Institute of Engineering in Life Sciences, Section IV: Biomolecular Separation Science, Karlsruhe Institute of Technology (KIT), 76131, Karlsruhe, Germany.
Biotechnol Bioeng. 2014 Jan;111(1):104-14. doi: 10.1002/bit.25000. Epub 2013 Aug 12.
Since the first FDA approval of a PEGylated product in 1990, so called random PEGylation reactions are still used to increase the efficacy of biopharmaceuticals and represent the major technology of all approved PEG-modified drugs. However, the great influence of process parameters on PEGylation degree and the PEG-binding site results in a lack of reaction specificity which can have severe impact on the product profile. Consequently, reproducible and well characterized processes are essential to meet increasing regulative requirements resulting from the quality-by-design (QbD) initiative, especially for this kind of modification type. In this study we present a general approach which combines the simple chemistry of random PEGylation reactions with high throughput experimentation (HTE) to achieve a well-defined process. Robotic based batch experiments have been established in a 96-well plate format and were analyzed to investigate the influence of different PEGylation conditions for lysozyme as model protein. With common SEC analytics highly reproducible reaction kinetics were measured and a significant influence of PEG-excess, buffer pH, and reaction time could be investigated. Additional mono-PEG-lysozyme analytics showed the impact of varying buffer pH on the isoform distribution, which allowed us to identify optimal process parameters to get a maximum concentration of each isoform. Employing Micrococcus lysodeikticus based activity assays, PEG-lysozyme33 was identified to be the isoform with the highest residual activity, followed by PEG-lysozyme1 . Based on these results, a control space for a PEGylation reaction was defined with respect to an optimal overall volumetric activity of mono-PEG-lysozyme isoform mixtures.
自 1990 年首个聚乙二醇（PEG）产品获得美国食品药品监督管理局（FDA）批准以来，所谓的随机 PEG 化反应仍被用于提高生物制药的疗效，并且是所有已批准的 PEG 修饰药物的主要技术。然而，工艺参数对 PEG 化程度和 PEG 结合位点的巨大影响导致反应特异性缺乏，这可能对产品特性产生严重影响。因此，为了满足质量源于设计（QbD）倡议带来的日益严格的监管要求，特别是对于这种修饰类型，必须采用可重复且经过良好表征的工艺。在这项研究中，我们提出了一种通用方法，将随机 PEG 化反应的简单化学与高通量实验（HTE）相结合，以实现明确的工艺过程。我们在 96 孔板格式中建立了基于机器人的分批实验，并对其进行了分析，以研究不同的溶菌酶（作为模型蛋白）PEG 化条件对其的影响。采用常见的 SEC 分析，可高度重现地测量反应动力学，并可研究 PEG 过量、缓冲液 pH 值和反应时间等不同条件的影响。对不同缓冲液 pH 值对同工型分布的影响的进一步单 PEG-溶菌酶分析，使我们能够确定最佳的工艺参数，以获得每种同工型的最大浓度。利用基于微球菌溶壁酶的活性测定法，发现 PEG-溶菌酶 33 是具有最高残留活性的同工型，其次是 PEG-溶菌酶 1。基于这些结果，定义了 PEG 化反应的控制空间，以获得单 PEG-溶菌酶同工型混合物的最佳总体体积活性。
