Yamanie, Almir; (2024) Towards Upstream Process Development Using Mixed Induction Strategy and Continuous System in Cultivation of Komagataella phaffii. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

In recent years, the biopharmaceutical market has grown a lot because of rising demand and the fast growth of biotechnology in terms of new ways to treat a wide range of diseases. Using yeast, like Komagataella phaffii, as a platform for making biopharmaceuticals is becoming increasingly popular because it is more cost-effective, easier to scale, and more consistent product quality. However, the standard fed-batch cultivation process using a pure methanol induction strategy that is currently commonly used in the cultivation of K. phaffii may not be able to keep up with the demands due to challenges such as a large carbon footprint due to scale-up of cultivation to increase production, a higher risk of hazard for using large volumes of pure methanol, and extended downtime for batch preparation, which causes high operating and utility costs. Thus, a new way of combining the continuous cultivation process with methanol and sorbitol mixed induction strategies is needed to reduce the production size and the amount of methanol used. In this thesis, the impact of both a methanol/sorbitol mixed induction strategy and continuous cultivation process on K. phaffii fermentation in the production of KEX2 protease was studied and evaluated in terms of cell growth, product content, and volumetric yield (ug.mL-1), as compared with commonly used yeast-based cultivation method that uses pure methanol as an induction agent in fed-batch mode. Furthermore, a techno-economic feasibility study of the processes was compared in a pilot bioreactor and on simulated industrial scales. The standard pure methanol induction method was compared to the 50% (C-mol/C-mol) methanol with sorbitol mixed induction method in the fed-batch cultivation process for the production of KEX2 protease, which improved the specific growth by almost 2-fold while also increasing the KEX2 protease yield (mg) in one batch by 2.3-fold when 2 L of broth was harvested 24 hours after the start of the induction process. In addition, the impurities inside the supernatant after separating the cells from the broth were also found to be lower when using the mixed induction method when compared to the pure methanol induction method, increasing the KEX2 protease content by 7% of the total protein bands found from the SDS PAGE result. The continuous cultivation process of K. phaffii for the production of KEX2 protease was also done using pure methanol induction and a 50% (C-mol/C-mol) methanol with sorbitol mixed induction method using the data previously acquired from the fed-batch cultivation process. Using a mixed induction strategy with a continuous cultivation method created a synergistic effect due to the increase in specific growth rate, which resulted in an increased daily volume harvested. The biomass could be kept at 80 g.L-1 and 100 g.L-1 for pure methanol induction and mixed induction, respectively, and the process was kept for at least 240 hours and harvested every 24 hours. The final harvested volume in one batch when using the pure methanol and mixed induction methods was found to be 8 L and 13.5 L, respectively. It resulted in a yield (mg) of 8.6-fold increase in one “batch.” Also, the yield (mg) of continuous cultivation using a mixed induction strategy was compared to the yield of fed-batch cultivation processes in one batch, which showed an increase of 8.9 times for mixed induction and 21.3 times for pure methanol induction. A simulation of a 400-litre fed-batch cultivation system with the methanol induction strategy was used as a benchmark. Then, a simulation of a combination of continuous cultivation with the mixed induction strategy at the same scale and on a smaller scale was compared with the benchmark to determine its benefits and cost savings. The combination method allowed for a 10x reduction in production scale while maintaining similar output and time. Other benefits also included lower capital costs, operating costs, and a smaller carbon footprint.

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