Liu Jian-Zhong, Li Lei, Fang Wei-Jie
Institute of Drug Metabolism and Pharmaceutical Analysis, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, 310058, China.
Taizhou Institute of Zhejiang University, Taizhou, 317000, China.
Pharm Res. 2024 Sep;41(9):1893-1901. doi: 10.1007/s11095-024-03763-6. Epub 2024 Sep 4.
Size exclusion chromatography (SEC) is widely used to characterize molecular size variants of antibody drugs. However, SEC analysis is hindered by secondary interactions (or nonspecific interactions) between proteins and stationary phase packing, which result in poor column efficiency. Previous studies have reported that chaotropic salt can inhibit these interactions, but the corresponding applications of this aspect are relatively rare. Therefore, this study introduces a novel approach using sodium iodide (NaI) as a mobile-phase component in SEC and investigates the influence of the mobile-phase composition on secondary interactions.
SEC analysis was performed on one antibody-drug conjugate and four monoclonal antibodies (mAbs) using three different mobile-phase systems (i.e., sodium chloride/L-arginine hydrochloride/NaI mobile phases system) to compare the column efficiency. Subsequently, mAb-1 was used as a model to investigate the effects of these factors on secondary interactions by adjusting the ionic strength (salt concentration) and pH of the NaI mobile-phase system.
NaI exhibits superior column efficiency performance in the SEC analysis of most products. The ionic strength will affect nonideal electrostatic and hydrophobic interaction. An appropriate ionic strength can inhibit electrostatic interactions, while an excessive ionic strength increases hydrophobic interactions. pH primarily influences electrostatic interactions. Determining the appropriate pH necessitates consideration of the isoelectric point of the protein and the pH tolerance of the column.
In SEC analysis, using NaI as the salt component in the mobile phase reduces secondary interactions and improves column efficiency. This approach is advantageous for samples with intense secondary interactions and is a suitable alternative.
尺寸排阻色谱法（SEC）被广泛用于表征抗体药物的分子大小变体。然而，SEC分析受到蛋白质与固定相填料之间的二级相互作用（或非特异性相互作用）的阻碍，这导致柱效不佳。先前的研究报道，离液盐可以抑制这些相互作用，但这方面的相应应用相对较少。因此，本研究引入了一种在SEC中使用碘化钠（NaI）作为流动相组分的新方法，并研究了流动相组成对二级相互作用的影响。
使用三种不同的流动相系统（即氯化钠/盐酸L-精氨酸/NaI流动相系统）对一种抗体药物偶联物和四种单克隆抗体（mAb）进行SEC分析，以比较柱效。随后，以mAb-1为模型，通过调节NaI流动相系统的离子强度（盐浓度）和pH值，研究这些因素对二级相互作用的影响。
在大多数产品的SEC分析中，NaI表现出优异的柱效性能。离子强度会影响非理想的静电和疏水相互作用。适当的离子强度可以抑制静电相互作用，而过高的离子强度会增加疏水相互作用。pH主要影响静电相互作用。确定合适的pH值需要考虑蛋白质的等电点和色谱柱的pH耐受性。
在SEC分析中，使用NaI作为流动相中的盐组分可减少二级相互作用并提高柱效。这种方法对二级相互作用强烈的样品具有优势，是一种合适的替代方法。
