein,
the filtrate produced by the viral filtration in step (c) can
include a reduced level (e.g., up to 5% reduction, up to 10%
reduction, up to a 15% reduction, up to a 20% reduction, up to a
30% reduction, up to a 35% reduction, up to a 40% reduction, up to
a 45% reduction, up to a 50% reduction, up to a 55% reduction, up
to a 60% reduction, up to a 60% reduction, up to a 70% reduction,
up to a 75% reduction, up to a 80% reduction, up to a 85%
reduction, up to a 90% reduction, up to a 95% reduction, or up to a
99% reduction) in the level of soluble protein aggregates (e.g., as
compared to a level of soluble protein aggregates in a purified
recombinant antibody produced by a method that does not include a
viral filtration step performed using any of the methods described
herein (and optionally also does not include a pre-filtration step
prior to the viral filtration step), after a step of capturing the
recombinant protein (and optionally further after one or more
additional unit operations)).
[0119] The methods provided herein can also result in a purified
recombinant antibody that has a decreased level of immunogenicity
when administered to a subject (such as a human subject) (e.g., as
compared to the immunogenicity of a purified recombinant antibody
produced by a method that does not include a viral filtration step
performed using any of the methods described herein (and optionally
also does not include a pre-filtration step prior to the viral
filtration step), after a step of capturing the recombinant protein
(and optionally further after one or more additional unit
operations)).
[0120] The methods provided herein can further provide for a
reduced risk of virus filter fouling or contamination in a method
of purifying a recombinant protein and a method of manufacturing a
recombinant protein product or in a system used to perform the same
(e.g., as compared to a method that does not include a viral
filtration step performed using any of the methods described herein
(and optionally also does not include a pre-filtration step prior
to the viral filtration step), after a step of capturing the
recombinant protein (and optionally further after one or more
additional unit operations) or a system used to perform the
same).
Cells and Cell Culture
[0121] Cells including a nucleic acid encoding a recombinant
antibody can be used to produce the recombinant antibody (such as a
secreted recombinant antibody). In some examples, the nucleic acid
encoding the recombinant antibody is stably integrated into the
genome of the cell.
[0122] The cells used to produce the recombinant antibody can be
bacteria (e.g., gram negative bacteria), yeast (e.g., Saccharomyces
cerevisiae, Pichia pastoris, Hansenula polymorpha, Kluyveromyces
lactis, Schizosaccharomyces pombe, Yarrowia hpolytica, or Arxula
adeninivorans), or mammalian cells.
[0123] The mammalian cell used to produce the recombinant antibody
can be a cell that grows in suspension or an adherent cell.
Non-limiting examples of mammalian cells that can be cultured to
produce a recombinant antibody (e.g., any of the recombinant
antibodies described herein, such as eculizumab, samalizumab,
BNJ441, and BNJ383) include: Chinese hamster ovary (CHO) cells
(such as CHO DG44 cells or CHO-K1s cells), Sp2.0, myeloma cells
(such as NS/0 cells), B-cells, hybridoma cells, T-cells, human
embryonic kidney (HEK) cells (such as HEK 293E and HEK 293F),
African green monkey kidney epithelial cells (Vero) cells, and
Madin-Darby Canine (Cocker Spaniel) kidney epithelial cells (MDCK)
cells. In some examples where an adherent cell is used to produce a
recombinant antibody, the cell is cultured in the presence of a
plurality of microcarriers (such as microcarriers that include one
or more pores). Additional mammalian cells that can be cultured to
produce a recombinant antibody (such as a secreted recombinant
antibody) are known in the art. In some instances, the mammalian
cell is cultured in a bioreactor. In some embodiments, the
mammalian cell used to inoculate a bioreactor was derived from a
frozen cell stock or a seed train culture.
[0124] A nucleic acid encoding a recombinant antibody can be
introduced into a mammalian cell using a wide variety of methods
known in molecular biology and molecular genetics. Non-limiting
examples include transfection (e.g., lipofection), transduction
(e.g., lentivirus, adenovirus, or retrovirus infection), and
electroporation. In some instances, the nucleic acid is not stably
integrated into a chromosome of the mammalian cell (transient
transfection), while in others it is stably integrated into a
chromosome of the mammalian cell. Alternatively or in addition, the
nucleic acid can be present in a plasmid and/or in a mammalian
artificial chromosome (such as a human artificial chromosome).
Alternatively or in addition, the nucleic acid can be introduced
into the cell using a viral vector (such as a lentivirus,
retrovirus, or adenovirus vector). The nucleic acid can be operably
linked to a promoter sequence (such as a strong promoter, such as a
.beta.-actin promoter and CMV promoter, or an inducible promoter).
The nucleic acid can be operably linked to a heterologous promoter.
A vector including the nucleic acid can, if desired, also include a
selectable marker (such as a gene that confers hygromycin,
puromycin, or neomycin resistance to the mammalian cell).
[0125] As noted herein, the recombinant antibody can be a secreted
antibody that is released by the mammalian cell into the
extracellular medium. For example, a nucleic acid sequence encoding
a soluble recombinant antibody can include a sequence that encodes
a secretion signal peptide at the N-terminus of the recombinant
antibody, which is cleaved by an enzyme present in the mammalian
cell, and subsequently released into the extracellular medium (such
as the first and/or second liquid culture medium in a perfusion
cell culture or the first liquid culture medium and/or liquid feed
culture medium in a feed batch culture).
[0126] Any of the methods described herein can further include
culturing a mammalian cell including a nucleic acid encoding a
recombinant antibody under conditions sufficient to produce the
recombinant antibody (such as a secreted recombinant antibody).
Fed Batch Culturing
[0127] The culturing step in the methods described herein can
include fed batch culturing. As is known in the art, fed batch
culturing includes incremental (periodic) or continuous addition of
a feed culture medium to an initial cell culture, which includes a
first liquid culture medium, without substantial or significant
removal of the first liquid culture medium from the cell culture.
The cell culture in fed batch culturing can be disposed in a
bioreactor (e.g., a production bioreactor, such as a 10,000-L
production bioreactor). In some instances, the feed culture medium
is the same as the first liquid culture medium. The feed culture
medium may be either in a liquid form or a dry powder. In other
instances, the feed culture medium is a concentrated form of the
first liquid culture medium and/or is added as a dry powder. In
some embodiments, both a first liquid feed culture medium and a
different second liquid feed culture medium are added (e.g.,
continuously added) to the first liquid culture medium. In some
examples, the addition of the first liquid feed culture medium and
addition of the second liquid feed culture medium to the culture is
initiated at about the same time. In some examples, the total
volume of the first liquid feed culture medium and the second
liquid feed culture medium added to the culture over the entire
culturing period are about the same.
[0128] When the feed culture medium is added continuously, the rate
of addition of the feed culture medium can be held constant or can
be increased (e.g., steadily increased) over the culturing period.
A continuous addition of feed culture medium can start at a
specific time point during the culturing period (e.g., when the
mammalian cells reach a target viable cell density, e.g., a viable
cell density of about 1.times.10.sup.6 cells/mL, about
1.1.times.10.sup.6 cells/mL, about 1.2.times.10.sup.6 cells/mL,
about 1.3.times.10.sup.6 cells/mL, about 1.4.times.10.sup.6
cells/mL, about 1.5.times.10.sup.6 cells/mL, about
1.6.times.10.sup.6 cells/mL, about 1.7.times.10.sup.6 cells/mL,
about 1.8.times.10.sup.6 cells/mL, about 1.9.times.10.sup.6
cells/mL, or about 2.0.times.10.sup.6 cells/mL). In some
embodiments, the continuous addition of feed culture medium can be
initiated at day 2, day 3, day 4, or day 5 of the culturing
period.
[0129] In some embodiments, an incremental (periodic) addition of
feed culture medium can begin when the mammalian cells reach a
target cell density (e.g., about 1.times.10.sup.6 cells/mL, about
1.1.times.10.sup.6 cells/mL, about 1.2.times.10.sup.6 cells/mL,
about 1.3.times.10.sup.6 cells/mL, about 1.4.times.10.sup.6
cells/mL, about 1.5.times.10.sup.6 cells/mL, about
1.6.times.10.sup.6 cells/mL, about 1.7.times.10.sup.6 cells/mL,
about 1.8.times.10.sup.6 cells/mL, about 1.9.times.10.sup.6, or
about 2.0.times.10.sup.6 cells/mL). Incremental feed culture media
addition can occur at regular intervals (e.g., every day, every
other day, or every third day) or can occur when the cells reach
specific target cell densities (e.g., target cell densities that
increase over the culturing period). In some embodiments, the
amount of feed culture medium added can progressively increase
between the first incremental addition of feed culture medium and
subsequent additions of feed culture medium. The volume of a liquid
culture feed culture medium added to the initial cell culture over
any 24 hour period in the culturing period can be some fraction of
the initial volume of the bioreactor containing the culture or some
fraction of the volume of the initial culture.
[0130] For example, the addition of the liquid feed culture medium
(continuously or periodically) can occur at a time point that is
between 6 hours and 7 days, between about 6 hours and about 6 days,
between about 6 hours and about 5 days, between about 6 hours and
about 4 days, between about 6 hours and about 3 days, between about
6 hours and about 2 days, between about 6 hours and about 1 day,
between about 12 hours and about 7 days, between about 12 hours and
about 6 days, between about 12 hours and about 5 days, between
about 12 hours and about 4 days, between about 12 hours and about 3
days, between about 12 hours and about 2 days, between about 1 day
and about 7 days, between about 1 day and about 6 days, between
about 1 day and about 5 days, between about 1 day and about 4 days,
between about 1 day and about 3 days, between about 1 day and about
2 days, between about 2 days and about 7 days, between about 2 days
and about 6 days, between about 2 days and about 5 days, between
about 2 days and about 4 days, between about 2 days and about 3
days, between about 3 days and about 7 days, between about 3 days
and about 6 days, between about 3 days and about 5 days, between
about 3 days and about 4 days, between about 4 days and about 7
days, between about 4 days and about 6 days, between about 4 days
and about 5 days, between about 5 days and about 7 days, or between
about 5 days and about 6 days, after the start of the culturing
period.
[0131] The volume of a liquid feed culture medium added
(continuously or periodically) to the initial cell culture over any
24 hour period can be between 0.01.times. and about 0.3.times. of
the capacity of the bioreactor. In other embodiments, the volume of
a liquid feed culture medium added (continuously or periodically)
to the initial cell culture over any 24 hour period during the
culturing period can be between 0.02.times. and about 1.0.times. of
the volume of the initial cell culture. The total amount of feed
culture medium added (continuously or periodically) over the entire
culturing period can be between about 1% and about 40% of the
volume of the initial culture.
[0132] In some examples, two different feed culture media are added
(continuously or incrementally) during feed batch culturing. The
amount or volume of the first feed culture medium and the second
feed culture medium added can be substantially the same or can
differ. The first feed culture medium can be in the form of a
liquid and the second feed culture medium can be in the form of a
solid, or vice-versa. The first feed culture medium and the second
feed culture medium can be liquid feed culture media.
Perfusion Culturing
[0133] The culturing step in the methods described herein can be
perfusion culturing. As is known in the art, perfusion culturing
includes removing from a bioreactor (e.g., a production bioreactor)
a first volume of a first liquid culture medium, and adding to the
production bioreactor a second volume of a second liquid culture
medium, wherein the first volume and the second volume are
typically (but need not be) about equal. The mammalian cells are
retained in the bioreactor by some cell retention device or through
techniques known in the art, such as cell settling. Removal and
addition of culture media in perfusion culturing can be performed
simultaneously or sequentially, or in some combination of the two.
Further, removal and addition can be performed continuously, such
as at a rate that removes and replaces a volume of between 0.1% to
800%, between 1% and 700%, between 1% and 600%, between 1% and
500%, between 1% and 400%, between 1% and 350%, between 1% and
300%, between 1% and 250%, between 1% and 100%, between 100% and
200%, between 5% and 150%, between 10% and 50%, between 15% and
40%, between 8% and 80%, or between 4% and 30% of the capacity of
the bioreactor over an increment of time (such as over a 24-hour
increment of time).
[0134] The first volume of the first liquid culture medium removed
and the second volume of the second liquid culture medium added can
in some instances be held approximately the same over each 24-hour
period. As is known in the art, the rate at which the first volume
of the first liquid culture medium is removed (volume/unit of time)
and the rate at which the second volume of the second liquid
culture medium is added (volume/unit of time) can be varied and
depends on the conditions of the particular cell culture system.
The rate at which the first volume of the first liquid culture
medium is removed (volume/unit of time) and the rate at which the
second volume of the second liquid culture medium is added
(volume/unit of time) can be about the same or can be
different.
[0135] Alternatively, the volume removed and added can change by
gradually increasing over each 24-hour period. For example, the
volume of the first liquid culture medium removed and the volume of
the second liquid culture medium added within each 24-hour period
can be increased over the culturing period. The volume can be
increased a volume that is between 0.5% and about 20% of the
capacity of the bioreactor over a 24-hour period. The volume can be
increased over the culturing period to a volume that is about 25%
and about 150% of the capacity of the bioreactor or the first
liquid culture medium volume over a 24-hour period.
[0136] In some examples of the methods described herein, after the
first 48 to 96 hours of the culturing period, in each 24-hour
period, the first volume of the first liquid culture medium removed
and the second volume of the second liquid culture medium added is
about 10% and about 95%, about 10% and about 20%, about 20% and
about 30%, about 30% and about 40%, about 40% and about 50%, about
50% and about 60%, about 60% and about 70%, about 70% and about
80%, about 80% and about 90%, about 85% and about 95%, about 60%
and about 80%, or about 70% of the volume of the first liquid
culture medium.
[0137] Skilled practitioners will appreciate that the first liquid
culture medium and the second liquid culture medium can be the same
type of media. In other instances, the first liquid culture medium
and the second liquid culture medium can be different. The second
liquid culture medium may be more concentrated with respect to one
or more media components. In some embodiments, the first liquid
culture medium includes processed BSA, the second liquid culture
medium includes processed BSA, or both the first and the second
liquid culture medium include processed BSA.
[0138] The first volume of the first liquid culture medium can be
removed using any method, e.g., using an automated system. For
example, alternating tangential flow filtration may be used.
Alternatively, the first volume of the first liquid culture medium
can be removed by seeping or gravity flow of the first volume of
the first liquid culture medium through a sterile membrane with a
molecular weight cut-off that excludes the mammalian cell.
Alternatively, the first volume of the first liquid culture medium
can be removed by stopping or significantly decreasing the rate of
agitation for a period of at least 1 minute, at least 2 minutes, 3
minutes, 4 minutes, 5 minutes, 10 minutes, 15 minutes, 20 minutes,
25 minutes, 30 minutes, 40 minutes, 50 minutes, or 1 hour, and
removing or aspirating the first volume of the first liquid culture
medium from the top of the production bioreactor.
[0139] The second volume of the second liquid culture medium can be
added to the first liquid culture medium by a pump. The second
liquid culture medium can be added to the first liquid medium
manually, such as by pipetting or injecting the second volume of
the second liquid culture medium directly onto the first liquid
culture medium or in an automated fashion.
Liquid Culture Medium and Clarification
[0140] A fluid including a recombinant antibody, e.g., a liquid
culture medium including a recombinant antibody and that is
substantially free of cells, can be derived from any source. For
example, a liquid culture medium can be obtained from a recombinant
cell culture (such as a recombinant bacterial, yeast, or mammalian
cell culture). The liquid culture medium can be obtained from a
fed-batch mammalian cell culture (such as a fed-batch bioreactor
containing a culture of mammalian cells that secrete the
recombinant antibody) or a perfusion cell mammalian cell culture
(such as a perfusion bioreactor containing a culture of mammalian
cells that secrete the recombinant antibody). The liquid culture
medium can be a clarified liquid culture medium from a culture of
bacterial, yeast, or mammalian cells that secrete the recombinant
antibody.
[0141] Liquid culture medium obtained from a recombinant cell
culture can be clarified to obtain a liquid culture medium that is
substantially free of cells and that includes a recombinant
antibody (also called a clarified culture medium or clarified
liquid culture medium). Methods for clarifying a liquid culture
medium in order to remove cells are known in the art (such as
through the use of 0.2-.mu.m filtration and filtration using an
Alternating Tangential Flow (ATF.TM.) system or tangential flow
filtration (TFF)). Recombinant cells can be removed from liquid
culture medium using centrifugation and removing the supernatant or
by allowing the cells to settle to the gravitational bottom of a
container (such as a bioreactor) and removing the liquid culture
medium that is substantially free of cells. The liquid culture
medium can be obtained from a culture of recombinant cells (such as
recombinant bacteria, yeast, or mammalian cells) producing any of
the recombinant antibodies described herein.
[0142] The liquid culture medium including a recombinant antibody
or the liquid culture media used to culture a mammalian cell
including a nucleic acid encoding a recombinant antibody (such as
the first and second liquid culture medium in perfusion culturing
or the first liquid culture medium and the liquid feed culture
medium in fed batch culturing) can be any of the types of liquid
culture medium described herein or known in the art. For example,
any of the liquid culture media described herein can be selected
from the group of: animal-derived component free liquid culture
medium, serum-free liquid culture medium, serum-containing liquid
culture medium, chemically-defined liquid culture medium, and
protein-free liquid culture medium. In any of the processes
described herein, a liquid culture medium obtained from a culture
can be diluted by addition of a second fluid (such as a buffered
solution) before or after it is clarified and/or before the
recombinant antibody is captured.
[0143] The liquid culture medium that includes a recombinant
antibody and is substantially free of cells can be stored (such as
at a temperature below about 15.degree. C., below about 10.degree.
C., below about 4.degree. C., below about 0.degree. C., below about
-20.degree. C., below about -50.degree. C., below about -70.degree.
C., or below about -80.degree. C.) for at least or about 1 day, at
least or about 2 days, at least or about 5 days, at least or about
10 days, at least or about 15 days, at least or about 20 days, or
at least or about 30 days) prior to capturing the recombinant
antibody from the liquid culture medium. Alternatively, in some
examples, the recombinant antibody is captured from the liquid
culture medium directly from a bioreactor after a clarification
step.
Capturing the Recombinant Antibody
[0144] The methods provided herein include a step of capturing a
recombinant antibody from a fluid including the recombinant
antibody (such as a clarified liquid culture medium including the
secreted recombinant antibody or a clarified liquid culture medium
including the recombinant antibody that has been diluted with a
buffered solution).
[0145] As can be appreciated in the art, through performance of the
capturing step, the recombinant antibody can be partially purified
or isolated (e.g., at least or about 5%, e.g., at least or about
10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,
80%, 85%, 90%, or at least or about 95% pure by weight),
concentrated, and stabilized from one or more other components
present in a clarified liquid culture medium including the
recombinant antibody (such as culture medium proteins or one or
more other components (such as DNA, RNA, or other proteins) present
in or secreted from a mammalian cell). Typically, capturing is
performed using a resin that binds a recombinant antibody (such as
through the use of affinity chromatography). Non-limiting examples
of methods for capturing a recombinant antibody from a fluid
including the recombinant antibody (such as a clarified liquid
culture medium) are described herein and others are known in the
art. In the methods described herein, a recombinant antibody can be
captured from a fluid using at least one chromatography column
(such as any of the chromatography columns and/or capture
mechanisms described herein, such as affinity chromatography resin,
an anionic exchange chromatography resin, a cationic exchange
chromatography resin, a mixed-mode chromatography resin, a
molecular sieve chromatography resin, or a hydrophobic interaction
chromatography resin). The capturing step can be performed using a
chromatography resin that utilizes a protein A-binding capture
mechanism, an antibody- or antibody fragment-binding capture
mechanism, a substrate binding capture mechanism, and an antigen
binding capture mechanism. In some embodiments, the capturing
system can be a protein A-binding capturing mechanism or an
antigen-binding capturing mechanism (where the capturing antigen is
specifically recognized by the recombinant antibody). If the
recombinant enzyme includes a tag, the capturing system can be a
protein, metal chelate, or antibody (or antibody fragment) that
specifically binds to the tag present in the recombinant antibody.
Non-limiting examples of resins that can be used to capture a
recombinant antibody are described herein and additional resins are
known in the art. Non-limiting examples of resin that utilize a
protein A-binding capture mechanism is MABSELECT.TM. SURE.TM. resin
and Protein A Sepharose.TM. CL-4B (GE Healthcare).
[0146] Exemplary non-limiting sizes and shapes of the
chromatography column(s) that can be used to capture the
recombinant antibody are well known in the art. The liquid culture
medium fed (loaded) can include, for example, between about 0.05
mg/mL and about 100 mg/mL recombinant antibody, between about 0.1
mg/mL and about 90 mg/mL, between about 0.1 mg/mL and about 80
mg/mL, between about 0.1 mg/mL and about 70 mg/mL, between about
0.1 mg/mL and about 60 mg/mL, between about 0.1 mg/mL and about 50
mg/mL, between about 0.1 mg/mL and about 40 mg/mL, between about
0.1 mg/mL and about 30 mg/mL, between about 0.1 mg/mL and about 20
mg/mL, between 0.5 mg/mL and about 20 mg/mL, between about 0.1
mg/mL and about 15 mg/mL, between about 0.5 mg/mL and about 15
mg/mL, between about 0.1 mg/mL and about 10 mg/mL, or between about
0.5 mg/mL and about 10 mg/mL recombinant antibody.
[0147] As can be appreciated in the art, to capture the recombinant
antibody using the chromatography column(s), one must perform the
sequential chromatographic steps of loading, washing, eluting, and
regenerating the chromatography column(s) or chromatography
membrane(s).
[0148] Following the loading of the recombinant antibody onto the
at least one chromatographic column that includes a resin that is
capable of capturing the recombinant antibody, the at least one
chromatographic column or chromatographic membrane is washed with
at least one washing buffer. As can be appreciated in the art, the
at least one (such as two, three, or four) washing buffer is meant
to elute all proteins that are not the recombinant antibody from
the at least one chromatography column, while not disturbing the
interaction of the recombinant antibody with the resin.
[0149] Following washing, the recombinant antibody is eluted from
the at least one chromatographic column or chromatographic membrane
by passing an elution buffer through the at least one
chromatographic column or chromatographic membrane. Non-limiting
examples of elution buffers that can be used in these methods will
depend on the capture mechanism and/or the recombinant antibody.
For example, an elution buffer can include a different
concentration of salt (e.g., increased salt concentration), a
different pH (e.g., an increased or decreased salt concentration),
or a molecule that will compete with the recombinant antibody for
binding to the resin that is capable of performing the unit
operation of capturing. Examples of such elution buffers for each
exemplary capture mechanism described herein are well known in the
art.
[0150] Following elution of the recombinant antibody from the at
least one chromatographic column that includes a resin that is
capable of capturing the recombinant antibody, and before the next
volume of fluid including the recombinant antibody can be loaded
onto the at least one chromatographic column, the at least one
chromatography column or chromatographic membrane must be
equilibrated using an regeneration buffer.
Depth Filtration
[0151] The methods can include a unit operation of depth
filtration. Depth filtration includes flowing the recombinant
antibody through a depth filter to provide a filtrate that includes
the purified recombinant antibody and, e.g., is substantially free
of soluble protein aggregates. Any of the exemplary depth filters
or methods for depth filtration described herein can be used to
flow a recombinant antibody through a depth filter.
[0152] In some embodiments of the methods described herein, the
recombinant antibody is flowed through the depth filter in a fluid
having a pH of between about 4.0 and about 8.5, between about 4.0
and about 8.4, between about 4.0 and about 8.2, between about 4.0
and about 8.0, between about 4.0 and about 8.0, between about 4.0
and about 7.8, between about 4.0 and about 7.6, between about 4.0
and about 7.5, between about 4.0 and about 7.4, between about 4.0
and about 7.2, between about 4.0 and about 7.0, between about 4.0
and about 6.8, between about 4.0 and about 6.6, between about 4.0
and about 6.4, between about 4.0 and about 6.2, between about 4.0
and about 6.0, between about 4.0 and about 5.8, between about 4.0
and about 5.6, between about 4.0 and about 5.4, between about 4.0
and about 5.2, between about 4.0 and about 5.0, between about 4.0
and about 4.8, between about 4.0 and about 4.6, between about 4.0
and about 4.4, between about 4.0 and about 4.2, between about 4.2
and about 8.5, between about 4.2 and about 8.4, between about 4.2
and about 8.2, between about 4.2 and about 8.0, between about 4.2
and about 7.8, between about 4.2 and about 7.6, between about 4.2
and about 7.5, between about 4.2 and about 7.4, between about 4.2
and about 7.2, between about 4.2 and about 7.0, between about 4.2
and about 6.8, between about 4.2 and about 6.6, between about 4.2
and about 6.4, between about 4.2 and about 6.2, between about 4.2
and about 6.0, between about 4.2 and about 5.8, between about 4.2
and about 5.6, between about 4.2 and about 5.4, between about 4.2
and about 5.2, between about 4.2 and about 5.0, between about 4.2
and about 4.8, between about 4.2 and about 4.6, between about 4.2
and about 4.4, between about 4.4 and about 8.5, between about 4.4
and about 8.4, between about 4.4 and about 8.2, between about 4.4
and about 8.0, between about 4.4 and about 7.8, between about 4.4
and about 7.6, between about 4.4 and about 7.5, between about 4.4
and about 7.4, between about 4.4 and about 7.2, between about 4.4
and about 7.0, between about 4.4 and about 6.8, between about 4.4
and about 6.6, between about 4.4 and about 6.4, between about 4.4
and about 6.2, between about 4.4 and about 6.0, between about 4.4
and about 5.8, between about 4.4 and about 5.6, between about 4.4
and about 5.4, between about 4.4 and about 5.2, between about 4.4
and about 5.0, between about 4.4 and about 4.8, between about 4.4
and about 4.6, between about 4.6 and about 8.5, between about 4.6
and about 8.4, between about 4.6 and about 8.2, between about 4.6
and about 8.0, between about 4.6 and about 7.8, between about 4.6
and about 7.6, between about 4.6 and about 7.5, between about 4.6
and about 7.4, between about 4.6 and about 7.2, between about 4.6
and about 7.0, between about 4.6 and about 6.8, between about 4.6
and about 6.6, between about 4.6 and about 6.4, between about 4.6
and about 6.2, between about 4.6 and about 6.0, between about 4.6
and about 5.8, between about 4.6 and about 5.6, between about 4.6
and about 5.4, between about 4.6 and about 5.2, between about 4.6
and about 5.0, between about 4.6 and about 4.8, between about 4.8
and about 8.5, between about 4.8 and about 8.4, between about 4.8
and about 8.2, between about 4.8 and about 8.0, between about 4.8
and about 7.8, between about 4.8 and about 7.6, between about 4.8
and about 7.5, between about 4.8 and about 7.4, between about 4.8
and about 7.2, between about 4.8 and about 7.0, between about 4.8
and about 6.8, between about 4.8 and about 6.6, between about 4.8
and about 6.4, between about 4.8 and about 6.2, between about 4.8
and about 6.0, between about 4.8 and about 5.8, between about 4.8
and about 5.6, between about 4.8 and about 5.4, between about 4.8
and about 5.2, between about 4.8 and about 5.0, between about 5.0
and about 8.5, between about 5.0 and about 8.4, between about 5.0
and about 8.2, between about 5.0 and about 8.0, between about 5.0
and about 7.8, between about 5.0 and about 7.6, between about 5.0
and about 7.5, between about 5.0 and about 7.2, between about 5.0
and about 7.0, between about 5.0 and about 6.8, between about 5.0
and about 6.6, between about 5.0 and about 6.4, between about 5.0
and about 6.2, between about 5.0 and about 6.0, between about 5.0
and about 5.8, between about 5.0 and about 5.6, between about 5.0
and about 5.4, between about 5.0 and about 5.2, between about 5.2
and about 8.5, between about 5.2 and about 8.4, between about 5.2
and about 8.2, between about 5.2 and about 8.0, between about 5.2
and about 7.8, between about 5.2 and about 7.6, between about 5.2
and about 7.5, between about 5.2 and about 7.4, between about 5.2
and about 7.2, between about 5.2 and about 7.0, between about 5.2
and about 6.8, between about 5.2 and about 6.6, between about 5.2
and about 6.4, between about 5.2 and about 6.2, between about 5.2
and about 6.0, between about 5.2 and about 5.8, between about 5.2
and about 5.6, between about 5.2 and about 5.4, between about 5.4
and about 8.5, between about 5.4 and about 8.4, between about 5.4
and about 8.2, between about 5.4 and about 8.0, between about 5.4
and about 7.8, between about 5.4 and about 7.6, between about 5.4
and about 7.5, between about 5.4 and about 7.4, between about 5.4
and about 7.2, between about 5.4 and about 7.0, between about 5.4
and about 6.8, between about 5.4 and about 6.6, between about 5.4
and about 6.4, between about 5.4 and about 6.2, between about 5.4
and about 6.0, between about 5.4 and about 5.8, between about 5.4
and about 5.6, between about 5.6 and about 8.5, between about 5.6
and about 8.4, between about 5.6 and about 8.2, between about 5.6
and about 8.0, between about 5.6 and about 7.8, between about 5.6
and about 7.6, between about 5.6 and about 7.5, between about 5.6
and about 7.4, between about 5.6 and about 7.2, between about 5.6
and about 7.0, between about 5.6 and about 6.8, between about 5.6
and about 6.6, between about 5.6 and about 6.4, between about 5.6
and about 6.2, between about 5.6 and about 6.0, between about 5.6
and about 5.8, between about 5.8 and about 8.5, between about 5.8
and about 8.4, between about 5.8 and about 8.2, between about 5.8
and about 8.0, between about 5.8 and about 7.8, between about 5.8
and about 7.6, between about 5.8 and about 7.5, between about 5.8
and about 7.4, between about 5.8 and about 7.2, between about 5.8
and about 7.0, between about 5.8 and about 6.8, between about 5.8
and about 6.6, between about 5.8 and about 6.4, between about 5.8
and about 6.2, between about 5.8 and about 6.0, between about 6.0
and about 8.5, between about 6.0 and about 8.4, between about 6.0
and about 8.2, between about 6.0 and about 8.0, between about 6.0
and about 7.8, between about 6.0 and about 7.6, between about 6.0
and about 7.5, between about 6.0 and about 7.4, between about 6.0
and about 7.2, between about 6.0 and about 7.0, between about 6.0
and about 6.8, between about 6.0 and about 6.6, between about 6.0
and about 6.4, between about 6.0 and about 6.2, between about 6.2
and about 8.5, between 6.2 and about 8.4, between 6.2 and about
8.2, between 6.2 and about 8.0, between 6.2 and about 7.8, between
6.2 and about 7.6, between about 6.2 and about 7.5, between about
6.2 and about 7.4, between about 6.2 and about 7.2, between about
6.2 and about 7.0, between about 6.2 and about 6.8, between about
6.2 and about 6.6, between about 6.2 and about 6.4, between 6.4 and
about 8.5, between 6.4 and about 8.4, between 6.4 and about 8.2,
between 6.4 and about 8.0, between 6.4 and about 7.8, between 6.4
and about 7.6, between about 6.4 and about 7.5, between about 6.4
and about 7.4, between about 6.4 and about 7.2, between about 6.4
and about 7.0, between about 6.4 and about 6.8, between about 6.4
and about 6.6, between 6.6 and about 8.5, between 6.6 and about
8.4, between 6.6 and about 8.2, between 6.6 and about 8.0, between
6.6 and about 7.8, between 6.6 and about 7.6, between about 6.6 and
about 7.5, between about 6.6 and about 7.4, between about 6.6 and
about 7.2, between about 6.6 and about 7.0, between about 6.6 and
about 6.8, between 6.8 and about 8.5, between 6.8 and about 8.4,
between 6.8 and about 8.2, between 6.8 and about 8.0, between 6.8
and about 7.8, between 6.8 and about 7.6, between about 6.8 and
about 7.5, between about 6.8 and about 7.4, between about 6.8 and
about 7.2, between about 6.8 and about 7.0, between about 7.0 and
about 8.5, between about 7.0 and about 8.4, between about 7.0 and
about 8.2, between about 7.0 and about 8.0, between about 7.0 and
about 7.8, between about 7.0 and about 7.6, between about 7.0 and
about 7.5, between about 7.0 and about 7.4, between about 7.0 and
about 7.2, between about 7.2 and about 8.5, between about 7.2 and
about 8.4, between about 7.2 and about 8.2, between about 7.2 and
about 8.0, between about 7.2 and about 7.8, between about 7.2 and
about 7.6, between about 7.2 and about 7.5, between about 7.2 and
about 7.4, between 7.4 and about 8.5, between about 7.4 and about
8.4, between about 7.4 and about 8.2, between about 7.4 and about
8.0, between about 7.4 and about 7.8, between about 7.4 and about
7.6, between about 7.6 and about 8.5, between about 7.6 and about
8.4, between about 7.6 and about 8.2, between about 7.6 and about
8.0, between about 7.6 and about 7.8, between about 7.8 and about
8.5, between about 7.8 and about 8.4, between about 7.8 and about
8.2, between about 7.8 and about 8.0, between about 8.0 and about
8.5, between about 8.0 and about 8.3, between about 8.0 and about
8.2, between about 8.2 and about 8.5, between about 8.2 and about
8.4, or between about 8.3 and about 8.5.
[0153] The fluid including the recombinant antibody that is flowed
through a depth filter can include a concentration of recombinant
antibody between about 0.01 mg/mL and about 25 mg/mL (e.g., between
about 0.01 mg/mL and about 22.5 mg/mL, between about 0.01 mg/mL and
about 20.0 mg/mL, between about 0.01 mg/mL and about 17.5 mg/mL,
between about 0.01 mg/mL and about 15.0 mg/mL, between about 0.01
mg/mL and about 12.5 mg/mL, between about 0.01 mg/mL and about 10
mg/mL, between about 0.01 mg/mL and about 8 mg/mL, between about
0.01 mg/mL and about 6 mg/mL, between about 0.01 mg/mL and about 5
mg/mL, between about 0.01 mg/mL and about 4 mg/mL, between about
0.01 mg/mL and about 3.5 mg/mL, between about 0.01 mg/mL and about
3.0 mg/mL, between about 0.01 mg/mL and about 2.5 mg/mL, between
about 0.01 mg/mL and about 2.0 mg/mL, between about 0.01 mg/mL and
about 1.5 mg/mL, between about 0.01 mg/mL and about 1.0 mg/mL,
between about 0.01 mg/mL and about 0.5 mg/mL, between about 0.01
mg/mL and about 0.25 mg/mL, between about 0.01 mg/mL and about 0.1
mg/mL, between about 0.1 mg/mL and about 12.5 mg/mL, between about
0.1 mg/mL and about 10.0 mg/mL, between about 0.1 mg/mL and about
8.0 mg/mL, between about 0.1 mg/mL and about 6.0 mg/mL, between
about 0.1 mg/mL and about 5.0 mg/mL, between about 0.1 mg/mL and
about 4.0 mg/mL, between about 0.1 mg/mL and about 3.5 mg/mL,
between about 0.1 mg/mL and about 3.0 mg/mL, between about 0.1
mg/mL and about 2.5 mg/mL, between about 0.1 mg/mL and about 2.0
mg/mL, between about 0.1 mg/mL and about 1.5 mg/mL, between about
0.1 mg/mL and about 1.0 mg/mL, between about 0.1 mg/mL and about
0.5 mg/mL, or between about 0.1 mg/mL and about 0.25 mg/mL).
[0154] In some embodiments, a fluid including the recombinant
antibody is flowed through the depth filter at a flow rate of
between about 25 L/m.sup.2/h and about 400 L/m.sup.2/h, between
about 25 L/m.sup.2/h and about 390 L/m.sup.2/h, between about 25
L/m.sup.2/h and about 380 L/m.sup.2/h, between about 25 L/m.sup.2/h
and about 360 L/m.sup.2/h, between about 25 L/m.sup.2/h and about
340 L/m.sup.2/h, between about 25 L/m.sup.2/h and about 320
L/m.sup.2/h, between about 25 L/m.sup.2/h and about 300
L/m.sup.2/h, between about 25 L/m.sup.2/h and about 280
L/m.sup.2/h, between about 25 L/m.sup.2/h and about 260
L/m.sup.2/h, between about 25 L/m.sup.2/h and about 240
L/m.sup.2/h, between about 25 L/m.sup.2/h and about 220
L/m.sup.2/h, between about 25 L/m.sup.2/h and about 200
L/m.sup.2/h, between about 25 L/m.sup.2/h and about 180
L/m.sup.2/h, between about 25 L/m.sup.2/h and about 160
L/m.sup.2/h, between about 25 L/m.sup.2/h and about 140
L/m.sup.2/h, between about 25 L/m.sup.2/h and about 120
L/m.sup.2/h, between about 25 L/m.sup.2/h and about 100
L/m.sup.2/h, between about 25 L/m.sup.2/h and about 80 L/m.sup.2/h,
between about 25 L/m.sup.2/h and about 60 L/m.sup.2/h, between
about 25 L/m.sup.2/h and about 40 L/m.sup.2/h, between about 25
L/m.sup.2/h and about 35 L/m.sup.2/h, between about 40 L/m.sup.2/h
and about 400 L/m.sup.2/h, between about 40 L/m.sup.2/h and about
380 L/m.sup.2/h, between about 40 L/m.sup.2/h and about 360
L/m.sup.2/h, between about 40 L/m.sup.2/h and about 340
L/m.sup.2/h, between about 40 L/m.sup.2/h and about 320
L/m.sup.2/h, between about 40 L/m.sup.2/h and about 300
L/m.sup.2/h, between about 40 L/m.sup.2/h and about 280
L/m.sup.2/h, between about 40 L/m.sup.2/h and about 260
L/m.sup.2/h, between about 40 L/m.sup.2/h and about 240
L/m.sup.2/h, between about 40 L/m.sup.2/h and about 220
L/m.sup.2/h, between about 40 L/m.sup.2/h and about 220
L/m.sup.2/h, between about 40 L/m.sup.2/h and about 200
L/m.sup.2/h, between about 40 L/m.sup.2/h and about 180
L/m.sup.2/h, between about 40 L/m.sup.2/h and about 160
L/m.sup.2/h, between about 40 L/m.sup.2/h and about 140
L/m.sup.2/h, between about 40 L/m.sup.2/h and about 120
L/m.sup.2/h, between about 40 L/m.sup.2/h and about 100
L/m.sup.2/h, between about 40 L/m.sup.2/h and about 80 L/m.sup.2/h,
between about 40 L/m.sup.2/h and about 60 L/m.sup.2/h, between
about 40 L/m.sup.2/h and about 50 L/m.sup.2/h, between about 60
L/m.sup.2/h and about 400 L/m.sup.2/h, between about 60 L/m.sup.2/h
and about 380 L/m.sup.2/h, between about 60 L/m.sup.2/h and about
360 L/m.sup.2/h, between about 60 L/m.sup.2/h and about 340
L/m.sup.2/h, between about 60 L/m.sup.2/h and about 320
L/m.sup.2/h, between about 60 L/m.sup.2/h and about 300
L/m.sup.2/h, between about 60 L/m.sup.2/h and about 280
L/m.sup.2/h, between about 60 L/m.sup.2/h and about 260
L/m.sup.2/h, between about 60 L/m.sup.2/h and about 240
L/m.sup.2/h, between about 60 L/m.sup.2/h and about 220
L/m.sup.2/h, between about 60 L/m.sup.2/h and about 200
L/m.sup.2/h, between about 60 L/m.sup.2/h and about 180
L/m.sup.2/h, between about 70 L/m.sup.2/h and about 150
L/m.sup.2/h, between about 70 L/m.sup.2/h and about 180
L/m.sup.2/h, between about 60 L/m.sup.2/h and about 160
L/m.sup.2/h, between about 60 L/m.sup.2/h and about 140
L/m.sup.2/h, between about 60 L/m.sup.2/h and about 120
L/m.sup.2/h, between about 60 L/m.sup.2/h and about 100
L/m.sup.2/h, between about 60 L/m.sup.2/h and about 80 L/m.sup.2/h,
between about 80 L/m.sup.2/h and about 400 L/m.sup.2/h, between
about 80 L/m.sup.2/h and about 380 L/m.sup.2/h, between about 80
L/m.sup.2/h and about 360 L/m.sup.2/h, between about 80 L/m.sup.2/h
and about 340 L/m.sup.2/h, between about 80 L/m.sup.2/h and about
320 L/m.sup.2/h, between about 80 L/m.sup.2/h and about 300
L/m.sup.2/h, between about 80 L/m.sup.2/h and about 280
L/m.sup.2/h, between about 80 L/m.sup.2/h and about 260
L/m.sup.2/h, between about 80 L/m.sup.2/h and about 240
L/m.sup.2/h, between about 80 L/m.sup.2/h and about 220
L/m.sup.2/h, between about 80 L/m.sup.2/h and about 200
L/m.sup.2/h, between about 80 L/m.sup.2/h and about 180
L/m.sup.2/h, between about 80 L/m.sup.2/h and about 160
L/m.sup.2/h, between about 80 L/m.sup.2/h and about 140
L/m.sup.2/h, between about 80 L/m.sup.2/h and about 120
L/m.sup.2/h, between about 80 L/m.sup.2/h and about 100
L/m.sup.2/h, between about 100 L/m.sup.2/h and about 400
L/m.sup.2/h, between about 100 L/m.sup.2/h and about 380
L/m.sup.2/h, between about 100 L/m.sup.2/h and about 360
L/m.sup.2/h, between about 100 L/m.sup.2/h and about 340
L/m.sup.2/h, between about 100 L/m.sup.2/h and about 320
L/m.sup.2/h, between about 100 L/m.sup.2/h and about 300
L/m.sup.2/h, between about 100 L/m.sup.2/h and about 280
L/m.sup.2/h, between about 100 L/m.sup.2/h and about 260
L/m.sup.2/h, between about 100 L/m.sup.2/h and about 240
L/m.sup.2/h, between about 100 L/m.sup.2/h and about 220
L/m.sup.2/h, between about 100 L/m.sup.2/h and about 200
L/m.sup.2/h, 100 L/m.sup.2/h and about 180 L/m.sup.2/h, between
about 100 L/m.sup.2/h and about 160 L/m.sup.2/h, between about 100
L/m.sup.2/h and about 140 L/m.sup.2/h, between about 100
L/m.sup.2/h and about 120 L/m.sup.2/h, between about 120
L/m.sup.2/h and about 400 L/m.sup.2/h, between about 120
L/m.sup.2/h and about 380 L/m.sup.2/h, between about 120
L/m.sup.2/h and about 360 L/m.sup.2/h, between about 120
L/m.sup.2/h and about 340 L/m.sup.2/h, between about 120
L/m.sup.2/h and about 320 L/m.sup.2/h, between about 120
L/m.sup.2/h and about 300 L/m.sup.2/h, between about 120
L/m.sup.2/h and about 280 L/m.sup.2/h, between about 120
L/m.sup.2/h and about 260 L/m.sup.2/h, between about 120
L/m.sup.2/h and about 240 L/m.sup.2/h, between about 120
L/m.sup.2/h and about 220 L/m.sup.2/h, between about 120
L/m.sup.2/h and about 200 L/m.sup.2/h, between about 120
L/m.sup.2/h and about 180 L/m.sup.2/h, between about 120
L/m.sup.2/h and about 160 L/m.sup.2/h, between about 120
L/m.sup.2/h and about 140 L/m.sup.2/h, between about 140
L/m.sup.2/h and about 400 L/m.sup.2/h, between about 140
L/m.sup.2/h and about 380 L/m.sup.2/h, between about 140
L/m.sup.2/h and about 360 L/m.sup.2/h, between about 140
L/m.sup.2/h and about 340 L/m.sup.2/h, between about 140
L/m.sup.2/h and about 320 L/m.sup.2/h, between about 140
L/m.sup.2/h and about 300 L/m.sup.2/h, between about 140
L/m.sup.2/h and about 280 L/m.sup.2/h, between about 140
L/m.sup.2/h and about 260 L/m.sup.2/h, between about 140
L/m.sup.2/h and about 240 L/m.sup.2/h, between about 140
L/m.sup.2/h and about 220 L/m.sup.2/h, between about 140
L/m.sup.2/h and about 200 L/m.sup.2/h, between about 140
L/m.sup.2/h and about 180 L/m.sup.2/h, between about 140
L/m.sup.2/h and about 160 L/m.sup.2/h, between about 160
L/m.sup.2/h and about 400 L/m.sup.2/h, between about 160
L/m.sup.2/h and about 380 L/m.sup.2/h, between about 160
L/m.sup.2/h and about 360 L/m.sup.2/h, between about 160
L/m.sup.2/h and about 340 L/m.sup.2/h, between about 160
L/m.sup.2/h and about 320 L/m.sup.2/h, between about 160
L/m.sup.2/h and about 300 L/m.sup.2/h, between about 160
L/m.sup.2/h and about 280 L/m.sup.2/h, between about 160
L/m.sup.2/h and about 260 L/m.sup.2/h, between about 160
L/m.sup.2/h and about 240 L/m.sup.2/h, between about 160
L/m.sup.2/h and about 220 L/m.sup.2/h, between about 160
L/m.sup.2/h and about 200 L/m.sup.2/h, between about 160
L/m.sup.2/h and about 180 L/m.sup.2/h, between about 180
L/m.sup.2/h and about 400 L/m.sup.2/h, between about 180
L/m.sup.2/h and about 380 L/m.sup.2/h, between about 180
L/m.sup.2/h and about 360 L/m.sup.2/h, between about 180
L/m.sup.2/h and about 340 L/m.sup.2/h, between about 180
L/m.sup.2/h and about 320 L/m.sup.2/h, between about 180
L/m.sup.2/h and about 300 L/m.sup.2/h, between about 180
L/m.sup.2/h and about 280 L/m.sup.2/h, between about 180
L/m.sup.2/h and about 260 L/m.sup.2/h, between about 180
L/m.sup.2/h and about 240 L/m.sup.2/h, between about 180
L/m.sup.2/h and about 220 L/m.sup.2/h, between about 180
L/m.sup.2/h and about 200 L/m.sup.2/h, between about 200
L/m.sup.2/h and about 400 L/m.sup.2/h, between about 200
L/m.sup.2/h and about 380 L/m.sup.2/h, between about 200
L/m.sup.2/h and about 360 L/m.sup.2/h, between about 200
L/m.sup.2/h and about 340 L/m.sup.2/h, between about 200
L/m.sup.2/h and about 320 L/m.sup.2/h, between about 200
L/m.sup.2/h and about 300 L/m.sup.2/h, between about 200
L/m.sup.2/h and about 280 L/m.sup.2/h, between about 200
L/m.sup.2/h and about 260 L/m.sup.2/h, between about 200
L/m.sup.2/h and about 240 L/m.sup.2/h, between about 200
L/m.sup.2/h and about 220 L/m.sup.2/h, between about 220
L/m.sup.2/h and about 400 L/m.sup.2/h, between about 220
L/m.sup.2/h and about 380 L/m.sup.2/h, between about 220
L/m.sup.2/h and about 360 L/m.sup.2/h, between about 220
L/m.sup.2/h and about 340 L/m.sup.2/h, between about 220
L/m.sup.2/h and about 320 L/m.sup.2/h, between about 220
L/m.sup.2/h and about 300 L/m.sup.2/h, between about 220
L/m.sup.2/h and about 280 L/m.sup.2/h, between about 220
L/m.sup.2/h and about 260 L/m.sup.2/h, between about 220
L/m.sup.2/h and about 240 L/m.sup.2/h, between about 240
L/m.sup.2/h and about 400 L/m.sup.2/h, between about 240
L/m.sup.2/h and about 380 L/m.sup.2/h, between about 240
L/m.sup.2/h and about 360 L/m.sup.2/h, between about 240
L/m.sup.2/h and about 340 L/m.sup.2/h, between about 240
L/m.sup.2/h and about 320 L/m.sup.2/h, between about 240
L/m.sup.2/h and about 300 L/m.sup.2/h, between about 240
L/m.sup.2/h and about 280 L/m.sup.2/h, between about 240
L/m.sup.2/h and about 260 L/m.sup.2/h, between about 260
L/m.sup.2/h and about 400 L/m.sup.2/h, between about 260
L/m.sup.2/h and about 380 L/m.sup.2/h, between about 260
L/m.sup.2/h and about 360 L/m.sup.2/h, between about 260
L/m.sup.2/h and about 340 L/m.sup.2/h, between about 260
L/m.sup.2/h and about 320 L/m.sup.2/h, between about 260
L/m.sup.2/h and about 300 L/m.sup.2/h, between about 260
L/m.sup.2/h and about 280 L/m.sup.2/h, between about 280
L/m.sup.2/h and about 400 L/m.sup.2/h, between about 280
L/m.sup.2/h and about 380 L/m.sup.2/h, between about 280
L/m.sup.2/h and about 360 L/m.sup.2/h, between about 280
L/m.sup.2/h and about 340 L/m.sup.2/h, between about 280
L/m.sup.2/h and about 320 L/m.sup.2/h, between about 280
L/m.sup.2/h and about 300 L/m.sup.2/h, between about 300
L/m.sup.2/h and about 400 L/m.sup.2/h, between about 300
L/m.sup.2/h and about 380 L/m.sup.2/h, between about 300
L/m.sup.2/h and about 360 L/m.sup.2/h, between about 300
L/m.sup.2/h and about 340 L/m.sup.2/h, between about 300
L/m.sup.2/h and about 320 L/m.sup.2/h, between about 320
L/m.sup.2/h and about 400 L/m.sup.2/h, between about 320
L/m.sup.2/h and about 380 L/m.sup.2/h, between about 320
L/m.sup.2/h and about 360 L/m.sup.2/h, between about 320
L/m.sup.2/h and about 340 L/m.sup.2/h, between about 340
L/m.sup.2/h and about 400 L/m.sup.2/h, between about 340
L/m.sup.2/h and about 380 L/m.sup.2/h, between about 340
L/m.sup.2/h and about 360 L/m.sup.2/h, between about 360
L/m.sup.2/h and about 400 L/m.sup.2/h, between about 360
L/m.sup.2/h and about 380 L/m.sup.2/h, or between about 380
L/m.sup.2/h and about 400 L/m.sup.2/h, to selectively retain
soluble protein aggregates, such as protein dimers and higher
protein oligomers (such as soluble recombinant antibody
aggregates). This filtration step is performed using a depth filter
including a filtration media of, for example, silica, one or more
layers of a fibrous media, one or more layers of charged or surface
modified microporous membranes, or a small bed of chromatography
media. A depth filter can have, e.g., a membrane surface area of
between about 10 cm.sup.2 and about 32000 cm.sup.2, between about
10 cm.sup.2 and about 31000 cm.sup.2, between about 10 cm.sup.2 and
about 30000 cm.sup.2, between about 10 cm.sup.2 and about 29000
cm.sup.2, between about 10 cm.sup.2 and about 28000 cm.sup.2,
between about 10 cm.sup.2 and about 27000 cm.sup.2, between about
10 cm.sup.2 and about 26000 cm.sup.2, between about 10 cm.sup.2 and
about 25000 cm.sup.2, between about 10 cm.sup.2 and about 24000
cm.sup.2, between about 10 cm.sup.2 and about 23000 cm.sup.2,
between about 10 cm.sup.2 and about 22000 cm.sup.2, between about
10 cm.sup.2 and about 21000 cm.sup.2, between about 10 cm.sup.2 and
about 20000 cm.sup.2, between about 10 cm.sup.2 and about 19000
cm.sup.2, between about 10 cm.sup.2 and about 18000 cm.sup.2,
between about 10 cm.sup.2 and about 17000 cm.sup.2, between about
10 cm.sup.2 and about 16000 cm.sup.2, between about 10 cm.sup.2 and
about 15000 cm.sup.2, between about 10 cm.sup.2 and about 14000
cm.sup.2, between about 10 cm.sup.2 and about 13000 cm.sup.2,
between about 10 cm.sup.2 and about 12000 cm.sup.2, between about
10 cm.sup.2 and about 11000 cm.sup.2, between about 10 cm.sup.2 and
about 10000 cm.sup.2, between about 10 cm.sup.2 and about 9000
cm.sup.2, between about 10 cm.sup.2 and about 8000 cm.sup.2,
between about 10 cm.sup.2 and about 7000 cm.sup.2, between about 10
cm.sup.2 and about 6000 cm.sup.2, between about 10 cm.sup.2 and
about 5000 cm.sup.2, between about 10 cm.sup.2 and about 4000
cm.sup.2, between about 10 cm.sup.2 and about 3000 cm.sup.2,
between about 10 cm.sup.2 and about 2000 cm.sup.2, between about 10
cm.sup.2 and about 1500 cm.sup.2, between about 10 cm.sup.2 and
about 1020 cm.sup.2, between about 10 cm.sup.2 and about 1000
cm.sup.2, between about 10 cm.sup.2 and about 500 cm.sup.2, between
about 10 cm.sup.2 and about 75 cm.sup.2, between about 100 cm.sup.2
and about 25000 cm.sup.2, between about 100 cm.sup.2 and about
24000 cm.sup.2, between about 100 cm.sup.2 and about 23000
cm.sup.2, between about 100 cm.sup.2 and about 22000 cm.sup.2,
between about 100 cm.sup.2 and about 21000 cm.sup.2, between about
100 cm.sup.2 and about 20000 cm.sup.2, between about 100 cm.sup.2
and about 19000 cm.sup.2, between about 100 cm.sup.2 and about
18000 cm.sup.2, between about 100 cm.sup.2 and about 17000
cm.sup.2, between about 100 cm.sup.2 and about 16000 cm.sup.2,
between about 100 cm.sup.2 and about 15000 cm.sup.2, between about
100 cm.sup.2 and about 14000 cm.sup.2, between about 100 cm.sup.2
and about 13000 cm.sup.2, between about 100 cm.sup.2 and about
12000 cm.sup.2, between about 100 cm.sup.2 and about 11000
cm.sup.2, between about 100 cm.sup.2 and about 10000 cm.sup.2,
between about 1000 cm.sup.2 and about 9000 cm.sup.2, between about
100 cm.sup.2 and about 8000 cm.sup.2, between about 100 cm.sup.2
and about 7000 cm.sup.2, between about 100 cm.sup.2 and about 6000
cm.sup.2, between about 100 cm.sup.2 and about 5000 cm.sup.2,
between about 100 cm.sup.2 and about 4000 cm.sup.2, between about
100 cm.sup.2 and about 3000 cm.sup.2, between about 100 cm.sup.2
and about 2000 cm.sup.2, between about 100 cm.sup.2 and about 1000
cm.sup.2, between about 100 cm.sup.2 and about 500 cm.sup.2,
between about 500 cm.sup.2 and about 25000 cm.sup.2, between about
500 cm.sup.2 and about 24000 cm.sup.2
, between about 500 cm.sup.2 and about 23000 cm.sup.2, between
about 500 cm.sup.2 and about 22000 cm.sup.2, between about 500
cm.sup.2 and about 21000 cm.sup.2, between about 500 cm.sup.2 and
about 20000 cm.sup.2, between about 500 cm.sup.2 and about 19000
cm.sup.2, between about 500 cm.sup.2 and about 18000 cm.sup.2,
between about 500 cm.sup.2 and about 17000 cm.sup.2, between about
500 cm.sup.2 and about 16000 cm.sup.2, between about 500 cm.sup.2
and about 15000 cm.sup.2, between about 500 cm.sup.2 and about
14000 cm.sup.2, between about 500 cm.sup.2 and about 13000
cm.sup.2, between about 500 cm.sup.2 and about 12000 cm.sup.2,
between about 500 cm.sup.2 and about 11000 cm.sup.2, between about
500 cm.sup.2 and about 10000 cm.sup.2, between about 500 cm.sup.2
and about 9000 cm.sup.2, between about 500 cm.sup.2 and about 8000
cm.sup.2, between about 500 cm.sup.2 and about 7000 cm.sup.2,
between about 500 cm.sup.2 and about 6000 cm.sup.2, between about
500 cm.sup.2 and about 5000 cm.sup.2, between about 500 cm.sup.2
and about 4000 cm.sup.2, between about 500 cm.sup.2 and about 3000
cm.sup.2, between about 500 cm.sup.2 and about 2000 cm.sup.2,
between about 500 cm.sup.2 and about 1000 cm.sup.2, between about
1000 cm.sup.2 and about 25000 cm.sup.2, between about 1000 cm.sup.2
and about 24000 cm.sup.2, between about 1000 cm.sup.2 and about
23000 cm.sup.2, between about 1000 cm.sup.2 and about 22000
cm.sup.2, between about 1000 cm.sup.2 and about 21000 cm.sup.2,
between about 1000 cm.sup.2 and about 20000 cm.sup.2, between about
1000 cm.sup.2 and about 19000 cm.sup.2, between about 1000 cm.sup.2
and about 18000 cm.sup.2, between about 1000 cm.sup.2 and about
17000 cm.sup.2, between about 1000 cm.sup.2 and about 16000
cm.sup.2, between about 1000 cm.sup.2 and about 15000 cm.sup.2,
between about 1000 cm.sup.2 and about 14000 cm.sup.2, between about
1000 cm.sup.2 and about 13000 cm.sup.2, between about 1000 cm.sup.2
and about 12000 cm.sup.2, between about 1000 cm.sup.2 and about
11000 cm.sup.2, between about 1000 cm.sup.2 and about 10000
cm.sup.2, between about 1000 cm.sup.2 and about 9000 cm.sup.2,
between about 1000 cm.sup.2 and about 8000 cm.sup.2, between about
1000 cm.sup.2 and about 7000 cm.sup.2, between about 1000 cm.sup.2
and about 6000 cm.sup.2, between about 1000 cm.sup.2 and about 5000
cm.sup.2, between about 1000 cm.sup.2 and about 4000 cm.sup.2,
between about 1000 cm.sup.2 and about 3000 cm.sup.2, between about
1000 cm.sup.2 and about 2000 cm.sup.2, between about 5000 cm.sup.2
and about 25000 cm.sup.2, between about 5000 cm.sup.2 and about
24000 cm.sup.2, between about 5000 cm.sup.2 and about 23000
cm.sup.2, between about 5000 cm.sup.2 and about 22000 cm.sup.2,
between about 5000 cm.sup.2 and about 21000 cm.sup.2, between about
5000 cm.sup.2 and about 20000 cm.sup.2, between about 5000 cm.sup.2
and about 19000 cm.sup.2, between about 5000 cm.sup.2 and about
18000 cm.sup.2, between about 5000 cm.sup.2 and about 17000
cm.sup.2, between about 5000 cm.sup.2 and about 16000 cm.sup.2,
between about 5000 cm.sup.2 and about 15000 cm.sup.2, between about
5000 cm.sup.2 and about 14000 cm.sup.2, between about 5000 cm.sup.2
and about 13000 cm.sup.2, between about 5000 cm.sup.2 and about
12000 cm.sup.2, between about 5000 cm.sup.2 and about 11000
cm.sup.2, between about 5000 cm.sup.2 and about 10000 cm.sup.2,
between about 5000 cm.sup.2 and about 9000 cm.sup.2, between about
5000 cm.sup.2 and about 8000 cm.sup.2, between about 5000 cm.sup.2
and about 7000 cm.sup.2, between about 5000 cm.sup.2 and about 6000
cm.sup.2, between about 10000 cm.sup.2 and about 25000 cm.sup.2,
between about 10000 cm.sup.2 and about 24000 cm.sup.2, between
about 10000 cm.sup.2 and about 23000 cm.sup.2, between about 10000
cm.sup.2 and about 2200 cm.sup.2, between about 10000 cm.sup.2 and
about 21000 cm.sup.2, between about 10000 cm.sup.2 and about 20000
cm.sup.2, between about 10000 cm.sup.2 and about 19000 cm.sup.2,
between about 10000 cm.sup.2 and about 18000 cm.sup.2, between
about 10000 cm.sup.2 and about 17000 cm.sup.2, between about 10000
cm.sup.2 and about 16000 cm.sup.2, between about 10000 cm.sup.2 and
about 15000 cm.sup.2, between about 10000 cm.sup.2 and about 14000
cm.sup.2, between about 10000 cm.sup.2 and about 13000 cm.sup.2,
between about 10000 cm.sup.2 and about 12000 cm.sup.2, between
about 10000 cm.sup.2 and about 11000 cm.sup.2, between about 15000
cm.sup.2 and about 25000 cm.sup.2, between about 15000 cm.sup.2 and
about 24000 cm.sup.2, between about 15000 cm.sup.2 and about 23000
cm.sup.2, between about 15000 cm.sup.2 and about 22000 cm.sup.2,
between about 15000 cm.sup.2 and about 21000 cm.sup.2, between
about 15000 cm.sup.2 and about 20000 cm.sup.2, between about 15000
cm.sup.2 and about 19000 cm.sup.2, between about 15000 cm.sup.2 and
about 18000 cm.sup.2, between about 15000 cm.sup.2 and about 17000
cm.sup.2, between about 15000 cm.sup.2 and about 16000 cm.sup.2,
between about 20000 cm.sup.2 and about 25000 cm.sup.2, between
about 20000 cm.sup.2 and about 24000 cm.sup.2, between about 20000
cm.sup.2 and about 23000 cm.sup.2, between about 20000 cm.sup.2 and
about 22000 cm.sup.2, between about 20000 cm.sup.2 and about 21000
cm.sup.2, or about 25 cm.sup.2. In some examples, two or more depth
filters are fluidly connected to a manifold in order to increase
the amount of recombinant antibody flowed through a depth filter at
one or more steps in a purification process.
[0155] The step of flowing the recombinant antibody through a depth
filter can result in substantially complete removal of soluble
protein aggregates. For example, the step of flowing the
recombinant antibody through a depth filter can provide a filtrate
that includes the purified recombinant antibody and is
substantially free (such as about or at least 90% free, about or at
least 90.5% free, about or at least 91.0% free, about or at least
91.5% free, about or at least 92.0% free, about or at least 92.5%
free, about or at least 93.0% free, about or at least 93.5% free,
about or at least 94.0%, about or at least 94.5% free, about or at
least 95.0% free, about or at least 95.5% free, about or at least
96.0% free, about or at least 96.5% free, about or at least 97.0%
free, about or at least 97.5% free, about or at least 98.0% free,
about or at least 98.5% free, about or at least 99.0% free, about
or at least 99.5% free, or about or at least 99.8% free). In some
embodiments, the depth filter provides a filtrate that includes the
purified recombinant antibody and no detectable soluble protein
aggregates.
[0156] Methods for detecting the level or amount of protein
aggregates are known in the art. For example, size exclusion
chromatography, native (non-denaturing) gel chromatography,
analytical ultracentrifugation (AUC), field-flow fractionation
(FFF), and dynamic light scattering (DLS) can be used to detect the
amount of soluble protein aggregates are present in the depth
filter filtrate.
[0157] In one embodiment of the methods, a constant pressure mode
of filtration or a constant flow mode of operation is used. A fluid
including a recombinant antibody can be retained by a pressurized
reservoir and pumped through a depth filter by the pressure in the
reservoir. The fluid is subjected to a normal flow mode of
filtration with the aggregates being retained by the depth filter
and an aggregate-free fluid is discharged as the filtrate. The
filtrate can be passed through a conduit for downstream processing,
such as one or more unit operations. By operating in this manner,
soluble protein aggregates are retained by the depth filter.
Alternatively, a pump located between the reservoir and the depth
filter could be used to create constant pressure and maintain
constant flow through the depth filter. The fluid including a
recombinant antibody is subjected to a normal flow mode of
filtration with the aggregates being retained by the depth filter
and an aggregate-free fluid discharged as the filtrate from the
depth filter. The filtrate can be passed through a conduit for
further downstream processing.
[0158] Non-limiting depth filters that can be used to remove
aggregates are described herein and additional depth filters that
can be used are known in the art. Representative suitable depth
filters include those formed from fibrous media formed of silica,
cellulosic fibers, synthetic fibers or blends thereof, such as
CUNO.RTM. Zeta PLUS.RTM. Delipid filters (3M, St. Paul, Minn.),
CUNO.RTM.Emphaze AEX filters (3M, St. Paul, Minn.), CUNO.RTM.
90ZA08A filters (3M, St. Paul, Minn.), CUNO.RTM. DELI08A Delipid
filters (3M, St. Paul, Minn.), Millipore X0HC filters (EMD
Millipore, Billerica, Mass.), MILLISTAK.RTM. pads (EMD Millipore,
Billerica, Mass.), microporous membranes that are either charged or
have a surface chemistry (such as hydrophilicity or hydrophobicity,
or a positive or negative charge as are taught by U.S. Pat. Nos.
5,629,084 and 4,618,533) made from a material selected from the
group consisting of regenerated cellulose, polyethersulfone,
polyarylsulphone, polysulfone, polyimide, polyamide or
polyvinylidenedifluoride (PVDF), such as charged DURAPORE.RTM.
membrane, hydrophobic DURAPORE.RTM. membrane, hydrophobic
AERVENT.RTM. membrane and INTERCEPT.TM. Q quaternary charged
membrane, all available from EMD Millipore, Billerica, Mass.
One or More Unit Operations
[0159] Some embodiments of any of the methods described herein
include, between the step of capturing and the step of performing
viral filtration (e.g., using any of the methods of performing
viral filtration described herein), the step of performing one or
more (e.g., two, three, four, or five) unit operations on the
recombinant antibody, e.g., one or more unit operations selected
from the group of filtering (e.g., ultrafiltration/diafiltration to
concentrate the recombinant antibody in a fluid), purifying the
recombinant antibody, polishing the recombinant antibody, viral
inactivation, depth filtration, adjusting (e.g., increasing or
decreasing) one or both of the pH and ionic concentration of a
fluid including the recombinant antibody, and pre-filtration. Some
embodiments of any of the methods described herein include, between
the step of capturing and the step of viral filtration, the step of
performing one or more (e.g., two, three, four, or five) unit
operations on the recombinant antibody, e.g., one or more unit
operations from the group of ultrafiltration/diafiltration to
concentrate a recombinant protein in a fluid, ion exchange
chromatography, hydrophobic interaction chromatography, polishing
the recombinant protein, viral inactivation, adjustment of pH,
adjustment of ionic strength, adjustment of both pH and ionic
strength of a fluid including the recombinant protein, depth
filtration, and pre-filtration. In some embodiments of any of the
methods described herein, the methods include between the capturing
step and the step of viral filtration, performing the sequential
unit operations of polishing (e.g., by performing hydrophobic
interaction chromatography), viral inactivation,
ultrafiltration/diafiltration to concentrate the recombinant
antibody, depth filtration, and pre-filtration (e.g., using a
Sartorius Virosart.RTM. Max pre-filter).
[0160] Some embodiments of any of the methods described herein
further include performing one or more (e.g., two, three, four, or
five) unit operations before the capturing step, e.g., one or more
unit operations selected from the group of clarifying a culture
medium, filtration (e.g., ultrafiltration/diafiltration to
concentrate a recombinant antibody in a fluid), viral inactivation,
purifying, and adjusting (e.g., increasing or decreasing) one or
both of the pH and ionic concentration of a fluid including the
recombinant antibody. Some embodiments of any of the methods
described herein further include performing one or more (e.g., two,
three, four, or five) unit operations before the capturing step,
e.g., one or more unit operations selected from the group of
ultrafiltration/diafiltration to concentrate the recombinant
antibody in a fluid, ion exchange chromatography, hydrophobic
interaction chromatography, polishing the recombinant antibody,
viral inactivation, adjustment of pH, adjustment of ionic strength,
and adjustment of both pH and ionic strength of a fluid including
the recombinant antibody. In some embodiments, the methods further
include, prior to the capturing step, the sequential steps of
clarification of culture media, ultrafiltration/diafiltration to
concentrate the recombinant antibody, and viral inactivation.
[0161] Some embodiments further include performing one or more unit
operations after the step of viral inactivation, e.g., one or more
unit operations selected from the group of purifying the
recombinant antibody, polishing the recombinant antibody, adjusting
(e.g., increasing or decreasing) one or both of the pH and ionic
concentration of a fluid including the purified recombinant
antibody, or passing the fluid through an additional virus filter.
In some embodiments of any of the methods described herein, the
unit operation of viral filtration occurs immediately following the
step of flowing the recombinant antibody through the depth filter
or immediately following the step of flowing the recombinant
antibody through a pre-filter.
Purifying and Polishing the Recombinant Protein
[0162] The methods described herein can include a step of purifying
the recombinant antibody using at least one chromatography column
that can be used to perform the unit operation of purifying a
recombinant protein. The methods described herein can include a
step of polishing the recombinant antibody using at least one
chromatography column or chromatographic membrane that can be used
to perform the unit operation of polishing the recombinant
protein.
[0163] The at least one chromatography column for purifying the
recombinant antibody can include a resin that utilizes a capture
mechanism (such as any of the capture mechanisms described herein
or known in the art), or a resin that can be used to perform anion
exchange, cation exchange, or molecular sieve chromatography. The
at least one chromatography column or chromatographic membrane for
polishing the recombinant antibody can include a resin can be used
to perform anion exchange, cation exchange, or molecular sieve
chromatography (such as any of the exemplary resins for performing
anion exchange, cation exchange, or molecular sieve chromatography
known in the art).
[0164] The size, shape, and volume of the at least one
chromatography column for purifying the recombinant antibody,
and/or the size and shape of the at least one chromatography column
or chromatographic membrane for polishing the recombinant antibody
can any of combination of the exemplary sizes, shapes, and volumes
of chromatography columns or chromatographic membranes described
herein or known in the art. Purifying or polishing a recombinant
antibody can, e.g., include the steps of loading, washing, eluting,
and equilibrating the at least one chromatography column or
chromatographic membrane used to perform the unit of operation of
purifying or polishing the recombinant antibody. Typically, the
elution buffer coming out of a chromatography column or
chromatographic membrane used for purifying includes the
recombinant antibody. Typically, the loading and/or wash buffer
coming out of a chromatography column or chromatographic membrane
used for polishing includes the recombinant antibody.
[0165] For example, the size of the chromatography column for
purifying the recombinant antibody can have a volume of, e.g.,
between about 1.0 mL and about 650 L (e.g., between about 5.0 mL
and about 600 L, between about 5.0 mL and about 550 L, between
about 5.0 mL and about 500 L, between about 5.0 mL and about 450 L,
between about 5.0 mL and about 400 L, between about 5.0 mL and
about 350 L, between about 5.0 mL and about 300 L, between about
5.0 mL and about 250 L, between about 5.0 mL and about 200 L,
between about 5.0 mL and about 150 L, between about 5.0 mL and
about 100 L, between about 5.0 mL and about 50 L, between about 5.0
mL and about 10 L, between about 5.0 mL and about 1.0 L, between
about 5.0 mL and about 900 mL, between about 5.0 mL and about 800
mL, between about 5.0 mL and about 700 mL, between about 5.0 mL and
about 600 mL, between about 5.0 mL and about 500 mL, between about
5.0 mL and about 400 mL, between about 5.0 mL and about 300 mL,
between about 5.0 mL and about 200 mL, between about 5.0 mL and
about 180 mL, between about 5.0 mL and about 160 mL, between about
5.0 mL and about 140 mL, between about 5.0 mL and about 120 mL,
between about 5.0 mL and about 100 mL, between about 5.0 mL and
about 80 mL, between about 5.0 mL and about 60 mL, between about
5.0 mL and about 40 mL, between about 5.0 mL and about 30 mL, or
between about 5.0 mL and about 25 mL).
[0166] The linear flow rate of the fluid including the recombinant
antibody as it is loaded onto the at least one chromatography
column for purifying the recombinant antibody can be, e.g., between
50 cm/hour and about 600 cm/hour, between about 50 cm/hour and
about 550 cm/hour, between about 50 cm/hour and about 500 cm/hour,
between about 50 cm/hour and about 450 cm/hour, between about 50
cm/hour and about 400 cm/hour, between about 50 cm/hour and about
350 cm/hour, between about 50 cm/hour and about 300 cm/hour,
between about 50 cm/hour and about 250 cm/hour, between about 50
cm/hour and about 200 cm/hour, between about 50 cm/hour and about
150 cm/hour, or between about 50 cm/hour and about 100 cm/hour
(e.g., for a chromatography column have a diameter of between about
100 cm and about 200 cm). The concentration of the recombinant
antibody loaded onto the chromatography column for purifying the
recombinant antibody can be, e.g., between about 0.05 mg/mL and
about 90 mg/mL recombinant antibody (e.g., between about 0.1 mg/mL
and about 90 mg/mL, between about 0.1 mg/mL and about 80 mg/mL,
between about 0.1 mg/mL and about 70 mg/mL, between about 0.1 mg/mL
and about 60 mg/mL, between about 0.1 mg/mL and about 50 mg/mL,
between about 0.1 mg/mL and about 40 mg/mL, between about 0.1 mg/mL
and about 30 mg/mL, between about 0.1 mg/mL and about 20 mg/mL,
between 0.5 mg/mL and about 20 mg/mL, between about 0.1 mg/mL and
about 15 mg/mL, between about 0.5 mg/mL and about 15 mg/mL, between
about 0.1 mg/mL and about 10 mg/mL, or between about 0.5 mg/mL and
about 10 mg/mL recombinant antibody). The resin in the at least one
chromatography column for purifying can be an anion exchange or
cation exchange chromatography resin. The resin in the at least one
chromatography column or chromatographic membrane that is used to
perform the unit operation of purifying can be a cationic exchange
resin.
[0167] Following the loading of the recombinant antibody, the at
least one chromatographic column or chromatographic membrane is
washed with at least one washing buffer. As can be appreciated in
the art, the at least one (e.g., two, three, or four) washing
buffer is meant to elute all proteins that are not the recombinant
protein from the at least one chromatography column, while not
disturbing the interaction of the recombinant antibody with the
resin or otherwise eluting the recombinant antibody.
[0168] The wash buffer can be passed through the at least one
chromatography column at a linear flow rate of, e.g., between 50
cm/hour and about 600 cm/hour, between about 50 cm/hour and about
550 cm/hour, between about 50 cm/hour and about 500 cm/hour,
between about 50 cm/hour and about 450 cm/hour, between about 50
cm/hour and about 400 cm/hour, between about 50 cm/hour and about
350 cm/hour, between about 50 cm/hour and about 300 cm/hour,
between about 50 cm/hour and about 250 cm/hour, between about 50
cm/hour and about 200 cm/hour, between about 50 cm/hour and about
150 cm/hour, or between about 50 cm/hour and about 100 cm/hour
(e.g., for a chromatography column have a diameter of between about
100 cm and about 200 cm). The volume of wash buffer used (such as
the combined total volume of wash buffer used when more than one
wash buffer is used) can be between about 1.times. column volume
(CV) and about 10.times. CV, between about 1.times. CV and about
9.times. CV, about 1.times. CV and about 8.times. CV, about
1.times. CV and about 7.times. CV, about 1.times. CV and about
6.times. CV, about 2.times. CV and about 10.times. CV, about
3.times. CV and about 10.times. CV, about 4.times. CV and about
10.times. CV, about 2.5.times. CV and about 5.0.times. CV, about
5.times. CV and about 10.times. CV, or about 5.times. CV and about
8.times. CV). The total time of the washing can be between about 2
minutes and about 5 hours (e.g., between about 5 minutes and about
4.5 hours, between about 5 minutes and about 4.0 hours, between
about 5 minutes and about 3.5 hours, between about 5 minutes and
about 3.0 hours, between about 5 minutes and about 2.5 hours,
between about 5 minutes and about 2.0 hours, between about 5
minutes and about 1.5 hours, between about 10 minutes and about 1.5
hours, between about 10 minutes and about 1.25 hours, between about
20 minutes and about 1.25 hours, between about 30 minutes and about
1 hour, between about 2 minutes and 10 minutes, between about 2
minutes and 15 minutes, or between about 2 minutes and 30
minutes).
[0169] Following washing of the at least one chromatographic column
for purifying the recombinant antibody, the recombinant antibody is
eluted by passing an elution buffer through the column. The elution
buffer can be passed through the column that can be used to perform
the unit operation of purifying the recombinant antibody at a liner
flow rate of, e.g., between about 25 cm/hour and about 600 cm/hour,
between about 25 cm/hour and about 550 cm/hour, between about 25
cm/hour and about 500 cm/hour, between about 25 cm/hour and about
450 cm/hour, between about 25 cm/hour and about 400 cm/hour,
between about 25 cm/hour and about 350 cm/hour, between about 25
cm/hour and about 300 cm/hour, between about 25 cm/hour and about
250 cm/hour, between about 25 cm/hour and about 200 cm/hour,
between about 25 cm/hour and about 150 cm/hour, or between about 25
cm/hour and about 100 cm/hour (e.g., for a chromatography column
have a diameter of between about 100 cm and about 200 cm). The
volume of elution buffer used to elute the recombinant antibody
from each the at least one chromatographic column for purifying the
recombinant antibody can be between about 1.times. column volume
(CV) and about 10.times. CV, between about 1.times. CV and about
9.times. CV, between about 1.times. CV and about 8.times. CV,
between about 1.times. CV and about 7.times. CV, about 1.times. CV
and about 6.times. CV, about 1.times. CV and about 5.times. CV,
about 1.times. CV and about 4.times. CV, about 2.times. CV and
about 10.times. CV, about 3.times. CV and about 10.times. CV, about
4.times. CV and about 10.times. CV, about 5.times. CV and about
10.times. CV, or about 5.times. CV and about 9.times. CV. The total
time of the eluting can be between about 5 minutes and about 3
hours, between about 5 minutes and about 2.5 hours, between about 5
minutes and about 2.0 hours, between about 5 minutes and about 1.5
hours, between about 5 minutes and about 1.5 hours, between about 5
minutes and about 1.25 hours, between about 5 minutes and about
1.25 hours, between about 5 minutes and about 1 hour, between about
5 minutes and about 40 minutes, between about 10 minutes and about
40 minutes, between about 20 minutes and about 40 minutes, or
between about 30 minutes and 1.0 hour. Non-limiting examples of
elution buffers that can be used in these methods will depend on
the resin and/or the therapeutic antibody. For example, an elution
buffer can include a different concentration of salt (e.g.,
increased salt concentration), a different pH (e.g., an increased
or decreased salt concentration), or a molecule that will compete
with the recombinant antibody for binding to the resin. Examples of
such elution buffers for each of the exemplary capture mechanisms
described herein are well known in the art.
[0170] Following the elution, and before the next volume of fluid
including a recombinant antibody can be loaded onto the at least
one chromatographic column, the at least one chromatography column
or chromatographic membrane must be equilibrated using a
regeneration buffer. The regeneration buffer can be passed through
the chromatography column at a linear flow rate of, e.g., between
about 25 cm/hour and about 600 cm/hour, between about 25 cm/hour
and about 550 cm/hour, between about 25 cm/hour and about 500
cm/hour, between about 25 cm/hour and about 450 cm/hour, between
about 25 cm/hour and about 400 cm/hour, between about 25 cm/hour
and about 350 cm/hour, between about 25 cm/hour and about 300
cm/hour, between about 25 cm/hour and about 250 cm/hour, between
about 25 cm/hour and about 200 cm/hour, between about 25 cm/hour
and about 150 cm/hour, or between about 25 cm/hour and about 100
cm/hour (e.g., for a chromatography column have a diameter of
between about 100 cm and about 200 cm). The volume of regeneration
buffer used for equilibration can be, e.g., between about 1.times.
column volume (CV) and about 10.times. CV, between about 1.times.
CV and about 9.times. CV, between about 1.times. CV and about
8.times. CV, between about 1.times. CV and about 7.times. CV,
between about 1.times. CV and about 6.times. CV, between about
2.times. CV and about 10.times. CV, between about 3.times. CV and
about 10.times. CV, between about 2.times. CV and about 5.times.
CV, between about 2.5.times. CV and about 7.5.times. CV, between
about 4.times. CV and about 10.times. CV, between about 5.times. CV
and about 10.times. CV, or between about 5.times. CV and about
10.times. CV. The concentration of recombinant antibody in a fluid
used to perform the unit operation of purifying the recombinant
antibody can be between about 0.05 mg/mL and about 90 mg/mL,
between about 0.1 mg/mL and about 90 mg/mL, between about 0.1 mg/mL
and about 80 mg/mL, between about 0.1 mg/mL and about 70 mg/mL,
between about 0.1 mg/mL and about 60 mg/mL, between about 0.1 mg/mL
and about 50 mg/mL, between about 0.1 mg/mL and about 40 mg/mL,
between about 2.5 mg/mL and about 7.5 mg/mL, between about 0.1
mg/mL and about 30 mg/mL, between about 0.1 mg/mL and about 20
mg/mL, between 0.5 mg/mL and about 20 mg/mL, between about 0.1
mg/mL and about 15 mg/mL, between about 0.5 mg/mL and about 15
mg/mL, between about 0.1 mg/mL and about 10 mg/mL, or between about
0.5 mg/mL and about 10 mg/mL recombinant antibody.
[0171] The at least one chromatography column or chromatography
membrane that can be used to perform the unit operation of
polishing the recombinant antibody can include a resin that can be
used to perform cation exchange, anion exchange, hydrophobic,
mixed-mode, or molecular sieve chromatography. As can be
appreciated in the art, polishing can include the steps of loading,
chasing, and regenerating the chromatography column or
chromatographic membrane. For example, when the steps of loading,
chasing, and regenerating are used to perform the polishing, the
recombinant antibody does not bind the resin in the at least one
chromatography column or chromatography membrane, and the
recombinant protein is eluted from the chromatography column or
chromatographic membrane in the loading and chasing steps, and the
regenerating step is used to remove any impurities from the
chromatography column or chromatographic membrane. Exemplary linear
flow rates and buffer volumes to be used in each of the loading,
chasing, and regenerating steps are described below.
[0172] The size, shape, and volume of the chromatography column or
chromatography membrane for polishing the recombinant antibody can
any of combination of the exemplary sizes, shapes, and volumes of
chromatography columns or chromatographic membranes described
herein. For example, the size of the at least one chromatography
column or chromatographic membrane can have a volume between about
2.0 mL and about 650 L, between about 2.0 mL and about 600 L,
between about 2.0 mL and about 550 L, between about 2.0 mL and
about 500 L, between about 2.0 mL and about 450 L, between about
2.0 mL and about 400 L, between about 2.0 mL and about 350 L,
between about 2.0 mL and about 300 L, between about 2.0 mL and
about 250 L, between about 2.0 mL and about 200 L, between about
2.0 mL and about 150 L, between about 2.0 mL and about 100 L,
between about 2.0 mL and about 50 L, between about 2.0 mL and about
25 L, between about 2.0 mL and about 10 L, between about 2.0 L and
about 5 L, between about 2.0 mL and about 2 L, between about 2.0 mL
and about 1 L, between about 2.0 mL and about 800 mL, between about
2.0 mL and about 600 mL, between about 2.0 mL and about 400 mL,
between about 2.0 mL and about 200 mL, between about 2.0 mL and
about 180 mL, between about 2.0 mL and about 160 mL, between about
2.0 mL and about 140 mL, between about 2.0 mL and about 120 mL,
between about 2.0 mL and about 100 mL, between about 2.0 mL and
about 80 mL, between about 2.0 mL and about 60 mL, between about
2.0 mL and about 40 mL, between about 2.0 mL and about 40 mL,
between about 2.0 mL and about 30 mL, between about 5.0 mL and
about 30 mL, between about 2.0 mL and about 25 mL, between about
2.0 mL and about 10 mL, or between about 2.0 mL and about 5 mL. The
at least one chromatography column can also be described in terms
of its diameter. For example, the at least one chromatography
column provided herein can have a diameter of between about 1 cm
and about 200 cm, between about 1 cm and about 180 cm, between
about 1 cm and about 160 cm, between about 1 cm and about 140 cm,
between about 1 cm and about 120 cm, between about 1 cm and about
100 cm, between about 1 cm and about 80 cm, between about 1 cm and
about 60 cm, between about 1 cm and about 40 cm, between about 1 cm
and about 20 cm, or between about 1 cm and about 10 cm. The linear
flow rate of the fluid including the recombinant antibody as it is
loaded onto the chromatography column or chromatographic membrane
can be between about 25 cm/hour and about 600 cm/hour, between
about 25 cm/hour and about 550 cm/hour, between about 25 cm/hour
and about 500 cm/hour, between about 25 cm/hour and about 450
cm/hour, between about 25 cm/hour and about 400 cm/hour, between
about 25 cm/hour and about 350 cm/hour, between about 25 cm/hour
and about 300 cm/hour, between about 25 cm/hour and about 250
cm/hour, between about 25 cm/hour and about 200 cm/hour, between
about 25 cm/hour and about 150 cm/hour, or between about 25 cm/hour
and about 100 cm/hour (e.g., for a chromatography column have a
diameter of between about 100 cm and about 200 cm). The amount of
recombinant protein loaded per mL of resin can be between about 5
mg/mL and about 250 mg/mL, between about 5 mg/mL and about 200
mg/mL, between about 5 mg/mL and about 150 mg/mL, between about 5
mg/mL and about 100 mg/mL, between about 5 mg/mL and about 80
mg/mL, between about 5 mg/mL and about 60 mg/mL, between about 5
mg/mL and about 40 mg/mL, between about 5 mg/mL and about 20 mg/mL,
between about 5 mg/mL and about 15 mg/mL, or between about 5 mg/mL
and about 10 mg/mL. The resin in the chromatography column or
chromatographic membrane for polishing can be an anion exchange or
cation exchange resin. The resin can be, e.g., a cationic exchange
resin.
[0173] Following the loading step, a chasing step is performed. For
example, a chase buffer can be passed through the at least one
chromatography membrane or chromatographic membrane to collect the
recombinant antibody that does not substantially bind to the column
or membrane). In these examples, the chase buffer can be passed
through the column or membrane at a linear flow rate of between
about 25 cm/hour and about 600 cm/hour, between about 25 cm/hour
and about 550 cm/hour, between about 25 cm/hour and about 500
cm/hour, between about 25 cm/hour and about 450 cm/hour, between
about 25 cm/hour and about 400 cm/hour, between about 25 cm/hour
and about 350 cm/hour, between about 25 cm/hour and about 300
cm/hour, between about 25 cm/hour and about 250 cm/hour, between
about 25 cm/hour and about 200 cm/hour, between about 25 cm/hour
and about 150 cm/hour, or between about 25 cm/hour and about 100
cm/hour (e.g., for a chromatography column have a diameter of
between about 100 cm and about 200 cm). The volume of chase buffer
used can be between about 1.times. column volume (CV) and about
20.times. CV, between about between about 1.times. CV and about
15.times. CV, between about 5.times. CV and about 20.times. CV,
between about 1.times. CV and about 14.times. CV, about 1.times. CV
and about 13.times. CV, about 1.times. CV and about 12.times. CV,
about 1.times. CV and about 11.times. CV, about 2.times. CV and
about 11.times. CV, about 3.times. CV and about 11.times. CV, about
4.times. CV and about 11.times. CV, about 2.5.times. CV and about
5.0.times. CV, about 5.times. CV and about 11.times. CV, or about
5.times. CV and about 10.times. CV. The total time of the chasing
can be between about 2 minutes and about 3 hours, between about 2
minutes and about 2.5 hours, between about 2 minutes and about 2.0
hours, between about 2 minutes and about 1.5 hours, between about 2
minutes and about 1.25 hours, between about 2 minute and about 5
minutes, between about 2 minute and about 10 minutes, between about
2 minutes and about 4 minutes, between about 30 minutes and about 1
hour, between about 2 minutes and 15 minutes, or between about 2
minutes and 30 minutes. The combined concentration of recombinant
antibody present in the filtrate coming through the column in the
loading step and the chasing step can be between about 0.1 mg/mL
and about 250 mg/mL recombinant antibody, between about 0.1 mg/mL
and about 200 mg/mL recombinant antibody, between about 0.1 mg/mL
and about 150 mg/mL recombinant antibody, between about 0.1 mg/mL
and about 100 mg/mL recombinant antibody, between about 0.1 mg/mL
and about 80 mg/mL recombinant antibody, between about 0.1 mg/mL
and about 70 mg/mL recombinant antibody, between about 0.1 mg/mL
and about 60 mg/mL recombinant antibody, between about 0.1 mg/mL
and about 50 mg/mL recombinant antibody, between about 0.1 mg/mL
and about 40 mg/mL recombinant antibody, between about 2.5 mg/mL
and about 7.5 mg/mL recombinant antibody, between about 0.1 mg/mL
and about 30 mg/mL recombinant antibody, between about 0.1 mg/mL
and about 20 mg/mL recombinant antibody, between 0.5 mg/mL and
about 20 mg/mL recombinant antibody, between about 0.1 mg/mL and
about 15 mg/mL recombinant antibody, between about 0.5 mg/mL and
about 15 mg/mL recombinant antibody, between about 0.1 mg/mL and
about 10 mg/mL recombinant antibody, between about 0.5 mg/mL and
about 10 mg/mL recombinant antibody, or between about 1 mg/mL and
about 5 mg/mL recombinant antibody.
[0174] Following the chasing step and before the next volume of
fluid is loaded, the column or membrane must be regenerated using a
regeneration buffer. Regeneration buffer can be passed through the
column or membrane for polishing at a linear flow rate of between
about 25 cm/hour and about 600 cm/hour, between about 25 cm/hour
and about 550 cm/hour, between about 25 cm/hour and about 500
cm/hour, between about 25 cm/hour and about 450 cm/hour, between
about 25 cm/hour and about 400 cm/hour, between about 25 cm/hour
and about 350 cm/hour, between about 25 cm/hour and about 300
cm/hour, between about 25 cm/hour and about 250 cm/hour, between
about 25 cm/hour and about 200 cm/hour, between about 25 cm/hour
and about 150 cm/hour, or between about 25 cm/hour and about 100
cm/hour. The volume of regeneration buffer used to regenerate can
be between about 1.times. column volume (CV) and about 20.times.
CV, between about 1.times. CV and about 15.times. CV, between about
5.times. CV and about 20.times. CV, between about 1.times. CV and
about 14.times. CV, about 1.times. CV and about 13.times. CV, about
1.times. CV and about 12.times. CV, about 1.times. CV and about
11.times. CV, about 2.times. CV and about 11.times. CV, about
3.times. CV and about 11.times. CV, about 4.times. CV and about
11.times. CV, about 2.5.times. CV and about 5.0.times. CV, about
5.times. CV and about 11.times. CV, or about 5.times. CV and about
10.times. CV.
[0175] In other examples, the one or more chromatography column(s)
and/or chromatographic membranes used to perform the unit operation
of polishing include a resin that selectively binds or retains
impurities present in a fluid including the recombinant antibody,
and instead of regenerating the one or more column(s) and/or
membrane(s), the one or more column(s) and/or membrane(s) are
replaced (such as with a similar column or membrane) once the
binding capacity of the resin in the one or more column(s) and/or
membrane(s) has been reached or is substantially close to being
reached.
Inactivation of Viruses and Viral Filtration
[0176] The unit operation of inactivating viruses present in a
fluid including the recombinant antibody can be performed using a
chromatography column, a chromatography membrane, or a holding tank
that is capable of incubating a fluid including the recombinant
antibody at a pH of between about 3.0 to 5.0, between about 3.5 and
about 4.5, between about 3.5 and about 4.25, between about 3.5 and
about 4.0, between about 3.5 and about 3.8, or about 3.75 for a
period of at least 25 minutes, a period of between about 30 minutes
to 1.5 hours, a period of between about 30 minutes to 1.25 hours, a
period of between about 0.75 hours to 1.25 hours, or a period of
about 1 hour.
[0177] The unit operation of viral filtration can be performed
using any of the methods of performing viral filtration described
herein.
Adjusting the pH and/or Ionic Concentration
[0178] Some methods described herein can include one or more steps
of adjusting (e.g., increasing or decreasing) the pH and/or ionic
concentration of a fluid including the recombinant antibody. As
described herein, the pH and/or ionic concentration of a fluid
including the recombinant antibody can be adjusted (e.g., increased
or decreased) by adding a buffer to the fluid (e.g., through the
use of an in-line buffer adjustment reservoir).
Formulating the Purified Recombinant Antibody
[0179] Some embodiments of any of the methods described herein
further include a step of formulating the recombinant antibody into
a pharmaceutical composition. For example, formulating can include
adding a pharmaceutically acceptable excipient to the purified
recombinant antibody (e.g., produced by any of the methods of
purifying or manufacturing a recombinant antibody described
herein). Formulating can include mixing a pharmaceutically
acceptable excipient with the purified recombinant antibody.
Examples of pharmaceutically acceptable excipients (e.g.,
non-naturally occurring pharmaceutically acceptable excipients) are
well known in the art. In some embodiments, the purified
recombinant antibody is formulated for intravenous, intraarterial,
subcutaneous, intraperitoneal, or intramuscular administration.
EXAMPLES
[0180] Several general protocols are described below, which may be
used in any of the methods described herein and do not limit the
scope of the invention described in the claims.
Example 1
Effect of Different Pre-Filters, pH, Arginine Concentration, and
Sodium Chloride Concentration on Viral Filtration of BNJ441
Antibody
[0181] A set of experiments were performed in order to test the
effect of different pre-filters, pH, arginine concentration, and
sodium chloride concentration on viral filtration. In these
experiments, a variety of different fluids having a pH of between
5.5 to 7.6, a sodium chloride concentration of between 65 mM to 300
mM, an L-arginine concentration of 0 or 50 mM, and between 2.91
mg/mL to 3.54 mg/mL of BNJ441 monoclonal antibody that have been
pre-filtered with either a 0.1 .mu.m pre-filter or a Sartorius
Virosart.RTM. Max pre-filter, where each flowed through a
Virosart.RTM. CPV virus filter. The flux decay and the flow through
(g/m.sup.2) of the Virosart.RTM. CPV filter as each fluid was
flowed through the Virosart.RTM. CPV filter was determined.
[0182] The data show that better Virosart.RTM. CPV virus filter
throughput was observed for fluids that were prefiltered using a
Sartorius Virosart.RTM. Max pre-filter and also generally fluids
having a pH of 5.5 and not including any L-arginine or fluids
having a pH of 7.6 and including 25 mM arginine (see, FIGS. 1 and
2). Statistical analysis was used to predict the throughput at 50%
flow decay for a Virosart.RTM. CPV virus filter (FIG. 3) using the
parameters listed in FIG. 4. The data in FIG. 4 also show that the
type of pre-filter significantly effects the virus filter
throughput. The data in FIG. 5 show that the average throughput of
the virus filter at 50% flow decay is increased in fluids that have
been pre-filtered using a Sartorius Virosart.RTM. Max pre-filter
(as compared to fluids that have been pre-filtered using a 0.1
.mu.m-filter).
[0183] FIG. 6 shows the relationship of the virus filter throughput
at 50% flow decay to the hydrodynamic radius of the BNJ441
monoclonal antibody. The data in FIG. 6 shows that as the
hydrodynamic radius decreases, the throughput of the virus filter
increases.
[0184] FIG. 7 shows the throughput of the virus filter at 50% flow
decay for fluids having a pH of 5.5, 6.5, or 7.5. These data show
that fluids having a pH of 5.5 have better throughput through a
virus filter as compared to fluids having a pH of 6.5 or pH 7.5,
with fluids having a pH of 6.5 having the lowest throughput through
the virus filter.
[0185] FIG. 8 shows the relationship of the virus filter throughput
at 50% flow decay to the concentration of stabilizing agent in the
fluid. These data show that generally the virus filter throughput
increases as the concentration of stabilizing agent in the fluid
increases. The data in FIGS. 9 and 10 show that there is no
significant relationship of the virus filter throughput at 50% flow
decay to the concentration of sodium chloride or the percentage of
protein aggregates in the fluid, respectively. The data in FIG. 11
show that, as the average particle concentration of the fluid
increases, the throughput of the virus filter at 50% flow decay
decreases.
[0186] FIG. 12 shows the effect of the concentration of sodium
chloride at pH 5.5, 6.5, or 7.6 on the throughput of the virus
filter at 50% flow decay. These data show that increasing sodium
chloride concentrations from 50 mM to 300 mM increase the virus
filter throughput at pH 5.5, while increasing sodium chloride
concentrations from 50 mM to 300 mM decreases the virus filter
throughput at pH 7.6.
[0187] FIG. 13 shows the effect of the concentration of sodium
chloride at pH 5.5, 6.5, or 7.6 on the percentage of protein
aggregates. The data show that increasing sodium chloride
concentrations at pH 5.5 results in an increase in the percentage
of protein aggregates, while increasing sodium chloride
concentrations at pH 7.6 results in a decrease in the percentage of
protein aggregates.
[0188] FIG. 14 shows the effect of the concentration of sodium
chloride, the pH of the fluid, and the type of pre-filter used (0.1
.mu.m pre-filter or Sartorius Virosart.RTM. Max pre-filter) on the
virus filter throughput at 50% flow decay. These data show that
fluids pre-filtered with a Sartorius Virosart.RTM. Max pre-filter
have higher virus throughput values as compared to fluids
pre-filtered with a 0.1 .mu.m pre-filter. For fluids pre-filtered
using a Sartorius Virosart.RTM. Max pre-filter, increases in sodium
chloride concentration correlated with increased virus filter
throughput at pH 5.5, while increases in sodium chloride
concentration correlated with decreased virus filter throughput at
pH 7.6.
[0189] The data in FIG. 15 show that at pH 5.5, increasing
concentrations of sodium chloride in the fluid correlate with a
decrease in the hydrodynamic radius of the BNJ441 monoclonal
antibody, while at pH 7.6, increasing concentrations of sodium
chloride in the fluid only modestly correlate with a decrease in
the hydrodynamic radius of the BNJ441 monoclonal antibody.
[0190] The data in FIG. 16 show that the hydrodynamic radius of the
BNJ441 monoclonal antibody generally increases with the increasing
pH of the fluid. The data in FIG. 17 show the relationship between
the percentage of aggregates in the fluids with the sodium chloride
concentrations in the fluids.
[0191] FIG. 18 shows the optimal concentrations of L-arginine that
should be included in a fluid including BNJ441 monoclonal antibody
to achieve a virus filter flow rate of greater than 750 g/m.sup.2
(the non-shaded area in the graph).
[0192] These data show indicate that for fluids including an
antibody having at least one histidine in a CDR of the light or
heavy chain (such as the BNJ441 monoclonal antibody), that a pH of
between about 5.0 and about 6.7 improves the virus filter
throughput, and optionally, that adjusting (e.g., increasing or
decreasing) the fluid to a stabilizing agent concentration of
between about 0 mM and about 25 mM can further increase virus
filter throughput. The data also indicate that for fluids including
an antibody having at least one histidine in a CDR of the light or
heavy chain (such as the BNJ441 monoclonal antibody) and having a
pH of between about 6.7 and about 8.5, that adjusting the
stabilizing agent concentration of the fluid to between about 10 mM
and about 100 mM will improve the virus filter throughput. In
addition, for all fluids, the data show that the use of a Sartorius
Virosart.RTM. Max pre-filter improves the virus filter
throughput.
Example 2
Effect of pH on Viral Filtration of BNJ441 Monoclonal Antibody
[0193] A set of experiments were performed to test the effect of
the pH of a fluid including 4 mg/mL BNJ441 monoclonal antibody on
viral filtration performed using an Asahi Planova BioEx or an Asahi
20N viral filter. Each tested fluid contained 65 mM sodium chloride
and had a pH of between 7 and 8.5 (see, Table 1 below).
TABLE-US-00001 TABLE 1 Tested Fluids including BNJ441 Monoclonal
Antibody Salt Product Concentration Concentration Filter pH (mM)
(mg/mL) Bio EX 7 65 4 Bio EX 7.75 65 4 Bio EX 8.5 65 4 20N 7.75 65
4
[0194] The data in FIGS. 19-22 show that the best flux to
throughput was achieved when a fluid having a pH of 7.75 and
containing 65 mM sodium chloride and 4 mg/mL BNJ441 monoclonal
antibody was flowed through an Asahi 20N viral filter.
Example 3
Statistical Analysis to Determine Factors Important for Viral
Filtration of Samalizumab
[0195] Statistical analyses were performed to determine what
parameters (factors) were most important for throughput of
samalizumab through a Sartorius Virosart.RTM. CPV virus filter. The
fluids tested in these experiments included between 5 mg/mL to 15
mg/mL of samalizumab and between 75 mM to 300 mM sodium chloride,
and had a pH of between 5 and 6. The fluids tested in this
experiment are listed in Table 2.
TABLE-US-00002 TABLE 2 Tested Fluids including Samalizumab Salt
Product Concentration Concentration pH (mM) (mg/mL) 5 75 5 5 75 15
5 300 5 5 300 15 5.5 190 10 6 75 5 6 75 15 6 300 5 6 300 15
[0196] The relationship between the percentage flux decay and the
throughput of the Virosart.RTM. CPV virus filter when each fluid
was flowed through the Virosart.RTM. CPV virus filter is shown in
FIG. 23. Statistical analyses was performed to test the
relationship of the pH of each fluid, the sodium chloride
concentration of each fluid, and the samalizumab concentration of
each fluid on the virus filter throughput (FIG. 24). The
statistical analyses show that virus filter throughput increases as
pH is increases between pH 5 and pH 6, that virus filter throughput
increases as the sodium concentration increases between 75 mM and
300 mM, and that virus filter throughput decreases as the
samalizumab concentration increases between 5 mg/mL and 15
mg/mL.
Example 4
Statistical Analyses to Determine Factors Important for Viral
Filtration of BNJ383 Monoclonal Antibody
[0197] Statistical analyses were performed to determine what
parameters (factors) were most important for throughput of BNJ383
monoclonal antibody through a Sartorius Virosart.RTM. CPV virus
filter or a Virosart HF virus filter. The fluids tested in these
experiments included 10 mg/mL of BNJ383 monoclonal antibody and
between 80 mM to 300 mM sodium chloride, and had a pH of between 7
and 8.5. The fluids tested in this experiment are listed in Table
3.
TABLE-US-00003 TABLE 3 Tested Fluids including BNJ383 Monoclonal
Antibody Viral Salt Product Filter Concentration Concentration Type
pH (mM) (mg/mL) HF 7 80 10 HF 8.5 80 10 HF 7.75 190 10 HF 7 300 10
HF 8.5 300 10 CPV 7.75 80 10
[0198] The data show that the best flux decay as compared to
throughput is achieved when a fluid including 10 mg/mL BNJ383
monoclonal antibody and 80 mM sodium chloride, and having a pH of 7
was flowed through a Virosart HF virus filter (FIG. 25).
Statistical analyses of the data for each fluid show that virus
filter throughput decreases as pH in the fluid increases between 7
and 8.5, and that virus filter throughput decreases a sodium
chloride concentration increases between 80 mM to 300 mM (FIG.
26).
Example 5
Effect of Different Pre-filters on Viral Filtration of
Eculizumab
[0199] A set of experiments was performed to test the effect of
pre-filtration using a number of different pre-filters on
downstream viral filtration of a fluid including 7.1 mg/mL
eculizumab and 80 mM sodium chloride, and having a pH of 6.5, using
a Virosart.RTM. CPV viral filter. The different pre-filters tested
were: a Millipore 0.5/0.2 .mu.m and 0.5/0.1 .mu.m pre-filter, a
Sartorius Virosart.RTM. Max pre-filter, a Sartopore 2 pre-filter,
Sartobind STIC pre-filter, Sartobind Q pre-filter, Sartobind HIC
Phenyl pre-filter, or a Sartobind S pre-filter.
[0200] The data show that the best flux decay as compared to
throughput of the virus filter occurred when a fluid including 7.1
mg/mL eculizumab and 80 mM sodium chloride, and having a pH of 6.5
was flowed through a Sartorius Virosart.RTM. Max pre-filter prior
to flowing the fluid through a Virosart.RTM. CPV virus filter (FIG.
27). The data also show that passing a fluid including 7.1 mg/mL
eculizumab and 80 mM sodium chloride, and having a pH of 6.5
through a Sartorius Virosart.RTM. Max pre-filter also significantly
reduced the percentage of soluble protein aggregates in fluid
before it is flowed into the Virosart.RTM. CPV virus filter (FIG.
28). The data also show that flowing a fluid including XX mg/mL
eculizumab and 80 mM sodium chloride, and having a pH of 6.5
through a Sartorius Virosart.RTM. Max pre-filter also reduces the
insoluble particle concentration in the fluid (compare the data for
Q1 Pool to the data for Sartorius Virosart.RTM. Max Pool) (FIG.
29).
[0201] These data show that flowing a fluid including eculizumab
through a pre-filter (e.g., a pre-filtering including a polyamide
membrane, such as Sartorius Virosart.RTM. Max pre-filter) reduces
the concentration of soluble protein aggregates and insoluble
particles in the fluid, and increases the throughput of viral
filtration (as the fluid is subsequently flowed through a virus
filter, such as a Virosart.RTM. CPV virus filter).
Example 6
Study of Virosart.RTM. CPV Virus Filter Lot-to-Lot Variability
[0202] A set of experiments was performed to evaluate the
lot-to-lot variability of the Virosart.RTM. CPV virus filter on the
throughput of eculizumab. In these experiments, three different
lots of the 5 cm.sup.2 Virosart.RTM. CPV virus filter were tested
using the same loading material.
Materials and Methods
[0203] The starting material for these experiments was prepared by
performing the following steps: capturing eculizumab from a
concentrated clarified culture medium using Protein A
chromatography, performing low pH viral inactivation, performing
ultrafiltration and diafiltration, and performing Q Sepharose
chromatography. The starting material was then flowed through a
pre-filter in each experiment, (i.e., immediately before being
flowed through a Virosart.RTM. CPV virus filter).
[0204] In the experiments, 218 mL of the starting material was
filtered across a 3.5 cm.sup.2 Millipore 0.5/0.2 .mu.m pre-filter
(SHC), and a 0.5 .mu.m/0.1 .mu.m pre-filter (SHR). One hundred mL
of the resulting filtrate was filtered across a 5 cm.sup.2
Virosart.RTM. CPV virus filter to determine the filter throughput
of eculizumab in 20 mM sodium phosphate, 80 mM sodium chloride, pH
6.50. The remaining 100 mL was processed over another lot of
Virosart.RTM. CPV virus filter until the flux declined to .about.0%
of the initial value (FIG. 30). The process was performed two
additional times to evaluate three different lots of Virosart.RTM.
CPV virus filters in duplicate. Table 4 lists the processing
conditions for the virus filter testing. The virus filter flux,
flux decay, and feed pressure were monitored. The starting material
and the viral filtrate were assessed for protein concentration
using absorbance at 280 nm, and level of soluble protein
aggregates.
TABLE-US-00004 TABLE 4 Process Conditions for Testing Lot- to-Lot
Variability in Viral Filters Parameter Experimental Condition
Equilibration buffer 20 mM sodium phosphate, 80 mM sodium chloride,
pH 6.50 Pre-Filters area 3.5 cm.sup.2 CPV Filter Area 5 cm.sup.2
Pre-Filter Load (L/m.sup.2) 623 (218 mL) Virus filter load
(L/m.sup.2) 200 Load volume 100 mL Feed pressure (psi) 27-33 Buffer
Chase 12.5 mL
Results
[0205] The data in FIGS. 31 and 32 show that there is significant
lot-to-lot variability in the flux of Virosart.RTM. CPV virus
filters, and also significant variability in Virosart.RTM. CPV
virus filters from the same lot.
Example 7
Study of Effect of Pre-Filtering Loading Parameters on the
Throughput of Downstream Virus Filter
[0206] A set of experiments was performed to test the effect of
pre-filter loading parameters on the throughput of a downstream
Virosart.RTM. CPV virus filter.
Materials and Methods
[0207] The starting material for these experiments was prepared by
performing the following steps: capturing eculizumab from a
concentrated clarified culture medium using Protein A
chromatography, performing low pH viral inactivation, performing
ultrafiltration and diafiltration, and performing Q Sepharose
chromatography. The starting material was pre-filtered using a 3.5
cm.sup.2 Millipore 0.5/0.2 .mu.m pre-filter (SHC), and a 3.5
cm.sup.2 0.5 .mu.m/0.1 .mu.m pre-filter (SHR) under two different
conditions: 622 L/m.sup.2 (218 mL flowed through the 3.5 cm.sup.2
SHC and 3.5 cm.sup.2 SHR) or 311 L/m.sup.2 (109 mL through the 3.5
cm.sup.2 SHC and 3.5 cm.sup.2 SHR), and then 100 mL of each
filtrate (including 20 mM sodium phosphate, 80 mM sodium chloride,
pH of 6.5) was passed through a 5 cm.sup.2 Virosart.RTM. CPV virus
filter at 200 L/m.sup.2, with a feed pressure of 27-33 psi, and a
buffer chase of 12.5 mL. Each experiment was performed in
duplicate. The flux over time of the Virosart.RTM. CPV virus filter
was measured.
Results
[0208] The data in FIGS. 33 and 34 show that there was no
significant difference in the throughput of the Virosart.RTM. CPV
virus filters when pre-filters were loaded at 311 L/m.sup.2 or 622
L/m.sup.2.
Example 8
Study of Effect of Feed Pressure on Throughput of Virus Filters
[0209] A set of experiments was performed to determine the effect
of feed pressure on throughput of Virosart.RTM. CPV virus
filters.
Materials and Methods
[0210] The starting material for these experiments was prepared by
performing the following steps: capturing eculizumab from a
concentrated clarified culture medium using Protein A
chromatography, performing low pH viral inactivation, performing
ultrafiltration and diafiltration, and performing Q Sepharose
chromatography. The starting material was filtered using a 3.5
cm.sup.2 Millipore 0.5/0.2 .mu.m pre-filter (SHC), and a 3.5
cm.sup.2 0.5 .mu.m/0.1 .mu.m pre-filter (SHR), or a Sartorius
Virosart.RTM. Max pre-filter (5 cm.sup.2) to yield a including
including 20 mM sodium phosphate, 80 mM sodium chloride, pH of 6.5,
which was then further filtered in-line with a 5 cm.sup.2
Virosart.RTM. CPV virus filter, with a feed pressure of 15 psi or
30 psi for SHC/SHR pre-filter runs and 30 psi for the Virosart Max
Pre-filter run. Each experiment was performed in duplicate. The
flux over time of the Virosart.RTM. CPV virus filter was
measured.
Results
[0211] The data in FIGS. 35 and 36 show that there is no
significant difference in the throughput of the Virosart.RTM. CPV
virus filter when it is loaded using different inlet pressures, and
that there is a significant improvement in Virosart.RTM. CPV virus
filter throughput when a Sartorius Virosart.RTM. Max pre-filter is
used prior to the Virosart.RTM. CPV virus filter. The viral
filtration operation time was significantly reduced with the
Virosart.RTM. Max pre-filter.
Example 8
Effect of Protein Concentration During Pre-Filtration on Throughput
of Downstream Viral Filter
[0212] A set of experiments was performed to test the effect of
protein concentration during pre-filtration on the throughput of an
immediately downstream Virosart.RTM. CPV virus filter.
Materials and Methods
[0213] The starting material for these experiments was prepared by
performing the following steps: capturing eculizumab from a
concentrated clarified culture medium using Protein A
chromatography, performing low pH viral inactivation, performing
ultrafiltration and diafiltration, and performing Q Sepharose
chromatography. The starting material (218 mL) having concentration
of eculizumab of 4 mg/mL or 8 mg/mL was loaded at 623 L/m.sup.2
onto a 3.5 cm.sup.2 Millipore 0.5/0.2 .mu.m pre-filter (SHC), and a
3.5 cm.sup.2 0.5 .mu.m/0.1 .mu.m pre-filter (SHR), or a Sartorius
Virosart.RTM. Max pre-filter (5 cm.sup.2), and then 100 mL of each
filtrate (including 20 mM sodium phosphate, 80 mM sodium chloride,
pH of 6.5) was passed through a 5 cm.sup.2 Virosart.RTM. CPV virus
filter at 200 L/m.sup.2, with a feed pressure of 27-33 psi, and a
buffer chase of 12.5 mL. The flux over time of the Virosart.RTM.
CPV virus filter was measured.
Results
[0214] The data in FIGS. 37 and 38 show that a significant increase
in the volumetric throughput of the Virosart.RTM. CPV virus filter
occurs when the pre-filter is loaded with a lower concentration of
eculizumab, and that there is a significant improvement in
Virosart.RTM. CPV virus filter throughput when a Sartorius
Virosart.RTM. Max pre-filter is used prior to the Virosart.RTM. CPV
virus filter.
Example 9
Purification of Eculizumab using Pre-Filtration Prior to Viral
Filtration
[0215] A set of experiments was performed to test the effect of
different pre-filters on a process for purifying eculizumab that
includes the use of a pre-filter immediately prior to viral
filtration.
Materials and Methods
[0216] The starting material for these experiments was prepared by
performing the following steps: capturing eculizumab from a
concentrated clarified culture medium using Protein A
chromatography, performing low pH viral inactivation, performing
ultrafiltration and diafiltration, and performing Q Sepharose
chromatography. The starting material was loaded with a pressure of
30 psi and at 623 L/m.sup.2 onto a 3.5 cm.sup.2 Millipore 0.5/0.2
.mu.m pre-filter (SHC), and a 3.5 cm.sup.2 0.5 .mu.m/0.1 .mu.m
pre-filter (SHR), or a Sartorius Virosart.RTM. Max pre-filter (5
cm.sup.2), and each filtrate (including 20 mM sodium phosphate, 80
mM sodium chloride, pH of 6.5) was then passed through a 5 cm.sup.2
Virosart.RTM. CPV virus filter at 200 L/m.sup.2, with a feed
pressure of 30 psi, and 25 mL of a chase buffer was flowed through
the Virosart.RTM. CPV virus filter 60 minutes after the last of the
pre-filter eluate has been flowed through the Virosart.RTM. CPV
virus filter. During filtration with the Virosart.RTM. CPV virus
filter, if an 80% flux decay was reached, the filtration was paused
for 60 minutes and the pre-filter eluate loaded onto the
Virosart.RTM. CPV virus filter was diluted 4-fold in buffer. The
flux over time of the Virosart.RTM. CPV virus filter was
measured.
Results
[0217] The data in FIGS. 39 and 40 show that an improved throughput
of the Virosart.RTM. CPV virus filter was achieved when a Sartorius
Virosart.RTM. Max pre-filter was used prior to the Virosart.RTM.
CPV virus filter, and that the pause and dilution used when a 80%
flux decay was reached, was not necessary when a Sartorius
Virosart.RTM. Max pre-filter was used prior to the Virosart.RTM.
CPV virus filter. In contrast, in one of the two experiments where
a 3.5 cm.sup.2 Millipore 0.5/0.2 .mu.m pre-filter (SHC), and a 3.5
cm.sup.2 0.5 .mu.m/0.1 .mu.m pre-filter (SHR) was used as a
pre-filter, an 80% flux decay was reached, and it was necessary to
use the 60 minute pause and four-fold dilution (FIGS. 39 and 40;
Run 1--SHC/SHR).
Example 10
Use of Depth Filtration between Different Steps of an Eculizumab
Purification Process
[0218] A set of experiments was performed to test the effect of
performing depth filtration between different steps of a
recombinant protein purification process.
Materials and Methods
[0219] A depth filtration step was inserted between different steps
of starting recombinant protein purification process. The starting
recombinant protein purification process uses the steps of
preparing a clarified culture medium, concentrating the recombinant
protein using ultrafiltration/diafiltration, solvent/detergent
viral inactivation, capturing the recombinant protein using protein
A chromatography (capture), polishing the recombinant protein,
performing ultrafiltration/diafiltration, performing viral
filtration, performing final polishing of the recombinant protein,
and performing ultrafiltration/diafiltration (shown in FIG. 41A). A
depth filtration step that includes flowing a fluid including the
recombinant protein through a CUNO Delipid filter was performed
between two different steps in the starting recombinant protein
purification process (see, FIGS. 41B to 41D). The impact of
performing the depth filtration step in each tested process was
determined by detecting the flux in a downstream Virosart.RTM. CPV
virus filter and detecting the level of soluble protein aggregates
and insoluble protein particles in the final purified recombinant
protein at the end of the tested purification process.
[0220] In a second set of experiments, three different depth
filters were tested for their ability to reduce host cell protein
levels. One of the three tested filters was a depth filter having
anionic and hydrophobic properties (Filter 2, which is a CUNO
Delipid filter). Alexion 1210 (also called BNJ441) was purified
using an eculizumab purification process as shown in FIG. 41D,
where the depth filter was the depth filter having anionic and
hydrophobic properties (Filter 2), and where the depth filtration
step was performed using original conditions or under conditions
optimized to maximize impurity removal while maintaining greater
than 85% yield of eculizumab. The depth filter having anionic and
hydrophobic properties (Filter 2) was also tested to determine if
the level of Murine Minute Virus (MMV) and Xenotrophic Murine
Leukemia Virus (XMuLV) in a fluid could be reduced by flowing the
fluid through the depth filter.
Results
[0221] The data in FIGS. 42 and 43 show that a significant
improvement in aggregate removal and particulate content was
observed when depth filtration was performed immediately prior to
virus filtration. The data in FIG. 44A and 44B show that a depth
filter having anionic and hydrophobic properties results in
significant removal of host cell protein and soluble protein
aggregates (Filter 2). The data in Table 5 show the result of
performing depth filtration using a depth filter having anionic and
hydrophobic properties on the level of MMV and XMuLV.
TABLE-US-00005 TABLE 5 Viral Clearance Results for Adsorptive Depth
Filtration Virus Log.sub.10 Reduction MVM 0.72 .+-. 0.33 X-MuLV
1.46 .+-. 0.20
Other Embodiments
[0222] It is to be understood that while the invention has been
described in conjunction with the detailed description thereof, the
foregoing description is intended to illustrate and not limit the
scope of the invention, which is defined by the scope of the
appended claims. Other aspects, advantages, and modifications are
within the scope of the following claims.
Sequence CWU 1
1
40110PRTArtificial SequenceBNJ383 Heavy chain CDR3BNJ383 Heavy
chain CDR3 1Gly His Ile Phe Ser Asn Tyr Trp Ile Gln 1 5 10
217PRTArtificial SequenceBNJ441 Heavy chain CDR2 2Glu Ile Leu Pro
Gly Ser Gly His Thr Glu Tyr Thr Glu Asn Phe Lys 1 5 10 15
Asp313PRTArtificial SequenceBNJ441 Heavy chain CDR3 3Tyr Phe Phe
Gly Ser Ser Pro Asn Trp Tyr Phe Asp Val 1 5 10 4122PRTArtificial
SequenceBNJ441 Heavy chain variable region 4Gln 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 His Ile Phe Ser Asn Tyr 20 25 30 Trp Ile
Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45
Gly Glu Ile Leu Pro Gly Ser Gly His Thr Glu Tyr Thr Glu Asn Phe 50
55 60 Lys Asp Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val
Tyr65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Ala Arg Tyr Phe Phe Gly Ser Ser Pro Asn Trp
Tyr Phe Asp Val Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser
Ser 115 120 5448PRTArtificial SequenceBNJ441 Heavy chain 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 His Ile Phe Ser Asn Tyr 20
25 30 Trp Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp
Met 35 40 45 Gly Glu Ile Leu Pro Gly Ser Gly His Thr Glu Tyr Thr
Glu Asn Phe 50 55 60 Lys Asp Arg Val Thr Met Thr Arg Asp Thr Ser
Thr Ser Thr Val Tyr65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu
Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Tyr Phe Phe Gly Ser
Ser Pro Asn Trp Tyr Phe Asp Val Trp 100 105 110 Gly Gln Gly Thr Leu
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe
Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr 130 135 140 Ala
Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr145 150
155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
Val Val Thr 180 185 190 Val Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr
Thr Cys Asn Val Asp 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp
Lys Thr Val Glu Arg Lys Cys 210 215 220 Cys Val Glu Cys Pro Pro Cys
Pro Ala Pro Pro Val Ala Gly Pro Ser225 230 235 240 Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255 Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro 260 265 270
Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275
280 285 Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val
Val 290 295 300 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
Lys Glu Tyr305 310 315 320 Lys Cys Lys Val Ser Asn Lys Gly Leu Pro
Ser Ser Ile Glu Lys Thr 325 330 335 Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val Tyr Thr Leu 340 345 350 Pro Pro Ser Gln Glu Glu
Met Thr Lys Asn Gln Val Ser Leu Thr Cys 355 360 365 Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380 Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp385 390 395
400 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser
405 410 415 Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Leu His
Glu Ala 420 425 430 Leu His Ser His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser Leu Gly Lys 435 440 445 611PRTArtificial SequenceBNJ441 Light
chain CDR1 6Gly Ala Ser Glu Asn Ile Tyr Gly Ala Leu Asn 1 5 10
77PRTArtificial SequenceBNJ441 Light chain CDR2 7Gly Ala Thr Asn
Leu Ala Asp1 5 89PRTArtificial SequenceBNJ441 Light chain CDR3 8Gln
Asn Val Leu Asn Thr Pro Leu Thr1 5 9107PRTArtificial SequenceBNJ441
Light chain variable region 9Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys
Gly Ala Ser Glu Asn Ile Tyr Gly Ala 20 25 30 Leu Asn Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ala
Thr Asn Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Asn Val Leu Asn Thr Pro Leu
85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105
10214PRTArtificial SequenceBNJ441 Light chain 10Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val
Thr Ile Thr Cys Gly Ala Ser Glu Asn Ile Tyr Gly Ala 20 25 30 Leu
Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40
45 Tyr Gly Ala Thr Asn Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly
50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
Gln Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Asn Val Leu
Asn Thr Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro
Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys
Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp
Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln145 150 155 160 Glu
Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170
175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr
Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 1110PRTArtificial
SequenceEculizumab Heavy chain CDR1 11Gly Tyr Ile Phe Ser Asn Tyr
Trp Ile Gln 1 5 10 1217PRTArtificial SequenceEculizumab Heavy chain
CDR2 12Glu Ile Leu Pro Gly Ser Gly Ser Thr Glu Tyr Thr Glu Asn Phe
Lys 1 5 10 15 Asp1313PRTArtificial SequenceEculizumab Heavy chain
CDR3 13Tyr Phe Phe Gly Ser Ser Pro Asn Trp Tyr Phe Asp Val 1 5 10
14122PRTArtificial SequenceEculizumab Heavy chain variable region
14Gln 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 Ile Phe Ser Asn
Tyr 20 25 30 Trp Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu
Glu Trp Met 35 40 45 Gly Glu Ile Leu Pro Gly Ser Gly Ser Thr Glu
Tyr Thr Glu Asn Phe 50 55 60 Lys Asp Arg Val Thr Met Thr Arg Asp
Thr Ser Thr Ser Thr Val Tyr65 70 75 80 Met Glu Leu Ser Ser Leu Arg
Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Tyr Phe Phe
Gly Ser Ser Pro Asn Trp Tyr Phe Asp Val Trp 100 105 110 Gly Gln Gly
Thr Leu Val Thr Val Ser Ser 115 120 15448PRTArtificial
SequenceEculizumab Heavy chain 15Gln 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 Ile Phe Ser Asn Tyr 20 25 30 Trp Ile Gln Trp
Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Glu
Ile Leu Pro Gly Ser Gly Ser Thr Glu Tyr Thr Glu Asn Phe 50 55 60
Lys Asp Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr65
70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Arg Tyr Phe Phe Gly Ser Ser Pro Asn Trp Tyr
Phe Asp Val Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser
Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Cys
Ser Arg Ser Thr Ser Glu Ser Thr 130 135 140 Ala Ala Leu Gly Cys Leu
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr145 150 155 160 Val Ser Trp
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala
Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185
190 Val Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp
195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg
Lys Cys 210 215 220 Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val
Ala Gly Pro Ser225 230 235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met Ile Ser Arg 245 250 255 Thr Pro Glu Val Thr Cys Val
Val Val Asp Val Ser Gln Glu Asp Pro 260 265 270 Glu Val Gln Phe Asn
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285 Lys Thr Lys
Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val 290 295 300 Ser
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr305 310
315 320 Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys
Thr 325 330 335 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
Tyr Thr Leu 340 345 350 Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln
Val Ser Leu Thr Cys 355 360 365 Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu Trp Glu Ser 370 375 380 Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro Val Leu Asp385 390 395 400 Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser 405 410 415 Arg Trp
Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 435
440 445 1611PRTArtificial SequenceEculizumab Light chain CDR1 16Gly
Ala Ser Glu Asn Ile Tyr Gly Ala Leu Asn 1 5 10 177PRTArtificial
SequenceEculizumab Light chain CDR2 17Gly Ala Thr Asn Leu Ala Asp1
5 189PRTArtificial SequenceEculizumab Light chain CDR3 18Gln Asn
Val Leu Asn Thr Pro Leu Thr1 5 19107PRTArtificial
SequenceEculizumab Light chain variable region 19Asp Ile Gln Met
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg
Val Thr Ile Thr Cys Gly Ala Ser Glu Asn Ile Tyr Gly Ala 20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35
40 45 Tyr Gly Ala Thr Asn Leu Ala Asp Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Asn Val
Leu Asn Thr Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys 100 105 20214PRTArtificial SequenceEculizumab Light chain
20Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1
5 10 15 Asp Arg Val Thr Ile Thr Cys Gly Ala Ser Glu Asn Ile Tyr Gly
Ala 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
Leu Leu Ile 35 40 45 Tyr Gly Ala Thr Asn Leu Ala Asp Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser Ser Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr
Cys Gln Asn Val Leu Asn Thr Pro Leu 85 90 95 Thr Phe Gly Gln Gly
Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val
Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr
Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135
140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser
Gln145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr
Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly
Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys
210 215PRTArtificial SequenceBNJ383 Heavy chain CDR1 21Asp Tyr Ser
Met Asp1 5 2217PRTArtificial SequenceBNJ383 Heavy chain CDR2 22Ala
Ile His Leu Asn Thr Gly Tyr Thr Asn Tyr Asn Gln Lys Phe Lys 1 5 10
15 Gly2311PRTArtificial SequenceBNJ383 Heavy chain CDR3 23Gly Phe
Tyr Asp Gly Tyr Ser Pro Met Asp Tyr 1 5 10 24120PRTArtificial
SequenceBNJ383 Heavy chain variable region 24Gln 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 Phe Thr Asp Tyr 20 25 30 Ser
Met Asp Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40
45 Gly Ala Ile His Leu Asn Thr Gly Tyr Thr Asn Tyr Asn Gln Lys Phe
50 55 60 Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr
Val Tyr65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Phe Tyr Asp Gly Tyr Ser Pro
Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Thr Val Thr Val Ser Ser
115 120 25446PRTArtificial SequenceBNJ383 Heavy chain 25Gln 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 Phe Thr Asp Tyr 20 25
30 Ser Met Asp Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45 Gly Ala Ile His Leu Asn Thr Gly Tyr Thr Asn Tyr Asn Gln
Lys Phe 50 55 60
Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr65
70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Arg Gly Phe Tyr Asp Gly Tyr Ser Pro Met Asp
Tyr Trp Gly Gln 100 105 110 Gly Thr Thr Val Thr Val Ser Ser Ala Ser
Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Cys Ser Arg
Ser Thr Ser Glu Ser Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys
Asp Tyr Phe Pro Glu Pro Val Thr Val Ser145 150 155 160 Trp Asn Ser
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185
190 Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys
195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys
Cys Val 210 215 220 Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly
Pro Ser Val Phe225 230 235 240 Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr Cys Val Val Val
Asp Val Ser Gln Glu Asp Pro Glu Val 260 265 270 Gln Phe Asn Trp Tyr
Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285 Lys Pro Arg
Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val 290 295 300 Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys305 310
315 320 Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile
Ser 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
Leu Pro Pro 340 345 350 Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu Val 355 360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn Gly 370 375 380 Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro Val Leu Asp Ser Asp385 390 395 400 Gly Ser Phe Phe
Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp 405 410 415 Gln Glu
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 435 440 445
2615PRTArtificial SequenceBNJ383 Light chain CDR1 26Arg Ala Ser Glu
Ser Val Asp Ser Tyr Gly Asn Ser Phe Met His 1 5 10 15
277PRTArtificial SequenceBNJ383 Light chain CDR2 27Arg Ala Ser Asn
Leu Glu Ser1 5 289PRTArtificial SequenceBNJ383 Light chain CDR3
28Gln Gln Ser Asn Glu Asp Pro Tyr Thr1 5 29112PRTArtificial
SequenceBNJ383 Light chain variable region 29Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val
Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Asp Ser Tyr 20 25 30 Gly
Asn Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro 35 40
45 Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ser Gly Val Pro Ser
50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser65 70 75 80 Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys
Gln Gln Ser Asn 85 90 95 Glu Asp Pro Tyr Thr Phe Gly Gly Gly Thr
Lys Val Glu Ile Lys Arg 100 105 110 30218PRTArtificial
SequenceBNJ383 Light chain 30Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys
Arg Ala Ser Glu Ser Val Asp Ser Tyr 20 25 30 Gly Asn Ser Phe Met
His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro 35 40 45 Lys Leu Leu
Ile Tyr Arg Ala Ser Asn Leu Glu Ser Gly Val Pro Ser 50 55 60 Arg
Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser65 70 75
80 Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Asn
85 90 95 Glu Asp Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile
Lys Arg 100 105 110 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro
Ser Asp Glu Gln 115 120 125 Leu Lys Ser Gly Thr Ala Ser Val Val Cys
Leu Leu Asn Asn Phe Tyr 130 135 140 Pro Arg Glu Ala Lys Val Gln Trp
Lys Val Asp Asn Ala Leu Gln Ser145 150 155 160 Gly Asn Ser Gln Glu
Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 165 170 175 Tyr Ser Leu
Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180 185 190 His
Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 195 200
205 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215
319PRTArtificial SequenceSamalizumab Heavy chain CDR1 31Tyr Ser Phe
Thr Asp Tyr Ile Ile Leu1 5 3217PRTArtificial SequenceSamalizumab
Heavy chain CDR2 32His Ile Asp Pro Tyr Tyr Gly Ser Ser Asn Tyr Asn
Leu Lys Phe Lys 1 5 10 15 Gly338PRTArtificial SequenceSamalizumab
Heavy chain CDR3 33Ser Lys Arg Asp Tyr Phe Asp Tyr1 5
34117PRTArtificial SequenceSamalizumab Heavy chain variable domain
34Gln Val Gln Leu Gln Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala 1
5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asp
Tyr 20 25 30 Ile Ile Leu Trp Val Arg Gln Asn Pro Gly Lys Gly Leu
Glu Trp Ile 35 40 45 Gly His Ile Asp Pro Tyr Tyr Gly Ser Ser Asn
Tyr Asn Leu Lys Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Ala Asp
Gln Ser Thr Thr Thr Ala Tyr65 70 75 80 Met Glu Leu Ser Ser Leu Arg
Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Gly Arg Ser Lys Arg
Asp Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr 100 105 110 Leu Thr Val
Ser Ser 115 35443PRTArtificial SequenceSamalizumab Heavy chain
35Gln Val Gln Leu Gln Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala 1
5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asp
Tyr 20 25 30 Ile Ile Leu Trp Val Arg Gln Asn Pro Gly Lys Gly Leu
Glu Trp Ile 35 40 45 Gly His Ile Asp Pro Tyr Tyr Gly Ser Ser Asn
Tyr Asn Leu Lys Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Ala Asp
Gln Ser Thr Thr Thr Ala Tyr65 70 75 80 Met Glu Leu Ser Ser Leu Arg
Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Gly Arg Ser Lys Arg
Asp Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr 100 105 110 Leu Thr Val
Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala
Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys 130 135
140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
Ser145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
Thr Val Pro Ser Ser Asn 180 185 190 Phe Gly Thr Gln Thr Tyr Thr Cys
Asn Val Asp His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Thr
Val Glu Arg Lys Cys Cys Val Glu Cys Pro 210 215 220 Pro Cys Pro Ala
Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro225 230 235 240 Pro
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 245 250
255 Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn
260 265 270 Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
Pro Arg 275 280 285 Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser
Val Leu Thr Val 290 295 300 Leu His Gln Asp Trp Leu Asn Gly Lys Glu
Tyr Lys Cys Lys Val Ser305 310 315 320 Asn Lys Gly Leu Pro Ser Ser
Ile Glu Lys Thr Ile Ser Lys Ala Lys 325 330 335 Gly Gln Pro Arg Glu
Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu 340 345 350 Glu Met Thr
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 355 360 365 Tyr
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 370 375
380 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
Phe385 390 395 400 Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg
Trp Gln Glu Gly 405 410 415 Asn Val Phe Ser Cys Ser Val Met His Glu
Ala Leu His Asn His Tyr 420 425 430 Thr Gln Lys Ser Leu Ser Leu Ser
Leu Gly Lys 435 440 3611PRTArtificial SequenceSamalizumab Light
chain CDR1 36Lys Ala Ser Gln Asp Ile Asn Ser Tyr Leu Ser 1 5 10
377PRTArtificial SequenceSamalizumab Light chain CDR2 37Arg Ala Asn
Arg Leu Val Asp1 5 389PRTArtificial SequenceSamalizumab Light chain
CDR3 38Leu Gln Tyr Asp Glu Phe Pro Tyr Thr1 5 39107PRTArtificial
SequenceSamalizumab Light chain variable region 39Asp Ile Gln Met
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Ile Gly 1 5 10 15 Asp Arg
Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Ser Tyr 20 25 30
Leu Ser Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35
40 45 Tyr Arg Ala Asn Arg Leu Val Asp Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser
Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Leu Gln Tyr
Asp Glu Phe Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu
Ile Lys 100 105 40214PRTArtificial SequenceSamalizumab Light chain
40Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Ile Gly 1
5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Ser
Tyr 20 25 30 Leu Ser Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys
Leu Leu Ile 35 40 45 Tyr Arg Ala Asn Arg Leu Val Asp Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu
Thr Ile Ser Ser Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr
Cys Leu Gln Tyr Asp Glu Phe Pro Tyr 85 90 95 Thr Phe Gly Gly Gly
Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val
Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr
Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135
140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser
Gln145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr
Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly
Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys
210
* * * * *