Study On The Pilot Fermentation Process Of Recombinant Human Serum Albumin In Protease Knockout Pichia Pastoris

Human Serum Albumin (HSA) is a widely used and market demand for playingclinical drug, which is widely used in the treatment of plasma volume expanderbleeding, shock, burns, cancerclinical treatment of liver disease, which takes normalosmotic pressure of the blood and the promotion of hydrophilic molecules transport.All current HSA used as a medicine are purified from human blood plasma, becausesources of plasma and placental are limited, production can only meet a little part ofthe pharmaceutical market, and due to the impact of viruses and other pathogenicmicro-organisms, the security can not be guaranteed. Therefore, developing anefficient and safe process for HSA production has become a hot topic ofbiopharmaceutical industry.With the rapid development of genetic engineering techniques, a variety of proteindrugs has been successfully expressed in E. coli, yeast, mammalian cells, the recombinantbiotechnology increases production, reduces low production costs, and improves safety,so dozens of recombinant protein drugs have been used in clinical treatment. In the pastfew decades, many investigators have made a large of effort for recombinant expressionof HSA, all agreed that Pichia pastoris is the most suitable for the HSA's industrializedproduction, and carried out extensive and in-depth research for relevant fermentationprocess, many technical difficulties are overcome. However, the degradation of HSA inthe fermentation process has not yet been resolved, which has become the key technicalbottleneck in the industrialization process of HSA.Here, we carried out a comparable study of protease-deficient Pichia pastorisstrain fermentation process for HSA. To combine molecular biology techniques andfermentation process, the study has screened and obtained the yeast strain that hashigh production, low-degradation of HSA expression. All details about the study aredescribed as follows:The First Part: the investigation on obtaining protease-deficient strains for rHSA and building relevant preliminary pilot fermentation processAccording to the coden usage of Pichia pastoris, we redesigned HSA's genesequence, and transform it into Pichia yeast strains of GS115NC, GS115pep4-,GS115prb1-respectively, then study the difference of growth, rHSA expression anddegradation between three yeast strains.The experimental results show that, compared with rHSA GS115NC, rHSAGS115pep4-has no significant difference in cell growth and rHSA expression, theproduction reached700μg/L in the scale of the shake-flask, which meet demand ofpilot scale fermentation, hence knock-out protease A did not interfere expression ofrHSA; by the contrast to the first two strains, rHSA GS115prb1-grow more slowly,and its rHSA expression was only1/2of the two strains', suggesting that knock-outprotease B has affected the growth and rHSA expression seriously; As is shown inWestern blotting, in the case of the similar rHSA expression, rHSA degradation ofrHSA GS115pep4-strain was less than of the one of rHSA GS115NC, indicating thatknock-out protease A could reduce the degradation rate of rHSA in the fermentationsupernatant. In the preliminary study on the pilot fermentation process, it was foundthat BMGY culture medium was too rich, cell growth too fast, but low production, notsuitable as a pilot fermentation scale medium.The Second Part: developing a medium suitable for the rHSA protease-deficientengineering strain in the shake flask scale fermentationThe main objective of this part is to develop a medium suitable for the rHSAprotease-deficient engineering strain in the shake flask scale fermentation, todetermine the rHSA expression of key components for the design of the pilotfermentation medium.In the contrast to BMGY medium, to prepare the8kinds of BSM salt mediumwith different components and measure rHSA expression, the results proved that inBMGY medium and basic salt medium (1/4BSM+2%of pancreatic of peptone+0.2%the Biotin+0.1M phosphate buffer), the expression of rHSA was highestamong of them, BMGY belongs to an enrichment medium, it suits for shake flask and seed preparation, basic salt medium is designed for the pilot fermentation originally,therefore in this part BSM was used as a basis of optimizing medium.Orthogonal experiment (4factors and3levels) was designed for optimizingmedium based on basic salt medium (+0.2%1/4BSM+2%of pancreatic peptone ofthe Biotin+0.1M phosphate buffer). It is determined that the results of rHSA GS115NC and of rHSA GS115pep4-optimal medium parameters is BSM salt1/6, pH6.0,tryptone peptone2g, Biotin40μg; rHSA GS115prb1-optimal medium parameters isBSM salt1/6, pH6.0, tryptone peptone4g, Biotin80μg.The Third Part: the pilot fermentation process for the rHSA protease deficientstrains and relevant rough treatment process of the fermentation brothIn this part, we studied the fermentation process of the rHSA protease-deficientstrains in the pilot scale, rHSA expression was measured through the process offermentation, the parameters of normal strains and two protease strains werecompared in the accumulation of biomass, rHSA expression, degradation rate, andbuild an efficient rough treatment process of the fermentation broth.The pilot fermentation process results of3strains further confirmed that therHSA GS115prb1-strain's protein expression was lower than that of rHSA GS115NC and of rHSA GS115pep4-, among of them, the degradation band and ratio ofrHSA GS115pep4-both were lower than those of rHSA GS115NC. All findingssuggested that knock-out protease A is a excellent strategy for obtaining highexpressed, low degraded rHSA Pichia pastoris strain.Using hollow fiber microfiltration and ultrafiltration, the fermentationsupernatant was separated and enrichment, they combined an efficient system for thetreatment of the fermentation broth, which also make a solid basis for followingpurification.