During a monoclonal antibody (mAb) process development, host cell protein (HCP) levels are typically monitored by ELISA to ensure adequate clearance.  2D gel electrophoresis and Western blotting may also be used in combination with ELISA testing to improve understanding of HCP species present and the impact of the purification process on HCP distribution.  Because purified mAb products are often highly concentrated (e.g. typically 50-150 mg/mL) and typically contain only very low (ppm) levels of HCP, product interference can hinder 2D gel analysis even after significant production depletion using conventional chromatography techniques. To address this problem, we have previously presented the use of mAb affinity-based product depletion procedures to remove the mAb product, maximize HCP recovery and concentrate HCPs to detectable levels. However, mAb affinity-based product depletion procedures present difficulties in ensuring complete HCP recovery in a representative distribution especially if the mAb was originally purified by protein A chromatography.  Other practical limitation is the long processing time and potential HCP loss from a multi-step preparation with having to pre-dilute and re-concentrate the recovered HCP pool. An alternative product depletion procedure has been developed using hexapeptide libraries to address the limitations of the previous approach. As the hexapeptides do not preferentially bind mAb over HCP species, simultaneous concentration and separation can achieve better HCP recovery using much smaller resin volumes. Due to the diversity in the peptide library, more HCP species are likely to be captured and identified during mass spectrometry analysis even if present at only trace levels in the starting sample.. This is independent of whether Protein A chromatography was used in the manufacturing process. The feasibility of using hexapeptide product depletion to monitor HCP distribution at different points in a mAb purification process will be presented, including considerations of reproducibility of results and resin reusability.

Recovery and concentration of low level HCPs from purified monoclonal antibody products using affinity hexapeptide resin libraries

247th National Meeting (2014)

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