Pichia pastoris fermentation optimization: energy state and testing a growth-associated model.

Area of Science:

• Biotechnology
• Bioprocess Engineering
• Molecular Biology

Background:

• Recombinant protein production in Pichia pastoris is crucial for therapeutic manufacturing.
• Existing growth-associated models did not accurately predict ovine interferon-tau (rOvIFN-tau) yields.
• A phenomenon termed 'decoupling' was observed, where product yield decreased after a certain fermentation time.

Purpose of the Study:

• To optimize the production of recombinant ovine interferon-tau (rOvIFN-tau) using Pichia pastoris.
• To investigate and overcome the decoupling phenomenon limiting rOvIFN-tau production.
• To develop a robust and stable fermentation process for preclinical and clinical material.

Main Methods:

• Applied a growth-associated model to Pichia pastoris fermentation for rOvIFN-tau production.
• Investigated the relationship between culture parameters (growth rate, time on methanol, culture density) and product yield.
• Identified a correlation between decoupling and cellular energy state drop during beta-galactosidase expression.
• Reformulated the growth medium and implemented decoupling as a constraint to extend production time.

Main Results:

• rOvIFN-tau yields followed a polynomial, not linear, relationship with growth.
• Decoupling was linked to time on methanol, not cell density.
• Product accumulation extended to 46.8+/-2.4 hours post-medium reformulation.
• Product yield increased from 203 to 337 mg/L, with coefficient of variation decreasing from 67.9% to 23.3%.
• Total yield improved by 210%, from 557+/-357 mg to 1,172+/-388 mg.

Conclusions:

• The decoupling phenomenon is a critical factor limiting recombinant protein production in Pichia pastoris.
• Optimizing fermentation by managing decoupling and medium composition enhances rOvIFN-tau yield and process robustness.
• The developed process is suitable for manufacturing preclinical and clinical active material.