Development of an o-pthalaldehyde (OPA) assay to measure protein content in Ricin Vaccine E. coli (RVEc™)

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.