U.S. patent application number 12/910301 was filed with the patent office on 2011-02-17 for antibody purification by protein a and ion exchange chromatography.
This patent application is currently assigned to Lonza Biologics plc.. Invention is credited to Julian BONNERJEA, Anna Preneta.
Application Number | 20110040075 12/910301 |
Document ID | / |
Family ID | 9953825 |
Filed Date | 2011-02-17 |
United States Patent
Application |
20110040075 |
Kind Code |
A1 |
BONNERJEA; Julian ; et
al. |
February 17, 2011 |
ANTIBODY PURIFICATION BY PROTEIN A AND ION EXCHANGE
CHROMATOGRAPHY
Abstract
A novel method for selectively removing leaked protein A from
antibody purified by means of protein A affinity chromatography is
disclosed.
Inventors: |
BONNERJEA; Julian; (High
Wycombe, GB) ; Preneta; Anna; (Walton-on-Thames,
GB) |
Correspondence
Address: |
NIXON & VANDERHYE, PC
901 NORTH GLEBE ROAD, 11TH FLOOR
ARLINGTON
VA
22203
US
|
Assignee: |
Lonza Biologics plc.
Slough
GB
|
Family ID: |
9953825 |
Appl. No.: |
12/910301 |
Filed: |
October 22, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11419306 |
May 19, 2006 |
7847071 |
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12910301 |
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11210669 |
Aug 25, 2005 |
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11419306 |
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PCT/EP2004/002041 |
Mar 1, 2004 |
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11210669 |
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60464973 |
Apr 24, 2003 |
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60604464 |
Aug 26, 2004 |
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Current U.S.
Class: |
530/387.3 ;
530/388.1; 530/389.5 |
Current CPC
Class: |
C07K 1/22 20130101; B01D
15/363 20130101; C07K 16/00 20130101; B01D 15/362 20130101; B01D
15/3809 20130101 |
Class at
Publication: |
530/387.3 ;
530/389.5; 530/388.1 |
International
Class: |
C07K 1/22 20060101
C07K001/22 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 28, 2003 |
GB |
0304576.2 |
Claims
1. Method of purifying an antibody, preferably an IgG antibody,
comprising the steps of: firstly, purifying an antibody by means of
protein A affinity chromatography wherein the protein A is a native
protein A or a functional derivative thereof, secondly, loading the
purified antibody on an anion exchange material under conditions
which allow for binding of the protein A or its derivative and
thirdly, collecting at least 70% of the amount of antibody loaded
onto the anion exchange material in the flow-through of the ion
exchanger whilst a contaminant protein A is bound to the ion
exchange material.
2. Method according to claim 1, characterized in the method
comprises, after the first anion exchanger step, the step of
further purifying said antibody, preferably allowing of removal of
aggregated antibody.
3. Method according to claim 2, characterized in the step of
further purifying said antibody by removal of aggregated
antibody.
4. Method according to claim 1, characterized in that the protein A
is a recombinant protein A that is engineered such as to allow of
single-point attachment to a column material.
5. Method according to claim 4, characterized in that the
recombinant protein A is attached by at least 50% via a thioether
bond from a cysteine to the chromatographic support material of the
protein A affinity chromatography.
6. Method according to claim 1, characterized in that the protein A
or its functional derivative is reduced to a concentration of <1
ng/mg IgG in the flow-through of the ion-exchanger.
7. Method according to claim 1, characterized in that the antibody
has a pI of at least 6.5 or above.
8. Method according to claim 1, characterized in that the antibody
is a monoclonal antibody, preferably an IgG antibody wherein the
IgG antibody may be chimeric or CDR-grafted IgG antibody.
9. Method according to claim 8, characterized in that the IgG
antibody is at least in those portions of the antibody that are
relevant for binding to a protein A, preferably in its Fc portion
that is relevant for binding to protein A, more preferably in the
C.gamma.2-C.gamma.3 interface region of IgG comprising the binding
sites for protein A, of such species origin and IgG subclass origin
which origins allow for high affinity binding to protein A.
10. Method according to claim 9, characterized in that the IgG
antibody is selected from the group comprising human IgG1, IgG2 and
IgG4 with regard to the Fc portion of the antibody.
11. Method according to claim 8, characterized in that the antibody
is harvested from a cell culture prior to purifying the antibody by
means of protein A affinity chromatography.
12. Method according to claim 11, characterized in that the
antibody is harvested from a mammalian cell culture.
13. Method according to claim 11, characterized in that the
antibody that is to be purified by means of protein A affinity
chromatography is not treated as to inactivate proteases,
preferably is not in admixture with at least one protease
inhibitor.
14. Method according to claim 12, characterized in that the
antibody that is to be purified by means of protein A affinity
chromatography is not treated as to inactivate proteases,
preferably is not in admixture with at least one protease
inhibitor.
15. Method of purifying an antibody comprising the steps of:
firstly, purifying an antibody by means of protein A affinity
chromatography wherein the protein A is a native protein A or a
functional derivative thereof, secondly, loading the purified
antibody on a first anion exchange material under conditions which
allow for binding of the protein A or its derivative, thirdly,
collecting the antibody loaded onto the anion exchange material in
the flow-through of the ion exchanger whilst a contaminant protein
A is bound to the ion exchange material, and further purifying the
antibody by loading on, binding to and eluting it from a second ion
exchanger.
16. Method according to claim 15, characterized in that at least
70% of the amount of antibody loaded onto the first anion exchange
material are recovered in the flow-through.
17. Method according to one claim 16, characterized in that the
antibody has a pI of at least 7.5 or above.
18. Method according to claim 16, characterized in that the second
ion exchanger is a cation exchanger.
19. Method according to claim 14, characterized in that the
purified antibody is monomeric antibody and that the second ion
exchange step allows of removal of aggregated antibody.
20. Method according to claim 1 wherein said anion exchange
material is a ceramic matrix anion exchange material.
Description
[0001] The present application is a continuation of application
Ser. No. 11/419,306, filed May 19, 2006 (allowed--which published
as US 2006-0194953 A1 on Aug. 31, 2006), which is a continuation of
Ser. No. 11/210,669, filed Aug. 25, 2005 (abandoned), which is a
continuation-in-part application of International Application No.
PCT/EP2004/002041, filed 1 Mar. 2004, which claims benefit of U.S.
Provisional application Ser. No. 60/464,973, filed 24 Apr. 2003,
and GB 0304576.2 filed 28 Feb. 2003; Ser. No. 11/210,669
additionally claims benefit of U.S. Provisional application Ser.
No. 60/604,464, filed 26 Aug. 2004, the entire contents of each of
which is hereby incorporated by reference.
SUMMARY OF THE INVENTION
[0002] The present invention relates to the field of antibody
purification in biotechnological production. It is an object of the
present invention to describe a novel process for purification of
such antibody.
[0003] Protein A chromatography is widely used in industrial
manufacturing of antibodies since allowing for almost complete
purification of antibodies, that is usually IgG, in a single step
from cell culture supernatants. Protein A affinity columns
inevitably are subject to some degree of leakage of ligand from the
column upon repeated runs. Partly, this may be due to proteolytic
clipping of protein A from the column; in industrial manufacture of
antibody for pharmaceutical applications, no protease inhibitor
cocktails may be added for regulatory reasons. Unfortunately, this
protein A or protein A fragment contaminants retain their affinity
for IgG and are difficult to remove from the purified antibody due
to ongoing complex formation. Removal is mandatory since protein A
which is a bacterial protein will elicit an unwanted immune
response; further, model complexes formed by adding protein A to
monomeric IgG have been reported to activate Fc-bearing leukocytes
and the complement system to generate oxidant and anaphylatoxin
activity in vitro (Balint et al., Cancer Res. 44, 734, 1984).
[0004] The commercialisation of recombinant Protein A species as
set forth in U.S. Pat. No. 6,399,750 which recombinant species is
attached to the column matrix via a single thioester bond allowed
for higher capacity protein A columns. As a concomitant
disadvantage, the leakage rate of such recombinant Protein A
matrices is often drastically increased in contrast to many
traditional, multi-point attached natural Protein A matrices
obtained by CNBr coupling. Protein A contaminant removal should
therefore proceed without concomitant removal of complexed IgG.
[0005] Balint et al. (ibd.) demonstrated that such IgG-Protein
complexes can be separated from uncomplexed IgG by gel filtration.
Low through-put and loss in antibody yield are the disadvantages of
this method.
[0006] U.S. Pat. No. 4,983,722 teaches selective separation of
contaminating protein A from a protein-A purified antibody
preparation by absorbing the mixture to an anion exchanger material
and to separate both components by sequentially eluting the
antibodies and protein A under to conditions of increasing ionic
strength. This resolution method is highly dependent on the pI of
the antibody which is specific and highly variable for a given
antibody. Further, throughput is limited by the steepness of the
salt gradient required for obtaining separation.
BRIEF DESCRIPTION OF THE FIGURES
[0007] FIG. 1 shows the result of pretreatment by means of
non-reducing 10% SDS-PAGE for a staphylococcal protein A standard
(lane 1: native protein A; lane 2: after pretreatment) and pure,
uncoupled Streamline.TM. recombinant protein A (provided by
courtesy of Pharmacia, now Amersham-Biosciences; lane 4: native
recombinant protein A; lane 5: after pretreatment).
[0008] FIG. 2 shows the levels of leaked recombinant protein A in
antibody eluates from Streamline.TM. recombinant protein A
chromatography with single-point attached protein A in thioether
linkage.
[0009] FIG. 3 further provides data on insubstantially reduced
leakage of contaminant protein A during repeated runs of the
protein A affinity chromatography.
[0010] FIG. 4 shows results of fractionation of aggregates during
step elution of the antibody peak, as described further herein.
[0011] FIG. 5 shows outline of separation schemes, as further
described herein.
DETAILED DESCRIPTION OF THE INVENTION
[0012] It is an object of the present invention to devise another
method for separating protein A or protein A fragments from
antibody, preferably an IgG, which method avoids the disadvantages
of the prior art. According to the present invention, such object
is solved according to the independent claims 1 and 9.
[0013] According to the present invention, a method of purifying an
antibody is devised which method comprises the steps of: firstly,
purifying an antibody by means of protein A affinity chromatography
wherein the protein A is a native protein A or a functional
derivative thereof.
[0014] Secondly, loading the purified antibody on an ion exchange
material under conditions which allow for binding of the protein A
or its functional derivative and thirdly, collecting the antibody,
preferably collecting at least 70%, more preferably collecting at
least 80%, most preferably collecting at least 90% of the amount of
antibody loaded onto the ion exchange material in the flow-through
of the ion exchanger whilst any contaminant protein A or protein A
derivative is bound to the ion exchange material.
[0015] Protein A is a cell surface protein found in Staphylococcus
aureus. It has the property of binding the Fc region of a mammalian
antibody, in particular of IgG class antibodies. Within a given
class of antibodies, the affinity slightly varies with regard to
species origin and antibody subclass or allotype (reviewed in
Surolia, A. et al., 1982, Protein A: Nature's universal, antibody',
TIBS 7, 74-76; Langone et al., 1982, Protein A of staphylococcus
aureus and related immunoglobulin receptors, Advances in Immunology
32:157-252). Protein A can be isolated directly from cultures of S.
aureus that are secreting protein A or is more conveniently
recombinantly expressed in E. coli (Lofdahl et al., 1983, Proc.
Natl. Acad. Sci. USA 80:697-701). Its use for purification of
antibodies, in particular monoclonal IgG, is amply described in the
prior art (e.g. Langone et al., supra; Hjelm et al, 1972; FEBS
Lett. 28: 73-76). For use in protein A affinity chromatography,
protein A is coupled to a solid matrix such as crosslinked,
uncharged agarose (Sepharose, freed from charged fraction of
natural agarose), trisacryl, crosslinked dextrane or silica-based
materials. Methods for such are commonly known in the art, e.g.
coupling via primary amino functions of the protein to a
CNBr-activated matrix. Protein A binds with high affinity and high
specificity to the Fc portion of IgG, that is the
C.gamma.2-C.gamma.3 interface region of IgG as described in Langone
et al., 1982, supra. In particular, it binds strongly to the human
allotypes or subclasses IgG1, IgG2, IgG3 and the mouse allotypes or
subclasses IgG2a, IgG2b, IgG3. Protein A also exhibits an affinity
for the Fab region of immunoglobulins that are encoded by the
V.sub.H gene family, V.sub.H III (Sasso et al., 1991, J. Immunol,
61: 3026-3031; Hilson et al., J. Exp. Med., 178: 331-336 (1993)).
The sequence of the gene coding for protein A revealed two
functionally distinct regions (Uhlen et al., J. Biol. Chem., 259:
1695-1702 (1984); Lofdahl et al., Proc. Nutl. Acad. Sci. (USA), 80:
697-701 (1983)). The amino-terminal region contains five highly
homologous IgG-binding domains (termed E, D, A, B and C), and the
carboxy terminal region anchors the protein to the cell wall and
membrane. All five IgG-binding domains of protein A bind to IgG via
the Fc region, involving e.g. in human IgG-Fc residues 252-254,
433-435 and 311, as shown for the crystal structure in Deisenhofer
et al. (1981, Biochemistry 20: 2361-2370) and in Sauer-Eriksson et
al. (1995, Structure 3: 265-278) in case of the B-domain of protein
A. The finding of two essentially contiguous main binding sites in
the Fc portion has been confirmed in the NMR-solution study of
Gouda et al., 1998, Biochemsitry 37: 129-136. In principle, each of
the IgG-binding domains A to E of protein A is sufficient for
binding to the Fc-portion of an IgG.
[0016] Further, certain alleles of the VH3 family in man have been
found to mediate optionally binding of human Ig by protein A
(Ibrahim et al., 1993, J. Immunol. 151:3597-3603; V-region mediated
binding of human Ig by protein A). In the context of the present
application, in another, separate object of the present invention,
everything that has been said applying to Fc-region binding of
antibody to protein A applies likewise to the binding of antibodies
via such VH3 family protein A-binding allele in case that the
Fc-region of such antibody did not allow on itself for
high-affinity protein A binding. It may be considered an equivalent
embodiment of the principal, Fc-based method of the present
invention; the latter is further described in the subsequent
sections.
[0017] An IgG antibody according to the present invention is to be
understood as an antibody of such allotype that it can be bound to
protein A in a high-affinity mode. Further, apart from the Fc
portions of the antibody that are relevant for binding to protein
A, such antibody must not correspond to a naturally occurring
antibody. In particular in its variable chain regions portions, it
can be an engineered chimeric or CDR-grafted antibody as are
routinely devised in the art. An IgG-antibody according to the
present invention is to be understood as an IgG-type antibody, in
short.
[0018] A functional derivative of protein A according to the
present invention is characterized by a binding constant of at
least K=10.sup.-8 M, preferably K=10.sup.-9 M for the Fc portion of
mouse IgG2a or human IgG1. An interaction compliant with such value
for the binding constant is termed `high affinity binding` in the
present context. Preferably, such functional derivative of protein
A comprises at least part of a functional IgG binding domain of
wild-type protein A which domain is selected from the natural
domains E,D,A,B, C or engineered muteins thereof which have
retained IgG binding functionality. An example of such is the
functional 59 aminoacid `Z`-fragment of domain B of protein A which
domain may be used for antibody purification as set forth in U.S.
Pat. No. 6,013,763. Preferably, however, an antibody binding
fragment according to the present invention comprises at least two
intact Fc binding domains as defined in this paragraph. An example
of such are the recombinant protein A sequences disclosed e.g. in
EP-282 308 and EP-284 368, both from Repligen Corporation.
[0019] Alone or in combination with a protein A or a functional
protein A-fragment or derivative as defined in the preceding
sections, further preferred are protein A fragments that are
engineered to allow of single-point attachment. Single point
attachment means that the protein moiety is attached via a single
covalent bond to a chromatographic support material of the protein
A affinity chromatography. Such single-point attachment by means of
suitably reactive residues which further are ideally placed at an
exposed amino acid position, namely in a loop, close to the N- or
C-terminus or elsewhere on the outer circumference of the protein
fold. Suitable reactive groups are e.g. sulfhydryl or amino
functions. More preferably, such recombinant protein A or
functional fragment thereof comprises a cysteine in its amino acid
sequence. Most preferably, the cysteine is comprised in a segment
that consists of the last 30 amino acids of the C-terminus of the
amino acid sequence of the recombinant protein A or functional
fragment thereof. In a further preferred embodiment of such type,
the recombinant protein A or functional fragment thereof is
attached by at least 50% via a thioether sulphur bond to the
chromatographic support or matrix material of the protein
A-affinity chromatography medium. An example of such an embodiment
is described e.g. in U.S. Pat. No. 6,399,750 from Pharmacia and is
commercially available under the brandnames of Streamline.TM. or
MabSelect.TM. from Amersham-Biosciences, depending on the nature of
the support matrix used. In the present context, thioether is to be
understood narrowly as a --S-- bonding scheme irrespective of
chemical context, deviating in this regard from normal chemical
language; it is possible, for instance, that said --S-- `thioether`
bridge according to the present invention is part of a larger
functional group such as e.g. a thioester or a mixed acetal,
deviating in this regard in the context of the present application
from the reacitivity-based normal language of chemists. Preferably,
the thioether bridge is a thioether bridge in its ordinary, narrow
chemical meaning. Such bridging thioether group can be e.g.
generated by reacting the sulfhydryl-group of a cysteine residue of
the protein A with an epoxide group harbored on the activated
chromatographic support material. With a terminal cysteine residue,
such reaction can be carried out under conditions suitable as to
allow only for coupling of an exposed, unique sulfhydryl group of a
protein as to result in single-point attachment of such protein
only.
[0020] In a particularly preferred embodiment, the protein A or
functional protein A derivative according to the present invention
is the recombinant protein A disclosed in U.S. Pat. No. 6,399,750
which comprises a juxtaterminal, engineered cysteine residue and
is, preferably by at least 50%, more preferably by at least 70%,
coupled to the chromatographic support material through the sulphur
atom of said cysteine residue as the sole point of attachment.
Further preferred, such coupling has been achieved by means of
epoxide mediated activation, more preferably either by means of
1,4-bis-(2,3-epoxypropoxy) butane activation of e.g. an agarose
matrix such as Sepharose Fast Flow (agarose beads crosslinked with
epichlorohydrin, Amersham Biosciences, UK) or by means of
epichlorohydrin activation of e.g. an agarose matrix such as
Sepharose FF. Further preferred in combination with afore said
preferred embodiment according to this paragraph is that the first
ion exchanger is an anion exchanger, in particular a quaternary
amine-based anion exchanger such as Sepharose Q.TM. FF
(Amersham-Biosciences/Pharmacia), most preferably it is an anion
exchanger having the functional exchanger group Q coupled to a
matrix support which group Q is N,N,N-Trimethylamino-methyl, most
preferably the anion exchanger is Sepharose Q.TM. FF from
Pharmacia/Amersham Biosciences. The quarternary amino group is a
strong exchanger which further is not susceptible to changes in pH
of the loading/wash buffer. The fast flow exchanger matrix is based
on 45-165 .mu.m agarose beads having a high degree of crosslinking
for higher physical stability; further sepharose is devoid of the
charged, sulfated molecule fraction of natural agarose and does not
allow for unspecific matrix adsorption of antibody, even under
condition of high antibody loads. An example of such an embodiment
can be found in the experimental section.
[0021] A contaminant protein A according to the present invention
is any type of functional, IgG binding offspring of a protein A or
a functional derivative thereof as defined above which is obtained
upon eluting bound antibody from a protein A affinity
chromatography column. Such contaminant protein A species may
result e.g. from hydrolysis of peptide bonds which is very likely
to occur by means of enzyme action in particular in industrial
manufacturing. Protein A chromatography is applied as an early step
in downstream processing when the crudely purified, fresh product
solution still harbors considerable protease activity. Dying cells
in the cell culture broth or cells disrupted in initial
centrifugation or filtration steps are likely to have set free
proteases; for regulatory purposes, supplementation of the cell
culture broth with protease inhibitors prior or in the course of
downstream processing is usually not accomplished, in contrast to
biochemical research practice. Examples are
Phenyl-methyl-sulfonyl-chloride (PMSF) or .epsilon.-caproic acid.
Such chemical agents are undesirable as an additives in the
production of biopharmaceuticals. It is further possible that
recombinant functional derivatives or fragments of protein A are
less protease resistant than wild-type protein A, depending on the
tertiary structure of the protein fold. Amino acid segments linking
individual IgG binding domains might be exposed once the total
number of binding domains is reduced. Interdomain contacts may
possible contribute to the stability of domain folding. It might
also be that binding of antibody by protein A or said functional
derivatives thereof influences or facilitates susceptibility to
protease action, due to conformational changes induced upon binding
of the antibody. Again, wild-type or full length protein A or
functional, engineered fragments thereof might behave differently.
Preferably, contaminant protein A according to the present
invention still is functional, IgG binding protein and thus is
associated with the protein A-purified antibody when loaded onto
the subsequent ion exchange separation medium according to the
present invention. The high-affinity based association of
contaminant protein A with the purified antibody is the reason why
it is difficult to efficiently separate contaminant protein A from
purified antibody.
[0022] Preferably, according to the present invention the antibody
sought to be purified is harvested from a cell culture prior to
purifying the antibody be means of protein A affinity
chromatography. More preferably, said cell culture is a mammalian
cell culture. Mammalian cells have large compartments called
lysosomes harboring degradating enyzmes which are disrupted upon
cell death or harvest. In particular, said cell culture may be a
myeloma cell culture such as e.g. NS0 cells (Galfre, G. and
Milstein, C. Methods Enzymology, 1981, 73, 3). Myeloma cells are
plasmacytoma cells, i.e. cells of lymphoid cell lineage. An
exemplary NSO cell line is e.g. cell line ECACC No. 85110503,
freely available from the European Collection of Cell Cultures
(ECACC), Centre for Applied Microbiology & Research, Salisbury,
Wiltshire SP4 0JG, United Kingdom. NS0 have been found able to give
rise to extremely high product yields, in particular if used for
production of recombinant antibodies. In return, NSO cells have
been found to give reproducibly rise to much higher levels of
contaminant protein A than other host cell types at least with
certain protein A affinity chromatography systems employing
recombinant, shortened fragments of wild-type protein A which
recombinant protein A is possibly single-point attached protein A.
An example of such is Streamline.TM. rProtein A affinity
chromatography resin (Amersham Biosciences; essentially thioester
single-point attached recombinant protein A as described in U.S.
Pat. No. 6,399,750). Levels of about or in excess of 1000 ng
contaminant protein A/mg antibody could be obtained with
Streamline.TM. rProtein A affinity columns. The method of the
present invention distinguishes from the prior art in efficiently
reducing contaminant protein A from such elevated levels to <1
ng/mg antibody in a single, fast purification step, that is with a
purification factor of about 1000.times..
[0023] Further preferred is, alone or in combination with the
preceding paragraph, that the antibody that is to be purified by
means of protein A affinity chromatography is not treated as to
inactivate proteases at or after harvest, more preferably is not in
admixture with at least one exogenously supplemented protease
inhibitor after harvest. Most preferably, said protease inhibitor
is selected from the group consisting of PMSF, specific proteinase
inhibiting peptides as described in Laskowski et al., 1980, Protein
inhibitors of proteinases, Ann. Rev. Biochem. 49, 593-626, and
epsilon-caproic acid.
[0024] Operation of protein A affinity chromatography has been
widely described in the technical literature and does not need to
be further described. Another example apart from the above cited is
e.g. Duhamel et al., J. Immunological Methods 31, (1979) 211-217,
pH Gradient elution of human IgG1, IgG2 and IgG4 from protein
A-Sepharose.
[0025] Preferably, the contaminant protein A is reduced to a
concentration of <10 ng/mg antibody, more preferably <4 ng/mg
antibody, most preferably <1 ng/mg antibody in the flow-through
of the first ion-exchanger, wherein antibody is preferably to be
understood as to refer to IgG. The Elisa assay method for
validation of these threshold values is described in detail in the
experimental section; it should be noted that acidification of the
sample to a pH.ltoreq.4, preferably in the presence of a mild
detergent, is crucial for accurate determination of the amount of
leaked protein A. It goes without saying that his is threshold is
to be understood such as that the loading capacity of the first
ion-exchanger for protein A binding is never exceeded, leading
inevitably to break-through of contaminant protein A. A suitable
Elisa-based method for assaying protein A or protein A fragments is
described in U.S. Pat. No. 4,983,722. Suitable anti-protein A
antibodies are commercially available, e.g. from Sigma-Aldrich. In
particular when using derivatives of protein A which derivatives
have been engineered to harbor additional sulfhydryl groups, proper
maintenance of the protein standard is important. It may be
important to verify the monomeric character of such pure protein A
derivative used as a standard for quantification of the test
sample, since covalent di- or multimers formed via --S--S-- bridges
could lead to wrong results. Verification can be easily achieved by
SDS-PAGE analysis under reducing and non-reducing conditions, as is
customary in the art. Reduction of such protein A
derivative-standard solution by means of DTT or
beta-mercaptoethanol helps accordingly to circumvent errors of
measurement in the ELISA-technique.
[0026] Further preferred, in the method according to the present
invention at least 70%, more preferably at least 80%, most
preferably at least 90% of the antibody loaded onto the first ion
exchanger can be recovered in the flow-through of the
ion-exchanger. Preferably, and disregarding glycoforms and eventual
processing variants of the same antibody, there is only type of
species antibody present in the mixture that is going to be
purified by means of protein A affinity and subsequent ion exchange
chromatography according to the present invention. For instance,
when purifying a human or human-mouse or primate or primatized IgG
antibody according to the present invention, no bovine IgG as may
be carried over from serum in serum-supplemented cell culture is
present. To put it differently, preferably the method of the
present invention is applied to curde, unpurified antibody
harvested from serum-free cell culture.
[0027] The first ion exchanger according to the present invention
is an anion exchanger resin; protein A can be bound by both types
of resin as described (EP-289 129 B1). The first ion exchanger or
anion exchanger can be operated in the column mode at a certain
flow rate or in batch operation mode, by submerging the ion
exchange resin into the mildly agitated sample solution and further
exchanging liquid media by filtration subsequently. According to
the present invention and taking into account the pI of a given
antibody, it is possible to define suitable conditions of pH and
ionic strength for loading the first ion exchanger, which
conditions result in retaining the antibody in the flow through
whilst the protein A contaminant is bound and thus removed from the
antibody. As has been said before, the method according to the
present invention allows of faster separation of antibody from
contaminant protein A. Examples of functional groups of such first,
anion exchanger that are attached to a matrix support are e.g.
primary, secondary, and particularly tertiary or quaternary animo
groups such as aminoethyl, diethylaminoethyl, trimethylaminoethyl,
trimethylaminomethyl and diethyl-(2-hydroxypropyl)-aminoethyl.
Suitable chromatographic support matrixes for the anion exchanger
are known in the art. Examples are agarose-based resins and beads,
dextran beads, polystyrene beads and polystyrene/divinyl-benzene
resins. Most preferably, the ion exchanger is a quaternary
amine-based anion exchanger mounted on an agarose matrix such as
e.g. Sepharose CL-6B or Sepharose Fast Flow (FF) from
Amersham-Biosciences/Pharmacia. An example of such is Sepharose
Q.TM. from Amersham-Biosciences/Pharmacia. Further preferred in
conjunction with the use of a first anion exchanger is that the
antibody according to the present invention is a monoclonal
antibody that has an isoelectric point (pI) which is at least two
pH units above, that is it is more basic than, the pI of the
protein A used in the preceding protein A affinity chromatography
step; e.g. whereas native protein A has a pI of about 5.0,
Streamline recombinant protein A has a pI of about 4.5. Preferably,
the antibody according to the present invention is a monoclonal
antibody that has an isoelectric point (pI) which is at least 6.5
or above, more preferably is 7.0 or above, most preferably has an
pI of at least 7.5 or above. It should be noted that this refers to
the pI of the actually harvested and purified antibody, not the pI
that can be simply predicted from the amino acid sequence alone.
The actually purified antibody molecule may have undergone further
modifications of the polypeptide backbone such as glycosylation,
which modifications may add charged moieties and thus may have
changed the pI of the molecule. Upon determination of pI for
product antibody by means of isoelectric focusing (IEF), the
microheterogenity of posttranslational processing of the antibody
protein, e.g. a monoclonal antibody protein, leads to a wider
pI-range for individual product antibody glycoprotein molecules,
them resembling a smear in an IEF gel rather than a single band and
thus a specific numeric value for at least the majority of product.
According to the present invention, in such afore mentioned
preferred embodiment, the `pI of an antibody` refers to that share
of antibody product molecules whose pI is within the preferred
range of pI as specified above. All further definitions of this
description, such as the %-proportion of antibody recovered after a
given purification step, refer to said pI-compliant share of
antibody only.
[0028] Preferably, the pH of buffer used for loading and running
the first ion exchanger is set as to put opposing total charge on
the antibody and the protein A or protein A contaminant to be
separated by means of the ion exchanger in a flow-through mode
according to the present invention, taking the pI's of antibody and
protein A or protein A derivative into account. However, this does
pertain to average pI value as determinable by experimental means;
hence having regard to glycoforms of antibody, this doesn't mean
that a smaller share of glycoforms might be close or be at pI.
According to the present invention, it is less desirable to use a
buffer pH set at the pI of either of these in view of optimum
recovery of antibody and separation from contaminant protein A.
[0029] The mode of operation of a first anion exchanger according
to the present invention requires buffer exchange of the acidic or
neutralized eluate from the protein A affinity chromatography step
with the equilibration buffer of the first anion exchanger.
Equilibration buffer and loading buffer are identical in the method
of the present invention. Commonly employed ultrafiltration devices
such as sold by Amicon or Millipore can be expediently used for
that purpose; those avoid the dilution effects whilst using e.g.
low molecular weight porous filtration matrices such as Sephadex
G-25. The equilibration buffer according to the present invention
preferably has a salt concentration of a displacer salt such as
e.g. sodium chloride in the range of 1 to 150 mM, more preferably
of from 5 to 110 mM, most preferably of from 20 to 100 mM salt. The
pH of the equilibration buffer is preferably in the range of pH 6.5
to pH 9.0, more preferably is in the range of pH 7.5 to pH 8.5,
most preferably is in the range of pH 7.9 to pH 8.4. It should be
kept in mind that N-terminal amino function of a protein has a pKs
value of about 9.25, thus binding of contaminant protein A and any
other already negatively charged protein to an anion exchanger will
get stronger at more basic pH; for a given application, the pH of
the loading buffer might need fine tuning for optimal
discrimination of binding and non-binding for a given pair of
antibody and contaminant protein A having differing pI values and
different content of cysteine and histidine side chains which may
contribute to changes in charge within the selected ranges of pH.
Further, a more basic pH interferes with protein A-antibody
interactions as will do any increase in ionic strength; likewise,
ionic strength needs fine tuning to balance prevention of binding
of antibody with the need to abolish binding of contaminant protein
A. It goes without saying for the skilled artisan that the ionic
strength of the buffer is usually inversely correlated with the pH
value; the more strongly protein A gets bound to the anion
exchanger depending on pH, the more salt is tolerated for
preventing binding of antibody and for interfering with potential
protein A-antibody interactions. Thus, the above given ranges for
pH and displacer salt thus are to be understood as to be
correlated: The lower the pH, the less salt is found permissible
within the above given preferred ranges for working the invention.
Further salt freight is added by the pH buffering substance,
further increasing the ionic strength of the solution. The ionic
strength can be determined by measuring the conductivity of the
equilibration buffer. The term `conductivity` refers to the ability
of an aqueous solution to conduct an electric current between two
electrodes measures the total amount of ions further taking charge
and ion motility into account. Therefore, with an increasing amount
of ions present in the aqueous solution, the solution will have a
higher conductivity. The unit of measurement for conductivity is
mS/cm (milliSiemens/cm), and can be measured using a commercially
available conductivity meter, e.g. from Topac Inc. (Hingham,
Mass./U.S.A.) or Honeywell. In the context of the present
application, all numerical values pertain to the specific
conductivity at 25.degree. C. Preferably, the loading or
equilibration buffer for the first anion exchange step has a
conductivity 0.5-5 mS/cm, more preferably of from 1-3 mS/cm, most
preferably of from 1.25-2.5 mS/cm. Ideally, it has a conductivity
of about 2 mS/cm. Examples of suitable buffer salts can be found in
Good, N.E. (1986, Biochemistry 5:467-476). E.g. Tris.HCl buffer or
a sodium hydrogen phosphate buffer as customarily employed are
suitable buffering substances. The concentration of the buffer
substance is customarily in the range of e.g. 10-40 mM buffer salt.
Amongst potential anion species useful for devising a buffer, those
having lower specific strength of anion elution as compared to
chloride, which property of low elution strength is approximately
inversely correlated with the density of ionic charge and is
approximately proportional to the ionic size, are preferred.
Empirical comparisons of strength of anionic elution are tabulated
in the standard textbooks of biochemistry. More preferably, the
buffer substance according to the present invention is a phosphate
buffer. Hydrogenphosphate has a low elution strength, in particular
if employed at a pH at or below pH 8, and further excels by
particularly low chaotropic properties.
[0030] In a further preferred embodiment, the first anion exchanger
is a ceramic matrix-anion exchanger such as the Biosepra-branded
HyperD.RTM. anion exchangers, more preferably a ceramic
matrix-anion exchanger having a quaternary ammonium (=quaternary
amine-based) ionic, matrix bonded functional group. These are
extremely useful for purification at a therapeutic scale. Most
preferably, the quaternary, ceramic anion exchanger is a Q-ceramic
matrix anion exchanger such as, and particularly preferred, the
Q-HyperD.RTM. anion exchanger resin sold by Ciphergen Biosystems
Ltd., Guildford/Surrey, UK under the `Biosepra` trademark. The
above and below mentioned preferred embodiments on pI of antibody,
protein load and buffer pH are also preferred in combination with
this embodiment, with the exception of the preferred conductivity
of buffer when using Q-Hyper D.RTM. material being at best 0.5-2
mS/cm, more preferably being in the range of 0.6-1.7 mS/cm, most
preferably at about 1 to 1.5 mS/cm and in particular when using
Q-Hyper D.RTM.-F ion exchanger. This conductivity ensures best
purification result in view of deriching contaminant protein A or
fragments thereof from the product protein. The Ceramic HYPERD
sorbents are made using a rigid porous bead, which is coated and
permeated with a functionalized hydrogel. This gives the beads
outstanding rigidity and flow performance, as well as exceptional
mass transfer and dynamic properties. The Ceramic HYPERD sorbents
are very easy to use. Their relatively high density makes them easy
to pack and use in very large columns. The complete lack of
shrinking or swelling eliminates the need for repeated
packing/unpacking of columns. Today, columns in excess of 500
liters are used for preparative chromatography of molecules for
therapeutic use. The Ceramic HYPERD ion exchangers are also
available in a 50 .mu.m grade (F grade) for preparative processes,
with their high capacity and lower back pressure the 50 grade is
perfect for capture processes and general downstream processing.
The ceramic nature of the bead makes it chemically very stable and
it can be cleaned using most commonly used cleaning agents,
including 0.5 M NaOH.
[0031] Whereas batch mode operation is possible, column operation
mode is preferred for the first anion exchanger step. In that case,
a flow rate of about 10 to 60 ml/h can be advantageously employed.
The loading concentration of antibody loaded can favorably be in
the range of 10 to 30 mg antibody/ml exchange resin. It goes
without saying that the use of extremely diluted samples would give
rise to decreased yield of antibody, as is known to the skilled
person. The antibody sought to be purified is collected in the
low-through of the loading operation including about one column
volume of wash with the same equilibration buffer. The pH of the
flow-through may be adjusted to neutral pH for improving stability
and preventing aggregation and/or precipitation of antibody
protein.
[0032] After the first anion exchanger, the antibody is ready for
use in applications or may be deemed to require further polishing
by customary purification methods. In a further preferred
embodiment, the first ion exchange step is followed by a second ion
exchange step in which second step the antibody is loaded and bound
by the second ion exchange medium and is eluted with a buffer other
than the loading buffer, by means of increased salt and/or pH, as
an essentially monomeric, non-aggregated antibody. `Essentially`
means less than 5% in this context. Preferably, alone or in
combination with a preferred embodiment described in the preceding
sections, the second ion exchanger is a cation exchanger. Such
combination of a protein A chromatography step followed by a first
anion exchanger and a second cation exchanger step is novel. It is
well known that most trace contaminant proteins from cell culture
broth have much lower pI values than antibodies, in particular IgG
antibodies; cation exchange will therefore allow of efficient
removal both of aggregated antibody and potential infectious agents
such as virus capsids as well as of protein contaminants other than
antibody. Due to speedy operation, highly efficient recovery of
antibody after loading, binding to and elution from the co and high
capacity of loading, it allows also of repeated, cyclic operation
with a single batch of antibody with additive effect of the
purification factor achieved in a single round of binding and
elution. Preferably, the pH of the loading buffer is about pH 4 to
7, more preferably pH 4.01 to 6, most preferably pH 4.02 to 5.5.
Further preferred, the antibody is eluted from the cation exchanger
with a salt gradient in the range of from 0.1 to 1.2 M salt,
wherein the salt preferably is an alkaline metal salt, more
preferably a lithium, potassium or sodium salt. Preferably, elution
takes place at a pH of from pH 7 to 8 in order to have maximum
aggregate removal and minimal damage to antibody due to acidic
conditions. Optionally preferred, the elution takes place at a an
acid pH of from pH 4 to 7, more preferably 4.01 to 6 for maximizing
removal of contaminant protein A; levels as low as <0.4 ng/mg
antibody can be realized in this way. This second cation exchanger
step renders traditional gel filtration moot whilst allowing of
high-capacity as well as fast operation as is typical for ion
exchangers. Ion exchangers support loads of 10-30 mg antibody/ml
resin. In a particularly preferred embodiment, the purification
method of a first anion exchanger and a second cation exchanger
step in the aftermath of protein A chromatography renders clinical
grade antibody in the absence of a further, terminal size exclusion
chromatography (SEC) step which SEC step would have a molecular
weight cut off suitable for separating antibody aggregates and/or
antibody-protein A complexes from monomeric antibody such as an
normal IgG.
[0033] On a general note, the method of the present invention can
not be exploited for antibodies that have been raised against
protein A-borne epitopes. Such antibodies are disclaimed, though
this is an obvious limitation to the skilled artisan.
[0034] The most appealing feature of the method of the present
invention is that purifying antibody via an anion exchanger in a
non-binding or flow-through mode, the capacity of the column is not
all limiting the through-put of material; the capacity is only
decisive with regard to minor amounts of contaminant protein A
retain. This saves a lot of processing time and material resources
whilst allowing for very efficient removal of protein A
contaminant.
Experiments
1. Protein A Elisa
[0035] Numerous Elisas for testing of protein A or recombinant
protein A have been described (see U.S. Pat. No. 4,983,722 and
references described in there). For all work described below, a
simple sandwich Elisa was used in which capture anti-protein A
antibody coated on a flat-bottomed 96 well microtiter plate
(Nunc.TM.) retains the protein A. Bound protein A is then detected
an a biotinylated anti-protein A detection antibody, which allows
for further binding of streptavidin conjugated horseradish
peroxidase (Amersham #RPN 1231). Commercially available
anti-protein A rabbit antibody (raised against the natural S.
aureus protein A) for capture is available from Sigma-Aldrich
(#P-3775). It was this antibody which was used through-out this
study. The detection rabbit antibody was equally purchased from
Sigma-Aldrich (#3775). After coating the protein by unspecific
adsorption process, the coated protein is used to retain protein
A-specific protein A capture antibody which capture antibody is
further detected with bioinylated rabbit anti-protein A and
streptavidin-horseradish peroxidase. Tetramethyl benzidine is used
as the chromogenic substrate. Samples of unknown concentration are
read off against a standard curve using the very parent-protein A
or -protein A derivative of the contaminant protein A sought to be
detected. Coating at acidic pH as well as proper preparation of the
standard has proven important. In particular for recombinant
protein A's engineered to carry additional cysteine residue such as
e.g. Streamline protein A.TM. (Amersham Biosciences, formerly
Pharmacia), the standard solution was found to require pretreatment
with a reducing sulfhydryl agent to ensure monomeric state of the
protein standard solution.
[0036] Wild-type protein A standard, in contrast, is commercially
available from a number of companies, e.g.
Sigma-Aldrich/Switzerland (#P6031) or Pharmacia (#17-0770-01) and
does not require such pretreatment. For the below described
experiments observing leakage of contaminant protein A from
Streamline.TM. matrix, samples of unconjugated recombinant protein
A obtained from the manufacturer were used as a standard.
1.1 Pretreatment of Cys-Enriched Protein A-Standard
[0037] Pure recombinant protein A-Cys as commercially in the
Streamline.TM. protein A affinity chromatography (Amersham
Biosciences) column material was obtained freeze-dried from
Pharmacia/Amersham Biosciences. Up to 20 mg/ml protein were
dissolved in 0.1M Tris pH 8 containing 0.5 M NaCl, 1 mM EDTA and 20
mM dithioerythritol, incubated for 15-30 min. at room temperature
and desalted with a disposable PD-10 gel filtration column
(Amersham Biosciences). All buffers used for handling the standard
solution before coating should be N.sub.2-treated to prevent
oxidation of the thiol groups. Preparation of the protein standard
was carried out at best immediately prior to use of the standard
for coating the microtiter plates. Optionally, a 1 mg/ml stock
solution was prepared and kept at -65.degree. C. in a freezer;
after thawing, monomeric character of protein A was assayed from an
aliquot loaded on non-reducing SDS-PAGE. The concentration of
protein standard was determined by Bradford assay (Bradford et al.,
1976, Anal. Biochem. 72:248-254; Splittgerber et al., 1989, Anal.
Biochem. 179:198-201) as well as by automated amino acid analysis.
The result of such pretreatment is shown in FIG. 1 by means of
non-reducing 10% SDS-PAGE for a staphylococcal protein A standard
(lane 1: native protein A; lane 2: after pretreatment) and pure,
uncoupled Streamline.TM. recombinant protein A (provided by
courtesy of Pharmacia, now Amersham-Biosciences; lane 4: native
recombinant protein A; lane 5: after pretreatment). Lane 1 is a
molecular weight marker with the corresponding molecular masses
being denoted on the vertical axis. The recombinant protein A from
Pharmacia harboring an additional Cys residue shifts after
reduction to lower molecular weight; a monomeric band at about 34
kD is preserved and much more intense, stemming obviously from
dissociation of disulfide bridged dimers.
1.2 Elisa
1.2.1 Preparation of Sample
[0038] By two dilution steps, 1: 200.000 dilution of the 1 mg/ml
protein A standard stock solution was prepared to provide the top
standard at 50 ng/ml. Thereof, dilutions down to 0.2 ng/ml were
prepared for assaying the standard curve. Further, the dilutions of
the standard (`spinking solutions`) were used for spiking of
duplicate product samples to be tested in order to exclude presence
of interfering substances in the sample.
[0039] For final product sample testing, every sample is divided
into 2 equal volumes of 500 .mu.l. One is spiked with the 1000
ng/ml spiking solution, or the 10 .mu.g/ml solution if appropriate,
to give a final protein A content of 10 ng protein A per mg of
antibody. The other half is spiked with the same volume of sample
buffer; thus the dilution factor of the product sample due to
spiking is accounted for. Both types of preparation will be
referred to as `spiked sample` in the following. The sample buffer
was made up from 7.51 g Glycine (base), 5.84 g NaCl, 0.5 ml Triton
X-100 to a volume of 1 L with deionized or bidestillated water.
[0040] For optimal accuracy measurements, the antibody
concentrations in the samples were pre-determined by customary
Elisa's well known in the art. A further standard solution was
spiked with an equal amount of a known standard antibody of
comparable constant region affinity for protein A, to determine
efficiency of the acidification step and to unravel any potential
systematic error introduced by antibody binding to and thus
scavenging protein A from capture in the assay.
[0041] Acidification: To 450 .mu.l of spiked sample or standard is
added 200 .mu.l of 0.2 M citrate/0.05% Triton X-100 buffer at pH
3.0. All samples were done in triplicate. Further, dilutions of
sample were prepared and tested in triplicate since the assay works
optimal for antibody concentrations being in the range of 1 mg/ml
and 0.2 mg/ml. The acidification step is crucial in the present
assay to liberate contaminant protein A or A fragments which were
otherwise bound to the excess of antibody present in the sample
solution.
1.2.2 Coating of Microtiter Plates with Antibody
[0042] Coating buffer was made up from 1.59 g/L Na.sub.2CO.sub.3,
2.93 g/L NaHCO.sub.3 and 0.20 g/L sodium azide. The pH of the
buffer was adjusted to pH 9.6. Add 100 .mu.l antibody solution per
well comprising antibody in an amount sufficient as not to show
saturation for the protein A standard. Cover plate with plastic
film and place in humidity chamber. Incubate at 37.degree. C.
overnight for approximately 18 hours. Rinse all wells 3 times with
at least 300 .mu.l washing buffer [NaCl 5.8 g/L, Na.sub.2HPO.sub.4
1.15 g/L, NaH.sub.2PO.H.sub.2O 0.26 g/L, EDTA 3.7 g/L, Tween-20 0.2
g/L, butanol 10 ml/L, pH 7.2], and tap dry. Add 250 .mu.l blocking
buffer [coating buffer with 0.5% casein hammarsten] to each well
and incubate for 2 hours at ambient temperature on a benchtop
orbital shaker (speed 120 rpm). Rinse all wells three times with at
least 300 .mu.l washing buffer, and tap dry.
1.2.3 Incubation of Sample and Detection
[0043] Plate out standards and samples including any spiked samples
with 100 .mu.l/well. Cover plate with plastic film and incubate for
90 minutes at ambient temperature on an orbital benchtop shaker.
Rinse all wells three times with at least 300 .mu.l washing buffer,
and tap dry. Dilute biotinylated rabbit anti-protein A at the
previously determined optimal dilution. Add 100 .mu.l/well, cover
plate with plastic film and incubate for 90 minutes at ambient
temperature on an orbital shaker. Repeat rinsing.
[0044] Dilute strepavidin-horseradish peroxidase at the
previously-determined optimal dilution using conjugate buffer
[Na.sub.2HPO.sub.4 1.15 g/L, NaCl 5.84 g/L, NaH2PO.sub.4.H20 0.26
g/L, EDTA 3.73 g/L, Triton X-100 0.05% (v/v), pH 7.2]. Add 100
.mu.l/well, cover plate in plastic film and incubate for 45 minutes
at ambient temperature on an orbital shaker. Repeat rinsing. Add
100 .mu.l freshly-prepared tetramethyl-benzidine (TMB, ICN product
number #980502) substrate solution. The substrate solution is
prepared like this: A stock solution is prepared by dissolving 10
mg TMB in 1 ml DMSO. 10 .mu.l of that stock, further 10 .mu.l of
H.sub.2O.sub.2 are added to a 2.05% (w/w) sodium acetate aqueous
solution that was adjusted to pH 6.0 with 0.5 M citric acid. It
goes without saying that all water used for preparing any reagent
of the assay is of highest quality, that is deionized ultrapure or
at least bidestillated water.
[0045] The substrate solution is incubated at ambient temperature
for 8-11 minutes on a shaker. The reaction is then stopped by
adding 50 .mu.l per well of stopping solution [13%
H.sub.2SO.sub.4]. Within 10 min. after addition of the stopping
solution, the absorbance of the wells at wavelength 450 nm is
determined on a plate-reading spectrophotometer.
[0046] The detection limit for such Elisa is 0.2 ng/ml Protein A,
with a working range of from 0.2 to 50 ng/ml. The interassay
variability is less than 10%.
[0047] FIG. 2 shows the levels of leaked recombinant protein A in
antibody eluates from Streamline.TM. recombinant protein A
chromatography with single-point attached protein A in thioether
linkage. The cycle number refers to repeated use after elution with
1 M sodium chloride and re-equilibration. Whereas leakage from cell
culture broth from hybridoma cell culture was typically in the
order of 500 ppm, other cell types gave levels as high as 1000 ppm.
An overview on the rate of leakage from differently sourced
matrices is given in Table 1; chromatography was performed
according to manufacturer' instruction.
TABLE-US-00001 TABLE 1 Typical Working Coupling leakage Capacity
Flow Rate Matrix Supplier chemistry p.p.m (mgml.sup.-1)
(cmh.sup.-1) Native Protein A Amersham- Multi -point 10-20 5-20
30-300 Sepharose 4FF Biosciences attached CNBr rmp Protein A
Amersham- Multi point 10-20 5-20 30-300 Sepharose Biosciences
attached Poros A High Applied Multi point 10-50 10 500-1000
Capacity Biosystems attached Protein A Biosepra Multi point Up to
300 10-20 200-500 Ceramic HyperD attached rProtein A Amersham-
Single point 50-1000 20-40 30-300 Sepharose Biosciences attached
Thioether linkage MabSelect Amersham- Single point 50-1000 20-40
500 Biosciences attached Thioether linkage STREAMLINE Amersham-
Single point 50-1000 20-40 200-400 rProtein A Biosciences attached
Thioether linkage
[0048] FIG. 3 further provides data on insubstantially reduced
leakage of contaminant protein A during repeated runs of the
protein A affinity chromatography with the same affinity matrix
material; wild-type protein A multipoint-attached Sepharose 4 FF
(Amersham-Biosciences) was repeatedly used as described in section
2.1 below and the level of contaminant protein A in the eluate,
before any further processing of eluate, was determined by Elisa as
described above.
2. Protein A and Sepharose Q chromatography 2.1 Protein A Affinity
Chromatography with Streamline.TM.
[0049] Cell culture supernatant from a NS0 myeloma cell culture was
crudely purified by centrifugation and in depth filtration and
concentrated by ultrafiltration; ultrafiltration was also used to
exchange the culture fluid to PBS pH 7.5. The titer of the antibody
#5 produced by the cells was 0.2 mg/ml, a total of 1 L
buffer-exchanged supernatant was loaded. The pI of the monoclonal
antibody #5 was 8.5. The protein A Streamline.TM. column (5.0 ml
volume) was previously equilibrated with 10 column volumes of 50 mM
glycine/glycinate pH 8.8, 4.3 M NaCl; flow rate was at 200 cm/h.
For loading, the column was operated at a flow rate of 50
cmh.sup.-1; loading capacity was about 20 mg/ml matrix material).
Before elution, the column was washed with at least 10 column
volumes of glycine equilibration buffer supplemented with
additional 200 mM NaCl and 0.1% Tween-20. Elution was achieved with
elution buffer made up of 0.1 M glycine/HCl pH 4.0 buffer.
Immediately after elution, fractions of eluate comprising the
antibody peak were neutralized with an adequate aliquot of 0.5 M
TrisHCl pH 7.5 and buffer exchanged with an Amicon diafiltration
device with loading/equilibration buffer (10 mM Tris/HCl pH 8.0, 50
mM NaCl) of the present invention for the subsequent anion
exchanger step for preventing longer exposure to acidic pH.
[0050] The antibody concentration and the contaminant protein A
concentration were determined as described above. The level of
contaminant protein A in the eluate amounted to 1434 ng/mg antibody
before and amounted to 1650 ng/mg antibody after diafiltration. The
recovery of antibody based on the titer of the buffer exchanged
supernatant solution prior to loading was 81%; the concentration of
antibody in the diafiltrated solution was 3.6 mg/ml.
2.2 Q-Sepharose FF Anion Exchange Step in Non-Binding Mode
[0051] The purified antibody from section 2.1 was further processed
as described: A 5.0 ml Q-Sepharose FF column (Amersham-Biosciences)
was packed 10 ml of 0.1 M NaOH, followed by 2 column volumes of 0.1
M Tris pH 8, and equilibrated in 10 column volumes of 10 mM Tris pH
8/50 mM NaCl, at a flow rate of 75 cm/h. After equilibration, the
flow rate was reduced to 50 cm/h. 6 ml of the diafiltrated antibody
solution was loaded onto the column and the flow-through was
collected for further processing; the flow-through was continued to
be collected until, after having loaded the column with the initial
6 ml and having continued thereafter with pure loading or
equilibration buffer 10 mM Tris pH 8, 50 mM NaCl, the absorption of
the flow-through monitored at 280 nm was back to baseline. The
total recovery of antibody in the flow-through was 23 mg antibody
(87%). Determination of the level of contaminant protein A resulted
in <3 ng/mg antibody. Further processing of this Q-Sepharose
purified antibody batch by gel filtration (size exclusion
chromatography, SEC) over Sephacryl S-300 in 10 mM Phosphate pH
7.0, 140 mM NaCl buffer at a flow rate of 10 cm/h with a loading
ratio of 15 mg antibody per ml gel was found not change this trace
contaminant protein A level substantially any more. By experience,
SEC may be used to further reduce levels of about 30-100 ng/mg
contaminant protein A to about 1-5 ng/mg. Thus SEC has a very low
purification factor with regard to trace amounts of protein A,
possibly accounting for affinity interactions in between antibody
and contaminant A. However, due to the unavoidable dilution of
sample and slow processing with allows for same decay of the
antibody protein, SEC will allow for 70% recovery only of the
amount of antibody loaded. This means SEC will unavoidably result
in loss of material whilst requiring much time.
[0052] The Q-Sepharose column was recycled for further use by
separate elution in 2M NaCl and further equilibration as described
above.
3. Protein A and Sepharose Q Purification with Subsequent Cation
Exchange Step
[0053] In a further experiment, the antibody from exp. 2.2.
purified in a non-binding mode by Q-Sepharose anion exchange was
used. Instead of testing a further, final SEC purification step,
the antibody harvested in the flow-through of the Sepharose Q
column was subjected to a second cation exchanger step with a
SP-Sepharose FF (SP=Sulphopropyl-) matrix from
Amersham-Biosciences. The SP-Sepharose FF allowed of a flow rate of
100 cm/h with a reproducible yield of 93% antibody after loading,
washing and elution of the antibody from the cation exchanger.
[0054] For loading, the pH of the antibody solution obtained after
Sepharose Q purification step was adjusted to pH 4.5-5.0 with 50 mM
acetate buffer pH 4.5. The loading capacity was set with 10 mg/ml
matrix material at a conductivity of load of 17 mS/cm. The 50 mM
acetate buffer was further used for washing to baseline. A 50 mM Na
acetate pH 4.5, 1 M NaCl high salt buffer was used for elution of
antibody; monomeric antibody eluted first, whereas aggregates used
to elute in the tail fractions at high ionic strength. Use of a
less steep salt gradient by implementation of a salt gradient in
the elution buffer before pumping on the column is equally
feasible; direct application of a high-salt buffer results in less
diluted antibody and consequently more precisely sampling and
shorter times of residence in the acidic solution. After elution,
the acidic buffer was quickly exchanged for PBS pH 7.5. The level
of contaminant protein A in the pooled eluate was determined with
<0.4 ng/mg antibody, the antibody was shown to be >99%
monomeric by means of size exclusion HPLC.
4. Streamline.TM. Protein a Affinity Chromatography with
Custom-Made, Multipoint-Attached Protein A
[0055] This multipoint-attached Streamline.TM. protein A-affinity
matrix was custom made and supplied by Pharmacia Biotech (now
Amersham-Pharmacia). It was made up by the manufacturer by coupling
the same 34 kD Streamline.TM.-type recombinant protein A having a
terminal Cys residue to the same Sepharose matrix material, but
used traditional CNBr chemistry for activation and coupling instead
of epoxide-mediated activation and selective reaction conditions
for coupling of --SH groups only (see product information from
manufacturer). The method of exp. 2.1 was repeated and the level of
contaminant protein A was determined with 353 ng/mg antibody. This
means that the mode of coupling of the protein A to the matrix
material partly accounts for increased protein leakage from
high-capacity, single-point attached recombinant protein A affinity
matrices; the modifications in amino acid sequence introduced into
such recombinant protein A as compared to full-length wild-type
protein A contribute considerably to increased protein leakage,
too.
5. Parallel Comparison of Methods: Comparison with Miles Method
(U.S. Pat. No. 4,983,722)
[0056] The Miles Patent (U.S. Pat. No. 4,983,722) claims that
binding DEAE Sepharose used as a second chromatography step with a
salt gradient (0.025M to 0.25M NaCl) for elution can reduce the
leached Protein A content in the eluate to less than 15 ng/mg
antibody (range of protein A was 0.9 to 14 ng/mg of antibody).
TABLE-US-00002 TABLE 2 Comparison of Protein A residues in eluate
samples of 6A1 Antibody purified on single and multipoint attached
Protein A affinity matrices Protein A levels Matrix Sample (ng/mg)
rProtein A Sepharose Protein A eluate 20.2 (single point attached)
rmp Protein A Sepharose Protein A eluate 2.16 (multi-point
attached) Native Protein A Protein A eluate <2.0 Sepharose
(multipoint attached)
[0057] The aim of these experiments was to confirm these results
using MabSelect (new single point attached rProtein A matrix) with
a lower pI antibody (pI 6.5-7.5), and to directly compare the
non-binding Q-Sepharose method (using different
equilibration/loading buffers) with the Miles Patent method.
Method Applied:
[0058] The purification of 6A1 antibody (pI 6.5-7.5) included two
chromatography steps consisting of MabSelect Protein A step
followed by Q-Sepharose anion exchange chromatography
(non-binding), or DEAE Sepharose chromatography (binding) step. See
L0 9007 and L0 9375
MabSelect Protein A Chromatography:
TABLE-US-00003 [0059] Column matrix Mab Select recombinant Protein
A (single point attached rPA) Column dimensions 1.6 cm internal
diameter .times. 15 cm bed height Column volume 30 mL Operational
flow rate 500 cm/hr (16.80 mL/min) Clean 6M guanidine HCL (2 column
volumes) Loading capacity 35 mg/ml matrix Equilibration 50 mM
glycine/glycinate pH 8.0/250 mM NaCL (8 column volumes) Post load
wash 50 mM glycine/glycinate pH 8.0/250 mM NaCL (8 column volumes)
Elution buffer 100 mM glycine pH 3.50 (6 column volumes) Wash 100
mM Citric acid pH 2.1 (2 column volumes)
[0060] The culture supernatant containing 6A1 antibody was purified
on a MabSelect column (30 ml), connected to an AKTA FPLC system.
The conditions used were as described in the table above. The
antibody was eluted using 0.1M glycine pH 3.5. Following elution
the eluate pH was adjusted to pH 7.0, and then the eluate sample
was divided into 5 aliquots; each aliquot was then diafiltered into
a different buffer for anion exchange chromatography.
[0061] The first aliquot was diafiltered into 50 mMTrisHCl pH8/75
mMNaC1 for Q-Sepharose chromatography run 1. The second aliquot was
diafiltered into 50 mMTrisHCl pH8/100 mMNaC1 for Q-Sepharose
chromatography Run 2. The third aliquot was diafiltered into 20 mM
sodium phosphate pH6.5/80 mM NaCl for Q-Sepharose chromatography
Run 3. Aliquots four and five were buffer exchanged into 25 mMTris
HCl pH 8.0/25 mMNaC1 for evaluation of binding DEAE Sepharose
method described in Miles patent. The difference between Runs 4
& 5 is that in Run 4 the main peak was collected as one
fraction and diafiltered into standard phosphate buffered saline
prior to analysis whereas in Run 5, the elution peak was
fractionated and dialysed into a phosphate buffer prepared as
described in the Miles Patent.
[0062] The conditions for each of the five column runs are
described below:
Q-Sepharose Chromatography: Run 1
TABLE-US-00004 [0063] Column matrix Q-Sepharose Fast Flow Column
dimensions 1.6 cm internal diameter .times. 8 cm bed height Column
volume 16 mL Column preparation Packed in 0.1 M Sodium Hydroxide at
150 cm/hr Operational flow rate 100 cm/hr (3.35 mL/min) Clean 0.1M
Sodium Hydroxide (2 column volumes) Loading capacity 15 mg/ml
matrix Equilibration 50 mM TrisHCl pH 8.0/75 mM NaCl (8 column
volumes) Post load wash 50 mM TrisHCl pH 8.0/75 mM NaCl (5 column
volumes) Strip buffer 2 M Sodium Chloride (2 column volumes) Wash
0.1M Sodium Hydroxide (2 column volumes)
Q-Sepharose Chromatography: Run 2
TABLE-US-00005 [0064] Column matrix Q-Sepharose Fast Flow Column
dimensions 1.6 cm internal diameter .times. 8 cm bed height Column
volume 16 mL Column preparation Packed in 0.1 M Sodium Hydroxide at
150 cm/hr Operational flow rate 100 cm/hr (3.35 mL/min) Clean 0.1M
Sodium Hydroxide (2 column volumes) Loading capacity 7.5 mg/ml
matrix Equilibration 50 mM TrisHCl pH 8.0/100 mM NaCl (8 column
volumes) Post load wash 50 mM TrisHCl pH 8.0/100 mM NaCl (5 column
volumes) Strip buffer 2 M Sodium Chloride (2 column volumes) Wash
0.1M Sodium Hydroxide (2 column volumes)
Q-Sepharose Chromatography: Run 3
TABLE-US-00006 [0065] Column matrix Q-Sepharose Fast Flow Column
dimensions 1.6 cm internal diameter .times. 8 cm bed height Column
volume 16 mL Column preparation Packed in 0.1 M Sodium Hydroxide at
150 cm/hr Operational flow rate 100 cm/hr (3.35 mL/min) Clean 0.1M
Sodium Hydroxide (2 column volumes) Loading capacity 7.5 mg/ml
matrix Equilibration 20 mM Sodium phosphate pH 6.5/80 mM NaCl Post
load wash 20 mM Sodium phosphate pH 6.5/80 mM NaCl (5 column
volumes) Strip buffer 2M Sodium Chloride (2 column volumes) Wash
0.1M Sodium Hydroxide (2 column volumes)
DEAE Sepharose: Run 4
TABLE-US-00007 [0066] Column matrix DEAE Sepharose Column
dimensions 1.6 cm internal diameter .times. 8 cm bed height Column
volume 16 mL Column preparation Packed in equilibration buffer at
150 cm/hr Operational flow rate 100 cm/hr (3.35 mL/min) Clean 0.1M
Sodium Hydroxide (2 column volumes) Loading capacity 7.5 mg/ml
matrix Equilibration 25 mM TrisHCl pH 8.6/25 mM NaCl (8 column
volumes) Post load wash 25 mM TrisHCl pH 8.6/25 mM NaCl (5 column
volumes) Elution buffer 25 mM TrisHCl pH 8.6/25 mM NaCl To 25 mM
TrisHCl pH 8.6/250 mM NaCl (10 column volumes) Wash 2M Sodium
Chloride (2 column volumes)
DEAE Sepharose Binding Method: Run 5 (Miles Method)
TABLE-US-00008 [0067] Column matrix DEAE Sepharose Column
dimensions 1.6 cm internal diameter .times. 8 cm bed height Column
volume 16 mL Column preparation Packed in equilibration buffer at
150 cm/hr Operational flow rate 100 cm/hr (3.35 mL/min) Clean 0.1M
Sodium Hydroxide (2 column volumes) Loading capacity 7.5 mg/ml
matrix Equilibration 25 mM TrisHCl pH 8.6/25 mM NaCl (8 column
volumes) Post load wash 25 mM TrisHCl pH 8.6/25 mM NaCl (5 column
volumes) Elution buffer 25 mM TrisHCl pH 8.6/25 mM NaCl To 25 mM
TrisHCl pH 8.6/250 mM NaCl (10 column volumes) Wash 2M Sodium
Chloride (2 column volumes)
[0068] The properties of the different buffers used in this study
are shown in Table 3.
[0069] Eluate samples generated from the 5 ion exchange runs were
assayed for Protein A levels in the rPA ELISA. The results are
shown in Table 4.
TABLE-US-00009 TABLE 3 Buffers used in this study Equilibration Run
Conductivity Buffer Number (ms/cm) Resin pH 50 mM TrisHCl pH 1
10.74 Q-Sepharose 8.00 8.0/75 mM NaCl (non-binding) 50 mM TrisHCl
pH 2 12.85 Q-Sepharose 8.01 8.0/100 mM NaCl (non-binding) 20 mM
Sodium 3 10.20 Q-Sepharose 6.50 phosphate pH 6.5/ (non-binding) 80
mM NaCl 25 mM TrisHCl pH 4/5 3.35 DEAE- 8.60 8.6/25 mM NaCl
Sepharose (binding) 25 mM TrisHCl pH 4/5 24.54 DEAE- 8.61 8.6/250
mM NaCl* Sepharose (binding) *Gradient elution buffer
[0070] Fractions across the elution profile of DEAE-Sepharose Run 5
(Miles method) were collected and analysed in the rProtein A ELISA;
the results are shown in Table 5.
TABLE-US-00010 TABLE 4 rProtein A ELISA Results: rProtein A
Antibody Elution levels concentration % Volumes Sample ID (ng/mg)
(mg/ml) Recovery (CV's)* Q-Sepharose <0.4 1.42 82 4.5 eluate Run
1 Q-Sepharose 2.94 1.49 70 3.5 eluate Run 2 Q-Sepharose 0.73 1.86
85 3.4 eluate Run 3 DEAE Sepharose 1.72 2.16 75 2.5 eluate pool Run
4 (pool of all fractions) DEAE Sepharose 1.55 1.83 73 3 eluate pool
(Miles Method) Run 5 (pool of fractions 2 to 6) *Where CV's denotes
column volumes
TABLE-US-00011 TABLE 5 Levels of rProtein A in Eluate fractions
across the elution peak obtained during binding DEAE-Sepharose
separation (Miles Method); Run 5. Fraction rProtein A levels
Absorbance Number (ng/mg) (A.sub.280) 1 3.33 0.018 2 0.4 0.108 3
0.4 0.22 4 0.4 0.169 5 2.01 0.092 6 16.7 0.042 7 6.38 0.016
[0071] The highest recovery (85%) and best clearance of rProtein A
for this antibody (6A1; pI 6.5-7.5) was obtained under non-binding
conditions on Q-Sepharose using 20 mM sodium phosphate pH 6.5/80 mM
NaCl buffer (corresponding to Run 3). Run 1 also showed good
recovery (82%) and rProtein A clearance however, the elution volume
for this run was significantly higher than expected for a
non-binding method; suggesting partial retardation of the antibody
on the column in this buffer system. Increasing the NaCl
concentration (Run 2) resulted in lower rProtein A clearance, hence
the buffer system used in Run 3 was more appropriate for this
antibody. It has been our previous observation that the buffer
system used in Run 1 is more appropriate for high pI antibodies and
that used in Run 3 is particularly useful for neutral or slightly
acidic antibodies. These experiments were done at similar
capacities (7.5 mg/ml resin) we would expect to be able to use this
non-binding method at much higher capacities (>30 mg/ml). We
would expect this non-binding method to be applicable to many anion
exchangers for example Q-Hyper D in addition to anion exchange
membrane adsorbers (such as Mustang Q, Intercept Q and Sartobind
Q). We would also expect this process to be more applicable to
large scale production compared to the Miles method as higher
capacities etc can be applied.
[0072] In the case of Run 5 (Miles method), fractionation of
rProtein A was observed across the main elution peak as shown in
table 5. Careful pooling of fractions is therefore required to
ensure good clearance of rProtein A. This had an impact on recovery
(73%) and even in this case did not give as good clearance as
obtained with the non-binding method. For the Miles method
therefore it is more difficult to achieve good clearance and high
recovery for cell lines/antibodies in cases where very high leakage
is observed (such as that commonly obtained with single point
attached matrices)
[0073] The data from Run 5 is representative of the results
obtained by and the conditions described in the Miles patent.
[0074] An overview of method comparisons and the data obtained is
shown in Table 6, below.
TABLE-US-00012 TABLE 6 Summary of rProtein A Levels at Different
Stages of Antibody Purification. ##STR00001## *All examples carried
out with 7.5 mg/ml loading of exchanges or 15 mg/ml loading
capacity: Concentration and diafiltration (50 mM TrisHcl/75 m MNaCl
pH 8.00) & Anion Exchange Q (<0.4) (50 mM TrisHcl/75 m MNaCl
pH 8.00) Note: Levels of rProtein A are shown in brackets ng/mg;
note that not all NSO clonal cell lines supernatants give similar
contamination levels of protein A.
6. Purification of a High pI Antibody
[0075] A high pI antibody (pI 9.0-9.3) was purified using Protein A
Affinity Chromatography (MabSelect--single point attached
recombinant Protein A matrix), followed by Q-Sepharose anion
exchange chromatography (under non binding conditions; for removal
of trace contaminants) followed by SP-Sepharose cation exchange
chromatography (under binding conditions for removal of
aggregates).
##STR00002##
Experimental Materials and Methods
MabSelect Protein A Chromatography:
TABLE-US-00013 [0076] Column matrix Mab Select recombinant Protein
A (single point attached rPA) Column dimensions 1.6 cm internal
diameter .times. 15 cm bed height Column volume 30 mL Operational
flow rate 500 cm/hr (16.80 mL/min) Clean 6M guanidine HCL (2 column
volumes) Loading capacity 35 mg/ml matrix Equilibration 50 mM
glycine/glycinate pH 8.0/250 mM NaCL (8 column volumes) Post load
wash 50 mM glycine/glycinate pH 8.0/250 mM NaCL (8 column volumes)
Elution buffer 100 mM glycine pH 3.50 (6 column volumes) Wash 100
mM Citric acid pH 2.1 (2 column volumes)
[0077] Culture supernatant containing high pI antibody was purified
on a MabSelect Protein A Affinity column (30 ml), connected to an
AKTA FPLC system. The conditions used were as described in the
table above. The antibody was eluted using 0.1M glycine pH 3.5.
Following elution, the eluate was held at pH 3.69 (no adjustment
required) for 60 min (low pH virus inactivation step), and then
neutralised to pH 8 using 2 M Tris Base. Three cycles on Protein A
were performed; product recovery was determined by A280 nm and is
shown in Table 7 for each cycle.
TABLE-US-00014 TABLE 7 % Recovery on Mab Select Protein A Column
Cycle Number % Recovery 1 81 2 81 3 80
[0078] After MabSelect Protein A chromatography the eluates from
each of the three cycles were pooled together and buffer exchanged
into 25 mM Tris pH 8.0 (Q-Sepharose equilibration buffer) using an
Amicon stirred cell concentrator fitted with 10 kDa Millipore
membrane.
Q-Sepharose Chromatography:
TABLE-US-00015 [0079] Column matrix Q-Sepharose Fast Flow Column
dimensions 1.6 cm internal diameter .times. 15 cm bed height Column
volume 30 mL Column preparation Packed in 0.1 M Sodium Hydroxide at
225 cm/hr Operational flow rate 150 cm/hr (5.0 mL/min) Clean 0.1M
Sodium Hydroxide (2 column volumes) Loading capacity 40 mg/ml
matrix Equilibration 20 mM Tris pH 8.0 (8 column volumes) Post load
wash 20 mM Tris pH 8.0 (5 column volumes) Strip buffer 20 mM Tris
pH 8.0/2M NaCl (2 column volumes) Wash 0.1M Sodium Hydroxide (2
column volumes)
[0080] 40 ml of concentrated/diafiltered MabSelect Protein A eluate
was loaded on Q-Sepharose column at a loading capacity of 40 mg/ml
matrix. The column was operated in a non-binding mode and the
unbound fraction containing the antibody was collected. The
recovery on this step was 69% by A.sub.280. This is slightly lower
than obtained under these conditions for this antibody and may be
due inaccurate estimation of the load volume due to hold up volume
in the FPLC sample pump.
[0081] Following Q-Sepharose chromatography, the unbound fraction
was concentrated to 13.98 mg/ml and diafiltered into SP-Sepharose
equilibration buffer (25 mM Sodium acetate pH5.0/25 mM NaCl) using
an Amicon stirred cell fitted with 10 kDa Millipore
Ultrafilteration membrane.
SP-Sepharose Chromatography:
TABLE-US-00016 [0082] Column matrix Q-Sepharose Fast Flow Column
dimensions 1.6 cm internal diameter .times. 15 cm bed height Column
volume 30 mL Column preparation Packed in 0.1 M Sodium Hydroxide at
150 cm/hr Operational flow rate 100 cm/hr (3.35 mL/min) Clean 0.1M
Sodium Hydroxide (2 column volumes) Loading capacity 10 mg/ml
matrix Equilibration 25 mM Sodium acetate pH 5.00/25 mM NaCl (8
column volumes) Post load wash 25 mM Sodium acetate pH 5.00/25 mM
NaCl (6column volumes) Elution 25 mM Sodium acetate pH 5.00/186 mM
NaCl (25column volumes) Strip buffer 25 mM Sodium acetate pH
5.00/2M NaCl (2column volumes) Wash 0.1M Sodium Hydroxide (2 column
volumes)
[0083] 24 ml of buffer exchanged Q-Sepharose eluate was loaded onto
the SP-Sepharose column at a loading capacity of 10 mg/ml matrix.
The column was operated in a binding mode; the eluate was collected
as fractions. Fractions across the elution profile were analysed by
GP-HPLC to determine aggregate levels results and are shown in
Table 8. Samples following each chromatography step were collected
and analysed for rProtein A residues, results are presented in
Table 9.
TABLE-US-00017 TABLE 8 rProtein A ELISA Results after each
chromatography step Antibody rProtein A concentration Sample ID
levels(ng/mg) (mg/ml) MabSelect Protein A 2.64 46.7 eluate (after
conc/diaf) Q-Sepharose eluate <4 8.10 SP-Sepharose eluate <4
1.79 pool F(1-16)
TABLE-US-00018 TABLE 9 GP-HPLC Analysis of SP-Sepharose fractions
Absorbance Sample ID % Aggregates (A.sub.280) SP eluate pool F(1-3)
0.57 11.2 SP eluate pool F(4-6) 1.10 2.7 SP eluate pool F(7-9) 2.07
0.655 SP eluate pool F(10-12) 2.12 0.351 SP eluate pool F(13-15)
2.56 0.208
[0084] Conclusion: Fractionation of aggregates was observed during
step elution of the antibody peak; see FIG. 4, with the
aggregate-enriched fractions eluting later (on the tail end of the
elution peak) compared to non-aggregate containing fractions. The
tail fractions can be omitted from the main pool to obtain 99%
monomeric pool and still have high recovery (>95%).
7. Purification Using Ceramic Q-HyperD.RTM. F als a First Anion
Exchanger
[0085] Q-HyperD.RTM. F (Biosepra-Brand of chromatographic supports)
was purchased from Ciphergen Biosystems Ltd., Guildford, UK. The
processing of a pI 8-9 antibody expressed from NSO cells by
Mab-Select Protein A affinity chromatography was conducted
essentially as described in example 5. Further essentially as
described in example 5 (for Runs 1-3), Q anion exchange
chromatography in flow-through mode was then applied to the Protein
A-affinity column eluate except that Q-Sepharose, except for a
comparative run, was replaced by Q-Hyper DF (Biosepra.RTM.) under
varying conditions of buffer salt, buffer pH and conductivity. The
respective conditions are outlined in the scheme according to FIG.
5; applying a very low conductivity of less than 2 mS/cm, namely at
about 1.26 mS/cm, proved superior with regard to deriching
contaminating protein A to the utmost extend possible and achieving
results equal to those obtainable with Sepharose Q material.
However, for large scale industrial manufacture, the ceramic HyperD
material offers advantages in view of life time, robustness and
compressibility (processing time, flow rate). Hence conductivity is
a very important parameter to be tested and optimized for different
column materials.
* * * * *