ABSTRACT
UNLABELLED: The purpose of this work was to investigate the susceptibility of an aluminum adjuvant and an aluminum-adjuvanted native outer membrane vesicle (nOMV) vaccine formulation to freeze/thaw-induced agglomeration using static light scattering and micro-flow Imaging analysis; and to evaluate the use of propylene glycol as a vaccine formulation excipient by which freeze/thaw-induced agglomeration of a nOMV vaccine formulation could be mitigated. Our results indicate that including 7% v/v propylene glycol in an nOMV containing aluminum adjuvanted vaccine formulation, mitigates freeze/thaw-induced agglomeration. LAY ABSTRACT: We evaluated the effect of freeze-thawing on an aluminum adjuvant and an aluminum adjuvanted native outer membrane vesicle (nOMV) vaccine formulation. Specifically, we characterized the freeze/thaw-induced agglomeration through the use of static light scattering, micro-flow imaging, and cryo-electron microscopy analysis. Further, we evaluated the use of 0-9% v/v propylene glycol as an excipient which could be included in the formulation for the purpose of mitigating the agglomeration induced by freeze/thaw. The results indicate that using 7% v/v propylene glycol as a formulation excipient is effective at mitigating agglomeration of the nOMV vaccine formulation, otherwise induced by freeze-thawing.
Subject(s)
Bacterial Outer Membrane Proteins/immunology , Biopharmaceutics/methods , Excipients/chemistry , Light , Meningococcal Vaccines/chemistry , Microfluidic Analytical Techniques , Neisseria meningitidis/immunology , Propylene Glycol/chemistry , Scattering, Radiation , Technology, Pharmaceutical/methods , Adjuvants, Immunologic/chemistry , Aluminum Hydroxide/chemistry , Cryoelectron Microscopy , Drug Compounding , Drug Stability , Freezing , Particle Size , Phosphates/chemistry , Protein Aggregates , Spectrophotometry, UltravioletABSTRACT
The toxicity of Clostridium difficile large clostridial toxin B (TcdB) can be reduced by many orders of magnitude by a combination of targeted point mutations. However, a TcdB mutant with five point mutations (referred to herein as mTcdB) still has residual toxicity that can be detected in cell-based assays and in-vivo mouse toxicity assays. This residual toxicity can be effectively removed by treatment with formaldehyde in solution. Storage of the formaldehyde-treated mTcdB as a liquid can result in reversion over time back to the mTcdB level of toxicity, with the rate of reversion dependent on the storage temperature. We found that for both the "forward" mTcdB detoxification reaction with formaldehyde, and the "reverse" reversion to toxicity reaction, mouse toxicity correlated with several biochemical assays including anion exchange chromatography retention time and appearance on SDS-PAGE. Maintenance of a low concentration of formaldehyde prevents reversion to toxicity in liquid formulations. However, when samples with 0.016% (v/v) formaldehyde were lyophilized and stored at 37 °C, formaldehyde continued to react with and modify the mTcdB in the lyophilized state. Lyophilization alone effectively prevented reversion to toxicity for formaldehyde-treated, formaldehyde-removed mTcdB samples stored at 37 °C for 6 months. Formaldehyde-treated, formaldehyde-removed lyophilized mTcdB showed no evidence of reversion to toxicity, appeared stable by several assays, and was immunogenic in mice, even after storage for 6 months at 37 °C.
Subject(s)
Bacterial Proteins/toxicity , Bacterial Toxins/toxicity , Bacterial Vaccines/toxicity , Formaldehyde/metabolism , Toxoids/toxicity , Animals , Bacterial Proteins/chemistry , Bacterial Proteins/immunology , Bacterial Toxins/chemistry , Bacterial Toxins/immunology , Bacterial Vaccines/chemistry , Bacterial Vaccines/immunology , Bacterial Vaccines/radiation effects , Chromatography, Ion Exchange , Drug Storage , Electrophoresis, Polyacrylamide Gel , Female , Freeze Drying , Mice, Inbred C57BL , Mutant Proteins/chemistry , Mutant Proteins/immunology , Mutant Proteins/toxicity , Temperature , Time Factors , Toxoids/chemistry , Toxoids/immunologyABSTRACT
TECHNICAL ABSTRACT: The purpose of this work was to investigate a potential mechanism for the inhibition of tungsten-mediated monoclonal antibody (mAb) biophysical modifications and sub-visible particle formation. A 1 mg/mL mAb formulated in 150 mM NaCl, 20 mM histidine, pH 6.0, was incubated with 1, 37, and 100 ppm of tungsten polyanions in the form of sodium tungstate both in the presence and absence of the anionic surfactant and chelating agent diethylene triamine pentaacetic acid (DTPA) or the cationic surfactant cetyl trimethyl ammonium bromide (CTAB) for 24 h at 25 °C. Assays including pH, UV-Vis spectroscopy, size exclusion chromatography, intrinsic tryptophan/tyrosine fluorescence, and micro-flow imaging were performed to assess the impact on short-term mAb stability and aggregation. We conclude that the use of micromolar concentrations of the formulation excipient and cationic surfactant CTAB equivalent to the anticipated tungsten concentration in solution effectively inhibits loss of protein concentration, fragmentation, changes in intrinsic fluorescence intensity, and the formation of sub-visible particles. LAY ABSTRACT: The purpose of this work was to investigate a potential mechanism for the inhibition of tungsten-mediated monoclonal antibody (mAb) biophysical modifications and sub-visible particle formation. A mAb formulation was incubated with tungsten polyanions in the presence and absence of the anionic surfactant and chelating agent diethylene triamine pentaacetic acid (DTPA) or the cationic surfactant cetyl trimethyl ammonium bromide (CTAB). Formulation was characterized by pH, UV-Vis spectroscopy, size exclusion chromatography, intrinsic tryptophan/tyrosine fluorescence, and micro-flow imaging. We conclude that the formulation excipient and cationic surfactant CTAB effectively inhibits biophysical modifications and sub-visible particle formation.
