Interfacing Pichia pastoris cultivation with expanded bed adsorption

Area of Science:

• Biotechnology
• Protein Purification
• Bioprocess Engineering

Background:

• Interfacing Pichia pastoris fed-batch cultivation with expanded bed adsorption (EBA) is challenging due to high cell density and salt concentrations.
• Traditional methods often lead to increased proteolysis and lower product purity.

Purpose of the Study:

• To develop a modified cultivation technique for improved integration of Pichia pastoris fed-batch cultivation with EBA.
• To optimize the recovery and purification of a CBM-CALB fusion protein.

Main Methods:

• Reduced and regulated medium salt concentration (conductivity ~8 mS/cm) during fed-batch cultivation.
• Dilution of culture broth to reduce viscosity before EBA loading.
• Application of EBA for one-step recovery and purification of CBM-CALB fusion protein.

Main Results:

• Lowered dead cell percentage (1% vs 3.5%) and reduced contaminating proteins.
• No proteolysis detected; product constituted 87% of total supernatant protein at cultivation end.
• EBA achieved 98% product yield, ~90% purity, and 2.1 g/L x h productivity with a breakthrough capacity of 12.4 g/L.

Conclusions:

• The modified cultivation technique significantly enhances the interfacing of Pichia pastoris fed-batch processes with EBA.
• This approach enables efficient, high-purity recovery of fusion proteins with minimal degradation.
• The optimized process demonstrates high yield, purity, and productivity for CBM-CALB purification.