Protein A based chromatographic work flows are often used for the purification of recombinant monoclonal antibodies. This is because Protein A binds recombinant antibodies selectively and efficiently even in complex cell culture supernatants, usually removing more than 99% of product impurities in a single step. However, Protein A resins have two major disadvantages, high cost and low resin stability. Alternatively, the application of non-affinity resins for the capture step followed by one or two resins with orthogonal characteristics enable the effective removal of contaminants such as host cell proteins (HCP), DNA and antibody aggregates, as well as improve the process economics.<br/>Here we have evaluated the purification of the recombinant antibody - Rituximab using three alternate purification schemes. These include a cation exchange (CEX) resin for capture purification, followed by further purification with anion-exchange (AEX), hydrophobic cation-exchange (mixed mode) and hydroxyapatite (mixed mode) media. Design of experiment (DoE) was applied to optimize the chromatographic conditions of two of the non-affinity resins and was an important tool to enhance the performance of all of the purification workflows investigated. The success of each purification scheme was monitored by quantifying Rituximab HCP and DNA, and through functional assays. We identified that a combination of CEX – AEX – mixed mode resins produced the most economical workflow giving &gt;99% purity and 75% yield of Rituximab. HCP levels were reduced by four log fold and DNA contamination was below the detection limit after the final step. Pharmacokinetic bridging assay suggests that the purified monoclonal antibody behaved in a similar way to a Rituximab standard, showing no significant loss of structure or function throughout purification. We therefore propose that a non-affinity based purification workflow for Rituximab provides the best process economics with uncompromised results.

Purification of recombinant monoclonal antibody – Rituximab Biosimilar – with alternate non-affinity based chromatographic processes

2019 Spring National Meeting

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