Recovery of Chinese hamster ovary host cell proteins for proteomic analysis

Abstract

Identification and characterization of Chinese hamster ovary (CHO) host cell protein (HCP) impurities by proteomic techniques can aid bioprocess design and lead to more efficient development and improved biopharmaceutical manufacturing operations. Recovery of extracellular CHO HCP for proteomic analysis is particularly challenging due to the relatively low protein concentration and complex composition of media. We developed optimized protocols that improve proteome capture for CHO HCP. Eleven precipitation protocols were screened for protein recovery and optimized for a subset of the precipitants by a design of experiments approach. Because total protein recovery cannot describe physicochemical characteristics of proteins or detect non-protein agents, which may interfere with proteomic methods, a subset of precipitation conditions were compared by two-dimensional electrophoresis and liquid chromatography coupled with mass spectrometry, with optimized recovery shown to differ between the two proteomic methods. This work demonstrates broadly applicable methods that can be applied as initial steps to optimize sample preparation of any sample type for proteomic analysis and presents optimized precipitation protocols for extracellular CHO HCP recovery, which can vary appreciably between gel-based and shotgun proteomic methods.

Knockout of a difficult‐to‐remove CHO host cell protein, lipoprotein lipase, for improved polysorbate stability in monoclonal antibody formulations

“…Extracellular CHO HCPs were precipitated from HCCF with methanol as described previously (Valente et al, 2014) and residual detergent was removed by DetergentOUT™ GBS10–800 detergent removal kit (G-Biosciences, St. Louis, MO, USA) according to the manufacturer's protocol. Trypsin digestion was performed as described previously (Valente et al, 2014).…”

Comprehensive tracking of host cell proteins during monoclonal antibody purifications using mass spectrometry

“…This methodology appears well suited as an information-rich, generic method to monitor HCPs in the biopharmaceutical industry and to provide valuable information for process developers at the individual HCP level. Because we wished to provide quantitative information on HCPs in actual process pools in the presence of mAb product, HCP enrichment techniques 21 were not applicable. In this study, we utilized MS E to comprehensively identify and quantify residual HCPs in purification pools through the initial steps of typical mAb platform processes.…”

Toward the complete characterization of host cell proteins in biotherapeutics via affinity depletions, LC-MS/MS, and multivariate analysis

“…[4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] In this methodology, proteins are first digested enzymatically. The resulting peptides are then separated (usually by liquid chromatography), and subsequently detected by mass/ charge via a mass spectrometer.…”