Department of Pharmaceutical and Pharmacological Sciences, Via Marzolo 5, 30131 Padova, Italy.
Department of Pharmaceutical and Pharmacological Sciences, Via Marzolo 5, 30131 Padova, Italy.
Eur J Pharm Biopharm. 2024 Aug;201:114387. doi: 10.1016/j.ejpb.2024.114387. Epub 2024 Jun 27.
Monoclonal antibodies (mAbs) are an essential class of therapeutic proteins for the treatment of cancer, autoimmune and rare diseases. During their production, storage, and administration processes, these proteins encounter various stressors such as temperature fluctuations, vibrations, and light exposure, able to induce chemico-physical modifications to their structure. Viral inactivation is a key step in downstream processes, and it is achieved by titration of the mAb at low pH, followed by neutralization. The changes of the pH pose a significant risk of unfolding and subsequent aggregation to proteins, thereby affecting their manufacturing. This study aims to investigate whether a combined exposure to light during the viral inactivation process can further affect the structural integrity of Ipilimumab, a mAb primarily used in the treatment of metastatic melanoma. The biophysical and biochemical characterization of Ipilimumab revealed that pH variation is a considerable risk for its stability with irreversible unfolding at pH 2. The threshold for Ipilimumab denaturation lies between pH 2 and 3 and is correlated with the loss of the protein structural cooperativity, which is the most critical factor determining the protein refolding. Light has demonstrated to exacerbate some local and global effects making pH-induced exposed regions more vulnerable to structural and chemical changes. Therefore, specific precautions to real-life exposure to ambient light during the sterilization process of mAbs should be considered to avoid loss of the therapeutic activity and to increase the yield of production. Our findings underscore the critical role of pH optimization in preserving the structural integrity and therapeutic efficacy of mAbs. Moreover, a detailed conformational study on the structural modifications of Ipilimumab may improve the chemico-physical knowledge of this effective drug and suggest new production strategies for more stable products under some kind of stress conditions.
单克隆抗体（mAbs）是治疗癌症、自身免疫性疾病和罕见病的重要一类治疗蛋白。在生产、储存和给药过程中，这些蛋白质会遇到各种应激源，如温度波动、振动和光照，这些应激源能够诱导其结构发生化学物理修饰。病毒灭活是下游工艺中的关键步骤，通过将 mAb 在低 pH 值下滴定，然后中和来实现。pH 值的变化会对蛋白质的变性和随后的聚集产生重大风险，从而影响其生产。本研究旨在探讨在病毒灭活过程中同时暴露于光线下是否会进一步影响伊匹单抗（Ipilimumab）的结构完整性，伊匹单抗主要用于治疗转移性黑色素瘤。对伊匹单抗的生物物理和生化特性进行了表征，结果表明 pH 值的变化对其稳定性有很大的风险，在 pH 值为 2 时会发生不可逆的变性。伊匹单抗变性的阈值在 pH 值 2 和 3 之间，与蛋白质结构协同性的丧失相关，这是决定蛋白质重折叠的最关键因素。光照已被证明会加剧一些局部和整体效应，使 pH 诱导暴露区域更容易发生结构和化学变化。因此，在 mAbs 的灭菌过程中，应考虑针对实际环境光照的特定预防措施，以避免治疗活性的丧失，并提高生产产量。我们的研究结果强调了 pH 值优化在保护 mAbs 结构完整性和治疗功效方面的关键作用。此外，对伊匹单抗结构修饰的详细构象研究可能会提高对这种有效药物的化学物理知识，并为在某些应激条件下生产更稳定的产品提出新的生产策略。
