Abstract
Eight antimicrobial preservatives used in parenteral multidose formulations (thimerosal, 2-phenoxy ethanol, phenol, benzyl alcohol, m-cresol, chlorobutanol, methyl paraben, propyl paraben) were examined for their effects on the storage stability (4 °C, 25 °C) of an Alhydrogel® (AH) adjuvanted formulation of the non-replicating rotavirus vaccine (NRRV) recombinant P[4] protein antigen. The stability of AH-adsorbed P[4] was monitored for antigen-antibody binding, conformational stability, and antigen-adjuvant interaction via competitive ELISA, DSC, and SDS-PAGE, respectively. There was an unexpected correlation between increasing storage stability of the AH-adsorbed P[4] and preservative hydrophobicity (log P) (e.g., the parabens and chlorobutanol were least destabilizing). We used hydrogen exchange-mass spectrometry (HX-MS) to better understand the destabilizing effects of temperature and preservative on backbone flexibility of AH-adsorbed P[4]. Thimerosal addition immediately increased the backbone flexibility across much of the AH-adsorbed P[4] protein backbone (except the N-terminal P2 region and residues G17-Y38), and further increase in P[4] backbone flexibility was observed after storage (4 °C, 4 weeks). HX-MS analysis of AH-adsorbed P[4] stored for 4 weeks at 25 °C revealed structural alterations in some regions of the epitope involved in P[4] specific mAb binding. These combined results are discussed in terms of a generalized workflow for multi-dose vaccine formulation development for recombinant protein antigens.
Highlights
Immunization programs in low- and middle-income countries (LMICs), those supported by international organizations such as UNICEF and GAVI, rely on multi-dose vaccine vials due to their cost benefits.1,2 The multi-dose vaccine formulations offer several cost benefits over single-dose formats including: (1) reduced packaging costs since a single vial contains multiple doses; (2) reduced distribution costs for storage and transportation; and (3) lower disposal costs for contaminated medical waste.1 Multi-dose vaccine presentations are appropriate in settings where the vaccine cold-chain capacity is limited by reducing space requirements.3Multi-dose parenteral formulations require use of antimicrobial preservatives (APs) to prevent the growth of microorganisms that might be introduced accidently during multiple drawings
The P[4] antigen without polysorbate 80 (PS-80) was used for hydrogen exchange-mass spectrometry (HX-MS) studies while P[4] antigen with PS80 was used for storage stability studies
AH-adsorbed P[4] § TH samples were deuterium labelled for 3600 s, and P[4] was desorbed from aluminum using quench buffer and immediately frozen for subsequent analysis in HX-MS analysis
Summary
Immunization programs in low- and middle-income countries (LMICs), those supported by international organizations such as UNICEF and GAVI, rely on multi-dose vaccine vials due to their cost benefits. The multi-dose vaccine formulations offer several cost benefits over single-dose formats including: (1) reduced packaging costs since a single vial contains multiple doses; (2) reduced distribution costs for storage and transportation; and (3) lower disposal costs for contaminated medical waste. Multi-dose vaccine presentations are appropriate in settings where the vaccine cold-chain capacity is limited by reducing space requirements.3Multi-dose parenteral formulations require use of antimicrobial preservatives (APs) to prevent the growth of microorganisms that might be introduced accidently during multiple drawings. Immunization programs in low- and middle-income countries (LMICs), those supported by international organizations such as UNICEF and GAVI, rely on multi-dose vaccine vials due to their cost benefits.. The multi-dose vaccine formulations offer several cost benefits over single-dose formats including: (1) reduced packaging costs since a single vial contains multiple doses; (2) reduced distribution costs for storage and transportation; and (3) lower disposal costs for contaminated medical waste.. Multi-dose vaccine presentations are appropriate in settings where the vaccine cold-chain capacity is limited by reducing space requirements.. Multi-dose parenteral formulations require use of antimicrobial preservatives (APs) to prevent the growth of microorganisms that might be introduced accidently during multiple drawings. The most commonly used preservatives in multi-dose vaccine formulations include thimerosal, 2-phenoxy ethanol, and phenol.. The most commonly used preservatives in multi-dose vaccine formulations include thimerosal, 2-phenoxy ethanol, and phenol. Thimerosal (TH)
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