Centre for Research and Knowledge Transfer in Biotechnology, University of Zagreb, Rockefellerova 10, 10000 Zagreb, Croatia.
Institute of Immunology, Inc., Rockefellerova 10, 10000 Zagreb, Croatia.
Molecules. 2022 Mar 3;27(5):1665. doi: 10.3390/molecules27051665.
Quality control of human immunoglobulin formulations produced by caprylic acid precipitation necessitates a simple, rapid, and accurate method for determination of residual caprylic acid. A high-performance liquid chromatography method for that purpose was developed and validated. The method involves depletion of immunoglobulins, the major interfering components that produce high background noise, by precipitation with acetonitrile (1:1, /). Chromatographic analysis of caprylic acid, preserved in supernatant with no loss, was performed using a reverse-phase C18 column (2.1 × 150 mm, 3 μm) as a stationary phase and water with 0.05% TFA-acetonitrile (50:50, /) as a mobile phase at a flow rate of 0.2 mL/min and run time of 10 min. The developed method was successfully validated according to the ICH guidelines. The validation parameters confirmed that method was linear, accurate, precise, specific, and able to provide excellent separation of peaks corresponding to caprylic acid and the fraction of remaining immunoglobulins. Furthermore, a 2 fractional factorial design was applied in order to test the robustness of developed method. As such, the method is highly suitable for the quantification of residual caprylic acid in formulations of human immunoglobulins for therapeutic use, as demonstrated on samples produced by fractionation of convalescent anti-SARS-CoV-2 human plasma at a laboratory scale. The obtained results confirmed that the method is convenient for routine quality control.
需要一种简单、快速和准确的方法来测定辛酸残留量，以控制用辛酸沉淀法生产的人免疫球蛋白制剂的质量。为此开发并验证了一种高效液相色谱法。该方法通过用乙腈（1:1，/）沉淀来去除免疫球蛋白等主要干扰成分，这些成分会产生很高的背景噪声。辛酸被保留在没有损失的上清液中，用反相 C18 柱（2.1×150mm，3μm）作为固定相，以含有 0.05%TFA-乙腈（50:50，/）的水作为流动相，在流速为 0.2mL/min 下进行色谱分析，运行时间为 10min。该方法根据 ICH 指南成功进行了验证。验证参数表明该方法具有线性、准确性、精密度、特异性，能够提供对辛酸和剩余免疫球蛋白部分的峰的出色分离。此外，还应用了 2 阶分数因子设计来测试所开发方法的稳健性。因此，该方法非常适合用于治疗用人免疫球蛋白制剂中残留辛酸的定量，这在实验室规模的从恢复期抗 SARS-CoV-2 人血浆中进行的分级分离中得到了验证。所得结果证实该方法方便用于常规质量控制。
