Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
  • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
  • C07K1/113—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides without change of the primary structure
  • C07K1/1133—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides without change of the primary structure by redox-reactions involving cystein/cystin side chains
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/575—Hormones
  • C07K14/61—Growth hormone [GH], i.e. somatotropin

Definitions

• This invention relates generally to a method for recovering recombinant proteins in their natural and bioactive forms from solutions containing inactive protein.
• heterologous DNA segments that encode for a particular protein are inserted into host microorganisms using recombinant technology.
• heterologous proteins such as insulin, somatotropins, interleukins, interferons, somatomedins, and the like can be produced.
• heterologous proteins produced by transformant microorganisms are frequently not biologically active because they do not fold into the proper tertiary structure when transcribed within the microorganism.
• the heterologous proteins tend to form aggregates which are recognizable within the cell as "inclusion bodies" (also sometimes referred to a
• inclusion bodies and/or “protein granules”
• inclusion bodies also may be caused by the formation of covalent intermolecular disulfide bonds which link together several protein molecules to form insoluble complexes.
• the inclusion bodies generally contain mostly heterologous proteins and a small fraction of contaminating host microorganism proteins .
• U.S. Patent No. 4,985,544 to Yokoo et al. discloses a process for reactivating a cysteine- containing protein in its natural form from inclusion bodies .
• the method comprises solubilizing the protein in the presence of both a denaturing agent and reducing agent, removing the reducing agent, oxidizing the protein, and thereafter removing the denaturing agent, causing the protein to refold to its native conformation.
• SDS, urea, guanidine hydrochloride, acids and alkalis are disclosed as possible denaturing agents, while suggested reducing agents include monovalent thiols such as ⁇ -mercaptoethanol, cysteine, glutathione and dithiothreitol (DTT). Among the listed reducing agents, DTT is particularly preferred.
• the oxidation step may be carried out by air-oxidation with aeration.
• Yokoo _ a____ illustrate the use of urea and DTT as the denaturing agent and reducing agent of choice, respectively.
• the disadvantage of the process in Yokoo et. a . is that it requires the delicate step of selectively and completely removing the reducing agent in the presence of the denaturing agent, preferably by gel filtration, prior to the oxidation step.
• U.S. Patent No. 4,518,526 to K. Olson relate to the dissolution and purification of refractile heterologous proteins, wherein the protein is subjected to a denaturing solution, which may include SDS as a possible denaturing agent, and is thereafter refolded by diluting the denaturing solution, optionally in the presence of a reducing agent. Included as a possible reducing agent is ⁇ -mercaptoethanol.
• U.S. Patent No. 4,766,205 to P. Ghosh-Dastidar relates to a method for producing biologically active native conformations in proteins derived from natural and recombinant sources containing multiple disulfide bonds .
• concentration of the reducing agent is decreased concurrent with the introduction of disulfide containing adduct-forming compounds.
• the disulfide containing compounds react with the reduced cysteine residues to form stable intermediate adducts concurrent with the removal of the reducing agent.
• the patent discloses mono-, di-, or poly-functional sulfhydryl- group containing agents such as ⁇ -mercaptoethanol or dithiothreitol as suitable reducing agents .
• the most preferred reducing agent is ⁇ -mercaptoethanol .
• Suitable disulfide adduct forming compounds include cystamine, oxidized glutathione, cystine, sodium sulfite salts and the like.
• the native cystine disulfide bonds can be reformed and the protein refolded to its native conformation in the presence of a mild oxidizing/reducing environment which comprises a weak reducing agent and a weak oxidizing agent in the presence of a suitable pH.
• Weak reducing agents suitable for such treatment include cysteine, while weak oxidizing agents suitable for such treatment include atmospheric oxygen.
• the present invention teaches a novel method for improving the yields of a biologically active protein obtained from a solution containing inactive protein.
• the inactive protein can be in the form of either insoluble inclusion bodies or oxidized, misfolded and aggregated waste protein.
• the method of the invention begins by diluting the inactive protein with a first alkaline buffer solution.
• the diluted inactive protein is treated with SDS as a denaturing agent and L-cysteine as a reducing agent to simultaneously solubilize and reduce the protein.
• concentration of both the SDS and L-cysteine in the solution containing the solubilized and reduced protein is decreased by a partial diafiltration against a second alkaline buffer solution free of both a denaturing agent and a reducing agent.
• the denatured and reduced protein then is oxidized to form disulfide bonds which correspond to those present in the naturally occurring bioactive protein.
• the solution comprising inactive protein is diluted with a first alkaline buffer solution such that the protein concentration is about 0.1 - 1.0 mg/ml.
• the diluted inactive protein thereafter is treated with about 0.05% to about 1.0% SDS and about 5 mM to about 25 mM L-cysteine.
• the concentrations of both SDS and L-cysteine in the solution containing the solubilized and reduced protein are decreased by about 70% to about 90% by diafiltration against about 0.5 to about 3 volumes of a second alkaline buffer solution which is free of both a denaturing agent and a reducing agent.
• the denatured and reduced protein then is air oxidized to form bonds corresponding to those present in the naturally occurring bioactive protein.
• the method of the present invention can be used to efficiently and economically enhance the recovery yields of bioactive proteins produced in the form of insoluble, biologically inactive inclusion bodies in transformant microorganisms, i.e. microorganisms which have been transformed with recombinant DNA vectors that direct the expression of genes coding for heterologous proteins.
• the method of the present invention can be used to convert biologically inactive, oxidized, misfolded and aggregated waste protein into a useful product, in particular a desired protein in its natural and bioactive form.
• the present invention further can be used to successfully recover oxidized monomers directly from inclusion bodies.
• Waste protein refers to the inactive, oxidized, misfolded and aggregated proteins formed along with a properly folded and biologically active protein after oxidizing a solubilized and reduced solution of inclusion body proteins as prescribed by recovery techniques known in the art.
• the waste protein is produced due to non-covalent aggregation and/or either intermolecular or intramolecular covalent polymerization of the recovered inclusion body proteins.
• the present invention can be used to recycle the waste protein to produce a correctly folded monomer product.
• Bioactive proteins obtainable using the method of this invention include animal somatotropins such as bovine, porcine, avian, ovine or human somatotropins. It is to be understood that reference herein to proteins generally or to specific proteins such as bovine and porcine somatotropins (bST and pST, respectively) is not intended to be restricted to molecules which contain the full amino acid sequence of the natural protein. Rather, it is also intended to include fragments of the protein having various portions of the sequence deleted and proteins or fragments thereof having various substitutions or modifications in their natural sequences (i.e. ana " -jues) which do not destroy the biological activity of i molecules.
• animal somatotropins such as bovine, porcine, avian, ovine or human somatotropins. It is to be understood that reference herein to proteins generally or to specific proteins such as bovine and porcine somatotropins (bST and pST, respectively) is not intended to be restricted to molecules which contain the full amino acid sequence of
• the first step of the method comprises diluting inactive protein with a first alkaline buffer solution so as to lower the protein concentration and adjust the pH of the protein solution.
• the preferred buffer is a carbonate buffer consisting of sodium carbonate (Na 2 C0 3 ) and sodium bicarbonate (NaHC0 3 ) on an equal weight basis, which typically has a concentration preferably about 35 mM to about 60 mM, more preferably about 40 mM to about 50 mM, and most preferably about 46 mM.
• the pH of the first buffer solution preferably is about 9 to about 11, more preferably about 9.6 to about 10, and most preferably about 9.8.
• the protein concentration after the buffer dilution preferably is about 0.1 to about 1.0 mg/ml, and more preferably about 0.3 to about 0.6 mg/ml. This may require a dilution by a factor of from about 1:1.1 to about 1:40, typically about 1:5 when the inactive protein is in the form of an oxidized, misfolded and aggregated waste protein, and about 1:40 when the inactive protein is in the form of an insoluble inclusion body.
• the initial dilution step creates a favorable environment for the present method.
• the concentration of the protein appears to strongly affect the efficiency of the subsequent dissolution/reduction and reoxidation steps.
• the dilution causes a decrease in the protein concentration which increases the distances between protein molecules. In particular, the decreased protein concentration minimizes both covalent and non-covalent intermolecular interactions.
• the diluted solution thus serves to promote the proper intramolecular interactions resulting in correct disulfide bond formation.
• the diluted inactive protein next is treated with SDS as a denaturing agent and L-cysteine as a reducing agent to simultaneously solubilize and reduce the desired protein.
• SDS is added to disrupt non-covalent interactions while L-cysteine is added to disassociate covalently mismatched intermolecular and intramolecular disulfide bonds into thiol groups.
• SDS desirably has a concentration of about 0.05% to about 1.0%, and most preferably about 0.2% by weight per volume, while the concentration of L-cysteine preferably is about 5 mM to about 25 mM, and most preferably about 15 mM.
• DTT dithiothreitol
• ⁇ ME ⁇ -mercaptoethanol
• glutathiones are widely used to control reoxidation of reduced proteins.
• ⁇ ME (20 mM) was used as the reducing agent in the present method, it often adversely affected refolding by forming rather stable refolding intermediates. The refolding intermediates, along with significant amounts of residual reduced monomer (RM) often remained even after 48 hours of oxidation.
• RM residual reduced monomer
• cysteine L-cysteine
• cysteine is a natural amino acid and is prepared as stable, dry crystals which are readily soluble in aqueous solutions at alkaline pH.
• cysteine is available in large quantity as FCC grade and is readily oxidized to cystine, a recognized oxidizing agent, especially at an alkaline pH (pKa of thiol group is 8.3) as occurs in the claimed process. Any residue problem, therefore, can be minimized. Also, the presence of cystine improves the subsequent oxidation of the protein solution.
• the amount of time needed for the above reaction to occur depends on whether the inactive protein is in the form of an insoluble inclusion body or an oxidized, misfolded and aggregated waste protein.
• a reaction time of 2 to 3 hours typically is needed whereas, for waste protein, 45 to 60 minutes generally are sufficient.
• the concentration of SDS and L-cysteine in the solubilized and reduced protein- containing solution is decreased by diafiltration, more particularly a partial diafiltration, typically against about 0.5 to about 3 volumes, preferably about 1 volume, of a second alkaline buffer solution free of both a denaturing agent and a reducing agent.
• the second alkaline buffer solution has a buffer salt concentration preferably about 35 mM to about 60 mM, more preferably about 40 mM to about 50 mM, and most preferably about 46 mM, and a pH of about 9 to about 11, more preferably about 9.6 to about 10, and most preferably about 9.8.
• the partial diafiltration decreases the concentration of both SDS and L-cysteine by about 70 to about 90 percent, preferably by about 80 percent, prior to the oxidation step. As shown by
• Example 4 diafiltration against a buffer volume higher than that taught for the claimed process will result in significantly lower yields of the desired, oxidized protein monomer.
• the advantage of diafiltration is that the concentration of low molecular weight substances such as SDS and L-cysteine can be reduced, while the concentration of higher molecular weight substances such as proteins is unaffected.
• the resultant solubilized and reduced protein monomers are next oxidized in the presence of air, preferably for about 24 hours, into correctly folded, oxidized monomers, containing disulfide bonds which correspond to those present in the naturally occurring bioactive protein.
• an oxidizing agent is not required, because at an alkaline pH, as present in the claimed process, cysteine spontaneously oxidizes to cystine, a known oxidizing agent.
• the resultant solution can be passed through an anion exchange column containing resins such as Amberlite ® IRA-400 to remove any residual SDS.
• the eluate containing aggregate-derived- monomers then can be subjected to subsequent processing steps, as desired.
• EXAMPLE 1 A waste protein solution containing both covalent and non-covalent aggregates of porcine somatotropin
• PM10 10,000 Dalton molecular weight cutoff
• the solubilized and reduced pST-containing solution then was subjected to ambient air oxidation for about 24 hours, followed by passing the solution through an Amerlite ® IRA-400 column to remove residual SDS.
• the eluate containing aggregate-derived-monomers then was subjected to subsequent processing steps, including ultrafiltration and chromatography, to produce purified monomeric pST.
• Figures 1A-E show high pressure liquid chromatography (HPLC) chromatograms for each step of the present method.
• Figure 1A represents the waste protein solution diluted by a factor of 1:5.
• Figure IB shows that after 1 hour of 0.2% SDS denaturation and 15 mM cysteine reduction, the peak representing oxidized monomer (OM) which originally was present in Figure 1A disappeared while a larger peak representing reduced monomer (RM) emerged. This indicated that both aggregates and oxidized monomers of pST present in the waste solution had been effectively reduced.
• OM oxidized monomer
• RM reduced monomer
• FIG. 2A represents two chromatograms obtained by Gel Permeation Chromatography (GPC), one of which was performed prior to the aggregate recycling process of Example 1 and the other which was performed after it.
• the solid line represents the undiluted waste protein solution, while the dotted line represents the eluate of product obtained from an A berlite ® IRA-400 column.
• the numbers reported in Figure 2B represent the GPC monomer (GM) in parts per million (ppm) and the ratio of the polymer pST or high molecular weight impurities to monomer pST. This ratio, called the P/M ratio, is used as a measure of the purity of the sample. The smaller the P/M ratio, the purer the sample.
• GPC Gel Permeation Chromatography
• FIGS 2A and 2B show that when a waste protein solution containing 174 ppm GM, wirh large amounts of pST aggregate and a P/M ratio of 42.4 was treated according to the aggregate recycling process of Example 1, the GM increased 10-fold to 1,695 ppm while the P/M ratio decreased to 4.1. This dramatic change in GM and P/M resulted from the conversion of pST aggregate into pST monomer.
• EXAMPLE 3 Inclusion bodies containing delta 7-pST (pST lacking the first 7 amino acids at the N-terminus of the amino acid sequence) were isolated from E.. coli strain HB101 which had been cultured under delta 7- pST-producing conditions as disclosed in U.S.P. 4,788,144. The cells were centrifuged out of the fermentor beer and resuspended in 0.1 M sodium phosphate buffer, pH 7.8, 20mM EDTA and lysed by passing twice through a Manton-Gaulin homogenizer at 8,000-10,000 psi.
• the crude inclusion bodies again were centrifuged out and washed twice more with 0.176 M sodium phosphate buffer, pH 7.5, lOmM EDTA, followed each time by centrifugation.
• the washed inclusion bodies were diluted by a factor of 1:40 with a 46 mM carbonate buffer solution, i.e., 21 mM sodium carbonate and 25 mM sodium bicarbonate, pH 9.8.
• 0.2% (w/v) SDS and 15 mM L-cysteine were added causing dissolution and reduction of the inclusion bodies .
• the mixture was agitated at ambient temperature for 2 and 1/4 hour.
• HPLC analysis indicated RM to be 16.8 g/L whereas OM was absent.
• the mixture was next diafiltered through a PM10 membrane against 1 volume of another 46 mM carbonate buffer solution at ambient temperature. HPLC analysis showed the diafiltrate contained 14.4 g/L RM and 0.96 g/L OM.
• OM increased substantially to 10.0 g/L and RM decreased to 0.52 g/L. This indicated the oxidation was practically completed.
• EXAMPLE 4 A waste protein solution, containing both covalent and non-covalent aggregates as well as oxidized monomers of pST, was diluted by a factor of 1:5 with a 46 mM carbonate buffer solution, i.e., 21 mM sodium carbonate and 25 mM sodium bicarbonate, pH 9.8. The OM concentration of the diluted waste protein solution was 0.26 g/L by HPLC. Next, 0.16% (w/v) SDS and 13 mM L- cysteine were added causing the dissolution and reduction of the waste solution. The mixture was agitated at ambient temperature for 3 hours, then diafiltered through a PM10 membrane against 15 volumes of another 46 mM carbonate buffer solution at ambient temperature, to completely remove both SDS and L- cysteine.
• a 46 mM carbonate buffer solution i.e., 21 mM sodium carbonate and 25 mM sodium bicarbonate, pH 9.8.
• the OM concentration of the diluted waste protein solution was 0.26 g/
• EXAMPLE 5 A waste protein solution containing both covalent and non-covalent aggregates as well as oxidized monomers of pST was diluted by a factor of 1:5 with a 46 mM carbonate buffer solution, i.e., 21 mM sodium carbonate and 25 mM sodium bicarbonate, pH 9.8. HPLC analysis indicated that the OM concentration of the diluted waste protein solution was 0.15 g/L.

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• 1993
  • 1993-07-01 WO PCT/US1993/006351 patent/WO1994001453A1/en not_active Application Discontinuation
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