Department of Analytical & Formulation Sciences, Amgen Inc., 1201 Amgen Court West, Seattle, WA 98119-3105, USA.
J Chromatogr B Analyt Technol Biomed Life Sci. 2010 Apr 1;878(11-12):868-76. doi: 10.1016/j.jchromb.2010.02.003. Epub 2010 Feb 11.
A new cation-exchange high-performance liquid chromatography (HPLC) method that separates fragment antigen-binding (Fab) and fragment crystallizable (Fc) domains generated by the limited proteolysis of monoclonal antibodies (mAbs) was developed. This assay has proven to be suitable for studying complex degradation processes involving various immunoglobulin G1 (IgG1) molecules. Assignment of covalent degradations to specific regions of mAbs was facilitated by using Lys-C and papain to generate Fab and Fc fragments with unique, protease-dependent elution times. In particular, this method was useful for characterizing protein variants formed in the presence of salt under accelerated storage conditions. Two isoforms that accumulated during storage were readily identified as Fab-related species prior to mass-spectrometric analysis. Both showed reduced biological activity likely resulting from modifications within or in proximity of the complementarity-determining regions (CDRs). Utility of this assay was further illustrated in the work to characterize light-induced degradations in mAb formulations. In this case, a previously unknown Fab-related species which populated upon light exposure was observed. This species was well resolved from unmodified Fab, allowing for direct and high-purity fractionation. Mass-spectrometric analysis subsequently identified a histidine-related degradation product associated with the CDR2 of the heavy chain. In addition, the method was applied to assess the structural organization of a noncovalent IgG1 dimer. A new species corresponding to a Fab-Fab complex was found, implying that interactions between Fab domains were responsible for dimerization. Overall, the data presented demonstrate the suitability of this cation-exchange HPLC method for studying a wide range of covalent and noncovalent degradations in IgG1 mAbs.
开发了一种新的阳离子交换高效液相色谱（HPLC）方法，用于分离单克隆抗体（mAb）有限蛋白水解产生的片段抗原结合（Fab）和片段可结晶（Fc）结构域。该方法已被证明适用于研究涉及各种免疫球蛋白 G1（IgG1）分子的复杂降解过程。通过使用赖氨酸 C 和木瓜蛋白酶生成具有独特的、依赖于蛋白酶的洗脱时间的 Fab 和 Fc 片段，有助于将共价降解分配到 mAb 的特定区域。特别是，该方法对于在加速储存条件下存在盐的情况下形成的蛋白质变体的特征描述非常有用。在进行质谱分析之前，很容易将储存过程中积累的两种同工型鉴定为 Fab 相关物质。这两种同工型的生物活性都降低了，可能是由于 CDRs 内或附近的修饰所致。该方法在 mAb 制剂中光诱导降解的特征描述工作中得到了进一步的应用。在这种情况下，观察到在暴露于光后出现的一种以前未知的 Fab 相关物质。该物质与未修饰的 Fab 很好地分离，允许直接进行高纯度的分级分离。随后的质谱分析鉴定出与重链 CDR2 相关的组氨酸降解产物。此外，该方法还用于评估非共价 IgG1 二聚体的结构组织。发现了一种对应于 Fab-Fab 复合物的新物质，表明 Fab 结构域之间的相互作用是导致二聚化的原因。总体而言，所提供的数据表明，该阳离子交换 HPLC 方法适用于研究 IgG1 mAb 中的广泛的共价和非共价降解。
