Therapeutic protein expression platform of microbial system

A number of expression systems have been developed for the production of pharmaceutical products. Pichia pastoris and Escherichia coli expression system operate in our lab and express antibody fragment (scFv), cytokine, protein base adjuvant and vaccine and process enzyme. The expression platform are consisted of three part, first is strain generation , the second is fermentation process development in 250 ml fermentor and the last is process scale-up to 5 litter fermentor. Please click Additional Files below to see the full abstract

Gram level scFv expression platform of Phichi pastoris

The methylotrophic yeast Pichia pastoris secretion expression system has been developed for the antibody fragments (scFv) production platform. The platform includes three technology platforms, the first one is strain generation, the second is fermentation process development in 250 ml fermentor and the last is process scale up to 5 L. A recombinant scFv went through clone generation, include signal peptide tool box, normally yield 2.5 mg/L titer in deep well. Through the fermentation process development of induction medium composition and feeding strategy by Eppendorf Dasgip parallel 250 ml mini fermentor. During induction step, feeding 100% methanol as induction medium can only produce less than 50 mg/L scFv while feeding methanol-sorbitol mixture can significant increase the production yield to 306 mg/L in five days, about 6-folds increase in productivity. With the supply of additional nitrogen source during glycerol feeding step or at induction step, higher scFv production with 510 mg/L can be achieved. Thus, following the medium composition optimization, the production titer was improved 10 folds in 250 ml mini-fermentor stage. Moreover, when we switched the induction medium feeding strategy from DO-stat to the stepwise feeding, the titer increased form 510 mg/L to ~1000 mg/L and yielded another 2- folds improvement. During medium composition and feeding strategy optimization at 250 ml mini fermentor scale, the production titer could increase 20 folds. Overall, the production titer increased 400 folds from cell line generation to 250 ml fermentation parameter optimization. Furthermore, the process parameter can be scale-up to 5 L fernentor achieving \u3e 1 g/L. Recent progress to include BIP in the expression vector gave at least 2 fold improvement in scFv titer in shake flask, the new clone will be optimized in our established 250 ml and 5 L fermentation platform Please click Additional Files below to see the full abstract

Gram Level scFv expression platform of Pichia pastoris

The methylotrophic yeast Pichia pastoris secretion expression system has been developed for the antibody fragments (scFv) production platform. The platform includes three technology platforms, the first one is strain generation, the second is fermentation process development in 250 ml fermentor and the last is process scale up to 5 L. A recombinant scFv went through clone generation, include signal peptide tool box, normally yield 2.5 mg/L titer in deep well. Through the fermentation process development of induction medium composition and feeding strategy by Eppendorf Dasgip parallel 250 ml mini fermentor. During induction step, feeding 100% methanol as induction medium can only produce less than 50 mg/L scFv while feeding methanol-sorbitol mixture can significant increase the production yield to 306 mg/L in five days, about 6-folds increase in productivity. With the supply of additional nitrogen source during glycerol feeding step or at induction step, higher scFv production with 510 mg/L can be achieved. Thus, following the medium composition optimization, the production titer was improved 10 folds in 250 ml mini-fermentor stage. Moreover, when we switched the induction medium feeding strategy from DO-stat to the stepwise feeding, the titer increased form 510 mg/L to ~1000 mg/L and yielded another 2- folds improvement. During medium composition and feeding strategy optimization at 250 ml mini fermentor scale, the production titer could increase 20 folds. Overall, the production titer increased 400 folds from cell line generation to 250 ml fermentation parameter optimization. Furthermore, the process parameter can be scale-up to 5 L fernentor achieving \u3e 1 g/L. Recent progress to include BIP in the expression vector gave at least 2 fold improvement in scFv titer in shake flask, the new clone will be optimized in our established 250 ml and 5 L fermentation platform. Please click Additional Files below to see the full abstract

Engineering of Escherichia coli protein expression process development

It almost 30% protein drugs are expression by Escherichia coli, because of rapid growth and high production yield. We have developed E.coli base system for recombinant protein expression, scFv, Fab and vaccine. In this study we introduce example about process development for nutrient components selection. Shaker flasks were used for different nitrogen and carbon components screening by DoE. Seven media formulations for E. coli fermentation were used in this study. By changing nitrogen and carbon source ratio, product titer of target protein could be optimized, at least 1.4 folds increased. The best result from shaker flask was used in 250 mL parallel fermenter and pH, dissolved oxygen, feeding/induction strategy were evaluated. The processes from seed culture to harvest only require 64 hours. The optimized time was reduced to 32 hours. The result showed that both target protein expression and cell density value were comparable, but the total process time was significantly reduced by half Please click Additional Files below to see the full abstract