Method of refolding an interleukin-2 (IL-2) protein
Conjugates of an interleukin-2 (“IL-2”) moiety and one or more nonpeptidic, water-soluble polymers are provided. Typically, the non-peptidic, water-soluble polymer is poly(ethylene glycol) or a derivative thereof. Also provided, among other things, are compositions comprising conjugates, methods of making conjugates, methods of administering compositions to an individual, nucleic acid sequences, expression systems, host cells, and methods for preparing IL-moieties.
1. A method of refolding an interleukin-2 protein, the method comprising:
(i) placing a solution comprising a denatured interleukin-2 protein dissolved in 6 M guanidine, 100 mM Tris buffer at pH 8, and having a Cu2+ concentration of 0.1 mM within a dialysis bag having a molecular weight pore size of 3.5 kilodaltons, wherein the interleukin-2 protein has an amino acid sequence of SEQ ID NO:3; (ii) placing the interleukin-2 protein-containing dialysis bag into a reservoir containing an aqueous solution comprising 4.8 M guanidine and 0.1M Tris pH8 buffer; (iii) allowing the interleukin-2 protein-containing dialysis bag from (ii) to equilibrate in the reservoir; and (iv) slowly further reducing the concentration of guanidine in the reservoir by adding water to the reservoir over a period of several hours to thereby provide the interleukin-2 protein as a refolded interleukin-2 protein, wherein steps (i)-(iv) are carried out at 4° C.
2. The method of claim 1 , wherein the interleukin-2 protein-containing dialysis bag from step (iii) is allowed to equilibrate in the reservoir for three hours.
3. The method of claim 1 , wherein step (iv) is carried out over a period of from about 6 hours to about 15 hours to thereby reduce the concentration of guanidine in the reservoir to 2 molar.
4. The method of claim 3 , wherein step (iv) is carried out over a period selected from 6 hours, 10 hours, and 15 hours.
5. The method of claim 1 , further comprising (v) isolating the refolded interleukin-2 protein by removing precipitates by centrifugation and collecting the supernatant containing the refolded interleukin-2 protein.
6. The method of claim 5 , further comprising purifying the isolated refolded interleukin-2 protein by column chromatography to obtain purified refolded interleukin-2 protein monomer.
7. The method of claim 6 , wherein the chromatography is selected from ion exchange chromatography and hydrophobic interaction chromatography (HIC).
8. The method of claim 7 , wherein the column chromatography is ion exchange chromatography.
9. The method of claim 6 , comprising adding the purified refolded interleukin-2 protein monomer to a formulation buffer to form an interleukin-2 protein formulation.
10. The method of claim 9 , wherein the adding step comprises dialysis or ultra-filtration.
11. The method of claim 9 , wherein the formulation buffer comprises 10 mM sodium acetate and 5% trehalose at pH 4.5.
12. The method of claim 1 , wherein the interleukin-2 protein in step (i) is recombinant interleukin-2, prepared by expression in E. coli.
13. The method of claim 12 , wherein the recombinant interleukin-2 is expressed as inclusion bodies.