(B-79) Scale Up of Mastocytoma Cells for the polysaccharide drug, Heparin Production in Bioreactors

Introduction:: Introduction: Heparin is the most widely used carbohydrate based anticoagulant drug worldwide, primarily used for surgeries and kidney dialysis in patients. Concerns arising from the 2008 adulteration of animal-derived heparin from China combined with increasing interest in using heparin to treat inflammatory and infectious diseases, have increased the demand for heparin from other sources. Although heparin is naturally produced in the intracellular granules of mast cells found in the intestines and lungs of many animals, mast cells are difficult to culture at industrial scales. Attempts to produce heparin from bacterial sources and engineered mammalian sources have shown potential, but scalability remains a concern. In this work, we describe the scale up of engineered cell cultures to small development-scale bioreactors using bioprocess formulations

Materials and Methods:: Materials and Methods: Genetically engineered murine mastocytoma cells¹ tailored for heparin production were thawed and inoculated from shake-flask cultures into Eppendorf DASbox 250mL bioreactors with a working volume of 200ml at a seeding density of 0.2M cells/ml in suspension culture. Bioprocess parameters such as lipid, vitamin, amino acid and glucose feeds, pH control, agitation and oxygen sparging rates were optimized to increase the cell viability and viable cell density (VCD). The glucose concentration was maintained at 3g/L and pH was maintained at 7.0 by addition of CO₂ or sodium bicarbonate. The bioreactors were harvested when the viability dropped below 60%; cells were centrifuged, and supernatant analyzed for osmolality and various metabolites – glucose, lactate, glutamine, glutamate, ammonium, and potassium, essential and non-essential amino acids, and lipids (using GC-MS). The cell pellets were frozen at -80 °C for assay of heparin anticoagulant activity.

Results, Conclusions, and Discussions:: Results: In this study, small-scale bioreactor suspension cultures of genetically engineered mastocytoma cells are described. Maximum VCD was ~10M cells/ml using a glucose feed to maintain 3g/L.VCD started to decrease around day 7. Metabolite analysis revealed that glutamine and glutamate decreased during the culture while ammonium levels increased. Lactate levels increased, particularly during oxygen limitation. Most amino acids showed decreased levels by day 7 of culture.


Conclusion: In this work, we have demonstrated the bioprocess optimizations using genetically engineered murine mastocytoma cells in bioreactors. The maximum VCD reached was with glucose feeding, sparging at 0.06 VVM and agitation at 125 RPM. Amino acid supplementation starting at day 7 or at the beginning of culture needs to be explored further as part of bioprocess optimization. Future work will focus on analyzing lipid depletion and determining the anticoagulant activity of heparin, setting the stage for production of a bioengineered heparin.




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References (Optional): : 1. B.E. Thacker, et al., Multiplex genome editing of mammalian cells for producing recombinant heparin, Metabolic Engineering, 70: 155-165 (2022) doi: 10.1016/j.ymben.2022.01.002