Characterizing and Minimizing Aggregation and Particle Formation of Three Recombinant Fusion-Protein Bulk Antigens for Use in a Candidate Trivalent Rotavirus Vaccine

Figures

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  Figure 1. Colloidal stability assessment and comparison of the 3 NRRV antigens after shake stressed for 6 h. (a) Light-scattering as measured by OD350 values, and (b) total subvisible particles (2-100 μm) per mL by MFI. Results from unstressed samples (0 h) are subtracted from stressed samples (6 h). Size-distribution analysis of shake stressed P[8] antigen in base buffer (10 mM PBS pH 7.2) were then monitored as soluble aggregates/fragments and protein-loss quantification by (c) SEC, and (d) SV-AUC analysis. Submicron and subvisible particle analysis by (e) resonant mass measurement, and (f) micro–flow imaging microscopy, respectively. Error bars represent 1 SD from 3 separate measurements. Refer Table 1 for comparative data of similar shake studies for 1.5 h with P[4], P[6], and P[8] antigens.

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  Figure 2. Morphology and structural analysis of protein within aggregates/particles formed for P[8] antigen after shake stress in base buffer. (a) Representative particle images recorded by micro–flow imaging microscopy, (b) optical microscopic image of an isolated protein particle/aggregate, (c) secondary structure analysis of protein in isolated insoluble protein particles/aggregates by FTIR microscopy, (d) higher order structure integrity analysis of unstressed and stressed protein in the supernatant (S) and pellet (P) fractions by ANS fluorescence spectroscopy, and inter-molecular disulfide bond analysis of unstressed and stressed protein in S and P fractions by (e) nonreduced and (f) reduced SDS-PAGE (lane 1—S unstressed, lanes 2,3,4—S stressed, lane 5—MW marker, lane 6—P unstressed, and lanes 7,8,9—P stressed). (g) Relative solubility assessment of 3 NRRV antigens using ammonium sulfate precipitation assay, error bars represent 1SD from triplicate experiments.

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  Figure 3. Morphology, higher-order structure and chemical composition of aggregates/particles formed for P[4] (top panel) and P[6] (bottom panel) antigens after shake stress in base formulation. (a and g) Representative particle images recorded by micro–flow imaging microscopy, (b and h) representative optical microscopic image of an isolated protein particle, (c and i) secondary structure analysis of unstressed protein in solution (control) and isolated particle from stressed sample by FTIR microscopy, (d and j) higher-order structure integrity analysis of unstressed and stressed protein in the supernatant (S) and pellet (P) fractions by ANS fluorescence spectroscopy. Inter-molecular disulfide bond analysis of unstressed and stressed protein in S and P fractions by (e and k) nonreduced and (f and l) reduced SDS-PAGE (lane 1—S unstressed, lanes 2,3,4—S stressed, lane 5—MW marker, lane 6—P unstressed, and lanes 7,8,9—P stressed).

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  Figure 4. Excipient screening against agitation stress of P[8] protein antigen. (a) Total subvisible particles, and (b) OD350 values of 0.15 mg/mL P[8] solution after shake stressed for 6 h in base buffer (10 mM PBS pH 7.2; black bar, highlighted in box) and in base buffer containing different excipients. Excipients are rank-ordered from lowest to highest total subvisible particles or OD350 value suggesting highest to lowest stability. Excipients in green, orange, gray, and red resulted in large increase, moderate increase, no effect, and decrease in stability, respectively. Error bars represent 1 SD from triplicate experiments. Refer to Supplemental Figure S1 for similar studies with down-selected excipients for P[4] and P[6] antigens.

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  Figure 5. Effect of excipient concentrations and combinations as well as different buffer types and pH conditions on shaking induced aggregation propensity of P[8]. OD350 value of P[8] samples stressed for 6 h minus time zero samples with different (a) excipients concentrations, (b) excipients combination, and (c) different buffer types and pH conditions. Error bars represent 1 SD from triplicate experiments.

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  Figure 6. Effects of top excipient combinations and solution conditions on shaking induced aggregation propensity of NRRV antigens as measured by OD350 analysis. (a) P[4] antigen, and (b) P[6] antigen with and without indicated excipients. (c) Shaking-induced aggregation propensity of P[4] in the presence of 150 mM NaCl with and without indicated excipients. Error bars represent 1 SD from triplicate experiments.

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  Figure 7. Excipient screening against thermal stress of P[8] protein antigen. OD350 studies of 0.1 mg/mL P[8] solution subjected to thermal stress from 10° C to 90° C in base buffer (10 mM PBS pH 7.2) and in base buffer containing different excipients. Average delta temperature (ΔT) value at which OD350 reaches 0.1 absorbance unit is shown and error bars represent 1SD from triplicate experiments. Excipients are rank ordered from lowest to highest ΔT value suggesting lowest to highest stability. Excipients in green, orange, gray, and red resulted in large increase, moderate increase, no effect, and decrease in stability, respectively. The inset shows OD350 versus temperature plots of a representative stabilizing @@@ (green line) and destabilizing (red line) excipient as compared to P[8] alone (black line) in base buffer.

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  Figure 8. Comparison of candidate bulk formulations (F2-F9) versus current bulk formulation (F1), of individual NRRV antigens against (a) thermal stress, (b) shaking stress, and (c) freeze-thaw stress as measured by UV-visible spectroscopy analysis. Error bars represent 1 SD from triplicate experiments. Refer to Table 2 for the composition and osmolality of each candidate formulation. *No protein loss was observed for P[6], **no protein loss was oberved for P[4] and P[6].

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  Figure 9. Effects of PS-80 concentration during freeze-thaw and shaking stresses on physical stability of NRRV antigens in current bulk formulation. (a) OD350 value after shaking each antigen for 6 h at 0.15 mg/mL protein concentration. Percent protein loss after 5 FT cycles at (b) 0.15 mg/mL and (c) 0.4 mg/mL protein concentration. Subvisible particle distribution analysis of P[8] antigen as measured by MFI after 0, 1, and 5 FT cycles in (d) current (1 mM sodium phosphate, 150 mM NaCl, pH 7.2) bulk formulation and 2 candidate bulk formulations, (e) 1 mM sodium phosphate, 150 mM NaCl, 0.05% PS80, pH 7.2, and (f) 10 mM histidine, 150 mM NaCl, 0.05% PS80, pH 6.8. Error bars represent 1 SD from triplicate experiments.