Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues – Somatostatin ; related peptides
Reexamination Certificate
1999-07-16
2001-10-23
Borin, Michael (Department: 1631)
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
Somatostatin ; related peptides
C530S421000, C514S806000
Reexamination Certificate
active
06307013
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates generally to the purification of proteins produced by recombinant DNA technologies. More particularly, it concerns the clarification of protein suspensions containing soluble and insoluble components via flocculation with anionic polymers.
The use of flocculating agents has been described in several industrial settings, including the biotechnology industry. For example, U.S. Pat. No. 5,047,511 describes the use of cationic flocculating agents in the recovery of recombinant somatotropin protein from a protein solution containing soluble high molecular weight contaminating proteins. This involved the selective precipitation of contaminating high molecular weight proteins by adding a cationic flocculant containing quaternary ammonium groups and then recovering the somatotropin from the solution.
Somatotropins, also known as growth hormones, are polypeptides produced and secreted by cells of the pituitary gland. These proteins are known to be effective in promoting pre-adult skeletal growth and meat production of beef cattle and swine, and can be produced reliably and inexpensively in large quantities by recombinant DNA technology. In addition, they are known to affect a variety of metabolic processes including the stimulation of lactation, improvements of the efficiency of converting feed to meat or milk, lipid-mobilizing effects, and others.
Recombinant DNA technology provides a means for the large scale production of heterologous proteins of interest in bacterial host cells. In the case of somatotropin, a growth hormone, the protein is sequestered in inclusion bodies within the cytoplasm of the host cells. The inclusion bodies can be recovered from the host cell culture by disrupting the cell so as to release the inclusion bodies, and thereafter collecting the inclusion bodies as a solid pellet by differential centrifugation. The inclusion bodies are solubilized in an aqueous solution of a suitable chaotropic agent such as urea or guanidine hydrochloride at an alkaline pH and subsequently naturized by contact with a mild oxidizing agent to form intramolecular disulfide bonds and to refold the protein into its biologically active, native conformation. Methods for the solubilization and naturation of somatotropin protein produced by
E. coli
bacteria are described in U.S. Pat. Nos. 4,511,502 and 4,652,630, each of which is incorporated herein by reference.
The somatotropin refold solution obtained from the naturation step (as described for example in U.S. Pat. Nos. 4,511,502 and 4,652,630) comprises an aqueous solution of somatotropin monomers, dimers and higher oligomers, along with residue and other debris from the host cells. Of these, the somatotropin monomer is the desired biologically active agent. U.S. Pat. No. 5,182,369, the disclosure of which is incorporated herein by reference, describes the selective precipitation of somatotropin dimer and higher oligomers together with residual host cell proteins and other contaminating substances from a pH-adjusted somatotropin refold solution, leaving the desired somatotropin monomer as the primary soluble constituent of the suspension.
Once the somatotropin oligomers and other contaminants have been selectively precipitated using this approach, it is necessary to remove the precipitated proteins and other insoluble contaminants from the suspension in order to obtain somatotropin monomers of the desired purity. Such liquid/solid separations as those required for this purification step are employed in most industrial biotechnological processes and are frequently accomplished via centrifugation and/or filtration procedures.
Flocculating agents can be employed to improve liquid/solid separations by aggregating the solids that are present in a protein suspension, thereby increasing the particle size of the solids (for review, see Halverson and Panzer, 1980). An increase in particle size is particularly beneficial in centrifugation and sedimentation applications where the particle sedimentation velocity is proportional to the square of the particle radius. The increased sedimentation velocity that results from larger particle sizes can improve productivity in any type of liquid/solid separation where particle sedimentation velocity is a factor.
SUMMARY OF THE INVENTION
This invention broadly concerns the separation of soluble proteins from insoluble contaminants via flocculation. More particularly, it relates to the use of anionic polymeric flocculants in the separation and recovery of somatotropin proteins.
Therefore, in accordance with one aspect of the present invention, there is provided a method for separating an aqueous protein suspension of soluble somatotropin monomer and insoluble contaminants by adding to the suspension an anionic polymer in an amount and under conditions effective to cause the flocculation of the insoluble contaminants. The flocculated, insoluble, material can be easily separated from the soluble somatotropin monomer to recover a clarified supernatant of soluble somatotropin monomer.
In accordance with another aspect of the invention, there is provided a method for the recovery of somatotropin monomer which comprises
obtaining a mixture of somatotropin proteins comprising somatotropin monomer and somatotropin oligomer in aqueous solution at a pH greater than about 7;
producing a protein suspension containing soluble somatotropin monomer by precipitating a major portion of the somatotropin oligomer from the solution while maintaining a major portion of somatotropin monomer in solution by reducing the pH of the solution to less than about 6.5;
adding to the protein suspension containing soluble somatotropin monomer an anionic polymer in an amount and under conditions effective to cause the flocculation of the precipitated proteins; and
separating the flocculated material from the solution of somatotropin monomer.
In accordance with another aspect of the invention, there is provided an aqueous protein suspension comprising somatotropin monomers, somatotropin oligomers (oligomer as used herein refers to dimers as well as other multimeric forms of the protein), and an anionic polymer.
Suitable anionic polymers used in accordance with the method of this invention include but are not limited to polyacrylamides, particularly those having charge densities in the range of about 1 to about 20%, and polysaccharides such as starch.
This invention provides an improved means for separating somatotropin monomer from the insoluble contaminants that are selectively precipitated from the monomer during recovery of the recombinant protein. Flocculation of the insoluble contaminants increases their particle sizes and therefore their sedimentation velocities, providing improved productivity in liquid/solid separations.
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F.A.P Garcia, (Chapter 12—Protein Purification), InRecovery Process for Biological Materialsedited by John F. Kennedy and Joaquin M. S. Cabral, 1993 John Wiley & Sons Ltd, pp. 355-367.
Halverson, F, Panzer, H. P. Kirk-Othmer Encycl. Chem. Technol., 3rd Ed. (1980), vol. 10, 489-523. Editor(s): Grayson, Martin; Eckroth, David. Publisher: Wiley, New York, N.Y.*
Database CaPlus, DN 97:180612, EP 57273, Nov. 1981.
Beck George R.
Borin Michael
Howrey Simon Arnold & White , LLP
Monsanto Technology LLC
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