title: Identification and monitoring of host cell proteins by mass spectrometry combined with high performance immunochemistry testing.
abstract: Biotherapeutics are often produced in non-human host cells like Escherichia coli, yeast, and various mammalian cell lines. A major focus of any therapeutic protein purification process is to reduce host cell proteins to an acceptable low level. In this study, various E. coli host cell proteins were identified at different purifications steps by HPLC fractionation, SDS-PAGE analysis, and tryptic peptide mapping combined with online liquid chromatography mass spectrometry (LC-MS). However, no host cell proteins could be verified by direct LC-MS analysis of final drug substance material. In contrast, the application of affinity enrichment chromatography prior to comprehensive LC-MS was adequate to identify several low abundant host cell proteins at the final drug substance level. Bacterial alkaline phosphatase (BAP) was identified as being the most abundant host cell protein at several purification steps. Thus, we firstly established two different assays for enzymatic and immunological BAP monitoring using the cobas® technology. By using this strategy we were able to demonstrate an almost complete removal of BAP enzymatic activity by the established therapeutic protein purification process. In summary, the impact of fermentation, purification, and formulation conditions on host cell protein removal and biological activity can be conducted by monitoring process-specific host cell proteins in a GMP-compatible and high-throughput (> 1000 samples/day) manner.
authors: Pharma Development, Roche Diagnostics GmbH, Penzberg, Germany.
authors: Katrin
authors: K
authors: Bomans
authors: Antje
authors: A
authors: Lang
authors: Veronika
authors: V
authors: Roedl
authors: Lisa
authors: L
authors: Adolf
authors: Kyrillos
authors: K
authors: Kyriosoglou
authors: Katharina
authors: K
authors: Diepold
authors: Gabriele
authors: G
authors: Eberl
authors: Michael
authors: M
authors: Mølhøj
authors: Ulrike
authors: U
authors: Strauss
authors: Christian
authors: C
authors: Schmalz
authors: Rudolf
authors: R
authors: Vogel
authors: Dietmar
authors: D
authors: Reusch
authors: Harald
authors: H
authors: Wegele
authors: Michael
authors: M
authors: Wiedmann
authors: Patrick
authors: P
authors: Bulau
mesh_terms: D000469:Alkaline Phosphatase / Q000032:analysis / Q000302:isolation & purification; D001691:Biological Therapy; D002846:Chromatography, Affinity; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000166:cytology* / Q000201:enzymology; D029968:Escherichia coli Proteins / Q000032:analysis* / Q000302:isolation & purification; D007120:Immunochemistry; D013058:Mass Spectrometry
chemical_list: D029968:Escherichia coli Proteins; D000469:Alkaline Phosphatase