Chromatography is widely applied for protein purification. A variety of resins and columns are used by process development analytics to separate proteins based on different mechanisms, including affinity capture, ion exchange, hydrophobic interaction, and mixed mode. While these conventional schemes are sufficient to purify platform monoclonal antibodies (mAbs), more tools are required to support process development for non-platform proteins. Although reverse-phase chromatography (RPC) in general is not suitable for mAb manufacturing processes due to the addition of organic solvents, it can serve as a powerful purification approach as part of protein analytical assays. Herein, we present two case studies to demonstrate the implementation of RPC to support no-platform process development.<br/>1. To support the clone selection for a non-mAb protein in cell line development, we performed a small scale high throughput AEX purification for 24 clones. The purified samples needed to be tested by peptide mapping for protein sequence variation and by Rapifluor-UPLC for N-glycan characterization assays. However, ELISA results indicated that the purified samples contain host cell protein (HCP) levels at approximately 10%, which caused significant interference to both peptide mapping and N-glycan analysis. These samples were further polished by RPC to reduce HCP levels. We demonstrated that protein quality and assay reliability were not impacted by the RPC polishing step.<br/>2. A non-mAb protein which doesn’t bind to Protein A generated an analytical challenge to accurately measure the titer in clarified bulks. Cell line development needed to screen 6000 samples with titers between 0.01 and 0.5 mg/mL. To address this challenge a 3-min run time reverse-phase UPLC assay was established, which provided the accuracy, sensitivity and throughput required.<br/>In conclusion, to support process development of non-platform proteins, process development analytics should consider all separation mechanisms. The uncommon reverse-phase chromatography could serve as a powerful purification approach and analytical assay.

Implementation of reverse-phase chromatography (RPC) to support non-platform process development analytics

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.