| Human serum albumin (HSA) is the most abundant protein component contained in plasma and has many functions. HSA has wide applications, such as pharmaceuticals under various clinical conditions, an excipient or stabilizer in other biotechnology based products, a component in cell culture media, and so on. Large scale production of recombination human serum albumin (rHSA) which is as a substitute for the HSA originating in plasma (pHSA) has developed. For the problem in separation and purification of rHSA from high density fermentation broths such as complex process, difficulties in solid-liquid separation and removal of impurities,some technical problems were explored from the following aspects.Firstly, the extraction ability of different ethanol/salt aqueous two-phase systems (ATPS) was investigated. The ethanol/dipotassium hydrogen phosphate system showed the highest recovery for rHSA. When the concentration of rHSA changed from lg/L to 30g/L, and temperature was from 4℃to 30℃, aqueous two-phase extraction (ATPE) of rHSA exhibited high extraction efficiency. And ATPE could recover rHSA directly from a crude culture medium. The recovery (Y) of rHSA reached up to 108.13%when the system was composed of 20%(w/w) ethanol and18%(w/w) dipotassium hydrogen phosphate. Take suitable volume ratio of the top to bottom phase (R) into consideration, extrations which ATPS was composed of 18%(w/w) ethanol and 20%(w/w) dipotassium hydrogen phosphate were scaled up from 0.01 to 73L successfully.The average recovery of rHSA at different scales was 100.4%,and average removal ratios of cells was up to 99%. Simultaneously, partial protein impurities and 87.2%polysaccharides were eliminated, proteinase A (PrA) activity was decreased by 24.4%, proteinase B(PrB) activity was partially inhibited, and thus the stability of rHSA was improved. ATPE has integrated solid-liquid separation, extraction, concentration and primary purification into a single step. Moreover, recycle of phase components were reaserched. More than 80% of ethanol in the top phase and 80% dipotassium hydrogen phosphate in the bottom phase can be recovered.Secondly, according to the characteristics of the top phase, the downstream purification process of rHSA has been established. It included vacuum distillation for removing ethanol, purification by Phenyl SepharoseTM hydrophobic interaction chromatography(HIC), DEAE anion exchange chromatography for removal of polymers,. When the equilibrium solution contained 1.2mol/L K2HPO4, HIC condition was optimum.The recovery of HIC attained 74.9%, and reduced SDS-PAGE analysis showed that rHSA was electrophoretic pure. Moreover, purified rHSA remained stable for a long time because PrA activity was decreased to 9.18% compared with the supernatant, and PrB might be removed.Finally, purity, impurities content, secondary structure and molecular characteristics of purified rHSA has been analyzed. The HPLC purity and A350/A280 of rHSA attained 99.77% and 0.028 respectively. Meanwhile, the content of polysaccharides was reduced to 2.98ug/mg, and PrA activities was 2.93% compared with the supernatant. Furthermore, purified rHSA was essentially identical with pHSA in molecular weight and secondary structure.Integration process of hydrophilic organic solvents/inorganic salts ATPE and chromatography technology has provided a simple process for the separation and purification of rHSA to a high purity level, which has advantages of short time, high yield and removal rate of impurity, and produces large-scale industrial prospect.
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