U.S. patent application number 15/864378 was filed with the patent office on 2018-08-09 for purification of proteins using hydrophobic interaction chromatography.
This patent application is currently assigned to AbbVie Inc.. The applicant listed for this patent is AbbVie Inc.. Invention is credited to Matthew Omon Herigstad, Stephen Ming-Teh Lu, Natarajan Ramasubramanyan, Linda E. Rich.
Application Number | 20180222938 15/864378 |
Document ID | / |
Family ID | 52395622 |
Filed Date | 2018-08-09 |
United States Patent
Application |
20180222938 |
Kind Code |
A1 |
Herigstad; Matthew Omon ; et
al. |
August 9, 2018 |
PURIFICATION OF PROTEINS USING HYDROPHOBIC INTERACTION
CHROMATOGRAPHY
Abstract
The present invention is directed to methods for purifying a
protein of interest, e.g., an antibody, from a sample comprising
the protein of interest and at least one impurity, e.g., an
aggregate, by employing a hydrophobic interaction chromatography
(HIC) method that allows for binding of both the protein of
interest and the at least one impurity under strong binding
conditions. The present invention is based, at least in part, on
the finding that both flow through and bind-elute techniques can be
combined to achieve greater purification and recovery of a protein
of interest, e.g., an antibody, under isocratic wash conditions and
strong binding conditions.
Inventors: |
Herigstad; Matthew Omon;
(Wayland, MA) ; Rich; Linda E.; (Worcester,
MA) ; Lu; Stephen Ming-Teh; (Worcester, MA) ;
Ramasubramanyan; Natarajan; (Westborough, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
AbbVie Inc. |
North Chicago |
IL |
US |
|
|
Assignee: |
AbbVie Inc.
North Chicago
IL
|
Family ID: |
52395622 |
Appl. No.: |
15/864378 |
Filed: |
January 8, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14567570 |
Dec 11, 2014 |
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15864378 |
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14077574 |
Nov 12, 2013 |
8946395 |
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14567570 |
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61893131 |
Oct 18, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 1/36 20130101; C07K
1/16 20130101; C07K 16/241 20130101; C07K 1/20 20130101; C07K 1/22
20130101; C07K 1/18 20130101; C07K 1/165 20130101 |
International
Class: |
C07K 1/20 20060101
C07K001/20; C07K 1/36 20060101 C07K001/36; C07K 1/22 20060101
C07K001/22; C07K 1/18 20060101 C07K001/18; C07K 16/24 20060101
C07K016/24; C07K 1/16 20060101 C07K001/16 |
Claims
1. A method for producing a preparation comprising a protein of
interest and having a reduced level of at least one impurity, said
method comprising: (a) contacting a sample comprising the protein
of interest and at least one impurity, to a hydrophobic interaction
chromatography (HIC) media, in the presence of a load buffer such
that (i) a portion of the protein of interest binds to the HIC
media and (ii) a substantial portion of the at least one impurity
binds to the HIC media; (b) collecting a flow through fraction
comprising the protein of interest unbound to the HIC media; (c)
washing the HIC media with a wash buffer that is substantially the
same as the load buffer such that a substantial portion of the
protein of interest bound to the HIC media is released from the
media; and (d) collecting a wash fraction comprising the protein of
interest released from the HIC media, wherein each of the flow
through and wash fractions comprise the protein of interest and
have a reduced level of the at least one impurity.
2. The method of claim 1, wherein the portion of the protein of
interest binds to the HIC media at (a) a Kp of greater than 10, 15,
20, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170,
180, 190, 200, 220, 250 or 300; or (b) at a Kp of greater than
20.
3. The method of claim 1, wherein the protein of interest is
adalimumab.
4. The method of claim 1, wherein a substantial portion of the
impurity bound to the HIC media remains bound upon washing with the
wash buffer.
5. The method of claim 1, wherein the flow through and/or wash
fractions are substantially free of the at least one impurity.
6. The method of claim 1, wherein the at least one impurity is an
aggregate of the protein of interest, optionally, a multimer, a
dimer, a trimer, a tetramer, an oligomer or other high molecular
weight species.
7. The method of claim 1, wherein the protein of interest is
adalimumab and the at least one impurity is an aggregate of
adalimumab, optionally, selected from the group consisting of
multimer 1, multimer 2 and multimer 3.
8. The method of claim 1, wherein the impurity is a process-related
impurity or a product-related substance.
9. The method of claim 8, wherein the process-related impurity is
selected from the group consisting of a host cell protein, a host
cell nucleic acid, a media component, and a chromatographic
material.
10. The method of claim 8, wherein the product-related substance is
selected from the group consisting of a charge variant, an acidic
variant, a basic variant, a lysine variant species, an aggregate of
the protein of interest, a fragment of the protein of interest, an
Fc fragment of the protein of interest, a Fab fragment of the
protein of interest, a modified protein, a deamidated protein, and
a glycosylated protein.
11. The method of claim 1, wherein the impurity is an acidic
species (AR), optionally, selected from the group consisting of
AR1, AR2, a charge variant, a structure variant, a fragmentation
variant, a process-related impurity and a product-related
impurity.
12. The method of claim 1, wherein the protein of interest is an
antibody or antigen-binding fragment thereof, a soluble protein, a
membrane protein, a structural protein, a ribosomal protein, an
enzyme, a zymogen, an antibody molecule, a humanized antibody or
antigen-binding portion thereof, a human antibody or
antigen-binding portion thereof, a chimeric antibody or
antigen-binding portion thereof, a multivalent antibody, a cell
surface receptor protein, a transcription regulatory protein, a
translation regulatory protein, a chromatin protein, a hormone, a
cell cycle regulatory protein, a G protein, a neuroactive peptide,
an immunoregulatory protein, a blood component protein, an ion gate
protein, a heat shock protein, an antibiotic resistance protein, a
functional fragment of any of the preceding proteins, an
epitope-containing fragment of any of the preceding proteins, and
combinations thereof.
13. The method of claim 1, further comprising repeating steps
(a)-(d) at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15
or 20 times using the flow through fraction, wash fraction, or
combination thereof having a reduced level of the at least one
impurity
14. The method of claim 1, wherein the flow through fraction and
the wash fraction are combined.
15. The method of claim 1, wherein (a) the portion of the protein
of interest that binds to the HIC media is at least about 20%, at
least about 30%, at least about 40%, at least about 50%, at least
about 60%, at least about 70%, at least about 80% or at least about
90% of the protein of interest in the sample; (b) the substantial
portion of the protein of interest released from the HIC media upon
washing with the wash buffer is at least about 20%, at least about
30%, at least about 40%, at least about 50%, at least about 60%, at
least about 70%, at least about 80%, at least about 90% or about
100% of the amount of protein of interest bound to the HIC media;
(c) the accumulative yield of the protein of interest in the flow
through fraction and/or wash fraction is at least about 35%, at
least about 40%, at least about 45%, at least about 50%, at least
about 55%, at least about 60%, at least about 65%, at least about
70%, at least about 75%, at least about 80%, at least about 85%, at
least about 90%, at least about 95%, or about 100%; (d) the
accumulative yield of the protein of interest in any one flow
through fraction and/or wash fraction is at least about 4%, at
least about 10%, at least about 20%, at least about 30%, at least
about 40%, at least about 50%, at least about 55%, at least about
60%, at least about 65%, at least about 70%, at least about 75%, at
least about 80%, at least about 85, at least about 90%, at least
about 95% or about 100%; (e) the substantial portion of the at
least one impurity that binds to the HIC media is at least about
50%, at least about 55%, at least about 60%, at least about 65%, at
least about 70%, at least about 75%, at least about 80%, at least
about 85%, at least about 90%, at least about 95% or about 100% of
the at least one impurity in the sample; (f) the reduced level of
the at least one impurity of the flow through fraction and/or wash
fraction is at least about 50%, at least about 55%, at least about
60%, at least about 65%, at least about 70%, at least about 75%, at
least about 80%, at least about 85%, at least about 90%, at least
about 95% or about 100% of the at least one impurity in the sample;
(g) the accumulative aggregate reduction of the at least one
impurity in any one flow through fraction and/or wash fraction is
at least about 0.1%, at least about 0.2%, at least about 0.5%, at
least about 1.0%, at least about 2.0%, at least about 3.0%, at
least about 4.0%, at least about 5.0%, at least about 10.0%, or at
least about 20.0%; (h) the accumulative aggregate reduction of the
at least one impurity in the flow through fraction and/or wash
fraction is at least about 0.1%, at least about 0.2%, at least
about 0.5%, at least about 1.0%, at least about 2.0%, at least
about 3.0%, at least about 4.0%, at least about 5.0%, at least
about 10.0%, or at least about 20.0%; and/or (i) the level of the
at least one impurity is reduced by at least 60%, at least 70%, at
least 80%, at least 90%, or at least 95% of the at least one
impurity in the sample.
16. The method of claim 1, wherein (a) the at least one impurity
binds to the HIC media at a Kp of greater than 250, greater than
300, greater than 400, greater than 500, greater than 600, greater
than 700, greater than 800, greater than 900, or greater than 1000;
(b) the protein of interest and the at least one impurity have a Kp
ratio less than 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3 or 1:2; (c)
the Kd for the binding of the protein of interest to the HIC media
is at least about 0.2, at least about 0.3, at least about 0.4, at
least about 0.5, or at least about 0.6; (d) the Kd for the binding
of the at least one impurity to the HIC media is less than or equal
to about 0.001, about 0.005, about 0.01, about 0.02, about 0.05,
about 0.1, about 0.15 or about 0.2; (e) the Kd for the binding of
the protein of interest to the HIC media is less than 50, 45, 40,
35, 30, 25, 20, 15, 10 or 5 times the Kd for the binding of the at
least one impurity to the HIC media; (f) the protein of interest
has a Qmax of at least about 20, at least about 30, at least about
40, at least about 50, at least about 60 or at least about 100;
and/or (g) the at least one impurity has a Qmax of at least about
2, at least about 5, at least about 10, at least about 20, at least
about 30 or at least about 40.
17. The method of claim 1, wherein the HIC media either (i)
comprises at least one hydrophobic ligand, optionally selected from
the group consisting of alkyl-, aryl-ligands, and butyl, hexyl,
phenyl, octyl, or polypropylene glycol ligands; and/or (ii) is
selected from the group consisting of CaptoPhenyl, Phenyl
Sepharose.TM. 6 Fast Flow with low or high substitution, Phenyl
Sepharose.TM. High Performance, Octyl Sepharose.TM. High
Performance, Fractogel.TM. EMD Propyl, Fractogel.TM. EMD Phenyl,
Macro-Prep.TM. Methyl, Macro-Prep.TM. t-Butyl, WP HI-Propyl
(C3).TM., Toyopearl.TM. ether, Toyopearl.TM. phenyl, Toyopearl.TM.
butyl, ToyoScreen PPG, ToyoScreen Phenyl, ToyoScreen Butyl,
ToyoScreen Hexyl, HiScreen Butyl FF, HiScreen Octyl FF, and Tosoh
Hexyl.
18. The method of claim 1, wherein (a) the load buffer and/or wash
buffer comprise a salt selected from the group consisting of a
sulfate salt, a citrate salt, ammonium sulfate, sodium sulfate,
sodium chloride, ammonium chloride, sodium bromide and a
combination thereof; (b) the load buffer and/or wash buffer
comprise a cation selected from the group consisting of Ba2+, Ca2+,
Mg2+, Li+, Cs+, Na+, K+, Rb+, NH4+ and a combination thereof; (c)
the load buffer and/or wash buffer comprise an anion selected from
the group consisting of PO43-, SO42-, CH3CO3-, Cl-, Br-, NO3-,
ClO4-, I-, SCN- and a combination thereof; (d) the load buffer
and/or wash buffer comprise a salt having a concentration of
between about 50 mM and 2000 mM; (e) the load buffer and/or wash
buffer have a pH between about 4.0 and 8.5 or between about 5.0 and
7.0; (f) the load buffer and/or wash buffer have a pH of about 4.0,
about 4.5, about 5.0, about 5.5, about 6, about 6.5, about 7.0,
about 7.5, about 8.0, or about 8.5; (g) the load buffer and/or wash
buffer are the same; (h) the load buffer and/or wash buffer are
substantially the same; and/or (i) the salt concentration and/or
the pH of the wash buffer are within about 20%, 15%, 10% or 5% of
the salt concentration and/or pH of the loading buffer.
19. The method of claim 1, wherein (a) about 100 g to about 800 g
of the sample are contacted per one liter of HIC media; (b) about
0.2 g to about 120 g of the at least one impurity is contacted per
one liter of HIC media; (c) the sample has a protein concentration
of about 2 mg/ml to about 50 mg/ml; (d) the sample has a protein of
interest concentration of about 2 mg/ml to about 50 mg/ml; and/or
(e) the concentration of the at least one impurity in the sample is
about 0.01 to about 5.0 mg/ml.
20. The method of claim 1, (a) wherein a precursor sample
comprising the protein of interest has been subjected to affinity
chromatography to generate the sample; and/or (b) further
comprising subjecting the preparation comprising a protein of
interest and having a reduced level of one impurity to affinity
chromatography, optionally wherein affinity chromatography is
performed using affinity chromatographic media selected from the
group consisting of Protein A, G, A/G, L media, and MabSuRe Protein
A media.
21. The method of claim 1, (a) wherein a precursor sample
comprising the protein of interest has been subjected to ion
exchange chromatography to generate the sample; and/or (b) further
comprising subjecting the preparation comprising a protein of
interest and having a reduced level of one impurity to ion exchange
chromatography, optionally wherein ion exchange chromatography is
performed using ion exchange chromatography media selected from the
group consisting of a cation exchange media and an anion exchange
media.
22. The method of claim 1, (a) wherein a precursor sample
comprising the protein of interest has been subjected to mixed mode
chromatography to generate the sample; and/or (b) further
comprising subjecting the preparation comprising a protein of
interest and having a reduced level of one impurity to mixed mode
chromatography; optionally wherein the mixed mode chromatography is
performed using CaptoAdhere resin.
23. The method of claim 1, (a) wherein a precursor sample
comprising the protein of interest has been subjected to a
filtration step to generate the sample; and/or (b) further
comprising subjecting the preparation comprising a protein of
interest and having a reduced level of one impurity to a filtration
step; optionally, wherein the filtration step is selected from the
group consisting of a depth filtration step, a nanofiltration step,
an ultrafiltration step, and an absolute filtration step, or a
combination thereof.
24. The method of claim 1, wherein the HIC media has a dynamic
binding capacity of at least about 2 g, at least about 5 g, at
least about 10 g, at least about 20 g, at least about 30 g, at
least about 40 g, at least about 50 g, at least about 60 g, at
least about 70 g, at least about 90 g, or at least about 100 g of
sample per one liter of media.
25. A method for producing a preparation comprising adalimumab and
having a reduced level of at least one aggregate, said method
comprising: (a) contacting a sample comprising adalimumab and at
least one aggregate, to a HIC media, in the presence of a load
buffer such that (i) a portion of the adalimumab in the sample
binds to the HIC media and (ii) a substantial portion of the at
least one aggregate binds to the HIC media; (b) collecting a flow
through fraction comprising the adalimumab unbound to the HIC
media; (c) washing the HIC media with a wash buffer that is
substantially the same as the load buffer such that a substantial
portion of the adalimumab bound to the HIC media is released from
the media; and (d) collecting a wash fraction comprising the
adalimumab released from the HIC media, wherein each of the flow
through and wash fractions comprise adalimumab and have a reduced
level of the at least one aggregate.
26. The method of claim 25, wherein adalimumab binds to the HIC
media (a) at a Kp of greater than 10, 15, 20, 50, 60, 70, 80, 90,
100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 250 or
300; or (b) at a Kp of greater than 20.
27. The method of claim 25, wherein the aggregate is multimer 1,
multimer 2 or multimer 3.
28. (canceled)
29. A pharmaceutical composition comprising a low-aggregate
composition of adalimumab and a pharmaceutically acceptable
carrier.
30. The pharmaceutical composition of claim 29, wherein the
composition comprises less than 5%, 4%, 3%, 2.5%, 2.4%, 2.3%, 2.2%,
2.1%, 2%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.5%, 0.1% of
aggregates.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 14/567,570, filed Dec. 11, 2014, which is a
continuation of U.S. patent application Ser. No. 14/077,574, filed
Nov. 12, 2013, now U.S. Pat. No. 8,946,395, issued Feb. 3, 2015,
which is related and claims priority to U.S. Provisional
Application No. 61/893,131, filed Oct. 18, 2013, the entire
contents of each of which are hereby incorporated herein by
reference.
FIELD OF INVENTION
[0002] The instant invention relates to the field of protein
production and purification, and in particular to compositions and
processes for reducing the levels of impurities, including
process-related impurities (e.g., host cell proteins and media
components) and/or product-related substances (e.g., product charge
variants, aggregates, and fragments).
BACKGROUND OF THE INVENTION
[0003] Hydrophobic interaction chromatography (HIC) is a
purification technique that exploits the interaction of HIC media
with hydrophobic regions present on a protein of interest, such as
an antibody, and/or those present on an impurity to separate a
protein of interest present in a sample mixture. HIC is often
utilized in either a bind-elute mode, in which the protein of
interest remains bound to HIC media until eluted during an elution
phase, or a flow through mode, in which the protein of interest
flows through the column while the impurity binds to the media.
[0004] Recently, a chromatographic method termed "weak partitioning
mode" has been described for the purification of proteins (U.S.
Pat. No. 8,067,182). According to U.S. Pat. No. 8,067,182, this
method allows for the binding of both product and impurity and is
defined by a intermediate partition coefficient (Kp) for the
product. Compared to the flow-through mode, in which the Kp for the
product is typically low (e.g., <0.1), thereby allowing the
product to flow through the column while the impurity is bound, and
the bind-elute mode, in which the Kp for the product is typically
high (e.g., >20), thereby allowing the product to remain bound
until eluted during an elution phase, in the weak portioning mode,
the Kp for the product is in the range of 0.1-20.
[0005] Importantly, U.S. Pat. No. 8,067,182 teaches the criticality
of this Kp range. Specifically, U.S. Pat. No. 8,067,182 teaches
that Kp values greater than 20 result in a decreased load challenge
at the point of contaminant breakthrough as the product begins to
compete with the contaminant for binding sites on the media. In
addition, U.S. Pat. No. 8,067,182 teaches that Kp values greater
than 20 result in decreased product recovery in that the isocratic
wash conditions are not effective at washing the bound product off
the column in a reasonable number of wash volumes. Accordingly,
U.S. Pat. No. 8,067,182, stresses the criticality of a Kp range to
achieve desired purification (see columns 9 and 10).
[0006] When applied to HIC, the weak partitioning mode described in
U.S. Pat. No. 8,067,182 requires an even more narrow Kp range. As
set forth in Example 4, weak partitioning for HIC required a Kp
less than 10. Patentees report that HIC performance deteriorates
with respect to both contaminant reduction and product recovery at
stronger binding conditions.
SUMMARY OF THE INVENTION
[0007] The present invention is directed to methods for purifying a
protein of interest, e.g., an antibody, from a sample including the
protein of interest and at least one impurity, e.g., an aggregate,
by employing a novel hydrophobic interaction chromatography (HIC)
method. The present invention is based, at least in part, on the
finding that both flow through and bind-elute techniques can be
combined to achieve greater purification and recovery of a protein
of interest. Moreover, the present invention is predicated, at
least in part, on the surprising finding that such methodology can
be employed under isocratic wash conditions and at stronger binding
conditions than previously appreciated, for example, at a Kp
greater than 10, so as to achieve greater purification and
recovery.
[0008] In one aspect, the present invention is directed to a method
for producing a preparation including a protein of interest and
having a reduced level of at least one impurity, said method
comprising: (a) contacting a sample including the protein of
interest and at least one impurity, to a hydrophobic interaction
chromatography (HIC) media, in the presence of a load buffer such
that (i) a portion of the protein of interest binds to the HIC
media and (ii) a substantial portion of the at least one impurity
binds to the HIC media; (b) collecting a flow through fraction
including the protein of interest unbound to the HIC media; (c)
washing the HIC media with a wash buffer that is substantially the
same as the load buffer such that a substantial portion of the
protein of interest bound to the HIC media is released from the
media; and (d) collecting a wash fraction including the protein of
interest released from the HIC media, wherein each of the flow
through and wash fractions include the protein of interest and have
a reduced level of the at least one impurity.
[0009] In various embodiments, the portion of the protein of
interest binds to the HIC media at a Kp of greater than 10, 15, 20,
50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180,
190, 200, 220, 250 or 300. For example, in various embodiments, the
portion of the protein of interest binds to the HIC media at a Kp
of greater than 10, the portion of the protein of interest binds to
the HIC media at a Kp of greater than 20, or the portion of the
protein of interest binds to the HIC media at a Kp of greater than
100.
[0010] In a particular embodiment, the protein of interest is
adalimumab.
[0011] In one embodiment, a substantial portion of the impurity
bound to the HIC media remains bound upon washing with the wash
buffer. In one embodiment, the flow through and/or wash fractions
are substantially free of the at least one impurity.
[0012] In one embodiment, the at least one impurity is an aggregate
of the protein of interest, for example, selected from the group
consisting of a multimer, a dimer, a trimer, a tetramer, an
oligomer and other high molecular weight species. In a particular
embodiment, the protein of interest is adalimumab and the at least
one impurity is an aggregate of adalimumab. For example, the
aggregate may be selected from the group consisting of multimer 1,
multimer 2 and multimer 3.
[0013] In another embodiment, the impurity is a process-related
impurity or a product-related substance. For example, the impurity
may be a process-related impurity selected from the group
consisting of a host cell protein, a host cell nucleic acid, a
media component, and a chromatographic material. Alternatively, the
impurity may be a product-related substance selected from the group
consisting of a charge variant, an aggregate of the protein of
interest, a fragment of the protein of interest and a modified
protein.
[0014] In a particular embodiment the impurity is an acidic or
basic variant, for example, of adalimumab. In a particular
embodiment, the basic variant is a lysine variant species, for
example, an antibody, or antigen-binding portion thereof, having
heavy chains with either zero, one or two C-terminal lysines. In
another embodiment, the impurity is an acidic species (AR), for
example, selected from the group consisting of a charge variant, a
structure variant, a fragmentation variant, a process-related
impurity and a product-related impurity. In a particular
embodiment, the acidic species is AR1 and the charge variant is a
deamidation variant, a glycation variant, an afucosylation variant,
a MGO variant and/or a citric acid variant. In another embodiment,
the acidic species is AR1 and the structure variant is a
glycosylation variant and/or an acetonation variant. In yet another
embodiment, the acidic species is AR1 and the fragmentation variant
is a Fab fragment variant, a C-terminal truncation variant or a
variant missing a heavy chain variable domain. In yet a further
embodiment, the acidic species is AR2 and the charge variant
comprises a deamidation variant and/or glycation variant.
[0015] In a particular embodiment, the impurity is a fragment such
as an Fc or a Fab fragment. In another embodiment, the impurity is
a modified protein such as a deamidated protein or glycosylated
protein.
[0016] In one embodiment, the protein of interest is an antibody or
antigen-binding fragment thereof, a soluble protein, a membrane
protein, a structural protein, a ribosomal protein, an enzyme, a
zymogen, an antibody molecule, a cell surface receptor protein, a
transcription regulatory protein, a translation regulatory protein,
a chromatin protein, a hormone, a cell cycle regulatory protein, a
G protein, a neuroactive peptide, an immunoregulatory protein, a
blood component protein, an ion gate protein, a heat shock protein,
an antibiotic resistance protein, a functional fragment of any of
the preceding proteins, an epitope-containing fragment of any of
the preceding proteins, and combinations thereof.
[0017] In a particular embodiment, the protein of interest is an
antibody or antigen-binding fragment thereof such as a humanized
antibody or antigen-binding portion thereof, a human antibody or
antigen-binding portion thereof, a chimeric antibody or
antigen-binding portion thereof, or a multivalent antibody. In one
embodiment, the antibody, or antigen-binding fragment thereof,
comprises a heavy chain constant region selected from the group
consisting of IgG1, IgG2, IgG3, IgG4, IgM, IgA and IgE constant
regions. In another embodiment, the antibody, or antigen-binding
fragment thereof is selected from the group consisting of a Fab
fragment, a F(ab')2 fragment, a single chain Fv fragment, an SMIP,
an affibody, an avimer, a nanobody, and a single domain
antibody.
[0018] In one embodiment, the methods of the invention further
include repeating steps (a)-(d) at least 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15 or 20 times using the flow through fraction,
wash fraction, or combination thereof having a reduced level of the
at least one impurity. In certain embodiments, the flow through
fraction and the wash fraction are combined.
[0019] In one embodiment, the portion of the protein of interest
that binds to the HIC media is at least about 20%, at least about
30%, at least about 40%, at least about 50%, at least about 60%, at
least about 70%, at least about 80% or at least about 90% of the
protein of interest in the sample. Alternatively or in combination,
the substantial portion of the protein of interest released from
the HIC media upon washing with the wash buffer is about at least
20%, at least about 30%, at least about 40%, at least about 50%, at
least about 60%, at least about 70%, at least about 80%, at least
about 90% or about 100% of the amount of protein of interest bound
to the HIC media. Alternatively or in combination, the substantial
portion of the at least one impurity that binds to the HIC media is
at least about 50%, at least about 55%, at least about 60%, at
least about 65%, at least about 70%, at least about 75%, at least
about 80%, at least about 85%, at least about 90%, at least about
95% or about 100% of the at least one impurity in the sample.
[0020] In certain embodiments, the accumulative yield of the
protein of interest in the flow through fraction and/or wash
fraction is at least about 35%, at least about 40%, at least about
45%, at least about 50%, at least about 55%, at least about 60%, at
least about 65%, at least about 70%, at least about 75%, at least
about 80%, at least about 85%, at least about 90%, at least about
95%, or about 100%. Alternatively or in combination, the
accumulative yield of the protein of interest in any one flow
through fraction and/or wash fraction is at least about 4%, at
least about 10%, at least about 20%, at least about 30%, at least
about 40%, at least about 50%, at least about 55%, at least about
60%, at least about 65%, at least about 70%, at least about 75%, at
least about 80%, at least about 85, at least about 90%, at least
about 95% or about 100%. Alternatively or in combination, the
reduced level of the at least one impurity of the flow through
fraction and/or wash fraction is at least about 50%, at least about
55%, at least about 60%, at least about 65%, at least about 70%, at
least about 75%, at least about 80%, at least about 85%, at least
about 90%, at least about 95% or about 100% of the at least one
impurity in the sample.
[0021] In certain embodiments, the accumulative aggregate reduction
of the at least one impurity in any one flow through fraction
and/or wash fraction is at least about 0.1%, at least about 0.2%,
at least about 0.5%, at least about 1.0%, at least about 2.0%, at
least about 3.0%, at least about 4.0%, at least about 5.0%, at
least about 10.0%, or at least about 20.0%. Alternatively or in
combination, the accumulative aggregate reduction of the at least
one impurity in the flow through fraction and/or wash fraction is
at least about 0.1%, at least about 0.2%, at least about 0.5%, at
least about 1.0%, at least about 2.0%, at least about 3.0%, at
least about 4.0%, at least about 5.0%, at least about 10.0%, or at
least about 20.0%.
[0022] In certain embodiments, the at least one impurity binds to
the HIC media at a Kp of greater than 250, greater than 300,
greater than 400, greater than 500, greater than 600, greater than
700, greater than 800, greater than 900, or greater than 1000. In
certain embodiments, the protein of interest and the at least one
impurity have a Kp ratio less than 1:10, 1:9, 1:8, 1:7, 1:6, 1:5,
1:4, 1:3 or 1:2.
[0023] In certain embodiments, the K.sub.d for the binding of the
protein of interest to the HIC media is at least about 0.2, at
least about 0.3, at least about 0.4, at least about 0.5, or at
least about 0.6. Alternatively or in combination, the K.sub.d for
the binding of the at least one impurity to the HIC media is less
than or equal to about 0.001, about 0.005, about 0.01, about 0.02,
about 0.05, about 0.1, about 0.15 or about 0.2. In particular
embodiments, the K.sub.d for the binding of the protein of interest
to the HIC media is less than 50, 45, 40, 35, 30, 25, 20, 15, 10 or
5 times the K.sub.d for the binding of the at least one impurity to
the HIC media.
[0024] In certain embodiments, the protein of interest has a Qmax
of at least about 20, at least about 30, at least about 40, at
least about 50, at least about 60 or at least about 100. In certain
embodiments, the at least one impurity has a Qmax of at least about
2, at least about 5, at least about 10, at least about 20, at least
about 30 or at least about 40.
[0025] In certain embodiments, the HIC media comprises at least one
hydrophobic ligand. For example, the HIC media may be selected from
the group consisting of alkyl-, aryl-ligands, and combinations
thereof. For example, the HIC media may be selected from the group
consisting of butyl, hexyl, phenyl, octyl, or polypropylene glycol
ligands. In a particular embodiment, the HIC media is selected from
the group consisting of CaptoPhenyl, Phenyl Sepharose.TM. 6 Fast
Flow with low or high substitution, Phenyl Sepharose.TM. High
Performance, Octyl Sepharose.TM. High Performance, Fractogel.TM.
EMD Propyl, Fractogel.TM. EMD Phenyl, Macro-Prep.TM. Methyl,
Macro-Prep.TM. t-Butyl, WP HII-Propyl (C3).TM., Toyopearl.TM.
ether, Toyopearl.TM. phenyl, Toyopearl.TM. butyl, ToyoScreen PPG,
ToyoScreen Phenyl, ToyoScreen Butyl, ToyoScreen Hexyl, HiScreen
Butyl FF, HiScreen Octyl FF, and Tosoh Hexyl. In one embodiment,
the HIC media is a column.
[0026] In various embodiments, the load buffer and/or wash buffer
comprise a salt selected from the group consisting of ammonium
sulfate, sodium sulfate, sodium chloride, ammonium chloride, sodium
bromide or a combination thereof. In a particular embodiment, the
load buffer and the wash buffer include a sulfate salt, a citrate
salt, or a combination thereof. For example, the sulfate salt may
be ammonium sulfate or sodium sulfate. In certain embodiments, the
citrate salt is sodium citrate. In various embodiments, the load
buffer and/or the wash buffer comprise a cation selected from the
group consisting of Ba.sup.2+, Ca.sup.2+, Mg.sup.2+, Li.sup.+,
Cs.sup.+, Na.sup.+, K.sup.+, Rb.sup.+, and NH.sub.4.sup.+, and/or
an anion selected from the group consisting of 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.-, and SCN.sup.- or a
combination thereof.
[0027] In one embodiment, the salt has a concentration of between
about 50 mM and 2000 mM. In a particular embodiment, the load
buffer and the wash buffer have a pH between about 4.0 and 8.5 or
between about 5.0 and 7.0. In certain embodiments, the load buffer
and the wash buffer have a pH of about 4.0, about 4.5, about 5.0,
about 5.5, about 6, about 6.5, about 7.0, about 7.5, about 8.0, or
about 8.5.
[0028] In one embodiment, the load buffer and the wash buffer are
the same. In one embodiment, the load buffer and the wash buffer
are substantially the same. For example, the salt concentration
and/or the pH of the wash buffer may be within about 20%, 15%, 10%
or 5% of the salt concentration, and/or pH of the loading
buffer.
[0029] In certain embodiments, about 100 g to about 800 g of the
sample are contacted per one liter of HIC media. Alternatively or
in combination, about 0.2 g to about 120 g of the at least one
impurity is contacted per one liter of HIC media. In certain
embodiments, the sample has a protein concentration of about 2
mg/ml to about 50 mg/ml. In certain embodiments, the sample has a
protein of interest concentration of about 2 mg/ml to about 50
mg/ml. Alternatively or in combination, the concentration of the at
least one impurity in the sample is about 0.01 to about 5.0
mg/ml.
[0030] In various embodiments, the level of the at least one
impurity is reduced by at least 60%, at least 70%, at least 80%, at
least 90%, or at least 95% of the at least one impurity in the
sample.
[0031] In one embodiment, the at least one impurity is a host cell
protein. For example, the host cell protein may be reduced by at
least 0.25, at least 0.5, at least 0.75, at least 1.0, at least
1.25 or at least 1.5 log reduction fraction.
[0032] In one embodiment, the HIC media has a dynamic binding
capacity of at least about 2 g, at least about 5 g, at least about
10 g, at least about 20 g, at least about 30 g, at least about 40
g, at least about 50 g, at least about 60 g, at least about 70 g,
at least about 90 g, or at least about 100 g of sample per one
liter of media.
[0033] In one embodiment, a precursor sample including the protein
of interest has been subjected to affinity chromatography to
generate the sample. Alternatively or in combination, the
preparation including a protein of interest and having a reduced
level of one impurity is subjected to affinity chromatography. In
such embodiments, affinity chromatography may be performed using
affinity chromatographic media selected from the group consisting
of Protein A, G, A/G, L media, and MabSuRe Protein A media.
[0034] In one embodiment, a precursor sample including the protein
of interest has been subjected to ion exchange chromatography to
generate the sample. Alternatively or in combination, the
preparation including a protein of interest and having a reduced
level of one impurity is subjected to ion exchange chromatography.
In such embodiments, ion exchange chromatography may be performed
using ion exchange chromatography media selected from the group
consisting of (i) a cation exchange media, for example, comprising
carboxymethyl (CM), sulfoethyl(SE), sulfopropyl(SP), phosphate(P)
or sulfonate(S) ligands, and (ii) an anion exchange media, for
example, comprising diethylaminoethyl (DEAE), quaternary aminoethyl
(QAE) or quaternary amine (Q) group ligands.
[0035] In one embodiment, a precursor sample including the protein
of interest has been subjected to mixed mode chromatography to
generate the sample. Alternatively or in combination, the method
involves subjecting the preparation including a protein of interest
and having a reduced level of one impurity to mixed mode
chromatography, for example, using CaptoAdhere resin.
[0036] In one embodiment, a precursor sample including the protein
of interest has been subjected to a filtration step to generate the
sample. Alternatively or in combination, the method involves
subjecting the preparation including a protein of interest and
having a reduced level of one impurity to a filtration step, for
example, a depth filtration step, a nanofiltration step, an
ultrafiltration step, and an absolute filtration step, or a
combination thereof.
[0037] In one aspect, the present invention is directed to a
pharmaceutical composition including the preparation produced by
any of the foregoing methods.
[0038] In another aspect, the present invention is directed to a
method for producing a preparation including adalimumab and having
a reduced level of at least one aggregate, by (a) contacting a
sample of adalimumab and at least one aggregate, to a HIC media, in
the presence of a load buffer such that (i) a portion of the
adalimumab in the sample binds to the HIC media and (ii) a
substantial portion of the at least one aggregate binds to the HIC
media; (b) collecting a flow through fraction of the adalimumab
unbound to the HIC media; (c) washing the HIC media with a wash
buffer that is substantially the same as the load buffer such that
a substantial portion of the adalimumab bound to the HIC media is
released from the media; and (d) collecting a wash fraction of the
adalimumab released from the HIC media, wherein each of the flow
through and wash fractions comprise adalimumab and have a reduced
level of the at least one aggregate.
[0039] In one embodiment of the foregoing method, adalimumab binds
to the HIC media at a Kp of greater than 10, 15, 20, 50, 60, 70,
80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220,
250 or 300. For example, adalimumab binds to the HIC media at a Kp
of greater than 10. Alternatively, adalimumab binds to the HIC
media at a Kp of greater than 20. Alternatively, adalimumab binds
to the HIC media at a Kp of greater than 90.
[0040] In a particular embodiment, the aggregate is multimer 1,
multimer 2 or multimer 3.
[0041] In various embodiments, the sample includes between 200 g
and 700 g protein per liter of HIC media. In certain embodiments,
the HIC media is selected from the group consisting of GE
CaptoPhenyl, Tosoh Hexyl, GE Butyl FF, Butyl, Hexyl, Phenyl, Octyl,
GE Butyl FF, PPG. In certain embodiments, the load buffer and the
wash buffer include ammonium sulfate, sodium sulfate, sodium
citrate, or a combination thereof. Alternatively or in combination,
the pH of the load buffer and the wash buffer is between 5 and 7.
Alternatively or in combination, the salt concentration of the load
buffer and the wash buffer is between about 150 mM and 1000 mM.
[0042] In another aspect, the present invention provides a
pharmaceutical composition comprising a low-aggregate composition
and a pharmaceutical acceptable carrier.
[0043] In one aspect, the present invention provides a
pharmaceutical composition comprising a preparation of adalimumab
produced by the foregoing methods and a pharmaceutically acceptable
carrier. In another aspect, the present invention provides a
pharmaceutical composition comprising a low-aggregate composition
of adalimumab and a pharmaceutically acceptable carrier. For
example, the composition may include less than 5%, 4%, 3%, 2.5%,
2.4%, 2.3%, 2.2%, 2.1%, 2%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.5%,
0.1% of aggregates, e.g., MM1, MM2 and MM3. Alternatively, the
composition may include less than 2%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%,
1%, 0.5%, 0.1% of aggregates, e.g., MM1, MM2 and MM3.
Alternatively, the composition may include less than 1%, 0.5%, 0.1%
of aggregates, e.g., MM1, MM2 and MM3.
BRIEF DESCRIPTIONS OF THE DRAWINGS
[0044] FIG. 1 depicts a size exclusion chromatography (SEC)
chromatogram used to determine the molecular weight distribution of
a sample of adalimumab. In combination with multi-angle light
scattering (MALS) analysis (data not shown), the apparent molecular
weight of each peak was determined and identified as a multimer or
the reference standard as indicated. Multimer 1 (MM1), Multimer 2
(MM2) and Multimer 3 (MM3) were identified as depicted.
[0045] FIGS. 2A-2B depicts schematic chromatograms for two modes of
chromatographic operation: bind-elute mode (FIG. 2A) and
flow-through mode (FIG. 2B). In the bind-elute mode, there is
strong binding of the protein of interest and the impurity. Elution
conditions are chosen to selectively elutes the protein of
interest. In the flow-through mode there is weak binding of the
product and strong binding of the impurity.
[0046] FIG. 3 depicts selection of operating conditions appropriate
for an antibody:media:buffer combination. A sample was loaded at 20
g/L and a linear gradient elution was performed over 20 CVs to
identify the salt concentration at the monomer and aggregate peak.
The salt concentration at or near the elution peak of the monomer
is the concentration at which the monomer is eluted from the HIC
media.
[0047] FIG. 4 depicts a process chromatogram for the HIC
purification of Adalimumab, wherein a GE CaptoPhenyl column was
equilibrated at 1.1 M AmSO.sub.4 pH 7.0 (Tris/Acetate) for 10 CVs,
Adalimumab was prepared at 1.1 M AmSO.sub.4 and loaded to the
column at 20 g-protein/L of media. The column was then washed with
10 CVs of the equilibration buffer and a linear gradient from 1.1 M
to 0 M AmSO.sub.4 pH 7.0 (Tris/Acetate) over 20 CVs was performed.
See Example 1.
[0048] FIG. 5 depicts a process chromatogram for the HIC
purification of Adalimumab, wherein a GE CaptoPhenyl column was
equilibrated with 400 mM NaCit pH 5.6 for 10 CVs, Adalimumab was
prepared at 400 mM NaCit pH 5.6 and then loaded to the column at
500 g-protein/L-media. Finally, the column was washed with 7 CVs of
the equilibration buffer. See Example 1.
[0049] FIG. 6 depicts results of an experiment wherein a feed
stream was serially diluted to cover a range of load concentrations
from 4 to 15 mg/mL and loaded at 500 g/L to a CaptoPhenyl column in
400 mM NaCit pH 5.6. The results indicate the impact that the
concentration of loaded protein can have on aggregate reduction.
See Example 7.
[0050] FIG. 7 depicts the effect of aggregate load concentration on
dynamic binding capacity and aggregate clearance. The column is
conditioned and loaded at different sample load concentrations. The
flow-through is fractionated to determine the product quality at
different times during the load and breakthrough. Using protein
mass and product quality for each of the collected fractions, the
accumulative impurity (e.g., aggregate) can be calculated. The
accumulative impurity of the preparation is reduced when the
concentration of the aggregate in the load is reduced, even when
the total load is unchanged (e.g., 500 g/L). See Example 13.
[0051] FIGS. 8A-8C depicts the effect of overall load protein
concentration in the sample. The column is conditioned and loaded
at different sample load concentrations. The flow through is
fractionated to determine the product quality at different times
during the load and breakthrough (FIG. 8A). Using protein mass and
product quality for each of the collected factions, the
accumulative aggregate impurity can be calculated. The accumulative
aggregate impurity of the preparation is reduced when the protein
concentration of the sample is reduced. The Equilibrium Binding
Isotherms for both the monomer and aggregate show that for all of
the loading conditions (FIG. 8B and FIG. 8C), the monomer was in
the non-linear part of its binding isotherm (e.g., equilibrium
binding capacity is independent of monomer concentration), and the
aggregate was in or near the linear part of its binding isotherm
(e.g., equilibrium binding capacity is dependent on aggregate
concentration). Aggregate dynamic binding capacity=f(C.sub.o, t).
See Example 13.
[0052] FIG. 9 depicts the modulation of the recovery-yield for a
given target impurity clearance by diluting the load material to a
specific range. See Example 13.
[0053] FIG. 10 depicts dynamic binding capacity (DBC),
conventionally measured at 10% breakthrough, as greater than the
equilibrium binding capacity (EBC), based on the data presented in
FIG. 5. See Example 1.
[0054] FIG. 11 depicts a dynamic binding capacity of >75 g/L.
Following an isocratic wash and regeneration step, the remaining
protein bound to the resin is <35 g/L.
[0055] FIGS. 12A-12B depicts determination of Apparent Binding
Capacity (FIG. 12A), and Actual Binding Capacity (FIG. 12B). See
Example 12.
[0056] FIGS. 13A-13B depict the results of experiments wherein
aliquots of resin are incubated with a load covering a range of
protein concentrations at room temperature for 3 hours, after which
the protein solution is then removed, and replaced with
equilibration buffer (Wash simulation) and incubated at room
temperature for 3 hours (repeated, Wash II). After each incubation,
the concentration of the protein solution is measured and used to
calculated the amount of protein ((FIG. 13A) monomer D2E7, (i.e.,
Adalimumab), and (FIG. 13B) aggregate D2E7) bound to the resin (g
protein/L resin) and plotted against the concentration of the
protein solution at the end of the incubation (e.g., equilibrium).
See Example 11.
[0057] FIGS. 14A-14B depict the results outlined in FIGS. 13A-13B,
highlighting the fact that at initial equilibrium a significant
amount of monomer/aggregate is bound to the resin. However, after
the protein solution is replaced with equilibration buffer (see
arrow), the monomer desorbs from the resin and back into solution,
whereas the aggregate remains bound. See Example 11.
[0058] FIGS. 15A-15B depict a determination of the binding monomer
and aggregate D2E7 (based on data provided in FIGS. 13A-13B) by
fitting the experimental equilibrium binding data to the Langmuir
Isotherm using the equation: q=(q.sub.max.times.
C.sub.equil)/(K.sub.a+C.sub.equil); where q=amount of protein bound
to resin [=] g/L-resin; q.sub.max=maximum amount of protein bound
to resin [=] g/L-resin; C.sub.equil=solution concentration of
protein [=] g/L-soln; and K.sub.a=equilibrium dissociation
constant. See Example 11.
[0059] FIG. 16 depicts the comparison of Apparent and Actual bound
protein under flow conditions (partial partitioning) as a function
of salt concentration. Binding of the protein of interest is
significant >10 g/L. The majority (>65%) of this monomer
bound during the load desorbs during the isocratic wash (i.e.,
reversibly bound). The mass balance of the impurity demonstrates
irreversible binding. See Example 12.
DETAILED DESCRIPTION OF THE INVENTION
[0060] The present invention is directed to methods for purifying a
protein of interest, e.g., an antibody, from a sample comprising
the protein of interest and at least one impurity, e.g., an
aggregate, by employing a novel hydrophobic interaction
chromatography (HIC) method. The present invention is based, at
least in part, on the finding that both flow through and bind-elute
techniques can be combined to achieve greater purification and
recovery of a protein of interest, e.g., an antibody. Moreover, the
present invention is predicated, at least in part, on the
surprising finding that such methodology can be employed under
isocratic wash conditions and at stronger binding conditions than
previously appreciated, for example, at a Kp greater than 10 or at
a Kp greater than 20, so as to achieve greater purification and
recovery.
[0061] In one aspect, the present invention provides a method for
producing a preparation including a protein of interest, e.g., an
antibody such as adalimumab, and having a reduced level of at least
one impurity, e.g., an aggregate, by (a) contacting a sample
including the protein of interest and at least one impurity, to a
hydrophobic interaction chromatography media, in the presence of a
load buffer such that (i) a portion of the protein of interest
binds to the hydrophobic interaction chromatography (HIC) media and
(ii) a substantial portion of the at least one impurity binds to
the HIC media; (b) collecting a flow through fraction including the
protein of interest unbound to the HIC media; (c) washing the HIC
media with a wash buffer that is substantially the same as the load
buffer such that a substantial portion of the protein of interest
bound to the HIC media is released from the media; and (d)
collecting a wash fraction including the protein of interest
released from the HIC media, wherein each of the flow through and
wash fractions include the protein of interest and have a reduced
level of the at least one impurity. In a particular embodiment, the
portion of the protein of interest binds to the HIC media at a Kp
of greater than 10. In another embodiment, the portion of the
protein of interest binds to the HIC media at a Kp of greater than
20. In another embodiment, the portion of the protein of interest
binds to the HIC media at a Kp of greater than 100.
[0062] In certain embodiments, the purification strategies of the
present invention may include one or more chromatography and/or
filtration steps to achieve a desired degree of purification prior
to exposure of the sample comprising the protein of interest, e.g.,
an antibody such as adalimumab, to the HIC media. For example, in
certain embodiments, such pre-HIC chromatography step(s) can
include one or more steps of chromatography and/or filtration. In
one embodiment the chromatography is ion exchange chromatography,
mixed mode chromatography, and/or affinity chromatography. In
another embodiment, the filtration step is depth filtration,
nanofiltration, ultrafiltration and/or absolute filtration. In
certain embodiments, the purification strategies of the present
invention may include one or more additional chromatography and/or
filtration steps after the HIC purification step. For example, in
certain embodiments, such post-HIC chromatography step(s) can
include one or more steps of chromatography and/or filtration. In
one embodiment, the chromatography is ion exchange chromatography,
mixed mode chromatography, and/or affinity chromatography. In
another embodiment, the filtration step is depth filtration,
nanofiltration, ultrafiltration and/or absolute filtration.
[0063] In addition, in certain embodiments, the present invention
is directed toward pharmaceutical compositions comprising one or
more proteins of interest purified by methods described herein. In
a particular embodiment, the present invention is directed to a
pharmaceutical composition comprising adalimumab and having a
reduced level of aggregates.
Definitions
[0064] Unless otherwise defined herein, scientific and technical
terms used in connection with the present invention shall have the
meanings that are commonly understood by those of ordinary skill in
the art. The meaning and scope of the terms should be clear,
however, in the event of any latent ambiguity, definitions provided
herein take precedent over any dictionary or extrinsic definition.
Further, unless otherwise required by context, singular terms, for
example, those characterized by "a" or "an", shall include
pluralities, e.g., one or more impurities. In this application, the
use of "or" means "and/or", unless stated otherwise. Furthermore,
the use of the term "including," as well as other forms of the
term, such as "includes" and "included", is not limiting. Also,
terms such as "element" or "component" encompass both elements and
components comprising one unit and elements and components that
comprise more than one unit unless specifically stated
otherwise.
[0065] As used herein, the term "sample", refers to a liquid
composition including the protein of interest and one or more
impurities. In a particular embodiment, the sample is a "clarified
harvest", referring to a liquid material containing a protein of
interest, for example, an antibody of interest such as adalimumab,
that has been extracted from cell culture, for example, a
fermentation bioreactor, after undergoing centrifugation to remove
large solid particles and subsequent filtration to remove finer
solid particles and impurities from the material.
[0066] In various embodiments, the sample may be partially
purified. For example, the sample may have already been subjected
to any of a variety of art recognized purification techniques, such
as chromatography, e.g., ion exchange chromatography, mixed mode
chromatography, and/or affinity chromatography, or filtration,
e.g., depth filtration, nanofiltration, ultrafiltration and/or
absolute filtration.
[0067] The term "precursor sample", as used herein refers to a
liquid composition containing the protein of interest and,
optionally, one or more impurities, either derived from the
clarified harvest, or a partially purified intermediate sample that
is subject to a purification or treatment step prior to being
subjected to HIC. Impurities in a precursor sample may be derived
from the production, purification or treatment of the protein of
interest prior to subjecting the resulting sample to HIC.
[0068] The term "protein of interest", as used herein refers to a
target protein present in a sample, purification of which is
desired. In various embodiment, the protein of interest is an
antibody or antigen-binding fragment thereof, a soluble protein, a
membrane protein, a structural protein, a ribosomal protein, an
enzyme, a zymogen, an antibody molecule, a cell surface receptor
protein, a transcription regulatory protein, a translation
regulatory protein, a chromatin protein, a hormone, a cell cycle
regulatory protein, a G protein, a neuroactive peptide, an
immunoregulatory protein, a blood component protein, an ion gate
protein, a heat shock protein, an antibiotic resistance protein, a
functional fragment of any of the preceding proteins, an
epitope-containing fragment of any of the preceding proteins, and
combinations thereof. In a particular embodiment, the protein of
interest is a monomer.
[0069] In a particular embodiment, the protein of interest is an
antibody, or an antigen binding portion thereof. 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. The term "antibody",
as used herein, also includes alternative antibody and
antibody-like structures, such as, but not limited to, dual
variable domain antibodies (DVD-Ig).
[0070] 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., hIL-12,
hTNF.alpha., or hIL-18). 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 may be part of a larger
immunoadhesion molecule, formed by covalent or non-covalent
association of the antibody 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.
[0071] 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.
[0072] 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.
[0073] 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 (AbbVie).
[0074] 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
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. 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. The nucleic acid sequence of the
light chain variable region is set forth in SEQ ID NO:9. The
nucleic acid sequence of the heavy chain variable region is set
forth in SEQ ID NO:10. The full length amino acid sequence of the
light chain is set forth as SEQ ID NO:11 and the full length amino
acid sequence of the heavy chain is set forth as SEQ ID NO:12.
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, 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.
[0075] 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.sup.-7 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 even more preferably, 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. The term "Koff", as used
herein, is intended to refer to the off rate constant for
dissociation of an antibody from the antibody/antigen complex.
[0076] The term "impurity", as used herein refers to any foreign or
objectionable molecule, including a biological macromolecule such
as a DNA, an RNA, or a protein other than the protein of interest
being purified. Exemplary impurities include, for example, protein
variants, such as aggregates, high molecular weight species, low
molecular weight species and fragments, and deamidated species;
host cell proteins; proteins that are part of an absorbent used for
affinity chromatography (e.g. Protein A); endotoxins; and
viruses.
[0077] The methods of the invention serve to generate a preparation
comprising a protein of interest and having a reduced level of
impurity. As used herein a "reduced level of impurity" refers to a
composition comprising reduced levels of an impurity as compared to
the levels of the impurity in the sample prior to purification by
the methods of the present invention. In another embodiment, the
methods of the invention generate a preparation comprising a
protein of interest and having a reduced level of total impurity.
As used herein a "reduced level of total impurity" refers to a
composition comprising reduced levels of total impurity as compared
to the levels of the impurity in the sample prior to purification
by the methods of the present invention. In one embodiment, a
preparation having a reduced level of total impurity is free of
impurities or substantially free of impurities.
[0078] The present invention is further directed to low impurity
compositions and methods of generating the same, for example, low
impurity compositions of adalimumab. The term "low impurity
composition," as used herein, refers to a composition comprising a
protein of interest, wherein the composition contains less than
about 15% total impurities. For example, a low impurity composition
may contain about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1.5%, 1.4%,
1.3%, 1.2%, 1.1%, 1%, 0.5%, or less total impurities. In a
particular embodiment, a low impurity composition comprises about
5%, 4%, 3%, 2.5%, 2.4%, 2.3%, 2.2%, 2.1%, 2%, 1.5%, 1.4%, 1.3%,
1.2%, 1.1%, 1%, 0.5%, 0.1%, or less total impurities.
[0079] The term "non-low impurity composition," as used herein,
refers to a composition comprising a protein of interest, which
contains more than about 15% total impurity. For example, a non-low
impurity composition may contain about 15%, 16%, 17%, 18%, 19%,
20%, 21%, 22%, 23%, 24%, 25%, or more total impurities.
[0080] In one embodiment, a low impurity 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 impurity composition. In one embodiment, the low
impurity 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 impurity 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
impurity 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.
[0081] As used herein, the term "increased cartilage penetration"
refers to increased penetration of cartilage in vivo by a low
impurity composition as compared to a non-low impurity composition
comprising the same antibody or antigen binding portion
thereof.
[0082] As used herein, the term "reduced cartilage destruction"
refers to measurable decrease in destruction of cartilage tissue in
vivo by a low impurity composition as compared to a non-low
impurity 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 impurity composition as compared to a non-low
aggregate 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 for further description of the TNF-Tg197
model of arthritis).
[0083] In another embodiment, a low impurity composition comprising
an antibody, or antigen-binding portion thereof, exhibits increased
protection against the development of arthritic scores and/or
histopathology scores as compared to a non-low impurity 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.
[0084] In another embodiment, a low impurity 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 impurity composition. In another embodiment, a low
impurity composition comprising an anti-TNF.alpha. antibody, or
antigen-binding portion thereof, exhibits increased TNF.alpha.
affinity as compared to a non-low impurity composition.
[0085] 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,
ulcerative colitis, active axial spondyloarthritis (active axSpA)
and non-radiographic axial spondyloarthritis (nr-axSpA). Disorders
in which TNF.alpha. activity is detrimental are set forth in U.S.
Pat. No. 6,090,382 and also in the Humira.RTM. Prescribing
Information, the contents of each 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.
[0086] In a particular embodiment, the impurity is a
process-related impurity. As used herein, the term "process-related
impurity," refers to impurities that are present in a composition
comprising a protein of interest 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 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.
[0087] In one embodiment, the impurity is a host cell protein. The
term "host cell protein" (HCP), as used herein, is intended to
refer to non-protein of interest proteinaceous impurities derived
from host cells, for example, host cells used to produce the
protein of interest.
[0088] In one embodiment, the impurity is a host cell nucleic acid.
The term "host cell nucleic acids", as used herein, is intended to
refer to nucleic acids derived from host cells, for example, host
cells used to produce the protein of interest.
[0089] In a particular embodiment, the impurity is a
product-related substance. As used herein, the term
"product-related substance" refers to variants of the protein of
interest formed during manufacturing and/or storage of the protein
of interest. Specific examples of product-related substances
include degradants of the protein, truncated forms of the protein,
high molecular weight species, low molecular weight species,
fragments of the protein, modified forms of the protein, including
deamidated, isomerized, mismatched S--S linked, oxidized or altered
conjugate forms (e.g., glycosylation, phosphorylation), aggregates
including dimers and higher multiples of the protein of interest,
and charge variants.
[0090] In a particular embodiment, the impurity is an aggregate. As
used herein, the term "aggregate" refers to agglomeration or
oligomerization of two or more individual molecules of the protein
of interest to form, for example, dimers, trimers, tetramers,
oligomers and other high molecular weight species. Protein
aggregates can be soluble or insoluble. In a particular embodiment,
the aggregate is a multimer of adalimumab. In a particular
embodiment, the aggregate is a dimer of adalimumab. In another
embodiment, the aggregate is a trimer of adalimumab. In another
embodiment, the aggregate is a tetramer of adalimumab.
[0091] In certain embodiments, the sample can comprise more than
one type of aggregate. For example, but not by way of limitation,
the nature of the aggregates and total aggregate composition can be
identified based on chromatographic residence time. For example,
FIG. 1 depicts a size exclusion chromatography (SEC) chromatogram
used to determine the molecular weight distribution of a sample of
adalimumab.
[0092] As set forth therein, the total aggregate species associated
with the expression of adalimumab can be divided into multimer 1
(MM1), multimer 2 (MM2) and multimer 3 (MM3). In various
embodiments, the methods of the present invention serve to reduce
the levels of one of MM1, MM2 or MM3. In another embodiment, the
methods of the present invention serve to reduce the levels of MM1
and MM2. In another embodiment, the methods of the present
invention serve to reduce the levels of MM1 and MM3. In another
embodiment, the methods of the present invention serve to reduce
the levels of MM2 and MM3. In yet another embodiment, the methods
of the present invention serve to reduce the levels of MM1, MM2 and
MM3.
[0093] In one embodiment, the methods of the invention generate a
preparation comprising a protein of interest and having a reduced
level of aggregate. As used herein, a "reduced level of aggregate"
refers to a composition comprising reduced levels of an aggregate
as compared to the levels of the aggregate in the sample prior to
purification by the methods of the present invention. In one
embodiment, a preparation having a reduced level of aggregate is
free of the aggregate or substantially free of the aggregate. In
another embodiment, the methods of the invention generate a
preparation comprising a protein of interest and having a reduced
level of total aggregate. As used herein a "reduced level of total
aggregate" refers to a composition comprising reduced levels of
total aggregate as compared to the levels of the impurity in the
sample prior to purification by the methods of the present
invention. In one embodiment, a preparation having a reduced level
of total aggregate is free of aggregates or substantially free of
the aggregates.
[0094] The present invention is further directed to low aggregate
compositions and methods of generating the same, for example, low
aggregate compositions of adalimumab. The term "low aggregate
composition," as used herein, refers to a composition comprising a
protein of interest, wherein the composition contains less than
about 15% total aggregates. For example, a low aggregate
composition may contain about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%,
1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.5%, or less total aggregates.
In a particular embodiment, a low aggregate composition comprises
about 5%, 4%, 3%, 2.5%, 2.4%, 2.3%, 2.2%, 2.1%, 2%, 1.5%, 1.4%,
1.3%, 1.2%, 1.1%, 1%, 0.5%, 0.1%, or less total aggregates.
[0095] In one embodiment, a low aggregate composition of adalimumab
can comprise about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2.5%, 2.4%,
2.3%, 2.2%, 2.1%, 2%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.5%, 0.1%,
or less of MM1, or 0.0% of MM1. In another embodiment, a low
aggregate composition of adalimumab can comprise about 10%, 9%, 8%,
7%, 6%, 5%, 4%, 3%, 2.5%, 2.4%, 2.3%, 2.2%, 2.1%, 2%, 1.5%, 1.4%,
1.3%, 1.2%, 1.1%, 1%, 0.5%, 0.1%, or less of MM2, or 0.0% of MM2.
In another embodiment, a low aggregate composition of adalimumab
can comprise about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2.5%, 2.4%,
2.3%, 2.2%, 2.1%, 2%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.5%, 0.1%,
or less of MM3, or 0.0% of MM3.
[0096] The term "non-low aggregate composition," as used herein,
refers to a composition comprising a protein of interest, which
contains more than about 15% total aggregates. For example, a
non-low aggregate composition may contain about 15%, 16%, 17%, 18%,
19%, 20%, 21%, 22%, 23%, 24%, 25%, or more total aggregates. In one
embodiment, a non-low aggregate composition of adalimumab can
comprise about 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%,
25%, or more of MM1. In another embodiment, a non-low aggregate
composition or adalimumab can comprise about 15%, 16%, 17%, 18%,
19%, 20%, 21%, 22%, 23%, 24%, 25%, or more of MM2. In another
embodiment, a non-low aggregate composition or adalimumab can
comprise about 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%,
25%, or more of MM3.
[0097] In one embodiment, a low aggregate 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 aggregate composition. In one embodiment, the low
aggregate 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 aggregate
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 aggregate 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.
[0098] In another embodiment, a low aggregate composition
comprising an antibody, or antigen-binding portion thereof,
exhibits increased protection against the development of arthritic
scores and/or histopathology scores as compared to a non-low
aggregate composition when administered to an animal model of
arthritis, e.g., the TNF-Tg197 model of arthritis, as described
above.
[0099] In another embodiment, a low aggregate 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 aggregate composition. In another embodiment,
a low aggregate composition comprising an anti-TNF.alpha. antibody,
or antigen-binding portion thereof, exhibits increased TNF.alpha.
affinity as compared to a non-low aggregate composition.
[0100] In a particular embodiment, the impurity is a fragment of
the protein of interest. The term "fragment" as used herein refers
to any truncated form of a protein of interest, resulting from, for
example, dissociation of a peptide chain, or enzymatic and/or
chemical modifications.
[0101] In a particular embodiment, the impurity is a charge
variant. The term "charge variant", as used herein, refers to the
full complement of product variants including, but not limited to
acidic species, and basic species (e.g., Lys variants). In certain
embodiments, such variants can include product aggregates and/or
product fragments, to the extent that such aggregation and/or
fragmentation results in a product charge variation as seen in an
analytical technique used for that purpose.
[0102] 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 nucleic acids, chromatographic materials, and
media components.
[0103] 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 residence time. For example, the total acidic
species associated with the expression of adalimumab can be divided
into a first acidic species region (AR1) and a second acidic
species region (AR2).
[0104] 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.
Other acidic variants such as host cells and unknown species may
also be present. In another embodiment, AR2 can comprise, for
example, charge variants such as glycation variants and deamidation
variants. Other acidic variants such as host cells and unknown
species may also be present.
[0105] 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. 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. 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.
[0106] Acidic species may also include process-related
impurities.
[0107] 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 are not present in the population of
proteins directly after preparation, but are formed 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.
[0108] In addition, a low AR composition can be lyophilized to
partially or completely inhibit the formation of storage-derived
AR.
[0109] The term "low acidic species 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. For example, a low acidic species composition
may contain about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1.5%, 1.4%,
1.3%, 1.2%, 1.1%, 1%, 0.5%, or less acidic species. In one
embodiment, a low acidic species composition can comprise about
10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2.5%, 2.4%, 2.3%, 2.2%, 2.1%, 2%,
1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.5%, 0.1%, or less of AR1, or
0.0% of AR1. In another embodiment, a low acidic species
composition can comprise about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%,
2.5%, 2.4%, 2.3%, 2.2%, 2.1%, 2%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%,
0.5%, 0.1%, or less of AR2, or 0.0% of AR2. In a preferred
embodiment, a low acidic species composition comprises about 5%,
4%, 3%, 2.5%, 2.4%, 2.3%, 2.2%, 2.1%, 2%, 1.5%, 1.4%, 1.3%, 1.2%,
1.1%, 1%, 0.5%, 0.1%, or less acidic species. In one embodiment, a
low acidic species composition comprises about 0.1% or less AR1 and
about 3% or less AR2. In another preferred embodiment, a low acidic
species composition comprises about 1% or 0.1% or less AR1. In
still another preferred embodiment, a low acidic species
composition comprises about 3% or less AR2. In another preferred
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.
[0110] 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 15% acidic
species. For example, a non-low acidic species composition may
contain about 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%,
25%, or more acidic species. In one embodiment, a non-low acidic
species composition can comprise about 15%, 16%, 17%, 18%, 19%,
20%, 21%, 22%, 23%, 24%, 25%, or more of AR1. In another
embodiment, a non-low acidic species composition can comprise about
15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, or more of
AR2.
[0111] 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.
[0112] In another embodiment, a low AR composition comprising an
antibody, or antigen-binding portion thereof, exhibits increased
protection against the development of 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 described above.
[0113] 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.
[0114] In another embodiment, the impurity is a 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 variants can be detected, for example, by weak cation
exchange chromatography (WCX) of the expression product of a host
cell expressing the antibody, or antigen-binding portion thereof.
With respect specifically to adalimumab, three main basic lysine
variant species have been identified, i.e., Lys 0, Lys 1, and Lys
2.
[0115] The term "load buffer", as used herein refers to a salt
solution passed through the HIC media upon contacting the sample
with the HIC media. In certain embodiments, the load buffer is
passed through the HIC media simultaneously or substantially
simultaneously with passage of the sample through the HIC media. In
certain embodiments, the load buffer is combined with the sample
prior to passage through the HIC media.
[0116] The term "flow through fraction", as used herein refers to
the liquid that passes through without binding the hydrophobic
column upon contacting the sample with the HIC media during the
load cycle. According to the methods of the present invention, the
flow through fraction includes protein of interest that does not
bind to the HIC media. The flow through fraction may also include
load buffer that passes through the HIC media during the load cycle
and/or a portion of the impurity that does not bind to the HIC
media.
[0117] The term "wash buffer", as used herein refers to a salt
solution passed through the HIC media during the wash cycle.
[0118] The term "wash fraction", as used herein refers to the
liquid eluted from the column upon washing the HIC media with the
wash buffer. According to the methods of the present invention, the
wash fraction includes protein of interest that is released from
the HIC media upon exposure to the wash buffer. The wash fraction
may also include wash buffer that passes through the HIC media
during the wash cycle and/or a portion of the impurity that does
not bind to the HIC media.
[0119] The term "isocratic", as used herein, refers to wash and
load conditions which are identical or vary only slightly in terms
of, for example, the nature of the buffer, the salt concentration,
the pH, and the temperature. In particular embodiments, the wash
and load conditions are substantially the same, for example, the
salt concentration and/or the pH of the wash buffer are identical
to or are adjusted to within about 20%, about 15%, about 10%, or
about 5% of the salt concentration, and/or pH of the loading
buffer. In a particular embodiment, the wash and load conditions
are identical.
[0120] The term "load challenge", as used herein refers to the
total mass of sample (e.g., protein of interest and at least one
protein) loaded onto the column in chromatography applications or
applied to the resin in batch binding, measured in units of mass of
product per unit volume of resin.
[0121] As used herein, the term "dynamic binding capacity" refers
to the amount of total protein that binds to the HIC media upon
breakthrough of 10% of the total protein load.
[0122] As used herein, the term "apparent binding capacity" refers
to the amount of protein of interest that binds to the HIC media
upon breakthrough of 10% of the total protein load, in reversible
HIC binding applications.
[0123] As used herein, the term "actual binding capacity" refers to
the amount of protein of interest that remains bound to the
chromatographic media under isocratic wash conditions.
[0124] As used herein, the term "equilibrium binding capacity"
refers to the maximum amount of total protein that can be bound
under certain conditions.
[0125] As used herein, the term "partition coefficient" (Kp) refers
to the equilibrium ratio of the concentration of protein of
interest adsorbed to the HIC media to the concentration of protein
of interest in the solution comprising the unbound protein of
interest, under specified conditions of pH and solution
composition. The partition coefficient Kp corresponds to the slope
of the protein of interest adsorption isotherm at very low solution
concentrations. Kp can be calculated from the Qmax (maximum
capacity of the HIC media for the protein of interest) and Kd
(dissociation constant for the HIC media-protein of interest
interaction) as follows: Kp=Q/C=Qmax/Kd.
Protein Purification
Protein Purification Generally
[0126] The present invention provides a method for producing a
preparation including a protein of interest, e.g., an antibody, and
having a reduced level of at least one impurity, e.g., an
aggregate, by contacting a sample including the protein of interest
and at least one impurity, to a hydrophobic interaction
chromatography media.
[0127] In certain embodiments, the compositions of the present
invention include, but are not limited to, a preparation comprising
a protein of interest having a reduced level of at least one
impurity. For example, but not by way of limitation, the present
invention is directed to preparations of adalimumab having a
reduced level of at least one impurity, for example, aggregate.
Such preparations having a reduced level of at least one impurity
address the need for improved product characteristics, including,
but not limited to, product stability, product safety and product
efficacy. In further embodiments, compositions of the present
invention include pharmaceutical compositions comprising the
preparation produced by the methods of the invention (e.g., protein
of interest having a reduced level of the at least on impurity) and
a pharmaceutically acceptable carrier.
[0128] In certain embodiments, the purification process of the
invention begins at the separation step when the protein of
interest has been produced using production methods described above
and/or by alternative production methods conventional in the art.
Once a clarified solution or sample comprising the protein of
interest has been obtained, separation of the protein of interest
from at least one impurity, such as process-related impurities,
e.g., other proteins produced by the cell, as well as any
product-related substances, e.g., charge variants and/or size
variants (aggregates and fragments), can be performed using a HIC
separation step, or a combination of a HIC separation step and one
or more purification techniques, including filtration and/or
affinity, ion exchange, and/or mixed mode chromatographic step(s),
as outlined herein.
Primary Recovery
[0129] In certain embodiments, the initial steps of the
purification methods of the present invention involve the
clarification and primary recovery of protein of interest, for
example, antibody, following production. In certain embodiments,
the primary recovery will include one or more centrifugation steps
to separate the protein of interest from cells and cell debris.
Centrifugation of the protein containing composition can be run 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.
[0130] In certain embodiments, the primary recovery will include
the use of one or more depth filtration steps to clarify the sample
and thereby aid in purifying the protein 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. Non-limiting examples of depth
filters that can be used in the context of the instant invention
include the Millistak+ XOHC, FOHC, DOHC, 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.
[0131] 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. 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, solvent/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 in U.S. Pat. No. 4,534,972. In certain
embodiments of the present invention, the sample is exposed to
detergent viral inactivation during the primary recovery phase. In
other embodiments, the sample may be exposed to low pH inactivation
during the primary recovery phase.
[0132] In those embodiments where viral reduction/inactivation is
employed, the sample can be adjusted, as needed, for further
purification steps. For example, following low pH viral
inactivation, the pH of the sample 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.
Hydrophobic Interaction Chromatography
[0133] The instant invention features methods for producing a
preparation comprising a protein of interest (e.g., the
anti-TNF.alpha. antibody adalimumab, or a fragment thereof) having
a reduced level of at least one impurity, for example, aggregate,
from a sample comprising the protein of interest and at least one
impurity by contacting the sample with HIC media.
[0134] According to the present invention, HIC purification of a
protein of interest comprises reversible binding of the protein of
interest and binding of one or more impurities through hydrophobic
interaction with hydrophobic moieties attached to a solid matrix
support (e.g., agarose). The hydrophobic interaction between
molecules results from the tendency of a polar environment to
exclude non-polar (i.e., hydrophobic) molecules. HIC relies on this
principle of hydrophobicity of molecules (i.e., the tendency of a
given protein to bind adsorptively to hydrophobic sites on a
hydrophobic adsorbent body) to separate biomolecules based on their
relative strength of interaction with the hydrophobic moieties
(see, e.g., U.S. Pat. No. 4,000,098 and U.S. Pat. No. 3,917,527
which are herein incorporated by reference in their entirety). An
advantage of this separation technique is its non-denaturing
characteristics and the stabilizing effects of salt solutions used
during loading, washing and or eluting.
[0135] Hydrophobic interaction chromatography employs the
hydrophobic properties of molecules (e.g., proteins, polypeptides,
lipids) to achieve separation of even closely-related molecules.
Hydrophobic groups on the molecules interact with hydrophobic
groups of the media or the membrane. In certain embodiments, the
more hydrophobic a molecule is, the stronger it will interact with
the column or the membrane. Thus, HIC steps, such as those
disclosed herein, can be used to remove a variety of impurities,
for example, process-related impurities (e.g., DNA) as well as
product-related species (e.g., high and low molecular weight
product-related species, such as protein aggregates and
fragments).
[0136] In one aspect, the present invention provides a method for
producing a preparation including a protein of interest, e.g., an
antibody, and having a reduced level of at least one impurity,
e.g., an aggregate, by (a) contacting a sample including the
protein of interest and at least one impurity, to a hydrophobic
interaction chromatography media, in the presence of a load buffer
such that (i) a portion of the protein of interest binds to the
hydrophobic interaction chromatography (HIC) media and (ii) a
substantial portion of the at least one impurity binds to the HIC
media; (b) collecting a flow through fraction including the protein
of interest unbound to the HIC media; (c) washing the HIC media
with a wash buffer that is substantially the same as the load
buffer such that a substantial portion of the protein of interest
bound to the HIC media is released from the media; and (d)
collecting a wash fraction including the protein of interest
released from the HIC media, wherein each of the flow through and
wash fractions include the protein of interest and have a reduced
level of the at least one impurity. In a particular embodiment, the
portion of the protein of interest binds to the HIC media at a Kp
of greater than 10. In a particular embodiment, the portion of the
protein of interest binds to the HIC media at a Kp of greater than
20. In a particular embodiment, the portion of the protein of
interest binds to the HIC media at a Kp of greater than 100.
[0137] In another aspect, the present invention provides a method
for producing a preparation including a protein of interest, e.g.,
an antibody, and having a reduced level of at least one impurity,
e.g., an aggregate, by (a) contacting a sample including the
protein of interest and at least one impurity, to a HIC media, in
the presence of a load buffer such that (i) a portion of the
protein of interest binds to the HIC media and (ii) a substantial
portion of the at least one impurity binds to the HIC media;
collecting a flow through fraction including the protein of
interest unbound to the HIC media; (c) washing the HIC media with a
wash buffer that is substantially the same as the load buffer such
that a substantial portion of the protein of interest bound to the
HIC media is released from the media; and (d) collecting a wash
fraction including the protein of interest released from the HIC
media, wherein either (i) the substantial portion of the at least
one impurity binds to the HIC media at a Kp greater than 200 and/or
(ii) the protein of interest and the at least on impurity have a Kp
ratio less than 1:7; and wherein each of the flow through and wash
fractions include the protein of interest and have a reduced level
of the at least one impurity.
[0138] In yet another aspect, the present invention provides a
method for producing a preparation including a protein of interest,
e.g., an antibody, and having a reduced level of at least one
impurity, e.g., an aggregate, by (a) contacting a sample including
the protein of interest and at least one impurity, to a HIC media,
in the presence of a load buffer such that (i) a portion of the
protein of interest binds to the HIC media, and (ii) a substantial
portion of the at least one impurity binds to the HIC media; (b)
collecting a flow through fraction including the protein of
interest unbound to the HIC media; (c) washing the HIC media with a
wash buffer that is substantially the same as the load buffer such
that a substantial portion of the protein of interest bound to the
HIC media is released from the media; and (d) collecting a wash
fraction including the protein of interest released from the HIC
media, wherein the K.sub.d for the binding of the protein of
interest to the HIC media is less than 60, 50, 45, 40, 35, 30, 25,
20, 15, 10, 5 or 2 times the K.sub.d for the binding of the at
least one impurity to the HIC media, and wherein each of the flow
through and wash fractions include the protein of interest and have
a reduced level of the at least one impurity.
[0139] According to the present invention, a portion of the protein
of interest reversibly binds to the HIC media while a portion of
the protein of interest flows through to form a flow through
fraction which has a reduced level of impurity. The portion of the
protein of interest that binds to the HIC media binds reversibly in
that the bound protein of interest may be released therefrom under
isocratic conditions, for example, by use of a wash buffer that is
substantially the same as the load buffer. In contrast, a
substantial portion of the at least impurity in the sample binds
the HIC media upon loading and a substantial portion thereof
remains bound upon washing the HIC media with the wash buffer.
[0140] The present invention is based, at least in part, on the
finding that such reversible binding can be achieved at relatively
high binding strength. For example, contrary to the teachings of
U.S. Pat. No. 8,067,182 which teaches weak partitioning binding of
a product, i.e., at a Kp of between 0.1 and 20 and less than 10 for
HIC, according to the methods of the present invention, the protein
of interest may bind at higher Kp levels so as to achieve higher
purification and greater recovery of the protein of interest. For
example, in a particular embodiment, the protein of interest binds
to the HIC media at a Kp of greater than 10, 15, 20, 50, 60, 70,
80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220,
250, 300, 400 or 500. In one embodiment, the protein of interest
binds to the HIC media at a Kp greater than 10. In another
embodiment, the protein of interest binds to the HIC media at a Kp
greater than 20. In another embodiment, the protein of interest
binds to the HIC media at a Kp of greater than 90.
[0141] According to the present invention, the impurity binds at a
higher strength and, thus to a greater degree to the HIC media,
thereby allowing for selective release of the bound protein of
interest upon wash. For example, in particular embodiments, the at
least one impurity binds to the HIC media at a Kp of greater than
200, greater than 250, greater than 300, greater than 400, greater
than 500, greater than 600, greater than 700, greater than 800,
greater than 900, greater than 1000, or greater than 2000. In a
specific embodiment, the at least one impurity binds to the HIC
media at a Kp of greater than 600.
[0142] In a further embodiment, the protein of interest and the at
least one impurity have a Kp ratio less than 1:10, less than 1:9,
less than 1:8, less than 1:7, less than 1:6, less than 1:5, less
than 1:4, less than 1:3 or less than 1:2. In a specific embodiment,
the protein of interest and the at least one impurity have a Kp
ratio less than 1:7.
[0143] The relative strength of binding may also be assessed by
determining K.sub.d, the dissociation constant for the
media-protein of interest interaction, or the media-impurity
interaction. In one embodiment of the invention, the K.sub.d for
the binding of the protein of interest to the HIC media is at least
about 0.2, at least about 0.3, at least about 0.4, at least about
0.5, at least about 0.6, at least about 0.7, or at least about 0.8.
In a preferred embodiment, the K.sub.d for the binding of the
protein of interest to the HIC media is at least about 0.4.
[0144] In another embodiment of the invention, the K.sub.d of the
binding of the at least one impurity to the HIC media is less than
or equal to about 0.001, about 0.005, about 0.01, about 0.02, about
0.05, about 0.1, about 0.15, about 0.2, about 0.3, about 0.4, about
0.5, or about 1.0. In a particular embodiment the K.sub.d for the
at least one impurity is less than or equal to about 0.01.
[0145] In another embodiment of the invention, the K.sub.d for the
binding of the protein of interest to the HIC media is less than
60, 50, 45, 40, 35, 30, 25, 20, 15, 10, 5 or 2 times the K.sub.d
for the binding of the at least one impurity to the HIC media.
[0146] The relative binding capacity of the protein of interest may
also be assessed by determining Qmax, the maximum capacity of the
media for the protein of interest, or for the at least one
impurity. In one embodiment of the invention, the protein of
interest has a Qmax of at least about 20, at least about 30, at
least about 40, at least about 50, at least about 60, at least
about 100, at least about 250, or at least about 500. In a
preferred embodiment, the protein of interest has a Qmax of at
least about 40.
[0147] In another embodiment, the at least one impurity has a Qmax
of at least about 2, at least about 5, at least about 10, at least
about 20, at least about 30, at least about 40, at least about 50,
at least about 75, or at least about 100. In a preferred
embodiment, the at least one impurity has a Qmax of at least about
5.
[0148] In performing the HIC separation, the sample is contacted
with the HIC media, e.g., using a batch purification technique or
using a column or membrane chromatography or monolithic material
(referred to as HIC media or resin). For example, in the context of
chromatographic separation, a chromatographic apparatus, commonly
cylindrical in shape, is employed to contain the chromatographic
support media (e.g., HIC media) prepared in an appropriate buffer
solution. Once the chromatographic material is added to the
chromatographic apparatus, a sample containing the protein of
interest, e.g., an antibody, and the protein of interest is
contacted to the chromatographic material in the presence of a
loading buffer to allow binding of a portion of the protein of
interest and a substantial portion of the impurity to the HIC
media. A portion of the protein of interest in the sample binds to
the HIC media while a portion of the protein interest flows
through, forming a flow through fraction having a reduced level of
impurity which is collected.
[0149] In one embodiment, the portion of the protein of interest
that binds to the HIC media is at least about 20%, at least about
30%, at least about 40%, at least about 50%, at least about 60%, at
least about 70%, at least about 80%, at least about 90%, at least
about 95% or at least about 98% of the amount of the protein of
interest in the sample.
[0150] Alternatively or in combination, the substantial portion of
the at least one impurity that binds to the HIC media is at least
about 60%, at least about 65%, at least about 70%, at least about
75%, at least about 80%, at least about 85%, at least about 90%, at
least about 95%, at least about 98% or about 100% of the level of
the at least one impurity in the sample.
[0151] Alternatively or in combination, the portion of the protein
of interest that flows through without binding to the HIC media is
at least about 20%, at least about 30%, at least about 40%, at
least about 50%, at least about 60%, at least about 70%, at least
about 80%, at least about 90%, at least about 95% or at least about
98% of the amount of the protein of interest in the sample.
[0152] The media is then subjected to a wash buffer, thereby
allowing for a portion of the bound protein of interest to release
from the HIC media in a wash fraction which is collected, while a
substantial portion of the impurity remains bound to the HIC media.
After loading, the column can be regenerated with water and cleaned
with caustic solution to remove the bound impurities before next
use.
[0153] In one embodiment, the substantial portion of the protein of
interest released from the HIC media upon washing with the wash
buffer is at least about at least about 50%, at least about 60%, at
least about 70%, at least about 80%, at least about 90%, at least
about 95%, at least about 98%, or about 100% of the amount of
protein of interest bound to the HIC media.
[0154] Alternatively or in combination, the substantial portion of
the impurity that remains bound to the HIC media is at least about
50%, at least about 60%, at least about 65%, at least about 70%, at
least about 75%, at least about 80%, at least about 85%, at least
about 90%, at least about 95%, at least about 98% or about 100% of
the impurity bound to the HIC media during the load cycle.
[0155] In order to achieve the desired reversible binding of the
protein of interest and the comparable strong binding of the at
least one impurity, appropriate selection of resin, buffer,
concentration, pH and sample load is required. Techniques to
identify optimal conditions for achieving such desired binding
profile are set forth in the Examples below.
[0156] Hydrophobic interactions are strongest at high salt
concentration (and hence the ionic strength of the anion and cation
components). Adsorption of the protein of interest 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. In various embodiments, the salt concentration may be in
the range of, for example, about 50 mM to about 5000 mM, about 100
mM to about 4000 mM, about 1000 mM to about 4000 mM, about 50 mM to
about 2000 mM, depending, in part, on the salt type and HIC
adsorbent. In one embodiment the salt concentration is about 50 mM,
about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM,
about 80 mM, about 85 mM, about 90 mM, about 100 mM, about 200 mM,
about 300 mM, about 400 mM, about 500 mM, about 600 mM, about 700
mM, about 800 mM, about 900 mM, about 1000 mM, about 1200 mM, about
1400 mM, about 1600 mM, about 1800 mM or about 2000 mM.
[0157] 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.-.
[0158] In certain embodiments, the anionic part of the salt is
chosen from among sulfate, citrate, chloride, or a mixture thereof.
In certain embodiments, the cationic part of the salt is chosen
from among ammonium, sodium, potassium, or a mixture thereof. 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
and about 2 M ammonium sulfate or between about 1 and 4 M NaCl are
useful. In another embodiment, the load buffer and the wash buffer
comprise a salt of the Hofmeister series or lyotropic series of
salts.
[0159] In one embodiment, the load buffer and the wash buffer
comprise a sulfate salt, a citrate salt, or a combination thereof.
In another embodiment, the sulfate salt in ammonium sulfate. In
another embodiment, the sulfate salt is a sodium sulfate. In yet
another embodiment, the citrate salt is sodium citrate. In certain
embodiments, the load and/or wash buffer may be comprised of at
least 2 salts.
[0160] In certain embodiments, the HIC adsorbent material is
composed of a chromatographic backbone with pendant hydrophobic
interaction ligands. For example, but not by way of limitation, the
HIC media can be composed of convective membrane media with pendent
hydrophobic interaction ligands, convective monolithic media with
pendent hydrophobic interaction ligands, and/or convective filter
media with embedded media containing the pendant hydrophobic
interaction ligands.
[0161] In certain embodiments, the HIC adsorbent material can
comprise a base matrix (e.g., derivatives of cellulose,
polystyrene, synthetic poly amino acids, synthetic polyacrylamide
gels, cross-linked dextran, cross-linked agarose, synthetic
copolymer material or even a glass surface) to which hydrophobic
ligands (e.g., alkyl, aryl and combinations thereof) are coupled or
covalently attached using difunctional linking groups such as
--NH--, --S--, --COO--, etc. The hydrophobic ligand may be
terminated in a hydrogen but can also terminate in a functional
group such as, for example, NH.sub.2, SO.sub.3H, PO.sub.4H.sub.2,
SH, imidazoles, phenolic groups or non-ionic radicals such as OH
and CONH.sub.2. In one embodiment, the HIC media comprises at least
one hydrophobic ligand. In another embodiment, the hydrophobic
ligand is selected from the group consisting of butyl, hexyl,
phenyl, octyl, or polypropylene glycol ligands.
[0162] One, non-limiting, example of a suitable HIC media comprises
an agarose media or a membrane functionalized with phenyl groups
(e.g., a Phenyl Sepharose.TM. from GE Healthcare or a Phenyl
Membrane from Sartorius). Many HIC medias are available
commercially. Examples include, but are not limited to, Tosoh
Hexyl, CaptoPhenyl, 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);
Toyopearl.TM. ether, phenyl or butyl (TosoHaas, PA); ToyoScreen
PPG, ToyoScreen Phenyl, ToyoScreen Butyl, and ToyoScreen Hexyl are
a rigid methacrylic polymer bead. GE HiScreen Butyl FF and HiScreen
Octyl FF are high flow agarose based beads.
[0163] In one embodiment, the HIC media has a dynamic binding
capacity of at least about 2 g, at least about 5 g, at least about
10 g, at least about 20 g, at least about 30 g, at least about 40
g, at least about 50 g, at least about 60 g, at least about 70 g,
at least about 80 g, at least about 90 g, at least about 100 g, or
at least about 200 g of sample per one liter of media.
[0164] Because the pH selected for any particular purification
process must be compatible with protein stability and activity,
particular pH conditions may be specific for each application. A
high or low pH may serve to weaken hydrophobic interactions and
retention of proteins changes.
[0165] The pH of the HIC purification process is dependent, in
part, on the pH of the buffers used to load, equilibrate and or
wash the chromatographic resin or media.
[0166] Accordingly, in one embodiment, the pH of any of the buffers
is between about 4.0 and 8.5. In a further embodiment, the pH of
any of the buffers is between about 5.0 and 7.0. In one embodiment,
the pH of any of the buffers may be about 4.0, about 4.5, about
5.0, about 5.5, about 6.0, about 6.5, about 7.0, about 7.5, about
8.0, or about 8.5. In a preferred embodiment the pH of any of the
buffers is 5.0. In a related embodiment the pH of any of the
buffers is 5.6. In yet another embodiment, the pH of any of the
buffers is 7.0.
[0167] In certain embodiments, the load challenge of the sample
comprising the protein of interest and at least one impurity is
adjusted to a total protein load to the column of between about 50
and 1000 g/L, or between about 250 and 700 g/L, or between about
350 and 500 g/L of HIC media. In certain embodiments, the protein
concentration of the load challenge is adjusted to a total protein
concentration of about 0.5 and 50 g/L, or between about 1 and 20
g/L, or between about 3 and 10 g/L. In one embodiment the load
challenge is about 50 g, about 100 g, about 150 g, about 200 g,
about 250 g, about 300 g, about 350 g, about 400 g, about 450 g,
about 500 g, about 550 g, about 600 g, about 650 g, about 700 g,
about 750 g, about 800 g, about 850 g, about 900 g, about 950 g, or
about 1000 g of sample per one liter of HIC media. In a particular
embodiment, the load challenge of the sample is 200 g/L. In another
embodiment, the load challenge is 350 g/L. In yet another
embodiment, load challenge of the sample is 500 g/L. In yet another
embodiment, the load challenge of the sample is 700 g/L.
[0168] In another embodiment, the load challenge for the impurity
alone is about 0.1 g, about 0.2 g, about 0.3 g, about 0.4 g, about
0.5 g, about 0.6 g, about 0.7 g, about 0.8 g, about 0.9 g, about
1.0 g, about 1.5 g, about 2.0 g, about 2.5 g, about 3.0 g, about
3.5 g, about 4.0 g, about 4.5 g, or about 5.0 g of the at least one
impurity per one liter of HIC media.
[0169] In certain embodiments, impurity (e.g., aggregate)
concentration is measured and used as a parameter for controlling
impurity clearance in the present invention. For example, but not
by way of limitation, the data presented in the Examples below,
demonstrates that impurity concentration influences the impurity
reduction by hydrophobic interaction chromatography. Thus, in
certain embodiments, the at least one impurity concentration is
adjusted from about 0.5 to 0.1 g/L, to about 0.1 to 0.05 g/L or to
below 0.05 g/L. In another embodiment, the at least one impurity
contacting the HIC media has a concentration of about 0.01, about
0.02, about 0.03, about 0.04, about 0.05, about 0.06, about 0.07,
about 0.08, about 0.09, about 0.1, about 0.15, about 0.2, about
0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about
0.9, about 1.0, about 2.0, about 3.0, about 4.0 or about 5.0
g/L.
[0170] In certain embodiments, protein of interest concentration
(e.g., antibody monomer) is measured and used as a parameter for
controlling impurity (e.g., aggregate) clearance in the present
invention. For example, but not by way of limitation, the data
presented in the Examples below demonstrates that control of the
concentration of the protein of interest can be used to achieve
improved impurity clearance. Thus, in certain embodiments, the
protein of interest concentration is adjusted from about 15 to 8
g/L, to about 8 to 4 g/L or to below 4 g/L. In another embodiment,
the protein of interest contacting the HIC media has a
concentration of less than about 1, about 2, about 3, about 4,
about 5, about 6, about 7, about 8, about 9, about 10, about 15,
about 20, about 25, about 30, about 35, about 40 about 45, about
50, or about 55 g/L.
[0171] In certain embodiments, protein of interest (e.g., antibody
monomer) and impurity (e.g., aggregate) concentration is measured
and used as a parameter for controlling impurity clearance in the
present invention. For example, but not by way of limitation, the
data presented in the Examples demonstrates that control of the
protein of interest and monomer concentrations within certain
ranges can be used to achieve improved impurity clearance. Thus, in
certain embodiments, the protein of interest concentration is
adjusted from about 20 to 15 g/L, about 15 to 8 g/L or to below 4
g/L and the impurity concentration is adjusted to 0.5 to 0.1 g/L,
about 0.1 to 0.05 g/L or to below 0.05 g/L to achieve impurity
reduction in the present invention. In another embodiment, the
protein of interest contacting the HIC media has a concentration of
about 1, about 2, about 3, about 4, about 5, about 6, about 7,
about 8, about 9, about 10, about 15, about 20, about 25, about 30,
about 35, about 40 about 45, or about 55 g/L and the at least one
impurity contacting the HIC media has a concentration of less than
about 0.01, about 0.02, about 0.03, about 0.04, about 0.05, about
0.06, about 0.07, about 0.08, about 0.09, about 0.1, about 0.15,
about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7,
about 0.8, about 0.9, or about 1.0 g/L.
[0172] In another embodiment, the sample contacting the HIC media
has a concentration of about 1, about 2, about 3, about 4, about 5,
about 6, about 7, about 8, about 9, about 10, about 15, about 20,
about 25, about 30, about 35, about 40 about 45, or about 55
g/L.
[0173] In one embodiment, the at least one impurity is an aggregate
of the protein of interest, for example, selected from the group
consisting of a dimer, a trimer, a tetramer, an oligomer and other
high molecular weight species. In a particular embodiment, the
protein of interest is adalimumab and the at least one impurity is
an aggregate of adalimumab. For example, the aggregate may be
selected from the group consisting of multimer 1, multimer 2 and
multimer 3.
[0174] In another embodiment, the impurity is a process-related
impurity or a product-related substance. For example, the impurity
may be a process-related impurity selected from the group
consisting of a host cell protein, a host cell nucleic acid, a
media component, and a chromatographic material. Alternatively, the
impurity may be a product-related substance selected from the group
consisting of a charge variant, an aggregate of the protein of
interest, a fragment of the protein of interest and a modified
protein.
[0175] In a particular embodiment the impurity is an acidic or
basic variant, for example, of adalimumab. In a particular
embodiment, the basic variant is a lysine variant species, for
example, an antibody, or antigen-binding portion thereof, having
heavy chains with either zero, one or two C-terminal lysines. In
another embodiment, the impurity is an acidic species (AR), for
example, selected from the group consisting of a charge variant, a
structure variant, a fragmentation variant, a process-related
impurity and a product-related impurity. In a particular
embodiment, the acidic species is AR1 and the charge variant is a
deamidation variant, a glycation variant, an afucosylation variant,
a MGO variant and/or a citric acid variant. In another embodiment,
the acidic species is AR1 and the structure variant is a
glycosylation variant and/or an acetonation variant. In yet another
embodiment, the acidic species is AR1 and the fragmentation variant
is a Fab fragment variant, a C-terminal truncation variant or a
variant missing a heavy chain variable domain. In yet a further
embodiment, the acidic species is AR2 and the charge variant
comprises a deamidation variant and/or glycation variant.
[0176] In a particular embodiment, the impurity is a fragment such
as an Fc or a Fab fragment. In another embodiment, the impurity is
a modified protein such as a deamidated protein or glycosylated
protein.
[0177] In certain embodiments, HIC chromatographic fractions are
collected during the load and/or wash cycles and are combined after
appropriate analysis to provide a protein preparation that contains
the reduced level of impurities. In certain embodiments, the flow
through fraction is combined with certain wash fractions to improve
the yield of the process while still achieving the desired, e.g.,
reduced level of impurities in the preparation.
[0178] Additionally, the flow through or wash fractions, or
combination thereof may be contacted with HIC media again to
further purify the sample. In various embodiments, the method may
be repeated at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20
times.
[0179] In certain embodiments, spectroscopy methods such as UV,
NIR, FTIR, Fluorescence, Raman may be used to monitor levels of
impurities such as aggregates and 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
aggregates in the pooled material collected from the HIC methods of
the present invention. In certain embodiments, on-line, at-line or
in-line monitoring methods can be used either on the wash 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, thereby facilitating product quality
control.
[0180] In one embodiment, the reduced level of the at least one
impurity of the flow through fractions and/or the wash fractions is
at least about 60%, at least about 65%, at least about 70%, at
least about 75%, at least about 80%, at least about 85%, at least
about 90%, at least about 95%, at least about 98% or about 100% of
the amount of the at least on impurity, e.g., aggregate or host
cell protein in the sample.
[0181] In another embodiment, the impurity is a host cell protein
and is reduced by at least 0.25, at least 0.5, at least 0.75, at
least 1.0, at least 1.25, at least 1.5, at least 1.75, at least
2.0, or at least 5.0 LFR.
[0182] In another embodiment, the accumulative aggregate reduction
of the at least one impurity in any one flow through fraction
and/or wash fraction collected during the preparation is at least
about 0.1%, at least about 0.2%, at least about 0.5%, at least
about 1.0%, at least about 2.0%, at least about 3.0%, at least
about 4.0%, at least about 5.0%, at least about 10%, at least about
20%, or at least about 50%.
[0183] In another embodiment, the accumulative aggregate reduction
of the at least one impurity in the flow through fraction and the
wash fractions is at least about 0.1%, at least about 0.2%, at
least about 0.5%, at least about 1.0%, at least about 2.0%, at
least about 3.0%, at least about 4.0%, at least about 5.0%, at
least about 10.0%, or at least about 20.0%.
[0184] In another embodiment, the accumulative yield of the protein
of interest in the flow through fraction and in the wash fraction
is at least about 35%, at least about 40%, at least about 45%, at
least about 50%, at least about 55%, at least about 60%, at least
about 65%, at least about 70%, at least about 75%, at least about
80%, at least about 85%, at least about 90%, at least about 95%, or
about 100%.
[0185] In yet another embodiment, the accumulative yield of the
protein of interest in any one flow through fraction or wash
fraction is at least about 4%, at least about 10%, at least about
20%, at least about 30%, at least about 40%, at least about 50%, at
least about 55%, at least about 60%, at least about 65%, at least
about 70%, at least about 75%, at least about 80%, at least about
85, at least about 90%, at least about 95% or about 100%.
Complementary Purification Techniques
[0186] In certain embodiments, a combination of HIC and at least
one of AEX (anion exchange chromatography) and CEX (cation exchange
chromatography) and MM (mixed-mode chromatography) methods can be
used to prepare preparations of protein of interest having a
reduced level of impurity, 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 a particularly 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 (ultrafiltration/diafiltration) steps so as to achieve the
desired product quality, ion concentration, and/or viral
reduction.
Affinity Chromatography
[0187] In certain embodiments, a precursor sample is subjected to
affinity chromatography to purify the protein of interest, prior to
the methods of the present invention. Alternatively or in addition,
the wash and/or flow through fractions generated by the methods of
the present invention can be subjected to affinity chromatography
to further purify the protein of interest. As noted above, certain
embodiments of the present invention will employ one or more
affinity chromatography steps prior to the HIC purification step,
while others will employ an affinity chromatography step after or
both before and after the HIC purification step. In certain
embodiments, the affinity chromatography media is a Protein A, G,
A/G, or L media, although alternative affinity chromatography
medias are known in the art. There are a variety of commercial
sources for Protein A media. Suitable medias include, but are 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.
[0188] In certain embodiments, 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. 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.
[0189] The Protein A eluate may be 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.
[0190] 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 are not
limited to, Millistak+ XOHC, FOHC, DOHC, AlHC, and B1HC Pod filters
(EMD Millipore), or Zeta Plus 30ZA/60ZA, 60ZA/90ZA, delipid, VR07,
and VRO5 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.
Ion Exchange Chromatography
[0191] In certain embodiments, a precursor sample is subjected to
ion exchange chromatography to purify the protein of interest,
prior to the methods of the present invention. Alternatively or in
addition, the wash and/or flow through fractions generated by the
methods of the present invention can be subjected to ion exchange
chromatography to further purify the protein of interest. As noted
above, certain embodiments of the present invention will employ one
or more ion exchange chromatography steps prior to the HIC
purification step, while others will employ an ion exchange
chromatography step after or both before and after the HIC
purification step.
[0192] As used herein, ion exchange separations includes any method
by which two substances are separated based on the difference in
their respective ionic charges, either on the protein of interest
and/or chromatographic material as a whole or locally on specific
regions of the protein of interest and/or chromatographic material,
and thus can employ either cationic exchange material or anionic
exchange material.
[0193] The use of a cationic exchange material versus an anionic
exchange material 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 HIC step, or a cationic exchange step prior to the use of an
HIC 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 either prior to or
subsequent to the HIC step.
[0194] In performing the separation, the sample containing the
protein of interest (e.g., an antibody or antigen-binding fragment
thereof) can be contacted with the ion 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 HIC.
[0195] Ion exchange chromatography separates molecules based on
differences between the local charges of the proteins of interest
and the local charges of the chromatographic material. A packed
ion-exchange chromatography column or an ion-exchange membrane
device can be operated in a bind-elute mode, a flow-through, or a
hybrid mode. 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 ion 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). The column is then regenerated before next use.
[0196] Anionic or cationic substituents may be attached to matrices
in order to form anionic or cationic supports for chromatography.
Non-limiting examples of anionic exchange substituents include
diethylaminoethyl (DEAE), quaternary aminoethyl (QAE) and
quaternary amine (Q) groups. Cationic substituents include
carboxymethyl (CM), sulfoethyl (SE), sulfopropyl (SP), phosphate
(P) and sulfonate (S). Cellulose ion exchange medias such as
DE23.TM., DE32.TM., DE52.TM., CM-23.TM., CM-32.TM., and CM-52.TM.
are available from Whatman Ltd. Maidstone, Kent, U.K.
SEPHADEX.RTM.-based and -locross-linked ion exchangers are also
known. For example, DEAE-, QAE-, CM-, and SP-SEPHADEX.RTM. and
DEAE-, Q-, CM- and S-SEPHAROSE.RTM. and SEPHAROSE.RTM. Fast Flow,
and Capto.TM. S are all available from GE Healthcare. Further, both
DEAE and CM derivitized ethylene glycol-methacrylate copolymer such
as TOYOPEARL.TM. DEAE-650S or M and TOYOPEARL.TM. CM-650S or M are
available from Toso Haas Co., Philadelphia, Pa., or Nuvia S and
UNOSphere.TM. S from BioRad, Hercules, Calif., Eshmuno.RTM. S from
EMD Millipore, Billerica, Calif.
Mixed Mode Chromatography
[0197] In certain embodiments, a precursor sample is subjected to
mixed mode chromatography to purify the protein of interest, prior
to the HIC methods of the present invention. Alternatively or in
addition, the wash and/or flow through fractions generated by the
methods of the present invention can be subjected to mixed mode
chromatography to further purify the protein of interest. As noted
above, certain embodiments of the present invention will employ one
or more mixed mode chromatography steps prior to the HIC
purification step, while others will employ a mixed mode
chromatography step after or both before and after the HIC
purification step.
[0198] Mixed mode chromatography is chromatography that utilizes a
mixed mode media, such as, but not limited to CaptoAdhere available
from GE Healthcare. Such a media comprises a mixed mode
chromatography ligand. In certain embodiments, such a ligand refers
to a ligand that is capable of providing at least two different,
but co-operative, sites which interact with the substance to be
bound. One of these sites gives an attractive type of charge-charge
interaction between the ligand and the protein of interest. The
other site typically gives 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. The mixed mode functionality can
give a different selectivity compared to traditional anion
exchangers. For example, CaptoAdhere is designed for post-Protein A
purification of monoclonal antibodies, where removal of leached
Protein A, aggregates, host cell proteins, nucleic acids and
viruses from monoclonal antibodies is performed in flow-through
mode (the antibodies pass directly through the column while the
contaminants are adsorbed). Mixed mode chromatography ligands are
also known as "multimodal" chromatography ligands.
[0199] In certain embodiments, the mixed mode chromatography media
is comprised of mixed mode 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.
Viral Filtration
[0200] In certain embodiments, a precursor sample is subjected to
viral filtration to purify the protein of interest, prior to the
HIC methods of the present invention. Alternatively or in addition,
the wash and/or flow through fractions generated by the methods of
the present invention can be subjected to viral filtration to
further purify the protein of interest. As noted above, certain
embodiments of the present invention will employ one or more viral
filtration steps prior to the HIC purification step, while others
will employ viral filtration after or both before and after the HIC
purification step.
[0201] Viral filtration is a dedicated viral reduction step in the
entire purification process. This step is usually performed as a
post chromatographic polishing step. 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.
Ultrafiltration/Diafiltration
[0202] In certain embodiments, a precursor sample is subjected to
ultrafiltration and/or diafiltration to purify the protein of
interest, prior to the HIC methods of the present invention.
Alternatively or in addition, the wash and/or flow through
fractions generated by the methods of the present invention can be
subjected to ultrafiltration and/or diafiltration to further purify
the protein of interest. As noted above, certain embodiments of the
present invention will employ one or more ultrafiltration and/or
diafiltration steps prior to the HIC purification step, while
others will employ ultrafiltration and/or diafiltration after or
both before and after the HIC purification step.
[0203] 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). A preferred 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 while antibodies are retained
behind the filter.
[0204] Diafiltration is a method of using ultrafilters to remove
and exchange salts, sugars, and non-aqueous solvents, to separate
free from bound species, to remove low molecular-weight material,
and/or to cause the rapid change of ionic and/or pH environments.
Microsolutes are removed most efficiently by adding solvent to the
solution being ultrafiltered at a rate approximately equal to the
ultratfiltration rate. This washes microspecies from the solution
at a constant volume, effectively purifying the retained antibody.
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 antibody preparations.
Exemplary Purification Strategies
[0205] 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.
[0206] Additionally, the HIC methodology as described herein is
utilized to further purify the protein of interest. As set forth
herein, such methods involve (a) contacting a sample including the
protein of interest and at least one impurity, to a hydrophobic
interaction chromatography (HIC) media, in the presence of a load
buffer such that (i) a portion of the protein of interest binds to
the HIC media, for example, at a Kp of at least 10, 20 or 100 and
(ii) a substantial portion of the at least one impurity binds to
the HIC media; (b) collecting a flow through fraction including the
protein of interest unbound to the HIC media; (c) washing the HIC
media with a wash buffer that is substantially the same as the load
buffer such that a substantial portion of the protein of interest
bound to the HIC media is released from the media; and (d)
collecting a wash fraction including the protein of interest
released from the HIC media, wherein each of the flow through and
wash fractions include the protein of interest and have a reduced
level of the at least one impurity.
[0207] Examples of buffers that can be used in the context of both
the loading and wash steps of the present invention include, but
are not limited to, the following: about 0.1 M to about 0.6 M
sodium citrate (NaCit), pH 5.6; or about 0.5 M to about 1.1 M
ammonium sulfate (AmSO.sub.4), pH 7.0 as well as buffers
substantially the same, in that any differences result in
insubstantial changes to the binding of impurities, but do not
substantially affect the ability to wash and release antibody
product. Such buffers can span a range of varying
"hydrophobicities" based on the rationales discussed in above.
[0208] In certain embodiments, the HIC media employed in the HIC
step is CaptoPhenyl (GE) resin. In certain embodiments, the
CaptoPhenyl (GE) resin is buffer exchanged into 0.4 M sodium
citrate (NaCit), pH 5.6, and then distributed in 100 .quadrature.L
aliquots into microcentrifuge tubes. Each tube is then challenged
with 2 mL of antibody produce source material, e.g., a partially
purified cell culture harvest sample, in 0.4 M NaCit, pH 5.6, at a
range of concentrations from 0.5-15.0 mg/mL and incubated for 3
hours at room temperature with mixing. The resin is allowed to
settle and the supernatant removed and replaced with 1 mL of fresh
0.4 M NaCit, pH 5.6, buffer and incubated for 2 hours at room
temperature with mixing. This step was repeated one more time.
[0209] In alternative embodiments, the CaptoPhenyl (GE) HIC resin
can be packed in 1.0 cm.times.10.0 cm (OmniFit) columns. Antibody
product HIC-load can be prepared by diluting the source material,
e.g., a partially purified cell culture harvest sample, with a 1.2
M stock solution of sodium citrate (NaCit), pH 5.6, to final
concentration in the range of 0.3 to 0.5 M NaCit, pH 5.6.
CaptoPhenyl columns can then be equilibrated with 7 column volumes
(CVs) of a NaCit buffer, pH 5.6, corresponding to the load
concentration. The antibody product solution can then be loaded to
the column in the range of 200-500 g/L, after which the column is
washed with 20 CVs of the wash buffer. The column can then be
regenerated (3 CVs f 25 mM sodium phosphate/20% (v/v) isopropyl
alcohol, pH 6.5), cleaned in place (3 CVs 1M NaOH, 60 min hold),
and stored (5 CVs of 25 mM sodium phosphate/20% (v/v) isopropyl
alcohol, pH 6.5). The released solution from the column can be
fractionated during the entire run and used to monitor the
breakthrough of both the protein of interest, e.g., antibody
product monomer, as well as impurities, e.g., aggregates and host
cell protein (HCP).
[0210] Such HIC purification steps can be preceded by affinity
chromatography, for example, but not limited to, the use of Protein
A-base affinity chromatography. There are several commercial
sources for Protein A media. One suitable media is MabSelect.TM.
from GE Healthcare. An example of a suitable column packed with
MabSelect.TM. is a column about 1.0 cm diameter.times.about 21.6 cm
long (.about.17 mL bed volume). This size column can be used for
bench scale. This 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 commercial production. Regardless of
the column, the column can be packed using a suitable media such as
MabSelect.TM..
[0211] In certain aspects, the Protein A column can be equilibrated
with a suitable buffer prior to sample loading. An example of a
suitable buffer is a Tris/NaCl buffer, pH of about 6 to 8, and in
certain embodiments about 7.2. A specific example of suitable
conditions is 25 mM Tris, 100 mM NaCl, pH 7.2. Following this
equilibration, the sample can be loaded onto the column. Following
the loading of the column, the column can be washed one or multiple
times using, e.g., the equilibrating buffer. Other washes including
washes employing different buffers can be used before eluting the
column. For example, the column can be washed using one or more
column volumes of 20 mM citric acid/sodium citrate, 0.5 M NaCl at
pH of about 6.0. This wash can optionally be followed by one or
more washes using the equilibrating buffer. The Protein A column
can then be eluted using an appropriate elution buffer. An example
of a suitable elution buffer is an acetic acid/NaCl buffer, pH
around 3.5. Suitable conditions are, e.g., 0.1 M acetic acid, pH
3.5. The eluate can be monitored using techniques well known to
those skilled in the art. For example, the absorbance at OD.sub.280
can be followed. Column eluate can be collected starting with an
initial deflection of about 0.5 AU to a reading of about 0.5 AU at
the trailing edge of the elution peak. The elution fraction(s) of
interest can then be prepared for further processing. For example,
the collected sample can be titrated to a pH of about 5.0 using
Tris (e.g., 1.0 M) at a pH of about 10. Optionally, this titrated
sample can be filtered and further processed.
[0212] In certain embodiments, the HIC purification step can also
be preceded by an ion exchange chromatography step. The ion
exchange purification step can occur before, after, or in place of
an affinity chromatography step. In certain embodiments, where a
Protein A step precedes the ion exchange step, a Protein A eluate
is purified using a cation exchange column. In certain embodiments,
the equilibrating buffer used in the cation exchange column is a
buffer having a pH of about 5.0. An example of a suitable buffer is
about 210 mM sodium acetate, pH 5.0. Following equilibration, the
column is loaded with sample prepared from HIC purification step
above. The column is packed with a cation exchange media, such as
CM Sepharose.TM. Fast Flow from GE Healthcare. The column is then
washed using the equilibrating buffer. The column is next subjected
to an elution step using a buffer having a greater ionic strength
as compared to the equilibrating or wash buffer. For example, a
suitable elution buffer can be about 790 mM sodium acetate, pH 5.0.
The antibodies will be eluted and can be monitored using a UV
spectrophotometer set at OD.sub.280nm. In a particular example,
elution collection can be from upside 3 OD.sub.280nm to downside 8
OD.sub.280nm. It should be understood that one skilled in the art
may vary the conditions and yet still be within the scope of the
invention.
[0213] In certain embodiments where a Protein A step precedes an
ion exchange step, a Protein A eluate is purified using an anion
exchange column. A non-limiting example of a suitable column for
this step is a 60 cm diameter.times.30 cm long column whose bed
volume is about 85 L. The column is packed with an anion exchange
media, such as Q Sepharose.TM. Fast Flow from GE Healthcare. The
column can be equilibrated using about seven column volumes of an
appropriate buffer such as Tris/sodium chloride. An example of
suitable conditions is 25 mM Tris, 50 mM sodium chloride at pH 8.0.
A skilled artisan may vary the conditions but still be within the
scope of the present invention. The column is loaded with the
collected sample from the HIC purification step outlined above. In
another aspect, the column is loaded from the eluate collected
during cation exchange. Following the loading of the column, the
column is washed with the equilibration buffer (e.g., the
Tris/sodium chloride buffer). The flow-through comprising the
antibodies can be monitored using a UV spectrophotometer at
OD.sub.280nm. This anion exchange step reduces process related
impurities such as nucleic acids like DNA, and host cell proteins.
The separation occurs due to the fact that the antibodies of
interest do not substantially interact with nor bind to the solid
phase of the column, e.g., to the Q Sepharose.TM., but many
impurities do interact with and bind to the column's solid phase.
The anion exchange can be performed at about 12.degree. C.
[0214] In certain embodiments, the cation exchange or anion
exchange eluate, depending on which ion exchange step is employed,
or employed first, is next filtered using, e.g., a 16 inch Cuno.TM.
delipid filter. This filtration, using the delipid filter, can be
followed by, e.g., a 30-inch 0.45/0.2 .mu.m Sartopore.TM. bi-layer
filter cartridge. The ion exchange elution buffer can be used to
flush the residual volume remaining in the filters and prepared for
ultrafiltration/diafiltration.
[0215] In order to accomplish the ultratfiltration/diafiltration
step, the filtration media is prepared in a suitable buffer, e.g.,
20 mM sodium phosphate, pH 7.0. A salt such as sodium chloride can
be added to increase the ionic strength, e.g., 100 mM sodium
chloride. This ultrafiltration/diafiltration step serves to
concentrate the anti-IL-12, anti-TNF.alpha., or anti-IL-18
antibodies, remove the sodium acetate and adjust the pH. Commercial
filters are available to effectuate this step. For example,
Millipore manufactures a 30 kD molecular weight cut-off (MWCO)
cellulose ultrafilter membrane cassette. This filtration procedure
can be conducted at or around room temperature.
[0216] In certain embodiments, the sample from the capture
filtration step above is subjected to a second ion exchange
separation step. In certain embodiments, this second ion exchange
separation will involve separation based on the opposite charge of
the first ion exchange separation. For example, if an anion
exchange step is employed after HIC purification, the second ion
exchange chromatographic step may be a cation exchange step.
Conversely, if the HIC purification step was followed by a cation
exchange step, that step would be followed by an anion exchange
step. In certain embodiments the first ion exchange eluate can be
subjected directly to the second ion exchange chromatographic step
where the first ion exchange eluate is adjusted to the appropriate
buffer conditions. Suitable anionic and cationic separation
materials and conditions are described above.
[0217] In certain embodiments, a mixed mode chromatography step
will precede the HIC chromatography step, thereby forming a mixed
mode chromatography sample that can be exposed to the HIC media in
the HIC chromatography step. Examples of mixed mode medias include,
but are not limited to: CaptoAdhere (GE Healthcare), PPA-HyperCel
(Pall Life Sciences), and HEA-HyperCel (Pall Life Sciences). In
certain embodiments, the mixed mode chromatography step is a
CaptoAdhere chromatography step. In certain embodiments, the mixed
mode chromatography sample is further subject to a filtration step.
Filters well known to those skilled in the art can be used in this
embodiment. In one aspect, the filtration step is a nanofiltration
step. In certain embodiments, a depth filtration step follows a
filtration step.
[0218] In certain embodiments of the invention, the wash and/or
flow through fractions from the hydrophobic chromatography step are
subjected to filtration for the removal of viral particles,
including intact viruses, if present. A non-limiting example of a
suitable filter is the Ultipor DV50.TM. filter from Pall
Corporation. Other viral filters can be used in this filtration
step and are well known to those skilled in the art. The HIC eluate
is passed through a pre-wetted filter of about 0.1 .mu.m and a
2.times.30-inch Ultipor DV50.TM. filter train at around 34 psig. In
certain embodiments, following the filtration process, the filter
is washed using, e.g., the HIC wash buffer in order to remove any
antibodies retained in the filter housing. The filtrate can be
stored in a pre-sterilized container at around 12.degree. C.
[0219] In a certain embodiments, the filtrate from the above is
again subjected to ultrafiltration/diafiltration. This step is
important if a practitioner's end point is to use the antibody in
a, e.g., pharmaceutical formulation. This process, if employed, can
facilitate the concentration of antibody, removal of buffering
salts previously used and replace it with a particular formulation
buffer. In certain embodiments, continuous diafiltration with
multiple volumes, e.g., two volumes, of a formulation buffer is
performed. A non-limiting example of a suitable formulation buffer
is 5 mM methionine, 2% mannitol, 0.5% sucrose, pH 5.9 buffer (no
Tween). Upon completion of this diavolume exchange the antibodies
are concentrated. Once a predetermined concentration of antibody
has been achieved, then a practitioner can calculate the amount of
10% Tween that should be added to arrive at a final Tween
concentration of about 0.005% (v/v).
[0220] 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 ion exchange 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).
[0221] In certain embodiments the unbound flow through and wash
fractions can be further fractionated and a combination of
fractions providing a target protein of interest purity can be
pooled.
[0222] In certain embodiments the protein concentration can be
adjusted to achieve a differential partitioning behavior between
the protein of interest and the impurities such that the purity
and/or yield can be further improved.
[0223] 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.
[0224] In certain embodiments the column temperature, can be
independently varied to improve the separation efficiency and/or
yield of any particular purification step.
[0225] In certain embodiments, the loading and washing buffers can
be different or composed of mixtures of chemicals, while achieving
similar "hydrophobic interaction" behavior such that the above
novel separation can be effected.
[0226] In certain embodiments, the loading and washing buffers can
be different, in terms of ionic strength or pH, while remaining
substantially the same in function in terms of the washout of the
protein of interest achieved during the wash step.
[0227] In certain embodiments, the loading & washing steps can
be controlled by in-line, at-line or off-line measurement of the
impurity levels, either in the column effluent, or the collected
pool or both, so as to achieve the protein of interest quality
and/or yield.
[0228] 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.
[0229] 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.
[0230] In certain embodiments, the separation can be performed on
any type of HIC media such as membranes, monoliths or depth filters
that have hydrophobic interaction characteristics.
[0231] Mixed mode media can also be employed to enable this method,
provided the same functionality is achieved by appropriately
adjusting the column loading and/or washing conditions.
Methods of Assaying Sample Purity
Assaying Aggregates
[0232] In certain embodiments, the levels of product-related
substances, such as aggregates, in either the initial sample or the
flow through and/or wash fractions following the HIC steps of the
present invention are analyzed. For example, but not by way of
limitation, the aggregates present in the Adalimumab process
samples can be quantified according to the following methods.
[0233] Aggregates may be measured using a size exclusion
chromatographic (SEC) method whereby molecules are separated based
on size and/or molecular weight such that larger molecules elute
earlier from the column. For example, but not by way of limitation,
a SEC columns useful for the detection of aggregates include:
TSK-gel G3000SWxL, 5 .mu.m, 125 .ANG., 7.8.times.300 mm column
(Tosoh Bioscience), TSK-gel Super SW3000, 4 .mu.m, 250 .ANG.,
4.6.times.300 mm column (Tosoh Bioscience), or Zorbax GF450 column
(Agilent Technologies). A further example of an SEC column for
analysis of monomers and aggregates is the MAbPac.TM. SEC-1 (Thermo
Scientific) column which may be used under non-denaturing
conditions, in both high- and low-salt mobile phases, and with
volatile eluents. In certain embodiments, the aforementioned
columns are used along with an Agilent or a Shimazhu HPLC system.
In a particular embodiment of SEC, aggregates may be quantified
using a Zorbax GF450 column on an Agilent HPLC system.
[0234] 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.
[0235] The elution profile may be further analyzed using multiangle
laser light-scattering (MALS), to determine the apparent molecular
weight of each peak, and allow identification as a dimer, tetramer,
or other high molecular weight species (FIG. 1). The elution
profile may also be further analyzed using sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). For example,
the fraction is mixed with either a non-reducing or reducing
denaturing sample buffer, treated for two minutes at 98.degree. C.
in an Eppendorf Thermomixer Confort, then loaded in a 5%
polyacrylamide tris-HCL gel alongside pre-stained broad range
molecular weight markers. Electrophoresis is performed using a
buffer comprising 0.3% (w/v) Tris, 1.44% (w/v) glycine and 0.1%
SDS, pH 8.3. Separation is performed at a constant current of 100 V
and at maximally 50 mA for about 1 hour, followed by staining of
the gel. In another embodiment, the aggregates may be analyzed and
the molecular weight determined using high performance-size
exclusion chromatography followed by native electrospray ionization
time-of-flight mass spectrometry (ESI-TOF MS). Further methods for
assaying levels of aggregates are provided in the Examples
below.
Assaying Host Cell Protein
[0236] The present invention also provides methods for determining
the residual levels of host cell protein (HCP) concentration in the
initial sample or the flow through and/or wash fractions following
the HIC steps of the present invention. As described above, HCPs
are desirably excluded from the final preparation. Exemplary HCPs
include proteins originating from the source of the protein of
interest production. Failure to identify and sufficiently remove
HCPs from the target protein of interest may lead to reduced
efficacy and/or adverse subject reactions when administered in a
therapeutic setting.
[0237] As used herein, the term "HCP ELISA" refers to an ELISA
where the antibody used in the assay is specific to the HCPs
produced from cells, e.g., CHO cells, used to generate the protein
of interest. The antibody may be produced according to conventional
methods known to those of skill in the art. For example, the
antibody may be produced using HCPs obtained by sham production and
purification runs, i.e., the same cell line used to produce the
protein of interest is used, but the cell line is not transfected
with antibody DNA. In an exemplary embodiment, the antibody is
produced using HPCs similar to those expressed in the cell
expression system of choice, i.e., the cell expression system used
to produce the protein of interest.
[0238] 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 embodiments, 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.
[0239] HCP ELISA may be used for determining the level of HCPs in
preparation or fraction, such as a wash fraction or a flow-through
obtained using the process described above. The present invention
also provides a preparation comprising a protein of interest,
wherein the composition has no detectable level of HCPs as
determined by an HCP Enzyme Linked Immunosorbent Assay ("ELISA").
In one embodiment, the protein of interest is adalimumab.
Assaying Charge and Size Variants
[0240] In certain embodiments, the levels of product-related
substances, such as acidic species and other charge variants, in
the chromatographic samples produced using the techniques described
herein are analyzed. For example, but not by way of limitation, the
acidic species and other charge variants present in the Adalimumab
process samples can be quantified according to the following
methods. Cation exchange chromatography was performed on a Dionex
ProPac WCX-10, Analytical column 4 mm.times.250 mm (Dionex, CA). An
Agilent 1200 HPLC system was used as the HPLC. The mobile phases
used were 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). 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)
was used with detection at 280 nm.
[0241] 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 G3000SWxL, 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.
Antibody Generation
[0242] Antibodies to be purified by the methods of the present
invention 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. Although somatic cell hybridization procedures are
preferred, in principle, other techniques for producing monoclonal
antibody can be employed e.g., viral or oncogenic transformation of
B lymphocytes.
[0243] In certain embodiments, the animal system for preparing
hybridomas is the murine system. Hybridoma production is a
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.
[0244] An antibody can be, in certain embodiments, a human, a
chimeric, or a humanized antibody. Humanized antibodies of the
present disclosure 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.).
[0245] 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).
[0246] 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 tranchromosome, 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 the
antibodies of this disclosure.
[0247] In certain embodiments, the antibodies of this disclosure
are recombinant human antibodies, which 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 Antibod 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.
[0248] Human monoclonal antibodies of this disclosure 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.
[0249] The antibodies or antigen-binding portions thereof 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.
Antibody Production
[0250] To express an antibody of the invention, DNAs encoding
partial or full-length light and heavy chains 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. 6,914,128, the entire teaching of which
is incorporated herein by reference.) In this context, the term
"operatively linked" is intended to mean that an antibody gene 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 antibody
gene. The expression vector and expression control sequences are
chosen to be compatible with the expression host cell used. The
antibody light chain gene and the antibody heavy chain gene can be
inserted into a separate vector or, more typically, both genes are
inserted into the same expression vector. The antibody genes are
inserted into an expression vector by standard methods (e.g.,
ligation of complementary restriction sites on the antibody gene
fragment and vector, or blunt end ligation if no restriction sites
are present). Prior to insertion of the antibody or
antibody-related light or heavy chain sequences, the expression
vector may already carry antibody constant region sequences.
Additionally or alternatively, the recombinant expression vector
can encode a signal peptide that facilitates secretion of the
antibody chain from a host cell. The antibody chain gene can be
cloned into the vector such that the signal peptide is linked
in-frame to the amino terminus of the antibody chain 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).
[0251] In addition to the antibody chain genes, a recombinant
expression vector of the invention can carry one or more regulatory
sequence that controls the expression of the antibody chain 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 antibody chain 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.
[0252] In addition to the antibody chain genes and regulatory
sequences, a recombinant expression vector of the invention may
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).
[0253] An antibody 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.
[0254] For expression of the light and heavy chains, the expression
vector(s) encoding the heavy and light chains 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
antibodies 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 antibody. Prokaryotic expression of antibody genes has been
reported to be ineffective for production of high yields of active
antibody (Boss and Wood (1985) Immunology Today 6:12-13, the entire
teaching of which is incorporated herein by reference).
[0255] 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.
[0256] 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.
[0257] Suitable host cells for the expression of 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.
[0258] Suitable mammalian host cells for expressing the recombinant
antibodies of 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), NSO myeloma cells, COS cells and SP2 cells. When
recombinant expression vectors encoding antibody 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.
[0259] Host cells are transformed with the above-described
expression or cloning vectors for antibody production and cultured
in conventional nutrient media modified as appropriate for inducing
promoters, selecting transformants, or amplifying the genes
encoding the desired sequences.
[0260] The host cells used to produce an antibody 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.
[0261] Host cells can also be used to produce portions of intact
antibodies, such as 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 of this invention. 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 the
antigen to which the putative antibody of interest binds. 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 one to
which the putative antibody of interest binds, 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.
[0262] In a suitable system for recombinant expression of an
antibody of the invention, a recombinant expression vector encoding
both the antibody heavy chain and the antibody light chain is
introduced into dhfr-CHO cells by calcium phosphate-mediated
transfection. Within the recombinant expression vector, the
antibody heavy and light chain genes are each operatively linked to
CMV enhancer/AdMLP promoter regulatory elements to drive high
levels of transcription of the genes. 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 antibody heavy and light
chains and intact 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 antibody from
the culture medium.
[0263] When using recombinant techniques, the antibody can be
produced intracellularly, in the periplasmic space, or directly
secreted into the medium. In one aspect, if the antibody 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 antibody 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.
[0264] Prior to the process of the invention, procedures for
purification of antibodies from cell debris initially depend on the
site of expression of the antibody. Some antibodies can be secreted
directly from the cell into the surrounding growth media; others
are made intracellularly. For the latter antibodies, 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.
Methods of Treatment Using the Low Impurity Compositions of the
Invention
[0265] The low impurity compositions, for example, low aggregate
compositions, of the invention may be used to treat any disorder in
a subject for which the therapeutic protein of interest (e.g., an
antibody or an antigen binding portion thereof) comprised in the
composition is appropriate for treating.
[0266] A "disorder" is any condition that would benefit from
treatment with the protein of interest. 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 impurity composition may be administered to treat a
disorder in which TNF.alpha. activity is detrimental.
[0267] 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.
[0268] 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 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.
[0269] 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, a1 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 and osteoarthritis.
[0270] 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.
[0271] 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.
[0272] In one embodiment, the invention provides a method of
administering a low impurity 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..
[0273] The low impurity 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 impurity
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.
[0274] 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.
[0275] An exemplary, non-limiting range for a therapeutically or
prophylactically effective amount of a low impurity 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 impurity 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.
[0276] 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.
Pharmaceutical Formulations Containing the Low Impurity
Compositions of the Invention
[0277] The present invention further provides preparations and
formulations comprising low impurity compositions, for example, low
aggregate compositions, of the invention. It should be understood
that any of the proteins of interest, such as antibodies and
antibody fragments described herein, including proteins of interest
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 a protein of interest, such
as an antibody, it is understood that such a protein of interest
may be a protein having any one or more of the characteristics of
the proteins of interest described herein. In one embodiment, the
antibody is an anti-TNF.alpha. antibody, or antigen-binding portion
thereof.
[0278] In certain embodiments, the low impurity compositions, for
example, low aggregate 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 proteins of interest
(e.g., 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.
[0279] The low impurity compositions, for example, low aggregate
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 impurity compositions, for example, low
aggregate compositions, of the invention is a liquid formulation.
In another embodiment, a formulation of the low impurity
compositions, for example, low aggregate compositions, of the
invention is a lyophilized formulation. In a further embodiment, a
formulation of the low impurity compositions, for example, low
aggregate compositions, of the invention is a reconstituted liquid
formulation. In one embodiment, a formulation of the low impurity
compositions, for example, low aggregate compositions, of the
invention is a stable liquid formulation. In one embodiment, a
liquid formulation of the low impurity compositions, for example,
low aggregate 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 impurity compositions, for example, low aggregate compositions,
of the invention is an aqueous formulation wherein the aqueous
carrier is distilled water.
[0280] The formulations of the low impurity compositions, for
example, low aggregate compositions, of the invention comprise a
protein of interest (e.g., an antibody) in a concentration
resulting in a w/v appropriate for a desired dose. The protein of
interest 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.
[0281] In a specific embodiment, a formulation of the low impurity
compositions, for example, low aggregate 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 protein
of interest (e.g., an antibody) of the invention.
[0282] In one embodiment, the concentration of protein of interest
(e.g., 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.
[0283] In a specific embodiment, a formulation of the low impurity
compositions, for example, low aggregate 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 a protein of
interest (e.g., an antibody).
[0284] The formulations of the low impurity compositions, for
example, low aggregate compositions, of the invention comprising a
protein of interest (e.g., 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
compounds are suitably present in combination in amounts that are
effective for the purpose intended.
[0285] The formulations of the low impurity compositions, for
example, low aggregate compositions, of the invention may be
prepared for storage by mixing the protein of interest (e.g.,
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).
[0286] 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
[0287] The formulations of the low impurity compositions, for
example, low aggregate 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 protein of interest (e.g., antibody) to be used in the
formulation.
[0288] 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).
[0289] 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.
[0290] In certain embodiments, the formulation of the low impurity
compositions, for example, low aggregate 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.
[0291] The formulations of the low impurity compositions, for
example, low aggregate 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%.
[0292] 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%.
[0293] In a specific embodiment, the formulations of the low
impurity compositions, for example, low aggregate 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.
[0294] In certain embodiments, a formulation of the low impurity
compositions, for example, low aggregate 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.
[0295] A formulation of the low impurity compositions, for example,
low aggregate 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.
[0296] A formulation of the low impurity compositions, for example,
low aggregate 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.
[0297] The formulations of the low impurity compositions, for
example, low aggregate 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
iso stearamidopropyl-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%.
[0298] The formulations of the low impurity compositions, for
example, low aggregate 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.
[0299] 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.
[0300] 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 protein of interest (e.g., an
antibody), as well known those in the art or as described
herein.
[0301] In one embodiment, the low impurity compositions, for
example, low aggregate 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 Prescribing Information" for
HUMIRA.RTM. (adalimumab) Injection (Revised January 2008) the
contents of which are hereby 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.
[0302] It will be understood by one skilled in the art that the
formulations of the low impurity compositions, for example, low
aggregate 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 isotonicity of the formulation. Tonicity modifiers
suitable for this invention include, but are not limited to,
saccharides, salts and amino acids.
[0303] In certain embodiments, the formulations of the low impurity
compositions, for example, low aggregate 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.
[0304] The concentration of any one component or any combination of
various components, of the formulations of the low impurity
compositions, for example, low aggregate compositions, of the
invention is adjusted to achieve the desired tonicity of the final
formulation. For example, the ratio of the carbohydrate excipient
to protein of interest (e.g., 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 protein of interest (e.g., antibody) may be from about 100 moles
to about 1000 moles of carbohydrate excipient to about 1 mole of
protein of interest, or from about 200 moles to about 6000 moles of
carbohydrate excipient to about 1 mole of protein of interest, or
from about 100 moles to about 510 moles of carbohydrate excipient
to about 1 mole of protein of interest, or from about 100 moles to
about 600 moles of carbohydrate excipient to about 1 mole of
protein of interest.
[0305] 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.
[0306] In one embodiment the formulations of the low impurity
compositions, for example, low aggregate 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
proteins of interest (e.g., 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.
[0307] When used for in vivo administration, the formulations of
the low impurity compositions, for example, low aggregate
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 protein of interest (e.g., 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 proteins of
interest (e.g., antibodies), such as those disclosed herein,
ordinarily will be stored in lyophilized form or in solution. It is
contemplated that sterile compositions comprising proteins of
interest (e.g., 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.
[0308] In one embodiment, a formulation of the low impurity
compositions, for example, low aggregate 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.
[0309] 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.
[0310] 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.
[0311] 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.
[0312] In one embodiment, the molar ratio of a lyoprotectant (e.g.,
trehalose) and protein of interest (e.g., 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 protein of interest 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.
[0313] A "reconstituted" formulation is one which has been prepared
by dissolving a lyophilized protein of interest (e.g., antibody)
formulation in a diluent such that the protein of interest 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 protein of
interest and, in certain embodiments of the invention, may be one
which is suitable for intravenous administration.
[0314] 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.
[0315] In certain embodiments, a formulation of the low impurity
compositions, for example, low aggregate compositions, of the
invention is a lyophilized formulation comprising a protein of
interest (e.g., 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 protein of interest 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 a protein of
interest 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 protein of interest 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 a protein of interest 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 protein
of interest may be recovered by reconstituting a lyophilized cake
generated from said formulation.
[0316] In one embodiment, a reconstituted liquid formulation may
comprise a protein of interest (e.g., antibody) at the same
concentration as the pre-lyophilized liquid formulation.
[0317] In another embodiment, a reconstituted liquid formulation
may comprise a protein of interest (e.g., 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 a protein of interest than the
pre-lyophilized liquid formulation.
[0318] In yet another embodiment, a reconstituted liquid
formulation may comprise a protein of interest (e.g., 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 a protein of interest
than the pre-lyophilized liquid formulation.
[0319] The pharmaceutical formulations of the low impurity
compositions, for example, low aggregate compositions, of the
invention are typically stable formulations, e.g., stable at room
temperature.
[0320] The terms "stability" and "stable" as used herein in the
context of a formulation comprising a protein of interest (e.g., an
antibody) of the invention refer to the resistance of the protein
of interest 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 protein
of interest 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 a protein of interest of
the invention in PBS.
[0321] Therapeutic formulations of the low impurity compositions,
for example, low aggregate 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 protein of interest (e.g., antibody)
and the particular therapeutic effect to be achieved, and (b) the
limitations inherent in the art of compounding such a protein of
interest for the treatment of sensitivity in individuals.
[0322] Therapeutic compositions of the low impurity compositions,
for example, low aggregate 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 proteins of interest
(including fragments of the protein of interest) are formulated for
intravenous administration. In certain other embodiments, the
proteins of interest (e.g., antibodies), including fragments of the
proteins of interest (e.g., antibody fragments) are formulated for
local delivery to the cardiovascular system, for example, via
catheter, stent, wire, intramyocardial delivery, intrapericardial
delivery, or intraendocardial delivery.
[0323] Formulations of the low impurity compositions, for example,
low aggregate 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. 20040042972).
[0324] 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.
[0325] Actual dosage levels of the active ingredients in the
pharmaceutical compositions of the low impurity compositions, for
example, low aggregate 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.
[0326] In certain embodiments, the proteins of interest (e.g.,
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 proteins
of interest (e.g., antibodies) of the invention may be delivered
locally to the brain to mitigate the risk that the blood brain
barrier slows effective delivery.
[0327] In certain embodiments, the low impurity compositions, for
example, low aggregate compositions, of the invention may be
administered with medical devices known in the art. For example, in
certain embodiments a protein of interest (e.g., antibody) or a
fragment of protein of interest (e.g., 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.
[0328] The efficient dosages and the dosage regimens for the
reduced level of at least one impurity 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.
Alternative Formulations Containing the Low Impurity Compositions
of the Invention
Alternative Aqueous Formulations
[0329] The invention also provides a low impurity composition, for
example a low aggregate composition, formulated as an aqueous
formulation comprising a protein of interest and water, as
described in U.S. Pat. No. 8,420,081, the contents of which are
hereby incorporated by reference. In these aqueous formulations,
the protein of interest 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 of interest in water without the requirement for additional
excipients, increased concentrations of protein of interest without
the need for additional excipients to maintain solubility of the
protein of interest, and low osmolality. These also have
advantageous storage properties, as the proteins of interest 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 of interest concentrations and
repeated freeze/thaw processing steps. In one embodiment,
formulations described herein include high concentrations of
proteins of interest such that the aqueous formulation does not
show significant opalescence, aggregation, or precipitation.
[0330] In one embodiment, an aqueous low impurity composition
comprising a protein of interest, e.g., an antibody, 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 of
interest concentration of at least about 10 .mu.g/mL, an osmolality
of no more than about 30 mOsmol/kg, and/or the protein of interest
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.
[0331] 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.
[0332] In another embodiment, low impurity 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.
[0333] 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 of interest, and no ionic
excipients (e.g., salts, free amino acids).
[0334] In certain embodiments, the aqueous formulation as described
herein comprise a low impurity composition comprising a protein of
interest 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 of interest concentration,
e.g., the concentration of the protein of interest 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.
[0335] The concentration of the aqueous formulation as described
herein is not limited by the protein of interest 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 of
interest, 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 of interest 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.
[0336] The aqueous formulation as described herein may be
characterized by the hydrodynamic diameter (D.sub.h) of the
proteins of interest in solution. The hydrodynamic diameter of the
protein of interest 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 of interest
are notably lower than protein of interest 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, 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.
[0337] In one embodiment, the D.sub.h of the protein of interest in
the aqueous formulation is smaller relative to the D.sub.h of the
same protein of interest in a buffered solution, irrespective of
protein of interest concentration. Thus, in certain embodiments, a
protein of interest 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 of interest 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 of interest
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 of interest in
PBS in at the given concentration; at least 60% less than the
D.sub.h of the protein of interest in PBS at the given
concentration; at least 70% less than the D.sub.h of the protein of
interest in PBS at the given concentration; or more than 70% less
than the D.sub.h of the protein of interest 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%.
[0338] In one aspect, the aqueous formulation includes the protein
of interest at a dosage of about 0.01 mg/kg-10 mg/kg. In another
aspect, the dosages of the protein of interest 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.
Alternative Solid Unit Formulations
[0339] The invention also provides a low impurity composition of
the invention formulated as a stable composition of a protein of
interest, e.g., an antibody, or antigen binding portion thereof,
and a stabilizer, referred to herein as solid units, as described
in Attorney Docket No. 117813-31001, the contents of which are
hereby incorporated by reference herein.
[0340] Specifically, it has been discovered that despite having a
high proportion of sugar, the solid units comprising the low
impurity compositions 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 (e.g., the solid units comprising the low impurity
compositions of the invention do not require storage in a sealed
container) and maintain long-term physical and chemical stability
of the protein of interest without significant degradation and/or
aggregate formation. Moreover, despite having a high proportion of
sugar, the solid units comprising the low impurity compositions of
the invention remain free-flowing when stored under ambient
conditions, e.g., room temperature and humidity, for extended
periods of time, and yet are easily dissolved in an aqueous
solvent, e.g., water (e.g., the solid units require minimal mixing
when contacted with a solvent for reconstitution). Furthermore, the
solid units comprising the low impurity compositions of the
invention may be prepared directly in a device for patient use.
These properties, when compared to existing techniques which
require a vial containing a lyophilized protein of interest
provided as a cake (which may not stabilize a protein of interest
for extended periods of time), a separate vial for a diluent, one
or more sterile syringes, and several manipulation steps, thus
provides alternative approaches for reconstitution since the solid
units comprising the low impurity compositions of the invention may
be provided, e.g., in a dual chambered cartridge, to make
reconstitution invisible during patient delivery. Furthermore, the
solid units comprising the low impurity compositions of the
invention are versatile in that they can be readily and easily
adapted for numerous modes of administration, such as parenteral
and oral administration.
[0341] As used herein, the term "solid unit," refers to a
composition which is suitable for pharmaceutical administration and
comprises a protein of interest, e.g., an antibody or peptide, and
a stabilizer, e.g., a sugar. The solid unit comprising the low
impurity compositions of the invention has a structural rigidity
and resistance to changes in shape and/or volume. In one
embodiment, the solid unit comprising the low impurity compositions
of the invention is obtained by freeze-drying a pharmaceutical
formulation of a therapeutic protein of interest. The solid unit
comprising the low impurity compositions of the invention 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
.quadrature.l to about 20 .quadrature.l. In another embodiment, the
solid unit is not obtained using spray drying techniques, e.g., the
solid unit is not a powder or granule.
[0342] As used herein, the phrase "a plurality of solid units"
refers to a collection or population of solid units comprising the
low impurity compositions of the invention, wherein the collection
comprises two or more solid units having a substantially uniform
shape, e.g., sphere, and/or volume distribution. A substantially
uniform size distribution is intended to mean that the individual
shapes and/or volumes of the solid units comprising the low
impurity compositions of the invention are substantially similar
and not greater than a 10% standard deviation in volume. For
example, a plurality of solid units which are spherical in shape
would include a collection of solid units having no greater than
10% standard deviation from an average volume of the spheres. In
one embodiment, the plurality of solid units is free-flowing.
Kits and Articles of Manufacture Comprising the Low Impurity
Compositions of the Invention
[0343] Also within the scope of the present invention are kits
comprising the low impurity 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
protein of interest (e.g., 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 a protein of interest
(e.g., an antibody) of the invention.
[0344] 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 proteins of interest 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.
[0345] The invention also provides a pharmaceutical pack or kit
comprising one or more containers filled with a liquid formulation
or lyophilized formulation of a protein of interest (e.g., 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.
[0346] 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, US Patents 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.
[0347] In certain embodiments, kits comprising proteins of interest
(e.g., 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 protein of interest (e.g.,
antibody) of the invention. Additional containers may be included
that contain, e.g., diluents and buffers, control proteins of
interest (e.g., 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.
[0348] 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 a protein of interest (e.g., an antibody) that is
suitable for parenteral administration. In another embodiment, the
unit dosage form is provided as a sterile lyophilized powder
comprising a protein of interest (e.g., an antibody) that is
suitable for reconstitution.
[0349] 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.
[0350] 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.
[0351] 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 a protein of interest (e.g., an
antibody). The packaging material includes instruction means which
indicate how that said protein of interest (e.g., antibody) can be
used to prevent, treat and/or manage one or more symptoms
associated with a disease or disorder.
[0352] 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-TNFa 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; and 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.
EXAMPLES
General Materials and Methods
[0353] Except where noted, the materials and methods described in
connection with the instant example were also employed in Examples
1-3, below.
Chromatography Method
[0354] Pre-packed media columns were used in the following
experiments, except where specified. The column was equilibrated in
a buffer system with appropriate pH and conductivity. The column
load was prepared from Protein A affinity chromatography eluates or
concentrated CEX chromatography elutes by buffer exchange (if the
eluates were with different buffer components from the mixed mode
target buffer system) or addition of the stock solutions and/or
water to obtain the target pH and conductivity as specified (if the
eluates were with the same buffer components as the mixed mode
target buffer system). The prepared load material was filtered and
loaded on the column according to the target load amount (g
protein/L media) as specified followed by washing with the
equilibration buffer or buffer similar to equilibration buffer with
volumes as specified. The column Flow Through/Wash were collected
as fractions or as a pool. HIC column was cleaned with 20%
Isopropyl Alcohol solution. 1M NaOH solution was used for column
cleaning.
Buffer Preparation Method
[0355] Buffers were prepared targeting a specific salt
concentration in a buffered system, and titrating to a specific pH
with the conjugate acid or base. For example, an 800 mM Ammonium
Sulfate (AmSO.sub.4) pH 7.0 solution was made by dissolving AmSO4
salt in a 20 mM Tris-Acetate buffered solution, titrating with
acetate, and subsequently bringing up to volume with water to
achieve the desired AmSO.sub.4 concentration. Load samples were
prepared targeting a specific salt concentration by addition of
concentrated salt solution in a buffered system, and titrating to a
specific pH with the conjugate acid or base. For example, an 800 mM
AmSO4 pH 7.0 load was made by mixing the load in a 1:1 ratio with a
1600 mM AmSO4 pH 7.0 stock buffer in a 40 mM Tris-Acetate, and
subsequently titrating with Tris or acetate to achieve a final pH
7.0.
Size Exclusion Chromatography
[0356] The molecular weight distribution of collected samples were
quantified according to the following methods. Size exclusion
chromatography (SEC) was performed using a TSK-gel G3000SWxL, 5
.mu.m, 125 .ANG., 7.8.times.300 mm column (Tosoh Bioscience) on an
HP Agilent HPLC system. Injections were made under isocratic
elution conditions using a mobile phase of 200 mM sodium sulfate,
100 mM sodium phosphate, pH 6.8, and detected with absorbance at
214 nm. Quantification is based on the relative area of detected
peaks.
Host Cell Protein (HCP) ELISA
[0357] HCP assay is based on process specific antigen based ELISA.
Sample dilutions were applied to achieve readings within the
calibration range. The limit of quantitation of the assay is 0.625
ng/mL.
UV Spectroscopy A.sub.280
[0358] UV A280 was used to determine protein concentrations for the
samples post protein A elution. The assay was performed on an
Agilent UV Spectrophotometer following the method. The protein
concentration was determined using Beer-Lambert's Law,
A=.epsilon.lc, where A is Absorbance, c is the extinction
coefficient, 1 is the path length, and c is the concentration. The
absorbance was taken at 280 nm, the path length was 1 cm, and the
extinction coefficients were 1.39 for Adalimumab, 1.38 for mAb B,
and 1.43 for mAb C.
Example 1: Determining Operating Conditions Appropriate for an
MAB:Media:Buffer Combination
[0359] The demonstration of the current invention for a specific
antibody & media is provided in this example, and consists of:
1) Choosing a salt concentration that allows product and impurities
to bind at a given pH; 2) Loading a small amount of protein to the
column and then performing a linear gradient elution by decreasing
the salt concentration; 3) Determining salt concentration range in
which the protein elutes from the HIC media.
[0360] In this example, adalimumab and GE CaptoPhenyl were chosen.
The column was equilibrated at 1.1 M AmSO.sub.4 pH 7.0
(Tris/Acetate) for 10 CVs. Adalimumab was prepared at 1.1 M
AmSO.sub.4 and loaded to the column at 20 g-protein/L of resin. The
column was washed with 10 CVs of the equilibration buffer. A linear
gradient from 1.1M to 0M AmSO.sub.4 pH 7.0 (Tris/Acetate) over
20CVs was performed. The process chromatogram is shown in FIG.
4.
[0361] This process can be repeated for any given mAb-media
combination for a given buffer system. Table 1 shows the DOE
parameters determined using the method described above for
adlimumab in AmSO4 pH 7.0 (Tris/acetate) for 3 different HIC
adsorbents.
TABLE-US-00001 TABLE 1 Example Experimental Design Scope determined
from LGE with different resins Adlimumab - Ammonium Sulfate pH 7.0
(Tris/Acetate) Resin Buffer Concentration Range Tosoh Hexyl 250-750
mM GE CaptoPhenyl 300-650 mM GE Butyl FF 800-950 mM
[0362] In practicing the current invention, the aggregate reduction
desired can be achieved by appropriate pooling of the load and wash
fractions. By collecting and subsequently determining the product
quality of each fraction throughout the load and wash, the
accumulative aggregate reduction and accumulative yield can be
calculated using the weighted averages up to a given fraction.
Additionally, the instantaneous yield can be estimated by comparing
the protein recovered against the total protein loaded to the
column at a given fraction. Sample calculations are shown
below:
[0363] Sample Calculation A: Accumulative Yield Up to a Given
Fraction
Accumulative Yield = Accumulated Protein Mass Recovered up to
Fraction Total Mass Protein Load ##EQU00001##
[0364] Sample Calculation B: Accumulative Aggregate Reduction Up to
a Given Fraction
Accumulative Aggregate reduction = Load Agg % - Accumulated
Aggregate Mass Recovered up to Fraction Accumulated Total Protein
Mass Recovered up to Fraction ##EQU00002##
[0365] Sample Calculation C: Instantaneous Yield Up to a Given
Fraction
Instantaneous Yield = Accumulated Protein Mass Recovered up to
Fraction Total Protein Mass Loaded to Column at Fraction
##EQU00003##
[0366] The demonstration of the current invention for a specific
antibody & resin is provided in this example, and consists of
[0367] 1. For a given salt concentration and optionally pH and HIC
media. [0368] 2. Loading the HIC media in excess of the dynamic
binding capacity for the product for the given condition. [0369] 3.
Washing the column with a buffer containing a similar salt
concentration and optionally pH used for the equilibration and
loading steps. [0370] 4. Collecting fractions throughout the
loading and wash steps and subsequently determining the product
quality profile (e.g. Aggregate, HCP etc.)
[0371] In this example, adalimumab and GE CaptoPhenyl were chosen.
The experiment was performed at 400 mM sodium citrate (NaCit) pH
5.6. The column was equilibrated with 400 mM NaCit pH 5.6 for 10
CVs. Adalimumab was prepared at 400 mM NaCit pH 5.6 and loaded to
the column at 500 g-protein/L-resin. The column was washed with 7
CVs of the equilibration buffer. The process chromatogram is shown
in FIG. 5. Fractions were collected and analyzed for product
quality and the accumulative yield and accumulative aggregate
reduction calculated, shown in Table 2. From this example, it is
clear to one skilled in the art to determine a run condition which
delivers a targeted product quality and/or step yield.
[0372] This general approach is used to evaluate the performance
for a given operating condition for any resin/mAb/buffer
combination.
TABLE-US-00002 TABLE 2 Accumulative Yield and Aggregate Reduction
from FIG. 5 Fraction Load Accumulative Recovery Accumulative
.DELTA.Agg D1 8 g/L 0% 0.82% D2 45 g/L 4% 0.77% D3 82 g/L 12% 0.71%
D4 119 g/L 19% 0.67% D5 156 g/L 26% 0.62% D6 193 g/L 33% 0.56% D7
231 g/L 41% 0.51% E1 268 g/L 48% 0.47% E2 305 g/L 55% 0.43% E3 342
g/L 62% 0.40% E4 379 g/L 70% 0.37% E5 416 g/L 77% 0.34% E6 454 g/L
84% 0.32% E7 491 g/L 91% 0.29% F1 500 g/L 93% 0.29% F2 WASH 99%
0.28% F3 WASH 100% 0.28% F4 WASH 101% 0.29% F5 WASH 101% 0.29%
Example 2: Demonstration of Aggregate Reduction with HIC Resins
[0373] This data set is compiled to demonstrate the aggregate
reduction achieved with six different HIC adsorbents. Each resin
was evaluated with a 500 g/L load of adalimumab at a NaCit
concentration near, and slightly higher than, the peak elution
concentration determined from the process outlined in Example 1.
Table 3 outlines the results from these experiments.
TABLE-US-00003 TABLE 3 Effect of HIC Resins on Aggregate Reduction
of Adalimumab HIC Resin NaCit, pH 5.6 .DELTA.Agg Yield Butyl 400 mM
1.5% 99.8% 450 mM 1.2% 85.7% Hexyl 240 mM 1.2% 93.9% 300 mM 1.1%
100.9% Phenyl 400 mM 1.5% 96.5% 450 mM 1.2% 90.7% Octyl 350 mM 0.4%
98.5% 400 mM 0.1% 103.3% GE Butyl FF 550 mM 1.2% 88.1% 600 mM 1.7%
83.0% PPG 450 mM 0.2% 97.5% 600 mM 1.0% 38.1%
Example 3: Demonstration of Aggregate Reduction with Other
Antibodies, MAB B and MAB C
[0374] Aggregate reduction technology of the current invention has
been demonstrated with multiple antibodies using HIC adsorbents.
Antibodies have different hydrophobic properties, leading to
interaction behavior on a HIC column that differs from one antibody
to another. Therefore the impact of salt type and concentration is
different for each antibody.
[0375] Table 4 and Table 5, presented below, provide the data
obtained for mAb B and mAB C. The data clearly demonstrates that
the aggregate reduction technology is effective for alternatives to
adalimumab.
TABLE-US-00004 TABLE 4 Aggregate reduction for mAb B, pI ~9.1 HIC
Resin AmSO4, pH 5.0 .DELTA.Agg Yield Hexyl 370 mM 0.8% 100% 710 mM
0.6% 93% Phenyl 340 mM 0.6% 95% 790 mM 0.5% 95% Butyl 840 mM 0.6%
99% 1000 mM 0.6% 96%
TABLE-US-00005 TABLE 5 Aggregate reduction for mAb C, pI ~7.0 HIC
Resin AmSO4, pH 5.0 .DELTA.Agg Yield Hexyl 80 mM 5.0% 89.0% 330 mM
4.5% 99.8% Phenyl 130 mM 3.5% 92.8% 480 mM 2.9% 92.8% Butyl 690 mM
5.2% 93.5% 880 mM 5.4% 87.9%
Example 4: Demonstration of Aggregate Reduction with Different Salt
Concentrations--Adalimumab
[0376] Ion concentration is a key variable in the performance of
hydrophobic interaction chromatography. For every combination of
antibody/resin/pH there is a range of ion concentrations that
provide aggregate reduction; the strategy outlined in Example 1.
can be followed to determine the aggregate reduction and the
corresponding recovery for each salt concentration.
[0377] Table 6, below, shows the effect of salt concentration on
aggregate reduction and step yield. In this example CaptoPhenyl and
adalimumab were chosen, and evaluated at a loading of 200-500 g/L
in NaCit pH 5.6 at the concentration specified. The data
demonstrates that the aggregate reduction can be effectively
achieved over a range of salt concentrations, and that the salt
concentration and column loading can be balanced to achieve a
desired step yield and final product quality
TABLE-US-00006 TABLE 6 Effect of Ion Concentration on Aggregate
Reduction NaCit pH 5.6 Load Yield .DELTA.Agg 300 mM 200 g/L 92%
0.59% 350 g/L 96% 0.33% 500 g/L 97% 0.24% 400 mM 200 g/L 90% 0.76%
350 g/L 94% 0.43% 500 g/L 96% 0.35% 500 mM 200 g/L 85% 1.09% 350
g/L 91% 0.97% 500 g/L 94% 0.86%
Example 5: Demonstration of Aggregate Reduction with Different
Buffer Systems with Adalimumab
[0378] In addition to the salt concentration, the salt anion and
cation types are key variables in hydrophobic interaction
chromatography. The invention has been demonstrated with ammonium
sulfate, sodium sulfate, and sodium citrate. As one skilled in the
art would appreciate the optimal salt concentration and optionally
pH are different for each salt type and was derived by using the
strategy outlined in Example 1. Table 7 shows the data of aggregate
reduction and corresponding recovery for the different anion/cation
types and different HIC adsorbents.
TABLE-US-00007 TABLE 7 Effect of Anion/Cation Type Aggregate
Reduction Resin Buffer System Load Yield .DELTA.Agg CaptoPhenyl 630
mM AmSO4 pH 7.0 300 g/L 95% 2.1% 300 mM AmSO4 pH 7.0 300 g/L 99%
1.1% 425 mM NaSO4 pH 7.0 300 g/L 95% 1.9% 240 mM NaSO4 pH 7.0 300
g/L 101% 1.1% 500 mM NaCit pH 5.6 350 g/L 91% 1.0% 300 mM NaCit pH
5.6 350 g/L 96% 0.2% Tosoh Hexyl 725 mM AmSO4 pH 7.0 300 g/L 94%
1.7% 275 mM AmSO4 pH 7.0 300 g/L 103% 0.9% 460 mM NaSO4 pH 7.0 300
g/L 97% 0.7% 180 mM NaSO4 pH 7.0 300 g/L 101% 0.6% 440 mM NaCit pH
5.6 300 g/L 87% 0.5% 150 mM NaCit pH 5.6 300 g/L 97% 0.5% Butyl FF
800 mM AmSO4 pH 7.0 300 g/L 100% 0.7% 1000 mM AmSO4 pH 7.0 300 g/L
94% 1.6% 750 mM NaSO4 pH 7.0 300 g/L 96% 1.8% 700 mM NaSO4 pH 7.0
300 g/L 101% 1.7% 700 mM NaCit pH 5.6 300 g/L 98% 1.6% 600 mM NaCit
pH 5.6 300 g/L 95% 1.5%
Example 6: Demonstration of Aggregate Reduction with Different
Loading
[0379] Furthermore, the strategy outlined in Example 1. to reduce
aggregates through careful control of ion concentration, ion type,
HIC adsorbent, and pH can be applied to various ranges of protein
loading. Aggregate reduction for a range of protein loadings (e.g.
250-700 g/L) for CaptoPhenyl using a 400 mM NaCit pH 5.6 buffer is
shown in Table 8, displaying a robust aggregate reduction across an
expansive loading range.
TABLE-US-00008 TABLE 8 Impact of Column loading Load Yield
.DELTA.Agg .DELTA.Agg/LoadAgg 250 g/L 95% 0.29% 87% 500 g/L 100%
0.25% 77% 700 g/L 100% 0.21% 65%
Example 7: Demonstration of Aggregate Reduction with Different Load
Concentration--Adalimumab
[0380] In addition to the strategy outlined in Example 6. to reduce
aggregates through careful control of ion concentration, ion type,
and HIC adsorbent, it has been shown that the concentration of the
load protein can have an effect on aggregate reduction. In this
example, a feed stream was serial diluted to cover a range of load
concentrations from 4 to 15 mg/mL and loaded at 500 g/L to a
CaptoPhenyl column in 400 mM NaCit pH 5.6. The effect of decreasing
the concentration of the load protein is shown in FIG. 6.
Example 8: Demonstration of HCP Reduction in Addition to Aggregate
Reduction
[0381] HIC chromatography can also be effective in reducing host
cell protein (HCP) levels. In the present invention, it has been
demonstrated that HCP levels can be effectively reduced under
operating conditions selected for aggregate reduction.
[0382] Table 9 shows HCP removal achieved along with aggregate
reduction. The data clearly shows that other process related
substances/impurities can be achieved using the current invention
on the HIC adsorbents, and hence functions as an effective
polishing step in the large scale purification of monoclonal
antibodies.
TABLE-US-00009 TABLE 9 HCP Removal during HIC Chromatography HCP
NaCit pH 5.6 Load Yield .DELTA.Agg Load Pool 300 mM 200 g/L 92%
0.59% 1398 ng/mg NA 350 g/L 96% 0.33% 150 ng/mg 500 g/L 97% 0.24%
348 ng/mg 200 g/L 99% 0.34% 38 ng/mg 5 ng/mg 400 mM 200 g/L 90%
0.76% 1599 ng/mg 104 ng/mg 350 g/L 94% 0.43% 148 ng/mg 500 g/L 96%
0.35% 350 ng/mg 350 g/L 97% 0.35% 38 ng/mg 6 ng/mg 500 mM 200 g/L
85% 1.09% 1528 ng/mg 169 ng/mg 350 g/L 91% 0.97% 203 ng/mg 500 g/L
94% 0.86% 301 ng/mg 500 g/L 87% 0.35% 38 ng/mg 11 ng/mg
Example 9: Demonstration of Impact of Dynamic and Equilibrium
Binding
[0383] In the HIC-based separation strategies described herein, the
measured dynamic binding capacity (DBC), which is conventionally
measured at 10% breakthrough, was found to be greater than the
amount of protein that remained bound after washing the column
(a.k.a equilibrium binding capacity, EBC) with a buffer with
similar pH and salt concentration to the equilibration and load
conditions. For example, but not by way of limitation, FIG. 10
shows an example of the DBC and EBC for the data presented in FIG.
5. In addition, Table 10 shows effect of salt type, concentration,
and HIC resin on DBC and EBC values for Adalimumab.
TABLE-US-00010 TABLE 10 Comparison of DBC and EBC values for
Adilmumab Resin Buffer System .DELTA.Agg DBC EBC CaptoPhenyl 630 mM
AmSO4 pH 7.0 2.1% 27 g/L 16 g/L 300 mM AmSO4 pH 7.0 1.1% 6 g/L 4
g/L 425 mM NaSO4 pH 7.0 1.9% 22 g/L 15 g/L 240 mM NaSO4 pH 7.0 1.1%
6 g/L 4 g/L Butyl FF 1000 mM AmSO4 pH 7.0 1.6% 17 g/L 11 g/L 800 mM
AmSO4 pH 7.0 0.7% 4 g/L 4 g/L 750 mM NaSO4 pH 7.0 1.8% 29 g/L 13
g/L 700 mM NaSO4 pH 7.0 1.7% 22 g/L 11 g/L 700 mM NaCit pH 5.6 1.6%
39 g/L 24 g/L 600 mM NaCit pH 5.6 1.5% 17 g/L 11 g/L
Example HIC 10: Combinations of Hic with Alternative Separation
Strategies
[0384] The methods described herein for reducing aggregates using
HIC can be used as an independent operation or in combination with
other process steps that provide additional aggregate reduction or
those providing additional complementary and supplementary
purification. Data for specific separation strategies is provided
in Tables 11 and 12. For example, but not by way of limitation, the
following process combinations can be used:
[0385] 1. Affinity.fwdarw.HIC
[0386] 2. Affinity.fwdarw.AEX.fwdarw.HIC
[0387] 3. Affinity.fwdarw.Mixed Mode.fwdarw.HIC
TABLE-US-00011 TABLE 11 Aggregate reduction with different source
materials HCP Load Source Buffer Condition Load Yield .DELTA.Agg
LRF Protein A 400 mM NaCit pH 5.6 500 g/L 96% 1.49% NA Eluate 450
mM NaCit pH 5.6 500 g/L 91% 1.22% NA ProteinA/ 300 mM NaCit pH 5.6
200 g/L 92% 0.59% 1.0 AEX FTW 400 mM NaCit pH 5.6 350 g/L 94% 0.43%
1.0 500 mM NaCit pH 5.6 500 g/L 94% 0.86% 0.7 ProteinA/ 300 mM
NaCit pH 5.6 200 g/L 99% 0.34% 0.8 Mixed Mode 400 mM NaCit pH 5.6
350 g/L 97% 0.35% 0.8 FTW 500 mM NaCit pH 5.6 500 g/L 87% 0.35%
0.5
TABLE-US-00012 TABLE 12 Complete Process Train with Protein A
Capture - AR, HMW and HCP reduction % HMW Process Yield (%)
reduction HCP LRF Clarified Harvest 97.00% n/a n/a Prt-A Eluate
Pool 69.60% n/a 1.87 Viral Inactivated 99.70% 0.07 0.39 Filtrate MM
FT pool 91.90% 0.83 1.63 HIC FT-pool 98.50% 0.23 0.46 VF(FT)
Filtrate 96.10% No reduction 0.1 BDS (FT) 103.80% No reduction
0.13
Example 11: Hybrid HIC Binding Mechanism
[0388] By estimating the partitioning coefficient K.sub.p, it can
be demonstrated that certain strategies described in the instant
application do not fall under the category of "Weak-Partitioning
(WP)" or "Flow-Through Overload (FT)" modes as those are described
in the art, e.g., US2007/0060741. For example, FIGS. 13A-13B depict
the results of experiments wherein aliquots of resin are incubated
with a load covering a range of protein concentrations at room
temperature for 3 hours, after which the protein solution is then
removed, and replaced with equilibration buffer (Wash simulation)
and incubated at room temperature for 3 hours (repeated, Wash II).
After each incubation, the concentration of the protein solution is
measured and used to calculated the amount of protein ((A) monomer
D2E7, a.k.a. Adalimumab, and (B) aggregate D2E7) bound to the resin
(g protein/L resin) and plotted against the concentration of the
protein solution at the end of the incubation (e.g. equilibrium).
FIGS. 14A-14B depict the results outlined in FIGS. 13A-13B,
highlighting the fact that at initial equilibrium a significant
amount of monomer/aggregate is bound to the resin. However, after
the protein solution is replaced with equilibration buffer (see
arrow), the monomer de-sorbs from the resin and back into solution,
whereas the aggregate remains bound.
[0389] FIGS. 15A-15B depict a determination of the binding monomer
and aggregate D2E7 (based on data provided in FIGS. 13A-13B) by
fitting the experimental equilibrium binding data to the Langmuir
Isotherm using the equation: q=(q.sub.max.times.C.sub.equil)
(K.sub.a+C.sub.equil); where q=amount of protein bound to resin [=]
g/L-resin; q.sub.max=maximum amount of protein bound to resin [=]
g/L-resin; C.sub.equil=solution concentration of protein [=]
g/L-soln; and K.sub.d=equilibrium dissociation constant.
[0390] By fitting the experimental data, the q.sub.max and K.sub.d
for the monomer and the aggregates can be calculated.
Species _ Q max [ mg / mL ] _ K d [ mg / mL ] _ _ Monomer 41.9 0.47
Aggregate 6.0 0.01 ##EQU00004##
[0391] Significantly, q.sub.max for both monomer/aggregate and the
K.sub.d values (i.e. strength of binding) are similar to those of
strong hydrophobic interactions, therefore it is not expected for
this interaction to be "reversible." In addition, by calculating
K.sub.p where:
Species _ Q max [ mg / mL ] _ K d [ mg / mL ] _ K p .ident. Q C
.apprxeq. Q max K d _ Monomer 41.9 0.47 90 Aggregate 6.0 0.01 600
##EQU00005##
it is apparent that the instant technique does not fall within the
category of flow-through (where K.sub.p.ltoreq.1) or weak
portioning (where K.sub.p=1-10), but rather fall within the
category of bind-elute (where K.sub.p.gtoreq.10).
Example 12: Determination of Binding Capacity at Saturation
[0392] The following protocol exemplifies the determination of (i)
apparent binding capacity, i.e., the binding capacity at saturation
(when outlet protein concentration equals inlet protein
concentration) under flow conditions and (ii) the actual binding
capacity, i.e., the amount of protein that remains bound after an
isocratic wash.
[0393] A column packed with resin containing a hydrophobic
interaction ligand was equilibrated with a buffer at a given salt
concentration and pH. A protein load in the same buffer condition
as the equilibration solution was loaded to the column until the
protein breaks through the column, and the protein concentration at
the effluent of the column was equal to the protein concentration
at the inlet of the column (i.e., saturated). The column was then
washed with the equilibration solution until the protein
concentration at the effluent was effectively zero. The remaining
protein bound to the column was then eluted with a buffer condition
that will cause the protein to desorb from the resin.
[0394] Taking into account the void volume of the column and
chromatography system, one can calculate the amount of protein
bound to the column at the saturation point by integrating the area
above the breakthrough curve at the effluent of the column (FIGS.
12A-12B). After the isocratic wash, one can calculate the protein
that remained bound to the resin by integrating the area under the
curve of the elution peak.
[0395] The differences between these two values is the `reversible`
binding capacity, which is significant when compared to the binding
capacity observed at the saturation point (e.g., "apparent binding
capacity"). This difference is also a function of the salt
concentration, which is shown in FIG. 16. FIG. 16 is a comparison
of apparent and actual bound protein under flow conditions. Binding
of the antibody during loading is significant (>10 g/l). The
majority (>65%) of the antibody monomer bound during load
desorbs during the isocratic wash (i.e., reversibly bound). The
mass balance of the impurity demonstrates irreversible binding.
Example 13: Determination of Binding Capacity at Saturation
[0396] A column was conditioned and loaded, as described in Example
12, at different inlet protein concentrations. In these
experiments, the flow-through fractionated to determine the product
quality at different times during the loading and breakthrough.
Using the protein mass and product quality for each of the
fractions, the accumulative impurity (e.g., aggregate) breakthrough
can be calculated using the weighted average:
Accumulative % Aggregates = ( Sum of total aggregate mass up to
fraction i ) ( Sum of total protein mass up to fraction i )
##EQU00006##
[0397] This calculation can be plotted for each successive fraction
(FIG. 8A) and used to compare different loading conditions. The
Equilibrium Binding Isotherms for both the monomer and aggregate
show that for all of the loading conditions (FIGS. 8B-8C), the
monomer was in the non-linear part of its binding isotherm (e.g.,
equilibrium binding capacity is independent of monomer
concentration), and the aggregate was in or near the linear part of
its binding isotherm (e.g., equilibrium binding capacity is
dependent on aggregate concentration). The clearance of the
aggregate improves by decreasing the overall load protein
concentration, even though this results in the resin having a lower
binding capacity for the aggregate.
[0398] In FIG. 7, material from a single source was serially
diluted to three different protein concentrations and subsequently
loaded to a column to 500 g/L. This data clearly demonstrates that
diluting the load material resulted in a better aggregate
clearance, even though the same amount of impurity was loaded in
each case. This is non-intuitive, especially when considering that
diluting the load protein concentration results in a lower overall
binding capacity for the impurity as the impurity is in the linear
range of the equilibrium binding isotherm and therefore the binding
capacity decreases linearly with concentration.
[0399] FIG. 9 is a re-plot of the same data as in FIG. 7 to
demonstrate that, for a given target impurity clearance, the
recovery-yield can be modulated by diluting the load material to a
specific range.
Sequence CWU 1
1
121107PRTArtificial Sequenceadalimumab 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 Pro65 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
Sequenceadalimumab 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 Tyr65 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 Sequenceadalimumab light chain variable
region CDR3VARIANT9Xaa = Thr or Ala 3Gln Arg Tyr Asn Arg Ala Pro
Tyr Xaa1 5 412PRTArtificial Sequenceadalimumab heavy chain variable
region CDR3VARIANT12Xaa = Tyr or Asn 4Val Ser Tyr Leu Ser Thr Ala
Ser Ser Leu Asp Xaa 1 5 10 57PRTArtificial Sequenceadalimumab light
chain variable region CDR2 5Ala Ala Ser Thr Leu Gln Ser1 5
617PRTArtificial Sequenceadalimumab 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 Gly711PRTArtificial Sequenceadalimumab light chain
variable region CDR1 7Arg Ala Ser Gln Gly Ile Arg Asn Tyr Leu Ala 1
5 10 85PRTArtificial Sequenceadalimumab heavy chain variable region
CDR1 8Asp Tyr Ala Met His1 5 9321DNAArtificial Sequenceadalimumab
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
Sequenceadalimumab 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 Sequenceadalimumab light chain 11Asp Ile Gln
Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15Asp
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 Pro65 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 Gln145 150 155
160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys
Val Tyr 180 185 190Ala 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 Sequenceadalimumab 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
* * * * *