Evaluating Pulses and Modified Fed-batch Feeding of Methanol to Increase Expression Level of Human Insulin Precursor in Pichia Pastoris High-Density Cultivation

Abstract

Production of sufficient insulin at a more affordable price is necessary. The increase in the number of people living with diabetes puts more burden on healthcare and the economy. P. pastoris is a promising host to produce human insulin precursors at a high yield in minimal medium and secretes low levels of endogenous protein impurities. Production of the precursor involves several parameters, including glycerol concentration, culture density, methanol concentration, and medium composition. This study evaluated the effect of those parameters on protein expression in the flask culture. Subsequently, fermentation in the bioreactor was carried on according to the information obtained from flask culture. Methanol feeding to induce protein expression was undertaken by pulses and fed-batch modes. The fed-batch method was modified from a standard technique by incorporating constant flow rates with variable feed concentrations. Cell density was determined based on optical density measurement at 600 nm and dry cell weight. Tricine SDS-PAGE and reversed-phase HPLC conducted protein analysis. The pulse feeding produced higher precursor concentrations at ~445 mg/L than modified fed-batch feeding at ~267 mg/L. However, the modified fed-batch feeding can be an alternative to producing human insulin precursors when a standard fed-batch feeding with variable flow rates and 100% (v/v) methanol feed is difficult to apply.

Expression of recombinant human insulin precursor by Pichia pastoris in a 10 liter bioreactor

“…In the previous study, when methanol was pulse-fed at the same final concentration and working volume, the HIP produced was lower (286 mg/L) than in the current research using a continuous feeding strategy [8]. In addition, previous continuous feeding of diluted MeOH at a flow rate higher than the current research resulted in lower DCW and HIP concentrations, approximately 20 g/L and 267 mg/L, respectively [7]. However, methanol fed at a flow rate of 9.999 mL/h was too much for this strain.…”

“…The flow rate was 1 mL/min, the running time was 25 minutes, the temperature column was 25 °C, and the injection volume was 10 µL. Samples were eluted with mobile phase A (0.1% v/v TFA in water) and mobile phase B (0.1% v/v TFA in acetonitrile) as follows: 15-30% B (0-12 minutes); 30-100% B (12-18 min) [7]. The eluted fraction was monitored at 214 nm.…”

“…Our recombinant strain P.pastoris X33/pD902-IP clone 4 (CL4) was stable and expressed human insulin precursor (HIP) in the flask scale [6]. The expression of HIP in the bioreactor 2 Liter using pulse and modified methanol fed had been studied [7]. Moreover, the expression of HIP in a 10 Liter bioreactor using a pulse-fed methanol strategy has also been studied [8].…”