Novo Nordisk A/S, Biopharmaceutical Research Unit, Novo Nordisk Park 1, DK-2760 Måløv, Denmark; Lund University, Department of Chemistry, Division of Biotechnology, Getingevägen 60, SE-221 00 Lund, Sweden.
Novo Nordisk A/S, Biopharmaceutical Research Unit, Novo Nordisk Park 1, DK-2760 Måløv, Denmark.
J Biotechnol. 2014 Mar 10;173:76-85. doi: 10.1016/j.jbiotec.2013.12.015. Epub 2014 Jan 18.
Site selective chemical modification is a preferred method, employed to prolong the circulation half-life of biopharmaceuticals. Cysteines have been used as attachment point for such modification, however, to be susceptible for chemical modification the involved thiol must be in its reduced form. Proteins often contain disulfides, which aid to maintain their tertiary structure and therefore must remain intact. Thus, methods for selectively reducing cysteine residues, introduced through site-directed mutagenesis, are of interest. In this study a macroporous, polymeric monolith was designed for selectively reducing a single cysteine residue inserted in recombinant human growth hormone (hGH). Advantages of such a material are the circumvention of the need to remove the reducing agent after reaction, as well as milder reduction conditions and a concomitant lower risk of reducing the native disulfides. The designed monolith showed very high capacity towards the selective reduction of an unpaired cysteine residue in a recombinant hGH variant. Factors influencing the selectivity and rate of reaction were investigated and it was found that monolith thiol loading, and buffer pH had an effect on the rate of reduction, whereas hGH variant concentration and buffer conductivity influenced both rate of reduction and selectivity. The developed system constitutes the basis for the development of a scalable platform for selective reduction of a capped cysteine residue in hGH.
位点选择性化学修饰是一种首选的方法，用于延长生物制药的循环半衰期。半胱氨酸已被用作此类修饰的附着点，但是，为了易受化学修饰的影响，所涉及的巯基必须处于还原形式。蛋白质通常含有二硫键，这些二硫键有助于维持其三级结构，因此必须保持完整。因此，人们对通过定点突变引入的选择性还原半胱氨酸残基的方法很感兴趣。在这项研究中，设计了一种大孔聚合物整体柱，用于选择性还原重组人生长激素（hGH）中插入的单个半胱氨酸残基。这种材料的优点是避免了在反应后需要去除还原剂，以及还原条件更温和，同时降低了还原天然二硫键的风险。设计的整体柱对重组 hGH 变体中单一对半胱氨酸残基的选择性还原表现出非常高的能力。研究了影响选择性和反应速率的因素，发现整体柱巯基载量和缓冲液 pH 值对半胱氨酸还原的速率有影响，而 hGH 变体浓度和缓冲液电导率对半胱氨酸还原的速率和选择性都有影响。所开发的系统为在 hGH 中选择性还原封闭半胱氨酸残基的可扩展平台的开发奠定了基础。
