Combined optimization of N‐terminal site‐specific PEGylation of recombinant hirudin using response surface methodology and kinetic analysis |
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| Authors: | Xudong Wang Shuchang Wang Duotao Pan Kairong Qin Hengli Yuan Fengying Zhang Yundong Sun Zhilong Xiu |
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| Affiliation: | 1. School of Life Science and Biotechnology, Dalian University of Technology, Dalian, P.R. China;2. Department of Biomedical Engineering, Dalian University of Technology, Dalian, P.R. China;3. State Key Laboratory Cultivating Base for Long‐acting Bio‐medical Research of Jiangsu Province, Jiangsu Hansoh Pharmaceutical Group Co. LTD., Lianyungang, P.R. China |
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| Abstract: | In this study, a combined optimization method was developed to optimize the N‐terminal site‐specific PEGylation of recombinant hirudin variant‐2 (HV2) with different molecular weight mPEG‐propionaldehyde (mPEG‐ALD), which is a multifactor‐influencing process. The HV2‐PEGylation with 5 kDa mPEG‐ALD was first chosen to screen significant factors and determine the locally optimized conditions for maximizing the yield of mono‐PEGylated product using combined statistical methods, including the Plackett–Burman design, steepest ascent path analysis, and central composition design for the response surface methodology (RSM). Under the locally optimized conditions, PEGylation kinetics of HV2 with 5, 10, and 20 kDa mPEG‐ALD were further investigated. The molar ratio of polyethylene glycol to HV2 and reaction time (the two most significant factors influencing the PEGylation efficiency) were globally optimized in a wide range using kinetic analysis. The data predicted by the combined optimization method using RSM and kinetic analysis were in good agreement with the corresponding experiment data. PEGylation site analysis revealed that almost 100% of the obtained mono‐PEGylated‐HV2 was modified at the N‐terminus of HV2. This study demonstrated that the developed method is a useful tool for the optimization of the N‐terminal site‐specific PEGylation process to obtain a homogeneous mono‐PEGylated protein with desirable yield. |
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| Keywords: | Kinetic analysis Optimization PEGylation Recombinant hirudin Response surface methodology |
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