Proteomic analysis of host cell protein dynamics in the supernatant of Fc‐fusion protein‐producing CHO DG44 and DUKX‐B11 cell lines in batch and fed‐batch cultures

Abstract

Chinese hamster ovary (CHO) cells are the most widely used host cell lines for the commercial production of therapeutic proteins including Fc-fusion proteins. During the culture of recombinant CHO (rCHO) cells, host cell proteins (HCPs), secreted from viable cells and released from dead cells, accumulate extracellularly, potentially impairing product quality. In this study, the HCPs that accumulated extracellularly in batch and fed-batch cultures of Fc-fusion protein-producing rCHO cell lines (DG-Fc and DUKX-Fc) were identified and quantified using nanoflow liquid chromatography-tandem mass spectrometry (LC-MS/MS), followed by gene ontology and functional analysis. When the proteome database of Cricetulus griseus was used as a reference to identify the HCPs, more HCPs were identified for DG-Fc (1632 HCPs in batch culture and 1733 HCPs in fed-batch culture) than for DUKX-Fc (1114 HCPs in batch culture and 1002 HCPs in fed-batch culture). Clustering analysis of HCPs, which were classified into four clusters according to their concentration profiles during culture, showed that the concentration profiles of HCPs affecting the quality of Fc-fusion proteins correlated with changes in Fc-fusion protein quality. Taken together, the dataset of HCPs obtained in this study using the two different rCHO cell lines provides insights into the determination of appropriate target proteins to be removed during the culture and purification steps so as to ensure good Fc-fusion protein quality. Biotechnol. Bioeng. 2017;114: 2267-2278. © 2017 Wiley Periodicals, Inc.

Experience with host cell protein impurities in biopharmaceuticals

“…Recently proteomics approaches using LC‐MS/MS have offered an orthogonal method for HCP detection and quantification . This has allowed cases where specific HCPs can be identified and quantified as individual proteins.…”

“…Recently proteomics approaches using LC-MS/MS have offered an orthogonal method for HCP detection and quantification. 11,12 This has allowed cases where specific HCPs can be identified and quantified as individual proteins. One advantage of LC-MS/MS information is that, once the HCP identified, then the specific strategies for its removal and risk assessment can be undertaken.…”

S-Trap eliminates cell culture media polymeric surfactants for effective proteomic analysis of mammalian cell bioreactor supernatants

“…The organic solvent used here was a 1:1 mixture of methanol and acetone [44][45] . All methods have been previously shown to eliminate detergents from diverse samples [24][25][46][47][48] , and organic solvent precipitation [61][62][63][64] and FASP 56-60 have been previously used to prepare bioreactor supernatant samples for LC-MS/MS proteomics. We found that all three methods could remove the polymer(s) from the samples and that SP3 and S-Trap were better than precipitation ( Fig.…”

Understanding the mechanism of copurification of “difficult to remove” host cell proteins in rituximab biosimilar products

“…In upstream processing, the cell culture platform is increasingly adopting high cell densities and perfusion cell culture modes for biotherapeutic production . These newer modes impact a particular subset of HCPs, as evidenced from a cell cultivation operated in batch and fed‐batch mode that resulted in expression of common HCPs in both modes on Day 3 (equivalent cell densities), but subsequently led to higher expression of few select HCPs in fed‐batch mode . In addition, it is seen that the difficult to remove HCPs exhibited near equivalent affinities to the biosimilar, except for the sulfated glycoprotein.…”