Product Development, Bristol-Myers Squibb, 1 Squibb Dr., New Brunswick, NJ, USA.
Product Development, Bristol-Myers Squibb, 1 Squibb Dr., New Brunswick, NJ, USA.
J Pharm Biomed Anal. 2020 May 10;183:113178. doi: 10.1016/j.jpba.2020.113178. Epub 2020 Feb 14.
Monoclonal antibodies are heterogeneous in nature and may contain numerous variants with differences in size, charge, and hydrophobicity, which may impact clinical efficacy, immunogenicity, and safety. Characterization of antibody variants is necessary to build structure-function correlation and establish a proper control strategy. Isolation and enrichment of variants by conventional chromatographic peak fractionation is labor-intensive and time-consuming. The instability of fractions during isolation and subsequent characterization may also be a concern. Hence, it is desirable to analyze antibody variants in an online and real-time manner. Here we demonstrate a 2D-LC methodology - multiple heart-cutting IEC-SEC- as an investigational tool to facilitate a charge variant characterization study. Both IEC modes - anion exchange (AEX) and cation exchange (CEX) chromatography are discussed. Using this approach, direct bridging of size and charge variants of an antibody molecule was achieved without offline peak fractionation. It was observed that antibody aggregates elute late on both the AEX and CEX columns, presumably due to secondary hydrophobic interactions. Additionally, we overcame the solvent mismatch issue and developed a 2D SEC-IEC method to confirm the bridging results. This is the first reported SEC-IEC 2D-LC application for the characterization of antibody size and charge variants.
单克隆抗体在性质上具有异质性，可能包含许多在大小、电荷和疏水性方面存在差异的变体，这些差异可能会影响临床疗效、免疫原性和安全性。为了建立结构-功能相关性并制定适当的对照策略，有必要对抗体变体进行特征描述。通过传统的色谱峰分级分离来分离和富集变体既费力又费时。在分离过程中以及随后的特性描述期间，馏分的不稳定性也可能令人担忧。因此，希望能够在线和实时地分析抗体变体。在这里，我们展示了一种 2D-LC 方法——多次心脏切割 IEC-SEC-，作为一种研究工具，以促进电荷变体特征研究。讨论了两种 IEC 模式——阴离子交换（AEX）和阳离子交换（CEX）色谱。使用这种方法，无需离线峰分级即可直接桥接抗体分子的大小和电荷变体。观察到抗体聚集物在 AEX 和 CEX 柱上都很晚才洗脱，推测是由于二级疏水性相互作用所致。此外，我们克服了溶剂不匹配问题，并开发了一种 2D SEC-IEC 方法来确认桥接结果。这是首次报道 SEC-IEC 2D-LC 用于抗体大小和电荷变体特征描述的应用。
