Basu Amartya, Yang Karen, Wang Maoliang, Liu Sam, Chintala Ramesh, Palm Thomas, Zhao Hong, Peng Ping, Wu Dechun, Zhang Zhenfan, Hua Jack, Hsieh Ming-Ching, Zhou John, Petti Gerald, Li Xiguang, Janjua Ahsen, Mendez Magda, Liu Jun, Longley Clifford, Zhang Zhihua, Mehlig Mary, Borowski Virna, Viswanathan Manickam, Filpula David
Enzon Pharmaceuticals, 20 Kingsbridge Road, Piscataway, New Jersey 08854-3969, USA.
Bioconjug Chem. 2006 May-Jun;17(3):618-30. doi: 10.1021/bc050322y.
Recombinant interferon-beta-1b (IFN-beta-1b) is used clinically in the treatment of multiple sclerosis. In common with many biological ligands, IFN-beta-1b exhibits a relatively short serum half-life, and bioavailability may be further diminished by neutralizing antibodies. While PEGylation is an approach commonly employed to increase the blood residency time of protein therapeutics, there is a further requisite for molecular engineering approaches to also address the stability, solubility, aggregation, immunogenicity and in vivo exposure of therapeutic proteins. We investigated these five parameters of recombinant human IFN-beta-1b in over 20 site-selective mono-PEGylated or multi-PEGylated IFN-beta-1b bioconjugates. Primary amines were modified by single or multiple attachments of poly(ethylene glycol), either site-specifically at the N-terminus, or randomly on the 11 lysines. In two alternate approaches, site-directed mutagenesis was independently employed in the construction of designed IFN-beta-1b variants containing either a single free cysteine or lysine for site-specific PEGylation. Optimization of conjugate preparation with 12 kDa, 20 kDa, 30 kDa, and 40 kDa amine-selective PEG polymers was achieved, and a comparison of the structural and functional properties of the IFN-beta-1b proteins and their PEGylated counterparts was conducted. Peptide mapping and MALDI-TOF mass spectrometric analysis confirmed the attachment sites of the PEG polymer. Independent biochemical and bioactivity analyses, including antiviral and antiproliferation bioassays, circular dichroism, capillary electrophoresis, flow cytometric profiling, reversed phase and size exclusion HPLC, and immunoassays demonstrated that the functional activities of the designed IFN-beta-1b conjugates were maintained, while the formation of soluble or insoluble aggregates of IFN-beta-1b was ameliorated. Immunogenicity and pharmacokinetic studies of selected PEGylated IFN-beta-1b compounds in mice and rats demonstrated both diminished IgG responses, and over 100-fold expanded AUC exposure relative to the unmodified protein. The results demonstrate the capacity of this macromolecular engineering strategy to address both pharmacological and formulation challenges for a highly hydrophobic, aggregation-prone protein. The properties of a lead mono-PEGylated candidate, 40 kDa PEG2-IFN-beta-1b, were further investigated in formulation optimization and biological studies.
重组干扰素β-1b（IFN-β-1b）在临床上用于治疗多发性硬化症。与许多生物配体一样，IFN-β-1b的血清半衰期相对较短，并且中和抗体可能会进一步降低其生物利用度。虽然聚乙二醇化是一种常用的增加蛋白质治疗剂血液停留时间的方法，但分子工程方法还有进一步的必要，以解决治疗性蛋白质的稳定性、溶解性、聚集性、免疫原性和体内暴露问题。我们研究了20多种位点选择性单聚乙二醇化或多聚乙二醇化IFN-β-1b生物共轭物中重组人IFN-β-1b的这五个参数。通过在N端位点特异性或在11个赖氨酸上随机连接聚乙二醇单链或多链来修饰伯胺。在两种替代方法中，分别采用定点诱变构建含有单个游离半胱氨酸或赖氨酸用于位点特异性聚乙二醇化的设计IFN-β-1b变体。实现了用12 kDa、20 kDa、30 kDa和40 kDa胺选择性聚乙二醇聚合物优化共轭物制备，并对IFN-β-1b蛋白及其聚乙二醇化对应物的结构和功能特性进行了比较。肽图分析和基质辅助激光解吸电离飞行时间质谱分析证实了聚乙二醇聚合物的连接位点。独立的生化和生物活性分析，包括抗病毒和抗增殖生物测定、圆二色性、毛细管电泳、流式细胞术分析、反相和尺寸排阻高效液相色谱以及免疫测定表明，设计的IFN-β-1b共轭物的功能活性得以维持，同时IFN-β-1b可溶性或不溶性聚集体的形成得到改善。对选定的聚乙二醇化IFN-β-1b化合物在小鼠和大鼠中的免疫原性和药代动力学研究表明，IgG反应降低，相对于未修饰的蛋白，AUC暴露增加了100多倍。结果表明，这种大分子工程策略有能力应对高度疏水、易于聚集的蛋白质在药理学和制剂方面的挑战。对一种主要的单聚乙二醇化候选物40 kDa PEG2-IFN-β-1b的特性在制剂优化和生物学研究中进一步进行了研究。
