| Therapeutic recombinant proteins are produced by the recombinant DNA technology. As a human protein or its mutant proteins, can compensate for deficiencies of certain body functions or increase the body protein function. The safety of therapeutic recombinant proteins was significantly higher than small molecule drugs. for some diseases, therapeutic recombinant proteins play an irreplaceable role and become an important driving force in its development. The core technology of large-scale production of recombinant pharmaceutical proteins is the purification of target product. But the process will lead to contamination of chromatographic resin easily and become the bottleneck restricting industrial production. Therefore, the contamination control of chromatographic column has a very important economic and practical value. In this study, contamination mechanism and washing and pretreatment methods of chromatographic resin were studied. In order to restore the chromatography performance of contaminated resin and increase its anti- contamination ability to reduce the costs of separation and purification of recombinant proteins effectively, the technologies of new regeneration and anti- contamination were established. Main tasks are as follows:1. Recombinant bovine milk as the experimental model system, the column pressure, ligand density and loading of SP Sepharose FF were studied. By before-after comparison of chromatography, the risks of contamination that loading was droped and the column pressure was increased were characterized.2. The interaction mechanism of proteins, lipids and lactose in recombinant bovine milk with resin was studied by detecting species of hangovers and calculateing their content in the contamination process. The adsorption mechanism between resin and whey components varied. The lipid in milk can clog the column or adhere to the resin through hydrophobic interaction, leading to an increase in column pressure. Some casein molecules were found to adsorb onto the resin through electrostatic interaction, therefore the adsorption capacity was decreased. There was no direct interaction between lactose and the resin in the chromatorgraphic process. 3. Effect of different factors upon regeneration was studied through different methods of regeneration for the contaminated ion exchange chromatography resin. The study of regeneration strategy showed that:in order to maximizing the exchange capacity of resin,0.2 M NaOH can clean the residual lipids and proteins in the column thoroughly. NaOH as regeneration agent, residual proteins can be washed and removed greatly at low regenerant concentrations, high shake Speed and low-temperature heating. Prolonged time interval between protein purification and column regeneration could enhance the undesirable interaction between the contaminants and resin. When more than 12 hours after the interval, the regeneration efficiency lower. The exchange capacity of the resin is difficult to be restored effectively if resin would not be regenerated in time after 2 chromatography cycles. High regeneration temperature is harm for the elution of hangovers and column regeneration. Room temperature or low temperature heating (≤30℃) can regenerate best.4. HBsAg protein as a representative of poly-subunit, effect of the pretreatment of HSA and Hansenula polymorpha cells fragmentation upon improveing anti-contamination performance of ion exchang chromatography column was studied. The result showed that:the pretreatment of HSA and a small amount of Hansenula polymorpha cells fragmentation will increase the dynamic adsorption loading and improve the ability of anti-contamination. But the former's anti-pollution effects more significant than the latter. A combination of both pretreatment can improve anti-pollution better than alone. Relatively large number of Hansenula polymorpha cells fragmentation will reduce the loading and lead to column contamination. |
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