U.S. patent application number 13/445962 was filed with the patent office on 2012-08-09 for process for the purification of fc-containing proteins.
This patent application is currently assigned to MERCK SERONO SA. Invention is credited to ALEX EON-DUVAL, CELINE TEPPET.
Application Number | 20120202974 13/445962 |
Document ID | / |
Family ID | 37758645 |
Filed Date | 2012-08-09 |
United States Patent
Application |
20120202974 |
Kind Code |
A1 |
EON-DUVAL; ALEX ; et
al. |
August 9, 2012 |
PROCESS FOR THE PURIFICATION OF FC-CONTAINING PROTEINS
Abstract
The invention relates to a process for the purification of an
Fc-containing protein based on cation exchange chromatography.
Inventors: |
EON-DUVAL; ALEX;
(VILLENEUVE, CH) ; TEPPET; CELINE; (ST. PIERRE
D'ENTREMONT, FR) |
Assignee: |
MERCK SERONO SA
COINSINS, VAUD
CH
|
Family ID: |
37758645 |
Appl. No.: |
13/445962 |
Filed: |
April 13, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12521789 |
Jun 30, 2009 |
8168185 |
|
|
PCT/EP2008/050501 |
Jan 17, 2008 |
|
|
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13445962 |
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Current U.S.
Class: |
530/387.1 |
Current CPC
Class: |
C07K 1/18 20130101; A61P
35/00 20180101; A61P 37/02 20180101; A61P 31/04 20180101 |
Class at
Publication: |
530/387.1 |
International
Class: |
C07K 1/18 20060101
C07K001/18; C07K 16/00 20060101 C07K016/00 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 17, 2007 |
EP |
07000859.4 |
Claims
1. A method for separating and purifying an Fc-containing protein
from a fluid comprising at least a cation exchange chromatography
purification step comprising: (a) binding an Fc protein containing
fluid comprising an Fc-containing protein to a cation exchange
chromatography resing; (b) washing the cation chromatography resin
with a buffer at a pH about 1 unit below the isoelectric point of
the Fc-containing protein, the buffer having a conductivity of
about 2 to 6 mS/cm; (c) eluting the Fc-containing protein with a
buffer at a pH about 1 unit below the isoelectric point of the
Fc-containing protein with an increasing salt gradient; and (d)
applying the eluent of step (c) to an anionic exchange
chromatography matrix or a hydrophobic interaction chromatography
matrix.
2. The method according to claim 1, wherein the method comprises
applying the eluted Fc-containing protein to an anion exchange
chromatography matrix, eluting the Fc-containing protein from the
anion exchange chromatography matrix, applying the eluted Fc
containing protein to a hydrophobic interaction chromatography
matrix and eluting the Fc-containing protein from the hydrophobic
interaction chromatography matrix.
3. The method according to claim 1, wherein the method comprises
applying the eluted Fc-containing protein to a hydrophobic
interaction chromatography matrix, eluting the Fc-containing
protein from the hydrophobic interaction chromatography matrix,
applying the eluted Fc-containing protein to an anion exchange
chromatography matrix and eluting the Fc-containing protein from
the anion exchange chromatography resin.
4. The method according to claim 1, wherein the binding of the
Fc-containing protein in step (a) is carried out at pH below 5.
5. The method according to claim 1, wherein the Fc-containing
protein is diluted in water to a conductivity of less than 4 mS/cm
at about pH 7.0 prior to its binding to the cation exchange resin
in step (a).
6. The method according to claim 1, wherein the washing in step (b)
is carried out at a pH from about 7 to about 8.5 at a conductivity
of about 2 to 6 mS/cm.
7. The method according to claim 1, wherein the washing in step (b)
is carried out with a phosphate buffer at about pH 8, having a
conductivity of about 3.5 mS/cm.
8. The method according to claim 1, wherein the Fc-containing
protein is eluted from the cation exchange resin with an increasing
salt gradient at a conductivity ranging from about 2 to about 15
mS/cm at a pH of about 7 to about 8.5.
9. The method according to claim 1, wherein the Fc-containing
protein is eluted from the cation exchange resin with an increasing
NaCl gradient ranging from about 0 to about 150 mM at a pH ranging
from about 7 to about pH 8.5.
10. The method according to claim 1, wherein cutting out the tail
of the elution peak in step (c) is performed.
11. The method according to claim 1, wherein the cation exchange
resin in step (a) is a strong cation exchange resin.
12. The method according to claim 11, wherein the strong cation
exchange resin is Fractogel EMD SE Hicap (M) resin.
13. The method according to claim 11, wherein the resin is loaded
at about pH 4, at a conductivity of about 15 mS/cm and at a dynamic
capacity of about 40 to 47 g of Fc-containing protein per liter of
packed cation exchange resin.
14. The method according to claim 1, wherein the eluate of the
cation exchange resin resulting from step (c) has an HCP level of
less than 10,000 ppm or of less than 5,000 ppm.
15. The method according to claim 1, wherein the eluate of the
cation exchange resin resulting from step (c) has an aggregate
level of less than 1%.
16. The method according to claim 1, wherein the eluate of the
cation exchange resin resulting from step (c) has levels of
incomplete Fc-containing protein that are undetectable by SDS-PAGE
under non-reducing conditions and silver staining when loading 1
mcg of Fc-containing protein.
17. The method according to claim 1, wherein the incomplete
Fc-containing protein fragment comprises free antibody heavy and/or
light chains.
18. The method according to claim 1, wherein the Fc-containing
fluid is clarified harvest.
19. The method according to claim 1, wherein the Fc-containing
protein has an isoelectric point between about 7.5 and about
9.5.
20. The method according to claim 1, further comprising formulating
the purified Fc-containing protein into a pharmaceutical
composition.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. Ser. No.
12/521,789, filed Jun. 30, 2009, now U.S. Pat. No. 8,168,185, which
is the U.S. national stage application of International Patent
Application No. PCT/EP2008/050501, filed Jan. 17, 2008, which
claims the benefit of U.S. Provisional Patent Application No.
60/886,376, filed Jan. 24, 2007, the disclosures of which are
hereby incorporated by reference in their entireties, including all
figures, tables and amino acid or nucleic acid sequences.
FIELD OF THE INVENTION
[0002] The present invention is in the field of protein
purification. More specifically, it relates to the purification of
Fc-containing proteins. The method comprises at least a step of
purification via cation exchange chromatography.
BACKGROUND OF THE INVENTION
[0003] Proteins have become commercially important as drugs that
are generally called "biologicals". One of the greatest challenges
is the development of cost effective and efficient processes for
purification of proteins on a commercial scale. While many methods
are now available for large-scale preparation of proteins, crude
products, such as cell culture supernatants, contain not only the
desired product but also impurities, which are difficult to
separate from the desired product. Although cell culture
supernatants of cells expressing recombinant protein products may
contain less impurities if the cells are grown in serum-free
medium, the host cell proteins (HCPs) still remain to be eliminated
during the purification process. Additionally, the health
authorities request high standards of purity for proteins intended
for human administration.
[0004] A number of chromatographic systems are known that are
widely used for protein purification.
[0005] Ion exchange chromatography systems are used for separation
of proteins primarily on the basis of differences in charge.
[0006] Anion exchangers can be classified as either weak or strong.
The charge group on a weak anion exchanger is a weak base, which
becomes de-protonated and, therefore, loses its charge at high pH.
DEAE-sepharose is an example of a weak anion exchanger, where the
amino group can be positively charged below pH.about.9 and
gradually loses its charge at higher pH values. Diethylaminoethyl
(DEAE) or diethyl-(2-hydroxy-propyl)aminoethyl (QAE) have chloride
as counter ion, for instance. A strong anion exchanger, on the
other hand, contains a strong base, which remains positively
charged throughout the pH range normally used for ion exchange
chromatography (pH 1-14). Q-sepharose (Q stands for quaternary
ammonium) is an example for a strong anion exchanger.
[0007] Cation exchangers can also be classified as either weak or
strong. A strong cation exchanger contains a strong acid (such as a
sulfopropyl group) that remains charged from pH 1-14; whereas a
weak cation exchanger contains a weak acid (such as a carboxymethyl
group), which gradually loses its charge as the pH decreases below
4 or 5. Carboxymethyl (CM) and sulphopropyl (SP) have sodium as
counter ion, for example.
[0008] Hydrophobic interaction chromatography (HIC) is used to
separate proteins on the basis of hydrophobic interactions between
the hydrophobic moieties of the protein and insoluble, immobilized
hydrophobic groups on the matrix. Generally, the protein
preparation in a high salt buffer is loaded on the HIC column. The
salt in the buffer interacts with water molecules to reduce the
salvation of the proteins in solution, thereby exposing hydrophobic
regions in the protein which are then adsorbed by hydrophobic
groups on the matrix. The more hydrophobic the molecule, the less
salt is needed to promote binding. Usually, a decreasing salt
gradient is used to elute proteins from a column. As the ionic
strength decreases, the exposure of the hydrophilic regions of the
protein increases and proteins elute from the column in order of
increasing hydrophobicity.
[0009] Hydrophobic charge induction chromatography (HCIC) is
another mode of chromatography based on the pH dependent behavior
of heterocyclic ligands that ionize at low pHs. While adsorption on
this mode of chromatography occurs via hydrophobic interactions,
desorption is facilitated by lowering the pH to produce charge
repulsion between the ionizable ligand and the bound protein (e.g.
sorbent MEP Hypercel from Biosepra).
[0010] Yet a further way of purifying proteins is based on the
affinity of a protein of interest to another protein that is
immobilized to a chromatography resin. Examples for such
immobilized ligands are the bacterial cell wall proteins Protein A
and Protein G, having specificity to the Fc portion of certain
immunoglobulins. Although both Protein A and Protein G have a
strong affinity for IgG antibodies, they have varying affinities to
other immunoglobulin classes and isotypes as well.
[0011] Affinity chromatography on protein A allows the clearance of
more than 99.5% of the impurities such as host cell proteins
(HCPs), DNA, viruses, incomplete forms of the antibodies in only
one step. However, the major disadvantage of this purification
technique is the cost of the resin. It is approximately 30 times
more expensive than ion exchange resins and can represent nearly
35% of the total cost of the raw material used for large scale
purification. Protein A resin also presents some stability problems
as Protein A residues, which are potentially immunogenic, are found
in the eluate and need therefore to be cleared. Protein A resin is
also difficult to sanitize as the ligand is easily denatured by
common sanitization solutions like sodium hydroxide and this
represents a major problem in production in the event of
contamination as re-use of the resin may be detrimentally
affected.
[0012] Combinatorial chemistry has enabled the synthesis of a wide
variety of ligands which can mimic the action of protein A e.g. the
triazine derivatives that mimic the Phe-132, Tyr-133 dipeptide
binding site in the hydrophobic core structure of Protein A
(marketed as MAbsorbent A1P, A2P, and A3P by Prometic).
[0013] A further way of purifying antibodies uses affinity ligands
developed by making use of Camelidae heavy chain antibody fragments
(CAPTURESELECT products from The Bio Affinity Company).
[0014] In the field of antibody purification, Follman and Fahrner
(2004) have determined that the same host cell protein removal
obtained with a process incorporating Protein A chromatography can
be achieved using a process with no affinity chromatography steps.
They identified three non-affinity purification processes including
hydrophobic interaction chromatography, anion-exchange
chromatography and cation-exchange chromatography that remove CHOPs
(Chinese Hamster Ovary Cell Proteins) to levels comparable to the
traditional Protein A process (J Chromatogr A. 2004. Jan 23;
1024(1-2):79-85); WO 03/102132A2). They also disclose a method for
protein purification that involves the combination of non-affinity
chromatography and high performance tangential flow filtration
(HPTFF). After a first purification (capture) step on cation
exchange chromatography the host cell protein content was about
14,000 ppm.
[0015] Antibodies, or immunoglobulins (Igs) consist of light chains
and heavy chains linked together by disulphide bonds. The first
domain located at the amino terminus of each chain is variable in
amino acid sequence, providing the vast spectrum of antibody
binding specificities. These domains are known as variable heavy
(VH) and variable light (VL) regions. The other domains of each
chain are relatively invariant in amino acid sequence and are known
as constant heavy (CH) and constant light (CL) regions.
[0016] The major classes of antibodies are IgA, IgD, IgE, IgG and
IgM; and these classes may be further divided into subclasses
(isotypes). For example, the IgG class has four subclasses, namely,
IgG.sub.1, IgG.sub.2, IgG.sub.3, and IgG.sub.4.
[0017] The differences between antibody classes are derived from
differences in the heavy chain constant regions, containing between
1 and 4 constant domains (CH1-CH4), depending on the immunoglobulin
class. A so-called hinge region is located between the CH1 and CH2
domains. The hinge region is particularly sensitive to proteolytic
cleavage; such proteolysis yields two or three fragments depending
on the precise site of cleavage. The part of the heavy chain
constant region containing the CH2 and CH3 domains, optionally
together with the hinge region, is also called the "Fc" part of the
immunoglobulin. Antibodies are thus Fc-containing proteins.
[0018] Several antibodies that are used as therapeutic proteins are
known. Examples for recombinant antibodies on the market are for
instance: Abciximab, Rituximab, Basiliximab, Daclizumab,
Palivizumab, Infliximab, Trastuzumab, Alemtuzumab, Adalimumab,
Cetuximab, Efalizumab, Ibritumomab, Bevacizumab, or Omalizumab.
[0019] Another type of Fc-containing proteins is the so-called
Fc-fusion proteins. Fc-fusion proteins are chimeric proteins
consisting of the effector region of a protein, such as the Fab
region of an antibody or the binding region of a receptor, fused to
the Fc region of an immunoglobulin that is frequently an
immunoglobulin G (IgG). Fc-fusion proteins are widely used as
therapeutics as they offer advantages conferred by the Fc region,
such as: [0020] The possibility of purification using protein A or
protein G affinity chromatography with affinities which vary
according to the IgG isotype. Human IgG.sub.1, IgG.sub.2 and
IgG.sub.4 hind strongly to Protein A and all human IgGs including
IgG.sub.3 bind strongly to Protein G; [0021] An increased half-life
in the circulatory system, since the Fc region binds to the salvage
receptor FcRn which protects from lysosomal degradation; [0022]
Depending on the medical use of the Fc-fusion protein, the Fc
effector functions may be desirable. Such effector functions
include antibody-dependent cellular cytotoxicity (ADCC) through
interactions with Fc receptors (Fc.gamma.Rs) and
complement-dependent cytotoxicity (CDC) by binding to the
complement component 1q (C1q). IgG isoforms exert different levels
of effector functions. Human IgG.sub.1 and IgG.sub.3 have strong
ADCC and CDC effects while human IgG.sub.2 exerts weak ADCC and CDC
effects. Human IgG.sub.4 displays weak ADCC and no CDC effects.
[0023] Serum half-life and effector functions can be modulated by
engineering the Fc region to increase or reduce its binding to
FcRn, Fc.gamma.Rs and C1q respectively, depending on the
therapeutic use intended for the Fc-fusion protein.
[0024] In ADCC, the Fc region of an antibody binds to Fc receptors
(Fc.gamma.Rs) on the surface of immune effector cells such as
natural killers and macrophages, leading to the phagocytosis or
lysis of the targeted cells.
[0025] In CDC, the antibodies kill the targeted cells by triggering
the complement cascade at the cell surface. IgG isoforms exert
different levels of effector functions increasing in the order of
IgG.sub.4<IgG.sub.2<IgG.sub.1.ltoreq.IgG.sub.3. Human
IgG.sub.1 displays high ADCC and CDC, and is the most suitable for
therapeutic use against pathogens and cancer cells:
[0026] Under certain circumstances, for example when depletion of
the target cell is undesirable, abrogating effector functions is
required. On the contrary, in the case of antibodies intended for
oncology use, increasing effector functions may improve their
therapeutic activity (Carter et al., 2006)
[0027] Modifying effector functions can thus be achieved by
engineering the Fc region to either improve or reduce binding of
Fc.gamma.Rs or the complement factors.
[0028] The binding of IgG to the activating (Fc.gamma.RI,
Fc.gamma.RIIa, Fc.gamma.RIIIa and Fc.gamma.RIIIb) and inhibitory
(Fc.gamma.RIIb) Fc.gamma.Rs or the first component of complement
(C1q) depends on residues located in the hinge region and the CH2
domain. Two regions of the CH2 domain are critical for Fc.gamma.Rs
and complement C1q binding, and have unique sequences in IgG.sub.2
and IgG.sub.4. For instance, substitution of IgG.sub.2 residues at
positions 233-236 into human IgG.sub.1 greatly reduced ADCC and CDC
(Armour et al., 1999 and Shields et al., 2001).
[0029] Numerous mutations have been made in the CH2 domain of IgG
and their effect on ADCC and CDC was tested in vitro (Shields et
al., 2001, Idusogie et al., 2001 and 2000, Steurer et al., 1995).
In particular, a mutation to alanine at E333 was reported to
increase both ADCC and CDC (Idusogie et al., 2001 and 2000).
[0030] Increasing the serum half-life of a therapeutic antibody is
another way to improve its efficacy, allowing higher circulating
levels, less frequent administration and reduced doses. This can be
achieved by enhancing the binding of the Fc region to neonatal FcR
(FcRn). FcRn, which is expressed on the surface of endothelial
cells, binds the IgG in a pH-dependent manner and protects it from
degradation. Several mutations located at the interface between the
CH2 and CH3 domains have been shown to increase the half-life of
IgG.sub.1 (Hinton et al., 2004 and Vaccaro et al., 2005).
[0031] The following Table 1 summarizes some known mutations of the
IgG Fc-region (taken from Invivogen's website).
TABLE-US-00001 Engineered IgG Fc Isotype Mutations Properties
Potential Benefits Applications hIgG1e1 human T250Q/M428L Increased
Improved localization Vaccination; IgG1 plasma half- to target;
increased therapeutic life efficacy; reduced dose use or frequency
of administration hIgG1e2 human M252Y/S254T/ Increased Improved
localization Vaccination; IgG1 T256E + plasma half- to target;
increased therapeutic H433K/N434F life efficacy; reduced dose us or
frequency of administration hIgG1e3 human E233P/L234V/ Reduced
Reduced adverse Therapeutic IgG1 L235A/.DELTA.G236 + ADCC and
events use without A327G/A330S/ CDC cell depletion P331S hIgG1e4
human E333A Increased Increased efficacy Therapeutic IgG1 ADCC and
use with cell CDC depletion hIgG2e1 human K322A Reduced Reduced
adverse Vaccination; IgG2 CDC events therapeutic use
[0032] Given the therapeutic utility of Fc-containing proteins,
particularly antibodies and Fc-fusion proteins, there is a need for
significant amounts of highly purified protein that is adequate for
human administration. Effective purification processes are suitable
for large-scale purification of Fc-containing proteins.
SUMMARY OF THE INVENTION
[0033] The present invention is based on the development of a
cation exchange chromatography step for the purification of
Fc-containing proteins.
[0034] Therefore, in a first aspect, the invention relates to a
method for separating and purifying an Fc-containing protein from a
fluid, comprising at least a cation exchange chromatography
purification step comprising the steps of: [0035] a. Binding the
Fc-containing protein to a cation exchange resin; [0036] b. Washing
the cation exchange resin with a buffer at a pH about 1 unit below
the isoelectric point of the Fc-containing protein, the buffer
having a conductivity of about 2 to 6 mS/cm; and [0037] c. Eluting
the Fc-containing protein with a buffer at a pH about 1 unit below
the isoelectric point of the Fc-containing protein with an
increasing salt gradient.
[0038] According to the method of the invention, the eluate of the
cation exchange chromatography step can be subjected to one or more
further purification steps selected from anion exchange
chromatography and hydrophobic interaction chromatography.
[0039] This process is preferably used for purifying Fc-containing
proteins selected from antibodies and Fc-fusion proteins.
[0040] It has been surprisingly shown that the HCP level in the
eluate of the cation exchange chromatography step was less than
10,000 ppm or of less than 5,000 ppm and the level of the
aggregates level was reduced to less than 1%.
[0041] It has further been shown that the wash step (b) allowed
removal of incomplete Fc-containing protein fragments such as e.g.
incomplete antibody fragments consisting of free heavy chains or
free light chains. Therefore the second aspect of the invention
relates to the use of a cation exchange chromatography for
capturing an Fc-containing protein from a fluid, preferably
clarified cell culture supernatent, wherein, following binding of
the Fc-containing protein to the cation exchange resin, the resin
is washed with a buffer at a pH of about 1 unit below the
isoelectric point of the Fc-containing protein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0042] FIG. 1: shows the chromatographic profile of the cation
exchange chromatography described in Example 1. 1--Load, 2--Wash,
3--Elution in a NaCl gradient, 4--Regeneration/Sanitisation,
5--Re-equilibration.
[0043] Elution at pH 5: NaCl gradient from 0 to 1 M, pH 6: NaCl
gradient from 0 to 0.8 M, pH 7: NaCl gradient from 0 to 0.6 M, pH
8: NaCl gradient from 0 to 0.45 M.
[0044] FIG. 2: shows a non-reduced silver stained SDS-PAGE of
different fractions produced during the cation exchange
chromatography capture step described in Example 2.
[0045] Lane 1: Molecular weight markers
[0046] Lane 2: Standard anti-CD25 rhAb
[0047] Lane 3: Antibody anti-CD25 harvest
[0048] Lane 4: Antibody anti-CD25 harvest adjusted to pH4
[0049] Lane 5: Flow-through
[0050] Lane 6: wash
[0051] Lane 7: Elution peak
[0052] Lane 8: Elution peak tail
[0053] FIG. 3: Shows the chromatographic profile of the cation
exchange chromatography described in Example 2. (a) Conductivity
(mS/cm), (b) OD at 280 nm, (c) Buffer B1 (%). 1--Load, 2--Wash,
3--Elution, 4--Regeneration, 5--Sanitisation,
6--Re-equilibration.
[0054] FIG. 4: shows a non-reduced silver stained SDS-PAGE--Steps 2
and 3 of the three step purification processes described in
Examples 3 and 4.
[0055] Lane 1--Molecular weight markers
[0056] Lane 2--Standard anti-CD25 rhAb
[0057] Lane 3--AEX Flow-through (step 2, Process 1)
[0058] Lane 4--HIC Eluate (step 3, Bulk from Process 1)
[0059] Lane 5--HIC Eluate (step 2, Process 2)
[0060] Lane 6--AEX Flow-through (step 3, Bulk from Process 2)
[0061] FIG. 5: LabChip 90 Electropherogram. Dotted line: Bulk from
process 1. Plain line: Bulk from process 2. A: light chain
(.about.25 kDa), B: heavy chain (.about.50 kDa), C: anti-CD25
rhAb.
BRIEF DESCRIPTION OF THE SEQUENCE LISTING
[0062] SEQ ID NO 1: Anti-CD25 rhAb light chain variable region
(VH).
[0063] SEQ ID NO 2: Anti-CD25 rhAb heavy chain variable region
(VL).
[0064] SEQ ID NO 3: CDR1 of anti-CD25 rhAb heavy chain variable
region.
[0065] SEQ ID NO 4: CDR2 of anti-CD25 rhAb heavy chain variable
region.
[0066] SEQ ID NO 5: CDR3 anti-CD25 rhAb heavy chain variable
region.
[0067] SEQ ID NO 6: CDR1 of the anti-CD25 rhAb light chain variable
region.
[0068] SEQ ID NO 7: CDR2 of the anti-CD25 rhAb light chain variable
region.
[0069] SEQ ID NO 8: CDR3 of the anti-CD25 rhAb light chain variable
region.
DETAILED DESCRIPTION OF THE INVENTION
[0070] The present invention is based on the development of a
purification method based on a cation exchange chromatography step
that can significantly reduce the amount or extent of impurities
such as incomplete Fc-containing protein fragments, aggregates and
host cell proteins (HCPs) that may be present in a fluid or
composition of an Fc-containing protein.
[0071] The invention therefore relates to a method for separating
and purifying an Fc-containing protein from a fluid, comprising at
least a cation exchange chromatography step comprising the steps
of: [0072] a. Binding the Fc-containing protein to a cation
exchange resin; [0073] b. Washing the cation exchange resin with a
buffer at a pH about 1 unit below the isoelectric point of the
Fc-containing protein, the buffer having a conductivity of about 2
to 6 mS/cm; and [0074] c. Eluting the Fc-containing protein with a
buffer at a pH about 1 unit below the isoelectric point of the
Fc-containing protein with an increasing salt gradient.
[0075] This purification step will be referred to herein as cation
exchange chromatography step (CEX).
[0076] The fluid comprising the Fc-containing protein may be any
composition or preparation, such as e.g. a body fluid derived from
a human or animal, or a fluid derived from a cell culture, such as
e.g. a cell culture supernatant or cell culture harvest. Preferably
it is clarified cell culture harvest. It may also be a fluid
derived from another purification step, such as e.g. the eluate or
flow-through from a capture step or any other suitable purification
step preceding the cation exchange chromatography step.
[0077] In accordance with the present invention, a fluid comprising
an Fc-containing protein is first subjected to cation-exchange
chromatography. The fluid may preferably be cell culture material,
e.g. solubilised cells, more preferably cell culture supernatant.
The term "cell culture supernatant", as used herein, refers to a
medium in which cells are cultured and into which proteins are
secreted provided they contain appropriate cellular signals,
so-called signal peptides. It is preferred that the Fc-containing
protein expressing cells are cultured under serum-free culture
conditions. Thus, preferably, the cell culture supernatant is
devoid of animal-serum derived components. Most preferably, the
cell culture medium is chemically defined medium.
[0078] Preferably, the protein purified according to the invention
is a Fc-containing protein such as, e.g. an antibody, more
preferably a human, humanized or chimeric antibody comprising human
constant regions, preferably an IgG1 antibody, it can also
preferably be an Fc-fusion protein. Fc-containing proteins are
chimeric proteins consisting of the effector region of a protein,
such as e.g. the Fab region of an antibody or the binding region of
a receptor, fused to the Fc region of an immunoglobulin that is
frequently an immunoglobulin G (IgG).
[0079] The cation exchange chromatography according to the method
of the present invention may be used in a purification method
having one or more additional steps. The additional steps may
precede or follow the cation exchange chromatography step.
Preferably they follow the cation exchange chromatography step.
More preferably, they are selected from, anion exchange
chromatography (AEX) and hydrophobic interaction chromatography
(HIC).
[0080] Therefore in a preferred embodiment, the eluate of the
purification step on cation exchange chromatography is subjected to
a further purification step selected from anion exchange
chromatography or hydrophobic interaction chromatography.
[0081] In a further preferred embodiment, the method according to
the invention comprises, further to the cation exchange
chromatography step, two purification steps on anion exchange
chromatography and hydrophobic interaction chromatography, in
either order.
[0082] The flow-through of the anion exchange chromatography is
preferably collected. Hence, the method of the invention may
comprise cation exchange chromatography, anion exchange
chromatography and hydrophobic interaction chromatography or cation
exchange chromatography, hydrophobic interaction chromatography and
anion exchange chromatography steps. One or more further
purification steps may precede or follow the method of the
invention, if required.
[0083] Before loading the fluid comprising an Fc-containing protein
on the cation-exchange chromatography, the fluid is preferably
either adjusted to a pH of less than 5, preferably about 4 or as an
alternative diluted with water to a conductivity of less than about
4 mS/cm at about pH7. This is essential to allow binding of the
Fc-containing protein to the cation-exchange resin.
[0084] The pH of less than 5 may e.g. be at about 5.0, 4.9, 4.8,
4.7, 4.6, 4.5, 4.4, 4.3, 4.2, 4.1, 4.0, 3.9, 3.8, 3.7, 3.6, 3.5,
3.4, 3.3, 3.2, 3.1, 3.0, 2.9, 2.8, 2.7, 2.6, 2.5, 2.4, 2.3, 2.2,
2.1 or at about 2.0.
[0085] The conductivity of less than 4 mS/cm can be e.g. 4.0, 4.9,
4.8, 4.7, 4.6, 4.5, 4.4, 4.3, 4.2, 4.1, 3.0, 2.9, 2.8, 2.7, 2.6,
2.5, 2.4, 2.3, 2.2, 2.1, 2.0 or 1.9 mS/cm. It is preferably at
about 2.8 mS/cm.
[0086] Adjustment of pH to about 4 is preferred since it is easily
performed by addition of concentrated acetic acid without
increasing the load volume significantly. In addition, dynamic
capacity is high when using Fractogel SE Hicap as the
cation-exchange resin (40 to 50 g of human IgG1 per liter of packed
resin).
[0087] In step (b) of the cation exchange chromatography according
to the invention, the cation exchange resin is washed with a buffer
having a conductivity of about 2 to about 6 mS/cm and at a pH about
one pH unit below the isoelectric point of the Fc-containing
protein.
[0088] The buffer in step (b) may e.g. have a conductivity of about
2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4,
3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8,
4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9 or 6 mS/cm.
[0089] In a further preferred embodiment, the cation exchange
column is washed in step (b) with a buffer at a pH ranging from
about 7 to about 8.5 at a conductivity of about 2 to 6 mS/cm. The
pH may e.g. be at about 7.0, 7.05, 7.1, 7.15, 7.2, 7.25, 7.3, 7.35,
7.4, 7.45, 7.5, 7.55, 7.6, 7.65, 7.7, 7.75, 7.8, 7.85, 7.9, 7.95,
8, 8.05, 8.1, 8.15, 8.2, 8.25, 8.3, 8.35, 8.4, 8.45 or about
8.5.
[0090] In a most preferred embodiment, the cation exchange resin in
step (b) is washed with a phosphate buffer at about pH 8, having a
conductivity of about 3.5 mS/cm.
[0091] In a further preferred embodiment, the washing step is
carried out in a buffer comprising about 10 to about 30, preferably
20 mM sodium phosphate. The buffer may e.g. comprise 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29
or 30 mM sodium phosphate.
[0092] In step (c) of the cation exchange chromatography according
to the invention, the Fc-containing protein is eluted from the
cation exchange resin at a pH about 1 unit below the isoelectric
point of the Fc-containing protein with an increasing salt
gradient.
[0093] The elution of the Fc-containing protein may be carried out
using any suitable salt e.g. NaCl or KCl. As increasing NaCl salt
gradient is preferred.
[0094] The increasing salt gradient according to the method of the
invention is preferably a shallow gradient.
[0095] Preferably, the Fc-containing protein is eluted from the
cation exchange resin with an increasing salt gradient at a
conductivity ranging from about 2 to about 15 mS/cm at a pH of
about 7 to about 8.5. The conductivity gradient ranging from about
2 to about 15 mS/cm may be generated by an increase in sodium
chloride concentration from 0 mM to about 150 mM. The pH is
maintained constant during the gradient and may be between 7.0 and
8.5.
[0096] In a preferred embodiment, the Fc-containing protein is
eluted from the cation exchange resin at pH ranging from about 7.0
to about 8.5 with an increasing salt gradient buffer ranging from
about 0 to about 150 mM NaCl. The increasing salt gradient buffer
can e.g. range from about 0 to about 155, 0 to 145, 5 to 145, 5 to
150, 5 to 155, 10 to 145, 10 to 150 or about 10 to about 155 mM
NaCl.
[0097] The pH of the elution buffer can be at about 7.0, 7.1, 7.15,
7.2, 7.25, 7.3, 7.35, 7.4, 7.45, 7.5, 7.55, 7.6, 7.65, 7.7, 7.75,
7.8, 7.85, 7.9, 7.95, 8, 8.05, 8.1, 8.15, 8.2, 8.25, 8.3, 8.35,
8.4, 8.45, or about 8.5.
[0098] In a further preferred embodiment, the Fc-containing protein
in step (c) is eluted from the cation exchange resin with a
gradient of conductivity at a pH of about one pH unit below the
isoelectric point of the Fc-containing protein.
[0099] Elution of Fc-containing protein is monitored by the
absorbance at 280 nm and fractions are collected during the
descending phase of the peak of absorbance. Fractions are then
pooled so as to avoid aggregates and HCPs in the tail of the peak
of elution, this is referred herein as "cutting out of the tail".
The tail of the peak of elution may present a distinct shoulder
which may preferably be removed from the main peak. Alternatively,
an isocratic elution can be performed with buffer at a conductivity
and pH that will prevent the elution of aggregates and HCPs.
Preferably, the Fc-containing protein is eluted in a buffer with an
increasing NaCl gradient from about 0 to about 150 mM of NaCl at
about pH 8.
[0100] In a further preferred embodiment, the elution in step (c)
is carried out in a buffer selected from sodium phosphate, Tris or
HEPES.
[0101] In a preferred embodiment of the invention, cutting out the
tail of the elution peak is performed in step (c) of the cation
exchange chromatography step.
[0102] The cation exchange chromatography may be carried out on any
suitable cation exchange resin, such as e.g. weak or strong cation
exchangers as explained above in the Background of the
Invention.
[0103] Preferably, the cation exchange resin used in the cation
exchange chromatography is a strong cation exchange resin. A column
commercially available under the name Fractogel EMD SE Hicap (M)
(from Merck) is an example of a cation exchange resin that is
particularly suitable in the context of the present method.
[0104] In a preferred embodiment, the cation exchange resin is
loaded with clarified cell culture supernatant adjusted to pH 4 by
addition of concentrated acetic acid and after removal of
precipitated material by centrifugation or filtration. In another
embodiment, the cation exchange resin is loaded with cell culture
material adjusted to pH 4 by addition of concentrated acetic acid
and after removal of precipitated material and cell debris by
centrifugation or filtration. In a further preferred embodiment,
the resin Fractogel EMD SE Hicap is loaded with the fluid
comprising the Fc-containing protein adjusted to a pH at about pH 4
and a conductivity of about 15 mS/cm at a dynamic capacity of 40 to
47 g of Fc-containing protein per liter of packed cation exchange
resin. The conductivity of the fluid at about 15 mS/cm can be e.g.
15.9, 15.8, 15.7, 15.6, 15.5, 15.5, 15.4, 15.3, 15.2, 15.1, 15,
14.9, 14.8, 14.7, 14.6, 14.5, 14.6, 14.7, 14.6 or 14.5 mS/cm.
[0105] In a preferred embodiment of the invention, the
Fc-containing fluid loaded on the cation exchange resin in step (a)
may be clarified harvest (i.e. clarified cell culture
supernatant).
[0106] The cation-exchange chromatography is preferably used as a
capture step, and thus serves for purification of the Fc-containing
protein, in particular to the reduction, decrease or elimination,
of host cell proteins, Fc-containing protein aggregates and
incomplete fragments of the Fc-containing protein, and for
concentration of the Fc-containing protein preparation.
[0107] The term "incomplete Fc-containing protein" or "incomplete
Fc-containing protein fragments", as used herein, is meant to
encompass any part of the Fc-containing protein to be purified in
accordance with the present invention, which is derived from the
immunoglobulin constant domain or domains without comprising
complete further domains. Thus, if the Fc-containing protein
comprises immunoglobulin variable domains, incomplete Fc-containing
protein fragment does not contain significant portions of the
variable domains. If the Fc-containing protein is an Fc-fusion
protein, incomplete Fc-containing protein does not contain
significant portions of the therapeutic moiety of the Fc-fusion
protein. If the Fc-containing protein is an antibody, incomplete
Fc-containing fragments are polypeptides comprising only part of
the target antibody amino acid sequence. These fragments may arise
from the incomplete synthesis of the target antibody, from the
cleavage of one or more internal peptide bonds or from the absence
of disulphide bridges between independent subunits resulting in,
for example, free heavy chain or free light chain for
antibodies.
[0108] In accordance with the present invention, cation exchange
chromatography can preferably be used for elimination or reduction
of HCPs in the range of 20 to 350 fold i.e. 20, 40, 60, 80, 100,
120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340 fold.
Thus, the eluate of the cation exchange resin resulting from step
(c) has an HCP level of less than 10,000 ppm or less than 9,500 ppm
or less than 9,000 ppm or less than 8,500 ppm or 8,000 ppm or less
than 7,500 ppm or less than 7,000 ppm or less than 6,500 ppm or
less than 6,000 ppm or less than 5,500 or less than 5,000 ppm or
less than 4,500 ppm or less than 4,000 ppm.
[0109] The cation-exchange chromatography of the invention has the
further advantage of reducing aggregate levels by about up to 10
fold. Therefore in a preferred embodiment, the eluate of the cation
exchange column has an aggregate level of less than 1% or less than
0.9% or 0.8% or less than 0.7% or less than 0.6% or less than 0.5%
or less than 0.5% or less than 0.4% or less than 0.3% or less than
0.2% or less than 0.1%.
[0110] In addition, the cation exchange chromatography of the
invention reduces the levels of incomplete Fc-containing proteins
to below detection levels as determined by SDS-PAGE. Therefore, in
a preferred embodiment of the invention, the eluate of the cation
exchange chromatography has levels of incomplete Fc-containing
protein, that are undetectable by SDS-PAGE under non-reducing
conditions and silver staining when loading 1 mcg of Fc-containing
protein. The incomplete Fc-containing protein preferably comprises
free antibody heavy and/or light chains.
[0111] The term "aggregates", as used herein, is meant to refer to
protein aggregates, and encompasses multimers of the Fc-containing
protein to be purified, e.g. resulting in high molecular weight
aggregates.
[0112] In a highly preferred embodiment, the method of the
invention is used as a first step of a purification scheme of an
Fc-containing protein comprising the following steps: [0113] i.
Subjecting a fluid comprising said Fc-containing protein and
adjusted to a pH of less than 5 or diluted with water until the
conductivity is less than 4 mS/cm to cation-exchange chromatography
according to the method of the invention; [0114] ii. Subjecting the
eluate of step (i) to Anion exchange chromatography or hydrophobic
interaction chromatography; [0115] iii. Subjecting the eluate or
flow-through of step (ii) to Hydrophobic interaction or Anion
exchange chromatography.
[0116] In accordance with the present invention, the eluate from
the cation exchange chromatography step or from the hydrophobic
interaction chromatography step can be subjected further to an
anion exchange chromatography. The anion exchange chromatography
may be carried out on any suitable anion exchange resin, such as
e.g. weak or strong anion exchangers as explained above in the
Background of the Invention. Preferably, the anion exchange
chromatography is carried out on a strong anion exchange resin. A
resin commercially available under the name Poros 50 HQ (from
Applied Biosystems) is an example of an anion exchange resin that
is particularly suitable for the anion exchange chromatography
according to the present method.
[0117] The anion exchange column is also preferably equilibrated
with an appropriate buffer.
[0118] Preferably, the eluate from a preceding step is diluted or
dialysed into an appropriate loading buffer before loading it on
the anion exchange column. The anion exchange column is also
preferably equilibrated with the loading buffer. An appropriate
equilibration/loading/washing buffer is e.g. sodium phosphate
ranging from about 5 to about 25 mM.
[0119] From about 5 to 25 mM, the buffer concentration may e.g. be
at about 5, 10, 15, 20, 25 mM. A preferred conductivity for the
loading buffer is in the range of about 1.0 to about 4.5 mS/cm e.g.
2, 2.5, 3, 3.5, 4 or 4.5 mS/cm.
[0120] A suitable pH for the loading buffer range is about 0.5 to 1
unit below the pI. Suitable pH values range from 7.0 to 9.0,
preferably from about 7.5 to about 9.0, e.g. about 7.5, 7.55, 7.6,
7.65, 7.7, 7.75, 7.8, 7.85, 7.9, 7.95, 8.0, 8.05, 8.1, 8.15, 8.2,
8.25, 8.3, 8.35, 8.4, 8.45, 8.5, 8.55, 8.6, 8.65, 8.7, 8.75, 8.8,
8.85, 8.9 or 8.95.
[0121] An appropriate equilibration/loading/washing buffer may e.g.
be sodium phosphate at a concentration of about 5 mM and a pH at
about 8.5. The load material is dialysed or diafiltered against
such buffer or as an alternative it is diluted with water to a
conductivity of about 1, mS/cm. In the frame of the present
invention, the flow-through (also called break-through) of the
anion exchange chromatography, comprising the Fc-containing protein
of interest, is collected.
[0122] In accordance with the present invention, the eluate from
the cation exchange chromatography step or the flow through from
the anion exchange chromatography step is then subjected to
hydrophobic interaction chromatography. The hydrophobic interaction
chromatography may be carried out on any suitable hydrophobic
interaction chromatography resin. Two resins commercially available
under the name Phenyl Sepharose 6 Fast Flow High sub and Phenyl
Sepharose HP (from GE Healthcare) are examples of HIC resins that
are particularly suitable for the hydrophobic interaction
chromatography step according to the present method.
[0123] The hydrophobic interaction chromatography column is
preferably equilibrated with an appropriate equilibration
buffer.
[0124] Preferably, the eluate from a preceding step is diluted,
dialysed or diafiltered into an appropriate loading buffer before
loading it on the hydrophobic interaction chromatography column
e.g. the flow through form the annion exchange chromatography can
preferably be diluted into a loading buffer. Prior to its dilution
into a loading buffer, the eluate from the cation exchange
chromatography bstep is preferably first diafiltered into about 100
mM sodium phosphate at about pH 7.0 and concentrated at about 2 to
4 fold.
[0125] An appropriate loading buffer is e.g. a buffer consisting of
sodium phosphate at 100 mM and sodium sulfate (Na.sub.2SO.sub.4) at
0.5 to 0.6M. Suitable pH values for the
equilibration/washing/loading buffer range from about 5.0 to about
8.0, preferably from about 6.5 to about 7.5, e.g. at about 6.5,
6.55, 6.6, 6.65, 6.7, 6.75, 6.8, 6.85, 6.9, 6.95, 7.0, 7.05, 7.1,
7.15, 7.2, 7.25, 7.3, 7.35, 7.4, 7.45 or 7.5. Other anti-chaotropic
salts than sodium sulphate may be used such as for example ammonium
sulphate ((NH.sub.4).sub.2SO.sub.4) at about 1.0 to 1.2M.
Alternatively, sodium chloride (NaCl) can also be used at a
concentration of about 3.5 to 4M.
[0126] After loading, the column is washed with an appropriate wash
buffer, and the Fc-containing protein is then eluted from the HIC
resin with an appropriate elution buffer. The elution from the HIC
column can be isocratic or gradient elution.
[0127] An appropriate equilibration/wash buffer can e.g. be 100 mM
sodium phosphate at pH 7 containing 0.5 to 0.6M Na.sub.2SO.sub.4 or
1.0 to 1.2M (NH.sub.4).sub.2SO.sub.4 or 3.5 to 4.0M NaCl.
[0128] The elution from the HIC column can be isocratic or gradient
elution. An appropriate elution buffer for the isocratic elution
comprises about 5 to about 25, preferably 10, 15 or 20 mM sodium
phosphate. When gradient elution is performed, the Fc-containing
protein is eluted from the HIC resin with a decreasing salt
gradient buffer consisting of about 0.5M to 0M Na.sub.2SO.sub.4 or
about 1.0 to 0M (NH.sub.4).sub.2SO.sub.4 or about 4 to 0M NaCl in
about 100 mM to about 10 mM sodium phosphate.
[0129] In the frame of the present invention, the eluate of the
HIC, comprising the Fc-containing protein of interest, is being
collected.
[0130] In a preferred embodiment of the invention, the
Fc-containing protein has an isoelectric point (pI) between about
7.5 and about 9.5. The pI can be e.g. about 7.5, 7.6, 7.7, 7.8,
7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2,
9.3, 9.4 or about 9.5.
[0131] The volume of the resin, the length and diameter of the
column to be used, as well as the dynamic capacity and flow-rate to
be used in the various purification steps of the invention depend
on several parameters such as the volume of fluid to be treated,
concentration of protein in the fluid to be subjected to the
process of the invention, etc. Determination of these parameters
for each step is well within the average skills of the person
skilled in the art.
[0132] In a preferred embodiment of the present purification
process, one or more ultrafiltration steps are performed.
Ultrafiltration is useful for removal of small organic molecules
and salts in the eluates resulting from previous chromatographic
steps, to equilibrate the Fc-containing protein in a suitable
buffer, or to concentrate the Fc-containing protein to the desired
concentration. Such ultrafiltration may e.g. be performed by the
technique known as tangential flow filtration (TFF) on membranes,
with pore sizes allowing the removal of components having molecular
weights below 5, 10, 15, 20, 25, 30 or more kDa.
[0133] In a further preferred embodiment, the Fc-containing protein
purified according to the method of the invention comprises an
Immunoglobulin (Ig) constant region, most preferably human constant
region.
[0134] The term "Fc-containing protein", as used herein, also
refers to any protein having at least one immunoglobulin constant
domain selected from the CH1, hinge, CH2, CH3, CH4 domain, or any
combination thereof, and preferably a hinge, CH2 and CH3 domain.
The immunoglobulin constant domain may be derived from any of IgG,
IgA, IgE, IgM, or combination or isotype thereof. Preferably, it is
IgG, such as e.g. IgG.sub.1, IgG.sub.2, IgG.sub.3 or IgG.sub.4.
More preferably, it is IgG.sub.1.
[0135] In a preferred embodiment, the Fc-containing protein
comprises an immunoglobulin variable region, e.g. one or more heavy
chain variable domains and/or one or more light chain variable
domains. Preferably, the Fc-containing protein contains one or two
heavy chain variable domains. More preferably, the Fc-containing
protein additionally contains one or two light chain constant
and/or variable domains.
[0136] The term "Fc-containing protein", as used herein, is meant
to encompass proteins, in particular therapeutic proteins,
comprising an immunoglobulin-derived moiety, which will be called
herein the "Fc-moiety", and a moiety derived from a second,
non-immunoglobulin protein, which will be called herein the
"therapeutic moiety", irrespective of whether or not treatment of
disease is intended. The recombinant polypeptide fused to the
Fc-moiety may correspond to any polypeptide of interest, in
particular for polypeptides for which cellular secretion and/or
production in a cell is desired.
[0137] Fc-fusion proteins are also Fc-containing proteins that are
preferably subjected to the method of the invention.
[0138] The Fc-moiety may be derived from a human or animal
immunoglobulin (Ig) that is preferably an IgG. The IgG may be an
IgG.sub.1, IgG.sub.2, IgG.sub.3 or IgG.sub.4. The Fc-moiety may
comprise all or a part of the constant region domains of an
immunoglobulin. It is preferred that the Fc-moiety comprises at
least a CH.sub.2 and CH.sub.3 domain. It is further preferred that
the Fc-moiety comprises the Ig hinge region, the CH.sub.2 and the
CH.sub.3 domain. Particularly It is preferred that the Fc-moiety
comprises the IgG CH.sub.2 and the CH.sub.3 domain, with or without
the hinge region.
[0139] The Fc-containing protein of the invention may be a monomer
or dimer. The Fc-containing protein may also be a "pseudo-dimer",
containing a dimeric Fc-moiety (e.g. a dimer of two
disulfide-bridged hinge-CH.sub.2--CH.sub.3 constructs), of which
only one is fused to a therapeutic moiety. The Fc-containing
protein may be a heterodimer, containing two different therapeutic
moieties, or a homodimer, containing two copies of a single
therapeutic moiety. Preferably, the Fc-fusion protein is a dimer.
It is also preferred that the Fc-containing protein of the
invention is a homodimer.
[0140] In accordance with the present invention, the Fc-moiety may
also be modified in order to modulate effector functions. For
instance, the following Fc mutations, according to EU index
positions (Kabat et al., 1991), can be introduced if the Fc-moiety
is derived from IgG.sub.1: [0141] T250Q/M428L [0142]
M252Y/S254T/T256E+H433K/N434F [0143]
E233P/L234V/L235A/.DELTA.G236+A327G/A330S/P331S [0144] E333A;
K322A.
[0145] Further Fc mutations may e.g. be the substitutions at EU
index positions selected from 330, 331 234, or 235, or combinations
thereof. An amino acid substitution at EU index position 297
located in the CH.sub.2 domain may also be introduced into the
Fc-moiety in the context of the present invention, eliminating a
potential site of N-linked carbohydrate attachment. The cysteine
residue at EU index position 220 may also be replaced with a serine
residue, eliminating the cysteine residue that normally forms
disulfide bonds with the immunoglobulin light chain constant
region.
[0146] The therapeutic moiety of the Fc-containing protein may e.g.
be or be derived from EPO, TPO, Growth Hormone, Interferon-alpha,
Interferon-beta, Interferon-gamma, PDGF-beta, VEGF, IL-1alpha,
IL-1beta, IL-2, IL-4, IL-5, IL-8, IL-10, IL-12, IL-18, IL-18
binding protein, TGF-beta, TNF-alpha, or TNF-beta.
[0147] The therapeutic moiety the Fc-containing protein may also be
derived from a receptor, e.g. a transmembrane receptor, preferably
be or be derived from the extracellular domain of a receptor, and
in particular a ligand binding fragment of the extracellular part
or domain of a given receptor. Examples for therapeutically
interesting receptors are CD2, CD3, CD4, CD8, CD11a, CD14, CD18,
CD20, CD22, CD23, CD25, CD33, CD40, CD44, CD52, CD74, CD80, CD86,
CD147, CD164, IL-2 receptor, IL-4 receptor, IL-6 receptor, IL-12
receptor, IL-18 receptor subunits (IL-18R-alpha, IL-18R-beta), EGF
receptor, MIF receptor, VEGF receptor, integrin alpha 4 10 beta 7,
the integrin VLA4, B2 integrins, TRAIL receptors 1, 2, 3, and 4,
RANK, RANK ligand, epithelial cell adhesion molecule (EpCAM),
intercellular adhesion molecule-3 (ICAM-3), CTLA4 (which is a
cytotoxic T lymphocyte-associated antigen), Fc-gamma-1 receptor,
HLA-DR 10 beta, HLA-DR antigen, L-selectin, a fragment of a
receptor belonging to the TNFR superfamily such as, e.g., a
fragment derived from the extracellular domain of TNFR1 (p55),
TNFR2 (p75), OX40, Osteoprotegerin, CD27, CD30, CD40, RANK, DR3,
Fas ligand, TRAIL-R1, TRAIL-R2, TRAIL-R3, TAIL-R4, NGFR, AITR,
BAFFR, BCMA or TACT.
[0148] Therapeutic Fc-fusion proteins, i.e. Fc-fusion proteins
intended for treatment or prevention of disease of an animal or
preferably for human treatment or administration, are especially
suitable for use in the frame of the invention, to be purified in
accordance with the invention.
[0149] Most preferably, said Fc-fusion protein comprises either a
fragment of the TACI receptor (see e.g. WO 02/094852) or a fragment
of IFN-beta (see e.g. WO 2005/001025).
[0150] In a preferred embodiment of the invention, the
Fc-containing protein that can be purified according to the
invention is an antibody. Preferably, said antibody is a monoclonal
antibody. The antibody may be a chimeric antibody, a humanized
antibody or a human antibody. The antibody may either be produced
in a host cell transfected with one, two or more polynucleotides
coding for the antibody or produced from a hybridoma.
[0151] As used herein, the term "antibody" refers to a
Fc-containing protein wherein the therapeutic moiety comprises at
least one variable domain of an immunoglobulin (Ig). Preferred
immunoglobulins are mammalian immunoglobulins. More preferred
immunoglobulins are camelid immunoglobulins. Even more preferred
immunoglobulins are rodent immunoglobulins, in particular from rat
or mouse. Most preferred immunoglobulins are primate
immunoglobulins, in particular human immunoglobulins.
[0152] The term "antibody" refers to an immunoglobulin or fragment
thereof, and encompasses any polypeptide comprising an
antigen-binding site. The term includes, but is not limited to,
polyclonal, monoclonal, monospecific, polyspecific, non-specific,
humanized, human, chimeric, single-chain, synthetic, recombinant,
hybrid, mutated, grafted, or in vitro generated antibodies. The
antibody may be selected from any of the known antibody classes,
for example, IgA, IgG, IgD, IgE, IgM. The antibody may be a
monomer, dimer, or multimer such as a trimer, or pentamer.
[0153] An "antibody" refers to a glycoprotein comprising at least
two heavy (H) chains and two light (L) chains inter-connected by
disulfide bonds, or an antigen binding portion thereof. Each heavy
chain is comprised of a heavy chain variable region (abbreviated
herein as VH) and a heavy chain constant region. Each light chain
is comprised of a light chain variable region (abbreviated herein
as VL) and a light chain constant region. The VH and VL regions
retain the binding specificity to the antigen and can be further
subdivided into regions of hypervariability, termed complementarity
determining regions (CDR) The CDRs are interspersed with regions
that are more conserved, termed framework regions (FR). Each VH and
VL is composed of three CDRs and four framework regions, arranged
from amino-terminus to carboxy-terminus in the following order:
FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the
heavy and light chains contain a binding domain that interacts with
an antigen.
[0154] Examples of antibodies that can be purified in accordance
with the present invention are antibodies directed against a
protein selected from the group consisting of CD3 (e.g. OKT3,
NI-0401), CD11a (e.g. efalizumab), CD4 (e.g. zanolimumab, TNX-355),
CD20 (e.g. ibritumomab tiuxetan, rituximab, tositumomab,
ocrelizumab, ofatumumab, IMMU-106, TRU-015, AME-133, GA-101), CD23
(e.g. lumiliximab), CD22 (e.g. epratuzumab), CD25 (e.g.
basiliximab, daclizumab), the epidermal growth factor receptor
(EGFR) (e.g. panitumumab, cetuximab, zalutumumab, MDX-214), CD30
(e.g. MDX-060), the cell surface glycoprotein CD52 (e.g.
alemtuzumab), CD80 (e.g. galiximab), the platelet GPIIb/IIIa
receptor (e.g. abciximab), TNF alpha (e.g. infliximab, adalimumab,
golimumab), the interleukin-6 receptor (e.g. tocilizumab),
carcinoembryonic antigen (CEA) (e.g. 99 mTc-besilesomab),
alpha-4/beta-1 integrin (VLA4) (e.g. natalizumab), alpha-5/beta-1
integrin (VLA5) (e.g. volociximab), VEGF (e.g. bevacizumab,
ranibizumab), immunoglobulin E (IgE) (e.g. omalizumab), HER-2/neu
(e.g. trastuzumab), the prostate specific membrane antigen (PSMA)
(e.g. 111In-capromab pendetide, MDX-070), CD33 (e.g. gemtuzumab
ozogamicin), GM-CSF (e.g. KB002, MT203), GM-CSF receptor (e.g.
CAM-3001), EpCAM (e.g. adecatumumab), IFN-gamma (e.g. NI-0501),
IFN-alpha (e.g. MEDI-545/MDX-1103), RANKL (e.g. denosumab),
hepatocyte growth factor (e.g. AMG 102), IL-15 (e.g. AMG 714),
TRAIL (e.g. AMG 655), insulin-like growth factor receptor (e.g. AMG
479, R1507), IL-4 and IL13 (e.g. AMG 317), BAFF/BLyS receptor 3
(BR3) (e.g. CBI), CTLA-4 (e.g. ipilimumab).
[0155] Preferably, the antibodies that can be purified in
accordance with the present invention are antibodies directed
against a protein selected from the group consisting of CD3, CD4,
CD11a, CD25, IFN-gamma, EpCAM, TACI.
[0156] Most preferably, said antibody is selected from the group
consisting of an anti-CD4 antibody (see e.g. WO 97/13852), an
anti-CD11a antibody (see e.g. WO 98/23761) and an anti-CD25
antibody (see e.g. WO 2004/045512).
[0157] In a preferred embodiment, the antibody to be purified is
anti-CD25 rhAb of the IgG1 subclass having a human heavy chain
variable region comprising the amino acid sequence as set forth in
SEQ ID NO: 1 and human kappa light chain variable region comprising
the amino acid sequence as set forth in SEQ ID NO: 2, or
conservative sequence modifications thereof.
[0158] In yet a further preferred embodiment, the antibody is anti
CD-25 antibody comprising (i) VH CDR1 of SEQ ID NO: 3, the VH CDR2
of SEQ ID NO: 4 and the VH CDR3 of SEQ ID NO: 5 and VL CDR1 of SEQ
ID NO: 6, the VL CDR2 of SEQ ID NO: 7 and the VL
[0159] CDR3 of SEQ ID NOS: 8; or (ii) conservative sequence
modifications of any one of the sequences defined in (i).
[0160] Antibodies directed against TNF, Blys, or Interferon-.gamma.
are further examples of therapeutically interesting antibodies.
[0161] If the protein purified according to the process of the
invention is intended for administration to humans, it is
advantageous to include one or more steps of virus removal in the
process.
[0162] In order to facilitate storage or transport, for instance,
the material may be frozen and thawed before and/or after any
purification step of the invention.
[0163] In accordance with the present invention, the recombinant
Fc-containing protein may be produced in eukaryotic expression
systems, such as yeast, insect, or mammalian cells, resulting in
glycosylated Fc-containing proteins.
[0164] In accordance with the present invention, it is most
preferred to express the Fc-containing protein in mammalian cells
such as animal cell lines, or in human cell lines. Chinese hamster
ovary cells (CHO) or the murine myeloma cell line NS0 are examples
of cell lines that are particularly suitable for expression of the
Fc-containing protein to be purified. The Fc-containing protein can
also preferably be produced in human cell lines, such as e.g. the
human fibrosarcoma HT1080 cell line, the human retinoblastoma cell
line PERC6, or the human embryonic kidney cell line 293, or a
permanent amniocyte cell line as described e.g. in EP 1 230
354.
[0165] If the Fc-containing protein to be purified is expressed by
mammalian cells secreting it, the starting material of the
purification process of the invention is cell culture supernatant,
also called harvest or crude harvest. If the cells are cultured in
a medium containing animal serum, the cell culture supernatant also
contains serum proteins as impurities.
[0166] Preferably, the Fc-containing protein expressing and
secreting cells are cultured under serum-free conditions. The
Fc-containing protein may also be produced in a chemically defined
medium. In this case, the starting material of the purification
process of the invention is serum-free cell culture supernatant
that mainly contains host cell proteins as impurities. If growth
factors are added to the cell culture medium, such as insulin, for
example, these proteins will be eliminated during the purification
process as well.
[0167] In order to create soluble, secreted Fc-containing protein,
that are released into the cell culture supernatant, either the
natural signal peptide of the therapeutic moiety of the
Fc-containing protein is used, or preferably a heterologous signal
peptide, i.e. a signal peptide derived from another secreted
protein being efficient in the particular expression system used,
such as e.g. the bovine or human Growth Hormone signal peptide, or
the immunoglobulin signal peptide.
[0168] Conservative sequence modifications of any or conservative
amino acid substitutions may include synonymous amino acids within
a group which have sufficiently similar physicochemical properties
that substitution between members of the group will preserve the
biological function of the molecule (Grantham, 1974).
[0169] The Fc-containing protein to be purified in accordance with
the present invention, may also be modified at functional groups
which occur as side chains on the residues or the N- or C-terminal
groups, by means known in the art. Such modified Fc-containing
proteins and are included in the invention as long as they do not
destroy the activity of the protein which is substantially similar
to the activity of the unmodified Fc-containing protein as defined
above, and do not confer toxic properties on compositions
containing it.
[0170] For example, Fc-containing protein can e.g. be conjugated to
polymers in order to improve the properties of the protein, such as
the stability, half-life, bioavailability, tolerance by the human
body, or immunogenicity. To achieve this goal, the Fc-containing
protein may be linked e.g. to polyethylene glycol (PEG). PEGylation
may be carried out by known methods, described in WO 92/13095, for
example.
[0171] In a second aspect, the invention relates to the use of a
cation exchange chromatography for capturing an Fc-containing
protein from a fluid wherein, following binding of the
Fc-containing protein to the cation exchange resin, the resin is
washed with a buffer at a pH of about 1 unit below the isoelectric
point of the Fc-containing protein. The Fc-containing protein is
preferably eluted from the resin in a salt gradient. In addition,
cutting out the tail of the elution peak can be performed.
[0172] The invention further relates to a protein purified by the
purification method according to the invention. In the following,
such protein is also called "purified Fc-containing protein".
[0173] Such purified Fc-containing protein is preferably highly
purified Fc-containing protein. Highly purified Fc-containing
protein is determined e.g. by the presence of a single band in a
silver-stained, non-reduced SDS-PAGE-gel after loading of protein
in the amount of 2 mcg per lane. Purified Fc-containing protein may
also be defined as eluting as a single peak in HPLC.
[0174] The Fc-containing protein preparation obtained from the
purification process of the invention may contain less than 20% of
impurities, preferably less than 10%, 5%, 3%, 2% or 1% of
impurities, or it may be purified to homogeneity, i.e. being free
from any detectable proteinaceous contaminants.
[0175] Purified Fc-containing protein may be intended for
therapeutic use, in particular for administration to human
patients. If purified Fc-containing protein is administered to
patients, it is preferably administered systemically, and
preferably subcutaneously or intramuscularly, or topically, i.e.
locally. Rectal or intrathecal administration may also be suitable,
depending on the specific medical use of purified Fc-containing
protein.
[0176] For this purpose, in a preferred embodiment of the present
invention, the purified Fc-containing protein may be formulated
into pharmaceutical composition, i.e. together with a
pharmaceutically acceptable carrier, excipients or the like.
[0177] The definition of "pharmaceutically acceptable" is meant to
encompass any carrier, which does not interfere with effectiveness
of the biological activity of the active ingredient and that is not
toxic to the host to which it is administered. For example, for
parenteral administration, the active protein(s) may be formulated
in a unit dosage form for injection in vehicles such as saline,
dextrose solution, serum albumin and Ringer's solution.
[0178] The active ingredients of the pharmaceutical composition
according to the invention can be administered to an individual in
a variety of ways. The routes of administration include
intradermal, transdermal (e.g. in slow release formulations),
intramuscular, intraperitoneal, intravenous, subcutaneous, oral,
intracranial, epidural, topical, rectal, and intranasal routes. Any
other therapeutically efficacious route of administration can be
used, for example absorption through epithelial or endothelial
tissues or by gene therapy wherein a DNA molecule encoding the
active agent is administered to the patient (e.g. via a vector),
which causes the active agent to be expressed and secreted in vivo.
In addition, the protein(s) according to the invention can be
administered together with other components of biologically active
agents such as pharmaceutically acceptable surfactants, excipients,
carriers, diluents and vehicles.
[0179] For parenteral (e.g. intravenous, subcutaneous,
intramuscular) administration, the active protein(s) can be
formulated as a solution, suspension, emulsion or lyophilized
powder in association with a pharmaceutically acceptable parenteral
vehicle (e.g. water, saline, dextrose solution) and additives that
maintain isotonicity (e.g. mannitol) or chemical stability (e.g.
preservatives and buffers). The formulation is sterilized by
commonly used techniques.
[0180] The therapeutically effective amounts of the active
protein(s) will be a function of many variables, including the type
of Fc-containing protein, the affinity of the Fc-containing protein
for its ligand, the route of administration, the clinical condition
of the patient.
[0181] A "therapeutically effective amount" is such that when
administered, the Fc-containing protein results in inhibition of
its ligand of the therapeutic moiety of the Fc-fusion protein, as
explained above.
[0182] The dosage administered, as single or multiple doses, to an
individual will vary depending upon a variety of factors, including
pharmacokinetic properties of the Fc-containing protein, the route
of administration, patient conditions and characteristics (sex,
age, body weight, health, size), extent of symptoms, concurrent
treatments, frequency of treatment and the effect desired.
Adjustment and manipulation of established dosage ranges are well
within the ability of those skilled in the art, as well as in vitro
and in vivo methods of determining the inhibition of the natural
ligand of the therapeutic moiety in an individual. Purified
Fc-containing protein may be used in an amount of about 0.001 to
100 mg/kg or about 0.01 to 10 mg/kg or body weight, or about 0.1 to
5 mg/kg of body weight or about 1 to 3 mg/kg of body weight or
about 2 mg/kg of body weight.
[0183] In further preferred embodiments, the purified Fc-containing
protein maybe administered daily or every other day or three times
per week or once per week. The daily doses are usually given in
divided doses or in sustained release form effective to obtain the
desired results. Second or subsequent administrations can be
performed at a dosage which is the same, less than or greater than
the initial or previous dose administered to the individual. A
second or subsequent administration can be administered during or
prior to onset of the disease.
[0184] The present invention further relates to the use of cation
exchange chromatography for the reduction of the concentration of
HCPs, aggregates and incomplete Fc-containing protein fragments in
a composition comprising an Fc-containing protein.
[0185] In a preferred embodiment, the HCP levels are reduced to
less than 10,000 ppm or less than 9,500 ppm or less than 9,000 ppm
or less than 8,500 ppm or 8,000 ppm or less than 7,500 ppm or less
than 7,000 ppm or less than 6,500 ppm or less than 6,000 ppm or
less than 5,500 or less than 5,000 ppm or less than 4,500 ppm or
less than 4,000 ppm. Aggregate level are reduced to less than 1% or
less than 0.9% or 0.8% or less than 0.7% or less than 0.6% or less
than 0.5% or less than 0.5% or less than 0.4% or less than 0.3% or
less than 0.2% or less than 0.1%. Levels of incomplete
Fc-containing proteins such as free heavy and/or free light chains
are reduced to below detection levels as determined by SDS-PAGE
under non-reducing conditions and silver staining with a load of 1
mcg Fc-containing protein.
[0186] Having now fully described this invention, it will be
appreciated by those skilled in the art that the same can be
performed within a wide range of equivalent parameters,
concentrations and conditions without departing from the spirit and
scope of the invention and without undue experimentation.
[0187] While this invention has been described in connection with
specific embodiments thereof, it will be understood that it is
capable of further modifications. This application is intended to
cover any variations, uses or adaptations of the invention
following, in general, the principles of the invention and
including such departures from the present disclosure as come
within known or customary practice within the art to which the
invention pertains and as may be applied to the essential features
hereinbefore set forth as follows in the scope of the appended
claims.
[0188] All references cited herein, including journal articles or
abstracts, published or unpublished U.S. or foreign patent
application, issued U.S. or foreign patents or any other
references, are entirely incorporated by reference herein,
including all data, tables, figures and text presented in the cited
references. Additionally, the entire contents of the references
cited within the references cited herein are also entirely
incorporated by reference.
[0189] Reference to known method steps, conventional methods steps,
known methods or conventional methods is not in any way an
admission that any aspect, description or embodiment of the present
invention is disclosed, taught or suggested in the relevant
art.
[0190] The foregoing description of the specific embodiments will
so fully reveal the general nature of the invention that others
can, by applying knowledge within the skill of the art (including
the contents of the references cited herein), readily modify and/or
adapt for various application such specific embodiments, without
undue experimentation, without departing from the general concept
of the present invention. Therefore, such adaptations and
modifications are intended to be within the meaning a range of
equivalents of the disclosed embodiments, based on the teaching and
guidance presented herein. It is to be understood that the
phraseology or terminology herein is for the purpose of description
and not of limitation, such that the terminology or phraseology of
the present specification is to be interpreted by the skilled
artisan in light of the teachings and guidance presented herein, in
combination with the knowledge of one of ordinary skill in the
art.
EXAMPLES
Purification of Recombinant Antibodies from Serum-Free CHO Cell
Supernatant
List of Abbreviations Frequently Used Throughout the Examples
[0191] Ab: Antibody [0192] AEX: anion-exchange chromatography
[0193] BV: bed volume [0194] CEX: cation-exchange chromatography
[0195] CHO: Chinese Hamster Ovary [0196] Cond.: Conductivity [0197]
ELISA: Enzyme-Linked ImmunoSorbent Assay [0198] HCP: Host Cell
Protein [0199] HIC: hydrophobic interaction chromatography. [0200]
K: potassium [0201] kD: kilo Dalton [0202] Na: sodium [0203] NaAc:
Sodium Acetate [0204] NaCl: Sodium chloride [0205] SE-HPLC:
Size-Exclusion High Performance Liquid Chromatography (Ab
Aggregates quantification) [0206] ppm: parts per million [0207] rh:
Recombinant human [0208] RT: Room Temperature [0209] SDS-PAGE:
Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis [0210]
SE-HPLC: Size-Exclusion High Performance Liquid Chromatography
[0211] UV: Ultra-Violet
Equipment
[0211] [0212] Akta explorer 100 (GE Healthcare) [0213] Fraction
Collector Frac-950 (GE Healthcare) [0214] XK16 chromatography
column, 1.6 cm diameter (GE Healthcare) [0215] 0.66 cm
chromatography column (Omnifit) [0216] Digital Balance PM6100
(Mettler) [0217] 712 Conductometer (Metrohm) [0218] 713 pH meter
(Metrohm)
Example 1
Capture Step--Cation-Exchange Chromatography--Elution
Conditions
[0219] Cation exchange chromatography was used for the capture of
an anti-CD25 recombinant human monoclonal antibody (anti-CD25 rhAb)
produced in CHO cells. The objective of this experiment was to
evaluate the effect of the pH of the elution buffer on the yield
and the purity (i.e. content of HCPs) of the capture step. Five
different elution conditions were tested according to the following
protocol:
[0220] Starting material was clarified harvest of Anti-CD25 rhAb
having human .quadrature.1 heavy chain variable region comprising
the amino acid sequence as set forth in SEQ ID NO: 1 and human
kappa light chain variable regions comprising the amino acid
sequence as set forth in SEQ ID NO: 2. The molecular weight of the
human monoclonal antibody expressed in CHO cells under serum-free
conditions was of about 150 kilodalton (kDa) and an isoelectric
point (pI) of approximately 9. All the operations were performed at
room temperature and the flow rate was kept constant at 100 cm/h.
The UV signal at 280 nm was recorded at all time.
[0221] Column
[0222] Fractogel EMD SE Hicap (M) resin (Merck) was packed into a
1.4 ml volume column of 0.66 cm diameter having a bed height of 4
cm.
[0223] Buffers
[0224] A1=20 mM citrate/phosphate at pH 5.0, pH 6.0, pH 7.0, or pH
8.0
[0225] A2=0.5 M NaOH
[0226] B1=20 mM citrate/phosphate+1 M NaCl, pH 5.0, 6.0, 7.0 or
8.0
[0227] Equilibration
[0228] The column was equilibrated with at least 10 BV of the
adequate Buffer A1.
[0229] Loading
[0230] 70 ml of anti-CD25 rhAb harvest at a titer of about 1 g/L,
first adjusted to pH 4.5 by the addition of concentrated acetic
acid and 0.22 .quadrature.m filtered (cond. 15.0 mS/cm). The load
capacity was 47 mg of anti-CD25 rhAb as determined by Biacore assay
per ml of packed resin.
[0231] Wash Step
[0232] The column was washed with at least 10 BV of the adequate
Buffer A1.
[0233] Elution
[0234] The column was eluted in a linear NaCl gradient (see table
1, column 1) with 25 BV of buffer A1 to buffer B1 at pH 5.0, 6.0,
7.0 or 8.0 followed by 5 BV of buffer B1. 1.4 ml fractions were
collected.
[0235] Regeneration & Sanitisation
[0236] The column was regenerated with 5 BV of Buffer A2.
[0237] Re-Equilibration
[0238] The column was re-equilibrated with at least 5 BV of the
adequate Buffer A1.
[0239] Results
[0240] The elution of the anti-CD25 rhAb from the capture column
was realized by an increasing NaCl gradient in the conditions set
forth below. An isocratic elution (pH and salt concentration
constant) was also tested.
TABLE-US-00002 TABLE 1 Elution results from the capture step using
different elution conditions Anti-CD25 rhAb Anti-CD25 Elution
concentration rhAb % HCPs in the Buffer (B1) Sample type (mg/L)
Yield (%) HCP (ppm) fraction collected Starting Material 951 261705
Salt Elution Peak 846 4 176067 2.4 Gradient Shoulder 0 to 1M
Elution Peak 12600 79 97584 29.6 NaCl pH Peak Tail 865 13 665618
32.4 5.0 Salt Wash shoulder <0.5 0 <861600 0.0 Gradient Wash
Peak <0.5 0 <120448000 1.5 0 to 0.8M Start Elution Peak 169 1
722062 2.0 NaCl pH Elution Peak 10600 111 44464 18.9 6.0 Elution
Peak tail 195 2 3381101 31.8 Salt Wash Peak 3.8 0 76642447 7.0
Gradient Start Elution Peak 76.8 0 517266 0.6 0 to 0.6M Elution
Peak 6410 94 46859 16.9 NaCl pH Elution Peak tail 288 3 1700708
19.7 7.0 Salt Wash Peak 14.9 0 13281208 6.4 Gradient Start Elution
Peak 467 2 71422 0.5 0 to 0.45M Elution Peak 5780 85 20502 6.7 NaCl
Elution Peak tail 276 4 1428406 22.2 pH 8.0 Isocratic Wash Peak
13.5 0 18414815 6.0 0.35M Elution Peak 9160 96 76591 28.2 NaCl
Elution Peak tail <0.5 <0.1 <31008000 7.5 pH 8.0
[0241] FIG. 1 shows overlapping chromatograms of the wash and
elution step experiments at the conditions shown in Table 1 (except
for the isocratic elution).
[0242] As shown in Table 1 above, the antibody yield obtained for
all elution conditions (salt gradient or isocratic), was greater
than 79%. The wash step at pH 8 followed by gradient elution
resulted in a purity of 20500 ppm HCPs in the elution peak, a level
4.7 times lower than the one obtained at pH 5 i.e. 97600 ppm. These
results correlate with the largest wash peak in FIG. 1 that
corresponds to the conditions at pH 8. At the same pH (Table 1),
the use of a gradient rather than isocratic elution allowed the
elimination of more HCPs. This is because HCPs were removed not
only during the wash step (6.4%) but also in the tail of the
elution peak (22.2%).
Conclusion
[0243] Wash and elution conditions were optimized to maximize the
recovery of product while providing significant HCP clearance.
Example 2
Capture Step--Cation-Exchange Chromatography--"Scale-Up"
[0244] The optimized elution conditions from example 1 were used to
capture Anti-CD25 rhAb using a column scaled-up from IA to 20
ml.
[0245] Starting material was clarified harvest of Anti-CD25 rhAb
expressed in CHO cells cultured under serum-free conditions. All
the operations were performed at room temperature and the flow rate
was kept constant at 150 cm/h. The UV signal at 280 nm was recorded
at all time.
[0246] Column
[0247] Fractogel EMD SE Hicap (M) resin (Merck) was packed into a
20 ml volume column of 1.6 cm diameter having a bed height of 10
cm.
[0248] Buffers
[0249] A1=100 mM NaAc+128 mM NaCl, pH 4.0, Conductivity 14.7
mS/cm
[0250] A2=0.5 M NaOH+2 M KCl
[0251] A3=20 mM phosphate, pH 8.0
[0252] B1=20 mM phosphate+1 M NaCl, pH 8.0
[0253] Equilibration
[0254] The column was equilibrated with at least 10 BV of Buffer A
1 (or until the target conductivity of 14.7 mS/cm and pH 4.0.+-.0.1
are reached).
[0255] Loading
[0256] Prior to loading, the anti-CD25 rhAb clarified harvest at a
titer of 1 g/L was first adjusted to pH 4.0 by the addition of
concentrated acetic and 0.22 .quadrature.m filtered. The column was
loaded at 80% of its dynamic capacity i.e. 36.7 mg of anti-CD25
rhAb per ml of packed resin with anti-CD25 rhAb adjusted harvest
with a conductivity of 15.0 mS/cm.
[0257] Wash Step
[0258] The column was washed with 20 BV of Buffer A3.
[0259] Elution
[0260] The column was eluted in a concentration gradient of Buffer
B1 from 0 to 15% over 25 BV (i.e. 0 to 150 mM NaCl in 20 mM
phosphate pH8). 15 ml fractions were collected.
[0261] Regeneration
[0262] The column was regenerated with 5 BV of Buffer B1 at 100%
(i.e. 1 M NaCl).
[0263] Sanitisation
[0264] Then, the column was sanitised with at least 3 BV of Buffer
A2 (0.5 M NaOH+2 M KCl) in up-flow mode. After 1 hour of incubation
the column was rinsed with 2 BV of Buffer A2.
[0265] Re-Equilibration
[0266] The column was re-equilibrated with at least 5 V of Buffer
A1
[0267] Results
[0268] The results in terms of antibody yield, HCP and aggregate
clearance are shown in Table 2:
TABLE-US-00003 TABLE 2 HCPs Biacore Elimination Ab yield HCPs
factor % Aggregates Sample type (%) (ppm) (clearance) HCPs ( %)
Harvest 724905 Harvest adjusted 93 1122812 to pH 4 Flow-through 0
5947984 0.2 1.1 Wash 3 7593091 0.1 25.3 0.0 Elution peak 89 9462
118.7 0.9 0.2 Elution peak tail 5 133265 8.4 0.7 7.6
[0269] This capture step was optimized by the selection of the
Fractogel EMD SE Hicap resin on the basis of its capacity at 5%
breakthrough of 47 mg/ml at pH 4.0 (results not shown) and the
conditions of wash in 20 mM sodium phosphate pH 8 and elution in a
NaCl gradient at pH 8.0 allow a better elimination of HCPs. The
adjustment of the pH of clarified harvest to 4.0, necessary to
maximize the load capacity, caused the formation of an important
precipitate which was removed by filtration on a 0.22 .mu.m filter.
However, despite the precipitate, the recovery of anti-CD25 rhAb
was 93%.
[0270] In the chromatogram of the capture step (FIG. 3) a
substantial peak of absorbance at 280 nm (i.e. protein) was
observed during the wash step. The SDS-PAGE profile of FIG. 2,
shows that the wash step resulted in the removal of low
molecular-weight proteins including the free heavy chain and the
free light chain of the antibody (bands at approximately 50 and 25
kDa respectively) as well as HCPs. These 2 bands are absent in the
elution peak (lane 7). The wash step allowed removal of antibody
fragments (including free light and free heavy chains) as well as
HCPs (see Table 2).
[0271] As shown in FIG. 2, lane 7, the product of the elution is
relatively pure as there is only one main hand at 150 kDa, which
corresponds to the anti-CD25 rhAb.
[0272] The antibody yield after the capture step was 89% (Table 2).
With the wash step and by cutting out the elution peak tail, the
HCP levels were reduced by a factor of 119 compared to the
clarified harvest adjusted to pH 4.0 to a final level of less than
10,000 ppm. Finally, the level of aggregates in the capture eluate
was 0.2%. The tail of the elution peak showed a distinct shoulder
(see chromatogram), which contains high levels of aggregates and
HCPs and if this fraction is not pooled with the eluate fraction, a
product of high purity is obtained.
Conclusion
[0273] With the conditions developed for the capture step on CEX,
the following impurities have been reduced to very low levels:
[0274] HCPs (<10,000 ppm)
[0275] aggregates (<1%)
[0276] antibody fragments such as heavy chain and light chain
(undetected by SDS-PAGE under non-reducing conditions and silver
staining).
Example 3
Three Step Purification Process: CEX-AEX-HIC (Process 1)
[0277] A three step purification process was developed for the
purification of recombinant antibodies. The first step, the capture
step on CEX, was followed by an AEX and HIC step in 2 possible
orders: CEX-AEX-HIC or CEX-HIC-AEX, In this Example, the CEX
capture step was followed by AEX and HIC steps.
3.1 Step 1: Cation Exchange Chromatography
[0278] Capture step as described in Example 2.
3.2 Step 2: Anion Exchange Chromatography
[0279] Starting Material
[0280] The eluate from the capture step on CEX (Example 2),
dialysed into a suitable loading buffer (5 mM sodium phosphate pH
8.5), was used as a starting material for the anion exchange
chromatography.
[0281] Column
[0282] Poros 50 HQ resin (Applied Biosystems) was packed to 20 ml
volume in a column of 10 cm bed height and 1.6 cm diameter.
[0283] All the operations were performed at room temperature and
the flow rate was kept constant at 150 cm/h. The UV signal at 280
nm was recorded at all time.
[0284] Buffers [0285] A1=5 mM phosphate, pH 8.5, Cond. 1.1 mS/cm
[0286] A2=0.5 M NaOH [0287] A3=0.5 M phosphate, pH 8.5
[0288] Equilibration
[0289] The column was equilibrated with at least 10 BV of Buffer A1
(5 mM phosphate, pH 8.5 or until the target conductivity of 1.1
mS/cm and pH 8.5.+-.0.1 are reached).
[0290] Loading, Washing and Concomitant Collection of Anti-CD25
rhAb in the Flow-Through
[0291] The column was loaded with post capture material at a
concentration of 1.5 g/L, in 5 mM phosphate buffer, at pH 8.5 (pH
at 8.5.+-.0.1, conductivity at 1.1.+-.1 mS/cm). The column was then
washed with 10 BV of Buffer A1. The flow-through and wash fractions
were collected.
[0292] Elution
[0293] The column was eluted with 5 BV of buffer A3.
[0294] Sanitisation
[0295] The column was sanitised with 5 BV of buffer A2.
[0296] Pre-Equilibration
[0297] The column was pre-equilibrated with 5 BV of buffer A3.
[0298] Re-Equilibration
[0299] The column was re-equilibrated with 5 BV of buffer A1.
3.3 Step 3: Hydrophobic Interaction Chromatography.
[0300] Starting Material
[0301] The starting material used for this purification step was
anion-exchange chromatography flow-through (see Example 3.2).
[0302] Column
[0303] Phenyl Sepharose 6 Fast Flow High sub resin (GE Healthcare)
was packed to 1.4 ml volume in a column of 0.66 cm diameter and a
bed height of 4 cm.
[0304] All the operations were performed at room temperature and
the flow rate was kept constant at 100 cm/h. The UV signal at 280
nm was recorded at all time.
[0305] Buffers [0306] A1=100 mM phosphate, pH 7.0 [0307] A2=0.5 M
NaOH [0308] A3=10 mM phosphate, pH 7.0 [0309] B1=100 mM phosphate+1
M Na.sub.2SO.sub.4, pH 7.0
[0310] Equilibration
[0311] The column was equilibrated with 10 BV of a mix between
buffer A 1 and buffer B1 (50% each).
[0312] Loading
[0313] The column was loaded with the anion exchange chromatography
flow-through of Example 3.2 diluted twice in buffer B1. The column
was loaded at 80% capacity (i.e. 16.3 mg of anti-CD25 rhAb per ml
of packed resin).
[0314] Wash Step
[0315] The column was washed with 5 BV of a mix between buffer A1
and buffer B1 (50% each).
[0316] Elution
[0317] The column was eluted in a concentration gradient of Buffer
B1 from 50 to 0% over 20 BV. 1 BV fractions were collected followed
by a wash with 5 BV of buffer A3.
[0318] Sanitisation and Regeneration
[0319] Sanitisation with 5 BV of buffer A2. After 1 hour of
incubation the column was rinsed with 3 BV of water.
[0320] Re-Equilibration
[0321] The column was re-equilibrated with 5 BV of a mix between
buffer A1 and buffer B1 (50% each).
Example 4
Three Step Purification Process: CEX-HIC-AEX (Process 2)
[0322] In this process, the CEX capture step of Example 2 was
followed by a HIC and finally by an AEX step. The same protocols as
the ones described in Example 3.2 and Example 3.3 were followed
with the exception of a few different parameters.
4.1 Step 1: Cation Exchange Chromatography
[0323] Capture step as described in Example 2
4.2 Step 2: Hydrophobic Interaction Chromatography (HIC)
[0324] The eluate from the capture step on CEX (Example 2) was
diafiltered in 100 mM phosphate buffer at pH 7.0 and concentrated
(about 4 fold). The steps described in Example 3.3 above were then
followed with the following differences: column size, elution
(isocratic instead of gradient).
[0325] Column
[0326] Phenyl Sepharose 6 Fast Flow High sub resin (GE Healthcare)
was packed to 20 ml volume in a column of 1.6 cm diameter and a bed
height of 10 cm.
[0327] Loading
[0328] The column was loaded with the eluate from the CEX capture
step diafiltered and diluted twice in buffer B1 (100 mM phosphate+1
M Na.sub.2SO.sub.4) at pH 7.0. The column was loaded at 80%
capacity (i.e. 16.3 mg of anti-CD25 rhAb per ml of packed
resin).
[0329] Elution
[0330] The column was eluted with 15 BV of buffer A3 and 15 ml
fractions were collected.
4.2 Step 3: Anion Exchange Chromatography
[0331] As the last step of the process, the AEX was realized at a
smaller scale and at a lower flow rate (i.e. 100 cm/h). The step of
example 3.2 above was followed except for a few differences:
[0332] Column
[0333] Poros 50 HQ resin (Applied Biosystems) was packed to 1.4 ml
volume in a column of 4 cm bed height and 0.66 cm diameter.
[0334] Loading
[0335] The column was loaded with the eluate from the HIC step
(Example 4.2) at a concentration of 2.4 g/L, dialysed into 5 mM
phosphate buffer, at pH 8.5 (pH at 8.5.+-.0.1, conductivity at
1.1.+-.1 mS/cm).
Results (Examples 3 and 4)
[0336] Two processes for the purification of antibodies were
tested. In both cases, the capture step on CEX was followed by 2
chromatography steps in one of the following sequences: AEX-HIC
(Process 1) or HIC-AEX (Process 2). The results of the processes in
terms of antibody yield, HCPs and aggregates are shown in Table 3
below:
TABLE-US-00004 TABLE 3 Ab yield by Ab yield by HCPs HCPs clearance
Aggregates Sample type OD 280 nm (%) Biacore (%) (ppm) factor (x)
(%) Process 1 CEX-AEX-HIC Harvest 72905 Harvest adjusted to pH 4 98
1089084 0.7 Step 1: CEX Eluate 88 7397 147.2 0.2 Step 2: AEX
flow-through 95 91 542 13.7 0.3 Step 3: HIC Eluate 71 83 19 28.9
0.0 Global process Yield 58.2 58012 Process 2 CEX-HIC-AEX Harvest
724905 Harvest adjusted to pH 4 98 1089084 0.7 Step 1: CEX Eluate
88 7397 147.2 0.2 Step 2: HIC Eluate 84 2949 2.5 0.1 Step 3: AEX
flow-through 88 99 15 196.6 0.1 Global process Yield 63.7 72615
[0337] The global process yield as measured by OD at 280 nm is
approximately 58% for process 1 and approximately 64% for process 2
(Table 3). In both cases less than 20 ppm of HCPs was obtained in
the final bulk (Note: the value of HCPs of the capture eluate
differs from the value in Table 2 as 2 different CEX eluates were
mixed together). The final aggregate content for both processes is
below 0.1%. FIG. 4 (SDS-PAGE analysis) shows that process 1 (Lane
4) gave a final bulk of a purity equivalent to the Ab standard
(Lane 2) (presence of a band of very attenuated light chain). For
process 2, bands representing the free light and free heavy chains
were visible. These results were confirmed by the electopherogram
in FIG. 5 (LabChip 90 analysis), where the main peak observed
corresponds to the purified antibody. The product obtained by
process 1 is free from free heavy chain (see peak B, dotted line).
The values in Table 4 below confirm that the concentration of free
heavy and free light chain are very low (<1%):
TABLE-US-00005 TABLE 4 Free Light Free Heavy chain (~25 chain (~50
kDa) kDa) Process 1 bulk 0.7% 0.1% Process 2 bulk 0.5% 0.3%
[0338] In addition, DNA levels in the purified bulk from process 1
(8.9 .mu.g per mg of Ab) are equivalent to those obtained with the
process Protein A affinity-CEX-AEX (9.4 .mu.g per mg of Ab) (not
shown).
Conclusion
[0339] The purity (HCPs, aggregates, incomplete antibody fragments,
DNA) obtained in the purified bulk produced with process 1
consisting of the sequence CEX-AEX-HIC is equivalent to that of the
process consisting of the sequence Protein A affinity-CEX-AEX.
Overall Conclusion
[0340] It has been shown that with the conditions developed for the
capture step for antibodies on CEX, very low levels of HCPs
(<10,000 ppm) and aggregates (<1%) could be obtained.
Antibody fragments such as free heavy chain and free light chain
were significantly reduced and undetectable by SDS-PAGE
analysis.
[0341] This optimized capture step gives an antibody of high purity
for HCPs, aggregates and antibody fragments at a high yield
(>90%). In addition, the high dynamic capacity of the capture
column when loading clarified harvest at pH4 (47 g/L) makes this
step very cost-effective.
[0342] The three step process with the addition of an AEX and a HIC
steps gives final material of purity comparable to a process with
affinity chromatography on Protein A with respect to HCPs,
aggregates, DNA and antibody fragments but at a lower cost (data
not shown).
[0343] The three step process (according to either process1 or 2)
resulted in highly purified anti-CD25 rhAb with an overall
reduction of aggregates to less than 0.1%, overall reduction of
HCPs to 15 to 20 ppm and an overall reduction of free light and
heavy chains to less than 1%.
REFERENCES
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[0346] 3. Follman D K, Fahrner R L. (2004). Factorial screening of
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[0347] 4, Grantham, R. (1974). Amino acid difference formula to
help explain protein evolution. Science 185, 862-864. [0348] 5.
Hinton P R, Johlfs M G, Xiong J M, Hanestad K, Ong K C, Bullock C,
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Engineered human IgG antibodies with longer serum half-lives in
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98/23761
Sequence CWU 1
1
81127PRTHomo sapiens 1Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly
Gly Thr Phe Ser Arg Tyr 20 25 30Ile Ile Asn Trp Val Arg Gln Ala Pro
Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Arg Ile Ile Pro Ile Leu Gly
Val Glu Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr
Ala Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser
Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Lys Asp
Trp Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110Val Ser
Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120
1252119PRTHomo sapiens 2Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu
Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser
Gln Ser Val Ser Ser Tyr 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly
Gln Ala Pro Arg Leu Leu Ile 35 40 45Tyr Gly Ala Ser Ser Arg Ala Thr
Gly Ile Pro Asp Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe
Thr Leu Thr Ile Ser Arg Leu Glu Pro65 70 75 80Glu Asp Phe Ala Val
Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro Leu 85 90 95Thr Phe Gly Gly
Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110Pro Ser
Val Phe Ile Phe Pro 11535PRTHomo sapiens 3Arg Tyr Ile Ile Asn1
5417PRTHomo sapiens 4Arg Ile Ile Pro Ile Leu Gly Val Glu Asn Tyr
Ala Gln Lys Phe Gln1 5 10 15Gly56PRThomo sapiens 5Lys Asp Trp Phe
Asp Tyr1 5611PRThomo sapiens 6Arg Ala Ser Gln Ser Val Ser Ser Tyr
Leu Ala1 5 1077PRThomo sapiens 7Gly Ala Ser Ser Arg Ala Thr1
589PRThomo sapiens 8Gln Gln Tyr Gly Ser Ser Pro Leu Thr1 5
* * * * *