Conjugation site heterogeneity causes variable electrostatic properties in Fc conjugates.
Conjugation site heterogeneity causes variable electrostatic properties in Fc conjugates.
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DOI：
10.1021/bc4000564
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发表时间：
2013-06-19
影响因子：
4.7
通讯作者：
Laurence, Jennifer S.
中科院分区：
文献类型：
--
作者：
Boylan, Nicholas J.;Zhou, Wen;Proos, Robert J.;Tolbert, Thomas J.;Wolfe, Janet L.;Laurence, Jennifer S.
免疫偶联物，包括抗体-药物偶联物和fc偶联物，代表了一种正在临床发展的快速增长的治疗方法。尽管它们越来越受欢迎，但免疫偶联物的高内在异质性往往使开发过程复杂化并限制了它们的广泛应用。特别是，免疫偶联电荷变体表现出明显不同的胶体稳定性、溶解度、药代动力学和组织分布。电荷变异体是由于降解而自发产生的，并且取决于药物类型、连接体和偶联位点，是通过药物偶联产生的。裸抗体的静电变化通常会导致性能差，因此由于降解引起的电荷变化对控制至关重要。电荷特性对于生产性能良好的adc同样重要。基于电荷的分析方法，如等电聚焦和离子交换色谱，能够探测免疫偶联药物产品中潜在的复杂性。尽管这些方法的效用，只有少数发表的报告基于电荷的检测应用于免疫偶联物。在本研究中，我们试图确定化学共轭对fc共轭物的静电性能的影响。为了尽量减少翻译后修饰（如脱酰胺）的影响，在将荧光探针Alexa Fluor 350偶联到赖氨酸残基的侧链之前，分离出单个Fc电荷变体。通过各种分析技术（包括等电聚焦和离子交换色谱）来评估所得的fc共轭物，以确定其电荷性质。
Immunoconjugates, including antibody-drug conjugates and Fc-conjugates, represent a rapidly growing class of therapeutics undergoing clinical development. Despite their growing popularity, the high intrinsic heterogeneity of immunoconjugates often complicates the development process and limits their widespread application. In particular, immunoconjugate charge variants exhibit markedly different colloidal stabilities, solubilities, pharmacokinetics and tissue distributions. Charge variants arise spontaneously due to degradation and, depending on the type of drug, linker and conjugation site, through drug conjugation. Electrostatic changes in naked antibodies often result in poor performance characteristics, and therefore charge alterations due to degradation are critical to control. Charge properties are expected to be equally important to producing well-behaved ADCs. Charge-based methods of analysis, such as isoelectric focusing and ion exchange chromatography, are capable of probing the underlying complexities within immunoconjugate drug products. Despite the utility of these methods, there are only a few published reports of charge-based assays applied to immunoconjugates. In the present study, we sought to identify the effects of chemical conjugation on the electrostatic properties of Fc-conjugates. In order to minimize the effects of post-translational modifications (e.g. deamidation), a single Fc charge variant was isolated prior to conjugation of a fluorescent probe, Alexa Fluor 350, to the side chains of lysine residues. The resulting Fc-conjugates were assessed by a variety of analytical techniques, including isoelectric focusing and ion exchange chromatography, to determine their charge properties.
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