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Contamination Mechanism and Controlling Strategies of Chromatographic Resin in Separation Process for Therapeutic Recombinant Proteins

Author: SunXiYan
Tutor: QinPeiYong
School: Beijing University of Chemical Technology
Course: Microbial and Biochemical Pharmacy
Keywords: Recombinant proteins Ion exchange medium Pollution Mechanism Regeneration strategy Anti-pollution
CLC: X502
Type: Master's thesis
Year: 2011
Downloads: 47
Quote: 0
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Abstract


Recombinant pharmaceutical proteins refers to a protein produced using recombinant DNA technology. Examples of the human protein or mutants thereof, can make up for some of the in vivo functional protein defects or to increase the body protein function. Drug safety of recombinant protein was significantly higher than the small-molecule drug treatment irreplaceable role in certain diseases has become an important driving force of the recombinant protein drug development. However, the separation and purification of the target product of the core technology of the large-scale production of recombinant pharmaceutical proteins, the process can easily result in the contamination of the medium has become the bottleneck of restricting industrialized production ramp. Pollution control column has a very important economic and practical value. Investigated through the chromatographic medium pollution mechanism, as well as washing and pretreatment mode, to establish a new type of media regeneration and fouling process, thus effectively restored pollution medium chromatography performance, improve the contamination resistance of the medium reduce the recombinant protein was isolated and purified process costs. Major work is as follows: 1. Recombinant milk for experimental model systems investigated media SP Sepharose FF column pressure, the ligand density and load, characterized by chromatography before and after pollution hazard: the media adsorption load reduction, column pressure. 2 calculated by detecting the type and quantity of the residual component chromatography column under different processing conditions, revealing the three major components (proteins, lipids, and lactose), recombinant bovine milk in a pollution process and medium interaction mechanism. Whey adsorption mechanism between the respective components and the medium of the same, there is a certain degree of hydrophobic interaction between the lipid and the medium, but the majority of the lipid particles to plug the gap between media particles through adhesion, resulting Column pressure; part of the casein by electrostatic interactions ligand sites occupy the medium, resulting in the exchange capacity of the medium decreases; no direct interaction in the between lactose same medium. 3 through recombinant bovine chromatography pollution after ion exchange medium for the regeneration of the different ways to investigate the influence of various factors on the regeneration effect. Regeneration strategy visits 0.2 MNaOH can be thoroughly cleaned of the column residual lipids and protein to maximize the recovery medium exchange capacity; preferred regenerant NaOH as the residual component proteins at low concentrations regenerant, high oscillation speed maximize the elution and the case of low temperature heating can be removed; adsorption between the residual component with the medium with the chromatography - the regeneration time interval is gradually increased when the time interval is more than 12 hours after the regeneration effect is greatly reduced; continuous should be promptly after 2 chromatography media regeneration, or the medium exchange capacity is difficult to obtain effective recovery; elevated regeneration temperature is not conducive to the regeneration of the elution and column chromatography of the residual component, heating at room temperature or low temperature (≤ 30 ° C) can get a better regeneration effect. 4 represents the poly subunit protein of HBsAg, anti-pollution performance examine HSA and Hansenula broken liquid pretreatment to improve the chromatography column. The study found that, HSA and a small amount of the yeast lysate pre-treatment will increase the load of the dynamic adsorption of the chromatography column, having improved dielectric anti-pollution effect, but the antifouling effect of the former is more significant than the latter; combination pretreatment chromatography The column against contamination capabilities better than used alone; representing the the broken liquid pretreatment column will reduce the adsorption of the medium load caused by the contamination of the column.

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