Developability assessments and analytical characterization of recombinant protein antigens formulated as low-cost, multi-dose, aluminum-adjuvanted vaccines

Vaccination is an important public health initiative for controlling, preventing, and in some cases eradicating, infectious diseases. Despite improvements in global vaccine coverage, many children worldwide, especially those residing in low- and middle-income countries (LMICs), still do not have access to routine and new vaccines. Improvements in vaccine production can facilitate vaccine access by addressing two major limitations: the costs associated with vaccine manufacturing and challenges associated with maintaining a consistent supply. Traditional vaccines consist of live attenuated or killed pathogens which can be difficult to manufacture at large scale. There is a promising trend, however, towards developing vaccines consisting of recombinant subunit proteins antigens. This newer approach not only offers advantages of improved safety and efficacy, but also enhance manufacturing scalability and lower costs compared to traditional approaches. This Ph.D. dissertation work focuses on potential ways to reduce the cost of recombinant subunit protein vaccines from a formulation perspective. In this work, a vaccine formulation developability assessment workflow was established to rapidly screen recombinant protein antigen variants using minimal material to identify optimally stable, low-cost vaccine candidates targeted for use in LMICs. This workflow includes two formulation approaches to reduce vaccine costs: (1) use of adjuvants to enhance immune responses resulting in antigen dose sparing, and (2) a multi-dose presentation (i.e., multiple doses per vial) to reduce packaging, distribution, and storage costs. This Ph.D. thesis work also aimed to better understand the compatibility of antimicrobial preservatives with recombinant protein vaccine antigens using a variety of analytical methods. The goal is to enable more rationale design and development of low-cost, multi-dose vaccine formulations. As described in Chapter 2, a two-step vaccine formulation developability assessment workflow was established to rapidly screen variants of recombinant protein antigens to be formulated as adjuvanted, multi-dose vaccines. A series of rationally designed site-directed variants of recombinant non-replicating rotavirus (NRRV) P[8] protein antigen were used as the model system to establish this developability assessment workflow. In stage 1 of developability assessment, which requires as little as ~1 mg of recombinant protein, a suite of key analytical techniques was used to assess and compare physicochemical properties of P[8] protein variants to assess primary structure, conformational stability, relative solubility, and binding to a P[8] specific antibody. Additionally, compatibility of these P[8] variants with preservatives required for multi-dose formulations was also evaluated. Based on the results from stage 1, promising P[8] variants were down selected and the impact of key formulation conditions (e.g., presence or absence of preservative thimerosal and the aluminum-adjuvant Alhydrogel®) on storage stability was examined using differential scanning calorimetry (DSC) and antibody binging (ELISA) in stage 2. Correlations of stage 1 developability data with stage 2 storage stability studies were observed for aluminum-adsorbed P[8] antigens. These results were confirmed using a second NRRV protein antigen, P[4]. This two-stage vaccine formulation developability assessment workflow can be used to better inform and optimize formulation design for a wide variety of recombinant protein antigens with long-term goal of rapidly and cost-efficiently identifying low-cost, multi-dose, aluminum-adjuvanted vaccine formulations for use in LMICs. Multi-dose vaccine formulations typically contain antimicrobial preservatives (APs) to prevent microbial contamination during multiple drawings from the same vial. Such preservatives, however, are known to destabilize protein antigens and can reduce the stability and potency of a vaccine. In Chapter 3, based on literature, a working mechanism by which the vaccine preservative thimerosal destabilizes the NRRV P[4] antigen is presented. A combination of biophysical characterization assays and hydrogen exchange mass spectrometry (HX-MS) was used to evaluate the effect of thimerosal on the conformational stability, antibody binding, and backbone flexibility of the parent and a cysteine to serine mutant (C173S) of the NRRV P[4] antigen. The single cysteine residue of P[4] antigen forms a partially reversible complex with ethyl mercury, a degradation product of thimerosal in aqueous solution. This P[4]-thimerosal interaction increased backbone flexibility of P[4] within the helical region surrounding the cysteine residue, leading to more global destabilization as detected by HX-MS. The modification of cysteine to serine mitigated the destabilizing effect of thimerosal on P[4] structure as demonstrated by HX-MS and biophysical studies, however, this substitution led to some decrease in the inherent conformational stability of the protein. Epitope mapping studies using HX-MS demonstrated involvement of same helical region of P[4] surrounding the cysteine residue in antigen-antibody binding. In Chapter 4, we further explored the interaction of the NRRV P[4] antigen with various preservatives, focusing on other preservatives used in multidose parenteral formulations of small molecule, protein-based drugs, and vaccines. First, we studied the impact of a total of eight different preservatives on the storage stability of an Alhydrogel® (AH)-adsorbed formulation of P[4] antigen. The real time and accelerated stability profile of AH-adsorbed P[4] protein was determined for antigen-antibody binding, conformational stability, and antigen-adjuvant interaction as measured by competitive ELISA, DSC, and SDS-PAGE, respectively. Interestingly, at their in-use concentrations, there was a correlation between increasing storage stability of the AH-adsorbed P[4] antigen and hydrophobicity (log P) values of the preservatives. For example, the most hydrophobic preservative examined, propyl paraben, resulted in the least destabilization of AH-adsorbed P[4] followed by methyl paraben and chlorobutanol. We then used HX-MS to better understand the destabilizing effects of storage temperature (4°C vs 25°C) and thimerosal (TH) on backbone flexibility of AH-adsorbed P[4] antigen. At time zero, TH addition increased the backbone flexibility across most of the P[4] molecule (except the N-terminal P2 region and residues G17-Y38), and these TH-induced alterations in the P[4] structure appeared to irreversible as indicated by further increase in backbone flexibility after four weeks of storage at 4°C. HX-MS analysis of AH-adsorbed P[4] stored at accelerated temperature (25°C) for four weeks revealed structural changes in some regions of the epitope involved in the binding to P[4] specific mAb, which correlated with a decrease in antibody binding as measured by ELISA. To the best of our knowledge, this is the first publication using HX-MS analysis to investigate the structural integrity and stability of a recombinant protein antigen adsorbed to an aluminum-adjuvant. The results from these studies will guide future formulation development work to enable a low-cost, multi-dose formulation of the AH-adjuvanted trivalent NRRV vaccine candidate.

Grantor dc:publisher

University of Kansas

Year dc:date.issued

2021

Author dc:creator

• Sawant, Nishant

Advisor dc:contributor.advisor

• Volkin, David B

• Pharmaceutical sciences
• Fashion
• Developability
• Hydrogen exchange-Mass spectrometry
• Rotavirus

Statement dc:rights

• Copyright held by the author.

Language dc:language.iso

en

Dc Identifier Other

http://dissertations.umi.com/ku:17963

OAI identifier oai:identifier

oai:kuscholarworks.ku.edu:1808/36555