More similar than different: Host cell protein production using three null CHO cell lines

Abstract

To understand the diversity in the cell culture harvest (i.e., feedstock) provided for downstream processing, we compared host cell protein (HCP) profiles using three Chinese Hamster Ovary (CHO) cell lines in null runs which did not generate any recombinant product. Despite differences in CHO lineage, upstream process, and culture performance, the cell lines yielded similar cell-specific productivities for immunogenic HCPs. To compare the dynamics of HCP production, we searched for correlations between the time-course profiles of HCP (as measured by multi-analyte ELISA) and those of two intracellular HCP species, phospholipase B-like 2 (PLBL2) and lactate dehydrogenase (LDH). Across the cell lines, proteins in the day 14 supernatants analyzed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) showed different spot patterns. However, subsequent analysis by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) indicated otherwise: the total number of peptides and proteins identified were comparable, and 80% of the top 1,000 proteins identified were common to all three lines. Finally, to assess the impact of culture viability on extracellular HCP profiles, we analyzed supernatants from a cell line whose viability dropped after day 10. The amounts of HCP and PLBL2 (quantified by their respective ELISAs) as well as the numbers and major populations of HCPs (identified by LC-MS/MS) were similar across days 10, 14, and 17, during which viabilities declined from ∼80% to <20% and extracellular LDH levels increased several-fold. Our findings indicate that the CHO-derived HCPs in the feedstock for downstream processing may not be as diverse across cell lines and upstream processes, or change as dramatically upon viability decline as originally expected. In addition, our findings show that high density CHO cultures (>10(7) cells/mL)-operated in fed-batch mode and exhibiting high viabilities (>70%) throughout the culture duration-can accumulate a considerable amount of immunogenic HCP (∼1-2 g/L) in the extracellular environment at the time of harvest (day 14). This work also demonstrates the potential of using LC-MS/MS to overcome the limitations associated with ELISA and 2D-PAGE for HCP analysis.

Toward the complete characterization of host cell proteins in biotherapeutics via affinity depletions, LC-MS/MS, and multivariate analysis

“…These results suggest that, while final drug product HCP profiles are influenced by the innate identities and abundances of the host cell, many important differences exist. Given the findings herein plus recent results that showed high similarity between proteomic profiles of different null CHO cell lines and upstream processes, 44 variations in downstream processes are likely the primary cause of the significant differences we observed for final drug product HCP profiles as certain proteins will be more effectively purified than others (depending on the downstream process employed).…”

Influence of Escherichia coli chaperone DnaK on protein immunogenicity

“…Hence, bacterial HCPs have the potential to influence the immunogenicity of the biotherapeutic by having adjuvant‐like activity. HCP contamination is also an issue for mammalian cell expression systems 6, 12, 13. However, non‐mammalian HCPs are more likely to pose a risk in patients, compared with their mammalian counterparts.…”

A modular and adaptive mass spectrometry-based platform for support of bioprocess development toward optimal host cell protein clearance

“…PLBL2 was identified and quantified in HCCF, with the Top 3 strategy 19 using a 4-point calibration curve of spiked internal standard protein (bovine carbonic anhydrase II). Table 1 shows the quantitation results obtained by DIA SWATH MS and by a PLBL2-specific ELISA 25 in five HCCF samples. Results showed that the measured PLBL2 levels by DIA SWATH MS quantification agreed well with those from an orthogonal analysis by PLBL2-specific ELISA ( Table 1).…”

“…This molar concentration was then normalized by the MW ratio of the HCP to the bovine carbonic anhydrase II to calculate the ppm level of these HCPs. Similarly, PLBL2 molar concentration was determined using the 4-point standard curve generated from the Top 3 peak area data of bovine carbonic anhydrase II, which was then converted to the mg/mL to compare with ELISA 25 results. Carbonic anhydrase II peptides used for quantification were unique to the bovine sequence of the protein.…”