Integrated strategy for deep profiling of host cell proteins in downstream processing of therapeutic monoclonal antibodies: Novel approach to isolate and digest host cell proteins.

Host cell proteins (HCPs) are process-related impurities generated during the production of biopharmaceuticals, which may contaminate the final product unless they are efficiently removed. Due to their potential impact on product safety, quality and efficacy, regulatory authorities require removal of HCPs during processing down to trace amounts in final manufactured biopharmaceuticals. The current standard method for detecting HCPs is enzyme-linked immunosorbent assay (ELISA), which should reveal the total amount of HCPs. A necessary orthogonal technique to get more granular information on HCPs is obtained by application of liquid chromatography-mass spectrometry (LC-MS) techniques that permit identification and quantification of individual HCPs. However, differences in sample preparation methods and MS acquisition techniques have led to discrepancies in detected HCPs between studies, which may compromise product safety, quality and efficacy. To address this issue, we have developed a novel and reproducible workflow for isolation, digestion, and mass spectrometry detection of HCPs that is applicable to downstream process characterization of therapeutic monoclonal antibodies (mAbs). This article describes a rapid and efficient workflow for the isolation, digestion and identification of HCPs. For the first time, Fc-receptor (FcγRIIIa) affinity chromatography is employed to isolate the HCP fraction from the mAb. Next, the HCPs are precipitated with acetone and digested using a newly developed "single-pot" method that improves digestion performance and prevents sample loss of problematic low-abundant HCPs. The new HCP isolation method outperforms protein A affinity chromatography for monitoring problematic HCPs.