Bioprocess and downstream optimization of recombinant human growth hormone in Pichia pastoris

Research in Pharmaceutical Sciences
Saeed AzadiRoya Solaimanian

Abstract

The methylotrophic yeast Pichia pastoris is a well-established expression host, which is often used in the production of protein pharmaceuticals. This work aimed to evaluate the effect of various concentrations of ascorbic acid in mixed feeding strategy with sorbitol/methanol on productivity of recombinant human growth hormone (r-hGH). The relevant concentration of ascorbic acid (5, 10, or 20 mmol) and 50 g/L sorbitol were added in batch-wise mode to the medium at the beginning of induction phase. The rate of methanol addition was increased stepwise during the first 12 h of production and then kept constant. Total protein and r-hGH concentrations were analyzed and the results compared with sorbitol/methanol feeding using one-way analysis of variance. Moreover, an effective clarification process using activated carbon was developed to remove process contaminants like pigments and endotoxins. Finally, a three-step chromatographic process was applied to purify the product. According to the obtained results, addition of 10 mmol ascorbic acid to sorbitol/methanol co-feeding could significantly increase cell biomass (1.7 fold), total protein (1.14 fold), and r-hGH concentration (1.43 fold). One percent activated carbon could signific...Continue Reading

References

Feb 27, 2010·Journal of Visualized Experiments : JoVE·Maria WeidnerSteven J Hallam
Dec 11, 2013·International Journal of Cell Biology·Suzana AulićGiuseppe Legname
Nov 14, 2016·Brazilian Journal of Microbiology : [publication of the Brazilian Society for Microbiology]·Angela Faustino JozalaAdalberto Pessoa

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Methods Mentioned

BETA
electrophoresis
flow
enzyme-linked immunosorbent assay
ELISA
protein assay

Software Mentioned

Chemstation
GraphPad
Unicorn
GraphPad Prism
TotalLab

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