U.S. patent application number 16/601121 was filed with the patent office on 2020-06-11 for methods of purifying bispecific antibodies.
The applicant listed for this patent is NovImmune SA. Invention is credited to Romain DABRE, Jean-Francois DEPOISIER, Nicolas FOUQUE, Egbert MUELLER, Judith VAJDA, Keith WILSON.
Application Number | 20200181287 16/601121 |
Document ID | / |
Family ID | 55527587 |
Filed Date | 2020-06-11 |
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United States Patent
Application |
20200181287 |
Kind Code |
A1 |
FOUQUE; Nicolas ; et
al. |
June 11, 2020 |
METHODS OF PURIFYING BISPECIFIC ANTIBODIES
Abstract
The invention relates to the purification of bispecific
antibodies carrying a different specificity for each binding site
of the immunoglobulin molecule from a mixture of monospecific
antibodies. The bispecific antibodies are composed of a single
heavy chain and two different light chains, one containing a Kappa
constant domain and the other a Lambda constant domain. This
invention in particular relates to the isolation of these
bispecific antibodies from mixtures that contain monospecific
antibodies having two Kappa light chains or portions thereof and
monospecific antibodies having two Lambda light chains or portions
thereof. The invention also provides the methods of efficiently
purifying these bispecific antibodies.
Inventors: |
FOUQUE; Nicolas; (Cernex,
FR) ; DEPOISIER; Jean-Francois; (Mont Saxonnex,
FR) ; WILSON; Keith; (Gwent, GB) ; VAJDA;
Judith; (Leonberg, DE) ; MUELLER; Egbert;
(Darmstadt, DE) ; DABRE; Romain; (Darmstadt,
DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
NovImmune SA |
Geneva |
|
CH |
|
|
Family ID: |
55527587 |
Appl. No.: |
16/601121 |
Filed: |
October 14, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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15068916 |
Mar 14, 2016 |
10457749 |
|
|
16601121 |
|
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62132782 |
Mar 13, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 1/16 20130101; C07K
16/248 20130101; C07K 1/20 20130101; C07K 16/249 20130101; C07K
2317/32 20130101; C07K 2317/31 20130101; C07K 16/00 20130101; C07K
1/36 20130101; C07K 16/2866 20130101; C07K 1/165 20130101; C07K
1/22 20130101; C07K 2317/52 20130101; C07K 16/468 20130101 |
International
Class: |
C07K 16/46 20060101
C07K016/46; C07K 1/36 20060101 C07K001/36; C07K 1/22 20060101
C07K001/22; C07K 1/20 20060101 C07K001/20; C07K 1/16 20060101
C07K001/16; C07K 16/24 20060101 C07K016/24; C07K 16/28 20060101
C07K016/28; C07K 16/00 20060101 C07K016/00 |
Claims
1. A method of purifying a bispecific antibody from a mixture of
antibodies, the method comprising the steps of: (a) providing a
mixed antibody composition that comprises (i) at least one
bispecific antibody with a different specificity in each combining
site and two copies of a single heavy chain polypeptide, a first
light chain with a kappa constant region, and a second light chain
with a lambda constant region (.kappa..lamda.-body); (ii) at least
one monospecific antibody having two lambda light chains or
portions thereof (.lamda. mono-Ab); and (iii) at least one
monospecific antibody having two kappa light chains or portions
thereof (.kappa. mono-Ab); (b) providing a separation means; (c)
contacting the separation means with the mixed antibody composition
under conditions that allow for differential binding to the
separation means by the .kappa..lamda.-body as compared to the
binding to the separation means by the .kappa. mono-Ab and the
.lamda. mono-Ab; and (d) eluting the .kappa..lamda.-body, the
.kappa. mono-Ab, and the .lamda. mono-Ab from the separation means
under conditions that allow for preferential detachment of the
.kappa..lamda.-body from the separation means as compared to
detachment of .kappa. mono-Ab and of the .lamda. mono-Ab from the
separation means.
2. The method of claim 1, wherein the separation means is a resin,
a membrane, a magnetic bead, a particle or a monolith.
3. The method of claim 1, wherein the binding conditions comprise a
variation in pH level, salt level, or both pH level and salt
level.
4. The method of claim 1, wherein the elution conditions comprise a
step variation in pH level, salt level, both pH level and salt
level, Hofmeister ion level, both pH and Hofmeister ion level,
buffer concentration, buffer composition, both buffer concentration
and composition, and combinations thereof.
5. The method of claim 1, wherein the separation means is a mixed
mode chromatography resin.
6. The method of claim 5, wherein the separation means is a
TOYOPEARL.RTM. MX-Trp 650M resin.
7. The method of claim 4, wherein the separation means is a mixed
mode chromatography resin.
8. The method of claim 7, wherein the separation means is a
TOYOPEARL.RTM. MX-Trp 650M resin.
9. The method of claim 1, wherein the separation means is a
hydrophobic interaction chromatography resin.
10. The method of claim 9, wherein the separation means is a
TOYOPEARL.RTM. Butyl 600M resin.
11. The method of claim 4, wherein the separation means is a
hydrophobic interaction chromatography resin.
12. The method of claim 11, wherein the separation means is a
TOYOPEARL.RTM. Butyl 600M resin.
13. The method of claim 1, wherein step (a) comprises performing
affinity chromatography on a biological sample to provide the mixed
antibody composition.
14. The method of claim 13, wherein the biological sample is cell
supernatant.
15. The method of claim 14, wherein the cell is transfected with a
.kappa..lamda. bispecific expression vector comprising one .gamma.1
heavy chain cDNA sequence, one .kappa. light chain cDNA sequence,
and one .lamda. cDNA sequence.
16. The method of claim 13, wherein the affinity chromatography is
Protein A chromatography.
17. The method of claim 13, wherein the affinity chromatography is
not Protein A chromatography.
18. The method of claim 13, wherein the separation means comprises
a combination of a hydrophobic interaction chromatography resin and
a mixed mode chromatography resin.
19. The method of claim 18, wherein the separation means comprises
use of a hydrophobic interaction chromatography resin followed by
use of a mixed mode chromatography resin.
20. The method of claim 19, wherein the hydrophobic interaction
chromatography resin comprises a TOYOPEARL.RTM. Butyl 600M resin,
and wherein the mixed mode chromatography resin comprises a
TOYOPEARL.RTM. MX-Trp 650M resin.
21. The method of claim 18, wherein the separation means comprises
use of a mixed mode chromatography resin followed by use of a
hydrophobic interaction chromatography resin.
22. The method of claim 21, wherein the mixed mode chromatography
resin comprises a TOYOPEARL.RTM. MX-Trp 650M resin, and wherein the
hydrophobic interaction chromatography resin comprises a
TOYOPEARL.RTM. Butyl 600M resin.
Description
RELATED APPLICATIONS
[0001] This application is a continuation application of U.S.
application Ser. No. 15/068,916, filed on Mar. 14, 2016, which
claims priority to and benefit of U.S. Provisional Application No.
62/132,782, filed Mar. 13, 2015 the contents of which are
incorporated herein by reference in their entirety.
FIELD OF THE INVENTION
[0002] The invention relates to the purification of bispecific
antibodies carrying a different specificity for each binding site
of the immunoglobulin molecule from a mixture of monospecific
antibodies. The bispecific antibodies are composed of a single
heavy chain and two different light chains, one containing a Kappa
constant domain and the other a Lambda constant domain. This
invention in particular relates to the isolation of these
bispecific antibodies from mixtures that contain monospecific
antibodies having two Kappa light chains or portions thereof and
monospecific antibodies having two Lambda light chains or portions
thereof. The invention also provides the methods of efficiently
purifying these bispecific antibodies.
BACKGROUND OF THE INVENTION
[0003] An antibody is composed of four polypeptides: two heavy
chains and two light chains. The antigen binding portion of an
antibody is formed by the light chain variable domain (VL) and the
heavy chain variable domain (VH). At one extremity of these domains
six loops form the antigen binding site and also referred to as the
complementarity determining regions (CDR). Three CDRs are located
on the VH domain (H1, H2 and H3) and the three others are on the VL
domain (L1, L2 and L3). During B cell development a unique
immunoglobulin region is formed by somatic recombination known as
V(D)J recombination. The variable region of the immunoglobulin
heavy or light chain is encoded by different gene segments. The
heavy chain is encoded by three segments called variable (V),
diversity (D) and joining (J) segments whereas the light chain
variable is formed by the recombination of only two segments V and
J. A large number of antibody paratopes can be generated by
recombination between one of the multiple copies of the V, D and J
segments that are present in the genome. The V segment encodes the
CDR1 and CDR2 whereas the CDR3 is generated by the recombination
events. During the course of the immune response further diversity
is introduced into the antigen binding site by a process called
somatic hypermutation (SHM). During this process point mutations
are introduced in the variable genes of the heavy and light chains
and in particular into the regions encoding the CDRs. This
additional variability allows for the selection and expansion of B
cells expressing antibody variants with improved affinity for their
cognate antigen.
[0004] The vast majority of immunoglobulins are bivalent and
monospecific molecules carrying the same specificity on both arms
as they are composed of two identical heavy chain polypeptides and
two identical light chain polypeptides. However, it was recognized
very early during the development of hybridoma technology that
hybrid hybridomas can be created by a fusion event between two
hybridomas (Suresh M R et al., Methods Enzymol 1986; 121: 210-228).
These `quadromas` express two different heavy and two different
light chains and therefore produce a variety of different antibody
species resulting from the random pairing of the heavy and light
chains. Amongst these different species, bispecific antibodies
(bsAbs) are generated, carrying a different specificity on each
arm. Another naturally occurring exception is the immunoglobulin of
the IgG4 isotype that is able to undergo heavy chain exchange due
to a less stable dimerization mediated by the hinge region of that
isotype (van der Neut Kolfschoten M et al., Science. 2007
317(5844):1554-7). Although this exchange seems to happen in vivo,
its biological significance remains unclear.
[0005] Monospecific antibodies have emerged as a successful and
attractive class of molecules for therapeutic intervention in
several areas of human disease. However, targeting or neutralizing
a single protein is not always sufficient to achieve efficacy in
certain diseases which limits the therapeutic use of monospecific
antibodies. It is increasingly clear that in a number of
indications neutralizing one component of a biological system is
not sufficient to achieve efficacy. One solution to this problem is
the co-administration of several monospecific antibodies. This
approach is however complicated by regulatory aspects if the
antibodies to be combined have not been previously approved
individually. Moreover, combination approaches are also costly from
a manufacturing perspective. Accordingly, there exists a need for
antibodies and therapeutics that enable targeting of multiple
antigens with a single molecule.
SUMMARY OF THE INVENTION
[0006] The invention allows for the purification of bispecific
antibodies that are undistinguishable in sequence from standard
antibodies. The unmodified nature of the purified antibodies
provides them with favorable manufacturing characteristics similar
to standard monospecific antibodies.
[0007] The methods provided herein are useful for purifying a
variety of bispecific antibodies particularly the bispecific
antibodies referred to herein as ".kappa..lamda.-bodies" (kappa
lambda-bodies), which have a common IgG heavy chain and two
different light chains, one having a kappa (.kappa.) constant
region and the other having a lambda (.lamda.) constant region,
that drive specificity for two independent targets. The methods
provided herein are useful from purifying these bispecific
.kappa..lamda.-bodies from mixtures that contain monospecific
antibodies having two Kappa light chains or portions thereof, also
referred to herein as ".kappa. monospecific antibodies" or ".kappa.
mono-Abs," and monospecific antibodies having two Lambda light
chains or portions thereof, also referred to herein as ".lamda.
monospecific antibodies" or ".lamda. mono-Abs."
[0008] The bispecific antibodies thereof to be purified can be
generated using any of a variety of methods. For example, the
bispecific antibodies and can be generated by (i) isolating two
antibodies having different specificities and sharing the same
variable heavy chain domain but different variable light chains,
for example by using antibody libraries having a fixed heavy chain
or transgenic animals containing a single VH gene; (ii) fusing the
variable heavy chain domain to the constant region of a heavy
chain, fusing one light chain variable domain to a Kappa constant
domain, and fusing the other variable light chain domain to a
Lambda constant domain; and (iii) co-expressing the three chains in
a host cell or cell line, for example, mammalian cells and/or
mammalian cell lines, leading to the assembly and secretion in the
supernatant of a mixture of three antibodies: two monospecific
antibodies and one bispecific antibody carrying two different light
chains. In some antibodies produced using this method, at least a
first portion of the first light chain is of the Kappa type and at
least a portion of the second light chain is of the Lambda type. In
some antibodies produced using this method, the first light chain
includes at least a Kappa constant region. In some antibodies
produced using this method, the first light chain further includes
a Kappa variable region. In some antibodies produced using this
method, the first light chain further includes a Lambda variable
region. In some antibodies produced using this method, the second
light chain includes at least a Lambda constant region. In some
antibodies using this method, the second light chain further
includes a Lambda variable region. In some antibodies using this
method, the second light chain further includes a Kappa variable
region. In some antibodies produced using this method, the first
light chain includes a Kappa constant region and a Kappa variable
region, and the second light chain includes a Lambda constant
region and a Lambda variable region. In some antibodies produced
using this method, the constant and variable framework region
sequences are human.
[0009] The bispecific antibodies made using this method or any
other suitable method known in the art are purified using standard
chromatography techniques used for antibody purification. The
bispecific antibodies generated using this method or any other
suitable method known in the art can also be purified using other
separation techniques, such as by way of non-limiting and
non-exhaustive example, membrane filtration techniques and protein
precipitation techniques. In a preferred embodiment, the bispecific
antibody (or antibodies) is purified using multimodal
chromatography, also known as mixed mode chromatography, or using
hydrophobic interaction chromatography.
[0010] The invention provides methods of purifying a bispecific
antibody from a mixture of antibodies by (a) providing a mixed
antibody composition that comprises at least one bispecific
antibody with a different specificity in each combining site and
two copies of a single heavy chain polypeptide, a first light chain
with a kappa constant region, and a second light chain with a
lambda constant region (.kappa..lamda.-body); and one or more of
the following: (i) at least one monospecific antibody having two
lambda light chains or portions thereof (.lamda. mono-Ab); and/or
(ii) at least one monospecific antibody having two kappa light
chains or portions thereof (.kappa. mono-Ab); (b) providing a
separation means; (c) contacting the separation means with the
mixed antibody composition under conditions that allow for
differential binding to the separation means by the
.kappa..lamda.-body as compared to the binding to the separation
means by the .kappa. mono-Ab and/or the .lamda. mono-Ab; and (d)
eluting the .kappa..lamda.-body, .kappa. mono-Ab, and/or the
.lamda. mono-Ab from the separation means under conditions that
allow for preferential detachment of the .kappa..lamda.-body from
the separation means as compared to detachment of .kappa. mono-Ab
and/or of the .lamda. mono-Ab from the separation means.
[0011] In some embodiments, the mixed antibody composition includes
at least one .kappa..lamda.-body and at least one .lamda. mono-Ab.
In some embodiments, the mixed antibody composition includes at
least one .kappa..lamda.-body and .kappa. mono-Ab. In some
embodiments, the mixed antibody composition includes at least the
following: (i) at least one .kappa..lamda.-body; (ii) at least one
.lamda. mono-Ab; and (iii) .kappa. mono-Ab.
[0012] In some embodiments, the methods use a single separation
means to separate bispecific .kappa..lamda.-bodies from .kappa.
mono-Abs and/or .lamda. mono-Abs by differentially binding each of
the three antibody species. In some embodiments, the methods use a
single separation means to separate bispecific
.kappa..lamda.-bodies from .kappa. mono-Abs and/or .lamda. mono-Abs
through differential elution of each of the three antibody species
from the separation means. In some embodiments, the methods use a
single separation means to separate bispecific
.kappa..lamda.-bodies from .kappa. mono-Abs and/or .lamda. mono-Abs
by differentially binding each of the three antibody species
followed by differential elution of each of the three antibody
species from the separation means.
[0013] In some embodiments, purification of the .kappa..lamda.-body
is performed by sequential binding to affinity chromatography
followed by hydrophobic interaction chromatography. In some
embodiments, purification of the .kappa..lamda.-body is performed
by sequential binding to Protein A chromatography followed by
hydrophobic interaction chromatography. In some embodiments, the
affinity chromatography is Protein A chromatography. In some
embodiments, the affinity chromatography is any art-recognized
affinity chromatography technique other than Protein A
chromatography, such as, by way of non-limiting example,
chromatography techniques based on the use of Protein A mimetics or
other affinity proteins. In some embodiments, the affinity
chromatography, e.g., Protein A chromatography or any
art-recognized affinity chromatography technique other than Protein
A chromatography, is performed on a biological sample. In some
embodiments, the biological sample is cell supernatant. In some
embodiments, the cell is transfected with a .kappa..lamda.
bispecific expression vector that includes at least one .gamma.1
heavy chain cDNA sequence, one .kappa. light chain cDNA sequence,
and one .lamda. cDNA sequence.
[0014] In some embodiments, purification of the .kappa..lamda.-body
is performed by sequential binding to affinity chromatography
followed by multimodal chromatography, also known as mixed mode
chromatography. In some embodiments, purification of the
.kappa..lamda.-body is performed by sequential binding to Protein A
chromatography followed by multimodal chromatography. In some
embodiments, the affinity chromatography is Protein A
chromatography. In some embodiments, the affinity chromatography is
any art-recognized affinity chromatography technique other than
Protein A chromatography, such as, by way of non-limiting example,
chromatography techniques based on the use of Protein A mimetics or
other affinity proteins. In some embodiments, the affinity
chromatography, e.g., Protein A chromatography or any
art-recognized affinity chromatography technique other than Protein
A chromatography, is performed on a biological sample. In some
embodiments, the biological sample is cell supernatant. In some
embodiments, the cell is transfected with a .kappa..lamda.
bispecific expression vector that includes at least one .gamma.1
heavy chain cDNA sequence, one .kappa. light chain cDNA sequence,
and one .lamda. cDNA sequence.
[0015] In some embodiments, purification of the .kappa..lamda.-body
is performed by sequential binding to affinity chromatography
followed by hydrophobic interaction chromatography (HIC) followed
by multi modal (mixed mode) chromatography. In some embodiments,
the affinity chromatography is Protein A chromatography. In some
embodiments, the affinity chromatography is any art-recognized
affinity chromatography technique other than Protein A
chromatography, such as, by way of non-limiting example,
chromatography techniques based on the use of Protein A mimetics or
other affinity proteins. In some embodiments, the affinity
chromatography, e.g., Protein A chromatography or any
art-recognized affinity chromatography technique other than Protein
A chromatography, is performed on a biological sample. In some
embodiments, the biological sample is cell supernatant. In some
embodiments, the cell is transfected with a .kappa..lamda.
bispecific expression vector that includes at least one .gamma.1
heavy chain cDNA sequence, one .kappa. light chain cDNA sequence,
and one .lamda. cDNA sequence.
[0016] In some embodiments, purification of the .kappa..lamda.-body
is performed by sequential binding to Protein A chromatography
followed by multi modal (mixed mode) chromatography followed by
hydrophobic interaction chromatography (HIC). In some embodiments,
the affinity chromatography, e.g., Protein A chromatography or any
art-recognized affinity chromatography technique other than Protein
A chromatography, is performed on a biological sample. In some
embodiments, the biological sample is cell supernatant. In some
embodiments, the cell is transfected with a .kappa..lamda.
bispecific expression vector that includes at least one .gamma.1
heavy chain cDNA sequence, one .kappa. light chain cDNA sequence,
and one .lamda. cDNA sequence.
[0017] In some embodiments, the separation means is a resin, a
membrane, a magnetic bead, a particle or a monolith.
[0018] In some embodiments, the separation means is multimodal
chromatography, also known as mixed mode chromatography. In some
embodiments, the separation means is hydrophobic interaction
chromatography.
[0019] In some embodiments, the separation means is a mixed mode
chromatography resin. In some embodiments, the mixed mode
chromatography resin is a TOYOPEARL.RTM. MX-Trp 650M resin (Tosoh
Bioscience LLC). TOYOPEARL.RTM. MX-Trp-650M is based on the
methacrylic polymer backbone of TOYOPEARL.RTM. media and uses
tryptophan as the active ligand.
[0020] In some embodiments, the separation means is a hydrophobic
interaction chromatography resin. In some embodiments, the
hydrophobic interaction chromatography resin is a TOYOPEARL.RTM.
Butyl 600M resin (Tosoh Bioscience LLC). TOYOPEARL.RTM. Butyl 600M
resin is based on the methacrylic polymer backbone of
TOYOPEARL.RTM. media and includes a butyl ligand.
[0021] In some embodiments, the separation means is a combination
of at least two resins. In some embodiments, the separation means
is a combination of at least two mixed mode chromatography resins.
In some embodiments, the separation means is a combination of more
than two mixed mode chromatography resins, e.g., three or more,
four or more, and/or five or more mixed mode chromatography resins.
In some embodiments, the separation means is a combination of at
least two hydrophobic interaction chromatography resins. In some
embodiments, the separation means is a combination of more than two
hydrophobic interaction chromatography resins, e.g., three or more,
four or more, and/or five or more hydrophobic interaction
chromatography resins. In some embodiments, the separation means is
a combination of at least one mixed mode chromatography resin and
at least one hydrophobic interaction chromatography resin.
[0022] In some embodiments, the separation means includes the use
of a mixed mode chromatography resin followed by the use of a
hydrophobic interaction chromatography resin. In some embodiments,
the separation means includes the use of a TOYOPEARL.RTM. MX-Trp
650M resin (Tosoh Bioscience LLC) followed by the use of a
TOYOPEARL.RTM. Butyl 600M resin (Tosoh Bioscience LLC).
[0023] In some embodiments, the separation means includes the use
of a hydrophobic interaction chromatography resin followed by the
use of a mixed mode chromatography resin. In some embodiments, the
separation mean includes the use of a TOYOPEARL.RTM. Butyl 600M
resin (Tosoh Bioscience LLC) followed by the use of a
TOYOPEARL.RTM. MX-Trp 650M resin (Tosoh Bioscience LLC).
[0024] In some embodiments, the binding and/or elution conditions
include a step variation in the pH level and/or a step variation in
conductivity corresponding to salt concentration variation. In some
embodiments, the binding and/or elution conditions include a step
variation in the inorganic salt concentration such as sodium
chloride (NaCl) concentration or the concentration of other
inorganic salts such as by way of non-limiting and non-exhaustive
example, inorganic salt combinations from the Hofmeister series of
ions, for example, a sulfate. In some embodiments, the methods
include the step of varying the concentration of ammonium sulfate
for binding and/or elution. In some embodiments, the methods
include the further step of determining the purity and proportions
of bispecific antibody, .kappa. mono-Ab and/or .lamda. mono-Ab in
the eluted fraction. This step can be accomplished using any of a
variety of art-recognized techniques, such as by way of
non-limiting and non-exhaustive example, hydrophobic
interaction-high performance liquid chromatography (HIC-HPLC), ion
exchange-high performance liquid chromatography (IEX-HPLC), cation
exchange-high performance liquid chromatography (CEX-HPLC) or
reverse phase-high performance liquid chromatography (RP-HPLC).
[0025] The Examples provided herein demonstrates the feasibility of
using a higher salt or a lower salt step elution to preferentially
elute bispecific antibody from the TOYOPEARL.RTM. MX-Trp-650M mixed
mode chromatography or the hydrophobic interaction resin
TOYOPEARL.RTM. Butyl 600M resin over .kappa. mono-Ab and/or .lamda.
mono-Ab, and additionally, the feasibility of using a combination
of mixed mode chromatography and hydrophobic interaction
chromatography. For example, a lower salt step elution, e.g.,
lowering the concentration of ammonium sulfate, is used to
preferentially elute bispecific antibody from the hydrophobic
interaction resin TOYOPEARL.RTM. Butyl 600M resin over .kappa.
mono-Ab and/or .lamda. mono-Ab.
BRIEF DESCRIPTION OF THE DRAWINGS
[0026] FIGS. 1A, 1B, and 1C are a series of schematic
representations of the structure of different .kappa..lamda.-body
bispecific antibodies composed of two copies of a unique heavy
chain polypeptide and two different light chain polypeptides. FIG.
1A depicts a Kappa variable domain fused to a Kappa constant domain
and a Lambda variable domain fused to Lambda constant domain. FIG.
1B depicts Kappa variable domains fused to a Kappa constant domain
and a Lambda constant domain. FIG. 1C depicts Lambda variable
domains fused to a Kappa constant domain and a Lambda constant
domain.
[0027] FIG. 2 is an illustration depicting that the expression of
tri-cistronic expression vector in CHO cells gives rise to three
antibody products with a theoretical 25:50:25 ratio.
[0028] FIG. 3A is a graph depicting a representative UV absorbance
trace profile of TOYOPEARL.RTM. Butyl 600M using buffer step
gradient elution.
[0029] FIG. 3B is graph depicting an illustration of TOYOPEARL.RTM.
Butyl 600M elution fractions analysis using CEX-HPLC.
[0030] FIG. 3C is a graph depicting a representative UV absorbance
trace profile of TOYOPEARL.RTM. MX-Trp 650 M using NaCl step
gradient elution.
[0031] FIG. 3D is an illustration depicting non-reduced and reduced
SDS-PAGE analysis of TOYOPEARL.RTM. MX-Trp 650M fractions.
[0032] FIG. 3E is a graph depicting HIC-HPLC analysis of
TOYOPEARL.RTM. MX-Trp 650M fractions.
[0033] FIG. 4A is a graph depicting a representative UV absorbance
trace profile of TOYOPEARL.RTM. MX-Trp 650M using NaCl step
gradient elution obtained at larger column scale.
[0034] FIG. 4B is a graph depicting an illustration of
TOYOPEARL.RTM. MX-Trp 650M elution fractions analysis using
CEX-HPLC.
[0035] FIG. 5A is a graph depicting a representative UV absorbance
trace profile of TOYOPEARL.RTM. Butyl 600M using NaCl step gradient
elution at larger column scale.
[0036] FIG. 5B is a graph depicting an illustration of
TOYOPEARL.RTM. Butyl 600M elution fractions analysis using
CEX-HPLC.
DETAILED DESCRIPTION
[0037] The present invention provides methods of purifying
bispecific antibodies that are identical in structure to a human
immunoglobulin. This type of molecule is composed of two copies of
a unique heavy chain polypeptide, a first light chain variable
region fused to a constant Kappa domain and second light chain
variable region fused to a constant Lambda domain. Each combining
site displays a different antigen specificity to which both the
heavy and light chain contribute. The light chain variable regions
can be of the Lambda or Kappa family and are preferably fused to a
Lambda and Kappa constant domain, respectively. This is preferred
in order to avoid the generation of non-natural polypeptide
junctions. However it is also possible to obtain bispecific
antibodies of the invention by fusing a Kappa light chain variable
domain to a constant Lambda domain for a first specificity and
fusing a Lambda light chain variable domain to a constant Kappa
domain for the second specificity (FIGS. 1A-1C). The bispecific
antibodies described herein are also referred to as IgG
.kappa..lamda. antibodies or ".kappa..lamda. bodies," a fully human
bispecific IgG format. This .kappa..lamda.-body format allows the
affinity purification of a bispecific antibody that is
indistinguishable from a standard monospecific antibody, e.g., a
standard IgG molecule, therefore, favorable as compared to previous
formats.
[0038] The locations and/or arrangements of the Kappa light chain
and the Lambda light chain (or portions thereof) shown in these
figures are not intended to be limiting. Those of ordinary skill in
the art will appreciate that the Kappa light chain and the Lambda
light chain (or portions thereof) can also be arranged so as to
produce the mirror-image of the bispecific antibodies shown in
FIGS. 1A-1C. Those of ordinary skill in the art will also
appreciate that the bispecific antibodies that are represented in a
full IgG format in FIGS. 1A-1C can also be generated using other
immunoglobulin isotypes or in other immunoglobulin formats such as
F(ab').sub.2.
[0039] The .kappa..lamda.-bodies are generated by identifying two
antibody Fv regions (each composed by a variable light chain and
variable heavy chain domain) having different antigen specificities
that share the same heavy chain variable domain.
[0040] The .kappa..lamda.-bodies to be purified using the methods
of the invention are generated using any of a variety of methods
for generating antibodies. Numerous methods have been described for
the generation of antibodies and fragments thereof. (See, e.g.,
Antibodies: A Laboratory Manual, Harlow E, and Lane D, 1988, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,
incorporated herein by reference). Fully human antibodies are
antibody molecules in which the sequence of both the light chain
and the heavy chain, including the CDRs 1 and 2, arise from human
genes. The CDR3 region can be of human origin or designed by
synthetic means. Such antibodies are termed "human antibodies" or
"fully human antibodies" herein. Human monospecific antibodies can
be prepared by using the trioma technique; the human B-cell
hybridoma technique (see Kozbor, et al., 1983 Immunol Today 4: 72);
and the EBV hybridoma technique to produce human monoclonal
antibodies (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND
CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96). Human antibodies
may be utilized and may be produced by using human hybridomas (see
Cote, et al., 1983. Proc Natl Acad Sci USA 80: 2026-2030) or by
transforming human B-cells with Epstein Barr Virus in vitro (see
Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY,
Alan R. Liss, Inc., pp. 77-96).
[0041] In some embodiments, the .kappa..lamda.-bodies to be
purified are generated, for example, using antibody libraries in
which the heavy chain variable domain is the same for all the
library members and thus the diversity is confined to the light
chain variable domain. Such libraries are described, for example,
in PCT Publication No. WO 2010/135558 and PCT Publication No. WO
2011/084255, each of which is hereby incorporated by reference in
its entirety. However, as the light chain variable domain is
expressed in conjunction with the heavy variable domain, both
domains can contribute to antigen binding. To further facilitate
the process, antibody libraries containing the same heavy chain
variable domain and either a diversity of Lambda variable light
chains or Kappa variable light chains can be used in parallel for
in vitro selection of antibodies against different antigens. This
approach enables the identification of two antibodies having a
common heavy chain but one carrying a Lambda light chain variable
domain and the other a Kappa light chain variable domain that can
be used as building blocks for the generation of a bispecific
antibody in the full immunoglobulin format of the invention. The
bispecific antibodies to be purified using the methods of the
invention can be of different isotypes and their Fc portion can be
modified in order to alter the bind properties to different Fc
receptors and in this way modify the effectors functions of the
antibody as well as it pharmacokinetic properties. Numerous methods
for the modification of the Fc portion have been described and are
applicable to antibodies of the invention. (See for example Strohl,
W R, "Optimization of Fc-mediated Effector Functions of Monoclonal
Antibodies," Curr. Opin. Biotechnol., 2009 (6):685-91; U.S. Pat.
No. 6,528,624; U.S. Patent Application Publication No.
2009/0191199). The methods of the invention can also be used to
purify bispecific antibodies and antibody mixtures in a
F(ab').sub.2 format that lacks the Fc portion.
[0042] Preferably, the .kappa..lamda.-bodies to be purified have
been optimized for the co-expression of the common heavy chain and
two different light chains into a single cell to allow for the
assembly of a bispecific antibody of the invention. If all the
polypeptides get expressed at the same level and get assembled
equally well to form an immunoglobulin molecule then the ratio of
monospecific (same light chains) and bispecific (two different
light chains) should be 50%. However, it is likely that different
light chains are expressed at different levels and/or do not
assemble with the same efficiency. Furthermore, light chains that
escape assembly into an intact IgG molecule may be secreted into
the cell culture supernatant as "free-light chains". Means to
modulate the relative expression of the different polypeptides to
compensate for their intrinsic expression characteristics or
different propensities to assemble with the common heavy chain
include, by way of non-limiting examples, the use of promoter(s)
with variable strength(s), the use of internal ribosome entry sites
(IRES) featuring different efficiencies or other types of
regulatory elements that can act at transcriptional or
translational levels as well as acting on mRNA stability. The
modulation of the expression can also be achieved by multiple
sequential transfections of cells to increase the copy number of
individual genes expressing one or the other light chain and thus
modify their relative expressions.
[0043] The co-expression of the heavy chain and two light chains
generates a mixture of three different antibodies secreted into the
cell culture supernatant: two monospecific bivalent antibodies and
one bispecific bivalent antibody. The latter has to be purified
from the mixture to obtain the .kappa..lamda.-body of interest.
Multi modal chromatography or mixed mode chromatography facilitates
the purification of the .kappa..lamda.-body due to various
mechanisms of interactions such as, by way of non-limiting example,
ion exchange characteristics and hydrophobic characteristics, which
confer high binding capacities and allow efficient purification of
bispecific antibodies, including purification of
.kappa..lamda.-bodies. The multi modal or mixed mode chromatography
methods are efficient because multiple modes of chromatography are
utilized simultaneously. Hydrophobic chromatography facilitates the
purification of the .kappa..lamda.-body due to the hydrophobic
characteristics which allow the efficient purification of specific
antibodies. The combination of multi modal or mixed mode
chromatography followed by hydrophobic chromatography facilitates
the purification of the .kappa..lamda.-body due to multiple
mechanisms of interactions applied sequentially, thus allowing even
more efficient purification of bispecific antibodies than either
mechanism alone.
[0044] The co-expression of the three chains led to the assembly of
three different antibodies: two monospecific and one bispecific
antibodies. Their theoretical relative ratios should be 1:1:2
provided the expression levels and assembly rates are similar for
both light chains. The bispecific antibodies were purified using
Protein A affinity chromatography procedure followed by either
multi modal chromatography or hydrophobic chromatography or Protein
A affinity chromatography followed by multi modal chromatography
and hydrophobic chromatography.
[0045] Previous approaches to produce and purify bispecific
antibody formats aimed at forcing the production of a homogenous
bispecific molecule using different antibody engineering approaches
were done at the expense of productivity, scalability and stability
of the product. The methods described herein provide efficient
means to purify bispecific antibodies.
[0046] In contrast to previous approaches to produce and purify
bispecific antibody formats, the methods provided herein use a
single separation means to separate bispecific
.kappa..lamda.-bodies from .kappa. mono-Abs and/or .lamda.
mono-Abs, by either differentially binding each of the three
antibody species or through differential elution of each of the
three antibody species from the separation means.
[0047] The methods provided herein are the first to use processes
such as mixed mode chromatography and/or hydrophobic interaction
chromatography and/or a combination of both these chromatography
methods to separate bispecific antibodies having two different
light chains, one containing a Kappa constant domain and the other
a Lambda constant domain from monospecific antibodies having two
Kappa light chains or portions thereof and monospecific antibodies
having two Lambda light chains or portions thereof. In contrast,
previous approaches such as, e.g., those in PCT Publication No. WO
2013/088259, were designed to remove intact, full length bispecific
antibodies from non-intact antibodies such as the free light chains
shown in FIG. 2. Thus, the methods provided herein are advantageous
over previous approaches.
EXAMPLES
Example 1: Purification of Bispecific Antibodies Utilizing
Hydrophobic Interaction Chromatography
[0048] The .kappa..lamda.-body is a novel bispecific IgG format
that includes a common IgG1 heavy chain and two different light
chains that drive specificity for two independent targets. In order
to allow for an efficient purification protocol applicable to large
scale industrial processes, the format requires that one light
chain contains a .kappa. constant region whilst the other contains
a .lamda. constant region. (See FIGS. 1A-1C).
[0049] In order to produce .kappa..lamda.-body, the common heavy
chain and two light chains are expressed in CHO cells using a
triple gene expression vector. This vector format allows for the
construction of three products: monospecific .kappa. antibody
(.kappa. mono-Ab), bispecific .kappa..lamda.-body and monospecific
.lamda. antibody (.lamda. mono-Ab). The theoretical product ratio
is 25:50:25. (See FIG. 2).
[0050] In these studies, purification of this .kappa..lamda.-body
format is performed by sequential binding to Protein A affinity
chromatography followed by the hydrophobic interaction resin
TOYOPEARL.RTM. Butyl 600M.
[0051] The studies provided herein demonstrate the successful
separation of .kappa..lamda.-body from monospecific lambda and
monospecific kappa antibodies (mono-Abs) using buffer step elution
chromatography.
[0052] Start Material:
[0053] The clarified 25 L wave bag fermentation supernatant of a
CHO cell transfected with a .kappa..lamda. bispecific expression
vector (containing one .gamma.1 heavy chain cDNA, one .kappa. light
chain cDNA and one .lamda. light chain cDNA) was used as the
starting material for Protein A chromatography followed by
hydrophobic interaction chromatography.
[0054] Step:
[0055] .kappa..lamda.-body bispecific IgG antibody was purified
using hydrophobic interaction chromatography (HIC) media (Tosoh
Bioscience). After dilution 1:1 of the sample in 100 mM sodium
phosphate 1M ammonium sulfate pH 7.0 buffer (equilibration buffer),
the column was loaded at 10 mg/mL. After a wash step with
equilibration buffer (5 column volumes), a step-elution was
performed using a 10 mM Sodium Phosphate pH 7.0 buffer (60% and 75%
in two sequential steps) (FIG. 3A). The eluted fractions were
collected and analyzed by UV absorbance measurement at 280 nm
(using a NanoDrop UV-Vis spectrophotometer, Thermo Scientific) in
order to determine product recovery. Cation exchange performance
liquid chromatography (CEX-HPLC) was performed in order to
determine the ability of the purification process to separate the
.kappa..lamda.-body bispecific IgG from the two monospecific
antibody by-products (FIG. 3B).
Example 2: Purification of Bispecific Antibodies Utilizing
Multimodal Mixed Mode Chromatography
[0056] As described in Example 1, the .kappa..lamda.-body is a
novel bispecific IgG format that includes a common IgG1 heavy chain
and two different light chains that drive specificity for two
independent targets. In order to allow for an efficient
purification protocol applicable to large scale industrial
processes, the format requires that one light chain contains a
.kappa. constant region whilst the other contains a .lamda.
constant region. (See FIGS. 1A-1C).
[0057] In order to produce .kappa..lamda.-body, the common heavy
chain and two light chains are expressed in CHO cells using a
triple gene expression vector. This vector format allows for the
construction of three products: monospecific .kappa. antibody
(.kappa. mono-Ab), bispecific .kappa..lamda.-body and monospecific
.lamda. antibody (.lamda. mono-Ab). The theoretical product ratio
is 25:50:25. (See FIG. 2).
[0058] In these studies, purification of this .kappa..lamda.-body
format is performed by sequential binding to Protein A affinity
chromatography followed by the mixed mode chromatography resin
TOYOPEARL.RTM. MX-Trp 650 M.
[0059] The studies provided herein demonstrate the successful
separation of .kappa..lamda.-body from monospecific lambda and
monospecific kappa antibodies (mono-Abs) using NaCl step elution
chromatography.
[0060] Start Material:
[0061] The clarified 25 L wave bag fermentation supernatant of a
CHO cell transfected with a .kappa..lamda. bispecific expression
vector (containing one .gamma.1 heavy chain cDNA, one .kappa. light
chain cDNA and one .lamda. light chain cDNA) was used as the
starting material for Protein A chromatography followed by multi
modal (mixed mode) interaction chromatography.
[0062] Step:
[0063] .kappa..lamda.-body bispecific IgG antibody was purified
using multi modal (mixed mode) chromatography media (Tosoh
Bioscience). After column loading at 25 mg/mL and a wash step with
100 mM Sodium Phosphate, pH 6.0. (5 column volumes), a NaCl
step-elution was performed using a 100 mM Sodium Phosphate pH 6.0
buffer (15% and 100% of 500 mM NaCl buffer in two sequential steps
(FIG. 3C). The flow through and eluted fractions were collected and
analyzed by absorbance measurement at 280 nm (using a NanoDrop
UV-Vis spectrophotometer, Thermo Scientific) in order to determine
product recovery, reduced and non-reduced SDS-PAGE (using
Invitrogen Novex NuPAGE 12-well 4-20% gradient gels following
manufacturer's guidelines) in order to determine the purity and
composition of the samples (FIG. 3D) and hydrophobic
interaction-high performance liquid chromatography (HIC-HPLC) (FIG.
3E); in order to determine the ability of the purification process
to separate the .kappa..lamda.-body bispecific IgG from the two
monospecific antibody by-products.
[0064] As shown by the UV absorbance trace (red) in FIG. 3C, the
step elutions applied to the TOYOPEARL.RTM. MX-Trp 650M
chromatography mixed mode resin allowed for the sequential
separation of three fractions. Reduced and non-reduced SDS-PAGE
analysis of fractions collected during the mixed mode purification,
shown in FIG. 3D revealed the high purity of the eluted fraction
(2.sup.nd peak) at 15% of NaCl containing the .kappa..lamda.-body
whereas the monospecific .lamda. mono-Ab and .kappa. mono-Ab IgGs
were separated and collected in the non-retained fraction (1.sup.st
peak) for the .lamda..lamda. and the 100% NaCl step fraction
(3.sup.rd peak) for the .kappa..kappa. respectively. The three
fractions were further characterized by HIC-HPLC analysis and
subsequent integration of the peak areas of the HIC-HPLC
chromatograms (FIG. 3E). The results summarized in Table 1 were in
accordance with the SDS-PAGE analysis, demonstrating the high
purity of the .kappa..lamda.-body (96%) in the 2.sup.nd eluted
fraction at 15% NaCl.
TABLE-US-00001 TABLE 1 UV peak integration of HIC-HPLC analysis of
TOYOPEARL .RTM. MX Trp-650M collected bound fractions Fractions
.kappa. mono-Ab % .lamda. mono-Ab % .kappa..lamda.-body % Flow
through 0 99.5 0.5 Step 15% 2 2 96 Step 100% 85 15 0
Example 3: Purification of Bispecific Antibodies Utilizing Multi
Modal Mixed Mode Chromatography Followed by Hydrophobic
Chromatography
[0065] As described in Example 1, the .kappa..lamda.-body is a
novel bispecific IgG format that includes a common IgG1 heavy chain
and two different light chains that drive specificity for two
independent targets. In order to allow for an efficient
purification protocol applicable to large scale industrial
processes, the format requires that one light chain contains a
.kappa. constant region whilst the other contains a .lamda.
constant region. (See FIGS. 1A-1C).
[0066] In order to produce .kappa..lamda.-body, the common heavy
chain and two light chains are expressed in CHO cells using a
triple gene expression vector. This vector format allows for the
construction of three products: monospecific .kappa. antibody
(.kappa. mono-Ab), bispecific .kappa..lamda.-body and monospecific
.lamda. antibody (.lamda. mono-Ab). The theoretical product ratio
is 25:50:25. (See FIG. 2).
[0067] In this example, purification of this .kappa..lamda.-body
format is performed by sequential binding to Protein A affinity
chromatography followed multi modal (mixed mode) chromatography by
the TOYOPEARL.RTM. MX-Trp 650M mixed mode resin followed by
hydrophobic interaction chromatography using the TOYOPEARL.RTM.
Butyl 600M resin.
[0068] Step:
[0069] Protein A affinity eluate containing .kappa..lamda.-body
bispecific IgG antibody was purified using mixed mode
chromatography media (Tosoh Bioscience) followed by hydrophobic
interaction chromatography (HIC) media (Tosoh Bioscience). The
TOYOPEARL.RTM. Butyl 600M column was loaded with the eluted sample
purified with the mixed mode column (corresponding to fraction 2 in
FIG. 4A) and diluted 1:1 in 100 mM sodium phosphate 1M ammonium
sulfate pH 7.0 buffer) and after a wash step, a buffer step elution
was performed to reduce the level of ammonium sulfate (FIG. 5A).
The eluted fractions were collected and analyzed by UV absorbance
measurement at 280 nm (using a NanoDrop UV-Vis spectrophotometer,
Thermo Scientific) in order to determine product recovery. cation
exchange-high performance liquid chromatography (CEX-HPLC) was
performed in order to determine the ability of the purification
process to separate the .kappa..lamda.-body bispecific IgG from the
two monospecific antibody by-products (FIG. 5B).
[0070] As shown by the UV absorbance trace (red) in FIG. 4A, the
step elutions applied to the TOYOPEARL.RTM. MX-Trp 650M mixed mode
chromatography resin allowed for the sequential separation of three
fractions. CEX-HPLC analysis of fractions collected during the
mixed mode purification confirmed the high purity of the
eluted--.kappa..lamda.-body (FIG. 4B). The main fraction (pool 2)
eluted at 20% NaCl in acetate pH 6.0 buffer was further loaded onto
the TOYOPEARL.RTM. butyl 600M hydrophobic interaction
chromatography resin. The results are depicted in FIG. 5A.
[0071] As shown by the UV absorbance trace (red) in FIG. 5A, the
step elutions applied to the TOYOPEARL.RTM. Butyl 600M hydrophobic
interaction chromatography resin allowed for the sequential
separation of two fractions. CEX-HPLC (FIG. 5B) analysis of
fractions confirmed the separation and purity of the
.kappa..lamda.-body eluted in the 2.sup.nd fraction in a
step-elution performed using a 10 mM Sodium Phosphate pH 7.0 buffer
(75%) and the remaining .kappa..kappa.-monospecific eluted in the
first fraction in a step-elution performed using a 10 mM Sodium
Phosphate pH 7.0 buffer (54%). The high purity of the main fraction
(pool 2) corresponding to the .kappa..lamda.-body was measured to
be >95%.
[0072] The data presented in these working examples demonstrates
the feasibility of using a multimodal (mixed mode) chromatography
or hydrophobic interaction chromatography or combination of
multimodal (mixed mode) chromatography and hydrophobic interaction
chromatography to purify bispecific antibodies from an IgG mixture,
including .kappa..lamda.-bodies.
[0073] HIC-HPLC Method:
[0074] In order to determine the relative proportions of the
.lamda. mono-Ab, .kappa. mono-Ab and the .kappa..lamda.-body in a
sample mixture, a HIC-HPLC (hydrophobic interaction
chromatography-high performance liquid chromatography) assay using
a Dionex ProPac HIC-10 column was used. A descending gradient
between 85 to 25% of ammonium sulfate was applied onto the column
after the loading of the sample in order to elute the 3 species
with high resolution, the .kappa. mono-Ab eluting first, followed
by the .kappa..lamda.-body and finally the .lamda. mono-Ab. Peak
area integration of the UV trace monitored at 280 nm was performed
in order to determine the amount of each species.
[0075] CEX-HPLC Method:
[0076] This cation exchange-high performance liquid chromatography
(CEX-HPLC) method was used to determine the proportions of
monospecific and bispecific antibody in purified samples. The
CEX-HPLC method allows for the separation of protein variants
according to their charge distribution. Samples were prepared to
load 50 .mu.g onto A BioMab NP5-SS column (Agilent) and a linear
gradient of 10 mM sodium phosphate, 500 mM NaCl, pH 6.5 (from 0% to
100% NaCl concentration) at a flow rate of 0.8 mL/min was applied
in order to separate the different antibody products. UV detection
at 214 nm was employed to monitor sample elution. The three
populations were identified (according to reference standards) and
analyzed according to their percentage relative area. The
percentage of each isoform was determined by calculating the peak
area of each component relative to the total peak area.
OTHER EMBODIMENTS
[0077] While the invention has been described in conjunction with
the detailed description thereof, the foregoing description is
intended to illustrate and not limit the scope of the invention,
which is defined by the scope of the appended claims. Other
aspects, advantages, and modifications are within the scope of the
following claims.
* * * * *