Tangential flow filtration based protein refolding methods
Provided herein are methods for refolding proteins that are denatured. Exemplary methods comprise solubilizing the denatured protein with a denaturing agent, e.g., a chaotropic agent, and renaturing the protein using a buffer exchanging system, e.g., tangential flow filtration (TFF).
1. A method for refolding a denatured protein, e.g., that is present in inclusion bodies (IBs), comprising
(i) providing the denatured protein that is present in IBs, wherein the protein comprises a tenth fibronectin type III ( 10 Fn3) domain and an Fc region;
(ii) suspending denatured protein in a suspension solution, to obtain a composition comprising suspended denatured protein;
(iii) combining the composition comprising suspended denatured protein with a solubilization buffer that comprises a denaturing agent and has a pH in the range of 7 to 10, wherein the denaturing agent is guanidine or guanidinium, to obtain a first protein composition comprising solubilized denatured protein;
(iv) diafiltering the first protein composition comprising solubilized denatured protein with 2-4 diavolumes of a refold buffer that comprises arginine and has a pH in the range of 9 to 11 to obtain a second protein composition comprising partially refolded protein; and
(v) incubating the second protein composition comprising partially refolded protein with a refold/oxidizing buffer that comprises arginine and glutathione and has a pH in the range of 9 to 11 to obtain a third protein composition comprising the protein in a refolded state.
2. The method of claim 1 , wherein step (v) comprises diafiltering the second protein composition with 2-6 diavolumes of a refold/oxidizing buffer.
3. The method of claim 1 , wherein diafiltering with refold buffer takes between 0.5 to 3 hours.
4. The method of claim 2 , wherein the diafiltering with the refold/oxidizing buffer takes between 0.5 to 3 hours.
5. The method of claim 1 , wherein the denatured protein is suspended in a suspension solution at a ratio of weight (grams) of denatured protein:volume (ml) of suspension solution of 1:1-10.
6. The method of claim 1 , wherein the composition comprising solubilized denatured protein is not diluted prior to diafiltering.
7. The method of claim 1 , wherein the composition comprising solubilized denatured protein is filtered prior to diafiltering.
8. The method of claim 1 , wherein the third protein composition comprises 1-10 mg/ml of protein.
9. The method of claim 1 , wherein the protein in refolded state has an efficiency of recovery is at least about 70%.
10. The method of claim 1 , wherein the denatured protein comprises at least one disulfide bond.
11. The method of claim 1 , wherein the solubilization buffer, refold buffer, and/or refold/oxidizing buffer comprises Tris.