Ziv-aflibercept (ziv-AFL) is a complex fusion protein which is widely used in hospitals for the treatment of colorectal metastatic cancer. Charge variants are critical attributes for assessing post-transitional modifications (PMTs) that have to be controlled during the development and manufacture of these proteins and until their administration to patients. Cation exchange (CEX) chromatography is a charge-sensitive analytical method that is well suited for analysing charge variants in proteins. The aim of this paper is to analyse the charge variants of ziv-AFL in the medicine (Zaltrap®) when fresh and when degraded. Two CEX chromatographic methods were compared for this purpose. The former was an adaptation of the method used in the first published study in which charge variants were analysed via pH gradient elution using volatile, low ionic strength buffers with direct coupling to high-resolution Orbitrap mass spectrometry. The second method was developed and optimized during our research using the salt-mediated pH gradient mode and classical non-volatile, high ionic strength buffers which were incompatible with direct coupling with mass detection. Fresh and controlled degraded samples of ziv-AFL were used to evaluate the capacity of both CEX chromatographic strategies for detecting charge variants in ziv-AFL. In the controlled degradation study the samples of the medicine were subjected to three stress factors: temperature of 60 °C for three hours, freeze/thaw process -two cycles-, and exposure to light for twelve hours. The CEX chromatographic method with non-volatile salts in the mobile phase enabled better detection of charge variants degraded ziv-AFL samples than the method using volatile salts with lower ionic strength. In addition, the complexity of the mass spectra data generated made it impossible to identify the multicharge variant species of ziv-AFL. Although charge variants were not separated in ziv-AFL fresh sample, our results indicate that the method with non-volatile salts in the mobile phase could be used to characterize and track changes in the charge variant UV chromatographic profile of ziv-AFL in fresh and degraded samples, even though it cannot be coupled to a mass detector and there is therefore no information about mass. The increase of basic protein degraded compounds were the most important degradation pattern detected in ziv-AFL (Zaltrap®).

中文翻译：

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通过阳离子交换液相色谱法对齐夫-阿夫西普的完整电荷变化分析，作为概念证明：流动相中挥发性和非挥发性盐之间的比较。

Ziv-aflibercept（ziv-AFL）是一种复杂的融合蛋白，在医院中广泛用于治疗结直肠转移癌。电荷变异体是评估过渡蛋白修饰（PMT）的关键属性，在这些蛋白的开发和生产过程中以及直至向患者给药之前，必须对其进行控制。阳离子交换（CEX）色谱法是一种电荷敏感的分析方法，非常适合分析蛋白质中的电荷变体。本文的目的是分析新鲜时和降解时药物（Zaltrap®）中ziv-AFL的电荷变化。为此，比较了两种CEX色谱方法。前者是对第一个发表的研究中使用的方法的改编，在该研究中，使用挥发性有机物通过pH梯度洗脱分析了电荷变体，低离子强度缓冲液，可直接与高分辨率Orbitrap质谱联用。第二种方法是在我们的研究过程中使用盐介导的pH梯度模式和经典的非挥发性，高离子强度缓冲液开发和优化的，这些缓冲液与直接结合质量检测不兼容。新鲜和受控降解的ziv-AFL样品用于评估两种CEX色谱策略检测ziv-AFL中电荷变异的能力。在受控降解研究中，药物样品受到三个应力因素的影响：温度为60°C，时间为三个小时，冷冻/融化过程为两个周期，暴露于光线下为十二小时。与使用离子强度较低的挥发性盐的方法相比，在流动相中使用非挥发性盐的CEX色谱方法能够更好地检测降解的ziv-AFL样品的电荷变体。此外，所生成质谱数据的复杂性使得无法识别ziv-AFL的多电荷变体种类。尽管未在ziv-AFL的新鲜样品中分离出电荷变体，但我们的结果表明，在流动相中使用非挥发性盐的方法可用于表征和跟踪ziv-AFL在新鲜和未加工样品中的电荷变体UV色谱图的变化。降级的样本，即使它不能耦合到质量检测器，因此也没有有关质量的信息。