WO2005077973A1

WO2005077973A1 - Purification of recombinant human proteins

Info

Publication number: WO2005077973A1
Application number: PCT/IN2005/000045
Authority: WO
Inventors: Sripad Gunwar; Murali Tummuru; Hemanth Nandigala
Original assignee: VIRCHOW BIOTECH PRIVATE Ltd
Current assignee: VIRCHOW BIOTECH PRIVATE Ltd
Priority date: 2004-02-11
Filing date: 2005-02-11
Publication date: 2005-08-25
Anticipated expiration: 2006-08-11

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 proteins can be obtained. The method employs RP-HPLC as primary step using polystyrene based wide pore matrix wherein unfold monomer form of recombinant protein was isolated and used for refolding in a novel way. This process enables the monomer to be converted to active folded form. Depending on the protein, the folded form was further purified by either gel filtration or ion exchange chromatography wherein greater than 98% purity is obtained which meets the criteria for therapeutic proteins.

Description

PURIFICATION OF RECOMBINANT HUMAN PROTEINS.

Filed of the invention:

The present invention provides a simple and economical method for the purification of recombinant proteins free from host proteins, wherein at least 90- 95% pure recombinant proteins can be obtained.

Background of the Invention

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 deoxyribonudeic 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 ('outside' the cell in the culture medium). They can be produced using a variety of organisms like E.coii; S.cerevisiae; P.pastoris etc. The present invention relates specifically to endocellular rDNA derived biomolecules produced in E.coii as 'inclusion 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. Chrαmatography 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.

References

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Objective of the Invention

The present invention relates to improved RP-HPLC methods for purifying recombinant proteins using simple and novel refolding procedures circumventing the use of complex refolding procedures and purification protocols. 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 performance Liquid Chromatography (RP-HPLC) as a purification and subsequently refolding step in the production of highly pure refolded active rDNA derived proteins.

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.

Brief Summary of the Invention:

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.coii) like cationic chromatography, gel filtration (or even reversed phase HPLC for that 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 to purification of endocellular recombinant DNA molecules produced in E.coii.

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 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 monomer form of recombinant protein was isolated and used for refolding in a novel way. This process enables the monomer to be converted to active folded form in a simplified unique step 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.coii. Detailed Description of the invention:

In one aspect, the present invention relates to improved RP-HPLC methods for purifying recombinant proteins using simple and novel refolding procedures circumventing the use of complex refolding procedures and purification protocols.

The RP-HPLC methods comprise using wide pore preparative polystyrene hydrophobic matrix and solvent system consisting of but not limited to acetonitrile; isopropanol; ethano! 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; ethano! or methanol. The volume of elution is preferably balanced with the amount of sample loaded on to the column.

After obtaining 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.

The process has been demonstrated 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 1 b.

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, nonhealing 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 ceils, fibroblasts and glial cells. Furthermore PDGF 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 wound 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 a! 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., E. 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

The E.coii was used for the manufacture contains an expression vector incorporating the gene for expression of recombinant proteins. E coli cells containing recombinant protein were harvested after fermentation, centrifuged, washed and 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 gradient centrifugation at 6000 rpm and inclusion body pellet was washed with distilled water and stored at -70 deg.C til! further use.

The human recombinant protein was extracted with 6M Guanidine Hydrochloride from inclusion body and loaded on to reverse phase polystyrene column (Resource 30 RPC) equilibrated with 30% Acetonitrile containing 0.1% TFA. The column was washed thoroughly with the starting buffer and the bound protein is eluted using 20- 80% Acetonitrile over 60 min. The monomeric form of recombinant protein is analyzed by SDS-PAGE. The pure monomer pooled fractions were concentrated and used for subsequent ^"purposes. The pooled fraction was diluted ten fold or higher in Tris CI buffer and left overnight for refolding. The refolded solution was concentrated by ultrafiltration and diafiltered extensively. The diafiltered sample was passed through 0.2u filter is almost 95% pure and has the activity comparing to the WHO standards. This protein is taken up for subsequent specific purifications depending on the nature of the biomolecule.

The method method herein described in the above examples is not limited to the examples given and is used for a variety of similar rDNA derived molecules like Recombinant Human lnterleukin-2.

Claims

1. A method for purification of human recombinant proteins wherein a polystyrene reversed phase column was used to purify unfolded recombinant protein using the unfolded monomeric protein loaded on to this column comprising monomer of recombinant protein was eluted and a organic acid is used.

2. A method of purification according to claim 1 wherein a completely folded form of recombinant proteins of at least 90- 95% purity is obtained in a few simple steps.

3. The method according to claim 1 , wherein monomer of recombinant protein was eluted using 0-100% linear gradient of organic solvent like but not limited to acetonitrile; isopropanol; ethanol or methanol

4. The method according to claim 1 , wherein the organic acid is 0.1 %-0.3%, trifluroacetic acid or any other organic acid like but not limited to acetic acid or hepta fluorobutaric acid was used,

5. The method according to claim 1 , wherein monomer was collected and refolded by diluting to ten fold or higher in TRIS or any other neutral buffer with pH 7-8 at 20 mM-100mM concentration,

6. The method according to claim 1 , wherein the protein concentration was maintained between 10ug-200ug during refolding

7. The method according to claim 1 , wherein refolded protein was concentrated and diafiltered to give at least 90%-95% folded protein.

8. The method of according to claim 1 , wherein, refolded, diafiltered and concentrated was purified recombinant protein further by which at least 98% pure was obtained which the specific activity higher than the WHO standards. The method according to claim 1 , wherein the method is not limited to the examples given and is used for a variety of similar rDNA derived molecules like Recombinant Human lnterleukin-2.