Comprehensive assessment of host cell protein expression after extended culture and bioreactor production of CHO cell lines

Abstract

The biomanufacturing industry is advancing toward continuous processes that will involve longer culture durations and older cell ages. These upstream trends may bring unforeseen challenges for downstream purification due to fluctuations in host cell protein (HCP) levels. To understand the extent of HCP expression instability exhibited by Chinese hamster ovary (CHO) cells over these time scales, an industrywide consortium collaborated to develop a study to characterize age-dependent changes in HCP levels across 30, 60, and 90 cell doublings, representing a period of approximately 60 days. A monoclonal antibody (mAb)-producing cell line with bulk productivity up to 3 g/L in a bioreactor was aged in parallel with its parental CHO-K1 host. Subsequently, both cell types at each age were cultivated in an automated bioreactor system to generate harvested cell culture fluid (HCCF) for HCP analysis.More than 1500 HCPs were quantified using complementary proteomic techniques, two-dimensional electrophoresis (2DE) and liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). While up to 13% of proteins showed variable expression with age, more changes were observed when comparing between the two cell lines with up to 47% of HCPs differentially expressed. A small subset (50 HCPs) with age-dependent expression were previously reported to be problematic as high-risk and/or difficult-to-remove impurities; however, the vast majority of these were downregulated with age. Our findings suggest that HCP expression changes over this time scale may not be as dramatic and pose as great of a challenge to downstream processing as originally expected but that monitoring of variably expressed problematic HCPs remains critical.

Generation of reference cell lines, media, and a process platform for CHO cell biomanufacturing

“…The qP of the VRC01 cell line drops at each increment of 30 generations (Hamaker et al, 2022). These previously published results suggest that cryopreservation and production experiments should be limited to early generation cultures.…”

“…Recent publications have highlighted the downregulation of host cell proteins with cell age (Hamaker et al, 2022), safe harbor locations for genetic editing (Hilliard & Lee, 2021), and the impact of ammonia stress on glycosylation (Synoground et al, 2021) within this cell line. Maintenance of production stability during long‐term culture is also an important parameter for biopharmaceutical manufacturing and has been previously studied in the VRC01 cell line (Hamaker et al, 2022). The qP of the VRC01 cell line drops at each increment of 30 generations (Hamaker et al, 2022).…”

“…Maintenance of production stability during long‐term culture is also an important parameter for biopharmaceutical manufacturing and has been previously studied in the VRC01 cell line (Hamaker et al, 2022). The qP of the VRC01 cell line drops at each increment of 30 generations (Hamaker et al, 2022). These previously published results suggest that cryopreservation and production experiments should be limited to early generation cultures.…”

“…In conjunction with the work presented here, the VRC01 cell line has a growing body of literature as a reference platform for the CHO biopharmaceutical industry. Recent publications have highlighted the downregulation of host cell proteins with cell age (Hamaker et al, 2022), safe harbor locations for genetic editing (Hilliard & Lee, 2021), and the impact of ammonia stress on glycosylation (Synoground et al, 2021) within this cell line. Maintenance of production stability during long-term culture is also an important parameter for biopharmaceutical manufacturing and has been previously studied in the VRC01 cell line (Hamaker et al, 2022).…”

Analytical characterization of host‐cell‐protein‐rich aggregates in monoclonal antibody solutions

“…LC-MS/MS analysis was performed as described previously. 51 was performed using sequential windowed acquisition of all theoretical fragment ion spectra (SWATH) with a full MS1 scan and 64 variable MS/MS windows for standard digestions and 32 variable MS/MS windows for native digestions. [52][53][54] Database searches were performed using the Paragon algorithm in the Sciex ProteinPilot software (v5.1) as described previously, 51,55 using a local copy of the CH RefSeq 2019 database supplemented with ADH, retention time calibrants, and common contaminants.…”