Title of Invention | PURIFICATION OF RECOMBINANT HUMAN PROTEINS |
---|---|
Abstract | The present invention provides a simple and economical method for the purification of some recombinant proteins free from host proteins, wherein at least 90- 95% pure recombinant active proteins can be obtained. The method employs RP-HPLC as a primary step using polystyrene based wide pore matrix wherein unfolded protein form of recombinant protein was isolated and used for refolding in a novel way. This process enables the inactive recombinant protein be converted to active folded form. Depending on the protein, the folded form was further purified by either gel filtration or ion exchange chromatography or any other proprietary chromatographic techniques wherein greater than 98% purity is obtained which meets the criteria for therapeutic proteins. |
Full Text | Rapid developments in biotechnology, such as genetic engineering and downstream processing have effectively made possible the production of recombinant protein molecules. As the world moves towards more biological protein based therapies and as more companies produce recombinant deoxyribonucleic acid (rDNA) derived molecules the efficiency of the process assumes significance. In addition to (the ideal culture conditions and better expression systems, the importance of simplification of protein purification technology for the production of recombinant protein molecules in commercially viable quantities is necessitated today than ever before. A decrease in the number of purification steps, improvements of the yields, preservation of the biological activity, use of techniques strictly selected on the basis of their simplicity, high performance and reproducibility, and possible use of automated techniques are major objectives in defining conditions. Biomolecules produced by rDNA technology can be endo or Intracellular proteins ('inside' the cell); Periplasmic (part of the cell wall) or extracellular proteins ('outside5 the cell in the culture-medium). They can be produced using a variety of organisms like E.coli; S.cerevisiae; P.pastdris etc. The present invention relates specifically to endocellular rDNA derived biomolecules produced in E.coli as 'inclusion bodies'or 'refractile' bodies. Endocellular proteins have to be purified from a cell extract containing a complex mixture of molecules (proteins, lipids, nucleic acids) that result from cell lysis. Then isolation, identification, and purification of the factor under investigation require a more or less complex technology that includes extraction, centrifugation and various separation techniques. This calls for different chromatographic techniques when the expected protein has to be highly purified. Chromatography is of greatest interest because it produces very good separation and opens way to many prospects. It is based essentially upon the various interactions between the molecules to be separated and the stationary phase constituting the support. These different techniques can be used separately or in combination. Brief Summary of the Invention Inclusion bodies containing the protein of interest can be produced as a general flow chart as listed in Figure 1. The present invention is limited to the use of Reversed Phase High ■» s performance Liquid Chromatography (RP-HPLC) as a purification and subsequently combined with refolding step in the production of highly pure refolded active rDNA derived proteins. Ion exchange and gel filtration are widely used in commercial production of recombinant molecules. These are known techniques that practically grew up with protein chemistry. These previous methods used for the purification of rDNA derived proteins (either from yeast or E.coli) likel cationic chromatography, gel filtration (or even reversed phase HPLC for tjiat-matter) resulted in yields that were low. Also the purified protein sometimes had clips or cleavages, which were still held together by disulfide bonds. In addition, further refolding parameters like different pH, oxidation and reduction systems using mercaptoethanol, DTT, glutathione and affinity chromatography like heparin sepharose column were also necessitated making the purification process lengthy and complex. Reversed phase was developed primarily for the analysis of small molecules and its potential was mostly unrealized until the introduction of wide pore RP adsorbents in the 1980's. Even after this its use was mostly limited to analytical scale due to the misconceptions about its practicality in the process scale and due to the apprehensions of loss of biological activity especially for recombinant proteins. This loss of biological activity is often attributed to the solvents used. Though many a commercial chromatographic processes use RP today, it is mainly for what are referred colloquially as 'polishing' steps in the tertiary or end stage purification process. It is evident to those skilled in the art that this technique has not been used in the initial stages especially with respect Uo purification of endocellular recombinant DNA molecules produced in E.coli. Thus there is need in the art of purification some rDNA derived molecules from E.Coli where in simplified quick procedure is followed leading to highly pure form with good t activity and the procedure should be ensured free of problems of purification and refolding. The present invention provides such possibility over other related methods. It is aim of this invention to show that this technique can used in the initial stages to provide a simple and trouble free method of purification of some recombinant DNA derived proteins free from host proteins wherein at least 90- 95% pure recombinant proteins can.be obtained. The method employs RP-HPLC using polystyrene based wide pore matrix wherein unfolded protein form of recombinant protein was isolated and used for refolding in a novel way. This process enables protein to be converted to active folded form in a simplified unique step which is combined with RPHPLC method that greatly increases the yield and reduce the need for elaborate multi stage processes. This process has demonstrated in three different molecules but is not limited in any way to these three molecules. It is the claim of this invention that this process can be used for a purification of range of rDNA derived inclusion body proteins produced in E.coli. .i i Description of Drawings: Figure 1 is the flow c&iit of the process. Detailed Description of the invention The figure 1 submitted along with description shows the flow chart of the method of the purification of recombinant human proteins wherein the white coloured marked steps is the inventive steps related to the present description. In one aspect, the present invention relates to improved RP-HPLC methods for purifying recombinant proteins and subsequent using simple and novel refolding procedures circumventing the use of complex refolding procedures and purification protocols. This invention shows that this technique can be used in the initial stages to provide a simple and trouble free method of purification of some recombinant DNA derived proteins free from host proteins wherein at least 90- 95% pure recombinant proteins can be obtained. The method employs RP-HPLC using polystyrene based wide pore matrix wherein unfolded protein form of recombinant protein was isolated and used for refolding in a novel wayJ? This process enables protein to be converted to active folded form in a simplified unique step which is combined with RPHPLC method that greatly increases the yield and reduce the need for elaborate multi stage processes. \ ■ ' • The RP-HPLC methods comprise using wide pore preparative polystyrene hydrophobic \ matrix and solvent system consisting of but not limited to acetonitrile; isopropanol; ethanol or methanol as an organic modifier and an organic acid like but not limited to trifluoroacetic; acetic acid; hepta fluorobutaric acid etc. The bonded phase is wide pore polystyrene/divinylbenzene matrix with a pore size of 30um or more preferably 30um using trifluoroacetic as organic acid. The organic acid can also be acetic acid or hepta fluorobutaric acid etc. The concentration can be 0.1% to 0.3% preferably in the pH range 3-7 more preferably pH 6. The elution can be isocratic or gradient, preferably gradient either linear or nonlinear, wherein the concentration of solvent is 0-100% preferably 20-80%. The solvent consists of but is not limited to acetonitrile; isopropanol; ethanol or methanol. The volume of elution is preferably balanced with the amount of sample loaded on to the column. The unfolded recombinant protein eluted from RPHPLC column is diluted ten fold or higher keeping a protein concentration in the range of 10-200 ug/ml using buffers preferably in the range PH 3-7. The refolded protein was concentrated, diafiltered depending upon the size of the protein and concentrated. The protein was further purified in order to obtain greater than 98 % pure. * *l After obtaujijig the refolded form of the protein subsequent steps of the purification are undertaken with specific consideration of the protein in particular to obtain >98% pure protein. f - ' ■ * 11 i The process has been demonstrated1 in,,at least three rDNA derived biomolecules, like but not limited to recombinant human platelet derived growth factor-B; recombinant human interferon alpha 2b; recombinant human interferon gamma lb. A skin wound is defined as a breach in the continuity of any body tissue caused by a minimal direct; injury to the skin. There are many instances where a quick closure of the wounded skin will promote a! beneficial response. Chronic, non healing wounds have plagued healthcare practitioners for decades. In this regard PDGF-BB has been shown to be active in promoting wound healing in several animal models. Human platelet-derived growth, factor has been shown to be the major mitogenic protein in serum for mesenchymal derived cells. A number of studies reported that platelet extracts or purified PDGF-BB induces either cell multiplication or DNA synthesis in cultured smooth muscle cells, fibroblasts and glial cells. Furthermore PDGF BB is a potent chemoattractant for cells that are responsive to it as a mitogen. This is also somewhat unusual in that mitogens generally do not also act as chemotactic agents. PDGF-BB has therapeutic applications for the treatment of injuries, which require the proliferation of fibroblasts or smooth muscle cells to heal. In this regard PDGF-BB has been shown to be active in promoting wbund healing in several animal models. Lynch et al. disclose the use of insulin like growth factor (IGF-1) and purified PDGF-BB to promote healing of dermal wounds in pigs. These two growth factors showed a synergestic effect in promoting the healing. Lynch et al also found that c combination PDGF-BB and IGF-1 promotes bone and cementum formation in a dog model of periodontitis. In addition Greenhalgh et al demonstrated enhanced healing of full-thickness skin wounds in genetically diabetic mice treated with recombinant PDGF-BB as compared to control animals. Thomason et al disclose that recombinant PDGF-BB accelerates the gain in tensile strength of healing skin wounds in rats and promotes wound healing in diabetic rats. Interferons are proteins naturally occurring in the body, which has antiviral, anti proliferative and immunoregulatory activity. In particular, it inhibits replication of a variety of RNA- and DNA-containing viruses, inhibits the growth of malignant cells, affects the expression of a variety of oncogenes and activates natural killer cells. Recombinant human interferon alpha is used for the treatment of hairy cell leukemia, AIDS-related ^Kaposi's sarcoma and chronic hepatitis B and C. Before recombinant DNA technology, interferon alpha (leukocyte interferon) used to be prepared from natural sources, human leukocyte. Today, recombinant interferons are prepared from microbial source, e.g!, £. coli and therefore, after their isolation from the micro organism or from the culture 'medium they are initially contaminated by a series of microbial impurities presence of which is prohibitive for a therapeutic use of the proteins produced this way. Recombinant DNA derived preparations should be highly pure and homogeneous with required biological activity. The purification of the recombinant material, therefore, plays a particularly important role. Example 1-Recombinant Human Interferon Alpha 2b (rhIFNa2b) The E.C6U was used for the manufacture of incorporating the gene for expression of rhIFNa2b in a suitable vecfor, E coli cells containing rhIFNa2b were harvested after fermentation, centrifuged, washed and lysed by sonication. The inclusion bodies were recovered by centriftigation and washed with buffers containing urea, high salt and detergent. Finally, the inclusion bodies were washed with distilled water. The rhIFNa2b was extracted with 6M GuanidineCl and diluted by 50 fold dilution in 100 mM Tris-HCl5 10 mM EDTA, pH 8.0 buffer containing 0.1 M L-arginine. The diluted solution was concentrated by ultrafiltration using lOkD cutoff membrane and loaded on to reverse phase polystyrene column equilibrated with 0.1% TFA solution. The bound protein is eluted using 30-60% acetonitrile over 60 min. The recombinant rhINF-lb eluting at 40% acetonitrile is pooled, diluted to 10 fold and diafiltered and used for subsequent purposes. The diafiltered sample was passed through 0.2u filter and the turbidity was removed. The diafiltered sample is almost 90% pure and has the activity comparing to the WHO standards. The diafiltered solution was fractionated on Superdex 75 to remove any aggregates. , • V,' 1 Example 2- Recombinant Human Interferon gamma lb (rhlFNylb) \ . The E.Coli wris used for the manufacture incorporating the gene for expression rhlFNylb in a suitable vector. E coli cells containing rhlFNylb were harvested after fermentation, centrifuged, washed and lysed by sbnicator/ bead beater. The inclusion bodies were recovered by cpntrifugation and washed with buffer containing detergent and urea. Finally, the inclusion bodies were purified by sucrose centriftigation at 6000 rpm and inclusion body pellet was washed with distilled water and stored at -70 deg C till further use. The rhlFNylb was extracted with 6M Gdn.Cl from inclusion body and loaded on to reverse phase polystyrene coltimnand equilibrated with 0% acetonitrile containing 0.1% TFA. The column was washed thoroughly with the starting buffer and the bound protein is eluted using 20-60% acetonitrile over 60 min. The monomelic form of rhlFNylb eluting at 40% acetonitrile is pooled, after analysis by SDS-PAGE. The pure monomer pooled fractions were concentrated and used for purposes. The pooled fraction was diluted ten fold in tris CI (50mM), pH 7.0 buffer and left couple of hours for 12 h for refolding. The refolded solution was concentrated by ultrafiltration using lOkD cutoff membrane to ten fold and diafiltered extensively. The diafiltered sample was passed through 0.2u filter and the turbidity was removed. The diafiltered sample is almost >98% pure dimer and has the activity comparing to the WHO standards. The diafiltered solution was left overnight and the fractionated on Sephacryl S-100 HR to remove any monomelic fraction. Even preferably S-Sepharose at pH 5.0 also can be used for further purification. Example 3- Recombinant Human Platelet derived Growth Factor-BB (rhPDGF-BB) E.Coli was used for the manufacture incorporating the gene for expression rhPDGF-BB in a suitable vector. E coli cells containing recombinant PDGF-BB were harvested after fermentation, centrifuged, washed arid lysed by sonicator/bead beater. The inclusion bodies were recovered by centrifugation and washed with buffer containing detergent. Finally, the inclusion bodies' were purified by sucrose centrifugation at 6000 rpm and inclusion body pellet was washed with distilled water and stored at -70 deg,C till further use.. The human r-PDGF-BB protein was extracted with 6M Gdn.Cl from inclusion body and loaded on to reverse phase polystyrene column and equilibrated with 30% Acetonitrile containing 0.1% TFA. The column was washed thoroughly with the starting buffer and the bound protein is eluted using 30-80% Acetonitrile over 60 min. The monomelic form of rhPDGF-BB eluting at 40% Acetonitrile is pooled, after analysis by SDS-PAGE. The pure monomer pooled fractions were concentrated and used for subsequent purposes. The pooled fraction was diluted ten fold in Tris CI (50mM), pH 8.0 buffer and left overnight for refolding. The refolded solution was concentrated by ultrafiltration using 1 OkD cutoff membrane to ten fold and diafiltered extensively. The diafiltered sample was passed through 0.2u filter and the turbidity was removed. The diafiltered sample is almost 95% pure dimer and has the activity comparing to the WHO standards. The diafiltered solution was left overnight and the fractionated on Sephacryl S-100 HR to remove any monomelic or highmolecular weight fraction. Even preferably S-Sepharose at pH 5.0 also can be used for further purification. We Claim, 1. A method for purification of human recombinant proteins wherein a polystyrene reversed phase column was used to purify recombinant protein comprising an unfolded protein with a linear gradient 0-100% of organic solvents containing 0.1- 0.3% organic acids and subsequently folded to active protein by steps linked to the RPHPLC method, the said refolding was performed by dilution to ten fold or higher by keeping the protein concentration around 10-200 micro gram per milliliter to obtain 90-95% purity, the said protein was concentrated, diafiltered and was further purified to obtain greater than 95% purity. 2. A method for purification of human recombinant proteins as per claim 1, wherein organic solvents used are acetonitrile, isopropanol, ethanol or methanol but not limited to these solvents. 3. A method for purification of human recombinant proteins as per claim 1, wherein organic acids used are triflouroacetic acid or any other organic acid like but not limited to acetic acid or heptaflouro butane acid. |
---|
107-che-2004-claims duplicate.pdf
107-che-2004-claims original.pdf
107-che-2004-correspondnece-others.pdf
107-che-2004-correspondnece-po.pdf
107-che-2004-description(complete) duplicate.pdf
107-che-2004-description(complete) original.pdf
Patent Number | 201898 | ||||||||
---|---|---|---|---|---|---|---|---|---|
Indian Patent Application Number | 107/CHE/2004 | ||||||||
PG Journal Number | 05/2007 | ||||||||
Publication Date | 02-Feb-2007 | ||||||||
Grant Date | 01-Sep-2006 | ||||||||
Date of Filing | 11-Feb-2004 | ||||||||
Name of Patentee | M/S. VIRCHOE BIOTECH PVT. LTD | ||||||||
Applicant Address | PLOT NO.4, S.V.CO-OPERATIVE INDUSTRIAL ESTATE, IDA, JEEDIMETLA, HYDERABAD | ||||||||
Inventors:
|
|||||||||
PCT International Classification Number | G01N30/00 | ||||||||
PCT International Application Number | N/A | ||||||||
PCT International Filing date | |||||||||
PCT Conventions:
|