Proteomic Analysis of Host Cell Protein Dynamics in the Culture Supernatants of Antibody-Producing CHO Cells

Abstract

Chinese hamster ovary (CHO) cells are the most common cell line used for the production of therapeutic proteins including monoclonal antibodies (mAbs). Host cell proteins (HCPs), secreted and released from lysed cells, accumulate extracellularly during the cultures of recombinant CHO (rCHO) cells, potentially impairing product quality. In an effort to maintain good mAb quality during the cultures, HCPs accumulated extracellularly in batch and fed-batch cultures of a mAb-producing rCHO cell line were identified and quantified by nanoflow liquid chromatography-tandem mass spectrometry, followed by their gene ontology and functional analysis. Due to higher cell concentration and longer culture duration, more HCPs were identified and quantitated in fed-batch culture (2145 proteins identified and 1673 proteins quantified) than in batch culture (1934 proteins identified and 1486 proteins quantified). Clustering analysis of HCPs showed that the concentration profiles of HCPs affecting mAb quality (Lgmn, Ctsd, Gbl1, and B4galt1) correlated with changes in mAb quality attributes such as aggregation, charge variants, and N-glycosylation during the cultures. Taken together, the dataset of HCPs obtained in this study provides insights into determining the appropriate target proteins to be removed during both the cultures and purification steps for ensuring good mAb quality.

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

“…In most cases, all the proteases mentioned above were identified at a higher concentration in the culture supernatants of fed‐batch cultures than those of batch cultures for both DG‐Fc and DUKX‐Fc cell lines. The same trend was observed for the rCHO‐K1 cell line (Park et al, ). This result suggests that an increased concentration of proteases is in part responsible for the increased degradation of Fc‐fusion proteins.…”

“…Among the nine HCPs commonly identified in DG‐Fc and DUKX‐Fc, Ywhae, Lgmn, Prdx1, and Cfl1 were also identified at all sampling times in both batch and fed‐batch cultures of the rCHO‐K1 cell line (Park et al, ). Interestingly, Lyz that is the most abundant HCP in both DG‐Fc and DUKX‐Fc was not identified in the culture supernatants of the rCHO‐K1 cell line (Park et al, ), suggesting that the HCP concentration profiles of rCHO cell lines during culture depend on their CHO host cell lines. These HCPs are secretory proteins located either in the extracellular region or the cytoplasm, indicating that a significant portion of HCPs in the culture supernatants was secreted from viable cells.…”

Proteomics in biomanufacturing control: Protein dynamics of CHO‐K1 cells and conditioned media during apoptosis and necrosis

“…For example, proteomics analysis of CHO cells undergoing apoptosis during prolonged cultivation or upon transition from exponential to stationary phase resulted in major changes in proteins related to the regulation of stress response, energy metabolism, and cell growth (Carlage et al, 2012;Wei et al, 2011). Complementary results were obtained in proteomics studies of HCPs in conditioned media at the end of batch-and fed-batch CHO cultures and galectin-1 was identified as a marker for early stress in CHO culture supernatants (Park et al, 2017;Woolley & Al-Rubeai, 2009). However, as most studies to date focus on either the cellular or the conditioned media proteome and a particular type of cell death, direct comparison between cellular and conditioned media proteome and different cell death mechanisms is difficult.…”

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.…”