Please use this identifier to cite or link to this item: https://idr.l3.nitk.ac.in/jspui/handle/123456789/12667
Title: Production and Optimization of Site-Specific monoPEGylated Uricase Conjugates Using mPEG-Maleimide Through RP HPLC Methodology
Authors: Nanda, P.
JagadeeshBabu, P.E.
Raju, J.R.
Issue Date: 2016
Citation: Journal of Pharmaceutical Innovation, 2016, Vol.11, 4, pp.279-288
Abstract: Purpose: Uricase (Uc), a therapeutic enzyme, is widely used in its PEGylated form to treat hyperuricemia and is largely manufactured by means of random/first generation PEGylation approach. Currently available randomly PEGylated uricase conjugates exhibit inadequacies like reduced uricolytic activity, risk of inducing immunogenic reactions, lack of selectivity, and molecular heterogeneity. In the present study, site-specific/second generation PEGylation strategy involving modification of specific and rare amino acids by means of terminally functionalized PEG polymers was applied. Methods: Uricase was conjugated with methoxypolyethyelenglycol-maleimide (mPEG-mal) by means of thiol PEGylation to synthesize monoPEGylated uricase conjugates. For enhancing the yield of monoPEGylated uricase conjugates, response surface methodology was employed to determine the yield of monoPEGylated conjugates using reverse phase high performance liquid chromatography. Using the optimized conditions, the developed method was validated for the production of monoPEGylated uricase conjugates which were further purified by size exclusion fast protein liquid chromatography (SE-FPLC). The molecular weights of the purified conjugates were determined by sodium dodecyl sulfide polyacrylamide gel electrophoresis (SDS-PAGE). Results: The optimum values of reaction conditions were determined as 1:12 concentration ratio of Uc to mPEG-mal, 2.76 kDa as mPEG-mal molecular weight and 3.55 mM EDTA concentration which resulted in a very high conjugate yield of 95.16 %. The conjugate synthesized using the optimized method retained a residual uricolytic activity of 84 % and a thiol group modification extent of 68.3 %. Conclusion: The PEGylation reaction was optimized using OVAT and statistical methods. Using the optimized conditions very high yield of conjugates were obtained and RP HPLC method was used to quantify the PEGylated uricase. 2016, Springer Science+Business Media New York.
URI: http://idr.nitk.ac.in/jspui/handle/123456789/12667
Appears in Collections:1. Journal Articles

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