Powers Thomas W, Mullins Elise K, Zhang Kun, Binder Joseph J, Friese Olga, Runnels Herbert A, Thompson Lawrence C
Analytical Research and Development, Biotherapeutic Pharmaceutical Sciences, Pfizer Inc., Chesterfield, MO, United States.
Cancer Vaccines and Immunotherapeutics, Pfizer Inc., San Diego, CA, United States.
Front Bioeng Biotechnol. 2022 Apr 5;10:753481. doi: 10.3389/fbioe.2022.753481. eCollection 2022.
Physicochemical tests represent important tools for the analytical control strategy of biotherapeutics. For adenoviral modalities, anion-exchange high performance liquid chromatography (AEX-HPLC) represents an important methodology, as it is able to simultaneously provide information on viral particle concentration, product purity and surface charge in a high-throughput manner. During product development of an adenoviral-based therapeutic, an accelerated stability study was performed and showed changes in each of the AEX-HPLC reportable attributes. These changes also correlated with a decrease in product infectivity prompting a detailed characterization of the impurity and mechanism of the surface charge change. Characterization experiments identified the impurity to be free hexon trimer, suggesting that capsid degradation could be contributing to both the impurity and reduced particle concentration. Additional mass spectrometry characterization identified deamidation of specific hexon residues to be associated with the external surface charge modification observed upon thermal stress conditions. To demonstrate a causal relationship between deamidation and surface charge changes observed by AEX-HPLC, site-directed mutagenesis experiments were performed. Through this effort, it was concluded that deamidation of asparagine 414 was responsible for the surface charge alteration observed in the AEX-HPLC profile but was not associated with the reduction in infectivity. Overall, this manuscript details critical characterization efforts conducted to enable understanding of a pivotal physicochemical test for adenoviral based therapeutics.
物理化学测试是生物治疗药物分析控制策略的重要工具。对于腺病毒载体形式，阴离子交换高效液相色谱法（AEX-HPLC）是一种重要的方法，因为它能够以高通量方式同时提供病毒颗粒浓度、产品纯度和表面电荷的信息。在一种基于腺病毒的治疗药物的产品开发过程中，进行了加速稳定性研究，结果显示AEX-HPLC可报告的每个属性都发生了变化。这些变化还与产品感染性的降低相关，这促使对杂质和表面电荷变化的机制进行详细表征。表征实验确定杂质为游离六邻体三聚体，这表明衣壳降解可能是杂质和颗粒浓度降低的原因。额外的质谱表征确定特定六邻体残基的脱酰胺作用与热应激条件下观察到的外部表面电荷修饰有关。为了证明AEX-HPLC观察到的脱酰胺作用与表面电荷变化之间的因果关系，进行了定点诱变实验。通过这项工作得出结论，天冬酰胺414的脱酰胺作用是AEX-HPLC图谱中观察到的表面电荷改变的原因，但与感染性降低无关。总体而言，本文详细介绍了为理解基于腺病毒的治疗药物的关键物理化学测试而进行的关键表征工作。
