Advances in Bioprocess Development

A continuing challenge in process development is meeting the ever changing needs of new products and technologies in a safe and cost-effective manner. Processes where the source is a living organism are associated with unique challenges that are not usually seen with traditional chemical synthesis. Even within the chemical synthesis space, bioprocessing techniques can enable new chemistries or products that are not accessible via conventional chemical processing, often at far less extreme operating conditions. This session will provide examples of new advances in bioprocessing applications as well as the use of bioprocessing techniques to increase the versatility of process development efforts.

Session Chairs:

• David Couling, Corteva
• Yamini Krishnan, Corteva

Tentative Schedule:

Abstracts:

Longevity Studies to Evaluate the Performance and Operating Conditions of a Continuous Enzymatic Packed Bed Reactor

Rick Ray, Corteva Agriscience

The production of a novel agricultural active ingredient requires the enzymatic resolution of a racemic alcohol intermediate to yield an enantio-enriched chiral alcohol for subsequent downstream processing. The enzymatic resolution of this racemic alcohol was readily carried out using the immobilized enzyme Purolite LifetechTM CalB Immo Plus in a packed bed reactor. A key manufacturing consideration was that the immobilized enzyme should be reused for a minimum of fifteen cycles at 10 wt% resin loading relative to the racemic substrate to be considered cost competitive, amounting to just 6.6 g CalB Immo Plus utilized per kg of substrate. To validate this manufacturing assumption, enzyme longevity studies were carried out for the resolution of the racemic alcohol in a packed bed reactor. The longevity studies revealed that the enzymatic resolution could be carried out for > 15 batches while maintaining high enantioselectivity (≥ 95% ee), though there  was a measurable logarithmic decrease in enzyme activity over sequential batches. This decrease in activity contributed to prolonged reaction times in later batches, though the loss in productivity was within an acceptable range for manufacturing considerations. The findings from the longevity studies provided opportunities to optimize the reaction conditions and maximize the efficiency of the lipase usage.

Exometabolome Profiling Reveals Activation of the Carnitine Buffering Pathway in Fed-Batch Cultures of CHO Cells Co-Fed with Glucose and Lactic Acid

Keegan Orzechowski, GlaxoSmithKline; Juan Carlos Aon, GlaxoSmithKline

Adjustments to CHO cell physiology were recently observed during implementation of a Raman spectroscopy-based glucose and lactate control strategy. To further understand how these cells, under monoclonal antibody (mAb) production conditions, utilized the extra lactic acid fed, we performed a comprehensive semi-quantitative and time-dependent analysis of the exometabolome. This study focused on the CHO cell’s metabolic shift from the fifth day of culture. We compared relative levels of extracellular metabolites in the absence or presence of a 2 g/L lactic acid setpoint while glucose was kept at 4 g/L. Our hypothesis is that extra lactic acid would supply more pyruvate, favoring oxidative phosphorylation. We subsequentially uncovered several carnitine derivatives as biomarkers of the simultaneous activation of TCA anaplerotic pathways as well as a carbon-buffering pathway. CHO cells exhibited a balance between intermediates from (i) amino acid catabolism, (ii) fatty acid β-oxidation, and (iii) pyruvate from glycolysis and lactic acid; and the secretion of their intermediate carnitine derivatives. In addition, 3-hydroxy-methyl-glutaric acid (HMG) and mevalonate syntheses were found as biomarkers of alternative acyl group removal. Together, under a limited capacity to assimilate the surplus of acyl-CoA groups as well as an ability to maintain the acyl-CoA:free CoA ratio for proper and continuous functioning of the TCA cycle, CHO cells activate the carnitine-buffering system, HMG, and mevalonate pathways. 

Opportunities and Challenges in the Biocatalytic Process Development of APIs

Rakeshwar Bandichhor, Dr. Reddy's Laboratories

Higher Process Intensity (PMI) of a pharmaceutical process is due to stoichiometric use of reagents, disproportionate use of solvent and water. Catalytic process development always leads to efficient processes with lower PMI. Biocatalysis in pharmaceutical development is viewed as one of the most promising toolboxes to manufacture APIs with high efficiency. There will be case studies highlighting opportunities and challenges in the process development of pharmaceuticals.