Differential effects of bioreactor process variables and purification on the human recombinant lysosomal enzyme β-glucuronidase produced from Chinese hamster ovary cells
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β-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 β-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.
KeywordsEnzyme replacement therapy Lysosomal storage disorders Parallel bioreactors Glycan content Mannose-6-phosphate β-Glucuronidase Chinese hamster ovary cells Critical quality attributes
The authors thank Sarah Johnson and Rukman DeSilva of the Office of Biotechnology Products/CDER for the critical reading and comments on the manuscript. All authors acknowledge the critical reading, feedback, and support by Celia Cruz.
HP, SAK, BG, GZ, and EJFB performed the experiments and wrote the manuscript. MA and TJ wrote the manuscript. CNM conceptualized the work, performed the experiments, and wrote the manuscript.
This study was intramurally funded by the Center for Drug Evaluation and Research, USFDA, for “Improved Understanding of Bioprocessing” and “Product Quality and Biopharmaceutics of Complex Dosage Forms.” HP, SAK, BG, GZ, and EJFB were recipients of ORISE fellowships from CDER.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflicts of interest.
This article does not contain any studies with human participants or animals performed by any of the authors.
This article reflects the views of the authors and should not be construed to represent the FDA’s views or policies.
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