Title of Invention	A PROCESS FOR FOLDING OF RECOMBINANT INSULIN PRECURSOR
Abstract	The present invention reveals a new process for the efficient folding of S- sulphonated recombinant human insulin precursor wherein the folding mixture consists of the insulin precursor protein as sulphonates and uses denaturant, reductant, organic solvent, additives and a buffer.

Full Text

THE PATENTS ACT, 1970 COMPLETE SPECIFICATION Section 10 "A PROCESS FOR FOLDING OF RECOMBINANT INSULIN PRECURSOR” Unichem Laboratories Ltd., an Indian company of Mahalaxmi Chambers, 22 Bhulabhai Desai Road, Mumbai - 400026, Maharashtra, India The following specification particularly describes and ascertains the nature of this invention and the manner in which it is to be performed: 1 FIELD OF THE INVENTION This invention describes a new process for the folding of S-sulphonated recombinant human insulin precursor protein. BACKGROUND OF THE INVENTION Human insulin consists of two peptide chains linked together by three disulphide bonds. The two-peptide chains are referred to as chain-A and chain-B. The A-chain has 21 and B-chain has 30 amino acid residues. Out of the three-disulphide bonds, two of them are inter-chain (A7=BZ &A20-B19) and one is intra-chain (A6-A11). The folding of recombinant insulin precursor expressed in E.coli to form the correct disulfide linkage is an important step in the generation of biologically active insulin. Therefore, the technology is being constantly up-graded to improve the yield during the folding process. In general, the recombinant insulin precursor produced is a single prepro polypeptide chain consisting of four distinct units of amino acid sequence. At the N-terminal region, the precursor peptide is designed to harbor an affinity tag (peptide showing affinity to a ligand) in fusion with the N-terminal amino acid of the B-chain of insulin. The B-chain and A-chain of insulin are joined by a C-peptide. Instead of the 35 amino acids normally found in the C-peptide of the natural human pro-insulin, the recombinant version has 2 or more amino acids in the C-peptide region. In the invitro folding of the above-mentioned precursor two distinct approaches are possible. One approach is to purify the denaturant solubilized protein by affinity chromatography and then to fold to form the native structure with proper disulphide bridges. After this, the tag portion and the intervening C-peptide amino acids are removed. In the alternate approach, the tag region is first 2 removed by enzymatic or by chemical fragmentation before initiating the folding of the pro insulin. Following folding, the intervening C-peptide is removed by enzymatic processing. The invitro folding of recombinant insulin precursor depends on a number of factors such as the concentration of the protein, the denaturant concentration, the pH of the buffer and the temperature and the nature of the C-peptide. It may also need the presence of other smaller ligands to improve the yield of correctly folded peptide. The problems normally encountered in achieving this are many. The critical step in the folding of insulin precursor is the formation of correct disulfide bridges. There are two cysteine residues next to each other in the A-chain at positions 6 .and 7. It is vital that the cysteine residue at position 7 of A-chain form the disulfide bridge with the cysteine residue at position 7 of the B-chain. But the proximal cysteine residue at position 6 in the A-chain is also likely to form the bond with its counterpart at position 7 in B-chain. When this happens it yields an unwanted product. Yet another problem is the possibility of forming the intermolecular disulfide bridge. Moreover the propensity of insulin to readily form aggregates through hydrophobic interaction makes the task of invitro folding even more difficult at protein concentrations above 100 microgram per milliliter. US Patent No.4430266 describes a method to produce porcine proinsulin from the corresponding s-sulphonate precursor. The A and B chains are joined by a C-peptide having the following sequences, Arg-X-Arg and Arg-Arg-X-Lys Arg where X is at least one amino acid residue. In this patent the effect of protein 3 concentration on the recovery of proinsulin has been studied. It clearly states that at protein concentrations of 0.1 mg/mL to 0.3 mg/mL the recovery of proinsulin is about 78% to 46% respectively, while at 1.0 mg/mL concentration the recovery is only 12%. US Patent No. 5952461 describes a method where the Human proinsulin S-sulphonate was purified by ion exchange chromatography and used for studying the effect protein concentration on the folding yield. This proinsulin has a formula R-B-X-A, where R is an amino acid residue or a peptide which is degradable enzymatically or chemically and X is a linkage of an amino group of A-1 in the insulin A-chain and a carboxyl group of B-30 in insulin B-chain which can be separated from the A-chain or B-chain enzymatically or chemically. At lower protein concentrations of 0.1 mg/mL the yield was 95% but at 1 mg/mL the yield was 53%. However folding of S-sulafonated human recombinant proinsulin having Lys-Arg as the C-peptide at a protein concentration of 1 mg/mL was tested and much lesser yield was found than that reported in US Patent No. 5952461 for the folding of proinsulin construct. In US Patent No. 56633204, refolding of misfolded Insulin-like Growth Factor-1 (IGF-1) which is a single chain protein having 70 aminoacid residues, was disclosed using different additives like alkaline and alkali earth metals and organic solvents like alcohols, and aprotic solvents. It demonstrates the advantage of using the organic solvents and salts for folding at higher protein concentrations. We hereby describe a method for efficient folding of a S-sulfonated human recombinant proinsulin having Lys-Arg as the C-peptide. By following this method effective folding at higher protein and denaturant concentrations and reduction in the folding volumes was achieved. To our knowledge the method described in the invention has not been reported for the folding of the above mentioned S-sulphonated human recombinant proinsulin.. 4 SUMMARY OF THE INVENTION: The invention seeks to obviate the above drawbacks. Accordingly, the invention provides for a process for folding of a purified sulphonated precursor form of recombinant human insulin polypeptide comprising the step of solubilization of the purified protein precursor in a buffer containing a denaturant agent, reduction of the sulphonated precursor with a thiol reagent, treatment of the reduced precursor solubilized in a denaturant with organic solvent, oxidative folding of the reduced protein in buffer containing denaturant and organic solvent, purification of the folded protein The sulphonated precursor is of the single chain composition like B-chain-C-A-chain and is in the form such as His tag-B chain-Lys-Arg-A chain. The precursor may also consist of a longer C- peptide region than the short Lys-Arg residue and peptide other than His-tag as a N-terminal fusion to the B-chain. The recombinant S-sulphonated Human insulin precursor obtained from the above steps is solubilised in tris-HCI buffer of 20 mM to 0.3M preferably 0.1M and pH of 8.0 to 11.0 preferably 8.5 containing a denaturant so that the protein concentration prior to reduction is 0.5 mg/mL to 6 mg/mL preferably of 1mg/mL to 3mg/ml_. The denaturant can be urea of 4M to 8M concentration, preferably 8M concentration or guanidine hydrochloride of 1M to 7M preferably of 6M concentration. Other denaturants like guanidine thiocyanate and thiourea can also be used. The solution of S-sulphonated precursor protein in a buffer containing a denaturant is then reduced using a aqueous solution of the sulfhydryl reducing 5 agent. The reducing agent can be 1,4 Dithiothreitol (DTT) of final concentration of 1 mM to 10 mM preferably 2 mM or tris(2-carboxyethyl) phosphine hydrochloride (TCEP) of final concentration of 0.5mM to 5 mM preferably of 2mM concentration or 2-mercaptoethanol (2ME) of final concentration of 1 mM to 10 mM preferably of 5 mM concentration or 1,4 Dithioerythritol (DTE) of 1mM to 10 mM concentration preferably of 2 mM concentration. The temperature for reduction can be from 4°C to 37°C preferably 25°C. The duration of reduction can be from 1min to 5h preferably for 1h duration. The reduced" protein is then diluted with the folding buffer so that the final denaturant concentration is diluted to 0.3 M to 3M preferably 2M to 2.5M. The folding buffer consists of a buffer, an organic solvent and an additive. The buffer can be glycine-NaOH of 20 mM to 0.6M or Tris-HCI of 20 mM to 0.6M preferably 0.3M glycine buffer. The pH of the above mentioned buffers can be from pH 8.0 to pH 11.5 preferably pH 11.0. The temperature of the above buffer can be from 4°C to 37°C preferably 25°C. The final protein concentration after dilution with the folding buffer is in the range of 0.1 to 2.0 mg/mL, but more preferably in the range of 0.5-1.5 mg/mL. The organic solvent can be any of the alcohols like ethanol, methanol, isopropanol, n-butanol or glycols like glycerol, polyethylene glycol, ethylene glycol, propylene glycol or dioxanes like 1,3 dioxane, 1,4 dioxane or solvents like acetonitrile, tetrahedrofuran. The preferable solvent is ethanol and acetonitrile. The organic solvents can be used individually or in combination. The percentage of organic solvent in the folding buffer can be from 5% to 45% (v/v). The additives can be any of the metal chelating agents like EDTA and EGTA of 0.3 mM to 3mM concentration in the dilution buffer preferably of 2mM concentration. 6 The dilution buffer is added to the reduced protein solution with gentle stirring and the stirring is continued for 4h to 96h preferably for 24h. The temperature of the folding mixture can be from 4°C to 30°C preferably 4 °C to15°C. The progress of folding is monitored by HPLC using reverse phase column (C4 or C18) using a gradient programme of water-acetonitrile with 0.1% trifluroacetic acid and a photo diode array (PDA) detector. About 20 \ig of the protein was injected from the folding solution. The chromatograms are integrated at l280nm. DETAILED DESCRIPTION Provided below is the detailed description of the most preferred embodiment of the invention. The invention will become apparent from the following description of the embodiments set forth hereinafter. The examples are provided to better explain the present invention to those of ordinary skill in the art. The embodiments presented in the examples can be modified variously. Thus, the scope of the present invention should be construed not limited to the embodiments to be described herein. Gene Construct The gene for insulin precursor was assembled from chemically synthesized oligonucleotides. These oligos were annealed and ligated to give the proinsulin gene corresponding to B-chain-Lys-Arg-A-chain. This gene after appropriate restriction digestion was inserted into a pET vector. This plasmid harboring human insulin precursor gene was used to transform E.coli host. The transformed cells were checked for expression of the recombinant precursor after induction with the inducer isopropylthiogalactoside. The expression of the precursor protein was in the range of 8-10 per cent of the total protein. 7 The expressed precursor was 8.3kD in size composed of N-terminal His-tag in fusion with proinsulin peptide of the composition B-chain-Lys-Arg -A-chain. A methionine residue was located at the junction between His-tag peptide and the B-chain. Extraction and purification of Recombinant human insulin precursor expressed in E.coli.: The protein expressed was in the inclusion bodies. The recombinant protein was solubilized using 6M guanidine hydrochloride in 0.05 M Tris-HCI pH 8.0. The protein solution was centrifuged to remove the insoluble materials. The. supernatant was subjected to sulfitolysis and then subjected to treatment with acetonitrile/isopropyl alcohol/ethanol to give a 30 percent vol/vol solution. The precipitated materials were removed by centrifugation. The clear supernant was passed through the Ni-NTA affinity column. The column was washed with 3-5 volumes of 0.05M Tris-HCI pH 8.0 containing 3M guanidine hydrochloride to remove the nonspecifically bound proteins. The column was then washed with 3 volumes of 0.05M Tris-HCI pH 6.5 containing 3M guanidine hydrochloride. It was then subjected to elution of the precursor protein following the standard protocol. The eluted protein fractions were monitored at 280nm and then fractions containing the protein were pooled. Removal of the His -tag sequence The protein was precipitated by acidifying the eluted fraction with TCA (10% final concentration). The precipitated protein was removed by centrifugation and washed with 25 % ethanol. The pellet was resolubilized in 70% formic acid and subjected to CNBr treatment to remove the His -tag portion from the precursor. The mixture is lyophilized to remove the unreacted reagent and formic acid. The peptide was solubilized in a 0.1 M Tris-HCI buffer pH 8.5 containing either 6M guanidine hydrochloride or 8M urea and passed through Ni-NTA column to remove the His tag peptide. The peptide without the tag was used for folding. 8 Folding of the truncated precursor The protein in the denaturant was diluted suitably to a protein concentration of 0.3 to 3.0 mg per milliliter using the buffer containing the corresponding denaturant. The denaturant concentration was maintained at 3-8M. The sulphonated protein was reduced by addition of 2 equivalents of DTT per sulphonated group. The reduction was allowed to proceed for 5 min. The pH was adjusted to 10- 11 by adding 0.3M glycine-NaOH. Following which organic solvent (alcohol/acetonitrile) was added to give a final concentration 10-30%. The final concentration of the protein in the reaction mixture was kept at 0.1 to 1.5 mg per milliliter. The oxidative folding was allowed to proceed at 4° C. The process of folding was monitored at different time periods by HPLC using C-4 reverse phase column. The reaction was essentially complete in about 16hrs. The alcohol used was methanol, ethanol, propanol, butanol and isopropyl alcohol. Of these, ethanol and isopropyl alcohol gave maximum yield of the product. Methanol and butanol were less effective in the folding of insulin precursor. However combination of ethanol with 1, 4 dioxane gave good yield. Folding of the precursor without the removal of the His-tag Similar to the folding of the truncated precursor, intact precursor could also be carried out under similar conditions. After the folding, the precursor is purified by binding on to Ni-NTA column and then is subjected to CNBr cleavage to remove the His tag portion of the molecule. While the present invention has been described with respect to the most preferred embodiment, it will be apparent to those skilled in the art that various changes and modifications may be made without departing from the scope of the invention as illustrated in the examples below and defined in the following claims. 9 The following examples are provided to illustrate the different embodiments of the invention: Example-1: Folding of sulphonated insulin precursor protein with ethanol in the folding buffer: The solution of precursor protein S-sulphonate in buffer containing 0.1 M Tris HCI (pH 8.0) and 8M urea (90 mg of protein in 30 mL of buffer) was reduced by adding the reducing agent (60uL of 1M stock) to make the final reductant concentration to 2 mM and kept for 1h at 25 °C. To the reduced protein solution was added with gentle stirring the folding buffer consisting of 0.3M Glycine buffer (30 mL, pH 11.0), 65% (v/v) of ethanol (30 mL) and EDTA (180mL of 0.5M stock) and the pH adjusted to 11.0 using 10M NaOH. The contents were stirred gently at 8 °C for 48h. The progress of folding was monitored by HPLC. After the folding was complete the pH of the solution was dropped to 4.0 using acetic acid. The yield of the folded precursor was 45 % (w/w). Example-2: Folding of sulphonated insulin precursor protein with isopropanol in the folding buffer: The solution of precursor protein S-sulphonate in buffer containing 0.1 M Tris HCI (pH 8.0) and 8M urea (90 mg of protein in 30 mL of buffer) was reduced by adding the reducing agent (60uL of 1M stock) to make the final reductant concentration to 2 mM and kept for 1h at 25 °C. To the reduced protein solution was added with gentle stirring the folding buffer consisting of 0.3M Glycine buffer (30mL, pH 11.0), 65% (v/v) of isopropyialcohol (30 mL) and EDTA (180uL of 0.5M stock) and the pH adjusted to 11.0 using 10M NaOH. The contents were stirred gently at 8°C. The progress of folding was monitored by HPLC. After the folding was complete the pH of the solution was dropped to 4.0 using acetic acid. The yield of the folded precursor was 30 % (w/w). The yield of the folded precursor was 24% (w/w) when 30% (v/v) of n-butanol was used instead of 65% (v/v) isopropyl alcohol in the process and was 14% (v/v) when 65% (v/v) methanol was used instead of 65% (v/v) isopropyl alcohol 10 Example-3: Folding of sulphonated insulin precursor protein with combination of ethanol and 1,4 dioxane in the folding buffer: The solution of precursor protein S-sulphonate in buffer containing 0.1 M Tris HCI (pH 8.0) and 8M urea (30 mg of protein in 10 mL of buffer) was reduced by adding the reducing agent (20mL of 1M stock) to make the final reductant concentration to 2 mM and kept for 1h at 25 °C. To the reduced protein solution was added with gentle stirring the folding buffer consisting of 0.3M Glycine buffer (10 mL, pH 11.0), 65% (v/v) of ethanol with 5% (v/v) of 1, 4 Dioxane (10 mL) and EDTA (60uL of 0.5M stock) and the pH adjusted to 11.0 using 10M NaOH. The contents were stirred gently at 8 °C. The progress of folding was monitored by HPLC. After the folding was complete the pH of the solution was dropped to 4.0 using acetic acid. The yield of the folded precursor was 40% (w/w). Example-4: Folding of sulphonated insulin precursor protein by diluting first with the folding buffer followed by reduction with 1,4 dithiothreitol: To the solution of precursor protein S-sulphonate in buffer containing 0.1 M Tris HCI (pH 8.0) and 8M urea (25mg of protein in 10mL of buffer) was added with gentle stirring the folding buffer consisting of 0.3M Glycine buffer (10 mL, pH 11.0), 65% (v/v) of ethanol (10mL) and EDTA (60uL of 0.5M stock) and the pH adjusted to 11.0 using 10M NaOH. The reducing agent DTT (60uL of 1M stock) was added to make the final reductant concentration to 2 mM. The contents were stirred gently at 8 °C. The progress of folding was monitored by HPLC. After the folding was complete the pH of the solution was dropped to 4.0 using acetic acid. The yield of the folded precursor was 45% (w/w). Example-5: Folding of sulphonated insulin precursor protein by diluting first with the folding buffer followed by reduction with 2-mercaptoethanol: To the solution of precursor protein S-sulphonate in buffer containing 0.1 M Tris HCI (pH 8.0) and 8M urea (25mg of protein in 10mL of buffer) was added with gentle stirring the folding buffer consisting of 0.3M Glycine buffer (10mL, pH 11 11.0), 65% (v/v) of alcohol (10mL) and EDTA (60ml_ of 0.5M stock) and the pH adjusted to 11.0 using 10M NaOH. The reducing agent 2ME (105ml_ of 1M stock) was added to make the final reductant concentration to 3.5 mM. The contents were stirred gently at 8 °C. The progress of folding was monitored by HPLC. After the folding was complete the pH of the solution was dropped to 4.0 using acetic acid. The yield of the folded precursor was 45% (w/w). 12 We Claim: 1. A process for folding the recombinant human insulin precursor protein comprising the steps of: a. sulfitolysing the precursor protein by a cysteine sulphonating agent, b. solubilising the sulphonated precursor protein in a buffer solution containing a denaturant to an appropriate protein concentration, c. reducing the denatured protein solution using a sulfhydryl reducing agent of appropriate concentration for an appropriate duration, d. folding the reduced denatured protein by adding a folding buffer of a suitable pH and temperature and incubating for an appropriate duration, e. acidifying the solution after completion of the folding to an appropriate pH, f. purifying the folded protein by reverse phase column chromatography, g. drying the purified fraction to obtain the purified folded recombinant human insulin precursor. 2. The process as claimed in claim 1 wherein said sulphonated precursor comprises of a single chain composition of B-chain-C peptide-A-chain insulin protein. 3. The process as claimed in claim 2 wherein said C- peptide region of the precursor consists of two or more amino acid residues. 4. The process as claimed in claim 3 wherein said C- peptide region of the precursor consists of Lysine and Arginine amino acid residues. 5. The process as claimed in claim 1 wherein the precursor consists of N-terminal fusion peptide to the B-chain. 6. The process as claimed in claim 5 wherein the N-terminal fusion peptide to the B-chain is His tag. 7. The process as claimed in claim 1 wherein said sulphonated precursor composition is His tag-B chain-Lys.Arg-A chain. 8. The process as claimed in claim 1 wherein said buffer solution in step (b) is 0.02M to 0.3M Tris-HCI or Glycine-NaOH of pH 8.0 to 11.0. 9. The process as claimed in claim 1 wherein said denaturant in step (b) is selected from a group comprising of urea, guanidine-hydrochloride, thiourea and guanidine thiocyanate. 13 10. The process as claimed in claim 9 wherein said denaturant concentration is 1M to 8M urea. 11. The process as claimed in claim 9 wherein said denaturant concentration is 1M to 6M Guanidine hydrochloride 12. The process as claimed in claim 1 wherein said protein concentration in step (b) is 0.5mg/mL to 6 mg/mL. 13. The process as claimed in claim 1 wherein said reducing agent in step (c) is selected from a group comprising of 1,4 Dithiothreitol (DTT), 1,4 Dithioerythritol (DTE), tris(2-carboxyethyl) phosphine hydrochloride (TCEP) and 2-mercaptoethanol. 14. The process as claimed in claim 1 wherein said reducing agent concentration in step (c) is 1mM to l0mM. 15. The process as claimed in claim 1 wherein said reducing agent concentration in step (c) is 0.5mM to 5mM. 16.The process as claimed in claim 1 wherein said duration of reduction in step (c) is 1min to 5h. 17.The process as claimed in claim 1 wherein said folding buffer of step (d) comprises of a suitable buffer salt, an organic' solvent/s and additives at an appropriate pH. 18.The a process as claimed in claim 17 wherein suitable buffer salt is Tris-HCl and/or Glycine-NaOH of 0.02M to 0.6M concentration. -19.The process as claimed in claim 17 wherein said pH of the buffer is 8.0 to 11.5. 20.The process as claimed in claim 17 wherein said organic solvent in the folding buffer is selected from a group comprising of ethanol, methanol, isopropanol, n-butanol, actonitrile, 1,4 dioxane, 1,3 dioxane, tetrahedrafuran, ethylene glycol, propylene glycol and polyethylene glycol. 21. The process as claimed in claim 20 wherein the organic solvents are used individually or in combination maintaining the final percentage of organic solvent in the folding buffer. 22. The process as claimed in claim 17 wherein the amount of organic solvent in the folding buffer is 5% to 45% (v/v). 23. The process as claimed in claim 17 wherein said additive in the folding buffer is EDTA or EGTA of 0.3 to 3mM concentration. 24. The process as claimed in claim 17 wherein said pH of the protein solution after dilution with the folding buffer is 8.0 to 11.5. 25.The process as claimed in claim 1 wherein the concentration of protein in the solution after dilution with the folding buffer is 0.3mg/ml_ to 2mg/mL. 26.The process as claimed in claim 1 wherein said temperature for folding in step (d) is 4°C to 30°C. 14 27. The process as claimed in claim 1 wherein the duration of step (d) is 4h to 96h. 28. The process as claimed in claim 1 wherein said step (g) of drying is by freeze-drying. 29. The process as claimed in any of the above claims wherein the step of addition of reducing agent is carried out after the protein solution is diluted with the folding buffer. 30.A process for folding the recombinant human insulin precursor protein substantially as herein described with reference to accompanying examples. 15 ABSTRACT A PROCESS FOR FOLDING OF RECOMBINANT INSULIN PRECURSOR The present invention reveals a new process for the efficient folding of S-sulphonated recombinant human insulin precursor wherein the folding mixture consists of the insulin precursor protein as sulphonates and uses denaturant, reductant, organic solvent, additives and a buffer. 16

Documents:

892-mum-2005-abstract (complete).doc

892-mum-2005-abstract (complete).pdf

892-MUM-2005-ABSTRACT(17-9-2013).pdf

892-mum-2005-claims (complete).doc

892-mum-2005-claims (complete).pdf

892-MUM-2005-CLAIMS(AMENDED)-(17-9-2013).pdf

892-MUM-2005-CLAIMS(AMENDED)-(27-10-2014).pdf

892-MUM-2005-CORRESPONDENCE(12-8-2014).pdf

892-MUM-2005-CORRESPONDENCE(17-9-2013).pdf

892-MUM-2005-CORRESPONDENCE(23-5-2014).pdf

892-MUM-2005-CORRESPONDENCE(26-11-2012).pdf

892-MUM-2005-CORRESPONDENCE(28-7-2005).pdf

892-MUM-2005-CORRESPONDENCE(30-7-2008).pdf

892-mum-2005-correspondence-received-ver-070905.pdf

892-mum-2005-correspondence-received-ver-080905.pdf

892-mum-2005-correspondence-received-ver-310706.pdf

892-mum-2005-correspondence-received.pdf

892-mum-2005-description (complete).pdf

892-MUM-2005-DESCRIPTION(PROVISIONAL)-(2-8-2005).pdf

892-mum-2005-form 1(17-12-2004).pdf

892-MUM-2005-FORM 1(17-9-2013).pdf

892-MUM-2005-FORM 1(26-11-2012).pdf

892-MUM-2005-FORM 1(9-9-2005).pdf

892-MUM-2005-FORM 18(30-7-2008).pdf

892-MUM-2005-FORM 2(PROVISIONAL)-(2-8-2005).pdf

892-MUM-2005-FORM 2(TITLE PAGE)-(17-9-2013).pdf

892-MUM-2005-FORM 2(TITLE PAGE)-(COMPLETE)-(31-7-2006).pdf

892-MUM-2005-FORM 2(TITLE PAGE)-(PROVISIONAL)-(2-8-2005).pdf

892-mum-2005-form-1.pdf

892-mum-2005-form-13.pdf

892-mum-2005-form-2 (complete).doc

892-mum-2005-form-2 (complete).pdf

892-mum-2005-form-2 (provisional).doc

892-mum-2005-form-26.pdf

892-mum-2005-form-3.pdf

892-mum-2005-form-5.pdf

892-MUM-2005-MARKED COPY(17-9-2013).pdf

892-MUM-2005-MARKED COPY(27-10-2014).pdf

892-MUM-2005-POWER OF ATTORNEY(17-9-2013).pdf

892-MUM-2005-REPLY TO EXAMINATION REPORT(17-9-2013).pdf

892-MUM-2005-REPLY TO HEARING(27-10-2014).pdf

892-MUM-2005-SPECIFICATION(AMENDED)-(17-9-2013).pdf

892-MUM-2005-SPECIFICATION(AMENDED)-(27-10-2014).pdf