INTRODUCTION
Aspects of this application relate to purification of proteins using chromatographic methods. In embodiments  purification is conducted at low pH values. In particular embodiments  the application relates to purification processes comprising protein A chromatography and anion exchange chromatography  wherein a protein A chromatography eluate is further purified by anion exchange chromatography at similar pH values  or at pH values less than or equal to 6.
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 heterologous 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. The presence of these contaminants is a potential health risk  and hence their removal from final products 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 FDA Office of Biologics Research and Review  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 final products is mandatory and poses a significant challenge in the development of methods for the purification of proteins.
Protein purification is frequently a multistep process  wherein different chromatographic steps are performed sequentially to yield a final purified product. For purification of monoclonal antibodies  protein A chromatography is one of the widely used methods and can be 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.) have been 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. Blaint et. al. have 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 WI. 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 losses of antibody or require substantial pH adjustment of the sample prior to a chromatography step. The 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.
Improved processes for purifying proteins are needed.

SUMMARY
In aspects  the application describes purification methods comprising multiple chromatographic steps  wherein in embodiments a low pH eluate from protein A chromatography is further purified by anion exchange chromatography at about the same pH  or at pH values less than or equal to 6.

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 2.
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 3.

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 load washing of a chromatography column. The desired protein in the flow-through 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 to chromatographic methods wherein a desired protein is bound to a chromatography resin when loaded onto a resin column and is subsequently eluted using an elution buffer. The desired protein is collected in 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 any immunoglobulins isolated from various sources  such as murine  human  recombinant  etc  truncated antibodies  chimeric  humanized  or pegylated antibodies  isotypes  allotypes  and alleles of immunoglobulin genes. The term antibody as used herein also refers to fusion proteins which contain an immunoglobulin moiety.
The term “low pH” or “acidic pH” as used herein refers to a pH less than or equal to 4.5.
In the purification of monoclonal antibodies (“MAbs”)  protein A chromatography is a common method  as highly purified MAbs can be obtained due to the high specificity and binding between protein A ligand and the Fc region of the antibody. However  as discussed earlier  a disadvantage of protein A chromatography is the leaching of protein A and its fragments in 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 proteins. However  the anion exchange step is performed at neutral to alkaline pH values  necessitating pH adjustment of input material. 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 0.5 M 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. The present application describes a process that  in embodiments  virtually eliminates the need of pH adjustment in the purification process  thereby minimizing its impact on process efficiency and product stability.
An aspect of the present application provides methods for antibody purification  embodiments comprising:
1. A first purification step using protein A chromatography  wherein the antibody is eluted at low pH values.
2. A second purification step using anion exchange chromatography that is performed in the flow-through mode  wherein eluate obtained from the first step is loaded onto the anion exchange resin at pH values less than or equal to 6.

In embodiments  the antibody is eluted in the first purification step at pH values about 3.5.
In embodiments  the antibody is loaded onto the anion exchange resin at pH values about 4.
In embodiments  the anion exchange resin is loaded at a pH of 6
An aspect of the present application provides methods for antibody purification  embodiments comprising:
1. A first purification step using protein A chromatography  wherein the antibody is eluted at low pH values.
2. A second purification step using anion exchange chromatography performed in the flow-through mode  wherein eluate obtained from the first step is loaded onto the anion exchange resin without substantial adjustment of pH (viz. within a range of ± 0.2 pH values).
In embodiments  the antibody is eluted in the first purification step at pH values about 3.3 to about 4.5 and loaded onto the anion exchange resin at pH values about 3.3 to about 4.5.
In embodiments  the anion exchange chromatography step is followed by a cation exchange chromatography step in a bind-elute mode  wherein the flow-through from the anion exchange chromatography step is loaded onto the cation exchange resin at pH values less than or equal to 6.
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). In embodiments  fresh (i.e.  not previously used) protein A chromatographic resin may be used to obtain a feed stream for the second chromatographic step. After washing with loading buffer and intermediate wash  the elution is carried out at low pH values.
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.
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.
Examples of buffering agents used in the buffer solutions include  but are not limited to  TRIS  phosphate  citrate  and acetate salts  or derivatives thereof.
The protein A leachates can be analyzed using protein A ELISA and the purified antibody can be analyzed 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 (CCCB) was loaded onto the protein A chromatography column (Prosep vA Ultra  VL44x250  205 mL) that was pre-equilibrated with 5 column volumes (CV) of equilibration buffer (50 mM Tris  150 mM NaCl  pH 7.5). The column was then washed with 5 CV of equilibration buffer. 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  pH 3.5.
Fig. 1 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 protein A chromatography resin.

EXAMPLE 2
Anion exchange chromatography
The protein A eluate obtained from Example 1 was incubated at pH 3.5 and 25°C for 30 minutes for viral inactivation  and the pH was adjusted to 4.0. The sample was then filtered through 0.8/0.2 µm membrane filter and loaded onto an anion exchange resin (Q-Sepharose FF  VL32x250  80 mL)  pre-equilibrated with 5-20 CV of an equilibration buffer (200 mM acetate buffer  pH 4.0). This was followed by a wash with 5 CV of equilibration buffer  and the flow-through during loading and washing steps was collected (corresponding to peak A in Fig. 2) and analyzed for percentage reduction in contaminants or impurities.
Alternatively  protein A eluate may be loaded at pH 3.5  5.0 or 6.0 onto an anion exchange resin pre-equilibrated with 5-20 CV of equilibration buffer at pH 3.5  5.0 or 6.0. The resin is washed with 5 CV of equilibration buffer and the load and wash flow-through collected.
Figs. 2 and 3 are illustrations of chromatograms from the procedure described in this example  wherein the anion exchange resin is loaded at pH 4.0 and 6.0  respectively. The line marked “Cond” represents the increase in conductivity in mS/cm. “FT” represents the flow-through obtained.

EXAMPLE 3
Cation exchange chromatography
The flow-through obtained from Example 2 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 4.0). This was followed by washing with 30 CV of wash buffer (35 mM phosphate buffer pH 6.0). The bound antibody was then 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 the flow-through obtained from Example 2 may be loaded at pH 3.5  5.0 or 6.0 onto the cation exchange resin pre-equilibrated with 10 CV of equilibration buffer at pH 3.5  5.0 or 6.0. The resin is washed with 10-30 CV of wash buffer (35 mM Phosphate buffer  pH 6.0). The bound antibody is eluted using a conductivity gradient (2.5 mS/cm to 7 mS/cm) with a phosphate buffer (35 to 80 mM  pH 6.0).
Figs. 4 and 5 are illustrations of chromatograms from the procedure described in this example  wherein the cation exchange resin is loaded at pH values of 4 and 6  respectively. The line marked “Cond” represents the increase in conductivity in mS/cm. “Buffer Conc.” represents the concentration of phosphate buffer during the chromatography run  where 100% corresponds to a buffer concentration of 80 mM.
Table 1 shows the percentage reductions in the impurities in the anion exchange flow-through and cation exchange eluate.
Table 1
Purification Step Impurity % Reduction After Protein A Chromatography
Load Solution
pH 4 Load Solution pH 6
Anion exchange chromatography Protein A leachates 30 80
Host cell proteins 30 BD
Cation exchange chromatography Protein A leachates BD BD
Host cell proteins BD BD
BD: Below detection limit.

WE CLAIM:
1. A process for purifying an antibody  comprising:
a) purifying using protein A chromatography  wherein the antibody is eluted at low pH; and
b) purifying using anion exchange chromatography performed in the flow-through mode  wherein eluate obtained from step a) is loaded onto an anion exchange resin at pH values less than or equal to 6.

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 and loaded onto the anion exchange resin at pH values about 3.3 to about 6.

3. A process according to claim 1  wherein the antibody is eluted in step a) at pH values about 3.5 and loaded onto the anion exchange resin at pH values about 3.5 to about 6.

4. A process according to claim 1  wherein the antibody is loaded onto the anion exchange resin at pH values about 4.

5. A process according to claim 1  wherein the antibody is loaded onto the anion exchange resin at pH values about 6.

6. A process according to claim 1  wherein the anion exchange chromatography is followed by a cation exchange chromatography step in a bind-elute mode  and wherein the flow-through from the anion exchange chromatography step is loaded onto a cation exchange resin at pH values less than or equal to 6.

7. A process for purifying an antibody  comprising:
a) purifying using protein A chromatography  wherein the antibody is eluted at low pH values; and
b) purifying using anion exchange chromatography  performed in the flow-through mode  wherein eluate obtained from step a) is loaded on to the anion exchange resin without substantial adjustment of pH.