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Study on the Preparation of a Novel Cross-linked Chitosan Suitable for the Separation of Antibody

Author: CuiGuoYan
Tutor: YingGuoQing；YiYu
School: Zhejiang University of Technology
Course: Microbial and Biochemical Pharmacy
Keywords: chitosan reverse phase suspension IMAC hCG
CLC: R943
Type: Master's thesis
Year: 2011
Downloads: 55
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Abstract

In order to find the substitution of the expensive polysaccharide supporter, such as glucan and agarose, investigation was carried out to elucidate the influence of process parameters on the characteristics of chitosan microspheres produced by reverse phase suspension cross-linked polymerization. The iminodiacetic acid (IDA) was attached onto chitosan microspheres activated by epichlorohydrin under basic condition. HCG was used as testing samples. The effects of various factors on antibody preparation were studied in this experiment. The experimental results are as follows:First, research on the optimal preparation conditions of porous chitosan microspheres. The results indicated that the optimal preparation conditions of porous chitosan microspheres, included:2%(v/v) ethanoic acid,3%(w/v) chitosan,2.5 mL 25%(w/v) glutaraldehyde,350 r/min stirring speed, and 2.5 mol/L NaOH, the ratio of O/W(liquid paraffin /chitosan solution) was 1:1.5.Second, the iminodiacetic acid (IDA) was attached onto chitosan microspheres activated by epichlorohydrin under basic condition. The results indicated that the optimal epoxy density above 0.15 mmol/g gel was achieved at 10%(φ) epichiorohydrin as activating accelerator in the solution consisting of 0.6 mol/L NaOH and 40% DMSO. The study on the linkaging of IDA demonstrated that the support was synthesized in the solution composed of 0.6 mol/L IDA and 2.0 mol/L NaOH at 60℃for 6 h, whose asoption of Cu2+ was up to 172.79 mg/g gel. The adsorption performance of BSA was consistent with the curves of Langmuir adsorption characteristics, and the maximum adsorption qm was up to 60.61 mg/g, KD was up to 1.39.After backfilling the solid phase in chromatographic column, the unchelated solid phase was obtained; copper ion was chosen to chelate with IDA, the immobilized metal ion solid phases were prepared. The purification of mAb was studied by using IMAC (Cu). The sample was loaded on the IMAC column using a phosphate buffer(pH8.0,20 mM PB)containing 0.5 M NaCl, followed by elution with a pH stepwise elution.With this method, purity up to 61% was achieved with recovery of 57%.

Study on the Preparation of a Novel Cross-linked Chitosan Suitable for the Separation of Antibody

Abstract

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