Bradford Powell United States Army Medical Research Institute of Infectious Diseases Office: 301-619-4933 FAX: 301-619-2152 E-mail: bradford.powell@det.amedd.army.mil Job Title: Principal Investigator Ph.D. in Biochemistry from the University of California at Davis

Speaker: Bradford Powell, Bacteriology Division, United States Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Frederick, MD 21702

Topic: Analysis of Anthrax Protective Antigen Isoforms by LC-ESI-MS/MS

Place: Building 549, Auditorium, NCI at Frederick, Frederick, MD

Time: Tuesday, June 8, 2004, at 2:00 PM

Abstract: Recombinant protective antigen protein (rPA) is the active pharmaceutical ingredient of the next generation anthrax vaccine recommended by the U. S. Army for advanced product development. Two major isoforms and several minor isoforms of purified rPA are observed that share the expected molecular weight of 83,200 but differ incrementally in apparent charge as viewed by native PhastGel and two-dimensional PAGE (2DE). The presence of isoaspartate in purified rPA was measured with a protein methyl transferase assay since isoaspartate is a sentinel end product of amino acid deamidation, a common pathway for protein chemical degradation. Pharmaceutical grade rPA protein contained 20 pmol of isoaspartate per pmol of protein, in agreement with possible complete deamidation of up to 13 of the 67 total asparagine residues. Analysis of tryptic peptides by liquid chromatography-tandem ion trap mass spectrometry, which achieved 71 % coverage of the amino acid sequence, revealed six asparagine residues to be partially deamidated and also indicated no other amino acid modification in appreciable amount. The extent of deamidation at these six asparagines was estimated by comparing peak areas of extracted ions surrounding the native and deamidated peptides, accounting for overlapping C13 isotope envelopes where discernable, and found to range from 6 % to 51 % for rPA from vials. Applying this approach to gel-purified isoforms, deamidation ratios of the three most discernable asparagines were estimated on protein excised from native PhastGel and 2-DE spots. Percent deamidation among these asparagines increased with isoform net negative charge, and the two major isoforms showed 60 % and 85 % deamidation at Asn537 by both separations. We conclude that asparagine deamidation is the dominant cause of micro-charge heterogeneity for rPA protein, and suggest that deamidation ratios of labile asparagines in the two major isoforms may serve as markers of manufacture consistency and product stability.