Preparation and characterization of PEGyated Concanavalin A for affinity chromatography with improved stability

• Published in: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences Vol. 879; no. 20; pp. 1732 - 1740
• Main Authors: Wen, Zhenzhen, Niemeyer, Bernd
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 15.06.2011
• Subjects: Adsorbents; Adsorption; Affinity adsorption; affinity chromatography; Binding; binding capacity; biocatalysis; bioprocessing; carbohydrate binding; chemical bonding; Chloroform - chemistry; Chromatography, Affinity - methods; Colorimetry; Concanavalin A; Concanavalin A - chemistry; Concanavalin A - metabolism; drug therapy; ethylene glycol; Glucose oxidase; Glucose Oxidase - chemistry; Immobilized Proteins - chemistry; Immobilized Proteins - metabolism; Linear Models; Methanol - chemistry; Molecular weight; MPEG encoders; Ninhydrin - chemistry; PEGylation; polyethylene glycol; Polyethylene Glycols - analysis; Polyethylene Glycols - chemistry; propionic acid; Protein Stability; Reproducibility of Results; Separation; solvents; sorption isotherms; Stability; Temperature; Glucose oxidase; C; PVPAA; Con A; HRP; Protein stability; DP; Concanavalin A; M; Affinity adsorption; TC; Ads; mPEG-SPA; PEGylation; GOD
• ISSN: 1570-0232; 1873-376X
• DOI: 10.1016/j.jchromb.2011.04.018

In order to improve its stability, immobilized Concanavalin A (Con A) on Toyopearl adsorbents was conjugated with monomethoxy poly(ethylene glycol) succinimidyl propionate (mPEG-SPA) with different molecular weight. A colorimetric method using ninhydrin is proposed to determine the degree of PEGylation; this method has proved to be easy applicable and reproducible. The PEGylation reaction was studied in detail to elucidate how parameters such as molar ratio of mPEG-SPA to Con A and molecular weight of mPEG-SPA affect the degree of PEGylation. The adsorption isotherms of glucose oxidase (GOD) onto native and PEGylated Con A adsorbents showed that the modification did not alter substantially the specificity of the carbohydrate binding ability of Con A. However, the binding capacity for GOD was slightly reduced probably due to the steric hindrance caused by mPEG chains. Adsorption kinetic studies revealed a lower adsorption rate after PEGylation which was attributed to the steric effect. The dynamic adsorption capacity for modified Con A depended very much on the degree of PEGylation and the molecular weight of mPEG derivatives. The adsorption capacity could be highly preserved for Toyopearl Con A modified by mPEG2k (90% of the original adsorption capacity) even with a degree of PEGylation up to 20% (the ratio of primary amino groups of PEGylated immobilized Con A to that of native immobilized Con A). Studies show that the binding capacity of PEGylated Con A was highly preserved under mild process conditions. PEGylated Con A also exhibited obviously higher stability against more stressful conditions such as the exposure to organic solvents and high temperatures. Conjugation of Con A with mPEG2k provided better adsorption performance thus has greater potential for application in affinity separation processes compared with mPEG5k. The fact that PEGylation stabilizes the properties of Con A may greatly expand the range of applications of unstable proteins to bioprocessing (e.g. biocatalysis and downstream separation) as well as other protein applications (e.g. medication, industrial use, etc.).