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: CQA; Vaccine; antigenicity; conformational stability; freezing/thawing; neuraminidase; recombinant.

Figure 1.

Freeze thaw (F/T) induced rNA conformational changes that impact physiochemical properties and functional activities. The rNA in NaPBS was subjected to 1, 3 and 5 F/T cycles and the impact was assessed by SEC, extrinsic fluorescence, and Tamiflu binding. (a) SEC profiles show a change from ‘closed’ to ‘open’ tetramer conformation after F/T stress. Changes in physiochemical properties were also observed in terms of (b) % Closed tetramer by SEC (n = 2), (c) Reduction in melting temperature Tm (n = 2) and (d) Reduced functional activity in terms of Tamiflu-binding (n = 3).

Figure 2.

HDX-MS data of 5X FT rNA in NaPBS buffer mapped onto three-dimensional structure. Highlighted region (red) represents peptides that showed increase in deuterium uptake and is also known as the 150 loop which undergoes conformational change during substrate binding.

Figure 3.

The freeze thaw-induced rNA conformational change is abolished when switching from NaPBS to KP buffer. The rNA in either KP buffer pH 7.4 or NaPBS pH 7.0 underwent 5X F/T cycles and (a) % Closed tetramer (n = 2), (b) Melting temperature Tm (n = 2), and (c) Tamiflu Binding (n = 4) were measured. Error bars represent the standard deviation from the mean.

Figure 4.

Acidic pH is necessary but not sufficient to trigger conformational change and closed tetramer is stabilized in KP-10% sucrose. Four rNA formulations in different buffers were analyzed (vertical axis). The % closed tetramer by SEC was monitored at ambient temperature, before (red bars) and after (blue bars) a single F/T cycle. Closed tetramer loss occurred following F/T in NaPBS pH 7.0 and monoNaPS pH 4.6. Tetramer was conserved in formulations with 10% sucrose.

Figure 5.

Antigenicity and % closed tetramer are potential critical quality attributes (pCQAs). Accelerated stability studies were initiated using rNA in KP-10% sucrose buffer. Samples from 3 months timepoint at 5°C and 37°C incubation and 1 week at 45°C were tested for (a) % closed tetramer by SEC (n = 2), (b) Tamiflu-binding (n = 1), (c) Antigenicity by ELISA (n = 2), and (d) immunogenicity, with symbols indicating NAI titer of individual animals, bars represent geometric mean titer (GMT) with 95% CI of 5–6 animals/group (* p < 0.05, Tukey’s multiple comparison test). All measurements decreased with increasing temperature and % closed tetramer by SEC and antigenicity by ELISA were considered pCQAs.

Figure 6.

The rNA vaccine in KP-10% sucrose is predicted and verified to be stable for at least 24 months at 2–8°C. AKTS prediction software used 6 months accelerated stability data for (a) % Closed tetramer by SEC and (b) Antigenicity by ELISA to predict rNA stability for at least 24 months at 2–8°C. Stability data points up to 6 months were used for prediction, marked as filled circles from 5°C (black), 25°C (red), 37°C (blue), and 45°C (green). Solid line is the prediction mean; broken lines are the upper and lower 95% prediction intervals (PI). Real time data at 5°C on day 270 (9 M), 365 (12 M), 540 (18 M), and 730 (24 M) are bold open circles, showing no significant upward or downward trend, aligning with the prediction mean, and well within the PI for both assays.