Fermentation, Recovery and Purification of the Hc Fragment of the Botulinum Neurotoxin from Pichia Pastoris.

摘要

This report documents the continued research and development of BoNTC He, BoNTE Hc and BoNTF Hc. A new approach to fermentation of BoNTC Hc was developed that incorporated a mixed feed of glycerol and methanol during the induction. It was determined that the optimum growth rate for expression of BoNTC Hc during methanol induction was 0.015 h(-1) and the range was very limited. The growth rate range was extended to 0.025 h(-1) by supplementing the methanol feed rate with a feed of glycerol. BoNTE Hc research focused only on purification development and a four step process produced over 98% pure BoNTE Hc based on SDS-PAGE. Two steps that were critical to the purification process, a batch capture step using anion exchange as BoNTE Hc would not completely bind (40 to 50% in column flow through) under dynamic conditions. A batch process was able to capture nearly 95% of BoNTE Hc. The last step was, hydrophobic interaction chromatography removed a 17 kD Pichia pasrtoris protein that was also problematic with BoNTA Hc. Key to the HIC step was addition of 5% glycerol to the buffer system. BoNTF Hc research focused on optimizing a process transferred to UN-L from USAMRIID through Covance.