Formulation development of a stable influenza recombinant neuraminidase vaccine candidate

ABSTRACT

Current influenza vaccines could be augmented by including recombinant neuraminidase (rNA) protein antigen to broaden protective immunity and improve efficacy. Toward this goal, we investigated formulation conditions to optimize rNA physicochemical stability. When rNA in sodium phosphate saline buffer (NaPBS) was frozen and thawed (F/T), the tetrameric structure transitioned from a “closed” to an “open” conformation, negatively impacting functional activity. Hydrogen deuterium exchange experiments identified differences in anchorage binding sites at the base of the open tetramer, offering a structural mechanistic explanation for the change in conformation and decreased functional activity. Change to the open configuration was triggered by the combined stresses of acidic pH and F/T. The desired closed conformation was preserved in a potassium phosphate buffer (KP), minimizing pH drop upon freezing and including 10% sucrose to control F/T stress. Stability was further evaluated in thermal stress studies where changes in conformation were readily detected by ELISA and size exclusion chromatography (SEC). Both tests were suitable indicators of stability and antigenicity and considered potential critical quality attributes (pCQAs). To understand longer-term stability, the pCQA profiles from thermally stressed rNA at 6 months were modeled to predict stability of at least 24-months at 5°C storage. In summary, a desired rNA closed tetramer was maintained by formulation selection and monitoring of pCQAs to produce a stable rNA vaccine candidate. The study highlights the importance of understanding and controlling vaccine protein structural and functional integrity.

KEYWORDS:

• Vaccine
• neuraminidase
• freezing/thawing
• recombinant
• CQA
• antigenicity
• conformational stability

Acknowledgments

We acknowledge many scientific and technical staff who supported testing at different locations including, Shilin Cheung and Cindy Xin Li for early recognition of open/closed tetramer in Toronto, Avanti Karkhanis and Jin Su for antigenicity ELISA development in Toronto, Marie-Julie Diana and Romain Pizzato who supported antibody characterization in Marcy l’Etoile, Timothy Farrell and Clint McDaniel who supported mouse immunogenicity studies and performed ELLA in Cambridge, Massachusetts. Finally, Swetha Vengadesh for editorial support.

Disclosure statement

This work was funded by Sanofi. At the time of preparation all authors were employees of Sanofi and may hold shares and/or stock options in the company.

Authors’ contribution

B.L., N.R., R.H.B., and S.F.A. were responsible for overall design of this study. S.Z. and D.A.J. carried out HDX-MS data analysis, and interpretation. L.S. was responsible for SEC data analysis and interpretation. M.M. was responsible for ELISA data analysis and interpretation. I.V.U. and G. C-G were responsible for the immunogenicity studies, data analysis and interpretation. M.H. and K.Y.Y. F were responsible for the AKTS design, data analysis and interpretation. R.H.B., B.L. and SFA wrote the paper and contributed to paper preparation. All authors contributed to and reviewed and gave approval to the final version of the paper.

Supplementary data

Supplemental data for this article can be accessed on the publisher’s website at https://doi.org/10.1080/21645515.2024.2304393.

Additional information

Funding

The work was funded by Sanofi.