INTRODUCTION
Aspects of the application relate to methods of purifying antibodies using chromatographic methods. In embodiments  the application describes antibody purification methods comprising multiple chromatographic steps  wherein a low pH eluate from a protein A chromatography is further purified without a requirement for substantial pH adjustment.
Large scale purification of proteins remains a significant challenge in the biopharmaceutical industry as efficient and cost-effective methods are required to achieve desired yields and purity levels. Therapeutic proteins are primarily produced by recombinant DNA technology  i.e.  by cloning and expression of a heterologus gene in prokaryotic or eukaryotic systems. However  proteins expressed by recombinant DNA methods are typically associated with contaminants such as host cell proteins (“HCP”)  host cell DNA (“HCD”)  viruses  etc. In addition  there is significant microheterogeneity in the expression of the desired protein  in the form of charged variants (typically acidic  lower pI variants and basic  higher pI variants).The presence of these contaminants  including undesirable charged variants  are a potential health risk  and hence their removal from a final product is a regulatory requirement. Thus  drug regulatory agencies such as United States Food and Drug administration (“FDA”) require that biopharmaceuticals be free from impurities  both product related (aggregates or degradation products) and process related (media components  HCP  DNA  chromatographic media used in purification  endotoxins  viruses  etc). See  Office of Biologics Research and Review  Food and Drug Administration  Points to consider in the production and testing of new drugs and biologicals produced by recombinant DNA technology (Draft)  1985. Thus  elimination of impurities and contaminants from a final product is mandatory and poses a significant challenge in the development of methods for the purification of proteins.
Protein purification is usually a multistep process  wherein different chromatographic steps are run sequentially to yield a final purified product. For purification of monoclonal antibodies  protein A chromatography is one of the more widely used methods  and usually is the first step in antibody purification. This is a type of affinity chromatography wherein separation is affected by means of a resin tagged with protein A (Hjelm H. et.al.  FEBS lett. 1972; 28  73-76; Langone JJ.  Adv Immunol  1982; 32  157-252). The various aspects of Protein A chromatography (protein A and its variants  chromatographic medium  etc.) are described in U.S. Patent Nos. 6 013 763 and 6 399 750  and European Patent Application Publication Nos. 282308 and 284368.
A disadvantage of protein A chromatography is the leaching of Protein A and its fragments from the chromatographic resin and its contamination of the eluate. Since protein A is of bacterial origin (obtained from Staphylococcus aureus)  it’s removal is necessary to avoid undesirable immune responses. It has been shown that IgG can form complexes with protein A that may activate Fc bearing leukocytes and complement system to generate oxidant and anaphylatoxin activity in vitro (Balint J. et.al.  Cancer Res. 1984; 44  734-743). Further  protein A has also been linked with toxicity (Bensinger  W.I. et. al.  J. Biol Resp. Modif. 1984; 3  347; Messeschimdt GL. et. al.  J. Biol. Resp. Modif. 1984; 3  325; Terman D.S. and Bertram  J.H.  Eur. J. Cancer Clin. Oncol. 1985; 21  1115 and Ventura G.J et. al.  Cancer Treat. Rep. 1987; 71  411). Thus  subsequent purification steps are required to remove protein A leachates  as well as residual host cell proteins  host cell DNA  etc. to meet regulatory requirements.
The literature discloses various methods for purification of crude or partially purified samples. Balint et. al. describe the use of gel filtration for separating uncomplexed antibodies from IgG-protein A complexes (Balint et.al.  Cancer Res 1984; 44  734-743). U.S. Patent No. 4 983 722  European Patent Application Publication No. 1601697 and U.S. Patent Application Publication No. 2007/0292442 describe the use of ion exchange chromatography for purification of antibodies. However  these methods either result in considerable loss of antibody or require substantial pH adjustment of the sample prior to a subsequent chromatography step. This change in pH is achieved by addition of a high molarity base that compromises process efficiency as a result of volume dilution and mixing efficiency  as well as product stability due to localized pH surge. The impact on product stability is of particular significance as it leads to significant product loss due to denaturation  precipitation  and aggregation.
There remains a need for efficient multi-step separation processes for antibodies.

SUMMARY
In aspects  the application describes antibody purification methods  comprising multiple chromatographic steps wherein a low pH eluate from a protein A chromatography is further purified without the need of substantial pH adjustment.

BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 is an illustration of a chromatogram from the procedure of Example 1.
Fig. 2 is an illustration of a chromatogram from the procedure of Example 2.
Fig. 3 is an illustration of a chromatogram from the procedure of Example 5.
Fig. 4 is an illustration of a chromatogram from the procedure of Example 3.
Fig. 5 is an illustration of a chromatogram from the procedure of Example 5.
Fig. 6 is an illustration of a chromatogram from the procedure of Example 5.

DETAILED DESCRIPTION
The term “flow-through mode” as used herein refers to chromatographic methods wherein a desired protein is obtained in the flow-through liquid during loading or post-loading washing of a chromatography column. A desired protein in the flow-through liquid may be collected as various fractions and pooled together or can be collected as a single fraction.
The term “bind-elute mode” as used herein refers chromatographic methods wherein a desired protein is bound to the chromatography resin when loaded onto a column and is eluted subsequently using an elution buffer. The desired protein is collected in an elution liquid and may be collected as a single fraction or as various fractions that are pooled together.
The term “antibody” as used herein refers to an immunoglobulin that is composed of four polypeptide chains consisting of two light and two heavy chains  as well as immunoglobulins isolated from various sources  such as murine  human  recombinant  etc.  truncated antibodies  chimeric  humanized  or pegylated antibody isotypes  allotypes  and alleles of immunoglobulin genes. The term antibody also refers to fusion proteins that contain an immunoglobulin moiety.
In the purification of monoclonal antibodies (“MAbs”)  protein A chromatography is a commonly used method  as highly purified MAbs can be obtained due to the high specificity and binding between a protein A ligand and a Fc region of the antibody. However  as discussed earlier  a disadvantage of protein A chromatography is the leaching of protein A and its fragments into the eluate. Hence  further purification steps are required for the removal of protein A and/or its fragments  as well as residual host cell proteins  endotoxins  and host cell DNA.
U.S. Patent No. 4 983 722 and European Patent Application Publication No. 1601697 describe the use of anion exchange chromatography for the purification of antibodies. However  the anion exchange step is performed at neutral to alkaline pH values  necessitating substantial pH adjustment of the antibody sample. For instance  in US 4 983 722 the protein A eluate is diafiltered against a DEAE equilibration buffer at pH 8.6  while in EP 1601697 the acidic protein A eluate is neutralized with a high molarity buffer  such as 0.5M TrisHCl pH 7.5  and diafiltered with binding/equilibration buffer at pH 8.0 prior to the next chromatographic step. Likewise  U.S. Patent Application Publication No. 2007/0292442 describes the use of two ion exchange resins for the purification of antibodies  wherein the pH of the first eluate is adjusted before loading onto the second ion exchange resin. The pH adjustment greatly compromises both the process efficiency and the product stability. Hence a process involving no  or minimal  pH adjustment will be a better alternative to the current methods.
In aspects  the present application describes processes that reduce the need of pH adjustment in a multistep purification process  thereby increasing process efficiency and product stability  in addition to achieving effective separation of acidic and basic variants of monoclonal antibodies.
In an aspect  the application provides methods for purifying antibodies  embodiments comprising:
1) A first purification step using protein A chromatography  wherein an antibody is eluted at particular pH values.
2) A second purification step using cation exchange chromatography that is performed in the bind-elute mode  wherein eluate obtained from step 1) is loaded onto a cation exchange resin without substantial adjustment of pH (viz. within a range of ± 0.2 pH values).

In embodiments  an antibody is eluted in purification step 1) at pH values about 3.5 to 6 and loaded onto a cation exchange resin at pH values about 3.5 to 6.
In embodiments  an antibody is eluted in purification step 1) at pH values about 3.5 and loaded onto a cation exchange resin at pH values about 3.5.
In an aspect  the application provides methods for purifying antibodies  embodiments comprising:
1) A first purification step using protein A chromatography  wherein an antibody is eluted at a first pH.
2) A second purification step using cation exchange chromatography performed in the bind-elute mode  wherein eluate obtained from step 1) is loaded onto a cation exchange resin at a second pH  and the elution is carried out at a third pH.
3) A third purification step using anion exchange chromatography performed in the flow-through mode  wherein eluate from step 2) is loaded onto an anion exchange resin without substantial adjustment of pH (viz. within a range of ± 0.2 pH values).
In embodiments  the first  second  and third pH values may be same or different.
In embodiments  the first and second pH values may be similar.
In embodiments  the first and second pH values may be different.
The protein A chromatographic resin used may be any protein A or variant or a functional fragment thereof coupled to any chromatographic support. In embodiments  the protein A resin is Prosep vA Ultra® (from Millipore)  wherein animal-free protein A is immobilized on porous glass. In embodiments  fresh (i.e.  not previously used) protein A chromatographic resin may be used to obtain a feed stream for a second chromatographic step. After washing with loading buffer and intermediate wash  the elution is carried out at low pH such as from pH 2.5 to about pH 4.5
Cation exchange chromatographic step mentioned in the embodiments may be carried out using any weak or strong cation exchange chromatographic resin or a membrane which could function as a weak or a strong cation exchanger. Commercially available cation exchange resins include  but are not limited to  those having a sulfonate based group e.g.  MonoS  MiniS  Source 15S and 30S  SP Sepharose Fast Flow  SP Sepharose High Performance from GE Healthcare  Toyopearl SP-650S and SP-650M from Tosoh  S-Ceramic Hyper D  from Pall Corporation or a carboxymethyl based group e.g.  CM Sepharose Fast Flow from GE Healthcare  Macro-Prep CM from BioRad  CM-Ceramic Hyper D  from Pall Corporation  Toyopearl CM-650S  CM-650M and CM-650C from Tosoh. In embodiments of the invention  a weak cation exchange resin  such as CM Ceramic Hyper D F® (Pall Corporation) is used; this is made using rigid porous beads that are coated with functionalized hydrogel.
Anion exchange chromatography mentioned in the embodiments may be carried out using any weak or strong anion exchange chromatographic resin or a membrane which could function as a weak or a strong anion exchanger. Commercially available anion exchange resins include  but are not limited to  DEAE cellulose  Poros PI 20  PI 50  HQ 10  HQ 20  HQ 50  D 50 from Applied Biosystems  MonoQ  MiniQ  Source 15Q and 3OQ  Q  DEAE and ANX Sepharose Fast Flow  Q Sepharose high Performance  QAE SEPHADEX and FAST Q SEPHAROSE from GE Healthcare  Macro-Prep DEAE and Macro-Prep High Q from Biorad  Q-Ceramic Hyper D  DEAE-Ceramic Hyper D  from Pall Corporation. In embodiments of the invention  a strong anion exchange resin  such as Q- Sepharose Fast Flow® (GE Healthcare Life Sciences) is used. This resin is made using a highly cross-linked  6 % agarose matrix attached to -O-CH2CHOHCH2OCH2CHOHCH2N+(CH3)3 functional group.
Examples of buffering agents used in the buffer solutions include  but are not limited to  TRIS  phosphate  citrate  and acetate salts  or derivatives thereof.
Protein A leachates can be analysed using protein A ELISA and purified antibodies can be analysed using protein A high performance liquid chromatography.
Certain specific aspects and embodiments of the application are more fully described by reference to the following examples  being provided only for purposes of illustration. These examples should not be construed as limiting the scope of the application in any manner.

EXAMPLE 1
Protein A chromatography
An anti-VEGF antibody was cloned and expressed in a CHO cell line as described in U.S. Patent No. 7 060 269  which is incorporated herein by reference. The cell culture broth containing the expressed antibody was harvested  clarified and subjected to protein A affinity chromatography as described below.
The clarified cell culture broth was loaded onto a protein A chromatography column (Prosep vA ultra  VL44x250  205 mL) that was pre-equilibrated with 5 column volumes (“CV”) of 50 mM Tris  150 mM NaCl  pH 7.5 buffer. The column was then washed with 5 CV of the equilibration buffer (50 mM Tris  150 mM NaCl  pH 7.5). This was followed by a wash with 5 CV of 50 mM Tris  750 mM NaCl  pH 7.5 buffer and a final wash with 25 mM Tris at pH 7.5. The bound antibody was eluted using the low pH buffer 200 mM acetate buffer  pH 3.5.
Figure 1 is an illustration of a chromatogram from the procedure as described in this example. The line marked “Cond” represents the increase in conductivity in mS/cm. Peak A  represents the eluate obtained from protein A chromatography resin.

EXAMPLE 2
Cation exchange chromatography
The eluate obtained from the protein A chromatography procedure described in Example 1 was loaded onto a cation exchange resin (CM Ceramic Hyper D F  VL44x250  304 mL) pre-equilibrated with 10 CV of equilibration buffer (200 mM acetate buffer  pH 3.5). This was followed by washing with 30 CV of wash buffer (35 mM Phosphate buffer pH 6.0). The bound antibody was eluted using a conductivity gradient (2.5 mS/cm to- 7 mS/cm) with a phosphate buffer (35 mM to 80 mM  pH 6.0).
Figure 2 is an illustration of a chromatogram from the procedure as described in this example. The line marked “Cond” represents the increase in conductivity in mS/cm. “Buffer Conc.” represents the concentration of phosphate buffer during the chromatographic run  where 100% corresponds to a buffer concentration of 80 mM. Peak A represents the acidic variant of the antibody  and Peak B represents the more basic (desired) variant of the antibody. The consistency of elution (Peak B) in multiple runs is shown.

EXAMPLE 3
Anion exchange chromatography
The eluate obtained from the cation exchange chromatography procedure described in Example 2 was loaded onto an anion exchange resin (Q-Sepharose FF  VL32x250  80 mL) pre-equilibrated with 5-20 CV of an equilibration buffer (67 mM phosphate buffer  pH 6). This was followed by a post load wash with 5 CV of equilibration buffer and the load and wash flow-through was collected.
Figure 4 is an illustration of a chromatogram from the procedure described in this example. The line marked “Cond” represents the increase in conductivity in mS/cm. “FT” represents the flow-through obtained. Impurity levels in the cation exchange chromatography eluate and anion exchange chromatography flow through are described in Table 1.
Table 1
Sample Impurity Concentration Step Recovery (%) Monomer (%)
Host Cell Proteins (ppm) Host Cell DNA (ng/mL) Protein A leachate (ppm)
Clarified cell culture broth 50170 296 -- 100
Protein A eluate (eluate pH 3.5) 5.0 ND 23.3 97
Cation Exchange chromatography eluate (load pH 3.5) BDL < 0.19 BDL 53 99.8
Anion Exchange chromatography flow through (load pH 6.0) BDL < 0.19 BDL 97
BDL: Below detection limit.
ppm: parts per million.
ND: not determined.

EXAMPLE 4
Protein A chromatography
Clarified cell culture broth from Example 1 was loaded onto a protein A chromatography resin (Prosep vA ultra  VL44x250  205 mL) that was pre-equilibrated with 5 CV of a 50 mM Tris  150 mM NaCl  pH 7.5 buffer. The column was then washed with 5 CV of an equilibration buffer (50 mM Tris  150 mM NaCl  pH 7.5). This was followed by a wash with 5 CV of a 50 mM Tris  750 mM NaCl  pH 7.5 buffer and a final wash with 25 mM Tris at pH 7.5. The bound antibody was eluted using the low pH buffer 200 mM acetate buffer  pH 3.5.

EXAMPLE 5
Cation exchange chromatography
The eluate obtained from protein A chromatography step described in Example 4 was adjusted to pH 6.0 and loaded onto a cation exchange resin (CM-Ceramic Hyper D F  VL44x250  304 mL) pre-equilibrated with 10 CV of equilibration buffer (35 mM phosphate buffer pH 6.0). This was followed by washing with 10 CV of equilibration buffer. The bound antibody was eluted using a conductivity gradient (2.5 mS/cm to 7 mS/cm) with a phosphate buffer (35 mM to 80 mM  pH 6.0). Alternatively  elution was done using a step-gradient with less than 3 to 5 CV of phosphate buffer  pH 6.2 wherein the concentration of the buffer was increased in steps from 35 mM to 80 mM  or from 35 mM to 90 mM.
Figure 3 is an illustration of a chromatogram from the procedure described in this example. The line marked “Cond” represents the increase in conductivity in mS/cm. “Buffer Conc.” represents the concentration of phosphate buffer during the chromatographic run  where 100% corresponds to a buffer concentration of 80 mM. Peak A represents the eluate obtained from the linear increase in conductivity.
Figure 5 is an illustration of a chromatogram from the procedure described in this example. The line marked “Cond” represents the increase in conductivity in mS/cm. Peak A represents the eluate obtained from the step increase in conductivity from 35 mM buffer to 80 mM buffer.
Figure 6 is an illustration of a chromatogram from the procedure described in this example. The line marked “Cond” represents the increase in conductivity in mS/cm. Peak A represents the eluate obtained from a step increase in conductivity from 35 mM buffer to 90 mM buffer.

EXAMPLE 6
Anion exchange chromatography
The eluate obtained from cation exchange chromatography described in Example 5 was loaded onto an anion exchange resin (Q-Sepharose FF  VL32x250  80 mL) pre-equilibrated with 5-20 CV of an equilibration buffer (67 mM phosphate buffer  pH 6). This was followed by a post load wash with 5 CV of equilibration buffer and the load and wash flow-through collected. Impurities levels in the cation exchange chromatography eluate and anion exchange chromatography flow-through are described in Table 2.
Table 2
Sample Impurity Concentration
Host Cell Proteins Protein A Leachates
Clarified cell culture broth 106551 ppm -
Protein A eluate 2.24 ppm 12.96 ppm
Cation exchange chromatography (load pH 6) eluate BDL BDL
Anion exchange chromatography (load pH 6.0) BDL BDL
BDL: Below detection limit.
ppm: parts per million.
WE CLAIM:
1. A process for purifying antibodies comprising:
a. purifying using protein A chromatography  wherein the antibody is eluted at a particular pH value; and
b. purifying using cation exchange chromatography in the bind-elute mode  wherein an eluate from step a) is loaded onto a cation exchange resin without substantial adjustment of pH.
2. A process according to claim 1  wherein the antibody is eluted in
step a) at pH values about 3.3 to about 4.5.
3. A process according to claim 1  wherein the antibody is eluted in step a) at a pH value about 3.5.
4. A process for purifying antibodies  comprising:
a. purifying using protein A chromatography  wherein an antibody is eluted at a first pH value;
b. purifying using cation exchange chromatography performed in the bind-elute mode  wherein eluate obtained from step a) is loaded onto a cation exchange resin at a second pH value  and elution is carried out at a third pH value; and
c. purifying using anion exchange chromatography performed in the flow-through mode  wherein eluate from step b) is loaded on to an anion exchange resin without substantial adjustment of pH.
5. A process according to claim 4  wherein the first and second pH values are similar.
6. A process according to claim 4  wherein the first and second values are different.
7. A process according to claim 4  wherein the second and third pH values are similar.
8. A process according to claim 4  wherein the first pH value is about 3.5
9. A process according to claim 4  wherein the second pH value is about 6 to about 8.
10. A process according to claim 4  wherein the second pH value is about 6.
11. A process according to claim 4  wherein the third pH value is about 6 to about 8.
12. A process according to claim 4  wherein the third pH value is about 6.
13. A process according to claim 4  wherein elution performed in step b uses a linear or a step-wise increase of eluting fluid conductivity.