Formulation development and comparability studies with an aluminum-salt adjuvanted SARS-CoV-2 spike ferritin nanoparticle vaccine antigen produced from two different cell lines

The development of safe and effective second-generation COVID-19 vaccines to improve affordability and storage stability requirements remains a high priority to expand global coverage. In this report, we describe formulation development and comparability studies with a self-assembled SARS-CoV-2 spike ferritin nanoparticle vaccine antigen (called DCFHP), when produced in two different cell lines and formulated with an aluminum-salt adjuvant (Alhydrogel, AH). Varying levels of phosphate buffer altered the extent and strength of antigen-adjuvant interactions, and these formulations were evaluated for their (1) in vivo performance in mice and (2) in vitro stability profiles. Unadjuvanted DCFHP produced minimal immune responses while AH-adjuvanted formulations elicited greatly enhanced pseudovirus neutralization titers independent of ~100%, ~40% or ~10% of the DCFHP antigen adsorbed to AH. These formulations differed, however, in their in vitro stability properties as determined by biophysical studies and a competitive ELISA for measuring ACE2 receptor binding of AH-bound antigen. Interestingly, after one month of 4°C storage, small increases in antigenicity with concomitant decreases in the ability to desorb the antigen from the AH were observed. Finally, we performed a comparability assessment of DCFHP antigen produced in Expi293 and CHO cells, which displayed expected differences in their N-linked oligosaccharide profiles. Despite consisting of different DCFHP glycoforms, these two preparations were highly similar in their key quality attributes including molecular size, structural integrity, conformational stability, binding to ACE2 receptor and mouse immunogenicity profiles. Taken together, these studies support future preclinical and clinical development of an AH-adjuvanted DCFHP vaccine candidate produced in CHO cells.

Data were collected using FelixGX software. Fluorescence emission spectra of 0.1 mg/mL DCFHP were recorded using an excitation wavelength of 295 nm (>95% Trp) and monitoring emission from 310-390 nm with a step size of 1 nm and an integration time of 1 s. Differential Scanning Calorimetry-DCFHP samples were diluted to 0.2 mg/mL in 20mM Tris, 100mM NaCl, 5% sucrose, pH 7.5 and loaded in a DSC autosampler tray held at 4°C. DSC was performed using an Auto-VP capillary differential scanning calorimeter (Malvern, Northhampton, MA). Sample and reference cells pressurized with N2 at ~65 psi. Two water-water scans were taken prior to the first reference scan. Samples were heated from 10-100°C using a scan rate of 60°C/hr, a pre-scan thermostat of 15 min, no feedback gain, and a filtering period of 10 points/s. Reference subtraction, baseline correction, and concentration normalization were performed using the instrument software. Since the baseline noise was significantly increased in the Alhydrogel containing scans, these scans were subjected to a 38-point Savitzky-Golay smoothing filter.

Sedimentation Velocity Analytical Ultracentrifugation-Sedimentation velocity (SV-
AUC) experiments were performed using an Optima analytical ultracentrifuge equipped with a scanning ultraviolet-visible optical system (Beckman Coulter, Indianapolis, IN.) A rotor speed of 7,000 rpm, rotor temperature of 20°C, UV detection at 280 nm, and a scan frequency of 60 seconds were used. DCFHP samples (at 0.2 mg/ml) in formulation buffer (20mM Tris, 150mM NaCl, 5% Sucrose, pH 7.5) alone were loaded into Beckman charcoal-epon two sector cells with 12 mm centerpieces and either sapphire or quartz windows and ultracentrifugation was performed for 800 total scans. Sedimentation data were analyzed using Sedfit (Peter Schuck, NIH) using a continuous c(s) model in the range of 0 to 100 svedbergs. A resolution of 300 points per distribution and a confidence level of 0.95 were applied to all fits. Baseline, radial independent noise, and time independent noise were fit, while the meniscus and bottom positions were set manually. The c(s) distributions were imported into Origin 2018 (OriginLab, Northampton, MA) for peak integrations and graph generation.
Dynamic Light scattering-Dynamic light scattering was performed using a Wyatt DynaPro 3 plate reader. Twenty-five microliters were loaded in each well of a 384 well plate (Corning) at a final protein concentration of 0.1 mg/mL DCFHP. After brief centrifugation, DLS was performed at 25°C with automatic laser attenuation enabled. All DLS data was corrected for solution viscosity and temperature.
Peptide Mapping Analysis-Prior to LCMS analysis, N-glycans from DCFHP were removed following an overnight 37°C incubation with EndoH or PNGaseF (New England Biolabs). DCFHP peptides were then generated using a commercial kit (S-Trap TM micro kit, Protifi LLC). Briefly, 20 µg of DCFHP was reduced with TCEP, heat denatured (10 min at 98°C), and then alkylated with methyl methanethiolsulfonate (MMTS). DCFHP was then treated for 2 hrs at in 96-well black microplates (Greiner Bio-One). Assay kinetics buffer (Quality Biological, 1X PBS pH 7.2 + 0.05% PS-80) was used for all baseline, dissociation, and reference wells as well as for diluting the ACE2-Fc receptor to loading concentration of 10 mcg/mL, and DCFHP at a starting concentration of 25 mcg/mL, followed by a 7-point 1:2 serial dilution in the kinetics buffer.
Biosensors were hydrated for ~10 min in kinetics buffer prior to the run. A typical run comprised of a baseline step (60 sec), followed by loading step (300 sec), another baseline step (60 sec), association step (250 sec), and lastly, dissociation step (1800 sec) with shake speed maintained at 1000 rpm throughout the experiment. Data analysis was performed using Octet Data Analysis software (v 10.0, Forte Bio). Following reference subtraction, baseline alignment, inter-step correction, and data processing with Savitzky-Golay filtering. Since there was negligible dissociation, Kd calculations were not possible.
Competitive ACE2 ELISA-DCFHP samples were diluted to 25-50 mcg/mL in 20mM Tris, 150mM NaCl, 5% Sucrose, pH 7.5 (in solution and AH-adsorbed) and were incubated in casein blocking buffer (Thermo-Fisher) with 0.05% Tween-20 such that the blocking buffer made up 1/3 of the final volume for one hour at room temperature with end over end rotation. Samples were then transferred to 96-well PCR plates (Thermo-Fisher) and 1:2 serial dilutions were made across the plates followed by incubation with ACE2-Fc receptor [3] (Institute for Protein Design, Seattle, Washington) at 0.04 mcg/ml. Plates were sealed using strip caps and incubated overnight at ambient temperature with gentle end over end rotation. Plates were then centrifuged at 1,600 x Where qe and Ce are solid phase and liquid phase equilibrium concentrations, respectively. Qmax is maximum (monolayer) binding capacity. KL represents the Langmuir isotherm constant, which represents binding strength [5].
Mice Immunization studies-Balb/C mice were procured from Jackson Laboratories (Bar  Table S3). Blood samples were collected from the immunized mice in a micro tube containing Z-Gel (Sarstedt AG and Company) via bleeding of retro-orbital plexus. Serum sample was isolated by centrifugation of the collection tube at 10,000 x g for 5 min and stored at -80 o C. Viral neutralizing titers of the serum samples were determined by the method described below.