LARGE-SCALE DEVELOPMENT OF DISPLACEMENT HPLC FOR SEPARATION OF PEPTIDES AND PROTEINS

THE RESEARCH PROPOSED FOR PHASE I FUNDING IS TO DETERMINE THE FEASIBILITY OF SCALING UP HPLC SEPARATIONS IN THE DISPLACEMENT MODE OF CHROMATOGRAPHY FOR THE PRODUCTION OF PHARMACEUTICAL GRADE SYNTHETIC PEPTIDES AND RECOMBINANT PROTEINS. IN CERTAIN CASES, HPLC IS THE ONLY LARGE SCALE PROCESS STEP THAT YIELDS THE PURITY LEVELS REQUIRED FOR HUMAN THERAPEUTIC USE. CONVENTIONAL ELUTION CHROMATOGRAPHY CAN BE CONVENIENTLY SCALED UP TO THE LEVELS REQUIRED IN MANUFACTURING, BUT IS EXPENSIVE, POORLY UTILIZES COLUMN CAPACITY AND IS DIFFICULT TO REPRODUCIBLE OPERATE. THE DISPLACEMENT MODE IS PERFORMED UNDER CONDITIONS SIMILAR TO CONVENTIONAL HPLC OPERATIONS, BUT INSTEAD OF A GRADIENT IN ELUENT STRENGTH THE FEED COMPONENTS ARE DISPLACED FROM THE COLUMN BY A CAREFULLY CHOSEN SOLUTION CONTAINING A COMPONENTTHAT IS MORE STRONGLY ADSORBED THAN ANY OF THE FEED COMPONENTS. THE PURIFIED PRODUCT EXITS THE COLUMN IN ADJACENT BANDS OF INDIVIDUAL COMPONENTS JUST AHEAD OF THE DISPLACER FRONT. CONCENTRATIONS AND COLUMN LOADINGS IN THISMODE OF CHROMATOGRAPHY ARE TYPICALLY TEN- TO ONE HUNDRED-FOLD HIGHER THAN IN ELUTION CHROMATOGRAPHY, AND SO DISPLACEMENT MORE FULLY EXPLOITS THE COLUMN AND EQUIPMENT CAPACITY. THESE FACTORS, TOGETHER WITH SIMPLIFIED CONTROL ASPECTS, MAKE DISPLACEMENT CHROMATOGRAPHY A PROMISING MANUFACTURING STEP. IN ORDER TO MAKE A COMMERCIALLY VIABLE PROCESS STEP, THE RESEARCH PLANNED FOR PHASE I WILL IDENTIFYDISPLACERS FOR REVERSED PHASE DISPLACEMENT PURIFICATION PROCEDURES OF PEPTIDES AND PROTEINS, AND FOR DISPLACEMENT OF PROTEINS ON HYDROPHOBIC INTERACTION CHROMATOGRAPHY (HIC) SYSTEMS. THE MOTIVE GOAL OF THIS RESEARCH AIMS AT DISCOVERING CONDITIONS APPROPRIATE TO PURIFICATION OF BIOTECHNOLOGY PRODUCTS AND TRANSFERRING THE TECHNIQUE FROM THE ACADEMIC TO THE MANUFACTURING SETTING.