Identification and Quantification of a Problematic Host Cell Protein to Support Therapeutic Protein Development

Monitoring of residual host cell proteins (HCPs) in therapeutic protein is essential to ensure product quality, safety and efficacy. Despite the development of advanced mass spectrometry techniques and optimized workflows, identifying and quantifying all problematic HCPs present at low levels remain challenging. Here, we developed a practical, effective strategy for the identification and quantification of low abundance HCPs, which facilitates the improvement of downstream purification process to eliminate potentially problematic HCPs. A case study of using this strategy to investigate a problematic HCP is presented. Initially, a commonly used native digestion approach coupled with UPLC-MS/MS was applied for HCP profiling, wherein several lipases and proteases were identified in a monoclonal antibody named mAb1 in early stages of purification process development. A highly active lipase, liver carboxylesterase (CES), was found to be responsible for polysorbate 80 degradation. To facilitate process improvement, after the identification of CES, we developed a highly sensitive LC-MS/MS-MRM assay with a lower limit of quantification of 0.05 ppm for routine monitoring of the CES in mAb1 produced through the different processes. This workflow was applied in low-level lipase identification and absolute quantification, which facilitated the investigation of polysorbate degradation and downstream purification improvement to further remove the problematic HCP. The current MRM method increased the sensitivity of HCP quantification by over 10-fold that in previously published studies, thus meeting the needs for quantification of problematic HCPs at sub-ppm to ppb levels during drug development. This workflow could be readily adapted to the detection and quantification of other problematic HCPs present at extremely low levels in therapeutic protein drug candidates.