Effect of Formulation Variables on the Stability of a Live, Rotavirus (RV3-BB) Vaccine Candidate using in vitro Gastric Digestion Models to Mimic Oral Delivery

Figures

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  Fig. 1. Experimental outline of the two in vitro digestion models used to examine RV3-BB stability under conditions to mimic in vivo oral delivery. (a) Forced degradation, low pH model used to assess buffering capacity of various excipients and to screen for RV3-BB stabilizers, and (b) sequential-addition model to evaluate RV3-BB stability under conditions that better mimic oral administration in infants. RV3-BB stability was determined by RT-qPCR infectivity assays as described in text. SGF - simulated gastric fluid, WFI - water for injection, DMEM - Dulbecco's Modified Eagle Medium.

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  Fig. 2. pH stability profile of RV3-BB as measured by infectivity RT-qPCR, (a) RV3-BB viral titer values (Log (FFU/mL)) and (b) RV3-BB titer losses (log loss vs. unstressed control). Samples were incubated for 1 h, 37 °C in the presence and absence of 2000 U/mL pepsin in 100 mM succinate-phosphate buffer (from pH 3.0 to 6.0). RV3-BB samples were prepared at a titer of 6.6 (Log (FFU/mL)) and stability data are presented as the mean ± SD (n = 4).

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  Fig. 3. Effect of five different ANC excipients on accelerated storage stability showing RV3-BB titer losses in different formulations as measured by infectivity RT-qPCR assays. RV3-BB in vitro potency losses after 1 week at 25 °C (vs unstressed control samples stored at −80 °C) were evaluated both in the presence and absence of 30% (w/v) sucrose in a phosphate buffer, pH 7.0 prepared at a titer of 6.6 (Log (FFU/mL)). The effect of the addition of five excipients on RV3-BB storage stability was determined at indicated concentrations: (a) Succinate, (b) Acetate, (c) Adipate, (d) Malic Acid, and (e) Citrate. ∗ Citrate resulted in the complete loss of RV3-BB titer in the no sucrose control at −80 °C. The data are presented as the mean ± SD (n = 3).

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  Fig. 4. Effect of Mylanta® and succinate on the stability of RV3-BB in the sequential-addition in vitro digestion model as measured by infectivity RT-qPCR, (a) Mylanta® (0–2 mL) and (b) succinate (0–400 mM). RV3-BB infectivity titer losses (log loss vs. unstressed control without HCl) after 1 h, 37 °C are presented as the mean ± SD (n = 4). RV3-BB formulation (60% (w/v) sucrose, 0.01% (w/v) PEG, 25% (v/v) DMEM, in a phosphate buffer at pH 7.8) was used in case of Mylanta® addition, while same formulation with 0–400 mM succinate was used in the case of succinate addition. All experiments were performed in absence of infant formula (Enfamil®) and prepared at a titer of 6.6 (Log (FFU/mL).

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  Fig. 5. Effect of infant formula (Enfamil®) addition on RV3-BB stability in the sequential-addition in vitro digestion model as measured by infectivity RT-qPCR, (a) candidate formulation A (containing 400 mM succinate), (b) candidate formulation B (same as formulation A but without succinate). RV3-BB infectivity titer losses (log loss vs. unstressed control without HCl) after 1 h, 37 °C are presented as the mean ± SD (n = 4). Formulations contained a titer of 6.6 (Log (FFU/mL)) with 60% (w/v) sucrose, 0.01% (w/v) PEG, 25% (v/v) DMEM, in a phosphate buffer pH 7.8, either in the presence of 400 mM succinate (Formulation A) or without succinate (Formulation B).

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  Fig. 6. Parameters of Design of Experiments model (RSM-CCD model) used to assess stability of RV3-BB infectivity titers and final solution pH in the sequential-addition in vitro digestion model. Parity plots showing statistically significant (p ≤ 0.05) correlation between experimentally measured values vs. DOE model predicted values for (a) RV3-BB stability (log loss) as measured by infectivity RT-qPCR, and (b) final solution pH. Panel (c) shows significant experimental factors affecting RV3-BB potency losses and final pH responses. (d) Experimentally measured RV3-BB in vitro potency log losses vs. final solution pH from RSM-CCD matrix showing rapid RV3-BB log titer loss below pH 5.0.