U.S. patent application number 16/242113 was filed with the patent office on 2019-06-27 for polypeptide separation methods.
This patent application is currently assigned to Zepteon, Incorporated. The applicant listed for this patent is Zepteon, Incorporated. Invention is credited to Austin Wayne Boesch, Glen Reed Bolton.
Application Number | 20190194248 16/242113 |
Document ID | / |
Family ID | 46634526 |
Filed Date | 2019-06-27 |
![](/patent/app/20190194248/US20190194248A1-20190627-D00001.png)
United States Patent
Application |
20190194248 |
Kind Code |
A1 |
Bolton; Glen Reed ; et
al. |
June 27, 2019 |
Polypeptide Separation Methods
Abstract
The present disclosure provides methods and compositions for
separating polypeptide glycoforms using a medium that includes an
Fc receptor. In certain embodiments, a medium includes an Fc
receptor which comprises an extracellular portion of an Fc gamma
RIII receptor.
Inventors: |
Bolton; Glen Reed; (Boston,
MA) ; Boesch; Austin Wayne; (Somerville, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Zepteon, Incorporated |
Boston |
MA |
US |
|
|
Assignee: |
Zepteon, Incorporated
Boston
MA
|
Family ID: |
46634526 |
Appl. No.: |
16/242113 |
Filed: |
January 8, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13554501 |
Jul 20, 2012 |
10221210 |
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16242113 |
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61509746 |
Jul 20, 2011 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 1/22 20130101 |
International
Class: |
C07K 1/22 20060101
C07K001/22 |
Claims
1. A method of enriching for afucosylated immunoglobulin C (IgG),
comprising: (a) providing a medium comprising an immunoglobulin Fc
receptor, wherein the Fc receptor comprises an extracellular domain
of an Fc gamma RIII receptor or Fc gamma RIV receptor, wherein the
Fc gamma RIII receptor comprises an amino acid sequence having at
least 85% identity to an amino acid sequence as set forth in
residues 21-208 of SEQ ID NO: 1, wherein F176 is changed to V176,
or an amino acid sequence as set forth in residues 20-208 of SEQ ID
NO: 2; (b) contacting the medium with a load fluid, wherein the
load fluid comprises IgG, under conditions in which the IgG binds
to the immunoglobulin Fc receptor, wherein the IgG comprises an
immunoglobulin Fc receptor binding moiety that is monomeric,
wherein the load fluid comprises a plurality of glycoforms of the
IgG, and wherein the Fc receptor preferentially binds to monomeric
afucosylated glycoforms of the IgG with an affinity that is at
least 2 fold greater than the affinity with which the Fc receptor
binds to fucosylated glycoforms of the IgG; (c) contacting the
medium with an elution solution under conditions in which the bound
IgG elutes from the medium; and (d) recovering the bound IgG that
elutes from the medium, thereby producing an eluate, wherein the
percentage of afucosylated glycoforms of the IgG present in the
eluate is at least 2-fold greater than the percentage of
afucosylated glycoforms of the IgG present in the load fluid prior
to being contacted with the medium, thereby enriching for
afucosylated IgG.
2. The method of claim 1, wherein the load fluid comprises serum
selected from the group consisting of: serum of an immunized
subject, serum of a subject who has been exposed to an infectious
agent, serum of a subject who has developed immunity to an
infectious agent, and serum of a naive subject.
3. The method of claim 2, wherein the serum is from a subject who
has been exposed to an infectious agent or from a subject who has
developed immunity to an infectious agent.
4. The method of claim 3, wherein a biological activity of the IgG
present in the eluate is altered relative to the activity of the
IgG in the load fluid.
5. The method of claim 4, wherein the biological activity comprises
therapeutic efficacy of the IgG against the infectious agent, and
wherein the therapeutic efficacy of the IgG against the infectious
agent is increased.
6. The method of claim 5, wherein the infectious agent is a
virus.
7. The method of claim 6, wherein the virus is an RNA virus.
8. The method of claim 4, wherein the biological activity is
antibody-dependent cell-mediated cytotoxicity (ADCC), and wherein
ADCC is increased.
9. The method of claim 4, wherein the biological activity is
antibody-dependent cellular phagocytosis (ADCP), and wherein ADCP
is increased.
10. The method of claim 1, wherein the load fluid comprises IgG1
IgG2, IgG3, and IgG4.
11. The method of claim 10, wherein the bound IgG that elutes from
the medium is IgG1 and/or IgG3.
12. The method of claim 1, wherein the Fc receptor comprises an
extracellular domain of an Fc gamma RIII receptor comprising an
amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99%
identity to an amino add sequence as set forth in residues 21-208
of SEQ ID NO: 1, wherein F176 is changed to V176, or an amino add
sequence as set forth in residues 20-208 of SEQ ID NO: 2.
13. The method of claim 1, wherein the Fc receptor is a full-length
Fc gamma RIII receptor or full-length Fc gamma RIV receptor.
14. The method of claim 5, further comprising producing a
pharmaceutical composition from the IgG in the eluate.
15. A method of treating a viral infection, comprising
administering to a subject in need thereof the pharmaceutical
composition of claim 14.
16. A method of enriching for one or more immunoglobulin G (IgG)
glycoforms, comprising: (a) providing a medium comprising an
immunoglobulin Fc receptor, wherein the Fc receptor comprises an
extracellular domain of an Fc gamma RIII receptor or Fc gamma RIV
receptor, wherein the Fc gamma RIII receptor comprises an amino add
sequence having at feast 85% identity to an amino add sequence as
set forth in residues 21-208 of SEQ ID NO: 1, wherein F176 is
changed to V178, or an amino add sequence as set forth in residues
20-208 of SEQ ID NO: 2; (b) contacting the medium with a load
fluid, wherein the load fluid comprises IgG, under conditions in
which the IgG binds to the immunoglobulin Fc receptor, wherein the
IgG comprises an immunoglobulin Fc receptor binding moiety, wherein
the load fluid comprises a plurality of glycoforms of the IgG, and
wherein the Fc receptor preferentially binds one or more of the
glycoforms of the IgG; (c) contacting the medium with an elution
solution under conditions in which the bound IgG elutes from the
medium; and (d) recovering the bound IgG that elutes from the
medium, thereby producing an eluate comprising one or more IgG
glycoforms.
17. The method of claim 16, wherein the IgG is IgG1 and/or
IgG3.
18. A medium comprising an Fc receptor coupled to a solid support,
wherein the Fc receptor comprises an extracellular binding domain
of an Fc gamma RIII receptor or Fc gamma RIV receptor, and wherein
the medium maximizes antibody binding capacity of the Fc
receptor.
19. The medium of claim 18, wherein the medium is equilibrated for
about 1-2 hours with a solution that has a pH between about 24.
20. The medium of claim 18, wherein the Fc receptor comprises an
extracellular binding domain of an Fc gamma RIII receptor
comprising an amino add sequence as set forth in residues 21-192 of
SEQ ID NO: 1, wherein F176 is changed to V176, or an amino add
sequence as set forth in residues 20-208 SEQ ID NO: 2.
Description
RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application No. 61/509,746, filed Jul. 20, 2011, which is
incorporated herein by reference in its entirety.
BACKGROUND
[0002] Most membrane and secretory proteins are glycosylated. In
many cases, the presence and characteristics of oligosaccharides
impact folding, stability, location, ligand interaction, and
biological activity of glycoproteins. For example, antibodies
typically have complex N-linked oligosaccharides. These can be
highly heterogeneous due to variations in levels of fucose,
mannose, galactose, N-acetylglucosamine, and/or sialic acid in
complex oligosaccharide chains (Jefferis, Trends Pharm. Sci.
30(7):356-362, 2009).
[0003] Serum and recombinant antibodies typically contain a mixture
of glycoforms. Certain antibody glycoforms have been observed to
have a higher affinity for Fc receptors on leukocytes such as Fc
gamma RI, Fc gamma RII, Fc gamma RIII, and C1q, which in turn can
after effector function. Antibodies with oligomannose-type
oligosaccharides display enhanced antibody dependent cell mediated
cytotoxicity (ADCC) and reduced C1q binding (Crispin et al., J.
Mol. Biol. 387:1061-1066, 2009). Removal of galactosylation reduces
C1q binding and binding to other Fc receptors (Crispin et al.,
supra; Kobata, Biochim. Biophys. Acta 1780:472-478, 2008). Terminal
sialic acids have been shown to reduce the affinity of antibodies
for Fc gamma receptors (Jefferis, Nat. Rev. Drug. Disc. 8:226-234,
2009; Walsh et al., Nat. Biotech. 24(10):1241-1252, 2006).
[0004] Antibody forms lacking fucose on the primary core
N-acetylglucosamine have increased affinity for Fc gamma RIIIa as
compared to core-fucosylated forms, and also have an increased
ability to trigger ADCC (Jefferis, Exp. Opin. Biol. Ther.
7(9)1401-1413, 2007; Okazaki et al., J. Mol. Biol. 336:1239-1249,
2004; Shibata-Koyama et al., Glycobiol. 19(2): 126-134, 2009).
Afucosylated forms have comparable affinity for antigen, C1q, Fc
gamma RI, the neonatal Fc receptor (FcRn) and slightly higher
affinity for Fc gamma RIIa and Fc gamma RIIb, as compared to
fucosylated forms (Jefferis, Exp. Opin. Biol. Ther. 7(9)1401-1413,
2007; Satoh et al., Exp. Opin. Biol. Ther. 6(11):1161-1173, 2006;
Kobata, supra). Afucosylated forms of rituximab and trastuzumab
have enhanced in vitro and ex vivo ADCC (Jefferis, Exp. Opin. Biol.
Ther. 7(9):1401-1413, 2007; Jefferis, Trends Pharm. Sci.
30(7):356-362, 2009; Satoh, supra). Fc gamma RI, Fc gamma RII, Fc
gamma RIII, and C1q receptors have been reported to interact with
the hinge or hinge proximal region of Fc polypeptides. The
increased affinity of afucosylated IgG Fc for Fc gamma RIII may be
due to a conformational change in the Fc that reduces steric
inhibition of binding (Kobata, supra; Satoh, supra).
SUMMARY
[0005] The present disclosure provides, inter alia, methods and
compositions for separating polypeptide glycoforms. In various
embodiments of methods provided herein, polypeptides having Fc
receptor binding moieties are separated using Fc receptor media
that preferentially bind one or more polypeptide glycoforms.
Separation of polypeptide glycoforms has a variety of applications
such as in preparative methods, e.g., for preparing compositions
having a desired glycoform profile, for enriching for particular
glycoforms that possess desired biological and/or therapeutic
activity, and analytical methods, e.g., to permit characterization
of polypeptide preparations.
[0006] Glycosylation is implicated in function, stability, and
other important aspects of polypeptide biochemistry. Benefits
offered by the present technology may impact development,
production, and use (e.g., therapeutic use) of polypeptide
products. The ability to separate glycoforms allows greater control
over product variable quality attributes, in turn facilitating
consistent manufacturing, clinical/regulatory analysis, and, in
some cases, impacting therapeutic efficacy.
[0007] Accordingly, in one aspect, the present disclosure provides
a method of separating polypeptide glycoforms in a load fluid. The
method includes, for example: (a) providing a medium comprising an
immunoglobulin Fc receptor; (b) contacting the medium with a load
fluid comprising a polypeptide under conditions in which the
polypeptide binds to the immunoglobulin Fc receptor, wherein the
polypeptide comprises an immunoglobulin Fc receptor binding moiety,
wherein the load fluid comprises a plurality of glycoforms of the
polypeptide, and wherein the Fc receptor preferentially binds one
or more of the glycoforms; (c) contacting the medium with an
elution solution under conditions in which bound polypeptide elutes
from the medium; and (d) recovering bound polypeptide that elutes
from the medium, thereby producing an eluate.
[0008] In some embodiments, an Fc receptor binds to a first
glycoform with an affinity which is at least 2 fold, 6 fold, 10
fold, 20 fold, 30 fold greater, 40 fold, 50 fold, 100 fold, or 150
fold greater than the affinity with it binds to another
glycoform.
[0009] In some embodiments, a method includes contacting the medium
with one or more wash solutions, prior to contacting the medium
with the elution solution.
[0010] In various embodiments, a method includes recovering
polypeptide that flows through the medium.
[0011] In some embodiments, a medium is loaded at 1-3000% (e.g.,
5-1900%, 5-100%, or 100-1900%) of its polypeptide capacity. A
medium can be loaded at a capacity that allows selective depletion
from the flow through of glycoforms that bind to the medium. For
example, in some embodiments, levels of certain glycoforms, e.g.,
glycoforms having reduced levels of fucose (e.g., afucosylated
glycoforms), in the flow through fraction are less than 95%, 90%,
75%, 50%, 25%, 10%, or 5% of the levels in the load material.
[0012] A load fluid can be from a variety of sources. In some
embodiments, a load fluid comprises cell culture medium (e.g.,
crude or filtered cell culture medium, cell culture medium
comprising cells, or cell culture medium from which cells are
removed). In some embodiments, a load fluid comprises a fluid that
has been purified by one or more of Ion exchange chromatography
(e.g., cation exchange, anion exchange), protein A chromatography,
UF/DF, virus reduction filtration, hydrophobic interaction
chromatography, hydroxyapatite chromatography, mixed mode
chromatography, lectin chromatography, or a combination thereof. In
other embodiments, a load fluid comprises a pharmaceutical drug
product or drug substance.
[0013] An immunoglobulin Fc receptor binding moiety can include,
for example, an immunoglobulin Fc region. In some embodiments, an
Fc receptor binding moiety includes a native immunoglobulin Fc
region, or a portion or variant thereof that retains ability to
bind to an Fc receptor.
[0014] In some embodiments, the immunoglobulin Fc receptor is
selected from the group consisting of: Fc gamma RIIIa V176, Fc
gamma RIIIa F176, Fc gamma RIIIb NA1, Fc gamma RIIIb NA2, Fc gamma
RIIa H131, Fc gamma RIIa R131, Fc gamma RIIb 1232, and Fc gamma
RIIb T232.
[0015] Methods can be used to separate glycoforms of a variety of
polypeptides. In some embodiments, a polypeptide separated using a
method comprises an antibody (e.g., a monoclonal antibody, a human
monoclonal antibody, a human IgG, a human IgG1). In some
embodiments, a polypeptide comprises an Fc fusion protein (e.g., an
Fc fusion protein having a human Fc region). Polypeptides for
separation according to methods herein include single domain
antibodies, maxibodies, minibodies, intrabodies, small modular
immunopharmaceuticals (SMIPs), IgG-scFv bispecific antibodies,
antibody-peptide conjugates, antibody-drug conjugates, and Fc
receptor binding polypeptides on a virus or virus capsid.
[0016] In some embodiments, a polypeptide for separation is
produced in a mammalian cell. In some embodiments, a polypeptide is
produced in a fungal cell (e.g., Pichia pastoris), an insect cell,
or a plant cell. In some embodiments, a polypeptide is produced in
a CHO cell (e.g., GS-CHO, CHO-K1, or CHO-K1SV), an NS0 cell, or an
Sp2/0 cell.
[0017] An Fc receptor is one which preferentially binds to one or
more glycoforms. In some embodiments, an Fc receptor preferentially
binds to a glycoform having reduced fucose (e.g., afucosylated
glycoforms, e.g., glycoforms having reduced core N-fucose).
[0018] In some embodiments, an Fc receptor preferentially binds to
polypeptide glycoforms having increased high mannose
oligosaccharides.
[0019] In some embodiments, an Fc receptor is glycosylated. For
example, the Fc can be N-glycosylated.
[0020] In some embodiments, an Fc receptor includes an Fc binding
portion of an Fc gamma RIII polypeptide (e.g., an Fc gamma RIIIa
polypeptide, or an Fc gamma RIIIb polypeptide) or an Fc gamma RIV
polypeptide.
[0021] For example, the Fc receptor includes an extracellular
domain of an Fc gamma RIII polypeptide or an Fc gamma RIV
polypeptide; e.g., an Fc receptor comprises a sequence at least 85%
identical to amino acid residues 21-209 of SEQ ID NO:1. In some
embodiments, an extracellular domain of a Fc gamma RIII polypeptide
is a V176 allotype.
[0022] In some embodiments, an Fc receptor comprises a full length
Fc gamma RIII polypeptide.
[0023] In various embodiments of a method provided herein, a load
fluid comprises a first glycoform that preferentially binds to the
Fc receptor, and the percentage of the first glycoform in the
eluate is increased by at least 20%, relative to the load fluid,
e.g., the percentage of the first glycoform in the eluate is
increased by at least 50%, 100%, 2-fold, 5-fold, 10-fold, or
20-fold, relative to the bad fluid.
[0024] A method can enrich for a glycoform such that the eluate
contains at least a given percentage. For example, in some
embodiments, the percentage of the first glycoform in the eluate is
at least 5%, 10% 20%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,
or 80%.
[0025] In some embodiments, the first glycoform is a glycoform
having reduced fucose (e.g., an afucosylated glycoform).
[0026] In some embodiments, the first glycoform is a glycoform
having reduced sialylation (e.g., an asialylated glycoform).
[0027] In some embodiments, the first glycoform is a galactosylated
glycoform.
[0028] In some embodiments, the first glycoform is a glycoform
having high mannose oligosaccharides.
[0029] An eluate can be collected or divided into fractions, one or
more of which may be enriched for a particular glycoform.
[0030] In some embodiments of a method, a load fluid includes a
second glycoform that does not preferentially bind to the Fc
receptor, and the percentage of the second glycoform in the eluate
is decreased, relative to the load fluid (e.g., wherein the second
glycoform is a fucosylated, sialylated, and/or high mannose
glycoform).
[0031] In some embodiments, a biological activity of polypeptide in
the eluate is altered (e.g., increased or decreased) relative to
the activity of the polypeptide in the load fluid. In some
embodiments, a biological activity includes antibody dependent cell
mediated cytotoxicity (ADCC). In some embodiments of a method, ADCC
is increased (e.g., ADCC is increased by at least 20%, 50%, 100%,
2-fold, 5-fold, 10-fold, 20-fold, or 50-fold).
[0032] In some embodiments, a biological activity of polypeptide
that has flowed through the medium is altered (e.g., increased or
decreased), relative to the activity of the polypeptide in the load
fluid.
[0033] Any of a variety of media can be used. In some embodiments,
a medium comprises beads, membranes, monoliths, a fiber matrix,
porous media, or a gel. In some embodiments, a medium comprises
agarose, cellulose, or dextran, ceramic, metal, glass, nylon,
Teflon, nylon, polycarbonate, polyacrylamide, polystyrene,
polypropylene, polyether sulfone, polyamide,
polytetrafluoroethylene, polysulfone, polyester, polyvinylidene
fluoride, a fluorocarbon (e.g.
poly(tetrafluoroethylene-co-perfluoro(alkyl vinyl ether)),
polyethylene, polyacrylate, or poly(azolactone).
[0034] An Fc receptor can be linked to a medium via a
crosslinker.
[0035] In some embodiments, an Fc receptor is linked to a medium
via a disulfide bond, metal chelation, cyanogen bromide, an NHS
linkage, a histidine tag, a glycidyl ether, an epoxy, a tresyl
chloride linkage, a tosyl chloride linkage, an EAH linkage, an ECH
linkage, an activated thiol linkage, or a thiopropyl linkage.
[0036] A medium comprises an Fc receptor, for example, at a
concentration of 0.01 to 15 mg/mL. In some embodiments, a medium
comprises an Fc receptor at a concentration between 2-10 mg/mL
(e.g., between 3-7.5 mg/mL, 3-5 mg/mL, or 5J.5 mg/mL).
[0037] In some embodiments, a medium is equilibrated with an
equilibration solution prior to contacting the medium with the load
fluid. In many embodiments, an equilibration solution includes a
buffer (e.g., Tris, MES, HEPES, Phosphate, Histidine) between 1 and
500 mM, and a salt (e.g., NaCl, CaCl.sub.2) between 0 and 2000 mM,
at a pH between 3.5 and 10.
[0038] In various embodiments, a load fluid comprises a polypeptide
at a concentration between 0.001 and 100 mg/mL. The amount of
polypeptide contacted with the medium can range from 0.1 to 25,000
mg polypeptide/mL medium.
[0039] In some embodiments, one or more wash solutions used in a
method include a buffer (e.g., Tris, MES, HEPES, Phosphate,
Histidine) between 1 and 500 mM, and a salt (e.g., NaCl,
CaCl.sub.2) between 0 and 2000 mM, and/or an additive (e.g.,
guanidine, urea, sucrose) and/or a solvent (e.g., ethanol,
acetonitrile, polyethylene glycol) at a pH between 3.5 and 10.
[0040] In some embodiments, an elution solution has a pH between 2
and 5 and or a salt (e.g., NaCl, CaCl.sub.2) between 0 and 2000 mM,
and/or an additive (e.g., guanidine, urea, sucrose) and/or a
solvent (e.g., ethanol, acetonitrile, polyethylene glycol).
[0041] In some embodiments, a medium is contacted with one or more
elution solutions under conditions in which a pH gradient is
applied (e.g., a pH gradient from pH 5.0 to pH 3.0).
[0042] A method can further include neutralizing an eluate.
[0043] A separation method can be used in conjunction with other
separation methods. Thus, in some embodiments, a method further
includes contacting an eluate with a second medium, and recovering
polypeptide that flows through, or is eluted from, the second
medium. An exemplary second medium can include an ion exchange
medium, a hydroxyapatite medium, a protein A medium, a hydrophobic
interaction medium, an immobilized metal affinity medium, a
synthetic medium (biomimetic), a lectin, or a combination
thereof.
[0044] A method can further include producing a pharmaceutical
composition from polypeptide in an eluate or from polypeptide that
has flowed through the medium.
[0045] A method can further include analyzing a characteristic of
polypeptide eluted from the medium. In some embodiments,
oligosaccharides from the polypeptide are analyzed (e.g., N-linked
oligosaccharides are analyzed by cleaving N-oligosaccharides from
the polypeptide, labeling the oligosaccharides, and detecting
labeled oligosaccharide species).
[0046] In some embodiments, a biological activity of polypeptide is
analyzed.
[0047] In some embodiments, one or more of toxicity, stability
(e.g., half life, shelf life), or efficacy are analyzed (e.g., as
compared to polypeptide in the load fluid, polypeptide that has
flowed through the medium, or as compared to a reference).
[0048] Analyzing can further include analyzing polypeptide in the
load fluid and/or polypeptide that has flowed through the medium
(e.g., for comparison with eluted polypeptide).
[0049] Alternatively, or in addition, a method can further include
analyzing polypeptide that has flowed through the medium.
[0050] In some embodiments, oligosaccharides on the polypeptide are
analyzed.
[0051] In some embodiments, one or more of toxicity, stability
(e.g., half life, shelf life), or efficacy are analyzed (e.g., as
compared to polypeptide in the load fluid, polypeptide that has
eluted from the medium, or as compared to a reference).
[0052] In some embodiments, a biological activity of polypeptide is
analyzed.
[0053] The present disclosure also features a composition including
a polypeptide recovered by a method described herein.
[0054] In another aspect, the present disclosure provides a method
that includes: (a) providing a medium comprising an Fc receptor,
wherein the Fc receptor comprises an Fc binding portion of an Fc
gamma RIII polypeptide; (b) contacting the medium with a load fluid
comprising a polypeptide under conditions in which the polypeptide
binds to the medium, wherein the polypeptide comprises an
immunoglobulin Fc region; (c) contacting the medium with an elution
solution under conditions in which bound polypeptide elutes from
the medium; and (d) recovering bound polypeptide that elutes from
the medium, thereby producing an eluate.
[0055] In some embodiments, an Fc receptor comprises an
extracellular domain of an Fc gamma RIII polypeptide.
[0056] The method can include additional features described
herein.
[0057] In some embodiments of the methods summarized above, the
load fluid may comprise serum IgG.
[0058] In a further aspect, the present disclosure provides a
medium comprising an Fc receptor linked to a solid support, wherein
the Fc receptor comprises an Fc binding portion of an Fc gamma RIII
polypeptide.
[0059] In some embodiments, a solid support comprises agarose,
cellulose, or dextran, ceramic, metal, glass, nylon, Teflon, nylon,
polycarbonate, polyacrylamide, polystyrene, polypropylene,
polyether sulfone, polyimide, polytetrafluoroethylene, polysulfone,
polyester, polyvinylidene fluoride, a fluorocarbon (e.g.
poly(tetrafluoroethylene-co-perfluoro(alkyl vinyl ether)),
polyethylene, polyacrylate, or poly(azolactone).
[0060] In some embodiments, a solid support comprises beads,
membranes, monoliths, a fiber matrix, porous media, or a gel.
[0061] In another aspect, the present disclosure provides a kit
that comprises (a) a medium comprising an immunoglobulin Fc
receptor linked to a solid support, wherein the immunoglobulin Fc
receptor comprises an Fc binding portion selected from the group
consisting of: an Fc binding portion of an Fc gamma RI polypeptide,
an Fc gamma RII polypeptide, an Fc gamma RIII polypeptide, and an
Fc gamma RIV polypeptide; and (b) instructions for use according to
any of the methods summarized above, such as the method, in
accordance with one aspect of the present disclosure, of separating
polypeptide glycoforms in a load fluid.
[0062] In some embodiments, the solid support of the kit comprises
agarose, cellulose, or dextran, ceramic, metal, glass, nylon,
Teflon, nylon, polycarbonate, polyacrylamide, polystyrene,
polypropylene, polyether sulfone, polyamide,
polytetrafluoroethylene, polysulfone, polyester, polyvinylidene
fluoride, a fluorocarbon (e.g.
poly(tetrafluoroethylene-co-perfluoro(alkyl vinyl ether)),
polyethylene, polyacrylate, or poly(azolactone). The solid support
may comprise beads, membranes, monoliths, a fiber matrix, porous
media, or a gel.
[0063] Details of certain embodiments of the present disclosure are
set forth in the accompanying drawing and the description below.
Other features, objects, and advantages of the present disclosure
will be apparent from the description and figures, and from the
claims. All cited patents, and patent applications and references
are incorporated by reference in their entireties for all
purposes.
BRIEF DESCRIPTION OF THE FIGURE
[0064] FIG. 1 is a set of graphs depicting glycan profiles of a
monoclonal antibody preparation (top panel) and the preparation
which has been eluted from an Fc gamma RIIIa receptor medium
(bottom panel). All peaks that have labels ending in `F` are
fucosylated and ail peaks have labels that do not end in `F` are
afucosylated.
Definitions
[0065] "Corresponding": As used herein, an amino acid (or
nucleotide) that is "corresponding" to an amino acid (or
nucleotide) in a reference sequence occupies a site that is
homologous to the site in the reference sequence. Corresponding
amino acids and nucleotides can be identified by alignment of
related sequences. Sequences can be compared to protein sequences
available in public databases using algorithms such as BLAST,
FASTA, ClustalW, which are well known to those skilled in the
art.
[0066] An "eluate", as used herein, refers to a fluid that has been
exposed to a medium and that has product eluted from the
medium.
[0067] An "immunoglobulin Fc receptor" or "Fc receptor", as used
herein, refers to a polypeptide that can interact with an
immunoglobulin Fc region (e.g., a native Fc region). An Fc receptor
typically binds to an Fc region with an affinity (K.sub.A) of at
least 10.sup.5 M.sup.-1 (e.g., at least 10.sup.6-10.sup.9
M.sup.-1). In some embodiments, an Fc receptor comprises an
extracellular portion of a native Fc gamma receptor, or a variant
thereof having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%
sequence identity to a native Fc gamma receptor.
[0068] An "immunoglobulin Fc receptor binding moiety" or "Fc
receptor binding moiety", as used herein, refers to a moiety
comprising amino acids that can interact with an immunoglobulin Fc
receptor. In some embodiments, an Fc receptor binding moiety
Includes an Fc region. An Fc region is a C-terminal region of an
immunoglobulin heavy chain, and generally comprises the last two
constant region domains of an immunoglobulin, e.g., an IgG, IgA, or
IgD, or the last three constant region domains of IgE or IgM.
Camelid antibodies lack light chains but have Fc regions that are
comparable to typical immunoglobulins. An Fc region can also
include a flexible hinge region N-terminal to the constant region
domains of an Immunoglobulin. A human IgG heavy chain Fc region is
usually defined to stretch from position C226 or P230 (according to
Kabat numbering; see Kabat et al. Sequences of Proteins of
Immunological Interest, 5.sup.th Ed. Public Health Service,
National Institutes of Health, Bethesda, Md. (1991)) to the
C-terminus of the molecule, although boundaries of an Fc region can
vary. Amino add and nucleic add sequences of numerous Fc regions
are known in the art.
[0069] In various embodiments, Fc receptor binding moieties include
native Fc domains (e.g., native human Fc domains) and portions and
variants thereof (e.g., variants having at least 80%, 85%, 90%,
95%, 96%, 97%, 98%, or 99% sequence identity to a native Fc domain)
that bind to an Fc receptor. Antibodies and Fc fusion proteins are
examples of polypeptides that include Fc receptor binding
impieties.
[0070] A "load", as used herein, refers to any material containing
a product of interest. A "load fluid" refers to a liquid containing
load material for contact with a medium (e.g., liquid for passage
through a column containing the medium). In some embodiments, a
load fluid is a cell culture medium. A cell culture medium may be
clarified (e.g., to remove cells or cell debris). In some
embodiments, a load fluid is a partially purified intermediate
derived from a chromatography step (e.g., a protein A
chromatography step). In some embodiments, a load fluid is a
purified preparation.
[0071] A "medium", as used herein, refers to any substance that can
be used as a support for an Fc receptor for separation of
polypeptide glycoforms.
[0072] "Preferentially binds", as used herein, e.g., to refer to Fc
receptor interactions with glycoforms of Fc receptor binding
moieties, means that the receptor binds to one glycoform more
readily than it would bind to another glycoform (e.g., to one
glycoform of a given polypeptide than to another glycoform of the
same polypeptide). A receptor which "preferentially binds" to a
given glycoform would more likely bind to that glycoform than to
another glycoform, even though such a receptor may bind to the
other glycoform. For example, a receptor may be considered to
preferentially bind to a glycoform if it binds a first glycoform of
a polypeptide with an affinity which is at least 50%, 100%, 2 fold,
5 fold, 10 fold, 20 fold, 30 fold, 40 fold, or 50 fold greater than
the affinity with which it binds to a second glycoform of the
polypeptide.
[0073] The term "epitope" refers to the site on an antigen or
hapten to which specific B cells and/or T cells respond. The term
is also used interchangeably with "antigenic determinant" or
"antigenic determinant site". Antibodies that recognize the same
epitope can be identified in a simple immunoassay showing the
ability of one antibody to block the binding of another antibody to
a target antigen.
[0074] The term "nucleic acid" encompasses DNA, RNA (e.g., mRNA,
tRNA), heteroduplexes, and synthetic molecules capable of encoding
a polypeptide and includes all analogs and backbone substitutes
such as PNA that one of ordinary skill in the art would recognize
as capable of substituting for naturally occurring nucleotides and
backbones thereof. Nucleic adds may be single stranded or double
stranded, and may be chemical modifications. The terms "nucleic
acid" and "polynucleotide" are used interchangeably. Because the
genetic code is degenerate, more than one codon may be used to
encode a particular amino add, and the present compositions and
methods encompass nucleotide sequences which encode a particular
amino add sequence.
[0075] Unless otherwise indicated, nucleic adds are written left to
right in 5' to 3' orientation; amino add sequences are written left
to right in amino to carboxy orientation, respectively.
[0076] As used herein, the term "amino add sequence" is synonymous
with the terms "polypeptide," "protein," and "peptide," and are
used interchangeably. Where such amino add sequences exhibit
activity, they may be referred to as an "enzyme." The conventional
one-letter or three-letter code for amino add residues are used
herein.
[0077] As used herein, a "synthetic" molecule is produced by in
vitro chemical or enzymatic synthesis rather than by an
organism.
[0078] As used herein, the term "expression" refers to the process
by which a polypeptide is produced based on the nucleic add
sequence of a gene. The process includes both transcription and
translation.
[0079] A "gene" refers to the DNA segment encoding a polypeptide or
RNA.
[0080] An "isolated" polynucleotide or polypeptide is one that is
substantially free of the materials with which it is associated in
its native environment. By substantially free, is meant at least
50%, advantageously at least 70%, more advantageously at least 80%,
and even more advantageously at least 90% free of these
materials.
[0081] An "isolated" nucleic add molecule is a nucleic add molecule
separate and discrete from the whole organism with which the
molecule is found in nature; or a nucleic add molecule devoid, in
whole or part, of sequences normally associated with it in nature;
or a sequence, as it exists in nature, but having heterologous
sequences in association therewith.
[0082] "Native" proteins or polypeptides refer to proteins or
polypeptides isolated from the source in which the proteins
naturally occur. "Recombinant" polypeptides refer to polypeptides
produced by recombinant DNA techniques; e.g., produced from cells
transformed by an exogenous DNA construct encoding the desired
polypeptide. "Synthetic" polypeptides include those prepared by
chemical synthesis as well as the synthetic antigens described
above.
Detailed Description of Certain Embodiments
[0083] Embodiments of the present invention may employ, unless
otherwise indicated, conventional techniques of chemistry,
molecular biology, microbiology, recombinant DNA and immunology,
which are within the capabilities of a person of ordinary skill in
the art. Such techniques are explained in the literature. See, for
example, J. Sambrook, E. F. Fritsch, and T. Maniatis, 1989,
Molecular Cloning: A Laboratory Manual, Second Edition, Books 1-3,
Cold Spring Harbor Laboratory Press; Ausubel, F. M. et al. (1995
and periodic supplements; Current Protocols in Molecular Biology,
ch. 9, 13, and 16, John Wiley & Sons, New York, N.Y.); B. Roe,
J. Crabtree, and A. Kahn, 1996, DNA Isolation and Sequencing:
Essential Techniques, John Wiley & Sons; M. J. Gait (Editor),
1984, Oligonucleotide Synthesis: A Practical Approach, IrI Press;
and, D. M. J. Lilley and J. E. Dahlberg, 1992, Methods of
Enzymology: DNA Structure Part A: Synthesis and Physical Analysis
of DNA Methods in Enzymology, Academic Press. Each of these general
texts is herein incorporated by reference.
Polypeptides for Separation
[0084] Methods and compositions provided herein can be used for
separation of polypeptide glycoforms. Polypeptides that can be
separated typically include an Fc receptor binding moiety (e.g., an
immunoglobulin Fc region or portion thereof that binds to Fc
receptor). In some embodiments, polypeptides for separation include
antibodies (e.g., monoclonal, polyclonal, chimeric, humanized, or
human antibodies), Fc fusion proteins, antibody-drug conjugates,
single domain antibodies, small modular immunopharmaceuticals
(MIPs), maxibodies, minibodies, intrabodies, scFv, IgG-scFv
bispecific antibodies, or Fc-containing minibodies. Other types of
polypeptides that bind to Fc receptors, Fc-receptor binding viral
polypeptides, can also be separated.
[0085] In various embodiments, methods and compositions provided
herein are used for separation of polypeptide glycoforms in
monoclonal antibody preparations. In some embodiments, a
therapeutic monoclonal antibody is separated. One class of
therapeutic polypeptides for which the present disclosure is
applicable is polypeptides whose therapeutic efficacy is thought to
rely, at least in part, on the ability to trigger ADCC or CDC. In
some embodiments, therapeutic polypeptides comprise human IgG
antibodies. In some embodiments, human IgG antibodies are IgG1. In
some embodiments, human IgG antibodies are IgG3. In some
embodiments, human IgG antibodies are IgG4. In some embodiments,
polypeptides comprise an Fc region of a human antibody, e.g. of a
human IgG antibody (e.g., an IgG1, IgG3, or IgG4 antibody). In some
embodiments, a polypeptide is a polypeptide (e.g., antibody) used
to treat a cancer.
[0086] Polypeptides (e.g., antibodies) for separation according to
methods provided herein include polypeptides that specifically bind
to one or more of the following target molecules (with examples of
polypeptides that bind these targets): the A.beta. fragment of
amyloid precursor protein (see, e.g., U.S. Pat. No. 7,625,560;
e.g., bapineuzumab); HER2/neu receptor (e.g., trastuzumab); CD20
(e.g., rituximab, ofatumumab, afutuzumab, tositumomab); B cell
activating factor (BAFF) (e.g., belimumab); TNF.alpha. (e.g.,
adalimumab, infliximab, etanercept, golimumab); CD52 (e.g.,
alemtuzumab); CD25 (e.g., basiliximab, daclizumab); VEGF (e.g.,
bevacizumab); EGFR (e.g., cetuximab, panitimumab, nimotuzumab);
CD11a (e.g., efalizumab); CD33 (e.g., gemtuzumab); CD3; alpha-4
integrin (e.g., natalizumab); IgE (e.g., omalizumab); GDF-8 (see,
e.g., U.S. Pat. Pub. No. 20040142382); IL-12R/IL-23R (p40) (e.g.,
ustekinumab); B7 (e.g., CTLA4-Ig, e.g., abatacept); complement C5
(e.g., eculizumab); platelet GpIIb/IIIa (e.g., abciximab);
phosphatidylserine (e.g., bavituximab); and pF-RSV
(palivizumab).
[0087] Polypeptides for separation according to methods described
herein can be from any of a number of sources including, but not
limited to, conditioned media derived from culturing a recombinant
cell line that expresses a polypeptide, cell extracts of
polypeptide-producing cells, serum (e.g., serum of an immunized
subject, serum of a subject who has been exposed to an infectious
agent, serum of a subject who has developed immunity to an
infectious agent (e.g., either by immunization or natural
exposure), serum of a nave subject, human serum), ascites fluid,
hybridoma or myeloma supernatants, commercially available
polypeptide preparations (e.g., drug product), and other sources.
In one embodiment of the present disclosure, partially purified
polypeptide from conditioned cell culture media of a variety of
polypeptide producing recombinant cells are separated.
[0088] In some embodiments, a polypeptide for separation or an Fc
receptor for use in separating the polypeptide according to a
method described herein is produced by expression in a recombinant
cell. Nucleotide sequences encoding the polypeptide for separation
or the Fc receptor may be inserted into vectors. The term "vector"
is widely used and understood by those of ordinary skill in the
art, and as used herein the term "vector" is used consistent with
its meaning to those of ordinary skill in the art. For example, the
term "vector" is commonly used by those ordinarily skilled in the
art to refer to a vehicle that allows or facilitates the transfer
of nucleic acid molecules from one environment to another or that
allows or facilitates the manipulation of a nucleic acid
molecule.
[0089] For example, a vector is a replicon, such as plasmid, phage
or cosmid, to which another DNA segment may be attached so as to
bring about the replication of the attached segment. A "replicon"
is any genetic element (e.g., plasmid, chromosome, virus) that
functions as an autonomous unit of DNA replication in vivo; i.e.,
capable of replication under its own control. An "origin of
replication" refers to those DNA sequences that participate in DNA
synthesis. An "expression control sequence" is a DNA sequence that
controls and regulates the transcription and translation of another
DNA sequence. A coding sequence is "operably linked" and "under the
control" of transcriptional and translational control sequences in
a cell when RNA polymerase transcribes the coding sequence into
mRNA, which is then translated into the protein encoded by the
coding sequence.
[0090] In general, expression vectors containing promoter sequences
which facilitate the efficient transcription and translation of the
inserted DNA fragment are used in connection with the host. The
expression vector typically contains an origin of replication,
promoter(s), terminator(s), as well as specific genes which are
capable of providing phenotypic selection in transformed cells.
When the polynucleotide encodes a polyprotein fragment,
advantageously, in the vector, an initiation codon (ATG) is placed
at 5' of the reading frame and a stop codon is placed at 3'. Other
elements for controlling expression may be present, such as
enhancer sequences, stabilizing sequences and signal sequences
permitting the secretion of the protein. The transformed hosts can
be fermented and cultured according to means known in the art to
achieve optimal cell growth.
[0091] Any vector that allows expression of the polypeptides of the
present disclosure may be used in accordance with embodiments of
the present invention. In certain embodiments, the polypeptides of
the present disclosure may be used in vitro (such as using
cell-free expression systems) and/or in cultured cells grown in
vitro. For such applications, any vector that allows expression of
the polypeptides in vitro and/or in cultured cells may be used.
[0092] A DNA "coding sequence" is a double-stranded DNA sequence
which is transcribed and translated into a polypeptide in vivo when
placed under the control of appropriate regulatory sequences. The
boundaries of the coding sequence are determined by a start codon
at the 5' (amino) terminus and a translation stop codon at the 3'
(carboxyl) terminus. A coding sequence can include, but is not
limited to, prokaryotic sequences, cDNA from eukaryotic mRNA,
genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and
even synthetic DNA sequences. A polyadenylation signal and
transcription termination sequence will usually be located 3' to
the coding sequence. A "cDNA" is defined as copy-DNA or
complementary-DNA, and is a product of a reverse transcription
reaction from an mRNA transcript.
[0093] Transcriptional and translational control sequences are DNA
regulatory sequences, such as promoters, enhancers, ribosome
binding sites, upstream regulatory domains, polyadenylation
signals, terminators, and the like, that provide for the expression
of a coding sequence in a host cell. A "cis-element" is a
nucleotide sequence, also termed a "consensus sequence" or "motif",
that interacts with other proteins which can upregulate or
downregulate expression of a specific gene locus. A "signal
sequence" can also be included with the coding sequence. This
sequence encodes a signal peptide, N-terminal to the polypeptide,
that communicates to the host cell and directs the polypeptide to
the appropriate cellular location. Signal sequences can be found
associated with a variety of proteins native to prokaryotes and
eukaryotes. Not all of these control sequences need always be
present in a recombinant vector so long as the desired gene is
capable of being transcribed and translated.
[0094] A "promoter sequence" is a DNA regulatory region capable of
binding RNA polymerase in a cell and initiating transcription of a
downstream (3' direction) coding sequence. The promoter sequence is
typically bounded at its terminus by the transcription initiation
site and extends upstream (5' direction) to include the minimum
number of bases or elements necessary to initiate transcription at
levels detectable above background. Within the promoter sequence is
a transcription initiation site, as well as protein binding domains
(consensus sequences) responsible for the binding of RNA
polymerase. Eukaryotic promoters often, but not always, contain
"TATA" boxes and "CAT" boxes. Prokaryotic promoters contain
Shine-Dalgarno sequences in addition to the -10 and -35 consensus
sequences.
[0095] As used herein, the terms "restriction endonucleases" and
"restriction enzymes" refer to enzymes which cut double-stranded
DNA at or near a specific nucleotide sequence.
[0096] "Recombinant DNA technology" refers to techniques for
uniting two heterologous DNA molecules, usually as a result of in
vitro ligation of DNAs from different organisms. Recombinant DNA
molecules are commonly produced by experiments in genetic
engineering. Synonymous terms include "gene splicing", "molecular
cloning" and "genetic engineering". The product of these
manipulations results in a "recombinant" or "recombinant
molecule".
[0097] A cell has been "transformed" or "transfected" with
exogenous or heterologous DNA when such DNA has been introduced
inside the cell. The transforming DNA may or may not be integrated
(covalently linked) into the genome of the cell. In prokaryotes,
yeast, and mammalian cells for example, the transforming DNA may be
maintained on an episomal element such as a vector or plasmid. With
respect to eukaryotic cells, a stably transformed cell is one in
which the transforming DNA has become integrated into a chromosome
so that it is inherited by daughter cells through chromosome
replication. This stability is demonstrated by the ability of the
eukaryotic cell to establish cell lines or clones comprised of a
population of daughter cells containing the transforming DNA. A
"clone" is a population of cells derived from a single cell or
ancestor by mitosis. A "cell line" is a clone of a primary cell
that is capable of stable growth in vitro for many generations. An
organism, such as a plant or animal, that has been transformed with
exogenous DNA is termed "transgenic".
[0098] A "heterologous" region of the DNA construct is an
identifiable segment of DNA within a larger DNA molecule that is
not found in association with the larger molecule in nature. Thus,
when the heterologous region encodes a mammalian gene, the gene
will usually be flanked by DNA that does not flank the mammalian
genomic DNA in the genome of the source organism. In another
example, the coding sequence is a construct where the coding
sequence itself is not found in nature (e.g., a cDNA where the
genomic coding sequence contains introns, or synthetic sequences
having codons different than the native gene). Allelic variations
or naturally-occurring mutational events do not give rise to a
heterologous region of DNA as defined herein. For example, a
polynucleotide, may be placed by genetic engineering techniques
into a plasmid or vector derived from a different source, and is a
heterologous polynucleotide. A promoter removed from its native
coding sequence and operatively linked to a coding sequence other
than the native sequence is a heterologous promoter.
[0099] As used herein, "fragment" or "portion" as applied to a gene
or a polypeptide, will ordinarily be at least 10 residues, more
typically at least 20 residues, and preferably at least 30 (e.g.,
50) residues in length, but less than the entire, intact sequence.
Fragments of these genes can be generated by methods known to those
skilled in the art, e.g., by restriction digestion of naturally
occurring or recombinant fiber or fibritin genes, by recombinant
DNA techniques using a vector that encodes a defined fragment of
the fiber or fibritin gene, or by chemical synthesis.
[0100] A wide variety of cells can be used to produce a recombinant
protein. Any cell that can be transformed with recombinant DNA to
express a protein of interest (e.g., a monoclonal antibody), can be
used in the methods of the present disclosure. Cells can be from a
variety of species, e.g., eukaryotic species, including plant,
yeast, nematode, worm, insect, amphibian, or mammal, for example,
human, primate, ovine, bovine, porcine, equine, feline, canine, or
rodent source. In particular embodiments, the cells are from human
or rodent. In particular embodiments, the cells are from hamster
(e.g., Chinese hamster ovary cells). Examples of mammalian cells
that may be used include BALB/c mouse myeloma line (NSO/I, ECACC
No: 85110503); SP2/0; Balb/c 3T3; human retinoblasts (PER.C6
(CruCell, Leiden, The Netherlands)); monkey kidney CV1 line
transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney
line (293 or 293 cells subcloned for growth in suspension culture,
Graham et al. J. Gen Virol., 36:59 (1977)); baby hamster kidney
cells (BHK, ATCC CCL 10); Chinese hamster ovary cells+/- DHFR (CHO,
Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216 (1980);
GS-CHO, CHO-K1, CHO-K1SV, CHO-DG44, CHO-DUKX, CHO-DUXB11, CHO-S);
mouse sertoli cells (TM4, Mather, Biol. Reprod., 23:243-251
(1980)); rat hybridoma YB2/0 (Shinkawa et al., J. Biol. Chem.
278:3466-3473, 2003); monkey kidney cells (CV1 ATCC CCL 70);
African green monkey kidney cells (VERO-76, ATCC CRL-1 587); human
cervical carcinoma cells (HeLa, ATCC CCL 2); canine kidney cells
(MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL
1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hap
G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI
cells (Mather et al., Annals N.Y. Acad. Sci., 383:44-68 (1982));
MRC 5 cells; FS4 cells; and a human hepatoma line (Hap G2). A
number of suitable cell lines can be obtained from depositories
such as the America Type Culture Collection (ATCC), Manassas, Va.
Examples of plant cells that may be used include Lemmna minor
(duckweed), Arabidopsis thalania, and Physcomitrella patens (moss)
cells. Examples of insect cells that may be used include Spodoptera
frugiperda (Sf9 and Sf21), Trichoplusia ni (Tni and BTI-Tn 5B1-4),
and Mamestra brassicae (Mb) cells. Useful fungal cells include
Pichia pastoris and Saccharomyces cerevisiae cells.
[0101] Methods described herein can permit selection and enrichment
of polypeptide glycoforms from an initial preparation having any
pattern of glycoform expression, without a need for selection of a
specialized host cell or use of atypical conditions or preparative
steps. However, it may, in some cases, be beneficial to apply
methods to separation of preparations in which a expression of a
glycoform is altered, e.g., so as to increase expression of a
desired glycoform and/or decrease expression of an unwanted
glycoform. Combinations of conditions and separation modes that
favor enrichment of a glycoform of interest may permit isolation of
glycoforms at higher levels of purity or with fewer steps than
previously possible. In some embodiments, glycoform expression is
altered by genetic manipulation of a host cell to have increased or
decreased expression of an enzyme that participates a glycosylation
pathway (e.g., an enzyme that catalyzes addition or trimming of
carbohydrate moieties). Such modifications can include
introduction, deletion, and/or knocking down of a gene encoding a
glycosyltransferase, e.g., an alpha-1,6-fucosyltransferase, or a
glycosidase. Cells lacking expression of
alpha-1,6-fucosyltransferase by way of disruption of the FUT8 gene
are described, e.g., in Yamani-Ohnuki et al., Biotechnol. Bioeng.
87(5):614-622, 2004 and WO2009/009086 Cell variants that produce
polypeptides having reduced fucosylation have been described (e.g.,
CHO Lec13 cells, Shields et al., J. Biol. Chem. 277(30)26733-26740,
2002; and CHO Ms704, Kande et al. Biotech. Bioeng. 94(4):680-688,
2006).
[0102] Lower eukaryotic (e.g., fungal cells can be engineered to
produce polypeptides with complex glycosylation characteristic of
mammalian cells. See, e.g., Hamilton et al., Science 313:1441-1443,
2006, which describes Pichia pastoris engineered to produce complex
terminally sialylated glycoproteins. See also WO2002/000879,
WO2004/074458, WO2004/074499, WO2004/074498, and WO2005/100584.
Plant cells (e.g., Lemna minor, or moss, can be engineered to
produce polypeptides having desired glycoforms, e.g., larking
plantlike glycosylation (see, e.g., Cox et al., Nat. Biotech.
24:1591-1597, 2006 and Nechansky et al., Mol. Immunol.
44:1815-1817, 2007).
[0103] In some embodiments, a polypeptide is produced in a cell
under conditions that alter glycosylation. For example, inclusion
of 10-600 nM manganese in cell culture medium can result in a more
extensive glycosylation pattern (U.S. Pat. Pub. 2008/0081356). In
some embodiments, a polypeptide is produced in a cell in the
presence of an agent that modulates glycosylation. For example, in
some embodiments, an inhibitor or agonist of a carbohydrate
modifying enzyme is used. In some embodiments, an inhibitor is a
nucleic acid antagonist (e.g., siRNA). Glycosylation of
polypeptides can be altered enzymatically in vitro (e.g., outside a
cell), e.g., by treatment with a glycosyltransferase or
glycosidase.
Binding Media
[0104] Fc Receptors
[0105] Fc receptors that can be used according to the present
disclosure include receptors that preferentially bind to one or
more polypeptide glycoforms. A receptor that preferentially binds
to one or more polypeptide glycoforms binds with an affinity that
is at least 50%, 100%, two fold (e.g., 5, 10, 20, 30, 40, 50, 75,
100, 150, or 200 fold) higher than an affinity with which it binds
to another glycoform of the polypeptide. In some embodiments, an Fc
receptor preferentially binds to glycoforms lacking or with reduced
laves of fucose (e.g., glycoforms with low levels or the absence of
core N-fucosylation; e.g., antibody glycoforms lacking fucose on
one or both heavy chains). For example, the Fc receptor can include
an extracellular portion of an Fc gamma RIII polypeptide (e.g., an
Fc gamma RIIIa polypeptide, or an Fc gamma RIIIb polypeptide). In
some embodiments, an Fc receptor preferentially binds glycoforms
with reduced sialic acid. In some embodiments, an Fc receptor
preferentially binds glycoforms containing high mannose. In some
embodiments, an Fc receptor preferentially binds galactosylated
glycoforms.
[0106] In certain further embodiments, the Fc receptor used
according to the present disclosure includes Fc receptors that bind
to polypeptide glycoforms lacking or with reduced levels of fucose
(e.g., glycoforms with low levels or the absence of core
N-fucosylation; e.g., antibody glycoforms lacking fucose on one or
both heavy chains) with a K.sub.D that is three times, four times,
five times, six times, seven times, eight times, nine times, or ten
times smaller than the K.sub.D of the Fc receptor for the
fucosylated form of the polypeptide. For examples of such binding
affinities of suitable Fc receptors for use in some embodiments of
the instant invention, see, e.g., Herbst, R et al. J. Pharmacol.
Exp. Ther. 335(1):213-222 (2010) and Shibata-Koyama, M et al. Exp.
Hematol. 37:309-321 (2007).
[0107] Accordingly, it has been surprisingly found by Applicants
that Fc receptors that bind to certain glycoform polypeptides, such
as afucosylated polypeptides, with K.sub.off rates of only about
five to ten fold higher than other glycoforms can be used in
affinity purification methods to purify the glycoform polypeptide,
such as, for example, purifying afucosylated polypeptide forms from
a mixture of different glycoform polypeptides. In a particular
embodiment, an Fc receptor such as Fc gamma RIIIa has a binding
affinity for afucosylated antibody forms that involves a K.sub.off
rate of the afucosylated antibody for the Fc receptor that doesn't
necessarily lend itself to affinity purification of the antibody,
such as, for example, by affinity chromatography involving binding
of the afucosylated antibody to the Fc receptor. In these
embodiments, the afucosylated polypeptide stays bound to the Fc
receptor such that it does not typically fall off during a wash
with, for example, PBS buffer, but will come off when exposed to
more stringent conditions, such as, for example, a pH gradient. For
examples of K.sub.off rates of Fc receptor and glycoform antibody
binding that surprisingly are amenable to purification by affinity
chromatography according to some embodiments of the instant
invention, see Li, P et al. J. Biol. Chem. 282(9):6210-6221
(2007).
[0108] Fc gamma RIIIa (also known as CD16a or FCGR3A protein) is a
type I membrane protein that is naturally expressed both as a
membrane protein and a soluble receptor produced by proteolytic
cleavage. Full length Fc gamma RIIIa has two Ig-like C2-type
domains in its extracellular region. Fc gamma RIIIa binds to the Fc
region of IgGs. The structural association of a human Fc gamma RIII
polypeptide with a human IgG1 Fc domain polypeptide has been
characterized (Sondermann et al., Nature 406:267-273, 2000). Fc
gamma RIIIa binds complexed, aggregated, and monomeric IgG. Fc
gamma RIIIa mediates antibody-dependent cellular toxicity and other
antibody-dependent responses such as phagocytosis. Depletion of
fucose from IgG1 enhances its affinity for Fc gamma RIIIa and
enhances its ability to cause ADCC (Okazaki et al., J. Mol. Biol.
336:1239-1249, 2004). In one study, an IgG1 bound to Fc gamma RIIIa
(V176 allotype) with a K.sub.a=1.87.times.10.sup.6 M.sup.-1, and a
defucosylated form of the IgG1 bound with a
K.sub.a=58.3.times.10.sup.6 M.sup.-1, as measured using a
BIAcore.TM. biosensor. Enhanced Fc gamma RIIIa affinity and ADCC
has also been observed for defucosylated IgG2, IgG3, and IgG4
(Jefferis, Trends Pharm. Sci. 30(7):356-362, 2009). The
oligosaccharide structure of Fc gamma Mile contributes to its
enhanced binding to defucosylated antibody.
[0109] An exemplary amino add sequence of a full length human Fc
gamma RIIIa polypeptide is shown in SEQ ID NO:1 (see also Acc. No.
P08637 of UniProt, on the world wide web at
uniprot.org/uniprot/P08637). The signal peptide is at about amino
adds 1-16. The extracellular domain is between amino adds 17-208.
Ig-like domains are found between amino adds 24-105 and 107-189.
Residues 230-254 are cytoplasmic. N-linked glycosylation may occur
at residues 56, 63, 92, 180, and 187. When an oligosaccharide at
position 180 is removed, the receptor has the same affinity for
fucosylated and afucosylated IgG Fc (Jefferis, Nat. Rev. Drug.
Disc. 8:226-234, 2009; Shibata-Koyama, supra). Removal of only the
glycosylation at position 63 of Fc gamma RIIIa increases its
affinity for afucosylated IgG. Removal of all glycosylation except
that at position 180 increases affinity for afucosylated IgG
(Shibata-Koyama, supra). Removal of all glycosylation except that
at positions 180 and 63 results in an affinity for afucosylated IgG
that is higher than the wild type Fc gamma RIIIa but lower than the
affinity with glycosylation only at position 180 (Shibata-Koyama,
supra).
TABLE-US-00001 TABLE 1 Exemplary full length Fc gamma RIIIa amino
acid sequence 10 20 30 40 50 60 MWQLLLPTAL LLLVSAGMRT EDLPKAVVFL
EPQWRVLEK DSVTLKCQGA YSPEDNSTQW 70 80 90 100 110 120 FHNESLISSQ
ASSYFIDAAT VDDSGEYRCQ TNLSTLSDPV QLEVHIGWLL LQAPRWVFKE 130 140 150
160 170 180 EDPIHLRCHS WKNTALHKVT YLQNGKGRKY FHHNSDFYIP KATLKDSGSY
FCRGLFGSKN 190 200 210 220 230 240 VSSETVNITI TQGLAVSTLS SFFPPGYQVS
FCLVMVLLFA VDTGLYFSVK TNIRSSTRDW 250 KDHKFKWRKD PQDK (SEQ. ID NO:
1)
[0110] Post-translational modifications of Fc gamma RIIIa include
high mannose and complex oligosaccharides (e.g., at residues 56,
63, 92, 180, and 187 of SEQ ID NO:1). Fc gamma RIIIa is
polymorphic. Examples of natural amino acid allotypes include
polypeptides having the following amino acid changes in SEQ ID
NO:1: L66H, L66R, G147D, Y158H, F176V, and F203S. Allotypes having
a valine at position 176 bind more strongly to IgG1, IgG3, and IgG4
than allotypes having a phenylalanine (Koene et al., Blood
90(3):1109-1114, 1997; Wu et al., J. Clin. Inv. 100(5):1059-1070,
1997). IgG1 is more efficient at mediating ADCC through Fc gamma
RIIIa V176-bearing cells than Fc gamma RIIIa F176-bearing cells
(Jeffries et al. Exp. Opin. Biol. Ther. 7(9):1401-1413, 2007).
[0111] Orthologs of human Fc gamma RIIIa have been identified,
e.g., in P. troglodytes (see GenBank. Acc. No. XP_01174052.1), M.
mulatta (see GenBank. Acc. No. NP_001041713.1), M. fascicularis
(see GenBank Acc. No. NP_001106117.1), P. anubis (see GenBank. Acc.
No. NP_001106117.1), M musculus (see GenBank Acc. No. NP_653142.1),
R. norvegicus (see GenBank Acc. No. NP_997486.1), B. Taurus (see
GenBank Acc. No. NP_001070870.1), C. lupus (see GenBank Acc. No.
XP_536141.2), Cereocebus torquatus atys (Red-crowned mangabey)
(Sooty mangabey) (see GenBank. Acc. No. DQ423376 mRNA. Translation:
ABD83656.1), Papio anubis (Olive baboon) (see GenBank Acc. No.
DQ423378 mRNA. Translation: ABD83658.1), and other species.
[0112] Fc receptors for use in media as described herein can
include an extracellular portion of an Fc gamma RIIIa polypeptide
that retains the ability to bind to a glycoform of an Fc-containing
polypeptide. For example, an Fc receptor can have a sequence at
least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the
extracellular domain of the sequence shown in SEQ ID NO:1, or a
sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical
to a portion thereof (e.g., a portion comprising amino acids
21-209, amino acids 17-208, amino acids 21-192, amino acids 21-169,
or amino acids 106-169 of SEQ ID NO:1). In various embodiments, an
Fc receptor has a sequence which differs from one of these portions
of an Fc gamma RIIIa sequence in at least 1 amino acid position,
but not more than 15 amino acid positions (e.g., the sequence
differs from amino acids 21-209 of SEQ ID NO:1 at 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid positions). In
various embodiments, an Fc receptor is glycosylated at a site
corresponding to N180 in SEQ ID NO:1. In various embodiments, an Fc
receptor lacks glycosylation at a site corresponding to N63 of SEQ
IIS NO:1 (e.g., due to substitution of an asparagines residue). In
various embodiments, an Fc receptor comprises amino acids 21-192 of
SEQ ID NO:1, wherein F176 is changed to V176. In various
embodiments, an Fc receptor comprises amino acids 21-192 of SEQ ID
NO:1, wherein N63 is substituted for another amino acid (e.g., to
glutamine, aspartic acid, or glutamic acid).
[0113] Fc gamma RIIIb (also known as CD16b or FCGR3B protein) is a
glycoprotein that shares sequence similarity with Fc gamma RIIIa.
Full length Fc gamma RIIIb has two Ig-like C2-type domains in its
extracellular region. Fc gamma RIIIb is naturally expressed as a
GPI-anchored form and a secreted form released by a proteolytic
cleavage. Fucosylation decreases the affinity of some antibodies
for Fc gamma RIIIb (Bruhns et al., Blood 113(16): 3716-3725,
2008).
[0114] An exemplary amino acid sequence of a full length human Fc
gamma Rift polypeptide is shown in SEQ ID NO:2 (see also Acc. No.
075015 of UniProt, on the world wide web at
uniprot.org/uniprot/O75015). The signal peptide is at about amino
acids 1-16. The extracellular domain is between amino acids 17-200.
Ig-like domains are found between amino acids 40-96 and 121-179.
Amino acids 201-233 may be removed in mature forms. N-linked
glycosylation may occur at residues 56, 63, 82, 92, 180, and 187.
Lipidation may occur at amino acid 200.
TABLE-US-00002 TABLE 2 Exemplary full length Fc gamma RIIIb amino
acid sequence 10 20 30 40 50 60 MWQLLLPTAL LLVSAGMRT EDLPKAVVFL
EFQWSVLEK DSVTLKCQGA YSPEDNSTQW 70 80 90 100 110 120 FHNESLISSQ
ASSYFIDAAT VNDSGEYRCQ TNLSTLSDPV QLEVHIGWLL LQAPRWVFKE 130 140 150
160 170 180 EDPIHLRCHS WKNTALHKVT YLQNGKDRKY FHHNSDFHIP KATLKDSGSY
FCRGLVGSKN 190 200 210 220 230 VSSETVNITI TQGLAVSTIS SFSPPGYQVS
FCLVMVLLFA VDTGLYFSVK TNI (SEQ. ID NO: 2)
[0115] Examples of natural amino acid allotypes include
polypeptides having the following amino acid changes in SEQ ID
NO:2: S36R, S65N, A78D, N82D, I106V. Other Fc gamma RIIIb allotypes
include NA1 and NA2.
[0116] Fc receptors for use in media as described herein can
include an extracellular portion of an Fc gamma RIIIb polypeptide
that retains the ability to bind to a glycoform of an Fc-containing
polypeptide. For example, an Fc receptor can have a sequence at
least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the
extracellular domain of the sequence shown in SEQ ID NO:2, or a
sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical
to a portion thereof (e.g., a portion comprising amino acids
20-208, amino acids 20-192, amino acids 21-169, or amino acids
106-169 of SEQ ID NO:2). In various embodiments, an Fc receptor has
a sequence which differs from one of these portions of an Fc gamma
RIIIb sequence in at least 1 amino acid position, but not more than
15 amino acid positions (e.g., the sequence differs from amino
acids 20-208 of SEQ ID NO:2 at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, or 15 amino acid positions). In various embodiments, an
Fc receptor is. glycosylated at a site corresponding to N180 in SEQ
ID NO:2. In various embodiments, an Fc receptor lacks glycosylation
at a site corresponding to N63 of SEQ ID NO:2 (e.g., due to
substitution of an asparagines residue). In one embodiment, an Fc
receptor comprises amino acids 20-192 of SEQ ID NO:2. In various
embodiments, an Fc receptor comprises amino acids 20-192 of SEQ ID
NO:2, wherein N63 is substituted for another amino acid (e.g., to
glutamine, aspartic acid, or glutamic acid).
[0117] Fc gamma RIV is another Fc receptor suitable for use in some
embodiments of the instant invention and is described in the review
article, Nimmerjahn, F and Ravetch, J V Immunity 24:19-28 (2006).
Examples of Fc gamma RIV protein sequences include those for mouse
(accession number, Q8R2R4), rat (accession number, Q6XPU4), and
macaque (accession number, Q8SPW2). As described in Nimmerjahn and
Ravetch, Fc gamma RIV requires gamma chain for its surface
expression, Fc gamma RIV is highly expressed on neutrophils,
monocytes, macrophages, and dendritic cells. It can be upregulated
by inflammatory stimuli such as LPS and TH-1 cytokines such as
IFN-gamma end can be downregulated by TH-2 cytokines such as IL-4
and IL-10 or TGF-beta. See Nimmerjahn, F and Revetch, JV Immunity
24:19-28 (2006). Mouse Fc gamma RIV has similar affinity properties
to human Fc gamma RIIIA in that the K.sub.D for afucosylated IgG is
approximately ten times smaller than the K.sub.D for fucosylated
antibodies. See Herbst, R et al. J. Pharmacol. Exp. Ther.
335(1):213-222 (2010). Thus, the Fc gamma RIV receptor is an
example of a suitable Fc receptor for enrichment of afucosylated
species when used as an affinity ligand according to the methods of
some embodiments of the instant invention.
[0118] Fc receptors for use in methods herein can be expressed in
mammalian cell or other types of cells (e.g., eukaryotic cells such
as yeast cells or insect cells; see exemplary host cells for
production of proteins above) that produce the receptor in a form
that retains an ability to bind to an Fc and to preferentially bind
a glycoform. In certain embodiments, the Fc receptor is produced in
prokaryotic cells, such as bacterial cells, such as E. coli. In
embodiments where the Fc receptor is produced in a prokaryotic cell
such as E. coil, the Fc receptor may be modified such that it
retains an ability to bind to an Fc and to preferentially bind a
glycoform but the Fc receptor itself is not glycosylated. Fc
receptors can also be obtained from commercial sources such as
R&D Systems (Minneapolis, Minn.) and Sino Biologicals, Inc.
(Beijing, China). Fc receptors can be expressed and/or modified
with a moiety to facilitate purification and/or coupling to a
medium. In some embodiments, a receptor has a peptide tag, e.g., a
polyhistidine tag or HA tag. In some embodiments, a receptor has
one or more amino acid residues at a terminus to facilitate
coupling. In some embodiments, a receptor has a recognition motif
for an enzyme that mediates coupling, such as an LPXTG (SEQ ID
NO:3) motif that is recognized by Staphylococcal Sortase A. In some
embodiments, a terminus (e.g., the N-terminus) includes a lysine or
cysteine. An amino acid linker that separates the terminal residue
from the Fc receptor can be included. A linker can be, for example,
1-20 amino acids long, e.g., 1, 2, 3, 5, 7, 9, 10, 12, 15, or 20
amino acids long. In various embodiments, a linker includes 3, 4,
or 5 adjacent glycine residues optionally followed by, or preceded
by, a serine residue.
[0119] A receptor may include at its N-terminus a KGGG (SEQ ID
NO:4) or CGGG (SEQ ID NO:5) motif.
[0120] In one example, an Fc receptor includes the following amino
acid sequence:
TABLE-US-00003 (SEQ ID NO: 6)
KGGGEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESL
ISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLELQAPRW
VFKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFYIPKATLKD
SGSYFCRGLVGSKNVSSETVNITITQGLAVSTISSFFFPGYQV
[0121] In another example, an Fc receptor includes the following
amino acid sequence:
TABLE-US-00004 (SEQ ID NO: 7)
CGGGEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESL
ISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRW
VFKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFYIPKATLKD
SGSYFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQV.
[0122] Methods and compositions of the present disclosure encompass
polypeptides and nucleic acids having the sequences specified, or
sequences substantially identical or similar thereto, e.g.,
sequences at least 85%, 90%, 95% identical or higher to the
sequence specified. In the context of an amino add sequence, the
term "substantially identical" is used herein to refer to a first
amino add that contains a sufficient or minimum number of amino add
residues that are i) identical to, or ii) conservative
substitutions of aligned amino add residues in a second amino add
sequence such that the first and second amino add sequences can
have a common structural domain and/or common functional activity.
For example, amino acid sequences that contain a common structural
domain having at least about 85%, 90%. 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98% or 99% identity to the sequence specified are termed
substantially identical.
[0123] In the context of nucleotide sequence, the term
"substantially identical" is used herein to refer to a first
nucleic add sequence that contains a sufficient or minimum number
of nucleotides that are identical to aligned nucleotides in a
second nucleic add sequence such that the first and second
nucleotide sequences encode a polypeptide having common functional
activity, or encode a common structural polypeptide domain or a
common functional polypeptide activity. For example, nucleotide
sequences having at least about 85%, 90%. 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98% or 99% identity to the sequence specified are termed
substantially identical.
[0124] Calculations of homology or sequence identity between
sequences (the terms are used interchangeably herein) are performed
as follows.
[0125] To determine the percent identity of two amino add
sequences, or of two nucleic add sequences, the sequences are
aligned for optimal comparison purposes (e.g., gaps can be
introduced in one or both of a first and a second amino add or
nucleic add sequence for optimal alignment and non-homologous
sequences can be disregarded for comparison purposes). In a
preferred embodiment, the length of a reference sequence aligned
for comparison purposes is at least 30%, preferably at least 40%,
more preferably at least 50%, 60%, and even more preferably at
least 70%, 80%, 90%, 100% of the length of the reference sequence.
The amino add residues or nucleotides at corresponding amino add
positions or nucleotide positions are then compared. When a
position in the first sequence is occupied by the same amino add
residue or nucleotide as the corresponding position in the second
sequence, then the molecules are identical at that position (as
used herein amino add or nucleic add "identity" is equivalent to
amino add or nucleic add "homology").
[0126] The percent identity between the two sequences is a function
of the number of identical positions shared by the sequences,
taking into account the number of gaps, and the length of each gap,
which need to be introduced for optimal alignment of the two
sequences.
[0127] The comparison of sequences and determination of percent
identity between two sequences can be accomplished using a
mathematical algorithm. In a preferred embodiment, the percent
identity between two amino add sequences is determined using the
Needleman and Wunsch (1970) J. Mol. Biol. 48:444-453) algorithm
which has been incorporated into the GAP program in the GCG
software package (available at http://www.gcg.com), using either a
Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14,
12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 8. In
yet another preferred embodiment, the percent identity between two
nucleotide sequences is determined using the GAP program in the GCG
software package (available at http://www.gcg.com), using a
NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and
a length weight of 1, 2, 3, 4, 5, or 6. A particularly preferred
set of parameters (and the one that should be used unless otherwise
specified) are a Blossum 62 scoring matrix with a gap penalty of
12, a gap extend penalty of 4, and a frameshift gap penalty of
5.
[0128] The percent identity between two amino acid or nucleotide
sequences can be determined using the algorithm of E. Meyers and W.
Miller ((1989) CABIOS, 4:11-17) which has been incorporated into
the ALIGN program (version 2.0), using a PAM120 weight residue
table, a gap length penalty of 12 and a gap penalty of 4.
[0129] The nucleic acid and protein sequences described herein can
be used as a "query sequence" to perform a search against public
databases to, for example, identify other family members or related
sequences. Such searches can be performed using the NBLAST and
XBLAST programs (version 2.0) of Altschul, et al. (1980) J. Mol.
Biol. 215:403-10. BLAST nucleotide searches can be performed with
the NBLAST program, score=100, wordlength=12 to obtain nucleotide
sequences homologous to nucleic add molecules of some embodiments
of the invention. BLAST protein searches can be performed with the
XBLAST program, score=50, wordlength=3 to obtain amino add
sequences homologous to protein molecules of some embodiments of
the invention. To obtain gapped alignments for comparison purposes.
Gapped BLAST can be utilized as described in Altschul et al.,
(1997) Nucleic Acids Res. 25:3389-3402. When utilizing BLAST and
Gapped BLAST programs, the default parameters of the respective
programs (e.g., XBLAST and NBLAST) can be used. See
http://www.ncbi.nlm.nih.gov.
[0130] Nucleic acid molecules corresponding to natural allelic
variants, homologs, orthologs, or other related sequences (e.g.,
paralogs) of the sequences described herein can be isolated based
on their homology to the nucleic adds encoding the amino add
sequences disclosed herein, for example by performing standard or
stringent hybridization reactions using all or a portion of the
known sequences as probes. Such methods for nucleic add
hybridization and cloning are well known in the art.
[0131] The homologs of the peptides as provided herein typically
have structural similarity with such peptides. A homolog of a
polypeptide includes one or more conservative amino add
substitutions, which may be selected from the same or different
members of the class to which the amino add belongs.
[0132] In one embodiment, the sequences may also have deletions,
insertions or substitutions of amino add residues which produce a
silent change and result in a functionally equivalent substance.
Deliberate amino add substitutions may be made on the basis of
similarity in polarity, charge, solubility, hydrophobicity,
hydrophilicity, and/or the amphipathic nature of the residues as
long as the secondary binding activity of the substance is
retained. For example, negatively charged amino adds include
aspartic add and glutamic acid; positively charged amino acids
include lysine end arginine; and amino acids with uncharged polar
head groups having similar hydrophilicity values include leucine,
isoleucine, valine, glycine, alanine, asparagine, glutamine,
serine, threonine, phenylalanine, and tyrosine.
[0133] Some embodiments of the present invention also encompass
conservative substitution (substitution and replacement are both
used herein to mean the interchange of an existing amino acid
residue with an alternative residue) that may occur e.g.,
like-for-like substitution such as basic for basic, acidic for
acidic, polar for polar, etc. Non-conservative substitution may
also occur e.g., from one class of residue to another or
alternatively involving the inclusion of unnatural amino acids such
as ornithine (hereinafter referred to as Z), diaminobutyric acid
ornithine (hereinafter referred to as B), norleucine ornithine
(hereinafter referred to as O), pyridylalanine, thienylalanine,
naphthylalanine and phenylglycine. Conservative substitutions that
may be made are, for example, within the groups of basic amino
acids (Arginine, Lysine and Histidine), acidic amino acids
(glutamic acid and aspartic acid), aliphatic amino acids (Alanine,
Valine, Leucine, isoleucine), polar amino acids (Glutamine,
Asparagine, Serine, Threonine), aromatic amino acids
(Phenylalanine, Tryptophan and Tyrosine), hydroxyl amino acids
(Serine, Threonine), large amino acids (Phenylalanine and
Tryptophan) and small amino acids (Glycine, Alanine).
[0134] Producing Fc Receptor Media
[0135] Fc receptors can be coupled to a medium (e.g., a solid
support) by any of a variety of techniques. In some embodiments, a
receptor is coupled to a medium by one or more covalent bonds,
e.g., formed by reaction of functional groups on the receptor or
the medium. Examples of functional groups include hydroxyl groups,
amine groups, thiol groups, thiopropyl groups, carbonyl groups,
N-hydroxysuccinimide (NHS) esters, epoxides (e.g.,
epichlorohydrin), carbonyldiimidazole (CDI) activated esters,
cyanogens (cyanogens bromide (CNBr)), N,N-disuccinimidylcarbonate
(DSC), or aldehydes. In some embodiments, an Fc receptor is coupled
via a disulfide bond. In some embodiments, an Fc receptor is
coupled to a medium using metal chelation. In some embodiments, an
Fc receptor is coupled using a histidine tag. Other compounds
useful for coupling include tosyl chloride, tresyl chloride, ECH
linkage (e.g., using ECH-Lysine Sepharose 4 Fast Flow, GE
Healthcare) or EAH linkage (e.g., using EAH-Sepharose.RTM. 4B, GE
Healthcare). In certain embodiments, an Fc receptor is coupled via
its C-terminus to a medium (e.g., a solid support). In certain
embodiments, an Fc receptor is coupled via its N-terminus to a
medium (e.g., a solid support).
[0136] A receptor can be directly coupled to a medium or indirectly
coupled via a linker. Any of a variety of linkers are Suitable for
coupling a medium to a receptor. In some embodiments, a linker
includes a chain of carbon, oxygen and nitrogen atoms, e.g., a
linear, branched, or cyclic chain, e.g., a chain having 1-30 carbon
atoms.
[0137] Methods of coupling polypeptides to media are described,
e.g., in WO 90/09237; Hermanson et al., Immobilized Affinity Ligand
Techniques, Academic Press, 1992; U.S. Pat. Nos. 5,260,373;
5,874,165; 3,932,557; 4,772,635; 4,210,723; 5,250,613; 5,543,054;
6,399,750; EP 1352957 A1, and WO 2004/074471.
[0138] In some embodiments, a receptor is coupled to a medium
comprising agarose having a functional group, such as CNBr or NHS,
and optionally, a linker. In some embodiments, a receptor is
coupled via primary amino groups to NHS-activated Sepharose.RTM. 4
Fast Row. In one exemplary method, a receptor is coupled by washing
the NHS-activated Sepharose.RTM. 4 Fast Row medium with 1 mM HCl,
mixing the medium with the receptor, allowing coupling to occur
(e.g., by incubation at room temperature for 1-4 hours), washing
the medium with 100 mM Iris to quench non-reacted groups.
[0139] Enzyme-mediated coupling can be used to link a receptor to a
medium (see, e.g., Chan et al., PLoS ONE 2(11):e1164, 2007). For
example, a receptor can be designed to Include a recognition motif
for a transpeptidase such as Staphylococcal Sortase A. Recognition
and cleavage at an LPXTG motif by Sortase A generates an
acyl-enzyme intermediate that is reactive with an available glycine
or aminomethylene. If a receptor having a recognition motif is
cleaved by Sortase A, it becomes linked to a medium having reactive
sites. In one example, beads modified to include one, two, three,
or four glycine residues are linked to receptors having a
C-terminal LPXTG motif by Sortase A-mediated ligation.
[0140] Suitable types of media include beads, membranes, matrices,
porous media, gels, plates, columns, and monoliths. A medium can be
comprised of a material such as agarose, cellulose, or dextran,
ceramic, metal, glass, nylon, Teflon, nylon, polycarbonate,
polyacrylamide, polystyrene, polypropylene, polyether sulfone,
polyamide, polytetrafluoroethylene, polysulfone, polyester,
polyvinylidene fluoride, a fluorocarbon, e.g.
poly(tetrafluoroethylene-co-perfluoro(alkyl vinyl ether)),
polyethylene, polyacrylate, or poly(azolactone).
[0141] An Fc receptor medium is coupled to a concentration of Fc
receptor that is sufficient for binding to an Fc-containing
polypeptide. In some embodiments, a medium has 0.01-15 mg/ml of an
Fc receptor (e.g., 0.1-15 mg/ml, or 1-15 mg/ml). In some
embodiments, a medium has between 2-10 mg/ml (e.g., between 3-7.5
mg/ml) of an Fc receptor.
Separation Methods
[0142] To separate polypeptide glycoforms, a load fluid is
contacted with an Fc receptor medium as described herein under
conditions in which polypeptide binds to the medium. The medium is
optionally washed, and is contacted with a solution that elutes
polypeptide that bound to the medium. In some embodiments,
polypeptide that elutes from the medium is recovered. In some
embodiments, polypeptide that flows through the medium (e.g.,
polypeptide that is not bound by the medium) is recovered.
[0143] A load fluid can be, for example, a cell culture medium
containing a polypeptide of interest (e.g., medium comprising
cells, or medium from which cells are removed), a cell extract,
serum, ascites, a purified or partially purified preparation from
cell culture, extract, serum, etc., or a formulated drug product or
drug substance. In a particular embodiment, the load fluid
comprises serum IgG. In some embodiments, a load fluid is an eluate
of a protein A column. A load fluid may contain a polypeptide at a
concentration between 0.001-500 mg/mL (e.g., 0.001-100 mg/ml,
0.001-30 mg/ml, 0.001-10 mg/ml). A polypeptide can be contacted
with a medium at a concentration of 0.1 mg polypeptide per 25,000
ml of medium.
[0144] Before contacting a medium with a load fluid, parameters
such as pH, ionic strength, and temperature may be adjusted if
necessary. A medium may be equilibrated by washing it with a
solution (e.g., a buffer for adjusting pH, ionic strength, etc., or
for the introduction of a detergent) to bring characteristics that
allow or facilitate binding and/or separation of a polypeptide.
[0145] In some embodiments, an Fc receptor medium is flushed and
equilibrated with one or more solutions prior to contact with a
polypeptide. Such solutions can include, for example, a buffer
(e.g., Tris, MES, HEPES, histidine, or phosphate, e.g., between
1-500 mM, 25-100 rnM, or 50 mM), and/or salt (e.g., NaCl,
NaPO.sub.4, sodium acetate, or CaCl.sub.2, e.g., between 0-2M, or
5-250 mM). The pH of an equilibration solution is generally between
3.5-10 (e.g., between pH 6.0-8.0). In some embodiments, an
equilibration buffer contains 10 mM to about 50 mM of Tris or MES
and 10-200 mM of a salt (e.g., 150 mM NaCl or 20 mM
CaCl.sub.2).
[0146] After contacting an Fc receptor medium with a load fluid,
the bound medium can be washed. Wash solutions can include a buffer
(e.g., Tris, MES, HEPES, phosphate, or histidine, e.g., between 1
and 500 mM), and/or salt (e.g., NaCl, CaCl.sub.2 NaPO.sub.4, or
sodium acetate, e.g., between 0 and 2 M), and/or an additive (e.g.
guanidine, urea, sucrose, arginine, or an arginine derivative),
and/or a solvent (e.g., ethanol, acetonitrile, or polyethylene
glycol). Wash solutions generally have a pH between 3.5 and 10
(e.g., a pH between 4.5-8.0).
[0147] In some embodiments, a medium is washed with the same
solution as used to equilibrate the medium. In some embodiments, a
medium is washed with 1 mM HEPES.
[0148] Polypeptides can be eluted from an Fc receptor medium using
a step or gradient change in pH, salt type, salt concentration,
solvent type, solvent concentration, displacer type, displacer
concentration, or a combination thereof. Proteins containing Fc
regions, other proteins, small polypeptides with homology to Fc
regions, detergents, hydrophobic solutes, polyelectrolytes, amino
acids, antibiotics, sugars, dextrans, ficolls, dendritic polymers
etc could be used as displacers. In general, to elute a polypeptide
from an Fc receptor medium, the medium is contacted with an elution
buffer. In some embodiments, an elution buffer contains of a salt
(e.g., NaCl or CaCl.sub.2, e.g., 0-2M, e.g., 10-100 mM). In some
embodiments, an elution buffer may contain glycine, acetic acid, or
citric acid (e.g., 20 mM-250 mM, e.g., 150 mM). An elution buffer
may also contain a buffer (e.g., HEPES, e.g., 10-100 mM). An
elution buffer may also contain acetic acid (e.g., 20 mM to about
50 mM), an additive (e.g. guanidine, urea, or sucrose), and/or a
solvent (e.g., ethanol, acetonitrile, polyethylene glycol, e.g.,
1-10% solvent, e.g., 5% solvent). The pH of the elution buffer may
range from about 2.0 to about 4.0. In some embodiments, pH can be
changed (e.g., gradually) to produce a gradient elution (e.g., a
gradient elution from pH 5.0 to pH 3.0). In one embodiment, the pH
of the elution buffer is about 3.0. An eluate can be neutralized,
e.g., by adjusting pH to 6.0-8.0 (in cases in which low pH is used
for elution) after recovery from the medium.
[0149] A medium may optionally be cleaned, i.e., stripped and
regenerated, after elution of a polypeptide. This can be 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.
[0150] Solution components may be adjusted according to the
knowledge of the person of ordinary skill in the art. Sample
solution composition ranges are provided in the Examples below. Not
all of the solutions or steps are necessary, but are provided for
illustration.
[0151] A separation using an Fc receptor medium may be performed
alone or in combination with other techniques. In some embodiments,
one or more processes are used to prepare load fluid, e.g., to
reduce the load challenge of contaminants or impurities. In some
embodiments, one or more processes are used to process an eluate or
flow-through of an Fc receptor medium.
[0152] Purification/separation techniques that can be practiced in
combination with Fc receptor methods described herein include depth
filtration, diafiltration, ultrafiltration, viral removal
filtration, protein A chromatography, protein G chromatography,
cation exchange chromatography, anion exchange chromatography, size
exclusion chromatography, hydrophobic interaction chromatography,
immobilized metal affinity chromatography, hydroxyapatite
chromatography, lectin chromatography, biomimetic affinity
chromatography, mixed mode chromatography, and combinations
thereof. Techniques herein can be practiced in column, membrane,
and/or expanded bed adsorption formats. In certain embodiments, one
or more chromatographic techniques are operated in a weak
partitioning mode (see U.S. Pub. No. 2007/0060741 and Kelley B D,
Tobler S A, Brown P, Coffman J L, Godavarti R, Iskra T, Switzer M,
Vunnum S. Biotechnol Bioeng. 2008 Oct. 15; 101(3): 553-66).
[0153] Commercially available protein A chromatography columns
include, for example, PROSEP-A.TM. (Millipore, U.K.), Protein A
Sepharose FAST FLOW.TM. (GE Healthcare, Piscataway, N.J.),
TOYOPEARL.TM. 650M Protein A (TosoHass Co., Philadelphia, Pa.), and
MabSelect.TM. columns (GE Healthcare, Piscataway, N.J.).
[0154] Anionic exchange resins that can be used include resins
having substituents such as diethylaminoethyl (DEAF),
trimethyalaminoethyl (TMAE), quaternary aminoethyl (QAE) and
quaternary amine (O) groups.
[0155] Cationic exchange resins that can be used include resins
having substituents such as carboxymethyl (CM), sulfoethyl (SE),
sulfopropyl (SP), phosphate (P) and sulfonate (S).
[0156] In some embodiments, a cellulosic ion exchange resin (e.g.,
DE23, DE32, DE52, CM-23, CM-32 or CM-52, available from Whatman
Ltd. Maidstone, Kent, U.K) is used. Sephadex-based and cross-linked
ion exchangers used for purification include, for example, DEAE-,
QAE-, CM-, and SP-Sephadex, and DEAE-, Q-, CM- and S-Sepharose, and
Sepharose (Amersham Biosciences, Piscataway, N.J.). DEAE and CM
derivatized ethylene glycol-methacrylate copolymer such as
TOYOPEARL.TM. DEAE-650S or M and TOYOPEARL.TM. CM-650S or M are
available from Toso Haas Co., Philadelphia, Pa.
[0157] In some embodiments, a hydrophobic interaction
chromatography (HIC) resin is used for purification. HIC separates
molecules based on hydrophobicity. Generally, sample molecules in a
high salt buffer are loaded onto the HIC resin. Salt in the buffer
interacts with water molecules to reduce the solution of the
molecules in solution, thereby exposing hydrophobic regions in the
sample molecules which are consequently absorbed by the HIC medium.
The more hydrophobic the molecule, the less salt needed to promote
binding. Binding interactions between the product molecules and a
HIC medium thus depend on conditions such as pH, ionic strength,
and salt concentrations of the medium. Commercially available HIC
resins that can be used include resins comprising a base matrix
(e.g., cross-linked agarose or synthetic copolymer material) to
which hydrophobic ligands (e.g., alkyl or aryl groups) are coupled.
Examples include Phenyl SEPHAROSE.TM., 6 FAST FLO.TM. (Pharmacia
LKB Biotechnology, AB, Sweden); Phenyl SEPHAROSE.TM. High
Performance (Pharmacia LKB Biotechnology, AB, Sweden); Octyl
SEPHAROSE.TM. High Performance (Pharmacia LKB Biotechnology, AB,
Sweden); Fractogel.TM. EMD Propyl or FRACTOGEL.TM. EMD Phenyl (E.
Merck, Germany); MACRO-PREP.TM. Methyl or MACRO-PREP.TM. t-Butyl
Supports (Bio-Rad, CA); WP HI-Propyl (C.sub.3).TM. (J. T. Baker,
N.J.); and TOYOPEARL.TM. ether, phenyl or butyl (TosoHaas, Pa.).
HIC can be performed in a weak partitioning mode.
[0158] In some embodiments hydroxyapatite chromatography is used
for purification. Hydroxyapatite chromatography utilizes an
insoluble hydroxylated calcium phosphate of the formula
[Ca.sub.10(PO.sub.4).sub.6(OH).sub.2], as both the matrix and the
ligand. Functional groups consist of pairs of positively charged
calcium Ions (C-sites) and clusters of negatively charged phosphate
groups (P-sites). Binding interactions between a product and a
hydroxyapatite medium depend on conditions such as the pH, ionic
strength, and excipient concentrations, such as phosphate
concentrations, calcium concentrations, arginine concentrations,
glycine concentrations, and HEPES concentrations of the medium.
Various hydroxyapatite chromatographic resins are available
commercially. Hydroxyapatite chromatography can be performed in a
weak partitioning mode.
[0159] In some embodiments, an immobilized metal affinity
chromatography (IMAC) resin is used for purification. IMAC is based
on the interaction between chelated transition metal ions
immobilized on a resin and imidazole side chains of histidine
residues on a tagged product of interest. Separation of molecules
occurs as a result of competition between the tagged product of
interest and counterligands for metal groups on the IMAC resin.
Binding interactions between a product and metal-charged IMAC
medium depend on conditions such as counterligand levels, such as
imidazole concentrations, and ionic strength of the medium. Various
IMAC resins are available commercially. IMAC can be performed in a
weak partitioning mode.
[0160] In some embodiments, lectin chromatography Is used for
purification (see, e.g., Tojo et al., Biol. Pharm. Bull.
32(9):1604-1608, 2009; and Shinkawa et al., supra).
Characterization of Polypeptides
[0161] Polypeptides separated using Fc receptor media described
herein can be analyzed for any type of characteristic, such as
glycan quantity and/or structure, stability (e.g., half life, shelf
life), toxicity, and biological activity. Polypeptides in any stage
of a separation process, e.g., eluates and/or flow through
portions, may be analyzed. Evaluation of glycoforms may be, for
example, by way of comparison of load fluid and eluate, or by way
of comparison of load fluid and flow through, or by way of
comparison of eluate to a reference sample.
[0162] Any available technique for detecting and characterizing
glycans can be applied. For example, glycan structure can be
analyzed by mass spectrometry (MS), chromatographic methods,
electrophoretic methods, nuclear magnetic resonance (NMR), and
combinations thereof. Chromatographic methods can include high
performance liquid chromatography, liquid chromatography, ultra
performance liquid chromatography, thin layer chromatography,
and/or amide column chromatography. MS can include tandem MS,
LC-MS, LC-MS/MS, matrix assisted laser desorption ionization MS
(MALDI-MS), and/or electron transfer dissociation MS (ETD-MS).
Electrophoretic methods can include capillary electrophoresis, gel
electrophoresis (e.g., with or without Western blotting to detect
glycan and/or polypeptide structures). NMR can include 1D-NMR,
2D-NMR, correlation spectroscopy NMR, heteronuclear single quantum
coherence NMR, heteronuclear multiple quantum coherence NMR,
rotational nuclear overhauser effect spectroscopy NMR, and/or
nuclear overhauser effect spectroscopy NMR.
[0163] Various techniques for analyzing glycans have been
described. See, e.g., Bigge et al. (Anal. Biochem. 230:229-238,
1995), which describes conditions for labelling fucosylated and
afucosylated glycans with 2-amino benzamide (2-AB) and
2-anthranilic acid (2-AA) for detection by chromatographic and mass
spectrophotometric means. See also Anamula et al., Anal. Biochem.
350(1):1-23, 2006; Townsend, R. R. Carbohydrate Analysis" High
Performance Liquid Chromatography and Capillary Electrophoresis.,
Ed. Z. El Rassl, pp 181-209, 1996; and Ruhaak et al., Anal Bioanal
Chem. 397(8):3457-81, 2010.
[0164] Polypeptides expressed in cells exhibit heterogeneous
glycosylation. Molecules of a given polypeptide may vary in the
number and type of a given sugar residue. The present disclosure
provides media and methods for separating glycoforms, e.g., to
enrich for polypeptides having increased or decreased levels of
particular residues. In some embodiments, a glycoform has "reduced"
levels of a particular sugar (e.g., fucose), if the number of
fucose residues are fewer than the number of residues on a
reference glycoform. In some embodiments, a reference glycoform is
a glycoform having an average or maximal number of residues of the
particular sugar observed on the polypeptide expressed in a given
cell type. For example, if an antibody polypeptide expressed in a
CHO cell has a maximum of two core fucose residues, a glycoform
having 0 or 1 core fucose has "reduced" levels.
[0165] Likewise, a glycoform has "increased" levels of a particular
sugar if the number of sugars are higher than the number of
residues on a reference glycoform. In some embodiments, a reference
glycoform is a glycoform having an average or minimal number of
residues of the particular sugar.
[0166] Detection of glycoforms for evaluation of separation can be
performed by any available means, and can involve analysis glycans
of any of load fluid, eluate, flow through, washes, or combinations
thereof.
[0167] In some embodiments, the percentage of a glycoform in an
eluate or flow through of an Fc receptor medium is altered (e.g.,
increased or decreased) by at least 20%, 50%, 100%, 2 fold, 5 fold,
10 fold, 20 fold, 50 fold, or 100 fold, relative to the load fluid.
In some embodiments, the proportion of a glycoform in an eluate or
flow through of an Fc receptor medium is increased to at least 10%,
20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the total.
For example, in some embodiments, separation of a recombinant
antibody sample using an Fc gamma RIII receptor medium described
herein results in an eluate having at least 40% afucosylated
material, whereas a sample that has not been subjected to
separation (e.g., load fluid) may typically have 5-7% afucosylated
mater (depending on the cell system for expression and other
factors).
[0168] Binding of polypeptides separated according to methods
provided herein can be analyzed. In some embodiments, binding to a
target protein (e.g., antibody binding to antigen) is analyzed. In
some embodiments, binding to an Fc receptor is analyzed. Methods
for evaluating binding interactions are known and include, for
example, ELISA, BIACore.TM. biosensor analysis, fluorescent
resonance energy transfer (FRET), and others.
[0169] Any available method for evaluating a biological activity of
a polypeptide may be employed. In some embodiments, a
glycoform-specific biological activity is evaluated. ADCC can be
analyzed by known methods (see, e.g., Shinkawa et al., supra).
Changes in relative proportion of one or more polypeptide
glycoforms as a result of separation using a medium herein may
result in a change in a biological activity, such as an increase in
ADCC, e.g., by at least 20%, 50%, 100%, 2 fold, 5 fold, 10 fold, 20
fold, 50 fold, or 100 fold, relative to activity of polypeptides in
a load fluid or a reference sample.
Products and Uses
[0170] Methods and compositions herein for separation of
polypeptides have numerous applications. Methods permit preparation
of polypeptides having a desired glycoform profile. In some
embodiments, methods are used to enrich for a glycoform of interest
(e.g., a glycoform having an enhanced biological activity). In some
embodiments, methods are used to normalize glycoform profiles
(e.g., between batches, or between molecules produced by different
processes). In some embodiments, methods are used to provide
glycoform pools that can be evaluated for any desired
characteristic, such as biological activity. The ability to control
glycoform profiles allows greater control and flexibility in
manufacturing processes. The present technology is an additional
tool for achieving batch-to-batch consistency for a given product.
It is also useful, e.g., for the comparison of products made by
different processes and/or different manufacturers. Furthermore, a
manufacturer can use the technology to match a quality attribute of
another manufacturer's product, e.g., in the development of a
biosimilar product, or to alter (e.g., increase or enhance) a
quality attribute in a follow-on product.
[0171] The present disclosure also relates to a product prepared
according to a method described herein. In some cases, it will be
desirable to further isolate and/or purify polypeptides isolated
according to the present disclosure and formulate them for
pharmaceutical use according to standard method. See for example
Protein Purification Principles and Practice 2nd Edition,
Springer-Verlag, New York, 1987; Higgins, S. J. and Hames, B. D,
(eds.), and Deutscher, M. P., Simon, M. I., Abelson, J. N. (eds.),
Guide to Protein Purification: Methods in Enzymology (Methods in
Enzymology Series, Vol 182), Academic Press, 1997, incorporated
herein by reference. One of ordinary skill in the art will
appreciate that the exact techniques used will vary depending on
the character of the polypeptide. Polypeptides having pharmacologic
activity will be useful in the preparation of pharmaceuticals.
These may be administered to a subject or may first be formulated
for delivery by any available route including, but not limited to
parenteral (e.g., intravenous), intradermal, subcutaneous, oral,
nasal, bronchial, opthalmic, transdermal (topical), transmucosal,
rectal, and vaginal.
[0172] A pharmaceutical composition of the product is formulated to
be compatible with its intended route of administration according
to methods known in the art, see for example, Remington: The
Science & Practice of Pharmacy, 19th ed., Williams &
Williams, (1995), and the Physician's Desk Reference, 52nd ed.,
Medical Economics, Montvale, N.J. (1988). In some embodiments, the
product is formulated using sterile water (e.g., SWFI), buffered
saline (e.g., phosphate buffered saline), polyol (e.g., glycerol,
propylene glycol, liquid polyethylene glycol), or suitable mixtures
thereof.
EXEMPLIFICATION
Example 1
[0173] This example describes preparation of an Fc gamma RIIIa
receptor medium and use of the medium with mAb1, a humanized IgG1
monoclonal antibody produced in CHO cells.
[0174] An extracellular domain of an Fc-gammaRIIIa-176V (valine)
allotype receptor having the following amino acid sequence was
transiently expressed in HEK-293 cells:
TABLE-US-00005 (SEQ ID NO: 8)
EDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLISSQ
ASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKE
EDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFYIPKATLKDSGSY
FCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQVHHHHHHHHHH
[0175] The receptor was expressed with a signal peptide that was
cleaved during cellular processing. A 10-his tag was expressed at
the C-terminus for purification, which was performed using a nickel
affinity column
[0176] The Fc-gamma receptor (1.25 ml in PBS at 0.85 mg/ml) was
added to 0.73 ml of activated NHS Sepharose.TM. 4 Fast Flow resin
(17-0906-01, GE) and allowed to react for 80 minutes at room
temperature. The reaction was quenched with 100 mM TRIS pH 8.3. The
coupling efficiency, determined using size exclusion
chromatography, was 100%. The resin was packed to 0.63 mL in a 0.5
cm internal diameter (ID) column (GE Healthcare) and flushed with
10 mls of 100 mM Tris pH 8.3 followed by 10 mls of 100 mM Acetate
pH 4.0 with 500 mM NaCl. This flushing was repeated twice and the
column was stored in 16% ethanol, 150 mM NaCl 50 mM Tris pH
7.7.
[0177] The column was flushed with 50 mM Tris 150 mM NaCl pH 7.2
and loaded at 0.25 ml/min (2.5 min residence time) with 50 ml of
0.34 mg/ml mAb1 in PBS buffer (27.0 mg protein/ml resin). The mAb1
material was prepared with a two column purification process in
which a protein A chromatography column and an anion exchange
chromatography column were used. After the mAb1 material was loaded
on the Fc receptor column, the column was flushed with 12 ml of PBS
at 0.5 ml/min and eluted with 50 mM HEPES pH 3.0 at 0.5 ml/min,
mAb1 was collected (0.1 mg in 7.5 ml), as determined by absorbance
at 280 nm and size exclusion chromatography.
[0178] To facilitate glycan analysis, the eluted mAb1 sample was
concentrated to 0.45 mg/ml in 115 microliters using 16.5
microliters of Protein A resin (GE MabSelect, 17-5199-03). The
Protein A concentration was performed in batch mode using a 96 well
plate, a PBS wash buffer, a 150 mM glycine elution buffer (pH 3.0),
and a MES neutralization buffer (pH 6.8).
[0179] To determine the percentage of non-fucosylated mAb1,
approximately 35 .mu.g was digested with PNGase F at 37.degree. C.
for 16 hours to release Fc-oligosaccharides. The released N-linked
oligosaccharides were labeled with 2-amino benzamide (2-AB) for 3
hours at 65.degree. C. Residual 2-AB was removed using acetone
precipitation.
Oligosaccharide analysis was performed using an Xbridge Amide
HILIC-HPLC at 45.degree. C. over a linear ammonium formate gradient
with fluorescence detection. The starting mAb1 material had 7%
non-fucosylated Fc. After enrichment using the Fc receptor column,
the mAb1 material had 61% non-fucosylated IgG Fc. FIG. 1 shows an
example of the typical oligosaccharide profile of mAb1 (top panel)
and the profile of mAb1 after enrichment using the Fc receptor
column (bottom panel). All peaks that have labels ending in `F` are
fucosylated and all peaks have labels that do not end in `F` in
FIG. 1 are afucosylated.
Example 2
[0180] The packed Fc receptor column described in Example 1 was
used to enrich the non-fucosylated glycoform of a second monoclonal
antibody, mAb2, which is a humanized IgG1 produced in CHO cells.
The mAb2 material was obtained from a ultrafiltration pool that
results from a purification process using the following: a protein
A chromatography column, an anion exchange chromatography column, a
virus-retaining filter (e.g., a Planova 20 filter (Asahi Kasai
Corporation, Tokyo, Japan)), and an ultrafilter.
[0181] The Fc receptor column was flushed with equilibration buffer
(50 mM MES with 20 mM CaCl.sub.2 at pH 6.5) and loaded at 0.25
ml/min (2.5 min residence time) with 50 ml of 1.80 mg/ml mAb2 in
equilibration buffer (142.9 mg protein/ml resin). The column was
then flushed with 12 ml of equilibration buffer at 0.5 ml/min and
eluted with 150 mM Glycine at pH 3.4 at as ml/min. mAb2 was
collected (0.12 mg in 9.25 ml), as determined by absorbance at 280
nm. The peak was brought to pH 6.6 with pH 8.8 1 M MES
neutralization buffer. To facilitate glycan analysis, the mAb2
sample was concentrated to 0.87 mg/ml in 100 microliters using an
Amicon Ultra-4 10,000 kD MWCO device ((Millipore UFC801024).
[0182] The percentage of non-fucosylated mAb2 was determined using
the method described in Example 1. The starting mAb2 material had
about 5% non-fucosylated Fc. After enrichment using the Fc receptor
column, the mAb2 material had 48.7% non-fucosylated IgG Fc.
[0183] A similar procedure as above was used to enrich a
significant amount of mAb2 using multiple cycles and running at a
lower load challenge to minimize process time and material waste.
The column was flushed with equilibration buffer (PBS) and loaded
at 0.25 ml/min (2.5 min residence time) with 1 ml of 1.89 mg/ml
mAb2 in equilibration buffer (3.0 mg protein/ml resin). The column
was then flushed with 12 ml of equilibration buffer at 0.5 ml/min
and eluted with 150 mM Glycine at pH 3.4 at 0.5 ml/min. This
procedure was repeated 39 times for a total of 40 cycles. The total
peak was brought to approximately pH 6.5 using 15% Of pH 10.4 1 M
MES neutralization buffer. A total of 4.4 mg of mAb2 was collected,
as determined by absorbance at 280 nm. To facilitate glycan
analysis, the mAb2 sample was concentrated to 2.3 mg/ml In 1.9 ml
using an Amicon Ultra-4 10,000 kD MWCO device (Millipore
UFC801024).
[0184] The percentage of non-fucosylated mAb2 was determined using
the method described in Example 1, The starting mAb2 material had
5.9% non-fucosylated and 2.3% mannosylated IgG Fc. After enrichment
using the Fc receptor column, the mAb2 material that bound to the
media had 41.8% non-fucosylated and 8.3% mannosylated IgG Fc. The
mAb2 material that flowed through the column had 4.3%
non-fucosylated and 2.0% mannosylated IgG Fc.
Example 3
[0185] A column packed with an Fc receptor medium was prepared as
described in Example 1. This medium was used to analyze binding
interactions between the Fc receptor and an Fc fusion protein
having a triple mutation, L234A, L235A, and G237A, which is known
to known to prevent induction of antibody dependent cellular
cytotoxicity (ADCC) (The 234, 235, 237 amino acid positions are
according to the Kabat numbering system). The Fc fusion protein
material was obtained from from a three column purification process
using the following: aprotein A chromatography column, an anion
exchange chromatography column and a mixed mode chromatography
column with cationic and HIC character.
[0186] The Fc receptor column was flushed with 50 mM MES 150 mM
NaCl pH 6.5 and loaded at 0.25 ml/min (3.0 min residence time) with
5 ml of 2.0 mg/ml Fc fusion in 50 mM MES 150 mM NaCl pH 6.5 (15.6
mg protein/mi resin). The column was then flushed with 9 ml of 50
mM MES 150 mM NaCl pH 6.5 at 1 ml/min and eluted with 12 ml of 150
mM Glycine pH 3.0 at 1 ml/min. The peak was neutralized with a 15%
spike of 1 M MES pH 10.4. This procedure was repeated with mAb2,
which does not have a corresponding triple mutation, followed by
the triple mutant Fc fusion protein again. Comparison of the
chromatograms measured by absorbance at 280 nm clearly showed that
the Fc fusion material in the low pH elutions did not produce a
significant peak relative to the non-mutated mAb2. Therefore the
triple mutant protein does not significantly bind to Fc gamma
RIIIA.
Example 4
[0187] This example describes preparation of an Fc gamma RIIa
receptor medium and use of the medium with mAb2.
[0188] Fc gamma RIIa receptors bind to IgG1 and other IgG
subclasses. An extracellular domain of an Fc gamma RIIa receptor
was obtained from R&D Systems, (Minneapolis, Minn.). The
receptor was expressed in NSO (murine myeloma)-derived cells and
contains a 10-his tag was expressed at the C-terminus. The portion
of the Fc gamma Rile receptor has the following amino acid sequence
(see also GenBank Accession # A35827):
TABLE-US-00006 (SEQ ID NO: 9) AAPPK AVLKLEPPWI NVLQEDSVTL
TCQGARSPES DSIQWFHNGN LIPTHTQPSY RFKANNNDSG EYTCQTGQTS LSDPVHLTVL
SEWLVLQTPH LEFQEGETIM LRCHSWKDKP LVKVTFFQNG KSQKFSHLDP TFSIPQANHS
HSGDYHCTGN IGYTLFSSKP VTITVQVPSM GSSSPMGI
[0189] The receptor was expressed with an N-terminal signal peptide
that is removed and the remaining portion of the molecule is the
extracellular domain (Ala 36 to Ile 218).
[0190] Fc gamma receptor (1.23 ml in PBS at 0,82 mg/ml) was added
to 0.82 ml of activated NHS Sepharose 4 Fast Row resin (17-0906-01,
GE Healthcare) and allowed to react for 60 minutes at room
temperature. The reaction was quenched with 100 mM TRIS pH 8.5. The
coupling efficiency, determined using size exclusion
chromatography, was 94%. The resin was packed to 0.72 mL in a 0.5
cm ID column (GE Healthcare) and flushed with 10 mls of 100 mM Tris
pH 8.5 followed by 10 mls of 100 mM Acetate pH 4.0 with 500 mM
NaCl. This was repeated twice and the column was stored in 16%
ethanol, 150 mM NaCl 50 mM Tris pH 7.7.
[0191] The column was flushed with PBS and loaded at 0.25 ml/min
(2.9 min residence time) with 5 ml of 2.0 mg/ml mAb2 in PBS buffer
(13.9 mg protein/ml resin). The column was then flushed with 9 ml
of PBS at 1 ml/min and eluted with 12 ml of 150 mM Glycine pH 2.6
at 1 ml/min. The peak was neutralized with a 15% spike of 1 M MES
pH 10.4. This procedure was repeated nine times and the ten peaks
were pooled to produce 95.4 ug of mAb2 as determined by absorbance
at 280 nm and size exclusion chromatography. To facilitate glycan
analysis, the mAb2 sample was concentrated to 0.51 mg/ml in 150
microliters using a 30 kD MWCO ultracentrifuge membrane.
[0192] To determine the glycan profile of mAb2, approximately 35
.mu.g was digested with PNGase F at 37.degree. C. for 16 hours to
release Fc-oligosaccharides. The released N-linked oligosaccharides
were labeled with 2-AB for 3 hours at 65.degree. C. Residual 2-AB
was removed using acetone precipitation. The oligosaccharide
analysis was done on a Xbridge Amide HILIC-HPLC at 45.degree. C.
over a linear ammonium formate gradient with fluorescence
detection. There was no alteration in the proportion of fucosylated
glycoforms in the sample compared to that of the load material.
Both the load and the eluted peak had about 5% fucosylated
material.
Example 5
[0193] This example describes preparation of an Fc gamma RIIb/c
receptor medium and use of the medium with mAb2.
[0194] Fc gamma RIIB/C receptors bind to IgG1 and other IgG
subclasses. An extracellular domain of an Fc gamma RIIb/c receptor
from R&D Systems (Minneapolis, Minn.) was obtained. The
receptor was expressed in ISO-derived cells and contained a
C-terminal 10-his tag, and has the following amino acid sequence
(see also GenBank Accession # P31994):
TABLE-US-00007 (SEQ ID NO: 10) APPKA VLKLEPQWIN VLQEDSVTLT
CRGTHSPESD SIQWFHNGNL IPTHTQPSYR FKANNNDSGE YTCQTGQTSL SDPVHLTVLS
EWLVLQTPHL EFQEGETIVL RCHSWKDKPL VKVTFFQNGK SKKFSRSDPN FSIPQANHSH
SGDYHCTGNI GYTLYSSKPV TITVQAP
[0195] The receptor was expressed with a signal peptide that is
cleaved off the N terminus, and the remaining portion of the
molecule is the extracellular domain (Ala46 to Pro 217).
[0196] Fc gamma receptor (0.87 ml in PBS at 1.16 mg/m1) was added
to 0.82 ml of activated NHS Sepharose 4 Fast Flow resin
(17-0906-01, GE) and allowed to react for 60 minutes at room
temperature. The reaction was quenched with 100 mM TRIS pH 8.5. The
coupling efficiency, determined using size exclusion
chromatography, was 95%. The resin was packed to 0.72 mL in a 0.6
cm ID column (GE Healthcare) and flushed with 10 mls of 100 mM Tris
pH 8.5 followed by 10 mls of 100 mM Acetate pH 4.0 with 500 mM
NaCl. This was repeated twice and the column was stored in 16%
ethanol, 150 mM NaCl 50 mM Tris pH 7.7.
[0197] The column was flushed with PBS and loaded at 0.25 ml/min
(2.9 min residence time) with 5 ml of 2.0 mg/ml mAb2 in PBS buffer
(13.9 mg protein/m1 resin). The column was then flushed with 9 ml
of PBS at 1 ml/min and eluted with 12 ml 150 mM Glycine pH 2.3 at 1
ml/min. The peak was neutralized with a 15% spike of 1 M MES pH
10.4. This procedure was repeated nine times and the ten peaks were
pooled to produce 93.0 ug of mAb2 as determined by absorbance at
280 nm and size exclusion chromatography. To allow glycan analysis,
the mAb2 sample was concentrated to 0.74 mg/ml in 100 microliters
using a 30 kD MWCO ultracentrifuge membrane.
[0198] To determine the glycan profile of mAb2, approximately 35
.mu.g was digested with PNGase F at 37.degree. C. for 16 hours to
release Fc-oligosaccharides. The released N-linked oligosaccharides
were labeled with 2-AB for 3 hours at 65.degree. C. Residual 2-AB
was removed using acetone precipitation. The oligosaccharide
analysis was done on a Xbridge Amide HILIC-HPLC at 45.degree. C.
over a linear ammonium formate gradient with fluorescence
detection. There was no alteration in the proportion of fucosylated
glycoforms in the sample compared to that of the load material.
Both the load and the &Wed peak had about 5% fucosylated
material.
Example 6
[0199] This example describes preparation of an Fc gamma RI
receptor medium and use of the medium with mAb2.
[0200] Fc gamma RI receptors bind to IgG1 and other IgG subclasses.
An extracellular domain of an Fc gamma RI receptor was obtain from
R&D Systems (Minneapolis, Minn.). This receptor, which was
expressed in NSO-derived cells and contained a C-terminal 6-his
tag, has the following amino acid sequence (see also GenBank Ace, #
P12314):
TABLE-US-00008 (SEQ ID NO: 11) QVDTT KAVITLQPPW VSVFQEETVT
LHCEVLHLPG SSSTQWFLNG TATQTSTPSY RITSASVNDS GEYRCQRGLS GRSDPIQLEI
HRGWLLLQVS SRVFTEGEPL ALRCHAWKDK LVYNVLYYRN GKAFKFFHWN SNLTILKTNI
SHNGTYHCSG MGKHRYTSAG ISVTVKELFP APVLNASVTS PLLEGNLVTL SCETKLLLQR
PGLQLYFSFY MGSKTLRGRN TSSEYQILTA RREDSGLYWC EAATEDGNVL KRSPELELQV
LGLQLPTP
[0201] The Fc receptor was expressed with a signal peptide that is
cleaved from the N terminus and the remaining molecule is the
extracellular domain less 4 residues on the C terminus (Gln16
Pro288).
[0202] Fc-gamma receptor (2.09 ml of in PBS at 0.483 mg/ml; was
added to 0.42 ml of activated NHS Sepharose 4 Fast Flow resin
(17-0906-01, GE) and allowed to react for 180 minutes at room
temperature. The reaction was quenched with 100 mM TRIS pH 8.5. The
coupling efficiency, determined using size exclusion
chromatography, was 95%. The resin was packed to 0.38 mL in a 0.5
cm ID GE healthcare column and flushed with 10 mls of 100 mM Tris
pH 8.5 followed by 10 mls of 100 rnM Acetate pH 4.0 with 500 mM
NaCl. This was repeated twice and the column was stored in 16%
ethanol, 150 mM NaCl 50 mM Tris pH 7.7.
[0203] The column was flushed with PBS and loaded at 0.15 all/min
(2.5 min residence time) with 5 ml of 2.0 mg/ml mAb2 in PBS buffer
(26,3 mg protein/mi resin). The column was then flushed with 9 ml
of PBS at 1 ml/min and eluted with 12 ml of 150 mM Glycine pH 3.0
and 6 ml of 150 mM Glycine pH 2.3 at 1 ml/min. The peak was
neutralized with a 15% spike of 1 M MES pH 10.4. The final pool
contained 38.6 ug of mAb2 as determined by absorbance at 280 nm. To
allow glycan analysis, the mAb2 sample was concentrated to 0.39
mg/ml in 100 microliters using a 30 kD MWCO ultracentrifuge
membrane.
[0204] To determine the glycan profile of mAb2, approximately 35
.mu.g was digested with PNGase F at 37.degree. C. for 16 hours to
release Fc oligosaccharides. The released N-linked oligosaccharides
were labeled with 2-AB for 3 hours at 5.degree. C. Residual 2-AB
was removed using acetone precipitation. The oligosaccharide
analysis was done on a Xbridge Amide HILIC-HPLC at 45.degree. C.
over a linear ammonium formate gradient with fluorescence
detection. There was no alteration in the proportion of fucosylated
glycoforms in the sample compared to that of the load material.
Both the load and the eluted peak had about 5% fucosylated
material.
Example 7
[0205] This example describes preparation of a C1q medium and use
of the medium with mAb2.
[0206] C1q is a complement protein that binds to IgG and IgM.
Native, human C1q prepared from serum was purchased in pure form
from EMD Biosciences, San Diego Calif. (CAS number 80295-33-6).
[0207] The C1q (0.85 ml in PBS at 1.17 mg/ml) was added to 0.72 ml
of activated NHS Sepharose 4 Fast Flow resin (17-0906-01, GE) and
allowed to react for 120 minutes at room temperature. The reaction
was quenched with 100 rnM TRIS pH 8.5. The coupling efficiency
could not be determined by size exclusion chromatography due to the
propensity of C1q to aggregate. The resin was packed to 0.64 mL in
a 0.5 cm ID column (GE Healthcare) and flushed with 10 mls of 100
mM Tris pH 8.5 followed by 10 mls of 100 mM Acetate pH 4.0 with 500
mM NaCl. This was repeated twice and the column was stored in 16%
ethanol, 150 mM NaCl 50 mM Tris pH 7.7.
[0208] The column was flushed with PBS and loaded at 0.25 ml/min
(2.6 min residence time) with 5 ml of 2.0 mg/ml mAb2 in PBS buffer
(15.6 mg protein/all resin). The column was then flushed with 9 ml
of PBS at 1 ml/min and eluted with 12 ml of 150 mM Glycine pH 3.0
at 1 ml/min. The peak was neutralized with a 15% spike of 1 M MES
pH 10.4. The final pool contained 7.5 ug of mAb2 as determined by
absorbance at 280 nm and size exclusion chromatography. Due to the
low amount of mass produced, glycan analysis was not performed.
Example 8
[0209] This example describes preparation of a OC SIGN medium and
use of the medium with mAb2.
[0210] Some literature indicates that DC-SIGN protein can
preferentially bind the sialated glycoforms of antibodies (DC-SIGN
and .alpha.2,6-sialylated IgG Fc interaction is dispensable for the
anti-inflammatory activity of IVIg on human dendritic cells, Bayry,
K Bansal, M D Kazatchkine, S V Kaveri; PNAS 2009 106 9 E24). A
recombinant human DC SIGN expressed in NSO-derived cells was
purchased in pure form from R&D Systems, The extracellular
portion (Lys62 Ala404) was expressed (see GenBank Acc. #
Q9NNX6).
[0211] DC SIGN (1 ml in PBS at 0.94 mg/ml) was added to 0.82 ml of
activated NHS Sepharose 4 Fast Flow resin (17-0906-01, GE) and
allowed to react for 60 minutes at room temperature. The reaction
was quenched with 100 mM IRIS pH 8.5. The coupling efficiency,
determined using size exclusion chromatography, was 95%. The resin
was packed to 0.74 mL in a 0.5 cm ID column (GE Healthcare) and
flushed with 10 mls of 100 mM Tris pH 8.5 followed by 10 mls of 100
mM Acetate pH 4.0 with 500 mM NaCl. This was repeated twice and the
column was stored in 16% ethanol, 150 mM NaCl 50 mM Tris pH
7.7.
[0212] The column was flushed with PBS and loaded at 0.25 ml/rein
(2.9 min residence time) with 5 ml of 2.0 mg/ml mAb2 in PBS buffer
(13.9 mg protein/mI resin). The column was then flushed with 9 ml
of PBS at 1 ml/min and eluted with 12 ml 150 mM Glycine pH 3.0 at 1
ml/min. The peak was neutralized with a 15% spike of 1 M MES pH
10.4. This procedure was repeated four times and the five peaks
were pooled to produce 128.0 ug of mAb2 as determined by absorbance
at 280 nm and size exclusion chromatography. To allow glycan
analysis, the mAb2 sample was concentrated to 1.02 mg/ml in 125
microliters using a 30 kD MWCO ultracentrifuge membrane.
[0213] To determine the glycan profile of mAb2, approximately 35
.mu.g was digested with PNGase F at 37.degree. C. for 16 hours to
release Fc-oligosaccharides. The released N-linked oligosaccharides
were labeled with 2-AB for 3 hours at 65.degree. C. Residual 2-AB
was removed using acetone precipitation. The oligosaccharide
analysis was done on a Xbridge Amide HILIC-HPLC at 45.degree. C.
over a linear ammonium formate gradient with fluorescence
detection. There was no alteration in the proportion of fucosylated
glycoforms in the sample compared to that of the load material.
Both the load and the eluted peak had about 5% fucosylated
material.
[0214] Given DC-SIGN has a Ca.sup.++ dependent binding domain, an
attempt was made to analyze whether calcium in the load could
affect binding of mAb2. The column was flushed with 50 mM MES 150
mM NaCl pH 6.5 20 mM CaCl.sub.2 and loaded at 0.25 ml/min (3.0 min
residence time) with 5 mi of 2,0 mg/ml mAb2 in 50 mM MES 150 mM
NaCl pH 6.5 20 mM CaCl.sub.2 (13.5 mg protein/ml resin). The column
was then flushed with 9 ml of 50 mM MES 150 mM NaCl pH 6.5 20 mM
CaCl.sub.2 at 1 ml/min. Three elutions were attempted: 6 ml of 50
mM MES 150 mM NaCl pH 6.5; 6 ml of 50 mM MES 150 mM NaCl pH 6.5 20
mM EDTA and 6 ml of 150 mM Glycine pH 2.3 at 1 ml/min. None of the
alternative elution conditions had a peak larger than the one
obtained with 150 mM Glycine pH 3.0. Thus, the presence of calcium
in the load, and variations in elution conditions did not appear to
affect binding or recovery in this study.
Example 9
[0215] This example describes preparation of an Fc gamma RIIIa
receptor medium and ligand density optimization using the medium
with mAb2.
[0216] Fc gamma RIIIa was obtained from R&D systems. The
receptor has the following sequence and a 6-His tag (see also
GenBank Acc. No. AAH17865, P08637 [UniParc]):
TABLE-US-00009 (SEQ ID NO: 12) GMRT EDLPKAVVFL EPQWYRVLEK
DSVTLKCQGA YSPEDNSTQW FHNESLISSQ ASSYFIDAAT VDDSGEYRCQ TNLSTLSDPV
QLEVHIGWLL LQAPRWVFKE EDPIHLRCHS WKNTALHKVT YLQNGKGRKY FHHNSDFYIP
KATLKDSGSY FCRGLFGSKN VSSETVNITI TQGLAVSTIS SFFPPGYQHHHHHH
[0217] The receptor was expression in an NSO-derived cell line. The
polypeptide does not have the signal peptide (residues 1-16) and
has extracellular residues Gly17-Gln208.
[0218] Microliter scale ligand coupling experiments were performed
to optimize the ligand density using Fc gamma RIIIA. Six aliquots
of 25 ul of activated NHS Sepharose.TM. 4 Fast Row resin
(17-0906-01, GE) were added to 800 ul wells of a 96 well plate with
a 0,45 urn polypropylene filter on the bottom (Seahorse Labware
F20023). The resin was washed and activated with 700 ul of 1 mM HCl
and spun down at 1200.times.G for 3 minutes. This was repeated for
a total of three wash cycles, Varying volumes of 0.66 ug/ul Fc
gamma RIIIA were added to the 25 ul of activated resin in each welt
to evaluate ligand densities between 1.5-9.6 ug-Fc gamma
RIIIA/ul-NHS resin including a control well where no Fc gamma RIIIA
was added (naive resin). During the reaction, the plate was
agitated on a Tecan Teshake at 1200 rpm for 2 hours at room
temperature.
[0219] After the reaction, the resin was washed with 700 ul of 100
mM Tris pH 8.3. This was repeated for a total of 2 cycles.
Following the Tris wash, 700 ul of 100 mM Acetate pH 4.0 was used.
Then 700 ul of 100 mM Tris pH 8.3 was added to each well again to
further quench the reaction and the plate was chilled to
2-8.degree. C. for storage. After approximately 15 hrs, the resin
was washed twice with 700 ul of 1 mM HEPES pH 7.3, mAb2 which was
previously enriched to approximately 45% a fucosylated species
using a larger scale 0.6 mL column packed with Fc gamma RIIIA
immobilized on activated NHS Sepharose.TM. 4 Fast Flow resin was
used as the load material for the batch-bind experiments.
Approximately 100 ul of 0,88 ug/ul mAb2 was added to each well and
the mixture was agitated on the Teshake at 1200 rpm for 20 minutes.
After the load step.sub.s the plate was spun down and the
flow-through was collected. The resin was then washed with 700 ul
of 1 mM HEPES pH 7.2. The resin was then eluted 3 times with 100 ul
of 150 mM Glycine pH 3.0. The load, flow-through, wash, and elution
fractions were collected with a 96 well plate and measured using UV
at 280 and 320 nm.
[0220] The results are shown in Table 3. As expected, the naive
resin did not exhibit significant protein binding. The mAb2
capacity of the micro well resin preparation was similar to that of
the larger resin volume preparation that was used in the packed
column (0.17 ug/ul vs. 0.20 ug/ul). The percent utilization was
calculated by first dividing the measured mAb2 capacity by the Fc
gamma RIIIA ligand density and then dividing this value by the
theoretical value at saturation capacity. If one antibody was bound
on each Fc gamma RIIIA molecule, then 3.5 ug of antibody would be
bound per ug of Fc gamma RIIIA. The best utilization was
approximately 10%. The optimal ligand density in terms of total
mAb2 capacity per eel of resin and in terms of percent utilization
is between 3-7.5 ug/ul. In some embodiments, a medium is prepared
and used so as to maximize capacity (e.g., to obtain the most
material from a load fluid). In some embodiments, a medium is
prepared and used to maximize utilization of Fc receptor. In some
embodiments, a medium is prepared and used so as to maximize both
capacity and utilization.
TABLE-US-00010 TABLE 3 Fc.gamma.RIIIA media optimization using
micro-liter resin preparation. ligand Cap density Capacity ug-mAb2/
% [ug/ul] Preparation [ug/mAb2/ul] ug-Fc Utilization 1.6 large
volume (ml) 0.17 0.11 3.1 1.5 micro well (ul) 0.20 0.13 3.8 3 micro
well (ul) 0.91 0.30 8.6 5 micro well (ul) 1.44 0.29 8.2 7.5 micro
well (ul) 1.63 0.22 6.2 9.5 micro well (ul) 1.34 0.14 4.0 0 (naive
micro well (ul) 0.04 N/A N/A resin)
Example 10
[0221] This example describes preparation of an Fc gamma RIIIb
receptor medium and use of the medium with mAb2.
[0222] Fc gamma RIIIB receptor binds to IgG1 and other IgG
subclasses. The extracellular domain of the Fc gamma RIIIB receptor
was obtained from R&D Systems. This receptor was expressed in
NSO-derived cells and includes a C-terminal 10-his tag. This
receptor has the following amino acid sequence (see also GenBank
Accession # O75015; the sequence includes Thr20-Gln208):
TABLE-US-00011 (SEQ ID NO: 13) T EDLPKAVVFL EPQWYSVLEK DSVTLKCQGA
YSPEDNSTQW FHNESLISSQ ASSYFIDAAT VNDSGEYRCQ TNLSTLSDPV QLEVHIGWLL
LQAPRWVFKE EDPIHLRCHS WKNTALHKVT YLQNGKDRKY FHHNSDFHIP KATLKDSGSY
FCRGLVGSKN VSSETVNITI TQGLAVSTIS SFSPPGYQ
[0223] Fc gamma receptor in PBS (0.54 ml of at 1.87 mg/ml was added
to 0.54 ml of activated NHS Sepharose 4 Fast Flow resin
(17-0906-01, GE) and allowed to react for $0 minutes at room
temperature. The reaction was quenched with 100 mM TRIS pH 8.5. The
resin was packed to 0.5 mL in a 0.5 cm ID column (GE Healthcare)
and flushed with 10 mls of 100 mM Tris pH 8.5 followed by 10 mls of
100 mM Acetate pH 4.0 with 500 mM NaCl. This was repeated twice and
the column was stored in 16% ethanol, 150 mM NaCl 50 MM Tris pH
7.7.
[0224] The column was flushed with PBS and loaded at 0.25 ml/min
(2.0 min residence time) with 5 ml of 1.96 mg/mi mAb2 in PBS buffer
(19.6 mg protein/ml resin). The column was then flushed with 9 ml
of PBS at 1 ml/min and eluted with 12 ml of 150 mM Glycine pH 3.0
at 1 ml/min. The peak was neutralized with a 15% spike of 1 M MES
pH 10.4. This procedure was repeated three times and the four peaks
were pooled to produce 78 ug of mAb2 as determined by absorbance at
280 nm. To allow glycan analysis, the mAb2 sample was concentrated
to 0.39 mg/ml in 200 microliters using a 30 kD MWCO ultracentrifuge
membrane.
[0225] To determine the glycan profile of mAb2, approximately 35
.mu.g was digested with PNGase F at 37.degree. C. for 16 hours to
release Fc-oligosaccharides. The released N-linked oligosaccharides
were labeled with 2-AB for 3 hours at 65.degree. C. Residual 2-AB
was removed using acetone precipitation. The oligosaccharide
analysis was done on a Xbridge Amide HILIC-HPLC at 45.degree. C.
over a linear ammonium formate gradient with fluorescence
detection. The starting mAb2 material had 5.9% non-fucosylated IgG.
After enrichment using the Fc receptor column, the mAb2 material
had about 39.3% non-fucosyleted IgG.
Example 11
[0226] This example describes preparation of an Fc gamma RIIIa
receptor medium and use of the medium with mAb2.
[0227] Fc gamma RIIIa was obtained from R&D systems. The
receptor has the following sequence and a 6-His tag (see also
GenBank Acc. No. AAH17865, P08637 [UniParc]):
TABLE-US-00012 (SEQ ID NO: 14) GMRT EDLPKAVVFL EPQWYRVLEK
DSVTLKCQGA YSPEDNSTQW FHNESLISSQ ASSYFIDAAT VDDSGEYRCQ TNLSTLSDPV
QLEVHIGWLL LQAPRWVFKE EDPIHLRCHS WKNTALHKVT YLQNGKGRKY FHHNSDFYIP
KATLKDSGSY FCRGLFGSKN VSSETVNITI TQGLAVSTIS SFFPPGYQHHHHHH
[0228] The receptor was expression in an NSO-derived cell line. The
polypeptide does not have the signal peptide (residues 1-16) and
has extracellular residues Gly17-Gln208.
[0229] The Fc-gamma receptor (1.3 ml in PBS at 0.81 mg/ml) was
added to 0.64 ml of activated NHS Sepharose.TM. 4 Fast Row resin
(17-0906-01, GE) and allowed to react for 65 minutes at room
temperature. The reaction was quenched with 100 mM TRIS pH 8.3. The
coupling efficiency, determined using size exclusion
chromatography, was 93%. The resin was packed to 0.55 mL in a 0.5
cm internal diameter (ID) column (GE Healthcare) and flushed with
10 mls of 100 mM Tris pH 8.3 followed by 10 mls of100 mM Acetate pH
4,0 with 500 mM NaCl. This flushing was repeated twice and the
column was stored in 16% ethanol, 160 mM NaCl 50 mM Tris pH
7,7.
[0230] The column was flushed with PBS at 1.0 ml/min and loaded at
0.15 ml/min (3.7 min residence time) with 1.0 ml of 0.77 mg/ml mAb2
in PBS buffer (1.4 mg protein/ml resin). The mAb2 material was
obtained from a conventional ultrafiltration pool that results from
a full purification process using the following: protein A
chromatography column, an anion exchange chromatography column, a
virus-retaining fitter (e.g., a Planova 20 filter (Asahi Kasei
Corporation, Tokyo, Japan)), and an ultrafilter.
[0231] The Fc receptor column was then flushed with 12 ml of PBS at
1.0 ml/min and eluted with 160 milt Glycine pH at 1.0 ml/min. This
procedure was repeated 28 times. The total elution pool had 1.8 mg
and the flow through 15.6 mg as determined by absorbance at 280
nm.
[0232] To determine the glycan profile of mAb2, approximately 35
.mu.g of IgG from the flow through pool was digested with PNGase F
at 37.degree. C. for 16 hours to release Fc-oligosaccharides. The
released N-linked oligosaccharides were labeled with 2-AB for 3
hours at 65.degree. C. Residual 2-AB was removed using acetone
precipitation. The oligosaccharide analysis was done on a Xbridge
Amide HILIC-HPLC at 45.degree. C. over a linear ammonium formate
gradient with fluorescence detection.
[0233] The starting mAb2 material had 5.4% non-fucosylated and 1.9%
mannosylated Fc. The Fc receptor column preferentially bound
non-fucosylated and mannosylated IgG Fc. The mAb2 flowing through
the column was depleted of non-fucosylated and mannosylated IgG Fc.
The mAb2 flow through material had 2.1% non-fucosylated and 1.1%
mannosylated IgG Fc. The glycan profile of the material that bound
to the Fc receptor column was not measured in this case.
[0234] 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 present disclosure
are described herein. The scope of the present disclosure is not
intended to be limited to the above Description. Alternative
methods and materials and additional applications will be apparent
to one of skill in the art, and are intended to be included within
the following claims:
Sequence CWU 1
1
161254PRTHomo sapiens 1Met Trp Gln Leu Leu Leu Pro Thr Ala Leu Leu
Leu Leu Val Ser Ala1 5 10 15Gly Met Arg Thr Glu Asp Leu Pro Lys Ala
Val Val Phe Leu Glu Pro 20 25 30Gln Trp Tyr Arg Val Leu Glu Lys Asp
Ser Val Thr Leu Lys Cys Gln 35 40 45Gly Ala Tyr Ser Pro Glu Asp Asn
Ser Thr Gln Trp Phe His Asn Glu 50 55 60Ser Leu Ile Ser Ser Gln Ala
Ser Ser Tyr Phe Ile Asp Ala Ala Thr65 70 75 80Val Asp Asp Ser Gly
Glu Tyr Arg Cys Gln Thr Asn Leu Ser Thr Leu 85 90 95Ser Asp Pro Val
Gln Leu Glu Val His Ile Gly Trp Leu Leu Leu Gln 100 105 110Ala Pro
Arg Trp Val Phe Lys Glu Glu Asp Pro Ile His Leu Arg Cys 115 120
125His Ser Trp Lys Asn Thr Ala Leu His Lys Val Thr Tyr Leu Gln Asn
130 135 140Gly Lys Gly Arg Lys Tyr Phe His His Asn Ser Asp Phe Tyr
Ile Pro145 150 155 160Lys Ala Thr Leu Lys Asp Ser Gly Ser Tyr Phe
Cys Arg Gly Leu Phe 165 170 175Gly Ser Lys Asn Val Ser Ser Glu Thr
Val Asn Ile Thr Ile Thr Gln 180 185 190Gly Leu Ala Val Ser Thr Ile
Ser Ser Phe Phe Pro Pro Gly Tyr Gln 195 200 205Val Ser Phe Cys Leu
Val Met Val Leu Leu Phe Ala Val Asp Thr Gly 210 215 220Leu Tyr Phe
Ser Val Lys Thr Asn Ile Arg Ser Ser Thr Arg Asp Trp225 230 235
240Lys Asp His Lys Phe Lys Trp Arg Lys Asp Pro Gln Asp Lys 245
2502233PRTHomo sapiens 2Met Trp Gln Leu Leu Leu Pro Thr Ala Leu Leu
Leu Leu Val Ser Ala1 5 10 15Gly Met Arg Thr Glu Asp Leu Pro Lys Ala
Val Val Phe Leu Glu Pro 20 25 30Gln Trp Tyr Ser Val Leu Glu Lys Asp
Ser Val Thr Leu Lys Cys Gln 35 40 45Gly Ala Tyr Ser Pro Glu Asp Asn
Ser Thr Gln Trp Phe His Asn Glu 50 55 60Ser Leu Ile Ser Ser Gln Ala
Ser Ser Tyr Phe Ile Asp Ala Ala Thr65 70 75 80Val Asn Asp Ser Gly
Glu Tyr Arg Cys Gln Thr Asn Leu Ser Thr Leu 85 90 95Ser Asp Pro Val
Gln Leu Glu Val His Ile Gly Trp Leu Leu Leu Gln 100 105 110Ala Pro
Arg Trp Val Phe Lys Glu Glu Asp Pro Ile His Leu Arg Cys 115 120
125His Ser Trp Lys Asn Thr Ala Leu His Lys Val Thr Tyr Leu Gln Asn
130 135 140Gly Lys Asp Arg Lys Tyr Phe His His Asn Ser Asp Phe His
Ile Pro145 150 155 160Lys Ala Thr Leu Lys Asp Ser Gly Ser Tyr Phe
Cys Arg Gly Leu Val 165 170 175Gly Ser Lys Asn Val Ser Ser Glu Thr
Val Asn Ile Thr Ile Thr Gln 180 185 190Gly Leu Ala Val Ser Thr Ile
Ser Ser Phe Ser Pro Pro Gly Tyr Gln 195 200 205Val Ser Phe Cys Leu
Val Met Val Leu Leu Phe Ala Val Asp Thr Gly 210 215 220Leu Tyr Phe
Ser Val Lys Thr Asn Ile225 23035PRTUnknownDescription of Unknown
Recognition motif peptideMOD_RES(3)..(3)Any amino acid 3Leu Pro Xaa
Thr Gly1 544PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 4Lys Gly Gly Gly154PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 5Cys
Gly Gly Gly16193PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 6Lys Gly Gly Gly Glu Asp Leu Pro Lys
Ala Val Val Phe Leu Glu Pro1 5 10 15Gln Trp Tyr Arg Val Leu Glu Lys
Asp Ser Val Thr Leu Lys Cys Gln 20 25 30Gly Ala Tyr Ser Pro Glu Asp
Asn Ser Thr Gln Trp Phe His Asn Glu 35 40 45Ser Leu Ile Ser Ser Gln
Ala Ser Ser Tyr Phe Ile Asp Ala Ala Thr 50 55 60Val Asp Asp Ser Gly
Glu Tyr Arg Cys Gln Thr Asn Leu Ser Thr Leu65 70 75 80Ser Asp Pro
Val Gln Leu Glu Val His Ile Gly Trp Leu Leu Leu Gln 85 90 95Ala Pro
Arg Trp Val Phe Lys Glu Glu Asp Pro Ile His Leu Arg Cys 100 105
110His Ser Trp Lys Asn Thr Ala Leu His Lys Val Thr Tyr Leu Gln Asn
115 120 125Gly Lys Gly Arg Lys Tyr Phe His His Asn Ser Asp Phe Tyr
Ile Pro 130 135 140Lys Ala Thr Leu Lys Asp Ser Gly Ser Tyr Phe Cys
Arg Gly Leu Val145 150 155 160Gly Ser Lys Asn Val Ser Ser Glu Thr
Val Asn Ile Thr Ile Thr Gln 165 170 175Gly Leu Ala Val Ser Thr Ile
Ser Ser Phe Phe Pro Pro Gly Tyr Gln 180 185 190Val7193PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
7Cys Gly Gly Gly Glu Asp Leu Pro Lys Ala Val Val Phe Leu Glu Pro1 5
10 15Gln Trp Tyr Arg Val Leu Glu Lys Asp Ser Val Thr Leu Lys Cys
Gln 20 25 30Gly Ala Tyr Ser Pro Glu Asp Asn Ser Thr Gln Trp Phe His
Asn Glu 35 40 45Ser Leu Ile Ser Ser Gln Ala Ser Ser Tyr Phe Ile Asp
Ala Ala Thr 50 55 60Val Asp Asp Ser Gly Glu Tyr Arg Cys Gln Thr Asn
Leu Ser Thr Leu65 70 75 80Ser Asp Pro Val Gln Leu Glu Val His Ile
Gly Trp Leu Leu Leu Gln 85 90 95Ala Pro Arg Trp Val Phe Lys Glu Glu
Asp Pro Ile His Leu Arg Cys 100 105 110His Ser Trp Lys Asn Thr Ala
Leu His Lys Val Thr Tyr Leu Gln Asn 115 120 125Gly Lys Gly Arg Lys
Tyr Phe His His Asn Ser Asp Phe Tyr Ile Pro 130 135 140Lys Ala Thr
Leu Lys Asp Ser Gly Ser Tyr Phe Cys Arg Gly Leu Val145 150 155
160Gly Ser Lys Asn Val Ser Ser Glu Thr Val Asn Ile Thr Ile Thr Gln
165 170 175Gly Leu Ala Val Ser Thr Ile Ser Ser Phe Phe Pro Pro Gly
Tyr Gln 180 185 190Val8199PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 8Glu Asp Leu Pro Lys Ala
Val Val Phe Leu Glu Pro Gln Trp Tyr Arg1 5 10 15Val Leu Glu Lys Asp
Ser Val Thr Leu Lys Cys Gln Gly Ala Tyr Ser 20 25 30Pro Glu Asp Asn
Ser Thr Gln Trp Phe His Asn Glu Ser Leu Ile Ser 35 40 45Ser Gln Ala
Ser Ser Tyr Phe Ile Asp Ala Ala Thr Val Asp Asp Ser 50 55 60Gly Glu
Tyr Arg Cys Gln Thr Asn Leu Ser Thr Leu Ser Asp Pro Val65 70 75
80Gln Leu Glu Val His Ile Gly Trp Leu Leu Leu Gln Ala Pro Arg Trp
85 90 95Val Phe Lys Glu Glu Asp Pro Ile His Leu Arg Cys His Ser Trp
Lys 100 105 110Asn Thr Ala Leu His Lys Val Thr Tyr Leu Gln Asn Gly
Lys Gly Arg 115 120 125Lys Tyr Phe His His Asn Ser Asp Phe Tyr Ile
Pro Lys Ala Thr Leu 130 135 140Lys Asp Ser Gly Ser Tyr Phe Cys Arg
Gly Leu Val Gly Ser Lys Asn145 150 155 160Val Ser Ser Glu Thr Val
Asn Ile Thr Ile Thr Gln Gly Leu Ala Val 165 170 175Ser Thr Ile Ser
Ser Phe Phe Pro Pro Gly Tyr Gln Val His His His 180 185 190His His
His His His His His 1959183PRTHomo sapiens 9Ala Ala Pro Pro Lys Ala
Val Leu Lys Leu Glu Pro Pro Trp Ile Asn1 5 10 15Val Leu Gln Glu Asp
Ser Val Thr Leu Thr Cys Gln Gly Ala Arg Ser 20 25 30Pro Glu Ser Asp
Ser Ile Gln Trp Phe His Asn Gly Asn Leu Ile Pro 35 40 45Thr His Thr
Gln Pro Ser Tyr Arg Phe Lys Ala Asn Asn Asn Asp Ser 50 55 60Gly Glu
Tyr Thr Cys Gln Thr Gly Gln Thr Ser Leu Ser Asp Pro Val65 70 75
80His Leu Thr Val Leu Ser Glu Trp Leu Val Leu Gln Thr Pro His Leu
85 90 95Glu Phe Gln Glu Gly Glu Thr Ile Met Leu Arg Cys His Ser Trp
Lys 100 105 110Asp Lys Pro Leu Val Lys Val Thr Phe Phe Gln Asn Gly
Lys Ser Gln 115 120 125Lys Phe Ser His Leu Asp Pro Thr Phe Ser Ile
Pro Gln Ala Asn His 130 135 140Ser His Ser Gly Asp Tyr His Cys Thr
Gly Asn Ile Gly Tyr Thr Leu145 150 155 160Phe Ser Ser Lys Pro Val
Thr Ile Thr Val Gln Val Pro Ser Met Gly 165 170 175Ser Ser Ser Pro
Met Gly Ile 18010172PRTHomo sapiens 10Ala Pro Pro Lys Ala Val Leu
Lys Leu Glu Pro Gln Trp Ile Asn Val1 5 10 15Leu Gln Glu Asp Ser Val
Thr Leu Thr Cys Arg Gly Thr His Ser Pro 20 25 30Glu Ser Asp Ser Ile
Gln Trp Phe His Asn Gly Asn Leu Ile Pro Thr 35 40 45His Thr Gln Pro
Ser Tyr Arg Phe Lys Ala Asn Asn Asn Asp Ser Gly 50 55 60Glu Tyr Thr
Cys Gln Thr Gly Gln Thr Ser Leu Ser Asp Pro Val His65 70 75 80Leu
Thr Val Leu Ser Glu Trp Leu Val Leu Gln Thr Pro His Leu Glu 85 90
95Phe Gln Glu Gly Glu Thr Ile Val Leu Arg Cys His Ser Trp Lys Asp
100 105 110Lys Pro Leu Val Lys Val Thr Phe Phe Gln Asn Gly Lys Ser
Lys Lys 115 120 125Phe Ser Arg Ser Asp Pro Asn Phe Ser Ile Pro Gln
Ala Asn His Ser 130 135 140His Ser Gly Asp Tyr His Cys Thr Gly Asn
Ile Gly Tyr Thr Leu Tyr145 150 155 160Ser Ser Lys Pro Val Thr Ile
Thr Val Gln Ala Pro 165 17011273PRTHomo sapiens 11Gln Val Asp Thr
Thr Lys Ala Val Ile Thr Leu Gln Pro Pro Trp Val1 5 10 15Ser Val Phe
Gln Glu Glu Thr Val Thr Leu His Cys Glu Val Leu His 20 25 30Leu Pro
Gly Ser Ser Ser Thr Gln Trp Phe Leu Asn Gly Thr Ala Thr 35 40 45Gln
Thr Ser Thr Pro Ser Tyr Arg Ile Thr Ser Ala Ser Val Asn Asp 50 55
60Ser Gly Glu Tyr Arg Cys Gln Arg Gly Leu Ser Gly Arg Ser Asp Pro65
70 75 80Ile Gln Leu Glu Ile His Arg Gly Trp Leu Leu Leu Gln Val Ser
Ser 85 90 95Arg Val Phe Thr Glu Gly Glu Pro Leu Ala Leu Arg Cys His
Ala Trp 100 105 110Lys Asp Lys Leu Val Tyr Asn Val Leu Tyr Tyr Arg
Asn Gly Lys Ala 115 120 125Phe Lys Phe Phe His Trp Asn Ser Asn Leu
Thr Ile Leu Lys Thr Asn 130 135 140Ile Ser His Asn Gly Thr Tyr His
Cys Ser Gly Met Gly Lys His Arg145 150 155 160Tyr Thr Ser Ala Gly
Ile Ser Val Thr Val Lys Glu Leu Phe Pro Ala 165 170 175Pro Val Leu
Asn Ala Ser Val Thr Ser Pro Leu Leu Glu Gly Asn Leu 180 185 190Val
Thr Leu Ser Cys Glu Thr Lys Leu Leu Leu Gln Arg Pro Gly Leu 195 200
205Gln Leu Tyr Phe Ser Phe Tyr Met Gly Ser Lys Thr Leu Arg Gly Arg
210 215 220Asn Thr Ser Ser Glu Tyr Gln Ile Leu Thr Ala Arg Arg Glu
Asp Ser225 230 235 240Gly Leu Tyr Trp Cys Glu Ala Ala Thr Glu Asp
Gly Asn Val Leu Lys 245 250 255Arg Ser Pro Glu Leu Glu Leu Gln Val
Leu Gly Leu Gln Leu Pro Thr 260 265 270Pro12198PRTHomo sapiens
12Gly Met Arg Thr Glu Asp Leu Pro Lys Ala Val Val Phe Leu Glu Pro1
5 10 15Gln Trp Tyr Arg Val Leu Glu Lys Asp Ser Val Thr Leu Lys Cys
Gln 20 25 30Gly Ala Tyr Ser Pro Glu Asp Asn Ser Thr Gln Trp Phe His
Asn Glu 35 40 45Ser Leu Ile Ser Ser Gln Ala Ser Ser Tyr Phe Ile Asp
Ala Ala Thr 50 55 60Val Asp Asp Ser Gly Glu Tyr Arg Cys Gln Thr Asn
Leu Ser Thr Leu65 70 75 80Ser Asp Pro Val Gln Leu Glu Val His Ile
Gly Trp Leu Leu Leu Gln 85 90 95Ala Pro Arg Trp Val Phe Lys Glu Glu
Asp Pro Ile His Leu Arg Cys 100 105 110His Ser Trp Lys Asn Thr Ala
Leu His Lys Val Thr Tyr Leu Gln Asn 115 120 125Gly Lys Gly Arg Lys
Tyr Phe His His Asn Ser Asp Phe Tyr Ile Pro 130 135 140Lys Ala Thr
Leu Lys Asp Ser Gly Ser Tyr Phe Cys Arg Gly Leu Phe145 150 155
160Gly Ser Lys Asn Val Ser Ser Glu Thr Val Asn Ile Thr Ile Thr Gln
165 170 175Gly Leu Ala Val Ser Thr Ile Ser Ser Phe Phe Pro Pro Gly
Tyr Gln 180 185 190His His His His His His 19513189PRTHomo sapiens
13Thr Glu Asp Leu Pro Lys Ala Val Val Phe Leu Glu Pro Gln Trp Tyr1
5 10 15Ser Val Leu Glu Lys Asp Ser Val Thr Leu Lys Cys Gln Gly Ala
Tyr 20 25 30Ser Pro Glu Asp Asn Ser Thr Gln Trp Phe His Asn Glu Ser
Leu Ile 35 40 45Ser Ser Gln Ala Ser Ser Tyr Phe Ile Asp Ala Ala Thr
Val Asn Asp 50 55 60Ser Gly Glu Tyr Arg Cys Gln Thr Asn Leu Ser Thr
Leu Ser Asp Pro65 70 75 80Val Gln Leu Glu Val His Ile Gly Trp Leu
Leu Leu Gln Ala Pro Arg 85 90 95Trp Val Phe Lys Glu Glu Asp Pro Ile
His Leu Arg Cys His Ser Trp 100 105 110Lys Asn Thr Ala Leu His Lys
Val Thr Tyr Leu Gln Asn Gly Lys Asp 115 120 125Arg Lys Tyr Phe His
His Asn Ser Asp Phe His Ile Pro Lys Ala Thr 130 135 140Leu Lys Asp
Ser Gly Ser Tyr Phe Cys Arg Gly Leu Val Gly Ser Lys145 150 155
160Asn Val Ser Ser Glu Thr Val Asn Ile Thr Ile Thr Gln Gly Leu Ala
165 170 175Val Ser Thr Ile Ser Ser Phe Ser Pro Pro Gly Tyr Gln 180
18514198PRTHomo sapiens 14Gly Met Arg Thr Glu Asp Leu Pro Lys Ala
Val Val Phe Leu Glu Pro1 5 10 15Gln Trp Tyr Arg Val Leu Glu Lys Asp
Ser Val Thr Leu Lys Cys Gln 20 25 30Gly Ala Tyr Ser Pro Glu Asp Asn
Ser Thr Gln Trp Phe His Asn Glu 35 40 45Ser Leu Ile Ser Ser Gln Ala
Ser Ser Tyr Phe Ile Asp Ala Ala Thr 50 55 60Val Asp Asp Ser Gly Glu
Tyr Arg Cys Gln Thr Asn Leu Ser Thr Leu65 70 75 80Ser Asp Pro Val
Gln Leu Glu Val His Ile Gly Trp Leu Leu Leu Gln 85 90 95Ala Pro Arg
Trp Val Phe Lys Glu Glu Asp Pro Ile His Leu Arg Cys 100 105 110His
Ser Trp Lys Asn Thr Ala Leu His Lys Val Thr Tyr Leu Gln Asn 115 120
125Gly Lys Gly Arg Lys Tyr Phe His His Asn Ser Asp Phe Tyr Ile Pro
130 135 140Lys Ala Thr Leu Lys Asp Ser Gly Ser Tyr Phe Cys Arg Gly
Leu Phe145 150 155 160Gly Ser Lys Asn Val Ser Ser Glu Thr Val Asn
Ile Thr Ile Thr Gln 165 170 175Gly Leu Ala Val Ser Thr Ile Ser Ser
Phe Phe Pro Pro Gly Tyr Gln 180 185 190His His His His His His
1951510PRTArtificial SequenceDescription of Artificial Sequence
Synthetic 10xHis tag 15His His His His His His His His His His1 5
10166PRTArtificial SequenceDescription of Artificial Sequence
Synthetic 6xHis tag 16His His His His His His1 5
* * * * *
References