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. 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. The HCPs that accumulated extracellularly in batch and fed‐batch cultures of rCHO cell lines were identified and quantified using LC‐MS/MS. The dataset of HCPs obtained in this study provides insights towards establishing effective methods to ensure high quality and stability of Fc‐fusion proteins in rCHO cell cultures.