The use of asymmetric cyclical chromatography to improve the recovery of low abundance proteins

Multi-column chromatography systems permits the development of purification schemes that are more efficient than batch chromatography.  Separations using feed streams with high concentrations of target molecule and high capacity resins have resulted in 60%-30% reductions in operating and resin costs using counter-current techniques.  The cyclical processes increase resin utilization while reducing process time and buffer volumes.  This approach, which uses multiple columns of the same type, is inefficient when the desired molecule is a small fraction of the total material in the feed and where the resin has a low binding capacity.  To overcome these limitations, an asymmetric cyclical process using a twin column system was devised for the production of fibronectin.  Fibronectin is 0.5% of the total protein in plasma and can be recovered by gelatin affinity chromatography followed by separation on heparin coated resins.  The binding capacity of gelatin resins was 2.5 mg/ml as compared to 25 mg/ml for heparin.  In the asymmetric process the fibronectin was captured on a gelatin column, eluted, in-line diluted and absorbed to a heparin column.  The absorption and elution from the gelatin column and absorption to the heparin column was repeated 10 times before the protein was eluted from the heparin column.  The asymmetric process was repeated for 6 cycles which yielded 150 mg of fibronectin from 938 ml of plasma at >98% purity using one 1 ml gelatin affinity column and one 1 ml heparin column.