Zinc supplementation improves the harvest purity of ß-glucuronidase from CHO cell culture by suppressing apoptosis.

The variability of trace metals in cell culture media is a potential manufacturing concern because it may significantly affect the production and quality of therapeutic proteins. Variability in trace metals in CHO cell culture has been shown to impact critical production metrics such as cell growth, viability, nutrient consumption, and production of recombinant proteins. To better understand the influence of excess supplementation, zinc and copper were initially supplemented with 50-µM concentrations to determine the impact on the production and quality of ß-glucuronidase, a lysosomal enzyme, in a parallel bioreactor system. Ethylenediaminetetraacetic acid (EDTA), a metal chelator, was included as another treatment to induce a depletion of trace metal bioavailability to examine deficiency. Samples were drawn daily to monitor cell growth and viability, nutrient levels, ß-glucuronidase activity, and trace zinc flux. Cell cycle analysis revealed the inhibition of sub-G0/G1 species in zinc supplemented cultures, maintaining higher viability compared to the control, EDTA-, and copper-supplemented cultures. Enzyme activity analysis in the harvests revealed higher specific activity of ß-glucuronidase in reactors supplemented with zinc. A confirmation run was conducted with supplementations of zinc at concentrations of 50, 100, and 150 µM. Further cell cycle analysis and caspase-3 analysis demonstrated the role of zinc as an apoptosis suppressor responsible for the enhanced harvest purity of ß-glucuronidase from zinc-supplemented bioreactors.

Differential effects of bioreactor process variables and purification on the human recombinant lysosomal enzyme ß-glucuronidase produced from Chinese hamster ovary cells.

ß-Glucuronidase is a lysosomal enzyme and a molecular model of a class of therapeutics approved as enzyme replacement therapies for lysosomal storage diseases. Understanding the effect of bioreactor process variables on the production and quality of the biologics is critical for maintaining quality and efficacy of the biotherapeutics. Here, we have investigated the effect of three process variables, in a head-to-head comparison using a parallel bioreactor system (n = 8), namely 0.25 mM butyrate addition, a temperature shift (from 37 to 32 °C), and a pH shift (from 7.0 to 6.7) along with a control (pH 7, temperature 37 °C, and no additive) on the production and quality of human recombinant ß-glucuronidase (GUS) by a Chinese hamster ovary (CHO) cell line. The study was performed as two independent runs (2 bioreactors per treatment per run; n ≤ 4). Although statistically not significant, protein production slightly increased with either 0.25 mM butyrate addition (13%) or pH shift (7%), whereas temperature shift decreased production (12%, not significant). Further characterization of the purified GUS samples showed that purification selectively enriched the mannose-6-phosphate (M6P)-containing GUS protein. Noticeably, a variation observed for the critical quality attribute (CQA) of the enzyme, namely M6P content, decreased after purification, across treatment replicates and, more so, across different treatments. The dimer content in the purified samples was comparable (~25%), and no significant discrepancy was observed in terms of GUS charge variants by capillary electrophoresis analysis. MALDI-TOF/TOF analysis of released N-glycans from GUS showed a minor variation in glycoforms among the treatment groups. Temperature shift resulted in a slightly increased sialylated glycan content (21.6%) when compared to control (15.5%). These results suggest that bioreactor processes have a differential effect, and better control is required for achieving improved production of GUS enzyme in CHO cells without affecting drastically its CQAs. However, the purification method allowed for enrichment of GUS with similar CQA profiles, regardless of the upstream treatments, indicating for the first time that the effect of slight alterations in upstream process parameters on the CQA profile can be offset with an effective and robust purification method downstream to maintain drug substance uniformity.