Abstract
This work describes the formulation design and development of a novel protein based adjuvant, a double mutant of heat labile toxin (dmLT), based on knowledge of the protein’s structural integrity and physicochemical degradation pathways. Various classes of pharmaceutical excipients were screened for their stabilizing effect on dmLT during exposure to thermal and agitation stresses as monitored by high throughput analytical assays for dmLT degradation. Sucrose, phosphate, sodium chloride, methionine and polysorbate-80 were identified as potential stabilizers that protected dmLT against either conformational destabilization, aggregation/particle formation or chemical degradation (e.g., Met oxidation and Lys glycation). Different combinations and concentrations of the selected stabilizers were then evaluated to further optimize dmLT stability while maintaining pharmaceutically acceptable ranges of solution pH and osmolality. The effect of multiple freeze-thaw (FT) cycles on the physical stability of candidate bulk formulations was also examined. Increasing the polysorbate-80 concentration to 0.1% in the lead candidate bulk formulation mitigated the loss of protein mass during FT. This formulation development study enabled the design of a new bulk formulation of the dmLT adjuvant and provides flexibility for future use in combination with a variety of different vaccine dosage forms with different antigens.
Highlights
Adjuvants are molecules/agents, that when properly formulated with certain antigen(s) in a pharmaceutical vaccine dosage form, enhance the desired immune response to the antigen(s) including elevated antibody or cellular responses, improved duration of vaccine protection, and/or reducing the required dose of an antigen [1,2,3]
Since thermal and agitation induced aggregation was identified as a major physical degradation pathway for dmLT (Toprani et al, 2017, submitted), assays used to monitor dmLT aggregation were used to identify stabilizing conditions and additives as a first step to design an optimized formulation
By starting the excipient screening with the goal of minimizing aggregation, we subsequently identified conditions/ excipients that would minimize the chemical degradation pathways such as Asn deamidation, Lys glycation and Met oxidation
Summary
Adjuvants are molecules/agents, that when properly formulated with certain antigen(s) in a pharmaceutical vaccine dosage form, enhance the desired immune response to the antigen(s) including elevated antibody or cellular responses, improved duration of vaccine protection, and/or reducing the required dose of an antigen [1,2,3]. The reasons for the failure of such novel adjuvants during the early stages of development is undesirable biological activity or side effects, but from a pharmaceutical perspective, poor compatibility with antigens and/ or instability during long-term storage leading to loss of potency [6]. In vivo animal studies are often used to monitor adjuvant structural integrity, while, sensitive biophysical and analytical assays can often help to better understand the structural characteristics and the instabilities of adjuvants and link them to the key critical quality attributes of a vaccine such as potency [7]. Since vaccine dosage forms are multicomponent and inherently complex in nature (e.g., biological antigens, adjuvants, excipients), it becomes essential as part of their development to study the factors that can affect their stability and potency.
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