SBIR-STTR Award

The process combining supercritical CO2 and co-sterilants produces strong synergistic microbicidal effects in relatively mild conditions. The mechanism of microbe inactivation is not well characterized, but the disruption of lipid membranes and mild oxidative damage appear to be involved. The process, which is also highly efficient against viruses, is considered to be very mild compared to other sterilization methods. Experimental evidence shows that damage to protein is limited, and that immunogenic epitopes can survive the process. In this project, NovaSterilis scientists and the group of Bryce Chackerian at the University of New Mexico will use both bacteriophages and recombinant Virus-Like Particle vaccines to optimize this sterilization process to obtain high virus inactivatin rates in combination with limited protein damage and strong epitope integrity. The process optimization will include varying sterilant concentrations, and exposure time, temperature and pressure in the supercritical environment. Viral and VLP particle integrity will be assessed by electron microscopy, gel electrophoresis and size exclusion chromatography. Protein damage analysis will include fragmentation, aggregation and carbonylation assays. Epitope integrity will be assessed by ELISA. Future development will further characterize the process with different types of animal viruses, study damage to proteins, nucleic acids and lipids, and demonstrate in vivo that scCO2-sterilized VLP vaccines still induce a strong immune response and protection against pathogen challenge. The developed process will be applicable to the production of inactivated vaccines, and the final sterilization of labile biopharmaceuticals and sub-unit vaccines. A better understanding of methods mitigating protein damage in the sterilization process will have broader applications, including protecting enzymes and growth factors in supercritical-based tissue engineering.

Public Health Relevance Statement:


Public Health Relevance:
There is a clear need for a gentle and non-toxic method for virus inactivation in labile products such as drugs, vaccines, medical devices and tissue allografts. Supercritical carbon dioxide combined with a peracetic acid co-sterilant has the potential to significantly improve the drug, medical device and tissue industries' ability to offer products with guaranteed final sterility.

Project Terms:
Allografting; Alzheimer's Disease; amnion; Animal Viruses; Animals; Area; Bacteria; Bacteriophages; base; Biological; Biological Assay; Biological Products; Biomanufacturing; Biopolymers; biothreat; Blood Vessels; bone; Carbon Dioxide; Cell Line; Cells; Data; Dermal; Development; Devices; DNA; Electron Microscopy; Emerging Communicable Diseases; Enterobacteria phage MS2; Environment; Enzyme-Linked Immunosorbent Assay; Enzymes; Epitopes; Future; gel electrophoresis; Goals; Growth Factor; Hybrids; Hydrogen Peroxide; Hypersensitivity; Immune response; immunogenic; Immunotherapy; improved; in vivo; Inactivated Vaccines; Industry; Killings; Left; Legal patent; Lipids; Liquid substance; Maintenance; Malignant Neoplasms; Measures; Medical Device; Membrane; Membrane Lipids; Methods; Microbe; microbicide; Modeling; Molds; Molecular Sieve Chromatography; New Mexico; Nucleic Acids; oxidation; oxidative damage; particle; pathogen; pathogenic bacteria; Peracetic Acid; Pericardial sac structure; Pharmaceutical Preparations; Phase; Plaque Assay; Positioning Attribute; pressure; Process; process optimization; Production; professor; Property; Protein Analysis; protein profiling; Proteins; public health relevance; Reagent; recombinant virus; Research; Research Personnel; Salmonella typhimurium; Sampling; Scientist; self assembly; Shipping; Ships; Sterility; Sterilization; success; Surface; Technology; Temperature; Tendon structure; Time; Tissue Banking; Tissue Banks; Tissue Engineering; Tissues; treatment effect; Universities; vaccine development; Vaccines; Vertebral column; Viral; viral RNA; Viral Vaccines; Virus; Virus Inactivation; Virus-like particle; Xenograft procedure; Yeasts

The process combining supercritical CO2 and co-sterilants produces strong synergistic microbicidal effects in relatively mild conditions. The mechanism of microbe inactivation is not well characterized, but the disruption of lipid membranes and mild oxidative damage appear to be involved. The process, which is also highly efficient against viruses, is considered to be very mild compared to other sterilization methods. Experimental evidence shows that damage to protein is limited, and that immunogenic epitopes can survive the process. In this project NovaSterilis will seek to optimize the supercritical CO2 sterilization process for broad and effective virucidal treatments while attempting to maintain the integrity of model proteins. The first goal is to fully validate the supercritical process against viruses as a method that could replace ?-radiations which fail at effective virus inactivation and damage sensitive products. The second goal will be to characterize and optimize the supercritical sterilization process for the sterilization of proteins. Proteins have become increasingly popular as vaccines, therapeutics, and in combination devices. Yet, their sterilization remains extremely challenging. In collaboration with the group of Bryce Chackerian at the University of New Mexico, and Dr. Stephen Eyles and Professor Igor Kaltashov at UMass-Amherst, the effect of supercritical sterilization on growth factors and recombinant vaccines will be studied with in vitro and in vivo functional assay, and will for the first time use state-of-the-art mass spectrometry to determine with great precision the effect of scCO2 itself or combined with a sterilant on the conformation and oxidation status of model proteins.

Public Health Relevance Statement:
Project Narrative There is a clear need for a gentle and non-toxic method for effective bacterial and virus inactivation in labile products such as drugs, vaccines, therapeutic proteins, combination devices and tissue allografts. Supercritical carbon dioxide combined with a peracetic acid co-sterilant has the potential to significantly improve the drug, medical device and tissue industries' ability to offer products with guaranteed final sterility. The project will seek to optimize this sterilization process against viruses and minimize damage to proteins.

Project Terms:
Address; adduct; Adopted; Affect; Allografting; Amino Acids; Area; Attention; Australia; Bacterial Spores; Bacteriophages; Biocompatible Materials; Biological Assay; biophysical properties; bone; Carbon Dioxide; carcinogenicity; Cell Line; Collaborations; Confidential Information; cost; crosslink; Data; Dehydration; Development; Devices; Epitopes; Ethylene Oxide; Genome; Goals; Growth Factor; Health; Hepatitis A; HIV; Human Papilloma Virus Vaccine; Human Papillomavirus; Human Resources; Immunization; immunogenic; improved; In Vitro; in vivo; Individual; Industry; innovation; International; link protein; Maintenance; Mammalian Cell; Mass Spectrum Analysis; Medical Device; Membrane Lipids; Methods; Microbe; microbicide; Modeling; Molecular Conformation; Mus; New Mexico; occupational hazard; Organism; oxidation; oxidative damage; particle; pathogen; Patients; Peracetic Acid; Pharmaceutical Preparations; Phase; Play; pressure; Process; professor; Property; protein structure; Proteins; Radiation; Recombinant Growth Factor; Recombinant Vaccines; Reproduction spores; Research; Residual state; Risk; Role; Safety; Sterility; Sterilization; Stress; Structural Protein; Surface; Suspensions; Technology; Testing; Therapeutic; therapeutic protein; Time; time use; Tissue Banks; Tissues; Universities; Vaccines; Validation; Viral; virucide; Virus; Virus Inactivation; Zika Virus