National Centre for Preclinical and Clinical Evaluation and Research of Drugs (FARVA), Istituto Superiore di Sanità, Viale, Regina Elena 299, 00161, Rome, Italy.
National Centre for the Control and Evaluation of Medicines (CNCF), Istituto Superiore di Sanità, Viale, Regina Elena 299, 00161, Rome, Italy.
Sci Rep. 2024 Nov 15;14(1):28087. doi: 10.1038/s41598-024-79108-5.
The success of therapeutic monoclonal antibodies (mAbs) and their biosimilars, highlights the challenge to control their purity, identity, and stability. For this purpose, among several orthogonal methodologies, imaged capillary Iso Electric Focusing (icIEF) is one of the leading techniques. Despite the isoelectric point (pI) being a univocal parameter relying on the protein's primary sequence and its post-translational modifications (PTMs), the current Charge Variants Profile Assessment (CVPA) carried out by cIEF relies on relative comparisons with corresponding reference standards, this is because the inconsistent outputs for the same sample across different instruments preclude the uniqueness of the measured parameters. We demonstrate that refining the current calibration approach in the iciEF method allows for obtaining more reliable and objective pIs, and a deeper understanding of the pH gradients along the capillaries. We are confident our advancements will enhance CVPA, by exploring the concept of univocal charge identity. This is crucial for constructing biobanks and developing algorithms to quickly identify divergences from the originators, thus ensuring drug quality, efficacy, and safety. Moreover, our method allows an experimental design optimized to minimize bias ("Unbiased" Experimental Design-UED) to study resolution as a multivariate function of different input variables. This endeavor aims to develop optimal methods tailored to specific pH ranges.
治疗性单克隆抗体（mAbs）及其生物类似药的成功，凸显了控制其纯度、身份和稳定性的挑战。为此，在几种正交方法中，成像毛细管等电聚焦（icIEF）是领先技术之一。尽管等电点（pI）是一个依赖于蛋白质一级序列及其翻译后修饰（PTMs）的唯一参数，但目前通过 cIEF 进行的电荷变异体分析（CVPA）依赖于与相应参比标准的相对比较，这是因为同一样品在不同仪器上的不一致输出排除了测量参数的独特性。我们证明，在 iciEF 方法中改进当前的校准方法可以获得更可靠和客观的 pI，并更深入地了解沿毛细管的 pH 梯度。我们有信心，通过探索单一电荷身份的概念，我们的进展将增强 CVPA。这对于构建生物库和开发算法以快速识别与原创者的差异至关重要，从而确保药物的质量、疗效和安全性。此外，我们的方法允许设计优化的实验设计，以最小化偏差（“无偏”实验设计-UED），从而研究不同输入变量的分辨率作为多元函数。这项工作旨在开发针对特定 pH 范围的最佳方法。
