Methods of reducing level of one or more impurities in a sample during protein purification

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A flow-through process for purifying a target molecule from a Protein A eluate comprising the steps of:(i) contacting the eluate recovered from a Protein A chromatography column with activated carbon, (ii) contacting the flow-through sample from step (i) with an anion exchange chromatography media, (iii) contacting the flow-through sample from step (ii) with a cation exchange chromatography media and (iv) obtaining the flow-through sample from step (iii) comprising the target molecule, wherein the eluate flows continuously through steps (i)-(iii) and wherein level of one or more impurities in the flow-through sample after step (iii) is lower than the level in the eluate in step (i). The present invention provides novel and improved protein purification processes which incorporate certain types of carbonaceous materials and result in effective and selective removal of certain undesirable impurities without adversely effecting the yield of the desired protein product.

Field of the Invention

The present invention relates to improved chromatography methods and methods of reducing the level of one or more impurities during protein purification.

Background

Chromatography is a dominant purification technique in the purification of biological materials, e.g., monoclonal antibodies.

Commonly used chromatography methods include one or more of affinity chromatography media, ion exchange chromatography media, hydrophobic interaction, hydrophilic interaction, size exclusion and mixed mode (i.e., combination of various chromatography interactions) chromatography. For example, for the purification of monoclonal antibodies, a typical purification process includes an initial Protein A affinity capture step followed by one or more ion exchange polishing steps, the purpose of which is to reduce the level of one or more impurities such as, e.g., host cell protein (HCP). Further, other chromatography techniques, such as: bind and elute hydrophobic interaction chromatography (HIC); flow-through hydrophobic interaction chromatography (FTHIC); flow-through anion-exchange chromatography (AEX); weak partitioning chromatography with cation-exchange, anion-exchange, or hydrophobic interaction reins; mixed mode chromatography techniques, e.g., bind and elute weak cation and anion exchange, bind and elute hydrophobic and ion exchange interaction and flow-through hydrophobic and ion exchange mixed mode interaction (FTMM), both of which can utilize resins such as Capto™ Adhere, Capto™ MMC, HEA Hypercel™, PPA Hypercel™, may be used. Additionally, hydrophobic charge induction (HCI) chromatography along with others and combinations of various techniques can be used for polishing.

Although, chromatography offers many advantages for protein purification on a smaller scale, on a large scale, packing of chromatography columns is not only labor and time intensive but also expensive. Further, fouling of chromatography columns is a common problem, resulting in a user having to dispose off columns, which is undesirable, especially due to the high cost of chromatography resins.

Recently, there has been a noticeable trend in the industry to try and reduce the number of steps in protein purification processes. Also, use of techniques for obtaining a higher expression titer using bioreactors is a rising trend in the industry. The combination of these two trends has resulted in more product being loaded onto a column, thereby resulting in increased burden of fairly expensive chromatography media as well as lower product purity, both of which are undesirable.

Summary of the Invention

The present invention is based, at least in part, on the surprising and unexpected discovery that certain materials (e.g., carbonaceous material such as activated carbon) can be incorporated into chromatography column based protein purification processes in a flow-through mode, resulting in reducing the burden of chromatography columns, and consequently increasing the life span of chromatography columns.

Further, the present invention is based on the surprising and unexpected discovery that carbonaceous material (e.g., activated carbon) can be used either upstream or downstream of a capture chromatography step to reduce the level of one or more impurities. In some embodiments according to the claimed methods, a sample is contacted with a carbonaceous material before a cation exchange (CEX) chromatography step. In other embodiments, a cation exchange (CEX) chromatography step is used before contacting a sample with a carbonaceous material. In yet other embodiments, a sample is contacted with a carbonaceous material after a Protein A affinity capture step. Alternatively, the Protein A affinity chromatography step may be used after contacting the sample with a carbonaceous material. In certain embodiments, the Protein A affinity capture step may be followed by an anion exchange (AEX) flow-through chromatography step and with or without a CEX chromatography bind/elute step. In still other embodiments, the carbonaceous material may be used after a non-affinity capture step (e.g., using CEX bind and elute chromatography as a capture step) and is followed by an AEX chromatography step.