Development of a candidate stabilizing formulation for bulk storage of a double mutant heat labile toxin (dmLT) protein based adjuvant

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.

Recombinant Subunit Rotavirus Trivalent Vaccine Candidate: Physicochemical Comparisons and Stability Evaluations of Three Protein Antigens

“…Descriptions of many of the analytical methods used in this work have been published previously, 21,28,29 and detailed descriptions for all methods in this work are provided in the Supplemental Methods section. These methods include intact mass spectrometry (MS), LC-MS peptide mapping, Fourier transform infrared (FTIR) spectroscopy, far-UV circular dichroism (CD) spectroscopy, fluorescence spectroscopy, SDS-PAGE, size-exclusion chromatography, sedimentation velocity analytical ultracentrifugation, reversed-phase (RP) ultra-high pressure chromatography, hydrophobic interaction chromatography (HIC), differential scanning calorimetry, extrinsic fluorescence spectroscopy (DSF), and OD 350 (turbidity) analysis.…”

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

“…A "base buffer" of 10 mM sodium phosphate, 150 mM NaCl, pH 7.2 was chosen for initial characterization and excipient screening based on our prior work with these antigens (see companion paper). In addition, details of the methods used have mostly been described previously [28][29][30] are provided in the Supplemental Methods section including visual appearance, turbidimetry, micro-flow imaging (MFI), UV-visible spectroscopy, resonant mass measurement, sedimentation velocity analytical ultracentrifugation (SV-AUC), size exclusion chromatography (SEC), SDS-PAGE, extrinsic 1-anilinonaphthalene-8-sulfonate (ANS) fluorescence spectroscopy, Fourier transform infrared (FTIR) spectroscopy and FTIR microscopy.…”

Structural Characterization and Physicochemical Stability Profile of a Double Mutant Heat Labile Toxin Protein Based Adjuvant

“…Based on these analyses, high-throughput assays for screening stabilizers can be developed and used as a key part of formulation development experiments of dmLT. 21 This approach has been extensively used for biophysical characterization and stabilization for a number of vaccine candidates. 32 …”

“…These results will not only enable characterization of improved manufacturing processes to produce future clinical lots of dmLT from an analytical comparability perspective but also facilitate monitoring lot-to-lot variability of dmLT made from the same process. In addition, this work provided the analytical tools for formulation development of stable candidate formulation of dmLT for bulk storage 21 as well as for future formulation development for its use with a wide variety of antigens as a final drug product.…”