METHODS OF PURIFYING SMALL MODULAR
IMMUNOPHARMACEUTICAL PROTEINS
REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional Patent Application No.
61/159,347, filed March 11, 2009, the contents of which are hereby incorporated by reference
in their entireties.
BACKGROUND OF THE INVENTION
[0002] Typically, when proteins are produced for pharmaceutical uses, contaminants
must be removed fi"om protein preparations before they can be used in diagnostic
applications, therapeutic applications, applied cell biology, and functional studies. For
example, protein preparations harvested from cultured cells often contain unwanted
components, such as high molecular weight (HMW) aggregates of the protein produced by
the cells. The high molecular weight aggregates can adversely affect product safety by
causing complement activation or anaphylaxis upon administration. Further, aggregates may
hinder manufacturing processes by causing decreased product yield, peak broadening, and
loss of activity.
[0003] Small modular immunopharmaceutical (SMIP^*^) proteins belong to a
relatively new class of pharmaceutical proteins as compared to antibodies and other
therapeutic proteins. Therefore, the purification of SMIP™* proteins is particularly
challenging due to lack of familiarity with this type of protein. In addition, SMIP^^ proteins
have a high propensity to aggregate. For example, the percentage of HMW aggregates in cell
culture may be as high as 50-60%.
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SUMMARY OF THE INVENTION
[0004] The present invention provides, among other things, effective methods of
purifying proteins containing HMW aggregates. The present invention encompasses the
discovery that small modular immunopharmaceutical proteins can be purified from protein
preparations containing high percentage of HMW aggregates (e.g., more than 50-60%) using
no more than three chromatography steps. Thus, inventive methods according to the
invention reduce the number of colunm steps resulting in significantly reduced process time
and improved product yield. The present invention is particularly useful for purifying small
modular immunopharmaceutical proteins. The methods of the invention may also be used to
purify other proteins, in particular, those proteins having a propensity to aggregate.
[0005] In one aspect, the present invention provides a method of purifying a small
modular immunopharmaceutical protein from a protein preparation containing high
molecular weight aggregates including a step of subjecting the protein preparation to
hydroxyapatite chromatography under an operating condition such that the purified small
modular immunopharmaceutical protein contains less than 4% aggregates (e.g., less than
3.5%, 3%, 2.5%, 2%, 1.5%, 1%, 0.8%, 0.6%, 0.5%, 0.4%, 0.2%, or 0.1%). In some
embodiments, a method according to the invention involves no more than 3 chromatography
steps.
[0006] In some embodiments, the operating condition includes eluting the small
modular immunopharmaceutical protein from a hydroxyapatite chromatography column in a
phosphate buffer. In some embodiments, the phosphate buffer is endotoxin-free. In some
embodiments, the phosphate buffer is depyrogenated. In some embodiments, the phosphate
buffer comprises sodium phosphate, potassium phosphate, and/or lithium phosphate. In some
embodiments, a suitable phosphate buffer contains sodium phosphate at a concentration
ranging from 1 mM to 50 mM. In some embodiments, a suitable phosphate buffer further
contains sodium chloride at a concentration ranging from 100 mM to 2.5 M. In some
embodiments, a suitable phosphate buffer contains sodium phosphate at a concentration
ranging fi-om 2 mM to 32 mM and sodium chloride at a concentration ranging from 100 mM
to 1.6 M. In some embodiments, a suitable phosphate buffer has a pH ranging fi-om 6.5 to
8.5.
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[0007] In some embodiments, the operating condition includes eluting the small
modular immunopharmaceutical protein from a hydroxyapatite chromatography column by a
NaCl gradient. In some embodiments, the operating condition includes eluting the small
modular immunopharmaceutical protein from a hydroxyapatite chromatography column by a
NaCl step elution method. In some embodiments, the operating condition includes eluting
the small modular immunopharmaceutical protein from a hydroxyapatite chromatography
column by a phosphate gradient (e.g., a linear phosphate gradient).
[0008] In some embodiments, the hydroxyapatite chromatography uses a column
containing ceramic hydroxyapatite Type I or Type II resins. In some embodiments, the
colunrn contains ceramic hydroxyapatite Type I resins. In some embodiments, the resins
suitable for the hydroxyapatite chromatography are 1 )j,m to 1,000 p.m in diameter. In some
embodiments, the resins suitable for the hydroxyapatite chromatography are 10 jxm to 100
p,m in diameter.
[0009] In some embodiments, the method further includes a step of purifying the
protein preparation by affinity chromatography before the step of hydroxyapatite
chromatography. In some embodiments, the affinity chromatography step uses a protein
absorbent that binds to a constant immunoglobulin domain. In some embodiments, the
affinity chromatography uses a protein absorbent that binds to a variable immunoglobulin
domain. In some embodiments, a protein absorbent suitable for the invention binds to a VH?
domain or a domain homologous to VH3 (e.g., a domain from the VH3 family). In some
embodiments, a protein absorbent suitable for the invention comprises protein A. In some
embodiments, the affinity chromatography step uses a MabSelect™^ rProtein A resin column.
In some embodiments, a method according to the invention further includes a step of adding
an additive (e.g., PEG and/or other nonionic organic polymers) to promote binding to protein
sorbents.
[0010] In some embodiments, the step of affinity chromatography comprises washing
an affinity chromatography column using a washing buffer comprising Hepes, sodium
chloride, calcium chloride, arginine, Tris, magnesium chloride, histidine, urea, imidazole, one
or more organic solvents (e.g., ethanol, methanol, propylene glycol, ethylene glycol,
propanol, isopropanol, and butanol), and/or detergents (e.g., ionic or nonionic). In some
embodiments, the step of affinity chromatography comprises eluting the small modular
immunopharmaceutical protein fh)m an affinity chromatography column using an elution
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buffer comprising Hepes, phosphoric acid, glycine, glycyiglycine, magnesium chloride, urea,
propylene glycol, ethylene glycol, one or more organic acids (e.g., acetic acid, citric acid,
formic acid, lactic acid, tartaric acid, malic acid, malonic acid, phthalic acid and salicyclic
acid), and/or arginine. In some embodiments, the elution buffer further comprises a salt
selected from the group consisting of sodium chloride, potassium chloride, calcium chloride,
magnesium chloride, and combinations thereof In some embodiments, the sah is at a
concentration ranging from 1 mM to 1 M. In certain embodiments, the salt is at a
concentration ranging fixim 1 mM to 500 mM. In certain embodiments, the salt is at a
concentration ranging fixjm 1 mM to 100 mM.
[0011] In some embodiments, a method according to the invention further comprises
a step of piuifying the protein preparation by anion exchange chromatography using an anion
exchange chromatography resin. In certain embodiments, a method according to the
invention further comprises a step of purifying the protein preparation by anion exchange
chromatography after the affinity chromatography but before the hydroxyapatite
chromatography step. In some embodiments, a method according to the invention further
comprises a step of adding an additive to enhance binding of the small modular
immunopharmaceutical protein and/or impurities to the anion exchange chromatography
resin. In some embodiments, the additive added induces precipitation of one or more
contaminants or impurities from the protein preparation. In some embodiments, the
precipitated contaminants are removed from the protein preparation by filtration. In some
embodiments, a suitable additive is or contains a nonionic organic polymer (e.g.,
polyethylene glycol (PEG), polypropylene glycol, cellulose, dextran, starch, and/or
poljrvinylpyrrolidone).
[0012] In some embodiments, the method fiuther comprises a step of applying the
protein preparation to a depth filter before the affinity or anion exchange chromatography.
[0013] In some embodiments, the method further comprises one or more filtration
steps. In some embodiments, the one or more filtration steps comprise a virus retaining
filtration step. In some embodiments, the one or more filtration steps comprise ultrafiltration
and/or diafiltration steps.
[0014] In some embodiments, the protein preparation is prepared from cultured
bacterial cells, mammalian cells, plant cells, yeast cells, insect cells, cell-free medium,
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transgenic animals or plants. In some embodiments, the protein preparation is a cell culture
medium preparation. In some embodiments, the culture medium preparation contains the
small modular immunopharmaceutical protein secreted from cultured cells. In certain
embodiments, the cultured cells are CHO cells. In certain embodiments, the culture medium
preparation is prepared from a large scale bioreactor. In some embodiments, the protein
preparation to be purified contains a cell extract. In some embodiments, the protein
preparation to be purified is prepared from inclusion bodies.
[0015] In another aspect, the present invention provides methods of purifying a small
modular immimopharmaceutical protein fix)m a protein preparation containing high
molecular weight aggregates by subjecting the protein preparation to (a) affinity
chromatography and/or ion exchange chromatography (e.g., one or two ion exchange
chromatography steps), and (b) hydroxyapatite chromatography under operating conditions
such that the purified small modular immunopharmaceutical protein contains less than 4%
(e.g., less than 3.5%, 3%, 2.5%, 2%, 1.5%, 1%, 0.8%, 0.6%, 0.5%, 0.4%, 0.2%, 0.1%)
aggregates. In some embodiments, the protein preparation is subjected to (al) affinity
chromatography, (a2) ion exchange chromatography, and (b) hydroxyapatite
chromatography. In some embodiments, the protein preparation is subjected to (al) cation
exchange chromatography, (a2) anion exchange chromatography, and (b) hydroxyapatite
chromatography. In some embodiments, the affinity chromatography is protein A
chromatography. In some embodiments, the ion exchange chromatography is anion or cation
exchange chromatography. In some embodiments, the ion exchange chromatography resin is
selected from the group consisting of Q Sepharose ' FF, Q Sepharose XL, DEAE
Sepharose™ FF, POROS® HQ50, Toyopearl® DEAE, Toyopearl® GigaCap Q-650M,
Toyopearl® DEAE-650M, Capto™ Q, Capto™ DEAE, and tentacle anion exchange
chromatography (e.g., Fractogel® TMAE HiCap (M)™, Fractogel® TMAE (S)™, or
Fractoprep® TMAE™). In some embodiments, the anion exchange chromatography resin is
a charged membrane adsorber (e.g.. Mustang® Q, Mustang® E, Sartobind® and/or
Chromasorb®). In some embodiments, the ion exchange chromatography resin is a charged
monolithic support (e.g., CIM®-DISK). In particular embodiments, the affinity
chromatography is MabSelect ^^ rProtein A affinity chromatography, the ion exchange
chromatography is tentacle anion exchange chromatography, and the hydroxyapatite
chromatography is Type I ceramic hydroxyapatite chromatography. In some embodiments, a
method according to the invention involves no more than 3 chromatography steps. In some
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embodiments, a method according to the present invention further includes a step of stripping
and/or regenerating one or more chromatography columns for reuse.
[0016] In some embodiments, the present invention can be used to purify a protein
preparation containing more than 5% (e.g., more than 10%, 20%, 30%, 40%, 50%, 60%,
70%, or more) high molecular weight aggregates. In some embodiments, the present
invention can be used to purify a protein preparation containing less than 70% (e.g., less than
60%, 50%, 40%, 30%, 20%, 15%, 10%, or 5%) high molecular weight aggregates. In some
embodiments, the present invention can be used to purify a protein preparation containing 4-
70% (e.g., 4-60%, 4-50%, 4-40%, 4-30%, 4-20%, 4-15%, 4-10%) high molecular weight
aggregates.
[0017] In some embodiments, the present invention is used to purify a small modular
immunopharmaceutical protein that binds specifically to CD20. In some embodiments, the
present invention is used to purify a small modular immunopharmaceutical protein that
comprises an amino acid sequence having at least 80% identity to any one of SEQ ID NOs; 1-
59 and 67-76.
[0018] In still another aspect, the present invention is used to purify a protein from a
protein preparation containing more than 20% (e.g., more than 25%, 30%, 35%, 40%, 45%,
50%, 55%, 60%, 70%, or more) high molecular weight aggregates including a step of
subjecting the protein preparation to hydroxyapatite chromatography under an operating
condition such that the purified protein contains less than 4% (e.g., less than 3.5%, 3%, 2.5%,
2%, 1.5%, 1%, 0.8%, 0.6%, 0.5%, 0.4%, 0.2%, or 0.1%) aggregates. In certain
embodiments, the protein preparation contains more than 60% high molecular weight
aggregates.
[0019] In certain embodiments, the operating condition comprises eluting the protein
from a hydroxyapatite chromatography column in a phosphate buffer. In some embodiments,
the phosphate buffer is endotoxin-free. In some embodiments, the phosphate buffer is
depyrogenated. In some embodiments, the phosphate buffer comprises sodium phosphate,
potassium phosphate, and/or Hthium phosphate. In some embodiments, the phosphate buffer
comprises sodium phosphate at a concentration ranging from 1 mM to 50 mM. In some
embodiments, the phosphate buffer further comprises sodium chloride at a concentration
ranging fi-om 100 mM to 2.5 M. In particular embodiments, the phosphate buffer comprises
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sodium phosphate at a concentration ranging from 2 mM to 32 mM and sodium chloride at a
concentration ranging from 100 mM to 1.6 M. In some embodiments, the phosphate buffer
has a pH ranging from 6.5 to 8.5.
[0020] In some embodiments, the protein to be purified contains a small modular
immunopharmaceutical polypeptide.
[0021] The present invention further provides a small modular
immunopharmaceutical protein purified using methods described herein. In addition, the
present invention provides pharmaceutical compositions comprising a small modular
immunopharmaceutical protein and a pharmaceutically acceptable carrier, wherein the small
modular immunopharmaceutical protein comprises less than 4% (e.g., less than 3.5%, 3%,
2.5%, 2%, 1.5%, 1%, 0.8%, 0.6%, 0.5%, 0.4%, 0.2%, or 0.1%) aggregates.
[0022] In this application, the use of "or" means "and/or" imless stated otherwise. As
used in this appUcation, the term "comprise" and variations of the term, such as "comprising"
and "comprises," are not intended to exclude other additives, components, integers or steps.
As used in this application, the terms "about" and "approximately" are used as equivalents.
Any numerals med in this application with or without about/approximately are meant to
cover any normal fluctuations appreciated by one of ordinary skill in the relevant art.
[0023] Other features, objects, and advantages of the present invention are apparent in
the detailed description, drawings and claims that follow. It should be understood, however,
that the detailed description, the drawings, and the claims, while indicating embodiments of
the present invention, are given by way of illustration only, not limitation. Various changes
and modifications within the scope of the invention will become apparent to those skilled in
the art.
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BRIEF DESCRIPTION OF THE DRAWING
[0024] The drawings are for illustration purposes only, not for limitation.
[0025] Figure 1 depicts an exemplary structure of an anti-CD20 small modular
immunopharmaceutical protein.
[0026] Figure 2 illustrates exemplary configurations of SMIP^'^ molecules that may
be in solution.
[0027] Figure 3A-3C illustrate that various domain-swapping mechanisms may lead
to the formation of high molecule weight aggregates of SMIP™* molecules, such as trimers,
tetramers or multimers.
[0028] Figure 4 depicts a schematic diagram illustrating an exemplary cell culture and
harvest procedure.
[0029] Figure 5 depicts exemplary daily titer measurements (^g/mL) of the
production bioreactor of TRU-015 produced by two different CHO cell clones over a 12-14
day culture period. Peak titer values were obtained between days 12 and 14 of production
bioreactor growth. Peak titer values ranged from 1500 to 3000 |i.g/mL.
[0030] Figure 6 depicts an exemplary design of high throughput screening using
batch binding mechanism.
[0031] Figure 7 depicts an exemplary design of Protein A column operation and high
throughput screening model.
[0032] Figure 8 depicts exemplary Protein A high-throughput screen results.
[0033] Figure 9. (A) Summary of exemplary variables tested in high-throughput
screens for ceramic hydroxyapatite chromatography. (B) Exemplary contour plot
demonstrating percent HMW compounds when varying concentrations of phosphate and
NaCl were used during cHA purification. (C) Exemplary HMW vs. Log Kp plot
demonstrating that at a Kp of approximately 10 (or log Kp of 1), most of the HMW
compounds have been removed from the sample.
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[0034] Figure 10 depicts an exemplary alternative screening using a cHA column and
a NaCl gradient elution for the development of the cHA chromatography step.
[0035] Figure 11 depicts an exemplary typical cHA chromatogram.
[0036] Figure 12 depicts an exemplary TRU-015 purification process.
[0037] Figure 13 depicts an exemplary comparison of reduction of amount of HMW
aggregates by MabSelect Protein A affinity chromatography with that by CEX.
[0038] Figure 14 depicts exemplary results illustrating protein product binding
capacities of CEX resins.
[0039] Figure 15 depicts exemplary results illustrating CEX peaks using 25 vs. 75
mg/mLr loading challenge.
[0040] Figure 16 depicts an exemplary result illustrating effective removal of HMW
using an AEX colunm. The collected pool was 88% pure with >95% yield of the
"monomeric" SMIP™ protein.
DETAILED DESCRIPTION OF THE INVENTION
[0041] The present invention, among other things, provides methods of purifying or
recovering proteins, in particular, small modular immunopharmaceutical proteins, from
protein preparations containing HMW aggregates and other impurities based on
hydroxyapatite chromatography. In some embodiments, the hydroxyapatite chromatography
is used in combination with affinity chromatography and/or ion exchange chromatography.
In some embodiments, inventive methods of the present invention fiirther include one or
more filtration steps to fiirther remove viral contaminants, to concentrate proteins, and/or
buffer exchange. In some embodiments, the methods of the invention have no more than
three chromatography steps (e.g., two chromatography steps, or three chromatography steps).
In some embodiments, the methods of the invention have no more than 3 filtration steps (e.g.,
two filtration steps, three filtration steps).
[0042] As described in the Examples section, the present inventors have discovered
suitable operating conditions for hydroxyapatite chromatography, affinity chromatography
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and/or ion exchange chromatography that allow effective removal of HMW aggregates and
other impurities (e.g., DNA, host cell protein, viruses, and other contaminants) from protein
preparations. In some embodiments, the percentage of HMW aggregates can be reduced
from more than 20% (e.g., 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70% or more) in a
starting preparation to less than 4% (e.g., less than 3.5%, 3.0%, 2.5%, 2.0%, 1.5%, 1.0%,
0.8%, 0.6%, 0.4%, 0.2%, 0.1%) in the purified protein product. In some embodiments, the
HMW aggregates in a starting preparation can be reduced by at least about 5 fold, or at least
about 10 fold, or at least about 20 fold, or at least about 30 fold, or at least about 40 fold, or at
least about 50 fold, or at least about 60 fold, or at least about 70 fold, or at least about 80 fold,
or at least about 90 fold, or at least about 100 fold. Additionally or alternatively, the
percentage of other contamination (e.g., HCP) in the purified protein is not more than about
10,000 ppm, or not more than about 5000 ppm, or not more than about 2500 ppm, or not
more than about 400 ppm, or not more than about 360 ppm, or not more than about 320 ppm,
or not more than about 280 ppm, or not more than about 240 ppm, or not more than about
200 ppm, or not more than about 160 ppm, or not more than about 140 ppm, or not more than
about 120 ppm, or not more than about 100 ppm, or not more than about 80 ppm, or not more
than about 60 ppm, or not more than about 40 ppm, or not more than about 30 ppm, or not
more than about 20 ppm, or not more than about 10 ppm.
[0043] In some embodiments, inventive methods according to the invention provide
at least 50%. recovery of the protein of interest (e.g., at least 55%,, 60%, 65%, 70%, 75%,
80%, 85%, 90%, or 95%). In some embodiments, the methods of the invention provide at
least 20% product yield (e.g., at least 22%, 24%, 26%, 28%, 30%, 32%, 34%, 36%, 38%,
40%, 42%, 44%, 46%, 48%, or 50%).
[0044] Various aspects of the invention are described in detail in the following
sections. The use of sections is not meant to limit the invention. Each section can apply to
any aspect of the invention. In this application, the use of "or" means "and/or" unless stated
otherwise.
Definitions
[0045] In order for the present invention to be more readily understood, certain terms
are first defined. Additional definitions for the following terms and other terms are set forth
throughout the specification.
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[0046] Absorbent: An absorbent is at least one molecule affixed to a solid support or
at least one molecule that is, itself, a solid, which is used to perform chromatography.
[0047] Affinity chromatography: Affinity chromatography is chromatography that
utilizes the specific, reversible interactions between biomolecules, for example, the ability of
Protein A to bind to an Fc portion of an IgG antibody, rather than the general properties of a
molecule, such as isoelectric point, hydrophobicity, or size, to effect chromatographic
separation. In practice, affinity chromatography involves using an absorbent, such as Protein
A affixed to a solid support, to chromatographically separate molecules that bind more or less
tightly to the absorbent. See Ostrove (1990) in Guide to Protein Purification, Methods in
Enzymology 182: 357-379, which is incorporated herein in its entirety.
[0048] Approximately: As used herein, the term "approximately" or "about," as
applied to one or more values of interest, refers to a value that is similar to a stated reference
value. In certain embodiments, the term "approximately" or "about" refers to a range of
values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%,
9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than)
of the stated reference value unless otherwise stated or otherwise evident from the context
(except where such number would exceed 100% of a possible value).
[0049] Bind-elute mode: The term "bind-elute mode" (also referred to as "binding
mode") refers to a product preparation separation technique in which at least one product
contained in the preparation binds to a chromatographic resin or medium. The bound product
in this mode is eluted during the elution phase.
[0050] Chromatography: Chromatography is the separation of chemically different
molecules in a mixture fi"om one another by percolation of the mixture through an absorbent,
which absorbs or retains different molecules more or less strongly. Molecules that are least
strongly absorbed to or retained by the absorbent are released fi^om the absorbent under
conditions where those more strongly absorbed or retained are not.
[0051] Constant immunoglobulin domain: A constant antibody immunoglobulin
domain is an immunoglobulin domain that is identical to or substantially similar to a CL, CHI,
CH2, CH3, or CH4 domain of human or animal origin. See e.g. Charles A Hasemann and J.
Donald Capra, Immunoglobulins: Structure and Function, in William E. Paul, ed.,
Fundamental Immunology, Second Edition, 209, 210-218 (1989), which is incorporated by
12
reference herein in its entirety. A CH2 or CH3 domain, or an immunoglobulin domain
substantially similar to CH2 or CH3 domain, is also referred to as the Fc portion of an antibody.
[0052] Contaminant or Impurity: A contaminant or an impurity refers to any foreign
or objectionable molecule, including a biological macromolecule such as a DNA, an RNA, or
a protein, other than the protein of interest being purified that is also present in a sample of
the protein of interest being purified. Impurities include, for example, protein variants, such
as aggregated proteins, high molecular weight species, low molecular weight species and
fragments, and deamidated species; other proteins from host cells that secrete the protein
being purified (host cell proteins); proteins that are part of an absorbent used for affinity
chromatography that may leach into a sample during prior purification steps, such as Protein
A; endotoxins; and viruses.
[0053] Flow-through mode: The term "flow-through mode" generally refers to a
product preparation separation technique in which at least one product contained in the
preparation is intended to flow through a chromatographic resin or medium, while at least
one potential contaminant or impurity binds to the chromatographic resin or medium. In
some embodiments, a flow-through mode is weak partitioning chromatography (WPC), in
which the product can bind weakly to the resin, while at least one potential contaminant or
impurity binds more preferentially to the chromatographic resin or medium. Typically, WPC
operates at a higher partition coefficient than in traditional flow-through mode, but at a
partition coefficient lower than a bind-and-elute mode. In weak partitioning, high recoveries
can be achieved with larger load challenges and short washes appUed following the load
phase.
[0054] Host cell proteins: Host cell proteins are proteins encoded by the naturallyoccurring
genome of a host cell into which DNA encoding a protein that is to be purified is
introduced. Host cell proteins may be contaminants of the protein to be purified, the levels of
which may be reduced by purification. Host cell proteins can be assayed for by any
appropriate method including gel electrophoresis and staining and/or ELISA assay, among
others.
[0055] Hydroxyapatite chromatography: Hydroxyapatite chromatography is
chromatography using ceramic hydroxyapatite as an absorbent. See e.g. Marina J. Gorbunoff
(1990), Protein Chromatography on Hydroxyapatite Columns, in Guide to Protein
13
Purification, Murray P. Deutscher, ed., Methods in Enzymology 182: 329-339, which is
incorporated herein in its entirety.
[0056] Load: The term "load" refers to any load material containing the product,
either derived from clarified cell culture or fermentation conditioned medium, or a partially
purified intermediate derived from a chromatography step. The term "load fluid" refers to a
liquid containing the load material, for passing through a medium under the operating
conditions of the invention.
[0057] Load challenge (LC): The term "load challenge" refers to the total mass of
product loaded onto the column in the load cycle of a chromatography step or applied to the
resin in batch binding, measured in units of mass of product per imit volume of resin.
[0058] Protein A: Protein A is a protein originally discovered in the cell wall of
Stapphylococcus that binds to an Fc portion or a variable domain of an antibody. In some
embodiments, Protein A binds to a domain from VH3 family (e.g., a VH3 domain of IgG
antibody). For purposes of the invention, "Protein A" is any protein identical or substantially
similar to Stapphylococcal Protein A, including commercially available and/or recombinant
forms of Protein A. For purposes of the invention, the biological activity of Protein A for the
purpose of determining substantial similarity is the capacity to bind to an Fc portion or a
variable domain (e.g., VH3) of IgG antibody.
[0059] Protein G: Protein G is a protein originally discovered in the cell wall of
Streptococcus that binds to an Fc portion or a variable domain of an antibody (e.g., IgG). In
some embodiments. Protein G binds to a domain from VH3 family (e.g., a VH3 domain of
IgG antibody). For purposes of the invention, "Protein G" is any protein identical or
substantially similar to Streptococcal Protein G, including commercially available and/or
recombinant forms of Protein G. For purposes of the invention, the biological activity of
Protein G for the pxirpose of determining substantial similarity is the capacity to bind to an Fc
portion or a variable domain (e.g., VH3) of IgG antibody.
[0060] Protein LG: Protein LG is a recombinant fixsion protein that binds to IgG
antibodies comprising portions of both Protein G (see definition above) and Protein L.
Protein L was originally isolated from the cell wall of Peptostreptococcus. Protein LG
comprises IgG binding domains from both Protein L and G. Vola et al. (1994) Cell. Biophys.
24-25: 27-36, which is incorporated herein in its entirety. For purposes of the invention,
14
"Protein LG" is any protein identical or substantially similar to Protein LG, including
commercially available and/or recombinant forms of Protein LG. For purposes of the
invention, the biological activity of Protein LG for the purpose of determining substantial
similarity is the capacity to bind to an IgG antibody.
[0061] Purify: To purify a protein means to reduce the amounts of foreign or
objectionable elements, especially biological macromolecules such as proteins or DNA, that
may be present in a sample of the protein. The presence of foreign proteins may be assayed
by any appropriate method including gel electrophoresis and staining and/or ELISA assay.
The presence of DNA may be assayed by any appropriate method including gel
electrophoresis and staining and/or assays employing polymerase chain reaction.
[0062] Variable antibody immunoglobulin domain: A variable antibody
immunoglobulin domain is an immunoglobulin domain that is identical or substantially
similar to a VL or a VH domain of human or animal origin. For purposes of the invention, the
biological activity of a variable antibody immunoglobulin domain for the purpose of
determining substantial similarity is antigen binding. In some embodiments, a variable
antibody immimoglobulin domain is a VH3 domain. A VH3 domain, as used herein refers to
VH3 itself, or any domain having homology to the VH3 domain.
Small modular immunopharmaceutical proteins
[0063] As used herein, a small modular immunopharmaceuticals (SMIP^ protein
refers to a protein that contains one or more of the following fused domains: a binding
domain, an immunoglobulin hinge region or a domain derived therefrom, and an effector
domain, which can be an immunoglobulin heavy chain Cm constant region or a domain
derived therefrom, and an immunoglobulin heavy chain CH3 constant region or a domain
derived therefrom. SMIP^M protein therapeutics are preferably mono-specific (i.e., they
recognize and attach to a single antigen target to initiate biological activity). The present
invention also relates to multi-specific and/or multi-valent molecules such as SCORPION''"^
therapeutics, which incorporate a SMIP^M protein and also have an additional binding domain
located C-terminally to the SMIP^M protein portion of the molecule. Preferably, the binding
domains of SCORPIOISF'^ therapeutics each bind to a different target. The domains of small
modular immunopharmaceuticals suitable for the present invention are, or are derived from,
15
polypeptides that are the products of human gene sequences, any other natural or artificial
sources, including genetically engineered and/or mutated polypeptides. Small modular
immunopharmaceuticals are also known as binding domain-inmiunoglobulin fiision proteins.
[0064] In some embodiments, a hinge region suitable for a small modular
immunopharmaceutical is derived fi"om an immunoglobulin such as IgGl, IgA, IgE, or the
like. For example, a hinge region can be a mutant IgGl hinge region polypeptide having
either zero, one or two cysteine residues.
[0065] A binding domain suitable for a small modular immunopharmaceutical protein
may be any polypeptide that possesses the ability to specifically recognize and bind to a
cognate biological molecule, such as an antigen, a receptor (e.g., CD20), or complex of more
than one molecule or assembly or aggregate.
[0066] Binding domains may include at least one immunoglobulin variable region
pol5T)eptide, such as all or a portion or fi^gment of a heavy chain or a light chain V-region,
provided it is capable of specifically binding an antigen or other desired target structure of
interest. In other embodiments, binding domains may include a single chain
immunoglobulin-derived Fv product, which may include all or a portion of at least one
inmiunoglobulin light chain V-region and all or a portion of at least one immunoglobulin
heavy chain V-region, and which fiirther comprises a linker fused to the V-regions.
[0067] The present invention can be applied to various small modular
immunopharmaceuticals. Exemplary small modular immunopharmaceuticals may target
receptors or other proteins, such as, CD3, CD4, CDS, CD 19, CD20 and CD34; members of
the HER receptor family such as the EOF receptor, HER2, HER3 or HER4 receptor; cell
adhesion molecules such as LFA-1, Mol, pl50,95, VLA-4, ICAM-1, VCAM, growth factors
such as VEGF; IgE; blood group antigens; flk2/flt3 receptor; obesity (OB) receptor; protein
C; EGFR, RAGE, P40, Dkkl, NOTCHl, IL-13, IL-21, IL-4, and IL-22, etc.
[0068] In some embodiments, the present invention is utilized to purify small modular
immunopharmaceuticals that specifically recognize CD20. An exemplary small modular
immunopharmaceutical protein that specifically binds CD20 is shown in Figure 1. As shown
in Figure 1, an anti-CD20 SMIP™^ protein is typically a recombinant homodimeric fiision
protein composed of three distinct domains: (1) a chimeric (murine/human) CD20 binding
domain including the variable heavy (VH) and light (VL) chain fragments cormected by an
16
amino acid linker (e.g., a 15-amino acid linker); (2) a modified human immunoglobulin (e.g.,
IgGl) hinge domain and, (3) an IgG effector domain such as the CH2 and CHS domains of
human IgGl.
[0069] Typically, an SMIP™* protein may exist in two distinctly associated
homodimeric forms, the major form, which is the predicted interchain disulfide linked
covalent homodimer (CD), and a homodimeric form that does not possess interchain disulfide
bonds (dissociable dimer, DD). The dissociable dimer is generally fully active. Typically, a
dimer has a theoretical molecular weight of approximately 106,000 daltons. SMIP^^ proteins
can also form multivalent complexes.
[0070] Typically, SMIP™* proteins are present as glycoproteins. For example, as
shown in Figure 1, an anti-CD20 SMIP^^ protein may be modified with oligosaccharides at
the N-linked glycosylation consensus sequence (e.g., ^^^NST) in the CH2 domain of each
protein chain (see Figure 1). SMIP™* proteins may also contain a core-fiicosylated asialoagalacto-
bianteimary A^-linked oligosaccharide (GOF); COOH-terminal Gly"*^*, and NH2-
terminal pyroglutamate on each chain (GOF/GOF). Two minor glycoforms, GIF/GOF and
GIF/GIF, and other expected trace-level A^-linked glycoforms may also present.
Additionally, low levels of a Core 1 O-glycan modification is also observed in the hinge
region of SMIP^^ proteins.
[0071] In some embodiments, the isoelectric point (pi or lEP) of SMIP^^ proteins
ranges firom approximately 7.0 to 9.0 (e.g., 7.2, 7.4, 7.6, 7.8, 8.0, 8.2, 8.4, 8.6, 8.8).
[0072] The present invention can be used to purify SMIP^^ proteins in various forms
as discussed herein (e.g., monomeric polypeptide, homodimer, dissociable dimer or
multivalent complexes). The present invention can be used to purify various modified
SMIP proteins, such as humanized SMIP , or chimeric SMIP proteins. As used herein,
the term "humanized SMIP proteins refers to SMIP proteins that include at least one
humanized immunoglobulin region (e.g., humanized inmiunoglobulin variable or constant
region). In some embodiments, a humanized SMIP^^ protein comprises a humanized
variable region that includes a variable framework region derived substantially from a human
immunoglobulin (e.g., a fully human FRl, FR2, FR3, and/or FR4), while maintaining targetspecific
one or more complementarity determining regions (CDRs) (e.g., at least one CDR,
two CDRs, or three CDRs). In some embodiments, a humanized SMIP™* protein comprises
17
one or more human or humanized constant regions (e.g., human immunoglobulin Cm and/or
CH3 domains). The term "substantially from a human immunoglobulin or antibody" or
"substantially human" means that, when aligned to a human immunoglobulin or
antibody amino sequence for comparison purposes, the region shares at least 80-90%,
preferably 90-95%, more preferably 95-99% identity (i.e., local sequence identity)
with the human framework or constant region sequence, allowing, for example, for
conservative substitutions, consensus sequence substitutions, germline substitutions,
backmutations, and the like. As used herein, the term "chimeric SMIP™' proteins"
refers to SMIP^'^ proteins whose variable regions derive from a first species and whose
constant regions derive from a second species. Chimeric SMIP^^ proteins can be
constructed, for example by genetic engineering, from immunoglobulin gene segments
belonging to different species. Humanized and chimeric SMIP^^ proteins are further
described in International AppUcation Publication No. WO 2008/156713, which is
incorporated by reference herein.
[0073] The present invention can also be used to purify SMIP^^ proteins with
modified glycosylation patterns and/or mutations to the hinge, CH2 and/or CH3 domains that
alter the effector functions. In some embodiments, SMIP™^ proteins may contain mutations
on adjacent or close sites in the hinge link region that affect affinity for receptor
binding. In addition, the invention can be used to purify fusion proteins including a small
modular immunopharmaceutical polypeptide or a portion thereof.
[0074] In some embodiments, the present invention can be used to purify SMIP^^
proteins that include an amino acid sequence of any one of SEQ ID NOs:l-76 (see the
Exemplary SMIP^^ Sequences section), or a variant thereof In some embodiments, the
present invention can be used to purify SMIP^^ proteins that contain a variable domain
having an amino acid sequence of any one of SEQ ID NOs: 1 -59 or a variant thereof. In some
embodiments, the present invention can be used to purify SMIP^^ proteins that contain a
variable domain having an amino acid sequence of any one of SEQ ID NOs: 1 -59 or a variant
thereof, a hinge region having an amino acid sequence of any one of SEQ ID NOs: 60-64 or a
variant thereof, and/or an immunoglobulin constant region having an amino acid sequence of
SEQ ID NO: 65 or 66 or a variant thereof In some embodiments, the present invention can
be used to purify SMIP^*^ proteins that have an amino acid sequence of any one of SEQ ID
NOs: 67-76, or a variant thereof.
18
[0075] As used herein, variants of a parent sequence include, but are not limited to,
amino acid sequences that are at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%,
99%, identical to the parent sequence. The percent identity of two amino acid sequences can
be determined by visual inspection and mathematical calculation, or more preferably, the
comparison is done by comparing sequence information using a computer program such as
the Genetics Computer Group (GCG; Madison, Wis.) Wisconsin package version 10.0
program, "GAP" (Devereux et al., 1984, Nucl. Acids Res. 12: 387) or other comparable
computer programs. The preferred default parameters for the 'GAP' program includes: (1)
the weighted amino acid comparison matrix of Gribskov and Burgess ((1986), Nucl. Acids
Res. 14: 6745), as described by Schwartz and DayhofiF, eds.. Atlas of Polypeptide Sequence
and Structure, National Biomedical Research Foundation, pp. 353-358 (1979), or other
comparable comparison matrices; (2) a penalty of 30 for each gap and an additional penalty
of 1 for each symbol in each gap for amino acid sequences; (3) no penalty for end gaps; and
(4) no maximum penalty for long gaps. Other programs used by those skilled in the art of
sequence comparison can also be used.
[0076] Additional small modular immunopharmaceuticals are further described in,
e.g., US Patent Publications 20030133939, 20030118592, 20040058445, 20050136049,
20050175614, 20050180970,20050186216, 20050202012, 20050202023,20050202028,
20050202534,20050238646, and 20080213273; International Patent Publications WO
02/056910, WO 2005/037989, and WO 2005/017148, which are all incorporated by reference
herein.
Protein Aggregation
[0077] Without wishing to be bound by any theory, it is contemplated that domain
swapping may be a protein aggregation mechanism. Domain swapping occurs when a
distinctly structured subsection of a protein (domain) physically exchanges with that of
another monomer to create a dimer or higher oligomers. In domain-swapped proteins, each
domain maintains native-like global structure that is comparable to its structure in the unswapped
monomer. When a folded protein, containing multiple domains, is stressed by low
pH, elevated temperature or shear force a partially folded or "open" conformation
(characterized by dissociated, but folded domains) can be induced. Some "open" molecules
19
refold to their native structure, by simple re-association of the folded domains. In some cases
(usually favored by higher protein concentrations) the domain re-association occurs between
two neighboring molecules, resulting in a domain-swapped dimer. Such inter-molecular
swapping may propagate, leading to larger aggregates. Typically, domain-swapped proteins
are non-covalently (but stably) associated molecules, having native-like domain folding and
inter-domain contacts. In such cases, multimeric proteins are held together by the very same
domain-domain interfaces that would normally exist intra-molecularly.
[0078] Prior to the purification process, SMIP^^ proteins contain a significant amount
(e.g., 20-60%) of HMW protein (aggregate). The excessive HMW content may be due to the
molecular structure of SMIPs . As shown in Figure 1, a typical SMIP dimer molecule
contains 2 identical single-chain-Fv regions, including VH and VL domains connected by a
flexible linker (e.g., GGGSGGGGSGGS (SEQ ID NO: 77)), which are fused to a human IgGl
Fc domain (Figure 1). Without wishing to be bound by any theory, SMIP^^ molecules may
be more susceptible to unfolding (open conformation of the Fv) and subsequent domain
swapping resulting in protein aggregation because of the flexible linker in each subunit.
[0079] According to studies using cryo-electron microscope, SMIP^^ molecules may
exist in, e.g., compact, mixed, stretched or diabody-like configurations in solution (Figure 2).
Without wishing to be bound by any theory, it is contemplated that some SMIP™ molecules
with stretched or open chains may attempt to refold to their native structure, by simple reassociation
of the folded domains. As shown in Figure 3 A, the domain re-association may
occur between two neighboring SMIP^^ molecules, resulting in a domain-swapped dimer.
Such inter-molecular swapping may propagate, leading to larger aggregates, such as trimers,
tetramers or multimers (see, Figures 3B and 3C).
Protein preparations
[0080] Protein preparations used with methods described herein can be obtained fi-om
any in vivo or in vitro protein expression systems. Exemplary sources for protein preparation
suitable for the invention include, but are not limited to, conditioned culture medium derived
from culturing a recombinant cell line that expresses a protein of interest, or from a cell
extract of, e.g., product-producing cells, bacteria, fungal cells, insect cells, transgenic plants
or plant cells, transgenic animals or animal cells, or serum of animals, ascites fluid,
20
hybridoma or myeloma supematants. Suitable bacterial cells include, but are not limited to,
Escherichia coli cells. Examples of suitable E. coli strains include: HBlOl, DH5a, GM2929,
JM109, KW251, NM538, NM539, and any E. coli strain that fails to cleave foreign DNA.
Suitable fungal host cells that can be used include, but are not limited to, Saccharomyces
cerevisiae, Pichia pastoris and Aspergillus cells. Suitable insect cells include, but are not
limited to, S2 Schneider cells, D. Mel-2 cells, SF9, SF21, High-5™, Mimic™ -SF9, MGl
and KCl cells. Suitable exemplary recombinant cell lines include, but are not limited to,
BALB/c mouse myeloma line, human retinoblasts (PER.C6), monkey kidney cells, human
embryonic kidney line (293), baby hamster kidney cells (BHK), Chinese hamster ovary cells
(CHO), mouse Sertoli cells, African green monkey kidney cells (VERO-76), human cervical
carcinoma cells (HeLa), canine kidney cells, buffalo rat liver cells, human lung cells, human
liver cells, mouse mammary tumor cells, TRI cells, MRC 5 cells, FS4 cells, and human
hepatoma line (Hep G2).
[0081] Proteins of interest can be expressed using various vectors (e.g., viral vectors)
known in the art and cells can be cultured under various conditions known in the art (e.g.,
fed-batch). Various methods of genetically engineering cells to produce proteins are well
known in the art. See e.g. Ausabel et al., eds. (1990), Current Protocols in Molecular Biology
(Wiley, New York).
[0082] Cells expressing SMIP^^ proteins may be cultured in various cell culture
media known in the art. Exemplary cell culture media may be based on DMEM,
DMEM/F12, Ham's F-10, Ham's F-12, F-12K, Medium 199, MEM, RPMI 1640, Ames',
BGJb, Click's, CMRL-1066, Fischers, GMEM, IMDM, L-15, McCoy's 5A Modified,
NCTC, Swik's S-77, Waymouth, William's Medium E. Suitable cell culture medium may be
serum free. In some embodiments, suitable cell culture medium may include serum/culture
medium additives including, but not limited to, fetal bovine serum, newborn bovine serum,
calf bovine serum, and adult bovine serum, chicken, goat, horse, porcine, rabbit, sheep,
human serum, serum replacement or bovine embryonic fluid. Suitable cell culture medium
may further include supplements and/or antibiotics including, but not limited to, L-Glutamine
Solution, L-Albany-L-Glutamine Solution, Non-essential Amino Acid Solution, Penicillin,
Streptomycin.
[0083] The present invention can be utilized to purify crude protein preparations. For
example, the present invention can be used to purify proteins directly from conditioned
21
culture medium containing proteins secreted from cultured cells. Conditioned culture
medium can be obtained from small scale cultures (e.g., shake flasks, wavebags), or from
seed bioreactors or production bioreactors (e.g., 250L, 600L, 2500L, or 6000L bioreactors).
In some embodiments, the present invention can be utilized to purify proteins expressed
intracellularly from crude cell lysates prepared from protein-containing cells. In some
embodiments, the present invention can be used to purify proteins from serum, milk or other
fluid containing protein of interest. In some embodiments, the present invention can be used
to purify proteins from partially purified preparations such as eluates or flow-through from
chromatography columns, or intermediate protein preparations from storage or formulation
processes.
[0084] In some embodiments, the present invention can be used to purify proteins that
are expressed in inclusion bodies (e.g., bacterial, viral, plant cell or any other types of
inclusion bodies). Proteins expressed in inclusion bodies typically form aggregates, which
pose challenges for purification. The present invention therefore can be particularly useful
for purifying proteins expressed in inclusion bodies. Purification of proteins from inclusion
bodies usually requires first extracting inclusion bodies from bacteria or other type of cells
followed by solubilizing the purified inclusion bodies. Various methods of inclusion body
extraction and solubilization are well known in the art and can be used in the present
invention. For example, strong denaturing agents (e.g., urea and guanidine hydrochloride),
altered pH and/or temperature, physical disruptions (e.g., sonication, etc.), among others can
be used to extract and/or solubilize inclusion bodies. Inclusion body extraction and/or
solubilization process may lead to mis-folded proteins. In some embodiments, inclusion
body extracts can be directly loaded to chromatography columns according to the present
invention. In some embodiments, the proteins extracted from inclusion bodies are first
subjected to a refolding process prior to chromatography steps described herein. In some
embodiments, a refolding process may include dialysis or dilution of the proteins into a
folding buffer. Various folding buffers are well known in the art and can be used in the
present invention.
[0085] In some embodiments, the present invention can be used to purify proteins
from preparations that contain various impurities including, but not limited to, undesirable
protein variants, such as aggregated proteins, e.g., high molecular weight species, low
molecular weight species and fragments, and deamidated species; other proteins from host
22
cells that secrete the protem being purified; host cell DNA; components from the cell culture
medium, molecules that are part of an absorbent used for affinity chromatography that leach
into a sample during prior purification steps, for example, Protein A and Protein G; an
endotoxin; a nucleic acid; a virus, or a fragment of any of the forgoing.
[0086] The present invention is particularly useful to remove HMW aggregates. In
some embodiments, starting protein preparations may contain at least 4%, 5%, 10%, 15%,
20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%
HMW aggregates. In some embodiments, starting protein preparations may contain less than
95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%,
15%, 10%, 5% HMW aggregates. In some embodiments, starting preparations may contain
HMW aggregates in a range of the above percentage combination (for example, about 4-95%,
5-70%, 10-60%, 4-30%, 4-25%, 4-20%, 4-15%, 4-10%, and any combinations of the above
identified percentages). As used herein, the term "high molecular weight (HMW)
aggregates" refers to an association of at least two protein monomers. For the purposes of
this invention, a monomer refers to the single unit of any biologically active form of the
protein of interest. For example, a monomer of a small modular immunopharmaceutical
protein can be a monomeric polypeptide, or a homodimer, or a dissociable dimer, or a unit of
multivalent complex of SMIP^^ protein. The association may be covalent, non-covalent,
disulfide, non-reducible crosslinking, or by other mechanism.
[0087] In some embodiments, appropriate protein preparations can be obtained by
harvest processing. As discussed in the Examples, conditioned medium can be harvested
from production bioreactors through centrifugation (e.g., by disc stack centriftigation (DSC)).
A centrifixgation step may separate cells and cell debris from conditioned medium containing
secreted proteins (e.g., SMIPs^. In some embodiments, after DSC, the contents can be
applied to a pad filtration apparatus, and then filters into a filtrate vessel or bag. In some
embodiments, a Hepes/EDTA buffer solution can be added to the filtrated concentrate pool to
reduce the generation of acidic species during the in-process hold period between the DSC
and the affinity chromatography step. In addition, protease inhibitors such as EDTA or
imidazole may be added to inhibit metalloprotease activity, certain serine protease or other
protease activities. In some embodiments, a suitable protease inhibitor may be added to a
protein preparation in an amount from about 0.001 fiM to about 100 mM. The protease
inhibitor(s) may be added to the protein preparations before and/or during protein A affinity
23
chromatography. Adding protease inhibitors (e.g., EDTA) may also reduce protein A
leaching. Other conditions such as temperature and pH may also be adjusted to reduce
protein A leaching. Methods and conditions for reducing protein A leaching are described in
details in US Publication No. 20050038231, which is incorporated by reference herein.
Methods of purification
[0088] Purification processes according to the invention involve one or more
chromatography steps (e.g., affinity chromatography, hydroxyapatite chromatography, or ion
exchange chromatography). In some embodiments, the purification methods of the invention
involve a step of hydroxyapatite chromatography. In some embodiments, the purification
methods of the invention involve a step of hydroxyapatite chromatography in combination of
affinity chromatography and/or ion exchange chromatography. In some embodiments, the
methods of the invention further include membrane filtration steps (e.g., ultrafiltration,
diafiltration, and/or final filtration). Exemplary purification processes are described in details
in the Examples section.
Affinity chromatography
[0089] The primary objectives of the affinity chromatography step include product
capture from starting preparations (e.g., cell-free conditioned medium, cell lysates, inclusion
body extracts, among others) and separation of protein of interest from process-derived
impurities (e.g., host cell DNA and host cell proteins, medium components, and adventitious
agents).
[0090] Thus, affinity chromatography suitable for the invention involves using an
absorbent that can bind to a SMIP^^ protein. For example, a suitable absorbent can be a
protein that binds to a constant antibody inununoglobulin domain. Suitable absorbents can be
Protein G, Protein LG, or Protein A. In some embodiments, a suitable absorbent is a protein
that binds to a variable antibody immimoglobulin domain (e.g., a VH3 domain or a domain
homologous to a VH3 domain). Absorbents can be affixed to any suitable solid support
including: agarose, sepharose, silica, collodion charcoal, sand, and any other suitable
24
material. Such materials are well known in the art. Any suitable method can be used to affix
an absorbent to the solid support. Methods for affixing proteins to suitable solid supports are
well known in the art. See e.g. Ostrove (1990), in Guide to Protein Purification, Methods in
Enzymology, 182: 357-371.
[0091] In some embodiments, a suitable affinity chromatography step may use a
Protein A chromatography column or a Protein G chromatography column. A Protein A
chromatography column can be, for example, PROSEP-A^^ (MiUipore, U.K.), Protein A
Sepharose FAST FLOW™ (GE Healthcare, Piscataway, N.J.), TOYOPEARL™ 650M
Protein A (TosoHass Co., Philadelphia, Pa.), or MabSelect™ Protein A column (GE
Healthcare, Piscataway, N.J.).
[0092] Before applying protein preparations to affinity chromatography columns, it
may be desirable to adjust parameters such as pH, ionic strength, and temperature and in
some instances the addition of substances of different kinds. Thus, it is an optional step to
perform an equilibration of an affinity chromatography column by washing it with a solution
(e.g., a buffer for adjusting pH, ionic strength, etc., or for the introduction of a detergent)
bringing suitable characteristics for binding and purification of the protein product.
[0093] In some embodiments, the Protein A column may be equilibrated using a
solution containing a sah, e.g., about 100 mM to about 150 mM sodium phosphate, about 100
mM to about 150 mM sodium acetate, and about 100 mM to about 150 mM NaCl. The pH of
the equilibration buffer may range from about 6.0 to about 8.0. In one embodiment, the pH
of the equilibration buffer is about 7.5. The equilibration buffer may also contain about 10
mM to about 50 mM Tris. In another embodiment, the buffer may contain about 20 mM Tris.
[0094] After a protein preparation is loaded, the bound column may be washed using
a wash solution. Suitable wash solutions may contain salt (e.g., Hepes, sodium chloride,
calcium chloride, magnesium chloride), arginine, histidine, Tris and/or other components. In
some embodiments, a wash solution suitable for the invention may contain arginine or an
arginine derivative. Suitable arginine derivative can be, but is not limited to, acetyl arginine,
agmatine, arginic acid, N-alpha-butyroyl-L-arginine, or N-alpha-pyvaloyl arginine. A
suitable concentration of arginine or arginine derivative in the wash solution is between about
0.1 M and about 2.0 M (e.g., 0.1 M, 0.4 M, 0.5 M, 1.0 M, 1.5 M, or 2.0 M), or between about
0.5 M and about 1.0 M (e.g., 0.5 M, 0.6 M, 0.7 M, 0.8 M, 0.9 M, or 1.0 M). The use of
25
arginine or arginine derivative in affinity chromatography is described in detailed in U.S.
Application Publication No. 2008/0064860, the disclosure of which is hereby incorporated by
reference. In some embodiments, a wash solution suitable for the invention may contain
imidazole or an imidazole derivative. In some embodiments, a suitable wash solution may
contain a chaotropic reagent (e.g., urea, sodium thiocynate, and/or guanidinium
hydrochloride). In some embodiments, a suitable wash solution may contain a hydrophobic
modifier (e.g., organic solvents including ethanol, methanol, propylene glycol, ethylene
glycol, hexaethylene glycol, propanol, butanol and isopropanol). In some embodiments, a
wash solution suitable for the invention may contain a detergent (e.g., nonionic detergent
and/or ionic detergent).
[0095] The pH of the wash solution is generally between about 4.5 and about 8.0, for
example, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5 and 8.0. In same cases, the pH of the wash solution is
greater than 5.0 and less than about 8.0, for example, 5.5, 6.0, 6.5, 7.0, 7.5 and 8.0. The wash
solution may contain 20 mM to 50 mM Tris (e.g., 20 mM, 30 mM, 40 mM or 50 mM).
[0096] The product may be eluted fi"om a washed column, e.g., a Protein A column,
by an elution buffer. Typically, a suitable elution buffer may contain Hepes, phosphoric acid,
glycine, glycylglycine, one or more organic acids (e.g., acetic acid, citric acid, formic acid,
lactic acid, tartaric acid, malic acid, malonic acid, phthalic acid, saUcyclic acid), and/or
arginine. A suitable elution buffer may fiuther contain a salt (e.g., sodium chloride,
potassium chloride, ammonium chloride, sodium acetate, potassium acetate, ammonium
acetate, calcium salts, and/or magnesium salts). A suitable salt concentration may range fi"om
0 mM to 1 M (e.g., 0 mM to 500 mM, 0 mM to 100 mM, 0 mM to 50 mM). In some
embodiments, a suitable elution buffer contains about 15 mM to about 100 mM NaCl. In
some embodiments, NaCl concentration in a elution buffer can be at 4 levels (e.g., 0 mM, 15
mM, 30 mM, and 50 mM). In other embodiments, an elution buffer may contain about 20
mM to about 200 mM arginine or arginine derivatives. In further embodiments, an elution
buffer may contain 20 mM to 200 mM glycine. The elution buffer may also contain about 20
mM to about 100 mM HEPES. The elution buffer may also contain about 25 mM to about
100 mM acetic acid. In some embodiments, the elution buffer may contain citric acid (e.g.,
about 10 mM to about 500 mM citric acid). In some embodiments, the elution buffer may
contain glycylglycine (e.g., about 10-50 mM, about 50-100 mM, or about 100-200 mM). In
some embodiments, a suitable elution buffer may contain a chaotropic reagent (e.g., urea,
26
sodium thiocynate, and/or guanidinium hydrochloride). In some embodiments, a suitable
elution buffer may contain alkyl glycol (e.g., ethylene glycol, propylene glycol, hexaethylene
glycol). The pH of the elution buffer may range from about 2.5 to about 4.0. In one
embodiment, the pH of the elution buffer is about 3.0.
[0097] Eluates from the affinity chromatography columns may be neutralized by
neutralization buffers. Suitable neutralization buffers may contain Tris, Hepes, and/or
imidazole.
[0098] After elution, the affinity chromatography columns may optionally be cleaned,
i.e., stripped and/or regenerated, after elution of the protein. This procedure is typically
performed regularly to minimize the building up of impurities on the surface of the solid
phase and/or to sterilize the matrix to avoid contamination of the product with
microorganisms. Stripped and/or regenerated columns may be used repeatedly.
[0099] Buffer components may be adjusted according to the knowledge of the person
of ordinary skill in the art. Sample buffer composition ranges are provided in the Examples
below. Not all of the buffers or steps are necessary, but are provided for illustration only. A
high throughput screen, as described in the Examples, may be used to efficiently optimize
buffer conditions for Protein A column chromatography.
Ion exchange chromatography
[0100] The primary objectives of the ion exchange chromatography step include
removal of process-derived impurities (e.g., leached protein A, host cell DNA and/or
proteins, adventitious agents) as well as product-related impurities such as HMW aggregates.
[0101] In some embodiments, ion exchange chromatography is used in combination
with affinity chromatography according to the invention. In some embodiments, ion
exchange chromatography (e.g., cation exchange and/or anion exchange chromatography)
can be used instead of affinity chromatography.
[0102] Various anionic or cationic substituents may be attached to matrices in order
to form anionic or cationic supports for chromatography. Anionic exchange substituents
include diethylaminoethyl (DEAE), trimethylaminoethyl acrylamide (TMAE), quaternary
27
aminoethyl (QAE) and quaternary amine (Q) groups. Cationic exchange substituents include
carboxymethyl (CM), sulfoethyl (SE), sulfopropyl (SP), phosphate (P) and sulfonate (S). Ion
exchange resins with polyethyleneimine functional groups, such as POROS® HQ50, are
available from Applied Biosystems, Foster City, CA. Exchange resins with an inunobilized
recombinant Protein A functional groups, such as POROS® A50, are available from Applied
Biosystems, Foster City, CA. Cellulosic ion exchange resins such as DE23, DE32, DE52,
CM-23, CM-32 and CM-52 are available from Whatman Ltd. Maidstone, Kent, U.K.
Sephadex-based and cross-linked ion exchangers are also known. For example, CAPTO Q,
DEAE-, QAE-, CM-, and SP-Sephadex, and DEAE-, Q-, CM- and S-Sepharose (e.g., DEAE
Sepharose FF, Q Sepharose FF and Q Sepharose XL), and Sepharose are all available from
GE Healthcare, Piscataway, N.J. Further, both DEAE and CM derivitized ethylene glycolmethaciylate
copolymer such as TOYOPEARL™ DEAE-650S or M, TOYOPEARL™ CM-
650S or M, TOYOPEARL™ GIGACAP Q-650, and TOYOPEARL™ GIGACAP CM-650
are available from Toso Haas Co., Philadelphia, Pa. Ion exchange monolithic
chromatographic supports, such as CIM®-DISK, may also be used in accordance with the
present invention and are available from Bia Separations, Austria. Ion exchange membrane
adsorbers, such as Mustang® Q and Mustang® E (Pall Corporation, New York), Sartobind® Q
(Sartorius Stedim Biotech S.A., France), and Chromasorb™' (Millipore, Massachusetts), may
also be used in accordance with the present invention.
[0103] In some embodiments, an anion exchange column is used. The anion
exchange column may be first equilibrated with a high salt buffer and then a low salt buffer
before being contacted with proteins. Typically, SMIPs'^ bind only weakly to the column,
which allows the majority of the product to flow through while impurities with a negative
charge, such as host cell DNA and HCPs, bind sfrongly and are retained. The column may
then washed with equilibration buffer to collect additional product weakly boxmd to the resin.
Once the product has been removed from the column, impurities can be stripped using a high
salt buffer. The resin can be regenerated, sanitized, and then stored in an ethanol solution.
[0104] In some embodiments, it is desirable to use an adsorptive depth filter before
the ion exchange chromatography to increase the impurity capacity and life time of resins
used in the ion exchange chromatography. For example, Fractogel® EMD TMAE Hi-Cap(M)
. resin is a strong anion exchanger with a synthetic methacrylate polymeric base. The pores
that are formed from intertwined polymer agglomerates have an approximate size of 800
28
Angstroms. The siuface is strongly hydrophilic due to the ether linkages in the polymer.
Long, linear polymer chains carry the functional ligands. These polymer chains are known as
tentacles. All tentacles are covalently attached to hydroxyl groups of the methacrylate
backbone. Additional tentacle resins, such as Fractogel® EMD TMAE (M), Fractogel® EMD
TMAE (S), and Fractoprep® TMAE, may also be used in accordance with the present
invention. Use of an adsorptive depth pre-filter can protect the TMAE column from
impurities in the protein load (e.g., the ProA peak pool). It is likely that these impurities can
exhaust or block the binding sites of the TMAE column, reducing resin capacity for
impurities. These impurities can be reduced by, for example, prefiltration through an
adsorptive depth filter or precipitation of protein.
[0105] After elution, the ion exchange chromatography columns may optionally be
cleaned, i.e., stripped and/or regenerated, after elution of the protein. This procedure is
typically performed regularly to minimize the building up of impurities on the surface of the
solid phase and/or to reduce the likelihood of contamination of the product with
microorganisms. In some embodiments, ion exchange columns are regenerated by treatment
with NaOH solution using concentrations ranging from 10 mM to 2M NaOH. Stripped
and/or regenerated columns may be used repeatedly.
[0106] As described above, in some embodiments, depth filtration may be used to
reduce impurities in a protein preparation. In some embodiments, depth filtration media is a
highly porous filter composed of cellulose fibers, diatomaceous earth, and a cationic resin
binder. The depth filter can remove impurities by sieving through the cellulose fibers, by
hydrophobic adsorption to the diatomaceous earth, and by ionic adsorption to the cationic
binder. A depth filter can be, for example, 0.5 cm, I cm, 1.5 cm, 2.0 cm thick.
[0107] In some embodiments, one or more additives can be added to a protein
preparation to induce precipitation and/or enhance protein adsorption to ion exchange
columns. In some embodiments, protein precipitation can be induced by additives to reduce
the amount of impurities. Various protein precipitation methods are known in the art and can
be used in the present invention. For example, proteins can be precipitated by salting out
(e.g., using a neutral salt). In some embodiments, proteins can be precipitated by addition of
organic solvents (e.g., methanol, ethanol).
29
[0108] In some embodiments, nonionic organic polymers can be used to promote
protein binding to sxirfaces and/or precipitation. Various nonionic organic polymers are
commercially available and can be used in the present invention. Examples include, but are
not limited to polyethylene glycol (PEG), polypropylene glycol, cellulose, dextran, starch,
and polyvinylpyrrolidone. In some embodiments, PEG is used as an additive. PEG with
various molecular weight can be used in the present invention. Suitable PEG may have an
average polymer molecular weight ranging from, e.g., about 100 to about 20,000 Daltons. In
some embodiments, suitable PEG may have an average weight between 200-12,000,400-
20,000,400-1000,200-1000,400-2000,1000-5000, 800-8,000,1000-10,000,2,000-12,000
Daltons. In some embodiments, exemplary PEG includes PEG having an average molecular
weight of, e.g., 200, 400, 800, 1000, 2,000, 4,000, 6,000, 8000, 10,000, 12,000, 14,000,
16,000, 18,000, 20,000 Daltons, etc. PEG can be added in various concentrations. Lower
molecular weight PEGs will generally require a higher concentration to achieve an effect
similar to higher molecular weight PEGs. Exemplary suitable PEG concentrations may range
from 0-25% (e.g., 0-6%, 0-9%, 0-12%, 0-15%, 0-18%, 0-20%, 3-9%, 3-15%, 6-12%, 6-20%,
or 6-25%). PEG or other organic polymers can be linear or branched polymers.
[0109] It is contemplated that binding or precipitation effects of PEG are generally
dependent on molecular weight of the protein. Typically, PEG effects are greater for larger
proteins. For example, lower concentrations of a given molecular weight of PEG are
generally used to enhance the binding of larger proteins (e.g., HMW aggregates) as well as
viruses compared to concentrations of PEG needed to result in the same amount of enhanced
binding of monomeric protein or LMW impurities. Thus, retention of aggregates, complexes,
and other large molecule contaminants will generally be enhanced to a greater degree than the
unaggregated forms of the proteins from which they are derived. Thus, PEG or other
nonionic polymer modification is particular useful for enhanced removal of impurities, in
particular those weak binding HMW aggregates, through weak partitioning chromatography.
In some embodiments, PEG may be added before anion exchange chromatography but after
the affinity chromatography step.
[0110] In some embodiments, the use of nonionic organic polymers for protein
precipitation can help reduce or eliminate protein denaturation as well as remove detergents
and other impurities. In some embodiments, additives (e.g., polyethylene glycols) can be
used to concentrate the product.
30
[01111 In some embodiments, the precipitate can be separated by centrifiigation,
filtration, or other separation methods known in the art. In some embodiments, the
precipitate contains contaminants, such as HMW aggregates. In some embodiments, it is
desirable to remove contaminant-containing precipitate (e.g., by filtration). In some
embodiments, SMIPs^^ are present in the precipitate. In some embodiments, it is desirable
to dissolve SMIPs^^ -containing precipitate in a resuspension buffer. In some embodiments,
the resuspension buffer has a pH and/or conductivity suitable for direct loading onto an ion
exchange column
[0112] A high throughput screen, as described in the Examples, may be used to
efficiently optimize buffer conditions for ion exchange chromatography.
Hydroxyapatite chromatography
[0113] The primary objectives of the ceramic hydroxyapatite (cHA) step are the
removal of high molecular weight (HMW) aggregates, leached Protein A, additives used to
promote precipitation or binding to absorbents (e.g., polyethylene glycol) and host cellderived
impurities, such as DN A and HCPs. cHA resins charged with phosphate around
neutral pH and low ionic strength can be used to bind both a monomer protein product (e.g., a
SMIP™*) and HMW aggregates. Since HMW aggregates bind more tightly to,the cHA resins
than monomers, the monomers can be selectively eluted using an elution buffer with suitable
ionic strength at slightly acidic to slightly basic pH. HMW aggregates can be optionally
subsequently washed off the resins using an even higher ionic strength and higher phosphate
concentration buffer at neutral pH. As described in the Examples, the present inventors have
developed cHA operating conditions that can effectively remove HMW aggregates from
protein preparations. In some embodiments, the percentage of HMW aggregates can be
reduced from more than 5% (e.g., 5%, 10%, 15%. 20%, 25%, 30%, 35%, 40%, 45%, 50%,
55%, 60%) in a load material to less than 4% (e.g., less than 3.5%, 3.0%, 2.5%, 2.0%, 1.5%,
1.0%, 0.8%, 0.6%, 0.4%, 0.2%, 0.1%) in the purified protein product. In some embodiments,
HMW aggregates can be reduced afl;er cHA chromatography, by at least about 2 fold, at least
about 5 fold, at least about 10 fold, at least about 20 fold, at least about 30 fold, at least about
40 fold, at least about 50 fold, at least about 60 fold, at least about 70 fold, at least about 80
fold, at least about 90 fold, or at least about 100 fold.
31
1. Hydroxyapatite resins
[0114] Various hydroxyapatite chromatographic resins are available commercially
and can be used for the invention. For example, the hydroxyapatite can be in a crystalline
form. In some embodiments, hydroxyapatites suitable for the invention may be those that are
agglomerated to form particles and sintered at high temperatures into a stable porous ceramic
mass. The particle size of the hydroxyapatite may vary widely, but a typical particle size
ranges from 1 nm to 1,000 i^m in diameter, and may be ftx)m 10 |j,m to 100 \im (e.g., 20 |4,m,
40 \im, 60 ^mi, or 80 |jm).
[0115] A number of chromatographic resins may be employed in the preparation of
cHA columns, the most extensively used are Type I and Type II hydroxyapatite. Type I has a
high protein binding capacity and better capacity for acidic proteins. Type I is particularly
suitable for the purification of small modular immunopharmaceutical proteins. Type II,
however, has a lower protein binding capacity, but has better resolution of nucleic acids and
certain proteins. The Type II material also has a very low affinity for albumin and is
especially suitable for the purification of many species and classes of immunoglobulins. The
choice of a particular hydroxyapatite type can be determined by the skilled artisan.
[0116] This invention may be used with hydroxyapatite resin that is loose, packed in a
column or in a continuous annual chromatograph. In one embodiment of the invention,
ceramic hydroxyapatite resin is packed in a column. The choice of column dimensions can
be determined by the skilled artisan. In some embodiments, a column diameter of at least 0.5
cm with a bed height of about 20 cm may be used for small scale purification. In some
embodiments, a column diameter of from about 35 cm to about 60 cm may be used. In some
embodiments, a column diameter of from 60 cm to 85 cm may be used. In some
embodiments, a slurry of ceramic hydroxyapatite resin in 200 mM Na2HP04 solution at pH
9.0 may be used to pack the column at a constant flow rate of about 4 cm/min or with gravity.
[0117] In some embodiments, the hydroxyapatite resins may optionally be cleaned,
i.e., stripped/or and regenerated, after elution of the protein. Stripped and/or regenerated
columns can be used repeatedly.
32
2. Operating Buffers and Conditions
[0118] Before contacting the hydroxyapatite resin with a load material, it may be
important to adjust parameters such as pH, ionic strength, and temperature and in some
instances the addition of substances of different kinds. Thus, it is an optional step to perform
an equilibration of the hydroxyapatite matrix by washing it with a solution (e.g., a buffer for
adjusting pH, ionic strength, etc., or for the introduction of a detergent) bringing the
necessary characteristics for purification of a protein of interest (e.g., SMIPs^^ protein).
[0119] In some embodiments, the hydroxyapatite matrix may be equilibrated using a
solution containing from 0.01 to 2.0 M NaCI at slightly basic to slightly acidic pH. In some
embodiments, an equilibration buffer may contain sodium phosphate, potassium phosphate,
and/or lithium phosphate. For example, an equilibration buffer may contain 1 to 20 mM
sodium phosphate (e.g., 1 to 10 mM sodiiun phosphate, 2 to 5 mM sodium phosphate, 2 mM
sodium phosphate, or 5 mM sodium phosphate). The equilibration buffer may contain 0.01
to 0.2 M NaCl (e.g., 0.025 to 0.1 M NaCl, 0.05 to 0.2 M NaCl, 0.05 to 0.1 M NaCl, 0.05 M
NaCl, or 0.1 M NaCl). The pH of the load buffer may range from 6.2 to 8.0 (e.g., 6.6 to 7.7,
6.5 to 7.5, 6.8, 7.0, 7.1, 7.2, or 7.3). The equilibration buffer may also contain 0 to 200 mM
arginine (e.g., 50 mM, 100 mM, 120 mM arginine, 140 mM, 160, or 180 mM arginine). The
equilibration buffer may contain 0 to 200 mM HEPES (e.g., 20 mM, 40 mM, 60 mM, 80
mM, 100 mM, 120 mM, 140 mM, 160 mM, 180 mM HEPES). More than one equilibration
step may be carried out.
[0120] Various protein preparations may be used as load materials (e.g., peak pools
from affinity chromatography, flow-through from ion exchange chromatography or raw
preparations). In some embodiments, a load may be buffer exchanged into an appropriate
loading buffer. For example, a protein preparation may be buffer exchanged into a loading
buffer containing 0.2 to 2.5 M NaCl at slightly acidic to slightly basic pH. For example, a
loading buffer may contain 1 to 20 mM sodium phosphate (e.g., 2 to 8 mM sodium
phosphate, 3 to 7 mM sodium phosphate, or 5 mM sodium phosphate). A loading buffer may
contain 0.01 to 0.2 M NaCl (e.g., 0.025 to 0.1 M NaCl, 0.05 to 0.2 M NaCl, 0.05 to 0.1 M
NaCl, 0.05 M NaCl, or 0.1 M NaCl). The pH of the loading buffer may range from 6.4 to 7.6
(e.g., from 6.5 to 7.0, or from 6.6 to 7.2).
33
[01211 Loading can be carried out by applying a protein preparation to a packed bed
column, a fluidized/expanded bed column containing the solid phase matrix, and/or mixing a
protein preparation with hydroxyapatite resins in a simple batch operation where the solid
phase matrix is mixed with the solution for a certain time.
[0122] After loading, the hydroxyapatite resins can be optionally washed using
washing buffer (e.g., a phosphate buffer) to remove loosely bound impurities. Washing
buffers that may be employed will depend on the nature of the hydroxyapatite resin and can
be determined by one of ordinary skill in the art.
[0123] The bound product may be eluted from the column after an optional washing
procedure. For effectively eluting monomers of SMIP^^ protein fix)m the colunm, the
present invention uses a high ionic strength phosphate buffer at slightly acidic to slightly
basic pH. In some embodiments, an elution buffer may contain sodium phosphate, potassium
phosphate, and/or lithium phosphate. For example, a suitable elution buffer may contain 1 to
100 mM sodium phosphate (e.g., 2 to 50 mM, 2 to 40 mM, 2 to 35 mM, 2 to 32 mM, 2 to 30
mM, 4 to 35 mM, 4 to 20 mM, 10 to 40 mM, 10 to 35 mM, 4 to 10 mM, or 2 to 6 mM
sodium phosphate). In some embodiments, a suitable elution buffer may contain 2 mM, 3
mM, 6 mM, 8 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50
mM, 55 mM, or 60 mM sodium phosphate.
[0124] A suitable elution buffer may also contain 0.01 to 2.5 M NaCl (e.g., 0.1 to 2.5
M, 0.1 to 2.0 M, 0.1 to 1.6 M, 0.1 to 1.2 M, 0.1 to 1.0 M, 0.1 to 0.8 M, 0.1 to 0.5 M, 0.2 to
2.5 M, 0.2 to 1.5 M, 0.2 to 1.2 M NaCl, 0.2 to 1.0 M, 0.2 to 0.8 M, 0.3 to 1.1 M, or 0.2 to 0.5
M NaCl). In some embodiments, a suitable elution buffer contains 10 mM, 50 mM, 100 mM,
150 mM, 200 mM, 250 mM, 300 mM, 350 mM, 375 mM, 400 mM, 425 mM, 450 mM, 500
mM, 1.0 M, 1.5 M, 2.0 M, or 2.5 M NaCl).
[0125] The pH of a suitable elution buffer may range fixjm 6.4 to 8.5 (e.g., 6.4 to 8.0,
6.4 to 7.8, 6.5 to 7.7, or 6.5 to 7.3). In some embodiments, the pH of a suitable elution buffer
may be 6.4, 65, 6.6, 6.8, 7.0, 7.2, 7.4, 7.6, 7.8, 8.0, 8.2, 8.4, or 8.5).
[0126] In some embodiments, elution buffers containing varied sah concentrations
may be used for elution of the bound product from the column in a continuous or stepwise
gradient.
34
[0127] Exemplary buffer and operating conditions are described in the Examples
section. High throughput screens, or ahemative screens (e.g., gradient elution screens), as
described in the Examples, may be used to efficiently optimize buffer and operating
conditions for hydroxyapatite chromatography.
[0128] TjT^ically, a binding mode cHA chromatography is used for the invention.
Alternatively or additionally, a flow-through mode can be used. In flow-through mode, a
protein preparation is typically buffer-exchanged into a suitable load buffer as described
herein. The protein preparation is then allowed to flow through a hydroxyapatite column,
while impurities such as HMW aggregates bind to the column. The column is optionally
subsequently washed to allow additional purified protein to flow through the column.
[0129] In combination binding/flow-through mode, the protein preparation is allowed
to flow through a hydroxyapatite column, with both protein monomer and HMW aggregates
binding initially. However, as the loading continues, incoming HMW aggregates are able to
bind more tightly than protein monomer and therefore displaces bound monomer.
Consequently, the displaced monomer flows through the column. The column is optionally
subsequently washed to allow additional displaced monomers to flow through the column.
[0130] In addition to the salts and buffers specifically discussed above,
chromatography and loading can occur in a variety of buffers and/or salts including sodium,
potassium, ammonium, magnesium, calcium, chloride, fluoride, acetate, phosphate, citrate
and/or Tris buffers. Specific examples of such buffers and salts are: Tris, sodium phosphate,
potassium phosphate, ammonium phosphate, sodiiun chloride, potassium chloride,
ammonium chloride, magnesium chloride, calcium chloride, sodixim fluoride, potassium
fluoride, ammonium fluoride, calcium fluoride, magnesium fluoride, sodium citrate,
potassium citrate, ammonium citrate, magnesium acetate, calcium acetate, sodium acetate,
potassium acetate, or ammonium acetate. A high throughput screen, as described in the
Examples, may be used to efficiently optimize buffer conditions for cHA chromatography.
[0131] In addition, various buffers and solutions described herein may be treated to
ensure free of endotoxin and/or exotoxin. In particular, if a purified protein preparation is
intended to be used for pharmaceutical and/or clinical purposes, it may be desirable to use
endotoxin- and/or exotoxin-free buffers. Various methods to remove endotoxins and/or
exotoxins from buffers or solutions are known in the art and can be used in the present
35
invention. For example, buffers and solutions can be depyrogenated. Depyrogenation may
be achieved by, e.g., acid-based hydrolysis, oxidation, heat, sodium hydroxide, among others.
Additional filtration steps
[0132] Additional membrane filtration steps may be used to reduce adventitious viral
and other contaminants, concentrate and/or buffer exchange. Various virus-retaining filters
can be used in the present invention including, but not limited to, Planova 20N virus retaining
filtration (VRP) and Planova 35N virus retaining filtration (VRF), among others. Various
ultrafiltration and/or diafiltration skids (e.g., molecular weight cut-off 10 kDa) can be used to
concentrate and/or buffer .exchange the process stream in the formulation buffer. The final
drug substance can be passed through, e.g., a single-use 0.2 pm filter, to remove any potential
adventitious microbial contaminants and particulate material.
Pharmaceutical compositions containing purified SMIP proteins
[0133] The purified protein preparations described herein can be formulated for
pharmaceutical uses. In some embodiments, pharmaceutical compositions according to the
invention may contain purified SMIP^^ proteins with less than 4% (e.g., less than 3.5%,
3.0%, 2.5%, 2.0%, 1.5%, 1.0%, 0.8%, 0.6%, 0.4%, 0.2,0.1%) HMW aggregates. In some
embodiments, pharmaceutical compositions according to the invention may contain purified
SMIP™ proteins with more than 70% (e.g., more than 75%, 80%, 85%, 90%, 92%, 94%,
96%, 98%, 99%) of the protein present in a biologically active monomer form.
[0134] Pharmaceutical compositions according to the invention may contain one or
more pharmaceutically acceptable carriers. Such pharmaceutically acceptable carriers are
intended to include any and all solvents, dispersion media, coatings, antibacterial and
antifungal agents, isotonic and absorption delaying agents, and the like, compatible with
pharmaceutical administration. The use of such media and agents for pharmaceutically active
substances is known in the art. Except insofar as any conventional media or agent is
incompatible with the active compound, use thereof in the compositions is contemplated.
Supplementary active compounds (identified according to the invention and/or known in the
36
art) also can be incorporated into the compositions by any of the methods well known in the
art of pharmacy/microbiology.
[0135] A pharmaceutical composition of the invention is formulated to be compatible
with its intended route of administration. Solutions or suspensions used for administration
can include components well known in the art, such as a sterile diluent such as water for
injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or
other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens;
antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as
ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents
for the adjustment of tonicity such as sodium chloride or dextrose, and/or acids or bases, such
as hydrochloric acid or sodium hydroxide, among others.
[0136] Formulations of the present invention suitable for administration can be in any
form known in the art. For example, suitable formulations for oral administration can be
capsules, gelatin capsules, sachets, tablets, troches, lozenges, powder, granules, a solution or
a suspension in an aqueous liquid or non-aqueous liquid, or an oil-in-water emulsion or a
water-in-oil emulsion, among others. The therapeutic can also be administered in the form of
a bolus, electuary or paste. As another example, suitable formulations for injectable use
include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for
the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous
administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor
ELTM (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). Formulations suitable
for intra-articular administration can be in the form of a sterile aqueous preparation of the
therapeutic which can be in microcrystalline form, for example, in the form of an aqueous
microcrystalline suspension. Liposomal formulations or biodegradable polymer systems can
also be used to present the therapeutic for both intia-articular and ophthalmic administration.
Formulations suitable for topical administiation, including eye treatment, include liquid or
semi-liquid preparations such as liniments, lotions, gels, applicants, oil-in-water or water-inoil
emulsions such as creams, ointments or pasts; or solutions or suspensions such as drops.
For inhalation treatments, such as for asthma, inhalation of powder (self-propelling or spray
formulations) dispensed with a spray can, a nebulizer, or an atomizer can be used. Such
formulations can be in the form of a finely comminuted powder for pulmonary administration
37
from a powder inhalation device or self-propelling powder-dispensing formulations [0109]
Systemic administration also can be by transmucosal or transdermal means.
[0137J According to the present invention, pharmaceutical compositions comprising
purified protein preparations can be administered to a mammalian host by any route. Thus,
as appropriate, administration can be oral or parenteral, e.g., intravenous, intradermal,
inhalation, transdermal (topical), transmucosal, and rectal administration.
[0138] The present invention, thus generally described, will be understood more
readily by reference to the following examples, which are provided by way of illustration and
are not intended to be limiting of the present invention.
EXAMPLES
Example 1. Cell Culture and Harvest
[0139] An anti-CD20 SMIP™ protein TRU-015 was produced using a recombinant
Chinese Hamster Ovary (CHO) cell line grown in suspension culture. An exemplary cell
culture and harvest process for the production of TRU-015 is illustrated in Figure 4. For all
the cell culture steps described herein, liquids added to the step were filtered at least once
through a 0.2 ^m filter prior to addition. The antifoam suspension, which cannot pass
through such fihers, was autoclaved prior to addition. Culture broth containing cells was not
filtered between steps.
[0140] Vials of cells containing CHO cell lines that express TRU-015 were thawed
and transferred to culture flasks containing pre-warmed, shake flask medium with 0.45 ^iM
methotrexate for selection pressure.
Cell Culture Expansion and Maintenance in Flasks and Wavebags
[0141] Cell cultures were initially expanded in disposable shake flasks (maximum
working volume 1 L) using a batch-refeed process. Each culture flask was incubated with
agitation under controlled temperature and CO2 atmosphere throughout a batch-refeed cycle.
At the end of a cycle, all or a portion of the culture was transferred to one or more other
38
shake flasks (or, if there is a sufficient number of cells, to wavebags - see below) and diluted
with shake flask medium to a pre-defined target initial cell density. After refeeding, each
diluted culture was returned to the incubator, where it was agitated throughout another batchrefeed
cycle. All cell culture medium used, up until the step of the terminal fed-batch culture,
optionally contained methotrexate to maintain selective pressure.
[0142] Culture expansion was continued in wavebags once a sufficient number of
cells were obtained by growth in shake flasks. The growth cycle and incubator conditions
were the same as for the flasks, except that wavebags were rocked instead of shaken. The
wavebag medium was the same as the shake flask medium.
[0143] Shake flask and wavebag cultures were continued in this fashion as long as
necessary, up until the specified maximum number of generations fi"om the thaw. Typically,
a seed train bioreactor was inoculated as soon as a sufficient number of cells were available
in the wavebags, and at least one wavebag was maintained as a backup to the seed train
bioreactors.
Cell Culture Expansion and Maintenance in Seed Train Bioreactors
[0144] Inoculum expansion was continued in seed train bioreactors. Seed bioreactor
medium was added to the bioreactor and supplemented with an autoclaved suspension of
antifoam in saline. Inoculum culture fiom the wavebags was added to a pre-defined target
cell density to start each batch-refeed passage. The culture was maintained with agitation
under controlled conditions throughout the passage, after which a portion was withdrawn and
used to inoculate the next bioreactor, or discarded if not needed. Temperature was controlled
at or near 37°C, dissolved oxygen (DO2) was controlled using sparged 0.2 ^im filtered air,
oxygen or a blend of both gases, and pH was controlled using carbonate solution (basic
titrant).
[0145] Each subsequent seed train bioreactor batch-refeed passage began with
retention of a portion of the culture fix)m the preceding cycle, dilution with seed bioreactor
medium and the addition of antifoam suspension. The seed train bioreactors and wave bags
were maintained in batch-refeed operations, both as needed to serve as backups to each other,
and to provide inocula for multiple production bioreactor batches. Once a sufficient number
of cells were available, the production bioreactor was inoculated.
39
Production Bioreactor
[0146] Conditioned medium containing TRU-015 were generated in a production
bioreactor using a terminal fed-batch process lasting 10 to 15 days. Inoculum culture from a
seed train bioreactor was added to initial production medium in the production bioreactor.
An antifoam suspension was added. The resulting culture was maintained with agitation
under controlled conditions throughout the batch. Approximately 4 days after inoculation,
the temperature set point was shifted from 37°C to 31°C. Throughout the production cell
culture process, DO2 was controlled using sparged 0.2 jim filtered air, oxygen or a blend, and
pH was controlled using carbonate solution (basic titrant). A concentrated feed medium
solution was also added during the fed-batch. Between 10 and 15 days after inoculation, the
entire volume of the production bioreactor culture was harvested. The harvest day was
chosen based on schedule considerations and/or on culture viability considerations.
[0147] Figure 5 shows an exemplary daily titer measurements (p.g/ml) of the
production bioreactor of TRU-015 produced by two different CHO cell clones over a 14 day
culture period. Peak titer values were obtained between days 12 and 14 of production
bioreactor growth. Peak titer values ranged from 1500 to 3000 ng/ml.
Harvest by Disc Stack Centrifugation (DSC)
[0148] Conditioned medium from the production bioreactor was harvested through a
disc-stacked centrifiige to yield clarified conditioned medium (CCM). One objective of the
DSC step was to separate CHO cells and cell debris from conditioned medium containing the
SMIP^^ protein. The contents of the bioreactor vessel were fed via pressure through the
DSC, then a pad filtration apparatus, and then 0.2 ^m filters into a filtrate vessel or bag.
Upon completion of the harvest processing, a HEPES/EDTA buffer solution was spiked into
the filtered cenfrate pool. One purpose of this spike was to reduce the generation of acidic
species during the in-process hold period between the DSC and subsequent steps. EDTA
may also inhibit protease activities and reduce protein A leaching. The DSC step was
operated at room temperature.
40
Example 2. High Throughput Screening of Chromatography Conditions
[0149] High throughput screens were used to develop optimal conditions for
purification process. Early high throughput screening of potential chromatography options
allows rapid identification of operating windows. Comparison of high throughput screening
results to database further narrows operating conditions. High throughput screening
minimizes the nimiber of column runs and in-process materials required and enables parallel
development efforts.
Protein A chromatography
[0150] The primary objectives of the Protein A chromatography step include product
capture from cell-free clarified conditioned medium and separation of TRU-015 from
process-derived impurities (e.g., host cell DNA and host cell proteins [HCPs], medium
components, and adventitious agents).
[0151] A high throughput screen was performed to optimize the Protein A column
conditions to increase product capture, impurity removal and minimize eluate precipitations.
An exemplary design of a high throughput screen using a batch binding mechanism is
illustrated in Figure 6 and an exemplary of Protein A column operation and high throughput
screen model is illustrated in Figure 7. As shown, different combinations of excipient wash,
elution and neutralization conditions were screened. In particular, the screen at least varied
levels of sodium chloride, calcium chloride, arginine, and Tris as wash excipients; HEPES,
acetic acid, and glycine as elution buffers; Tris, HEPES, and imidazole as neutralization
buffers; and sodium chloride concentration levels in elution (e.g., OmM, 15mM, 30mM, and
50mM).
[0152] The screen used filterplates containing 96 wells with each well having a
different condition. Each well contained about 50 ^il of resin and 300 |j,l of liquid. The resin
and liquid was mixed for about 20 minutes using Tecan Robot (Tecan US, Inc. 4022 Stirrup
Creek Drive Suite 310 Durham, NC 27703, USA) and the plates were centrifuged to collect
supernatant. The supernatant from each well was analyzed to determine the recovery of the
product, the amoimt of monomer and aggregates, and the presence of host cell proteins. For
example, UV absorbance at A280 was used to determine the overall protein concentration.
The turbidity was measured by absorbance at A320. The amoimt of monomer and aggregates
was measured by size exclusive HPLC. The host cell protein was characterized by ELISA.
41
[0153] Exemplary Protein A high throughput screen results are shown in Figure 8.
One exemplary favorable condition identified in this experiment included calcium wash,
acetic acid elution with sodium chloride, and HEPES neutralization (see Figure 8).
Ion Exchange Chromatography
[0154] The primary objectives of ion exchange chromatography include removal of
process-derived impurities (e.g., leached Protein A, host cell DNA and proteins, and
adventitious agents) as well as product-related impurities such as high molecular weight
(HMW) species. Similarly, high throughput screen was used to identify potential operating
conditions for anion exchange chromatography (AEX) conditions and cation exchange
chromatography (CEX) to remove impurities. Exemplary variables tested are shown in Table
1.
Table 1. High Throughput Screen Approach for AEX and CEX
Variable Resin Type T t H P^ ^^"^'^ Strength
7.0-8.75 10-210mM
(8 levels) (12 levels)
Typical Range ~""~"~~~"~"~ ^~~'~"~"~"~ —^.—^_^— . . _ ^ - ^ — ^ _ i ^ _ ^ _
PP„ ^ 4.5-6.5 20-300mM
(8 levels) (6 levels)
Ceramic Hvdroxvapatite (cHA) Chromatographv
[0155] The primary objectives of ceramic hydroxyapatite (cHA) chromatography
include the removal of high molecular weight (HMW) aggregates, leached Protein A, and
host cell-derived impurities, such as DNA and HCPs.
[0156] High throughput screens were also performed to optimize operating conditions
for ceramic hydroxyapatite chromatography. The screens at least varied pH, salts
concentrations and phosphate concentration. Exemplary variables are shown in Figure 9.
Exemplary results with respect to the removal of HMW aggregate are also shown in Figure 9.
42
Example 3. Development of the cHA Chromatography Step
[0157] A high throughput screen was able to qualitatively predict suitable monomer
recovery and HMW aggregates removal conditions in a column purification scheme. For
example, the high throughput screens identified that the cHA chromatography step was
effective in removing HMW aggregates. Approximate ranges of salt or buffer conditions
suitable for removing HMW aggregates were also predicted (see Figure 9). Alternative
screens may be used to further refine the conditions identified by high throughput screens.
[0158] An alternative screening using a cHA column and a sodium chloride gradient
elution was performed. An exemplary scheme and result is shown in Figure 10.
[0159] Potential elution buffers based on high throughput and alternative screenings
were further evaluated in the step-elution mode. For example, a Protein A column peak pool
with 60% HMW aggregates 6000 ppm HCP was purified using cHA columns with different
combinations of phosphate and NaCl concentrations as shown in Table 2.
Table 2. Exemplary Purification of Protein A Column Peak Pool
Run Phos.(mM) NaCl(mM) %HMW %REC HCP (ppm) ProA(ppm)
1 " 30 ' 50 " 0.2 88 954 <1
2 35 50 02 92 1529 <1
3 4 350 0^4 88 50 <1
4 I 4 I 375 I 0.5 I 86 I 42 <1
[0160] An anion exchange chromatography pool with 59% HMW and 290 ppm HCP
was purified using cHA columns with different combinations of phosphate and NaCl
concentrations as shown in Table 3.
Table 3. Exemplary Purification of Anion Exchange Chromatography Pool
Run Phos(mM) NaCl (mM) %HMW %REC HCP (ppm) ProA(ppm)
1 60 ~ 10 0.1 73 " 137 <1 "
2 40 50 05 80 198 <1
3 20 100 OJ 78 1£1 <1
4 I 10 I 200 I 1 I 82 I 32 <1
43
[0161] An exemplary typical cHA chromatography step developed based on the
experiments described herein is shown in Figure 11.
Example 4. Purification Process for TRU-015
[0162] Based on the experiments described above, purification processes of SMIP^^
proteins have been developed. An exemplary purification process of TRU-015 is illustrated
in Figure 12. This process includes three chromatographic steps and three membrane
filtration steps. All steps were performed at room temperature unless indicated otherwise.
[0163] Clarified cell-free conditioned medium prepared as described in Example 1
was first subject to MabSelect™^ Protein A affinity chromatography. A 17.7 L (30 cm
diameter x 25 cm height) MabSelect^*^ column with recombinant Protein A resin (GE
Healthcare, Piscataway, NJ) was used. The Protein A column was equilibrated with Hepesbuffered
saline and loaded with clarified conditioned mediimi. The loaded resin was washed
with a Hepes buffer containing calcium chloride to fiirther reduce the level of impurities,
followed by a wash containing a low concentration of Hepes buffer and sodium chloride.
The bound product was eluted from the colirnm with a low pH acetic acid buffer.
[0164] The product pool was held at pH < 4.1 for about 1.5 ± 0.5 hours at 18°C to
24°C. The low pH hold was designed to inactivate enveloped viruses. The elution pool was
then neutralized with a concentrated Hepes buffer. The resin was regenerated, sanitized, and
then stored in an ethanol solution.
[0165] A TMAE HiCap (M) column was equilibrated first with a high salt buffer and
then a low salt buffer. The column was loaded with the neutralized MabSelect rProtein A
peak. One or multiple neutralized MabSelect rProtein A peak pools were loaded onto the
TMAE HiCap (M) column. TRU-015 binds only weakly to the column, which allows the
majority of the product to flow through while impurities with a negative charge, such as host
cell DNA and HCPs, bind strongly and are retained. The TMAE HiCap (M) column was
then washed with equilibration buffer to collect additional product weakly bound to the resin.
Once the product was removed from the TMAE HiCap (M) column, impurities were stripped
using a high salt buffer. The resin was regenerated, sanitized, and then stored in an ethanol
solution.
44
[0166] A cHA column was first equilibrated with a high salt buffer and then followed
with a low sah buffer. The TMAE flow-through pool was then applied to the cHA column.
After loading, the column was washed with the low salt equilibration buffer and TRU-015
was recovered using a higher sah buffer (see Example 3). The HMW species and other
impurities were removed from the column at much higher salt and phosphate concentrations.
The column was regenerated and then stored in a sodium hydroxide solution.
[0167] A Pianova 20N virus retaining filtration (VRF) step provides a significant
level of viral clearance for assurance of product safety by removal of particles that may
represent potential adventitious viral contaminants. The single-use Piano va 20N VRF device
was equilibrated and loaded with the cHA product pool. The TRU-015 protein was collected
in the permeate stream. After the load was processed, an equilibration buffer flush was used
to recover additional product remaining in the system.
[0168] An ultrafiltration/diafiltration skid (molecular weight cut-off 10 kDa) was
used to concentrate and buffer exchange the process stream in the formulation buffer. After
equilibration, the load solution was initially concentrated approximately 8-fold and then
diafiltered at least 10-fold with formulation buffer (e.g., 20 mM L-histidine, 4% mannitol, 1%
sucrose, pH 6.0). Following further concentration, the pool was recovered from the system
with a formulation buffer flush.
[0169] The drug substance was passed through a single-use 0.2 jxm filter to remove
any potential adventitious microbial contaminants and particulate material.
[0170] Filtered TRU-015 drug substance was filled into, e.g., stainless steel vessels,
frozen, and stored at -50°C ± 10°C.
[0171] The column performance at each step was analyzed to determine the product
recovery and impurity removal efficiency. Exemplary results are summarized in Table 4.
45
Table 4. Exemplary summary of column performance (lab-scale)
Z I 0/ox^w I HCP (ppm) I ProA (ppm) I%Rec (POI)* I %Rec
Step %HMW (total)
Harvest 50-61 300,000 NA 95 95
Protein A 50-61 5,000-45,000 20-50 85-95 81-90
AEX 48-60 50-400 1-3 85-95 86-92
cHA <2 <100 <1 75-85 35-41
Total Process Recovery excluding VRF and UF/DF: 51-73 23-32
* POI: Product-of-Interest
[0172] As shown in Table 4, the cHLA chromatography step effectively removed most
of the HMW aggregates. The total process recovery for the product of interest ranged from
51-73% and the product yield was about 23-32%. The results shown in Table 4 were based
on lab-scale purification processes. Comparable results were obtained for clinical
manufacturing processes. For example, based on multiple clinical-scale processes, the
average yield was about 28%, and the percentage of HMW aggregates in purified product
was about 0.8% or less (reduced from 50-60% in the starting material). Compared to an
existing process, there is more than an 8-fold increase in productivity due to increases in
protein expression/harvest and purification yield.
Example 5. Pad filtration of Load Material
[0173] Load materials can be optionally subjected to pad filtration to increase the
capacity for impurities and life time of the anion exchange column. For example, the TMAE
Hi-cap resin is a strong anion exchanger with a synthetic methacrylate polymeric base. The
pores that are formed from intertwined polymer agglomerates have an approximate size of
800 Angstroms. The surface is strongly hydrophilic due to the ether linkages in the polymer.
Long, linear polymer chains known as tentacles, carry the functional ligands. The tentacles
are covalently attached to hydroxyl groups of the methacrylate backbone. Impurities can
exhaxist or block the binding surface of the TMAE column reducing capacity.
46
[0174] Use of an adsorptive depth pre-filter can protect a column, such as the TMAE
column, from the impurities in the load material (e.g., MabSelect^^ ProA peak pool). Depth
filtration media is typically a highly porous one cm thick filter composed of cellulose fibers,
diatomaceous earth, and a cationic resin binder. The depth filter can remove impurities by
sieving through the cellulose fibers, by hydrophobic adsorption to the diatomaceous earth,
and by ionic adsorption to the cationic binder.
[0175] During the purification of TRU-015, a Millistack AIHC PAD filter (Millipore,
Billerica, MA) was used to remove impurities from the MabSelect ProA peak pool before
loading to the TMAE column and improved the TMAE capacity by at least 2-fold. For
example, prefiltration of the product load with the adsorptive depth filter Millistack AIHC
provided an increase in the load challenge on a subsequent TMAE Hi-Cap resin column from
100 to 200 mg/mL, as indicated by the secondary breakthrough of contaminants. The AIHC
was run at a flux of 200 liters per square meter per hour and loaded to 200 liters per square
meter.
Example 6. Capture of SBI-087 Using Cation Exchange Chromatographv
[0176] This example demonstrates that CEX can be used to effectively capture
SMIP^'^ instead of affinity chromatography. It is contemplated that using CEX may have
various benefits including lower capture costs, possibly greater capacities than certain affinity
colimins, solid potential for HMW reduction and potential elimination of cHA or anion
exchange step, among others.
[0177] SBI-087 was used in this experiment. Load material for CEX can be acidified
using any suitable acid. Exemplary acidification conditions are shown in Table 5.
Table 5. Exemplary summary of load material acidification
Percent IM
Acetic Acid
Load pH addition (v/v)
4.25 20%
4^5 12%
4.75 7%
5 I 5%
47
[0178] Reduction of amount of HM W aggregates by MabSelect^^ Protein A affinity
chromatography was compared to that by CEX using a batch-binding method. As shown in
Figure 13, the removal of HMW aggregates by CEX is comparable or better than
MabSelect^^ affinity chromatography, indicating that CEX can be used to replace affinity
chromatography for removal of HMW aggregates fi-om a protein preparation.
[01791 Exemplary CEX steps include load and elute. Operating conditions for CEX
capture were optimized using high throughput screen methods. For example, two types of
CEX resins, GigaCap® and Capto^^ S, were used. Load challenge of 25 mg/mLr vs. 75
mg/mLr were lised. Load pH (adjusted with IM acetic acid) was 4.25, 4.5,4.75, or 5.0.
Elution conditions were as follows:
Elution (total mM Na+):
pH 5 (acetate): 100, 125, 150, 175 mM
pH6.5(MES): 40,65,90,115
pH8.0(Tris): 20,40,60,80
Assumes 50 mM buffer
[0180] Exemplary results showing binding capacities with 2 hour incubation at room
temperature are shown in Figure 14. It was also observed that higher or longer challenge can
lead to more LMW species in the eluted pool. Exemplary results illustrating CEX peaks
eluted from columns loaded with with 25 vs. 75 mg/mLr LC are shown in Figure 15.
Exemplary strip conditions utilized were 8M urea, 2M NaCl, pH 6. CEX resins can be
stripped and/or reused.
Example 7. Removal of HMW using Anion Exchange Chromatography
[0181] This example demonstrates that anion exchange chromatography can be used
to effectively remove HMW impurities from SMIP^^ preparation. In this experiment, SBI-
087 was used as an exemplary SMIP^^ protein.
[0182] An anion exchange chromatography step was developed using the high
throughput screen approach as outlined in Table 1, The chromatography conditions derived
fiiom that screen were employed in an exemplary run using a load containing Protein A peak
pool with 37% HMW. A packed column of Fractogel® TMAE HiCap was run in the weak
48
partitioning chromatography mode to a load challenge of 100 and 93 mg/mL, respectively.
Figure 16 shows an exemplary effective removal of HMW. The collected pool was 88% pure
with >95% yield of the "monomeric" SMIP^^. The post-load wash allowed for greater
recovery of "monomeric" SMIP™ protein.
[0183] These results demonstrated that the second column (e.g., AEX)
chromatography can effect substantial removal of HMW, which allows for more flexibility
and lower costs in developing and running the downstream cHLA step. In addition, these
results also indicate that it is feasible to develop a 2-column (e.g., protein A to AEX) process
to remove substantial amount of HMW impurities from protein preparation.
49
EXEMPLARY SMIP™ SEQUENCES
Italics: Linker sequence
Underline; CDR sequences
Construct Name
VK3 VH5
EIVLTOSFATLSLSPGERATLSCRASOSVSYIV
WYOOKPGOAPRLLIYAPSNLASGIPARFSGS
GSGTDFTLTISSLEPEDFAVYYCOOWSFNPPT
FGQGTKVEIKDGGGSGGGGSGGGGTGEVQLV
2Lml9- , OSGAEVKKPGESLKISCKGSGYSFTSYNMHW
^^"^^ 3 2«5ni3 VROMPGKGLEWMGAIYPGNGDTSYNOKFKG
QVTISADKSISTAYLQWSSLKASDTAMYYCAR
SYYSNSYWYFDLWGRGTLVTVSS
(SEQIDNO:!)
EIVLTOSPATLSLSPGERATLSCRASOSVSYMH
WYOOKPGOAPRLLIYAPSNLASGIPARFSGSGS
GTDFTLTISSLEPEDFAVYYCOOWSFNPPTFGO
GJKVElYiDGGGSGGGGSGGGGTGEVQLVQSGA
EVKKPGESLKISCKGSGYSFTSYNMHWVRQMP
18008 2Lm5 2H5 GKGLEWMGAIYPGNGDTSYNOKFKGOVTISA
DKSISTAYLQWSSLKASDTAMYYCAR
WYYSNSYWYFDLWGRGTLVTVSS
(SEQ ID N0:2)
EIVLTOSPATLSLSPGERATLSCRASOSVSYIV
WYOOKPGOAPRLLIYAPSNLASGIPARFSGSG
SGTDFTLTISSLEPEDFAVYYCOOWSFNPPTF
GQGTKVEIKDGGGSGGGGSGGGGTGEVQLW
2Lml9- ^ „ - OSGAEVKKPGESLKISCKGSGYSFTSYNMHW
3 VROMPGKGLEWMGAIYPGNGDTSYNOKFKG
QVTISADKSISTAYLQWSSLKASDTAMYYCAR
WYYSNSYWYFDLWGRGTLVTVSS
(SEQIDN0:3)
50
EIVLTOSPATLSLSPGERATLSCRASOSVSYIV
WYOOKPGOAPRLLIYAPSNLASGIPARFSGS
GSGTDFTLTISSLEPEDFAVYYCOOWSFNPPT
FGQGTKVEIKDGGGSGGGGSGGGGTGEVQLV
QSGAEVKXPGESLKISCKGSGYSFTSYNMHW
18009 2Lm5 2H5m3 VROMPGKGLEWMGAIYPGNGDTSYNOKFKG
QVTISADKSISTAYLQWSSLKASDTAMYYCA
RWYYSNSYWYFDLWGRGTLVTVSS
(SEQ ID N0:4)
EIVLTOSPATLSLSPGERATLSCRASOSVSSYMH
WYOOKPGOAPRLLIYAPSNLASGIPARFSGSGS
GTDFTLTISSLEPEDFAVYYCOOWSFNPPTFG
QGTKVEIKD(7GG5C7GC?G5GGG(7rGEVQLLES
^ ^ m ^ ->! «; ?m -x GGGLVOPGGSLRLSCAASGFTFSSYNMHWVR
2Lm5ZH3m3 ZLinS 2U3m3 OAPGKGLEWVSAIYPGNGPTSYNOKFKGRFT
ISRDNSKNTLYLQMNSLRAEDTAVYYCA
KSYYSNSYWYFDLWGRGTLVTVSS
(SEQIDN0:5)
Construct Name
VK3 VHl
EIVLTOSPATLSLSPGERATLSCRASSSVSSYMHW
YOOKPGOAPRLLIYAPSNLASGIPARFSGSGSGTD
FTLTISSLEPEDFAVYYCOOWSFNPPTFGOGTKV
EIKDGGG5GGGG5GGGG55QVQLVQSGAEVKKP
GASVKVSCKASGYTFTSYNMHWVROAPGOGLE
2L IHm WMGAIYPGNGDTSYNOKFKGRVTMTRDTSTST
VYMELSSLRSEDTAVYYCARSVYYSN.YWYFDL
WGRGTLVTVSS
(SEQIDN0:6)
EIVLTOSPATLSLSPGERATLSCRASSSVSYMIW
YOOKPGOAPRLLIYAISNLASGIPARFSGSGSGT
DFTLTISSLEPEDFAVYYCOOWISNPPTFGOGTK
VEIKZ)GGG5GGGG5GGGG55QVQLVQSGAEVK
KPGASVKVSCKASGYTFTSYNMHWVRQAPGQ
2Lm 2Hm GLEWMGAIYPGNGDTSYNOKFKGRVTMTRDT
STSTVYMELSSLRSEDTAVYYCAR
SVYYSN.YWYFDLWGRGTLVTVSS
(SEQ ID N0:7)
51
EIVLTOSPATLSLSPGERATLSCRASSSVSYMIW
YOOKPGOAPRLLIYAISNLASGIPARFSGSGSGT
DFTLTISSLEPEDFAVYYCOOWISNPPTFGOGTK
VEIKDGGGSGGGGSGGGGSSQWQLVQSGAEYK
KPGASVKVSCKASGYTFTSYNMHWVRQAPGQ
2Lm 2H GLEWMGAIYPGNGDTSYNOKFKGRVTMTRDT
STSTVYMELSSLRSEDTAVYYCAR
WYYSNSYWYFDLWGRGTLVTVSS
(SEQIDN0:8)
EIVLTOSPATLSLSPGERATLSCRASOSSVSYMH
WYOOKPGOAPRLLIYAPSNLASGIPARFSGSGS
GTDFTLTISSLEPEDFAVYYCOOWISNPPTFGOG
7KYEl¥J)GGGSGGGGSGGGGSSQyQLyQSGAE
VKKPGASVKVSCKASGYTFTSYNMHWVRQAP
2Linl 2Hm GOGLEWMGATyPGNGDTSYNOKFKGRVTMTRD
TSTSTVYMELSSLRSEDTAVYYCAR
SVYYSN.YWYFDLWGRGTLVTVSS
(SEQ ID N0:9)
EIVLTOSPATLSLSPGERATLSCRASOSSVSYMH
WYQQKPGQAPRLLIYAPSNLASGIPARFSGSGSG
TDFTLTISSLEPEDFAVYYCOOWISNPPTFGOGTK
VEIKDGGGSGGGGSGGGGSSQVQLVQSGAEWKKP
GASVKVSCKASGYTFTSYNMHWVRQAPGQGLEW
2Liiil 2H MGAIYPGNGDTSYNOKFKGRVTMTRDTSTSTVY
MELSSLRSEDTAVYYCARWYYSNSYWYFDLW
GRGTLVTVSS
(SEQ ID NO: 10)
EIVLTQSPATLSLSPGERATLSCRASOSVSYMIW
YQQKPGOAPRLLIYAISNLASGIPARFSGSGSGT
DFTLTISSLEPEDFAVYYCOOWSFNPPTFGOGTK
YEIKDGGGSGGGGSGGGGSSQVQLVQSGAEVK
KPGASVKVSCKASGYTFTSYNMHWVROA
2Lm2 2Hm PGOGLEWMGAIYPGNGDTSYNOKFKGRV
TMTRDTSTSTVYMELSSLRSEDTAVYYCA
RSVYYSN.YWYFDLWGRGTLVTVSS
(SEQ ID NO: 11)
EIVLTQSPATLSLSPGERATLSCRASSSVSYMI
WYOQKPGQAPRLLIYAISNLASGIPARFSGSG
- , SGTDFTLTISSLEPEDFAVYYCQOWTSNPPTF
GQGTKYEIKDGGGSGGGGSGGGGSSQWQLV
OSGAEVKKPGASVKVSCKASGYTFTSYNMH
WVROAPGOGLEWMGAIYPGNGDTSYNOKFKG
52
RVTMTRDTSTSTVYMELSSLRSEDTAVYYCA
RSVYYSN.YWYFDLWGRGTLVTVSS
(SEQIDN0:12)
EIVLTOSPATLSLSPGERATLSCRASOSVSSYMH
WYOOKPGOAPRLLIYAPSNLASGIPARFSGSGS
GTDFTLTISSLEPEDFAVYYCQOWTSNPPTFGO
GTKVEIKDGGGSGGGGSGGGGSSQWQLVQSGA
EVKKPGASVKVSCKASGYTFTSYNMHWVRQA
2Lin4 2Hin PGOGLEWMGAIYPGNGDTSYNOKFKGRVTMT
RDTSTSTVYMELSSLRSEDTAVYYCAR
SVYYSN.YWYFDLWGRGTLVTVSS
(SEQIDN0:13)
EIVLTOSPATLSLSPGERATLSCRASOSVSYMH
WYOOKPGOAPRLLIYAPSNLASGIPARFSGSG
SGTDFTLTISSLEPEDFAVYYCOOWSFNPPTFG
QGJKWEIKDGGGSGGGGSGGGGSSQVQLYQS
GAEVKKPGASVKVSCKASGYTFTSYNMHWV
2Lm5 2HI11 ROAPGOGLEWMGAIYPGNGDTSYNOKFKGRV
TMTRDTSTSTVYMELSSLRSEDTAVYYCA
RSVYYSN.YWYFDLWGRGTLVTVSS
(SEQIDN0:14)
EIVLTOSPATLSLSPGERATLSCRASOSVSYMH
WYOOKPGOAPRLLIYAPSNLASGIPARFSGSGS
GTDFTLTISSLEPEDFAVYYCOOWSFNPPTFGO
GTKVEIKDGGGSGGGGSGGGGSSQVQLVQSGA
EVKKPGASVKVSCKASGYTFTSYNMHWVRQA
2Lin5-l 2Hm3 PGOGLEWMGAIYPGNGDTSYNOKFKGRVTMT
RDTSTSTVYMELSSLRSEDTAVYYCAR
S.YYSNSYWYFDLWGRGTLVTVSS
(SEQIDN0:15)
EIVLTOSPATLSLSPGERATLSCRASOSVSYMH
WYOOKPGOAPRLLIYAPSNLASGIPARFSGSGS
GTDFTLTISSLEPEDFAVYYCOOWSFNPPTFGO
GTKWEIKDGGGSGGGGSGGGGSSQWQLWQSGA
EVKKPGASVKVSCKASGYTFTSYNMHWVROA
2Lm5-2 2Hin4 PGOGLEWMGAIYPGNGDTSYNOKFKGRVTMT
RDTSTSTVYMELSSLRSEDTAVYYCAR
V.YYSNSYWYFDLWGRGTLVTVSS
(SEQIDN0:16)
53
EIVLTOSPATLSLSPGERATLSCRASOSVSYMH
WYOOKPGOAPRLLIYAPSNLASGIPARFSGSGS
GTDFTLTISSLEPEDFAVYYCOOWSFNPPTFGO
GTKWEIKDGGGSGGGGSGGGGSSQVQLVQSGA
EVKKPGASVKVSCKASGYTFTSYNMHWVRQA
2Lin5-3 2Hm5 PGOGLEWMGAIYPGNGDTSYNOKFKGRVTMT
RDTSTSTVYMELSSLRSEDTAVYYCAR
SVYY.NSYWYFDLWGRGTLVTVSS
(SEQIDN0:17)
EIVLTOSPATLSLSPGERATLSCRASOSVSYMH
WYOOKPGOAPRLLIYAPSNLASGIPARFSGSG
SGTDFTLTISSLEPEDFAVYYCOOWTSNPPTF
GQGTKYEJKDGGGSGGGGSGGGGSSQVQLW
QSGAEVKKPGASVKVSCKASGYTFTSYNMH
2Lm6 2Hin WVROAPGOGLEWMGAIYPGNGDTSYNOtCFKG
RVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR
SVYYSN.YWYFDLWGRGTLVTVSS
(SEQIDN0:18)
EIVLTOSPATLSLSPGERATLSCRASOSVSYMH
WYOOKPGOAPRLLIYAPSNLASGIPARFSGSGS
GTDFTLTISSLEPEDFAVYYCOOWTSNPPTFGO
GTKYEIKDGGGSGGGGSGGGGSSQYQLWQSGA
EVKKPGASVKVSCKASGYTFTSYNMHWVRQA
2Lm6-l 2Hm3 PGOGLEWMGAIYPGNGDTSYNQKFKGRVTMT
RDTSTSTVYMELSSLRSEDTAVYYCAR
S.YYSNSYWYFDLWGRGTLVTVSS
(SEQIDN0:19)
EIVLTOSPATLSLSPGERATLSCRASOSVSYMH
WYQQKPGQAPRLLIYAPSNLASGIPARFSGSGS
GTDFTLTISSLEPEDFAVYYCOOWTSNPPTFGO
GTKYEIKDGGGSGGGGSGGGGSSQWQLWQSGA
EVKKPGASVKVSCKASGYTFTSYNMHWVROA
2Lm6-2 2Hm4 PGOGLEWMGAIYPGNGDTSYNQKFKGRVTMT
RDTSTSTVYMELSSLRSEDTAVYYCAR
V.YYSNSYWYFDLWGRGTLVTVSS
(SEQIDNO:20)
EIVLTOSPATLSLSPGERATLSCRASOSVSYMH
WYOOKPGOAPRLLIYAPSNLASGIPARFSGSG
2T fil 7H s SGTDFTLTISSLEPEDFAVYYCQOWTSNPPTFG
QGTKYEI¥iDGGGSGGGGSGGGGSSQYQLWQS
GAEVKKPGASVKVSCKASGYTFTSYNMHWV
RQAPGQGLEWMGAIYPGNGDTSYNOKFKGR
54
VTMTRDTSTSTVYMELSSLRSEDTAVYYCAR
SVYY.NSYWYFDLWGRGTLVTVSS
(SEQIDN0:21)
EIVLTOSPATLSLSPGERATLSCRASSSVSYMH
WYOOKPGOAPRLLIYATSNLASGIPARFSGSG
SGTDFTLTISSLEPEDFAVYYCOOWTSNPPTFG
QGTKWEIKDGGGSGGGGSGGGGSSQVQLYQS
GAEVKKPGASVKVSCKASGYTFTSYNMHWV
2Lm7 2Hm ROAPGOGLEWMGAIYPGNGDTSYNOKFKGR
VTMTRDTSTSTVYMELSSLRSEDTAVYYCAR
SVYYSN.YWYFDLWGRGTLVTVSS
(SEQIDNO:22)
EIVLTQSPATLSLSPGERATLSCRASSSVSYMI
WYOOKPGOAPRLLIYAISNLASGIPARFSGSG
SGTDFTLTISSLEPEDFAVYYCOOWISNPYTF
GQGTKVEIKDGGGSGGGGSGGGGSSQVQLY
QSGAEVKKPGASVKVSCBCASGYTFTSYNMH
2Lm8 2Hm WVROAPGOGLEWMGAIYPGNGDTSYNOKFKG
RVTMTRDTSTSTVYMELSSLRSEDTAVYYCA
RSVYYSN.YWYFDLWGRGTLVTVSS
(SEQIDNO:23)
EIVLTQSPATLSLSPGERATLSCRASSSVSYMI
WYQQKPGOAPRLLIYAISNLASGIPARFSGSG
SGTDFTLTISSLEPEDFAVYYCOOWISNPFTFG
QGTKWEIKDGGGSGGGGSGGGGSSQVQLVQS
GAEVKKPGASVKVSCKASGYTFTSYNMHWV
2Lm9 2Hin ROAPGOGLEWMGAIYPGNGDTSYNOKFKGR
VTMTRDTSTSTVYMELSSLRSEDTAVYYCAR
SVYYSN.YWYFDLWGRGTLVTVSS
(SEQIDNO:24)
EIVLTOSPATLSLSPGERATLSCRASSSVSYMI
WYOOKPGOAPRLLIYAISNLASGIPARFSGSG
SGTDFTLTISSLEPEDFAVYYCOOWISNPLTFG
QGTKVEIKDGGGSGGGGSGGGGSSQYQLWQS
GAEVKKPGASVKVSCKASGYTFTSYNMHWV
2L111IO 2Hin ROAPGOGLEWMGAIYPGNGDTSYNOKFKGR
VTMTEIDTSTSTVYMELSSLRSEDTAVYYCA
RSVYYSN.YWYFDLWGRGTLVTVSS
(SEQIDNO:25)
55
EIVLTOSPATLSLSPGERATLSCRASSSVSYMI
WYOOKPGOAPRLLIYAISNLASGIPARFSGSG
SGTDFTLTISSLEPEDFAVYYCOOWISNPITFG
QGTKVEIKDGGGSGGGGSGGGGSSQVQLVQS
GAEVKKPGASVKVSCKASGYTFTSYNMHWV
2Lmll 2Hin ROAPGOGLEWMGAIYPGNGDTSYNOKFKGR
VTMTRDTSTSTVYMELSSLRSEDTAVYYCAR
SVYYSN.YWYFDLWGRGTLVTVSS
(SEQIDNO:26)
EIVLTOSPATLSLSPGERATLSCRASOSVSYMH
WYOOKPGOAPRLLIYATSNLASGIPARFSGSG
SGTDFTLTISSLEPEDFAVYYCOOWSFNPPTFG
QGTKVEIKDGGGSGGGGSGGGGSSQVQLVQS
GAEVKKPGASVKVSCKASGYTFTSYNMHWV
2Linl2 2Hin ROAPGOGLEWMGAIYPGNGDTSYNOKFKGR
VTMTRDTSTSTVYMELSSLRSEDTAVYYCAR
SVYYSN.YWYFDLWGRGTLVTVSS
(SEQ ID NO:27)
EIVLTOSPATLSLSPGERATLSCRASOSVSYMH
WYOOKPGOAPRLLIYAPSNLASGIPARFSGSGS
GTDFTLTISSLEPEDFAVYYCOOWISNPPTFGOG
TKVEIKDGGG5GGGGSGGGGSSQVQLVQSGAE
VKKPGASVKVSCKASGYTFTSYNMHWVRQAP
2Lml3 2Hm GOGLEWMGAIYPGNGDTSYNOKFKGRVTMTR
DTSTSTVYMELSSLRSEDTAVYYCAR
SVYYSN.YWYFDLWGRGTLVTVSS
(SEQIDNO:28)
EIVLTOSPATLSLSPGERATLSCRASOSVSYMH
WYOOKPGOAPRLLIYATSNLASGIPARFSGSGS
GTDFTLTISSLEPEDFAVYYCOOWISNPPTFGO
GTKVEIKDGGGS'GGGGSGGGGS^QVQLVQSGA
EVKKPGASVKVSCKASGYTFTSYNMHWVRQA
2Lml4 2Hin PGOGLEWMGAIYPGNGDTSYNOKFKGRVTMT
RDTSTSTVYMELSSLRSEDTAVYYCAR
SVYYSN.YWYFDLWGRGTLVTVSS
(SEQIDNO:29)
EIVLTOSPATLSLSPGERATLSCRASOSVSYIHW
YOOKPGOAPRLLIYAPSNLASGIPARFSGSGSG
IH TDFTLTISSLEPEDFAVYYCOOWISNPPTFGOG
2l.inl5 2Hm TKVEIKDGGG5GGGG5GGGG55QVQLVQSGA
EVKKPGASVKVSCKASGYTFTSYNMHWVRO
APGOGLEWMGAIYPGNGDTSYNOKFKGRVT
56
MTRDTSTSTVYMELSSLRSEDTAVYYCAR
SVYYSN.YWYFDLWGRGTLVTVSS
(SEQIDNO:30)
EIVLTOSPATLSLSPGERATLSCRASSSVSYMH
WYOOKPGOAPRLLIYAPSNLASGIPARFSGSG
SGTDFTLTISSLEPEDFAVYYCQOWSFNPPTFG
QGTKWEIKDGGGSGGGGSGGGGSSQYQLYQS
GAEVKKPGASVKVSCKASGYTFTSYNMHWV
2Linl6 2Hm3 ROAPGOGLEWMGAIYPGNGDTSYNOKFKGR
VTMTRDTSTSTVYMELSSLRSEDTAVYYCAR
S.YYSNSYWYFDLWGRGTLVTVSS
(SEQIDN0:31)
EIVLTOSPATLSLSPGERATLSCRASOSVSYLS
WYOOKPGOAPRLLIYAPSNLASGIPARFSGSG
SGTDFTLTISSLEPEDFAVYYCOOWSFNPPTFG
QGTKVEIKDGGGSGGGGSGGGGSSQVQLVQS
GAEVKKPGASVKVSCKASGYTFTSYNMHWV
2Linl7-3 2Hm3 ROAPGOGLEWMGAIYPGNGDTSYNOKFKGR
VTMTRDTSTSTVYMELSSLRSEDTAVYYCA
S.YYSNSYWYFDLWGRGTLVTVSS
(SEQIDNO:32)
EIVLTOSPATLSLSPGERATLSCRASOSVSYLT
WYOOKPGOAPRLLIYAPSNLASGIPARFSGSG
SGTDFTLTISSLEPEDFAVYYCOOWSFNPPTFG
QGTKWEIKDGGGSGGGGSGGGGSSQYQLYQS
GAEVKKPGASVKVSCKASGYTFTSYNMHWV
2Lml7-4 2Hm3 ROAPGOGLEWMGAIYPGNGDTSYNOKFKGR
VTMTRDTSTSTVYMELSSLRSEDTAVYYCAR
S.YYSNSYWYFDLWGRGTLVTVSS
(SEQIDNO:33)
EIVLTOSPATLSLSPGERATLSCRASOSVSYLY
WYOOKPGOAPRLLIYAPSNLASGIPARFSGSGS
GTDFTLTISSLEPEDFAVYYCOOWSFNPPTFGO
GTKVEIKDGGGSGGGGSGGGGSSQYQLVQSGA
EVKKPGASVKVSCKASGYTFTSYNMHWVRQA
2Lml7-6 2Hm3 PGOGLEWMGAIYPGNGDTSYNOKFKGRVTMT
RDTSTSTVYMELSSLRSEDTAVYYCAR
S.YYSNSYWYFDLWGRGTLVTVSS
(SEQIDNO:34)
57
EIVLTOSPATLSLSPGERATLSCRASOSVSYLH
WYOOKPGOAPRLLIYAPSNLASGIPARFSGSG
SGTDFTLTISSLEPEDFAVYYCOOWSFNPPTFG
QGTKWEIKDGGGSGGGGSGGGGSSQYQLYQS
GAEVKKPGASVKVSCKASGYTFTSYNMHWV
2Lml7-8 2Hm3 ROAPGOGLEWMGAIYPGNGDTSYNOKFKGR
VTMTRDTSTSTVYMELSSLRSEDTAVYYCAR
S.YYSNSYWYFDLWGRGTLVTVSS
(SEQIDNO:35)
EIVLTOSPATLSLSPGERATLSCRASOSVSYLN
WYQQKPGQAPRLLIYAPSNLASGIPARFSGSG
SGTDFTLTISSLEPEDFAVYYCOOWSFNPPTF
GQGTKVEIKDGGGSGGGGSGGGGSSQVQLY
QSGAEVKKPGASVKVSCKASGYTFTSYNlVffl
ILmn- 2Hm3 WVROAPGOGLEWMGAIYPGNGDTSYNQKFK
GRVTMTRDTSTSTVYMELSSLRSEDTAVYYCA
RS.YYSNSYWYFDLWGRGTLVTVSS
(SEQIDNO:36)
EIVLTOSPATLSLSPGERATLSCRASOSVSYLA
WYQQKPGQAPRLLIYAPSNLASGIPARFSGSG
SGTDFTLTISSLEPEDFAVYYCOOWSFNPPTFG
QGTKYEIKDGGGSGGGGSGGGGSSQVQLVQS
GAEVKKPGASVKVSCICASGYTFTSYNMHWVR
TA ^^^^ OAPGOGLEWMGAIYPGNGDTSYNOKFKGRVT
^* MTRDTSTSTVYMELSSLRSEDTAVYYCAR
S.YYSNSYWYFDLWGRGTLVTVSS
(SEQIDNO:37)
EIVLTOSPATLSLSPGERATLSCRASSSVSYLA
WYOOKPGOAPRLLIYAPSNLASGIPARFSGSG
SGTDFTLTISSLEPEDFAVYYCOOWSFNPPTFG
QGTKVEIKDGGGSGGGGSGGGGSSQYQLYQS
GAEVKKPGASVKVSCKASGYTFTSYNMHWV
2Lml8-2 2Hm3 ROAPGOGLEWMGAIYPGNGDTSYNOKFKGR
VTMTRDTSTSTVYMELSSLRSEDTAVYYCAR
S.YYSNSYWYFDLWGRGTLVTVSS
(SEQIDNO:38)
EIVLTOSPATLSLSPGERATLSCRASSSVSYLN
WYQQKPGQAPRLLIYAPSNLASGIPARFSGS
2H ^ GSGTDFTLTISSLEPEDFAVYYCOOWSFNPPT
2Lml»-3 2kim5 YGQGTKYEIKDGGGSGGGGSGGGGSSQVQLW
OSGAEVKKPGASVKVSCKASGYTFTSYNMH
WVROAPGOGLEWMGAIYPGNGDTSYNOKFKG
58
RVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR
S.YYSNSYWYFDLWGRGTLVTVSS
(SEQIDNO:39)
EIVLTOSPATLSLSPGERATLSCRASSSVSYLD
WYOOKPGOAPRLLIYAPSNLASGIPARFSGSG
SGTDFTLTISSLEPEDFAVYYCOOWSFNPPTFG
QGJKYEIKDGGGSGGGGSGGGGSSQVQLYQS
GAEVKKPGASVKVSCKASGYTFTSYNMHWV
2Lml8-4 2Hm3 ROAPGOGLEWMGAIYPGNGDTSYNOKFKGR
VTMTRDTSTSTVYMELSSLRSEDTAVYYCAR
S.YYSNSYWYFDLWGRGTLVTVSS
(SEQIDNO:40)
EIVLTOSPATLSLSPGERATLSCRASSSVSYLSW
YOOKPGOAPRLLIYAPSNLASGIPARFSGSGSG
TDFTLTISSLEPEDFAVYYCOOWSFNPPTFGOG
TKYEIKDGGGSGGGGSGGGGSSQVQLVQSGAE
VKKPGASVKVSCKASGYTFTSYNMHWVRQAP
2Linl8-5 2Hm3 GOGLEWMGAIYPGNGDTSYNOKFKGRVTMTR
DTSTSTVYMELSSLRSEDTAVYYCAR
S.YYSNSYWYFDLWGRGTLVTVSS
(SEQIDN0:41)
EIVLTOSPATLSLSPGERATLSCRASSSVSYLHW
YOOKPGOAPRLLIYAPSNLASGIPARFSGSGSGT
DFTLTISSLEPEDFAVYYCOOWSFNPPTFGOGTK
YEIKDGGGSGGGGSGGGGSSQVQLVQSGAEYKK
jj PGASVKVSCKASGYTFTSYNMHWVRQAPGQGL
J" 2Hm3 EWMGAIYPGNGDTSYNOKFKGRVTMTRDTSTST
VYMELSSLRSEDTAVYYCAR
S.YYSNSYWYFDLWGRGTLVTVSS
(SEQIDNO:42)
EIVLTOSPATLSLSPGERATLSCRASOSVSYIPW
YQOKPGQAPRLLIYAPSNLASGIPARFSGSGSGT
DFTLTISSLEPEDFAVYYCOOWSFNPPTFGOGTK
VEIKDGGGSGGGGSGGGG5SQVQLVQSGAEVK
KPGASVKVSCKASGYTFTSYNMHWVRQAPGQG
2Lml9-l 2Hm3 LEWMGAIYPGNGDTSYNOKFKGRVTMTRDTST
STVYMELSSLRSEDTAVYYCAR
S.YYSNSYWYFDLWGRGTLVTVSS
(SEQIDNO:43)
59
EIVLTQSPATLSLSPGERATLSCRASOSVSYISW
YQQKPGQAPRLLIYAPSNLASGIPARFSGSGSG
TDFTLTISSLEPEDFAVYYCOOWSFNPPTFGOG
TKVEIKDGGGSGGGGSGGGGSSQWQLYQSGAE
VKKPGASVKVSCKASGYTFTSYNMHWVROAP
2Linl9-2 2Hm3 GQGLEWMGAIYPGNGDTSYNOKFKGRVTMTR
DTSTSTVYMELSSLRSEDTAVYYCAR
S.YYSNSYWYFDLWGRGTLVTVSS
(SEQIDNO:44)
EIVLTOSPATLSLSPGERATLSCRASOSVSYIVW
YQQKPGQAPRLLIYAPSNLASGIPARFSGSGSGT
DFTLTISSLEPEDFAVYYCOOWSFNPPTFGOGT
KWEIKDGGGSGGGGSGGGGSSQYQLVQSGAEY
KKPGASVKVSCKASGYTFTSYNMHWVRQAPG
2Lml9-3 2Hin3 OGLEWMGAIYPGNGDTSYNOKFKGRVTMTRD
TSTSTVYMELSSLRSEDTAVYYCAR
S.YYSNSYWYFDLWGRGTLVTVSS
(SEQIDNO:45)
EIVLTOSPATLSLSPGERATLSCRASOSVSYIAW
YOOKPGOAPRLLIYAPSNLASGIPARFSGSGSG
TDFTLTISSLEPEDFAVYYCOOWSFNPPTFGOG
TKVEIKDGGGSGGGGSGGGGSSQYQLVQSGAE
VKKPGASVKVSCKASGYTFTSYNMHWVRQAP
2Lml9-4 2Hm3 GOGLEWMGAIYPGNGDTSYNOKFKGRVTMTR
DTSTSTVYMELSSLRSEDTAVYYCAR
S.YYSNSYWYFDLWGRGTLVTVSS
(SEQ ID NO:46)
EIVLTOSPATLSLSPGERATLSCRASOSVSYITW
YOOKPGOAPRLLIYAPSNLASGIPARFSGSGSG
TDFTLTISSLEPEDFAVYYCOOWSFNPPTFGOG
TKWEl¥iDGGGSGGGGSGGGGSSQVQLVQSGAE
VKKPGASVKVSCKASGYTFTSYNMHWVRQAP
2Lml9-7 2Hm3 GOGLEWMGAPyPGNGDTSYNOKFKGRVTMTR
DTSTSTVYMELSSLRSEDTAVYYCAR
S.YYSNSYWYFDLWGRGTLVTVSS
(SEQIDNO:47)
EIVLTOSPATLSLSPGERATLSCRASOSVSYIIW
YQQKPGQAPRLLIYAPSNLASGIPARFSGSGSG
9W 1 TDFTLTISSLEPEDFAVYYCOOWSFNPPTFGOG
2Lml9-9 ZHinJ JKYEIKDGGGSGGGGSGGGGSSQYQLVQSGAE
VKKPGASVKVSCKASGYTFTSYNMHWVROAP
GOGLEWMGAIYPGNGDTSYNOKFKGRVTMTR
60
DTSTSTVYMELSSLRSEDTAVYYCAR
S.YYSNSYWYFPLWGRGTLVTVSS
(SEQIDNO:48)
ETVLTOSPATLSLSPGERATLSCRASOSVSYIPW
YOOKPGOAPRLLIYAPSNLASGIPARFSGSGSG
TDFTLTISSLEPEDFAVYYCOOWSFNPPTFGOG
TKVEIKDGGGSGGGGSGGGGSSQVQLWQSGAE
VKKPGASVKVSCKASGYTFTSYNMHWVRQAP
2Lml9- 2Hin3 GOGLEWMGAIYPGNGDTSYNOKFKGRVTMTR
^^ DTSTSTVYMELSSLRSEDTAVYYCAR
S.YYSNSYWYFDLWGRGTLVTVSS
(SEQ ID NO:49)
EIVLTOSPATLSLSPGERATLSCRASOSVSYINW
YOOKPGOAPRLLIYAPSNLASGIPARFSGSGSG
TDFTLTISSLEPEDFAVYYCOOWSFNPPTFGOG
IKYEIKDGGGSGGGGSGGGGSSQVQLVQSGAE
VKKPGASVKVSCKASGYTFTSYNMHWVRQAP
Td ^^^^ GOGLEWMGAIYPGNGDTSYNOKFKGRVTMTR
^^ DTSTSTVYMELSSLRSEDTAVYYCAR
S.YYSNSYWYFDLWGRGTLVTVSS
(SEQIDNO:50)
EIVLTOSPATLSLSPGERATLSCRASSSVSYISW
YOOKPGOAPRLLIYAPSNLASGIPARFSGSGSG
TDFTLTISSLEPEDFAVYYCOOWSFNPPTFGOG
TKVEIKDGGGSGGGGSGGGGSSQVQLVQSGAE
VKKPGASVKVSCKASGYTFTSYNMHWVRQAP
2Lin20-l 2Hm3 GOGLEWMGAIYPGNGDTSYNOKFKGRVTMTR
DTSTSTVYMELSSLRSEDTAVYYCAR
S.YYSNSYWYFDLWGRGTLVTVSS
(SEQIDN0:51)
EIVLTQSPATLSLSPGERATLSCRASSSVSYIAW
YOOKPGOAPRLLIYAPSNLASGIPARFSGSGSG
TDFTLTISSLEPEDFAVYYCOOWSFNPPTFGOG
TKVEIKDGGGSGGGG5GGGG55QVQLVQSGAE
VKKPGASVKVSCKASGYTFTSYNMHWVRQAP
2Lm20-2 2Hm3 GOGLEWMGAIYPGNGDTSYNOtCFKGRVTMTR
DTSTSTVYMELSSLRSEDTAVYYCAR
S.YYSNSYWYFDLWGRGTLVTVSS
(SEQIDNO:52)
61
EIVLTOSPATLSLSPGERATLSCRASSSVSYIVW
YOOKPGOAPRLLIYAPSNLASGIPARFSGSGSG
TDFTLTISSLEPEDFAVYYCOOWSFNPPTFGQG
TKVEIKDGGGSGGGGSGGGGSSQVQLWQSGAE
VKtCPGASVKVSCKASGYTFTSYNMHWVROAP
2Lm20-4 2Hm3 GOGLEWMGAIYPGNGDTSYNQKFKGRVTMTR
DTSTSTVYMELSSLRSEDTAVYYCAR
S.YYSNSYWYFDLWGRGTLVTVSS
(SEQIDNO:53)
EIVLTOSPATLSLSPGERATLSCRASSSVNYIYW
YOOKPGOAPRLLIYAPSNLASGIPARFSGSGSG
TDFTLTISSLEPEDFAVYYCOOWSFNPPTFGOG
TKVEIKDGGGSGGGGSGGGGSSQWQLVQSGAE
VKXPGASVKVSCKASGYTFTSYNMHWVROAP
2Lm20-8 2Hm3 GOGLEWMGAIYPGNGDTSYNOKFKGRVTMTR
DTSTSTVYMELSSLRSEDTAVYYCAR
S.YYSNSYWYFDLWGRGTLVTVSS
(SEQIDNO:54)
EIVLTOSPATLSLSPGERATLSCRASSSVSYIDW
YOOKPGOAPRLLIYAPSNLASGIPARFSGSGSG
TDFTLTISSLEPEDFAVYYCOOWSFNPPTFGOG
TKYEIKDGGGSGGGGSGGGGSSQWQLWQSGAE
VKKPGASVKVSCKASGYTFTSYNMHWVRQAP
ZLtnZO- 2Hni3 GOGLEWMGAIYPGNGDTSYNOKFKGRVTMTR
DTSTSTVYMELSSLRSEDTAVYYCAR
S.YYSNSYWYFDLWGRGTLVTVSS
(SEQIDNO:55)
EIVLTOSPATLSLSPGERATLSCRASSSVSYIIW
YOOKPGOAPRLLIYAPSNLASGIPARFSGSGSG
TDFTLTISSLEPEDFAVYYCOOWSFNPPTFGOG
TKYEIKDGGGSGGGGSGGGGSSQVQLWQSGAE
VKKPGASVKVSCKASGYTFTSYNMHWVRQAP
" 2Hm3 GOGLEWMGAIYPGNGDTSYNOKFKGRVTMTR
DTSTSTVYMELSSLRSEDTAVYYCAR
S.YYSNSYWYFDLWGRGTLVTVSS
(SEQIDNO:56)
EIVLTQSPATLSLSPGEEIATLSCRASSSVSYIYW
YQQKPGQAPRLLIYAPSNLASGIPARFSGSGSG
2Lm20- J TDFTLTISSLEPEDFAVYYCOOWSFNPPTFGOG
13 ^^ TKVEIKDGGGSGGGGSGGGGSSQWQLVQSGAE
VKKPGASVKVSCKASGYTFTSYNMHWVROAP
GOGLEWMGAryPGNGDTSYNOKFKGRVTMTR
62
DTSTSTVYMELSSLRSEDTAVYYCAR
S.YYSNSYWYFDLWGRGTLVTVSS
(SEQIDNO:57)
EIVLTOSPATLSLSPGERATLSCRASOSVSYMH
WYOOKPGOAPRLLIYAPSNLASGIPARFSGS
GSGTDFTLTISSLEPEDFAVYYCOOWSFNPPT
FGQGTKVEIKDGGGSGGGGSGGGGTGEVQLV
nfMaQ\ 7HS 1 QSGAEVKKPGESLKISCKGSGYSFTSYNMHW
(ISUUy) ZHSmJ VROMPGKGLEWMGAIYPGNGDTSYNOKFKG
QVTISADKSISTAYLQWSSLKASDTAMYYCA
RWYYSNSYWYFDLWGRGTLVTVSS
(SEQIDNO:58)
63
EIVLTOSPATLSLSPGERATLSCRASOSVSYMH
WYQQKPGQAPRLLIYAPSNLASGIPARFSGSGS
GTDFTLTISSLEPEDFAVYYCOOWSFNPPTFG
QGTKVEIKDGGGSGGGGSGGGGTGEVQLLES
m ^ GGGLVQPGGSLRLSCAASGFTFSSYNMHWVR
yn'X^\\ OAPGKGLEWVSAIYPGNGDTSYNOKFKGRFT
2H3m3) ISRDNSKNTLYLQMNSLRAEDTAVYYCA
KSYYSNSYWYFDLWGRGTLVTVSS
(SEQ ID NO:59)
DQEPKSCDKTHTSPPSS
^ ^ ^ CSSS (SEQ1DNO:60)
DQEPKSCDKTHTCPPCP
I o n ^'"8®
^ ^ WT (SEQIDN0:61)
DQEPKSCDKTHTSPPCS
I o n '''"Se
'^'^^ CSCS (SEQIDNO:62)
DQEPKSSDKTHTCPPCS
I o n ™"8®
^ ^ sees (SEQIDNO:63)
'Iso^n* s™e"e8®p
DQEPKSSDKTHXePPCP
(SEQIDNO:64)
APELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKF
N
• WYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKE
eH2eH YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQV
IgGl 3 SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS
WT KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQIDNO:65)
64
APELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKF
N
WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE
CH2CH YKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSRDELTJCNQV
igGi 3 SLTCLVKGFYPSDTAVEWESNGQPENNYKTTPPVLDSDGSFFLYS
P331S KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
( S E Q I D N 0 : 6 6 )
Exemplary Full Length
EIVLTQSPATLSLSPGERATLSCRASQSVSYIVWYQQKPGQAPRL
LIYAPSNLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQWS
FNPPTFGQGTKVEIBCDGGGSGGGGSGGGGTGEVQLVQSGAEVK
KPGESLKISCKGSGYSFTSYNMHWVRQMPGKGLEWMGAIYPGN
GDTSYNQKFKGQVTISADKSISTAYLQWSSLKASDTAMYYCARS
SEQ ID NO:67 YYSNSYWYFDLWGRGTLVTVSSDQEPKSSDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYV
DGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKC
KVSNKALPASIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLT
CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT
VDJCSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
EIVLTQSPATLSLSPGERATLSCRASSSVSYIVWYQQKPGQAPRLL
lYAPSNLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQWSF
NPPTFGQGTKVEIKDGGGSGGGGSGGGGSSQVQLVQSGAEVKK
PGASVKVSCKASGYTFTSYNMHWVRQAPGQGLEWMGAIYPGN
GDTSYNQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARS
SEQ ID NO:68 .YYSNSYWYFDLWGRGTLVTVSSDQEPKSSDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYV
DGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKC
KVSNKALPASIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLT
CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT
VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
QIVLSQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKPGSSPKP
WIYAPSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQ
WSFNPPTFGAGTKLELKDGGGSGGGGSGGGGSSQAYLQQSGAE
SVRPGASVKMSCKASGYTFTSYNMHWVKQTPRQGLEWIGAIYP
GNGDTSYNQKFKGKATLTVDKSSSTAYMQLSSLTSEDSAVYFCA
SEQ ID NO:69 RWYYSNSYWYFDVWGTGTTVTVSDQEPKSCDKTHTSPPCSAP
ELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNW
YVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEY
KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTBGSIQVS
LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK
LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
65
EIVLTQSPATLSLSPGERATLSCRASQSVSYIVWYQQKPGQAPRL
LIYAPSNLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQWS
FNPPTFGQGTKVEIKDGGGSGGGGSGGGGTGEVQLVQSGAEVK
KPGESLKISCKGSGYSFTSYNMHWVRQMPGKGLEWMGAIYPGN
GDTSYNQKFKGQVTISADKSISTAYLQWSSLKASDTAMYYCARS
SEQ ID NO:70 YYSNSYWYFDLWGRGTLVTVSSDQEPKSSDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYV
DGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKC
KVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLT
CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSJCLT
VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
EIVLTQSPATLSLSPGERATLSCRASSSVSYIVWYQQJCPGQAPRLL
lYAPSNLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQWSF
NPPTFGQGTKVEIKDGGGSGGGGSGGGGSSQVQLVQSGAEVKK
PGASVKVSCKASGYTFTSYNMHWVRQAPGQGLEWMGAIYPGN
GDTSYNQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARS
SEQ ID NO:71 .YYSNSYWYFDLWGRGTLVTVSSDQEPKSSDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYV
DGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKC
KVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLT
CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT
VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
EIVLTQSPATLSLSPGERATLSCRASSSVSYIDWYQQKPGQAPRLL
lYAPSNLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQWSF
NPPTFGQGTKVEIKDGGGSGGGGSGGGGSSQVQLVQSGAEVKK
PGASVKVSCKASGYTFTSYNMHWVRQAPGQGLEWMGAIYPGN
GDTSYNQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARS
SEQ ID NO:72 YYSNSYWYFDLWGRGTLVTVSSDQEPKSCDKTHTSPPSSAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYV
DGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKC
KVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLT
CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT
VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
EIVLTQSPATLSLSPGERATLSCRASSSVSYIVWYQQKPGQAPRLL
lYAPSNLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQWSF
NPPTFGQGTKVEIKDGGGSGGGGSGGGGSSQVQLVQSGAEVKK
PGASVKVSCKASGYTFTSYNMHWVRQAPGQGLEWMGAIYPGN
GDTSYNQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARS
SEQ ID NO:73 YYSNSYWYFDLWGRGTLVTVSSDQEPKSSDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYV
DGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKC
KVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLT
CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT
VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
66
EIVLTQSPATLSLSPGERATLSCRASQSVSYIVWYQQKPGQAPRL
LIYAPSNLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQWS
FNPPTFGQGTKVEIKDGGGSGGGGSGGGGTGEVQLVQSGAEVK
KPGESLKISCKGSGYSFTSYNMHWVRQMPGKGLEWMGAIYPGN
GDTSYNQBCFKGQVTISADKSISTAYLQWSSLKASDTAMYYCARV
SEQ ID NO:74 VYYSNSYWYFDLWGRGTLVTVSSDQEPKSCDKTHTSPPCSAPEL
LGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWY
VDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYK
CKVSNKALPASIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSL
TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL
TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
EIVLTQSPATLSLSPGERATLSCRASSSVSYMIWYQQKPGQAPRL
LIYAISNLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQWIS
NPLTFGQGTKVEIKDGGGSGGGGSGGGGSSQVQLVQSGAEVKK
PGASVKVSCKASGYTFTSYNMHWVRQAPGQGLEWMGAIYPGN
GDTSYNQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARS
SEQ ID NO:75 VYYSN.YWYFDLWGRGTLVTVSSDQEPKSCDKTHTCPPCPAPEL
LGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWY
VDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYK
CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSL
TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL
TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
EIVLTQSPATLSLSPGERATLSCRASSSVSYIIWYQQKPGQAPRLLI
YAPSNLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQWSFN
PPTFGQGTKVEIKDGGGSGGGGSGGGGSSQVQLVQSGAEVKKP
GASVKVSCKASGYTFTSYNMHWVRQAPGQGLEWMGAIYPGNG
DTSYNQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSY
SEQ ID NO:76 YSNSYWYFDLWGRGTLVTVSSDQEPKSCDKTHTSPPSSAPELLG
GPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVD
GVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCK
VSNKALPASIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCL
VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV
DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
67
EQUIVALENTS
[0184] The foregoing has been a description of certain non-limiting embodiments of
the invention. Those skilled in the art will recognize, or be able to ascertain using no more
than routine experimentation, many equivalents to the specific embodiments of the invention
described herein. Those of ordinary skill in the art will appreciate that various changes and
modifications to this description may be made without departing fixjm the spirit or scope of
the present invention, as defined in the following claims.
[0185] In the claims articles such as "a,", "an" and "the" may mean one or more than
one unless indicated to the contrary or otherwise evident fi"om the context. Claims or
descriptions that include "or" between one or more members of a group are considered
satisfied if one, more than one, or all of the group members are present in, employed in, or
otherwise relevant to a given product or process unless indicated to the contrary or otherwise
evident from the context. The invention includes embodiments in which exactly one member
of the group is present in, employed in, or otherwise relevant to a given product or process.
The invention also includes embodiments in which more than one, or all of the group
members are present in, employed in, or otherwise relevant to a given product or process.
Furthermore, it is to be understood that the invention encompasses all variations,
combinations, and permutations in which one or more limitations, elements, clauses,
descriptive terms, etc., from one or more of the claims or from relevant portions of the
description is introduced into another claim. For example, any claim that is dependent on
another claim can be modified to include one or more limitations found in any other claim
that is dependent on the same base claim. Furthermore, where the claims recite a
composition, it is to be understood that methods of using the composition for any of the
purposes disclosed herein are included, and methods of making the composition according to
any of the methods of making disclosed herein or other methods known in the art are
included, unless otherwise indicated or unless it would be evident to one of ordinary skill in
the art that a contradiction or inconsistency would arise. In addition, the invention
encompasses compositions made according to any of the methods for preparing compositions
disclosed herein.
[0186] Where elements are presented as lists, e.g., in Markush group format, it is to
be imderstood that each subgroup of the elements is also disclosed, and any element(s) can be
removed from the group. It is also noted that the term "comprising" is intended to be open
68
and permits the inclusion of additional elements or steps. It should be understood that, in
general, where the invention, or aspects of the invention, is/are referred to as comprising
particular elements, features, steps, etc., certain embodiments of the invention or aspects of
the invention consist, or consist essentially of, such elements, features, steps, etc. For
purposes of simplicity those embodiments have not been specifically set forth in haec verba
herein. Thus for each embodiment of the invention that comprises one or more elements,
features, steps, etc., the invention also provides embodiments that consist or consist
essentially of those elements, features, steps, etc.
[0187] Where ranges are given, endpoints are included. Furthermore, it is to be
understood that unless otherwise indicated or otherwise evident fi-om the context and/or the
imderstanding of one of ordinary skill in the art, values that are expressed as ranges can
assume any specific value within the stated ranges in different embodiments of the invention,
to the tenth of the unit of the lower limit of the range, unless the context clearly dictates
otherwise. It is also to be understood that unless otherwise indicated or otherwise evident
from the context and/or the understanding of one of ordinary skill in the art, values expressed
as ranges can assume any subrange within the given range, wherein the endpoints of the
subrange are expressed to the same degree of accuracy as the tenth of the unit of the lower
limit of the range.
[0188] In addition, it is to be understood that any particular embodiment of the
present invention may be explicitly excluded from any one or more of the claims. Any
embodiment, element, feature, application, or aspect of the compositions and/or methods of
the invention can be excluded from any one or more claims. For purposes of brevity, all of
the embodiments in which one or more elements, features, purposes, or aspects is excluded
are not set forth explicitly herein.
INCORPORATION OF REFERENCES
[0189] All publications and patent documents cited in this application are
incorporated by reference in their entirety to the same extent as if the contents of each
individual publication or patent document were incorporated herein.
[0190] What is claimed is:

1. A method of purifying a small modular immunopharmaceutical protein from a protein
preparation containing high molecular weight aggregates comprising a step of subjecting the
protein preparation to hydroxyapatite chromatography under an operating condition such that
the purified small modular immunopharmaceutical protein contains less than 4% aggregates.
2. The method of claim 1, wherein the method comprises no more than 3 chromatography
steps.
3. The method of claim 1 or 2, wherein the operating condition comprises eluting the small
modular immunopharmaceutical protein from a hydroxyapatite chromatography column in a
phosphate buffer.
4. The method of claim 3, wherein the phosphate buffer is endotoxin-free.
5. The method of claim 3 or 4, wherein the phosphate buffer is depyrogenated.
6. The method of claim 3, wherein the phosphate buffer comprises sodium phosphate,
potassium phosphate, and/or lithium phosphate.
7. The method of claim 3, wherein the phosphate buffer comprises sodium phosphate at a
concentration ranging from 1 mM to 50 mM.
8. The method of claim 3, wherein the phosphate buffer further comprises sodium chloride at
a concentration ranging from 100 mM to 2.5 M.
9. The method of claim 3, wherein the phosphate buffer comprises sodium phosphate at a
concentration ranging from 2 mM to 32 mM and sodium chloride at a concentration ranging
fix)ml00mMtol.6M.
10. The method of any one of claims 3-9, wherein the phosphate buffer has a pH ranging
fh)m 6.5 to 8.5.
11. The method of claim 1, wherein the operating condition comprises eluting the small
modular immunopharmaceutical protein from a hydroxyapatite chromatography column by a
NaCl gradient.
70
12. The method of claim 1, wherein the operating condition comprises eluting the small
modular immunopharmaceutical protein from a hydroxyapatite chromatography column by a
NaCl step elution method.
13. The method of claim 1, wherein the operating condition comprises eluting the small
modular inmiunopharmaceutical protein from a hydroxyapatite chromatography colunrn by a
phosphate gradient.
14. The method of claim 13, wherein the phosphate gradient is a linear gradient.
15. The method of claim 13, wherein the phosphate gradient is a step gradient.
16. The method of any one of claims 1-15, wherein the hydroxyapatite chromatography uses
a column containing ceramic hydroxyapatite Type I or Type II resins.
17. The method of claim 16, wherein the column contains ceramic hydroxyapatite Type I
resins.
18. The method of claim 16 or 17, wherein the resins are 1 |j,m to 1,000 (i.m in diameter.
19. The method of claim 16 or 17, wherein the resins are 10 ^m to 100 ^m in diameter.
20. The method of any one of the preceding claims, wherein the method further comprises a
step of purifying the protein preparation by affinity chromatography before the
hydroxyapatite chromatography.
21. The method of claim 20, wherein the affinity chromatography uses a protein absorbent
that binds to a constant immunoglobulin domain.
22. The method of claim 20, wherein the affinity chromatography lises a protein absorbent
that binds to a variable immunoglobulin domain.
23. The method of claim 22, wherein the protein absorbent binds to a VH3 domain.
24. The method of any one of claims 21-23, wherein the protein absorbent comprises protein
A.
25. The method of claim 24, wherein the affinity chromatography uses a MabSelect rProtein
A resin column.
71
26. The method of claim 20, wherein the step of affinity chromatography comprises washing
an affinity chromatography column using a washing buffer comprising Hepes, sodium
chloride, calcium chloride, arginine, Tris, magnesium chloride, histidine, urea, imidazole, one
or more organic solvents, ionic and/or nonionic detergents.
27. The method of claim 26, wherein the one or more organic solvents are selected fi"om the
group consisting of ethanol, methanol, propylene glycol, ethylene glycol, propanol,
isopropanol, butanol, and combinations thereof
28. The method of claim 20, wherein the step of affinity chromatography comprises eluting
the small modular immunopharmaceutical protein from an affinity chromatography column
using an elution buffer comprising Hepes, phosphoric acid, glycine, glycylglycine,
magnesium chloride, urea, propylene glycol, ethylene glycol, one or more organic acids,
and/or arginine.
29. The method of claim 28, wherein the one or more organic acids are selected from the
group consisting of acetic acid, citric acid, formic acid, lactic acid, tartaric acid, malic acid,
malonic acid, phthalic acid and salicyclic acid.
30. The method of claim 28, wherein the elution buffer further comprises a sah selected from
the group consisting of sodium chloride, potassium chloride, calcium chloride, magnesium
chloride, and combinations thereof
31. The method of claim 30, wherein the salt is at a concentration ranging fi-om 1 mM to 1
M.
32. The method of claim 30, wherein the sah is at a concentration ranging fi-om 1 mM to 500
mM.
33. The method of claim 30, wherein the sah is at a concentration ranging from 1 mM to 100
mM.
34. The method of any one of claims 20-33, wherein the method further comprises adding an
additive to promote binding to sorbents.
72
35. The method of claim 1, wherein the method further comprises a step of purifying the
protein preparation by anion exchange chromatography using an anion exchange
chromatography resin.
36. The method of any one of claims 20-35, wherein the method further comprises a step of
purifying the protein preparation by anion exchange chromatography after the affinity
chromatography but before the hydroxyapatite chromatography.
37. The method of claim 35 or 36, wherein the method further comprises a step of adding an
additive to enhance binding of the small modular immunopharmaceutical protein and/or
impurities to the anion exchange chromatography resin.
38. The method of claim 37, wherein the additive comprises a nonionic organic polymer.
39. The method of claim 38, wherein the nonionic organic polymer is polyethylene glycol
(PEG).
40. The method of any one of claims 35-39, wherein the method further includes a depth
filter step before the anion exchange chromatography.
41. The method of any one of the preceding claims, the method further comprises one or
more filtration steps.
42. The method of claim 41, wherein the one or more filtration steps comprise a virus
retaining filtration step.
43. The method of claim 41, wherein the one or more filtration steps comprise ultrafiltration
and/or diafiltration steps.
44. The method of any one of the preceding claims, wherein the method further comprises a
step of adding an additive to induce protein precipitation of one or more contaminants from
the protein preparation such that the small modular immunopharmaceutical protein can be
further separated from contaminates.
45. The method of claim 44, wherein the additive comprises a nonionic organic polymer.
46. The method of claim 45, wherein the nonionic organic polymer is polyethylene glycol
(PEG).
73
47. The method of claim 44-46, wherein the precipitation is induced before anion exchange
chromatography and wherein the method further comprises a step of removing precipitated
contaminants from the protein preparation by filtration.
48. The method of any one of the preceding claims, wherein the purified small modular
immunopharmaceutical protein contains less than 2% aggregates.
49. The method of any one of the preceding claims, wherein the purified small modular
iinmunopharmaceutical protein contains less than 1% aggregates.
50. The method of any one of the preceding claims, wherein the protein preparation contains
more than 10% high molecular weight aggregates.
51. The method of any one of the preceding claims, wherein the protein preparation contains
more than 20% high molecular weight aggregates.
52. The method of any one of the preceding claims, wherein the protein preparation contains
more than 30% high molecular weight aggregates.
53. The method of any one claims 1-47, wherein the protein preparation contains more than
60% high molecular weight aggregates.
54. The method of any one of claims 1-47, wherein the protein preparation contains less than
30% high molecular weight aggregates.
55. The method of any one of claims 1-47, wherein the protein preparation contains less than
20% high molecular weight aggregates.
56. The method of any one of claims 1-47, wherein the protein preparation contains less than
15% high molecular weight aggregates.
57. The method of any one of claims 1-47, wherein the protein preparation contains less than
10% high molecular weight aggregates.
58. The method of any one of claims 1-47, wherein the protein preparation contains less than
5% high molecular weight aggregates.
74
59. The method of any one of the preceding claims, wherein the small modular
immunopharmaceutical protein binds specifically to CD20.
60. The method of claim 59, wherein the small modular immunopharmaceutical protein
comprises an amino acid sequence having at least 80% identity to any one of SEQ ID NOs:l-
59 and 67-76.
61. The method of any one of the preceding claims, wherein the protein preparation is
prepared from cultured bacterial cells, mammalian cells, insect cells, plant cells, yeast cells,
cell-free medium, fransgenic animals or plants.
62. The method of any one of claims 1-60, wherein the protein preparation is a cell culture
medium preparation.
63. The method of claim 62, wherein the culture medium preparation comprises the small
modular inununopharmaceutical protein secreted from cultured cells.
64. The method of claim 63, wherein the cultured cells are CHO cells.
65. The method of claim 62, wherein the culture medium preparation is prepared from a
large scale bioreactor.
66. The method of any one of claims 1-61, wherein the protein preparation comprises a cell
extract.
67. The method of any one of claims 1-61, wherein the protein preparation is prepared from
inclusion bodies.
68. A method of purifying a small modular immunopharmaceutical protein from a protein
preparation containing high molecular weight aggregates, the method comprising subjecting
the protein preparation to (a) affinity chromatography and/or ion exchange chromatography,
and (b) hydroxyapatite chromatography under operating conditions such that the purified
small modular immimopharmaceutical protein contains less than 4% aggregates.
69. The method of claim 68, wherein the protein preparation is subjected to (al) affinity
chromatography, (a2) ion exchange chromatography, and (b) hydroxyapatite
chromatography.
75
70. The method of claim 68, wherein the protein preparation is subjected to (al) cation
exchange chromatography, (a2) anion exchange chromatography, and (b) hydroxyapatite
chromatography.
71. The method of any one of claims 68-70, wherein the method comprises no more than 3
chromatography steps.
72. The method of claim 68 or 69, wherein the affinity chromatography is protein A
chromatography.
73. The method of claim 68 or 69, wherein the ion exchange chromatography is anion
exchange chromatography using an anion exchange chromatography resin.
74. The method of claim 73, wherein the anion exchange chromatography resin is selected
from the group consisting of Q Sepharose FF, Q Sepharose XL, DEAE Sepharose FF,
POROS® HQ50, POROS® A50, Toyopearl® DEAE, Toyopearl® GigaCap Q-650M,
Toyopearr DEAE-650M, Capto'"^ Q, Capto'"^ DEAE, and tentacle anion exchange
chromatography.
75. The method of claim 73, wherein the anion exchange chromatography resin is a charged
membrane adsorber
76. The method of claim 75, wherein the anion exchange chromatography is selected from
the group consisting of Mustang® Q, Mustang® E, Sartobind® Q and Chromasorb^^.
77. The method^f claim 73, wherein the anion exchange chromatography resin is a charged
monolithic support.
78. The method of claim 77, wherein the anion exchange chromatography is CIM®-DISK.
79. The method of claim 69, wherein the affinity chromatography is MabSelect^^ rProtein A
affinity chromatography, the ion exchange chromatography is tentacle anion exchange
chromatography, and the hydroxyapatite chromatography is Type I ceramic hydroxyapatite
chromatography.
80. The method of claim 79, wherein the tentacle anion exchange chromatography is selected
from the group consisting of Fractogel® TMAE HiCap (M)™ Fractogel® TMAE (S)™, and
Fractoprep® TMAE™.
76
81. The method of any one of the preceding claims, wherein the method further comprises
stripping and/or regenerating one or more of the chromatography columns for reuse.
82. The method of any one of claims 68-81, wherein the purified small modular
immunopharmaceutical protein contains less than 2% aggregates.
83. The method of any one of claims 68-81, wherein the purified small modular
immunopharmaceutical protein contains less than 1% aggregates.
84. The method of any one of claims 68-81, wherein the protein preparation contains more
than 20% high molecular weight aggregates.
85. The method of claim 84, wherein the protein preparation contains more than 60% high
molecular weight aggregates.
86. The method of any one of claims 68-81, wherein the protein preparation contains less
than 30% high molecular weight aggregates.
87. The method of any one of claims 68-86, wherein the small modular
immunopharmaceutical protein binds specifically to CD20.
88. The method of claim 87, wherein the small modular immunopharmaceutical protein
comprises an amino acid sequence having at least 80% identity to SEQ ID NOs:l-59 and 67-
76.
89. A small modular immimopharmaceutical protein purified using a method of any one of
claims 1-88.
90. A method of purifying a protein from a protein preparation containing more than 20%
high molecular weight aggregates comprising a step of subjecting the protein preparation to
hydroxyapatite chromatography under an operating condition such that the purified protein
contains less than 4% aggregates.
91. The method of claim 90, wherein the protein preparation contains more than 60%o high
molecular weight aggregates.
92. The method of claim 90, wherein the operating condition comprises eluting the protein
from a hydroxyapatite chromatography column in a phosphate buffer.
77
• 93. The method of claim 92, wherein the phosphate buffer comprises sodium phosphate,
potassium phosphate, and/or lithium phosphate.
94. The method of claim 92, wherein the phosphate buffer comprises sodium phosphate at a
concentration ranging from 1 mM to 50 mM.
95. The method of claim 92, wherein the phosphate buffer further comprises sodium chloride
at a concentration ranging from 100 mM to 2.5 M.
96. The method of claim 92, wherein the phosphate buffer comprises sodium phosphate at a
concentration ranging from 2 mM to 32 mM and sodium chloride at a concenfration ranging
from 100 mM to 1.6 M.
97. The method of any one of claims 92-96, wherein the phosphate buffer has a pH ranging
from 6.5 to 8.5.
98. The method of claim 90, wherein the protein comprises a small modular
immunopharmaceutical polypeptide.
99. A pharmaceutical composition comprising a small modular immimopharmaceutical
! protein and a pharmaceutically acceptable carrier, wherein the small modular
immunopharmaceutical protein comprises less than 4% high molecular weight aggregates.
100. The pharmaceutical composition of claim 99, wherein the small modular
immimopharmaceutical protein comprises less than 3% high molecular weight aggregates.
•
101. The pharmaceutical composition of claim 99, wherein the small modular
immunopharmaceutical protein comprises less than 2% high molecular weight aggregates.
102. The pharmaceutical composition of claim 99, wherein the small modular
immunopharmaceutical protein comprises less than 1% high molecular weight aggregates.
...... L.
Of Atrano anoAnand Advocates
j Agent^loF^he Applicant
j 78
•