CA2074250A1 - Method for recovering recombinant proteins - Google Patents
Method for recovering recombinant proteinsInfo
- Publication number
- CA2074250A1 CA2074250A1 CA002074250A CA2074250A CA2074250A1 CA 2074250 A1 CA2074250 A1 CA 2074250A1 CA 002074250 A CA002074250 A CA 002074250A CA 2074250 A CA2074250 A CA 2074250A CA 2074250 A1 CA2074250 A1 CA 2074250A1
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- Prior art keywords
- solution
- molecular weight
- protein
- calcium
- group
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Classifications
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- 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
- C07K1/14—Extraction; Separation; Purification
- C07K1/30—Extraction; Separation; Purification by precipitation
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- 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 hormones [GH] (Somatotropin)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/06—Lysis of microorganisms
Abstract
A method for recovering a recombinant protein from a protein solution containing high molecular weight contaminating proteins by directly adding Group IIA metal salts to the solution in amounts sufficient to selectively precipitate the high molecular weight protein contaminants is disclosed. The high molecular weight precipitates are removed and the solution is further processed to remove low molecular weight contaminating proteins and other non-protein contaminants. The recombinant protein is subsequently recovered and further processed to produce a protein composition suitable for its intended use.
Description
2 ~ 7 ~ 0 BGE:m~:l9 M~HOD FOR RECOV~RING RB~OWBINANT PROT~INS
This invention relatas generally to methods for recovering rQcombin~nt proteinQ and particularly to a method for recovering recombinant proteins from protein solutions containing high molecular weight contaminating proteins.
Background of the Inven~ion Methods for producing recombinant proteins are well known in the art; heterologous DNA segments that encode for a particular protein are inserted into host microorganisms using re~ombinant DNA technology. By growing the transformant microorganisms under condition~ which induce the expression of proteins, heterologous proteins ~uch as insulin, somatotropins, interleukins, inter`ferons, somatomedins, and the like can be produced.
Unfortunately, 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 heterologouq proteins tend to form aggregates which are recognizable within the cell as l'inclusion bodies". These inclusion bodies may also be cau~ed by the formation of covalent intermolecular di~ulfide bonds which link together several protein molecules to form insoluble complexes. The inclusion 2~7~2~
bodies generally contain mostly heterologous proteins and a small fraction of contaminating host microorganism proteins.
Several processes have been developed to e~tract the inclusion bodies from the microorganisms and convert the heterologous proteins contained therein into proteins having native bioactivity consistent with the natural parent or non-recombinant proteins. These proces~es generally involvQ disrupting the microorganism cell, separating the inclusion bodiec from cell debris, solubilizing the inclusion body proteins in a denaturant/detergent which unfolds the protein, separating the heterologous inclusion body proteins from insoluble contaminan~s, removing the denaturant/detergent thereby allowing the heterologous proteins to refold into a bioactive tertiary conformation, and separating the protein from the contaminating proteins that remain in solution.
Several recombinant protein purification schemes following this genaral procedure are known in the art:
U~S. Patent Nos. 4,511,503 and 4,518,526 to Olson et al and U.S. Patent Nos. 4,511,502 and 4,620,948 to Builder et al disclose multi-step methods wherein (1) inclusion bodies are solubilized in a strong denaturant and a reducing agent, (2) insoluble contaminants are removed from the solubilized protein solution, (3) the strong denaturant is replaced with a weak denaturant, t4) the protein is allowed to refold assisted by oxidation of the sulfhydryl groups to disulfide bonds using molecular oxygen and a catalyst, typically metal cation~ or sodium tetrathionate, and (5) the protein is separated from other contaminating proteins by membrane separation techniques or chromatography procedures.
2t~7l 2~
Rausch et al, U.S. Pat. No. 4,677,196, incorporated herein by reference, discloses a particular method for purifying and activating proteins which is a variation of the general scheme described above. The method comprises solubili~ing the inclusion bodies in SDS, remo~ing the excass S~S from the solution using dialysis or other suitable technique, chroma~ographing the SDS-protein solution on an ion-retardation resin, and chromatographing the resulting solution on an anion-exchange re~in to recover the protein.
All these known procedures share a common problem~
The protein solution produced when the denaturant/detergent is removed contains the recombinant protein, low molecular weight contaminating proteins, non-protein contaminants, and high molecular weight contaminating proteins; the high molecular weight protein contaminants are often mostly dimers, oligomers and aggregates of the recombinant protein but al~o include non-recombinant protein~ from the cell digest. It is often difficult, time consuming, and expen~ive to separate the recombinant protein from these contaminants, particularly the recombinant protein dimers, oligomers and aggregates.
Chromatographic and membrane separation techniques may be effective for separating the recombinant proteins from the contaminants but are cumbersome, lengthy, expensive and often give low percentage yields for protein recovery.
New and improved methods for easily, quickly, and inexpensively recovering high yields of recombinant proteins from solutions containing high molecular weight protein contaminants are therefore needed~
2~ 2~0 Summary of the Invention It is, therefore, an ob~ect of the present invention to provide a new method for easily, quickly, and inexpensively recovering high yields of recombinant proteins from protein solutions containing high molecular weight contaminating proteins~
It is another ob~ect of the present invention to provide a method for removing high molecular weight contaminating proteins from a recombinant protein solution thereby allowing the easy, quick, and inexpensive recovery of the recombinant protein~
TheRe and other ob~ects are achieved by directly adding Group IIA metal salts to a solution containing high molecular weight contaminating proteins and a recombinant protein in amounts sufficient to selectively precipitate the high molecular weight protein contaminants. The compounds induce preferential precipitation of proteins having a molecular weight greater than about 1.5 times the molecular weight of the recombinant protein, particularly recombinant protein dimers, oligomers and aggregateq having a molecular weight greater than about 1.5 times the molecular weight of the recombinant protein. The precipitates are separated from the solution leaving the recombinant protein, low molecular weight contaminating proteins and other non-protein contaminants in solution. The recombinant protein is recovered from the solution using known techniques and processed to produce the desired protein product.
In the preferred embodiment, Group IIA metal salts are added directly to the solution in amounts sufficient to produce a 0.1-5% solution by volume. The high molecular weight contaminating proteins 2~7'12~
precipitate and are removed irom the solution by conventional means such as filtration, centrifugation, and the like. The resulting protein solution containing the recombinant protein, low molecular weight contaminating proteins and other non-protein contaminants is further processed using conventional techniques such as chromatograph~ to recovQr the recombinant protein.
Other ob~ects, advantagQs, and novel features of thQ present invention will becomQ apparent from the following detailed description of the invention.
Detailed De~c~iption of the Invention The term "recombinant protein`' as u~ed herein defines a protein produced by recomb`nant techniques which one desires to recover in a relatively pure form and includQs proteins having the amino acid sequence of native proteins and their analogs and muteins having substituted, deleted, replaced, or otherwise modified sequences.
The term `'recombinant somatotropin~ (rST) as used herein includes recombinant protein4 having the amino acid sequence of nati~e somatotropin, amino acid sequences substantially similar thereto, or an abbreviated sequence form thereof, and their analogs and muteins having qubstituted, deleted, replaced, or otherwise`modified sequences. In particular, rST as used herein includes a protein of the same sequence as native somatotropin (ST), but having amino acids deleted from its amino terminal end. Examples of such proteins include but are not limited to delta-7 recombinant porcine somatotropin, delta-4 recombinant bovine somatotropin, and the like.
2~7~2~
The term `'high molecular weight contaminating proteins~' or "high molecular weight protein contaminants" as used herein refers to proteins having a molecular weight greater than about 1.5 times the S molecular weight of the recombinant protein, particularly recombinant protein dimers, oligomers and aggregates having a molecular weight greater than about ~ times the molecular weight of the recombinant protein~
The term "low molecular weight contaminating proteins" or "low molecular weight protein contaminants" as used herein refers t~ proteins having a molecular weight less than about 1.5 times the molecular weight of the recombinant protein.
The term ~`non-protein contaminantq" as used herein refers to relatively low molecular weight substances such as precipitating agents, solubilizing agents, oxidizing agents, reducing agent~, and the like which are typically in a protein solution.
According to the present invention, a method is provided for recovering a recombinant protein from a protein solution containing high molecular weight contaminating proteins. The method comprises directly adding Group IIA metal salts to the solution containing high molecular weight contaminating proteins and the recombinant protein in amounts suf ficient to selectively precipitate the high molecular weight protein contaminants. The Group IIA metal salts preferentially induce the precipitation of proteins having a molecular weight greater than about 1.5 times the molecular weight of the recombinant protein, particularly recombinant protein dimers, oligomers and aggregates having a molecular weight greater than about 2 timeq the molecular weight of the recombinant 2~7d~0 protein. The method provides an improved method for easily, quickly, and inexpensively recovering high yields of recombinant proteins from solutions containing high molecular weight protein contaminants.
In ~he pre~erred embodiment, a method is provided ~or recovering recombinant somatotropins (molecular weight about 20,000) by directly adding Group IIA metal salts to soluti~ns containing high molecular weight contaminating protein~ and recombinant somatotropins in amounts sufficient to selectively precipitate the high molecular weight protein contaminants. Group IIA metal salts preferentially induce the precipitation of proteins having a molecular weight greater than about 1.5 times the molecular weight of th~ recombinant somatotropin (molecular weight greater than about 30,000), particularly recombinant somatotropin dimers, oligomers and aggregates having a molecular weight greater than about 2 times the molecular weight of the recombinant somatotropin (molecular weight about 40,000 and up). The present method, therefore, provides a method for separating the recombinant ~omatotropin from its bioinactive dimers, oligomers and aggregates.
Solutions containing a recombinant protein, non-protein contaminants, high molecular weight protein contaminants, and low molecular weight protein contaminantq useful in the pre~ent invention are obtained by m~thod~ known in the art. Typically, protein inclusion bodies which have been produced by recombinant microorganisms are processed to remove lipid~ and cell debris and the resulting relatively pure inclusion bodies containing recombinant protein and contaminating protein~, particularly high molecular weight recombinant protein dimers, oligomer~ and aggregates, are solubilized in a strong denaturant or 2~7~
detergent such as guanidine hydrochloride, sodium dodecyl sulfate (SDS), Triton, and the like.
The resulting protein solution is separated from any insoluble materials and the strong denaturant or detergent is removed to produce a protein solution containing the recombinant protein refolded into its native bioactive configuration, high molecular weight protein contaminants, low molecular weight contaminating proteins and other non-protein contaminants~ Such solutions typically contain from about 1-50 mg/ml total protein and from about 0~05-4 mg/ml recombinant protein~
The polymer to monomer (P/M) ratio of such a solution includes a mass ratio of ag~regates including void peak to monomer and is thus a measure of purity of the solution. The solution typically goe~ through a "prepurification" step where P/M ratio is reduced to about 0.5 or less. The solution can then be further purified and processed into a final product, typically using a DEAE Sepharose or other suitable column~ The present invention provide~ a method for removing the high molecular weight contaminating proteins while simultaneously achieving higher monomer recovery and a lower P/M ratio.
Group IIA metal salts are added to this solution according to the present invention to precipitate the high molecular weight contaminating proteins~ The high molecular weight contaminating proteins that precipitate upon addition of the Group IIA metal salts are removed from the solution by conventional means such as filtration, centrifugation, and the like~ The resulting protein solution containing the recombinant protein, low molecular weight contaminating proteins and other non-protein contaminants, if any, is further 2~71 ,~
processed, as needed, to remove low molecular weight contaminating proteins and other non-protein contaminants such as precipitating agents, solubilizing agents, oxidizing agents, reducing agents, and the like. Typically, such non`-pro~ein contaminants are removed by dialysis, chromatography, or other suitable means whereas the low molecular weight contaminating proteins are separated from the protein by ion-exchange or other form~ of chromatography~
The protein solution is further processed to produce 8 protein or protein composition suitable for its intended use, typically by lyophilization. These methods are well known in the art~
Group IIA metal salts useful in the present invention include all salts of the elements in Group IIA of the Periodic Table: beryllium, magnesium, calcium, strontium, barium and radium. Preferred compounds include salts of magnesium, calcium, barium and strontium.
Most preferably, the compounds of the present invention include the -~ulfate (SO,), chloride (C12), and nitrate (NO3) ~alt~ of magnesium, calcium, barium and strontium. Preferred compounds include calcium sulfate (caso4);`anhydrous CaSO~, CaS0~ hemihydrate, calcium nitrate (Ca(NO3)2), calcium lactate, magnesium sulfate (MgS0~), magnesium chloride (MgCl2), barium chloride (BaCl2 and strontium chloride (SrCl2).
Although the amount of Group IIA metal salts needed to induce precipitation varies depending on protein concentration, protein characteristic~, compound added, and the like, the Group IIA metal sa~ts are typically added to the solution in amounts sufficient to produce from about a 0~1-5~ by volume 2 ~ a solution of the compound, preferably from about a l-3 solution by volume.
Recombinant proteins recoverable using the method of the present invention can be any ~rotein having a molecular weight greater than about 5000 which are produced by recombinant microorganisms, typically in inclusion bodies. These include somatotropins, insulins, somatomedins, somatostatins, prolactins, placental lactogens, and the like~
Most preferably, recombinant somatotropins (molecular weight about 20,000) are recovered using the method of the present invention. The recombinant somatotropin can be a recombinant somatotropin from any species but are preferably bovine, porcine, avian, ovine, or human recombinant somatotropin, most preferably porcine or bovine recombinant somatotropin.
Methods for producing these recombinant proteins are well known in the art: For example, U.S. Patent Nos. 4,604,359 and 4,332,717 disclo~e methods for producing human recombinant somatotropin; U.S. Patent No. 4,431,739 discloses a method for producing recombinant somatotropins; E.P. Patent Application 0 104 920 discloses a method for producing recombinant porcine somatotropin; U.S. Patent No. 4,443,359 discloses a method for producing recombinant bovine somatotropin; Schoner, BiotechnoloqY, 3(2):151-54, discloses a method for producing recombinant somatotropin, and ~uell, Nucleic Acid Res., 13, 1923-38 (1985) disclose~ a method for producing recombinant somatomedin C.
Also, European Patent Application Publication No.
0 103 395 describes the construction of a transformant strain of E. coli containing a first plasmid which codes for delta 9 (Ser) bovine somatotropin 2~7~12~ ~
ll (~omatotropin less its 9 N-terminal amino acids and having an additional serine residue at the N-terminus) under the control of the lambda PL promoter-operator and which has a Shine-Dalgarno region derived from bacteriophage mu. The transformant also contains ~
second plasmid, pcI857, which codes for the production of the pcI857 temperature-sensitive repressor protein~
The repressor protein can be inactivated by raising the temperature to about 42C, thereby inducing expression of delta 9 (SQr) bovine somatotropin~ A transformant strain o~ this type, E~ coli HB101 (PL-mu-delta 9 (Ser) bovine somatotropin and pcI857) has been deposited with The American Type Culture Collection (ATCC), ~oc~ville, MD and assigned Accession No. 53030.
Construction of a similar transformant strain which codes for the production of delta 7 porcine somatotropin (porcine somatotropin less its fir~t 7 N-terminal amino acids) is de~cribed in European Patent Application Publication No. 0 104 920. A transformant strain of thi~ type, E. coli H~101 (PL-mu-delta 7 porcine somatotropin and pcI857), has been deposited with ATCC and a~signed Acces~ion No. 53031.
Strains 53030 and 53031 are prolific producer~ of delta 3 (Ser) bovine somatotropin and delta 7 porcine somatotropin, respectively~ In both instances, the expressed protein is se~uestered within the cell in the form of inqoluble, biologically inactive inclusion bodies which are visible under a microscope. Other methods for many similar protein~ are known in the art~
In the preferred embodiment, a recombinant somatotropin solution containing from about 1-50 mg/ml total protein and from about 0.05-2 mg/ml recombinant somatotropin i8 treated with from about 1-3% CaSO6 to precipitate the high molecular weight protein 12 2~7~
contaminants. The precipitate is removed by centrifugation and the recombinant somatotropin is recovered from the resulting solution using conventional means as described above~
Although the above recovery method is directed to recovering recombinant proteins, the method is equally applicable to separating and recovering non-recombinant proteins. For example, a solution containing a mixture o~ ~1) a '`useful or wanted protain~ 2) high molecular weight p.rotein~ (molecular weight greater than about 1.5 times the molecular weight of the useful protein) and (3) low molecular weight proteins (molecular weight 1Q8S than about 1.5 times the molecular weight of the useful protein) is treated according to the present invention to precipitate the high mo ecular weight proteins and thus separate the high molecular weight proteins from the useful protein and the low molecular weight proteins. The high molecular weight proteins are separated from the solution and discarded or further processed, as desired; the high molecular weight proteins can be recovered from the precipitate by redissolving the precipitate and recovering the proteins from the solution.
The useful protein is separated from the low molecular weight proteins by conventional means and further processed, if desired, to produce a protein product. The low molecular weight proteins which were separated from the useful protein are discarded or further processed, as desired. Typically, the low molecular weight proteins can be separated from the u~eful protein by chromatography or other means ~uitable for separating proteins having similar molecular weights. Many such protein ~eparation means 13 2 ?~) 7 2 ~ ~
are well known to skilled artisans and are equally applicable in the present invention.
The invention having been generally described, the following examples are given as particular embodiments of the invention and to demonstrate the practice and advantages thereof. It is understood that the examples are giVQn by way of illustration and are not intended to limit the specification or the claims to follow in any manner~ In particular, inclu~ion bodies used in the experiments were prepared from transformed E. Coli strains which produce delta-7 porcine somatotropin~
The inclusion bodies were isolated from E. Coli host strain HB101 transformed with a first plasmid (pL-mu-delta-7 pST) coding for delta-7 pST and a second plasmid (pcI857) coding for the temperature sensitive lambda phage repression protein. Many other strains of microorganisms produce inclusion bodies containing many types of recombinant proteins which will function in the present invention. Similarly, methods for growing thQse microorganisms to produce inclusion bodies are well known in the art.
Example 1 Recombinant porcine somatotropin (rpST) was recoverè~ from microorganism inclusion bodies by (1) dissolving the inclusion bodie~ in sodium dodecyl sulfate (SDS) in carbonate buffer (25 mM NaHC03 and 21 mN Na2C03), (2) removing insoluble contaminants from the solution, (3) adding an oxidizing agent for oxidation of rpsT, and (4) removing the SDS from the solution to allow the rpST to refold into its bioactive configuration. The resulting protein solution contained the rpST, high molecular weight protein contaminants, low molecular weight contaminating proteins and other non-protein contaminants.
14 2 ~ 71~2 CaSO~.2H~O was added at 2.5~ (w/v) concentration to the above refolded protein solution which contained 0.67 mg/ml of rpsT monomer with a P/M ratio of 5.1 (pH
9.8 at 22-25C) and mixed for 2 hours. The mixture was centrif~ged at about x5000 & for 10 minutes to recover the supernatant. The rpsT monomer and P/M ratio of the supernatant was analyzed by Superose-12 Fast Protein Liquid Chromatography. The result showed that the P~
ratio was reducQd to 0.3 and that the monom~r recovery was 83.0~. The dsta showed that the protein aggrega~e impurities and high molecular weight contaminating protQins were selectively precipitated by CaSO4~2H~O
while leaving the monomers in solution.
Example 2 Example 1 was repeated using other calcium salts:
anhydrous CaSO4, CaSO4 hemihydrate, calcium nitrate (Ca(NO3) 2 ) and calcium lactate. The result~ are shown in Table 1.
Referring to Table 1, the data shows that the aggregate impurities and high molecular weight contaminating proteins were ~electively precipitated by the addition of these calcium salts.
Example 3 Example 1 was repeated using alkaline metal salts of magnesium, barium and strontium. The results are shown in Table 2.
Referring to Table 2, the data shows that the aggregate impurities and high molecular weight contaminating protein~ were ~electively precipitated by these alkaline metal ~alts.
Example 4 CaCl2-2H2O was added at 0.4~ (w/v) concentration to the refolded protein solution in Example 1 which contained 0.4 mg/ml pST monomer with P/M ratio of 4.7 2~7~250 and mixed for about 30 minutes at room temperature (22-25C). The pH of the mixture was adjusteq to about 9.0 and the supernatant of the mixture was collected after centrifuging at xS,000 G for 10 minutes. The resulting S supernatant was analyzed by Superose-12 FPLC.
The result showed that the P~M ratio was reduced to 0.4 with a monomer recovery of 92.1~. This data shows that CaCl2~2~l2O precipitates the protein aggregates and high molecular weight contaminating proteins selectively leaving monomer~ in solution.
Example 5 The refolded protQin solution in Example 1 was approximately 2x concentrated and diafiltered against 80 mM ethanolamine buffer at pH 9.O. To the diafiltered protein solution, which contained 0~67 mg~ml rpsT monomer with P/M ratio of 3.5, CaCl2-2~2O was added at 0.4~ ~w/v) con~entration and mixed for 30 minuteq at 22-25C. The supernatant was separated by centrifuging at x5,000 G for 10 minutes. The supernatant was analyzed by Superose-12 FPLC.
The result showed the P/M ratio was reduced to O.22 and a monomer recovery was 91.5~. Thi~ data shows that CaCl2-2H2O selectively precipitates the protein aggregatè~ and high molecular weight contaminating proteins in an ethanolamine buffer ~ystem.
Obviously many modifications and variation~ of the prQsent invention are poqsible in light of the above teachings. It is therefore to be understood that within the scope of the appended claims the invention may be practiced otherwise than as specifically described.
2~7d~
T8blQ 1 Concentration Initial Final Monomer Calcium salt ~ (w/v)] P/M P/M Recovery(%) _____________________________________~________________ Anhydrous CaSO~ 2.5 3~0 0.03 87.3 CaSO~ hemihydrate 2.5 3.6 0.05 83~7 Calcium nitrate 2.5 3.3 0~2 91~0 Calcium lactate 2.S 4.1 0.06 72.S
_____________________________________~_________________ Table 2 Concentration Initial ~inal Monomer Salt ~ (w/v)] P/N P/M Recovery(%) _______________________________________________________ MgSO~-7H2O 2.5 3.0 0.1 71.0 MgCl2 1.0 3.0 0.3 92.0 BaCl2 1.6 3.7 0.5 50.0 srcl2~6H2o 1.22 3.7 o.o 43.0 _______________________________________________________
This invention relatas generally to methods for recovering rQcombin~nt proteinQ and particularly to a method for recovering recombinant proteins from protein solutions containing high molecular weight contaminating proteins.
Background of the Inven~ion Methods for producing recombinant proteins are well known in the art; heterologous DNA segments that encode for a particular protein are inserted into host microorganisms using re~ombinant DNA technology. By growing the transformant microorganisms under condition~ which induce the expression of proteins, heterologous proteins ~uch as insulin, somatotropins, interleukins, inter`ferons, somatomedins, and the like can be produced.
Unfortunately, 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 heterologouq proteins tend to form aggregates which are recognizable within the cell as l'inclusion bodies". These inclusion bodies may also be cau~ed by the formation of covalent intermolecular di~ulfide bonds which link together several protein molecules to form insoluble complexes. The inclusion 2~7~2~
bodies generally contain mostly heterologous proteins and a small fraction of contaminating host microorganism proteins.
Several processes have been developed to e~tract the inclusion bodies from the microorganisms and convert the heterologous proteins contained therein into proteins having native bioactivity consistent with the natural parent or non-recombinant proteins. These proces~es generally involvQ disrupting the microorganism cell, separating the inclusion bodiec from cell debris, solubilizing the inclusion body proteins in a denaturant/detergent which unfolds the protein, separating the heterologous inclusion body proteins from insoluble contaminan~s, removing the denaturant/detergent thereby allowing the heterologous proteins to refold into a bioactive tertiary conformation, and separating the protein from the contaminating proteins that remain in solution.
Several recombinant protein purification schemes following this genaral procedure are known in the art:
U~S. Patent Nos. 4,511,503 and 4,518,526 to Olson et al and U.S. Patent Nos. 4,511,502 and 4,620,948 to Builder et al disclose multi-step methods wherein (1) inclusion bodies are solubilized in a strong denaturant and a reducing agent, (2) insoluble contaminants are removed from the solubilized protein solution, (3) the strong denaturant is replaced with a weak denaturant, t4) the protein is allowed to refold assisted by oxidation of the sulfhydryl groups to disulfide bonds using molecular oxygen and a catalyst, typically metal cation~ or sodium tetrathionate, and (5) the protein is separated from other contaminating proteins by membrane separation techniques or chromatography procedures.
2t~7l 2~
Rausch et al, U.S. Pat. No. 4,677,196, incorporated herein by reference, discloses a particular method for purifying and activating proteins which is a variation of the general scheme described above. The method comprises solubili~ing the inclusion bodies in SDS, remo~ing the excass S~S from the solution using dialysis or other suitable technique, chroma~ographing the SDS-protein solution on an ion-retardation resin, and chromatographing the resulting solution on an anion-exchange re~in to recover the protein.
All these known procedures share a common problem~
The protein solution produced when the denaturant/detergent is removed contains the recombinant protein, low molecular weight contaminating proteins, non-protein contaminants, and high molecular weight contaminating proteins; the high molecular weight protein contaminants are often mostly dimers, oligomers and aggregates of the recombinant protein but al~o include non-recombinant protein~ from the cell digest. It is often difficult, time consuming, and expen~ive to separate the recombinant protein from these contaminants, particularly the recombinant protein dimers, oligomers and aggregates.
Chromatographic and membrane separation techniques may be effective for separating the recombinant proteins from the contaminants but are cumbersome, lengthy, expensive and often give low percentage yields for protein recovery.
New and improved methods for easily, quickly, and inexpensively recovering high yields of recombinant proteins from solutions containing high molecular weight protein contaminants are therefore needed~
2~ 2~0 Summary of the Invention It is, therefore, an ob~ect of the present invention to provide a new method for easily, quickly, and inexpensively recovering high yields of recombinant proteins from protein solutions containing high molecular weight contaminating proteins~
It is another ob~ect of the present invention to provide a method for removing high molecular weight contaminating proteins from a recombinant protein solution thereby allowing the easy, quick, and inexpensive recovery of the recombinant protein~
TheRe and other ob~ects are achieved by directly adding Group IIA metal salts to a solution containing high molecular weight contaminating proteins and a recombinant protein in amounts sufficient to selectively precipitate the high molecular weight protein contaminants. The compounds induce preferential precipitation of proteins having a molecular weight greater than about 1.5 times the molecular weight of the recombinant protein, particularly recombinant protein dimers, oligomers and aggregateq having a molecular weight greater than about 1.5 times the molecular weight of the recombinant protein. The precipitates are separated from the solution leaving the recombinant protein, low molecular weight contaminating proteins and other non-protein contaminants in solution. The recombinant protein is recovered from the solution using known techniques and processed to produce the desired protein product.
In the preferred embodiment, Group IIA metal salts are added directly to the solution in amounts sufficient to produce a 0.1-5% solution by volume. The high molecular weight contaminating proteins 2~7'12~
precipitate and are removed irom the solution by conventional means such as filtration, centrifugation, and the like. The resulting protein solution containing the recombinant protein, low molecular weight contaminating proteins and other non-protein contaminants is further processed using conventional techniques such as chromatograph~ to recovQr the recombinant protein.
Other ob~ects, advantagQs, and novel features of thQ present invention will becomQ apparent from the following detailed description of the invention.
Detailed De~c~iption of the Invention The term "recombinant protein`' as u~ed herein defines a protein produced by recomb`nant techniques which one desires to recover in a relatively pure form and includQs proteins having the amino acid sequence of native proteins and their analogs and muteins having substituted, deleted, replaced, or otherwise modified sequences.
The term `'recombinant somatotropin~ (rST) as used herein includes recombinant protein4 having the amino acid sequence of nati~e somatotropin, amino acid sequences substantially similar thereto, or an abbreviated sequence form thereof, and their analogs and muteins having qubstituted, deleted, replaced, or otherwise`modified sequences. In particular, rST as used herein includes a protein of the same sequence as native somatotropin (ST), but having amino acids deleted from its amino terminal end. Examples of such proteins include but are not limited to delta-7 recombinant porcine somatotropin, delta-4 recombinant bovine somatotropin, and the like.
2~7~2~
The term `'high molecular weight contaminating proteins~' or "high molecular weight protein contaminants" as used herein refers to proteins having a molecular weight greater than about 1.5 times the S molecular weight of the recombinant protein, particularly recombinant protein dimers, oligomers and aggregates having a molecular weight greater than about ~ times the molecular weight of the recombinant protein~
The term "low molecular weight contaminating proteins" or "low molecular weight protein contaminants" as used herein refers t~ proteins having a molecular weight less than about 1.5 times the molecular weight of the recombinant protein.
The term ~`non-protein contaminantq" as used herein refers to relatively low molecular weight substances such as precipitating agents, solubilizing agents, oxidizing agents, reducing agent~, and the like which are typically in a protein solution.
According to the present invention, a method is provided for recovering a recombinant protein from a protein solution containing high molecular weight contaminating proteins. The method comprises directly adding Group IIA metal salts to the solution containing high molecular weight contaminating proteins and the recombinant protein in amounts suf ficient to selectively precipitate the high molecular weight protein contaminants. The Group IIA metal salts preferentially induce the precipitation of proteins having a molecular weight greater than about 1.5 times the molecular weight of the recombinant protein, particularly recombinant protein dimers, oligomers and aggregates having a molecular weight greater than about 2 timeq the molecular weight of the recombinant 2~7d~0 protein. The method provides an improved method for easily, quickly, and inexpensively recovering high yields of recombinant proteins from solutions containing high molecular weight protein contaminants.
In ~he pre~erred embodiment, a method is provided ~or recovering recombinant somatotropins (molecular weight about 20,000) by directly adding Group IIA metal salts to soluti~ns containing high molecular weight contaminating protein~ and recombinant somatotropins in amounts sufficient to selectively precipitate the high molecular weight protein contaminants. Group IIA metal salts preferentially induce the precipitation of proteins having a molecular weight greater than about 1.5 times the molecular weight of th~ recombinant somatotropin (molecular weight greater than about 30,000), particularly recombinant somatotropin dimers, oligomers and aggregates having a molecular weight greater than about 2 times the molecular weight of the recombinant somatotropin (molecular weight about 40,000 and up). The present method, therefore, provides a method for separating the recombinant ~omatotropin from its bioinactive dimers, oligomers and aggregates.
Solutions containing a recombinant protein, non-protein contaminants, high molecular weight protein contaminants, and low molecular weight protein contaminantq useful in the pre~ent invention are obtained by m~thod~ known in the art. Typically, protein inclusion bodies which have been produced by recombinant microorganisms are processed to remove lipid~ and cell debris and the resulting relatively pure inclusion bodies containing recombinant protein and contaminating protein~, particularly high molecular weight recombinant protein dimers, oligomer~ and aggregates, are solubilized in a strong denaturant or 2~7~
detergent such as guanidine hydrochloride, sodium dodecyl sulfate (SDS), Triton, and the like.
The resulting protein solution is separated from any insoluble materials and the strong denaturant or detergent is removed to produce a protein solution containing the recombinant protein refolded into its native bioactive configuration, high molecular weight protein contaminants, low molecular weight contaminating proteins and other non-protein contaminants~ Such solutions typically contain from about 1-50 mg/ml total protein and from about 0~05-4 mg/ml recombinant protein~
The polymer to monomer (P/M) ratio of such a solution includes a mass ratio of ag~regates including void peak to monomer and is thus a measure of purity of the solution. The solution typically goe~ through a "prepurification" step where P/M ratio is reduced to about 0.5 or less. The solution can then be further purified and processed into a final product, typically using a DEAE Sepharose or other suitable column~ The present invention provide~ a method for removing the high molecular weight contaminating proteins while simultaneously achieving higher monomer recovery and a lower P/M ratio.
Group IIA metal salts are added to this solution according to the present invention to precipitate the high molecular weight contaminating proteins~ The high molecular weight contaminating proteins that precipitate upon addition of the Group IIA metal salts are removed from the solution by conventional means such as filtration, centrifugation, and the like~ The resulting protein solution containing the recombinant protein, low molecular weight contaminating proteins and other non-protein contaminants, if any, is further 2~71 ,~
processed, as needed, to remove low molecular weight contaminating proteins and other non-protein contaminants such as precipitating agents, solubilizing agents, oxidizing agents, reducing agents, and the like. Typically, such non`-pro~ein contaminants are removed by dialysis, chromatography, or other suitable means whereas the low molecular weight contaminating proteins are separated from the protein by ion-exchange or other form~ of chromatography~
The protein solution is further processed to produce 8 protein or protein composition suitable for its intended use, typically by lyophilization. These methods are well known in the art~
Group IIA metal salts useful in the present invention include all salts of the elements in Group IIA of the Periodic Table: beryllium, magnesium, calcium, strontium, barium and radium. Preferred compounds include salts of magnesium, calcium, barium and strontium.
Most preferably, the compounds of the present invention include the -~ulfate (SO,), chloride (C12), and nitrate (NO3) ~alt~ of magnesium, calcium, barium and strontium. Preferred compounds include calcium sulfate (caso4);`anhydrous CaSO~, CaS0~ hemihydrate, calcium nitrate (Ca(NO3)2), calcium lactate, magnesium sulfate (MgS0~), magnesium chloride (MgCl2), barium chloride (BaCl2 and strontium chloride (SrCl2).
Although the amount of Group IIA metal salts needed to induce precipitation varies depending on protein concentration, protein characteristic~, compound added, and the like, the Group IIA metal sa~ts are typically added to the solution in amounts sufficient to produce from about a 0~1-5~ by volume 2 ~ a solution of the compound, preferably from about a l-3 solution by volume.
Recombinant proteins recoverable using the method of the present invention can be any ~rotein having a molecular weight greater than about 5000 which are produced by recombinant microorganisms, typically in inclusion bodies. These include somatotropins, insulins, somatomedins, somatostatins, prolactins, placental lactogens, and the like~
Most preferably, recombinant somatotropins (molecular weight about 20,000) are recovered using the method of the present invention. The recombinant somatotropin can be a recombinant somatotropin from any species but are preferably bovine, porcine, avian, ovine, or human recombinant somatotropin, most preferably porcine or bovine recombinant somatotropin.
Methods for producing these recombinant proteins are well known in the art: For example, U.S. Patent Nos. 4,604,359 and 4,332,717 disclo~e methods for producing human recombinant somatotropin; U.S. Patent No. 4,431,739 discloses a method for producing recombinant somatotropins; E.P. Patent Application 0 104 920 discloses a method for producing recombinant porcine somatotropin; U.S. Patent No. 4,443,359 discloses a method for producing recombinant bovine somatotropin; Schoner, BiotechnoloqY, 3(2):151-54, discloses a method for producing recombinant somatotropin, and ~uell, Nucleic Acid Res., 13, 1923-38 (1985) disclose~ a method for producing recombinant somatomedin C.
Also, European Patent Application Publication No.
0 103 395 describes the construction of a transformant strain of E. coli containing a first plasmid which codes for delta 9 (Ser) bovine somatotropin 2~7~12~ ~
ll (~omatotropin less its 9 N-terminal amino acids and having an additional serine residue at the N-terminus) under the control of the lambda PL promoter-operator and which has a Shine-Dalgarno region derived from bacteriophage mu. The transformant also contains ~
second plasmid, pcI857, which codes for the production of the pcI857 temperature-sensitive repressor protein~
The repressor protein can be inactivated by raising the temperature to about 42C, thereby inducing expression of delta 9 (SQr) bovine somatotropin~ A transformant strain o~ this type, E~ coli HB101 (PL-mu-delta 9 (Ser) bovine somatotropin and pcI857) has been deposited with The American Type Culture Collection (ATCC), ~oc~ville, MD and assigned Accession No. 53030.
Construction of a similar transformant strain which codes for the production of delta 7 porcine somatotropin (porcine somatotropin less its fir~t 7 N-terminal amino acids) is de~cribed in European Patent Application Publication No. 0 104 920. A transformant strain of thi~ type, E. coli H~101 (PL-mu-delta 7 porcine somatotropin and pcI857), has been deposited with ATCC and a~signed Acces~ion No. 53031.
Strains 53030 and 53031 are prolific producer~ of delta 3 (Ser) bovine somatotropin and delta 7 porcine somatotropin, respectively~ In both instances, the expressed protein is se~uestered within the cell in the form of inqoluble, biologically inactive inclusion bodies which are visible under a microscope. Other methods for many similar protein~ are known in the art~
In the preferred embodiment, a recombinant somatotropin solution containing from about 1-50 mg/ml total protein and from about 0.05-2 mg/ml recombinant somatotropin i8 treated with from about 1-3% CaSO6 to precipitate the high molecular weight protein 12 2~7~
contaminants. The precipitate is removed by centrifugation and the recombinant somatotropin is recovered from the resulting solution using conventional means as described above~
Although the above recovery method is directed to recovering recombinant proteins, the method is equally applicable to separating and recovering non-recombinant proteins. For example, a solution containing a mixture o~ ~1) a '`useful or wanted protain~ 2) high molecular weight p.rotein~ (molecular weight greater than about 1.5 times the molecular weight of the useful protein) and (3) low molecular weight proteins (molecular weight 1Q8S than about 1.5 times the molecular weight of the useful protein) is treated according to the present invention to precipitate the high mo ecular weight proteins and thus separate the high molecular weight proteins from the useful protein and the low molecular weight proteins. The high molecular weight proteins are separated from the solution and discarded or further processed, as desired; the high molecular weight proteins can be recovered from the precipitate by redissolving the precipitate and recovering the proteins from the solution.
The useful protein is separated from the low molecular weight proteins by conventional means and further processed, if desired, to produce a protein product. The low molecular weight proteins which were separated from the useful protein are discarded or further processed, as desired. Typically, the low molecular weight proteins can be separated from the u~eful protein by chromatography or other means ~uitable for separating proteins having similar molecular weights. Many such protein ~eparation means 13 2 ?~) 7 2 ~ ~
are well known to skilled artisans and are equally applicable in the present invention.
The invention having been generally described, the following examples are given as particular embodiments of the invention and to demonstrate the practice and advantages thereof. It is understood that the examples are giVQn by way of illustration and are not intended to limit the specification or the claims to follow in any manner~ In particular, inclu~ion bodies used in the experiments were prepared from transformed E. Coli strains which produce delta-7 porcine somatotropin~
The inclusion bodies were isolated from E. Coli host strain HB101 transformed with a first plasmid (pL-mu-delta-7 pST) coding for delta-7 pST and a second plasmid (pcI857) coding for the temperature sensitive lambda phage repression protein. Many other strains of microorganisms produce inclusion bodies containing many types of recombinant proteins which will function in the present invention. Similarly, methods for growing thQse microorganisms to produce inclusion bodies are well known in the art.
Example 1 Recombinant porcine somatotropin (rpST) was recoverè~ from microorganism inclusion bodies by (1) dissolving the inclusion bodie~ in sodium dodecyl sulfate (SDS) in carbonate buffer (25 mM NaHC03 and 21 mN Na2C03), (2) removing insoluble contaminants from the solution, (3) adding an oxidizing agent for oxidation of rpsT, and (4) removing the SDS from the solution to allow the rpST to refold into its bioactive configuration. The resulting protein solution contained the rpST, high molecular weight protein contaminants, low molecular weight contaminating proteins and other non-protein contaminants.
14 2 ~ 71~2 CaSO~.2H~O was added at 2.5~ (w/v) concentration to the above refolded protein solution which contained 0.67 mg/ml of rpsT monomer with a P/M ratio of 5.1 (pH
9.8 at 22-25C) and mixed for 2 hours. The mixture was centrif~ged at about x5000 & for 10 minutes to recover the supernatant. The rpsT monomer and P/M ratio of the supernatant was analyzed by Superose-12 Fast Protein Liquid Chromatography. The result showed that the P~
ratio was reducQd to 0.3 and that the monom~r recovery was 83.0~. The dsta showed that the protein aggrega~e impurities and high molecular weight contaminating protQins were selectively precipitated by CaSO4~2H~O
while leaving the monomers in solution.
Example 2 Example 1 was repeated using other calcium salts:
anhydrous CaSO4, CaSO4 hemihydrate, calcium nitrate (Ca(NO3) 2 ) and calcium lactate. The result~ are shown in Table 1.
Referring to Table 1, the data shows that the aggregate impurities and high molecular weight contaminating proteins were ~electively precipitated by the addition of these calcium salts.
Example 3 Example 1 was repeated using alkaline metal salts of magnesium, barium and strontium. The results are shown in Table 2.
Referring to Table 2, the data shows that the aggregate impurities and high molecular weight contaminating protein~ were ~electively precipitated by these alkaline metal ~alts.
Example 4 CaCl2-2H2O was added at 0.4~ (w/v) concentration to the refolded protein solution in Example 1 which contained 0.4 mg/ml pST monomer with P/M ratio of 4.7 2~7~250 and mixed for about 30 minutes at room temperature (22-25C). The pH of the mixture was adjusteq to about 9.0 and the supernatant of the mixture was collected after centrifuging at xS,000 G for 10 minutes. The resulting S supernatant was analyzed by Superose-12 FPLC.
The result showed that the P~M ratio was reduced to 0.4 with a monomer recovery of 92.1~. This data shows that CaCl2~2~l2O precipitates the protein aggregates and high molecular weight contaminating proteins selectively leaving monomer~ in solution.
Example 5 The refolded protQin solution in Example 1 was approximately 2x concentrated and diafiltered against 80 mM ethanolamine buffer at pH 9.O. To the diafiltered protein solution, which contained 0~67 mg~ml rpsT monomer with P/M ratio of 3.5, CaCl2-2~2O was added at 0.4~ ~w/v) con~entration and mixed for 30 minuteq at 22-25C. The supernatant was separated by centrifuging at x5,000 G for 10 minutes. The supernatant was analyzed by Superose-12 FPLC.
The result showed the P/M ratio was reduced to O.22 and a monomer recovery was 91.5~. Thi~ data shows that CaCl2-2H2O selectively precipitates the protein aggregatè~ and high molecular weight contaminating proteins in an ethanolamine buffer ~ystem.
Obviously many modifications and variation~ of the prQsent invention are poqsible in light of the above teachings. It is therefore to be understood that within the scope of the appended claims the invention may be practiced otherwise than as specifically described.
2~7d~
T8blQ 1 Concentration Initial Final Monomer Calcium salt ~ (w/v)] P/M P/M Recovery(%) _____________________________________~________________ Anhydrous CaSO~ 2.5 3~0 0.03 87.3 CaSO~ hemihydrate 2.5 3.6 0.05 83~7 Calcium nitrate 2.5 3.3 0~2 91~0 Calcium lactate 2.S 4.1 0.06 72.S
_____________________________________~_________________ Table 2 Concentration Initial ~inal Monomer Salt ~ (w/v)] P/N P/M Recovery(%) _______________________________________________________ MgSO~-7H2O 2.5 3.0 0.1 71.0 MgCl2 1.0 3.0 0.3 92.0 BaCl2 1.6 3.7 0.5 50.0 srcl2~6H2o 1.22 3.7 o.o 43.0 _______________________________________________________
Claims (30)
1. A method for recovering a recombinant protein from a protein solution containing high molecular weight contaminating proteins and the recombinant protein, comprising:
directly adding Group IIA metal salts to the solution in amounts sufficient to selectively precipitate the high molecular weight contaminating proteins;
separating the precipitate from the solution; and recovering the recombinant protein from the solution.
directly adding Group IIA metal salts to the solution in amounts sufficient to selectively precipitate the high molecular weight contaminating proteins;
separating the precipitate from the solution; and recovering the recombinant protein from the solution.
2. The method of Claim 1 wherein the total protein concentration of the solution is from about 1-50 mg/ml and the recombinant protein concentration of the solution is from about 0.05-4 mg/ml.
3. The method of Claim 1 wherein the Group IIA
metal salts are added directly to the solution in amounts sufficient to produce a 0.1-10% solution by volume.
metal salts are added directly to the solution in amounts sufficient to produce a 0.1-10% solution by volume.
4. The method of Claim 1 wherein the Group IIA
metal salts are selected from the group consisting of beryllium, magnesium, calcium, strontium, barium and radium salts.
metal salts are selected from the group consisting of beryllium, magnesium, calcium, strontium, barium and radium salts.
5. The method of Claim 4 wherein the Group IIA
metal salts are selected from the group consisting of sulfate (SO4), chloride (Cl2), and nitrate (NO3) salts.
metal salts are selected from the group consisting of sulfate (SO4), chloride (Cl2), and nitrate (NO3) salts.
6. The method of Claim 1 wherein the Group IIA
metal qalts are selected from the group consisting of magnesium, calcium, strontium and barium salts.
metal qalts are selected from the group consisting of magnesium, calcium, strontium and barium salts.
7. The method of Claim 6 wherein the Group IIA
metal salts are selected from the group consisting of sulfate (SO4), chloride (Cl2), and nitrate (NO3) salts.
metal salts are selected from the group consisting of sulfate (SO4), chloride (Cl2), and nitrate (NO3) salts.
8. The method of Claim 1 wherein the Group IIA
metal salts are selected from the group consisting of anhydrous calcium sulfate (CaSO4), CaSO4 dihydrate, CaSO4 hemihydrate, calcium chlorider calcium chloride dihydrate, calcium nitrate (Ca(NO3)2), calcium lactate, calcium formate, magnesium sulfate (MgSO4), magnesium chloride (MgCl2), barium chloride (BaCl2) and strontium chloride (SrCl2).
metal salts are selected from the group consisting of anhydrous calcium sulfate (CaSO4), CaSO4 dihydrate, CaSO4 hemihydrate, calcium chlorider calcium chloride dihydrate, calcium nitrate (Ca(NO3)2), calcium lactate, calcium formate, magnesium sulfate (MgSO4), magnesium chloride (MgCl2), barium chloride (BaCl2) and strontium chloride (SrCl2).
9. The method of Claim 1 wherein the high molacular weight contaminating proteins have a molecular weight of greater than about 30,000 and the recombinant protein to be recovered has a molecular weight of about 20,000.
10. The method of Claim 1 wherein the recombinant protein is a somatotropin.
11. The method of Claim 10 wherein the recombinant somatotropin is bovine, porcine, avian, ovine or human recombinant somatotropin.
12. The method of Claim 11 wherein the recombinant somatotropin is porcine or bovine recombinant somatotropin.
13. A method for recovering recombinant somatotropin from a protein solution containing high molecular weight contaminating proteins and the recombinant somatotropin, comprising:
directly adding Group IIA metal salts to the solution in amounts sufficient to selectively precipitate the high molecular weight contaminating proteins;
separating the precipitate from the solution; and recovering the recombinant protein from the solution.
directly adding Group IIA metal salts to the solution in amounts sufficient to selectively precipitate the high molecular weight contaminating proteins;
separating the precipitate from the solution; and recovering the recombinant protein from the solution.
14. The method of Claim 13 wherein the total protein concentration of the solution is from about 1-50 mg/ml and the recombinant somatotropin concentration of the solution is from about 0.05-4 mg/ml.
15. The method of Claim 13 wherein the Group IIA
metal salts are added directly to the solution in amounts sufficient to produce a 0.1-10% solution by volume.
metal salts are added directly to the solution in amounts sufficient to produce a 0.1-10% solution by volume.
16. The method of Claim 13 wherein the Group IIA
metal salts aro selected from the group consisting of beryllium, magnesium, calcium, strontium, barium and radium salts.
metal salts aro selected from the group consisting of beryllium, magnesium, calcium, strontium, barium and radium salts.
17. The method of Claim 16 wherein the Group IIA
metal salts are sQlected from the group consisting of sulfate (SO4), chloride (Cl2), and nitrate (NO3) salts.
metal salts are sQlected from the group consisting of sulfate (SO4), chloride (Cl2), and nitrate (NO3) salts.
18. The method of Claim 13 wherein the Group IIA
metal salts are selected from the group consisting of magnesium, calcium, strontium and barium salts.
metal salts are selected from the group consisting of magnesium, calcium, strontium and barium salts.
19. The method of Claim 18 wherein the Group IIA
metal salts are selected from the group consisting of sulfate (SO4), chloride (Cl2), and nitrate (NO3) salts.
metal salts are selected from the group consisting of sulfate (SO4), chloride (Cl2), and nitrate (NO3) salts.
20. The method of Claim 13 wherein the Group IIA
metal salts aro selected from the group consiRting of anhydrous calcium sulfate (CASO4), CaSO4 dihydrate, CaSO4 hemihydrate, calcium nitrate (Ca(NO3)2), calcium chloride, calcium chloride dihydrate, calcium lactate, calcium formate, magnesium sulfate (MgSO4), magnesium chloride (MgCl2), barium chloride (BaCl2) and strontium chloride (SrCl2).
metal salts aro selected from the group consiRting of anhydrous calcium sulfate (CASO4), CaSO4 dihydrate, CaSO4 hemihydrate, calcium nitrate (Ca(NO3)2), calcium chloride, calcium chloride dihydrate, calcium lactate, calcium formate, magnesium sulfate (MgSO4), magnesium chloride (MgCl2), barium chloride (BaCl2) and strontium chloride (SrCl2).
21. The method of Claim 13 wherein the high molecular weight contaminating proteins have a molecular weight of greater than about 30,000.
22. A method for separating and recovering a protein from a protein solution containing the protein, high molecular weight proteins and low molecular weight proteins, comprising:
directly adding Group IIA metal salts to the solution in amounts sufficient to selectively precipitate the high molecular weight contaminating proteins;
separating the precipitate from the solution; and recovering the recombinant protein from the solution.
directly adding Group IIA metal salts to the solution in amounts sufficient to selectively precipitate the high molecular weight contaminating proteins;
separating the precipitate from the solution; and recovering the recombinant protein from the solution.
23. The method of Claim 22 wherein the total protein concentration of the solution is from about 1-50 mg/ml and the protein concentration of the solution is from about 0.05-4 mg/ml.
24. The method of Claim 22 wherein the Group IIA
metal salts are added directly to the solution in amounts sufficient to produce a 0.1-10% solution by volume.
metal salts are added directly to the solution in amounts sufficient to produce a 0.1-10% solution by volume.
25. The method of Claim 22 wherein the Group IIA
metal salts are selected from the group consisting of beryllium, magnesium, calcium, strontium, barium and radium salts.
metal salts are selected from the group consisting of beryllium, magnesium, calcium, strontium, barium and radium salts.
26. The method of Claim 25 wherein the Group IIA
metal salts are selected from the group consisting of sulfate (SO4), chloride (Cl2), and nitrate (NO3) salts.
metal salts are selected from the group consisting of sulfate (SO4), chloride (Cl2), and nitrate (NO3) salts.
27. The method of Claim 22 wherein the Group IIA
metal salts are selected from the group consisting of maqnesium, calcium, strontium and barium salts.
metal salts are selected from the group consisting of maqnesium, calcium, strontium and barium salts.
28. The method of Claim 27 wherein the Group IIA
metal salts are selected from the group consisting of sulfate (SO4), chloride (Cl2), and nitrate (NO3) salts.
metal salts are selected from the group consisting of sulfate (SO4), chloride (Cl2), and nitrate (NO3) salts.
29. The method of Claim 22 wherein the Group IIA
metal salts are selected from the group consisting of anhydrous calcium sulfate (CaSO4), CaSO4 dihydrate, CaSO4 hemihydrate, calcium nitrate (Ca(NO3)2), calcium chloride, calcium chloride dihydrate, calcium lactate, calcium formate, magnesium sulfate (MgSO4), magnesium chloride (MgCl2), barium chloride (BaCl2) and strontium chloride (SrCl2).
metal salts are selected from the group consisting of anhydrous calcium sulfate (CaSO4), CaSO4 dihydrate, CaSO4 hemihydrate, calcium nitrate (Ca(NO3)2), calcium chloride, calcium chloride dihydrate, calcium lactate, calcium formate, magnesium sulfate (MgSO4), magnesium chloride (MgCl2), barium chloride (BaCl2) and strontium chloride (SrCl2).
30. The method of Claim 22 wherein the high molecular weight contaminating proteins have a molecular weight of greater than about 30,000 and the protein to be recovered has a molecular weight of about 20,000.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US07/468,054 US4988798A (en) | 1990-01-22 | 1990-01-22 | Method for recovering recombinant proteins |
| US468,054 | 1990-01-22 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2074250A1 true CA2074250A1 (en) | 1991-07-23 |
Family
ID=23858261
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002074250A Abandoned CA2074250A1 (en) | 1990-01-22 | 1990-10-01 | Method for recovering recombinant proteins |
Country Status (7)
| Country | Link |
|---|---|
| US (1) | US4988798A (en) |
| EP (1) | EP0511959A1 (en) |
| JP (1) | JPH05503212A (en) |
| AU (1) | AU643712B2 (en) |
| BR (1) | BR9007992A (en) |
| CA (1) | CA2074250A1 (en) |
| WO (1) | WO1991010678A1 (en) |
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| US5109121A (en) * | 1990-01-22 | 1992-04-28 | Pitman-Moore, Inc. | Method for recovering recombinant proteins |
| GB9304399D0 (en) * | 1993-03-04 | 1993-04-21 | Smithkline Beecham Biolog | Novel process |
| US5760189A (en) * | 1995-06-02 | 1998-06-02 | Genetics Institute, Inc. | Protein recovery & purification methods |
| JP2000506145A (en) * | 1996-03-08 | 2000-05-23 | ノボ ノルディスク アクティーゼルスカブ | Crystallization of proteins by sulfide salts |
| HUE027196T2 (en) * | 2002-09-06 | 2016-10-28 | Genentech Inc | Process for protein extraction |
| WO2004022593A2 (en) * | 2002-09-09 | 2004-03-18 | Nautilus Biotech | Rational evolution of cytokines for higher stability, the cytokines and encoding nucleic acid molecules |
| US7998930B2 (en) | 2004-11-04 | 2011-08-16 | Hanall Biopharma Co., Ltd. | Modified growth hormones |
| FR2901796A1 (en) * | 2006-05-31 | 2007-12-07 | Lab Francais Du Fractionnement | PROCESS FOR EXTRACTING ONE OR MORE PROTEINS PRESENT IN MILK |
| EP2078039B1 (en) * | 2006-11-01 | 2017-09-13 | Biogen MA Inc. | Method of isolating biomacromolecules using low ph and divalent cations |
| JPWO2018235958A1 (en) * | 2017-06-23 | 2020-04-23 | Ｓｐｉｂｅｒ株式会社 | Protein purification method, protein solution production method, and protein molded article production method |
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| US3579495A (en) * | 1970-04-24 | 1971-05-18 | Diagnostic Data Inc | Isolation of orgotein from blood |
| US4683294A (en) * | 1985-04-03 | 1987-07-28 | Smith Kline Rit, S.A. | Process for the extraction and purification of proteins from culture media producing them |
| DE3580590D1 (en) * | 1985-05-29 | 1990-12-20 | Green Cross Corp | MANUFACTURING PROCESS FOR HETEROGENEOUS PROTEINS. |
| WO1987000553A1 (en) * | 1985-07-16 | 1987-01-29 | The Green Cross Corporation | Process for preparing hetero-protein |
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1990
- 1990-01-22 US US07/468,054 patent/US4988798A/en not_active Expired - Lifetime
- 1990-10-01 JP JP2514182A patent/JPH05503212A/en active Pending
- 1990-10-01 WO PCT/US1990/005543 patent/WO1991010678A1/en not_active Application Discontinuation
- 1990-10-01 CA CA002074250A patent/CA2074250A1/en not_active Abandoned
- 1990-10-01 EP EP90915267A patent/EP0511959A1/en not_active Ceased
- 1990-10-01 AU AU65311/90A patent/AU643712B2/en not_active Ceased
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| AU643712B2 (en) | 1993-11-25 |
| BR9007992A (en) | 1992-11-17 |
| JPH05503212A (en) | 1993-06-03 |
| US4988798A (en) | 1991-01-29 |
| EP0511959A1 (en) | 1992-11-11 |
| WO1991010678A1 (en) | 1991-07-25 |
| AU6531190A (en) | 1991-08-05 |
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