AlphaVax has adapted LC/MS assays to quantitate the vaccines it is producing for a range of infectious diseases and cancer. The company manufactures its vaccines using an alphavirus-like replicon particle (VPR) containing an RNA that encodes a protein of interest. The VPR is used to infect a cell monolayer in culture.
Following viral particle entry into a cell, the RNA is released and translated, resulting in accumulation of the desired antigenic protein in the cell. The cells are then lysed and digested with trypsin in preparation for MS analysis. The MS portion of the assay provides a means of quantifying the resulting tryptic peptides without the need to use isotopically labeled standards.
According to Jeremy Johnson, Ph.D., senior scientist, MS-based assay development does not depend on antibody-based reagents and permits rapid assay development without specific biologically derived reagents. Quantitation of the desired protein is accomplished by measuring the total ion count of a specific tryptic peptide present in cell lysate and correlating it against a standard curve generated from the equivalent synthetic peptide standard.
Dr. Johnson explained that the MS-based assay is a platform technology that can be used to screen vaccine candidates without the need to develop a full ELISA, thereby saving considerable time and money during early-phase product development. “Quantification based on peptide total ion count requires no additional experimental steps,” he added. “The direct correlation of total ion count to peptide concentration enables peptide and, therefore, protein quantitation. This method can be developed quickly with excellent specificity and a large dynamic range.”
AlphaVax will continue to use this protein assay method in product and process development. The data collected from its use will be utilized to support ELISA development as products mature. The availability of this data for material made in early development should be useful in supporting product/process consistency throughout the development cycle, Dr. Johnson explained.