U.S. patent application number 15/618385 was filed with the patent office on 2017-12-07 for low acidic species compositions and methods for producing and using the same using displacement chromatography.
The applicant listed for this patent is AbbVie Inc.. Invention is credited to Chris Chumsae, Germano Coppola, Chen Wang.
Application Number | 20170349654 15/618385 |
Document ID | / |
Family ID | 52826381 |
Filed Date | 2017-12-07 |
United States Patent
Application |
20170349654 |
Kind Code |
A1 |
Wang; Chen ; et al. |
December 7, 2017 |
LOW ACIDIC SPECIES COMPOSITIONS AND METHODS FOR PRODUCING AND USING
THE SAME USING DISPLACEMENT CHROMATOGRAPHY
Abstract
The present invention relates to low acidic species (AR)
compositions comprising a protein, e.g., an antibody, or
antigen-binding portion thereof, and methods for producing such low
AR compositions using displacement chromatography. Methods for
using such compositions to treat a disorder, e.g., a disorder in
which TNF.alpha. is detrimental, are also provided.
Inventors: |
Wang; Chen; (Shrewsbury,
MA) ; Coppola; Germano; (Shrewsbury, MA) ;
Chumsae; Chris; (North Andover, MA) |
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Applicant: |
Name |
City |
State |
Country |
Type |
AbbVie Inc. |
North Chicago |
IL |
US |
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|
Family ID: |
52826381 |
Appl. No.: |
15/618385 |
Filed: |
June 9, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14635505 |
Mar 2, 2015 |
9688752 |
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15618385 |
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14079076 |
Nov 13, 2013 |
9017687 |
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14635505 |
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14077576 |
Nov 12, 2013 |
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14079076 |
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61892833 |
Oct 18, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 14/525 20130101;
C07K 2317/565 20130101; C07K 16/065 20130101; C07K 16/241 20130101;
C07K 1/165 20130101; A61K 39/39591 20130101 |
International
Class: |
C07K 16/24 20060101
C07K016/24; C07K 16/06 20060101 C07K016/06; C07K 14/525 20060101
C07K014/525; A61K 39/395 20060101 A61K039/395 |
Claims
1. A method for producing a low acidic species composition
comprising an antibody, or antigen-binding portion thereof, the
method comprising: (a) contacting a first sample comprising the
antibody, or antigen-binding portion thereof, with a chromatography
media, wherein the antibody, or antigen-binding portion thereof,
binds to the chromatography media; (b) displacing the antibody, or
antigen-binding portion thereof, bound to the chromatography media
with a displacing buffer comprising at least one displacer
molecule; and (c) collecting a chromatography sample, wherein the
chromatography sample comprises a composition of the antibody, or
antigen-binding portion thereof, which contains less than about 10%
acidic species, thereby producing a low acidic species composition
comprising an antibody, or antigen-binding portion thereof.
2. The method of claim 1, wherein the pH of the displacing buffer
is lower than the isoelectric point of the antibody, or
antigen-binding portion thereof.
3. The method of claim 1, wherein the displacing buffer carries
positive charge and wherein the concentration of the displacer in
the displacing buffer is at least about 0.1 mM.
4. The method of claim 1, wherein the conductivity of the
displacing buffer is about 2 mS/cm to about 20 mS/cm.
5. The method of claim 1, wherein one displacing buffer is
used.
6. The method of claim 1, wherein a first displacing buffer and a
second displacing buffer are used, and wherein the first and second
displacing buffers comprise different concentrations of
displacer.
7. The method of claim 1, wherein the method is run in a linear
displacement chromatography mode, a two-step displacement
chromatography mode, or a multiple-step displacement chromatography
mode.
8. The method of claim 1, wherein the chromatography sample
comprises a reduced level of host cell proteins as compared to the
first sample.
9. The method of claim 1, wherein the chromatography sample
comprises a reduced level of one or more of charge variants,
structure variants or fragmentation variants as compared to the
first sample.
10. The method of claim 9, wherein the chromatography sample
comprises a reduced level of basic species as compared to the first
sample.
11. The method of claim 10, wherein the reduced level of basic
species comprise a reduced level of a lysine species Lys 0 as
compared to the first sample.
12. The method of claim 1, wherein the chromatography sample
comprises a reduced level of aggregates as compared to the first
sample.
13. The method of claim 1, wherein the chromatography sample
comprises a reduced level of antibody fragments as compared to the
first sample.
14. The method of claim 1, wherein the antibody, or antigen-binding
portion thereof, is an anti-TNF.alpha. antibody, or antigen-binding
portion thereof.
15. A low acidic species composition comprising an antibody, or
antigen-binding portion thereof, wherein the composition comprises
less than about 15% acidic species.
16. The composition of claim 15, wherein the acidic species
comprise a first acidic region (AR1) and a second acidic region
(AR2).
17. The composition of claim 15, wherein the composition comprises
about 5% or less acidic species (AR).
18. The composition of claim 15, wherein the composition comprises
about 3% or less acidic species (AR).
19. The composition of claim 16, wherein the composition comprises
about 0.1% or less AR1 and about 3% or less AR2.
20. The composition of claim 17, wherein the composition comprises
about 1.4% or less acidic species (AR).
21. The composition of claim 16, wherein the composition comprises
about 1.4% AR2 and about 0.0% AR1.
22. The composition of claim 15, wherein the composition exhibits
increased cartilage tissue penetration, increased TNF affinity,
reduced cartilage destruction, reduced bone erosion, reduced
synovial proliferation, reduced cell infiltration, reduced
chondrocyte death, or reduced proteoglycan loss as compared to a
non-low acidic species composition, or wherein the composition
exhibits increased protection against the development of arthritic
scores or histopathology scores as compared to a non-low acidic
species composition when administered to an animal model of
arthritis.
23. A low acidic species composition comprising an anti-TNF.alpha.
antibody, or antigen-binding portion thereof, comprising a light
chain variable region (LCVR) having a CDR1 domain comprising the
amino acid sequence of SEQ ID NO: 7, a CDR2 domain comprising the
amino acid sequence of SEQ ID NO: 5, and a CDR3 domain comprising
the amino acid sequence of SEQ ID NO: 3; and a heavy chain variable
region (HCVR) having a CDR1 domain comprising the amino acid
sequence of SEQ ID NO: 8, a CDR2 domain comprising the amino acid
sequence of SEQ ID NO: 6, and a CDR3 domain comprising the amino
acid sequence of SEQ ID NO: 4, wherein the composition comprises
less than about 10% acidic species (AR).
24. The composition of claim 23, wherein the anti-TNF.alpha.
antibody, or antigen-binding portion thereof, comprises a light
chain variable region comprising the amino acid sequence set forth
in SEQ ID NO: 1 and a heavy chain variable region comprising the
amino acid sequence set forth in SEQ ID NO: 2.
25. The composition of claim 23, wherein the anti-TNF.alpha.
antibody, or antigen-binding portion thereof, is adalimumab, or an
antigen binding-portion thereof.
26. The composition of claim 23, wherein the acidic species
comprise a first acidic region (AR1) and a second acidic region
(AR2), and wherein the composition comprises about 0.1% or less AR1
and about 3% or less AR2.
27. The composition of claim 23, wherein the composition exhibits
increased cartilage tissue penetration, increased TNF affinity,
reduced cartilage destruction, reduced bone erosion, reduced
synovial proliferation, reduced cell infiltration, reduced
chondrocyte death, or reduced proteoglycan loss as compared to a
non-low acidic species composition, or wherein the composition
exhibits increased protection against the development of arthritic
scores or histopathology scores as compared to a non-low acidic
species composition when administered to an animal model of
arthritis.
28. The composition of claim 23, wherein the acidic species are
product preparation-derived acidic species, cell-culture-derived
acidic species or storage-derived acidic species.
29. A method for treating a subject having a disorder in which
TNF.alpha. is detrimental, comprising administering to the subject
the composition of claim 15, thereby treating the subject having a
disorder in which TNF.alpha. is detrimental.
30. The method of claim 29, wherein the disorder in which
TNF.alpha. is detrimental is rheumatoid arthritis (RA), psoriasis,
psoriatic arthritis, ankylosing spondylitis, Crohn's Disease,
Ulcerative Colitis, juvenile idiopathic arthritis, active axial
spondyloarthritis or non-radiographic axial spondyloarthritis.
Description
RELATED APPLICATION INFORMATION
[0001] This application is a continuation of U.S. application Ser.
No. 14/635,505, filed on Mar. 2, 2015 which, in turn, is a
continuation of U.S. application Ser. No. 14/079,076, filed on Nov.
13, 2013, now U.S. Pat. No. 9,017,687, issued Apr. 28, 2015, which,
in turn, is a continuation of U.S. application Ser. No. 14/077,576,
filed on Nov. 12, 2013, now abandoned, which in turn claims
priority to U.S. Provisional Application Ser. No. 61/892,833, filed
on Oct. 18, 2013, the entire contents of each of which are
expressly incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] The production of compositions comprising proteins for
biopharmaceutical applications involves the use of upstream process
technologies (e.g., cell culture) and downstream process
technologies (e.g., protein purification) that are known to produce
proteins exhibiting varying levels of protein variants and
impurities within the composition. Such protein variants include,
but are not limited to, the presence of charge variants (e.g.,
basic variants and acidic species, including variants) and
process-related impurities. For example, in monoclonal antibody
(mAb) preparations, charge variants can be detected by various
methods, such as ion exchange chromatography, for example, WCX-10
HPLC (a weak cation exchange chromatography) or IEF (isoelectric
focusing). Because of their similar chemical characteristics to the
antibody product molecules of interest, reduction of charge
variants is a challenge in monoclonal antibody production.
[0003] Reduction of charge variants and/or product- or
process-related impurities is particularly advantageous in the
context of commercially produced recombinant biotherapeutics, as
they have the potential to impact numerous product characteristics,
including, but not limited to, product stability, product safety
and product efficacy. Accordingly, there remains a need in the art
for low acidic species compositions and high-efficiency methods of
producing protein compositions, e.g., antibodies, having low levels
of acidic species.
SUMMARY OF THE INVENTION
[0004] The present invention is based on the optimization of
displacement chromatography process technologies for protein
production, e.g., production of antibodies or antigen-binding
portions thereof, resulting in the production of compositions
comprising proteins that comprise low percentages of acidic
species. These low acidic species compositions have improved
therapeutic efficacy and improved biological properties, for
example, increased cartilage tissue penetration, reduced cartilage
destruction, reduced synovial proliferation, reduced bone erosion,
increased protection against the development of arthritis as
measured by arthritic scores and/or histopathology scores, reduced
cell infiltration, reduced proteoglycan loss, reduced chondrocyte
death, and/or increased TNF.alpha. affinity, as compared to a
non-low acidic species composition.
[0005] Displacement chromatography is a chromatographic separation
technology that involves the use of a displacer molecule to aid in
the separation of a mixture, e.g., an antibody-containing solution
derived from cell culture harvest. The displacer molecule is
conventionally selected to have a higher affinity for the
stationary phase (i.e., the chromatographic support) as compared to
the components present in the material to be separated. Due to its
higher affinity, the displacer molecule competes with protein
mixture components for the binding sites on the stationary phase.
Under appropriate conditions, the displacer induces the components
of the mixture to develop into consecutive zones of concentrated
and purified species in the order of decreasing binding affinity
ahead of the displacer front. This ordered displacement of the
components of the mixture results in the formation of a so-called
"displacement train." In contrast to traditional elution mode
chromatography, the displacement process takes advantage of the
nonlinearity of the adsorption isotherm, allowing for higher column
loading levels without compromising the purity and recovery of the
component of interest. Finally, washing of the displacement train
with the displacing buffer from the column allows for the component
of interest to be isolated by collecting (and pooling if necessary)
the proper fraction(s) of the displaced eluate.
[0006] Accordingly, in one aspect, the invention provides a method
for producing a low acidic species composition comprising an
antibody, or antigen-binding portion thereof, by contacting a first
sample comprising the antibody, or antigen-binding portion thereof,
with a chromatography media, wherein the antibody, or
antigen-binding portion thereof, binds to the chromatography media;
displacing the antibody, or antigen-binding portion thereof, bound
to the chromatography media with a displacing buffer comprising at
least one displacer molecule; and collecting a chromatography
sample, wherein the chromatography sample comprises a composition
of the antibody, or antigen-binding portion thereof, which contains
less than about 10% acidic species, thereby producing a low acidic
species composition comprising an antibody, or antigen-binding
portion thereof.
[0007] In one embodiment, the chromatography media is selected from
the group consisting of anion exchange adsorbent material, cation
exchange adsorbent material and mixed mode media. In another
embodiment, the cation exchange (CEX) adsorbent material is
selected from the group consisting of a CEX resin and a CEX
membrane adsorber. In another embodiment, the CEX resin is Poros XS
resin.
[0008] In another embodiment, the chromatography media is a mixed
mode media comprising cation exchange (CEX) and hydrophobic
interaction functional groups. In one embodiment, the mixed mode
media is Capto MMC resin. In another embodiment, the mixed mode
media is selected from the group consisting of a CEX-based mixed
mode resin and a CEX-based mixed mode membrane adsorber. In another
embodiment, the mixed mode media is selected from the group
consisting of CaptoMMC ImpRes, Nuvia cPrime, and Toyopearl Trp-650M
resins.
[0009] In one embodiment, the pH of the displacing buffer is lower
than the isoelectric point of the antibody, or antigen-binding
portion thereof. In another embodiment, the pH of the displacing
buffer is in the range of about 6.0 to about 8.0.
[0010] In one embodiment, the displacing buffer carries positive
charge and wherein the concentration of the displacer in the
displacing buffer is at least about 0.1 mM. In another embodiment,
the displacer is a quaternary ammonium salt and the concentration
of the displacer in the displacing buffer about 0.1 mM to about 10
mM. In another embodiment, the displacer is protamine sulfate and
the concentration of the protamine sulfate in the displacing buffer
is about 0.1 mM to 5 about mM.
[0011] In one embodiment, the conductivity of the displacing buffer
is about 2 mS/cm to about 20 mS/cm. In another embodiment, the
chromatography media is in a column, wherein the column length is
in the range of about 10 cm to about 30 cm, and wherein flow
residence time is in the range of about 5 min to about 25 min.
[0012] In one embodiment, one displacing buffer is used.
[0013] In another embodiment, a first displacing buffer and a
second displacing buffer are used, and wherein the first and second
displacing buffers comprise different concentrations of displacer.
In one embodiment, the first displacing buffer comprises a lower
displacer concentration of displacer than the second displacing
buffer. In one embodiment, the first displacing buffer comprises
about 0.5 mM Expell SP1.TM.. In another embodiment, the first
displacing buffer comprises about 0.25 mM protamine sulfate.
[0014] In one embodiment, the method is run in a two-step
displacement chromatography mode.
[0015] In another embodiment, the method is run in a multiple-step
displacement chromatography mode or a linear displacement
chromatography mode.
[0016] In one embodiment, the chromatography sample comprises a
reduced level of host cell proteins as compared to the first
sample. In another embodiment, the chromatography sample comprises
a reduced level of one or more of charge variants, structure
variants or fragmentation variants as compared to the first sample.
In one embodiment, the chromatography sample comprises a reduced
level of acidic species region AR1, and wherein the charge variants
comprise deamidation variants, glycation variants, afucosylation
variants, MGO variants or citric acid variants. In another
embodiment, the chromatography sample comprises a reduced level of
the acidic species region AR1, and wherein the structure variants
comprise glycosylation variants or acetonation variants. In one
embodiment, the chromatography sample comprises a reduced level of
the acidic species region AR1, and wherein the fragmentation
variants comprise Fab fragment variants, C-terminal truncation
variants or variants missing a heavy chain variable domain. In
another embodiment, the chromatography sample comprises a reduced
level of the acidic species region AR2, and wherein the charge
variants comprise deamidation variants or glycation variants.
[0017] In another embodiment, the chromatography sample comprises a
reduced level of basic species as compared to the first sample. In
one embodiment, the reduced level of basic species comprise a
reduced level of a lysine species Lys 0 as compared to the first
sample.
[0018] In another embodiment, the chromatography sample comprises a
reduced level of aggregates as compared to the first sample. In
another embodiment, the chromatography sample comprises a reduced
level of antibody fragments as compared to the first sample.
[0019] In one embodiment, the antibody, or antigen-binding portion
thereof, is an anti-TNF.alpha. antibody, or antigen-binding portion
thereof. In one embodiment, the antibody, or antigen-binding
portion thereof, has dissociates from human TNF.alpha. with a
K.sub.d of about 1.times.10.sup.-8 M or less and a K.sub.off rate
constant of 1.times.10.sup.-3 S.sup.-1 or less. In another
embodiment, the anti-TNF.alpha. antibody, or antigen-binding
portion thereof, comprises a light chain variable region (LCVR)
having a CDR1 domain comprising the amino acid sequence of SEQ ID
NO: 7, a CDR2 domain comprising the amino acid sequence of SEQ ID
NO: 5, and a CDR3 domain comprising the amino acid sequence of SEQ
ID NO: 3; and a heavy chain variable region (HCVR) having a CDR1
domain comprising the amino acid sequence of SEQ ID NO: 8, a CDR2
domain comprising the amino acid sequence of SEQ ID NO: 6, and a
CDR3 domain comprising the amino acid sequence of SEQ ID NO: 4. In
another embodiment, the anti-TNF.alpha. antibody, or
antigen-binding portion thereof, comprises a light chain variable
region comprising the amino acid sequence set forth in SEQ ID NO: 1
and a heavy chain variable region comprising the amino acid
sequence set forth in SEQ ID NO: 2. In another embodiment, the
anti-TNF.alpha. antibody, or antigen-binding portion thereof, is
adalimumab, or an antigen binding-portion thereof.
[0020] In another aspect, the present invention provides a low
acidic species (low AR) composition comprising an antibody, or
antigen-binding portion thereof, where the composition comprises
about 15% or less AR. In one aspect of this embodiment, the low AR
composition comprises about 14% or less AR, 13% or less AR, 12% or
less AR, 11% or less AR, 10% or less AR, 9% or less AR, 8% or less
AR, 7% or less AR, 6% or less AR, 5% or less AR, 4.5% or less AR,
4% or less AR, 3.5% or less AR, 3% or less AR, 2.5% or less AR, 2%
or less AR, 1.9% or less AR, 1.8% or less AR, 1.7% or less AR, 1.6%
or less AR, 1.5% or less AR, 1.4% or less AR, 1.3% or less AR, 1.2%
or less AR, 1.1% or less AR, 1% or less AR, 0.9% or less AR, 0.8%
or less AR, 0.7% or less AR, 0.6% or less AR, 0.5% or less AR, 0.4%
or less AR, 0.3% or less AR, 0.2% or less AR, 0.1% or less AR, or
0.0% AR, and ranges within one or more of the preceding. In one
aspect of this embodiment, the present invention provides a low AR
composition comprising an antibody, or antigen-binding portion
thereof, where the composition comprises about 0.0% to about 10%
AR, about 0.0% to about 5% AR, about 0.0% to about 4% AR, about
0.0% to about 3% AR, about 0.0% to about 2% AR, about 3% to about
5% AR, about 5% to about 8% AR, or about 8% to about 10% AR, or
about 10% to about 15% AR, and ranges within one or more of the
preceding.
[0021] In one embodiment, the low AR composition comprises a first
acidic species region (AR1) and a second acidic species region
(AR2). In one aspect of this embodiment, the low AR composition
comprises about 0.1% or less AR1 and about 3% or less AR2, or about
0.0% AR1 and about 1.4% or less AR2. In a related embodiment, the
low AR composition comprises about 15% or less AR1, 14% or less
AR1, 13% or less AR1, 12% or less AR1, 11% or less AR1, 10% or less
AR1, 9% or less AR1, 8% or less AR1, 7% or less AR1, 6% or less
AR1, 5% or less AR1, 4.5% or less AR1, 4% or less AR1, 3.5% or less
AR1, 3% or less AR1, 2.5% or less AR1, 2% or less AR1, 1.9% or less
AR1, 1.8% or less AR1, 1.7% or less AR1, 1.6% or less AR1, 1.5% or
less AR1, 1.4% or less AR1, 1.3% or less AR1, 1.2% or less AR1,
1.1% or less AR1, 1% or less AR1, 0.9% or less AR1, 0.8% or less
AR1, 0.7% or less AR1, 0.6% or less AR1, 0.5% or less AR1, 0.4% or
less AR1, 0.3% or less AR1, 0.2% or less AR1, 0.1% or less AR1, or
0.0% AR1, and ranges within one or more of the preceding. In one
aspect of this embodiment, the present invention provides a low AR
composition comprising an antibody, or antigen-binding portion
thereof, where the composition comprises about 0.0% to about 10%
AR1, about 0.0% to about 5% AR1, about 0.0% to about 4% AR1, about
0.0% to about 3% AR1, about 0.0% to about 2% AR1, about 3% to about
5% AR1, about 5% to about 8% AR1, or about 8% to about 10% AR1, or
about 10% to about 15% AR1, and ranges within one or more of the
preceding.
[0022] In one aspect of this embodiment, the low AR composition
comprises about 15% or less 3.5% or less AR2, 14% or less AR2, 13%
or less AR2, 12% or less AR2, 11% or less AR2, 10% or less AR2, 9%
or less AR2, 8% or less AR2, 7% or less AR2, 6% or less AR2, 5% or
less AR2, 4.5% or less AR2, 4% or less AR2, 3.5% or less AR2, 3% or
less AR2, 2.5% or less AR2, 2% or less AR2, about 1.9%, 1.8%, 1.7%,
1.6%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1.9% or less AR2, 1.8% or less
AR2, 1.7% or less AR2, 1.6% or less AR2, 1.5% or less AR2, 1.4% or
less AR2, 1.3% or less AR2, 1.2% or less AR2, 1.1% or less AR2, 1%
or less AR2, 0.9% or less AR2, 0.8% or less AR2, 0.7% or less AR2,
0.6% or less AR2, 0.5% or less AR2, 0.4% or less AR2, 0.3% or less
AR2, 0.2% or less AR2, 0.1% or less AR2, or 0.0% AR2, and ranges
within one or more of the preceding. In one aspect of this
embodiment, the present invention provides a low AR composition
comprising an antibody, or antigen-binding portion thereof, where
the composition comprises about 0.0% to about 10% AR2, about 0.0%
to about 5% AR2, about 0.0% to about 4% AR2, about 0.0% to about 3%
AR2, about 0.0% to about 2% AR2, about 3% to about 5% AR2, about 5%
to about 8% AR2, or about 8% to about 10% AR2, or about 10% to
about 15% AR2, and ranges within one or more of the preceding 0%,
0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2% or 0.1% AR2. In
another embodiment, the low AR composition comprises about 0%
AR2.
[0023] In another embodiment, the low AR composition, e.g., a low
AR composition of adalimumab, comprises about 1.4% or less AR. For
example, in one aspect of this embodiment, the low AR composition,
e.g., a low AR composition of adalimumab comprising about 1.4% or
less AR can comprise about 0.0% AR1 and about 1.4% or less AR2.
[0024] In one embodiment, the acidic species in the low AR
composition comprise one or more variants selected from the group
consisting of charge variants, structure variants, aggretation
variants and fragmentation variants. For example, in one
embodiment, the charge variants in the low AR composition are AR1
species and comprise, for example, deamidation variants, glycation
variants, afucosylation variants, MGO variants or citric acid
variants. In another embodiment, the structure variants in the low
AR composition are AR1 species and comprise, for example,
glycosylation variants or acetonation variants. In still another
embodiment, the fragmentation variants in the low AR composition
are AR1 and comprise, for example, Fab fragment variants,
C-terminal truncation variants or variants missing a heavy chain
variable domain.
[0025] In another embodiment, the acidic species in the low AR
composition are AR2 variants, and comprise, for example, charge
variants such as deamidation variants or glycation variants.
[0026] In another aspect, the present invention provides
compositions comprising an antibody, or antigen-binding portion
thereof, wherein the composition is substantially free of acidic
species such as process-related impurities, including, for example,
host cell proteins (HCPs), host cell nucleic acids, chromatographic
materials, and/or media components, as well as product related
impurities such as aggregates etc.
[0027] In one embodiment, the antibody, or antigen-binding portion
thereof, of the compositions disclosed herein is an anti-TNF.alpha.
antibody, or antigen-binding portion thereof. For example, in one
aspect of this embodiment, the anti-TNF.alpha. antibody, or
antigen-binding portion thereof, dissociates from human TNF.alpha.
with a K.sub.d of about 1.times.10.sup.-8 M or less and a K.sub.off
rate constant of 1.times.10.sup.-3 S.sup.-1 or less. In another
aspect of this embodiment, the anti-TNF.alpha. antibody, or
antigen-binding portion thereof, comprises a light chain variable
region (LCVR) having a CDR1 domain comprising the amino acid
sequence of SEQ ID NO: 7, a CDR2 domain comprising the amino acid
sequence of SEQ ID NO: 5, and a CDR3 domain comprising the amino
acid sequence of SEQ ID NO: 3; and a heavy chain variable region
(HCVR) having a CDR1 domain comprising the amino acid sequence of
SEQ ID NO: 8, a CDR2 domain comprising the amino acid sequence of
SEQ ID NO: 6, and a CDR3 domain comprising the amino acid sequence
of SEQ ID NO: 4.
[0028] In still another aspect of this embodiment, the
anti-TNF.alpha. antibody, or antigen-binding portion thereof,
comprises a light chain variable region comprising the amino acid
sequence set forth in SEQ ID NO: 1 and a heavy chain variable
region comprising the amino acid sequence set forth in SEQ ID NO:
2. In yet another aspect of this embodiment, the anti-TNF.alpha.
antibody, or antigen-binding portion thereof, is adalimumab, or an
antigen binding-portion thereof.
[0029] In one embodiment, the low AR composition of the invention
comprises adalimumab, and has a percentage of AR that is not the
same as the percentage of AR present in adalimumab formulated as
HUMIRA.RTM. as currently approved and described in the "Highlights
of HUMIRA.RTM. Prescribing Information" for HUMIRA.RTM.
(adalimumab) Injection (Revised January 2008), the contents of
which are hereby incorporated herein by reference.
[0030] In another embodiment, the low AR composition of the
invention comprises adalimumab, and has a percentage of AR that is
lower than the percentage of AR present in adalimumab formulated as
HUMIRA.RTM. as currently approved and described in the "Highlights
of HUMIRA.RTM. Prescribing Information" for HUMIRA.RTM.
(adalimumab) Injection (Revised January 2008), the contents of
which are hereby incorporated herein by reference.
[0031] In another embodiment, the present invention provides low AR
compositions comprising an anti-TNF.alpha. antibody, or
antigen-binding portion thereof, comprising a light chain variable
region (LCVR) having a CDR1 domain comprising the amino acid
sequence of SEQ ID NO: 7, a CDR2 domain comprising the amino acid
sequence of SEQ ID NO: 5, and a CDR3 domain comprising the amino
acid sequence of SEQ ID NO: 3; and a heavy chain variable region
(HCVR) having a CDR1 domain comprising the amino acid sequence of
SEQ ID NO: 8, a CDR2 domain comprising the amino acid sequence of
SEQ ID NO: 6, and a CDR3 domain comprising the amino acid sequence
of SEQ ID NO: 4, wherein the composition comprises less than about
10% AR. In one aspect of this embodiment, the anti-TNF.alpha.
antibody, or antigen-binding portion thereof, comprises a light
chain variable region comprising the amino acid sequence set forth
in SEQ ID NO: 1 and a heavy chain variable region comprising the
amino acid sequence set forth in SEQ ID NO: 2, wherein the
composition comprises less than about 10% AR. In another aspect of
this embodiment, the anti-TNF.alpha. antibody, or antigen-binding
portion thereof, is adalimumab, or an antigen binding-portion
thereof, and the composition comprises less than about 10% AR. In
one aspect of this embodiment, the low AR composition comprising an
anti-TNF.alpha. antibody, or antigen-binding portion thereof,
comprises about 0.1% or less AR1 and about 3% or less AR2, or about
0.0% AR1 and about 1.4% or less AR2.
[0032] In one embodiment, the acidic species in the low AR
composition comprising an antibody, or antigen-binding portion
thereof (e.g., an anti-TNF.alpha. antibody, or antigen binding
portion thereof, such as adalimumab) comprise one or more variants
selected from the group consisting of charge variants, structure
variants and fragmentation variants. For example, in one aspect of
this embodiment, the charge variants in the low AR composition are
AR1 species and comprise, for example, deamidation variants,
glycation variants, afucosylation variants, methylglyoxal (MGO)
variants or citric acid variants. For example, when the low AR
composition comprises adalimumab, the deamidation variants can
result from deamidation occurring at asparagine residues comprising
Asn393 and Asn329 of adalimumab and at glutamine residues
comprising Gln3 and Gln6. In another aspect of this embodiment,
when the low AR composition comprises adalimumab, the glycation
variants can result from glycation occurring at Lys98 and Lys151 of
adalimumab.
[0033] In another aspect of this embodiment, the structure variants
in the low AR composition comprising an antibody, or
antigen-binding portion thereof (e.g., an anti-TNF.alpha. antibody,
or antigen binding portion thereof, such as adalimumab) are AR1
species and comprise, for example, glycosylation variants or
acetonation variants.
[0034] In still another aspect of this embodiment, the
fragmentation variants in the low AR composition comprising an
antibody, or antigen-binding portion thereof (e.g., an
anti-TNF.alpha. antibody, or antigen binding portion thereof, such
as adalimumab), are AR1 species and comprise, for example, Fab
fragment variants, C-terminal truncation variants or variants
missing a heavy chain variable domain.
[0035] In another embodiment, the acidic species in the low AR
composition comprising an antibody, or antigen-binding portion
thereof (e.g., an anti-TNF.alpha. antibody, or antigen binding
portion thereof, such as adalimumab), are AR2 species, and comprise
charge variants, such as deamidation variants or glycation
variants. For example, when the low AR composition comprises
adalimumab, the deamidation variants can result from deamidation
occurring at asparagine residues comprising Asn393 and Asn329 of
adalimumab and at glutamine residues comprising Gln3 and Gln6. In
another aspect of this embodiment, when the low AR composition
comprises adalimumab, the glycation variants result from glycation
occurring at Lys98 and Lys151 of adalimumab.
[0036] In one embodiment, the percent of acidic species in a low AR
composition is determined using ion exchange chromatography, for
example WCX-10 HPLC. In another aspect of this embodiment, the
percent acidic species in a low AR composition is determined using
isoelectric focusing (IEF).
[0037] In one embodiment, the low AR compositions of the invention
comprise product preparation-derived acidic species. For example,
in one aspect of this embodiment, the acidic species are cell
culture-derived acidic species. In another aspect of this
embodiment, the acidic species of the low AR compositions are
storage-derived acidic species which are primarily generated when
stored under process, intermediate or shelf storage conditions
prior to use.
[0038] In still another embodiment, the invention provides low AR
compositions that further comprise a pharmaceutically acceptable
carrier.
[0039] In another aspect, the present invention provides methods
for treating a subject having a disorder in which TNF.alpha. is
detrimental, by administering to the subject a low AR composition
of the invention, e.g., a low AR adalimumab composition, thereby
treating the subject having a disorder in which TNF.alpha. is
detrimental. In one aspect of this embodiment, the disorder in
which TNF.alpha. is detrimental is selected from the group
consisting of rheumatoid arthritis (RA), psoriasis, psoriatic
arthritis, ankylosing spondylitis, juvenile idiopathic arthritis
(JIA), ulcerative colitis, Crohn's Disease, active axial
spondyloarthritis (active axSpA) and non-radiographic axial
spondyloarthritis (nr-axSpA).
[0040] The present invention is further illustrated by the
following detailed description and claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0041] FIG. 1 depicts (a) comparison of a desired and an undesired
displacement chromatogram for Adalimumab on Poros XS resin using
Expell SP1.TM.; (b) charge variants distribution in eluate
fractions derived from the undesired displacement chromatography
process for Adalimumab (Poros XS resin & Expell SP1.TM.)
[0042] FIG. 2 depicts CEX-HPLC chromatograms of Expell
SP1.TM.-displaced Adalimumab sample fractions.
[0043] FIG. 3 depicts the separation of Adalimumab charge variants
by Poros XS displacement chromatography using Expell SP1.TM..
[0044] FIG. 4 depicts the reduction of acidic species level in
Adalimumab by Poros XS displacement chromatography using Expell
SP1.TM..
[0045] FIG. 5 depicts the effect of Expell SP1.TM. concentration on
acidic species reduction in Adalimumab by Poros XS displacement
chromatography.
[0046] FIG. 6 depicts the effect of pH on acidic species reduction
in Adalimumab by Poros XS displacement chromatography using Expell
SP1.TM..
[0047] FIG. 7 depicts the reduction of acidic species level in
Adalimumab by Poros XS two-step displacement chromatography using
Expell SP1.TM..
[0048] FIG. 8 depicts the separation of Adalimumab size variants by
Poros XS displacement chromatography using Expell SP1.TM..
[0049] FIG. 9 depicts the separation of HCP in Adalimumab by Poros
XS displacement chromatography using Expell SP1.TM..
[0050] FIG. 10 depicts the separation of Adalimumab charge variants
by Poros XS displacement chromatography using protamine
sulfate.
[0051] FIG. 11 depicts the reduction of acidic species in
Adalimumab by Poros XS displacement chromatography using protamine
sulfate.
[0052] FIG. 12 depicts the effect of protamine sulfate
concentration on acidic species reduction in Adalimumab by Poros XS
displacement chromatography.
[0053] FIG. 13 depicts the effect of pH on acidic species reduction
in Adalimumab by Poros XS displacement chromatography using
protamine sulfate.
[0054] FIG. 14 depicts the reduction of acidic species in
Adalimumab by Poros XS two-step displacement chromatography using
protamine sulfate.
[0055] FIG. 15 depicts the reduction of acidic species in
Adalimumab on Poros XS using protamine sulfate linear gradient
displacement chromatography.
[0056] FIG. 16 depicts the separation of Adalimumab size variants
by Poros XS displacement chromatography using protamine
sulfate.
[0057] FIG. 17 depicts the separation of mAb X charge variants by
Poros XS displacement chromatography using Expell SP1.TM..
[0058] FIG. 18 depicts the reduction of acidic species in mAb X by
Poros XS displacement chromatography using Expell SP1.TM..
[0059] FIG. 19 depicts the effect of Expell SP1.TM. concentration
on acidic species reduction in mAb X by Poros XS displacement
chromatography.
[0060] FIG. 20 depicts the reduction of acidic species in mAb X by
Poros XS two-step displacement chromatography using Expell
SP1.TM..
[0061] FIG. 21 depicts the separation of mAb X charge variants by
Poros XS displacement chromatography using protamine sulfate.
[0062] FIG. 22 depicts the reduction of acidic species in mAb X by
Poros XS displacement chromatography using protamine sulfate.
[0063] FIG. 23 depicts the effect of protamine sulfate
concentration on acidic species reduction in mAb X by Poros XS
displacement chromatography.
[0064] FIG. 24 depicts the reduction of acidic species in mAb X by
Poros XS two-step displacement chromatography using protamine
sulfate.
[0065] FIG. 25 depicts the separation of mAb X size variants by
Poros XS displacement chromatography using protamine sulfate.
[0066] FIG. 26 depicts the separation of mAb Y charge variants by
Poros XS displacement chromatography using Expell SP1.TM..
[0067] FIG. 27 depicts the reduction of acidic species in mAb Y by
Poros XS displacement chromatography using Expell SP1.TM..
[0068] FIG. 28 depicts the separation of Adalimumab charge variants
by Capto MMC displacement chromatography using protamine
sulfate.
[0069] FIG. 29 depicts the reduction of acidic species in
Adalimumab by Capto MMC displacement chromatography using protamine
sulfate.
[0070] FIG. 30 depicts the separation of mAb X charge variants by
Capto MMC displacement chromatography using protamine sulfate.
[0071] FIG. 31 depicts the reduction of acidic species in mAb X by
Capto MMC displacement chromatography using protamine sulfate.
[0072] FIG. 32 depicts the effect of pH on acidic species reduction
in mAb X by Capto MMC displacement chromatography.
[0073] FIG. 33 depicts the AR Growth at 25.degree. C. of low and
high AR containing samples.
DETAILED DESCRIPTION OF THE INVENTION
[0074] The present invention is based on the identification and
optimization of displacement chromatography technologies for
protein production, e.g., production of proteins, e.g., antibodies
or antigen-binding portions thereof, e.g., adalimumab, resulting in
compositions that comprise low percentages of protein charge
variants, e.g., acidic species (AR), and basic species, and/or low
levels of product- and process-related impurities (e.g.,
aggregates, fragments, host cell proteins and media components). In
one embodiment, the displacement chromatography methods of the
invention can surprisingly be used in large-scale protein
purification processes due to superior resolution of closely
related species using practically relevant chromatography resins
and conditions. In one embodiment, the displacement chromatography
methods of the invention can surprisingly be used in large-scale
protein purification processes due to reduced buffer volume for a
given separation, thereby providing improved process
efficiency.
[0075] The compositions of the present invention exhibit increased
therapeutic efficacy when administered to a subject. For example,
compositions comprising anti-TNF.alpha. antibodies, or antigen
binding portions thereof, comprising low AR are capable of
increased therapeutic efficacy in the treatment and prevention of a
disorder in which TNF.alpha. is detrimental, e.g., rheumatoid
arthritis (RA), juvenile idiopathic arthritis (JIA), psoriasis,
psoriatic arthritis, ankylosing spondylitis, Crohn's disease, and
ulcerative colitis. Accordingly, the instant invention provides
compositions comprising proteins that comprise low AR and/or low
levels of product- and process-related impurities, and methods for
producing and using the same.
[0076] In one embodiment, the low AR compositions of the invention
comprise about 15% or less AR, 14% or less AR, 13% or less AR, 12%
or less AR, 11% or less AR, 10% or less AR, 9% or less AR, 8% or
less AR, 7% or less AR, 6% or less AR, 5% or less AR, 4.5% or less
AR, 4% or less AR, 3.5% or less AR, 3% or less AR, 2.5% or less AR,
2% or less AR, 1.9% or less AR, 1.8% or less AR, 1.7% or less AR,
1.6% or less AR, 1.5% or less AR, 1.4% or less AR, 1.3% or less AR,
1.2% or less AR, 1.1% or less AR, 1% or less AR, 0.9% or less AR,
0.8% or less AR, 0.7% or less AR, 0.6% or less AR, 0.5% or less AR,
0.4% or less AR, 0.3% or less AR, 0.2% or less AR, 0.1% or less AR,
or 0.0% AR, and ranges within one or more of the preceding. In one
aspect of this embodiment, the low AR compositions of the invention
comprise about 0.0% to about 10% AR, about 0.0% to about 5% AR,
about 0.0% to about 4% AR, about 0.0% to about 3% AR, about 0.0% to
about 2% AR, about 3% to about 5% AR, about 5% to about 8% AR, or
about 8% to about 10% AR, or about 10% to about 15% AR, and ranges
within one or more of the preceding.
[0077] In another embodiment, the low AR composition comprises a
first acidic species region (AR1) and a second acidic species
region (AR2). In one aspect of this embodiment, the low AR
composition comprises about 0.1% or less AR1 and about 3% or less
AR2. In another aspect of this embodiment, the low AR composition
comprises about 0.0% AR1 and about 1.4% or less AR2.
[0078] In another aspect of this embodiment, the low AR composition
comprises about 15% or less AR1, 14% or less AR1, 13% or less AR1,
12% or less AR1, 11% or less AR1, 10% or less AR1, 9% or less AR1,
8% or less AR1, 7% or less AR1, 6% or less AR1, 5% or less AR1,
4.5% or less AR1, 4% or less AR1, 3.5% or less AR1, 3% or less AR1,
2.5% or less AR1, 2% or less AR1, 1.9% or less AR1, 1.8% or less
AR1, 1.7% or less AR1, 1.6% or less AR1, 1.5% or less AR1, 1.4% or
less AR1, 1.3% or less AR1, 1.2% or less AR1, 1.1% or less AR1, 1%
or less AR1, 0.9% or less AR1, 0.8% or less AR1, 0.7% or less AR1,
0.6% or less AR1, 0.5% or less AR1, 0.4% or less AR1 or less, 0.3%
or less AR1 or less, 0.2% or less AR1 or less, 0.1% or less AR1, or
0.0% AR1, and ranges within one or more of the preceding. In one
aspect of this embodiment, the low AR compositions of the invention
comprise about 0.0% to about 10% AR1, about 0.0% to about 5% AR1,
about 0.0% to about 4% AR1, about 0.0% to about 3% AR1, about 0.0%
to about 2% AR1, about 3% to about 5% AR1, about 5% to about 8%
AR1, or about 8% to about 10% AR1, or about 10% to about 15% AR1,
and ranges within one or more of the preceding.
[0079] In yet another aspect of this embodiment, the low AR
composition comprises about 15% or less AR2, 14% or less AR2, 13%
or less AR2, 12% or less AR2, 11% or less AR2, 10% or less AR2, 9%
or less AR2, 8% or less AR2, 7% or less AR2, 6% or less AR2, 5% or
less AR2, 4.5% or less AR2, 4% or less AR2, 3.5% or less AR2, 3% or
less AR2, 2.5% or less AR2, 2% or less AR2, 1.9% or less AR2, 1.8%
or less AR2, 1.7% or less AR2, 1.6% or less AR2, 1.5% or less AR2,
1.4% or less AR2, 1.3% or less AR2, 1.2% or less AR2, 1.1% or less
AR2, 1% or less AR2, 0.9% or less AR2, 0.8% or less AR2, 0.7% or
less AR2, 0.6% or less AR2, 0.5% or less AR2, 0.4% or less AR2,
0.3% or less AR2, 0.2% or less AR2, 0.1% or less AR2, or 0.0% AR2,
and ranges within one or more of the preceding. In one aspect of
this embodiment, the low AR compositions of the invention comprise
about 0.0% to about 10% AR2, about 0.0% to about 5% AR2, about 0.0%
to about 4% AR2, about 0.0% to about 3% AR2, about 0.0% to about 2%
AR2, about 3% to about 5% AR2, about 5% to about 8% AR2, or about
8% to about 10% AR2, or about 10% to about 15% AR2, and ranges
within one or more of the preceding.
[0080] In another embodiment, the low AR composition, e.g., a low
AR composition of adalimumab, comprises about 1.4% or less AR. For
example, in one aspect of this embodiment, the low AR composition,
e.g., a low AR composition of adalimumab comprising about 1.4% or
less AR comprises about 0.0% AR1 and about 1.4% or less AR2.
[0081] In one embodiment, the protein is an antibody or antigen
binding portion thereof, such as the adalimumab antibody, or an
antigen binding portion thereof.
I. Definitions
[0082] In order that the present invention may be more readily
understood, certain terms are first defined.
[0083] As used herein, the terms "acidic species", "acidic region",
and "AR," refer to the variants of a protein, e.g., an antibody or
antigen-binding portion thereof, which are characterized by an
overall acidic charge. For example, in monoclonal antibody (mAb)
preparations, such acidic species can be detected by various
methods, such as ion exchange, for example, WCX-10 HPLC (a weak
cation exchange chromatography), or IEF (isoelectric focusing).
Acidic species of an antibody may include charge variants,
structure variants, and/or fragmentation variants. Exemplary charge
variants include, but are not limited to, deamidation variants,
afucosylation variants, methylglyoxal (MGO) variants, glycation
variants, and citric acid variants. Exemplary structure variants
include, but are not limited to, glycosylation variants and
acetonation variants. Exemplary fragmentation variants include any
truncated protein species from the target molecule due to
dissociation of peptide chain, enzymatic and/or chemical
modifications, including, but not limited to, Fc and Fab fragments,
fragments missing a Fab, fragments missing a heavy chain variable
domain, C-terminal truncation variants, variants with excision of
N-terminal Asp in the light chain, and variants having N-terminal
truncation of the light chain. Other acidic species variants
include variants containing unpaired disulfides, host cell
proteins, and host cell nucleic acids, chromatographic materials,
and media components.
[0084] In certain embodiments, a protein composition can comprise
more than one type of acidic species variant. For example, but not
by way of limitation, the total acidic species can be divided based
on chromatographic retention time of the peaks appearing, for
example, in a WCX-10 Weak Cation Exchange HPLC of the protein
preparation. FIG. 2 depicts a non-limiting example of such a
division wherein the total acidic species associated with the
expression of adalimumab is divided into a first acidic species
region (AR1) and a second acidic species region (AR2).
[0085] AR1 can comprise, for example, charge variants such as
deamidation variants, MGO modified species, glycation variants, and
citric acid variants, structural variants such as glycosylation
variants and acetonation variants, and/or fragmentation variants.
In another embodiment, AR2 can comprise, for example, charge
variants such as glycation variants and deamidation variants.
[0086] With respect, in particular, to adalimumab (and antibodies
sharing certain structural characteristics of adalimumab, e.g., one
or more CDR and/or heavy and light chain variable regions of
adalimumab), AR1 charge variants can comprise, but are not limited
to, deamidation variants, glycation variants, afucosylation
variants, MGO variants or citric acid variants. In one embodiment,
deamidation variants result from deamidation occurring at
asparagine residues comprising Asn393 and Asn329 and at glutamine
residues comprising Gln3 and Gln6. In another embodiment, the
glycation variants result from glycation occurring at Lys98 and
Lys151. AR1 structure variants can comprise, but are not limited
to, glycosylation variants or acetonation variants.
[0087] AR1 fragmentation variants can comprise Fc and Fab
fragments, fragments missing a Fab, fragments missing a heavy chain
variable domain, C-terminal truncation variants, variants with
excision of N-terminal Asp in the light chain, and variants having
N-terminal truncation of the light chain.
[0088] AR2 charge variants can comprise, but are not limited to,
deamidation variants or glycation variants, wherein the deamidation
variants can result from deamidation occurring at asparagine
residues comprising Asn393 and Asn329 and at glutamine residues
comprising Gln3 and Gln6, and the glycation variants can result
from glycation occurring at Lys98 and Lys151.
[0089] The term "acidic species" does not include process-related
impurities. The term "process-related impurity," as used herein,
refers to impurities that are present in a composition comprising a
protein but are not derived from the protein itself.
Process-related impurities include, but are not limited to, host
cell proteins (HCPs), host cell nucleic acids, chromatographic
materials, and media components. A "low process-related impurity
composition," as used herein, refers to a composition comprising
reduced levels of process-related impurities as compared to a
composition wherein the impurities were not reduced. For example, a
low process-related impurity composition may contain about 10%, 9%,
8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or less of process-related
impurities. In one embodiment, a low process-related impurity
composition is free of process-related impurities or is
substantially free of process-related impurities.
[0090] The acidic species may be the result of product preparation
(referred to herein as "preparation-derived acidic species"), or
the result of storage (referred to herein as "storage-derived
acidic species"). Preparation-derived acidic species are acidic
species that are formed during the preparation (upstream and/or
downstream processing) of the protein, e.g., the antibody or
antigen-binding portion thereof. For example, preparation-derived
acidic species can be formed during cell culture ("cell
culture-derived acidic species"). Storage-derived acidic species
are acidic species that may or may not be present in the population
of proteins directly after preparation, but are formed or generated
while the sample is being stored. The type and amount of
storage-derived acidic species can vary based on the formulation of
the sample. Formation of storage-derived acidic species can be
partially or completely inhibited when the preparation is stored
under particular conditions. For example, an aqueous formulation
can be stored at a particular temperature to partially or
completely inhibit AR formation. For example, formation or
storage-derived AR can be partially inhibited in an aqueous
formulation stored at between about 2.degree. C. and 8.degree. C.,
and completely inhibited when stored at -80.degree. C. In addition,
a low AR composition can be lyophilized or freeze-dried to
partially or completely inhibit the formation of storage-derived
AR.
[0091] The term "low acidic species composition," or "low AR
composition," as used herein, refers to a composition comprising an
antibody or antigen-binding portion thereof, wherein the
composition contains less than about 15% acidic species. As used
herein, the percent AR in the low AR composition refers to the
weight of the acidic species in a sample in relation to the weight
of the total antibodies contained in the sample. For example, the
percent AR can be calculated using weak cation exchange
chromatography such as WCX-10, as described herein and also in
Example 1 of U.S. Provisional Patent Application 61/893,068,
entitled "Low Acidic Species Compositions and Methods for Producing
the Same", Attorney Docket Number 117813-73901, filed on Oct. 18,
2013, the entire contents of which are expressly incorporated
herein by reference.
[0092] In one embodiment, a low AR composition of the invention may
comprise about 15% or less AR, 14% or less AR, 13% or less AR, 12%
or less AR, 11% or less AR, 10% or less AR, 9% or less AR, 8% or
less AR, 7% or less AR, 6% or less AR, 5% or less AR, 4.5% or less
AR, 4% or less AR, 3.5% or less AR, 3% or less AR, 2.5% or less AR,
2% or less AR, 1.9% or less AR, 1.8% or less AR, 1.7% or less AR,
1.6% or less AR, 1.5% or less AR, 1.4% or less AR, 1.3% or less AR,
1.2% or less AR, 1.1% or less AR, 1% or less AR, 0.9% or less AR,
0.8% or less AR, 0.7% or less AR, 0.6% or less AR, 0.5% or less AR,
0.4% or less AR, 0.3% or less AR, 0.2% or less AR, 0.1% or less AR,
or 0.0% AR, and ranges within one or more of the preceding. A low
AR composition of the invention may also comprise about 0.0% to
about 10% AR, about 0.0% to about 5% AR, about 0.0% to about 4% AR,
about 0.0% to about 3% AR, about 0.0% to about 2% AR, about 3% to
about 5% AR, about 5% to about 8% AR, or about 8% to about 10% AR,
or about 10% to about 15% AR, and ranges within one or more of the
preceding.
[0093] A low AR composition of the invention may comprise about 15%
or less AR1, 14% or less AR1, 13% or less AR1, 12% or less AR1, 11%
or less AR1, 10% or less AR1, 9% or less AR1, 8% or less AR1, 7% or
less AR1, 6% or less AR1, 5% or less AR1, 4.5% or less AR1, 4% or
less AR1, 3.5% or less AR1, 3% or less AR1, 2.5% or less AR1, 2% or
less AR1, 1.9% or less AR1, 1.8% or less AR1, 1.7% or less AR1,
1.6% or less AR1, 1.5% or less AR1, 1.4% or less AR1, 1.3% or less
AR1, 1.2% or less AR1, 1.1% or less AR1, 1% or less AR1, 0.9% or
less AR1, 0.8% or less AR1, 0.7% or less AR1, 0.6% or less AR1,
0.5% or less AR1, 0.4% or less AR1, 0.3% or less AR1, 0.2% or less
AR1, 0.1% or less AR1, or 0.0% AR1, and ranges within one or more
of the preceding. A low AR composition of the invention may also
comprise about 0.0% to about 10% AR1, about 0.0% to about 5% AR1,
about 0.0% to about 4% AR1, about 0.0% to about 3% AR1, about 0.0%
to about 2% AR1, about 3% to about 5% AR1, about 5% to about 8%
AR1, or about 8% to about 10% AR1, or about 10% to about 15% AR1,
and ranges within one or more of the preceding.
[0094] A low AR composition of the invention may also comprise
about 15% or less AR2, 14% or less AR2, 13% or less AR2, 12% or
less AR2, 11% or less AR2, 10% or less AR2, 9% or less AR2, 8% or
less AR2, 7% or less AR2, 6% or less AR2, 5% or less AR2, 4.5% or
less AR2, 4% or less AR2, 3.5% or less AR2, 3% or less AR2, 2.5% or
less AR2, 2% or less AR2, 1.9% or less AR2, 1.8% or less AR2, 1.7%
or less AR2, 1.6% or less AR2, 1.5% or less AR2, 1.4% or less AR2,
1.3% or less AR2, 1.2% or less AR2, 1.1% or less AR2, 1% or less
AR2, 0.9% or less AR2, 0.8% or less AR2, 0.7% or less AR2, 0.6% or
less AR2, 0.5% or less AR2, 0.4% or less AR2, 0.3% or less AR2,
0.2% or less AR2, 0.1% or less AR2, or 0.0% AR2, and ranges within
one or more of the preceding. A low AR composition of the invention
may also comprise about 0.0% to about 10% AR2, about 0.0% to about
5% AR2, about 0.0% to about 4% AR2, about 0.0% to about 3% AR2,
about 0.0% to about 2% AR2, about 3% to about 5% AR2, about 5% to
about 8% AR2, or about 8% to about 10% AR2, or about 10% to about
15% AR2, and ranges within one or more of the preceding.
[0095] In one embodiment, a low AR composition comprises between
about 0.0% and about 3% AR1. In another embodiment, a low AR
composition comprises about between about 0.0% and about 3% AR2. In
a preferred embodiment, a low acidic species composition comprises
about 3% or less AR2.
[0096] In another embodiment, the low AR composition comprises
about 1.4% or less AR. For example, in one embodiment, the
composition comprises about 1.4% AR2 and about 0.0% AR1.
[0097] In one embodiment, a low AR composition of the invention may
comprise about 15% or less, 14% or less, 13% or less, 12% or less,
11% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or
less, 5% or less, 4.5% or less, 4% or less, 3.5% or less, 3% or
less, 2.5% or less, 2% or less, 1.9% or less, 1.8% or less, 1.7% or
less, 1.6% or less, 1.5% or less, 1.4% or less, 1.3% or less, 1.2%
or less, 1.1% or less, 1% or less, 0.9% or less, 0.8% or less, 0.7%
or less, 0.6% or less, 0.5% or less, 0.4% or less, 0.3% or less,
0.2% or less, 0.1% or less, or 0.0% of one or more of a deamidation
variant, an afucosylation variant, an MGO variant, a glycation
variant, a citric acid variant, a glycosylation variant, an
acetonation variant, or a fragmentation variant, and ranges within
one or more of the preceding. In one aspect of this embodiment, a
low AR composition of the invention may also comprise about 0.0% to
about 10%, about 0.0% to about 5%, about 0.0% to about 4%, about
0.0% to about 3%, about 0.0% to about 2%, about 3% to about 5%,
about 5% to about 8%, or about 8% to about 10%, or about 10% to
about 15%, of one or more of a deamidation variant, an
afucosylation variant, an MGO variant, a glycation variant, a
citric acid variant, a glycosylation variant, an acetonation
variant, or a fragmentation variant, and ranges within one or more
of the preceding. For example, a low AR composition of the
invention may comprise less than 15% of a deamidation variant,
while each of the other acidic variants, alone or in combination,
are at a percentage that is greater than 15%.
[0098] The term "non-low acidic species composition," as used
herein, refers to a composition comprising an antibody or
antigen-binding portion thereof, which contains more than about 13%
acidic species. For example, a non-low acidic species composition
may contain about 16% or more, 17% or more, 18% or more, 19% or
more, 20% or more, 21% or more, 22% or more, 23% or more, 24% or
more, or 25% or more acidic species. In one embodiment, a non-low
acidic species composition can comprise about 5% or more, 6% or
more, 7% or more, 8% or more, 9% or more, 10% or more, 11% or more,
12% or more, 13% or more, 14% or more, 15% or more, 16% or more,
17% or more, 18% or more, 19% or more, 20% or more, 21% or more,
22% or more, 23% or more, 24% or more, or 25% or more of AR1. In
another embodiment, a non-low acidic species composition can
comprise about 10% or more, 11% or more, 12% or more, 13% or more,
14% or more, 15% or more, 16% or more, 17% or more, 18% or more,
19% or more, 20% or more, 21% or more, 22% or more, 23% or more,
24% or more, or 25% or more of AR2, and ranges within one or more
of the preceding.
[0099] In one embodiment, a low AR composition has improved
biological and functional properties, including increased efficacy
in the treatment or prevention of a disorder in a subject, e.g., a
disorder in which TNF.alpha. activity is detrimental, as compared
to a non-low acidic species composition. In one embodiment, the low
AR composition comprises an anti-TNF.alpha. antibody, or
antigen-binding portion thereof, such as adalimumab or a fragment
thereof. For example, in one embodiment, a low AR composition
comprising an antibody, or antigen-binding portion thereof,
exhibits increased cartilage penetration, decreased bone erosion,
and/or reduced cartilage destruction, as compared to a non-low
acidic species composition comprising the same antibody or antigen
binding portion thereof, when administered to a subject suffering
from a disorder in which TNF.alpha. activity is detrimental.
[0100] As used herein, the term "increased cartilage penetration"
refers to increased penetration of cartilage in vivo by a low AR
composition as compared to a non-low AR composition comprising the
same antibody or antigen binding portion thereof.
[0101] As used herein, the term "reduced cartilage destruction"
refers to measurable decrease in destruction of cartilage tissue in
vivo by a low AR composition as compared to a non-low AR
composition comprising the same antibody or antigen binding portion
thereof. As used herein, the term "decreased bone erosion" refers
to measurable decrease, in vivo, of the erosion of bone tissue by a
low AR composition as compared to a non-low acidic species
composition comprising the same antibody or antigen binding portion
thereof. For example, an in vivo model of a disease or disorder in
which TNF.alpha. activity is detrimental, e.g., a mouse model of
arthritis, can be used to measure cartilage penetration, bone
erosion, and/or cartilage destruction by a composition comprising
an anti-TNF.alpha. antibody or antigen binding portion thereof. One
non-limiting example of an art-recognized mouse model of arthritis
is the human TNF transgenic 197 mouse model of arthritis
(TNF-Tg197) (see Keffer, J. et al., EMBO J (1991) 10:4025-4031, the
contents of which are expressly incorporated herein by reference,
for further description of the TNF-Tg197 model of arthritis).
[0102] In another embodiment, a low AR composition comprising an
antibody, or antigen-binding portion thereof, exhibits increased
protection against the development of arthritis, as measured by
arthritic scores, and/or histopathology scores as compared to a
non-low acidic species composition when administered to an animal
model of arthritis, e.g., the TNF-Tg197 model of arthritis. As used
herein, "arthritic scores" refer to signs and symptoms of arthritis
in an animal model of arthritis. As used herein, "histopathology
scores" refer to radiologic damage involving cartilage and bone as
well as local inflammation.
[0103] In another embodiment, a low AR composition comprising an
antibody, or antigen-binding portion thereof, exhibits reduced
synovial proliferation, reduced cell infiltration, reduced
chondrocyte death, and/or reduced proteoglycan loss as compared to
a non-low acidic species composition. In another embodiment, a low
AR composition comprising an anti-TNF.alpha. antibody, or
antigen-binding portion thereof, exhibits increased TNF.alpha.
affinity as compared to a non-low acidic species composition.
[0104] As used herein, the term "a disorder in which TNF.alpha.
activity is detrimental" is intended to include diseases and other
disorders in which the presence of TNF.alpha. in a subject
suffering from the disorder has been shown to be or is suspected of
being either responsible for the pathophysiology of the disorder or
a factor that contributes to a worsening of the disorder.
Accordingly, a disorder in which TNF.alpha. activity is detrimental
is a disorder in which inhibition of TNF.alpha. activity is
expected to alleviate the symptoms and/or progression of the
disorder. Such disorders may be evidenced, for example, by an
increase in the concentration of TNF.alpha. in a biological fluid
of a subject suffering from the disorder (e.g., an increase in the
concentration of TNF.alpha. in serum, plasma, or synovial fluid of
the subject), which can be detected, for example, using an
anti-TNF.alpha. antibody as described above. There are numerous
examples of disorders in which TNF.alpha. activity is detrimental.
In one embodiment, the disorder in which TNF.alpha. activity is
detrimental is an autoimmune disorder. In one embodiment, the
autoimmune disorder is selected from the group consisting of
rheumatoid arthritis, juvenile idiopathic arthritis, rheumatoid
spondylitis, ankylosing spondylitis, psoriasis, osteoarthritis,
gouty arthritis, an allergy, multiple sclerosis, psoriatic
arthritis, autoimmune diabetes, autoimmune uveitis, nephrotic
syndrome, juvenile rheumatoid arthritis, Crohn's disease and
ulcerative colitis. Disorders in which TNF.alpha. activity is
detrimental are set forth in U.S. Pat. No. 6,090,382 and also in
the "Highlights of HUMIRA.RTM. Prescribing Information" for
HUMIRA.RTM. (adalimumab) Injection (Revised January 2008) the
entire contents of which are hereby incorporated herein by
reference. The use of TNF.alpha. antibodies and antibody portions
obtained using methods of the invention for the treatment of
specific disorders is discussed in further detail below.
[0105] The term "antibody" includes an immunoglobulin molecule
comprised of four polypeptide chains, two heavy (H) chains and two
light (L) chains inter-connected by disulfide bonds. Each heavy
chain is comprised of a heavy chain variable region (abbreviated
herein as HCVR or VH) and a heavy chain constant region (CH). The
heavy chain constant region is comprised of three domains, CH1, CH2
and CH3. Each light chain is comprised of a light chain variable
region (abbreviated herein as LCVR or VL) and a light chain
constant region. The light chain constant region is comprised of
one domain, CL. The VH and VL regions can be further subdivided
into regions of hypervariability, termed complementarity
determining regions (CDRs), interspersed with regions that are more
conserved, termed framework regions (FR). Each VH and VL is
composed of three CDRs and four FRs, arranged from amino-terminus
to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2,
FR3, CDR3, FR4.
[0106] The term "antigen-binding portion" of an antibody (or
"antibody portion") includes fragments of an antibody that retain
the ability to specifically bind to an antigen (e.g., in the case
of adalimumab, hTNF.alpha.). It has been shown that the
antigen-binding function of an antibody can be performed by
fragments of a full-length antibody. Examples of binding fragments
encompassed within the term "antigen-binding portion" of an
antibody include (i) a Fab fragment, a monovalent fragment
comprising the VL, VH, CL and CH1 domains; (ii) a F(ab')2 fragment,
a bivalent fragment comprising two Fab fragments linked by a
disulfide bridge at the hinge region; (iii) a Fd fragment
comprising the VH and CH1 domains; (iv) a Fv fragment comprising
the VL and VH domains of a single arm of an antibody, (v) a dAb
fragment (Ward et al., (1989) Nature 341:544-546, the entire
teaching of which is incorporated herein by reference), which
comprises a VH domain; and (vi) an isolated complementarity
determining region (CDR). Furthermore, although the two domains of
the Fv fragment, VL and VH, are coded for by separate genes, they
can be joined, using recombinant methods, by a synthetic linker
that enables them to be made as a single protein chain in which the
VL and VH regions pair to form monovalent molecules (known as
single chain Fv (scFv); see, e.g., Bird et al. (1988) Science
242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA
85:5879-5883, the entire teachings of which are incorporated herein
by reference). Such single chain antibodies are also intended to be
encompassed within the term "antigen-binding portion" of an
antibody. Other forms of single chain antibodies, such as diabodies
are also encompassed. Diabodies are bivalent, bispecific antibodies
in which VH and VL domains are expressed on a single polypeptide
chain, but using a linker that is too short to allow for pairing
between the two domains on the same chain, thereby forcing the
domains to pair with complementary domains of another chain and
creating two antigen binding sites (see, e.g., Holliger, P., et al.
(1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R. J., et
al. (1994) Structure 2:1121-1123, the entire teachings of which are
incorporated herein by reference). Still further, an antibody or
antigen-binding portion thereof may be part of a larger
immunoadhesion molecule, formed by covalent or non-covalent
association of the antibody or antibody portion with one or more
other proteins or peptides. Examples of such immunoadhesion
molecules include use of the streptavidin core region to make a
tetrameric scFv molecule (Kipriyanov, S. M., et al. (1995) Human
Antibodies and Hybridomas 6:93-101, the entire teaching of which is
incorporated herein by reference) and use of a cysteine residue, a
marker peptide and a C-terminal polyhistidine tag to make bivalent
and biotinylated scFv molecules (Kipriyanov, S. M., et al. (1994)
Mol. Immunol. 31:1047-1058, the entire teaching of which is
incorporated herein by reference). Antibody portions, such as Fab
and F(ab')2 fragments, can be prepared from whole antibodies using
conventional techniques, such as papain or pepsin digestion,
respectively, of whole antibodies. Moreover, antibodies, antibody
portions and immunoadhesion molecules can be obtained using
standard recombinant DNA techniques, as described herein. In one
aspect, the antigen binding portions are complete domains or pairs
of complete domains.
[0107] The terms "Kabat numbering" "Kabat definitions" and "Kabat
labeling" are used interchangeably herein. These terms, which are
recognized in the art, refer to a system of numbering amino acid
residues which are more variable (i.e., hypervariable) than other
amino acid residues in the heavy and light chain variable regions
of an antibody, or an antigen binding portion thereof (Kabat et al.
(1971) Ann. NY Acad, Sci. 190:382-391 and, Kabat, E. A., et al.
(1991) Sequences of Proteins of Immunological Interest, Fifth
Edition, U.S. Department of Health and Human Services, NIH
Publication No. 91-3242, the entire teachings of which are
incorporated herein by reference). For the heavy chain variable
region, the hypervariable region ranges from amino acid positions
31 to 35 for CDR1, amino acid positions 50 to 65 for CDR2, and
amino acid positions 95 to 102 for CDR3. For the light chain
variable region, the hypervariable region ranges from amino acid
positions 24 to 34 for CDR1, amino acid positions 50 to 56 for
CDR2, and amino acid positions 89 to 97 for CDR3.
[0108] The term "human antibody" includes antibodies having
variable and constant regions corresponding to human germline
immunoglobulin sequences as described by Kabat et al. (See Kabat,
et al. (1991) Sequences of proteins of Immunological Interest,
Fifth Edition, U.S. Department of Health and Human Services, NIH
Publication No. 91-3242). The human antibodies of the invention may
include amino acid residues not encoded by human germline
immunoglobulin sequences (e.g., mutations introduced by random or
site-specific mutagenesis in vitro or by somatic mutation in vivo),
e.g., in the CDRs and in particular CDR3. The mutations can be
introduced using the "selective mutagenesis approach." The human
antibody can have at least one position replaced with an amino acid
residue, e.g., an activity enhancing amino acid residue which is
not encoded by the human germline immunoglobulin sequence. The
human antibody can have up to twenty positions replaced with amino
acid residues which are not part of the human germline
immunoglobulin sequence. In other embodiments, up to ten, up to
five, up to three or up to two positions are replaced. In one
embodiment, these replacements are within the CDR regions. However,
the term "human antibody", as used herein, is not intended to
include antibodies in which CDR sequences derived from the germline
of another mammalian species, such as a mouse, have been grafted
onto human framework sequences.
[0109] The phrase "recombinant human antibody" includes human
antibodies that are prepared, expressed, created or isolated by
recombinant means, such as antibodies expressed using a recombinant
expression vector transfected into a host cell, antibodies isolated
from a recombinant, combinatorial human antibody library,
antibodies isolated from an animal (e.g., a mouse) that is
transgenic for human immunoglobulin genes (see, e.g., Taylor, L.
D., et al. (1992) Nucl. Acids Res. 20:6287-6295, the entire
teaching of which is incorporated herein by reference) or
antibodies prepared, expressed, created or isolated by any other
means that involves splicing of human immunoglobulin gene sequences
to other DNA sequences. Such recombinant human antibodies have
variable and constant regions derived from human germline
immunoglobulin sequences (see, Kabat, E. A., et al. (1991)
Sequences of Proteins of Immunological Interest, Fifth Edition,
U.S. Department of Health and Human Services, NIH Publication No.
91-3242). In certain embodiments, however, such recombinant human
antibodies are subjected to in vitro mutagenesis (or, when an
animal transgenic for human Ig sequences is used, in vivo somatic
mutagenesis) and thus the amino acid sequences of the VH and VL
regions of the recombinant antibodies are sequences that, while
derived from and related to human germline VH and VL sequences, may
not naturally exist within the human antibody germline repertoire
in vivo. In certain embodiments, however, such recombinant
antibodies are the result of selective mutagenesis approach or
back-mutation or both.
[0110] An "isolated antibody" includes an antibody that is
substantially free of other antibodies having different antigenic
specificities (e.g., an isolated antibody that specifically binds
hTNF.alpha. is substantially free of antibodies that specifically
bind antigens other than hTNF.alpha.). An isolated antibody that
specifically binds hTNF.alpha. may bind TNF.alpha. molecules from
other species. Moreover, an isolated antibody may be substantially
free of other cellular material and/or chemicals. A suitable
anti-TNF.alpha. antibody is adalimumab.
[0111] As used herein, the term "adalimumab," also known by its
trade name HUMIRA.RTM. (AbbVie) refers to a human IgG.sub.1
antibody that binds human tumor necrosis factor .alpha.
(TNF.alpha.). In general, the heavy chain constant domain 2 (CH2)
of the adalimumab IgG-Fc region is glycosylated through covalent
attachment of oligosaccharide at asparagine 297 (Asn-297). The
HUMIRA.RTM. Prescribing Information, the contents of each of which
are expressly incorporated by reference herein. The light chain
variable region of adalimumab is provided herein as SEQ ID NO:1,
and the heavy chain variable region of adalimumab is provided
herein as SEQ ID NO:2. The light chain of adalimumab is provided
herein as SEQ ID NO:11, and the heavy chain of adalimumab is
provided herein as SEQ ID NO:12. Adalimumab comprises a light chain
variable region comprising a CDR1 of SEQ ID NO:7, a CDR2 of SEQ ID
NO:5, and a CDR3 of SEQ ID NO:3. Adalimumab comprises a heavy chain
variable region comprising a CDR1 of SEQ ID NO:8, a CDR2 of SEQ ID
NO:6 and CDR3 of SEQ ID NO:4. Adalimumab is described in U.S. Pat.
Nos. 6,090,382; 6,258,562; 6,509,015; 7,223,394; 7,541,031;
7,588,761; 7,863,426; 7,919,264; 8,197,813; 8,206,714; 8,216,583;
8,420,081; 8,092,998; 8,093,045; 8,187,836; 8,372,400; 8,034,906;
8,436,149; 8,231,876; 8,414,894; 8,372,401, and PCT Publication No.
WO2012/065072, the entire contents of each which are expressly
incorporated herein by reference in their entireties. Adalimumab is
also described in the "Highlights of Prescribing Information" for
HUMIRA.RTM. (adalimumab) Injection (Revised January 2008) the
contents of which are hereby incorporated herein by reference.
[0112] In one embodiment, adalimumab dissociates from human
TNF.alpha. with a Kd of 1.times.10.sup.-8 M or less and a K.sub.off
rate constant of 1.times.10.sup.-3 s.sup.-1 or less, both
determined by surface plasmon resonance, and neutralizes human
TNF.alpha. cytotoxicity in a standard in vitro L929 assay with an
IC50 of 1.times.10-M or less. In another embodiment, adalimumab
dissociates from human TNF.alpha. with a K.sub.off of
5.times.10.sup.-4 s.sup.-1 or less, or with a K.sub.off of
1.times.10.sup.-4 s.sup.-1 or less. In still another embodiment,
adalimumab neutralizes human TNF.alpha. cytotoxicity in a standard
in vitro L929 assay with an IC50 of 1.times.10.sup.-8 M or less, an
IC50 of 1.times.10.sup.-9 M or less or an IC50 of
1.times.10.sup.-1.degree. M or less.
[0113] Analysis of adalimumab has shown that it has three main
basic variants (i.e., Lys 0, Lys 1, and Lys 2), referred to herein
as "lysine variant species." As used herein, the term "lysine
variant species" refers to an antibody, or antigen-binding portion
thereof, comprising heavy chains with either zero, one or two
C-terminal lysines. For example, the "Lys 0" variant comprises an
antibody, or antigen-binding portion thereof, with heavy chains
that do not comprise a C-terminal lysine. The "Lys 1" variant
comprises an antibody, or antigen-binding portion thereof, with one
heavy chain that comprises a C-terminal lysine. The "Lys 2" variant
comprises an antibody with both heavy chains comprising a
C-terminal lysine. Lysine variant species can be detected by
various methods, such as ion exchange, for example, by weak cation
exchange chromatography (such as WCX-10) of the expression product
of a host cell expressing the antibody, or antigen-binding portion
thereof. For example, but not by way of limitation, FIG. 2 depicts
analysis of adalimumab wherein the three lysine variants, as well
as the two acidic species regions, are resolved from each
other.
[0114] A composition of the invention may comprise more than one
lysine variant species of an antibody, or antigen-binding portion
thereof. For example, in one embodiment, the composition may
comprise a Lys 2 variant of an antibody, or antigen-binding portion
thereof. The composition may comprise a Lys 1 variant of an
antibody, or antigen-binding portion thereof. The composition may
comprise a Lys 0 variant of an antibody, or antigen-binding portion
thereof. In another embodiment, the composition may comprise both
Lys 1 and Lys 2, or Lys 1 and Lys 0, or Lys 2 and Lys 0 variants of
an antibody, or antigen-binding portion thereof. In another
embodiment, the composition may comprise all three lysine variant
species, i.e., Lys 0, Lys 1 and Lys 2, of an antibody, or
antigen-binding portion thereof.
[0115] In one embodiment, the invention comprises a composition
comprising an antibody, or antigen-binding portion thereof, wherein
the composition comprises less than about 50% lysine variant
species that lack a C-terminal lysine (Lys 0). In another
embodiment, the composition comprises less than about 49%, 48%,
47%, 46%, 45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%, 36%, 35%,
34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%,
21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11% 10%, 9%, 8%,
7%, 6%, 5%, 4%, 3%, 2% or 1% lysine variant species that lack a
C-terminal lysine ("Lys 0"). In another embodiment, the composition
comprises about 50% to about 0%, about 40% to about 10%, about 30%
to about 20%, about 40% to about 20%, or about 30% to about 15%
lysine variant species that lack a C-terminal lysine (Lys 0). In
one embodiment, the composition comprises 0% lysine variant species
that lack a C-terminal lysine (Lys 0). As used herein, the percent
lysine variant species in the composition refers to the weight of
the specific lysine variant species in a sample in relation to the
weight of the total lysine variant species sum (i.e., the sum of
Lys 0, Lys 1 and Lys 2) contained in the sample or composition. For
example, the percent lysine variant species can be calculated using
weak cation exchange chromatography such as WCX-10, as described
herein.
[0116] In another embodiment, the composition comprises less than
about 25% lysine variant species that have one C-terminal lysine
(Lys 1). In another embodiment, the composition comprises less than
about 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%,
12%, 11% 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% lysine variant
species that have one C-terminal lysine (Lys 1). In another
embodiment, the composition comprises about 25% to about 0%, about
20% to about 5%, about 15% to about 10%, about 20% to about 10%,
about 15% to about 5%, or about about 25% to about 5% lysine
variant species that have one C-terminal lysine (Lys 1). In one
embodiment, the composition comprises 0% lysine variant species
that have one C-terminal Lysine (Lys 1).
[0117] In another embodiment, the composition comprises at least
about 70% lysine variant species that have two C-terminal lysines
(Lys 2). In another embodiment, the composition comprises at least
about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,
82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98% or 99% lysine variant species that have two
C-terminal lysines (Lys 2). In one embodiment, the composition
comprises about 70% to about 100%, about 70% to about 90%, about
70% to about 80%, about 80% to about 100%, about 85% to about 100%,
about 90% to about 100%, about 95% to about 100%, about 80% to
about 90%, about 85% to about 95%, about 75% to about 85%, or about
97% to about 100% lysine variant species that have two C-terminal
lysines (Lys 2). In one embodiment, the composition comprises 100%
lysine variant species that have two C-terminal lysines (Lys
2).
[0118] As used herein, the term "upstream process technology," in
the context of protein, e.g., antibody, preparation, refers to
activities involving the production and collection of proteins
(e.g. antibodies) from cells (e.g., during cell culture of a
protein of interest). As used herein, the term "cell culture"
refers to methods for generating and maintaining a population of
host cells capable of producing a recombinant protein of interest,
as well as the methods and techniques for optimizing the production
and collection of the protein of interest. For example, once an
expression vector has been incorporated into an appropriate host,
the host can be maintained under conditions suitable for expression
of the relevant nucleotide coding sequences, and the collection and
purification of the desired recombinant protein.
[0119] When using the cell culture techniques of the instant
invention, the protein of interest can be produced intracellularly,
in the periplasmic space, or directly secreted into the medium. In
embodiments where the protein of interest is produced
intracellularly, the particulate debris, either host cells or lysed
cells (e.g., resulting from homogenization) can be removed by a
variety of means, including but not limited to, centrifugation or
ultrafiltration. Where the protein of interest is secreted into the
medium, supernatants from such expression systems can be first
concentrated using a commercially available protein concentration
filter, e.g., an Amicon.TM. or Millipore Pellicon.TM.
ultrafiltration unit.
[0120] As used herein, the term "downstream process technology"
refers to one or more techniques used after the upstream process
technologies to purify the protein, e.g., antibody, of interest.
For example, downstream process technology includes purification of
the protein product, using, for example, displacement
chromatography, affinity chromatography, including Protein A
affinity chromatography, ion exchange chromatography, such as anion
or cation exchange chromatography, hydrophobic interaction
chromatography, or displacement chromatography.
[0121] The phrase "isolated nucleic acid molecule," as used herein
in reference to nucleic acids encoding antibodies or antibody
portions (e.g., VH, VL, CDR3), e.g., those that bind hTNF.alpha.,
includes a nucleic acid molecule in which the nucleotide sequences
encoding the antibody or antibody portion are free of other
nucleotide sequences encoding antibodies or antibody portions that
bind antigens other than hTNF.alpha., which other sequences may
naturally flank the nucleic acid in human genomic DNA. Thus, e.g.,
an isolated nucleic acid of the invention encoding a VH region of
an anti-TNF.alpha. antibody contains no other sequences encoding
other VH regions that bind antigens other than, for example,
hTNF.alpha.. The phrase "isolated nucleic acid molecule" is also
intended to include sequences encoding bivalent, bispecific
antibodies, such as diabodies in which VH and VL regions contain no
other sequences other than the sequences of the diabody.
[0122] The phrase "recombinant host cell" (or simply "host cell")
includes a cell into which a recombinant expression vector has been
introduced. It should be understood that such terms are intended to
refer not only to the particular subject cell but to the progeny of
such a cell. Because certain modifications may occur in succeeding
generations due to either mutation or environmental influences,
such progeny may not, in fact, be identical to the parent cell, but
are still included within the scope of the term "host cell" as used
herein.
[0123] As used herein, the term "recombinant protein" refers to a
protein produced as the result of the transcription and translation
of a gene carried on a recombinant expression vector that has been
introduced into a host cell. In certain embodiments the recombinant
protein is an antibody, e.g., a chimeric, humanized, or fully human
antibody. In certain embodiments the recombinant protein is an
antibody of an isotype selected from group consisting of: IgG
(e.g., IgG1, IgG2, IgG3, IgG4), IgM, IgA1, IgA2, IgD, or IgE. In
certain embodiments the antibody molecule is a full-length antibody
(e.g., an IgG1 or IgG4 immunoglobulin) or alternatively the
antibody can be a fragment (e.g., an Fc fragment or a Fab
fragment).
[0124] The term "preparative scale," as used herein, refers to a
scale of purification operation that can be readily scaled-up and
implemented at large scale manufacturing while still providing
desired separation. For instance, one skilled in the field may
develop a process using, e.g., a 0.5 cm (i.d.).times.20 cm (L)
column in the lab, and transfer it to large scale production using,
e.g., a 30 cm (i.d.).times.20 cm (L) column packed with the same
resin and operated with the same set of buffers, same linear flow
rates (or residence times) and buffer volumes. In preparative scale
separation, column bed height is typically .ltoreq.about 30 cm and
column pressure drop .ltoreq.about 5 bar.
[0125] The phrase "displacing buffer", "displacer buffer",
"displacing wash buffer", or "displacer wash buffer", as used
herein, refers to a buffer that comprises a displacer molecule. The
phrase "displacer molecule", as used herein, refers to a molecule
employed to displace from the chromatographic support components of
the mixture to be separated. Selection of a particular displacer
molecule will, therefore, be dependent on the chromatographic
support employed as well as the protein system. Regardless of which
chromatographic support is employed, displacer molecules will
generally be selected such that they have a high affinity for the
support. However, in certain embodiments, a displacer molecule may
be selected that has a reduced affinity for the support, so long as
it retains the ability to induce a displacement train that includes
the protein of interest. In certain non-limiting embodiments, the
displacer molecule will be employed in the context of protein
separations in ion exchange chromatography and can be selected
from, but not limited to, the group consisting of:
polyelectrolytes; polysaccharides; low-molecular-mass dendrimers;
amino acids; peptide; antibiotics; and aminoglycosidepolyamines. In
certain embodiments the displacer is selected from, but not limited
to, the group consisting of: Expell SP1.TM. (for CEX and for mixed
mode); Expell Q3 (for anion-exchange chromatography (AEX) and for
mixed mode); Propel Q2 (for AEX and for mixed mode); and protamine
sulfate (for CEX and for mixed mode). Exemplary displacer molecules
are described in U.S. Pat. No. 7,632,409, WO 99/47574, WO 03074148,
WO2007/055896, WO 2007/064809; and U.S. Pat. No. 6,881,540.
[0126] As used herein, the term"aggregates" means agglomeration or
oligomerization of two or more individual molecules, including but
not limiting to, protein dimers, trimers, tetramers, oligomers and
other high molecular weight species. Protein aggregates can be
soluble or insoluble.
II. Low Acidic Species Compositions of the Invention
[0127] The present invention provides low AR compositions
comprising a protein, e.g., an antibody, or antigen-binding portion
thereof, such as adalimumab, where the composition comprises about
15% or less AR, 14% or less AR, 13% or less AR, 12% or less AR, 11%
or less AR, 10% or less AR, 9% or less AR, 8% or less AR, 7% or
less AR, 6% or less AR, 5% or less AR, 4.5% or less AR, 4% or less
AR, 3.5% or less AR, 3% or less AR, 2.5% or less AR, 2% or less AR,
1.9% or less AR, 1.8% or less AR, 1.7% or less AR, 1.6% or less AR,
1.5% or less AR, 1.4% or less AR, 1.3% or less AR, 1.2% or less AR,
1.1% or less AR, 1% or less AR, 0.9% or less AR, 0.8% or less AR,
0.7% or less AR, 0.6% or less AR, 0.5% or less AR, 0.4% or less AR,
0.3% or less AR, 0.2% or less AR, 0.1% or less AR, or 0.0% AR, and
ranges within one or more of the preceding. A low AR composition of
the invention may also comprise about 0.0% to about 10% AR, about
0.0% to about 5% AR, about 0.0% to about 4% AR, about 0.0% to about
3% AR, about 0.0% to about 2% AR, about 3% to about 5% AR, about 5%
to about 8% AR, or about 8% to about 10% AR, or about 10% to about
15% AR, and ranges within one or more of the preceding.
[0128] In one embodiment, a low AR composition of the invention may
comprise about 15% or less AR1, 14% or less AR1, 13% or less AR1,
12% or less AR1, 11% or less AR1, 10% or less AR1, 9% or less AR1,
8% or less AR1, 7% or less AR1, 6% or less AR1, 5% or less AR1,
4.5% or less AR1, 4% or less AR1, 3.5% or less AR1, 3% or less AR1,
2.5% or less AR1, 2% or less AR1, 1.9% or less AR1, 1.8% or less
AR1, 1.7% or less AR1, 1.6% or less AR1, 1.5% or less AR1, 1.4% or
less AR1, 1.3% or less AR1, 1.2% or less AR1, 1.1% or less AR1, 1%
or less AR1, 0.9% or less AR1, 0.8% or less AR1, 0.7% or less AR1,
0.6% or less AR1, 0.5% or less AR1, 0.4% or less AR1, 0.3% or less
AR1, 0.2% or less AR1, 0.1% or less AR1, or 0.0% AR1, and ranges
within one or more of the preceding. A low AR composition of the
invention may also comprise about 0.0% to about 10% AR1, about 0.0%
to about 5% AR1, about 0.0% to about 4% AR1, about 0.0% to about 3%
AR1, about 0.0% to about 2% AR1, about 3% to about 5% AR1, about 5%
to about 8% AR1, or about 8% to about 10% AR1, or about 10% to
about 15% AR1, and ranges within one or more of the preceding.
[0129] In another embodiment, a low AR composition of the invention
may also comprise about 15% or less AR2, 14% or less AR2, 13% or
less AR2, 12% or less AR2, 11% or less AR2, 10% or less AR2, 9% or
less AR2, 8% or less AR2, 7% or less AR2, 6% or less AR2, 5% or
less AR2, 4.5% or less AR2, 4% or less AR2, 3.5% or less AR2, 3% or
less AR2, 2.5% or less AR2, 2% or less AR2, 1.9% or less AR2, 1.8%
or less AR2, 1.7% or less AR2, 1.6% or less AR2, 1.5% or less AR2,
1.4% or less AR2, 1.3% or less AR2, 1.2% or less AR2, 1.1% or less
AR2, 1% or less AR2, 0.9% or less AR2, 0.8% or less AR2, 0.7% or
less AR2, 0.6% or less AR2, 0.5% or less AR2, 0.4% or less AR2,
0.3% or less AR2, 0.2% or less AR2, 0.1% or less AR2, or 0.0% AR2,
and ranges within one or more of the preceding. A low AR
composition of the invention may also comprise about 0.0% to about
10% AR2, about 0.0% to about 5% AR2, about 0.0% to about 4% AR2,
about 0.0% to about 3% AR2, about 0.0% to about 2% AR2, about 3% to
about 5% AR2, about 5% to about 8% AR2, or about 8% to about 10%
AR2, or about 10% to about 15% AR2, and ranges within one or more
of the preceding.
[0130] A low AR composition of the invention may further comprise
more than one lysine variant species of an antibody, or
antigen-binding portion thereof. For example, in a preferred
embodiment, the composition may comprise a Lys 2 variant of an
antibody, or antigen-binding portion thereof. The composition may
comprise a Lys 1 variant of an antibody, or antigen-binding portion
thereof. The composition may comprise a Lys 0 variant of an
antibody, or antigen-binding portion thereof. In another
embodiment, the composition may comprise both Lys 1 and Lys 2, or
Lys 1 and Lys 0, or Lys 2 and Lys 0 variants of an antibody, or
antigen-binding portion thereof. In another embodiment, the
composition may comprise all three lysine variant species, i.e.,
Lys 0, Lys 1 and Lys 2, of an antibody, or antigen-binding portion
thereof.
[0131] In one embodiment, a low AR composition of the invention
comprises less than about 50% lysine variant species that lack a
C-terminal lysine (Lys 0). In another embodiment, the composition
comprises less than about 55%, 54%, 53%, 52%, 51%, 50%, 49%, 48%,
47%, 46%, 45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%, 36%, 35%,
34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%,
21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11% 10%, 9%, 8%,
7%, 6%, 5%, 4%, 3%, 2% or 1% lysine variant species that lack a
C-terminal lysine ("Lys 0"). In another embodiment, the composition
comprises about 50% to about 0%, about 40% to about 10%, about 30%
to about 20%, about 40% to about 20%, or about 30% to about 15%
lysine variant species that lack a C-terminal lysine (Lys 0). In
one embodiment, the composition comprises 0% lysine variant species
that lack a C-terminal lysine (Lys 0).
[0132] In one embodiment, a low AR composition comprises less than
about 25% lysine variant species that have one C-terminal lysine
(Lys 1). In another embodiment, the composition comprises less than
about 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%,
12%, 11% 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% lysine variant
species that have one C-terminal lysine (Lys 1). In another
embodiment, the composition comprises about 25% to about 0%, about
20% to about 5%, about 15% to about 10%, about 20% to about 10%,
about 15% to about 5%, or about about 25% to about 5% lysine
variant species that have one C-terminal lysine (Lys 1). In one
embodiment, the composition comprises 0% lysine variant species
that have one C-terminal Lysine (Lys 1).
[0133] In another embodiment, a low AR composition of the invention
comprises at least about 70% lysine variant species that have two
C-terminal lysines (Lys 2). In another embodiment, the composition
comprises at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,
50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%,
79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% lysine variant species
that have two C-terminal lysines (Lys 2). In one embodiment, the
composition comprises about 70% to about 100%, about 70% to about
90%, about 70% to about 80%, about 80% to about 100%, about 85% to
about 100%, about 90% to about 100%, about 95% to about 100%, about
80% to about 90%, about 85% to about 95%, about 75% to about 85%,
or about 97% to about 100% lysine variant species that have two
C-terminal lysines (Lys 2). In one embodiment, the composition
comprises 100% lysine variant species that have two C-terminal
lysines (Lys 2).
[0134] As demonstrated herein, these low AR compositions of the
invention have improved biological properties. For example, the low
AR compositions of the invention are characterized by increased
cartilage tissue penetration, reduced cartilage destruction,
reduced synovial proliferation, reduced bone erosion, increased
protection against the development of arthritic scores and/or
histopathology scores, reduced cell infiltration, reduced
proteoglycan loss, reduced chondrocyte death, and/or increased
TNF.alpha. affinity, as compared to non-low acidic species
compositions. In addition, the compositions of the present
invention exhibit increased therapeutic efficacy when administered
to a subject.
[0135] In one embodiment, the protein in the low AR composition of
the invention is an antibody or antigen binding portion thereof.
For example, the antibody, or antigen binding portion thereof may
be an anti-TNF.alpha. antibody, or antigen binding portion thereof,
such as adalimumab, or an antigen binding portion thereof. In one
aspect of this embodiment, the antibody, or antigen binding portion
thereof, can comprise a light chain variable region comprising the
sequence set forth as SEQ ID NO:1, and a heavy chain variable
region comprising the sequence set forth as SEQ ID NO:2. In another
aspect of this embodiment, the antibody can comprise a light chain
variable region comprising a CDR1 having the sequence set forth as
SEQ ID NO:7, a CDR2 having the sequence set forth as SEQ ID NO:5,
and a CDR3 having the sequence set forth as SEQ ID NO:3. In another
aspect of this embodiment, the antibody can comprise a heavy chain
variable region comprising a CDR1 having the sequence set forth as
SEQ ID NO:8, a CDR2 having the sequence set forth as SEQ ID NO:6
and a CDR3 having the sequence set forth as SEQ ID NO:4.
[0136] The antibody, or antigen binding portion thereof, used in
the low AR compositions of the invention, may be a human,
humanized, or chimeric antibody.
[0137] The antibodies that can be used in the low AR compositions
of the present disclosure can be generated by a variety of
techniques, including immunization of an animal with the antigen of
interest followed by conventional monoclonal antibody methodologies
e.g., the standard somatic cell hybridization technique of Kohler
and Milstein (1975) Nature 256: 495. Somatic cell hybridization
procedures can be used. In principle, other techniques for
producing monoclonal antibody can be employed as well, including
viral or oncogenic transformation of B lymphocytes.
[0138] One exemplary animal system for preparing hybridomas is the
murine system. Hybridoma production is a very well-established
procedure. Immunization protocols and techniques for isolation of
immunized splenocytes for fusion are known in the art. Fusion
partners (e.g., murine myeloma cells) and fusion procedures are
also known.
[0139] An antibody used in the low AR compositions of the invention
can be a human, a chimeric, or a humanized antibody. Chimeric or
humanized antibodies used in the low AR compositions of the
invention can be prepared based on the sequence of a non-human
monoclonal antibody prepared as described above. DNA encoding the
heavy and light chain immunoglobulins can be obtained from the
non-human hybridoma of interest and engineered to contain
non-murine (e.g., human) immunoglobulin sequences using standard
molecular biology techniques. For example, to create a chimeric
antibody, murine variable regions can be linked to human constant
regions using methods known in the art (see e.g., U.S. Pat. No.
4,816,567 to Cabilly et al.). To create a humanized antibody,
murine CDR regions can be inserted into a human framework using
methods known in the art (see e.g., U.S. Pat. No. 5,225,539 to
Winter, and U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and
6,180,370 to Queen et al.).
[0140] In one non-limiting embodiment, the antibodies to be used in
the low AR compositions of the invention are human monoclonal
antibodies. Such human monoclonal antibodies can be generated using
transgenic or transchromosomic mice carrying parts of the human
immune system rather than the mouse system. These transgenic and
transchromosomic mice include mice referred to herein as the HuMAb
Mouse.RTM. (Medarex, Inc.), KM Mouse.RTM. (Medarex, Inc.), and
XenoMouse.RTM. (Amgen). The antibodies, or antigen-binding portions
thereof, used in the low AR compositions of the invention can also
be produced using the methods described in U.S. Pat. No. 6,090,382,
the entire contents of which is expressly incorporated herein by
reference.
[0141] Moreover, alternative transchromosomic animal systems
expressing human immunoglobulin genes are available in the art and
can be used to raise antibodies of the disclosure. For example,
mice carrying both a human heavy chain transchromosome and a human
light chain transchromosome, referred to as "TC mice" can be used;
such mice are described in Tomizuka et al. (2000) Proc. Natl. Acad.
Sci. USA 97:722-727. Furthermore, cows carrying human heavy and
light chain transchromosomes have been described in the art (e.g.,
Kuroiwa et al. (2002) Nature Biotechnology 20:889-894 and PCT
application No. WO 2002/092812) and can be used to raise antibodies
of this disclosure.
[0142] Recombinant human antibodies to be used in the low AR
compositions of the invention can be isolated by screening of a
recombinant combinatorial antibody library, e.g., a scFv phage
display library, prepared using human VL and VH cDNAs prepared from
mRNA derived from human lymphocytes. Methodologies for preparing
and screening such libraries are known in the art. In addition to
commercially available kits for generating phage display libraries
(e.g., the Pharmacia Recombinant Phage Antibody System, catalog no.
27-9400-01; and the Stratagene SurfZAP.TM. phage display kit,
catalog no. 240612, the entire teachings of which are incorporated
herein), examples of methods and reagents particularly amenable for
use in generating and screening antibody display libraries can be
found in, e.g., Ladner et al. U.S. Pat. No. 5,223,409; Kang et al.
PCT Publication No. WO 92/18619; Dower et al. PCT Publication No.
WO 91/17271; Winter et al. PCT Publication No. WO 92/20791;
Markland et al. PCT Publication No. WO 92/15679; Breitling et al.
PCT Publication No. WO 93/01288; McCafferty et al. PCT Publication
No. WO 92/01047; Garrard et al. PCT Publication No. WO 92/09690;
Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay et al. (1992)
Hum Antibody Hybridomas 3:81-85; Huse et al. (1989) Science
246:1275-1281; McCafferty et al., Nature (1990) 348:552-554;
Griffiths et al. (1993) EMBO J 12:725-734; Hawkins et al. (1992) J
Mol Biol 226:889-896; Clackson et al. (1991) Nature 352:624-628;
Gram et al. (1992) PNAS 89:3576-3580; Garrard et al. (1991)
Bio/Technology 9:1373-1377; Hoogenboom et al. (1991) Nuc Acid Res
19:4133-4137; and Barbas et al. (1991) PNAS 88:7978-7982; the
entire teachings of which are incorporated herein.
[0143] Human monoclonal antibodies to be used in the low AR
compositions of the invention can also be prepared using SCID mice
into which human immune cells have been reconstituted such that a
human antibody response can be generated upon immunization. Such
mice are described in, for example, U.S. Pat. Nos. 5,476,996 and
5,698,767 to Wilson et al.
[0144] In certain embodiments, the human antibodies to be used in
the low AR compositions of the invention are anti-TNF.alpha.
antibodies and antibody portions thereof, anti-TNF.alpha.-related
antibodies and antibody portions, and human antibodies and antibody
portions with equivalent properties to anti-TNF.alpha. antibodies,
such as high affinity binding to hTNF.alpha. with low dissociation
kinetics and high neutralizing capacity. In one aspect, the
invention provides low AR compositions containing an isolated human
antibody, or an antigen-binding portion thereof, that dissociates
from hTNF.alpha. with a Kd of about 1.times.10.sup.-8 M or less and
a Koff rate constant of 1.times.10.sup.-3 s.sup.-1 or less, both
determined by surface plasmon resonance. In specific non-limiting
embodiments, an anti-TNF.alpha. antibody to be used in the low AR
compositions of the invention competitively inhibits binding of
adalimumab to TNF.alpha. under physiological conditions. In one
embodiment, the low AR compositions of the invention comprise
adalimumab, or an antigen binding fragment thereof.
[0145] Antibodies or fragments thereof to be used in the low AR
compositions of the invention can be altered wherein the constant
region of the antibody is modified to reduce at least one constant
region-mediated biological effector function relative to an
unmodified antibody. To modify an antibody of the invention such
that it exhibits reduced binding to the Fc receptor, the
immunoglobulin constant region segment of the antibody can be
mutated at particular regions necessary for Fc receptor (FcR)
interactions (see, e.g., Canfield and Morrison (1991) J. Exp. Med.
173:1483-1491; and Lund et al. (1991) J. of Immunol. 147:2657-2662,
the entire teachings of which are incorporated herein). Reduction
in FcR binding ability of the antibody may also reduce other
effector functions which rely on FcR interactions, such as
opsonization and phagocytosis and antigen-dependent cellular
cytotoxicity.
[0146] To express an antibody or antigen-binding fragment thereof
to be used in the low AR compositions of the invention, DNAs
encoding the protein, such as DNAs encoding partial or full-length
light and heavy chains in the case of antibodies, are inserted into
one or more expression vector such that the genes are operatively
linked to transcriptional and translational control sequences.
(See, e.g., U.S. Pat. No. 6,090,382, the entire teaching of which
is incorporated herein by reference.) In this context, the term
"operatively linked" is intended to mean that a gene encoding the
protein of interest is ligated into a vector such that
transcriptional and translational control sequences within the
vector serve their intended function of regulating the
transcription and translation of the gene. The expression vector
and expression control sequences are chosen to be compatible with
the expression host cell used. In certain embodiments, the protein
of interest will comprising multiple polypeptides, such as the
heavy and light chains of an antibody. Thus, in certain
embodiments, genes encoding multiple polypeptides, such as antibody
light chain genes and antibody heavy chain genes, can be inserted
into a separate vector or, more typically, the genes are inserted
into the same expression vector. Genes are inserted into expression
vectors by standard methods (e.g., ligation of complementary
restriction sites on the gene fragment and vector, or blunt end
ligation if no restriction sites are present). Prior to insertion
of the gene or genes, the expression vector may already carry
additional polypeptide sequences, such as, but not limited to,
antibody constant region sequences. For example, one approach to
converting the anti-TNF.alpha. antibody or anti-TNF.alpha.
antibody-related VH and VL sequences to full-length antibody genes
is to insert them into expression vectors already encoding heavy
chain constant and light chain constant regions, respectively, such
that the VH segment is operatively linked to the CH segment(s)
within the vector and the VL segment is operatively linked to the
CL segment within the vector. Additionally or alternatively, the
recombinant expression vector can encode a signal peptide that
facilitates secretion of the protein from a host cell. The gene can
be cloned into the vector such that the signal peptide is linked
in-frame to the amino terminus of the gene. The signal peptide can
be an immunoglobulin signal peptide or a heterologous signal
peptide (i.e., a signal peptide from a non-immunoglobulin
protein).
[0147] In addition to protein coding genes, a recombinant
expression vector can carry one or more regulatory sequence that
controls the expression of the protein coding genes in a host cell.
The term "regulatory sequence" is intended to include promoters,
enhancers and other expression control elements (e.g.,
polyadenylation signals) that control the transcription or
translation of the protein coding genes. Such regulatory sequences
are described, e.g., in Goeddel; Gene Expression Technology:
Methods in Enzymology 185, Academic Press, San Diego, Calif.
(1990), the entire teaching of which is incorporated herein by
reference. It will be appreciated by those skilled in the art that
the design of the expression vector, including the selection of
regulatory sequences may depend on such factors as the choice of
the host cell to be transformed, the level of expression of protein
desired, etc. Suitable regulatory sequences for mammalian host cell
expression include viral elements that direct high levels of
protein expression in mammalian cells, such as promoters and/or
enhancers derived from cytomegalovirus (CMV) (such as the CMV
promoter/enhancer), Simian Virus 40 (SV40) (such as the SV40
promoter/enhancer), adenovirus, (e.g., the adenovirus major late
promoter (AdMLP)) and polyoma. For further description of viral
regulatory elements, and sequences thereof, see, e.g., U.S. Pat.
No. 5,168,062 by Stinski, U.S. Pat. No. 4,510,245 by Bell et al.
and U.S. Pat. No. 4,968,615 by Schaffner et al., the entire
teachings of which are incorporated herein by reference.
[0148] A recombinant expression vector may also carry one or more
additional sequences, such as a sequence that regulates replication
of the vector in host cells (e.g., origins of replication) and/or a
selectable marker gene. The selectable marker gene facilitates
selection of host cells into which the vector has been introduced
(see e.g., U.S. Pat. Nos. 4,399,216, 4,634,665 and 5,179,017, all
by Axel et al., the entire teachings of which are incorporated
herein by reference). For example, typically the selectable marker
gene confers resistance to drugs, such as G418, hygromycin or
methotrexate, on a host cell into which the vector has been
introduced. Suitable selectable marker genes include the
dihydrofolate reductase (DHFR) gene (for use in dhfr- host cells
with methotrexate selection/amplification) and the neo gene (for
G418 selection).
[0149] An antibody, or antibody portion, to be used in the low AR
compositions of the invention can be prepared by recombinant
expression of immunoglobulin light and heavy chain genes in a host
cell. To express an antibody recombinantly, a host cell is
transfected with one or more recombinant expression vectors
carrying DNA fragments encoding the immunoglobulin light and heavy
chains of the antibody such that the light and heavy chains are
expressed in the host cell and secreted into the medium in which
the host cells are cultured, from which medium the antibodies can
be recovered. Standard recombinant DNA methodologies are used to
obtain antibody heavy and light chain genes, incorporate these
genes into recombinant expression vectors and introduce the vectors
into host cells, such as those described in Sambrook, Fritsch and
Maniatis (eds), Molecular Cloning; A Laboratory Manual, Second
Edition, Cold Spring Harbor, N.Y., (1989), Ausubel et al. (eds.)
Current Protocols in Molecular Biology, Greene Publishing
Associates, (1989) and in U.S. Pat. Nos. 4,816,397 & 6,914,128,
the entire teachings of which are incorporated herein.
[0150] For expression of protein, for example, the light and heavy
chains of an antibody, the expression vector(s) encoding the
protein is (are) transfected into a host cell by standard
techniques. The various forms of the term "transfection" are
intended to encompass a wide variety of techniques commonly used
for the introduction of exogenous DNA into a prokaryotic or
eukaryotic host cell, e.g., electroporation, calcium-phosphate
precipitation, DEAE-dextran transfection and the like. Although it
is theoretically possible to express the proteins of the invention
in either prokaryotic or eukaryotic host cells, expression of
antibodies in eukaryotic cells, such as mammalian host cells, is
suitable because such eukaryotic cells, and in particular mammalian
cells, are more likely than prokaryotic cells to assemble and
secrete a properly folded and immunologically active protein.
Prokaryotic expression of protein genes has been reported to be
ineffective for production of high yields of active protein (Boss
and Wood (1985) Immunology Today 6:12-13, the entire teaching of
which is incorporated herein by reference).
[0151] Suitable host cells for cloning or expressing the DNA in the
vectors herein are the prokaryote, yeast, or higher eukaryote cells
described above. Suitable prokaryotes for this purpose include
eubacteria, such as Gram-negative or Gram-positive organisms, e.g.,
Enterobacteriaceae such as Escherichia, e.g., E. coli,
Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g.,
Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and
Shigella, as well as Bacilli such as B. subtilis and B.
licheniformis (e.g., B. licheniformis 41P disclosed in DD 266,710
published Apr. 12, 1989), Pseudomonas such as P. aeruginosa, and
Streptomyces. One suitable E. coli cloning host is E. coli 294
(ATCC 31,446), although other strains such as E. coli B, E. coli
X1776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325) are suitable.
These examples are illustrative rather than limiting.
[0152] In addition to prokaryotes, eukaryotic microbes such as
filamentous fungi or yeast are suitable cloning or expression hosts
for polypeptide encoding vectors. Saccharomyces cerevisiae, or
common baker's yeast, is the most commonly used among lower
eukaryotic host microorganisms. However, a number of other genera,
species, and strains are commonly available and useful herein, such
as Schizosaccharomyces pombe; Kluyveromyces hosts such as, e.g., K.
lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K.
wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum
(ATCC 36,906), K. thermotolerans, and K. marxianus; yarrowia (EP
402,226); Pichia pastoris (EP 183,070); Candida; Trichoderma reesia
(EP 244,234); Neurospora crassa; Schwanniomyces such as
Schwanniomyces occidentalis; and filamentous fungi such as, e.g.,
Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts such
as A. nidulans and A. niger.
[0153] Suitable host cells for the expression of glycosylated
proteins, for example, glycosylated antibodies, are derived from
multicellular organisms. Examples of invertebrate cells include
plant and insect cells. Numerous baculoviral strains and variants
and corresponding permissive insect host cells from hosts such as
Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito),
Aedes albopictus (mosquito), Drosophila melanogaster (fruitfly),
and Bombyx mori have been identified. A variety of viral strains
for transfection are publicly available, e.g., the L-1 variant of
Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV,
and such viruses may be used as the virus herein according to the
present invention, particularly for transfection of Spodoptera
frugiperda cells. Plant cell cultures of cotton, corn, potato,
soybean, petunia, tomato, and tobacco can also be utilized as
hosts.
[0154] Mammalian cells can be used for expression and production of
the recombinant protein used in the low AR compositions of the
invention, however other eukaryotic cell types can also be employed
in the context of the instant invention. See, e.g., Winnacker, From
Genes to Clones, VCH Publishers, N.Y., N.Y. (1987). Suitable
mammalian host cells for expressing recombinant proteins according
to the invention include Chinese Hamster Ovary (CHO cells)
(including dhfr-CHO cells, described in Urlaub and Chasin, (1980)
PNAS USA 77:4216-4220, used with a DHFR selectable marker, e.g., as
described in Kaufman and Sharp (1982) Mol. Biol. 159:601-621, the
entire teachings of which are incorporated herein by reference),
NS0 myeloma cells, COS cells and SP2 cells. When recombinant
expression vectors encoding protein genes are introduced into
mammalian host cells, the antibodies are produced by culturing the
host cells for a period of time sufficient to allow for expression
of the antibody in the host cells or secretion of the antibody into
the culture medium in which the host cells are grown. Other
examples of useful mammalian host cell lines are monkey kidney CV1
line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic
kidney line (293 or 293 cells subcloned for growth in suspension
culture, Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster
kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells/-DHFR
(CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980));
mouse sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980));
monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney
cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells
(HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34);
buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells
(W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse
mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al.,
Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells;
and a human hepatoma line (Hep G2), the entire teachings of which
are incorporated herein by reference.
[0155] Host cells are transformed with the above-described
expression or cloning vectors for protein production and cultured
in conventional nutrient media modified as appropriate for inducing
promoters, selecting transformants, or amplifying the genes
encoding the desired sequences.
[0156] The host cells used to produce a protein may be cultured in
a variety of media. Commercially available media such as Ham's
F10.TM. (Sigma), Minimal Essential Medium.TM. (MEM), (Sigma),
RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium.TM.
(DMEM), (Sigma) are suitable for culturing the host cells. In
addition, any of the media described in Ham et al., Meth. Enz.
58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), U.S.
Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469;
WO 90/03430; WO 87/00195; or U.S. Pat. No. Re. 30,985 may be used
as culture media for the host cells, the entire teachings of which
are incorporated herein by reference. Any of these media may be
supplemented as necessary with hormones and/or other growth factors
(such as insulin, transferrin, or epidermal growth factor), salts
(such as sodium chloride, calcium, magnesium, and phosphate),
buffers (such as HEPES), nucleotides (such as adenosine and
thymidine), antibiotics (such as gentamycin drug), trace elements
(defined as inorganic compounds usually present at final
concentrations in the micromolar range), and glucose or an
equivalent energy source. Any other necessary supplements may also
be included at appropriate concentrations that would be known to
those skilled in the art. The culture conditions, such as
temperature, pH, and the like, are those previously used with the
host cell selected for expression, and will be apparent to the
ordinarily skilled artisan.
[0157] Host cells can also be used to produce portions of intact
proteins, for example, antibodies, including Fab fragments or scFv
molecules. It is understood that variations on the above procedure
are within the scope of the present invention. For example, in
certain embodiments it may be desirable to transfect a host cell
with DNA encoding either the light chain or the heavy chain (but
not both) of an antibody. Recombinant DNA technology may also be
used to remove some or all of the DNA encoding either or both of
the light and heavy chains that is not necessary for binding to an
antigen. The molecules expressed from such truncated DNA molecules
are also encompassed by the antibodies of the invention. In
addition, bifunctional antibodies may be produced in which one
heavy and one light chain are an antibody of the invention and the
other heavy and light chain are specific for an antigen other than
the target antibody, depending on the specificity of the antibody
of the invention, by crosslinking an antibody of the invention to a
second antibody by standard chemical crosslinking methods.
[0158] In a suitable system for recombinant expression of a
protein, for example, an antibody, or antigen-binding portion
thereof, a recombinant expression vector encoding the protein, for
example, both an antibody heavy chain and an antibody light chain,
is introduced into dhfr-CHO cells by calcium phosphate-mediated
transfection. Within the recombinant expression vector, the protein
gene(s) are each operatively linked to CMV enhancer/AdMLP promoter
regulatory elements to drive high levels of transcription of the
gene(s). The recombinant expression vector also carries a DHFR
gene, which allows for selection of CHO cells that have been
transfected with the vector using methotrexate
selection/amplification. The selected transformant host cells are
cultured to allow for expression of the protein, for example, the
antibody heavy and light chains, and intact protein, for example,
an antibody, is recovered from the culture medium. Standard
molecular biology techniques are used to prepare the recombinant
expression vector, transfect the host cells, select for
transformants, culture the host cells and recover the protein from
the culture medium.
[0159] When using recombinant techniques, the protein, for example,
antibodies or antigen binding fragments thereof, can be produced
intracellularly, in the periplasmic space, or directly secreted
into the medium. In one aspect, if the protein is produced
intracellularly, as a first step, the particulate debris, either
host cells or lysed cells (e.g., resulting from homogenization),
can be removed, e.g., by centrifugation or ultrafiltration. Where
the protein is secreted into the medium, supernatants from such
expression systems can be first concentrated using a commercially
available protein concentration filter, e.g., an Amicon.TM. or
Millipore Pellicon.TM. ultrafiltration unit.
[0160] Some antibodies can be secreted directly from the cell into
the surrounding growth media; others are made intracellularly. For
antibodies made intracellularly, the first step of a purification
process typically involves: lysis of the cell, which can be done by
a variety of methods, including mechanical shear, osmotic shock, or
enzymatic treatments. Such disruption releases the entire contents
of the cell into the homogenate, and in addition produces
subcellular fragments that are difficult to remove due to their
small size. These are generally removed by differential
centrifugation or by filtration. Where the antibody is secreted,
supernatants from such expression systems are generally first
concentrated using a commercially available protein concentration
filter, e.g., an Amicon.TM. or Millipore Pellicon.TM.
ultrafiltration unit. Where the antibody is secreted into the
medium, the recombinant host cells can also be separated from the
cell culture medium, e.g., by tangential flow filtration.
Antibodies can be further recovered from the culture medium using
the antibody purification methods of the invention.
III. Preparation of Low AR Compositions Using Displacement
Chromatography
[0161] In certain embodiments, the low AR compositions of the
present invention may be produced using downstream process
technologies (e.g., displacement chromatography), following cell
culture of a protein.
[0162] The methods described herein for the production of
compositions comprising low AR and/or low process-related
impurities comprise the purification of a protein, such as an
antibody or antigen-binding portion thereof, using displacement
chromatography.
[0163] The methods described herein for the production of
compositions comprising low AR and/or low process-related
impurities also comprise the purification of a protein, such as an
antibody or antigen-binding portion thereof, using displacement
chromatography in combination with, for example, other types of
chromatography, such as multimodal (MM) chromatography, wherein the
MM media comprises both ion exchange and hydrophobic interaction
functional groups, and an aqueous salt solution. In one embodiment,
the same or substantially the same aqueous salt solution is used as
a loading buffer and a wash buffer.
[0164] In further embodiments, the methods described herein for the
production of compositions comprising low AR and/or low
process-related impurities comprise the purification of a protein,
such as an antibody or antigen-binding portion thereof, using
displacement chromatography in combination with chromatography
comprising an anion exchange (AEX) resin and an aqueous salt
solution. In one embodiment, the same or substantially the same
aqueous salt solution is used as a loading buffer and a wash
buffer.
[0165] In yet further embodiments, the methods described herein for
the production of compositions comprising low AR and/or low
process-related impurities comprise the purification of a protein,
such as an antibody or antigen-binding portion thereof, using
displacement chromatography in combination with chromatography
comprising a cation exchange (CEX) adsorbent resin and an aqueous
salt solution. In one embodiment, the same or substantially the
same aqueous salt solution is used as a loading buffer and a wash
buffer, and the target protein bound to the CEX adsorbent resin is
eluted with a buffer having a higher conductivity and/or pH than
the loading/wash buffer.
[0166] In still further embodiments, the methods described herein
for production of compositions comprising low AR and/or low
process-related impurities comprise the purification of a protein,
such as an antibody or antigen-binding portion thereof, using
displacement chromatography in combination with several media, for
example by using an anion exchange (AEX) resin, and chromatography
using a cation exchange (CEX) adsorbent resin, in a suitable
buffer, such as, for example, a Tris/Formate buffer system. In one
embodiment, the sample is purified affinity chromatography media,
e.g., Protein A, prior to the ion chromatography resins. For
example, in one embodiment, the methods described herein for
production of compositions comprising low AR comprise the exemplary
process reflected in FIG. 190 of U.S. Provisional Application No.
61/893,068, entitled "Low Acidic Species Compositions and Methods
for Producing and Using the Same", filed on Oct. 18, 2013, the
entire contents of which are expressly incorporated herein by
reference.
[0167] In one embodiment, the method for producing a low AR
composition comprising an antibody, or antigen binding portion
thereof, comprises contacting a first sample comprising the
antibody, or antigen binding portion thereof, to affinity
chromatography media in a load buffer (for example a low
concentration Tris/Formate buffer), and eluting the sample from the
affinity chromatography media as a first eluted sample, contacting
the first eluted sample to a first chromatography media, such as an
AEX chromatography resin, in a load buffer, and eluting the sample
from the AEX chromatography resin as a second eluted sample. The
second eluted sample is then contacted with a second chromatography
media, such as a CEX chromatography resin, in a load buffer, and
the sample is eluted from the CEX chromatography resin as a third
eluted sample. In one embodiment, the CEX chromatography resin is
eluted one, two, three or more times. In one embodiment, the
process optionally includes one or more intermediate filtration
steps, pH adjustment steps and inactivation steps.
[0168] In one embodiment, the displacement chromatography steps,
alone or in combination with other purification steps, produce a
low AR composition comprising an antibody, or antigen binding
portion thereof, which contains 15% or less AR, 14% or less AR, 13%
or less AR, 12% or less AR, 11% or less AR, 10% or less AR, 9% or
less AR, 8% or less AR, 7% or less AR, 6% or less AR, 5% or less
AR, 4.5% or less AR, 4% or less AR, 3.5% or less AR, 3% or less AR,
2.5% or less AR, 2% or less AR, 1.9% or less AR, 1.8% or less AR,
1.7% or less AR, 1.6% or less AR, 1.5% or less AR, 1.4% or less AR,
1.3% or less AR, 1.2% or less AR, 1.1% or less AR, 1% or less AR,
0.9% or less AR, 0.8% or less AR, 0.7% or less AR, 0.6% or less AR,
0.5% or less AR, 0.4% AR, 0.3% or less AR, 0.2% or less AR, 0.1% or
less AR, or 0.0% AR, and ranges within one or more of the
preceding. In one aspect of this embodiment, the low AR composition
of the invention comprises about 0.0% to about 10% AR, about 0.0%
to about 5% AR, about 0.0% to about 4% AR, about 0.0% to about 3%
AR, about 0.0% to about 2% AR, about 3% to about 5% AR, about 5% to
about 8% AR, or about 8% to about 10% AR, or about 10% to about 15%
AR, and ranges within one or more of the preceding.
[0169] In one embodiment, the displacement chromatography steps,
alone or in combination with other purification steps, produce a
low AR composition comprising an antibody, or antigen binding
portion thereof, which contains 15% or less AR1, 14% or less AR1,
13% or less AR1, 12% or less AR1, 11% or less AR1, 10% or less AR1,
9% or less AR1, 8% or less AR1, 7% or less AR1, 6% or less AR1, 5%
or less AR1, 4.5% or less AR1, 4% or less AR1, 3.5% or less AR1, 3%
or less AR1, 2.5% or less AR1, 2% or less AR1, 1.9% or less AR1,
1.8% or less AR1, 1.7% or less AR1, 1.6% or less AR1, 1.5% or less
AR1, 1.4% or less AR1, 1.3% or less AR1, 1.2% or less AR1, 1.1% or
less AR1, 1% or less AR1, 0.9% or less AR1, 0.8% or less AR1, 0.7%
or less AR1, 0.6% or less AR1, 0.5% or less AR1, 0.4% or less AR1,
0.3% or less AR1, 0.2% or less AR1, 0.1% or less AR1, or 0.0% AR1,
and ranges within one or more of the preceding. In one aspect of
this embodiment, the low AR composition of the invention comprises
about 0.0% to about 10% AR1, about 0.0% to about 5% AR1, about 0.0%
to about 4% AR1, about 0.0% to about 3% AR1, about 0.0% to about 2%
AR1, about 3% to about 5% AR1, about 5% to about 8% AR1, or about
8% to about 10% AR1, or about 10% to about 15% AR1, and ranges
within one or more of the preceding.
[0170] In one embodiment, the displacement chromatography steps,
alone or in combination with other purification steps, produce a
low AR composition comprising an antibody, or antigen binding
portion thereof, which contains 15% or less AR2, 14% or less AR2,
13% or less AR2, 12% or less AR2, 11% or less AR2, 10% or less AR2,
9% or less AR2, 8% or less AR2, 7% or less AR2, 6% or less AR2, 5%
or less AR2, 4.5% or less AR2, 4% or less AR2, 3.5% or less AR2, 3%
or less AR2, 2.5% or less AR2, 2% or less AR2, 1.9% or less AR2,
1.8% or less AR2, 1.7% or less AR2, 1.6% or less AR2, 1.5% or less
AR2, 1.4% or less AR2, 1.3% or less AR2, 1.2% or less AR2, 1.1% or
less AR2, 1% or less AR2, 0.9% or less AR2, 0.8% or less AR2, 0.7%
or less AR2, 0.6% or less AR2, 0.5% or less AR2, 0.4% or less AR2,
0.3% or less AR2, 0.2% or less AR2, 0.1% or less AR2, or 0.0% AR2,
and ranges within one or more of the preceding. In one aspect of
this embodiment, the low AR composition of the invention comprises
about 0.0% to about 10% AR2, about 0.0% to about 5% AR2, about 0.0%
to about 4% AR2, about 0.0% to about 3% AR2, about 0.0% to about 2%
AR2, about 3% to about 5% AR2, about 5% to about 8% AR2, or about
8% to about 10% AR2, or about 10% to about 15% AR2, and ranges
within one or more of the preceding.
[0171] Protein Purification Generally
[0172] Following upstream processing of a protein of interest,
downstream process technologies can be used to purify the protein.
For example, but not by way of limitation, once a clarified
solution or mixture comprising the protein of interest, for
example, an antibody or antigen binding fragment thereof, has been
obtained, separation of the protein of interest from the
process-related impurities, aggregates, fragments, and/or charge
variant species (e.g., acidic species and basic species) can be
effected using displacement chromatography, either alone or in
combination with different purification techniques, including, but
not limited to, affinity separation steps, ion exchange separation
steps, mixed mode separation steps, and hydrophobic interaction
separation steps, singularly or in combination. The separation
steps separate mixtures of proteins on the basis of their charge,
degree of hydrophobicity, or size, or any combination thereof,
depending on the particular form of separation, including
chromatographic separation. In one aspect of the invention,
separation is performed using chromatography, including cationic,
anionic, and hydrophobic interaction. Several different
chromatography resins are available for each of these techniques,
allowing accurate tailoring of the purification scheme to the
particular protein involved. Each of the separation methods result
in the protein traversing at different rates through a column, to
achieve a physical separation that increases as they pass further
through the column, or adhere selectively to the separation medium.
The proteins are then differentially eluted by different solvents.
In some cases, the antibody is separated from impurities when the
impurities preferentially adhere to the column and the antibody
less so, i.e., the desired antibody variant is present in the Flow
Through.
[0173] In certain embodiments, a low AR composition is produced
using displacement chromatography to identify the particular
conditions, e.g., displacers, displacer concentration, salt
concentration, pH, temperature, load amount and conditions, and
washing conditions, sufficient to elicit the desired fractionation
profile, e.g., fractionation of acidic species and lysine variants,
of a sample comprising the protein of interest and at least one
process-related impurity. In certain embodiments, the method
further comprises pooling the resulting fractions comprising the
desired low AR composition.
[0174] The purification process may begin at the separation step
after the antibody has been produced using upstream production
methods described herein and in U.S. Provisional Application No.
61/893,068, entitled "Low Acidic Species Compositions and Methods
for Producing and Using the Same", filed on Oct. 18, 2013, the
entire contents of which are incorporated herein by reference,
and/or by alternative production methods conventional in the art.
Once a clarified solution or mixture comprising the protein of
interest, e.g., an antibody, has been obtained, separation of the
protein of interest from process-related impurities, such as the
other proteins produced by the cell, as well as product-related
substances, such as acidic or basic variants, is performed.
[0175] In certain non-limiting embodiments, such separation is
performed using cation exchange (CEX), anion exchange (AEX), and/or
mixed mode (MM) chromatography. In certain embodiments, a
combination of one or more different purification techniques,
including affinity separation step(s), ion exchange separation
step(s), mixed-mode step(s), and/or hydrophobic interaction
separation step(s) can also be employed. Such additional
purification steps separate mixtures of proteins on the basis of
their charge, degree of hydrophobicity, and/or size. In one aspect
of the invention, such additional separation steps are performed
using chromatography, including hydrophobic, anionic or cationic
interaction (or a combination thereof). Numerous chromatography
resins are commercially available for each of these techniques,
allowing accurate tailoring of the purification scheme to the
particular protein involved. Each of the separation methods allow
proteins to either traverse at different rates through a column,
achieving a physical separation that increases as they pass further
through the column, or to adhere selectively to a separation resin
(or medium). The proteins are then differentially eluted using
different eluents. In some cases, the protein of interest is
separated from impurities when the impurities specifically adhere
to the column's resin and the protein of interest does not, i.e.,
the protein of interest is contained in the effluent, while in
other cases the protein of interest will adhere to the column's
resin, while the impurities and/or product-related substances are
extruded from the column's resin during a wash cycle.
[0176] Primary Recovery and Virus Inactivation
[0177] In certain embodiments, the initial steps of the
purification methods of the present invention involve the
clarification and primary recovery of antibody from a sample
matrix. In certain embodiments, the primary recovery will include
one or more centrifugation steps to separate the antibody product
from the cells and cell debris. Centrifugation of the sample can be
performed at, for example, but not by way of limitation,
7,000.times.g to approximately 12,750.times.g. In the context of
large scale purification, such centrifugation can occur on-line
with a flow rate set to achieve, for example, but not by way of
limitation, a turbidity level of 150 NTU in the resulting
supernatant. Such supernatant can then be collected for further
purification, or in-line filtered through one or more depth filters
for further clarification of the sample.
[0178] In certain embodiments, the primary recovery will include
the use of one or more depth filtration steps to clarify the sample
matrix and thereby aid in purifying the antibodies of interest in
the present invention. In other embodiments, the primary recovery
will include the use of one or more depth filtration steps post
centrifugation to further clarify the sample matrix. Non-limiting
examples of depth filters that can be used in the context of the
instant invention include the Millistak+X0HC, F0HC, D0HC, A1HC,
B1HC depth filters (EMD Millipore), Cuno.TM. model 30/60ZA, 60/90
ZA, VR05, VR07, delipid depth filters (3M Corp.). A 0.2 .mu.m
filter such as Sartorius's 0.45/0.2 .mu.m Sartopore.TM. bi-layer or
Millipore's Express SHR or SHC filter cartridges typically follows
the depth filters.
[0179] In certain embodiments, the primary recovery process can
also be a point at which to reduce or inactivate viruses that can
be present in the sample matrix. For example, any one or more of a
variety of methods of viral reduction/inactivation can be used
during the primary recovery phase of purification including heat
inactivation (pasteurization), pH inactivation, buffer/detergent
treatment, UV and .gamma.-ray irradiation and the addition of
certain chemical inactivating agents such as .beta.-propiolactone
or e.g., copper phenanthroline as described in U.S. Pat. No.
4,534,972. In certain embodiments of the present invention, the
sample matrix is exposed to detergent viral inactivation during the
primary recovery phase. In other embodiments, the sample matrix may
be exposed to low pH inactivation during the primary recovery
phase.
[0180] In those embodiments where viral reduction/inactivation is
employed, the sample mixture can be adjusted, as needed, for
further purification steps. For example, following low pH viral
inactivation, the pH of the sample mixture is typically adjusted to
a more neutral pH, e.g., from about 4.5 to about 8.5, prior to
continuing the purification process. Additionally, the mixture may
be diluted with water for injection (WFI) to obtain a desired
conductivity.
[0181] Affinity Chromatography
[0182] In certain embodiments, it will be advantageous to subject a
sample produced by the techniques of the instant invention to
affinity chromatography to further purify the protein of interest
away from acidic species before a displacement chromatography step.
In certain embodiments the chromatographic material is capable of
selectively or specifically binding to the protein of interest
("capture"). Non-limiting examples of such chromatographic material
include: Protein A, Protein G, chromatographic material comprising,
for example, an antigen bound by an antibody of interest, and
chromatographic material comprising an Fc binding protein. In
specific embodiments, the affinity chromatography step involves
subjecting the primary recovery sample to a column comprising a
suitable Protein A resin. In certain embodiments, Protein A resin
is useful for affinity purification and isolation of a variety of
antibody isotypes, particularly IgG1, IgG2, and IgG4. Protein A is
a bacterial cell wall protein that binds to mammalian IgGs
primarily through their Fc regions. In its native state, Protein A
has five IgG binding domains as well as other domains of unknown
function.
[0183] There are several commercial sources for Protein A resin.
One suitable resin is MabSelect.TM. from GE Healthcare. Suitable
resins include, but not limited to, MabSelect SuRe.TM., MabSelect
SuRe LX, MabSelect, MabSelect Xtra, rProtein A Sepharose from GE
Healthcare, ProSep HC, ProSep Ultra, and ProSep Ultra Plus from EMD
Millipore, MapCapture from Life Technologies. A non-limiting
example of a suitable column packed with MabSelect.TM. is an about
1.0 cm diameter x about 21.6 cm long column (.about.17 mL bed
volume). This size column can be used for small scale purifications
and can be compared with other columns used for scale ups. For
example, a 20 cm.times.21 cm column whose bed volume is about 6.6 L
can be used for larger purifications. Regardless of the column, the
column can be packed using a suitable resin such as
MabSelect.TM..
[0184] Protein A Affinity Chromatography
[0185] In certain embodiments, particularly where the protein of
interest is an antibody, the composition, e.g., a primary recovery
sample, is subjected to Protein A affinity chromatography before
displacement chromatography to produce the low AR compositions of
the invention. There are a variety of commercial sources for
Protein A resin. Suitable resins include, but not limited to,
MabSelect SuRe.TM., MabSelect SuRe LX, MabSelect, MabSelect Xtra,
rProtein A Sepharose from GE Healthcare, ProSep HC, ProSep Ultra,
and ProSep Ultra Plus from EMD Millipore, MapCapture from Life
Technologies.
[0186] The Protein A column can be equilibrated with a suitable
buffer prior to sample loading. Following the loading of the
column, the column can be washed one or multiple times using a
suitable sets of buffers. The Protein A column can then be eluted
using an appropriate elution buffer. For example, glycine-HCL or
citric acid can be used as an elution buffer. The eluate can be
monitored using techniques well known to those skilled in the art.
The eluate fractions of interest can be collected and then prepared
for further processing.
[0187] The Protein A eluate may subject to a viral inactivation
step either by detergent or low pH, provided this step is not
performed prior to the Protein A capture operation. A proper
detergent concentration or pH and time can be selected to obtain
desired viral inactivation results. After viral inactivation, the
Protein A eluate is usually pH and/or conductivity adjusted for
subsequent purification steps.
[0188] The Protein A eluate may be subjected to filtration through
a depth filter to remove turbidity and/or various impurities from
the antibody of interest prior to additional chromatographic
polishing steps. Examples of depth filters include, but not limited
to, Millistak+X0HC, F0HC, D0HC, A1HC, and B1HC Pod filters (EMD
Millipore), or Zeta Plus 30ZA/60ZA, 60ZA/90ZA, delipid, VR07, and
VR05 filters (3M). The Protein A eluate pool may need to be
conditioned to proper pH and conductivity to obtain desired
impurity removal and product recovery from the depth filtration
step.
[0189] The invention is not limited to capture of the protein of
interest using Protein A chromatography. A non-Protein A
chromatography capture step can also be carried out. For example,
cation exchange capture and non-chromatographic methods, such as
aqueous two phase extraction or precipitation, or other methods
known in the art, can be used,
[0190] Displacement Chromatography
[0191] In certain embodiments of the present invention, a protein
sample, e.g., a primary recovery sample from cell culture, a
Protein A eluate sample, or a sample having undergone one or more
of the other purification strategies outlined herein, is subjected
to displacement chromatography to produce the low AR compositions
of the invention. In certain embodiments, the displacer molecule is
selected to have a higher affinity for the stationary phase (i.e.,
the chromatographic support) than the components present in the
material to be separated. In certain embodiments, the displacer
induces the components of the mixture to develop into consecutive
zones of concentrated and purified species in the order of
decreasing binding affinity ahead of the displacer front (a
"displacement train"). In certain embodiments, the displacement
process allows for higher column loading levels (as compared to
conventional high-resolution chromatographic separations such as
bind and linear gradient elution mode) without compromising the
purity and recovery of the component of interest. In certain
embodiments, washing of the displacement train from the column
using the displacer solution allows for the component of interest
to be isolated by collecting (and pooling if necessary) the proper
fraction(s) of the displaced eluate. Along with acidic species,
other product-related substances, such as basic species, product
aggregates, and/or product fragments, and process-related
impurities, such as HCPs, can be selectively collected or reduced
using displacement chromatography.
[0192] Displacement chromatography in described, in general, in
Brgles et al., Journal of Chromatography A, 1218 (2011) 2389-2395;
Gajdosik et al., Journal of Chromatography A, 1239 (2012) 1-9;
Gerstner et al., Biotechnol. Prog., (1992), 8, 540-545; Kundu et
al., Analytical Biochemistry, 248, 111-116, (1997); and Vogt et
al., Journal of Chromatography A, 760 (1997) 125-137.
[0193] In certain embodiments, the displacer will be employed in
the context of an ion exchange, e.g., anion exchange or cation
exchange, or mixed mode chromatographic separation. A detailed
description of ion exchange chromatography and a listing of
exemplary chromatographic supports which can be employed in the
context of displacement chromatography are presented below. A
detailed description of mixed mode chromatography and a listing of
exemplary chromatographic supports which can be employed in the
context of displacement chromatography are also presented below. In
certain non-limiting embodiments, a cation exchange, an anion
exchange, or a mixed mode displacement chromatography step is
employed to effectively reduce product-related substances (e.g.,
acidic species and/or basic species such as lysine variant species)
from, e.g., a monoclonal antibody feed stream. In specific
embodiments, conventional (or relatively weak) binding conditions
can be employed and cationic molecules having high affinity for a
CEX, AEX, or multimodal ligand (such as Expell SP1.TM. and
protamine sulfate) can be employed to induce the formation of a
product-related substances displacement train. In certain of such
embodiments, the acidic species variant population is enriched in
the front followed by the main isoform, and, thereafter, the basic
population. Thus, in certain embodiments, exclusion of those
earlier fractions from the remainder eluate results in an
AR-reduced product. Alternatively, exclusion of the fractions
following the main isoform results in a lysine species
variant-reduced product. In certain embodiments, the fragments and
aggregates are reduced in an AR-reduced product. In certain
embodiments, the HCPs are reduced in an AR-reduced product.
[0194] In certain embodiments, the displacer concentration will be
selected from the range of about 0.1 mM to about 10 mM, or about
0.25 mM to about 10 mM. In certain embodiments, the displacer
concentration will be selected from a range of about 0.1 mM to
about 5 mM, or about 0.25 mM to about 3 mM. In certain embodiments,
the displacer concentration will be selected from a range of about
0.1 mM to about 5 mM, or about 0.25 mM to about 2 mM. In certain
embodiments, the displacer concentration will be selected from a
range of about 0.1 mM to about 2 mM, or about 0.25 mM to about 1
mM. In certain embodiments, the displacer concentration will be
selected from a concentration of about 0.1 mM to about 1 mM, or
about 0.25 mM to about 0.5 mM.
[0195] In certain embodiments the displacer is Expell SP1.TM. and
the displacer concentration will be selected from the range of
about 0.1 mM to about 10 mM, or about 0.25 mM to about 10 mM. In
certain embodiments, the displacer is protamine sulfate and the
displacer concentration will be selected from the range of about
0.1 mM to about 5 mM, or about 0.25 mM to about 5 mM.
[0196] In certain embodiments, a displacing buffer is used in
one-step displacement process. In certain embodiments, the total
volume of the one-step displacing buffer is in the range of about
20 CVs to about 50 CVs, or about 25 CVs to about 40 CVs, or about
30 CVs. In another embodiment, the total volume of the one-step
displacing buffer is about 15 CVs, about 20 CVs, about 25 CVs,
about 30 CVs, about 35 CVs, about 40 CVs, about 45 CVs, or about 50
CVs.
[0197] Although displacement chromatography conventionally employs
a displacer at a fixed concentration to achieve component
separation, an improved method using multiple displacing buffers is
also disclosed herein. For example, a two-step displacement method
is employed where a first displacer concentration is employed for a
certain initial number of column volumes (CVs) and a second,
higher, displacer concentration is employed for a subsequent number
of CVs. The total volume of the displacing buffers needed to
complete the displacement process is significantly (e.g., 25-45%)
less than that needed when using one displacing buffer in the
one-step displacement process in order to achieve comparable
separation performances. In certain embodiments, the first
displacer concentration is about 0.25 mM, about 0.3 mM, about 0.35
mM, about 0.4 mM, about 0.45 mM, or about 0.5 mM. In certain
embodiments, the second displacer concentration is about 0.5 mM,
about 1 mM, about 1.5 mM, about 2 mM, about 2.5 mM, about 3 mM,
about 3.5 mM, about 4 mM, about 4.5 mM, or about 5 mM.
[0198] In certain embodiments, a two-step displacement method is
employed where the first displacer concentration is employed for up
to about 10 CVs. In certain embodiments, the first displacer
concentration is employed for up to about 25 CVs. In another
embodiments, the first displacer concentration is employed for up
to about 10, 11 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24
or 25 CVs.
[0199] In certain embodiments, the second displacer concentration
is employed for up to about 10 CVs. In certain embodiments, the
second displacer concentration is employed for up to about 25 CVs.
In another embodiment, the second displacer concentration is
employed for up to about 10, 11 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24 or 25 CVs.
[0200] In certain embodiments, the total required displacing buffer
volume is about 13 CVs for a two-step displacement process. In
certain embodiments, the total required displacer buffer volume is
about 15 CVs for a two-step displacement process. In certain
embodiments, the total required displacer buffer volume is about 33
CVs for a two-step displacement process. In another embodiment, the
total required displacing buffer volume is about 10, 11, 12, 13,
14, 115, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,
30, 31, 32, 33, 34 or 35 CVs for a two-step displacement
process.
[0201] One of skill in the art would understand that further
reduction in required buffer volumes for each displacement step is
expected. In certain embodiments, multiple steps of increasing
displacer concentration are employed. As outlined in the Examples
section, below, incorporation of additional displacement
concentration steps into the purification strategy can allow for
unexpectedly efficient charge variant, product aggregate, product
fragment, and/or HCP clearance. For example, in one embodiment,
three, four, five, six, seven, eight, nine or ten displacement
concentration steps are used.
[0202] In certain embodiments, a linear gradient displacement
method is employed where an initial, low, displacer concentration
is followed by the addition of displacer at increasing
concentrations in accordance with a linear gradient. For example,
but not by way of limitation, the displacer concentration can range
from about 0 mM to about 1 mM over the course of about 40 CVs.
Again, as outlined in the Examples section, below, incorporation of
a linear displacer concentration gradient into the purification
strategy can allow for unexpectedly efficient charge variant,
product aggregate, product fragment, and/or HCP clearance.
[0203] In certain embodiments, a displacement buffer consisting of
two or more displacers is used. In certain embodiments, different
displacers are used in the multi-step displacement process. For
example, a displacement buffer consisting of two, three, four,
five, six, seven, eight, nine or ten displacers may be used.
Alternatively, two, three, four five, six, seven, eight, nine or
ten displacers may be used in a multi-step displacement
process.
[0204] In certain embodiments of the present invention, the pH of
the displacing wash buffer is below the pI of the protein of
interest. In certain embodiments, the pH of the displacing wash
buffer is in the range of about 5.0 to about 9.0, about 6.0 to
about 8.0, about 7.0 to about 7.7, or about 7.5 to about 7.7. In
another embodiment, the pH of the displacing wash buffer is about
5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3,
6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6,
7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, or
9.0.
[0205] In certain embodiments of the present invention, the
conductivity of the displacing wash buffer is between about 1 to
about 86 mS/cm, about 2 to about 20 mS/cm, about 2 to about 7
mS/cm, or about 5 to about 6.6 mS/cm. In another embodiment, the
conductivity of the displacing wash buffer is about 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35,
40, 45, 50, 55, 60, 65, 70, 75, 80, 85 or 86 mS/cm.
[0206] In certain embodiments of the present invention, the column
bed height is between about 10 cm to about 30 cm, about 15 cm to
about 25 cm, about 20 cm to about 30 cm, or about 25 cm. In another
embodiment, the column bed height is about 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,
33, 34 or 35 cm.
[0207] In certain embodiments of the present invention, the flow
residence time is between about 2 minutes to about 25 minutes,
about 5 minutes to about 20 minutes, about 10 minutes to about 20
minutes, or about 15 minutes to about 20 minutes. In another
embodiment, the flow residence time is about 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or
25 minutes.
[0208] In certain embodiments, the displacer buffer pH and
displacer concentration can affect the displacement profile and, as
a result, impact clearance of process-related impurities and/or
product-related substances, such as charge variants, in unexpected
ways. Thus, effective operating regimes with regard to the
reduction of process-related impurities and/or product-related
substances depend on the specific protein-resin-displacer system.
For example, but not by way of limitation, when a feed stream
containing Adalimumab is separated using displacement
chromatography, significant AR reduction (.DELTA.AR %) can be
achieved using a displacing buffer with pH in the range of 6-8 with
displacer concentration as low as 0.25-0.5 mM. In fact, as
described in Example 2 below, the extent of Adalimumab AR reduction
increases significantly as pH varies from 6.5 to 7.5, for example,
over a 6% decrease in AR level can be achieved at pH 7.5 with a
product yield .about.75%. In certain embodiments, as outlined in
the Examples presented below, the total AR level (%) in Adalimumab
product pool can be reduced by over 10% with an acceptable
processing yield (.gtoreq.75%) from a CEX displacement
chromatography process, or 4-7% from a mixed mode displacement
chromatography process. Similarly, for mAb X, FIG. 32 indicates
that .DELTA.AR % surprisingly increases from 3.3 to 6.5% as pH
varies from 7 to 7.7 in a mixed mode displacement chromatography
process.
[0209] In certain embodiments, conditions selected for reducing AR
are also capable of reducing process-related impurities and/or
other product-related substances. For example, but not by way of
limitation, conditions selected for AR reduction are also capable
of reducing process-related impurities, such as HCPs. In
additional, non-limiting examples, conditions selected for AR
reduction are also capable of reducing product-related impurities,
such as aggregates and/or fragments.
[0210] In certain embodiments, displacement chromatography can be
used as the sole method of purification of the protein of interest.
In certain embodiments, displacement chromatography can be used in
combination with other purification strategies, such as, but not
limited to, the alternative purification techniques described
herein, to reduce process-related impurities and/or other
product-related substances. In one embodiment, displacement
chromatography is used following Protein A affinity steps.
[0211] In certain embodiments, fractions are collected during the
displacement step and are combined (pooled) after appropriate
analysis to provide a protein preparation, which is also referred
to herein as a purified or partially-purified sample, that contains
a desired level of the protein of interest and which can include
one or more process-related impurities and/or other product-related
substances. In certain embodiments, one or more process monitoring
tools can be used in connection with the techniques described
herein to facilitate the identification of an effective product
pooling strategy. In certain embodiments, such monitoring can
include on-line or in-line process monitoring. For example, but not
by way of limitation, spectroscopy methods such as UV, NIR, FTIR,
Fluorescence, and Raman may be used to monitor levels of
product-related species, e.g., acidic species and lysine variants,
in an on-line, at line or in-line mode. These methods allow for the
production of data that can then be used to control the level of
product-related species in the pooled material collected. In
certain embodiments, specific signals arising from the chemical
modification of the proteins such as glycation, MGO modification,
deamidation, glycosylation may be specifically measurable by
spectroscopic methods through such in-line, on-line or at-line
methods, enabling real time or near-real time control of product
quality of the resulting product.
[0212] In certain embodiments, the purification and/or pooling
techniques described herein allow for the reduction of
process-related impurities and/or other product-related substances.
In certain embodiments, the purification and/or pooling techniques
described herein allow for reduction of process-related impurities
and the selective inclusion of particular product-related
substances. For example, but not by way of limitation, the
purification and/or pooling techniques described herein allow for
modulation of the concentration of product-related substances in
the purified sample, e.g., increasing or decreasing the amount of
AR and/or basic species. In certain embodiments, the concentration
of particular AR and/or basic species, e.g., Lys 0, Lys 1, and/or
Lys 2, are modulated (increased or decreased) in the purified
sample. In certain embodiments, such techniques can be used to
ensure product uniformity over the course of multiple production
runs.
[0213] Anion Exchange (AEX) Chromatography
[0214] In certain embodiments, the low AR compositions of the
invention are produced by subjecting a primary protein recovery
sample to at least one anion exchange separation step after the
above-described displacement chromatography step. In another
embodiment, the anion exchange step will occur before the
above-described displacement chromatography step. In one
embodiment, the anion exchange chromatography step will occur after
the above-described Protein A affinity and displacement
chromatography steps.
[0215] The use of an anionic exchange material versus a cationic
exchange material, such as those cation exchange materials
discussed in detail below, is based on the local charges of the
protein of interest in a given solution. Therefore, it is within
the scope of this invention to employ an anionic exchange step
prior to the use of a cationic exchange step, or a cationic
exchange step prior to the use of an anionic exchange step.
Furthermore, it is within the scope of this invention to employ
only an anionic exchange step, only an cationic exchange step, or
any serial combination of the two (including serial combinations of
one or both ion exchange steps with the other chromatographic
separation technologies described herein).
[0216] In performing the separation, the initial protein
composition can be contacted with the anion exchange material by
using any of a variety of techniques, e.g., using a batch
purification technique or a chromatographic technique.
[0217] For example, in the context of batch purification, anion
exchange material is prepared in, or equilibrated to, the desired
starting buffer. Upon preparation, or equilibration, a slurry of
the anion exchange material is obtained. The protein of interest,
e.g., antibody, solution is contacted with the slurry to allow for
protein adsorption to the anion exchange material. The solution
comprising the acidic species that do not bind to the AEX material
is separated from the slurry, e.g., by allowing the slurry to
settle and removing the supernatant. The slurry can be subjected to
one or more washing steps and/or elution steps.
[0218] In the context of chromatographic separation, a
chromatographic apparatus, commonly cylindrical in shape, is
employed to contain the chromatographic support material (e.g., AEX
material) prepared in an appropriate buffer solution. The
chromatographic apparatus, if cylindrical, can have a diameter of
about 5 mm to about 2 meters, and a height of 5 cm to 50 cm, and in
certain embodiments, particularly for large scale processing, a
height of .ltoreq.30 cm is employed. Once the chromatographic
material is added to the chromatographic apparatus, a sample
containing the protein of interest, e.g., an antibody, is contacted
to the chromatographic material to induce the separation. Any
portion of the solution that does not bind to the chromatographic
material, e.g., which may comprise, depending on the AEX material
being employed, the protein of interest, acidic species, is
separated from the chromatographic material by washing the material
and collecting fractions from column. The chromatographic material
can be subjected to one or more wash steps. If desired, the
chromatographic material can then be contacted with a solution
designed to desorb any components of the solution that have bound
to the chromatographic material.
[0219] In certain embodiments, a wash step can be performed in the
context of AEX chromatography using conditions similar to the load
conditions or alternatively by decreasing the pH and/or increasing
the ionic strength/conductivity of the wash in a step wise or
linear gradient manner. The resulting Flow Through and wash
fractions can be analyzed and appropriate fractions pooled to
achieve the desired reduction in charged variant species. In
certain embodiments, the aqueous salt solution used as both the
loading and wash buffer has a pH that at or near the isoelectric
point (pI) of the protein of interest. In certain embodiments the
pH is about 0 to 2 units higher or lower than the pI of the protein
of interest. In certain embodiments, it will be in the range of 0
to 0.5 units higher or lower. In certain embodiments, it will be at
the pI of the antibody.
[0220] In certain non-limiting embodiments, the anionic agent is
selected from the group consisting of acetate, formate, or
combinations thereof. In certain non-limiting embodiments, the
cationic agent is selected from the group consisting of Tris,
arginine, or combinations thereof. In one embodiment, the buffer
solution is a Tris/formate buffer. In another embodiment, the
buffer is selected from the group consisting of pyridine,
piperazine, L-histidine, Bis-tris, Bis-tris propane, imidazole,
N-Ethylmorpholine, TEA (triethanolamine), Tris, Morpholine,
N-Methyldiethanolamine, AMPD (2-amino-2-methyl-1,3-propanediol),
diethanolamine, ethanolamine, AMP (2-amino-2-methyl-1-propaol),
piperazine, 1,3-Diaminopropane and piperidine.
[0221] A packed anion-exchange chromatography column,
anion-exchange membrane device, anion-exchange monolithic device,
or depth filter media can be operated either in bind-elute mode,
flow-through mode, or a hybrid mode wherein the product exhibits
binding to the chromatographic material, yet can be washed from the
column using a buffer that is the same or substantially similar to
the loading buffer. In the bind-elute mode, the column or the
membrane device is first conditioned with a buffer with appropriate
ionic strength and pH under conditions where certain proteins will
be immobilized on the resin based matrix. For example, in certain
embodiments, during the feed load, the protein of interest will be
adsorbed to the resin due to electrostatic attraction. After
washing the column or the membrane device with the equilibration
buffer or another buffer with different pH and/or conductivity, the
product recovery is achieved by increasing the ionic strength
(i.e., conductivity) of the elution buffer to compete with the
solute for the charged sites of the anion exchange matrix. Changing
the pH and thereby altering the charge of the solute is another way
to achieve elution of the solute. The change in conductivity or pH
may be gradual (gradient elution) or stepwise (step elution). In
the flow-through mode, the column or the membrane device is
operated at selected pH and conductivity such that the protein of
interest does not bind to the resin or the membrane while the
acidic species will either be retained on the column or will have a
distinct elution profile as compared to the protein of interest. In
the context of this hybrid strategy, acidic species will bind to
the chromatographic material (or Flow Through) in a manner distinct
from the protein of interest, e.g., while the protein of interest
and certain aggregates and/or fragments of the protein of interest
may bind the chromatographic material, washes that preferentially
remove the protein of interest can be applied. The column is then
regenerated before next use.
[0222] Non-limiting examples of anionic exchange substituents
include diethylaminoethyl (DEAE), quaternary aminoethyl (QAE) and
quaternary amine (Q) groups. Additional non-limiting examples
include: Poros 50PI and Poros 50HQ, which are a rigid polymeric
bead with a backbone consisting of cross-linked
poly[styrene-divinylbenzene]; Capto Q Impres and Capto DEAE, which
are a high flow agarose bead; Toyopearl QAE-550, Toyopearl
DEAE-650, and Toyopearl GigaCap Q-650, which are a polymeric base
bead; Fractogel.RTM. EMD TMAE Hicap, which is a synthetic polymeric
resin with a tentacle ion exchanger; Sartobind STIC.RTM. PA nano,
which is a salt-tolerant chromatographic membrane with a primary
amine ligand; Sartobind Q nano; which is a strong anion exchange
chromatographic membrane; CUNO BioCap; which is a zeta-plus depth
filter media constructed from inorganic filter aids, refined
cellulose, and an ion exchange resin; and X0HC, which is a
depth-filter media constructed from inorganic filter aid,
cellulose, and mixed cellulose esters. The detailed information is
listed in Table A.
TABLE-US-00001 TABLE A List of AEX Adsorbent Properties Media
Particle/ AEX Adsorbent Vendor Type Ligand Type Pore Size Catalog
Number Poros PI Applied Resin Weak ~50 .mu.m 1-2459-11 Poros HQ
Biosystems Strong ~50 .mu.m 1-2559-11 Capto DEAE GE Weak ~90 .mu.m
17-5443-10 CaptoQ Impres Strong ~90 .mu.m 17-5316-10 QAE-550 Tosoh
Strong ~100 .mu.m 43271 DEAE-650 Weak ~65 .mu.m 43201 GigaCap Q-650
Strong ~75 .mu.m 21854 TMAE HiCap EMD/Millipore Strong ~40-90 .mu.m
1.16881.0013 Sartobind Sartorius Membrane Weak 3-5 .mu.m
92STPA42DN- STIC .RTM. PA Nano 11-A Sartobind Q Strong 3-5 .mu.m
92IEXQ42DN-11 Nano CUNO BioCap 3M Depth NA NA BC0025L60ZA05A 25
Filter X0HC Millipore NA NA MX0HC23CL3
[0223] In certain embodiments, the protein load of the mixture
comprising protein of interest is adjusted to a total protein load
to the column of between about 50 and 500 g/L, or between about 75
and 350 g/L, or between about 200 and 300 g/L. In certain
embodiments, the protein concentration of the load protein mixture
is adjusted to a protein concentration of the material loaded to
the column of about 0.5 and 50 g/L, between about 1 and 20 g/L, or
between 3 and 10 g/L. In certain embodiments, the protein
concentration of the load protein mixture is adjusted to a protein
centration of the material to the column of about 37 g/L.
[0224] In certain embodiments, additives such as poly ethylene
glycol, detergents, amino acids, sugars, chaotropic agents can be
added to enhance the performance of the separation, so as to
achieve better recovery or product quality.
[0225] In certain embodiments, including, but not limited to those
relating to adalimumab, the methods of the instant invention can be
used to selectively remove, significantly reduce, or essentially
remove all of AR in the Flow Through and wash fractions while
enriching for the same in the flow elution fraction, thereby
producing protein compositions that have reduced AR or are free of
AR. In certain embodiments relating to the purification of
adalimumab, the methods of the instant invention can be used to
selectively remove, significantly reduce, or essentially remove all
of AR1 charge variants in the Flow Through and wash fractions while
enriching for the same in the flow elution fraction, thereby
producing protein compositions that have reduced AR1 or are free of
AR1 variants. In certain embodiments relating to adalimumab, the
methods of the instant invention can be used to selectively remove,
significantly reduce, or essentially remove all of AR2 charge
variants in the flow-through and wash fractions while enriching for
the same in the flow elution fraction, thereby producing protein
compositions that have reduced AR2 or are free of AR2 variants.
[0226] In certain embodiments, including but not limited to those
relating to adalimumab, the methods of the instant invention can be
used to selectively remove, significantly reduce, or essentially
remove all of the MGO variants in the Flow Through and wash
fractions while enriching for the same in the elution fraction,
thereby producing protein compositions that have reduced MGO or are
free of MGO variants (for example, see U.S. Patent Application Ser.
No. 61/777,883, filed on Mar. 12, 2013). In certain embodiments,
including, but not limited to those relating to adalimumab, the
methods of the instant invention can be used to selectively remove,
significantly reduce, or essentially remove all of the glycated
variants (Schiff's base and permanently glycated forms) in the Flow
Through and wash fractions while enriching for the same in the
elution fraction, thereby producing protein preparations with
reduced or free of glycated variants.
[0227] In certain embodiments, the loading, pH, conductivity of the
AEX chromatography step, as well as elution pH conductivity, can be
modified to achieve a desired distribution of product-relates
substances (AR or lysine variants) For example, but not by way of
limitation, certain embodiments are directed to the modulation of
the lysine distribution of purified sample of a protein of
interest, e.g., increasing Lys 0 and decreasing Lys 1 and Lys 2. In
certain embodiments, the methods of the present invention allow for
the preparation of samples wherein the amount of Lys 0 is
decreased, while the amount of Lys 1 and/or Lys 2 is increased.
[0228] In certain embodiments, an AEX chromatographic separation
can be performed and combinations of fractions can be pooled to
achieve a combination of desired process-related impurity and/or
product-relates substance levels, in addition to, or in place of
merely modulating charge variant concentration.
[0229] Spectroscopy methods such as UV, NIR, FTIR, Fluorescence,
and Raman may be used to monitor levels of AR species in an
on-line, at-line or in-line mode, which can then be used to control
the level of charge variants, e.g., acidic species, in the pooled
material collected from the AEX effluent.
[0230] In certain embodiments, specific signals arising from the
chemical modification of the proteins such as glycation, MGO
modification, deamidation, glycosylation may be specifically
measurable by spectroscopic methods through such in-line, on-line
or at-line methods, enabling realtime or near-real time control of
product quality of the resulting product. In certain embodiments,
on-line, at-line or in-line monitoring methods can be used either
on the effluent line of the chromatography step or in the
collection vessel, to enable achievement of the desired product
quality/recovery. In certain embodiments, the UV signal can be used
as a surrogate to achieve an appropriate product quality/recovery,
wherein the UV signal can be processed appropriately, including,
but not limited to, such processing techniques as integration,
differentiation, moving average, such that normal process
variability can be addressed and the target product quality can be
achieved. In certain embodiments, such measurements can be combined
with in-line dilution methods such that ion
concentration/conductivity of the load/wash can be controlled by
feedback and hence facilitate product quality control.
[0231] In certain embodiments, a combination of AEX and CEX and MM
methods can be used to prepare product-related substance-modulated
materials, including certain embodiments where one technology is
used in a complementary/supplementary manner with another
technology. In certain embodiments, such a combination can be
performed such that certain sub-species are removed predominantly
by one technology, such that the combination provides the desired
final composition/product quality. In certain embodiments, such
combinations include the use of additional intervening
chromatography, filtration, pH adjustment, and/or UF/DF steps so as
to achieve the desired AR, product quality, ion concentration,
and/or viral reduction.
[0232] AEX chromatography can be used in conjunction with recycle
chromatography modes and continuous chromatography modes.
[0233] Cation Exchange (CEX) Chromatography
[0234] The low AR compositions of the invention can be produced by
subjecting the composition, e.g., a primary recovery sample, to at
least one cation exchange separation step after the above-described
displacement chromatography step. In another embodiment, the cation
exchange step will occur before the above-described displacement
chromatography step. In one embodiment, the cation exchange
chromatography step will occur after the above-described Protein A
affinity and displacement chromatography steps.
[0235] The use of a cationic exchange material versus an anionic
exchange material, such as those anionic exchange materials
discussed in detail above, is based on the local charges of the
protein of interest in a given solution. Therefore, it is within
the scope of this invention to employ a cationic exchange step
prior to the use of an anionic exchange step, or an anionic
exchange step prior to the use of a cationic exchange step.
Furthermore, it is within the scope of this invention to employ
only a cationic exchange step, only an anionic exchange step, or
any serial combination of the two (including serial combinations of
one or both ion exchange steps with the other chromatographic
separation technologies described herein).
[0236] In performing the separation, the initial protein mixture
can be contacted with the cation exchange material by using any of
a variety of techniques, e.g., using a batch purification technique
or a chromatographic technique, as described above in connection
with Protein A or AEX.
[0237] In certain embodiments, the aqueous salt solution used as
both the loading and wash buffer has a pH that is lower than the
isoelectric point (pI) of the protein of interest. In certain
embodiments, the pH is about 0 to 5 units lower than the pI of the
protein. In certain embodiments, it is in the range of 1 to 2 units
lower. In certain embodiments, it is in the range of 1 to 1.5 units
lower.
[0238] In certain embodiments, the concentration of the anionic
agent in aqueous salt solution is increased or decreased to achieve
a pH of between about 3.5 and 10.5, or between about 4 and 10, or
between about 4.5 and 9.5, or between about 5 and 9, or between
about 5.5 and 8.5, or between about 6 and 8, or between about 6.5
and 7.5. In certain embodiments, the concentration of anionic agent
is increased or decreased in the aqueous salt solution to achieve a
pH of 5, or 5.5, or 6, or 6.5, or 6.8, or 7.5. Buffer systems
suitable for use in the CEX methods include, but are not limited
to, tris formate, tris acetate, ammonium sulfate, sodium chloride
and sodium sulfate.
[0239] In certain embodiments, the conductivity and pH of the
aqueous salt solution is adjusted by increasing or decreasing the
concentration of a cationic agent. In certain embodiments, the
cationic agent is maintained at a concentration of between about
range of 20 mM to 500 mM, or between about 50 to 350 mM or between
about 100 to 300 mM or between about 100 to 200 mM.
[0240] In certain non-limiting embodiments, the cationic agent is
selected from the group consisting of sodium, Tris, tromethalmine,
ammonium, arginine, or combinations thereof. In certain
non-limiting embodiments, the anionic agent is selected from the
group consisting of formate, acetate, citrate, chloride anion,
sulphate, phosphate or combinations thereof.
[0241] A packed cation-exchange chromatography column or a
cation-exchange membrane device can be operated either in
bind-elute mode, flow-through mode, or a hybrid mode wherein the
product exhibits binding to the chromatographic material, yet can
be washed from the column using a buffer that is the same or
substantially similar to the loading buffer. The details of these
modes are outlined above.
[0242] Cationic substituents include carboxymethyl (CM), sulfoethyl
(SE), sulfopropyl (SP), phosphate (P) and sulfonate (S). Additional
cationic materials include, but are not limited to: Capto SP
ImpRes, which is a high flow agarose bead; CM Hyper D grade F;
which is a ceramic bead coated and permeated with a functionalized
hydrogel, 250-400 ionic groups .mu.eq/mL; Eshmuno S, which is a
hydrophilic polyvinyl ether base matrix with 50-100 .mu.eq/mL ionic
capacity; Nuvia C Prime, which is a hydrophobic cation exchange
media composed of a macroporous highly crosslinked hydrophilic
polymer matrix 55-75 .mu.eq/mL; Nuvia S, which has a UNOsphere base
matrix with 90-150 .mu.eq/mL ionic groups; Poros HS; which is a
rigid polymetic bead with a backbone consisting of cross-linked
poly[styrene-divinylbenzene]; Poros XS; which is a rigid polymetic
bead with a backbone consisting of cross-linked
poly[styrene-divinylbenzene]; Toyo Pearl Giga Cap CM 650M, which is
a polymeric base bead with 0.225 meq/mL ionic capacity; Toyo Pearl
Giga Cap S 650M which is a polymeric base bead; Toyo Pearl MX TRP,
which is a polymeric base bead. Detailed information concerning the
aforementioned materials is listed in Table B. It is noted that CEX
chromatography can be used with MM resins, described herein.
TABLE-US-00002 TABLE B Cationic Materials Particle Catalog Resin
Vendor Type Size Number Capto SP ImpRes GE Strong ~40 .mu.m
17-5468-10 CM Hyper D Pall Weak ~50 .mu.m 20050-027 Eshmuno S
Millipore Strong ~85 .mu.m 1.20078 Nuvia C Prime Biorad Mix ~70
.mu.m 156-3401 Mode Nuvia S Biorad Strong ~85 .mu.m 156-0315 Poros
HS Applied Weak ~50 .mu.m 13359-06 Biosystems Poros XS Applied
Strong ~50 .mu.m 4404337 Biosystems Toyo Pearl Giga Cap Tosoh Weak
~75 .mu.m 21946 CM 650M Toyo Pearl Giga Cap S Tosoh Strong ~75
.mu.m 21833 650M Toyo Pearl MX Trp Tosoh Mix ~75 .mu.m 22817 650M
Mode
[0243] In certain embodiments, the protein load of the mixture
comprising protein of interest is adjusted to a total protein load
to the column of between about 5 and 150 g/L, or between about 10
and 100 g/L, between about 20 and 80 g/L, between about 30 and 50
g/L, or between about 40 and 50 g/L. In certain embodiments, the
protein concentration of the load protein mixture is adjusted to a
protein concentration of the material loaded to the column of about
0.5 and 50 g/L, or between about 1 and 20 g/L.
[0244] In certain embodiments, additives such as poly ethylene
glycol, detergents, amino acids, sugars, chaotropic agents can be
added to enhance the performance of the separation, so as to
achieve better recovery or product quality.
[0245] In certain embodiments, including, but not limited to those
relating to adalimumab, the methods of the instant invention can be
used to selectively remove, significantly reduce, or essentially
remove all of AR in the Flow Through and wash fractions while
enriching for the same in the elution fraction, thereby producing
protein compositions that have reduced AR or are free of AR. In
certain embodiments relating to the purification of adalimumab, the
methods of the instant invention can be used to selectively remove,
significantly reduce, or essentially remove all of AR1 charge
variants in the Flow Through and wash fractions while enriching for
the same in the flow elution fraction, thereby producing protein
compositions that have reduced AR1 or are free of AR1 variants. In
certain embodiments relating to adalimumab, the methods of the
instant invention can be used to selectively remove, significantly
reduce, or essentially remove all of AR2 charge variants in the
flow-through and wash fractions while enriching for the same in the
flow elution fraction, thereby producing protein compositions that
have reduced AR2 or are free of AR2 variants.
[0246] In certain embodiments, including, but not limited to those
relating to adalimumab, the methods of the instant invention can be
used to selectively remove, significantly reduce, or essentially
remove all of the MGO variants in the elution fractions while
enriching for the same in the Flow Through and wash fractions,
thereby producing protein preparations with reduced or free of MGO
variants. In certain embodiments, including, but not limited to
those relating to adalimumab, the methods of the instant invention
can be used to selectively remove, significantly reduce, or
essentially remove all of the glycated variants (Schiff's base and
permanently glycated forms) in the elution fractions while
enriching for the same in the Flow Through and wash fractions,
thereby producing protein preparations with reduced or free of
glycated variants.
[0247] In certain embodiments, the loading, pH, conductivity of the
CEX chromatography step, as well as elution pH conductivity, can be
modified to achieve a desired distribution of acidic species. For
example, but not by way of limitation, certain embodiments are
directed to the modulation of the lysine distribution of a purified
sample of a protein of interest, e.g., increasing Lys 0 and
decreasing Lys 1 and Lys 2. In certain embodiments, the methods of
the present invention allow for the preparation of samples wherein
the amount of Lys 0 is decreased, while the amount of Lys 1 and/or
Lys 2 is increased.
[0248] In certain embodiments, a CEX chromatographic separation can
be performed and combinations of fractions can be pooled to achieve
a combination of desired process-related impurity and/or
product-relates substance levels, in addition to, or in place of
merely modulating charge variant concentration.
[0249] In certain embodiments, spectroscopy methods such as UV,
NIR, FTIR, Fluorescence, Raman may be used to monitor levels of
product-related charge variants, aggregates, low molecular weight
variants (e.g., fragments of the protein of interest) in an
on-line, at-line or in-line mode, which can then be used to control
the level of charge variants, e.g., acidic species, in the pooled
material collected from the CEX effluent. In certain embodiments,
specific signals arising from the chemical modification of the
proteins such as glycation, MGO modification, deamidation,
glycosylation may be specifically measurable by spectroscopic
methods through such in-line, on-line or at-line methods, enabling
realtime or near-real time control of product quality of the
resulting product. In certain embodiments, on-line, at-line or
in-line monitoring methods can be used either on the effluent line
of the chromatography step or in the collection vessel, to enable
achievement of the desired product quality/recovery. In certain
embodiments, the UV signal can be used as a surrogate to achieve an
appropriate product quality/recovery, wherein the UV signal can be
processed appropriately, including, but not limited to, such
processing techniques as integration, differentiation, moving
average, such that normal process variability can be addressed and
the target product quality can be achieved. In certain embodiments,
such measurements can be combined with in-line dilution methods
such that ion concentration/conductivity of the load/wash can be
controlled by feedback and hence facilitate product quality
control.
[0250] In certain embodiments, a combination of CEX and AEX and/or
MM methods can be used to prepare product-related
substance-modulated materials, including certain embodiments where
one technology is used in a complementary/supplementary manner with
another technology. In certain embodiments, such a combination can
be performed such that certain sub-species are removed
predominantly by one technology, such that the combination provides
the desired final composition/product quality. In certain
embodiments, such combinations include the use of additional
chromatography, filtration, pH adjustment, UF/DF steps so as to
achieve the desired product quality, AR, ion concentration, and/or
viral reduction.
[0251] CEX chromatography can be used in conjunction with recycle
chromatography and continuous chromatography modes.
[0252] Mixed Mode Chromatography
[0253] Mixed mode ("MM") chromatography may also be used to prepare
the low AR compositions of the invention. MM chromatography, also
referred to herein as "multimodal chromatography", is a
chromatographic strategy that utilizes a support comprising a
ligand that is capable of providing at least two different, and in
certain embodiments co-operative, sites that interact with the
substance to be bound. In certain embodiments, one of these sites
gives an attractive type of charge-charge interaction between the
ligand and the substance of interest and the other site provides
for electron acceptor-donor interaction and/or hydrophobic and/or
hydrophilic interactions. Electron donor-acceptor interactions
include interactions such as hydrogen-bonding, .pi.-.pi.,
cation-.pi., charge transfer, dipole-dipole, induced dipole
etc.
[0254] In certain embodiments, the resin employed for a mixed mode
separation is Capto Adhere. Capto Adhere is a strong anion
exchanger with multimodal functionality. Its base matrix is a
highly cross-linked agarose with a ligand (N-Benzyl-N-methyl
ethanol amine) that exhibits many functionalities for interaction,
such as ionic interaction, hydrogen bonding and hydrophobic
interaction. In certain embodiments, the resin employed for a mixed
mode separation is selected from PPA-HyperCel and HEA-HyperCel. The
base matrices of PPA-HyperCel and HEA-HyperCel are high porosity
cross-linked cellulose. Their ligands are Phenylpropylamine and
Hexylamine, respectively. Phenylpropylamine and Hexylamine offer
different selectivity and hydrophobicity options for protein
separations. Additional mixed mode chromatographic supports
include, but are not limited to, Nuvia C Prime, Toyo Pearl MX Trp
650M, and Eshmuno.RTM. HCX.
[0255] In certain embodiments, the mixed mode chromatography resin
is comprised of ligands coupled to an organic or inorganic support,
sometimes denoted a base matrix, directly or via a spacer. The
support may be in the form of particles, such as essentially
spherical particles, a monolith, filter, membrane, surface,
capillaries, etc. In certain embodiments, the support is prepared
from a native polymer, such as cross-linked carbohydrate material,
such as agarose, agar, cellulose, dextran, chitosan, konjac,
carrageenan, gellan, alginate etc. To obtain high adsorption
capacities, the support can be porous, and ligands are then coupled
to the external surfaces as well as to the pore surfaces. Such
native polymer supports can be prepared according to standard
methods, such as inverse suspension gelation (S Hjerten: Biochim
Biophys Acta 79(2), 393-398 (1964). Alternatively, the support can
be prepared from a synthetic polymer, such as cross-linked
synthetic polymers, e.g. styrene or styrene derivatives,
divinylbenzene, acrylamides, acrylate esters, methacrylate esters,
vinyl esters, vinyl amides etc. Such synthetic polymers can be
produced according to standard methods, see e.g. "Styrene based
polymer supports developed by suspension polymerization" (R
Arshady: Chimica e L'Industria 70(9), 70-75 (1988)). Porous native
or synthetic polymer supports are also available from commercial
sources, such as Amersham Biosciences, Uppsala, Sweden.
[0256] In certain embodiments, the protein load of the mixture
comprising protein of interest is adjusted to a total protein load
to the column of between about 50 and 750 g/L, or between about 75
and 500 g/L, or between about 100 and 300 g/L. In certain
embodiments, the protein concentration of the load protein mixture
is adjusted to a protein concentration of the material loaded to
the column of about 1 and 50 g/L, or between about 9 and 25
g/L.
[0257] In certain embodiments, additives such as poly ethylene
glycol, detergents, amino acids, sugars, chaotropic agents can be
added to enhance the performance of the separation, so as to
achieve better recovery or product quality.
[0258] In certain embodiments, including, but not limited to those
relating to adalimumab, the MM methods of the instant invention can
be used to selectively remove, significantly reduce, or essentially
remove all of AR in the Flow Through and wash fractions while
enriching for the same in the flow elution fraction, thereby
producing protein compositions that have reduced AR or are free of
AR. In certain embodiments relating to the purification of
adalimumab, the methods of the instant invention can be used to
selectively remove, significantly reduce, or essentially remove all
of AR1 charge variants in the Flow Through and wash fractions while
enriching for the same in the flow elution fraction, thereby
producing protein compositions that have reduced AR1 or are free of
AR1 variants. In certain embodiments relating to adalimumab, the
methods of the instant invention can be used to selectively remove,
significantly reduce, or essentially remove all of AR2 charge
variants in the flow-through and wash fractions while enriching for
the same in the flow elution fraction, thereby producing protein
compositions that have reduced AR2 or are free of AR2 variants.
[0259] In certain embodiments, including, but not limited to those
relating to adalimumab, the MM methods of the instant invention can
be used to selectively remove, significantly reduce, or essentially
remove all of the MGO variants in the Flow Through and wash
fractions while enriching for the same in the elution fraction,
thereby producing protein preparations with reduced or free of MGO
variants. In certain embodiments, including, but not limited to
those relating to adalimumab, the methods of the instant invention
can be used to selectively remove, significantly reduce, or
essentially remove all of the glycated variants (Schiff's base and
permanently glycated forms) in the Flow Through and wash fractions
while enriching for the same in the elution fraction, thereby
producing protein preparations with reduced or free of glycated
variants.
[0260] In certain embodiments, the loading, pH, conductivity of the
MM chromatography step, wash pH and conductivity, as well as
elution pH conductivity, can be modified to achieve a desired
distribution of acidic species. For example, but not by way of
limitation, certain embodiments are directed to the modulation of
the lysine distribution of a purified sample of a protein of
interest, e.g., increasing Lys 0 and decreasing Lys 1 and Lys 2. In
certain embodiments, the methods of the present invention allow for
the preparation of samples wherein the amount of Lys 0 is
decreased, while the amount of Lys 1 and/or Lys 2 is increased.
[0261] In certain embodiments, a MM chromatographic separation can
be performed and combinations of fractions can be pooled to achieve
a combination of desired process-related impurity and/or
product-relates substance levels, in addition to, or in place of
merely modulating charge variant concentration.
[0262] In certain embodiments, spectroscopy methods such as UV,
NIR, FTIR, Fluorescence, Raman may be used to monitor levels of AR
species in an on-line, at-line or in-line mode, which can then be
used to control the level of charge variants, e.g., acidic species,
in the pooled material collected from the MM effluent. In certain
embodiments, specific signals arising from the chemical
modification of the proteins such as glycation, MGO modification,
deamidation, glycosylation may be specifically measurable by
spectroscopic methods through such in-line, on-line or at-line
methods, enabling real time or near-real time control of product
quality of the resulting product. In certain embodiments, on-line,
at-line or in-line monitoring methods can be used either on the
effluent line of the chromatography step or in the collection
vessel, to enable achievement of the desired product
quality/recovery. In certain embodiments, the UV signal can be used
as a surrogate to achieve an appropriate product quality/recovery,
wherein the UV signal can be processed appropriately, including,
but not limited to, such processing techniques as integration,
differentiation, moving average, such that normal process
variability can be addressed and the target product quality can be
achieved. In certain embodiments, such measurements can be combined
with in-line dilution methods such that ion
concentration/conductivity of the load/wash can be controlled by
feedback and hence facilitate product quality control.
[0263] In certain embodiments, a combination of mixed mode and AEX
and CEX methods can be used to prepare the low AR compositions of
the invention, including certain embodiments where one technology
is used in a complementary/supplementary manner with another
technology. In certain embodiments, such a combination can be
performed such that certain sub-species are removed predominantly
by one technology, such that the combination provides the desired
final composition/product quality. In certain embodiments, such
combinations include the use of additional intervening
chromatography, filtration, pH adjustment, UF/DF steps so as to
achieve the desired product quality, AR, ion concentration, and/or
viral reduction.
[0264] MM chromatography can be used in conjunction with recycle
chromatography and continuous chromatography modes.
[0265] Continuous and Recycle Chromatography
[0266] Continuous and recycle chromatography modes can be used to
produce the low AR compositions of the invention, and are described
below. These methods result in significant improvements in recovery
of the protein, e.g., antibody, of interest while maintaining the
AR reduction levels. These continuous and recycle chromatography
modes are applicable to chromatography methods where (a) the low
acidic species component of interest is collected in the unbound
fraction during the chromatography (Flow Through/wash
chromatography) or (b) where the low acidic species component of
interest is first bound to the media and subsequently recovered by
washing the media with conditions that elute the bound
component.
[0267] Continuous and Recycle Chromatography--Flow Through/Wash
Chromatography
[0268] In the case where the low acidic species component of
interest is collected in the unbound fraction, the following
approach is employed which prevents loss of the material loaded on
the column.
[0269] In one embodiment, a recycle chromatography mode is used
wherein the column is loaded and the unbound fractions that results
in the target AR level are collected. Subsequently, instead of
regenerating the column and losing the product, the column is
washed under conditions that result in recovery of the product
remaining bound to the column. This product recovered under these
conditions contains significantly higher AR levels than the
original feed material. This wash fraction is adjusted to the
appropriate conditions to achieve the separation desired on
subsequent processing (typically similar conditions to the initial
preparation) and combined with the original feed material and
loaded on the column again (after preparing the column
appropriately for the next cycle). The amount of material prepared
for the next cycle, combining the wash fraction from the first
cycle and the fresh material is adjusted to the target loading
capacity for the column to achieve the desired separation
(typically similar to the capacity targeted for the first
cycle).
[0270] In performing the second cycle, a similar strategy is
employed, collecting the unbound fraction so as to achieve the
target AR level and then subsequently washing the column under
conditions to recover the product remaining on the column.
[0271] In one embodiment, this recycle chromatography mode is
continued until all the load materials are used. The number of
cycles can be controlled by designing the column size
appropriately.
[0272] In employing the recycle chromatography mode, the recovery
of the product loaded on the column is significantly improved while
achieving the target AR levels.
[0273] Several variations of the recycle chromatography mode can be
employed. In one embodiment, the fractions that are collected
targeting a certain AR level can be determined based on
predetermined criteria or based on at-line, off-line or on-line
analysis of the effluent of the column or the collected pool.
[0274] In another embodiment, the wash conditions used for the
first cycle can be adjusted to recover the desired amount of
product at the desired product quality, only limited by the
feasibility of preparing an appropriate load mixture for the
subsequent step. In one aspect of this embodiment, the wash
condition may be similar to the load condition. In another aspect
of this embodiment, the wash condition can be stringent to recover
all of the product species (desired and undesired) remaining on the
column.
[0275] In still another embodiment, the loading amount, the loading
conditions and the washing conditions used for the subsequent
cycles can be modified to achieve the desired purity, given that
that loading material for the subsequent cycles are likely to
contain higher levels of AR.
[0276] In another embodiment, the last cycle of the operation can
be performed under different conditions such that the target purity
and target recovery can be achieved to optimize overall recovery
and purity.
[0277] The methods for producing the low AR composition of the
invention can also be implemented in a continuous chromatography
mode. In this mode, at least two columns are employed (referred to
as a "first" column and a "second" column). In one embodiment, the
feed material is loaded onto the first column, and the unbound
fraction from the first column is collected such that the pool
material contains the target AR level. The column is then washed
under conditions that recover the remaining product. This material
is then dynamically diluted with appropriate solutions to achieve
the desired loading conditions, mixed with fresh feed material and
directed to the second column. The unbound fraction from the second
column is collected to achieve the target AR level. The second
column is then washed under conditions to recover the product and
diluted with appropriate solutions, mixed with fresh materials
dynamically and directed to the first column (which is prepared to
receive the load after regeneration/cleaning). In one embodiment,
this cycling is continued until all the load material is used. The
last cycle can be operated in a "typical" mode, with appropriate
adjustments to the load and wash conditions as necessary.
[0278] In certain embodiments this continuous chromatography mode
can be carried out such that the wash material containing the
higher AR levels can be directed back into the load tank after
appropriate dilution. This material can then be loaded subsequently
or concurrently onto the second column, such that the operation of
the two columns are not in tandem, reducing complexity of the
operation.
[0279] This continuous chromatography mode, while similar to the
recycle chromatography mode, can be carried out more efficiently,
and therefore has a reduced processing time.
[0280] For this continuous chromatography mode, several variations
can be employed. In one embodiment, the fractions that are
collected targeting a certain AR level can be determined based on
predetermined criteria or based on at-line, off-line or on-line
analysis of the effluent of the column or the collected pool.
[0281] In another embodiment, the wash conditions used for the
first cycle can be adjusted to recover the desired amount of
product at the desired product quality, only limited by the
feasibility of preparing an appropriate load mixture for the
subsequent step. In one aspect of this embodiment, the wash
conditions may be similar to the load conditions. In another aspect
of the embodiment, the wash conditions can be stringent to recover
all of the product species (desired and undesired) remaining on the
column.
[0282] In still another embodiment, the loading amount, the loading
conditions and the washing conditions used for the subsequent
cycles can be modified to achieve the desired purity, given that
that loading material for the subsequent cycles are likely to
contain higher levels of AR.
[0283] In another embodiment, the last cycle of the operation can
be performed under different conditions such that the target
purity/recovery can be achieved to optimize overall recovery and
and/or purity.
[0284] In one embodiment, the media choice for the recycle or
continuous modes can be one of many chromatographic resins with
pendant hydrophobic and anion exchange functional groups,
monolithic media, membrane adsorbent media or depth filtration
media.
[0285] In certain embodiments, membrane or depth filter based media
("convective media") can be used in the recycle or continuous
chromatography modes because selectivity of separation is not
required to be high given the fact that the less enriched portions
of the product are "recycled" while the pure fractions are
selectively pooled.
[0286] Continuous and Recycle Chromatography--Elution
Chromatography
[0287] In the elution mode of chromatography or separation, as
exemplified by the CEX technology for AR reduction, the conditions
are chosen for the load and wash steps such that the AR enriched
material is collected in the Flow Through and/or wash fractions,
while the AR reduced material is collected in the elution fraction.
In the typical implementation of the CEX technology, about 10 to
40% of the product (the desired charge variant) may be lost in the
Flow Through/Wash fractions. Two modes of operation, namely the
recycle chromatography mode and the continuous chromatography mode
provide improved recovery, while maintaining the target AR
levels.
[0288] In the recycle chromatography mode, the load material is, in
general, processed over multiple cycles. In implementing the
recycle chromatography mode, the load material is prepared such
that the eluate contains the target product purity or AR level.
Under these conditions, the AR enriched material is collected in
the Flow Through/wash fractions. This material is pooled and
additional fresh load material is added to achieve the appropriate
loading capacity for the next cycle of chromatography on the same
column. In particular, in one embodiment, the column is eluted
under conditions where the bound product (having low AR levels) is
recovered, and subsequently regenerated and equilibrated to prepare
for the next cycle.
[0289] In the next cycle, the combined load (Flow Through/wash from
cycle 1 above, as well as fresh material) is loaded to the target
capacity. The Flow Through/wash fractions are collected and pooled.
The column is eluted to obtain the second eluate, again containing
the target low AR composition. In one embodiment, this sequence is
continued until all the load materials are processed.
[0290] In another embodiment, by implementing the recycle
chromatography mode, the material that would otherwise be discarded
as AR enriched material is further purified to "recover" pure
protein product, thereby improving the overall recovery of the
protein. In one embodiment, the level of recovery depends on the
number of cycles employed.
[0291] For the recycle chromatography mode, several variations can
be employed. In one embodiment, the entire pool of the Flow
Through/wash fractions are preferably combined with fresh materials
to maximize recovery of the entire operation. However, a portion of
the flow through wash can be discarded to achieve higher purity or
efficiency. For example, in one embodiment, certain fractions
containing very high levels of AR species can be discarded. To
enable such selective pooling, off-line, in-line or at line methods
can be used to directly or indirectly measure the levels of AR.
[0292] In another embodiment, the loading amount and the conditions
for loading, washing and eluting can be modified for the second and
subsequent cycles to accommodate the higher levels of AR that will
be present in the loading pool.
[0293] In still another embodiment, the last cycle of the method
can be performed under conditions such that the target purity and
recovery can be achieved to optimize overall recovery and
purity.
[0294] A continuous chromatography mode provides additional
advantages in terms of time efficiency. In this mode of operation,
two or more columns are used. Specifically, as with the recycle
mode, an appropriate condition for the load capacity, load, wash
and elution conditions are chosen for the operation. The Flow
Through and wash fractions (or a portion thereof) is directed to
the load tank containing the fresh material. After completion of
the load and wash steps, the first column is eluted and
subsequently regenerated. Meanwhile, the second column is loaded
with the material that is a mix of fresh material and the wash and
Flow Through from the previous cycle. The wash and Flow Through
from the second column is again directed back to the load tank. The
second column is then eluted and regenerated. The first column is
then ready to be loaded and the cycle continues. This continuous
chromatography mode is efficient as the product is processed
continuously and the purified product is obtained in a
semi-continuous manner.
[0295] Several variations of the continuous chromatography mode can
be employed. In one embodiment, the entire pool of the Flow
Through/wash fractions is combined with fresh materials to maximize
recovery of the entire operation. However, a portion of the Flow
Through wash can be discarded to achieve higher purity or
efficiency. For example, certain fractions containing very high
levels of AR species can be discarded. To enable such selective
pooling, off-line, in-line or at line methods can be used to
measure directly or indirectly the levels of acidic species.
[0296] In another embodiment, the loading amount, conditions for
loading, washing and eluting can be modified for the second and
subsequent cycles to accommodate the higher levels of AR that will
be present in the loading pool.
[0297] In still another embodiment, the last cycle of the operation
can be performed under different conditions to optimize overall
recover and purity.
[0298] The recycle chromatography mode and the continuous
chromatography mode are not limited to use with any particular
chromatography resin. The media used for the recycle or continuous
modes can be one of many chromatographic resins with pendant
hydrophobic and anion exchange functional groups, monolithic media,
membrane adsorber media or depth filtration media.
[0299] In certain embodiments, membrane depth filter-based media
("convective media") can be used with the recycle or continuous
modes as the selectivity of separation is not required to be high
given the fact that the less enriched portions of the product are
"recycled" while the pure fractions are selectively pooled.
[0300] Recycle chromatography mode and the continuous
chromatography mode can be used inconjunction with AEX, CEX, or MM
chromatography methods, as described herein, to produce the low AR
compositions of the invention.
[0301] Hydrophobic Interaction Chromatography
[0302] The low AR compositions of the invention may also be
prepared using a hydrophobic interaction chromatography (HIC) step
in addition to the displacement chromatography step.
[0303] In performing the separation, the sample mixture is
contacted with the HIC material, e.g., using a batch purification
technique or using a column or membrane chromatography. Prior to
HIC purification it may be desirable to adjust the concentration of
the salt buffer to achieve desired protein binding to the resin or
the membrane.
[0304] Whereas ion exchange chromatography relies on the local
charge of the protein of interest for selective separation,
hydrophobic interaction chromatography employs the hydrophobic
properties of the proteins to achieve selective separation.
Hydrophobic groups on the protein interact with hydrophobic groups
of the resin or the membrane. The more hydrophobic a protein is the
stronger it will interact with the column or the membrane. Thus the
HIC step removes process-related impurities (e.g., HCPs) as well as
product-related substances (e.g., aggregates and fragments).
[0305] Like ion exchange chromatography, a HIC column or membrane
device can also be operated in product a bind-elute mode, a
flow-through, or a hybrid mode wherein the product exhibits binding
to the chromatographic material, yet can be washed from the column
using a buffer that is the same or substantially similar to the
loading buffer. The details of these modes are outlined above in
connection with AEX purification.
[0306] As hydrophobic interactions are strongest at high ionic
strength, this form of separation is conveniently performed
following salt elution step, such as those that are typically used
in connection with ion exchange chromatography. Alternatively,
salts can be added into a low salt level feed stream before this
step. Adsorption of the antibody to a HIC column is favored by high
salt concentrations, but the actual concentrations can vary over a
wide range depending on the nature of the protein of interest, salt
type and the particular HIC ligand chosen. Various ions can be
arranged in a so-called soluphobic series depending on whether they
promote hydrophobic interactions (salting-out effects) or disrupt
the structure of water (chaotropic effect) and lead to the
weakening of the hydrophobic interaction. Cations are ranked in
terms of increasing salting out effect as Ba.sup.2+; Ca.sup.2+;
Mg.sup.2+; Li.sup.+; Cs.sup.+; Na.sup.+; K.sup.+; Rb.sup.+;
NH.sub.4.sup.+, while anions may be ranked in terms of increasing
chaotropic effect as PO.sub.4.sup.3-; SO.sub.4.sup.2-;
CH.sub.3CO.sub.3.sup.-; Cl.sup.-; Br.sup.-; NO.sub.3.sup.-;
ClO.sub.4.sup.-; I.sup.-; SCN.sup.-.
[0307] In general, Na.sup.+, K.sup.+ or NH.sub.4.sup.+ sulfates
effectively promote ligand-protein interaction in HIC. Salts may be
formulated that influence the strength of the interaction as given
by the following relationship:
(NH.sub.4).sub.2SO.sub.4>Na.sub.2SO.sub.4>NaCl>NH.sub.4Cl>NaB-
r>NaSCN. In general, salt concentrations of between about 0.75 M
and about 2 M ammonium sulfate or between about 1 and 4 M NaCl are
useful.
[0308] HIC media normally comprise a base matrix (e.g.,
cross-linked agarose or synthetic copolymer material) to which
hydrophobic ligands (e.g., alkyl or aryl groups) are coupled. A
suitable HIC media comprises an agarose resin or a membrane
functionalized with phenyl groups (e.g., a Phenyl Sepharose.TM.
from GE Healthcare or a Phenyl Membrane from Sartorius). Many HIC
resins are available commercially. Examples include, but are not
limited to, Capto Phenyl, Phenyl Sepharose.TM. 6 Fast Flow with low
or high substitution, Phenyl Sepharose.TM. High Performance, Octyl
Sepharose.TM. High Performance (GE Healthcare); Fractogel.TM. EMD
Propyl or Fractogel.TM. EMD Phenyl (E. Merck, Germany);
Macro-Prep.TM. Methyl or Macro-Prep.TM. t-Butyl columns (Bio-Rad,
California); WP HI-Propyl (C3).TM. (J. T. Baker, New Jersey); and
Toyopearl.TM. ether, phenyl or butyl (TosoHaas, Pa.).
[0309] Viral Filtration
[0310] Viral filtration is a dedicated viral reduction step in the
entire purification process. This step is usually performed post
chromatographic polishing steps. Viral reduction can be achieved
via the use of suitable filters including, but not limited to,
Planova 20N.TM., 50 N or BioEx from Asahi Kasei Pharma,
Viresolve.TM. filters from EMD Millipore, ViroSart CPV from
Sartorius, or Ultipor DV20 or DV50.TM. filter from Pall
Corporation. It will be apparent to one of ordinary skill in the
art to select a suitable filter to obtain desired filtration
performance.
[0311] Ultrafiltration/Diafiltration
[0312] Certain embodiments of the present invention employ
ultrafiltration and diafiltration steps to further concentrate and
formulate the protein of interest, e.g., an antibody product, in
addition to the displacement chromatography steps. Ultrafiltration
is described in detail in: Microfiltration and Ultrafiltration:
Principles and Applications, L. Zeman and A. Zydney (Marcel Dekker,
Inc., New York, N.Y., 1996); and in: Ultrafiltration Handbook,
Munir Cheryan (Technomic Publishing, 1986; ISBN No. 87762-456-9).
One filtration process is Tangential Flow Filtration as described
in the Millipore catalogue entitled "Pharmaceutical Process
Filtration Catalogue" pp. 177-202 (Bedford, Mass., 1995/96).
Ultrafiltration is generally considered to mean filtration using
filters with a pore size of smaller than 0.1 .mu.m. By employing
filters having such small pore size, the volume of the sample can
be reduced through permeation of the sample buffer through the
filter membrane pores while proteins, such as antibodies, are
retained above the membrane surface.
[0313] Diafiltration is a method of using membrane filters to
remove and exchange salts, sugars, and non-aqueous solvents, to
separate free from bound species, to remove low molecular-weight
species, and/or to cause the rapid change of ionic and/or pH
environments. Micro solutes are removed most efficiently by adding
solvent to the solution being diafiltered at a rate approximately
equal to the permeate flow rate. This washes away microspecies from
the solution at a constant volume, effectively purifying the
retained protein of interest. In certain embodiments of the present
invention, a diafiltration step is employed to exchange the various
buffers used in connection with the instant invention, optionally
prior to further chromatography or other purification steps, as
well as to remove impurities from the protein preparations.
[0314] One of ordinary skill in the art can select appropriate
membrane filter device for the UF/DF operation. Examples of
membrane cassettes suitable for the present invention include, but
not limited to, Pellicon 2 or Pellicon 3 cassettes with 10 kD, 30
kD or 50 kD membranes from EMD Millipore, Kvick 10 kD, 30 kD or 50
kD membrane cassettes from GE Healthcare, and Centramate or
Centrasette 10 kD, 30 kD or 50 kD cassettes from Pall
Corporation.
[0315] Exemplary Purification Strategies
[0316] In certain embodiments, primary recovery can proceed by
sequentially employing pH reduction, centrifugation, and filtration
steps to remove cells and cell debris (including HCPs) from the
production bioreactor harvest. In certain embodiments, the present
invention is directed to subjecting a sample mixture from said
primary recovery to Protein A affinity followed by displacement
chromatography. Certain embodiments of the present invention will
include further purification steps. Examples of additional
purification procedures which can be performed prior to, during, or
following the displacement chromatography method include ethanol
precipitation, isoelectric focusing, reverse phase HPLC,
chromatography on silica, chromatography on heparin Sepharose.TM.,
further anion exchange chromatography and/or further cation
exchange chromatography, chromatofocusing, SDS-PAGE, ammonium
sulfate precipitation, hydroxylapatite chromatography, gel
electrophoresis, dialysis, and affinity chromatography (e.g., using
protein G, an antibody, a specific substrate, ligand or antigen as
the capture reagent).
[0317] Specific examples of such combinations of strategies is
presented below, with specific data relating to particular
combinations useful in the context of the instant invention
included in Examples 1-8 and in Tables 80-87 and 76-78 of U.S.
Provisional Application No. 61/893,068, entitled "Low Acidic
Species Compositions and Methods for Producing and Using the Same",
filed on Oct. 18, 2013, the entire contents of which are expressly
incorporated herein by reference.
[0318] In certain embodiments, the unbound Flow Through and wash
fractions can be further fractionated and a combination of
fractions providing a target product purity can be pooled.
[0319] In certain embodiments, the protein concentration can be
adjusted to achieve a differential partitioning behavior between
the antibody product and the product-related substances, such that
the purity and/or yield can be further improved. In certain
embodiments, the loading can be performed at different protein
concentrations during the loading operation to improve the product
quality/yield of any particular purification step.
[0320] In certain embodiments, the column temperature can be
independently varied to improve the separation efficiency and/or
yield of any particular purification step.
[0321] In certain embodiments, the loading and washing buffer
matrices can be different or composed of mixtures of chemicals,
while achieving similar "resin interaction" behavior such that the
above novel separation can be effected. For example, but not by way
of limitation, the loading and washing buffers can be different, in
terms of ionic strength or pH, while remaining substantially
similar in function in terms of the washout of the product achieved
during the wash step. In certain embodiments, additives such as
amino acids, sugars, PEG, etc can be added to the load or wash
steps to modulate the partitioning behavior to achieve the
separation efficiency and/or yield.
[0322] In certain embodiments, the loading & washing steps can
be controlled by in-line, at-line or off-line measurement of the
product related impurity/substance levels, either in the column
effluent, or the collected pool or both, so as to achieve the
target product quality and/or yield. In certain embodiments, the
loading concentration can be dynamically controlled by in-line or
batch or continuous dilutions with buffers or other solutions to
achieve the partitioning necessary to improve the separation
efficiency and/or yield.
IV. Methods of Assaying Sample Purity
[0323] Assaying Host Cell Protein
[0324] The present invention also provides methods for determining
the residual levels of host cell protein (HCP) concentration in the
low AR compositions of the invention. As described above, HCPs are
desirably excluded from the final target substance product.
Exemplary HCPs include proteins originating from the source of the
antibody production. Failure to identify and sufficiently remove
HCPs from the target antibody may lead to reduced efficacy and/or
adverse reactions in a subject.
[0325] As used herein, the term "HCP ELISA" refers to an ELISA
where the second antibody used in the assay is specific to the HCPs
produced from cells, e.g., CHO cells, used to generate the antibody
of interest. The second antibody may be produced according to
conventional methods known to those of skill in the art. For
example, the second antibody may be produced using HCPs obtained by
sham production and purification runs, i.e., the same cell line
used to produce the antibody of interest is used, but the cell line
is not transfected with antibody DNA. In an exemplary embodiment,
the second antibody is produced using HCPs similar to those
expressed in the cell expression system of choice, i.e., the cell
expression system used to produce the target antibody.
[0326] Generally, HCP ELISA comprises sandwiching a liquid sample
comprising HCPs between two layers of antibodies, i.e., a first
antibody and a second antibody. The sample is incubated during
which time the HCPs in the sample are captured by the first
antibody, for example, but not limited to goat anti-CHO, affinity
purified (Cygnus). A labeled second antibody, or blend of
antibodies, specific to the HCPs produced from the cells used to
generate the antibody, e.g., anti-CHO HCP Biotinylated, is added,
and binds to the HCPs within the sample. In certain embodiments the
first and second antibodies are polyclonal antibodies. In certain
aspects the first and second antibodies are blends of polyclonal
antibodies raised against HCPs. The amount of HCP contained in the
sample is determined using the appropriate test based on the label
of the second antibody.
[0327] HCP ELISA may be used for determining the level of HCPs in
an antibody composition, such as an eluate or flow-through obtained
using the process described above. The present invention also
provides a composition comprising an antibody, wherein the
composition has no detectable level of HCPs as determined by an HCP
Enzyme Linked Immunosorbent Assay ("ELISA").
[0328] Assaying Acidic Species (AR)
[0329] The levels of acidic species in the chromatographic samples
produced using the techniques described herein may be analyzed as
described in the Examples section. In certain embodiments a
CEX-HPLC method is employed. For example, but not by way of
limitation, cation exchange chromatography can be performed on a
Dionex ProPac WCX-10, Analytical column 4 mm.times.250 mm (Dionex,
Calif.). An Agilent 1200 HPLC system can then be used as the HPLC.
In certain embodiments, mobile phases such as 10 mM Sodium
Phosphate dibasic pH 7.5 (Mobile phase A) and 10 mM Sodium
Phosphate dibasic, 500 mM Sodium Chloride pH 5.5 (Mobile phase B)
can be used. In certain embodiments, a binary gradient (94% A, 6%
B: 0-20 min; 84% A, 16% B: 20-22 min; 0% A, 100% B: 22-28 min; 94%
A, 6% B: 28-34 min) can be used with detection at 280 nm. In
certain embodiments, quantitation is based on the relative area
percent of detected peaks. In certain embodiments, the peaks that
elute at relative residence time less than a certain time are
together represented as the acidic peaks.
[0330] Assaying Size Variants
[0331] In certain embodiments, the levels of aggregates, monomer,
and fragments in the chromatographic samples produced using the
techniques described herein are analyzed. In certain embodiments,
the aggregates, monomer, and fragments are measured using a size
exclusion chromatographic (SEC) method for each molecule. For
example, but not by way of limitation, a TSK-gel G3000SW.times.L, 5
.mu.m, 125 .ANG., 7.8.times.300 mm column (Tosoh Bioscience) can be
used in connection with certain embodiments, while a TSK-gel Super
SW3000, 4 .mu.m, 250 .ANG., 4.6.times.300 mm column (Tosoh
Bioscience) can be used in alternative embodiments. In certain
embodiments, the aforementioned columns are used along with an
Agilent or a Shimazhu HPLC system. In certain embodiments, sample
injections are made under isocratic elution conditions using a
mobile phase consisting of, for example, 100 mM sodium sulfate and
100 mM sodium phosphate at pH 6.8, and detected with UV absorbance
at 214 nm. In certain embodiments, the mobile phase will consist of
1.times.PBS at pH 7.4, and elution profile detected with UV
absorbance at 280 nm. In certain embodiments, quantification is
based on the relative area of detected peaks.
[0332] Any additional technique, such as mass spectroscopy, can be
used for assaying size variants.
V. Methods of Treatment Using the Low AR Compositions of the
Invention
[0333] The low AR compositions of the invention may be used to
treat any disorder in a subject for which the therapeutic protein
comprised in the composition is appropriate for treating. \
[0334] A "disorder" is any condition that would benefit from
treatment with the protein. This includes chronic and acute
disorders or diseases including those pathological conditions which
predispose the subject to the disorder in question. In the case of
an anti-TNF.alpha. antibody, or antigen binding portion thereof,
such as adalimumab, a therapeutically effective amount of the low
AR composition may be administered to treat a disorder in which
TNF.alpha. activity is detrimental.
[0335] A disorder in which TNF.alpha. activity is detrimental
includes a disorder in which inhibition of TNF.alpha. activity is
expected to alleviate the symptoms and/or progression of the
disorder. Such disorders may be evidenced, for example, by an
increase in the concentration of TNF.alpha. in a biological fluid
of a subject suffering from the disorder (e.g., an increase in the
concentration of TNF.alpha. in serum, plasma, synovial fluid, etc.
of the subject), which can be detected, for example, using an
anti-TNF.alpha. antibody.
[0336] TNF.alpha. has been implicated in the pathophysiology of a
wide variety of a TNF.alpha.-related disorders including sepsis,
infections, autoimmune diseases, transplant rejection and
graft-versus-host disease (see e.g., Moeller, A., et al. (1990)
Cytokine 2:162-169; U.S. Pat. No. 5,231,024 to Moeller et al.;
European Patent Publication No. 260 610 B1 by Moeller, A., et al.
Vasilli, P. (1992) Annu. Rev. Immunol. 10:411-452; Tracey, K. J.
and Cerami, A. (1994) Annu. Rev. Med. 45:491-503). Accordingly, the
low AR compositions or a low process-related impurity compositions
of the invention may be used to treat an autoimmune disease, such
as rheumatoid arthritis, juvenile idiopathic arthritis, or
psoriatic arthritis, an intestinal disorder, such as Crohn's
disease or ulcerative colitis, a spondyloarthropathy, such as
ankylosing spondylitis, or a skin disorder, such as psoriasis.
[0337] Disorders in which TNF.alpha. activity is detrimental are
well known in the art and described in detail in U.S. Pat. No.
8,231,876 and U.S. Pat. No. 6,090,382, the entire contents of each
of which are expressly incorporated herein by reference. In one
embodiment, "a disorder in which TNF.alpha. activity is
detrimental" includes sepsis (including septic shock, endotoxic
shock, gram negative sepsis and toxic shock syndrome), autoimmune
diseases (including rheumatoid arthritis, rheumatoid spondylitis,
osteoarthritis and gouty arthritis, allergy, multiple sclerosis,
autoimmune diabetes, autoimmune uveitis, nephrotic syndrome,
multisystem autoimmune diseases, lupus (including systemic lupus,
lupus nephritis and lupus cerebritis), Crohn's disease and
autoimmune hearing loss), infectious diseases (including malaria,
meningitis, acquired immune deficiency syndrome (AIDS), influenza
and cachexia secondary to infection), allograft rejection and graft
versus host disease, malignancy, pulmonary disorders (including
adult respiratory distress syndrome (ARDS), shock lung, chronic
pulmonary inflammatory disease, pulmonary sarcoidosis, pulmonary
fibrosis, silicosis, idiopathic interstitial lung disease and
chronic obstructive airway disorders (COPD), such as asthma),
intestinal disorders (including inflammatory bowel disorders,
idiopathic inflammatory bowel disease, Crohn's disease and Crohn's
disease-related disorders (including fistulas in the bladder,
vagina, and skin; bowel obstructions; abscesses; nutritional
deficiencies; complications from corticosteroid use; inflammation
of the joints; erythem nodosum; pyoderma gangrenosum; lesions of
the eye, Crohn's related arthralgias, fistulizing Crohn's
indeterminant colitis and pouchitis), cardiac disorders (including
ischemia of the heart, heart insufficiency, restenosis, congestive
heart failure, coronary artery disease, angina pectoris, myocardial
infarction, cardiovascular tissue damage caused by cardiac arrest,
cardiovascular tissue damage caused by cardiac bypass, cardiogenic
shock, and hypertension, atherosclerosis, cardiomyopathy, coronary
artery spasm, coronary artery disease, valvular disease,
arrhythmias, and cardiomyopathies), spondyloarthropathies
(including ankylosing spondylitis, psoriatic arthritis/spondylitis,
enteropathic arthritis, reactive arthritis or Reiter's syndrome,
and undifferentiated spondyloarthropathies), metabolic disorders
(including obesity and diabetes, including type 1 diabetes
mellitus, type 2 diabetes mellitus, diabetic neuropathy, peripheral
neuropathy, diabetic retinopathy, diabetic ulcerations, retinopathy
ulcerations and diabetic macrovasculopathy), anemia, pain
(including acute and chronic pains, such as neuropathic pain and
post-operative pain, chronic lower back pain, cluster headaches,
herpes neuralgia, phantom limb pain, central pain, dental pain,
opioid-resistant pain, visceral pain, surgical pain, bone injury
pain, pain during labor and delivery, pain resulting from burns,
including sunburn, post partum pain, migraine, angina pain, and
genitourinary tract-related pain including cystitis), hepatic
disorders (including hepatitis, alcoholic hepatitis, viral
hepatitis, alcoholic cirrhosis, al antitypsin deficiency,
autoimmune cirrhosis, cryptogenic cirrhosis, fulminant hepatitis,
hepatitis B and C, and steatohepatitis, cystic fibrosis, primary
biliary cirrhosis, sclerosing cholangitis and biliary obstruction),
skin and nail disorders (including psoriasis (including chronic
plaque psoriasis, guttate psoriasis, inverse psoriasis, pustular
psoriasis and other psoriasis disorders), pemphigus vulgaris,
scleroderma, atopic dermatitis (eczema), sarcoidosis, erythema
nodosum, hidradenitis suppurative, lichen planus, Sweet's syndrome,
scleroderma and vitiligo), vasculitides (including Behcet's
disease), and other disorders, such as juvenile rheumatoid
arthritis (JRA), endometriosis, prostatitis, choroidal
neovascularization, sciatica, Sjogren's syndrome, uveitis, wet
macular degeneration, osteoporosis, osteoarthritis, active axial
spondyloarthritis (active axSpA) and non-radiographic axial
spondyloarthritis (nr-axSpA).
[0338] As used herein, the term "subject" is intended to include
living organisms, e.g., prokaryotes and eukaryotes. Examples of
subjects include mammals, e.g., humans, dogs, cows, horses, pigs,
sheep, goats, cats, mice, rabbits, rats, and transgenic non-human
animals. In specific embodiments of the invention, the subject is a
human.
[0339] As used herein, the term "treatment" or "treat" refers to
both therapeutic treatment and prophylactic or preventative
measures. Those in need of treatment include those already with the
disorder, as well as those in which the disorder is to be
prevented.
[0340] In one embodiment, the invention provides a method of
administering a low AR composition comprising an anti-TNF.alpha.
antibody, or antigen binding portion thereof, to a subject such
that TNF.alpha. activity is inhibited or a disorder in which
TNF.alpha. activity is detrimental is treated. In one embodiment,
the TNF.alpha. is human TNF.alpha. and the subject is a human
subject. In one embodiment, the anti-TNF.alpha. antibody is
adalimumab, also referred to as HUMIRA.RTM..
[0341] The low AR compositions can be administered by a variety of
methods known in the art. Exemplary routes/modes of administration
include subcutaneous injection, intravenous injection or infusion.
In certain aspects, a low AR compositions may be orally
administered. As will be appreciated by the skilled artisan, the
route and/or mode of administration will vary depending upon the
desired results.
[0342] Dosage regimens may be adjusted to provide the optimum
desired response (e.g., a therapeutic or prophylactic response).
For example, a single bolus may be administered, several divided
doses may be administered over time or the dose may be
proportionally reduced or increased as indicated by the exigencies
of the therapeutic situation. In certain embodiments it is
especially advantageous to formulate parenteral compositions in
dosage unit form for ease of administration and uniformity of
dosage. Dosage unit form as used herein refers to physically
discrete units suited as unitary dosages for the mammalian subjects
to be treated; each unit comprising a predetermined quantity of
active compound calculated to produce the desired therapeutic
effect in association with the required pharmaceutical carrier. The
specification for the dosage unit forms of the invention are
dictated by and directly dependent on (a) the unique
characteristics of the active compound and the particular
therapeutic or prophylactic effect to be achieved, and (b) the
limitations inherent in the art of compounding such an active
compound for the treatment of sensitivity in individuals.
[0343] An exemplary, non-limiting range for a therapeutically or
prophylactically effective amount of a low AR composition of the
invention is 0.01-20 mg/kg, or 1-10 mg/kg, or 0.3-1 mg/kg. With
respect to low AR compositions comprising an anti-TNF.alpha.
antibody, or antigen-binding portion thereof, such as adalimumab,
an exemplary dose is 40 mg every other week. In some embodiments,
in particular for treatment of ulcerative colitis or Crohn's
disease, an exemplary dose includes an initial dose (Day 1) of 160
mg (e.g., four 40 mg injections in one day or two 40 mg injections
per day for two consecutive days), a second dose two weeks later of
80 mg, and a maintenance dose of 40 mg every other week beginning
two weeks later. Alternatively, for psoriasis for example, a dosage
can include an 80 mg initial dose followed by 40 mg every other
week starting one week after the initial dose.
[0344] It is to be noted that dosage values may vary with the type
and severity of the condition to be alleviated. It is to be further
understood that for any particular subject, specific dosage
regimens should be adjusted over time according to the individual
need and the professional judgment of the person administering or
supervising the administration of the compositions, and that dosage
ranges set forth herein are exemplary only and are not intended to
limit the scope or practice of the claimed composition.
VI. Pharmaceutical Formulations Containing the Low AR Compositions
of the Invention
[0345] The present invention further provides preparations and
formulations comprising the low AR compositions of the invention.
It should be understood that any of the antibodies and antibody
fragments described herein, including antibodies and antibody
fragments having any one or more of the structural and functional
features described in detail throughout the application, may be
formulated or prepared as described below. When various
formulations are described in this section as including an
antibody, it is understood that such an antibody may be an antibody
or an antibody fragment having any one or more of the
characteristics of the antibodies and antibody fragments described
herein. In one embodiment, the antibody is an anti-TNF.alpha.
antibody, or antigen-binding portion thereof.
[0346] In certain embodiments, the low AR compositions of the
invention may be formulated with a pharmaceutically acceptable
carrier as pharmaceutical (therapeutic) compositions, and may be
administered by a variety of methods known in the art. As will be
appreciated by the skilled artisan, the route and/or mode of
administration will vary depending upon the desired results. The
term "pharmaceutically acceptable carrier" means one or more
non-toxic materials that do not interfere with the effectiveness of
the biological activity of the active ingredients. Such
preparations may routinely contain salts, buffering agents,
preservatives, compatible carriers, and optionally other
therapeutic agents. Such pharmaceutically acceptable preparations
may also routinely contain compatible solid or liquid fillers,
diluents or encapsulating substances which are suitable for
administration into a human. The term "carrier" denotes an organic
or inorganic ingredient, natural or synthetic, with which the
active ingredient is combined to facilitate the application. The
components of the pharmaceutical compositions also are capable of
being co-mingled with the antibodies of the present invention, and
with each other, in a manner such that there is no interaction
which would substantially impair the desired pharmaceutical
efficacy.
[0347] The low AR compositions of the invention are present in a
form known in the art and acceptable for therapeutic uses. In one
embodiment, a formulation of the low AR compositions of the
invention is a liquid formulation. In another embodiment, a
formulation of the low AR compositions of the invention is a
lyophilized formulation. In a further embodiment, a formulation of
the low AR compositions of the invention is a reconstituted liquid
formulation. In one embodiment, a formulation of the low AR
compositions of the invention is a stable liquid formulation. In
one embodiment, a liquid formulation of the low AR compositions of
the invention is an aqueous formulation. In another embodiment, the
liquid formulation is non-aqueous. In a specific embodiment, a
liquid formulation of the low AR compositions of the invention is
an aqueous formulation wherein the aqueous carrier is distilled
water.
[0348] The formulations of the low AR compositions of the invention
comprise an antibody in a concentration resulting in a w/v
appropriate for a desired dose. The antibody may be present in the
formulation at a concentration of about 1 mg/ml to about 500 mg/ml,
e.g., at a concentration of at least 1 mg/ml, at least 5 mg/ml, at
least 10 mg/ml, at least 15 mg/ml, at least 20 mg/ml, at least 25
mg/ml, at least 30 mg/ml, at least 35 mg/ml, at least 40 mg/ml, at
least 45 mg/ml, at least 50 mg/ml, at least 55 mg/ml, at least 60
mg/ml, at least 65 mg/ml, at least 70 mg/ml, at least 75 mg/ml, at
least 80 mg/ml, at least 85 mg/ml, at least 90 mg/ml, at least 95
mg/ml, at least 100 mg/ml, at least 105 mg/ml, at least 110 mg/ml,
at least 115 mg/ml, at least 120 mg/ml, at least 125 mg/ml, at
least 130 mg/ml, at least 135 mg/ml, at least 140 mg/ml, at least
150 mg/ml, at least 200 mg/ml, at least 250 mg/ml, or at least 300
mg/ml.
[0349] In a specific embodiment, a formulation of the low AR
compositions of the invention comprises at least about 100 mg/ml,
at least about 125 mg/ml, at least 130 mg/ml, or at least about 150
mg/ml of an antibody of the invention.
[0350] In one embodiment, the concentration of antibody, which is
included in the formulation of the invention, is between about 1
mg/ml and about 25 mg/ml, between about 1 mg/ml and about 200
mg/ml, between about 25 mg/ml and about 200 mg/ml, between about 50
mg/ml and about 200 mg/ml, between about 75 mg/ml and about 200
mg/ml, between about 100 mg/ml and about 200 mg/ml, between about
125 mg/ml and about 200 mg/ml, between about 150 mg/ml and about
200 mg/ml, between about 25 mg/ml and about 150 mg/ml, between
about 50 mg/ml and about 150 mg/ml, between about 75 mg/ml and
about 150 mg/ml, between about 100 mg/ml and about 150 mg/ml,
between about 125 mg/ml and about 150 mg/ml, between about 25 mg/ml
and about 125 mg/ml, between about 50 mg/ml and about 125 mg/ml,
between about 75 mg/ml and about 125 mg/ml, between about 100 mg/ml
and about 125 mg/ml, between about 25 mg/ml and about 100 mg/ml,
between about 50 mg/ml and about 100 mg/ml, between about 75 mg/ml
and about 100 mg/ml, between about 25 mg/ml and about 75 mg/ml,
between about 50 mg/ml and about 75 mg/ml, or between about 25
mg/ml and about 50 mg/ml.
[0351] In a specific embodiment, a formulation of the low AR
compositions of the invention comprises between about 90 mg/ml and
about 110 mg/ml or between about 100 mg/ml and about 210 mg/ml of
an antibody.
[0352] The formulations of the low AR compositions of the invention
comprising an antibody may further comprise one or more active
compounds as necessary for the particular indication being treated,
typically those with complementary activities that do not adversely
affect each other. Such additional active compound/s is/are
suitably present in combination in amounts that are effective for
the purpose intended.
[0353] The formulations of the low AR compositions of the invention
may be prepared for storage by mixing the antibody having the
desired degree of purity with optional physiologically acceptable
carriers, excipients or stabilizers, including, but not limited to
buffering agents, saccharides, salts, surfactants, solubilizers,
polyols, diluents, binders, stabilizers, salts, lipophilic
solvents, amino acids, chelators, preservatives, or the like
(Goodman and Gilman's The Pharmacological Basis of Therapeutics,
12.sup.th edition, L. Brunton, et al. and Remington's
Pharmaceutical Sciences, 16th edition, Osol, A. Ed. (1999)), in the
form of lyophilized formulations or aqueous solutions at a desired
final concentration. Acceptable carriers, excipients, or
stabilizers are nontoxic to recipients at the dosages and
concentrations employed, and include buffers such as histidine,
phosphate, citrate, glycine, acetate and other organic acids;
antioxidants including ascorbic acid and methionine; preservatives
(such as octadecyldimethylbenzyl ammonium chloride; hexamethonium
chloride; benzalkonium chloride, benzethonium chloride; phenol,
butyl or benzyl alcohol; alkyl parabens such as methyl or propyl
paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and
m-cresol); low molecular weight (less than about 10 residues)
polypeptide; proteins, such as serum albumin, gelatin, or
immunoglobulins; hydrophilic polymers such as polyvinylpyrolidone;
amino acids such as glycine, glutamine, asparagine, histidine,
arginine, or lysine; monosaccharides, disaccharides, and other
carbohydrates including trehalose, glucose, mannose, or dextrins;
chelating agents such as EDTA; sugars such as sucrose, mannitol,
trehalose or sorbitol; salt-forming counter-ions such as sodium;
metal complexes (e.g., Zn-protein complexes); and/or non-ionic
surfactants such as TWEEN, polysorbate 80, PLURONICS.TM. or
polyethylene glycol (PEG).
[0354] The buffering agent may be histidine, citrate, phosphate,
glycine, or acetate. The saccharide excipient may be trehalose,
sucrose, mannitol, maltose or raffinose. The surfactant may be
polysorbate 20, polysorbate 40, polysorbate 80, or Pluronic F68.
The salt may be NaCl, KCl, MgCl.sub.2, or CaCl.sub.2
[0355] The formulations of the low AR compositions of the invention
may include a buffering or pH adjusting agent to provide improved
pH control. A formulation of the invention may have a pH of between
about 3.0 and about 9.0, between about 4.0 and about 8.0, between
about 5.0 and about 8.0, between about 5.0 and about 7.0, between
about 5.0 and about 6.5, between about 5.5 and about 8.0, between
about 5.5 and about 7.0, or between about 5.5 and about 6.5. In a
further embodiment, a formulation of the invention has a pH of
about 3.0, about 3.5, about 4.0, about 4.5, about 5.0, about 5.1,
about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7,
about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3,
about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9,
about 7.0, about 7.5, about 8.0, about 8.5, or about 9.0. In a
specific embodiment, a formulation of the invention has a pH of
about 6.0. One of skill in the art understands that the pH of a
formulation generally should not be equal to the isoelectric point
of the particular antibody to be used in the formulation.
[0356] Typically, the buffering agent is a salt prepared from an
organic or inorganic acid or base. Representative buffering agents
include, but are not limited to, organic acid salts such as salts
of citric acid, ascorbic acid, gluconic acid, carbonic acid,
tartaric acid, succinic acid, acetic acid, or phthalic acid; Tris,
tromethamine hydrochloride, or phosphate buffers. In addition,
amino acid components can also function in a buffering capacity.
Representative amino acid components which may be utilized in the
formulations of the invention as buffering agents include, but are
not limited to, glycine and histidine. In certain embodiments, the
buffering agent is chosen from histidine, citrate, phosphate,
glycine, and acetate. In a specific embodiment, the buffering agent
is histidine. In another specific embodiment, the buffering agent
is citrate. In yet another specific embodiment, the buffering agent
is glycine. The purity of the buffering agent should be at least
98%, or at least 99%, or at least 99.5%. As used herein, the term
"purity" in the context of histidine and glycine refers to chemical
purity of histidine or glycine as understood in the art, e.g., as
described in The Merck Index, 13.sup.th ed., O'Neil et al. ed.
(Merck & Co., 2001).
[0357] Buffering agents are typically used at concentrations
between about 1 mM and about 200 mM or any range or value therein,
depending on the desired ionic strength and the buffering capacity
required. The usual concentrations of conventional buffering agents
employed in parenteral formulations can be found in: Pharmaceutical
Dosage Form: Parenteral Medications, Volume 1, 2.sup.nd Edition,
Chapter 5, p. 194, De Luca and Boylan, "Formulation of Small Volume
Parenterals", Table 5: Commonly used additives in Parenteral
Products. In one embodiment, the buffering agent is at a
concentration of about 1 mM, or of about 5 mM, or of about 10 mM,
or of about 15 mM, or of about 20 mM, or of about 25 mM, or of
about 30 mM, or of about 35 mM, or of about 40 mM, or of about 45
mM, or of about 50 mM, or of about 60 mM, or of about 70 mM, or of
about 80 mM, or of about 90 mM, or of about 100 mM. In one
embodiment, the buffering agent is at a concentration of 1 mM, or
of 5 mM, or of 10 mM, or of 15 mM, or of 20 mM, or of 25 mM, or of
30 mM, or of 35 mM, or of 40 mM, or of 45 mM, or of 50 mM, or of 60
mM, or of 70 mM, or of 80 mM, or of 90 mM, or of 100 mM. In a
specific embodiment, the buffering agent is at a concentration of
between about 5 mM and about 50 mM. In another specific embodiment,
the buffering agent is at a concentration of between 5 mM and 20
mM.
[0358] In certain embodiments, the formulation of the low AR
compositions of the invention comprises histidine as a buffering
agent. In one embodiment the histidine is present in the
formulation of the invention at a concentration of at least about 1
mM, at least about 5 mM, at least about 10 mM, at least about 20
mM, at least about 30 mM, at least about 40 mM, at least about 50
mM, at least about 75 mM, at least about 100 mM, at least about 150
mM, or at least about 200 mM histidine. In another embodiment, a
formulation of the invention comprises between about 1 mM and about
200 mM, between about 1 mM and about 150 mM, between about 1 mM and
about 100 mM, between about 1 mM and about 75 mM, between about 10
mM and about 200 mM, between about 10 mM and about 150 mM, between
about 10 mM and about 100 mM, between about 10 mM and about 75 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 75 mM, between about 20 mM and about 50 mM, between about 20
mM and about 40 mM, or between about 20 mM and about 30 mM
histidine. In a further embodiment, the formulation comprises about
1 mM, about 5 mM, about 10 mM, about 20 mM, about 25 mM, about 30
mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 60
mM, about 70 mM, about 80 mM, about 90 mM, about 100 mM, about 150
mM, or about 200 mM histidine. In a specific embodiment, a
formulation may comprise about 10 mM, about 25 mM, or no
histidine.
[0359] The formulations of the low AR compositions of the invention
may comprise a carbohydrate excipient. Carbohydrate excipients can
act, e.g., as viscosity enhancing agents, stabilizers, bulking
agents, solubilizing agents, and/or the like. Carbohydrate
excipients are generally present at between about 1% to about 99%
by weight or volume, e.g., between about 0.1% to about 20%, between
about 0.1% to about 15%, between about 0.1% to about 5%, between
about 1% to about 20%, between about 5% to about 15%, between about
8% to about 10%, between about 10% and about 15%, between about 15%
and about 20%, between 0.1% to 20%, between 5% to 15%, between 8%
to 10%, between 10% and 15%, between 15% and 20%, between about
0.1% to about 5%, between about 5% to about 10%, or between about
15% to about 20%. In still other specific embodiments, the
carbohydrate excipient is present at 1%, or at 1.5%, or at 2%, or
at 2.5%, or at 3%, or at 4%, or at 5%, or at 10%, or at 15%, or at
20%.
[0360] Carbohydrate excipients suitable for use in the formulations
of the invention include, but are not limited to, monosaccharides
such as fructose, maltose, galactose, glucose, D-mannose, sorbose,
and the like; disaccharides, such as lactose, sucrose, trehalose,
cellobiose, and the like; polysaccharides, such as raffinose,
melezitose, maltodextrins, dextrans, starches, and the like; and
alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol
sorbitol (glucitol) and the like. In one embodiment, the
carbohydrate excipients for use in the present invention are chosen
from, sucrose, trehalose, lactose, mannitol, and raffinose. In a
specific embodiment, the carbohydrate excipient is trehalose. In
another specific embodiment, the carbohydrate excipient is
mannitol. In yet another specific embodiment, the carbohydrate
excipient is sucrose. In still another specific embodiment, the
carbohydrate excipient is raffinose. The purity of the carbohydrate
excipient should be at least 98%, or at least 99%, or at least
99.5%.
[0361] In a specific embodiment, the formulations of the low AR
compositions of the invention may comprise trehalose. In one
embodiment, a formulation of the invention comprises at least about
1%, at least about 2%, at least about 4%, at least about 8%, at
least about 20%, at least about 30%, or at least about 40%
trehalose. In another embodiment, a formulation of the invention
comprises between about 1% and about 40%, between about 1% and
about 30%, between about 1% and about 20%, between about 2% and
about 40%, between about 2% and about 30%, between about 2% and
about 20%, between about 4% and about 40%, between about 4% and
about 30%, or between about 4% and about 20% trehalose. In a
further embodiment, a formulation of the invention comprises about
1%, about 2%, about 4%, about 6%, about 8%, about 15%, about 20%,
about 30%, or about 40% trehalose. In a specific embodiment, a
formulation of the invention comprises about 4%, about 6% or about
15% trehalose.
[0362] In certain embodiments, a formulation of the low AR
compositions of the invention comprises an excipient. In a specific
embodiment, a formulation of the invention comprises at least one
excipient chosen from: sugar, salt, surfactant, amino acid, polyol,
chelating agent, emulsifier and preservative. In one embodiment, a
formulation of the invention comprises a salt, e.g., a salt
selected from: NaCl, KCl, CaCl.sub.2, and MgCl.sub.2. In a specific
embodiment, the formulation comprises NaCl.
[0363] A formulation of the low AR compositions of the invention
may comprise at least about 10 mM, at least about 25 mM, at least
about 50 mM, at least about 75 mM, at least about 80 mM, at least
about 100 mM, at least about 125 mM, at least about 150 mM, at
least about 175 mM, at least about 200 mM, or at least about 300 mM
sodium chloride (NaCl). In a further embodiment, the formulation
may comprise between about 10 mM and about 300 mM, between about 10
mM and about 200 mM, between about 10 mM and about 175 mM, between
about 10 mM and about 150 mM, between about 25 mM and about 300 mM,
between about 25 mM and about 200 mM, between about 25 mM and about
175 mM, between about 25 mM and about 150 mM, between about 50 mM
and about 300 mM, between about 50 mM and about 200 mM, between
about 50 mM and about 175 mM, between about 50 mM and about 150 mM,
between about 75 mM and about 300 mM, between about 75 mM and about
200 mM, between about 75 mM and about 175 mM, between about 75 mM
and about 150 mM, between about 100 mM and about 300 mM, between
about 100 mM and about 200 mM, between about 100 mM and about 175
mM, or between about 100 mM and about 150 mM sodium chloride. In a
further embodiment, the formulation may comprise about 10 mM, about
25 mM, about 50 mM, about 75 mM, about 80 mM, about 100 mM, about
125 mM, about 150 mM, about 175 mM, about 200 mM, or about 300 mM
sodium chloride.
[0364] A formulation of the low AR compositions of the invention
may also comprise an amino acid, e.g., lysine, arginine, glycine,
histidine or an amino acid salt. The formulation may comprise at
least about 1 mM, at least about 10 mM, at least about 25 mM, at
least about 50 mM, at least about 100 mM, at least about 150 mM, at
least about 200 mM, at least about 250 mM, at least about 300 mM,
at least about 350 mM, or at least about 400 mM of an amino acid.
In another embodiment, the formulation may comprise between about 1
mM and about 100 mM, between about 10 mM and about 150 mM, between
about 25 mM and about 250 mM, between about 25 mM and about 300 mM,
between about 25 mM and about 350 mM, between about 25 mM and about
400 mM, between about 50 mM and about 250 mM, between about 50 mM
and about 300 mM, between about 50 mM and about 350 mM, between
about 50 mM and about 400 mM, between about 100 mM and about 250
mM, between about 100 mM and about 300 mM, between about 100 mM and
about 400 mM, between about 150 mM and about 250 mM, between about
150 mM and about 300 mM, or between about 150 mM and about 400 mM
of an amino acid. In a further embodiment, a formulation of the
invention comprises about 1 mM, 1.6 mM, 25 mM, about 50 mM, about
100 mM, about 150 mM, about 200 mM, about 250 mM, about 300 mM,
about 350 mM, or about 400 mM of an amino acid.
[0365] The formulations of the low AR compositions of the invention
may further comprise a surfactant. The term "surfactant" as used
herein refers to organic substances having amphipathic structures;
namely, they are composed of groups of opposing solubility
tendencies, typically an oil-soluble hydrocarbon chain and a
water-soluble ionic group. Surfactants can be classified, depending
on the charge of the surface-active moiety, into anionic, cationic,
and nonionic surfactants. Surfactants are often used as wetting,
emulsifying, solubilizing, and dispersing agents for various
pharmaceutical compositions and preparations of biological
materials. Pharmaceutically acceptable surfactants like
polysorbates (e.g., polysorbates 20 or 80); polyoxamers (e.g.,
poloxamer 188); Triton; sodium octyl glycoside; lauryl-, myristyl-,
linoleyl-, or stearyl-sulfobetaine; lauryl-, myristyl-, linoleyl-
or stearyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine;
lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-,
myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine
(e.g., lauroamidopropyl); myristamidopropyl-, palmidopropyl-, or
isostearamidopropyl-dimethylamine; sodium methyl cocoyl-, or
disodium methyl oleyl-taurate; and the MONAQUA.TM. series (Mona
Industries, Inc., Paterson, N.J.), polyethyl glycol, polypropyl
glycol, and copolymers of ethylene and propylene glycol (e.g.,
PLURONICS.TM., PF68, etc.), can optionally be added to the
formulations of the invention to reduce aggregation. In one
embodiment, a formulation of the invention comprises Polysorbate
20, Polysorbate 40, Polysorbate 60, or Polysorbate 80. Surfactants
are particularly useful if a pump or plastic container is used to
administer the formulation. The presence of a pharmaceutically
acceptable surfactant mitigates the propensity for the protein to
aggregate. The formulations may comprise a polysorbate which is at
a concentration ranging from between about 0.001% to about 1%, or
about 0.001% to about 0.1%, or about 0.01% to about 0.1%. In other
specific embodiments, the formulations of the invention comprise a
polysorbate which is at a concentration of 0.001%, or 0.002%, or
0.003%, or 0.004%, or 0.005%, or 0.006%, or 0.007%, or 0.008%, or
0.009%, or 0.01%, or 0.015%, or 0.02%.
[0366] The formulations of the low AR compositions of the invention
may optionally further comprise other common excipients and/or
additives including, but not limited to, diluents, binders,
stabilizers, lipophilic solvents, preservatives, adjuvants, or the
like. Pharmaceutically acceptable excipients and/or additives may
be used in the formulations of the invention. Commonly used
excipients/additives, such as pharmaceutically acceptable chelators
(for example, but not limited to, EDTA, DTPA or EGTA) can
optionally be added to the formulations of the invention to reduce
aggregation. These additives are particularly useful if a pump or
plastic container is used to administer the formulation.
[0367] Preservatives, such as phenol, m-cresol, p-cresol, o-cresol,
chlorocresol, benzyl alcohol, phenylmercuric nitrite,
phenoxyethanol, formaldehyde, chlorobutanol, magnesium chloride
(for example, but not limited to, hexahydrate), alkylparaben
(methyl, ethyl, propyl, butyl and the like), benzalkonium chloride,
benzethonium chloride, sodium dehydroacetate and thimerosal, or
mixtures thereof can optionally be added to the formulations of the
invention at any suitable concentration such as between about
0.001% to about 5%, or any range or value therein. The
concentration of preservative used in the formulations of the
invention is a concentration sufficient to yield a microbial
effect. Such concentrations are dependent on the preservative
selected and are readily determined by the skilled artisan.
[0368] Other contemplated excipients/additives, which may be
utilized in the formulations of the invention include, for example,
flavoring agents, antimicrobial agents, sweeteners, antioxidants,
antistatic agents, lipids such as phospholipids or fatty acids,
steroids such as cholesterol, protein excipients such as serum
albumin (human serum albumin (HSA), recombinant human albumin
(rHA), gelatin, casein, salt-forming counterions such as sodium and
the like. These and additional known pharmaceutical excipients
and/or additives suitable for use in the formulations of the
invention are known in the art, e.g., as listed in "Remington: The
Science & Practice of Pharmacy", 21.sup.st ed., Lippincott
Williams & Wilkins, (2005), and in the "Physician's Desk
Reference", 60.sup.th ed., Medical Economics, Montvale, N.J.
(2005). Pharmaceutically acceptable carriers can be routinely
selected that are suitable for the mode of administration,
solubility and/or stability of an antibody, as well known those in
the art or as described herein.
[0369] In one embodiment, the low AR compositions of the invention
are formulated with the same or similar excipients and buffers as
are present in the commercial adalimumab (HUMIRA.RTM.) formulation,
as described in the "Highlights of HUMIRA.RTM. Prescribing
Information" for HUMIRA.RTM. (adalimumab) Injection (Revised
January 2008) the entire contents of which are expressly
incorporated herein by reference. For example, each prefilled
syringe of HUMIRA.RTM., which is administered subcutaneously,
delivers 0.8 mL (40 mg) of drug product to the subject. Each 0.8 mL
of HUMIRA.RTM. contains 40 mg adalimumab, 4.93 mg sodium chloride,
0.69 mg monobasic sodium phosphate dihydrate, 1.22 mg dibasic
sodium phosphate dihydrate, 0.24 mg sodium citrate, 1.04 mg citric
acid monohydrate, 9.6 mg mannitol, 0.8 mg polysorbate 80, and water
for Injection, USP. Sodium hydroxide is added as necessary to
adjust pH.
[0370] It will be understood by one skilled in the art that the
formulations of the low AR compositions of the invention may be
isotonic with human blood, wherein the formulations of the
invention have essentially the same osmotic pressure as human
blood. Such isotonic formulations will generally have an osmotic
pressure from about 250 mOSm to about 350 mOSm. Isotonicity can be
measured by, for example, using a vapor pressure or ice-freezing
type osmometer. Tonicity of a formulation is adjusted by the use of
tonicity modifiers. "Tonicity modifiers" are those pharmaceutically
acceptable inert substances that can be added to the formulation to
provide an isotonity of the formulation. Tonicity modifiers
suitable for this invention include, but are not limited to,
saccharides, salts and amino acids.
[0371] In certain embodiments, the formulations of the low AR
compositions of the invention have an osmotic pressure from about
100 mOSm to about 1200 mOSm, or from about 200 mOSm to about 1000
mOSm, or from about 200 mOSm to about 800 mOSm, or from about 200
mOSm to about 600 mOSm, or from about 250 mOSm to about 500 mOSm,
or from about 250 mOSm to about 400 mOSm, or from about 250 mOSm to
about 350 mOSm.
[0372] The concentration of any one component or any combination of
various components, of the formulations of the low AR compositions
of the invention is adjusted to achieve the desired tonicity of the
final formulation. For example, the ratio of the carbohydrate
excipient to antibody may be adjusted according to methods known in
the art (e.g., U.S. Pat. No. 6,685,940). In certain embodiments,
the molar ratio of the carbohydrate excipient to antibody may be
from about 100 moles to about 1000 moles of carbohydrate excipient
to about 1 mole of antibody, or from about 200 moles to about 6000
moles of carbohydrate excipient to about 1 mole of antibody, or
from about 100 moles to about 510 moles of carbohydrate excipient
to about 1 mole of antibody, or from about 100 moles to about 600
moles of carbohydrate excipient to about 1 mole of antibody.
[0373] The desired isotonicity of the final formulation may also be
achieved by adjusting the salt concentration of the formulations.
Pharmaceutically acceptable salts and those suitable for this
invention as tonicity modifiers include, but are not limited to,
sodium chloride, sodium succinate, sodium sulfate, potassium
chloride, magnesium chloride, magnesium sulfate, and calcium
chloride. In specific embodiments, formulations of the invention
comprise NaCl, MgCl.sub.2, and/or CaCl.sub.2. In one embodiment,
concentration of NaCl is between about 75 mM and about 150 mM. In
another embodiment, concentration of MgCl.sub.2 is between about 1
mM and about 100 mM. Pharmaceutically acceptable amino acids
including those suitable for this invention as tonicity modifiers
include, but are not limited to, proline, alanine, L-arginine,
asparagine, L-aspartic acid, glycine, serine, lysine, and
histidine.
[0374] In one embodiment the formulations of the low AR
compositions of the invention are pyrogen-free formulations which
are substantially free of endotoxins and/or related pyrogenic
substances. Endotoxins include toxins that are confined inside a
microorganism and are released only when the microorganisms are
broken down or die. Pyrogenic substances also include
fever-inducing, thermostable substances (glycoproteins) from the
outer membrane of bacteria and other microorganisms. Both of these
substances can cause fever, hypotension and shock if administered
to humans. Due to the potential harmful effects, even low amounts
of endotoxins must be removed from intravenously administered
pharmaceutical drug solutions. The Food & Drug Administration
("FDA") has set an upper limit of 5 endotoxin units (EU) per dose
per kilogram body weight in a single one hour period for
intravenous drug applications (The United States Pharmacopeial
Convention, Pharmacopeial Forum 26 (1):223 (2000)). When
therapeutic proteins are administered in amounts of several hundred
or thousand milligrams per kilogram body weight, as can be the case
with antibodies, even trace amounts of harmful and dangerous
endotoxin must be removed. In certain specific embodiments, the
endotoxin and pyrogen levels in the composition are less then 10
EU/mg, or less then 5 EU/mg, or less then 1 EU/mg, or less then 0.1
EU/mg, or less then 0.01 EU/mg, or less then 0.001 EU/mg.
[0375] When used for in vivo administration, the formulations of
the low AR compositions of the invention should be sterile. The
formulations of the invention may be sterilized by various
sterilization methods, including sterile filtration, radiation,
etc. In one embodiment, the antibody formulation is
filter-sterilized with a presterilized 0.22-micron filter. Sterile
compositions for injection can be formulated according to
conventional pharmaceutical practice as described in "Remington:
The Science & Practice of Pharmacy", 21.sup.st ed., Lippincott
Williams & Wilkins, (2005). Formulations comprising antibodies,
such as those disclosed herein, ordinarily will be stored in
lyophilized form or in solution. It is contemplated that sterile
compositions comprising antibodies are placed into a container
having a sterile access port, for example, an intravenous solution
bag or vial having an adapter that allows retrieval of the
formulation, such as a stopper pierceable by a hypodermic injection
needle. In one embodiment, a composition of the invention is
provided as a pre-filled syringe.
[0376] In one embodiment, a formulation of the low AR compositions
of the invention is a lyophilized formulation. The term
"lyophilized" or "freeze-dried" includes a state of a substance
that has been subjected to a drying procedure such as
lyophilization, where at least 50% of moisture has been
removed.
[0377] The phrase "bulking agent" includes a compound that is
pharmaceutically acceptable and that adds bulk to a lyo cake.
Bulking agents known to the art include, for example,
carbohydrates, including simple sugars such as dextrose, ribose,
fructose and the like, alcohol sugars such as mannitol, inositol
and sorbitol, disaccharides including trehalose, sucrose and
lactose, naturally occurring polymers such as starch, dextrans,
chitosan, hyaluronate, proteins (e.g., gelatin and serum albumin),
glycogen, and synthetic monomers and polymers.
[0378] A "lyoprotectant" is a molecule which, when combined with a
protein of interest (such as an antibody of the invention),
significantly prevents or reduces chemical and/or physical
instability of the protein upon lyophilization and subsequent
storage. Lyoprotectants include, but are not limited to, sugars and
their corresponding sugar alcohols; an amino acid such as
monosodium glutamate or histidine; a methylamine such as betaine; a
lyotropic salt such as magnesium sulfate; a polyol such as
trihydric or higher molecular weight sugar alcohols, e.g.,
glycerin, dextran, erythritol, glycerol, arabitol, xylitol,
sorbitol, and mannitol; propylene glycol; polyethylene glycol;
PLURONICS.TM.; and combinations thereof. Additional examples of
lyoprotectants include, but are not limited to, glycerin and
gelatin, and the sugars mellibiose, melezitose, raffinose,
mannotriose and stachyose. Examples of reducing sugars include, but
are not limited to, glucose, maltose, lactose, maltulose,
iso-maltulose and lactulose. Examples of non-reducing sugars
include, but are not limited to, non-reducing glycosides of
polyhydroxy compounds selected from sugar alcohols and other
straight chain polyalcohols. Examples of sugar alcohols include,
but are not limited to, monoglycosides, compounds obtained by
reduction of disaccharides such as lactose, maltose, lactulose and
maltulose. The glycosidic side group can be either glucosidic or
galactosidic. Additional examples of sugar alcohols include, but
are not limited to, glucitol, maltitol, lactitol and iso-maltulose.
In specific embodiments, trehalose or sucrose is used as a
lyoprotectant.
[0379] The lyoprotectant is added to the pre-lyophilized
formulation in a "lyoprotecting amount" which means that, following
lyophilization of the protein in the presence of the lyoprotecting
amount of the lyoprotectant, the protein essentially retains its
physical and chemical stability and integrity upon lyophilization
and storage.
[0380] In one embodiment, the molar ratio of a lyoprotectant (e.g.,
trehalose) and antibody molecules of a formulation of the invention
is at least about 10, at least about 50, at least about 100, at
least about 200, or at least about 300. In another embodiment, the
molar ratio of a lyoprotectant (e.g., trehalose) and antibody
molecules of a formulation of the invention is about 1, is about 2,
is about 5, is about 10, about 50, about 100, about 200, or about
300.
[0381] A "reconstituted" formulation is one which has been prepared
by dissolving a lyophilized antibody formulation in a diluent such
that the antibody is dispersed in the reconstituted formulation.
The reconstituted formulation is suitable for administration (e.g.,
parenteral administration) to a patient to be treated with the
antibody and, in certain embodiments of the invention, may be one
which is suitable for intravenous administration.
[0382] The "diluent" of interest herein is one which is
pharmaceutically acceptable (safe and non-toxic for administration
to a human) and is useful for the preparation of a liquid
formulation, such as a formulation reconstituted after
lyophilization. In some embodiments, diluents include, but are not
limited to, sterile water, bacteriostatic water for injection
(BWFI), a pH buffered solution (e.g., phosphate-buffered saline),
sterile saline solution, Ringer's solution or dextrose solution. In
an alternative embodiment, diluents can include aqueous solutions
of salts and/or buffers.
[0383] In certain embodiments, a formulation of the low AR
compositions of the invention is a lyophilized formulation
comprising an antibody of the invention, wherein at least about
90%, at least about 95%, at least about 97%, at least about 98%, or
at least about 99% of said antibody may be recovered from a vial
upon shaking said vial for 4 hours at a speed of 400 shakes per
minute wherein the vial is filled to half of its volume with the
formulation. In another embodiment, a formulation of the invention
is a lyophilized formulation comprising an antibody of the
invention, wherein at least about 90%, at least about 95%, at least
about 97%, at least about 98%, or at least about 99% of the
antibody may be recovered from a vial upon subjecting the
formulation to three freeze/thaw cycles wherein the vial is filled
to half of its volume with said formulation. In a further
embodiment, a formulation of the invention is a lyophilized
formulation comprising an antibody of the invention, wherein at
least about 90%, at least about 95%, at least about 97%, at least
about 98%, or at least about 99% of the antibody may be recovered
by reconstituting a lyophilized cake generated from said
formulation.
[0384] In one embodiment, a reconstituted liquid formulation may
comprise an antibody at the same concentration as the
pre-lyophilized liquid formulation.
[0385] In another embodiment, a reconstituted liquid formulation
may comprise an antibody at a higher concentration than the
pre-lyophilized liquid formulation, e.g., .about 2 fold, about 3
fold, about 4 fold, about 5 fold, about 6 fold, about 7 fold, about
8 fold, about 9 fold, or about 10 fold higher concentration of an
antibody than the pre-lyophilized liquid formulation.
[0386] In yet another embodiment, a reconstituted liquid
formulation may comprise an antibody of the invention at a lower
concentration than the pre-lyophilized liquid formulation, e.g.,
about 2 fold, about 3 fold, about 4 fold, about 5 fold, about 6
fold, about 7 fold, about 8 fold, about 9 fold or about 10 fold
lower concentration of an antibody than the pre-lyophilized liquid
formulation.
[0387] The pharmaceutical formulations of the low AR compositions
of the invention are typically stable formulations, e.g., stable at
room temperature.
[0388] The terms "stability" and "stable" as used herein in the
context of a formulation comprising an antibody of the invention
refer to the resistance of the antibody in the formulation to
aggregation, degradation or fragmentation under given manufacture,
preparation, transportation and storage conditions. The "stable"
formulations of the invention retain biological activity under
given manufacture, preparation, transportation and storage
conditions. The stability of the antibody can be assessed by
degrees of aggregation, degradation or fragmentation, as measured
by HPSEC, static light scattering (SLS), Fourier Transform Infrared
Spectroscopy (FTIR), circular dichroism (CD), urea unfolding
techniques, intrinsic tryptophan fluorescence, differential
scanning calorimetry, and/or ANS binding techniques, compared to a
reference formulation. For example, a reference formulation may be
a reference standard frozen at -70.degree. C. consisting of 10
mg/ml of an antibody of the invention in PBS.
[0389] Therapeutic formulations of the low AR compositions of the
invention may be formulated for a particular dosage. Dosage
regimens may be adjusted to provide the optimum desired response
(e.g., a therapeutic response). For example, a single bolus may be
administered, several divided doses may be administered over time
or the dose may be proportionally reduced or increased as indicated
by the exigencies of the therapeutic situation. It is especially
advantageous to formulate parenteral compositions in dosage unit
form for ease of administration and uniformity of dosage. Dosage
unit form as used herein refers to physically discrete units suited
as unitary dosages for the subjects to be treated; each unit
contains a predetermined quantity of active compound calculated to
produce the desired therapeutic effect in association with the
required pharmaceutical carrier. The specification for the dosage
unit forms of the invention are dictated by and directly dependent
on (a) the unique characteristics of the antibody and the
particular therapeutic effect to be achieved, and (b) the
limitations inherent in the art of compounding such an antibody for
the treatment of sensitivity in individuals.
[0390] Therapeutic compositions of the low AR compositions of the
invention can be formulated for particular routes of
administration, such as oral, nasal, pulmonary, topical (including
buccal and sublingual), rectal, vaginal and/or parenteral
administration. The formulations may conveniently be presented in
unit dosage form and may be prepared by any methods known in the
art of pharmacy. The amount of active ingredient which can be
combined with a carrier material to produce a single dosage form
will vary depending upon the subject being treated, and the
particular mode of administration. The amount of active ingredient
which can be combined with a carrier material to produce a single
dosage form will generally be that amount of the composition which
produces a therapeutic effect. By way of example, in certain
embodiments, the antibodies (including antibody fragments) are
formulated for intravenous administration. In certain other
embodiments, the antibodies (including antibody fragments) are
formulated for local delivery to the cardiovascular system, for
example, via catheter, stent, wire, intramyocardial delivery,
intrapericardial delivery, or intraendocardial delivery.
[0391] Formulations of the low AR compositions of the invention
which are suitable for topical or transdermal administration
include powders, sprays, ointments, pastes, creams, lotions, gels,
solutions, patches and inhalants. The active compound may be mixed
under sterile conditions with a pharmaceutically acceptable
carrier, and with any preservatives, buffers, or propellants which
may be required (U.S. Pat. Nos. 7,378,110; 7,258,873; 7,135,180;
7,923,029; and US Publication No. 2004-0042972 and
2004-0042971).
[0392] The phrases "parenteral administration" and "administered
parenterally" as used herein means modes of administration other
than enteral and topical administration, usually by injection, and
includes, without limitation, intravenous, intramuscular,
intraarterial, intrathecal, intracapsular, intraorbital,
intracardiac, intradermal, intraperitoneal, transtracheal,
subcutaneous, subcuticular, intraarticular, subcapsular,
subarachnoid, intraspinal, epidural and intrasternal injection and
infusion.
[0393] Actual dosage levels of the active ingredients in the
pharmaceutical compositions of the low AR compositions of the
invention may be varied so as to obtain an amount of the active
ingredient which is effective to achieve the desired therapeutic
response for a particular patient, composition, and mode of
administration, without being toxic to the patient. The selected
dosage level will depend upon a variety of pharmacokinetic factors
including the activity of the particular compositions of the
present invention employed, or the ester, salt or amide thereof,
the route of administration, the time of administration, the rate
of excretion of the particular compound being employed, the
duration of the treatment, other drugs, compounds and/or materials
used in combination with the particular compositions employed, the
age, sex, weight, condition, general health and prior medical
history of the patient being treated, and like factors well known
in the medical arts.
[0394] In certain embodiments, antibodies of the invention can be
formulated to ensure proper distribution in vivo. For example, the
blood-brain barrier (BBB) excludes many highly hydrophilic
compounds. To ensure that the therapeutic compounds of the
invention can cross the BBB (if desired), they can be formulated,
for example, in liposomes. For methods of manufacturing liposomes,
see, e.g., U.S. Pat. Nos. 4,522,811; 5,374,548; 5,399,331. The
liposomes may comprise one or more moieties which are selectively
transported into specific cells or organs, thus enhance targeted
drug delivery (see, e.g., V. V. Ranade (1989) J. Clin. Pharmacol.
29:685). Exemplary targeting moieties include folate or biotin
(see, e.g., U.S. Pat. No. 5,416,016); mannosides (Umezawa et al.,
(1988) Biochem. Biophys. Res. Commun. 153:1038); antibodies (P. G.
Bloeman et al. (1995) FEBS Lett. 357:140; M. Owais et al. (1995)
Antimicrob. Agents Chemother. 39:180); surfactant Protein A
receptor (Briscoe et al. (1995) Am. J. Physiol. 1233:134),
different species of which may comprise the formulations of the
invention, as well as components of the invented molecules; p120
(Schreier et al. (1994) J. Biol. Chem. 269:9090); see also K.
Keinanen; M. L. Laukkanen (1994) FEBS Lett. 346:123; J. J. Killion;
I. J. Fidler (1994) Immunomethods 4:273. In one embodiment of the
invention, the therapeutic compounds of the invention are
formulated in liposomes; in another embodiment, the liposomes
include a targeting moiety. In another embodiment, the therapeutic
compounds in the liposomes are delivered by bolus injection to a
site proximal to the desired area. When administered in this
manner, the composition must be fluid to the extent that easy
syringability exists. It must be stable under the conditions of
manufacture and storage and may be preserved against the
contaminating action of microorganisms such as bacteria and fungi.
Additionally or alternatively, the antibodies of the invention may
be delivered locally to the brain to mitigate the risk that the
blood brain barrier slows effective delivery.
[0395] In certain embodiments, the low AR compositions of the
invention may be administered with medical devices known in the
art. For example, in certain embodiments an antibody or antibody
fragment is administered locally via a catheter, stent, wire, or
the like. For example, in one embodiment, a therapeutic composition
of the invention can be administered with a needleless hypodermic
injection device, such as the devices disclosed in U.S. Pat. Nos.
5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824;
4,596,556. Examples of well-known implants and modules useful in
the present invention include: U.S. Pat. No. 4,487,603, which
discloses an implantable micro-infusion pump for dispensing
medication at a controlled rate; U.S. Pat. No. 4,486,194, which
discloses a therapeutic device for administering medicants through
the skin; U.S. Pat. No. 4,447,233, which discloses a medication
infusion pump for delivering medication at a precise infusion rate;
U.S. Pat. No. 4,447,224, which discloses a variable flow
implantable infusion apparatus for continuous drug delivery; U.S.
Pat. No. 4,439,196, which discloses an osmotic drug delivery system
having multi-chamber compartments; and U.S. Pat. No. 4,475,196,
which discloses an osmotic drug delivery system. Many other such
implants, delivery systems, and modules are known to those skilled
in the art.
[0396] The efficient dosages and the dosage regimens for the low AR
compositions of the invention depend on the disease or condition to
be treated and can be determined by the persons skilled in the art.
One of ordinary skill in the art would be able to determine such
amounts based on such factors as the subject's size, the severity
of the subject's symptoms, and the particular composition or route
of administration selected.
VII. Alternative Formulations Containing the Low AR Compositions of
the Invention
[0397] a. Aqueous Formulations
[0398] The invention also provides a low AR composition formulated
as an aqueous formulation comprising a protein and water, as
described in U.S. Pat. No. 8,420,081 and PCT Publication No.
WO2012/065072, the contents of which are hereby incorporated by
reference. In these aqueous formulations, the protein is stable
without the need for additional agents. This aqueous formulation
has a number of advantages over conventional formulations in the
art, including stability of the protein in water without the
requirement for additional excipients, increased concentrations of
protein without the need for additional excipients to maintain
solubility of the protein, and low osmolality. These also have
advantageous storage properties, as the proteins in the formulation
remain stable during storage, e.g., stored as a liquid form for
more than 3 months at 7.degree. C. or freeze/thaw conditions, even
at high protein concentrations and repeated freeze/thaw processing
steps. In one embodiment, formulations described herein include
high concentrations of proteins such that the aqueous formulation
does not show significant opalescence, aggregation, or
precipitation.
[0399] In one embodiment, an aqueous low AR composition comprising
a protein, e.g., an antibody, e.g., an anti-TNF.alpha. antibody or
antigen biding portion thereof, and water is provided, wherein the
formulation has certain characteristics, such as, but not limited
to, low conductivity, e.g., a conductivity of less than about 2.5
mS/cm, a protein concentration of at least about 10 .mu.g/mL, an
osmolality of no more than about 30 mOsmol/kg, and/or the protein
has a molecular weight (Mw) greater than about 47 kDa. In one
embodiment, the formulation has improved stability, such as, but
not limited to, stability in a liquid form for an extended time
(e.g., at least about 3 months or at least about 12 months) or
stability through at least one freeze/thaw cycle (if not more
freeze/thaw cycles). In one embodiment, the formulation is stable
for at least about 3 months in a form selected from the group
consisting of frozen, lyophilized, or spray-dried.
[0400] In one embodiment, the formulation has a low conductivity,
including, for example, a conductivity of less than about 2.5
mS/cm, a conductivity of less than about 2 mS/cm, a conductivity of
less than about 1.5 mS/cm, a conductivity of less than about 1
mS/cm, or a conductivity of less than about 0.5 mS/cm.
[0401] In another embodiment, low AR compositions included in the
formulation have a given concentration, including, for example, a
concentration of at least about 1 mg/mL, at least about 10 mg/mL,
at least about 50 mg/mL, at least about 100 mg/mL, at least about
150 mg/mL, at least about 200 mg/mL, or greater than about 200
mg/mL. In another embodiment, the formulation of the invention has
an osmolality of no more than about 15 mOsmol/kg.
[0402] The aqueous formulations described herein do not rely on
standard excipients, e.g., a tonicity modifier, a stabilizing
agent, a surfactant, an anti-oxidant, a cryoprotectant, a bulking
agent, a lyroprotectant, a basic component, and an acidic
component. In other embodiments of the invention, the formulation
contains water, one or more proteins, and no ionic excipients
(e.g., salts, free amino acids).
[0403] In certain embodiments, the aqueous formulation as described
herein comprise a low AR composition comprising a protein
concentration of at least 50 mg/mL and water, wherein the
formulation has an osmolality of no more than 30 mOsmol/kg. Lower
limits of osmolality of the aqueous formulation are also
encompassed by the invention. In one embodiment the osmolality of
the aqueous formulation is no more than 15 mOsmol/kg. The aqueous
formulation of the invention may have an osmolality of less than 30
mOsmol/kg, and also have a high protein concentration, e.g., the
concentration of the protein is at least 100 mg/mL, and may be as
much as 200 mg/mL or greater. Ranges intermediate to the above
recited concentrations and osmolality units are also intended to be
part of this invention. In addition, ranges of values using a
combination of any of the above recited values as upper and/or
lower limits are intended to be included.
[0404] The concentration of the aqueous formulation as described
herein is not limited by the protein size and the formulation may
include any size range of proteins. Included within the scope of
the invention is an aqueous formulation comprising at least 40
mg/mL and as much as 200 mg/mL or more of a protein, for example,
40 mg/mL, 65 mg/mL, 130 mg/mL, or 195 mg/ml, which may range in
size from 5 kDa to 150 kDa or more. In one embodiment, the protein
in the formulation of the invention is at least about 15 kD in
size, at least about 20 kD in size; at least about 47 kD in size;
at least about 60 kD in size; at least about 80 kD in size; at
least about 100 kD in size; at least about 120 kD in size; at least
about 140 kD in size; at least about 160 kD in size; or greater
than about 160 kD in size. Ranges intermediate to the above recited
sizes are also intended to be part of this invention. In addition,
ranges of values using a combination of any of the above recited
values as upper and/or lower limits are intended to be
included.
[0405] The aqueous formulation as described herein may be
characterized by the hydrodynamic diameter (D.sub.h) of the
proteins in solution. The hydrodynamic diameter of the protein in
solution may be measured using dynamic light scattering (DLS),
which is an established analytical method for determining the
D.sub.h of proteins. Typical values for monoclonal antibodies,
e.g., IgG, are about 10 nm. Low-ionic formulations may be
characterized in that the D.sub.h of the proteins are notably lower
than protein formulations comprising ionic excipients. It has been
discovered that the D.sub.h values of antibodies in aqueous
formulations made using the disfiltration/ultrafilteration (DF/UF)
process, as described in U.S. Pat. No. 8,420,081 and PCT
Publication No. WO2012/065072, using pure water as an exchange
medium, are notably lower than the D.sub.h of antibodies in
conventional formulations independent of protein concentration. In
one embodiment, antibodies in the aqueous formulation as described
herein have a D.sub.h of less than 4 nm, or less than 3 nm.
[0406] In one embodiment, the D.sub.h of the protein in the aqueous
formulation is smaller relative to the D.sub.h of the same protein
in a buffered solution, irrespective of protein concentration.
Thus, in certain embodiments, protein in an aqueous formulation
made in accordance with the methods described herein, will have a
D.sub.h which is at least 25% less than the D.sub.h of the protein
in a buffered solution at the same given concentration. Examples of
buffered solutions include, but are not limited to phosphate
buffered saline (PBS). In certain embodiments, proteins in the
aqueous formulation of the invention have a D.sub.h that is at
least 50% less than the D.sub.h of the protein in PBS in at the
given concentration; at least 60% less than the D.sub.h of the
protein in PBS at the given concentration; at least 70% less than
the D.sub.h of the protein in PBS at the given concentration; or
more than 70% less than the D.sub.h of the protein in PBS at the
given concentration. Ranges intermediate to the above recited
percentages are also intended to be part of this invention, e.g.,
about 55%, 56%, 57%, 64%, 68%, and so forth. In addition, ranges of
values using a combination of any of the above recited values as
upper and/or lower limits are intended to be included, e.g., about
50% to about 80%.
[0407] In one aspect, the aqueous formulation includes the protein
at a dosage of about 0.01 mg/kg-10 mg/kg. In another aspect, the
dosages of the protein include approximately 1 mg/kg administered
every other week, or approximately 0.3 mg/kg administered weekly. A
skilled practitioner can ascertain the proper dosage and regime for
administering to a subject.
[0408] b. "Solid Unit" Formulations
[0409] The present invention also provides a low AR composition of
the invention formulated as a stable composition of a protein,
e.g., particularly a therapeutic protein such as an antibody, or
antigen binding portion thereof, and a stabilizer, referred to
herein as solid units. These formulations are described, for
example, in U.S. Provisional Patent Application 61/893,123,
entitled "Stable Solid Protein Compositions and Methods of Making
Same", Attorney Docket Number 117813-31001, filed on Oct. 18, 2013,
the entire contents of which are expressly incorporated herein by
reference.
[0410] Specifically, it has been discovered that despite having a
high proportion of sugar relative to the protein, the solid units
of the invention maintain structural rigidity and resist changes in
shape and/or volume when stored under ambient conditions, e.g.,
room temperature and humidity, for extended periods of time. The
solid units of the invention remain free-flowing and are able to
maintain long-term physical and chemical stability of the protein
without significant degradation and/or aggregate formation. The
solid units of the invention have many advantages over the art,
including that they can be formulated for oral delivery and are
easily reconstituted in a diluent, such as water. Because the solid
units are readily dissolved, they may be used in dual chamber
delivery devices and may be prepared directly in a device for
patient use.
[0411] As used herein, the term "solid unit," refers to a
composition which is suitable for pharmaceutical administration and
comprises a protein, e.g., an antibody or peptide, and a
stabilizer, e.g., a sugar. The solid unit has a structural rigidity
and resistance to changes in shape and/or volume. In a preferred
embodiment, the solid unit is obtained by lyophilizing a
pharmaceutical formulation of a therapeutic protein. The solid unit
may be any shape, e.g., geometric shape, including, but not limited
to, a sphere, a cube, a pyramid, a hemisphere, a cylinder, a
teardrop, and so forth, including irregularly shaped units. In one
embodiment, the solid unit has a volume ranging from about 1 ml to
about 20 nil. In one embodiment, the solid unit is not obtained
using spray drying techniques, e.g., the solid unit is not a powder
or granule.
[0412] As used herein, the phrase "a plurality of solid units"
refers to a collection or population of solid units, wherein the
collection comprises two or more solid units having a substantially
uniform shape, e.g., sphere, and/or volume distribution. In one
embodiment, the plurality of solid units is free-flowing.
VIII. Kits and Articles of Manufacture Comprising the Low AR
Compositions of the Invention
[0413] Also within the scope of the present invention are kits
comprising the low AR compositions of the invention and
instructions for use. The term "kit" as used herein refers to a
packaged product comprising components with which to administer the
antibody, or antigen-binding portion thereof, of the invention for
treatment of a disease or disorder. The kit may comprise a box or
container that holds the components of the kit. The box or
container is affixed with a label or a Food and Drug Administration
approved protocol. The box or container holds components of the
invention which may be contained within plastic, polyethylene,
polypropylene, ethylene, or propylene vessels. The vessels can be
capped-tubes or bottles. The kit can also include instructions for
administering an antibody of the invention.
[0414] The kit can further contain one more additional reagents,
such as an immunosuppressive reagent, a cytotoxic agent or a
radiotoxic agent or one or more additional antibodies of the
invention (e.g., an antibody having a complementary activity which
binds to an epitope in the TNF.alpha. antigen distinct from a first
anti-TNF.alpha. antibody). Kits typically include a label
indicating the intended use of the contents of the kit. The term
label includes any writing, or recorded material supplied on or
with the kit, or which otherwise accompanies the kit.
[0415] The invention also provides a pharmaceutical pack or kit
comprising one or more containers filled with a liquid formulation
or lyophilized formulation of an antibody or antibody fragment
thereof of the invention. In one embodiment, a container filled
with a liquid formulation of the invention is a pre-filled syringe.
In a specific embodiment, the formulations of the invention are
formulated in single dose vials as a sterile liquid. For example,
the formulations may be supplied in 3 cc USP Type I borosilicate
amber vials (West Pharmaceutical Services--Part No. 6800-0675) with
a target volume of 1.2 mL. Optionally associated with such
container(s) can be a notice in the form prescribed by a
governmental agency regulating the manufacture, use or sale of
pharmaceuticals or biological products, which notice reflects
approval by the agency of manufacture, use or sale for human
administration.
[0416] In one embodiment, a container filled with a liquid
formulation of the invention is a pre-filled syringe. Any
pre-filled syringe known to one of skill in the art may be used in
combination with a liquid formulation of the invention. Pre-filled
syringes that may be used are described in, for example, but not
limited to, PCT Publications WO05032627, WO08094984, WO9945985,
WO03077976, U.S. Pat. No. 6,792,743, U.S. Pat. No. 5,607,400, U.S.
Pat. No. 5,893,842, U.S. Pat. No. 7,081,107, U.S. Pat. No.
7,041,087, U.S. Pat. No. 5,989,227, U.S. Pat. No. 6,807,797, U.S.
Pat. No. 6,142,976, U.S. Pat. No. 5,899,889, U.S. Pat. No.
7,699,811, U.S. Pat. No. 7,540,382, U.S. Pat. No. 7,998,120, U.S.
Pat. No. 7,645,267, and US Patent Publication No. US20050075611.
Pre-filled syringes may be made of various materials. In one
embodiment a pre-filled syringe is a glass syringe. In another
embodiment a pre-filled syringe is a plastic syringe. One of skill
in the art understands that the nature and/or quality of the
materials used for manufacturing the syringe may influence the
stability of a protein formulation stored in the syringe. For
example, it is understood that silicon based lubricants deposited
on the inside surface of the syringe chamber may affect particle
formation in the protein formulation. In one embodiment, a
pre-filled syringe comprises a silicone based lubricant. In one
embodiment, a pre-filled syringe comprises baked on silicone. In
another embodiment, a pre-filled syringe is free from silicone
based lubricants. One of skill in the art also understands that
small amounts of contaminating elements leaching into the
formulation from the syringe barrel, syringe tip cap, plunger or
stopper may also influence stability of the formulation. For
example, it is understood that tungsten introduced during the
manufacturing process may adversely affect formulation stability.
In one embodiment, a pre-filled syringe may comprise tungsten at a
level above 500 ppb. In another embodiment, a pre-filled syringe is
a low tungsten syringe. In another embodiment, a pre-filled syringe
may comprise tungsten at a level between about 500 ppb and about 10
ppb, between about 400 ppb and about 10 ppb, between about 300 ppb
and about 10 ppb, between about 200 ppb and about 10 ppb, between
about 100 ppb and about 10 ppb, between about 50 ppb and about 10
ppb, between about 25 ppb and about 10 ppb.
[0417] In certain embodiments, kits comprising antibodies of the
invention are also provided that are useful for various purposes,
e.g., research and diagnostic including for purification or
immunoprecipitation of protein of interest from cells, detection of
the protein of interest in vitro or in vivo. For isolation and
purification of a protein of interest, the kit may contain an
antibody coupled to beads (e.g., sepharose beads). Kits may be
provided which contain the antibodies for detection and
quantitation of a protein of interest in vitro, e.g., in an ELISA
or a Western blot. As with the article of manufacture, the kit
comprises a container and a label or package insert on or
associated with the container. The container holds a composition
comprising at least one antibody of the invention. Additional
containers may be included that contain, e.g., diluents and
buffers, control antibodies. The label or package insert may
provide a description of the composition as well as instructions
for the intended in vitro or diagnostic use.
[0418] The present invention also encompasses a finished packaged
and labeled pharmaceutical product. This article of manufacture
includes the appropriate unit dosage form in an appropriate vessel
or container such as a glass vial, pre-filled syringe or other
container that is hermetically sealed. In one embodiment, the unit
dosage form is provided as a sterile particulate free solution
comprising an antibody that is suitable for parenteral
administration. In another embodiment, the unit dosage form is
provided as a sterile lyophilized powder comprising an antibody
that is suitable for reconstitution.
[0419] In one embodiment, the unit dosage form is suitable for
intravenous, intramuscular, intranasal, oral, topical or
subcutaneous delivery. Thus, the invention encompasses sterile
solutions suitable for each delivery route. The invention further
encompasses sterile lyophilized powders that are suitable for
reconstitution.
[0420] As with any pharmaceutical product, the packaging material
and container are designed to protect the stability of the product
during storage and shipment. Further, the products of the invention
include instructions for use or other informational material that
advise the physician, technician or patient on how to appropriately
prevent or treat the disease or disorder in question, as well as
how and how frequently to administer the pharmaceutical. In other
words, the article of manufacture includes instruction means
indicating or suggesting a dosing regimen including, but not
limited to, actual doses, monitoring procedures, and other
monitoring information. Specifically, the invention provides an
article of manufacture comprising packaging material, such as a
box, bottle, tube, vial, container, pre-filled syringe, sprayer,
insufflator, intravenous (i.v.) bag, envelope and the like; and at
least one unit dosage form of a pharmaceutical agent contained
within said packaging material, wherein said pharmaceutical agent
comprises a liquid formulation containing an antibody. The
packaging material includes instruction means which indicate how
that said antibody can be used to prevent, treat and/or manage one
or more symptoms associated with a disease or disorder.
The present invention is further illustrated by the following
examples which should not be construed as limiting in any way. The
contents of all cited references, including literature references,
issued patents, and published patent applications, as cited
throughout this application are hereby expressly incorporated
herein by reference. It should further be understood that the
contents of all the figures and tables attached hereto are
expressly incorporated herein by reference. The entire contents of
the following applications are also expressly incorporated herein
by reference: U.S. Provisional Patent Application 61/893,123,
entitled "STABLE SOLID PROTEIN COMPOSITIONS AND METHODS OF MAKING
SAME", Attorney Docket Number 117813-31001, filed on Oct. 18, 2013;
U.S. Provisional Application Ser. No. 61/892,833, entitled "LOW
ACIDIC SPECIES COMPOSITIONS AND METHODS FOR PRODUCING THE SAME
USING DISPLACEMENT CHROMATOGRAPHY", Attorney Docket Number
117813-73602, filed on Oct. 18, 2013; U.S. Provisional Patent
Application 61/892,710, entitled "MUTATED ANTI-TNF.alpha.
ANTIBODIES AND METHODS OF THEIR USE", Attorney Docket Number
117813-73802, filed on Oct. 18, 2013; U.S. Provisional Patent
Application 61/893,068, entitled "LOW ACIDIC SPECIES COMPOSITIONS
AND METHODS FOR PRODUCING THE SAME", Attorney Docket Number
117813-73901, filed on Oct. 18, 2013; U.S. Provisional Patent
Application 61/893,088, entitled "MODULATED LYSINE VARIANT SPECIES
AND METHODS FOR PRODUCING AND USING THE SAME", Attorney Docket
Number 117813-74101, filed on Oct. 18, 2013; and U.S. Provisional
Patent Application 61/893,131, entitled "PURIFICATION OF PROTEINS
USING HYDROPHOBIC INTERACTION CHROMATOGRAPHY", Attorney Docket
Number 117813-74301, filed on Oct. 18, 2013.
IX. Examples
[0421] Three antibodies were used in connection with the studies
outlined below (Examples 1-8). Adalimumab antibody was generated
from cell culture processed using chemical defined medium (CDM) and
purified by a 4.4 cm (id.).times..about.20 cm (L) MabSelect SuRe
Protein A column. mAb X bulk drug substance was obtained from a
three-step large scale purification process. mAb Y antibody was
generated from a large scale manufacturing process and purified by
a MabSelect SuRe Protein A column. Adalimumab Protein A eluate was
in a buffer of .about.20 mM acetic acid at pH .about.4.2. The mAb X
was in a buffer containing .about.15 mM histidine, pH .about.6. The
mAb Y was in a buffer containing .about.10 mM sodium formate, pH
.about.4.2. Each mAb feed was conditioned to the targeted pH,
conductivity and concentration prior to the displacement
chromatography experiment.
[0422] The cationic displacers, Expell SP1.TM. and protamine
sulfate (from salmon sperm), were purchased from SACHEM Chemical
Company and Sigma Aldrich, respectively.
[0423] Poros XS CEX resin (Life Technologies) was packed in a 0.66
cm.times..about.25 cm column. The column was equilibrated with a
140 mM Tris/Acetate buffer or a 30 mM MES, 10 mM NaCl buffer at the
targeted pH and conductivity (Table 1). After equilibration, the
column was loaded with each pre-conditioned feed at a resin loading
level of .about.40 g/L followed by a 2 CV of equilibration buffer
wash. The displacing buffer, which consists of defined
concentration of Expell SP1.TM. or protamine sulfate in the
equilibration buffer, was flowed through the column to initiate the
displacement process. In standard one-step displacement wash
process, this step was continued for at least 30 CV at a flow rate
corresponding to 15 to 22 min residence time (RT) before column
regeneration and cleaning with a caustic solution consisting of 0.5
N NaOH and 0.5 M KCl. Alternatively, the displacement wash step
comprised two displacement buffers each flowing for defined
volumes, or a linear gradient flow from low to high concentration
displacer buffer. Sample fractions were collected at every 0.5 or 1
CV for protein concentration and quality analysis. The specific
processing conditions are detailed in Tables 1 and 2.
[0424] Capto MMC resin (GE Healthcare) was packed in a 0.66
cm.times..about.30 cm column. The column was equilibrated with a
140 mM Tris/Acetate buffer at the targeted pH and conductivity
(Table 3). After equilibration, the column was loaded with each
pre-conditioned feed at a resin loading level about 34 to 40 g/L
followed by a 2 CV equilibration buffer wash. The displacing
buffer, which consists of defined concentration of protamine
sulfate in the equilibration buffer, was flowed through the column
to initiate the displacement process. This step was continued for
30 CV at a flow rate corresponding to .about.22 min RT before
column regeneration and cleaning. Sample fractions were collected
at every 0.5 or 1 CV for protein concentration and quality
analysis. The specific processing conditions are detailed in Table
3.
[0425] The levels of acidic species and other charge variants in
the Adalimumab, mAb X and mAb Y samples were quantified using the
respective qualified CEX-HPLC method. For Adalimumab, a 4
mm.times.250 mm analytical Dionex ProPac WCX-10 column (Dionex,
Calif.) was used along with a Shimazhu HPLC system. The mobile
phases were 10 mM Sodium Phosphate dibasic pH 7.5 buffer (Mobile
phase A) and 10 mM Sodium Phosphate dibasic, 500 mM Sodium Chloride
pH 5.5 buffer (Mobile phase B). A binary gradient (6% B: 0 min;
6-16% B: 0-20 min; 16-100% B: 20-22 min; 100% B: 22-26 min; 100-6%
B: 26-28 min; 6% B: 28-35 min) was used with detection at 280 nm.
Quantitation was based on the relative area percentage of detected
peaks. The peaks that elute at residence time less than .about.7
min were together represented as the acidic peaks or AR region.
[0426] For mAb X, a 4 mm.times.250 mm analytical Dionex ProPac
WCX-10 column (Dionex, Calif.) was used along with a Shimazhu HPLC
system. The mobile phases were 20 mM MES, pH 6.5 buffer (Mobile
phase A) and 20 mM MES, 500 mM NaCl, pH 6.5 buffer (Mobile phase
B). A binary gradient (10% B: 0 min; 10-28% B: 1-46 min; 28-100% B:
46-47 min; 100% B: 47-52 min; 100-10% B: 52-53 min; 10% B: 53-58
min) was used with detection at 280 nm. Quantitation was based on
the relative area percentage of detected peaks. All peaks eluting
prior to the Main Isoform peak were summed as the acidic region,
and all peaks eluting after the Main peak were summed as the basic
region.
[0427] For mAb Y, a 4 mm.times.250 mm Dionex ProPac analytical
WCX-10 column (Dionex, Calif.) was used on a Shimazhu HPLC system.
The mobile phases were 20 mM MES, pH 6.2 (Mobile phase A) and 20 mM
MES, 250 mM NaCl, pH 6.2 (Mobile phase B). A binary gradient (1% B:
0-1 min; 1-25% B: 1-46 min; 25-100% B: 46-47 min; 100% B: 47-52
min; 100-1% B: 52-53 min; 1% B: 53-60 min) was used with detection
at 280 nm. Column temperature was set at 35.degree. C. Quantitation
was based on the relative area percentage of detected peaks. All
peaks eluting prior to the Main Isoform peak (but after 2 min
retention time) were summed as the acidic region, and all peaks
eluting after the Main peak were summed as the basic region.
[0428] The levels of aggregates, monomer and fragments in eluate
samples were measured using a SEC method for each molecule. For
Adalimumab and mAb Y, a TSK-gel G3000SW.times.L, 5 .mu.m, 125
.ANG., 7.8.times.300 mm column (Tosoh Bioscience) was used while a
TSK-gel Super SW3000, 4 .mu.m, 250 .ANG., 4.6.times.300 mm column
(Tosoh Bioscience) was used for mAb X along with an Agilent or a
Shimazhu HPLC system. For Adalimumab and mAb X, injections were
made under isocratic elution conditions using a mobile phase
consisting of 100 mM sodium sulfate and 100 mM sodium phosphate at
pH 6.8, and detected with UV absorbance at 214 nm. For mAb Y, the
mobile phase consists of 1.times.PBS at pH 7.4, and elution profile
detected with UV absorbance at 280 nm. Quantification is based on
the relative area of detected peaks.
[0429] An HCP ELISA assay was used to determine the HCP levels in
various samples and feeds for all three mAbs.
TABLE-US-00003 TABLE 1 Processing conditions for Poros XS one-step
displacement chromatography Displacer Equilibration/Wash/Displacing
buffer Conc. Buffer Conductivity Loading Molecule Displacer (mM)
System pH (mS/cm) Conditions Regeneration Adalim- Expell 0.5-3
Tris/ 6.7-7.8 5.4-6.6 pH ~7.5, ~6 2M NaCl umab SP1 Acetate mS/cm
.sup. 2-5 MES/ 6.1 2.1 pH 6.1, ~2 0.2M acetic acid NaCl mS/cm &
1M KCl Protamine 0.25-2 Tris/ 6.5-7.5 5.6-6.6 pH 7.5, 5.4-6.3 2M
NaCl, 6M Sulfate Acetate mS/cm Guanidine HCl mAb X Expell 0.5-2
Tris/ 6 6.2-6.5 pH 6, ~6 2M NaCl SP1 Acetate mS/cm Protamine
0.25-0.5 Tris/ 6 6.0-6.5 pH 6, 5.6-6.5 2M NaCl, 6M Sulfate Acetate
mS/cm Guanidine HCl mAb Y Expell 0.5-1 Tris/ 5 ~6 pH 5, 6.2 2M NaCl
SP1 Acetate mS/cm
TABLE-US-00004 TABLE 2 Processing conditions for Poros XS two-step
or linear gradient displacement chromatography Displacer
Equilibration/Wash/Displacing buffer Displacement Concentration
Buffer Conductivity Loading Molecule Displacer Method (mM) System
pH (mS/cm) Conditions Regeneration Adalim- Expell Two- (1): 0.5 mM,
Tris/ 7 ~6 pH 7.5, 2M NaCl umab SP1 step 25 CV; Acetate 6.1 mS/cm
(2): 2 mM, 20 CV Protamine Two- (1): 0.25 mM, Tris/ 7.5 5.5 pH 7.5,
2M NaCl, 6M Sulfate step 10 CV; Acetate 6.1 mS/cm Guanidine HCl
(2): 2 mM, 10 CV Expell Linear 0-1 mM Tris/ 7 ~6 pH 7.5, 2M NaCl
SP1 Gradient over 40 CV Acetate 6.0 mS/cm Protamine Linear 0-1 mM
Tris/ 7.5 ~6 pH 7.5, 2M NaCl, 6M Sulfate Gradient over 40 CV
Acetate 5.9 mS/cm Guanidine HCl mAb X Expell Two- (1): 0.5 mM,
Tris/ 6 6.1 pH 6, 2M NaCl SP1 step 22 CV; Acetate 6.3 mS/cm (2): 2
mM, 12 CV Protamine Two- (1): 0.35 mM, Tris/ 6 ~6 pH 6, 2M NaCl
Sulfate step 10 CV; Acetate 6.3 mS/cm (2): 0.5 mM, 10 CV
TABLE-US-00005 TABLE 3 Processing conditions for Capto MMC one-step
displacement chromatography Displacer Equilibration/Wash/Displacing
buffer Concentration Buffer Conductivity Loading Molecule Displacer
(mM) System pH (mS/cm) Conditions Regeneration CIP Adalim-
Protamine 0.25-0.5 Tris/ 7-7.5 ~6 pH 7.5, 2M NaCl, 6M 0.5N NaOH +
umab Sulfate Acetate 5.3-6.1 Guanidine HCl 0.5M KCl mS/cm mAb X
Protamine 0.25-0.5 Tris/ 7-7.7 ~6 pH 7-7.7, 2M NaCl, 6M Sulfate
Acetate 5.9-6.5 Guanidine HCl mS/cm mAb Y Protamine 0.25-0.5 Tris/
5-5.5 ~6.5 pH 5.5, 2M NaCl, 6M Sulfate Acetate 5.2-5.6 Guanidine
HCl mS/cm
Example 1. Displacement Chromatography Performances of Expell
SP1.TM. for Adalimumab on Poros XS Resin
[0430] Expell SP1.TM. is a low molecular weight quaternary ammonium
salt that exhibited pronounced displacement effect for Adalimumab
on Poros XS resin under selected sets of operating conditions. The
feed material used for this set of experiments contained about
20-25% total AR, of which 2-5% was AR1 and 18-20% AR2. The results
for this system are shown in the following sections.
[0431] A representative, desired displacement chromatographic
profile is shown in FIG. 1a (solid line). In this experiment, the
column was equilibrated with a pH 7 Tris/acetate buffer (6.4
mS/cm), loaded with a pre-adjusted protein A eluate feed (pH 7.5,
6.3 mS/cm, .about.3.4 g/L) to .about.40 g/L resin loading level,
followed by EQ buffer wash and then displacement process using 1 mM
Expell SP1.TM. in the pH 7 EQ buffer. The extended, square shape
UV280 "elution" profile indicated establishing a proper
displacement train and thus a degree of separation of the feed
components can be realized.
[0432] FIG. 2 illustrates the CEX-HPLC chromatograms for several
samples taken along this well-established displacement UV trace.
Clearly, the variant species were rearranged during the
displacement process according to their respective binding affinity
to the resin: AR1 was enriched in the foremost of the displacement
train followed by AR2, Lys 0, Lys 1 and Lys 2 in order.
[0433] FIG. 3 shows the distribution of each variant species in all
the collected sample fractions. The acidic species were enriched in
the earlier fractions compared to the Lys variants. By excluding
those earlier fractions the product pool AR level will be reduced
relative to that in the feed. This is reflected in FIG. 4 which
plots the reduction of total AR (i.e., AR1+AR2) and AR1 level
versus cumulative product yield. At a yield of .about.75%, the
total AR % was reduced by 11.7% and AR1% by 4.2% under this set of
condition. Along with the removal of AR species, the product pool
lysine variant species distribution profile was also modulated. As
shown in Table 4, below, the ratio of Lys 0 species to the lysine
variant sum decreased from 0.67 to 0.62; the ratio of Lys 1 species
to the lysine variant sum increased from 0.24 to 0.28; and the
ratio of Lys 2 species to the lysine variant sum increased from
0.08 to 0.1. The lysine prolife can be further altered by pooling
different fractions from the displacement chromatography
process.
[0434] Varying the processing conditions such as the buffer pH and
displacer concentration can modulate the shape of the displacement
chromatogram and hence the separation performance. In an extreme
case, the chromatogram more or less resembles the typical elution
"peak" profile without incurring the separation of variant species
(FIGS. 1A and 1B). Interestingly, this occurs at stronger binding
conditions; for instance, the conditions corresponding to FIG. 1B
is pH 6.1 and .about.2 mS/cm for equilibration, loading, wash, and
displacement. Without being bond by theory, the lack of variant
separation under such conditions may be due to the diminishing
difference in binding affinity of each species and thus the
selectivity by the displacer.
[0435] The effect of Expell SP1.TM. concentration on Adalimumab AR
reduction was measured in pH 7.5 Tris/Acetate buffer, as shown in
FIG. 5. The same equilibration/wash and feed loading conditions
were used for all the runs here. Increasing Expell SP1.TM.
concentration from 0.5 to 3 mM decreased AAR % from 8.9% to 3.5% at
similar product yield .about.75%. Controlling the Expell SP1.TM.
concentration within 2 mM will consistently achieve .gtoreq.6% AR %
reduction.
[0436] The effect of displacing buffer pH on AR reduction for
Adalimumab was measured at 1 mM Expell SP1.TM. concentration in the
Tris/Acetate buffer, as shown in FIG. 6. In this set of
experiments, the column was conditioned with an EQ buffer at the
respective displacing buffer pH, and then loaded with protein feed
at pH 7.5 and .about.6 mS/cm followed by a brief EQ buffer wash
before starting the displacement step. The buffer pH significantly
impacts AR clearance in pH range of 6-8. At similar yield
(.about.75%), the maximal reduction in AR level (.about.12%) is
seen at pH 7. Despite such pH-dependency, the majority of the
conditions here (pH 6.5 to 7.8) gave at least 5% AR removal in
final product pool.
[0437] In the aforementioned experiments, one displacing buffer was
used to achieve the protein variant separation. It was observed
that, relatively lower displacer concentration gives better
separation but tends to elongate the process due to substantial
increase in the required displacing buffer volume. For instance,
when using 0.5 mM Expell SP1.TM. in a pH 7 displacing buffer (Table
1), the displacement phase requires 44 column volumes (CV) of this
buffer for completion. To accelerate the operation without
affecting the acidic species separation, a two-step displacement
process was explored at this pH condition. In the example provided
here, the displacement process was started with 0.5 mM Expell
SP1.TM. at pH 7 and continued for 25 CV, followed by 20 CV of 2 mM
Expell SP1.TM. solution at the same pH. Under such conditions, the
protein displacement profile was completed in a total of 33 CV
which is 25% less than that required for one-step displacement
process, thus significantly shortening the process.
[0438] FIG. 7 shows the reduction of AR % versus product yield for
the aforementioned two-step displacement run. The net total AR
level in product pool was reduced by 6.6% at .about.75% yield. In
contrast to the conventional use of a single displacing solution
consisting of a single displacer at a defined concentration, herein
the AR clearance was achieved by excluding the AR-enriched early
fractions as induced by 0.5 mM Expell SP1.TM. displacement, while
the higher Expell SP1.TM. concentration was used to accelerate the
displacement of the remainder proteins off the solid phase. In
light of this unexpected similar product quality and yield results,
step-gradient displacement schemes are considered to be
advantageous over conventional strategies.
[0439] Besides the two-step displacement scheme, a linear gradient
displacement method was also tested for the Adalimumab charge
variant separation. As detailed in Table 2, after the feed loading
at pH 7.5 (.about.6 mS/cm), the column was briefly washed with the
equilibration buffer (pH 7, .about.6 mS/cm) and then started with a
40 CV linear gradient from the EQ buffer to a 1 mM Expell SP1.TM.
displacing buffer (which was made from the EQ buffer). Under such
condition, the displacement profile matured within this 40 CV
gradient. The product eluate was pooled by excluding the first a
few fractions. In this case, the net AR % decreased by 6.8% at a
product recovery of 72%.
[0440] Apart from acidic species, other product- or process-related
impurities can be effectively separated by Poros XS displacement
chromatography using Expell SP1.TM. as the displacer. FIG. 8 shows
the separation of aggregates, monomer and fragments in Adalimumab
sample fractions obtained from a one-step displacement experiment
using 1 mM Expell SP1.TM., pH 7 buffer. It should be noted that the
last two fractions from this run were not collected, therefore the
increased aggregate levels at the end of the displacement train was
not fully exemplified here. Interestingly, the early fractions
which contained elevated acidic species also showed enriched
aggregates, indicating that this population of aggregates may
consist of more acidic species, or the acidic species has higher
propensity to form aggregates. As summarized in Table 4, the
aggregate level in the product pool (at .about.75% yield) was
reduced from the feed level 1.16% to 0.11% and the fragment level
down to 0.04% along with significant reduction in the AR
concentration. In addition to the standard method, the linear
gradient displacement run also showed aggregate reduction from the
feed level of 0.9% to about 0.2% in final product.
[0441] FIG. 9 shows the distribution of HCP in the Adalimumab
displacement train coming off the Poros XS column. Relatively
higher level of HCP was observed at both ends of the train, due to
their diverse charge characteristics and associated binding
strength. The final product pool HCP level was reduced to 5 ng/mg
from the starting feed, representing approximately 50-fold
reduction.
TABLE-US-00006 TABLE 4 Step yield & product quality in
Adalimumab before and after Poros XS displacement chromatography
using Expell SP1 .TM. (pH 7, 1 mM Expell SP1 .TM.) Lys Yield AR1
AR2 Sum Lys 0 Lys 1 Lys 2 HMW Monomer LMW HCP % % % % % % % % % %
(ng/mg) Feed -- 4.3 17.8 77.9 52.5 19.0 6.5 1.16 98.57 0.27 267
Product 74 0.1 10.3 89.6 55.5 24.8 9.3 0.11 99.85 0.04 5 pool
Example 2. Displacement Chromatography Performance of Protamine
Sulfate for Adalimumab on Poros XS Resin
[0442] Protamine sulfate, a cationic peptide with molecule weight
.about.5.1 kD, was also evaluated as a cation exchange displacer
for Adalimumab on Poros XS resin under various operating
conditions. The feed material used for this set of experiments
contained about 17-24% total AR, of which 3-6% was AR1 and 14-19%
AR2. The results for this system are illustrated in the following
sections.
[0443] FIG. 10 shows the distribution of charge variant species in
sample fractions collected from a well established displacement
process induced by protamine sulfate. In this experiment, the
column was equilibrated with a pH 7.5 Tris/acetate buffer (5.6
mS/cm), loaded with a pre-adjusted protein A eluate feed (pH 7.5,
5.4 mS/cm, 5.2 g/L) to 39 g/L resin loading level, followed by a
brief EQ buffer wash and then displacement process using 0.5 mM
protamine sulfate dissolved in the pH 7.5 EQ buffer. Similar to the
Expell SP1.TM. displacement profile (FIG. 3), the charge variants
were enriched at different locations of the displacement train and
were peaked in the order of AR1, AR2, Lys0, Lys1 and Lys2. The
cumulative AR % reduction as a function of product yield is
illustrated in FIG. 11. A 6-8% decrease in the total AR level can
be obtained at a yield of 75-85% under this set of condition. The
actual levels of AR1, AR2 and total lysine variant species (i.e.,
Lys 0+Lys 1+Lys 2) for the feed and the final product pool are
shown in Table 5.
[0444] The effect of protamine sulfate concentration on Adalimumab
AR reduction was measured in pH 7.5 Tris/Acetate buffer, as shown
in FIG. 12. The same equilibration/wash and feed loading conditions
as described above were used for all the runs here. At similar
yield (.about.75%), the total AR % was reduced by approximately
7-8% when using 0.25 to 2 mM protamine sulfate. This broad
concentration range reflects the robustness of charge variant
separation by protamine sulfate displacement process.
[0445] The effect of displacing buffer pH on AR clearance for
Adalimumab was measured at 0.5 mM protamine sulfate concentration
in Tris/Acetate buffer. In this set of experiments, the column was
conditioned with an EQ buffer at the respective displacing buffer
pH, loaded with protein feed at pH 7.5 and .about.6 mS/cm followed
by a brief EQ buffer wash before starting the displacement phase.
As shown in FIG. 13, the extent of AR reduction increases
significantly as pH varies from 6.5 to 7.5. Over 6% decrease in AR
level can be achieved at pH 7.5 with a product yield
.about.75%.
[0446] The two-step displacement scheme was also tested with
protamine sulfate. In one experiment, the displacement process
consists of 10 CV of 0.25 mM protamine and 10 CV of 2 mM protamine
at pH 7.5 (Table 2). The protein displacement profile was completed
in a total of 13 CV, which is about 11 CV or almost 2 fold shorter
than that in the one-step displacement process with 0.25 mM
protamine sulfate. The reduction of AR % versus product yield is
shown in FIG. 14. The total AR level in product pool were reduced
by .about.8% at .about.75% yield, which is comparable to that
achieved by the one-step displacement process using 0.25 mM
protamine sulfate.
[0447] The linear gradient displacement scheme was also evaluated
with protamine sulfate on Poros XS resin for Adalimumab charge
variant separation. As summarized in Table 2, after the feed
loading at pH 7.5 (5.9 mS/cm), the column was briefly washed with
the equilibration buffer (pH 7.5, .about.6 mS/cm) and then started
with a 40 CV linear gradient from the EQ buffer to a 1 mM protamine
sulfate displacing buffer (which was made from the EQ buffer). FIG.
15 shows the cumulative AAR % versus yield from this run. At a
product yield of 75.6%, the total AR % was reduced from the feed
level of 21.3% to 12.1%.
[0448] Protamine sulfate displacement chromatography also
demonstrated significant clearance of aggregates, fragments and
HCP. FIG. 16 exemplifies the size variant profiles of Adalimumab
from the same experiment described above (i.e., 0.5 mM protamine
sulfate, pH 7.5, one-step displacement run). As expected, the
fragments were mostly enriched at the front while the aggregates
primarily resided at the back of the train. Similar to that shown
in FIG. 8, a subpopulation of the aggregates was also observed in
the displacement front; in addition, a portion of fragments was
noticed at the tail. Table 5 compares the levels of aggregates,
fragments and HCP in final product pool (at .about.75% yield)
relative to the feed.
TABLE-US-00007 TABLE 5 Step yield & product quality in
Adalimumab before and after Poros XS displacement chromatography
using protamine sulfate Lys Mono- HCP Yield AR1 AR2 Sum HMW mer LMW
(ng/ % % % % % % % mg) Feed -- 4.1 16.9 79.0 0.8 98.0 1.2 153
Product 73 1.3 13.1 84.7 0.3 99.6 0.1 14 pool
Example 3. Displacement Chromatography Performance of Expell
SP1.TM. for mAb X on Poros XS Resin
[0449] The displacement separation performance of Expell SP1.TM.
was assessed for mAb X on the Poros XS resin. A purified mAb X drug
substance was used in this study, which contained about 16-17%
acidic species and 12-14% basic species.
[0450] A representative set of mAb X charge variant separation
profiles are shown in FIGS. 17 and 18. In this experiment, the
Poros XS column was loaded with 40 g/L of mAb X at pH 6, 6 mS/cm
Tris/Acetate binding condition, and was displaced using 1 mM Expell
SP1.TM. in a pH 6, .about.6 mS/cm buffer. The specific conditions
are detailed in Table 1. Pronounced enrichment and separation of
acidic, main and basic species were achieved, with AR % reduced by
9.4% at 76% yield.
[0451] The effect of Expell SP1.TM. concentration on AR reduction
for mAb X was measured in the pH 6 Tris/Acetate buffer. As shown in
FIG. 19, increasing the Expell SP1.TM. concentration from 0.5 to 2
mM decreased the AAR % for mAb X from 9.8% at 81% yield to 7.9% at
69% yield.
[0452] The two-step displacement scheme was evaluated for mAb X. As
shown in Table 2, the displacement process comprised of 22 CV of
0.5 mM Expell SP1.TM. and 12 CV of 2 mM Expell SP1.TM. at pH 6. The
protein displacement profile was completed within 30 CV of total
displacing buffer volume, which was 30% less than that required for
one-step displacement separation. The reduction of AR % versus
product yield is shown in FIG. 20. The total AR % in product pool
was reduced by .about.9% at .about.75% yield, again comparable to
that obtained with one-step displacement process using 0.5 mM
Expell SP1.TM. buffer.
Example 4. Displacement Chromatography Performance of Protamine
Sulfate for mAb X on Poros XS Resin
[0453] Protamine sulfate was also evaluated for separating acidic
species for mAb X on Poros XS resin. The feed material for this set
of experiments contained about 12-16% acidic and 12-13% basic
species. The results for this system are shown in the following
sections.
[0454] A representative set of variant separation profiles are
shown in FIGS. 21 and 22. In this experiment, the Poros XS column
was loaded with .about.36 g/L of mAb X at pH 6, 6.5 mS/cm binding
condition, and was displaced using 0.25 mM protamine sulfate in a
pH 6, 6.5 mS/cm Tris/Acetate buffer. The specific conditions are
detailed in Table 1. Pronounced enrichment and separation of
acidic, main and basic species were achieved, with AR level reduced
by 6% at 75% yield.
[0455] The effect of protamine sulfate concentration on mAb X AR
reduction was measured in a pH 6 Tris/Acetate buffer, as shown in
FIG. 23. In this case, the protamine sulfate concentration strongly
affects the AR clearance in a relatively small protamine
concentration range (i.e., from 0.35 to 0.5 mM). Nevertheless, over
8% of AR reduction can be achieved at pH 6 for mAb X at acceptable
yield (.gtoreq.70%).
[0456] The two-step displacement scheme was evaluated for mAb X
with protamine sulfate. In this experiment, the displacement
process comprised of 10 CV of 0.35 mM Expell and 10 CV of 0.5 mM
Expell at pH 6 (see Table 2). The protein displacement profile was
completed in .about.15 CV of total displacing buffer volume,
representing .about.26% reduction of buffer volume relative to the
one-step displacement operation. The reduction of AR % versus
product yield is shown in FIG. 24. The product pool AR level was
reduced by .about.6% at .about.75% yield.
[0457] The mAb X BDS has about 0.74% of aggregates, which can be
further reduced during the protamine sulfate displacement process.
FIG. 25 shows the size variant profiles of mAb X as displaced by a
0.25 mM protamine sulfate, pH 6 buffer (i.e., the one-step
displacement run shown in FIGS. 21 and 22). The aggregates were all
enriched at the end of the displacement train, which differs from
the observations with Adalimumab. Using the same product pooling
strategy based on AR reduction, the monomer level was enhanced to
99.9% (Table 6).
TABLE-US-00008 TABLE 6 Step yield & product quality in mAb X
before and after Poros XS displacement chromatography using
protamine sulfate Yield % Acidic % Main % HMW % Monomer % Feed --
12.3 75.7 0.7 99.3 Product pool 79 4.7 80.8 0.1 99.9
Example 5. Displacement Chromatography Performance of Expell
SP1.TM. for mAb Y on Poros XS Resin
[0458] The displacement separation performance of Expell SP1.TM.
was further assessed for mAb Y on Poros XS resin. The mAb Y has a
pI of 7-7.5, much lower than Adalimumab and mAb X. An mAb Y protein
A eluate was used in this study, which contained about 22% acidic
species and 15% basic species.
[0459] An appropriate set of displacement conditions for mAb Y is
shown in Table 1. The equilibration, wash and displacement buffers
are all at pH 5 with conductivity around 6 mS/cm. The 0.5 mM Expell
SP1.TM. buffer generated the desired displacement profile. The
sample fractions from this run were analyzed by cation exchange
HPLC. FIGS. 26 and 27 indicate the distribution of charge variant
species and the cumulative AAR % versus product yield,
respectively. A 6.6% decrease in AR % was observed at 74% yield
under such condition.
Example 6. Displacement Chromatography Performance of Protamine
Sulfate for Adalimumab on Capto MMC (Multimodal) Resin
[0460] Capto MMC.TM. is a mixed mode resin based on weak
cation-exchange and hydrophobic interaction mechanism. Its
capability for acidic species and aggregates removal by
displacement chromatography was assessed here. The Adalimumab feed
material for this set of experiments contained about 20-21% total
AR. The results for protamine sulfate system are shown in the
following sections.
[0461] A representative set of variant separation profiles are
shown in FIGS. 28 and 29. In this experiment, the Capto MMC column
was equilibrated with a 140 mM Tris/acetate, pH 7 buffer
(.about.5.7 mS/cm), loaded with .about.34 g/L of Adalimumab at pH
7.5 and 5.3 mS/cm binding condition, briefly washed with EQ buffer
and then displaced with 0.35 mM protamine sulfate in the pH 7 EQ
buffer. A typical displacement chromatogram was generated under
such experimental condition. As shown in FIG. 10, Capto MMC also
showed enrichment of each variant in the train, yielding total AR
reduction of .about.4% at .about.75% yield. The shape of the AAR %
versus yield curve (FIG. 29) differs from that given by the Poros
XS resin, possibly due to stronger binding of each protein species
(related to secondary mode of interaction) by this mixed mode
ligand.
[0462] The buffer pH and protamine sulfate concentrations were
varied to assess the overall AR clearance by Capto MMC displacement
chromatography. Table 7 summarized the results for three runs.
Overall, 3-5% of AR reduction can be achieved for Adalimumab when
using protamine sulfate as a displacer for Capto MMC resin.
TABLE-US-00009 TABLE 7 AR removal for Adalimumab by Capto MMC
displacement chromatography using protamine sulfate Protamine Run
EQ/Displacing Conc. No. buffer pH Load pH (mM) Yield (%) .DELTA.AR
% 1 7 7 0.5 75 3.1 2 7 7.5 0.35 75 4.0 3 7.25 7.5 0.25 78 5.2
[0463] The clearance of aggregates by Capto MMC displacement
chromatography is illustrated in Table 8. The same operating
conditions as described for obtaining the results in Table 7 were
used here. The product pool monomer level was enhanced from 98.8%
in the feed to 99.4% with aggregates reduced from 1.0% to 0.5%.
TABLE-US-00010 TABLE 8 Step yield & product quality in
Adalimumab before and after Capto MMC displacement chromatography
using protamine sulfate Lys Yield AR1 AR2 Sum HMW Monomer LMW % % %
% % % % Feed -- 4.4 16.5 79.1 1.0 98.8 0.2 Product 75 2.5 14.4 83.1
0.5 99.4 0.1 pool
Example 7. Displacement Chromatography Separation of Protamine
Sulfate for mAb X on Capto MMC Resin
[0464] Protamine sulfate was evaluated for removing mAb X acidic
species on Capto MMC resin. The same feed material as shown in
Example 4 was used for this set of experiments. As detailed in
Table 3, the pH and protamine concentrations were varied in order
to generate desired displacement profile. One representative set of
working condition is to load Capto MMC column with 40 g/L of mAb X
at pH 7.5 and .about.6 mS/cm, and to use 0.25 mM protamine sulfate
in the pH 7.5, .about.6 mS/cm EQ buffer for displacement (Table 3).
The separation of charge variants and AR reduction as a function of
product recovery are shown in FIGS. 30 and 31, respectively. In
this case, 3-5% of AR reduction was resulted at product yield of
70-90%.
[0465] The effect of protamine sulfate concentration on mAb X AR
reduction by Capto MMC resin was measured in a pH 7, 6 mS/cm
Tris/Acetate buffer, as shown in Table 9. Varying the protamine
concentration from 0.25 mM to 0.5 mM had very little effect on AAR
% and product yield, which is quite different from the observations
with the Poros XS resin (FIG. 23). The mixed mode MMC resin gave
over 3% AR clearance under such selected conditions.
TABLE-US-00011 TABLE 9 Reduction of AR level by Capto MMC
displacement chromatography for mAb X at different protamine
sulfate concentrations Expell concentration (mM) Yield (%)
.DELTA.AR (%) 0.25 75 3.4 0.5 77 3.3
[0466] The effect of displacing buffer pH on AR clearance for mAb X
was measured at 0.25 mM protamine sulfate concentration in
Tris/Acetate buffer. In this set of experiments, the column was
conditioned with an EQ buffer at the respective displacing buffer
pH, loaded with protein feed at the same pH and .about.6 mS/cm
followed by a brief EQ buffer wash before starting the displacement
phase. As shown in FIG. 32, AAR % increases from 3.3 to 6.5% as pH
varies from 7 to 7.7.
[0467] The Capto MMC resin also removes aggregates during protamine
sulfate displacement chromatography. Table 10 shows the level of
charge and size variants of mAb X in product eluate when using 0.5
mM protamine sulfate, pH 7.5 (.about.6 mS/cm) displacing buffer for
separation. The product pool monomer level was enhanced from 98.7%
in the feed to 99.2% with aggregate and fragment levels reduced by
50% and 28%, respectively.
TABLE-US-00012 TABLE 10 Step yield & product quality for mAb X
before and after Capto MMC displacement chromatography using
protamine sulfate Yield Acidic Main HMW Monomer LMW % % % % % %
Feed -- 16.8 74.0 0.6 98.7 0.7 Product 77 12.4 71.8 0.3 99.2 0.5
pool
Example 8. Displacement Chromatography Separation of Protamine
Sulfate for mAb Y on Capto MMC Resin
[0468] Protamine sulfate was also evaluated for removing mAb Y
acidic species on Capto MMC resin. The same feed material as shown
in Example 5 was used for the experiments here. Two sets of
conditions were evaluated here. In one experiment, the
equilibration, wash and displacement buffers were adjusted to pH
5.5 and .about.6.5 mS/cm, and the displacement buffer contained
0.25 mM protamine sulfate. In the other experiment, all those
buffers were adjusted to pH 5, .about.6.5 mS/cm and the protamine
sulfate concentration was 0.5 mM. In both runs the feed was
adjusted to pH 5.5, 5.2-5.5 mS/cm and loaded to the Capto MMC
column at .about.40 g/L loading level. As shown in Table 11, both
sets of conditions resulted in AR % decrease by 5-7% in product
pool. In addition, significant aggregates and fragments reduction
was achieved with the same product pooling strategy.
TABLE-US-00013 TABLE 11 Step yield & product quality for mAb Y
before and after Capto MMC displacement chromatography using
protamine sulfate Mono- Condi- Yield Acidic Main HMW mer LMW tions
Sample % % % % % % pH 5.5, Feed -- 16.4 75.8 8.5 91.0 0.5 0.25 mM
Protamine Product 76 9.8 79.4 2.0 97.6 0.4 pool pH 5, Feed -- 20.9
72.8 6.3 93.2 0.5 0.5 mM Protamine Product 75 15.9 81.1 1.6 98.2
0.2 pool
Summary of Examples 1-8
[0469] Examples 1-8 above, demonstrate the use of cation exchange
and mixed mode displacement chromatography for effectively reducing
acidic species along with various other impurities from different
monoclonal antibody feed streams. Under appropriate (or relatively
weak) binding conditions, cationic molecules with high affinity for
a CEX or multimodal ligand (such as Expell SP1.TM. and protamine
sulfate) can induce the formation of charge variant displacement
train, wherein the acidic population is enriched in the front
followed by the main isoform, and, thereafter, the basic
population. Thus, in certain embodiments, exclusion of those
earlier fractions from the remainder eluate results in an
AR-reduced product. Alternatively, exclusion of the fractions
following the main isoform results in a Lys variant- or basic
species-reduced product.
[0470] Also demonstrated in the preceding experiments is the fact
that the operating pH and displacer concentration can strongly
affect the displacement profile and as a result the charge variant,
product aggregate, product fragment, and HCP clearance profile. The
selection of a particular operating regime with regard to charge
variant reduction depends, in general, on the specific
protein-resin-displacer system. For example, for Adalimumab,
significant AR reduction can be achieved using a displacing buffer
with pH in the range of 6-8 with displacer concentration as low as
0.25-0.5 mM. The total AR level (%) in Adalimumab product pool can
be reduced by over 10% with an acceptable processing yield
(.gtoreq.75%) from a CEX displacement chromatography process, or
4-7% from a mixed mode displacement chromatography process. Along
with acidic species, other product variants or process impurities
such as basic species, aggregates, fragments and HCP can be
selectively collected or reduced to meet the quality requirements.
In addition to the surprisingly effective preparative scale
standard one-step displacement operation, unconventional
displacement separation schemes are shown above to have unexpected
properties, including two-step displacement chromatography and
linear gradient displacement chromatography, which can
significantly reduce buffer volumes and shorten the processing time
without compromising the charge variant, product aggregate, product
fragment, and HCP clearance at a given yield target.
[0471] The instant invention provides a method for reducing acidic
species for a given protein of interest. For example, adalimumab
was prepared using a combination of AEX and CEX technologies to
produce a Low-AR and High-AR sample with a final AR of 2.5% and
6.9%, respectively. Both samples were incubated in a controlled
environment at 25.degree. C. and 65% relative humidity for 10
weeks, and the AR measured every two weeks. FIG. 23 shows the
growth of AR for each sample over the 10 week incubation. It is
evident from FIG. 23 the growth rate of AR is linear and similar
between both the Low-AR and High-AR samples. Based on these
results, the reduced AR compositions of the invention can be stored
3-fold longer before reaching the same AR level as the High--AR
sample. These surprising results are very beneficial for storage,
handling, and use of an antibody or other protein for therapeutic
use.
[0472] The present invention is not to be limited in scope by the
specific embodiments described herein. Indeed, various
modifications of the invention in addition to those described
herein will become apparent to those skilled in the art from the
foregoing description and the accompanying figures. Such
modifications are intended to fall within the scope of the appended
claims.
Sequence CWU 1
1
121107PRTArtificial SequenceDescription of Artificial Sequence
Synthetic adalimumab light chain variable region 1Asp 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 Gln Gly Ile Arg Asn Tyr 20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35
40 45 Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu Gln Pro 65 70 75 80 Glu Asp Val Ala Thr Tyr Tyr Cys Gln Arg Tyr
Asn Arg Ala Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys 100 105 2121PRTArtificial SequenceDescription of Artificial
Sequence Synthetic adalimumab heavy chain variable region 2Glu Val
Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20
25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45 Ser Ala Ile Thr Trp Asn Ser Gly His Ile Asp Tyr Ala
Asp Ser Val 50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala
Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu
Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Val Ser Tyr Leu Ser
Thr Ala Ser Ser Leu Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val
Thr Val Ser Ser 115 120 39PRTArtificial SequenceDescription of
Artificial Sequence Synthetic adalimumab light chain variable
region CDR3MOD_RES(9)..(9)Thr or Ala 3Gln Arg Tyr Asn Arg Ala Pro
Tyr Xaa 1 5 412PRTArtificial SequenceDescription of Artificial
Sequence Synthetic adalimumab heavy chain variable region
CDR3MOD_RES(12)..(12)Tyr or Asn 4Val Ser Tyr Leu Ser Thr Ala Ser
Ser Leu Asp Xaa 1 5 10 57PRTArtificial SequenceDescription of
Artificial Sequence Synthetic adalimumab light chain variable
region CDR2 5Ala Ala Ser Thr Leu Gln Ser 1 5 617PRTArtificial
SequenceDescription of Artificial Sequence Synthetic adalimumab
heavy chain variable region CDR2 6Ala Ile Thr Trp Asn Ser Gly His
Ile Asp Tyr Ala Asp Ser Val Glu 1 5 10 15 Gly 711PRTArtificial
SequenceDescription of Artificial Sequence Synthetic adalimumab
light chain variable region CDR1 7Arg Ala Ser Gln Gly Ile Arg Asn
Tyr Leu Ala 1 5 10 85PRTArtificial SequenceDescription of
Artificial Sequence Synthetic adalimumab heavy chain variable
region CDR1 8Asp Tyr Ala Met His 1 5 9321DNAArtificial
SequenceDescription of Artificial Sequence Synthetic adalimumab
light chain variable region 9gacatccaga tgacccagtc tccatcctcc
ctgtctgcat ctgtagggga cagagtcacc 60atcacttgtc gggcaagtca gggcatcaga
aattacttag cctggtatca gcaaaaacca 120gggaaagccc ctaagctcct
gatctatgct gcatccactt tgcaatcagg ggtcccatct 180cggttcagtg
gcagtggatc tgggacagat ttcactctca ccatcagcag cctacagcct
240gaagatgttg caacttatta ctgtcaaagg tataaccgtg caccgtatac
ttttggccag 300gggaccaagg tggaaatcaa a 32110363DNAArtificial
SequenceDescription of Artificial Sequence Synthetic adalimumab
heavy chain variable region 10gaggtgcagc tggtggagtc tgggggaggc
ttggtacagc ccggcaggtc cctgagactc 60tcctgtgcgg cctctggatt cacctttgat
gattatgcca tgcactgggt ccggcaagct 120ccagggaagg gcctggaatg
ggtctcagct atcacttgga atagtggtca catagactat 180gcggactctg
tggagggccg attcaccatc tccagagaca acgccaagaa ctccctgtat
240ctgcaaatga acagtctgag agctgaggat acggccgtat attactgtgc
gaaagtctcg 300taccttagca ccgcgtcctc ccttgactat tggggccaag
gtaccctggt caccgtctcg 360agt 36311214PRTArtificial
SequenceDescription of Artificial Sequence Synthetic adalimumab
light chain 11Asp 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 Gln
Gly Ile Arg Asn Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly
Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Thr Leu Gln
Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr
Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Val
Ala Thr Tyr Tyr Cys Gln Arg Tyr Asn Arg Ala Pro Tyr 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 Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys
Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys
Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr
His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg
Gly Glu Cys 210 12451PRTArtificial SequenceDescription of
Artificial Sequence Synthetic adalimumab heavy chain 12Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20 25
30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45 Ser Ala Ile Thr Trp Asn Ser Gly His Ile Asp Tyr Ala Asp
Ser Val 50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys
Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Val Ser Tyr Leu Ser Thr
Ala Ser Ser Leu Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr
Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125 Val Phe Pro Leu
Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140 Ala Leu
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155
160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
Thr Val 180 185 190 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys
Asn Val Asn His 195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys
Val Glu Pro Lys Ser Cys 210 215 220 Asp Lys Thr His Thr Cys Pro Pro
Cys Pro Ala Pro Glu Leu Leu Gly 225 230 235 240 Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255 Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270 Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280
285 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
290 295 300 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly 305 310 315 320 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
Leu Pro Ala Pro Ile 325 330 335 Glu Lys Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val 340 345 350 Tyr Thr Leu Pro Pro Ser Arg
Asp Glu Leu Thr Lys Asn Gln Val Ser 355 360 365 Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380 Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 400
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405
410 415 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
Met 420 425 430 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
Ser Leu Ser 435 440 445 Pro Gly Lys 450
* * * * *