Everest Incorporated

HIGH-YIELD, HIGH-PURITY EXTRACELLULAR VESICLE ISOLATION COLUMNS FOR HIGH-THROUGHPUT PLATE-BASED BIOMARKER DISCOVERY AND DIAGNOSIS - PROJECT SUMMARY IN THIS DIRECT-TO-PHASE II SBIR APPLICATION, EVEREST BIOLABS PROPOSES TO ADVANCE THE DEVELOPMENT OF INNOVATIVE PLATE-BASED METHODS AND MATERIALS FOR ISOLATING EXTRACELLULAR VESICLES (EVS) FROM BIOFLUIDS USING DUAL- MODE CHROMATOGRAPHY THAT COMBINES SIZE EXCLUSION CHROMATOGRAPHY WITH AFFINITY-BASED DEPLETION OF COMMON CONTAMINANTS. IMPACT: THIS PROJECT IS EXPECTED TO DELIVER THE FIRST HIGH-PERFORMANCE, HIGH-THROUGHPUT TOOL FOR USE WITH FULLY-AUTOMATED LIQUID HANDLING ROBOTS THAT CAN ISOLATE EVS WITH THE HIGH YIELD, HIGH PURITY, AND BROAD SIZE DISTRIBUTION REQUIRED TO FULLY REALIZE THE ENORMOUS POTENTIAL OF EVS IN RAPID, REPRODUCIBLE, AND COST-EFFECTIVE BIOMARKER DISCOVERY AND DIAGNOSTIC TESTING. VIRTUALLY ALL LIVING CELLS RELEASE EVS THAT DIFFUSE THROUGH EXTRACELLULAR SPACES, TRANSPORTING MATERIALS AND INFORMATION TO OTHER CELLS TO AFFECT THEIR FUNCTION. SOME OF THESE EVS ARE EVENTUALLY CIRCULATED IN THE BLOOD STREAM OR SHOW UP IN SALIVA, URINE, AND OTHER BIOFLUIDS. THE CONTENTS OF EVS, AS WELL AS THE PROTEINS EMBEDDED IN AND ATTACHED TO THE MEMBRANE SURFACE, PROVIDE A RICH SOURCE OF INFORMATION THAT CAN BE ANALYZED TO DETERMINE WHERE THE EVS ORIGINATED IN THE BODY AND ASSESS THE PHYSIOLOGICAL OR PATHOLOGICAL STATE OF THE ORIGINATING TISSUE. DESPITE ENORMOUS INTEREST IN LEVERAGING EVS TO DIAGNOSE ILLNESS, IDENTIFY PATIENTS WHO MAY BE MORE OR LESS RESPONSIVE TO A PARTICULAR TREATMENT, ASSESS THE EFFICACY OF A TREATMENT, OR EVEN DESIGN AND DELIVER NEW THERAPIES, THE TRANSLATION OF EVS INTO ROBUST RESEARCH AND DIAGNOSTIC TOOLS REQUIRES THE DEVELOPMENT OF RAPID, REPRODUCIBLE, AND SCALABLE METHODS FOR ISOLATING AND ANALYZING THEM. CURRENT METHODS FOR EV ISOLATION REQUIRE TRADE-OFFS BETWEEN EV YIELD AND EV PURITY, WHICH ARE BOTH CRITICAL FOR MAKING USE OF LOW-ABUNDANCE EVS TO STUDY OR DIAGNOSE ILLNESS IN SPECIFIC TISSUES. IN PRELIMINARY WORK, EVEREST BIOLABS ESTABLISHED PROOF-OF-CONCEPT FOR A MULTI-LAYER EV ISOLATION COLUMN THAT COMBINES A SIZE EXCLUSION CHROMATOGRAPHY LAYER WITH AN ADDITIONAL RESIN CONTAINING ANTIBODIES THAT SPECIFICALLY TARGET LIPOPROTEINS, A MAJOR CONTAMINANT OF EVS ISOLATED FROM PLASMA AND SERUM. THESE COLUMNS EXHIBITED ALMOST NO LOSS IN EV YIELD COMPARED TO SEC COLUMNS ALONE WHILE ELIMINATING > 99% OF LIPOPROTEINS. IN THIS DIRECT-TO-PHASE II SBIR APPLICATION, EVEREST BIOLABS PROPOSES TO BUILD ON THIS FINDING TO A) OPTIMIZE COLUMNS FOR CAPTURING THE THREE MAJOR CONTAMINANTS FOUND IN EV ISOLATES FROM PLASMA AND SERUM (I.E., ALBUMIN, IMMUNOGLOBULINS, AND LIPOPROTEINS) AND B) ADAPT THESE COLUMNS FOR USE IN A FULLY-AUTOMATED HIGH-THROUGHPUT 24- WELL PLATE-BASED EV ISOLATION METHOD. AIM 1. OPTIMIZE A DEPLETION-INTEGRATED EV ISOLATION COLUMN FOR ACHIEVING HIGH PURITY WHILE RETAINING HIGH YIELD. AIM 2. ADAPT MULTI-LAYER EV ISOLATION MATERIALS FOR A PLATE-BASED FORMAT. AIM 3. COMBINE AND VALIDATE THE OPTIMIZED DEPLETION CHEMISTRY AND MULTI-LAYER EV ISOLATION FORMAT IN A PLATE- BASED DESIGN FOR HIGH-PERFORMANCE, HIGH-THROUGHPUT EV ISOLATION.