The goal of this STTR Phase I project is to demonstrate the feasibility of developing the first universal affinity membrane for the rapid, selective, one-step purification of difficult-to-purify proteins. Despite the urgency for expanding the application of gene editing systems and accelerating vaccine and antibody test development, the rapid purification of critical proteins such as CRISPR-Cas9 and the SARS-COV2 spike protein with high yield, high purity, and high activity remains a difficult challenge. The proposed innovation will benefit public health and have a significant impact on the biopharmaceutical industry by addressing this challenge. The products of this innovation will be first-in-market universal affinity membrane chromatography columns based on TriAltusÂ’ ultrahigh affinity CL7/Im7 purification system. Research will be done in partnership with Clemson University and the University of Alabama at Birmingham. Purification of CRISPR-Cas9 protein from E. coli lysate will be used as a demonstration case for evaluating the performance of the affinity membrane innovation. The Specific Aims of this Phase I study are to (1) Synthesize and characterize Im7-activated affinity membranes and (2) Test prototype affinity membrane columns for capture purification of CRISPR-Cas9. In Specific Aim 1, we will establish the feasibility of synthesizing the affinity membranes by immobilizing four Im7 ligand variants at high density on polymer-grafted macroporous membranes. We will measure protein binding capacity, recovery, and column reusability in screening experiments using purified, CL7-tagged CRISPR-Cas9. In Specific Aim 2, we will quantify and benchmark performance for capture step purification of CRISPR-Cas9 from E. Coli lysate. We will measure Cas9 binding capacity, recovery, purity, and activity. In Phase II, the team plans to comprehensively evaluate the roles that synthesis conditions and membrane support structure play on binding capacity; delineate operating ranges that maximize productivity, recovery, purity, and activity; collaborate with a membrane technology company on research to establish a scalable process to produce membrane columns for external validation; and conduct field research with external partners and prospective customers. Commercialization will bring the first universal affinity membrane chromatography column to the market. Market entry for the new column products will be to research and development scientists. The columns will provide a universal platform for the rapid, cost-effective purification of tag-free proteins with high yield, purity, and activity. By simplifying purification protocols and reducing purification times, products derived from this membrane innovation will have a major impact in the race to develop new protein drugs, gene therapies, vaccines, and antibody test kits. Public Health Relevance Statement Public Health Relevance Statement The goal of this STTR Phase I project is to develop a universal affinity membrane purification technology based on TriAltusÂ’ ultra-high-affinity CL7/Im7 system. The proposed technology will benefit public health and have a significant impact on the biopharmaceutical industry as a universal platform for the rapid, one step purification of difficult-to-isolate proteins such as CRISPR-Cas9 enzyme and the spike protein from SARS- COV2, which is critically important for expanding the application of gene editing systems and accelerating vaccine and antibody test development.
Project Terms: Alabama ; Sickle Cell Anemia ; Hb SS disease ; HbSS disease ; Hemoglobin S Disease ; Hemoglobin sickle cell disease ; Hemoglobin sickle cell disorder ; sickle cell disease ; sickle cell disorder ; sickle disease ; sicklemia ; Biological Products ; Biologic Products ; Biological Agent ; biopharmaceutical ; biotherapeutic agent ; Biological Sciences ; Biologic Sciences ; Bioscience ; Life Sciences ; Malignant Neoplasms ; Cancers ; Malignant Tumor ; malignancy ; neoplasm/cancer ; Cells ; Cell Body ; Chromatography ; Cystic Fibrosis ; Mucoviscidosis ; Disease ; Disorder ; Pharmaceutical Preparations ; Drugs ; Medication ; Pharmaceutic Preparations ; drug/agent ; Engineering ; Enzymes ; Enzyme Gene ; Escherichia coli ; E coli ; E. coli ; gene therapy ; DNA Therapy ; Gene Transfer Clinical ; Genetic Intervention ; gene-based therapy ; genetic therapy ; genomic therapy ; Genes ; Goals ; Health ; Heart Diseases ; Cardiac Diseases ; Cardiac Disorders ; heart disorder ; Human ; Modern Man ; Immobilization ; orthopedic freezing ; Industry ; Ligands ; Pathology ; Play ; Polymers ; Production ; Productivity ; Proteins ; Public Health ; Race ; Racial Group ; Racial Stocks ; Diagnostic Reagent Kits ; diagnostic kit ; test kit ; Research ; research and development ; Development and Research ; R & D ; R&D ; Plant Resins ; resin ; Ribonucleoproteins ; Role ; social role ; Sales ; Technology ; Testing ; Time ; Universities ; Work ; protein E ; Measures ; Guide RNA ; gRNA ; Chimeric Proteins ; Chimera Protein ; Fusion Protein ; base ; density ; improved ; Phase ; Variant ; Variation ; residence ; residential building ; residential site ; Recovery ; Binding Proteins ; Ligand Binding Protein ; Ligand Binding Protein Gene ; Protein Binding ; bound protein ; Scientist ; Complex ; Protocol ; Protocols documentation ; System ; Best Practice Analysis ; Benchmarking ; interest ; membrane structure ; Membrane ; Performance ; protein purification ; Speed ; Structure ; novel technologies ; new technology ; Column Chromatography ; Drops ; Molecular Interaction ; Binding ; evaluate vaccines ; vaccine screening ; vaccine testing ; vaccine evaluation ; preventing ; prevent ; Address ; Affinity ; Data ; Small Business Technology Transfer Research ; STTR ; Validation ; Process ; Development ; developmental ; cost effective ; prospective ; innovation ; innovate ; innovative ; commercial application ; prototype ; commercialization ; public health relevance ; phase 1 study ; Phase I Study ; screening ; Clustered Regularly Interspaced Short Palindromic Repeats ; CRISPR ; CRISPR/Cas system ; Mendelian disorder ; Mendelian disease ; Mendelian genetic disorder ; monogenic disease ; monogenic disorder ; single-gene disease ; single-gene disorder ; genome editing ; genomic editing ; CRISPR/Cas technology ; CRISPR method ; CRISPR methodology ; CRISPR technique ; CRISPR technology ; CRISPR-CAS-9 ; CRISPR-based method ; CRISPR-based technique ; CRISPR-based technology ; CRISPR-based tool ; CRISPR/Cas method ; CRISPR/Cas9 ; CRISPR/Cas9 technology ; Cas nuclease technology ; Clustered Regularly Interspaced Short Palindromic Repeats method ; Clustered Regularly Interspaced Short Palindromic Repeats methodology ; Clustered Regularly Interspaced Short Palindromic Repeats technique ; Clustered Regularly Interspaced Short Palindromic Repeats technology ; experimental study ; experiment ; experimental research ; COVID-19 ; COVID19 ; CV-19 ; CV19 ; corona virus disease 2019 ; coronavirus disease 2019 ; SARS-CoV-2 spike protein ; 2019-nCoV S protein ; 2019-nCoV spike glycoprotein ; 2019-nCoV spike protein ; COVID-19 S protein ; COVID-19 spike glycoprotein ; COVID-19 spike protein ; COVID19 S protein ; COVID19 spike glycoprotein ; COVID19 spike protein ; SARS-CoV-2 S protein ; SARS-CoV-2 spike glycoprotein ; SARS-CoV2 S protein ; SARS-CoV2 spike glycoprotein ; SARS-CoV2 spike protein ; Severe acute respiratory syndrome coronavirus 2 S protein ; Severe acute respiratory syndrome coronavirus 2 spike glycoprotein ; Severe acute respiratory syndrome coronavirus 2 spike protein ; coronavirus disease 2019 S protein ; coronavirus disease 2019 spike glycoprotein ; coronavirus disease 2019 spike protein ; antibody test ; antibody based test ;
