Protein purification by ion exchange

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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 pl variants and basic, higher pl 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. The various aspects of Protein A chromatography are described in U.S. Pat. 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 , 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. Further, protein A has also been linked with toxicity. 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. Pat. 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.