Zhang Xin, Voronov Sergey, Mussa Nesredin, Li Zhengjian
Department of Biological Process Development, Bristol-Myers Squibb, USA.
Department of Biological Process Development, Bristol-Myers Squibb, USA.
Anal Biochem. 2017 Mar 15;521:1-7. doi: 10.1016/j.ab.2016.12.013. Epub 2017 Jan 6.
Imaged Capillary Isoelectric Focusing (icIEF) has been used as primary method for charge variants analysis of therapeutic antibodies and proteins [1], [9]. Proteins tend to precipitate around their pI values during focusing [14], which directly affects the reproducibility of their charge profiles. Protein concentration, focusing time and various supplementing additives are key parameters to minimize the protein precipitation and aggregation. Urea and sucrose are common additives to reduce protein aggregation, solubilize proteins in sample matrix and therefore improve assay repeatability [15]. However some proteins and antibodies are exceptions, we found urea and sucrose are not sufficient for a typical fusion protein (Fusion protein A) in icIEF assay and high variability is observed. We report a novel reagent, formamide, significantly improved reproducibility of protein charge profiles. Our results show formamide is a good supplementary reagent to reduce aggregation and stabilize proteins in isoelectric focusing. We further confirmed the method robustness, linearity, accuracy and precision after introducing the new reagent; extremely tight pI values, significantly improved method precision and sample on-board stability are achieved by formamide. Formamide is also proven to be equally functional to multiple antibodies as urea, which makes it an extra tool in icIEF method development.
成像毛细管等电聚焦（icIEF）已被用作治疗性抗体和蛋白质电荷变体分析的主要方法[1,9]。在聚焦过程中，蛋白质往往会在其等电点（pI）值附近沉淀[14]，这直接影响其电荷图谱的重现性。蛋白质浓度、聚焦时间和各种补充添加剂是使蛋白质沉淀和聚集最小化的关键参数。尿素和蔗糖是常见的添加剂，可减少蛋白质聚集，使蛋白质在样品基质中溶解，从而提高检测的重复性[15]。然而，一些蛋白质和抗体是例外情况，我们发现尿素和蔗糖对于icIEF检测中的一种典型融合蛋白（融合蛋白A）并不足够，并且观察到了高变异性。我们报道了一种新型试剂甲酰胺，它显著提高了蛋白质电荷图谱的重现性。我们的结果表明，甲酰胺是一种很好的补充试剂，可减少聚集并在等电聚焦中稳定蛋白质。在引入新试剂后，我们进一步证实了该方法的稳健性、线性、准确性和精密度；甲酰胺实现了极其紧密的pI值，显著提高了方法的精密度和样品在板上的稳定性。甲酰胺也被证明与尿素对多种抗体具有同等功能，这使其成为icIEF方法开发中的一个额外工具。
