Comprehensive Identification and Quantification of Host Cell Proteins by Mass Spectrometry

With the rapid expansion of biologics and biosimilars and the accompanying improvements in analytical technologies, there is increasing pressure to better characterize both the process and the final product. In contrast to traditional antibody-based methodologies, such as ELISA and Western blot, mass spectrometry can provide more detailed characterization and quantitation of individual host cell proteins (HCP) and other protein impurities, without affinity-based bias.

Caprion is applying its expertise in proteomics to provide comprehensive identification and quantitation of individual HCPs throughout process development and manufacturing. Caprion’s mass spectrometry-based HCP workflow begins with a discovery phase to detect and identify host cell proteins in any of multiple processing steps as well as in the final drug product. The method is applicable to DS generated in CHO, yeast or E. coli host cells. Two orthogonal fractionation methods are used to partition the DS from the lower abundance HCPs, thereby increasing coverage without removing the DS and any DS-bound impurities. The relative abundance of each HCP is determined in comparison to other HCPs within the same sample as well as between processing steps, manufacturing sites, host strains, culture media or other process variables of interest.

The discovery phase is followed by the development of a targeted multiple reaction monitoring (MRM) mass spectrometry assay, which utilizes heavy isotope-labeled peptide standards to accurately quantify and positively identify each of the host cell proteins of interest. Typically, up to 50 proteins are selected for this assay, based on their relative abundance in the discovery study as well as on their biology or likely immunogenicity. Since detection is protein sequence-based, the DS can be easily distinguished from related host proteins. The MRM assay can be deployed on a routine basis to provide more detailed and orthogonal characterization of HCPs, as needed during process development and scale-up.

Examples will be shown illustrating the value of this approach for:

• Identification and quantification of HCP in the low ppm range
• Demonstration of HCP clearance
• Assessing the impact of process changes
• Determining batch reproducibility
• Comparison of manufacturing sites

Speaker

Daniel Chelsky, Chief Scientific Officer, Caprion Proteomics

Dan brings to Caprion Proteomics deep industrial experience in applying the ProteoCartaTM proteomics platform to drug target and biomarker discovery. Prior to joining Caprion, Dan was President and General Manager of BioSignal Packard Inc. (a division of Perkin Elmer), where he oversaw development of novel technologies and reagents for drug discovery and genomics research, including the AlphaScreenTM and BRET2TM technologies and product lines. During his tenure at Pharmacopeia as Senior Director of Biology, Dan’s team identified a number of lead compounds from their in-house million member combinatorial chemistry libraries, resulting in multiple compounds in the clinic. Dan’s previous experience also includes positions at Onyx Pharmaceuticals and the DuPont Merck Pharmaceutical Company in cancer research.

Dan received a B.Sc. from UC Santa Barbara, his Ph.D. in Chemistry from the University of Oregon Institute of Molecular Biology and was an American Cancer Society fellow at UC Berkeley.

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