Quantification of the percentage of lymphocyte-binding components in polyclonal anti-thymocyte/lymphocyte globulin products

IF 3.1 3区医学 Q2 CHEMISTRY, ANALYTICAL

Journal of pharmaceutical and biomedical analysis Pub Date : 2025-09-26 DOI:10.1016/j.jpba.2025.117165

Wenqu Yin , Haoyong Zou , Yujuan Xu , Qinyu Yu , Guizhi Song , Xiaolu Zhou , Zeqiong Yu , Kuanhong Xu , Zhi Zhang , Zhi Wang , Xilin Nie , Peng Geng

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Abstract

The immunomodulatory and immunosuppressive properties of anti-thymocyte globulin (ATG)/anti-lymphocyte globulin (ALG) are primarily facilitated through the interaction between the lymphocyte-binding components of ATG/ALG (active ATG) and the antigens expressed on various immune cell populations. Nevertheless, the precise concentration and proportion of active ATG present in ATG/ALG preparations have yet to be definitively ascertained. In this study, we employed a methodology integrating flow cytometry and ELISA to quantify the percentage of active ATG in ATG/ALG preparations. The method was replicated six times to assess active ATG in ATG-Fresenius utilizing Jurkat cells, resulting in a coefficient of variation (CV) of 11.05 %. Analysis of 4 porcine immune plasma batches showed significant inter-batch variability in active ATG concentrations and total IgG concentrations. Analysis of 12 batches of porcine anti-thymocyte globulin (pATG) stock solutions revealed that the mean percentages and CV value for active ATG were 45.08 % and 23.58 %, respectively. Moreover, the biological activity assay demonstrated a strong correlation between the biological activity of pATG and the percentage of active ATG in the pATG preparation (r = 0.9283). In conclusions, we have developed an absolute quantification method for active ATG, which will facilitate precise pharmacokinetic analysis of active ATG and contribute to improving the potency testing and dosing regimens of ATG.

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多克隆抗胸腺细胞/淋巴细胞球蛋白产品中淋巴细胞结合成分百分比的定量分析

抗胸腺细胞球蛋白（anti-thymocyte globulin， ATG）/抗淋巴细胞球蛋白（anti-lymphocyte globulin， ALG）的免疫调节和免疫抑制特性主要是通过ATG/ALG的淋巴细胞结合成分（活性ATG）与各种免疫细胞群上表达的抗原相互作用来实现的。然而，ATG/ALG制剂中活性ATG的精确浓度和比例尚未明确确定。在这项研究中，我们采用了流式细胞术和ELISA相结合的方法来量化ATG/ALG制剂中活性ATG的百分比。利用Jurkat细胞对ATG- fresenius的活性ATG进行了6次重复评价，变异系数（CV）为11.05 %。对4批猪免疫血浆的分析显示，批次间活性ATG浓度和总IgG浓度存在显著差异。对12批猪抗胸腺细胞球蛋白（pATG）原液的分析表明，活性ATG的平均百分比和CV值分别为45.08% %和23.58 %。此外，生物活性测定表明，pATG的生物活性与pATG制剂中活性ATG的百分比有很强的相关性（r = 0.9283）。综上所述，我们建立了一种活性ATG的绝对定量方法，将有助于对活性ATG进行精确的药动学分析，并有助于改进ATG的效价检测和给药方案。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Journal of pharmaceutical and biomedical analysis 医学-分析化学

CiteScore

6.70

自引率

5.90%

发文量

588

审稿时长

37 days

期刊介绍： This journal is an international medium directed towards the needs of academic, clinical, government and industrial analysis by publishing original research reports and critical reviews on pharmaceutical and biomedical analysis. It covers the interdisciplinary aspects of analysis in the pharmaceutical, biomedical and clinical sciences, including developments in analytical methodology, instrumentation, computation and interpretation. Submissions on novel applications focusing on drug purity and stability studies, pharmacokinetics, therapeutic monitoring, metabolic profiling; drug-related aspects of analytical biochemistry and forensic toxicology; quality assurance in the pharmaceutical industry are also welcome. Studies from areas of well established and poorly selective methods, such as UV-VIS spectrophotometry (including derivative and multi-wavelength measurements), basic electroanalytical (potentiometric, polarographic and voltammetric) methods, fluorimetry, flow-injection analysis, etc. are accepted for publication in exceptional cases only, if a unique and substantial advantage over presently known systems is demonstrated. The same applies to the assay of simple drug formulations by any kind of methods and the determination of drugs in biological samples based merely on spiked samples. Drug purity/stability studies should contain information on the structure elucidation of the impurities/degradants.