Division of Bacteriology, U.S. Army Medical Research Institute of Infectious Diseases, 1425 Porter St, Ft Detrick, MD 21702, United States.
Division of Advanced Regulated Studies, U.S. Army Medical Research Institute of Infectious Diseases, 1412 Porter St, Ft Detrick, MD 21702, United States.
Vaccine. 2021 Jan 15;39(3):564-570. doi: 10.1016/j.vaccine.2020.12.008. Epub 2020 Dec 16.
A recombinant ricin vaccine from E. coli (RVEc™), was developed at the US Army Medical Research Institute of Infectious Diseases (USAMRIID) and assessed in an FDA sponsored Phase 1a clinical trial. At the maximum dosage, two of the study participants developed physiological responses that were elevated to the level of severe adverse reactions. To stay within safe dosing guidelines, the FDA recommended that an assay be developed to accurately quantify the recombinant protein content in the vaccine. The RVEc™ vaccine Final Drug Product (FDP) contains the adjuvant Alhydrogel®, which by its colloidal nature interferes with most conventional protein assay methods. We decided to develop an assay measuring RVEc™ FDP using o-pthalaldehyde (OPA) reagent. The OPA reagent reacts to the primary amines and lysine side chains of proteins in the presence of a thiol under alkaline conditions with a quantifiable fluorescent signature, but does not react with Alhydrogel®. Protein content in the RVEc™ FDP can be determined by comparing the fluorescence of the test sample to the fluorescence of a standard curve of defined concentration. Each phase of the assay was tested to optimize and simplify the assay procedure. The accuracy, specificity, reproducibility, and stability of the assay were evaluated. Results indicated that the optimized and modified OPA assay was simple and able to quantify antigen concentration from a standard curve in the 25 µg/mL-600 µg/mL range. The assay accuracy and coefficient of variation (CV) was 95% and less than 8%, respectively, when determining the ricin protein content in the 200 µg/mL vialed RVEc™ FDP. The assay was simple to perform and used conventional laboratory equipment. This assay could be adapted to measure the protein content in the FDP of other vaccines, but with the proviso that each step of the assay would need to be optimized for each antigen.
一种来自大肠杆菌的重组蓖麻毒素疫苗（RVEc™）由美国陆军传染病医学研究所（USAMRIID）开发，并在美国食品和药物管理局（FDA）赞助的 I 期临床试验中进行了评估。在最大剂量下，两名研究参与者的生理反应升高到严重不良反应的水平。为了遵守安全剂量指南，FDA 建议开发一种能够准确量化疫苗中重组蛋白含量的检测方法。RVEc™ 疫苗最终药物产品（FDP）含有佐剂 Alhydrogel®，其胶体性质会干扰大多数常规蛋白质检测方法。我们决定开发一种使用邻苯二醛（OPA）试剂测量 RVEc™ FDP 的检测方法。OPA 试剂在碱性条件下与巯基一起，与蛋白质的伯胺和赖氨酸侧链反应，产生可量化的荧光特征，但不与 Alhydrogel®反应。通过将测试样品的荧光与定义浓度的标准曲线的荧光进行比较，可以确定 RVEc™ FDP 中的蛋白质含量。该检测方法的每个阶段都经过测试，以优化和简化检测程序。评估了该检测方法的准确性、特异性、重现性和稳定性。结果表明，优化和改进的 OPA 检测方法简单，能够在 25µg/mL-600µg/mL 的范围内从标准曲线中定量抗原浓度。当确定 200µg/mL 装瓶的 RVEc™ FDP 中的蓖麻蛋白含量时，该检测方法的准确性和变异系数（CV）分别为 95%和小于 8%。该检测方法简单易用，使用常规实验室设备。该检测方法可以适应测量其他疫苗 FDP 中的蛋白质含量，但前提是需要为每个抗原优化检测方法的每个步骤。
