Effect of Aluminum Adjuvant and Preservatives on Structural Integrity and Physicochemical Stability Profiles of Three Recombinant Subunit Rotavirus Vaccine Antigens.
A nonreplicating rotavirus vaccine (NRRV) containing 3 recombinant fusion proteins adsorbed to aluminum adjuvant (Alhydrogel [AH]) is currently in clinical trials. The compatibility and stability of monovalent NRRV antigen with key components of a multidose vaccine formulation were examined using physicochemical and immunochemical methods. The extent and strength of antigen-adjuvant binding were diminished by increasing phosphate concentration, and acceptable levels were identified along with alternate buffering agents. Addition of the preservative thimerosal destabilized AH-adsorbed P2-VP8-P[8] as measured by differential scanning calorimetry. Over 3 months at 4°C, AH-adsorbed P2-VP8-P[8] was stable, whereas at 25°C and 37°C, instability was observed which was greatly accelerated by thimerosal addition. Loss of antibody binding (enzyme-linked immunosorbent assay) correlated with loss of structural integrity (differential scanning calorimetry, fluorescence spectroscopy) with concomitant nonnative disulfide bond formation (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and Asn deamidation (liquid chromatography -mass spectrometry peptide mapping). An alternative preservative (2-phenoxyethanol) showed similar antigen destabilization. Due to limited availability, only key assays were performed with monovalent P2-VP8-P[4] and P2-VP8-P[6] AH-adsorbed antigens, and varying levels of preservative incompatibility were observed. In summary, monovalent AH-adsorbed NRRV antigens stored at 4°C showed good stability without preservatives; however, future formulation development efforts are required to prepare a stable, preservative-containing, multidose NRRV formulation.
['Agarwal|Sanjeev|S|', 'Hickey|John M|JM|', 'McAdams|David|D|', 'White|Jessica A|JA|', 'Sitrin|Robert|R|', 'Khandke|Lakshmi|L|', 'Cryz|Stanley|S|', 'Joshi|Sangeeta B|SB|', 'Volkin|David B|DB|']
D000276:Adjuvants, Immunologic D000536:Aluminum Hydroxide D000956:Antigens, Viral D002021:Buffers D004339:Drug Compounding D004355:Drug Stability D006863:Hydrogen-Ion Concentration D011310:Preservatives, Pharmaceutical D011487:Protein Conformation D055550:Protein Stability D022243:Rotavirus Vaccines D013696:Temperature D013849:Thimerosal D022223:Vaccines, Subunit D014614:Vaccines, Synthetic D014764:Viral Proteins
adjuvant preservative stability rotavirus vaccine formulation thimerosal