U.S. patent application number 17/268719 was filed with the patent office on 2021-12-09 for method and chromatography system for determining amount and purity of a multimeric protein.
This patent application is currently assigned to REGENERON PHARMACEUTICALS, INC.. The applicant listed for this patent is REGENERON PHARMACEUTICALS, INC.. Invention is credited to Hanne BAK, Michael PERRONE, Audrey RODRIGUEZ, Andrew TUSTINA.
Application Number | 20210382065 17/268719 |
Document ID | / |
Family ID | 1000005822046 |
Filed Date | 2021-12-09 |
United States Patent
Application |
20210382065 |
Kind Code |
A1 |
PERRONE; Michael ; et
al. |
December 9, 2021 |
METHOD AND CHROMATOGRAPHY SYSTEM FOR DETERMINING AMOUNT AND PURITY
OF A MULTIMERIC PROTEIN
Abstract
The invention relates to a chromatography system and method for
assessing amount and/or purity of a multimeric protein in a sample,
wherein the chromatography system comprises two different affinity
chromatography matrices connected via a switch valve.
Inventors: |
PERRONE; Michael;
(Brookfield, CT) ; RODRIGUEZ; Audrey; (New York,
NY) ; TUSTINA; Andrew; (Millwood, NY) ; BAK;
Hanne; (New York, NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
REGENERON PHARMACEUTICALS, INC. |
Tarrytown |
NY |
US |
|
|
Assignee: |
REGENERON PHARMACEUTICALS,
INC.
Tarrytown
NY
|
Family ID: |
1000005822046 |
Appl. No.: |
17/268719 |
Filed: |
August 16, 2019 |
PCT Filed: |
August 16, 2019 |
PCT NO: |
PCT/US2019/046769 |
371 Date: |
February 16, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62719323 |
Aug 17, 2018 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 16/00 20130101;
C07K 1/22 20130101; G01N 2030/027 20130101; G01N 30/8624 20130101;
G01N 30/14 20130101; G01N 33/6854 20130101; C07K 2317/31
20130101 |
International
Class: |
G01N 33/68 20060101
G01N033/68; C07K 1/22 20060101 C07K001/22; C07K 16/00 20060101
C07K016/00; G01N 30/14 20060101 G01N030/14; G01N 30/86 20060101
G01N030/86 |
Claims
1. A method for quantifying an amount of a heterodimeric antibody
in a sample comprising a mixture of the heterodimeric antibody, a
first homodimeric antibody impurity, and a second homodimeric
antibody impurity, wherein the heterodimeric antibody and the first
impurity bind to a first affinity matrix and the second impurity
does not substantially bind to the first affinity matrix and binds
to a second affinity matrix, and wherein the heterodimeric antibody
has a lower affinity to the first affinity matrix than the first
homodimeric antibody impurity, said method comprising the steps of:
a. applying the sample to a chromatography system comprising the
first affinity matrix, the second affinity matrix, and a detector,
wherein the first affinity matrix is serially connected to the
second affinity matrix via a switch valve; b. eluting the second
impurity from the first affinity matrix onto the second affinity
matrix under a first set of conditions; c. switching the switch
valve to bypass the second affinity matrix, eluting the
heterodimeric antibody through the detector under a second set of
conditions, and determining the amount of the eluted heterodimeric
antibody; d. eluting the first impurity through the detector under
a third set of conditions and determining the amount of the eluted
first impurity; e. eluting the second impurity from the second
affinity matrix through the detector under the third set of
conditions, and determining the amount of the eluted second
impurity, and f. quantifying the amount of the heterodimeric
antibody in the sample.
2. The method of claim 1, wherein the heterodimeric antibody
comprises a first immunoglobulin CH3 domain and a second
immunoglobulin CH3 domain, wherein said first and second
immunoglobulin CH3 domains are different in their affinity to the
first affinity matrix, and wherein the sample comprises a mixture
comprising said heterodimeric antibody, a homodimeric antibody
comprising two first CH3 domains, and a homodimeric antibody
comprising two second CH3 domains.
3. The method of claim 2, wherein the second CH3 domain comprises
H435R and Y436F amino acid substitutions.
4. The method of claim 1, wherein the heterodimeric antibody is a
bispecific antibody.
5. The method of any claim 1, wherein the first affinity matrix
comprises protein A and the second affinity matrix comprises
protein G.
6. The method of claim 1, wherein the first set of conditions
comprises a first pH, the second set of conditions comprises a
second pH, and the third set of conditions comprises a third
pH.
7. The method of claim 6, wherein the second pH is lower than the
first pH, and the third pH is lower than the second pH.
8. The method of claim 6, wherein the first pH is from about pH 5.0
to about pH 7.4, the second pH is from about pH 4.3 to about pH 5.6
and the third pH is from about pH 2.0 to about pH 2.8.
9. The method of claim 1, wherein the first set of conditions, the
second set of conditions, and the third set of conditions comprise
a mobile phase modifier.
10. The method of claim 9, wherein the mobile phase modifier is a
salt buffer selected from LiCl, NaCl, KCl, MgCl.sub.2, and
CaCl.sub.2) buffer.
11. A method for quantifying an amount of a heterodimeric antibody
in a sample comprising a mixture of the heterodimeric antibody, a
first homodimeric antibody, and a second homodimeric antibody,
wherein the heterodimeric antibody and the first homodimeric
antibody bind to a protein A matrix and the second homodimeric
antibody does not substantially bind to the protein A matrix and
binds to a protein G matrix, said method comprising the steps of:
a. applying the sample to a chromatography system comprising the
protein A matrix, the protein G matrix, and a detector, wherein the
protein A matrix is serially connected to the protein G matrix via
a switch valve; b. eluting the second homodimeric antibody from the
protein A matrix onto the protein G matrix under a first set of
conditions; c. switching the switch valve to bypass the protein G
matrix, eluting the heterodimeric antibody through the detector
under a second set of conditions, and determining the amount of the
eluted heterodimeric antibody; d. eluting the first homodimeric
antibody through the detector under a third set of conditions and
determining the amount of the eluted first homodimeric antibody; e.
eluting the second homodimeric antibody from the protein G matrix
through the detector under the third set of conditions, and
determining the amount of the eluted second homodimeric antibody,
and f. quantifying the amount of the heterodimeric antibody in the
sample.
12. The method of claim 11, wherein the heterodimeric antibody
comprises FcFc*, the first homodimeric antibody comprises FcFc, and
the second homodimeric antibody comprises Fc*Fc*.
13. A chromatography system comprising a first affinity matrix, a
second affinity matrix, and a detector, wherein each of the first
affinity matrix, the second affinity matrix and the detector are
connected via a switch valve.
14. A chromatography system comprising (i) a protein A
chromatography column, (ii) a protein G chromatography column, and
(iii) a detector comprising an HPLC column equipped with a UV
detector, a charge aerosol detector, and/or a mass-spectrometer,
wherein each of the protein A chromatography column, the protein G
chromatography column and to the detector are connected via a
switch valve.
Description
FIELD OF INVENTION
[0001] The present invention relates to a chromatography system and
method for assessing an amount and/or purity of a multimeric
protein in a sample, wherein the chromatography system comprises
two different affinity chromatography matrices connected via a
switch valve.
BACKGROUND
[0002] Bispecific antibodies are antibodies that can simultaneously
and selectively bind to two different types of epitopes on the same
or different antigens. The binding of multiple targets with a
single molecule is an attractive therapeutic concept, especially in
the fields of oncology and autoimmune disease. The most widely used
application is in cancer immunotherapy, where bispecific antibodies
are engineered to simultaneously bind a cytotoxic cell and a target
such as a tumor cell to be destroyed. Additionally, targeting more
than one molecule can be useful to circumvent the regulation of
parallel pathways and avoid resistance to the treatment. Binding or
blocking multiple targets in a pathway can be beneficial to
stopping disease, as most conditions have complicated multifaceted
effects throughout the body.
[0003] Multiple bispecific antibody formats have been proposed and
are currently under development. One such format is based upon a
standard fully human IgG antibody having an improved
pharmacokinetic profile and minimal immunogenicity (see U.S. Pat.
No. 8,586,713, and WO2016/018740), shown schematically in FIG. 1. A
single common light chain and two distinct heavy chains combine to
form such bispecific. One of the heavy chains contains a
substituted Fc sequence (hereinafter "Fc*") that greatly reduces
binding of the Fc* to Protein A due to H435R/Y436F (by EU numbering
system; H95R/Y96F by IMGT exon numbering system) substitutions in
the CH3 domain. As a result of co-expression of the Fc* and Fc
heavy chains and the common light chain, three products are
created: two of which (FcFc and Fc*Fc*) are homodimeric with
respect to the heavy chains, and one of which (FcFc*) is the
desired heterodimeric bispecific product. The Fc* sequence allows
selective purification of the FcFc* bispecific product on
commercially available affinity chromatography columns, due to
intermediate binding affinity for Fc-binding proteins, such as
Protein A, compared to the high affinity FcFc heavy chain
homodimer, or the weakly binding Fc*Fc* homodimer.
[0004] Another antibody format is a so-called "one-arm" antibody
described in WO2013/166604, shown schematically in FIG. 2A. This
heterodimeric antibody consists, e.g., of two distinct heavy chains
and only one light chain. In one such example, the heavy chain
coupled to the light chain contains an Fc* sequence, while the
heavy chain without a light chain contains the regular Fc sequence.
The initial reaction mixture thus contains three products, two
homodimeric (FcFc and Fc*Fc*) and the desired heterodimeric FcFc*
product, which can be separated using affinity chromatography.
[0005] Yet another possible antibody format is an antibody
construct with a C-terminal single-chain variable fragment (ScFv),
shown schematically in FIG. 2B. These antibodies may be
monospecific or bispecific, and comprise a single common light
chain and two distinct heavy chains. In one example, one of the
heavy chain has the Fc* sequence and is coupled to a ScFv, while
the other heavy chain has the native Fc sequence and no ScFv. In
another example, the heavy chain with the ScFv construct has the
native Fc sequence, and the Fc* heavy chain does not have ScFv.
Additional examples, where an additional mutation that abrogates
binding to Affinity Columns is a mutation to the Heavy chain
Variable Region (VH) on the same chain with the Fc* mutation, are
provided in e.g. U.S. Pat. No. 9,493,563 (mutations in VH3 and Fc
described as IMGT 3, 5, 7, 20, 22, 26, 27, 79, 81, 84, 84.2, 85.1,
86, 90), which is incorporated by reference in its entirety. As in
the above examples, the three-component mixture of FcFc*
heterodimer and the FcFc and Fc*Fc* homodimers can be separated
using the differential binding affinity chromatography.
[0006] There is a need in the field of commercial scale production
of bispecific antibodies to assess the relative and absolute amount
and purity of the heterodimer in various stages of antibody
production and purification. For this, effective resolution between
the heterodimer, and the two homodimer impurities is desired.
Moreover, speed and efficiency of quality control measurements are
desired during the cell culture process as well as the purification
process of antibodies. The present invention addresses this and
other needs by providing a novel chromatography system and
method.
[0007] The foregoing discussion is presented solely to provide a
better understanding of nature of the problems confronting the art
and should not be construed in any way as an admission as to prior
art nor should the citation of any reference herein be construed as
an admission that such reference constitutes "prior art" to the
instant application.
SUMMARY OF THE INVENTION
[0008] Various non-limiting aspects and embodiments of the
invention are described below.
[0009] The present invention describes a novel chromatography
system comprising a switch valve and a method of quantitatively
assessing the amount and/or purity of the heterodimer fraction in a
sample.
[0010] In one aspect, the present invention provides a method for
quantifying an amount and/or purity of a protein in a sample
comprising a mixture of the protein, a first protein impurity, and
a second protein impurity, wherein the protein and the first
impurity bind to a first affinity matrix and the second impurity
does not substantially bind to the first affinity matrix and binds
to a second affinity matrix, said method comprising the steps
of:
[0011] a. applying the sample to a chromatography system comprising
the first affinity matrix, the second affinity matrix, and a
detector, wherein the first affinity matrix is serially connected
to the second affinity matrix via a switch valve;
[0012] b. eluting the second impurity from the first affinity
matrix onto the second affinity matrix under a first set of
conditions;
[0013] c. switching the switch valve to bypass the second affinity
matrix, eluting the protein through the detector under a second set
of conditions, and determining the amount of the eluted
protein;
[0014] d. eluting the first impurity through the detector under a
third set of conditions and determining the amount of the eluted
first impurity;
[0015] e. eluting the second impurity from the second affinity
matrix through the detector under the third set of conditions, and
determining the amount of the eluted second impurity, and
[0016] f. quantifying the amount and/or purity of the protein in
the sample.
[0017] In one embodiment the protein is a multimeric protein, e.g.
an antibody. In one embodiment, the protein is an antibody of
interest, and the first and second protein impurities are
multimeric proteins, e.g., antibodies that may or may not be
structurally related to the antibody of interest. In one
embodiment, the protein is a bispecific antibody, i.e., a
heterodimeric protein, the first protein impurity is a first
homodimeric protein, and the second protein impurity is a second
homodimeric protein. In some cases, the mixture of multimeric
proteins is produced by a plurality of eukaryotic cells, such as,
for example, Chinese hamster ovary (CHO) cells in a cell
culture.
[0018] In one embodiment, the protein has a lower affinity to the
first affinity matrix than the first impurity. In one embodiment
the protein is a heterodimeric protein, the first protein impurity
is a first homodimeric protein, and the second protein impurity is
a second homodimeric protein, the heterodimeric protein and the
first homodimeric protein bind to the first affinity matrix and the
second homodimeric protein does not substantially bind to the first
affinity matrix and binds to the second affinity matrix.
[0019] In one embodiment, the protein comprises a first
immunoglobulin CH3 domain and a second immunoglobulin CH3 domain,
wherein said first and second immunoglobulin CH3 domains are
different in their affinity to the first affinity matrix, and
wherein the sample comprises a mixture comprising said protein, a
protein comprising two first CH3 domains, and a protein comprising
two second CH3 domains.
[0020] In one embodiment, the second CH3 domain comprises H435R and
Y436F (by EU numbering system; H95R/Y96F by IMGT exon numbering
system) amino acid substitutions. In another embodiment, the second
CH3 domain comprises an H435R (by EU numbering system; H95R by IMGT
exon numbering system) amino acid substitution. In some
embodiments, the second CH3 domain comprising an H435R (by EU
numbering system; H95R by IMGT exon numbering system) amino acid
substitution and exhibits weak or no detectable binding to
Fc-binding ligands, such as protein A, protein G, protein L, or
derivatives thereof.
[0021] In one embodiment, the protein is an antibody. In one
embodiment, the protein is a bispecific antibody.
[0022] In one embodiment, the first affinity matrix comprises
protein A and the second affinity matrix comprises protein G.
[0023] In one embodiment, the first set of conditions comprises a
first pH, the second set of conditions comprises a second pH, and
the third set of conditions comprises a third pH.
[0024] In one embodiment, the second pH is lower than the first pH,
and the third pH is lower than the second pH. In one embodiment,
the first pH is from about pH 5.0 to about pH 7.4, the second pH is
from about pH 4.3 to about pH 5.6, and the third pH is from about
pH 2.0 to about pH 2.8.
[0025] In one embodiment, the first set of conditions, the second
set of conditions, and the third set of conditions comprise a
mobile phase modifier. In one embodiment, the mobile phase modifier
is a salt buffer selected from LiCl, NaCl, KCl, MgCl.sub.2, and
CaCl.sub.2) buffer.
[0026] In another aspect, a method for quantifying an amount and/or
purity of a heterodimeric protein in a sample is provided,
comprising a mixture of the heterodimeric protein, a first
homodimeric protein, and a second homodimeric protein, wherein the
heterodimeric protein and the first homodimeric protein bind to a
first affinity matrix and the second homodimeric protein does not
substantially bind to the first affinity matrix and binds to a
second affinity matrix matrix, said method comprising the steps
of:
a. applying the sample to a chromatography system comprising the
first affinity matrix, the second affinity matrix, and a detector,
wherein the first affinity matrix is serially connected to the
second affinity matrix via a switch valve; b. eluting the second
homodimeric protein from the first affinity matrix onto the second
affinity matrix under a first set of conditions; c. switching the
switch valve to bypass the second affinity matrix, eluting the
heterodimeric protein through the detector under a second set of
conditions, and determining the amount of the eluted protein; d.
eluting the first homodimeric protein through the detector under a
third set of conditions and determining the amount of the eluted
first impurity; e. eluting the second homodimeric protein from the
second affinity matrix through the detector under the third set of
conditions, and determining the amount of the eluted second
impurity, and f. quantifying the amount and/or purity of the
protein in the sample.
[0027] In still another aspect, a method for quantifying an amount
and/or purity of a heterodimeric protein in a sample is provided,
comprising a mixture of the heterodimeric protein, a first
homodimeric protein, and a second homodimeric protein, wherein the
heterodimeric protein and the first homodimeric protein bind to a
protein A matrix and the second homodimeric protein does not
substantially bind to the protein A matrix and binds to a protein G
matrix, said method comprising the steps of:
a. applying the sample to a chromatography system comprising the
protein A matrix, the protein G matrix, and a detector, wherein the
protein A matrix is serially connected to the protein G matrix via
a switch valve; b. eluting the second homodimeric protein from the
protein A matrix onto the protein G matrix under a first set of
conditions; c. switching the switch valve to bypass the protein G
matrix, eluting the heterodimeric protein through the detector
under a second set of conditions, and determining the amount of the
eluted protein; d. eluting the first homodimeric protein through
the detector under a third set of conditions and determining the
amount of the eluted first impurity; e. eluting the second
homodimeric protein from the protein G affinity matrix through the
detector under the third set of conditions, and determining the
amount of the eluted second impurity, and f. quantifying the amount
and/or purity of the protein in the sample.
[0028] In one embodiment, the heterodimeric protein comprises
FcFc*, the first homodimeric protein comprises FcFc, and the second
homodimeric protein comprises Fc*Fc*.
[0029] In another aspect, a chromatography system comprising a
first affinity matrix, a second affinity matrix, and a detector is
provided, wherein each of the first affinity matrix, the second
affinity matrix and the detector are connected via a switch
valve.
[0030] In another aspect, a chromatography system is provided
comprising (i) a protein A chromatography column, (ii) a protein G
chromatography column, and (iii) a detector comprising an HPLC
column equipped with a UV detector, a charge aerosol detector,
and/or a mass-spectrometer, wherein each of the protein A
chromatography column, the protein G chromatography column and to
the detector are connected via a switch valve.
[0031] These and other aspects of the present invention will become
apparent to those skilled in the art after a reading of the
following detailed description of the invention, including the
appended claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0032] FIG. 1 is a schematic representation of a bispecific
antibody format suitable for separating with the method of the
invention based upon a standard fully human IgG antibody having
single common light chain and two distinct heavy chains, one
comprising an Fc* mutation and one with a native (Fc) sequence.
Note one representative example is drawn, and that the Fc* mutation
may be incorporated into either the first or the second heavy
chain.
[0033] FIGS. 2A and 2B show schematic representations of two
additional antibody formats suitable for separation according to
embodiments of the invention: FIG. 2(A) shows a heterodimeric
"one-arm" antibody, which consists of two distinct heavy chains and
only one light chain, where one heavy chain does not contain a Fab
fragment (e.g. contains only the heavy chain constant domain). FIG.
2(B) shows two exemplary antibody constructs with a C-terminal
single-chain variable fragment (ScFv). The Fc* mutation may be
incorporated into either the first or the second heavy chain
(constant domain) polypeptide.
[0034] FIG. 3 depicts a titer chromatogram illustrating separation
of a mixture of a bispecific antibody and monomeric impurities
according to the method of the disclosure and utilizing the system
according to an embodiment of the disclosure.
[0035] FIGS. 4(A) and 4(B) depict schematic representations of
exemplary chromatography systems according to an embodiment of the
disclosure. In one exemplary system, shown in FIG. 4(A), an
autosampler is connected to a first affinity matrix (column), which
is connected to a second affinity matrix (column) and a detector
via a switch valve. In the shown configuration, the switch valve is
positioned to serially connect the first column with the second
column, and the eluent flows through the first column onto the
second column, and subsequently through the detector for
quantitation. In another exemplary system, shown in FIG. 4(B), an
autosampler is connected to a first affinity matrix (column), which
is connected to a second affinity matrix (column) via a switch
valve. The detector is connected via switch valve in a bypass (no
column) configuration. Herein, the switch valve either connects the
first column to the second column, which is further connected to
the detector, or bypasses the second column and connects the first
column directly to the detector.
[0036] FIG. 5 shows a bispecific antibody titer/purity setup
according to an embodiment of the disclosure. The schematic
representation depicts a solvent degasser, a solvent manager, a
sample manager, a column compartment manager, and a detector.
[0037] FIG. 6 depicts an exemplary chromatography system according
to an embodiment of the disclosure. In this exemplary column
compartment of the system, sample moves through to a number of
serially connected columns containing the first affinity matrix,
followed by a number of serially connected columns containing the
second affinity matrix. A switch valve connects first set of
columns to the second set of columns.
[0038] FIG. 7 represents an alternative schematic view of an
exemplary chromatography system according to an embodiment of the
disclosure.
DETAILED DESCRIPTION
[0039] Detailed embodiments of the present invention are disclosed
herein; however, it is to be understood that the disclosed
embodiments are merely illustrative of the invention that may be
embodied in various forms. In addition, each of the examples given
in connection with the various embodiments of the invention is
intended to be illustrative, and not restrictive. Therefore,
specific structural and functional details disclosed herein are not
to be interpreted as limiting, but merely as a representative basis
for teaching one skilled in the art to variously employ the present
invention.
[0040] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, particular methods and materials are now described.
[0041] As used in this specification and the appended claims, the
singular forms "a", "an", and "the" include plural references
unless the context clearly dictates otherwise. Thus, for example, a
reference to "a method" includes one or more methods, and/or steps
of the type described herein and/or which will become apparent to
those persons skilled in the art upon reading this disclosure.
[0042] Not-limiting examples of proteins suitable for separation
with methods according to the invention may include, without
limitation, heterodimeric antibodies, e.g., bispecific antibodies,
one-arm antibodies, and ScFv antibodies. Bispecific antibodies
generally comprise two different heavy chains, with each heavy
chain specifically binding a different epitope-either on two
different molecules (e.g., two different antigens or an antigen and
a T-cell receptor) or on the same molecule (e.g., on the same
antigen). If a bispecific antibody is capable of selectively
binding two different epitopes (a first epitope and a second
epitope), the affinity of the first heavy chain for the first
epitope will generally be at least one to two or three or four
orders of magnitude lower than the affinity of the first heavy
chain for the second epitope, and vice versa. The epitopes
recognized by the bispecific antibody can be on the same or a
different target (e.g., on the same or a different protein).
[0043] Bispecific antibodies can be made, for example, by combining
heavy chains that recognize different epitopes of the same antigen.
For example, nucleic acid sequences encoding heavy chain variable
sequences that recognize different epitopes of the same antigen can
be fused to nucleic acid sequences encoding different heavy chain
constant regions, and such sequences can be expressed in a cell
that expresses an immunoglobulin light chain. A typical bispecific
antibody has two heavy chains each having three heavy chain CDRs,
followed by (N-terminal to C-terminal) a CH1 domain, a hinge, a CH2
domain, and a CH3 domain, and an immunoglobulin light chain that
either does not confer antigen-binding specificity but that can
associate with each heavy chain, or that can associate with each
heavy chain and that can bind one or more of the epitopes bound by
the heavy chain antigen-binding regions, or that can associate with
each heavy chain and enable binding or one or both of the heavy
chains to one or both epitopes.
[0044] The phrase "Fc-containing protein" includes antibodies,
bispecific antibodies, immunoadhesins, and other binding proteins
that comprise at least a functional portion of an immunoglobulin
CH2 and CH3 region. A "functional portion" refers to a CH2 and CH3
region that can bind a Fc receptor (e.g., an Fc.gamma.R; or an
FcRn, i.e., a neonatal Fc receptor), and/or that can participate in
the activation of complement. If the CH2 and CH3 region contains
deletions, substitutions, and/or insertions or other modifications
that render it unable to bind any Fc receptor and also unable to
activate complement, the CH2 and CH3 region is not functional.
[0045] Fc-containing proteins can comprise modifications in
immunoglobulin domains, including where the modifications affect
one or more effector function of the binding protein (e.g.,
modifications that affect Fc.gamma.R binding, FcRn binding and thus
half-life, and/or CDC activity). Such modifications include, but
are not limited to, the following modifications and combinations
thereof, with reference to EU numbering of an immunoglobulin
constant region: 238, 239, 248, 249, 250, 252, 254, 255, 256, 258,
265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289,
290, 292, 293, 294, 295, 296, 297, 298, 301, 303, 305, 307, 308,
309, 311, 312, 315, 318, 320, 322, 324, 326, 327, 328, 329, 330,
331, 332, 333, 334, 335, 337, 338, 339, 340, 342, 344, 356, 358,
359, 360, 361, 362, 373, 375, 376, 378, 380, 382, 383, 384, 386,
388, 389, 398, 414, 416, 419, 428, 430, 433, 434, 435, 437, 438,
and 439.
[0046] For example, and not by way of limitation, the binding
protein is an Fc-containing protein and exhibits enhanced serum
half-life (as compared with the same Fc-containing protein without
the recited modification(s)) and have a modification at position
250 (e.g., E or Q); 250 and 428 (e.g., L or F); 252 (e.g., UY/F/W
or T), 254 (e.g., S or T), and 256 (e.g., S/R/Q/E/D or T); or a
modification at 428 and/or 433 (e.g., L/R/SI/P/Q or K) and/or 434
(e.g., H/F or Y); or a modification at 250 and/or 428; or a
modification at 307 or 308 (e.g., 308F, V308F), and 434. In another
example, the modification can comprise a 428L (e.g., M428L) and
434S (e.g., N434S) modification; a 428L, 2591 (e.g., V259I), and a
308F (e.g., V308F) modification; a 433K (e.g., H433K) and a 434
(e.g., 434Y) modification; a 252, 254, and 256 (e.g., 252Y, 254T,
and 256E) modification; a 250Q and 428L modification (e.g., T250Q
and M428L); a 307 and/or 308 modification (e.g., 308F or 308P).
[0047] The term "star substitution," "Fc*," and "HC*" includes any
molecule, immunoglobulin heavy chain, Fc fragment, Fc-containing
molecule and the like that contains a mutation that abrogates the
binding to bacterial proteins known to bind the Fc domain of
immunoglobulins, such as Protein A, Protein G, Protein L or
derivatives thereof (see for example SpA and SpA mimetic affinity
ligands described in Choe, W., et al. 2016, Materials 9, 994,
doi:10.3390/ma9120994, which is incorporated herein by reference).
Immunoglobulins or other Fc-containing proteins may, for example,
contain a modified sequence within the CH3 domain that greatly
reduces binding to Protein A, as described, e.g., in WO2016/018740
and U.S. Pat. No. 8,586,713. A mutation in the Fc domain may be
designated as the "star substitution" or Fc* throughout the
specification for ease of noting that one polypeptide of a dimer
contains a mutation, and one does not. Thus, Fc*Fc* denotes a
homodimer wherein both monomers comprise an Fc*, and FcFc*, or
Fc*Fc, denotes a heterodimer with respect to the Fc* substitution.
The terms FcFc* and Fc*Fc are used interchangeably herein.
[0048] The phrase "mobile phase modifier" includes moieties that
reduce the effect of, or disrupt, non-specific (i.e., non-affinity)
ionic and other non-covalent interactions between proteins. "Mobile
phase modifiers" include, for example, salts, ionic combinations of
Group I and Group II metals with acetate, bicarbonate, carbonate, a
halogen (e.g., chloride or fluoride), nitrate, phosphate, or
sulfate. A non-limiting illustrative list of "mobile phase
modifiers" includes beryllium, lithium, sodium, and potassium salts
of acetate; sodium and potassium bicarbonates; lithium, sodium,
potassium, and cesium carbonates; lithium, sodium, potassium,
cesium, and magnesium chlorides; sodium and potassium fluorides;
sodium, potassium, and calcium nitrates; sodium and potassium
phosphates; and calcium and magnesium sulfates.
[0049] "Mobile phase modifiers" also include chaotropic agents,
which weaken or otherwise interfere with non-covalent forces and
increase entropy within biomolecular systems. Non-limiting examples
of chaotropic agents include butanol, calcium chloride, ethanol,
guanidinium chloride, lithium perchlorate, lithium acetate,
magnesium chloride, phenol, propanol, sodium dodecyl sulfate,
thiourea, and urea. Chaotropic agents include salts that affect the
solubility of proteins. The more chaotropic anions include for
example chloride, nitrate, bromide, chlorate, iodide, perchlorate,
and thiocyanate. The more chaotropic cations include for example
lithium, magnesium, calcium, and guanidinium.
[0050] "Mobile phase modifiers" include those moieties that affect
ionic or other non-covalent interactions that, upon addition to a
pH gradient or step, or upon equilibration of a Protein A support
in a "mobile phase modifier" and application of a pH step or
gradient, results in a broadening of pH unit distance between
elution of a homodimeric IgG and a heterodimeric IgG (e.g., a
wild-type human IgG and the same IgG but bearing one or more
modifications of its CH3 domain as described herein). A suitable
concentration of a "mobile phase modifier" can be determined by its
concentration employing the same column, pH step or gradient, with
increasing concentration of "mobile phase modifier" until a maximal
pH distance is reached at a given pH step or pH gradient. "Mobile
phase modifiers" may also include non-polar modifiers, including
for example propylene glycol, ethylene glycol, and the like.
[0051] An affinity matrix is the solid support non-aqueous matrix
onto which an affinity protein, e.g., Protein A, Protein G, Protein
L, Protein Z, or recombinant derivatives thereof, adheres (Choe,
W., et al, 2016 supra). Such supports include agarose, sepharose,
glass, silica, polystyrene, nitrocellulose, charcoal, sand,
cellulose and any other suitable material. Such materials are well
known in the art. Any suitable method can be used to affix the
second protein to the solid support. Methods for affixing proteins
to suitable solid supports are well known in the art. See e.g.
Ostrove, in Guide to Protein Purification, Methods in Enzymology,
182: 357-371, 1990. Such solid supports, with and without
immobilized Protein A, are readily available from many commercial
sources including such as Vector Laboratory (Burlingame, Calif.),
Santa Cruz Biotechnology (Santa Cruz, Calif.), BioRad (Hercules,
Calif.), Amersham Biosciences (part of GE Healthcare, Uppsala,
Sweden), Pall (Port Washington, N.Y.) and EMD-Millipore (Billerica,
Mass.). Protein A immobilized to a pore glass matrix is
commercially available as PROSEP.RTM.-A (Millipore). The solid
phase may also be an agarose-based matrix. Protein A immobilized on
an agarose matrix is commercially available as, e.g., MABSELECT.TM.
(GE Amersham Biosciences). Affinity columns containing an
immunoglobulin- or Fc-binding protein may be manufactured by
affixing any of the SpA or mimetic SpA ligands to a solid
support.
[0052] As used herein, "affinity chromatography" is a
chromatographic method that makes use of the specific, reversible
interactions between biomolecules rather than general properties of
the biomolecule such as isoelectric point, hydrophobicity, or size,
to effect chromatographic separation. "Protein A affinity
chromatography" or "Protein A chromatography" refers to a specific
affinity chromatographic method that makes use of the affinity of
the IgG binding domains of Protein A for the Fc portion of an
immunoglobulin molecule. This Fc portion comprises human or animal
immunoglobulin constant domains CH2 and CH3 or immunoglobulin
domains substantially similar to these.
[0053] Protein A is a cell wall component produced by several
strains of Staphylococcus aureus which consists of a single
polypeptide chain. The Protein A gene product consists of five
homologous repeats attached in a tandem fashion to the pathogen's
cell wall. The five domains are approximately 58 amino acids in
length and denoted EDABC, each exhibiting immunoglobulin binding
activity (Tashiro M & Montelione G T (1995) Curr. Opin. Struct.
Biol., 5(4): 471-481. The five homologous immunoglobulin binding
domains fold into a three-helix bundle. Each domain is able to bind
proteins from many mammalian species, most notably IgGs (Hober S et
al., (2007) J. Chromatogr. B Analyt. Technol. Biomed. Life Sci.,
848(1): 40-47). Protein A binds the heavy chain of most
immunoglobulins within the Fc region but also within the Fab region
in the case of the human VH3 family (Jansson B et al, (1998) FEMS
Immunol. Med. Microbiol., 20(1): 69-78). Protein A binds IgG from
various species including human, mouse, rabbit, and guinea pig but
does not bind human IgG3 (Hober S et al., (2007) supra). The
inability of human IgG3 to bind Protein A can be explained by the
H435R and Y436F substitutions in the human IgG3 Fc region (EU
numbering, Jendeberg et al., (1997) J. Immunol. Methods, 201(1):
25-34). Besides IgG, Protein A also interacts with IgM and IgA.
[0054] The capacity of Protein A to bind antibodies with such high
affinity is the driving motivation for its industrial scale use in
biologic pharmaceuticals. Protein A used for production of
antibodies in bio-pharmaceuticals is usually produced recombinantly
in E. coli and functions essentially the same as native Protein A
(Liu H F et al., (2010) MAbs, 2(5): 480-499). Most commonly,
recombinant Protein A is bound to a stationary phase chromatography
resin for purification of antibodies. Optimal binding occurs at
pH8.2, although binding is also good at neutral or physiological
conditions (pH 7.0-7.6). Elution is usually achieved through pH
shift towards acidic pH (glycine-HCl, pH2.5-3.0). This effectively
dissociates most protein-protein and antibody-antigen binding
interactions without permanently affecting protein structure.
Nevertheless, some antibodies and proteins are damaged by low pH,
and in some cases it may be best to neutralize immediately after
recovery by addition of 1/10th volume of alkaline buffer such as 1
M Tris-HCl, pH 8.0 to minimize the duration of time in the low-pH
condition.
[0055] There are various commercially available Protein A
chromatography resins. The main differences between these media are
the support matrix type, Protein A ligand modification, pore size
and particle size. The differences in these factors give rise to
differences in compressibility, chemical and physical robustness,
diffusion resistance and binding capacity of the adsorbents (Hober
S et al., (2007), supra). Examples of Protein A chromatography
resins include but are not limited to the MabSelect SuRe.TM.
Protein A resin and MabSelect.TM. Protein A resin from GE
Healthcare, EconoPac Protein A column from BioRad, rProA, available
from Applied Biosystems, and POROS.RTM. A from Thermo Fisher, as
seen in the Examples.
[0056] Protein A, as used herein, encompasses native protein from
the cell wall of Staphylococcus aureus, Protein A produced by
recombinant or synthetic methods, and variants that retain the
ability to bind to an Fc region. Engineered Protein A may be for
example a Z-domain tetramer, a Y-domain tetramer, or an engineered
Protein A that lacks D and E domains. These engineered Protein A
exemplars are unable to bind (or bind with very low affinity if at
all) to the VH3 domain of an immunoglobulin, but can still bind to
the CH3 domains of IgG1, IgG2 and IgG4. In practice, Protein A
chromatography involves using Protein A immobilized to a solid
support. See Gagnon, Protein A Affinity Chromotography,
Purification Tools for Monoclonal Antibodies, pp. 155-198,
Validated Biosystems, 1996.
[0057] Protein G is a bacterial cell wall protein isolated from
group C and G Streptococci. DNA sequencing of native Protein G
isolated from different Streptococci identified immunoglobulin
binding domains as well as sites for albumin and cell surface
binding. Depending on the strain both the immunoglobulin binding
region and the albumin binding region consist of 2-3 independently
folding units (Tashiro M & Montelione G T (1995) Curr. Opin.
Struct. Biol., 5(4): 471-481). Protein G from strain G148 consists
of 3 albumin and immunoglobulin binding domains respectively
denoted ABD1, ABD2, and ABD3, and C1, C2, and C3 (Olsson A et al.,
(1987) Eur. J. Biochem., 168(2): 319-324.). Each immunoglobulin
binding domain denoted C1, C2, and C3 is approximately 55 residues
and separated by linkers of about 15 residues. All experimentally
solved 3D structures of Protein G immunoglobulin binding domains
show a highly compact globular structure without any disulfide
bridges or tightly bound prosthetic groups (Sauer-Eriksson A E et
al., (1995) Structure, 3(3): 265-278; Derrick J P & Wigley D B
(1992) Nature, 359(6397): 752-754; Derrick J P & Wigley D B
(1994) J. Mol. Biol., 243(5): 906-918; Lian L Y et al., (1994) Nat.
Struct. Biol., 1(6): 355-357). The structure comprises a
four-stranded beta-sheet made up of two anti-parallel beta-hairpins
connected by an alpha-helix.
[0058] Streptococcus strains from groups C and G show binding to
all human subclasses of IgG including IgG3 in contrast to Protein
A. Protein G also binds to several animal IgG including mouse,
rabbit, and sheep (Bjorck L & Kronvall G (1984) J. Immunol.,
133(2): 969-974; Akerstrom B et al., (1985) J. Immunol., 135(4):
2589-2592; Akerstrom B & Bjorck L (1986) J. Biol. Chem.,
261(22): 10240-10247; Fahnestock S R et al., (1986) J. Bacteriol.,
167(3): 870-880). Hence, Protein G exhibits a broader binding
spectrum to subclasses of different species compared to Protein A.
In addition, Protein G binds to the Fab portion of IgGs with high
affinity. The structure of the binding domain of streptococcal
Protein G has been determined both alone (by NMR, Lian L Y et al.,
(1994) supra), and in complex with an IgG1 Fab (by x-ray
crystallography, Derrick J P & Wigley D B (1992) supra and
Derrick J P & Wigley D B (1994) supra). All experimentally
solved 3D structures showed a binding within the CH1 domain of IgG
heavy chains.
[0059] The Protein G, as used herein, may be a naturally occurring
or modified Streptococcal Protein G, or it may be an engineered
Protein G. Engineered Protein G may comprise the B1 domain (aka
GB1) and may be conjugated or unconjugated. In another embodiment,
the second affinity matrix comprises a protein L ligand and its
derivatives affixed to a solid substrate. Similarly to Protein A,
recombinant Protein G produced in E. coli may be used to purify
antibodies. The albumin and cell surface binding domains have been
eliminated from recombinant Protein G to reduce non specific
binding and, therefore, can be used to separate IgG from crude
samples. Similarly to Protein A, recombinant Protein G is bound to
a stationary phase chromatography resin for purification of
antibodies. Optimal binding occurs at pH 5, although binding is
also good at pH 7.0-7.2; as for Protein A, elution is also achieved
through pH shift towards acidic pH (glycine-HCl, pH2.5-3.0).
Examples of Protein G chromatography resins include but are not
limited to the Protein G Sepharose.TM. 4 Fast Flow resin and
HiTrap.TM. Protein G HP column from GE Healthcare.
[0060] Similarly to Protein A, recombinant Protein G produced in E.
coli is routinely used to purify antibodies. The albumin and cell
surface binding domains have been eliminated from recombinant
Protein G to reduce nonspecific binding and, therefore, can be used
to separate IgG from crude samples. Similarly to Protein A,
recombinant Protein G is bound to a stationary phase chromatography
resin for purification of antibodies. Optimal binding occurs at pH
5, although binding is also good at pH 7.0-7.2; as for Protein A,
elution is also achieved through pH shift towards acidic pH
(glycine-HCl, pH2.5-3.0). Examples of Protein G chromatography
resins include but are not limited to the Protein G Sepharose.TM. 4
Fast Flow resin and HiTrap.TM. Protein G HP column from GE
Healthcare, rProG, available from Applied Biosystems, and
POROS.RTM. G from Thermo Fisher, as seen in the Examples.
[0061] Other proteins, such as Protein L, M1 Protein, and Protein
H, may also be used in the affinity chromatography of the present
invention. Protein L is an immunoglobulin binding protein that was
originally derived from the bacteria Peptostreptococcus magnus, but
is now produced recombinantly (Bjorck L (1988) J. Immunol., 140(4):
1194-1197; Kastern W et al., (1992) J. Biol. Chem., 267(18):
12820-12825). Protein L has the unique ability to bind through
kappa light chain interactions without interfering with an
antibody's antigen binding site (Nilson B H et al., (1993) J.
Immunol. Methods, 164(1): 33-40). This gives Protein L the ability
to bind a wider range of immunoglobulin classes and subclasses than
other antibody binding protein. Protein L will bind to all classes
of immunoglobulins (IgG, IgM, IgA, IgE and IgD). Protein L will
also bind single chain variable fragments (scFv) and Fab fragments
(Nilson B H et al., (1993) supra; Bottomley S P et al., (1995)
Bioseparation, 5(6): 359-367). Protein L binds the human variable
domains of kappa I, III, and IV subclasses and mouse kappa I
subclass (Nilson B H et al., (1992) supra). Examples of Protein L
chromatography resins include but are not limited to the Protein L
resin from Genescript as used in examples.
[0062] M1 Protein and Protein H are surface proteins simultaneously
present at the surface of certain strains of Streptococcus
pyogenes. Protein H has affinity for the Fc region of IgG (Akesson
P et al., (1990) Mol. Immunol., 27(6): 523-531; Akesson P et al.,
(1994) Biochem. J., 300 (Pt 3): 877-886). Protein H binds to the Fc
region of IgGs from human, monkeys and rabbits (Akesson P et al.,
(1990), supra; Frick I M et al., (1995) EMBO J., 14(8): 1674-1679).
M Proteins are also known to bind fibrinogen (Kantor F S (1965) J
Exp Med, 121: 849-859), and previous work has demonstrated that M1
Protein from the API strain also has affinity for albumin and
polyclonal IgG (Schmidt K H & Wadstrom T (1990) Zentralbl.
Bakteriol., 273(2): 216-228).
[0063] Affinity chromatography also includes media that can be used
to selectively bind and thus purify antibodies, fragments of
antibodies, or chimeric fusion proteins that contain immunoglobulin
domains and/or sequences. Antibodies include IgG, IgA, IgM, IgY,
IgD and IgE types. Antibodies also include single chain antibodies
such as camelid antibodies, engineered camelid antibodies, single
chain antibodies, single-domain antibodies, nanobodies, and the
like. Antibody fragments include VH, VL, CL, CH sequences. Antibody
fragments and fusion proteins containing antibody sequences include
for example F(ab')3, F(ab')2, Fab, Fc, Fv, dsFv, (scFv)2, scFv,
scAb, minibody, diabody, triabody, tetrabody, Fc-fusion proteins,
trap molecules, and the like (see Ayar et al., Methods 56 (2012):
116-129).
[0064] Such affinity chromatography media may contain ligands that
selectively bind antibodies, their fragments, and fusion proteins
contains those fragments. Such ligands include antibody binding
proteins, bacterially derived receptors, antigens, lectins or
anti-antibodies directed to the target molecule. the antibody
requiring purification. For example, camelid-derived affinity
ligands directed against any one or more of IgG-CH1, IgG-Fc,
IgG-CH3, IgG1, LC-kappa, LC-lambda, IgG3/4, IgA, IgM, and the like
may be used as affinity ligands (commercially available as
CAPTURESELECT chromatography resins, Life Technologies, Inc.,
Carlsbad, Calif.).
[0065] Techniques that ease the recovery of heterodimers from
homodimers based on a differential affinity of the heterodimers for
an affinity reagent have been described. The first example of
differential affinity technique involved the use of two different
heavy chains from two different animal species, wherein one of
which does not bind Protein A (Lindhofer H et al., (1995) J
Immunol., 155(1): 219-225). The same authors also described the use
of two different heavy chains originating from two different human
immunoglobulin isotypes (IGHG1 and IGHG3), one of which does not
bind Protein A (IGHG3; see U.S. Pat. No. 6,551,592 Lindhofer H et
al.). A variation of the latter technique has been described in
WO10/151792 (Davis S et al.) and involved the use of the two amino
acid substitutions H435R/Y436F described by Jendeberg et at
(Jendeberg et al., (1997) J. Immunol. Methods, 201(1): 25-34) to
greatly reduce the affinity for Protein A in one of the heterodimer
heavy chains.
[0066] As used herein, the term "detector" comprises a
chromatography column equipped with a means for detecting and/or
assessing components of a mixture being eluted off the
chromatography column. Two general types of detectors are known in
the art: destructive and non-destructive detectors. The destructive
detectors perform continuous transformation of the column effluent
(burning, evaporation or mixing with reagents) with subsequent
measurement of some physical property of the resulting material
(plasma, aerosol or reaction mixture). The non-destructive
detectors are directly measuring some property of the column eluent
(for example UV absorption) and thus affords for the further
analysis recovery. Examples of destructive detectors include
charged aerosol detector (CAD), flame ionization detector (FID),
aerosol-based detector (NQA), flame photometric detector (FPD),
atomic-emission detector (AED), nitrogen phosphorus detector (NPD),
evaporative light scattering detector (ELSD), mass spectrometer
(MS), electrolytic conductivity detector (ELCD), summon detector
(SMSD), and mira detector (MD). One example of non-destructive
detectors includes UV detectors, including fixed and variable
length UV detectors, including diode array detector (DAD) or
photodiode array (PDA) detector. UV absorption of the effluent may
be measured continuously at single or multiple wavelengths. Other
examples of non-destructive detectors include thermal conductivity
detector (TCD), fluorescence detector (FLR), electron capture
detector, photoionization detector (PID), and refractive index
detector (RI or RID). In one example, a DAD/UV detector may be
utilized to detect and quantify the eluate material flowing from
the column compartment of the system. Bispecific FcFc* antibody,
FcFc homodimer, and Fc*Fc* heterodimer selectively elute from the
chromatography system, and the signal is picked up by UV detection
at 280 nM. Any detector may be adapted to connect a temperature
control system, such as temperature controlled flow cells with
cooling functions to allow for better stability of protein
material.
[0067] The present invention may be set up as part of a
chromatography system, e.g. a commercially available chromatography
system, such as, e.g., an HPLC system available from Shimadzu
Corporation, Agilent Technologies, Waters Corporation, or the like.
In one non-limiting embodiment, such system comprises, inter alia,
a solvent and/or reagent holding unit, a solvent manager/pump, a
sample manager, a column compartment manager, and a detector unit.
One non-limiting embodiment is depicted in FIG. 5.
[0068] The holding unit houses solvents and buffers used in
chromatographic applications, and optionally comprises a solvent
degasser. In one embodiment, solvents and buffers pass through the
solvent degasser prior to flowing to the solvent manager.
[0069] The solvent manager is responsible for solvent delivery and
may include a computer platform configurable to address the needs
of the analytical system according to each particular embodiment in
order to improve separation and resolution, and may include, e.g.,
binary or quaternary gradient modules. The solvent manager may
control, inter alia, solvent rates, buffer compositions, and
pressure limits of the HPLC system.
[0070] The sample manager may optionally comprise a temperature
control unit capable of changing the temperature (e.g., cooling
and/or heating), or keeping the temperature of the sample constant
(by e.g., cooling the sample to a constant temperature) prior to
loading onto the columns. In one embodiment, samples are contained
in the sample manager compartment and kept at 4.degree. C. while
awaiting injection. Samples are brought into the autosampler where
a needle goes directly to sample indicated in the queue. In one
embodiment, the autosampler further comprises a pressure regulator
for handling overflow of injected solvent and/or sample. From the
autosampler needle, the sample then moves to the column
compartment.
[0071] The amount, or quantity, of a protein in a fraction may be
determined by eluting the protein through the detector and using
computational methods known in the art. The system may further
comprise a system controller unit, or other computer-aided
device.
[0072] The purity and/or quality control analysis is performed by
analyzing and quantifying the ratios of the three protein species
present in the sample. The purity of a protein in a mixture may be
calculated by determining the amount of each protein fraction and
calculating the ratio of the amount of the protein of interest to
the sum of the amounts of all proteins in the mixture. By way of
example, the purity of a heterodimeric protein in a mixture
comprising the heterodimeric protein and two or more protein
impurities may be quantified by determining the amount of each
protein fraction and calculating the ratio of the amount of the
heterodimeric protein to the sum of the amounts of the
heterodimeric protein and the two or more protein impurities.
[0073] The metric bispecific purity gives the percentage of
bispecific antibody as compared to total antibody amount, as
defined by Equation 1. By way of example, the purity of FcFc* in a
mixture comprising FcFc, FcFc*, and Fc*Fc* can be quantified as
follows:
Quantification .times. .times. of .times. .times. Purity .times.
.times. of .times. .times. a .times. .times. Bispecific .times.
.times. Antibody .times. .times. Purity .times. .times. of .times.
.times. FcFc * = amount .times. .times. of .times. .times. FcFc * (
amount .times. .times. of .times. .times. FcFc + amount .times.
.times. of .times. .times. FcFc * + amount .times. .times. .times.
of .times. .times. Fc * .times. Fc * ) Equation .times. .times. 1
##EQU00001##
[0074] A switch valve, a flow switch valve, or a flow control
valve, as used herein, is a means for directing, varying, or
cutting off the flow path of the eluent off a chromatography
column. The switch valve may be multi-way, e.g., two-way,
three-way, four-way, and the like, i.e. the switch valve is capable
of directing the flow to two, three, or more different receptacles.
Receptacles may be of any origin, e.g., chromatography columns,
affinity chromatography columns, detectors, or a waste disposal
bin. The change of the receptacles is achieved by switching the
switch valve between different positions.
[0075] By way of example, in a chromatography system a switch valve
may connect a first affinity matrix, a second affinity matrix, and
a detector in a serial or non-serial fashion. In non-limiting
embodiments, e.g. shown in FIGS. 5-7, a switch valve may serially
connect a first affinity matrix to a second affinity matrix. In
this exemplary system, one switch valve position would allow the
eluent to flow from the first affinity matrix to the second
affinity matrix and, subsequently, to the detector. Switching the
switch valve to another position would allow the eluent to flow
from the first affinity matrix directly to the detector, bypassing
the second affinity matrix. The eluent from the second affinity
matrix may flow to the detector with or without engaging the first
affinity matrix. The second affinity matrix may or may not be
directly connected to an autosampler. In another non-limiting
embodiment depicted in FIG. 4B, the eluent from the first affinity
matrix is able to flow directly to the detector, bypassing the
second affinity matrix.
[0076] In one aspect, the present invention describes a method of
quantitatively assessing the amount and/or purity of the
heterodimer fraction by utilizing a novel chromatography system
comprising a switch valve.
[0077] In one aspect, the present invention describes a method for
quantifying an amount and/or purity of a protein in a sample
comprising a mixture of the protein, a first protein impurity, and
a second protein impurity, wherein the protein and the first
impurity bind to a first affinity matrix and the second impurity
does not substantially bind to the first affinity matrix and binds
to a second affinity matrix, said method comprising the steps
of:
[0078] a. applying the sample to a chromatography system comprising
the first affinity matrix, the second affinity matrix, and a
detector, wherein the first affinity matrix is serially connected
to the second affinity matrix via a switch valve;
[0079] b. eluting the second impurity from the first affinity
matrix onto the second affinity matrix under a first set of
conditions;
[0080] c. switching the switch valve to bypass the second affinity
matrix, eluting the protein through the detector under a second set
of conditions, and determining the amount of the eluted
protein;
[0081] d. eluting the first impurity through the detector under a
third set of conditions and determining the amount of the eluted
first impurity;
[0082] e. eluting the second impurity from the second affinity
matrix through the detector under the third set of conditions, and
determining the amount of the eluted second impurity, and
[0083] f. quantifying the amount and/or purity of the protein in
the sample.
[0084] In one embodiment the protein is a multimeric protein, e.g.
an antibody. In one embodiment, the protein is an antibody of
interest, and the first and second protein impurities are
multimeric proteins, e.g., antibodies that may or may not be
structurally related to the antibody of interest. In one
embodiment, the protein is a bispecific antibody, i.e., a
heterodimeric protein, the first protein impurity is a first
homodimeric protein, and the second protein impurity is a second
homodimeric protein. In some cases, the mixture of multimeric
proteins is produced by a plurality of eukaryotic cells, such as,
for example, Chinese hamster ovary (CHO) cells in a cell
culture.
[0085] In one embodiment, the protein has a lower affinity to the
first affinity matrix than the first impurity. In one embodiment
the protein is a heterodimeric protein, the first protein impurity
is a first homodimeric protein, and the second protein impurity is
a second homodimeric protein, the heterodimeric protein and the
first homodimeric protein bind to the first affinity matrix and the
second homodimeric protein does not substantially bind to the first
affinity matrix and binds to the second affinity matrix.
[0086] In one embodiment, the protein comprises a first
immunoglobulin CH3 domain and a second immunoglobulin CH3 domain,
wherein said first and second immunoglobulin CH3 domains are
different in their affinity to the first affinity matrix, and
wherein the sample comprises a mixture comprising said protein, a
protein comprising two first CH3 domains, and a protein comprising
two second CH3 domains.
[0087] In one embodiment, the second CH3 domain comprises H435R and
Y436F (by EU numbering system; H95R/Y96F by IMGT exon numbering
system) amino acid substitutions.
[0088] In one embodiment, the first affinity matrix comprises a
protein A ligand and its derivatives affixed to a solid substrate.
In some cases, the substrate is a bead or particle, such that the
affinity matrix is a plurality of particles affixed with Protein A.
The Protein A may be a naturally occurring or modified
Staphylococcal Protein A, or it may be an engineered Protein A.
Engineered Protein A may be for example a Z-domain tetramer, a
Y-domain tetramer, or an engineered Protein A that lacks D and E
domains. These engineered Protein A exemplars are unable to bind
(or bind with very low affinity if at all) to the VH3 domain of an
immunoglobulin, but can still bind to the CH3 domains of IgG1, IgG2
and IgG4.
[0089] In one embodiment, the second affinity matrix comprises a
protein G ligand and its derivatives affixed to a solid substrate.
In some cases, the substrate is a bead or particle, such that the
affinity matrix is a plurality of particles affixed with Protein G.
The Protein G may be a naturally occurring or modified
Streptococcal Protein G, or it may be an engineered Protein G.
Engineered Protein G may comprise the B1 domain (aka GB1) and may
be conjugated or unconjugated. In another embodiment, the second
affinity matrix comprises a protein L ligand and its derivatives
affixed to a solid substrate.
[0090] In one embodiment, elution conditions may comprise a
particular pH range and a buffer comprising a mobile phase
modifier, e.g., a chaotropic agent. In one embodiment, the first
set of elution conditions for eluting the second impurity, e.g.,
the second homodimeric protein, comprises a first pH. In one
embodiment, the second set of elution conditions for eluting the
protein, e.g., the heterodimeric protein, comprises a second pH. In
one embodiment, the third set of elution conditions for eluting the
first impurity, e.g., the first homodimeric protein, comprises a
third pH. In one embodiment, the second pH may be lower than the
first pH. In one embodiment, the third pH may be lower than the
second pH. In one embodiment, the second pH may be lower than the
first pH, and the third pH may be lower than the second pH. In
another embodiment, the first pH may be greater than pH 5, or about
pH 5 to about pH 8, or about pH 5.2 to about pH 7.4, or pH 6.4. In
one embodiment, the second pH may be about pH 3.5 to about pH 6, or
about 3.8 to about 5.6. In one embodiment, the third pH may be less
than pH 4, or about pH 1.5 to about pH 3.6, or about pH 2.0 to
about pH 2.8, or about pH 2.2.
[0091] In one embodiment, the first, second, and third sets of
elution conditions comprise a suitable buffer, e.g., a citrate,
acetate, 4-Morpholineethanesulfonate (MES), phosphate, succinate,
and the likes, as well as combinations and mixtures thereof. In one
embodiment, the first, second, and third sets of elution conditions
comprise a chaotropic agent. The chaotropic agent can be a salt,
having a cation selected from lithium, magnesium, calcium, and
guanidinium, and an anion selected from chloride, nitrate, bromide,
chlorate, iodide, perchlorate, and thiocyanate. In one particular
embodiment, the chaotropic agent is CaCl.sub.2, for example 250-500
mM CaCl.sub.2. In another particular embodiment, the chaotropic
agent is MgCl.sub.2, for example 250-500 mM MgCl.sub.2.
[0092] In one embodiment, the heterodimer is a bispecific antibody.
Here, the first polypeptide comprises a CH3 domain that is capable
of binding to Protein A ("Fc") and the second polypeptide comprises
a CH3 domain that is not capable of binding to Protein A ("Fc*").
In some cases, the second polypeptide comprises a H435R/Y436F (by
EU numbering system; H95R/Y96F by IMGT exon numbering system)
substitution in its CH3 domain (a.k.a "Fc*" or "star
substitution"). Thus, in some embodiments, the first homodimer is a
monospecific antibody having two unsubstituted CH3 domains (i.e.,
FcFc); the second homodimer is a monospecific antibody having two
H435R/Y436F substituted CH3 domains (i.e., Fc*Fc*); and the
heterodimer is a bispecific antibody having one unsubstituted CH3
domain and one H435R/Y436F substituted CH3 domain (i.e.,
Fc*Fc).
[0093] In another aspect, the present invention describes a method
for quantifying an amount and/or purity of a heterodimeric protein
in a sample comprising a mixture of the heterodimeric protein, a
first homodimeric protein, and a second homodimeric protein,
wherein the heterodimeric protein and the first homodimeric protein
bind to a protein A matrix and the second homodimeric protein does
not substantially bind to the protein A matrix and binds to a
protein G matrix, said method comprising the steps of:
[0094] a. applying the sample to a chromatography system comprising
the protein A matrix, the protein G matrix, and a detector, wherein
the protein A matrix is serially connected to the protein G matrix
via a switch valve;
[0095] b. eluting the second homodimeric protein from the protein A
matrix onto the protein G matrix under a first set of
conditions;
[0096] c. switching the switch valve to bypass the protein G
matrix, eluting the heterodimeric protein through the detector
under a second set of conditions, and determining the amount of the
eluted protein;
[0097] d. eluting the first homodimeric protein through the
detector under a third set of conditions and determining the amount
of the eluted first impurity;
[0098] e. eluting the second homodimeric protein from the second
affinity matrix through the detector under the third set of
conditions, and determining the amount of the eluted second
impurity, and
[0099] f. quantifying the amount and/or purity of the protein in
the sample.
[0100] Differential binding of the first homodimer and the
heterodimer to the second affinity matrix can be manipulated by
changing inter alia the pH and/or ionic strength of a solution that
is passed over the affinity matrix. The addition of a chaotropic
agent to the solution enhances the elution each dimer species from
the second affinity matrix in non-overlapping fractions, thereby
increasing to purity of each dimer species. In one embodiment, the
first homodimer, e.g. Fc*Fc*, is eluted from the first affinity
matrix onto the second affinity matrix in a buffer having a first
pH. In one embodiment, the heterodimer, e.g. the FcFc* heterodimer,
is eluted from the first affinity matrix bypassing the second
affinity matrix directly to the detector in a buffer having a
second pH range. In one embodiment, the second homodimer, e.g. FcFc
is eluted from the first affinity matrix bypassing the second
affinity matrix directly to the detector in a buffer having a third
pH range. In one embodiment, the first homodimer, e.g. Fc*Fc*, is
eluted from the second affinity matrix onto the detector in a
buffer having a third pH. Here, the first pH range comprises a
higher pH than does the second pH range, and the second pH range
comprises a higher pH than does the third pH range.
[0101] In one embodiment, the first set of elution conditions for
eluting the second impurity, e.g., the second homodimeric protein,
comprises a first pH. In one embodiment, the second set of elution
conditions for eluting the protein, e.g., the heterodimeric
protein, comprises a second pH. In one embodiment, the third set of
elution conditions for eluting the first impurity, e.g., the first
homodimeric protein, comprises a third pH. In one embodiment, the
second pH may be lower than the first pH. In one embodiment, the
third pH may be lower than the second pH. In one embodiment, the
second pH may be lower than the first pH, and the third pH may be
lower than the second pH. In another embodiment, the first pH may
be greater than pH 5, or about pH 5 to about pH 8, or about pH 5.2
to about pH 7.4. In one embodiment, the second pH may be about pH 4
to about pH 5, or about 4.2 to about 5.0. In one embodiment, the
third pH may be less than pH 4, or about pH 2 to about pH 3.6, or
about pH 2.2 to about pH 2.8.
[0102] In one aspect, a method for determining a quantity and/or
purity of a FcFc* protein in a sample is disclosed, wherein said
FcFc* protein comprises a first immunoglobulin CH3 domain (Fc), a
fragment and/or a derivative thereof, and a second immunoglobulin
CH3 domain (Fc*), a fragment and/or a derivative thereof, wherein
said first and second immunoglobulin CH3 domains are different in
their affinity to a first protein affinity matrix, and wherein the
sample comprises a mixture comprising said FcFc* protein, a protein
comprising two first CH3 domains (FcFc protein), and a protein
comprising two second CH3 domains (Fc*Fc* protein), said method
comprising the steps of:
(a) applying the sample to the first protein affinity matrix under
a first set of conditions, wherein said FcFc* protein and said FcFc
protein bind to said first protein affinity matrix and said Fc*Fc*
protein does not substantially bind to said first protein affinity
matrix; (b) washing said first protein affinity matrix under the
first set of conditions; (c) applying the flow-through from step
(a) and the wash from step (b) to a second protein affinity matrix
under such set of conditions that the Fc*Fc* protein binds to said
second protein affinity matrix; (d) washing said second protein
affinity matrix under the same set of conditions as in step (c);
(e) eluting the Fc*Fc* protein from said second protein affinity
matrix and determining the amount of said eluted Fc*Fc* protein;
(f) eluting the remaining bound Fc*Fc* protein from said first
protein affinity matrix under a second set of conditions, and
determining the amount of said eluted Fc*Fc* protein; (g) eluting
the FcFc* protein bound to said first protein affinity matrix under
a third set of conditions, and determining the amount of said
eluted FcFc* protein; (h) eluting the FcFc protein bound to said
first protein affinity matrix under a fourth set of conditions, and
determining the amount of said eluted FcFc protein, and (i)
determining the quantity and/or purity of the FcFc* protein in the
sample, wherein step (d) and/or step (e) can be performed
simultaneously with, before or after steps (f)-(h).
[0103] In one aspect, a method for determining a quantity and/or
purity of a bispecific antibody (e.g., an FcFc* antibody) in a
sample is disclosed, wherein said FcFc* antibody comprises a first
immunoglobulin heavy chain (Fc) and a second immunoglobulin heavy
chain (Fc*) wherein said first and second immunoglobulin heavy
chains are different in their affinity to protein A, and wherein
the sample comprises a mixture comprising said FcFc* antibody, an
antibody comprising two first heavy chains (FcFc antibody), and an
antibody comprising two second heavy chains (Fc*Fc* antibody), said
method comprising the steps of:
(a) applying the sample to a protein A affinity column (protein A
column) under a first set of conditions, wherein said FcFc*
antibody and said FcFc antibody bind to said protein A column,
while said Fc*Fc* antibody does not substantially bind to said
protein A column, and wherein the protein A column is connected
through a switch valve to a protein G affinity column (protein G
column) so that the flow-through from the protein A column can be
directly applied to the protein G column, which protein G column is
fur-ther connected to an HPLC column; (b) washing said protein A
column under the first set of conditions with the switch valve in a
position so that the flow-through from the protein A column is
directly applied to the protein G column; (c) washing the protein G
column under the same conditions as in step (b); (d) eluting the
Fc*Fc* antibody from the protein G column and determining the
amount of said eluted Fc*Fc* antibody; (e) putting the switch valve
in the position disconnecting the protein A column from the protein
G column and connecting said protein A column with an HPLC column;
(f) eluting the remaining bound Fc*Fc* antibody from the protein A
column under a second set of conditions, and determining the amount
of said eluted Fc*Fc* antibody using; (g) eluting the FcFc*
antibody bound to said protein A column under a third set of
conditions, and determining the amount of said eluted FcFc*
antibody; (h) eluting the FcFc antibody bound to said protein A
column under a fourth set of conditions, and determining the amount
of said eluted FcFc antibody, and (i) determining the quantity
and/or purity of the FcFc* protein in the sample, wherein step (c)
and/or step (d) can be performed simultaneously with, before or
after steps (e)-(h).
[0104] In another aspect, a chromatography system for purifying,
analyzing, and/or assessing amount and/or purity of proteins is
provided. In one embodiment, the chromatography system comprises a
first affinity matrix, a second affinity matrix, and a detector,
wherein each of the first affinity matrix, the second affinity
matrix and the detector are connected via a switch valve. In one
embodiment, the first affinity matrix may be a protein A
chromatography column. In one embodiment, a second affinity matrix
may be a protein G or a protein L chromatography column.
[0105] In one embodiment, the detector may be an HPLC column
equipped with a UV detector, a charge aerosol detector, and/or a
mass-spectrometer. In one embodiment, the first affinity matrix and
the second affinity matrix are serially connected via a switch
valve.
[0106] In another embodiment, the first affinity matrix, the second
affinity matrix, and the detector are all serially connected via a
switch valve. In one embodiment, the first affinity matrix and the
second affinity matrix are serially connected via a switch valve,
but the detector is non-serially connected to the first affinity
matrix and the second affinity matrix.
[0107] In one embodiment, a chromatography system is provided
comprising (i) a protein A chromatography column, (ii) a protein G
chromatography column, and (iii) a detector comprising an HPLC
column equipped with a UV detector, a charge aerosol detector,
and/or a mass-spectrometer, wherein each of the protein A
chromatography column, the protein G chromatography column and to
the detector are connected via a switch valve.
EXAMPLES
[0108] The following examples illustrate specific aspects of the
instant description. The examples should not be construed as
limiting, as the examples merely provide specific understanding and
practice of the embodiments and their various aspects.
[0109] Chromatograpy experiments were performed using an HPLC
system, adapted to the configurations needed to perform the method
described herein. InfinityLab Quick Change valves are available
from Agilent Technologies. Examples of suitable valves include, but
are not limited to, Agilent Quick Change Valve G4231A/C, G4232C/D,
G4234A/C, G4236A/B, and G4238A/B. Non-limiting examples of valves
suitable for practicing the invention is provided at world wide web
agilent.com/cs/library/usermanuals/public/G4232-90009_ValveKit_TN_EN.pdf,
and also Acquity UPLC Systems with 2D Technology Capabilities
Guide, Revision A, Waters Corporation, 2012, each of which is
incorporated by reference herein in its entirety.
Example 1
[0110] The purity and quantity of a bispecific antibody in a
mixture comprising two contaminating homodimers was determined as
follows (titer chromatogram is depicted as FIG. 1). Two heavy chain
polypeptides (IgG4 Fc- and IgG4-Fc*-containing) and a common light
chain polypeptide were co-expressed in CHO cells. A sample of the
cell supernatant comprising the resulting mixture of homodimers and
heterodimer was subjected to high-speed centrifugation to eliminate
protein aggregates, and the supernatant was subjected to affinity
chromatography according to purification methods described in PCT
Publication No. WO2016/018740, published Feb. 4, 2016, hereby
incorporated by reference. A sample of the purification product,
containing FcFc* heterodimer and any impurity products, FcFc and
Fc*Fc* homodimers, were loaded onto a 3.times.0.1 mL POROS.RTM. A
20 .mu.m Protein A column (rProA, obtained from Applied Biosystems,
#2-1001-00) in pH 6.4 mobile phase containing 0.5 M NaCl. The
Protein A column was serially connected to a 2.times.0.1 mL
POROS.RTM. G 20 .mu.m Protein G column (rProG, obtained from
Applied Biosystems, #2-1002-00) and to a standard UV detector (2.0
mL/min flow rate, UV@280 nm peak detection) via a switch valve as
outlined in FIG. 4A.
[0111] A series of washes was applied to remove process-related
contaminants such as CHO DNA or host cell protein (HCP). The
mixture was eluted using pH 5.6 mobile phase containing 0.5M
CaCl.sub.2. Since Fc*Fc* homodimer has both Protein A binding sites
deleted from the Fc region, this product-related impurity was
expected to flow though the rProA onto the rProG, while the
bispecific FcFc* and FcFc homodimer was expected to be retained on
the rProA.
[0112] The switch valve was then switched to take rProG offline,
connecting rProA directly to the detector. The bispecific FcFc*
antibody was then selectively eluted from rProA at to the detector
using pH 3.8-5.6 (molecule specific) mobile phase containing 0.5M
calcium chloride, while the FcFc impurity was retained due to its
stronger binding relative to the bispecific FcFc* antibody. The
amount of FcFc* was calculated. Then, the FcFc impurity was
selectively eluted from rProA using pH 2.2 mobile phase containing
0.5M calcium chloride to the detector, and the amount of FcFc was
calculated.
[0113] The switch valve was then switched back to serially connect
rProG online, connecting rProA to rProG, and rProG to the detector.
The Fc*Fc* impurity was then eluted using pH 2.2 mobile phase
containing 0.5M calcium chloride to the detector, and the amount of
Fc*Fc* was calculated.
[0114] The amount and purity of FcFc* bispecific antibody was
determined by calculating the ratio of the FcFc* fraction to the
sum of the FcFc, FcFc*, and Fc*Fc* fractions.
[0115] Method flow rate, wash length, bispecific elution length,
and % Buffer C in the elution step of the method were continuous
factors that were studied and deemed probable to have an effect on
the recovery of all three antibody species. Table 1, below, shows
the parameters, their role, and the values studied in the
method.
TABLE-US-00001 TABLE 1 Bispecific Purity Robustness Factors Name
Role Values Load Flow Rate (mL/min) Continuous 0.5-2.5 Wash Length
(CV) Continuous 0-60 Bispecific Elution Length (CV) Continuous
10-130 % C Continuous 5-30 Isotype Categorical IgG1*, IgG4*A,
IgG4*B
[0116] Table 2, below, shows the various sets of run conditions for
the inventive methods of assessing purity and quantity of three
antibody samples: IgG* 1, IgG4*A and IgG4*B.
TABLE-US-00002 TABLE 2 Bispecific Purity Robustness Run Conditions
Bispecific Condition Flow Rate Wash Elution % # (mL/min) Length
(CV) Length (CV) Buffer C Isotype 1 0.5 0 10 5 IgG1* 2 0.5 0 10
17.5 IgG4* B 3 0.5 0 10 30 IgG4* A 4 0.5 0 130 5 IgG4* A 5 0.5 0
130 30 IgG1* 6 0.5 30 130 5 IgG4* B 7 0.5 60 10 5 IgG4* A 8 0.5 60
10 30 IgG1* 9 0.5 60 70 30 IgG4* B 10 0.5 60 130 5 IgG1* 11 0.5 60
130 30 IgG4* A 12 1.5 0 130 30 IgG4* B 13 1.5 30 10 5 IgG4* B 14
1.5 30 70 17.5 IgG4* A 15 1.5 60 130 5 IgG4* B 16 2.5 0 10 5 IgG4*
A 17 2.5 0 10 30 IgG1* 18 2.5 0 70 5 IgG4* B 19 2.5 0 130 5 IgG1*
20 2.5 0 130 30 IgG4* A 21 2.5 30 10 30 IgG4* B 22 2.5 30 130 17.5
IgG4* B 23 2.5 60 10 5 IgG1* 24 2.5 60 10 17.5 IgG4* B 25 2.5 60 10
30 IgG4* A 26 2.5 60 130 5 IgG4* A 27 2.5 60 130 30 IgG1*
[0117] As various changes can be made in the above-described
subject matter without departing from the scope and spirit of the
present invention, it is intended that all subject matter contained
in the above description, or defined in the appended claims, be
interpreted as descriptive and illustrative of the present
invention. Many modifications and variations of the present
invention are possible in light of the above teachings.
Accordingly, the present description is intended to embrace all
such alternatives, modifications, and variances which fall within
the scope of the appended claims.
[0118] All patents, applications, publications, test methods,
literature, and other materials cited herein are hereby
incorporated by reference in their entirety as if physically
present in this specification.
* * * * *