Method for purifying somatotropin monomers
AssigneeMonsanto Company
InventorsStephen B. Storrs, Jeffrey S. Schorey
Life of this patent
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Abstract
A method for purifying and recovering biologically active somatotropin monomers from refold solution following the solubilization and naturation of refractile bodies of host cells produced by recombinant DNA methodology. The purification process is based on the discovery that somatotropin monomers and somatotropin oligomers having overlapping isoelectric points may nevertheless be separated by selective precipitation over a narrow pH range. Host cell residues including proteins, pyrogens and other impurities present in the refold solution are effectively removed in the process. The purified somatotropin monomers recovered from solution after removing precipitated solids are suitable for parenteral application to target animals without further purification.
Claims40
A method for the purification and recovery of somatotropin monomer which comprises
- a. obtaining a mixture of somatotropin proteins comprising somatotropin monomer and somatotropin oligomer in aqueous solution containing a chaotropic agent selected from the group consisting of urea, guanidine hydrochloride, sodium thiocyanate and chaotropic detergents at a pH greater than about 7;
- b. preferentially precipitating a major portion of the somatotropin oligomer from said solution while maintaining a major portion of somatotropin monomer in solution by reducing the pH of said solution to less than about 6.5;
- and
- c. removing the precipitate to recover the somatotropin monomer remaining in solution.
A method for the purification and recovery of somatotropin monomer which comprises
- a. obtaining a mixture of proteins comprising somatotropin monomer, somatotropin oligomer and bacterial residue in aqueous solution containing a chaotropic agent selected from the group consisting of urea, guanidine hydrochloride, sodium thiocyanate and detergents at a pH greater than about 7;
- b. preferentially precipitating somatotropin oligomer and bacterial residue from said solution while maintaining a major portion of somatotropin monomer in solution by reducing the pH of said solution to less than about 6.5;
- and
- c. removing the precipitate to recover the purified somatotropin monomer remaining in solution.
A method for the separation and purification of somatotropin monomers which comprises
- a. obtaining a mixture of somatotropin monomer and somatotropin oligomers in aqueous solution with bacterial residues at a pH above about 7;
- b. reducing the pH of said solution to a first pH end point value of between about 4.0 and 5.5 effective to precipitate a substantial portion of the somatotropin oligmer and bacterial residues while a major portion of the somatotropin monomer remains in solution;
- c. removing the precipitate from the solution;
- d. increasing the pH of said solution to a second pH end point value of between about 5.5 and 8.5 effective to preferentially precipitate additional somatotropin oligomer and bacterial residues from solution while maintaining a major portion of somatotropin monomer in solution;
- and
- e. removing the precipitate to recover the purified somatotropin protein in solution.
A method for the separation of somatotropin proteins which comprises
- a. obtaining a mixture of somatotropin proteins comprising somatotropin monomers and somatotropin oligomers in aqueous solution at a pH greater than about 7;
- b. reducing the pH of said solution to less than about 4.5 while substantially maintaining said monomers and oligomers in solution;
- c. preferentially precipitating somatotropin oligomers from said solution while maintaining a major portion of somatotropin monomers in solution by increasing the pH of said solution to greater than about 5.5;
- and
- d. removing the precipitate to recover the somatotropin proteins remaining in solution.
A method for purifying and recovering somatotropin monomers from a mixture of somatotropin monomer, somatotropin oligomer and bacterial residue in aqueous solution containing a chaotropic agent selected from the group consisting of urea, guanidine hydrochloride, sodium thiocyanate and detergents at a pH outside the range of 4.0 to 8.5 which comprises adjusting the pH of said solution to an end point value within the range of 4.0 to 8.5,at which pH a major portion of the somatotropin oligomer and bacterial residues are precipitated while a major portion of the somatotropin monomer remains in solution, and removing the precipitate to recover the purified somatotropin monomer in solution.
Dependencies are inferred from the claim text.