Refolding of Recombinant Proteins (2)

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The invention provides a method for recovering and purifying refolded recombinant proteins from cell culture. In particular the invention provides a method of recovering a recombinant protein from prokaryotic host cells, e.g., bacterial cells. The processes of the invention are broadly applicable to recombinant proteins. In certain embodiments, the recombinant protein is a growth factor, e.g., vascular endothelial growth factor (VEGF). In one embodiment, the growth factor is VEGF165.

In one embodiment, a process includes: (a) isolating said recombinant protein from the prokaryotic cell culture; (b) solubilizing said protein in a first buffered solution, pH greater than 9, comprising a first chaotropic agent; (c) refolding said solubilized protein in a second buffered solution, pH>9 but ≦11, comprising a second chaotropic agent, two or more reducing agents and addition of air or oxygen for such a time and under such conditions that refolding of the recombinant protein occurs; and (d) recovering said refolded recombinant protein. In one embodiment, the first buffered solution and/or the second buffered solution further comprises arginine. In one embodiment, the first buffered solution comprises 1 M Urea, 300 mM arginine, 10 mM CHES, 5 mM EDTA, pH 11, final concentration. In another embodiment, the first buffered solution comprises 1 M Urea, 300 mM arginine, 10 mM TRIS, 5 mM EDTA, pH 11, final concentration. In one embodiment, the second buffered solution comprises two or more reducing agents, e.g., DTT and cysteine. In one embodiment, the second buffered solution comprises 1 M Urea, 15 mM cysteine, 2 mM DTT, 100 mM arginine, 10 mM CHES, 5 mM EDTA, pH 10, final concentration. In another embodiment, the second buffered solution comprises 1 M Urea, 15 mM cysteine, 0.5-2 mM DTT, 100 mM arginine, 10 mM TRIS, 5 mM EDTA, pH 10, final concentration.

In one embodiment, a process includes: (a) isolating said recombinant protein from the prokaryotic cell culture; (b) solubilizing and refolding said protein in a combo buffered solution, pH>9 but ≦11 with the addition of air or oxygen; and, (c) recovering said recombinant protein. In one embodiment, the combo buffered solution comprises 1 M Urea, 15 mM cysteine, 2 mM DTT, 100 mM arginine, 10 mM CHES, 5 mM EDTA, pH 10, final concentration. In another embodiment, the combo buffered solution comprises 1 M Urea, 15 mM cysteine, 0.5-2 mM DTT, 100 mM arginine, 10 mM TRIS, 5 mM EDTA, pH 10, final concentration.

The oxygen or air for the refold reaction can be provided by an air source or an oxygen enriched compressed gas supply. In one embodiment, a kLa of 0.004 min−1 is used, e.g., which represents a mixing rate of 200-400 rpm and sparging rate of 0.3 cc/min/L in a 2.5L vessel containing a marine type impeller. In other embodiments, kLz=0.01 min−1 or 0.1 min−1 are used to produce refolded protein.

The solubilization and/or refolding can be done at a variety of temperatures. In one embodiment, the incubation temperature for the solubilization and/or refolding is room temperature. The incubation time can vary according to the recombinant protein being recovered and refolded. In one embodiment, the recombinant protein is incubated in the first buffered solution for at least 1 hour, or 1 to 2 hours. In one embodiment, the solubilized protein is incubated in the second buffered solution for about 3 to 24 hours. In one embodiment, the isolated recombinant protein is incubated in the combo buffered solution for 3 to 24 hours.

The invention additionally provides processes and methods for refolding of recombinant proteins either alone or in connection with the recovery of the recombinant protein as described herein. In a particular embodiment, purification methods include clarifying the solution containing the recombinant protein and contacting said refolded recombinant protein in the clarified solution with a mixed mode support, a cationic chromatographic support, a first hydrophobic interaction chromatographic support, and optionally, a second hydrophobic chromatographic support or an ion exchange support; and selectively recovering or eluting the refolded recombinant protein from each support. In one embodiment, clarifying the solution comprises adding detergent to a final concentration of 1%, adjusting pH to about 8.5-9.5, incubating solution for 1 to 10 hours at 25-30° C., centrifuging the solution; and filtering the liquid recovered from the centrifugation step. In one embodiment, the pH is about 8.7. In another embodiment, the pH is about 9.0. It is contemplated that the steps for recovery steps can be performed in any order, e.g., sequentially or altering the order of the chromatographic supports. In certain embodiments of the invention, methods are provided for recovering and purifying refolded recombinant proteins from manufacturing or industrial scale cell culture.