Study Of High Efficient Ferment Technique And Purification Of Genetically Engineered Antibody Produced By Pichia Pastoris

Now antibody drug has become pillar industries of biotechnology drugs. By 2006, FDA approved 26 antibody drugs which can be used in vivo, including 21 therapeutic agents. In the world, the annual sales of antibody drugs in 1997 have increased from 310 million to 150 billion U.S. dollars in 2005. The percentage in biopharmaceutical industry has increased from one-fifth in 2000 to one-third in 2005.Genetically engineered antibody has been the current of antibody drug. In the world there were 100,000 antibodies which have been reported, including 1,000 genetically engineered antibodies. With the rapid development of genetically engineered antibody, the new methods and ways of diagnosis, prevention and treatment of major disease have been acquired. But the bottleneck of industrial development is the expression and purification of genetically engineered antibody. There are two expression systems of genetically engineered antibody: prokaryotic expression system, including secreted and inclusion, and eukaryotic expression system. Pichia pastoris has been applied for expression of genetically engineered antibody widely, because it not only has many eukaryotic expression system merits (post-translational modification, etc), but also is a simple, low-cost expression system. Based on anti-keratin antibody Fab fragment of target products, this study aimed at the establishment of efficient fermentation and purification process of genetically engineered antibody which produced by Pichia pastoris.The contents of this research have been conducted in the following three aspects:1).Optimization of anti-keratin Fab expression condition. Glycerine concentration, temperature and pH of three levels were investigated by orthogonal design to determine their influence's on cell growth. Induce opportunity, methanol concentration, temperature and pH of four levels were investigated by orthogonal design to determine their influence's on Fab production. 2). Optimization of fed-batch condition. The best condition which can lead to high cell density and Fab production were determined by screening different fed-batch strategies of glycerine, methanol and ammonium sulfate in 5L fermentor. 3).Establish and optimization of separation and purification condition. Firstly the protein characteristics were predicted by bioinformatics, and a suitable chromatography and media were determined. Then multiply parameters including equilibration, elution, and regeneration conditions, column scale, bed height, and flow rate, were compared and screened. Lastly, technology parameters which maintain high purity, high recovery and good bioactivity were determined.The results have been conducted in the following three aspects:1). In shake flask, best cultivation condition for Pichia pastoris were 1% glycerine, 29℃, pH6.5 with cell density of OD600=15.3, cell activity=95% and the best anti-keratin Fab expression condition were 1% methanol, 29℃, pH6.25 with antibody productor of 9mg/L. 2). The best timepoint for feeding was the bump of rapid upstroke of DO. In 5L fermentor, glycerine was add to fermentation broth with the flow-rate 9ml/(L/h) and ammonium sulfate at final concentration of 0.5g/L in cultivation period and methanol was add to fermentation broth with the flow-rate 6ml/(L/h) and ammonium sulfate at final concentration of 0.5g/L in induction period. Pichia pastoris cell density reached OD600≥250,cell activity=95% and anti-keratin Fab production reached 150mg/L. Compared with the shake flask cell density increased 17 times, Fab expression increased 16- times. 3). PPG-600M HIC was the most appropriate method investigated for the purification of Fab determined by bioinformatics analysis and screen experiment. The sodium phosphate buffer was more advantageous over Tris buffer for binding. For elution 20mM sodium phosphate (pH7.2) and 30%(2CVs)-60%(2CVs)-100%(2CVs) were chosen. The 6M urea was more effective cleaning agents than others. The best column bed height was 12.5cm. 10ml/min flow-rate was chosen for equilibration and 5ml/min for elution. The appropriate chromatography conditions were established and 85% recovery and purity larger than 90% and good bioactivity was achieved. The processes developed for the Fab fragment have been shown to be scalable further.In conclusion, firstly, the appropriate cultivation and induction conditions for preparation of anti- keratin Fab were optimized and laid a solid foundation for scale up fermentation. Secondly, the best feeding strategy of glycerine, methanol and ammonium sulfate was determined, and fed-batch technology was optimized. The cell density and Fab production was improved by 17 times and 16 times, respectively than that of shake flack. Thirdly, the appropriate chromatography conditions for anti-keratin Fab preparation were established and high recovery, purity and bioactivity were achieved. A high performance preparation technique platform for engineered antibody from Pichia pastoris has been established.