title: 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. In this article, we report the development of optimized protocols that improve proteome capture for CHO HCP. Eleven precipitation protocols were screened for protein recovery and optimized for a subset of precipitants by a design of experiments (DOE) approach. Because total protein recovery does not fully replicate a proteomics experiment, or detect non-protein agents that 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.
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, DE, USA; Delaware Biotechnology Institute, University of Delaware, Newark, DE, USA.
authors.forename: Kristin N
authors.initials: KN
authors.lastname: Valente
authors.forename: Amy K
authors.initials: AK
authors.lastname: Schaefer
authors.forename: Hannah R
authors.initials: HR
authors.lastname: Kempton
authors.forename: Abraham M
authors.initials: AM
authors.lastname: Lenhoff
authors.forename: Kelvin H
authors.initials: KH
authors.lastname: Lee
mesh_terms: D000818:Animals; D016466:CHO Cells / Q000737:chemistry*; D006224:Cricetinae; D003412:Cricetulus; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D057166:High-Throughput Screening Assays / Q000379:methods*; D020543:Proteome / Q000737:chemistry* / Q000302:isolation & purification*; D040901:Proteomics / Q000379:methods*; D012107:Research Design; D012997:Solvents
chemical_list: D020543:Proteome; D012997:Solvents
keywords: Chinese hamster ovary cells; Design of experiments optimization; Extracellular host cell proteins; Proteomics; Sample preparation
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