title: Quantitative definition and monitoring of the host cell protein proteome using iTRAQ - a study of an industrial mAb producing CHO-S cell line.
abstract: There are few studies defining CHO host cell proteins (HCPs) and the flux of these throughout a downstream purification process. Here we have applied quantitative iTRAQ proteomics to follow the HCP profile of an antibody (mAb) producing CHO-S cell line throughout a standard downstream purification procedure consisting of a Protein A, cation and anion exchange process. We used both 6 sample iTRAQ experiment to analyze technical replicates of three samples, which were culture harvest (HCCF), Protein A flow through and Protein A eluate and an 8 sample format to analyze technical replicates of four sample types; HCCF compared to Protein A eluate and subsequent cation and anion exchange purification. In the 6 sample iTRAQ experiment, 8781 spectra were confidently matched to peptides from 819 proteins (including the mAb chains). Across both the 6 and 8 sample experiments 936 proteins were identified. In the 8 sample comparison, 4187 spectra were confidently matched to peptides from 219 proteins. We then used the iTRAQ data to enable estimation of the relative change of individual proteins across the purification steps. These data provide the basis for application of iTRAQ for process development based upon knowledge of critical HCPs.
authors.affiliation: Centre for Molecular Processing and School of Biosciences, University of Kent, Canterbury, Kent, UK.
authors.forename: Lesley M
authors.initials: LM
authors.lastname: Chiverton
authors.affiliation: ChELSI Institute, Department of Chemical and Biological Engineering, University of Sheffield, Sheffield, UK.
authors.forename: Caroline
authors.initials: C
authors.lastname: Evans
authors.affiliation: ChELSI Institute, Department of Chemical and Biological Engineering, University of Sheffield, Sheffield, UK.
authors.forename: Jagroop
authors.initials: J
authors.lastname: Pandhal
authors.affiliation: ChELSI Institute, Department of Chemical and Biological Engineering, University of Sheffield, Sheffield, UK.
authors.forename: Andrew R
authors.initials: AR
authors.lastname: Landels
authors.affiliation: Pall Life Sciences, Portsmouth, UK.
authors.forename: Byron J
authors.initials: BJ
authors.lastname: Rees
authors.affiliation: Pall Life Sciences, Portsmouth, UK.
authors.forename: Peter R
authors.initials: PR
authors.lastname: Levison
authors.affiliation: ChELSI Institute, Department of Chemical and Biological Engineering, University of Sheffield, Sheffield, UK. p.c.wright@sheffield.ac.uk.
authors.forename: Phillip C
authors.initials: PC
authors.lastname: Wright
authors.affiliation: Centre for Molecular Processing and School of Biosciences, University of Kent, Canterbury, Kent, UK. c.m.smales@kent.ac.uk.
authors.forename: C Mark
authors.initials: CM
authors.lastname: Smales
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000302:isolation & purification*; D016466:CHO Cells; D002852:Chromatography, Ion Exchange; D006224:Cricetinae; D003412:Cricetulus; D007218:Industrial Microbiology / Q000379:methods; D040901:Proteomics / Q000379:methods*; D013205:Staphylococcal Protein A / Q000032:analysis*
chemical_list: D000911:Antibodies, Monoclonal; D013205:Staphylococcal Protein A
keywords: Bioprocessing; Chinese hamster ovary (CHO); Host cell proteins; Monoclonal antibody; iTRAQ