Purpose: The potential for treating a wide array of diseases with biologics is constantly increasing and biologics offer a great promise for treating congenital disorders. In such disorders, patients expressing a dysfunctional protein, or lacking the protein, can be treated with a functional protein, as is the case of enzymes for lysosomal storage disorders (LSDSs). An enzyme replacement therapy (ERT) for an LSD utilizes an intravenous infusion of a recombinantly produced functional lysosomal enzyme to replace the absent or mutant enzyme. The United States Food and Drug Administration regulates many ERTs used for treating several LSDs. As these ERTs are complex large molecules they necessitate an examination of all stages of production to ensure the quality and efficacy of the protein drugs. Typically, investigation must start with expression of recombinant proteins, with the effects of bioprocess parameters on cellular growth parameters, and protein production and quality. Furthermore, analysis must be performed to determine levels of impurities, including host cell DNA and protein. Downstream processes, such as formulation effects on protein stability and efficacy need to be understood as well. Here, we utilize Chinese Hamster Ovary (CHO) cells expressing human recombinant Cathepsin D (CatD), as another model of ERT protein production for LSDs, to examine the production aspects of an ERT development. CatD is a lysosomal aspartic protease (EC 18.104.22.168) and loss of its function is known to cause progressive neurodegeneration, loss of vision, epileptic symptoms and regressed growth and development. In this work, CatD is expressed under a tetracycline inducible promoter in CHO cells and provides an opportunity to examine production of an enzyme under inducible control. Here we examined the effects of adding a low concentration (0.25 mM) of sodium butyrate, which is a known histone deacetylase inhibitor that has been shown to increase recombinant protein production. Cell growth profiles, protein production and critical quality attributes (CQAs) of CatD were monitored. As CatD is a lysosomal enzyme, one CQA is the necessity to have mannose-6-phosphate (M6P) on enzyme glycans, which allows for intracellular targeting. Endogenous lysosomal enzymes typically bind to M6P-receptors in the Golgi, whereas ERT infusions would bind M6P-receptors on the cell surface, in both instances the receptor-enzyme complex is transported to lysosomes via vesicular trafficking.
Methods: In the first phase of this study, CHO cells were grown in chemically defined media in 1.2 L parallel bioreactors. Samples were taken daily to monitor metabolites, viability and protein production (estimated by enzymatic activity). Upon harvest, a quantitative enzymatic activity assay was used to estimate differences in protein production. To assess the effects of sodium butyrate treatment on CatD quality, the harvested protein was purified and subjected to multiple analyses, including enzyme specific activity measurements, capillary electrophoresis, mannose-6-phosphate quantification and qPCR for host cell DNA.
Results: During the production phase of this work, the monitored parameters of the parallel bioreactors indicate that the low concentration of 0.25 mM sodium butyrate treatment show no significant differences from control cells in cell density, viability (Fig 1), metabolites, or nutrient usage. Additional analysis shows a small, but not significant, increase in the specific productivity (enzymatic activity per cell per day) due to treatment (10.4% at harvest; Fig 2). Likewise, analysis of the purified protein indicates that 0.25 mM sodium butyrate treatment increases CatD production by 23.2%, but falls short of statistical significance (p=0.086; Fig 3, grey). Analysis of levels of M6P, a CQA, indicates a small but statistically insignificant difference between treated and control cells (Fig 3, red).
Conclusion: Examination of the bioreactor processes indicates that a 0.25 mM sodium butyrate treatment does not significantly alter cellular growth profiles. Estimates of protein production from enzymatic assays suggest that at 0.25 mM concentration sodium butyrate treatment alone is not sufficient to significantly increase CatD production, consistent with our experience with another LSD enzyme, β-glucuronidase (GUS). Analysis of the purified protein further supports this, and also shows that the small but statistically insignificant increase in CatD production is not at the expense of protein quality, as all examined CQAs remained comparable. These results indicate that low concentrations of sodium butyrate treatment may be used to slightly increase enzyme production without compromising quality, which nevertheless needs critical evaluation. Further work is required to determine if the treatment causes any differences in protein stability, efficacy or the required sub-cellular targeting.
Disclaimer: The conclusions reflect the views of the authors and should not be construed to represent FDA’s views or policies.
Bidesh Ghosh– FDA/CDER
Muhammad Ashraf– Supervisory Chemist, US Food and Drug Administration, Silver Spring, Maryland
Howard Anderson– FDA/CDER
Gibbes Johnson– FDA/CDER
Chikkathur Madhavarao– Biologist, US Food and Drug Administration, Silver Spring