A Combined LC-MS and Immunoassay Approach to Characterize Preservative-Induced Destabilization of Human Papillomavirus Virus-like Particles Adsorbed to an Aluminum-Salt Adjuvant.

During the multi-dose formulation development of recombinant vaccine candidates, protein antigens can be destabilized by antimicrobial preservatives (APs). The degradation mechanisms are often poorly understood since available analytical tools are limited due to low protein concentrations and the presence of adjuvants. In this work, we evaluate different analytical approaches to monitor the structural integrity of HPV16 VLPs adsorbed to Alhydrogel™ (AH) in the presence and absence of APs (i.e., destabilizing m-cresol, MC, or non-destabilizing chlorobutanol, CB) under accelerated conditions (pH 7.4, 50 °C). First, in vitro potency losses displayed only modest correlations with the results from two commonly used methods of protein analysis (SDS-PAGE, DSC). Next, results from two alternative analytical approaches provided a better understanding of physicochemical events occurring under these same conditions: (1) competitive ELISA immunoassays with a panel of mAbs against conformational and linear epitopes on HPV16 VLPs and (2) LC-MS peptide mapping to evaluate the accessibility/redox state of the 12 cysteine residues within each L1 protein comprising the HPV16 VLP (i.e., with 360 L1 proteins per VLP, there are 4320 Cys residues per VLP). These methods expand the limited analytical toolset currently available to characterize AH-adsorbed antigens and provide additional insights into the molecular mechanism(s) of AP-induced destabilization of vaccine antigens.

Multi-dose Formulation Development for a Quadrivalent Human Papillomavirus Virus-Like Particle-Based Vaccine: Part II- Real-time and Accelerated Stability Studies.

This work describes Part 2 of multi-dose formulation development of a Human Papillomavirus (HPV) Virus-Like Particle (VLP) based vaccine (see Part 1 in companion paper). Storage stability studies with candidate multi-dose formulations containing individual or combinations of seven different antimicrobial preservatives (APs) were performed with quadrivalent HPV VLP (6, 11, 16, 18) antigens adsorbed to aluminum-salt adjuvant (Alhydrogel®). Real-time (up to two years, 2-8°C) and accelerated (months at 25 and 40°C) stability studies identified eight lead candidates as measured by antigen stability (competitive ELISA employing conformational serotype-specific mAbs), antimicrobial effectiveness (modified European Pharmacopeia assay), total protein content (SDS-PAGE), and AP concentration (RP-UHPLC). The AH-adsorbed HPV18 VLP component was most sensitive to AP-induced destabilization. Optimal quadrivalent antigen storage stability while maintaining antimicrobial effectiveness was observed with 2-phenoxyethanol, benzyl alcohol, chlorobutanol, and 2-phenoxyethanol + benzyl alcohol combination. Interestingly, for single-AP containing multi-dose formulations, this rank-ordering of storage stability did not correlate with previously reported biophysical measurements of AP-induced antigen destabilization. Moreover, other APs (e.g., m-cresol, phenol, parabens) described by others for inclusion in multi-dose HPV VLP formulations showed suboptimal stability. These results suggest that each HPV VLP vaccine candidate (e.g., different serotypes, expression systems, processes, adjuvants) will require customized multi-dose formulation development.

Multi-Dose Formulation Development for a Quadrivalent Human Papillomavirus Virus-Like Particle-Based Vaccine: Part I - Screening of Preservative Combinations.

The development of multi-dose, subunit vaccine formulations can be challenging since antimicrobial preservatives (APs) often destabilize protein antigens. In this work, we evaluated Human Papillomavirus (HPV) Virus-Like Particles (VLPs) to determine if combining different APs used in approved parenteral products, each at lower concentrations than used alone, would maintain both antimicrobial effectiveness and antigen stability. To identify promising AP combinations, two different screening strategies were utilized: (1) empirical one-factor-at-a-time (OFAT) and (2) statistical design-of-experiments (DOE). Seven different APs were employed to screen for two- and three-AP combinations using high-throughput methods for antimicrobial effectiveness (i.e., microbial growth inhibition assay and a modified European Pharmacopeia method) and antigen stability (i.e., serotype-specific mAb binding to conformational epitopes of HPV6, 11, 16 VLPs by ELISA). The OFAT and DOE approaches were complementary, such that initial OFAT results (and associated lessons learned) were subsequently employed to optimize the combinations using DOE. Additional validation experiments confirmed the final selection of top AP-combinations predicted by DOE modeling. Overall, 20 candidate multi-dose formulations containing two- or three-AP combinations were down-selected. As described in Part 2 (companion paper), long-term storage stability profiles of aluminum-adjuvanted, quadrivalent HPV VLP formulations containing these lead candidate AP combinations are compared to single APs.

Analytical and Preformulation Characterization Studies of Human Papillomavirus Virus-Like Particles to Enable Quadrivalent Multi-Dose Vaccine Formulation Development.

Introducing multi-dose formulations of Human Papillomavirus (HPV) vaccines will reduce costs and enable improved global vaccine coverage, especially in low- and middle-income countries. This work describes the development of key analytical methods later utilized for HPV vaccine multi-dose formulation development. First, down-selection of physicochemical methods suitable for multi-dose formulation development of four HPV (6, 11, 16, and 18) Virus-Like Particles (VLPs) adsorbed to an aluminum adjuvant (Alhydrogel®, AH) was performed. The four monovalent AH-adsorbed HPV VLPs were then characterized using these down-selected methods. Second, stability-indicating competitive ELISA assays were developed using HPV serotype-specific neutralizing mAbs, to monitor relative antibody binding profiles of the four AH-adsorbed VLPs during storage. Third, concentration-dependent preservative-induced destabilization of HPV16 VLPs was demonstrated by addition of eight preservatives found in parenterally administered pharmaceuticals and vaccines, as measured by ELISA, dynamic light scattering, and differential scanning calorimetry. Finally, preservative stability and effectiveness in the presence of vaccine components were evaluated using a combination of RP-UHPLC, a microbial growth inhibition assay, and a modified version of the European Pharmacopoeia assay (Ph. Eur. 5.1.3). Results are discussed in terms of analytical challenges encountered to identify and develop high-throughput methods that facilitate multi-dose formulation development of aluminum-adjuvanted protein-based vaccine candidates.