U.S. patent application number 15/900461 was filed with the patent office on 2018-07-05 for method for the reduction of host cell proteins in affinity chromatography.
This patent application is currently assigned to Genentech, Inc.. The applicant listed for this patent is Genentech, Inc., Hoffmann-La Roche Inc.. Invention is credited to Ryan ERICKSON, Roberto FALKENSTEIN, Annika KLEINJANS, Wolfgang KOEHNLEIN, Carina KOPP, Paul MCDONALD, Klaus SCHWENDNER, Bernhard SPENSBERGER, Richard ST. JOHN, Benjamin TRAN, Michael WIEDMANN, Marc WONG, Frank ZETTL.
Application Number | 20180186832 15/900461 |
Document ID | / |
Family ID | 56802471 |
Filed Date | 2018-07-05 |
United States Patent
Application |
20180186832 |
Kind Code |
A1 |
MCDONALD; Paul ; et
al. |
July 5, 2018 |
Method for the reduction of host cell proteins in affinity
chromatography
Abstract
The current invention reports a method for purifying an antibody
by reducing the content of a host cell protein. The method employs
a wash step with a low conductivity aqueous solution in an affinity
chromatography.
Inventors: |
MCDONALD; Paul; (San
Francisco, CA) ; ST. JOHN; Richard; (Millbrae,
CA) ; WONG; Marc; (San Carlos, CA) ;
FALKENSTEIN; Roberto; (Muenchen, DE) ; KOEHNLEIN;
Wolfgang; (Benediktbeuern, DE) ; SCHWENDNER;
Klaus; (Weilheim, DE) ; SPENSBERGER; Bernhard;
(Eberfing, DE) ; WIEDMANN; Michael; (Penzberg,
DE) ; ZETTL; Frank; (Neuried, DE) ; KLEINJANS;
Annika; (Iffeldorf, DE) ; KOPP; Carina; (Pei
enberg, DE) ; TRAN; Benjamin; (San Mateo, CA)
; ERICKSON; Ryan; (Oakland, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Genentech, Inc.
Hoffmann-La Roche Inc. |
South San Francisco
Little Falls |
CA
NJ |
US
US |
|
|
Assignee: |
Genentech, Inc.
South San Francisco
CA
Hoffmann-La Roche Inc
Little Falls
NJ
|
Family ID: |
56802471 |
Appl. No.: |
15/900461 |
Filed: |
February 20, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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PCT/EP2016/069604 |
Aug 18, 2016 |
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15900461 |
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62208523 |
Aug 21, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 1/22 20130101; C07K
16/22 20130101; C07K 16/32 20130101; C07K 2317/14 20130101; C07K
16/2854 20130101; C07K 16/18 20130101; C07K 2317/31 20130101; C07K
16/36 20130101 |
International
Class: |
C07K 1/22 20060101
C07K001/22; C07K 16/28 20060101 C07K016/28; C07K 16/18 20060101
C07K016/18; C07K 16/32 20060101 C07K016/32; C07K 16/22 20060101
C07K016/22; C07K 16/36 20060101 C07K016/36 |
Claims
1. Use of a low conductivity aqueous solution in a wash step of a
protein A chromatography for reducing the content of a host cell
protein wherein the protein A chromatography is used to purify a
human IgG4 or IgG1 isotype antibody, wherein the low conductivity
aqueous solution has a conductivity value of about 0.5 mS/cm or
less.
2. Use according to claim 1, wherein the host cell protein is
phospholipase B-like 2 (PLBL2) or Clusterin.
3. Use according to any one of claims 1 to 2, wherein the low
conductivity aqueous solution comprises about 0.1 mM to about 8 mM
Tris.
4. Use according to any one of claims 1 to 2, wherein the low
conductivity aqueous solution comprises about 0.05 mM to about 2 mM
potassium phosphate.
5. Use according any one of claims 1 to 4, wherein the low
conductivity aqueous solution has a pH of about 7 or higher.
6. Use according to any one of claims 1 to 5, wherein the low
conductivity aqueous solution wash step is preceded or succeeded by
a high conductivity aqueous solution wash step.
7. Use according to claim 6, wherein the high conductivity aqueous
solution has a conductivity value of about 20 mS/cm or higher.
8. Use according to any one of claims 6 to 7, wherein the high
conductivity aqueous solution comprises Histidine.
9. Use according to any one of claims 1 to 8, wherein the human
IgG4 isotype antibody is an antibody against P-selectin or an
antibody against factor IXa and factor X or an antibody against
IL-13 or an antibody against amyloid beta.
10. Use according to any one of claims 1 to 8, wherein the human
IgG1 isotype antibody is an antibody against Influenza B or an
antibody against VEGF-A or an antibody against CD22 or a bispecific
antibody against HER3 and EGFR or an antibody against amyloid beta
or an antibody against Her2 or a bispecific antibody against Ang2
and VEGF-A or a bispecific antibody against carcinoembryonic
antigen (CEA) and CD3.
11. Method for producing a human IgG4 or IgG1 isotype antibody
comprising the steps of a) cultivating a cell comprising a nucleic
acid encoding a human IgG4 or IgG1 isotype antibody, b) recovering
the human IgG4 or IgG1 isotype antibody from the cell or the
cultivation medium, c) contacting the human IgG4 or IgG1 isotype
antibody with a protein A chromatography material, d) washing the
protein A chromatography material with a low conductivity aqueous
solution, wherein the low conductivity aqueous solution has a
conductivity value of about 0.5 mS/cm or less, e) recovering the
human IgG4 or IgG1 isotype antibody from the protein A
chromatography material and thereby producing the human IgG4 or
IgG1 isotype antibody.
12. Method for purifying a human IgG4 or IgG1 isotype antibody from
a sample comprising the steps of a) providing a sample comprising a
human IgG4 or IgG1 isotype antibody, b) purifying the human IgG4 or
IgG1 isotype antibody with a protein A chromatography method/step,
comprising washing the protein A chromatography material with a low
conductivity aqueous solution, wherein the low conductivity aqueous
solution has a conductivity value of about 0.5 mS/cm or less.
13. Method according to any one of claims 11 to 12, wherein the
amount of a host cell protein is reduced and wherein said host cell
protein is phospholipase B-like 2 (PLBL2) or Clusterin.
14. Method according to any one of claims 11 to 13, wherein the low
conductivity aqueous solution comprises about 0.1 mM to about 8 mM
Tris.
15. Method according to any one of claims 11 to 14, wherein the low
conductivity aqueous solution comprises about 0.05 mM to about 2 mM
potassium phosphate.
16. Method according to any one of claims 11 to 15, wherein the low
conductivity aqueous solution has a pH of about 7 or higher.
17. Method according to any one of claims 11 to 16, wherein the
method additionally comprises washing the affinity chromatography
material with a high conductivity aqueous solution and/or with a
medium conductivity aqueous solution before or after washing the
protein A chromatography material with low conductivity aqueous
solution.
18. Method according to claim 17, wherein the high conductivity
aqueous solution has a conductivity value of about 20 mS/cm or
higher.
19. Method according to claim 17, wherein the medium conductivity
aqueous solution has a conductivity value of from more than 0.5
mS/cm to less than 20 mS/cm.
20. Method according to any one of claims 17 to 19, wherein the
high or medium conductivity aqueous solution comprises
Histidine.
21. Method according to any one of claims 11 to 20, wherein the
human IgG4 isotype antibody is an antibody against P-selectin or an
antibody against factor IXa and factor X or an antibody against
IL-13 or an antibody against amyloid beta.
22. Method according to any one of claims 11 to 20, wherein the
human IgG1 isotype antibody is an antibody against Influenza B or
an antibody against VEGF-A or an antibody against CD22 or a
bispecific antibody against HER3 and EGFR or an antibody against
amyloid beta or an antibody against Her2 or a bispecific antibody
against Ang2 and VEGF-A or a bispecific antibody against
carcinoembryonic antigen (CEA) and CD3.
Description
[0001] The present invention relates to the field of purification
of polypeptides. The present invention in particular relates to the
reduction of host cell proteins like phospholipase B-like 2 (PLBL2)
or Clusterin in solutions containing antibodies.
BACKGROUND OF THE INVENTION
[0002] Proteins and especially immunoglobulins play an important
role in today's medical portfolio. For human application every
therapeutic protein has to meet distinct criteria. To ensure the
safety of biopharmaceutical agents to humans by-products
accumulating during the production process have to be removed
especially. To fulfill the regulatory specifications one or more
purification steps have to follow the manufacturing process. Among
other things, purity, throughput, and yield play an important role
in determining an appropriate purification process.
[0003] Different methods are well established and widespread used
for protein purification, such as affinity chromatography (e.g.
protein A or protein G affinity chromatography, single chain Fv
ligand affinity chromatography), ion exchange chromatography (e.g.
cation exchange (sulfopropyl or carboxymethyl resins), anion
exchange (amino ethyl resins) and mixed-mode ion exchange),
thiophilic adsorption (e.g. with beta-mercaptoethanol and other SH
ligands), hydrophobic interaction or aromatic adsorption
chromatography (e.g. with phenyl-sepharose, aza-arenophilic resins,
or m-aminophenylboronic acid), metal chelate affinity
chromatography (e.g. with Ni(II)- and Cu(II)-affinity material),
size exclusion chromatography, and electrophoretical methods (such
as gel electrophoresis, capillary electrophoresis).
[0004] For the purification of recombinantly produced
immunoglobulins often a combination of different column
chromatography steps is employed. During the purification
non-immunoglobulin contaminants such as host cell protein and host
cell DNA as well as endotoxins and viruses are depleted. Therefore,
generally an affinity chromatography step, like protein A affinity
chromatography is followed by one or more additional separation
steps. In general, high conductivity buffers are described to be
employed in wash steps of affinity chromatrography methods.
[0005] In U.S. Pat. No. 6,127,526 a method for purifying proteins
by Protein A chromatography is described which comprises the steps
of: (a) adsorbing the protein to Protein A immobilized on a solid
phase comprising silica or glass; (b) removing contaminants bound
to the solid phase by washing the solid phase with a hydrophobic
electrolyte solvent; and (c) recovering the protein from the solid
phase.
[0006] In WO2011/038894 a protein A chromatography method with a
pronounced depletion of host cell protein and DNA by specific wash
steps prior to the recovery of the immunoglobulin from the protein
A chromatographic material is reported.
[0007] In WO2013/177118 compositions and methods for the isolation
and purification of antibodies from a sample matrix are
reported.
[0008] In WO2013/033517 methods for separating a polypeptide of
interest (such as an antibody) from a virus are reported.
[0009] A method for purifying a protein, including one or more
chromatographic processes, in which an amino acid; or a dipeptide,
an oligopeptide, or a polyamino acid thereof is included in a
buffer solution used in at least one chromatographic process
(equilibration buffer, wash buffer, and elution buffer), thereby
purifying a high-purity protein with a very small quantity of the
impurity (e.g., polymers or host cell proteins) is reported in
EP2583973.
SUMMARY OF THE INVENTION
[0010] Herein is reported a method for the production of an
antibody with reduced content of host cell proteins by purifying
the antibody with an affinity chromatography step.
[0011] In more detail it has been found that by the method of the
current invention which uses a low conductivity aqueous solution in
a wash step of an affinity chromatography prior to the recovery of
an antibody from the chromatographic material, that the content of
certain host cell proteins in a solution comprising the antibody
can be reduced. Accordingly, it has been found that the content of
phospholipases (in particular phospholipase B-like 2 (PLBL2)) can
be reduced. It has been found that the PLBL2 content can be reduced
100-fold or more if the antibody is of the IgG4 isotype.
[0012] One aspect as reported herein is the use of a low
conductivity aqueous solution in a wash step of a protein A
chromatography for reducing the content of a host cell protein
wherein the protein A chromatography is used to purify a human IgG1
or a human IgG4 isotype antibody.
[0013] In one embodiment of this aspect the human IgG4 isotype
antibody is an antibody against P-selectin, or an bispecific
antibody against factor IXa and factor X, or an antibody against
IL-13, or an antibody against amyloid beta. In one embodiment of
this aspect the human IgG1 isotype antibody is an antibody against
Influenza B, or an antibody against VEGF-A, or an antibody against
CD22, or a bispecific antibody against HER3 and EGFR, or an
antibody against amyloid beta, or an antibody against Her2, or a
bispecific antibody against Ang2 and VEGF-A, or a bispecific
antibody against carcinoembryonic antigen (CEA) and CD3.
[0014] In one embodiment of this aspect the low conductivity
aqueous solution has a conductivity value of about 0.5 mS/cm or
less.
[0015] In one embodiment of this aspect the host cell protein is
phospholipase B-like 2 (PLBL2) or Clusterin.
[0016] In one embodiment of this aspect the low conductivity
aqueous solution comprises about 0.1 mM to about 8 mM Tris.
[0017] In one embodiment of this aspect the low conductivity
aqueous solution comprises about 0.05 mM to about 2 mM potassium
phosphate.
[0018] In one embodiment of this aspect the low conductivity
aqueous solution has a pH of about 7 or higher.
[0019] In one embodiment of this aspect the low conductivity
aqueous solution wash step is preceded or succeeded by a high
conductivity aqueous solution wash step.
[0020] In one embodiment of this aspect the high conductivity
aqueous solution has a conductivity value of about 20 mS/cm or
higher.
[0021] In one embodiment of this aspect an intermediate wash step
is performed with a medium conductivity aqueous solution between
the low conductivity aqueous solution wash step and the high
conductivity aqueous solution wash step.
[0022] In one embodiment of this aspect the medium conductivity
aqueous solution has a conductivity value of from more than 0.5
mS/cm to less than 20 mS/cm.
[0023] In one embodiment of this aspect the high (or medium)
conductivity aqueous solution comprises Histidine.
[0024] One aspect as reported herein is a method for producing a
human IgG4 or IgG1 isotype antibody comprising the steps of [0025]
a) cultivating a cell comprising a nucleic acid encoding a human
IgG4 or IgG1 isotype antibody, [0026] b) recovering the human IgG4
or IgG1 isotype antibody from the cell or the cultivation medium,
[0027] c) contacting the human IgG4 or IgG1 isotype antibody with a
protein A chromatography material, [0028] d) washing the protein A
chromatography material with a low conductivity aqueous solution,
[0029] e) recovering the human IgG4 or IgG1 isotype antibody from
the protein A chromatography material and thereby producing the
human IgG4 or IgG1 isotype antibody.
[0030] One aspect as reported herein is method for purifying a
human IgG4 or IgG1 isotype antibody from a sample comprising the
steps of [0031] a) providing a sample comprising a human IgG4 or
IgG1 isotype antibody, [0032] b) purifying the human IgG4 or IgG1
isotype antibody with a protein A chromatography method/step,
comprising washing the protein A chromatography material with a low
conductivity aqueous solution.
[0033] In one embodiment of all aspects the human IgG4 isotype
antibody is an antibody against P-selectin or a bispecific antibody
against factor IXa and factor X or an antibody against IL-13 or an
antibody against amyloid beta. In one embodiment of all aspects the
human IgG1 isotype antibody is an antibody against Influenza B or
an antibody against VEGF-A or an antibody against CD22 or a
bispecific antibody against HER3 and EGFR or an antibody against
amyloid beta or an antibody against Her2 or a bispecific antibody
against Ang2 and VEGF-A, or a bispecific antibody against
carcinoembryonic antigen (CEA) and CD3.
[0034] In one embodiment of all aspects the low conductivity
aqueous solution has a conductivity value of about 0.5 mS/cm or
less.
[0035] In one embodiment of all aspects the content of a host cell
protein is reduced and the (specific) host cell protein is
phospholipase B-like 2 (PLBL2) or Clusterin.
[0036] In one embodiment of all aspects the low conductivity
aqueous solution comprises about 0.1 mM to about 8 mM Tris.
[0037] In one embodiment of all aspects the low conductivity
aqueous solution comprises about 0.05 mM to about 2 mM potassium
phosphate.
[0038] In one embodiment of all aspects the low conductivity
aqueous solution has a pH of about 7 or higher.
[0039] In one embodiment of all method aspects the method
additionally comprises washing the affinity chromatography material
with a high conductivity aqueous solution and/or with a medium
conductivity aqueous solution before or after washing the protein A
chromatography material with low conductivity aqueous solution.
[0040] In one embodiment of all aspects the high conductivity
aqueous solution has a conductivity value of about 20 mS/cm or
higher.
[0041] In one embodiment of all aspects the medium conductivity
aqueous solution has a conductivity value of from more than 0.5
mS/cm to less than 20 mS/cm.
[0042] In one embodiment of all aspects the high or medium
conductivity aqueous solution comprises Histidine.
DETAILED DESCRIPTION OF THE INVENTION
[0043] Herein is reported an improved affinity chromatography
method and use comprising the washing of the affinity
chromatography material with a low conductivity aqueous
solution.
[0044] It has been found that specific host cell proteins can be
reduced with a wash step with a low conductivity aqueous solution,
when this wash step is used in an affinity chromatography step,
e.g. a protein A chromatrography step. The affinity chromatography
step is used in a purification or production method for antibodies.
The low conductivity aqueous solution wash step is particularly
effective to reduce the content of phospholipase B-like 2
(PLBL2).
[0045] One aspect as reported herein is the use of a low
conductivity aqueous solution in a wash step of an affinity
chromatography for reducing the content of a (specific) host cell
protein.
[0046] One aspect as reported herein is a method for producing a
human IgG isotype antibody comprising [0047] a) cultivating a cell
comprising a nucleic acid encoding the human IgG isotype antibody,
[0048] b) recovering the human IgG isotype antibody from the cell
or the cultivation medium, [0049] c) contacting (a solution
comprising) the human IgG isotype antibody with an affinity
chromatography material, [0050] d) washing the affinity
chromatography material with a low conductivity aqueous solution,
while at least 90% the bispecific antibody remains bound to the
affinity chromatography material, [0051] e) recovering the human
IgG isotype antibody from affinity chromatography material and
thereby producing the human IgG isotype antibody.
[0052] One aspect as reported herein is a method for purifying a
human IgG isotype antibody from a sample comprising the steps of
[0053] a) providing a (buffered aqueous) sample comprising a human
IgG isotype antibody, [0054] b) purifying the human IgG isotype
antibody with a affinity chromatography method/step, comprising
washing the affinity chromatography material with low conductivity
aqueous solution.
[0055] Recombinant polypeptides produced in CHO cells may be
purified according to the methods described herein to remove or
reduce levels of a host cell proteins.
[0056] Exemplary recombinant polypeptides include therapeutic
antibodies and immunoadhesins, including, without limitation,
antibodies, including antibody fragments, to one or more of the
following antigens: HER1 (EGFR), HER2 (e.g., trastuzumab,
pertuzumab), HER3, HER4, VEGF (e.g., bevacizumab, ranibizumab), MET
(e.g., onartuzumab), CD20 (e.g., rituximab, obinutuzumab,
ocrelizumab), CD22, CD11a, CD11b, CD11c, CD18, an ICAM, VLA-4,
VCAM, IL-17A and/or F, IgE (e.g., omalizumab), DRS, CD40,
Apo2L/TRAIL, EGFL7 (e.g., parsatuzumab), NRP1, integrin beta7
(e.g., etrolizumab), IL-13 (e.g., lebrikizumab), Abeta (e.g.,
crenezumab, gantenerumab), P-selectin (e.g., inclacumab), IL-6R
(e.g., tociluzumab), IFNa (e.g., rontalizumab), M1prime (e.g.,
quilizumab), mitogen activated protein kinase (MAPK), OX40L, TSLP,
Factor D (e.g., lampalizumab) and receptors such as: IL-9 receptor,
IL-5 receptor, IL-4receptor alpha, IL-13receptoralphal and
IL-13receptoralpha2, OX40, TSLP-R, IL-7R alpha (a co-receptor for
TSLP), IL17RB (receptor for IL-25), ST2 (receptor for IL-33), CCR3,
CCR4, CRTH2, FcepsilonRI and FcepsilonRII/CD23 (receptors for IgE).
Other exemplary antibodies include those selected from, and without
limitation, antiestrogen receptor antibody, anti-progesterone
receptor antibody, anti-p53 antibody, anticathepsin D antibody,
antiBcl-2 antibody, anti-E-cadherin antibody, anti-CA125 antibody,
anti-CA15-3 antibody, antiCA19-9 antibody, anti-c-erbB-2 antibody,
anti-P-glycoprotein antibody, anti-CEA antibody, Ki-67 antibody,
anti-PCNA antibody, anti-CD3 antibody, anti-CD4 antibody, anti-CD5
antibody, anti-CD7 antibody, anti-CD8 antibody, anti-CD9/p24
antibody, anti-CD10 antibody, anti-CD11c antibody, anti-CD13
antibody, anti-CD14 antibody, anti-CD15 antibody, anti-CD19
antibody, anti-CD23 antibody, anti-CD30 antibody, anti-CD31
antibody, anti-CD33 antibody, anti-CD34 antibody, anti-CD35
antibody, anti-CD38 antibody, anti-CD41 antibody, antiLCA/CD45
antibody, anti-CD45RO antibody, anti-CD45RA antibody, anti-CD39
antibody, anti-CD100 antibody, anti-CD95/Fas antibody, anti-CD99
antibody, anti-CD106 antibody, antiubiquitin antibody, anti-CD71
antibody, anti-c-myc antibody, anti-cytokeratins antibody,
antivimentins antibody, anti-HPV proteins antibody, anti-kappa
light chains antibody, anti-lambda light chains antibody,
anti-melanosomes antibody, anti-prostate specific antigen antibody,
antiS-100 antibody, anti-tau antigen antibody, anti-fibrin
antibody, anti-keratins antibody and antiTn-antigen antibody.
[0057] In some embodiments, exemplary antibodies include antibodies
to Abeta, antibodies to IL17 A/F and antibodies to CMV. Exemplary
anti-Abeta antibodies and methods of producing such antibodies have
been described previously, for example, in WO2008011348,
WO2007068429, WO2001062801, and WO2004071408. Exemplary anti-IL17
A/F antibodies and methods of producing such antibodies have been
described previously, for example, in WO 2009136286 and U.S. Pat.
No. 8,715,669. Exemplary anti-CMV antibodies, including
anti-CMV-MSL, and methods of producing such antibodies have been
described previously, for example, in WO 2012047732.
[0058] In some embodiments the affinity chromatography is used to
purify a human IgG isotype antibody. In some embodiments the
affinity chromatography is used to purify an IgG4 antibody. In one
embodiment the IgG4 isotype antibody is an antibody against
P-selectin or a (bispecific) antibody against factor IXa and factor
X or an antibody against IL-13 or an antibody against amyloid beta.
In some embodiments the affinity chromatography is used to purify
an IgG1 isotype antibody. In one embodiment the IgG1 isotype
antibody is an antibody against Influenza B or an antibody against
VEGF-A or an antibody against CD22 or an (bispecific) antibody
against HER3 and EGFR or an antibody against amyloid beta or an
antibody against Her2 or a bispecific antibody against Ang2 and
VEGF-A or a bispecific antibody against carcinoembryonic antigen
(CEA) and CD3.
[0059] One aspect as reported herein is a method for producing a
human IgG4 isotype antibody (containing solution) comprising [0060]
a) cultivating a cell comprising a nucleic acid encoding a human
IgG4 isotype antibody, [0061] b) recovering the human IgG4 isotype
antibody from the cell or the cultivation medium, [0062] c)
contacting the human IgG4 isotype antibody with an affinity
chromatography material, [0063] d) washing the affinity
chromatography material with a low conductivity aqueous solution,
[0064] e) recovering the human IgG4 isotype antibody from the
affinity chromatography material and thereby producing the human
IgG4 isotype antibody.
[0065] One aspect as reported herein is a method for producing an
IgG4 isotype antibody (containing solution) comprising [0066] a)
cultivating a cell comprising a nucleic acid encoding an IgG4
isotype antibody, [0067] b) recovering the IgG4 isotype antibody
from the cell or the cultivation medium, [0068] c) contacting the
IgG4 isotype antibody with an affinity chromatography material,
[0069] d) washing the affinity chromatography material with a low
conductivity aqueous solution, [0070] e) recovering the IgG4
isotype antibody from the affinity chromatography material and
thereby producing the IgG4 isotype antibody.
[0071] One aspect as reported herein is a method for purifying a
human IgG4 isotype antibody from a sample comprising the steps of
[0072] a) providing a sample comprising a human IgG4 isotype
antibody, [0073] b) purifying the human IgG4 isotype antibody with
a affinity chromatography method/step, comprising washing the
affinity chromatography material with a low conductivity aqueous
solution.
[0074] One aspect as reported herein is a method for purifying an
IgG4 isotype antibody from a sample comprising the steps of [0075]
a) providing a sample comprising an IgG4 isotype antibody, [0076]
b) purifying the IgG4 isotype antibody with a affinity
chromatography method/step, comprising washing the affinity
chromatography material with a low conductivity aqueous
solution.
[0077] It has been found that the content of a host cell protein
can be reduced if the conductivity of the aqueous solution used in
the wash step is low i.e a low conductivity aqueous solution is
used for washing. In one embodiment of all aspects the low
conductivity aqueous solution has a conductivity value of about 1
mS/cm or less. In one preferred embodiment of all aspects the low
conductivity aqueous solution has a conductivity value of about 0.5
mS/cm or less. In one embodiment the low conductivity aqueous
solution has a conductivity value of from about 0.03 .mu.S/cm to
about 0.5 mS/cm. In one embodiment the low conductivity aqueous
solution has a conductivity value of from about 0.05 .mu.S/cm to
about 0.35 mS/cm. In one embodiment of all aspects the low
conductivity aqueous solution is deionized water. For some
applications deionized water is not suitable to be used in a wash
step. In some embodiments the low conductivity aqueous solution is
not deionized water.
[0078] It has been found that a protein A affinity chromatography
can be used for the purposes as reported herein. In one preferred
embodiment of all aspects the affinity chromatography is a protein
A affinity chromatography. In one embodiment the protein A affinity
chromatography is selected from the group comprising MabSelectSure
affinity chromatography, ProSep vA affinity chromatography, Poros
Mab Capture A affinity chromatography, ProSep Ultra Plus affinity
chromatography, MabSelect SuRe LX, MabSelect, Eshmuno A, Toyopearl
AF-rProtein A-650F; Toyopearl AF-rProtein A HC-650HF). In one
embodiment the affinity chromatography is a protein G affinity
chromatography. In one embodiment the affinity chromatography is an
affinity chromatography that uses a recombinant protein as a
ligand, that means that the affinity chromatography is a
recombinant protein ligand affinity chromatography. In one
embodiment the affinity chromatography is an affinity
chromatography that uses a single chain Fv as a ligand, that means
that the affinity chromatography is a single chain Fv ligand
affinity chromatography. In one embodiment the affinity
chromatography comprises a mutated Protein A coupled to a
chromatography matrix or a fragment of Protein A coupled to a
chromatography matrix.
[0079] It has been found that the content of (specific) host cell
proteins can be reduced. It has been found that especially the
content of phospholipase B-like 2 (PLBL2) can be reduced. In one
embodiment the (specific) host cell protein is a Chinese hamster
ovary (CHO) host cell protein. In one preferred embodiment of all
aspects the (specific) host cell protein is phospholipase B-like 2
(PLBL2) or Clusterin. In one embodiment the (specific) host cell
protein is phospholipase B-like 2 (PLBL2).
[0080] It has been found that low conductivity aqueous solution may
comprise certain buffering substances e.g. Tris or potassium
phosphate in low amounts. In one embodiment the low conductivity
aqueous solution contains tris(hydroxymethyl)aminomethane (Tris).
In one embodiment the low conductivity aqueous solution comprises
about 0.1 mM to about 10 mM Tris. In one embodiment the low
conductivity aqueous solution comprises about 0.5 mM to about 6.5
mM Tris. In one embodiment the low conductivity aqueous solution
comprises about 2 mM Tris. In one embodiment the low conductivity
aqueous solution contains potassium phosphate. In one embodiment
the low conductivity aqueous solution comprises about 0.05 mM to
about 5 mM potassium phosphate. In one embodiment the low
conductivity aqueous solution comprises about 0.05 mM to about 2 mM
potassium phosphate. In one embodiment the low conductivity aqueous
solution comprises about 0.5 mM potassium phosphate.
[0081] It has been found that the effect of reducing the content of
a host cell protein is pronounced if the low conductivity aqueous
solution has a certain pH. In one embodiment the low conductivity
aqueous solution has a pH of about 7 or higher. In one embodiment
the low conductivity aqueous solution has a pH of about 7.5 or
higher. In one embodiment the low conductivity aqueous solution has
a pH of from about 7 to about 9.5. In one embodiment the low
conductivity aqueous solution has a pH of from about 7.5 to about
8.5. In one embodiment the low conductivity aqueous solution has a
pH of about 8. In one embodiment the low conductivity aqueous
solution has a pH of about 9.
[0082] It has been found that the effect of reducing the content of
a host cell protein can also be achieved if the pH of the low
conductivity aqueous solution is about 8.5 or higher and the low
conductivity aqueous solution has a conductivity value of about 1.2
mS/cm or less. In one embodiment the low conductivity aqueous
solution has a pH of about 8.5 or higher and the low conductivity
aqueous solution has a conductivity value of about 1.2 mS/cm or
less. In one embodiment the low conductivity aqueous solution has a
pH of about 8.5 or higher and the low conductivity aqueous solution
has a conductivity value of about 1 mS/cm or less. In one
embodiment low conductivity aqueous solution has a pH of about 8.5
or higher and the low conductivity aqueous solution comprises about
55 mM Tris or less. In one embodiment low conductivity aqueous
solution has a pH of about 8.5 or higher and the low conductivity
aqueous solution comprises about 30 mM Tris or less.
[0083] In one embodiment the low conductivity aqueous solution is
in the pH range of from pH 7 to less than pH 8.5 and has a
conductivity value of about 0.5 mS/cm or less and at a pH value of
8.5 or more a conductivity value of about 1.2 mS/cm or less.
[0084] It has been found that by the uses and the methods as
reported herein the content of host cell proteins like PLBL2 can be
reduced to a certain level, e.g. when compared to the load amount
of PLBL2 prior to a purification step like an affinity
chromatography step. In one embodiment the content of PLBL2 is
reduced at least 20-fold. In one embodiment the content of PLBL2 is
reduced at least 40-fold. In one embodiment the content of PLBL2 is
reduced at least 50-fold. In one embodiment the content of PLBL2 is
reduced at least 90-fold. In one embodiment the content of PLBL2 is
reduced at least 100-fold. In some cases the level of reduction is
even higher. In some embodiments the content of PLBL2 is reduced at
least 200-fold. In some embodiments the content of PLBL2 is reduced
at least 250-fold. In some embodiments the content of PLBL2 is
reduced at least 300-fold. In some embodiments the content of PLBL2
is reduced at least 400-fold. In some embodiments the content of
PLBL2 is reduced at least1000-fold. In one embodiment the content
of PLBL2 is reduced at least by 50%. In one embodiment the content
of PLBL2 is reduced at least by 66%. In one embodiment the content
of PLBL2 is reduced at least by 80%. In one embodiment the content
of PLBL2 is reduced at least by 90%. In one embodiment the content
of PLBL2 is reduced at least by 95%. In some embodiments the
content of PLBL2 is reduced to below 10 ng per mg of antibody. In
some embodiments the content of PLBL2 is reduced to below 5 ng per
mg of antibody. In some embodiments the content of PLBL2 is reduced
to below 2 ng per mg of antibody.
[0085] In the methods and the uses as reported herein further wash
steps can be employed with medium and/or high conductivity aqueous
solutions. In one embodiment the low conductivity aqueous solution
wash step is preceded or succeeded by a high conductivity aqueous
solution wash step. In one embodiment the high conductivity aqueous
solution has a conductivity value of about 20 mS/cm or higher. In
one embodiment the high conductivity aqueous solution has a
conductivity value of from about 20 mS/cm to about 100 mS/cm. In
one embodiment an intermediate wash step is performed with a medium
conductivity aqueous solution between the low conductivity aqueous
solution wash step and the high conductivity aqueous solution wash
step. In one embodiment the medium conductivity aqueous solution
has a conductivity value of from more than 0.5 mS/cm to less than
20 mS/cm.
[0086] It has been found that the host cell protein reducing effect
can be improved when the high or medium conductivity aqueous
solution further comprises an amino acid. In one embodiment the
high or medium conductivity aqueous solution comprises an amino
acid. In one embodiment the high or medium conductivity aqueous
solution comprises Histidine or Arginine. In one embodiment the
high or medium conductivity aqueous solution comprises Histidine.
In one embodiment the high or medium conductivity aqueous solution
comprises Histidine and Tris.
[0087] The methods and the uses as reported herein may include one
or more further chromatography steps. In one embodiment at least
one additional chromatography method/step is performed. In one
embodiment an additional ion exchange chromatography method/step is
performed. In one embodiment an additional anion exchange
chromatography method/step is performed. In one embodiment an
additional anion exchange chromatography method/step and an
additional cation exchange chromatography method/step are
performed.
[0088] It has been found that the use of a hydrophobic interaction
chromatography step may be omitted. In one embodiment the use or
the methods is without an hydrophobic interaction chromatography
method/step.
[0089] One aspect as reported herein is the use of a low
conductivity aqueous solution in a wash step of a protein A
chromatography for reducing the content of PLBL2 or Clusterin
wherein the protein A chromatography is used to purify an IgG4 or
IgG1 isotype, e.g., a human IgG4 or IgG1, antibody and wherein the
low conductivity aqueous solution has a conductivity value of about
0.5 mS/cm or less and a pH of about 7 or higher.
[0090] One aspect is the use of a low conductivity aqueous solution
in a wash step of a protein A chromatography for reducing the
content of PLBL2 or Clusterin wherein the protein A chromatography
is used to purify a human IgG4 or IgG1 isotype antibody and wherein
the low conductivity aqueous solution has a conductivity value of
about 0.5 mS/cm or less and a pH of about 7 or higher. In some
embodiments, the antibody is an IgG4 isotype antibody, e.g., an
antibody against P-selectin, or a bispecific antibody against
factor IXa and factor X, or an antibody against IL-13, or an
antibody against amyloid beta. In some embodiments, the antibody is
a IgG1 isotype antibody, e.g., an antibody against Influenza B, or
an antibody against VEGF-A, or an antibody against CD22, or a
bispecific antibody against HER3 and EGFR, or an antibody against
amyloid beta, or an antibody against Her2, or a bispecific antibody
against Ang2 and VEGF-A, or a bispecific antibody against
carcinoembryonic antigen (CEA) and CD3.
[0091] In an aspect, the present disclosure provides a method for
producing a human IgG4 or IgG1 isotype antibody comprising [0092]
a) cultivating a cell comprising a nucleic acid encoding the human
IgG4 or IgG1 isotype antibody, [0093] b) recovering the human IgG4
or IgG1 isotype antibody from the cell or the cultivation medium,
[0094] c) contacting the human IgG4 or IgG1 isotype antibody with a
protein A affinity chromatography material, [0095] d) washing the
protein A affinity chromatography material with a low conductivity
aqueous solution, [0096] e) recovering the human IgG4 or IgG1
isotype antibody from affinity chromatography material and thereby
producing the human IgG4 or IgG1 isotype antibody, wherein the low
conductivity aqueous solution has a conductivity value of about 0.5
mS/cm or less and a pH of about 7 or higher.
[0097] In an aspect, the present disclosure provides a method for
producing a human IgG4 or IgG1 isotype antibody comprising [0098]
a) cultivating a cell comprising a nucleic acid encoding the human
IgG4 or IgG1 isotype antibody, [0099] b) recovering the human IgG4
or IgG1 isotype antibody from the cell or the cultivation medium,
[0100] c) contacting the human IgG4 or IgG1 isotype antibody with a
protein A affinity chromatography material, [0101] d) washing the
protein A affinity chromatography material with a low conductivity
aqueous solution, [0102] e) recovering the human IgG4 or IgG1
isotype antibody from affinity chromatography material and thereby
producing the human IgG4 or IgG1 isotype antibody, wherein the low
conductivity aqueous solution has a conductivity value of about 0.5
mS/cm or less and a pH of about 7 or higher, and wherein the human
IgG4 isotype antibody is an antibody against P-selectin, or a
bispecific antibody against factor IXa and factor X, or an antibody
against IL-13, or an antibody against amyloid beta and wherein the
human IgG1 isotype antibody is an antibody against Influenza B, or
an antibody against VEGF-A, or an antibody against CD22, or a
bispecific antibody against HER3 and EGFR, or an antibody against
amyloid beta, or an antibody against Her2, or a bispecific antibody
against Ang2 and VEGF-A, or a bispecific antibody against
carcinoembryonic antigen (CEA) and CD3.
[0103] In an aspect, the present disclosure provides a method for
purifying a human IgG4 or IgG1 isotype antibody from a sample
comprising the steps of [0104] a) providing a sample comprising a
human IgG4 or IgG1 isotype antibody, [0105] b) purifying the human
IgG4 or IgG1 isotype antibody with a protein A affinity
chromatography method/step, comprising washing the protein A
affinity chromatography material with low conductivity aqueous
solution, wherein the low conductivity aqueous solution has a
conductivity value of about 0.5 mS/cm or less and a pH of about 7
or higher.
[0106] In an aspect, the present disclosure provides a method for
purifying a human IgG4 or IgG1 isotype antibody from a sample
comprising the steps of [0107] a) providing a sample comprising a
human IgG4 or IgG1 isotype antibody, [0108] b) purifying the human
IgG4 or IgG1 isotype antibody with a protein A affinity
chromatography method/step, comprising washing the protein A
affinity chromatography material with low conductivity aqueous
solution, wherein the low conductivity aqueous solution has a
conductivity value of about 0.5 mS/cm or less and a pH of about 7
or higher, and wherein the human IgG4 isotype antibody is an
antibody against P-selectin, or an antibody against factor IXa and
factor X, or an antibody against IL-13, or an antibody against
amyloid beta and wherein the human IgG1 isotype antibody is an
antibody against Influenza B, or an antibody against VEGF-A, or an
antibody against CD22, or a antibody against HER3 and EGFR, or an
antibody against amyloid beta, or an antibody against Her2, or a
bispecific antibody against Ang2 and VEGF-A, or a bispecific
antibody against carcinoembryonic antigen (CEA) and CD3.
[0109] In an aspect, the present disclosure provides a method for
producing a human IgG4 isotype antibody comprising [0110] a)
cultivating a cell comprising a nucleic acid encoding the human
IgG4 isotype antibody, [0111] b) recovering the human IgG4 isotype
antibody from the cell or the cultivation medium, [0112] c)
contacting the human IgG4 isotype antibody with a protein A
affinity chromatography material, [0113] d) washing the protein A
affinity chromatography material with a low conductivity aqueous
solution, [0114] e) recovering the human IgG4 isotype antibody from
affinity chromatography material and thereby producing the human
IgG4 isotype antibody, wherein the low conductivity aqueous
solution has a conductivity value of about 0.5 mS/cm or less and a
pH of about 7 or higher, and wherein the human IgG4 isotype
antibody is antibody against factor IXa and factor X.
[0115] In an aspect, the present disclosure provides a method for
purifying a human IgG4 isotype antibody from a sample comprising
the steps of [0116] a) providing a sample comprising a human IgG4
isotype antibody, [0117] b) purifying the human IgG4 isotype
antibody with a protein A affinity chromatography method/step,
comprising washing the protein A affinity chromatography material
with low conductivity aqueous solution, wherein the low
conductivity aqueous solution has a conductivity value of about 0.5
mS/cm or less and a pH of about 7 or higher, and wherein the human
IgG4 isotype antibody is an antibody against factor IXa and factor
X.
[0118] The terms "anti-P-selectin antibody" and "an antibody that
binds to P-selectin" or "antibody against P-selectin"refer to an
antibody that is capable of binding P-selectin with sufficient
affinity such that the antibody is useful as a diagnostic and/or
therapeutic agent in targeting P-selectin. In one embodiment, the
extent of binding of an anti-P-selectin antibody to an unrelated,
non-P-selectin protein is less than about 10% of the binding of the
antibody to P-selectin as measured, e.g., by ELISA or surface
plasmon resonance. In certain embodiments, an anti-P-selectin
antibody binds to an epitope of P-selectin that is conserved among
P-selectin from different species. The above also holds for the
terms "antibody against factor IXa and factor X" or "antibody
against IL-13" or "antibody against amyloid beta" or the like.
[0119] In some embodiments, the antibody against P-selectin is
inclacumab (IgG4 isotype) as described in WO 2005/100402 or SEQ ID
NO: 07 to 12. In some embodiments, the antibody is a bispecific
antibody against factor IXa and factor X, e.g., anti-FIXa/X
antibody (IgG4 isotype) as described in WO 2012/067176. In some
embodiments, the antibody is an antibody against Her2, e.g.,
trastuzumab (IgG1 isotype) as described in WO 1992/022653. In some
embodiments, the antibody is a bispecific antibody against
angiopoietin 2 (Ang2) and vascular endothelial growth factor A
(VEGF-A), e.g., vanucizumab (IgG1 isotype) as described in WO
2011/117329 or SEQ ID NO: 01 to 04. In some embodiments, the
antibody is an antibody against amyloid beta, e.g., gantenerumab
(IgG1 isotype) as described in WO 2003/070760 or SEQ ID NO: 05 to
06, or crenezumab (IgG4 isotype). In some embodiments, the antibody
is an antibody against CD22, an antibody against IL13 (e.g.,
lebrikizumab), a bispecific antibody against Her3 and EGFR (e.g.,
duligotuzumab), an antibody against VEGF-A (e.g., bevacizumab), and
an antibody against Influenza B. The terms VEGF or VEGF-A can be
used interchangeably herein.
[0120] As used herein, the term "binding" or "specifically binding"
refers to the binding of the antibody to an epitope of the antigen
in an in-vitro assay, preferably in a surface plasmon resonance
assay (SPR, BIAcore, GE-Healthcare Uppsala, Sweden). The affinity
of the binding is defined by the terms ka (rate constant for the
association of the antibody from the antibody/antigen complex),
k.sub.d (dissociation constant), and K.sub.D (k.sub.d/k.sub.a).
Binding or specifically binding means a binding affinity (K.sub.D)
of 10.sup.-7 mol/L or less.
[0121] The term "antibody" herein is used in the broadest sense and
encompasses various antibody structures, including but not limited
to monoclonal antibodies, polyclonal antibodies, multispecific
antibodies (e.g., bispecific antibodies), and antibody fragments so
long as they exhibit the desired antigen-binding activity.
[0122] An "antibody fragment" refers to a molecule other than an
intact antibody that comprises a portion of an intact antibody that
binds the antigen to which the intact antibody binds. Examples of
antibody fragments include but are not limited to Fv, Fab, Fab',
Fab'-SH, F(ab').sub.2; diabodies; linear antibodies; single-chain
antibody molecules (e.g. scFv); and multispecific antibodies formed
from antibody fragments. A Fab fragment is an antibody fragment
obtained by a papain digestion of a (full length/complete)
antibody.
[0123] Bispecific antibodies" are antibodies which have two
different antigen-binding specificities. The term "bispecific"
antibody as used herein denotes an antibody that has at least two
binding sites each of which bind to different epitopes.
[0124] The term "chimeric" antibody refers to an antibody in which
a portion of the heavy and/or light chain is derived from a
particular source or species, while the remainder of the heavy
and/or light chain is derived from a different source or
species.
[0125] The "class" of an antibody refers to the type of constant
domain or constant region possessed by its heavy chain. There are
five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and
several of these may be further divided into subclasses (isotypes),
e.g., IgG.sub.1, IgG.sub.2, IgG.sub.3, IgG.sub.4, IgA.sub.1, and
IgA.sub.2. The heavy chain constant domains that correspond to the
different classes of immunoglobulins are called .alpha., .delta.,
.epsilon., .gamma., and .mu., respectively.
[0126] The term "human IgG isotype antibody" denotes an antibody
that comprises a constant region that is derived from a human
wild-type IgG isotype, i.e. for example it may comprise a constant
region derived from a human IgG isotype with a mutation, e.g. an
P329G mutation (numbering according to Kabat).
[0127] The term "human IgG4 isotype antibody" denotes an antibody
that comprises a constant region that is derived from a human
wild-type IgG4 isotype, i.e. for example it may comprise a constant
region derived from a human IgG4 isotype with a mutation, e.g. an
an P329G mutation and/or S228P, L235E mutation (numbering according
to Kabat).
[0128] The term "Fc-region" herein is used to define a C-terminal
region of an immunoglobulin heavy chain that contains at least a
portion of the constant region. The term includes native sequence
Fc-regions and variant Fc-regions. In one embodiment, a human IgG
heavy chain Fc-region extends from Cys226, or from Pro230, to the
carboxyl-terminus of the heavy chain. However, the C-terminal
lysine (Lys447) or the C-terminal glycyl-lysine dipeptide
(Gly446Lys447) of the Fc-region may or may not be present. Unless
otherwise specified herein, numbering of amino acid residues in the
Fc-region or constant region is according to the EU numbering
system, also called the EU index, as described in Kabat, E. A. et
al., Sequences of Proteins of Immunological Interest, 5th ed.,
Public Health Service, National Institutes of Health, Bethesda, Md.
(1991), NIH Publication 91-3242.
[0129] "Framework" or "FR" refers to variable domain residues other
than hypervariable region (HVR) residues. The FR of a variable
domain generally consists of four FR domains: FR1, FR2, FR3, and
FR4. Accordingly, the HVR and FR sequences generally appear in the
following sequence in VH (or VL):
FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.
[0130] The terms "host cell", "host cell line", and "host cell
culture" are used interchangeably and refer to cells into which
exogenous nucleic acid has been introduced, including the progeny
of such cells. Host cells include "transformants" and "transformed
cells," which include the primary transformed cell and progeny
derived therefrom without regard to the number of passages. Progeny
may not be completely identical in nucleic acid content to a parent
cell, but may contain mutations. Mutant progeny that have the same
function or biological activity as screened or selected for in the
originally transformed cell are included herein. The term "cell"
includes cells which are used for the expression of nucleic acids.
In one embodiment the host cell is a CHO cell (e.g. CHO K1, CHO
DG44), or a BHK cell, or a NS0 cell, or a SP2/0 cell, or a HEK 293
cell, or a HEK 293 EBNA cell, or a PER.C6.RTM. cell, or a COS
cells. In another embodiment the cell is a CHO cell, or a BHK cell,
or a PER.C6.RTM. cell. As used herein, the expression "cell"
includes the subject cell and its progeny.
[0131] The term "washing" denotes the applying of a solution to an
affinity chromatography material in order to remove non
specifically bound polypeptides and non-polypeptide compounds from
the chromatography material, especially to remove host cell protein
and host cell DNA. The term "washing" does not encompass the
elution of bound material from an affinity chromatography
material.
[0132] Different methods are well established and widespread used
for protein recovery and purification, such as affinity
chromatography with microbial proteins (e.g. protein A or protein G
affinity chromatography) affinity chromatographie with a
recombinant protein as ligand (e.g. single chain Fv as ligand, e.g.
Kappa select), ion exchange chromatography (e.g. cation exchange
(carboxymethyl resins), anion exchange (amino ethyl resins) and
mixed-mode exchange), thiophilic adsorption (e.g. with
beta-mercaptoethanol and other SH ligands), hydrophobic interaction
or aromatic adsorption chromatography (e.g. with phenyl-sepharose,
aza-arenophilic resins, or m-aminophenylboronic acid), metal
chelate affinity chromatography (e.g. with Ni(II)- and
Cu(II)-affinity material), size exclusion chromatography, and
electrophoretical methods (such as gel electrophoresis, capillary
electrophoresis). These methods can be combined independently in
different embodiments as reported herein.
[0133] The term "protein A" denotes a protein A polypeptide either
obtained from a natural source or produced synthetically.
[0134] The term "protein A chromatography material" denotes an
inert solid phase to which a protein A is covalently linked.
[0135] In one embodiment the protein A chromatography material is
selected from MabSelectSure, ProSep vA, Mab Capture A, ProSep Ultra
Plus, Mab Select, Mab Select Xtra, Poros A, or ProSep A.
[0136] The term "high conductivity aquaeous solution" denotes an
aquaeous solution with a high conductivity value. The conductivity
value may be about 20 mS/cm or higher.
[0137] The term "medium conductivity aquaeous solution" denotes an
aquaeous solution with a medium conductivity value. The
conductivity value may be more than 0.5 mS/cm to less than 20
mS/cm.
[0138] The term "low conductivity aquaeous solution" denotes an
aquaeous solution with a low conductivity value. The conductivity
value may be about 0.5 mS/cm or less. The conductivity value may be
about 1.2 mS/cm or less, if the pH is about 8.5 or higher. The
conductivity values can be determined by standard methods known to
the person skilled in the art.
[0139] The following examples and sequences are provided to aid the
understanding of the present invention, the true scope of which is
set forth in the appended claims. It is understood that
modifications can be made in the procedures set forth without
departing from the spirit of the invention.
Specific Embodiments of the Invention
[0140] 1. Use of a low conductivity aqueous solution in a wash step
of an affinity chromatography for reducing the content of a host
cell protein. [0141] 2. Use according to embodiment 1, wherein the
affinity chromatography is used to purify a human IgG isotype
antibody. [0142] 3. Use according to embodiment 2, wherein the
affinity chromatography is used to purify a human IgG4 isotype
antibody or a human IgG1 isotype antibody. [0143] 4. Use according
to embodiment 3, wherein the affinity chromatography is used to
purify a human IgG4 isotype antibody or a human IgG1 isotype
antibody without a glycosylated glycosylation site in its Fab
fragment/with exactly one glycosylation site (at position Asn 297
numbering according to Kabat). [0144] 5. Use according to
embodiment 4, wherein the low conductivity aqueous solution has a
conductivity value of about 0.5 mS/cm or less. [0145] 6. Use
according to embodiment 5, wherein the low conductivity aqueous
solution has a conductivity value of from about 0.03 .mu.S/cm to
about 0.5 mS/cm. [0146] 7. Use according to embodiment 5, wherein
the low conductivity aqueous solution has a conductivity value of
from about 0.05 .mu.S/cm to about 0.35 mS/cm. [0147] 8. Use
according to any of embodiments 5 to 7, wherein the low
conductivity aqueous solution is not deionized water. [0148] 9. Use
according to any of the previous embodiments, wherein the affinity
chromatography is a protein A affinity chromatography or a Protein
G affinity chromatography or a single chain Fv ligand (KappaSelect)
affinity chromatography. [0149] 10. Use according to embodiment 9,
wherein the affinity chromatography is a protein A affinity
chromatography. [0150] 11. Use according to embodiment 10, wherein
the protein A affinity chromatography is selected from the group
comprising MabSelectSure affinity chromatography, ProSep vA
affinity chromatography, Poros Mab Capture A affinity
chromatography, ProSep Ultra Plus affinity chromatography,
MabSelect SuRe LX, MabSelect, Eshmuno A, Toyopearl AF-rProtein
A-650F; Toyopearl AF-rProtein A HC-650HF). [0151] 12. Use according
to any one of the previous embodiments, wherein said host cell
protein is a Chinese hamster ovary (CHO) host cell protein. [0152]
13. Use according to embodiment 12, wherein the host cell protein
is a phospholipase. [0153] 14. Use according to embodiment 13,
wherein the host cell protein is a phospholipase A, phospholipase
B, phospholipase C or phospholipase D.
[0154] 15. Use according to embodiments 12, 13 or 14, wherein the
host cell protein is phospholipase B-like 2 (PLBL2) [0155] 16. Use
according to embodiment 12, wherein the host cell protein is
phospholipase B-like 2 (PLBL2) or Clusterin. [0156] 17. Use
according to any of the preceding embodiments, wherein the low
conductivity aqueous solution contains
tris(hydroxymethyl)aminomethane (Tris). [0157] 18. Use according to
embodiment 17, wherein the low conductivity aqueous solution
comprises about 0.1 mM to about 10 mM Tris. [0158] 19. Use
according to embodiment 18, wherein the low conductivity aqueous
solution comprises about 0.1 mM to about 8 mM Tris. [0159] 20. Use
according to embodiment 19, wherein the low conductivity aqueous
solution comprises about 0.5 mM to about 6.5 mM Tris. [0160] 21.
Use according to embodiment 20, wherein the low conductivity
aqueous solution comprises about 2 mM Tris. [0161] 22. Use
according to any one of embodiments 17 to 21, wherein the low
conductivity aqueous solution contains potassium phosphate. [0162]
23. Use according to embodiment 22, wherein the low conductivity
aqueous solution comprises about 0.05 mM to about 5 mM potassium
phosphate. [0163] 24. Use according to embodiment 23, wherein the
low conductivity aqueous solution comprises about 0.05 mM to about
2 mM potassium phosphate. [0164] 25. Use according to embodiment
24, wherein the low conductivity aqueous solution comprises about
0.5 mM potassium phosphate. [0165] 26. Use according to any of the
preceeding embodiments, wherein the low conductivity aqueous
solution has a pH of about 7 or higher. [0166] 27. Use according to
embodiment 26, wherein the low conductivity aqueous solution has a
pH of about 7.5 or higher. [0167] 28. Use according to embodiment
27, wherein the low conductivity aqueous solution has a pH of from
about 7 to about 9.5. [0168] 29. Use according to embodiment 28,
wherein the low conductivity aqueous solution has a pH of from
about 7.5 to about 8.5. [0169] 30. Use according to embodiment 29,
wherein the low conductivity aqueous solution has a pH of about 8.
[0170] 31. Use according to any one of the preceeding embodiments,
wherein the low conductivity aqueous solution wash step is preceded
or succeeded by a high conductivity aqueous solution wash step.
[0171] 32. Use according to embodiment 31, wherein the low
conductivity aqueous solution wash step is preceded by a high
conductivity aqueous solution wash step. [0172] 33. Use according
to embodiment 31, wherein the high conductivity aqueous solution
has a conductivity value of about 20 mS/cm or higher. [0173] 34.
Use according to embodiment 33, wherein the high conductivity
aqueous solution has a conductivity value of from about 20 mS/cm to
about 100 mS/cm. [0174] 35. Use according to embodiment 31, wherein
an intermediate wash step is performed with a medium conductivity
aqueous solution between the low conductivity aqueous solution wash
step and the high conductivity aqueous solution wash step. [0175]
36. Use according to embodiment 35, wherein the medium conductivity
aqueous solution has a conductivity value of from more than 0.5
mS/cm to less than 20 mS/cm. [0176] 37. Use according to any one of
embodiments 33 to 36, wherein the high or medium conductivity
aqueous solution comprises an amino acid. [0177] 38. Use according
to embodiment 37, wherein the high or medium conductivity aqueous
solution comprises Histidine. [0178] 39. Use according to
embodiment 37, wherein the high or medium conductivity aqueous
solution comprises Histidine and Tris. [0179] 40. Use according to
any of the preceeding embodiments, wherein at least one additional
chromatography method/step is performed. [0180] 41. Use according
to embodiment 40, wherein an additional ion exchange chromatography
method/step is performed. [0181] 42. Use according to embodiment
41, wherein an additional anion exchange chromatography method/step
is performed. [0182] 43. Use according to embodiment 40, wherein an
additional cation exchange chromatography method/step is performed.
[0183] 44. Use according to embodiment 40, wherein an additional
anion exchange chromatography method/step and an additional cation
exchange chromatography method/step are performed. [0184] 45. Use
according to embodiment 40, wherein the use is without a
hydrophobic interaction chromatography method/step. [0185] 46. Use
according to any of the preceeding embodiments, wherein the human
IgG4 isotype antibody is an antibody against P-selectin or an
antibody against factor IXa and factor X or an antibody against
IL-13 or an antibody against amyloid beta. [0186] 47. Use according
to embodiments 1 to 45, wherein the human IgG1 isotype antibody is
an antibody against Influenza B or an antibody against VEGF-A or an
antibody against CD22 or an antibody against HER3 and EGFR or an
antibody against amyloid beta or an antibody against Her2 or an
antibody against Ang2 and VEGF-A or an antibody against
carcinoembryonic antigen (CEA) and CD3. [0187] 48. Method for
producing a human IgG isotype antibody comprising [0188] a)
cultivating a cell comprising a nucleic acid encoding the human IgG
isotype antibody, [0189] b) recovering the human IgG isotype
antibody from the cell or the cultivation medium, [0190] c)
contacting the human IgG isotype antibody with an affinity
chromatography material, [0191] d) washing the affinity
chromatography material with a low conductivity aqueous solution
[0192] e) recovering the human IgG isotype antibody from affinity
chromatography material and thereby producing the human IgG isotype
antibody. [0193] 49. Method for producing a human IgG4 isotype
antibody comprising [0194] a) cultivating a cell comprising a
nucleic acid encoding a human IgG4 isotype antibody, [0195] b)
recovering the human IgG4 isotype antibody from the cell or the
cultivation medium, [0196] c) contacting the human IgG4 isotype
antibody with an affinity chromatography material, [0197] d)
washing the affinity chromatography material with a low
conductivity aqueous solution [0198] e) recovering the human IgG4
isotype antibody from the affinity chromatography material and
thereby producing the human IgG4 isotype antibody. [0199] 50.
Method according to embodiment 48, wherein the human IgG isotype
antibody is a human IgG4 isotype antibody or a human IgG1 isotype
antibody. [0200] 51. Method according to embodiment 48, wherein the
human IgG isotype antibody is a human IgG4 isotype antibody or a
human IgG1 isotype antibody without a glycosylated glycosylation
site in its Fab fragment/with exactly one glycosylation site (at
position Asn 297 numbering according to Kabat). [0201] 52. Method
according to embodiment 48, wherein the low conductivity aqueous
solution has a conductivity value of about 0.5 mS/cm or less.
[0202] 53. Method according to embodiment 52, wherein the low
conductivity aqueous solution has a conductivity value of from
about 0.03 .mu.S/cm to about 0.5 mS/cm. [0203] 54. Method according
to embodiment 53, wherein the low conductivity aqueous solution has
a conductivity value of from about 0.05 .mu.S/cm to about 0.35
mS/cm. [0204] 55. Method according to embodiment 54, wherein the
low conductivity aqueous solution is not deionized water. [0205]
56. Method according to any one of embodiments 48 to 55, wherein
the affinity chromatography is a protein A affinity chromatography
or a Protein G affinity chromatography or a single chain Fv ligand
(KappaSelect) affinity chromatography. [0206] 57. Method according
to embodiment 56, wherein the affinity chromatography is a protein
A affinity chromatography. [0207] 58. Method according to
embodiment 56, wherein the protein A affinity chromatography is
selected from the group comprising MabSelectSure affinity
chromatography, ProSep vA affinity chromatography, Poros Mab
Capture A affinity chromatography, ProSep Ultra Plus affinity
chromatography, MabSelect SuRe LX, MabSelect, Eshmuno A, Toyopearl
AF-rProtein A-650F; Toyopearl AF-rProtein A HC-650HF). [0208] 59.
Method according to embodiment 48 or 49, wherein the content of a
host cell protein is reduced. [0209] 60. Method according to any
one of embodiments 59, wherein said host cell protein is a Chinese
hamster ovary (CHO) host cell protein. [0210] 61. Method according
to embodiment 60, wherein the host cell protein is a phospholipase.
[0211] 62. Method according to embodiment 61, wherein the host cell
protein is a phospholipase A, phospholipase B, phospholipase C or
phospholipase D. [0212] 63. Method according to any one of
embodiments 62, wherein the host cell protein is phospholipase
B-like 2 (PLBL2). [0213] 64. Method according to embodiment 60,
wherein the host cell protein is phospholipase B-like 2 (PLBL2) or
Clusterin. [0214] 65. Method according to any of embodiments 48 to
64, wherein the low conductivity aqueous solution contains
tris(hydroxymethyl)aminomethane (Tris). [0215] 66. Method according
to embodiment 65, wherein the low conductivity aqueous solution
comprises about 0.1 mM to about 10 mM Tris. [0216] 67. Method
according to embodiment 66, wherein the low conductivity aqueous
solution comprises about 0.1 mM to about 8 mM Tris. [0217] 68.
Method according to embodiment 67, wherein the low conductivity
aqueous solution comprises about 0.5 mM to about 6.5 mM Tris.
[0218] 69. Method according to embodiment 68, wherein the low
conductivity aqueous solution comprises about 2 mM Tris. [0219] 70.
Method according to embodiment 65 to 69, wherein the low
conductivity aqueous solution contains potassium phosphate. [0220]
71. Method according to embodiment 70, wherein the low conductivity
aqueous solution comprises about 0.2 mM to about 5 mM potassium
phosphate. [0221] 72. Method according to embodiment 71, wherein
the low conductivity aqueous solution comprises about 0.05 mM to
about 2 mM potassium phosphate. [0222] 73. Method according to
embodiment 72, wherein the low conductivity aqueous solution
comprises about 0.5 mM potassium phosphate. [0223] 74. Method
according to any of embodiments 48 to 73, wherein the low
conductivity aqueous solution has a pH of about 7 or higher. [0224]
75. Method according to embodiment 74, wherein the low conductivity
aqueous solution has a pH of about 7.5 or higher. [0225] 76. Method
according to embodiment 75, wherein the low conductivity aqueous
solution has a pH of from about 7 to about 9.5. [0226] 77. Method
according to embodiment 76, wherein the low conductivity aqueous
solution has a pH of from about 7.5 to about 8.5. [0227] 78. Method
according to embodiment 77, wherein the low conductivity aqueous
solution has a pH of about 8. [0228] 79. Method according to
embodiment 48 or 49, wherein the method additionally comprises
washing the affinity chromatography material with a high
conductivity aqueous solution before or after washing the affinity
chromatography material with low conductivity aqueous solution.
[0229] 80. Method according to embodiment 79, wherein the method
additionally comprises washing the affinity chromatography material
with a high conductivity aqueous solution before washing the
affinity chromatography material with low conductivity aqueous
solution. [0230] 81. Method according to embodiment 79, wherein the
method additionally comprises washing the affinity chromatography
material with a high conductivity aqueous solution and/or with a
medium conductivity aqueous solution before or after washing the
affinity chromatography material with low conductivity aqueous
solution. [0231] 82. Method according to embodiment 79, wherein the
method additionally comprises washing the affinity chromatography
material with a high conductivity aqueous solution and/or with a
medium conductivity aqueous solution before washing the affinity
chromatography material with low conductivity aqueous solution.
[0232] 83. Method according to any one of embodiments 79 to 82,
wherein the high conductivity aqueous solution has a conductivity
value of about 20 mS/cm or higher. [0233] 84. Method according to
embodiment 83, wherein the high conductivity aqueous solution has a
conductivity value of from about 20 mS/cm to about 100 mS/cm.
[0234] 85. Method according to any one of embodiments 81 to 82,
wherein the medium conductivity aqueous solution has a conductivity
value of from more than 0.5 mS/cm to less than 20 mS/cm. [0235] 86.
Method according to any one of embodiments 79 to 85, wherein the
high or medium conductivity aqueous solution comprises an amino
acid. [0236] 87. Method according to embodiment 86, wherein the
high or medium conductivity aqueous solution comprises Histidine.
[0237] 88. Method according to embodiment 86 or 87, wherein the
high or medium conductivity aqueous solution comprises Histidine
and Tris. [0238] 89. Method according to embodiment 48 or 49,
wherein at least one additional chromatography method/step is
performed after step e). [0239] 90. Method according to embodiment
89, wherein an additional ion exchange chromatography method/step
is performed after step e). [0240] 91. Method according to
embodiment 90, wherein an additional anion exchange chromatography
method/step is performed after step e). [0241] 92. Method according
to embodiment 90, wherein an additional cation exchange
chromatography method/step is performed after step e). [0242] 93.
Method according to embodiment 90, wherein an additional anion
exchange chromatography method/step and an additional cation
exchange chromatography method/step are performed after step e).
[0243] 94. Method according to embodiment 48 or 49, wherein the
method is without an hydrophobic interaction chromatography
method/step. [0244] 95. Method according to any of embodiments 49
to 94, wherein the human IgG4 isotype antibody is an antibody
against P-selectin or an antibody against factor IXa and factor X
or an antibody against IL-13 or an antibody against amyloid beta.
[0245] 96. Method according to any one of embodiments 48 or 50 to
94, wherein the human IgG1 isotype antibody is an antibody against
Influenza B or an antibody against VEGF-A or an antibody against
CD22 or a bispecific antibody against HER3 and EGFR or an antibody
against amyloid beta or an antibody against Her2 or a bispecific
antibody against Ang2 and VEGF-A or an antibody against
carcinoembryonic antigen (CEA) and CD3. [0246] 97. Method for
purifying a human IgG isotype antibody from a sample comprising the
steps of [0247] a) providing a sample comprising a human IgG
isotype antibody, [0248] b) purifying the human IgG isotype
antibody with a affinity chromatography method/step, comprising
washing the affinity chromatography material with low conductivity
aqueous solution. [0249] 98. Method according to embodiment 97,
wherein the human IgG isotype antibody is a human IgG4 isotype
antibody or a human IgG1 isotype antibody. [0250] 99. Method
according to embodiment 98, wherein the human IgG isotype antibody
is a human IgG4 isotype antibody or a human IgG1 isotype antibody
without a glycosylated glycosylation site in its Fab fragment/with
exactly one glycosylation site (at position Asn 297 numbering
according to Kabat).
[0251] 100. Method according to any one of embodiments 97 to 99,
wherein the low conductivity aqueous solution has a conductivity
value of about 0.5 mS/cm or less. [0252] 101. Method according to
embodiment 100, wherein the low conductivity aqueous solution has a
conductivity value of from about 0.03 .mu.S/cm to about 0.5 mS/cm.
[0253] 102. Method according to embodiment 101, wherein the low
conductivity aqueous solution has a conductivity value of from
about 0.05 .mu.S/cm to about 0.35 mS/cm. [0254] 103. Method
according to embodiment 102, wherein the low conductivity aqueous
solution is not deionized water. [0255] 104. Method according to
any one of embodiments 97 to 104, wherein the affinity
chromatography is a protein A affinity chromatography or a Protein
G affinity chromatography or a single chain Fv ligand (KappaSelect)
affinity chromatography. [0256] 105. Method according to embodiment
104, wherein the affinity chromatography is a protein A affinity
chromatography. [0257] 106. Method according to embodiment 105,
wherein the protein A affinity chromatography is selected from the
group comprising MabSelectSure affinity chromatography, ProSep vA
affinity chromatography, Poros Mab Capture A affinity
chromatography, ProSep Ultra Plus affinity chromatography,
MabSelect SuRe LX, MabSelect, Eshmuno A, Toyopearl AF-rProtein
A-650F; Toyopearl AF-rProtein A HC-650HF). [0258] 107. Method
according to any one of embodiments 97 to 106, wherein the content
of a host cell protein is reduced. [0259] 108. Method according to
embodiment 107, wherein said host cell protein is a Chinese hamster
ovary (CHO) host cell protein. [0260] 109. Method according to
embodiment 108, wherein the host cell protein is a phospholipase.
[0261] 110. Method according to embodiment 109, wherein the host
cell protein is a phospholipase A, phospholipase B, phospholipase C
or phospholipase D. [0262] 111. Method according to any one of
embodiments 110, wherein the host cell protein is phospholipase
B-like 2 (PLBL2). [0263] 112. Method according to embodiment 107,
wherein the host cell protein is phospholipase B-like 2 (PLBL2) or
Clusterin. [0264] 113. Method according to any one of embodiments
97 to 112, wherein the low conductivity aqueous solution contains
tris(hydroxymethyl)aminomethane (Tris). [0265] 114. Method
according to embodiment 113, wherein the low conductivity aqueous
solution comprises about 0.1 mM to about 10 mM Tris. [0266] 115.
Method according to embodiment 114, wherein the low conductivity
aqueous solution comprises about 0.1 mM to about 8 mM Tris. [0267]
116. Method according to embodiment 115, wherein the low
conductivity aqueous solution comprises about 0.5 mM to about 6.5
mM Tris. [0268] 117. Method according to embodiment 116, wherein
the low conductivity aqueous solution comprises about 2 mM Tris.
[0269] 118. Method according to any one of embodiments 113 to 117,
wherein the low conductivity aqueous solution contains potassium
phosphate. [0270] 119. Method according to embodiment 118, wherein
the low conductivity aqueous solution comprises about 0.2 mM to
about 5 mM potassium phosphate. [0271] 120. Method according to
embodiment 119, wherein the low conductivity aqueous solution
comprises about 0.2 mM to about 2 mM potassium phosphate. [0272]
121. Method according to embodiment 120, wherein the low
conductivity aqueous solution comprises about 0.5 mM potassium
phosphate. [0273] 122. Method according to any one of embodiments
97 to 121, wherein the low conductivity aqueous solution has a pH
of about 7 or higher. [0274] 123. Method according to embodiment
122, wherein the low conductivity aqueous solution has a pH of
about 7.5 or higher. [0275] 124. Method according to embodiment
123, wherein the low conductivity aqueous solution has a pH of from
about 7 to about 9.5. [0276] 125. Method according to embodiment
124, wherein the low conductivity aqueous solution has a pH of from
about 7.5 to about 8.5. [0277] 126. Method according to embodiment
125, wherein the low conductivity aqueous solution has a pH of
about 8. [0278] 127. Method according to any one of embodiments 97
to 126, wherein the method additionally comprises washing the
affinity chromatography material with a high conductivity aqueous
solution before or after washing the affinity chromatography
material with low conductivity aqueous solution. [0279] 128. Method
according to embodiment 127, wherein the method additionally
comprises washing the affinity chromatography material with a high
conductivity aqueous solution before washing the affinity
chromatography material with low conductivity aqueous solution.
[0280] 129. Method according to embodiment 127, wherein the method
additionally comprises washing the affinity chromatography material
with a high conductivity aqueous solution and/or with a medium
conductivity aqueous solution before or after washing the affinity
chromatography material with low conductivity aqueous solution.
[0281] 130. Method according to embodiment 127, wherein the method
additionally comprises washing the affinity chromatography material
with a high conductivity aqueous solution and/or with a medium
conductivity aqueous solution before washing the affinity
chromatography material with low conductivity aqueous solution.
[0282] 131. Method according to any one of embodiments 127 to 130,
wherein the high conductivity aqueous solution has a conductivity
value of about 20 mS/cm or higher. [0283] 132. Method according to
embodiment 131, wherein the high conductivity aqueous solution has
a conductivity value of from about 20 mS/cm to about 100 mS/cm.
[0284] 133. Method according to embodiment 129 or 130, wherein the
medium conductivity aqueous solution has a conductivity value of
from more than 0.5 mS/cm to less than 20 mS/cm. [0285] 134. Method
according to any one of embodiments 127 to 133, wherein the high or
medium conductivity aqueous solution comprises an amino acid.
[0286] 135. Method according to embodiment 134, wherein the high or
medium conductivity aqueous solution comprises Histidine. [0287]
136. Method according to embodiment 134, wherein the high or medium
conductivity aqueous solution comprises Histidine and Tris. [0288]
137. Method according to embodiment 97 or 98, wherein at least one
additional chromatography method/step is performed after step b).
[0289] 138. Method according to embodiment 137, wherein an
additional ion exchange chromatography method/step is performed
after step b). [0290] 139. Method according to embodiment 138,
wherein an additional anion exchange chromatography method/step is
performed after step b). [0291] 140. Method according to embodiment
138, wherein an additional cation exchange chromatography
method/step is performed after step b). [0292] 141. Method
according to embodiment 138, wherein an additional anion exchange
chromatography method/step and an additional cation exchange
chromatography method/step are performed after step b). [0293] 142.
Method according to any one of embodiments 97 to 141, wherein the
method is without an hydrophobic interaction chromatography
method/step. [0294] 143. Method according to embodiment 97 or 98,
wherein the human IgG4 isotype antibody is an antibody against
P-selectin or an antibody against factor IXa and factor X or an
antibody against IL-13 or an antibody against amyloid beta. [0295]
144. Method according to embodiment 97 or 98, wherein the human
IgG1 isotype antibody is an antibody against Influenza B or an
antibody against VEGF-A or an antibody against CD22 or an
(bispecific) antibody against HER3 and EGFR or an antibody against
amyloid beta or an antibody against Her2 or a bispecific antibody
against Ang2 and VEGF-A or a bispecific antibody against
carcinoembryonic antigen (CEA) and CD3. [0296] 145. Use of a low
conductivity aqueous solution in a wash step of a protein A
chromatography for reducing the content of a host cell protein
wherein the protein A chromatography is used to purify a human IgG4
or IgG1 isotype antibody, wherein the low conductivity aqueous
solution has a conductivity value of about 0.5 mS/cm or less.
[0297] 146. Method for producing a human IgG4 or IgG1 isotype
antibody comprising the steps of [0298] a) cultivating a cell
comprising a nucleic acid encoding a human IgG4 or IgG1 isotype
antibody, [0299] b) recovering the human IgG4 or IgG1 isotype
antibody from the cell or the cultivation medium, [0300] c)
contacting the human IgG4 or IgG1 isotype antibody with a protein A
chromatography material, [0301] d) washing the protein A
chromatography material with a low conductivity aqueous solution,
wherein the low conductivity aqueous solution has a conductivity
value of about 0.5 mS/cm or less, [0302] e) recovering the human
IgG4 or IgG1 isotype antibody from the protein A chromatography
material and thereby producing the human IgG4 or IgG1 isotype
antibody. [0303] 147. Method for purifying a human IgG4 or IgG1
isotype antibody from a sample comprising the steps of [0304] a)
providing a sample comprising a human IgG4 or IgG1 isotype
antibody, [0305] b) purifying the human IgG4 or IgG1 isotype
antibody with a protein A chromatography method/step, comprising
washing the protein A chromatography material with a low
conductivity aqueous solution, wherein the low conductivity aqueous
solution has a conductivity value of about 0.5 mS/cm or less.
[0306] 148. Use according to any of embodiments 5 to 7, wherein the
low conductivity aqueous solution is deionized water. [0307] 149.
Method according to embodiment 54, wherein the low conductivity
aqueous solution is deionized water.
DESCRIPTION OF THE SEQUENCE LISTING
[0308] SEQ ID NO: 01 variable heavy chain domain VH of <VEGF>
SEQ ID NO: 02 variable light chain domain VL of <VEGF> SEQ ID
NO: 03 variable heavy chain domain VH of <ANG-2> SEQ ID NO:
04 variable light chain domain VL of <ANG-2> SEQ ID NO: 05
variable heavy chain domain VH of anti-amyloid beta antibody (IgG1
isotype) SEQ ID NO: 06 variable light chain domain VL of
anti-amyloid beta antibody (IgG1 isotype) SEQ ID NO: 07 variable
heavy chain domain VH1 of anti-P-selectin antibody SEQ ID NO: 08
variable heavy chain domain VH2 of anti-P-selectin antibody SEQ ID
NO: 09 variable heavy chain domain VH3 of anti-P-selectin antibody
SEQ ID NO: 10 variable light chain domain VL1 of anti-P-selectin
antibody SEQ ID NO: 11 variable light chain domain VL2 of
anti-P-selectin antibody SEQ ID NO: 12 variable light chain domain
VL3 of anti-P-selectin antibody
EXAMPLE 1
Material and Methods
Antibodies
[0309] The current invention is exemplified using a number of
exemplary antibodies, including: an antibody against P-selectin
(anti-P-selectin antibody; inclacumab; IgG4 isotype) as described
in WO 2005/100402 or SEQ ID NO: 07 to SEQ ID NO: 12; a bispecific
antibody against factor IXa and factor X (anti-FIXa/X antibody;
IgG4 isotype) as described in WO 2012/067176; with an antibody
against Her2; a bispecific antibody against Ang2 and VEGF-A
(anti-Ang2/VEGF-A antibody; vanucizumab; IgG1 isotype) as described
in WO 2011/117329 or SEQ ID NO: 01 to SEQ ID NO: 04; an antibody
against amyloid beta (anti-amyloid beta antibody; gantenerumab;
IgG1 isotype) as described in WO 2003/070760 or SEQ ID NO: 05 to
SEQ ID NO: 06. Also included herein are a number of IgG1 antibodies
and IgG4 antibodies, as described in the examples below.
Detection Methods for Overall Host Cell Protein (HCP),
Phospholipase B-like 2 Protein (PLBL2) and Clusterin
[0310] a) CHO HCP Assay
[0311] The residual CHO HCP content in process samples is
determined by an electrochemiluminescence immunoassay (ECLIA) on
cobas e 411 immunoassay analyzer (Roche Diagnostics).
[0312] The assay is based on a sandwich principle using polyclonal
anti-CHO HCP antibody from sheep.
[0313] First incubation: Chinese hamster ovary host cell protein
(CHO HCP) from 15 .mu.L sample (neat and/or diluted) and a biotin
conjugated polyclonal CHO HCP specific antibody form a sandwich
complex, which becomes bound to streptavidin-coated microparticles
via interaction of biotin with streptavidin.
[0314] Second incubation: After addition of polyclonal CHO
HCP-specific antibody labeled with ruthenium complex
(Tris(2,2'-bipyridyl)ruthenium(II)-complex) a ternary sandwich
complex is formed on the microparticles.
[0315] The reaction mixture is aspirated into the measuring cell
where the microparticles are magnetically captured onto the surface
of the electrode. Unbound substances are then removed in a washing
step. Application of a voltage to the electrode then induces
chemiluminescent emission which is measured by a
photomultiplier.
[0316] The concentration of CHO HCP in the test sample is finally
calculated from a CHO
[0317] HCP standard curve of known concentration. [0318] b) CHO
PLBL2 Assay
[0319] The residual Chinese hamster ovary (CHO) Phospholipase
B-like 2 protein (PLBL2) content in process samples is determined
by an electrochemiluminescence immunoassay (ECLIA) on cobas e 411
immunoassay analyzer (Roche Diagnostics).
[0320] The assay is based on a sandwich principle using monoclonal
anti-CHO PLBL2 antibody from mouse.
[0321] In a first incubation step, CHO PLBL2 from 30 .mu.L sample
(neat and/or diluted), biotin labeled monoclonal CHO PLBL2-specific
antibody, and a monoclonal CHO PLBL2-specific antibody labeled with
a ruthenium complex (Tris(2,2'-bipyridyl)ruthenium(II)-complex)
form a sandwich complex.
[0322] In a second step after addition of streptavidin-coated
microparticles, the ternary complex becomes bound to the solid
phase via interaction of biotin and streptavidin.
[0323] The reaction mixture is aspirated into the measuring cell
where the microparticles are magnetically captured onto the surface
of the electrode. Unbound substances are then removed in a washing
step. Application of a voltage to the electrode then induces
chemiluminescence, which is measured by a photomultiplier.
[0324] The concentration of CHO PLBL2 in the test sample is finally
calculated from a CHO PLBL2 standard curve of known concentration.
[0325] c) Clusterin Assay
[0326] The residual Clusterin content in process samples is
determined by a commercial assay from Merck Millipore (GyroMark HT
Kit GYRCLU-37K) which was used according to the manufacturer's
instructions.
[0327] In brief, this assay is a Sandwich ELISA based,
sequentially, on: [0328] 1) binding of the rat Clusterin
biotinylated capture antibody to the streptavidin coated affinity
columns of the Bioaffy 1000 nL CD, [0329] 2) capture of rat
Clusterin molecules from samples to the anti Clusterin antibody,
[0330] 3) binding of a second dye-labeled anti Clusterin detection
antibody to the captured molecules, [0331] 4) quantification of the
rat Clusterin using the Gyrolab Evaluator.
EXAMPLE 2
Purification of an Anti-P-Selectin Antibody (IgG4 Isotype) in a
Protein A Chromatography
Antibody: Anti-P-Selectin
General Chromatography Conditions
[0332] Column resin: Protein A material "Mab Select SuRe"
(GE-Healthcare) O1 cm, Height: 20.1 cm, CV: 15.79 ml
[0333] Equipment: Akta Avant 150
[0334] Flow rate: 300 cm/h during all steps
[0335] A solution containing an anti-P-Selectin antibody, was
applied to a Protein A affinity column after equilibration (step 1)
of the column. Initial load of PLBL2 determined in solution
containing an anti-P-Selectin antibody: 335 ng PLBL2/mg of
antibody. Initial load of Clusterin determined in solution
containing an anti-P-Selectin antibody: 2874.8 ng Clusterin/mg of
antibody. Initial load of CHOP determined in solution containing an
anti-P-Selectin antibody: 100971 ng CHOP/mg of antibody.
[0336] The chromatographic steps were performed according to the
following general scheme:
Step 1: Equilibration:
[0337] Step 2: Load of antibody containing solution
Step 3: Wash I
Step 4: Wash II
Step 5: Wash III
[0338] Step 6: Wash IV (additional wash)
Step 7: Elution
[0339] After Elution from Protein A affinity column the protein was
determined by size exclusion chromatography (SEC) and
spectrophotometrically (OD) Analytics.
SEC:
[0340] Resin: TSK 3000 (Tosoh) [0341] Column: 300.times.7.8 mm
[0342] Flow rate: 0.5 ml/min [0343] Buffer: 200 mM potassium
phosphate containing 250 mM potassium chloride, adjusted to pH 7.0
[0344] Wavelength: 280 nm
OD:
[0344] [0345] Specific coefficient: 1.54 [0346] Wavelength: 280 nm
minus 320 nm
[0347] Specific buffer conditions for Protein A chromatography
(anti-P-Selectin antibody)
a) Control (wash with equilibration buffer only)
Step 1: Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0
Step 2: Load
Step 3: Wash I: 25 mM Tris, 25 mM NaCl, pH 7.0
Step 4: Wash II: 25 mM Tris, 25 mM NaCl, pH 7.0
Step 5: Wash III:
Step 6: Wash IV:
[0348] Step 7: Elution: 50 mM acetic acid, pH 4.0 b) low
conductivity wash (with Tris buffer only)
Step 1: Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0
Step 2: Load
Step 3: Wash I: 25 mM Tris, 25 mM NaCl, pH 7.0
Step 4: Wash II: 25 mM Tris, 25 mM NaCl, pH 7.0
Step 5: Wash III: 25 mM Tris, 25 mM NaCl, pH 7.0
Step 6: Wash IV: 2 mM Tris, pH 8.0
[0349] Step 7: Elution: 50 mM acetic acid, pH 4.0 c) low
conductivity wash (with potassium phosphate (KP) only)
Step 1: Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0
Step 2: Load
Step 3: Wash I: 25 mM Tris, 25 mM NaCl, pH 7.0
Step 4: Wash II: 25 mM Tris, 25 mM NaCl, pH 7.0
Step 5: Wash III: 25 mM Tris, 25 mM NaCl, pH 7.0
[0350] Step 6: Wash IV: 0.5 mM potassium phosphate, pH 8.0 Step 7:
Elution: 50 mM acetic acid, pH 4.0 d) high conductivity wash (with
Tris buffer only)
Step 1: Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0
Step 2: Load
Step 3: Wash I: 25 mM Tris, 25 mM NaCl, pH 7.0
Step 4: Wash II: 700 mM Tris, pH 7.2
Step 5: Wash III: 25 mM Tris, 25 mM NaCl, pH 7.0
Step 6: Wash IV:
[0351] Step 7: Elution: 50 mM acetic acid, pH 4.0 e) low
conductivity wash (with Tris buffer only; pH 6.0)
Step 1: Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0
Step 2: Load
Step 3: Wash I: 25 mM Tris, 25 mM NaCl, pH 7.0
Step 4: Wash II: 25 mM Tris, 25 mM NaCl, pH 7.0
Step 5: Wash III: 25 mM Tris, 25 mM NaCl, pH 7.0
Step 6: Wash IV: 2 mM Tris, pH 6.0
[0352] Step 7: Elution: 50 mM acetic acid, pH 4.0 f) high
conductivity wash (with Histidine (His)/Tris buffer only)
Step 1: Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0
Step 2: Load
Step 3: Wash I: 25 mM Tris, 25 mM NaCl, pH 7.0
Step 4: Wash II: 200 mM His/1000 mM Tris, pH 7.0
Step 5: Wash III: 25 mM Tris, 25 mM NaCl, pH 7.0
Step 6: Wash IV:
[0353] Step 7: Elution: 50 mM acetic acid, pH 4.0 g) low
conductivity Tris +high conductivity Histidine (His)/Tris
Step 1: Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0
Step 2: Load
Step 3: Wash I: 25 mM Tris, 25 mM NaCl, pH 7.0
Step 4: Wash II: 200 mM His/1000 mM Tris, pH 7.0
Step 5: Wash III: 25 mM Tris, 25 mM NaCl, pH 7.0
Step 6: Wash IV: 2 mM Tris, pH 8.0
[0354] Step 7: Elution: 50 mM acetic acid, pH 4.0 h) low
conductivity potassium phosphate (KP)+high conductivity Histidine
(His)/Tris
Step 1: Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0
Step 2: Load
Step 3: Wash I: 25 mM Tris, 25 mM NaCl, pH 7.0
Step 4: Wash II: 200 mM His/1000 mM Tris, pH 7.0
Step 5: Wash III: 25 mM Tris, 25 mM NaCl, pH 7.0
[0355] Step 6: Wash IV: 0.5 mM potassium phosphate, pH 8.0 Step 7:
Elution: 50 mM acetic acid, pH 4.0 i) low conductivity Tris+high
conductivity Tris
Step 1: Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0
Step 2: Load
Step 3: Wash I: 25 mM Tris, 25 mM NaCl, pH 7.0
Step 4: Wash II: 700 mM Tris, pH 7.2
Step 5: Wash III: 25 mM Tris, 25 mM NaCl, pH 7.0
Step 6: Wash IV: 2 mM Tris, pH 8.0
[0356] Step 7: Elution: 50 mM acetic acid, pH 4.0 j) low
conductivity Tris; pH 6.0+high conductivity Tris
Step 1: Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0
Step 2: Load
Step 3: Wash I: 25 mM Tris, 25 mM NaCl, pH 7.0
Step 4: Wash II: 700 mM Tris, pH 7.2
Step 5: Wash III: 25 mM Tris, 25 mM NaCl, pH 7.0
Step 6: Wash IV: 2 mM Tris, pH 6.0
[0357] Step 7: Elution: 50 mM acetic acid, pH 4.0
Results:
TABLE-US-00001 [0358] HCP total PLBL2 Clusterin Yield Run [ng/mg]
[ng/mg] [ng/mg] [%] a 218 105.9 27.4 93.44 b 105 0.8 11.2 94.61 c
114 0.7 11.8 86.67 d 48 14.7 23.2 89.33 e 155 18.7 53.8 107.3 f 106
2.9 21.6 84.9 g 83 0.4 11.8 85 h 91 0.4 9.1 80.34 i 90 0.4 15.7
84.92 j 141 1.5 53 106.9
EXAMPLE 3
Purification of an Anti-Amyloid Beta Antibody (IgG1 Isotype) in a
Protein A Chromatography
[0359] General conditions were according to the conditions
described in Example 2.
[0360] Antibody: anti-amyloid beta.
[0361] Initial load of PLBL2 determined in solution containing an
anti-amyloid beta antibody: 2019.7 ng PLBL2/mg of antibody. Initial
load of CHOP determined in solution containing an anti-amyloid beta
antibody: 578908 ng CHOP/mg of antibody.
[0362] Specific buffer conditions for Protein A chromatography
a) Control (wash with equilibration buffer only)
Step 1: Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0
Step 2: Load
Step 3: Wash I: 25 mM Tris, 25 mM NaCl, pH 7.0
Step 4: Wash II: 25 mM Tris, 25 mM NaCl, pH 7.0
Step 5: Wash III:
Step 6: Wash IV:
[0363] Step 7: Elution: 50 mM acetic acid, pH 4.0 b) low
conductivity wash (with Tris buffer only)
Step 1: Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0
Step 2: Load
Step 3: Wash I: 25 mM Tris, 25 mM NaCl, pH 7.0
Step 4: Wash II: 25 mM Tris, 25 mM NaCl, pH 7.0
Step 5: Wash III: 25 mM Tris, 25 mM NaCl, pH 7.0
Step 6: Wash IV: 2 mM Tris, pH 8.0
[0364] Step 7: Elution: 50 mM acetic acid, pH 4.0 c) high
conductivity wash (with Tris buffer only)
Step 1: Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0
Step 2: Load
Step 3: Wash I: 25 mM Tris, 25 mM NaCl, pH 7.0
Step 4: Wash II: 700 mM Tris, pH 7.2
Step 5: Wash III: 25 mM Tris, 25 mM NaCl, pH 7.0
Step 6: Wash IV:
[0365] Step 7: Elution: 50 mM acetic acid, pH 4.0 d) low
conductivity Tris+high conductivity Histidine (His)/Tris
Step 1: Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0
Step 2: Load
Step 3: Wash I: 25 mM Tris, 25 mM NaCl, pH 7.0
Step 4: Wash II: 200 mM His/1000 mM Tris, pH 7.0
Step 5: Wash III: 25 mM Tris, 25 mM NaCl, pH 7.0
Step 6: Wash IV: 2 mM Tris, pH 8.0
[0366] Step 7: Elution: 50 mM acetic acid, pH 4.0
TABLE-US-00002 HCP total PLBL2 Clusterin Yield Run [ng/mg] [ng/mg]
[ng/mg] [%] a 6828 17.3 n.d. 80.3 b 7794 17.8 n.d. 73.1 c 1595 1.7
n.d. 55.6 d 6132 2.3 n.d. 67.3
EXAMPLE 4
Purification of an Anti-Her2 Antibody (IgG1 Isotype) in a Protein A
Chromatography
[0367] General conditions were according to the conditions
described in Example 2.
[0368] Antibody: anti-Her2
[0369] Initial load of PLBL2 determined in solution containing an
anti-Her2 antibody: 1662.5 ng PLBL2/mg of antibody. Initial load of
CHOP determined in solution containing an anti-Her2 antibody:
727070 ng CHOP/mg of antibody.
[0370] Specific buffer conditions for Protein A chromatography
a) Control (wash with equilibration buffer only)
Step 1: Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0
Step 2: Load
Step 3: Wash I: 25 mM Tris, 25 mM NaCl, pH 7.0
Step 4: Wash II: 25 mM Tris, 25 mM NaCl, pH 7.0
Step 5: Wash III:
Step 6: Wash IV:
[0371] Step 7: Elution: 50 mM acetic acid, pH 4.0 b) low
conductivity wash (with Tris buffer only)
Step 1: Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0
Step 2: Load
Step 3: Wash I: 25 mM Tris, 25 mM NaCl, pH 7.0
Step 4: Wash II: 25 mM Tris, 25 mM NaCl, pH 7.0
Step 5: Wash III: 25 mM Tris, 25 mM NaCl, pH 7.0
Step 6: Wash IV: 2 mM Tris, pH 8.0
[0372] Step 7: Elution: 50 mM acetic acid, pH 4.0 c) high
conductivity wash (with Tris buffer only)
Step 1: Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0
Step 2: Load
Step 3: Wash I: 25 mM Tris, 25 mM NaCl, pH 7.0
Step 4: Wash II: 700 mM Tris, pH 7.2
Step 5: Wash III: 25 mM Tris, 25 mM NaCl, pH 7.0
Step 6: Wash IV:
[0373] Step 7: Elution: 50 mM acetic acid, pH 4.0 d) low
conductivity Tris+high conductivity Histidine (His)/Tris
Step 1: Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0
Step 2: Load
Step 3: Wash I: 25 mM Tris, 25 mM NaCl, pH 7.0
Step 4: Wash II: 200 mM His/1000 mM Tris, pH 7.0
Step 5: Wash III: 25 mM Tris, 25 mM NaCl, pH 7.0
Step 6: Wash IV: 2 mM Tris, pH 8.0
[0374] Step 7: Elution: 50 mM acetic acid, pH 4.0
TABLE-US-00003 HCP total PLBL2 Clusterin Yield Run [ng/mg] [ng/mg]
[ng/mg] [%] a 309 1.2 n.d. 85.5 b 227 1 n.d. 77 c 26 0.2 n.d. 70.9
d 42 0.5 n.d. 83.8
EXAMPLE 5
Purification of a Bispecific Anti-Ang2/VEGF-A Antibody (IgG1
Isotype) in a Protein A Chromatography
[0375] General conditions were according to the conditions
described in Example 2.
[0376] Antibody: anti-Ang2/VEGF-A
[0377] Initial load of PLBL2 determined in solution containing a
bispecific anti-Ang2/VEGF-A antibody: 919.7 ng PLBL2/mg of
antibody. Initial load of CHOP determined in solution containing an
anti-Ang2/VEGF-A: 682304 ng CHOP/mg of antibody.
[0378] Specific buffer conditions for Protein A chromatography
a) Control (wash with equilibration buffer only)
Step 1: Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0
Step 2: Load
Step 3: Wash I: 25 mM Tris, 25 mM NaCl, pH 7.0
Step 4: Wash II: 25 mM Tris, 25 mM NaCl, pH 7.0
Step 5: Wash III:
Step 6: Wash IV:
[0379] Step 7: Elution: 50 mM acetic acid, pH 4.0 b) low
conductivity wash (with Tris buffer only)
Step 1: Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0
Step 2: Load
Step 3: Wash I: 25 mM Tris, 25 mM NaCl, pH 7.0
Step 4: Wash II: 25 mM Tris, 25 mM NaCl, pH 7.0
Step 5: Wash III: 25 mM Tris, 25 mM NaCl, pH 7.0
Step 6: Wash IV: 2 mM Tris, pH 8.0
[0380] Step 7: Elution: 50 mM acetic acid, pH 4.0 c) high
conductivity wash (with Tris buffer only)
Step 1: Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0
Step 2: Load
Step 3: Wash I: 25 mM Tris, 25 mM NaCl, pH 7.0
Step 4: Wash II: 700 mM Tris, pH 7.2
Step 5: Wash III: 25 mM Tris, 25 mM NaCl, pH 7.0
Step 6: Wash IV:
[0381] Step 7: Elution: 50 mM acetic acid, pH 4.0 d) low
conductivity Tris+high conductivity Histidine (His)/Tris
Step 1: Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0
Step 2: Load
Step 3: Wash I: 25 mM Tris, 25 mM NaCl, pH 7.0
Step 4: Wash II: 200 mM His/1000 mM Tris, pH 7.0
Step 5: Wash III: 25 mM Tris, 25 mM NaCl, pH 7.0
Step 6: Wash IV: 2 mM Tris, pH 8.0
[0382] Step 7: Elution: 50 mM acetic acid, pH 4.0
TABLE-US-00004 HCP total PLBL2 Clusterin Yield Run [ng/mg] [ng/mg]
[ng/mg] [%] a 3035 1.0 n.d. 85.0 b 1707 0.8 n.d. 79.8 c 655 0.7
n.d. 52 d 1050 0.8 n.d. 92.3
EXAMPLE 6
Purification of a Bispecific Anti-FIXa/X Antibody (IgG4 Isotype) in
a Protein A Chromatography
[0383] Purification of anti-FIXa/X antibody was tested in two
different chromatography settings:
Setting 1
[0384] General conditions were according to the conditions
described in Example 2.
[0385] Antibody: anti-FIXa/X
[0386] Initial load of PLBL2 determined in solution containing an
anti-FIXa/X antibody: 557 ng PLBL2/mg of antibody. Initial load of
CHOP determined in solution containing an anti-FIXa/X: 387377 ng
CHOP/mg of antibody.
[0387] Specific buffer conditions for Protein A chromatography
a) high conductivity wash (with Tris buffer only)
Step 1: Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0
Step 2: Load
Step 3: Wash I: 25 mM Tris, 25 mM NaCl, pH 7.0
Step 4: Wash II: 700 mM Tris, pH 7.2
Step 5: Wash III: 25 mM Tris, 25 mM NaCl, pH 7.0
Step 6: Wash IV:
[0388] Step 7: Elution: 50 mM acetic acid, pH 4.0 b) low
conductivity Tris+high conductivity Histidine (His)/Tris
Step 1: Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0
Step 2: Load
Step 3: Wash I: 25 mM Tris, 25 mM NaCl, pH 7.0
Step 4: Wash II: 200 mM His/1000 mM Tris, pH 7.0
Step 5: Wash III: 25 mM Tris, 25 mM NaCl, pH 7.0
Step 6: Wash IV: 2 mM Tris, pH 8.0
[0389] Step 7: Elution: 50 mM acetic acid, pH 4.0
TABLE-US-00005 HCP total PLBL2 Clusterin Yield Run [ng/mg] [ng/mg]
[ng/mg] [%] a 1632 19.1 n.d. 79 b 2148 1.1 n.d. 77
Setting 2
General Chromatography Conditions
[0390] Column resin: Protein A material "Mab Select SuRe"
(GE-Healthcare) O1 cm, Height: 20.1 cm, CV: 15.79 ml
[0391] Equipment: Akta Avant 150
[0392] Flow rate: 300 cm/h during all steps
[0393] A solution containing an anti-FIXa/X antibody, was applied
to a Protein A affinity column after equilibration (step 1) of the
column.
[0394] Initial load of PLBL2 determined in solution containing an
anti-FIXa/X antibody: 557 ng PLBL2/mg of antibody.
[0395] The chromatographic steps were performed according to the
following general scheme:
Step 1: Equilibration:
[0396] Step 2: Load of antibody containing solution
Step 3: Wash I
Step 4: Wash II
[0397] Step 5: Wash III (additional wash)
Step 6: Elution
[0398] Specific buffer conditions for Protein A chromatography
a) high conductivity wash (with NaSO4 buffer only)
Step 1: Equilibration: 20 mM NaPO4, pH 7.5
Step 2: Load
Step 3: Wash I: 450 mM NaSO4, 20 mM NaAc, pH 4.8
Step 4: Wash II: 20 mM NaPO4, pH 7.5
Step 5: Wash III:
[0399] Step 6: Elution: 35 mM acetic acid, pH 4.0 b) low
conductivity wash (Tris 1 mM)+high conductivity wash (with
NaSO4)
Step 1: Equilibration: 20 mM NaPO4, pH 7.5
Step 2: Load
Step 3: Wash I: 450 mM NaSO4, 20 mM NaAc, pH 4.8
Step 4: Wash II: 20 mM NaPO4, pH 7.5
Step 5: Wash III: 1 mM Tris, pH 8.0
[0400] Step 6: Elution: 50 mM acetic acid, pH 4.0 c) low
conductivity wash (Tris 2 mM)+high conductivity wash (with
NaSO4)
Step 1: Equilibration: 20 mM NaPO4, pH 7.5
Step 2: Load
Step 3: Wash I: 450 mM NaSO4, 20 mM NaAc, pH 4.8
Step 4: Wash II: 20 mM NaPO4, pH 7.5
Step 5: Wash III: 2 mM Tris, pH 8.0
[0401] Step 6: Elution: 35 mM acetic acid, pH 4.0 d) low
conductivity wash (Tris 4 mM)+high conductivity wash (with
NaSO4)
Step 1: Equilibration: 20 mM NaPO4, pH 7.5
Step 2: Load
Step 3: Wash I: 450 mM NaSO4, 20 mM NaAc, pH 4.8
Step 4: Wash II: 20 mM NaPO4, pH 7.5
Step 5: Wash III: 4 mM Tris, pH 8.0
[0402] Step 6: Elution: 50 mM acetic acid, pH 4.0 e) low
conductivity wash (Tris 6 mM)+high conductivity wash (with
NaSO4)
Step 1: Equilibration: 20 mM NaPO4, pH 7.5
Step 2: Load
Step 3: Wash I: 450 mM NaSO4, 20 mM NaAc, pH 4.8
Step 4: Wash II: 20 mM NaPO4, pH 7.5
Step 5: Wash III: 6 mM Tris, pH 8.0
[0403] Step 6: Elution: 50 mM acetic acid, pH 4.0 f) low
conductivity wash (Tris 4 mM, pH 7.8)+high conductivity wash (with
NaSO4)
Step 1: Equilibration: 20 mM NaPO4, pH 7.5
Step 2: Load
Step 3: Wash I: 450 mM NaSO4, 20 mM NaAc, pH 4.8
Step 4: Wash II: 20 mM NaPO4, pH 7.5
Step 5: Wash III: 4 mM Tris, pH 7.8
[0404] Step 6: Elution: 50 mM acetic acid, pH 4.0 g) low
conductivity wash (Tris 4 mM, pH 8.2)+high conductivity wash (with
NaSO4)
Step 1: Equilibration: 20 mM NaPO4, pH 7.5
Step 2: Load
Step 3: Wash I: 450 mM NaSO4, 20 mM NaAc, pH 4.8
Step 4: Wash II: 20 mM NaPO4, pH 7.5
Step 5: Wash III: 4 mM Tris, pH 8.2
[0405] Step 6: Elution: 50 mM acetic acid, pH 4.0 h) low
conductivity wash (Tris 2 mM)+high conductivity wash (with
Histidine (His)/Tris 1 M)
Step 1: Equilibration: 20 mM NaPO4, pH 7.5
Step 2: Load
Step 3: Wash I: 200 mM His/1000 mM Tris, pH 7.0
Step 4: Wash II: 20 mM NaPO4, pH 7.5
Step 5: Wash III: 2 mM Tris, pH 8.0
[0406] Step 6: Elution: 35 mM acetic acid, pH 4.0 i) low
conductivity wash (Tris 2 mM)+high conductivity wash (Histidine
(His)/Tris 0.85 M)
Step 1: Equilibration: 20 mM NaPO4, pH 7.5
Step 2: Load
Step 3: Wash I: 200 mM His/850 mM Tris, pH 7.0
Step 4: Wash II: 20 mM NaPO4, pH 7.5
Step 5: Wash III: 2 mM Tris, pH 8.0
[0407] Step 6: Elution: 50 mM acetic acid, pH 4.0 j) low
conductivity wash (Tris 2 mM)+high conductivity wash (Histidine
(His)/Tris 0.7 M)
Step 1: Equilibration: 20 mM NaPO4, pH 7.5
Step 2: Load
Step 3: Wash I: 200 mM His/700 mM Tris, pH 7.0
Step 4: Wash II: 20 mM NaPO4, pH 7.5
Step 5: Wash III: 2 mM Tris, pH 8.0
[0408] Step 6: Elution: 50 mM acetic acid, pH 4.0 k) low
conductivity wash (Tris 2 mM)+high conductivity wash (Histidine
(His)/Tris 0.55 M)
Step 1: Equilibration: 20 mM NaPO4, pH 7.5
Step 2: Load
Step 3: Wash I: 200 mM His/550 mM Tris, pH 7.0
Step 4: Wash II: 20 mM NaPO4, pH 7.5
Step 5: Wash III: 2 mM Tris, pH 8.0
[0409] Step 6: Elution: 50 mM acetic acid, pH 4.0
TABLE-US-00006 HCP total PLBL2 Clusterin Yield Run [ng/mg] [ng/mg]
[ng/mg] [%] a 1518 204.2 n.d. 82 b 646 1 n.d. 73.8 c 737 1.2 n.d.
79 d 595 1.4 n.d. 78.5 e 685 1.8 n.d. 79.5 f 692 1.4 n.d. 78.2 g
707 1.1 n.d. 76.4 h 299 0.5 n.d. 79 i 140 0.4 n.d. 70 j 100 0.5
n.d. 71.9 k 112 0.7 n.d. 73
EXAMPLE 7
General Procedure/Conditions:
Mock Cell Culture Fluid
[0410] Null harvested cell culture fluid was produced using
non-transfected CHO-DP12 cells cultured in serum-free media.
Fermentation was performed at the 2 L-scale using a representative
cell culture process. At the end of 14 days of fermentation, cell
culture fluid was harvested via centrifugation and sterile
filtration. This harvested cell culture fluid (HCCF) was then
stored at -70.degree. C. until experimentation.
Purified PLBL2
[0411] Recombinant CHO PLBL2 with a C-terminal hexahistidine-tag
was expressed in 35 L-scale transient transfections and purified
from harvested cell culture fluid as previously described
(Vanderlaan et al., 2015). Purified PLBL2 was then formulated in a
PBS solution and stored at -70.degree. C. until
experimentation.
Purified Antibody
[0412] Recombinant humanized antibodies were expressed in CHO cells
and purified using column chromatography to ensure PLBL2
concentration was below 20 ng/mg. Prior to beginning each study,
each antibody was buffer-exchanged into PBS using PD-10 desalting
columns (GE Healthcare).
Preparation of Load Material for Protein A Chromatography
[0413] To normalize the population and abundance of host cell
proteins in the Protein A load across antibodies, purified
antibodies were diluted to the same concentration with PBS and
spiked into HCCF from a non-producing cell line to give a final
antibody titer of 5 g/L. A control was also prepared wherein PBS
was added instead of the purified antibody to evaluate non-specific
host cell protein binding to the Protein A resin in the absence of
antibody.
Packed-Bed Column Chromatography
[0414] All packed-bed column chromatography experiments were
performed using a 0.66 cm inner diameter by 20 cm bed height
MabSelect SuRe (GE Healthcare) Protein A resin column. For each
purification, the column was first equilibrated for 3 column
volumes (CVs) with 25 mM tris, 25 mM NaCl, pH 7.7 (equilibration
buffer). Then Protein A load was applied to a target load density
of 30 g antibody/L resin, after which the column was washed for 3
CVs with equilibration buffer, 3 CVs of different types of washing
buffers, and again with 3 CVs of equilibration buffer.
Subsequently, antibody was eluted at low pH with 0.1 to 0.15 M
acetic acid, and eluate pools were collected starting at 0.5 OD at
the beginning of the elution peak; pooling was terminated after 2.8
CVs. For the control run with PBS-spiked null HCCF, a 2.8 CV mock
elution pool was generated starting from 1 CV to 3.8 CVs after the
start of the elution phase. At the end of every run, each Protein A
eluate was then titrated to pH 5.0 using 1.5 M tris base. The
column was then cleaned with a 0.1 M sodium hydroxide solution. All
phases had a volumetric flow rate of 20 CV/h except for the load,
first equilibration wash, and elution phases, which had a flow rate
of 15 CV/h. [0415] A) Purification of an exemplary antibody (IgG4
isotype), Antibody A, in a protein A chromatography
[0416] Specific washing buffer conditions for purification of
Antibody A (IgG4 isotype) using the general procedure of Example 7
(as outlined above): [0417] a) 0.4 M Potassium Phosphate, pH 7.0
[0418] b) 25 mM Tris, 25 mM NaCl, pH 7.7 [0419] c) 0.75 M Arg--HCl,
pH 7.0 [0420] d) 0.6 M NaCl, pH 7.0 [0421] e) Deionized water
Results:
TABLE-US-00007 [0422] HCP total PLBL2 [ng/mg] [ng/mg] Load: 1067817
Load: 7668 a 1434 167 b 1440 107 c 331 40 d 1864 66 e 1204 31
[0423] B) Purification of an exemplary antibody (IgG1 isotype),
Antibody B, in a protein A chromatography
[0424] Specific washing buffer conditions for purification of
Antibody B (IgG1 isotype) using the general procedure of Example 7:
[0425] a) Deionized water
[0426] Results:
TABLE-US-00008 HCP total PLBL2 [ng/mg] [ng/mg] Load: 1067817 Load:
7668 a 2630 71
[0427] C) Purification of an exemplary antibody (IgG4 isotype),
Antibody C, in a protein A chromatography
[0428] Specific washing buffer conditions for purification of
Antibody C (IgG4 isotype) using the general procedure of Example 7:
[0429] a) 0.4 M Potassium Phosphate, pH 7.0 [0430] b) 25 mM Tris,
25 mM NaCl, pH 7.7 [0431] c) 0.75 M Arg--HCl, pH 7.0 [0432] d) 0.6
M NaCl, pH 7.0 [0433] e) Deionized water
[0434] Results:
TABLE-US-00009 HCP total PLBL2 [ng/mg] [ng/mg] Load: 1067817 Load:
7668 a 314 434 b 361 369 c 214 82 d 780 214 e 410 68
[0435] D) Purification of an exemplary antibody (IgG1 isotype),
Antibody D, in a protein A chromatography
[0436] Specific washing buffer conditions for purification of
Antibody D (IgG1 isotype) using the general procedure of Example 7:
[0437] a) Deionized water
TABLE-US-00010 [0437] HCP total PLBL2 [ng/mg] [ng/mg] Load: 1067817
Load: 7668 a 6427 28
[0438] E) Purification of an exemplary antibody (IgG1 isotype),
Antibody E, in a protein A chromatography
[0439] Specific washing buffer conditions for purification of
Antibody E (IgG1 isotype) using the general procedure of Example 7:
[0440] a) 0.4 M Potassium Phosphate, pH 7.0 [0441] b) 31 mM Tris,
pH 8.5 [0442] c) 55 mM Tris, pH 9.0 [0443] d) Deionized water
[0444] Results:
TABLE-US-00011 HCP total PLBL2 [ng/mg] [ng/mg] Load: 169706 Load:
333 a 1307 36 b 884 7 c 601 0.2 d 929 8
[0445] F) Purification of an exemplary antibody (IgG1 isotype),
Antibody F, in a protein A chromatography
[0446] Specific washing buffer conditions for purification of
Antibody F using the general procedure of Example 7: [0447] a) 25
mM Tris, pH 9.0
[0448] Results:
TABLE-US-00012 HCP total PLBL2 [ng/mg] [ng/mg] Load: 994582 Load:
1363 a 806 0.5
Sequence CWU 1
1
121123PRTArtificialvariable heavy chain domain VH of <VEGF>
bevacizumab 1Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln
Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr
Thr Phe Thr Asn Tyr 20 25 30 Gly Met Asn Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Trp Ile Asn Thr Tyr Thr
Gly Glu Pro Thr Tyr Ala Ala Asp Phe 50 55 60 Lys Arg Arg Phe Thr
Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala Tyr 65 70 75 80 Leu Gln Met
Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala
Lys Tyr Pro His Tyr Tyr Gly Ser Ser His Trp Tyr Phe Asp Val 100 105
110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120
2107PRTArtificialvariable light chain domain VL of <VEGF>
bevacizumab 2Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala
Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Gln
Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly
Lys Ala Pro Lys Val Leu Ile 35 40 45 Tyr Phe Thr Ser Ser Leu His
Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr
Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe
Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Thr Val Pro Trp 85 90 95 Thr
Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 3
128PRTArtificialvariable heavy chain domain VH of <ANG-2> E6Q
3Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1
5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly
Tyr 20 25 30 Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu
Glu Trp Met 35 40 45 Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn
Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp
Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg
Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Pro Asn
Pro Tyr Tyr Tyr Asp Ser Ser Gly Tyr Tyr Tyr 100 105 110 Pro Gly Ala
Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser 115 120 125
4108PRTArtificialvariable light chain domain VL of < ANG-2>
E6Q 4Gln Pro Gly Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly
Gln 1 5 10 15 Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser
Lys Ser Val 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro
Val Leu Val Val Tyr 35 40 45 Asp Asp Ser Asp Arg Pro Ser Gly Ile
Pro Glu Arg Phe Ser Gly Ser 50 55 60 Asn Ser Gly Asn Thr Ala Thr
Leu Thr Ile Ser Arg Val Glu Ala Gly 65 70 75 80 Asp Glu Ala Asp Tyr
Tyr Cys Gln Val Trp Asp Ser Ser Ser Asp His 85 90 95 Tyr Val Phe
Gly Thr Gly Thr Lys Val Thr Val Leu 100 105 5 126PRTHomo sapiens
5Gln Val Glu Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1
5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser
Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45 Ser Ala Ile Asn Ala Ser Gly Thr Arg Thr Tyr
Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Lys Gly
Asn Thr His Lys Pro Tyr Gly Tyr Val Arg Tyr 100 105 110 Phe Asp Val
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 6108PRTHomo
sapiens 6Asp Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser
Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser
Val Ser Ser Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly
Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Gly Ala Ser Ser Arg Ala
Thr Gly Val Pro Ala Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr
Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu 65 70 75 80 Pro Glu Asp Phe
Ala Thr Tyr Tyr Cys Leu Gln Ile Tyr Asn Met Pro 85 90 95 Ile Thr
Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 7 124PRTHomo
sapiens 7Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Ser Ser Tyr 20 25 30 Asp Met His Trp Val Arg Gln Ala Thr Gly
Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Thr Thr Ala Gly Asp
Thr Tyr Tyr Pro Gly Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser
Arg Glu Asn Ala Lys Asn Ser Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser
Leu Arg Ala Gly Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gly
Arg Ile Ser Met Asp Arg Gly Val Lys Asn Asn Trp Phe Asp 100 105 110
Pro Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 8124PRTHomo
sapiens 8Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Arg Pro
Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Ser Asn Tyr 20 25 30 Asp Met His Trp Val Arg Gln Ala Thr Gly
Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Thr Ala Ala Gly Asp
Ile Tyr Tyr Pro Gly Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser
Arg Glu Asn Ala Lys Asn Ser Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser
Leu Arg Ala Gly Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gly
Arg Tyr Ser Gly Ser Gly Ser Tyr Tyr Asn Asp Trp Phe Asp 100 105 110
Pro Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 9124PRTHomo
sapiens 9Gln Pro Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Val Ser Gly Asn Thr
Leu Thr Glu Leu 20 25 30 Ser Met His Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Met 35 40 45 Gly Gly Phe Asp Pro Glu Asn Gly
Glu Ala Ile Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met
Thr Ala Asp Thr Ser Thr Asp Thr Ala Tyr 65 70 75 80 Met Asp Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr
Asp Leu Ala Gly Gly Ser Asp Phe Tyr Tyr Tyr Gly Leu Asp 100 105 110
Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120
10107PRTHomo sapiens 10Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu
Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala
Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys
Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn
Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu
Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Asn Asn Trp Pro Leu 85 90
95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 11107PRTHomo
sapiens 11Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser
Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser
Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr
Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala
Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Leu 85 90 95 Thr Phe
Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 12107PRTHomo sapiens
12Ala Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1
5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser
Ala 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Ser Leu Glu Ser Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr
Cys Gln Gln Phe Asn Ser Tyr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly
Thr Lys Leu Glu Ile Lys 100 105
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