Impact of Polymer Bioconjugation on Protein Stability and Activity Investigated with Discrete Conjugates: Alternatives to PEGylation
Impact of Polymer Bioconjugation on Protein Stability and Activity Investigated with Discrete Conjugates: Alternatives to PEGylation
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DOI：
10.1021/acs.biomac.8b01020
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发表时间：
2018-11-01
影响因子：
6.2
通讯作者：
Hubbuch, Juergen
中科院分区：
文献类型：
--
作者：
Morgenstern, Josefine;Alvaradejo, Gabriela Gil;Hubbuch, Juergen
合成聚合物与蛋白质的共价连接，称为蛋白质聚合物共轭，是目前改善这些生物分子物理化学性质的主要途径之一。最常用的聚合物是聚乙二醇(PEG)，广泛的研究和临床记录证明了它在生物制药中的应用。然而，一方面，过敏反应或超敏反应的发生，以及聚乙二醇单抗的发现，另一方面，可控聚合技术和新型单体的兴起，推动了对生物偶联替代聚合物的寻找。本文报道了溶菌酶与聚N-丙烯酰吗啉(PNAM)和聚低聚乙二醇甲基醚甲基丙烯酸酯(POEGMA)的偶联物的合成、纯化和性质。特别是，通过高通量筛选方法，对具有不同共轭程度的共轭物种的剩余活性、聚集行为和溶解性进行了研究。我们的研究展示了评估通过分离的具有离散的连接度的物种而不是在批次水平上通过非位点特异性修饰获得的连接物的重要性。相对低摩尔质量聚合物的单价偶联物的活性与天然蛋白相当，甚至更高，而所有偶联物的蛋白质溶解性都有所改善。为了达到与聚乙二醇组分相当的溶解度效果，需要较高摩尔质量的PNAM和POEGMA。
Covalent attachment of synthetic polymers to proteins, known as protein polymer conjugation, is currently one of the main approaches for improving the physicochemical properties of these biomolecules. The most commonly employed polymer is polyethylene glycol (PEG), as evidenced by extensive research and clinical track records for its use in biopharmaceuticals. However, the occurrence of allergic reactions or hypersensitivity and the discovery of PEG antibodies, on the one hand, and the rise of controlled polymerization techniques and novel monomers, on the other hand, have been driving the search for alternative polymers for bioconjugation. The present study describes the synthesis, purification, and properties of conjugates of lysozyme with poly(N-acryloylmorpholine) (PNAM) and poly(oligoethylene glycol methyl ether methacrylate) (POEGMA). Particularly, conjugate species with distinct conjugation degrees are investigated for their residual activity, aggregation behavior, and solubility, by using a high-throughput screening approach. Our study showcases the importance of evaluating conjugates obtained by nonsite-specific modification through isolated species with discrete degrees of conjugation rather than on the batch level. Monovalent conjugates with relatively low molar mass polymers displayed equal or even higher activity than the native protein, while all conjugates showed an improved protein solubility. To achieve a comparable effect on solubility as with PEG, PNAM and POEGMA of higher molar masses were required.