Post-refolding stability considerations for optimization of in-vitro refolding: L-asparaginase as a case study

Area of Science:

• Biotechnology
• Enzyme Engineering
• Process Chemistry

Background:

• L-asparaginase is crucial in food and biopharmaceutical industries.
• Enzyme stability is a significant challenge in L-asparaginase manufacturing.
• Product loss due to instability impacts commercial biopharmaceutical processing.

Purpose of the Study:

• To develop a strategy for simultaneously improving refolding yield and stability of L-asparaginase.
• To enhance the overall process yield of L-asparaginase.
• To investigate the interplay between process intermediate stability and process design.

Main Methods:

• One Variable At a Time (OVAT) experiments identified key parameters: urea concentration (6 M), solubilized inclusion bodies (15 mg/ml), step dilution refolding, and pH (8.6).
• Design of Experiments (DOE) was employed for optimizing the refolding step.
• Stability assessments were conducted at room temperature and refrigerated conditions (2-8°C).

Main Results:

• A three-fold increase in enzyme recovery was achieved, with optimized conditions yielding 4.90 IU/ml compared to 1.26 IU/ml unoptimized.
• The optimized L-asparaginase process intermediate demonstrated stability for over a week at room temperature and 2-8°C.
• Unoptimized samples showed instability at room temperature after 72 hours, losing activity.

Conclusions:

• Process intermediate stability is a critical factor in optimizing labile biopharmaceutical production.
• Consideration of stability during process design is essential for maximizing enzyme recovery and ensuring product quality.
• The findings provide a pathway for more robust and efficient L-asparaginase manufacturing.