Optimized purification of mono-PEGylated lysozyme by heparin affinity chromatography using response surface methodology
Optimized purification of mono-PEGylated lysozyme by heparin affinity chromatography using response surface methodology
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DOI：
10.1002/jctb.5269
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发表时间：
2017-10-01
影响因子：
3.4
通讯作者：
Asenjo, Juan A.
中科院分区：
文献类型：
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作者：
Alberto Mejia-Manzano, Luis;Elena Lienqueo, M.;Asenjo, Juan A.
背景技术从聚乙二醇化中高效、受控且稳健地纯化缀合物在生物制药市场中的需求不断增长。一般来说，通过传统的色谱模式达到的收率和纯度并不是特别高或有效。迄今为止，亲和色谱几乎没有被探索过。目前的工作首次介绍了通过肝素亲和层析 (HAC) 从聚乙二醇化反应中一步纯化单聚乙二醇化溶菌酶。采用响应面方法 (RSM)，特别是 Box-Behnken 设计 (BBD) 来优化分离。 结果 聚乙二醇化和天然溶菌酶在肝素琼脂糖 6 Fast Flow 树脂上的蛋白质吸附由 Langmuir 等温线描述，显示对聚乙二醇化蛋白质的亲和力相对较低。根据实验设计，对于三个响应变量（产量、纯度和生产率），氯化钠 (NaCl) 线性梯度的最佳洗脱条件为：13 倍柱体积 (CV) 的梯度长度、0.8 mL min(-1) 的流速和 1 mg mL(-1) 的蛋白质负载。基于此优化，设计了一步梯度程序，实现了单聚乙二醇化溶菌酶的纯化，收率和纯度约为 100%，而线性梯度的收率和纯度分别为 92.7% 和 99.7%。生产力为c。 0.048 0.001 mg mL(-1) min(-1)，使用0.05 mol L-1 NaCl进行洗脱。结论 使用HAC首次以高收率和纯度从聚乙二醇化混合物中完全分离出单聚乙二醇化溶菌酶。应用响应面法 (RSM)，找到了满足多个要求的充分条件以及 NaCl 线性梯度的最佳条件。基于这一优化，设计了一步梯度程序，一步实现了单聚乙二醇化溶菌酶的纯化，与疏水相互作用层析（HIC）和阳离子交换层析（CEX）等其他层析模式相比，在时间、分辨率、产量和纯度方面具有优势。 (c) 2017年化学工业学会
BACKGROUNDThe efficient, controlled and robust purification of conjugates from PEGylation has a growing demand in the biopharmaceutical's market. In general, the yield and purity reached through the conventional chromatographic modes are not particularly high or efficient. Affinity chromatography has so far scarcely been explored. The present work introduces the purification of mono-PEGylated lysozyme from a PEGylation reaction by heparin affinity chromatography (HAC) for the first time in a single step. Response surface methodology (RSM), particularly a Box-Behnken design (BBD) was employed to optimize the separation.RESULTSProtein adsorption of PEGylated and native lysozyme on Heparin Sepharose 6 Fast Flow resin was described by Langmuir isotherms, showing a relatively low affinity for the PEGylated proteins. From the experimental design, optimal elution conditions in a linear gradient of sodium chloride (NaCl) for the three response variables (yield, purity and productivity) were: gradient length of 13 column volumes (CVs), flow at 0.8 mL min(-1) and protein load of 1 mg mL(-1). Based on this optimization, a step gradient procedure was designed that achieved the purification of mono-PEGylated lysozyme with approximately 100% yield and purity in comparison with 92.7% and 99.7% with the linear gradient. Productivity was c. 0.048 0.001 mg mL(-1) min(-1) using 0.05 mol L-1 NaCl for its elution.CONCLUSIONS Mono-PEGylated lysozyme was completely separated from a PEGylation mixture with high yield and purity using HAC for first time. Applying response surface methodology (RSM), adequate conditions for more than one requirement were found as well as optimal conditions for a linear gradient of NaCl. Based on this optimization a step gradient procedure was designed that achieved the purification of mono-PEGylated lysozyme in one step with advantages respect to time, resolution, yield and purity compared with other chromatographic modes such as hydrophobic interaction chromatography (HIC) and cation exchange chromatography (CEX). (c) 2017 Society of Chemical Industry