Department of Chemistry, Biosystems Research Complex, Clemson University, Clemson, SC 29634-0973, USA.
Department of Bioengineering, Biosystems Research Complex, Clemson University, Clemson, SC 29634-0973, USA.
J Chromatogr A. 2024 Mar 15;1718:464722. doi: 10.1016/j.chroma.2024.464722. Epub 2024 Feb 10.
Immunoglobulin G (IgG) is the most common monoclonal antibody (mAb) grown for therapeutic applications. While IgG is often selectively isolated from cell lines using protein A (ProA) chromatography, this is only a stepping stone for complete characterization. Further classification can be obtained from weak cation exchange chromatography (WCX) to determine IgG charge variant distributions. The charge variants of monoclonal antibodies can influence the stability and efficacy in vivo, and deviations in charge heterogeneity are often cell-specific and sensitive to upstream process variability. Current methods to characterize IgG charge variants are often performed off-line, meaning that the IgG eluate from the ProA separation is collected, diluted to adjust the pH, and then transferred to the WCX separation, adding time, complexity, and potential contamination to the sample analysis process. More recently, reports have appeared to streamline this separation using in-line two-dimensional liquid chromatography (2D-LC). Presented here is a novel, 2D-LC coupling of ProA in the first dimension (D) and WCX in the second dimension (D) chromatography. As anticipated, the initial direct column coupling proved to be challenging due to the pH incompatibility between the mobile phases for the two stages. To solve the solvent compatibility issue, a size exclusion column was placed in the switching valve loop of the 2D-LC instrument to act as a means for the on-line solvent exchange. The efficacy of the methodology presented was confirmed through a charge variant determination using the NIST monoclonal antibody standard (NIST mAb), yielding correct acidic, main, and basic variant compositions. The methodology was employed to determine the charge variant profile of IgG from an in-house cultured Chinese hamster ovary (CHO) cell supernatant. It is believed that this methodology can be easily implemented to provide higher-throughput assessment of IgG charge variants for process monitoring and cell line development.
免疫球蛋白 G（IgG）是最常用于治疗应用的常见单克隆抗体（mAb）。虽然 IgG 通常使用蛋白 A（ProA）层析法从细胞系中选择性分离，但这只是完全表征的一个起点。进一步的分类可以通过弱阳离子交换层析（WCX）来确定 IgG 电荷变异体分布。单克隆抗体的电荷变异体可以影响体内的稳定性和功效，并且电荷异质性的偏差通常是细胞特异性的，并且对上游工艺变异性敏感。目前用于表征 IgG 电荷变异体的方法通常是离线进行的，这意味着从 ProA 分离物中收集 IgG 洗脱液，稀释以调节 pH 值，然后转移到 WCX 分离物中，这会增加样品分析过程的时间、复杂性和潜在污染。最近，有报道称使用在线二维液相色谱（2D-LC）可以简化这种分离。本文介绍了一种新颖的 ProA 在第一维（D）和 WCX 在第二维（D）层析中的 2D-LC 偶联。正如预期的那样，由于两个阶段的流动相的 pH 值不兼容，最初的直接柱偶联证明是具有挑战性的。为了解决溶剂兼容性问题，在 2D-LC 仪器的切换阀环中放置了一个尺寸排阻柱，作为在线溶剂交换的手段。通过使用 NIST 单克隆抗体标准品（NIST mAb）进行电荷变异体测定，证明了所提出方法的有效性，得到了正确的酸性、主要和碱性变异体组成。该方法用于确定来自内部培养的中国仓鼠卵巢（CHO）细胞上清液的 IgG 的电荷变异体分布。相信这种方法可以很容易地实施，以提供更高通量的 IgG 电荷变异体评估，用于工艺监测和细胞系开发。
