U.S. patent application number 15/461198 was filed with the patent office on 2018-06-14 for immunoglobulin purification.
This patent application is currently assigned to Hoffmann-La Roche Inc.. The applicant listed for this patent is Hoffmann-La Roche Inc.. Invention is credited to Marc Pompiati, Andreas Schaubmar.
Application Number | 20180162930 15/461198 |
Document ID | / |
Family ID | 40668760 |
Filed Date | 2018-06-14 |
United States Patent
Application |
20180162930 |
Kind Code |
A1 |
Pompiati; Marc ; et
al. |
June 14, 2018 |
IMMUNOGLOBULIN PURIFICATION
Abstract
The current invention reports a method for purifying an
immunoglobulin, wherein the method comprises applying an aqueous,
buffered solution comprising an immunoglobulin in monomeric, in
aggregated, and in fragmented form to an anion exchange
chromatography material under conditions whereby the immunoglobulin
in monomeric form does not bind to the anion exchange material, and
recovering the immunoglobulin in monomeric form in the flow-through
from the anion exchange chromatography material, whereby the
buffered aqueous solution has a pH value of from 8.0 to 8.5. In one
embodiment the anion exchange chromatography material is a membrane
anion exchange chromatography material.
Inventors: |
Pompiati; Marc; (Penzberg,
DE) ; Schaubmar; Andreas; (Penzberg, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Hoffmann-La Roche Inc. |
Little Falls |
NJ |
US |
|
|
Assignee: |
Hoffmann-La Roche Inc.
Little Falls
NJ
|
Family ID: |
40668760 |
Appl. No.: |
15/461198 |
Filed: |
March 16, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13141306 |
Jun 21, 2011 |
9631008 |
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PCT/EP2009/009157 |
Dec 18, 2009 |
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15461198 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 16/065 20130101;
C07K 16/2803 20130101; A61K 39/39591 20130101; C07K 1/18 20130101;
C07K 16/2866 20130101 |
International
Class: |
C07K 16/06 20060101
C07K016/06; C07K 16/28 20060101 C07K016/28; A61K 39/395 20060101
A61K039/395; C07K 1/18 20060101 C07K001/18 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 22, 2008 |
EP |
08022236.7 |
Claims
1-4. (canceled)
5. A method for obtaining an immunoglobulin in monomeric form,
wherein the method comprises the following step: a. applying an
aqueous, buffered solution comprising an immunoglobulin in
monomeric and in aggregated form and/or immunoglobulin fragments to
an anion exchange chromatography material, wherein the aqueous,
buffered solution has a pH value of from pH 8.0 to pH 8.5, and
whereby the immunoglobulin depleted of immunoglobulin aggregates
and immunoglobulin fragments is recovered from the flow-through of
the anion exchange chromatography material and thereby an
immunoglobulin in monomeric form is obtained.
6. The method according to claim 5, characterized in that said
anion exchange chromatography material is a membrane anion exchange
chromatography material.
7. The method according to claim 5, characterized in that said
anion exchange chromatography material is a strong anion exchange
chromatography material.
8. The method according claim 5, characterized in that said method
comprises prior to step a) an additional protein A chromatography
step or a HCIC chromatography step.
9. The method according to claim 8, characterized in that said
anion exchange chromatography material is a membrane anion exchange
chromatography material.
10. The method according to claim 8, characterized in that said
anion exchange chromatography material is a strong anion exchange
chromatography material.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 13/141,306, filed Jun. 21, 2011, which is a National Stage
Entry of International Application No. PCT/EP2009/009157, having an
international filing date of Dec. 18, 2009, which claims priority
to and the benefit of European Application No. 08022236.7, filed
Dec. 22, 2008, the entire contents of which applications are hereby
incorporated by reference.
FIELD OF THE INVENTION
[0002] The current invention is in the field of purification of
polypeptides. It is reported a method for providing an
immunoglobulin in monomeric form by separating the immunoglobulin
in solution from impurities, especially from the immunoglobulin in
aggregated form and from immunoglobulin fragments.
BACKGROUND OF THE INVENTION
[0003] 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 for humans, nucleic acids,
viruses and host cell proteins, which could cause harm, have to be
removed especially. To meet regulatory specifications one or more
purification steps have to follow the fermentation process. Among
other things, purity, throughput, and yield play an important role
in determining an appropriate purification process.
[0004] Different methods are well established and widespread used
for protein purification, such as affinity chromatography with
microbial proteins (e.g. protein A or protein G 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) (see e.g. Vijayalakshmi, M. A., Appl. Biochem.
Biotech. 75 (1998) 93-102).
[0005] Necina, R., et al. (Biotechnol. Bioeng. 60 (1998) 689-698)
reported the capture of human monoclonal antibodies directly from
cell culture supernatants by ion exchange media exhibiting high
charge density. In WO 89/05157 a method is reported for the
purification of product immunoglobulins by directly subjecting the
cell culture medium to a cation exchange treatment. A one-step
purification of monoclonal IgG antibodies from mouse ascites is
described by Danielsson, A., et al., J. Immunol. Meth. 115 (1988),
79-88.
[0006] Mhatre, R., et al., J. Chrom. A 707 (1995) 225-231, explored
the purification of antibody Fab fragments by cation exchange
chromatography and pH gradient elution. WO 94/00561 reports human
monoclonal anti-rhesus antibodies and cell lines producing the
same. A method for purifying a polypeptide by ion exchange
chromatography is reported in WO 2004/024866 in which a gradient
wash is used to resolve a polypeptide of interest from one or more
contaminants. Schwarz, A., et al., Laborpraxis 21 (1997) 62-66,
report the purification of monoclonal antibodies with a
CM-HyperD-column. WO 2004/076485 reports a process for antibody
purification by protein A and ion exchange chromatography. In EP 0
530 447 a process for purifying IgG monoclonal antibodies by a
combination of three chromatographic steps is reported. The removal
of protein A from antibody preparations is reported in U.S. Pat.
No. 4,983,722.
[0007] Recombinant monoclonal antibody processes often employ
anion-exchange chromatography to bind trace levels of impurities
and potential contaminants such as DNA, host cell protein, and
virus, while allowing the antibody to flow through (Knudsen, H. L.,
et al., J. Chrom. A 907 (2001) 145-154).
[0008] WO 95/16037 reports the purification of anti-EGF-R/anti-CD3
bispecific monoclonal antibodies from hybrid hybridoma performed by
protein A and cation exchange chromatography. The separation of
antibody monomers from its multimers by use of ion exchange
chromatography is reported in EP 1 084 136. U.S. Pat. No. 5,429,746
relates to the application of hydrophobic interaction
chromatography combination chromatography to the purification of
antibody molecule proteins. An anionic modified microporous
membrane for use for the filtration of fluids, particular
parenteral or biological liquids contaminated with charged
particulates, is reported in U.S. Pat. No. 4,604,208. WO 03/040166
reports a membrane and a device designed for the removal of trace
impurities in protein containing streams.
[0009] A method for recovering a polypeptide is reported in U.S.
Pat. No. 6,716,598. In US 2006/0194953 a method is reported for
selectively removing leaked protein A from antibody purified by
means of protein A affinity chromatography. The separation of
protein monomers from aggregates by use of ion-exchange
chromatography is reported in WO 99/62936. Lynch, P. and Londo, T.,
Gen. Eng. News 11 (1997) 17, report a system for aggregate removal
from affinity-purified therapeutic-grade antibody. A two-step
purification of a murine monoclonal antibody intended for
therapeutic application in man is reported by Jiskoot, W., et al.,
J. Immunol. Meth. 124 (1989) 143-156.
SUMMARY OF THE INVENTION
[0010] The current invention comprises aspects in the field of
immunoglobulin purification. It has been found that an anion
exchange chromatography step, in which the immunoglobulin in
monomeric form can be obtained from an anion exchange material in a
flow-through mode, has to be performed in a narrow pH value range
of from e.g. pH 7.8 to pH 8.8. Surprisingly a small deviation from
this pH value range, e.g., to pH 7.0 or pH 9.0, abolishes this
effect. With the method according to the invention it is possible
to separate in a single step the immunoglobulin in monomeric form
from the immunoglobulin in aggregated form and from immunoglobulin
fragments.
[0011] One aspect is a method for obtaining an immunoglobulin in
monomeric form, wherein the method comprises the following step:
[0012] applying an aqueous, buffered solution comprising an
immunoglobulin in monomeric and in aggregated form and/or
immunoglobulin fragments to an anion exchange chromatography
material,
[0013] whereby the immunoglobulin depleted of immunoglobulin
aggregates and immunoglobulin fragments is recovered from the
flow-through or supernatant of the anion exchange chromatography
material, wherein the aqueous, buffered solution has a pH value of
from pH 7.8 to pH 8.8, and thereby an immunoglobulin in monomeric
form is obtained. In one embodiment the aqueous, buffered solution
has a pH value of from pH 8.0 to pH 8.5. In another embodiment the
anion exchange chromatography material is a membrane anion exchange
chromatography material. In a further embodiment the anion exchange
chromatography material is a strong anion exchange chromatography
material. In another embodiment the strong anion exchange
chromatography material is Q-sepharose.RTM., i.e. a cross-linked
agarose matrix (R) to which quaternary ammonium groups of the
formula
R--O--CH.sub.2CHOHCH.sub.2OCH.sub.2CHOHCH.sub.2N.sup.+(CH.sub.3).sub.3
are covalently bound. In still another embodiment the method
comprises as first step an additional protein A chromatography step
or an additional HCIC chromatography step or an additional ion
exchange chromatography step.
DETAILED DESCRIPTION OF THE INVENTION
[0014] The term "ion exchange material" or grammatical equivalents
thereof denotes an immobile matrix that carries covalently bound
charged substituents. For overall charge neutrality not covalently
bound counter ions are bound to the charged substituents by ionic
interaction. The "ion exchange material" has the ability to
exchange its not covalently bound counter ions for similarly
charged binding partners or ions of the surrounding solution.
Depending on the charge of its exchangeable counter ions the "ion
exchange material" is referred to as "cation exchange material" or
as "anion exchange material". Depending on the nature of the
charged group (substituent) the "ion exchange material" is referred
to, e.g. in the case of cation exchange materials, as sulfonic acid
or sulfopropyl resin (S), or as carboxymethyl resin (CM). Depending
on the chemical nature of the charged group/substituent the "ion
exchange material" can additionally be classified as strong or weak
ion exchange material, depending on the strength of the covalently
bound charged substituent. For example, strong cation exchange
materials have a sulfonic acid group, preferably a sulfopropyl
group, as charged substituent, weak cation exchange materials have
a carboxylic acid group, preferably a carboxymethyl group, as
charged substituent. Strong anion exchange materials have a
quarternary ammonium group, and weak anion exchange materials have
a diethylaminoethyl group as charged substituent.
[0015] The term "membrane" denotes both a microporous or
macroporous membrane. The membrane itself is composed of a
polymeric material such as, e.g. polyethylene, polypropylene,
ethylene vinyl acetate copolymers, polytetrafluoroethylene,
polycarbonate, poly vinyl chloride, polyamides (nylon, e.g.
Zetapore.TM., N.sub.66 Posidyne.TM.), polyesters, cellulose
acetate, regenerated cellulose, cellulose composites,
polysulphones, polyethersulfones, polyarylsulphones,
polyphenylsulphones, polyacrylonitrile, polyvinylidene fluoride,
non-woven and woven fabrics (e.g. Tyvek.RTM.), fibrous material, or
of inorganic material such as zeolithe, SiO.sub.2, Al.sub.2O.sub.3,
TiO.sub.2, or hydroxyapatite.
[0016] Ion exchange materials are available under different names
and from a multitude of companies such as e.g. cation exchange
resins Bio-Rex.RTM. (e.g. type 70), Chelex.RTM. (e.g. type 100),
Macro-Prep.RTM. (e.g. type CM, High S, 25 S), AG.RTM. (e.g. type
50W, MP) all available from BioRad Laboratories, WCX 2 available
from Ciphergen, Dowex.RTM. MAC-3 available from Dow chemical
company, Cellulose CM (e.g. type 23, 52), hyper-D, partisphere
available from Whatman plc., Amberlite.RTM. IRC (e.g. type 76, 747,
748), Amberlite.RTM. GT 73, Toyopearl.RTM. (e.g. type SP, CM, 650M)
all available from Tosoh Bioscience GmbH, CM 1500 and CM 3000
available from BioChrom Labs, SP-Sepharose.TM., CM-Sepharose.TM.
available from GE Healthcare, Poros resins available from
PerSeptive Biosystems, Asahipak ES (e.g. type 502C), CXpak P, IEC
CM (e.g. type 825, 2825, 5025, LG), IEC SP (e.g. type 420N, 825),
IEC QA (e.g. type LG, 825) available from Shoko America Inc., 50W
cation exchange resin available from Eichrom Technologies Inc., and
such as e.g. anion exchange resins like Dowex.RTM. 1 available from
Dow chemical company, AG.RTM. (e.g. type 1, 2, 4), Bio-Rex.RTM. 5,
DEAE Bio-Gel 1, Macro-Prep.RTM. DEAE all available from BioRad
Laboratories, anion exchange resin type 1 available from Eichrom
Technologies Inc., Source Q, ANX Sepharose.RTM. 4, DEAE
Sepharose.RTM. (e.g. type CL-6B, FF), Q Sepharose.RTM., Capto
Q.RTM., Capto S.RTM. all available from GE Healthcare, AX-300
available from PerkinElmer, Asahipak ES-502C, AXpak WA (e.g. type
624, G), IEC DEAE all available from Shoko America Inc.,
Amberlite.RTM. IRA-96, Toyopearl.RTM. DEAE, TSKgel DEAE all
available from Tosoh Bioscience GmbH, Germany. Membrane ion
exchange materials are available from different companies such as
membrane cation exchange materials Mustang.TM. C and Mustang.TM. S
available from Pall Corporation, Sartobind.TM. CM, Sartobind.TM. S
available from Sartorius, and anion exchange membranes, such as
Mustang.TM. Q available from Pall Corporation, Sartobind.TM. Q
available from Sartorius. In a membrane ion exchange material the
binding sites can be found at the flow-through pore walls and not
hidden within diffusion pores allowing the mass transfer via
convection rather than diffusion. In one embodiment the additional
chromatography step is a cation exchange chromatography step
employing a membrane cation exchange material selected from
Sartobind.TM. CM, or Sartobind.TM. S, or Mustang.TM. S, or
Mustang.TM. C. In another embodiment the anion exchange material is
a Q-type membrane anion exchange material or Q-type anion exchange
column.
[0017] A "polypeptide" is a polymer of amino acid residues joined
by peptide bonds, whether produced naturally or synthetically.
Polypeptides of less than about 20 amino acid residues are referred
to as "peptides."
[0018] A "protein" is a macromolecule comprising one or more
polypeptide chains or at least one polypeptide chain of more than
100 amino acid residues. A protein may also comprise non-peptidic
components, such as carbohydrate groups. Carbohydrates and other
non-peptidic substituents may be added to a protein by the cell in
which the protein is produced, and will vary with the type of cell.
Proteins are defined herein in terms of their amino acid backbone
structures; substituents such as carbohydrate groups are generally
not specified, but may be present nonetheless.
[0019] The term "immunoglobulin" and grammatical equivalents
thereof denotes a molecule consisting of two light polypeptide
chains and two heavy polypeptide chains. Each of the heavy and
light polypeptide chains comprises a variable region (generally the
amino terminal portion of the polypeptide chains), which contains a
binding domain for interaction with an antigen. Each of the heavy
and light polypeptide chains also comprises a constant region
(generally the carboxyl terminal portion of the polypeptide
chains), which may mediate the binding of the antibody to host
tissue or factors including various cells of the immune system,
some phagocytic cells and a first component (C1q) of the classical
complement system. In one embodiment the light and heavy
polypeptide chains are chains each consisting of a variable region,
i.e. V.sub.L or V.sub.H, and a constant region, i.e. of C.sub.L in
case of a light polypeptide chain, or of C.sub.H1, hinge, C.sub.H2,
C.sub.H3, and optionally C.sub.H4 in case of a heavy polypeptide
chain. The term "immunoglobulin" also refers to a protein
consisting of polypeptides encoded by immunoglobulin genes. The
recognized immunoglobulin genes include the different constant
region genes as well as the myriad immunoglobulin variable region
genes. Immunoglobulins may exist in a variety of forms.
Immunoglobulin fragments are e.g. Fv, Fab, and F(ab).sub.2 as well
as single chains (e.g. Huston, J. S., et al., Proc. Natl. Acad.
Sci. USA 85 (1988) 5879-5883; Bird et al., Science 242 (1988)
423-426; Hood et al., Immunology, Benjamin N. Y., 2nd edition
(1984); and Hunkapiller and Hood, Nature 323 (1986) 15-16). In one
embodiment of the method according to the invention the
immunoglobulin is a monoclonal immunoglobulin.
[0020] The term "immunoglobulin in monomeric form" and grammatical
equivalents thereof denotes an immunoglobulin molecule not
associated with a second immunoglobulin molecule, i.e. neither
covalently nor non-covalently bound to another immunoglobulin
molecule. The term "immunoglobulin in aggregated form" and
grammatical equivalents thereof denotes an immunoglobulin molecule
which is associated, either covalently or non-covalently, with at
least one additional immunoglobulin molecule or fragment thereof,
and which is eluted in a chromatography with a size exclusion
chromatography column before the immunoglobulin in monomeric form.
The term "in monomeric form" and grammatical equivalents thereof as
used within this application not necessarily denotes that 100% of
an immunoglobulin molecule are present in monomeric form. It
denotes that an immunoglobulin is essentially in monomeric form,
i.e. at least 90% of the immunoglobulin are in monomeric from, in
one embodiment at least 95% of the immunoglobulin are in monomeric
form, in another embodiment at least 98% of the immunoglobulin are
in monomeric form, in a further embodiment at least 99% of the
immunoglobulin are in monomeric form, and in still another
embodiment more than 99% of the immunoglobulin are in monomeric
form determined as peak area of a size exclusion chromatogram of
the immunoglobulin. The term "in monomeric and in
aggregated/fragmented form" denotes a mixture comprising at least
immunoglobulin molecules not associated with other immunoglobulin
molecules, immunoglobulin molecules associated with other
immunoglobulin molecules, and/or parts of other immunoglobulin
molecules. In this mixture neither the monomeric form nor the
aggregated form nor the fragmented form is present exclusively.
[0021] The term "100%" denotes that the amount of components other
than a specified component are below the detection limit of the
referred to analytical method under the specified conditions.
[0022] The terms "90%", "95%", "98%", "99%" denote no exact values
but values within the accuracy of the referred to analytical method
under the specified conditions.
[0023] The term "monomeric immunoglobulin depleted of
immunoglobulin aggregates and immunoglobulin fragments" denotes
that the monomeric immunoglobulin accounts in certain embodiments
for at least 90% by weight, at least 95% by weight, at least 98% by
weight, or at least 99% by weight. In turn the immunoglobulin
aggregates and immunoglobulin fragments account in certain
embodiments for not more than 10% by weight, not more than 5% by
weight, not more than 2% by weight, or not more than 1% by weight
of the preparation.
[0024] General chromatographic methods and their use are known to a
person skilled in the art. See for example, Chromatography,
5.sup.th edition, Part A: Fundamentals and Techniques, Heftmann, E.
(ed.), Elsevier Science Publishing Company, New York, (1992);
Advanced Chromatographic and Electromigration Methods in
Biosciences, Deyl, Z. (ed.), Elsevier Science BV, Amsterdam, The
Netherlands, (1998); Chromatography Today, Poole, C. F., and Poole,
S. K., Elsevier Science Publishing Company, New York, (1991);
Scopes, Protein Purification: Principles and Practice (1982);
Sambrook, J., et al. (eds.), Molecular Cloning: A Laboratory
Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, N.Y., 1989; Current Protocols in Molecular Biology,
Ausubel, F. M., et al. (eds), John Wiley & Sons, Inc., New
York.
[0025] For the purification of recombinantly produced
immunoglobulins often a combination of different chromatographical
steps is employed. Generally a protein A affinity chromatography is
followed by one or two additional separation steps. The final
purification step is a so called "polishing step" for the removal
of trace impurities and contaminants like residual HCP (host cell
protein), DNA (host cell nucleic acid), viruses, or endotoxins. For
this polishing step only often an anion exchange material in a
flow-through mode is used.
[0026] The term "flow-through mode" and grammatical equivalents
thereof denotes an operation mode of a purification method, in
which a solution containing a substance of interest, e.g. an
immunoglobulin in monomeric form, to be purified is brought in
contact with a stationary phase, in one embodiment a solid phase,
whereby the substance of interest does not bind to that stationary
phase. As a result the substance of interest is obtained either in
the flow-through (if the purification method is a chromatographical
method) or the supernatant (if the purification method is a batch
method). Substances not of interest, e.g. an immunoglobulin in
aggregated form and/or immunoglobulin fragments, which were also
present in the solution prior to the bringing into contact with the
stationary phase, bind to the stationary phase and are therewith
removed from the solution. This does not denote that 100% of the
substances not of interest are removed from the solution but
essentially 100% of the substances not of interest are removed, in
specific embodiments at least 50% of the substances not of interest
are removed from the solution, at least 75% of the substances not
of interest are removed the from solution, at least 90% of the
substances not of interest are removed from the solution, or more
than 95% of the substances not of interest are removed from the
solution as determined by the peak area of a size exclusion
chromatography.
[0027] The term "applying to" and grammatical equivalents thereof
denotes a partial step of a purification method, in which a
solution containing a substance of interest to be purified is
brought in contact with a stationary phase. This denotes that a)
the solution is added to a chromatographic device in which the
stationary phase is located, or b) that a stationary phase is added
to the solution. In case a) the solution containing the substance
of interest to be purified passes through the stationary phase
allowing for an interaction between the stationary phase and the
substances in solution. Depending on the conditions, such as e.g.
pH, conductivity, salt concentration, temperature, and/or flow
rate, some substances of the solution are bound to the stationary
phase and, thus, are removed from the solution. Other substances
remain in solution. The substances remaining in solution can be
found in the flow-through. The "flow-through" denotes the solution
obtained after the passage of the chromatographic device. In one
embodiment the chromatographic device is a column with
chromatography material, or in another embodiment a cassette with
membrane chromatography material. The substance of interest not
bound to the stationary phase can be recovered from the flow-though
by methods familiar to a person of skill in the art, such as e.g.
precipitation, salting out, ultrafiltration, diafiltration,
lyophilization, affinity chromatography, or solvent volume
reduction to obtain a concentrated solution. In case b) the
stationary phase is added, e.g. as a powder, to the solution
containing the substance of interest to be purified allowing for an
interaction between the stationary phase and the substances in
solution. After the interaction the stationary phase in removed,
e.g. by filtration, and the substance of interest not bound to the
stationary phase is obtained in the supernatant.
[0028] The term "does not bind to" and grammatical equivalents
thereof denotes that a substance of interest, e.g. an
immunoglobulin, remains in solution when brought in contact with a
stationary phase, e.g. a membrane ion exchange material. This does
not denote that 100% of the substance of interest remains in
solution but essentially 100% of the substance of interest remains
in solution, in specific embodiments at least 50% of the substance
of interest remains in solution, at least 65% of the substance of
interest remains in solution, at least 80% of the substance of
interest remains in solution, at least 90% of the substance of
interest remains in solution, or more than 95% of the substance of
interest remains in solution as determined by the peak area of a
size exclusion chromatography.
[0029] The term "buffered" denotes a solution, in which changes of
pH due to the addition or release of acidic or basic substances is
leveled by a buffer substance. Any buffer substance resulting in
such an effect can be used. In one embodiment pharmaceutically
acceptable buffer substances are used, such as e.g. phosphoric acid
and salts thereof, citric acid and salts thereof, morpholine,
2-(N-morpholino) ethanesulfonic acid and salts thereof, histidine
and salts thereof, glycine and salts thereof, or tris
(hydroxymethyl) aminomethane (TRIS) and salts thereof. In another
embodiment the buffer substance is selected from phosphoric acid
and salts thereof, citric acid and salts thereof, or histidine and
salts thereof. Optionally the buffered solution may comprise an
additional salt, such as e.g. sodium chloride, sodium sulphate,
potassium chloride, potassium sulfate, sodium citrate, or potassium
citrate.
[0030] The term "bind-and-elute mode" and grammatical equivalents
thereof denotes an operation mode of a purification method, in
which a solution containing a substance of interest to be purified
is brought in contact with a stationary phase, in one embodiment
with a solid phase, whereby the substance of interest binds to the
stationary phase. As a result the substance of interest is retained
on the stationary phase whereas substances not of interest are
removed with the flow-through or the supernatant. The substance of
interest is afterwards eluted from the stationary phase in a second
step and thereby recovered from the stationary phase with an
elution solution.
[0031] Thus, the current invention reports a method for obtaining
an immunoglobulin in monomeric form, wherein the method comprises
the following step: [0032] applying an aqueous, buffered solution
comprising an immunoglobulin in monomeric and in aggregated form
and/or immunoglobulin fragments to an anion exchange chromatography
material under conditions whereby the immunoglobulin does not bind
to the anion exchange chromatography material,
[0033] whereby the immunoglobulin in monomeric form is recovered
from the flow-through, and
[0034] wherein the aqueous, buffered solution has a pH value of
from pH 7.8 to pH 8.8.
[0035] The term "conditions under which the immunoglobulin in
monomeric form does not bind to the anion exchange chromatography
material" and grammatical equivalents thereof denotes conditions at
which an immunoglobulin in monomeric form is not bound by the anion
exchange chromatography material when brought in contact with the
anion exchange material. This does not denote that 100% of the
immunoglobulin in monomeric form is not bound but essentially 100%
of the immunoglobulin in monomeric form is not bound, in specific
embodiments at least 50% of the immunoglobulin in monomeric form is
not bound, at least 65% of the immunoglobulin in monomeric form is
not bound, at least 80% of the immunoglobulin in monomeric form is
not bound, at least 90% of the immunoglobulin in monomeric form is
not bound, or more than 95% of the immunoglobulin in monomeric form
is not bound to the anion exchange material as determined by the
peak area in a size exclusion chromatography. In one embodiment the
aqueous, buffered solution has a pH value of from pH 7.8 to pH 8.8.
In a further embodiment such a condition is a pH value of the
aqueous, buffered solution of from pH 8.0 to pH 8.5.
[0036] It has now surprisingly been found that an anion exchange
chromatography step, in which the immunoglobulin in monomeric form
can be obtained from the anion exchange material in a flow-through
mode, can be performed in a narrow pH value range of from pH 7.8 to
pH 8.8, in one embodiment of from pH 8.0 to pH 8.5. Surprisingly a
small deviation of this pH value range, e.g. to pH 7.0 or pH 9.0,
reduces this effect. With the method according to the invention it
is possible to separate in a single step the immunoglobulin in
monomeric form from the immunoglobulin in aggregated form and from
immunoglobulin fragments.
[0037] The method according to the invention can be employed as a
single step method or combined with other steps, such as, e.g., in
one embodiment with a protein A chromatography step or a
hydrophobic charge induction chromatography step.
[0038] In one embodiment the anion exchange chromatography material
is a membrane anion exchange chromatography material. It is also
advantageous e.g. to remove the bulk of the host cell proteins and
culture by-products in a foremost purification step employing an
affinity chromatography. The affinity chromatography may e.g. be a
protein A affinity chromatography, a protein G affinity
chromatography, a hydrophobic charge induction chromatography
(HCIC), or a hydrophobic interaction chromatography (HIC, e.g. with
phenyl-sepharose, aza-arenophilic resins, or m-aminophenylboronic
acid). In one embodiment the method according to the invention
comprises a protein A chromatography step or a HCIC chromatography
step prior to the anion exchange chromatography step.
[0039] In one embodiment of the method according to the invention,
wherein the method comprises more than one chromatography step,
prior to the application of a solution to one step (or to a
subsequent step) of the purification method, parameters, such as
e.g. the pH value or the conductivity of the solution, have to be
adjusted. In one embodiment the pH value of the aqueous, buffered
solution applied to the anion exchange chromatography material is
of from pH 7.8 to pH 8.8, in another embodiment of from pH 8.0 to
pH 8.5.
[0040] The following examples and figures 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.
DESCRIPTION OF THE FIGURES
[0041] FIG. 1A A solution containing an anti-CCR5 antibody was
adjusted to pH 7.0, 7.5 and 8.0 (fractions designated as pH x.x
load); 5 mg of protein were each brought in contact with a 15
cm.sup.2 membrane adsorber in flow-though mode (fractions
designated as pH x.x flow-through). The bound substances were
eluted with sodium chloride (fractions designated as pH x.x
elution). Fractions were analyzed by SDS-PAGE with Coomassie
brilliant blue staining.
[0042] FIG. 1B A solution containing an anti-CCR5 antibody was
adjusted to pH 8.5 and 9.0 (fractions designated as pH x.x. load);
5 mg of protein were each brought in contact with a 15 cm.sup.2
membrane adsorber in flow thought mode (fractions designated as pH
x.x flow-through). The bound substances were eluted with sodium
chloride (fractions designated as pH x.x elution). Fractions were
analyzed by SDS-PAGE with Coomassie-Staining.
[0043] FIG. 1C-1, FIG. 1C-2, FIG. 1C-3, FIG. 1C-4 Comparison of
load and flow-through fractions of an anti-CCR5 antibody containing
solution at pH 7.5 (FIG. 1C-1 and FIG. 1C-2) and load and
flow-through fraction at pH 8.5 (FIG. 1C-3 and FIG. 1C-4) by
analytical size exclusion chromatography. Aggregates and fragments
can be detected in the flow-through at pH 7.5, but not at pH
8.5.
[0044] FIG. 2A-1 and FIG. 2A-2 Overlay showing load and
flow-through fraction of a CD19 antibody at pH 7.5 (FIG. 2A-1) and
pH 8.5 (FIG. 2A-2). Aggregates can be detected in the flow-through
at pH 7.5, but not in the flow-through at pH 8.5.
[0045] FIG. 2B Table showing the removal of aggregates from
anti-CCR5 antibody and anti-CD19 antibody solutions. Reduction in
the flow-though is at pH values above pH 7.5, such as pH 8.5.
[0046] FIG. 3 Scale-up experiment: A solution containing an
anti-CCR5 antibody was adjusted to pH 8.5 and 25 mg were pumped
through a 75 cm.sup.2 membrane adsorber (fractions designated as pH
x.x flow-through). The bound substances were eluted with sodium
chloride (fractions designated as pH x.x. elution). Fractions were
analyzed by SDS-PAGE with Coomassie-Staining.
[0047] FIG. 4 A solution containing an anti-CCR5 antibody was
adjusted to pH 8.5 (fraction designated as pH 8.5 load) and 6 mg
protein were pumped through a 1 ml Q-Sepharose.RTM. fast-flow anion
exchange chromatography column (fractions designated as pH 8.5
flow-through). The column was eluted with sodium chloride
(fractions designated as pH 8.5. elution). Fractions were analyzed
by SDS-PAGE with Coomassie-Staining.
EXAMPLES
[0048] Materials and Methods:
[0049] Conditioned Protein A Eluate:
[0050] An anti-CCR5 antibody (hereinafter referred to as mAb CCR5,
see e.g. WO 2006/103100) and an anti-CD19 antibody (hereinafter
referred to as mAb CD19) were purified in a first step with a
protein A affinity chromatography.
[0051] The mAb CCR5 was eluted from the protein A column under
acidic conditions. Before further processing the pH value of the
fraction containing the immunoglobulin was adjusted by dialysis
against a buffered solution (e.g. tris (hydroxymethyl)
amino-methane (TRIS) or phosphate buffer) to pH values of 7.0, 7.5,
8.0, 8.5, and 9.0. This material is referred to in the following as
conditioned protein A eluate of mAb CCR5.
[0052] The mAb CD19 was eluted from the protein A column under
acidic conditions. Before further processing the pH value of the
fraction containing the immunoglobulin was adjusted by dialysis
against a buffered solution (e.g. tris (hydroxymethyl)
amino-methane (TRIS) or phosphate buffer) to a pH value of pH 8.5.
This material is referred to in the following as conditioned
protein A eluate of mAb CD19.
[0053] Analytical Methods:
[0054] Size Exclusion Chromatography: [0055] resin: TSK 3000
(Tosohaas) [0056] column: 300.times.7.8 mm [0057] flow rate: 0.5
ml/min [0058] buffer: 200 mM potassium phosphate buffer containing
250 mM potassium chloride, adjusted to pH 7.0 [0059] wavelength:
280 nm
[0060] SDS-PAGE:
[0061] LDS sample buffer, fourfold concentrate (4.times.): 4 g
glycerol, 0.682 g TRIS-Base, 0.666 g TRIS-hydrochloride, 0.8 g LDS
(lithium dodecyl sulfate), 0.006 g EDTA (ethylene diamin tetra
acetic acid), 0.75 ml of a 1% by weight (w/w) solution of Serva
Blue G250 in water, 0.75 ml of a 1% by weight (w/w) solution of
phenol red, add water to make a total volume of 10 ml.
[0062] The solution containing the immunoglobulin was centrifuged
to remove debris. An aliquot of the clarified supernatant was
admixed with 1/4 volumes (v/v) of 4.times. LDS sample buffer and
1/10 volume (v/v) of 0.5 M 1,4-dithiotreitol (DTT). Then the
samples were incubated for 10 min. at 70.degree. C. and protein
separated by SDS-PAGE. The NuPAGE.RTM. Pre-Cast gel system
(Invitrogen Corp.) was used according to the manufacturer's
instruction. In particular, 10% NuPAGE.RTM. Novex.RTM. Bis-TRIS
Pre-Cast gels (pH 6.4) and a NuPAGE.RTM. MOPS running buffer was
used.
Example 1
[0063] Conditioned protein A eluates of mAb CCR5 with pH 7.0, 7.5,
8.0, 8.5 and 9.0 were each adjusted to a concentration of 1 mg/ml.
5 ml of each solution was applied separately to a regenerated and
equilibrated (to the respective pH) Q-membrane adsorber (membrane
anion exchange material, membrane area: 15 cm.sup.2) in flow-though
mode with the help of a chromatographic system. The membrane was
afterwards washed with buffer of the correspondent pH. Bound
protein was eluted with a salt gradient at the correspondent pH
values.
[0064] It has been found that mAb CCR5 did not bind at pH 7.0 and
7.5 to the membrane adsorber. Slight binding was achieved between
pH 8.0 and 8.5. At pH 9.0 strong binding of the antibody appeared.
Analysis of the flow-through and elution fractions by size
exclusion chromatography and SDS-PAGE revealed a significant
removal of immunoglobulin aggregates and immunoglobulin fragments
from the flow-through at pH 8.0 and 8.5. No removal was visible at
pH 7.0 and 7.5 and high product losses due to matrix binding
occurred at pH 9.0. With conductivity driven elution at pH 8.0 and
pH 8.5 fractions enriched with immunoglobulin aggregates and
immunoglobulin fragments were obtained.
Example 2
[0065] Conditioned protein A eluates of mAb CD19 with pH 7.0, 7.5,
8.0, 8.5 and 9.0 were each adjusted to a concentration of 1 mg/ml.
5 ml of each solution was applied separately to a regenerated and
equilibrated (to the respective pH) Q-membrane adsorber (15
cm.sup.2) in flow-though mode with the help of a chromatographic
system. The membrane was afterwards washed with buffer of the
correspondent pH. Bound protein was eluted with a salt gradient at
the correspondent pH values.
[0066] It has been found that mAb CD19 did not bind at pH 7.0 and
7.5 to the membrane adsorber. Slight binding was achieved between
pH 8.0 and 8.5. At pH 9.0 strong binding of the immunoglobulin
appeared. Analysis of the flow-through and elution fractions by
size exclusion chromatography and SDS-PAGE revealed a significant
removal of immunoglobulin aggregates and immunoglobulin fragments
of the immunoglobulin from the product at pH 8.0 and 8.5. No
removal was visible at pH 7.0 and pH 7.5 and high product losses
due to matrix binding occurred at pH 9.0. With conductivity driven
elution at pH 8.0 and pH 8.5 fractions enriched with immunoglobulin
aggregates and immunoglobulin fragments were obtained.
Example 3
[0067] A protein A eluate of mAb CCR5 was conditioned at pH 8.5 and
adjusted to a concentration of 1 mg/ml.
[0068] 25 ml of the solution was applied to a regenerated and
equilibrated (to pH 8.5) Q-membrane adsorber (75 cm.sup.2 membrane
surface area) in flow-through mode with the help of a
chromatographic system. The membrane was afterwards washed with
buffer of pH 8.5. Bound protein was eluted with a salt gradient at
the correspondent pH values.
[0069] The result of example 1 could be reproduced at a 5-fold
larger scale. The flow-through was depleted from immunoglobulin
aggregates and immunoglobulin fragments. Both impurities could be
eluted from the membrane adsorber with a salt gradient.
Example 4
[0070] Conditioned protein A eluate of mAb CCR5 at pH 8.5 was
adjusted to a concentration of 1 mg/ml. 6 ml of the solution was
applied to a regenerated and equilibrated (to pH 8.5) Q
Sepharose.RTM. FF in flow-though mode with the help of a
chromatographic system. The sepharose was afterwards washed with
buffer of the correspondent pH. Bound protein was eluted with a
salt gradient at the correspondent pH values.
[0071] Analysis of the flow-through and the eluted fractions by
size exclusion chromatography and SDS-PAGE revealed a significant
removal of immunoglobulin aggregates and immunoglobulin fragments
of the immunoglobulin from the product at pH 8.5. With conductivity
driven elution at pH 8.0 and pH 8.5 fractions enriched with
immunoglobulin aggregates and immunoglobulin fragments were
obtained.
* * * * *