Title of Invention

AN IMPROVED PROCESS FOR THE PREPARATION OF ENANTIOMERICALLY PURE PHARMACEUTICAL GRADE D-METHIONINE FROM DL-METHIONINE

Abstract

Abstract An Improved Process for the Preparation of Enantiomerically Pure Pharmaceutical Grade D-Methionine from DL-Methionine The present invention describes a process for preparation of enantiomerically pure (>99.8%ee) pharmaceutical grade D-methionine (III) from DL-methionine (I).The process involves preparation of N-acyl-DL-methionine (II) from DL-methionine, its resolution by enzymes, subtilisin Carlsberg or subtilisin BPN or alpha chymotrypsin to give crude D-methionine. The invention also describes purification of crude D-methionine to pharmaceutical grade D-methionine

Full Text

An Improved Process for the Preparation of Enantiomerically Pure Pharmaceutical Grade D-Methionine from DL-Methionine The present invention describes a process for preparation of enantiomerically pure (>99.8%ee) pharmaceutical grade D-methionine from DL-methionine by enzymatic resolution of N-acyl-DL-methionine ester. The invention also describes a process for production of N-acyl-DL-methionine ester and purification of crude D-methionine to pharmaceutical grade D-methionine Background of the Invention Recently it is reported that D-methionine reduces the ototoxicity caused by drugs such as cisplatin, gentamycin, streptomycin, noise and radiation. It is also found to give excellent protection against toxicity of platinum containing anticancer drugs (US patent No. 6,265,386 ; US patent No. 6,187,817, both to Campbell Kathleen CM.; Campbell KC, Rybak LP, Meech RP, Hughes L., Hear Res. 1996, 102, 90-8; Cloven NG, Re A, McHale MT, Burger RA, DiSaia PJ, Rose GS, Campbell KC, Fan Hf Anticancer Res. 2000, 20, 4205-9; Ekborn A, Laurell G, Johnstrom P, Wallin I, Eksborg S, Ehrsson H. Hear Res. 2002,165, 53-61). Hence there is renewed interest in this molecule. Conventionally D-methionine is prepared in laboratory scale by two methods i) resolution of DL-methionine or its derivatives by diastereomeric salt formation using chiral bases/acids; ii) Enzymatic resolution. N-Formyl-DL-methionine was resolved using chiral base brucine to give N-formyl-D-methionine, which was acid hydrolyzed to give D-methionine (Jackson RW, Bolocj RJ, J. Biol. Chem, 1938,122,425; Spies J, J. Biol. Chem, 1950,182, 439). N-Acetyl-DL-methionine was resolved by chiral base (+) - a -fenchylamine to N-acetyl-D-methionine then further hydrolyzed to D-methionine. In the same publication DL-methionine was resolved to D-methionine by chiral acid (+)-a-bromocamphor-7i-sulfonic acid (Wheeler GP, lngersoll AW, J Am. Chem. Soc., 1951, 73, 4604). These chemical methods of resolution pose the following disadvantages: - Expensive chiral bases / acids are used and which are not available in quantities required for commercial scale - These processes do not provide good yields with high optical purity - Recovery of product by breaking the diastereomeric salt is always a problem in chemical resolution - Batch to batch inconsistency is a major problem in chemical resolutions and the operations are environmentally unfriendly In comparison to chemical methods, enzymatic resolution methods give clean products with minimal batch to batch variations and scale up problems. These are environmental friendly and provide products with good optical purity. D-methionine is prepared in laboratory scale by using enzymes such as acylase, D- amino acylase, amino oxidases and proteases. N-acetyl-DL-methionine was resolved using rat or hog kidney acylase to yield N-acetyl- D-methionine, which was further hydrolyzed to give D-methionine (Price EV, Gilbert, BJ, Greenstein JP, J. Biol. Chem, 1949, 179, 1169). Similarly using D-aminoacylase, N-acetyl - DL - methionine was resolved to D- methionine at milligram level (Moriguchi M, Ideta K, Appl. Environ Microbiol , 1988, 2767). These two methods have the following disadvantages - Hog or rat kidney acylases are expensive and also of animal origin hence are not suitable for large scale commercial production and also pose serious regulatory concerns. - D-acylase is very expensive and is not available in quantities required for commercial production - D-isomer obtained by this method is generally contaminated with L-isomer leading to lower optical purity. - Cobalt ion is required for acylase activity, which invariably imparts colors to the product and causes heavy metal contamination. - Acylases are slow acting enzymes in comparison to esterases and proteases DL-methionine was subjected to enzymatic resolution using L-amino acid oxidase to give D-methionine (Stumpf PK, Green DE, J. Biol. Chem, 1944,153, 387). This method is not suitable for commercial production as one isomer is completely destroyed and also amino oxidase is expensive and not available in quantities required for commercial production. N-carbobenzyloxy -DL-methionine and N-Benzoyl-DL-methionine were resolved to N-carbobenzyloxy-D-methionine anilide and N-Benzoly-D-methionine anilide by papain extract (Dekker CA, Fruton, JS, J. Biol. Chem, 1948,173, 3471) This method is not suitable for commercial production as it involves expensive nitrogen protecting groups, additional chemicals, aniline and cysteine. Moreover, papain is slow acting enzyme. Recently scientists of Tanabe Seiku (Furutani T, Furui M, Biotech Lett, 1999, 21, 1101; Furui, M, Toshiyuki F, Takeji S, Japanese Patent No. 09206089) reported preparation of D-methionine at laboratory scale (lOg) by enzymatic resolution using esterases, lipases and proteases. This procedure is not suitable for manufacture of pharmaceutical grade D-methionine for the following reasons. i) the method fails to produce D-methionine free of chlorides, sulfates, heavy metals and fails clarity and residue on ignition tests, ii) the method uses phosphate buffer, in buffered solutions small pH change is not indicated, hence monitoring the rate of hydrolysis on a pH indicator is not feasible iii) chiral purity is less (about 99%) iv) final work-up to isolate the material involves pH adjustments, which becomes difficult in buffered solutions particularly on manufacturing scale Thus, none of the available prior art procedures are suitable to produce enantiomerically pure (>99.8%, ee) and pharmaceutical grade D-methionine free of chlorides, sulfates, heavy metals and test for clarity and residue on ignition. Therefore, we focused on investigation to develop improved process for the preparation of enantiomerically pure (>99.8% ee), pharmaceutical grade D-methionine from DL- methionine. The present invention describes production scale robust process to afford enantiomerically pure (>99.8%, ee,) pharmaceutical grade D-methionine from DL- methionine. The main objective of present invention is to provide an improved process for preparation of enantiomerically pure (>99.8% ee) pharmaceutical grade D-methionine from DL- methionine. Another objective of present invention is to provide an improved process for the preparation of enantiomerically pure (>99.8% ee) pharmaceutical grade D-methionine from DL-methionine as regards to quality parameters such as clarity of solution, chlorides, sulfates, heavy metals and ash content and having 99.8% optical purity. Yet another objective of present invention is to provide an improved process for the preparation of enantiomerically pure (.99.8% ee) pharmaceutical grade D-methionine from DL-methionine which is simple, efficient, environmentally friendly and commercially viable. Summary of the Invention Accordingly the present invention provides an improved process for the preparation of enantiomerically pure (99.8%ee) pharmaceutical grade D-methionine which comprises. 1. one pot synthesis if N-acyl-DL-methionine ester from DL-methionine by treating with acylating reagent and alcohol 2. resolving N-acyl-DL-methionine ester to N-acyl-D-methionine ester by using proteases 3. acid hydrolysis of N-acyl-D-methionine ester to give crude D-methionine 4. purifying crude D-methionine to pharmaceutical grade enantiomerically pure (99.8%ee) D-methionine. Detailed Description of the Invention and its Preferred Embodiments This invention provides and especially efficient process for preparing D-methionine meeting to pharmaceutical grade as regards quality parameters such as clarity of solution, chlorides, sulfates, heavy metals and ash content and having >99.8% optical purity. In summary, this invention involves a production process for D-methionine comprising (1) one pot synthesis of N-acyl-DL-methionine ester from DL-methionine by treating with acid anhydrides and alcohols (2) resolving N-acyl-DL-methionine ester to N-acyl-D-methionine ester by proteases (3) acid hydrolysis of N-acyl-D-methionine ester to give crude D-methionine (4) purification of crude D-methionine to pharmaceutical grade as regards quality parameters such as clarity of the solution, chlorides, heavy metals and ash content and having >99.8%ee optical purity. N-Acyl-DL-methionine ester used in this invention is prepared from DL-methionine. The synthesis involves two transformations namely N-acylation and esterification of acylated amino acid. Preferably the acyl group is derived from fatty acids containing from 1 to 3 carbon atoms. More particularly, the acyl group will be preferably formyl, acetyl, propionoyl. These groups could be contributed from their anhydrides, chlorides. Acylation is done in presence of a bases in a range of solvents such as water, alcohols, aromatic/ aliphatic amines, halogenated hydrocarbons. The bases could be alkali or alkaline earth metal hydroxides, carbonates, bicarbonates or organic bases such as pyridines, aliphatic and aromatic amines. The most preferred bases are sodium hydroxide or potassium hydroxide and the most preferred acylating agent is acetic anhydride. The second transformation involves ester formation. The ester group can be derived from variety of alcohols containing from 1 to 6, preferably 1 to 3 carbon atoms. Especially suitable examples of ester groups are methyl, ethyl, n-propyl, isopropyl. An especially suitable example of acyl groups is acetyl. Racemic N-acetyl-DL-methionine methyl ester is most preferred for use in the process of this invention. Esterification can be done using mineral acids catalysts such as hydrochloric, sulfuric, phosphoric acids. The most preferred catalyst is sulfuric acid. N-Acyl-DL-methionine ester can be isolated by extracting with variety of solvents. During the extraction, in addition to desired N-acyl- DL-methionine ester some amount of un-esterified product, N-acyl- DL-methionine is also extracted, which gives a product with less than 95%ee optical purity and lower yields. In order to avoid this problem, we adopted method of isolating N-acyl-DL-methionine ester by neutralizing the excess acid. Neutralization can be done by using alkali metal or alkaline earth metal hydroxides, carbonates and bicarbonates. The preferred neutralizing agent is either potassium bicarbonate or sodium bicarbonates. In the literature N-acyl-DL-methionine ester is resolved to N-acyl-D-methionine ester using variety of proteases, lipases and esterases in buffered aqueous solution. Generally non-enzymatic hydrolysis is high in buffered solutions. In addition, monitoring the progress of the reaction on pH indicator is not possible, and pH adjustment at the final work-up is problematic. Hence in the present invention, enzymatic hydrolysis was carried out at pH 7.8 in aqueous solution or water and water miscible organic solvents. During the enzymatic hydrolysis, due to the liberation of free carboxylic acid group from N-acyl-L-methionine pH falls. To maintain pH, bases have to be used; these could be alkali or alkaline earth metal hydroxides, carbonates, and bicarbonates. Carbonates and hydroxides cause rapid changes in pH, and it may shoot pH to more than 10, at this pH non-enzymatic hydrolysis is higher. In order to decrease competing non-enzymatic hydrolysis solid potassium or sodium bicarbonate was used as pH controlling agent. Enzymatic hydrolysis is fast at the beginning of reaction i.e. in first half hour, later pH changes are and slow. Incomplete enzymatic hydrolysis is the main cause of decrease in the chiral purity; in order to complete hydrolysis, one strategy is to use enzyme in two or more lots. In the present invention, enzyme is added in two lots. Most of the enzymatic hydrolysis is complete during the first one hour itself, however small amount of un-hydrolyzed L-isomer causes chiral impurity problem. In order to complete resolution, after the addition of second lot of enzyme, the reaction was further conducted for additional 3-10h, preferably 3h. Enzymatic hydrolysis could be conducted at temperature of 20-60°C; the preferable temperature is 35-40°C. The suitable enzymes for resolution are sulfyhydral proteases papain, ficin or serine proteases from microorganism such as bacteria, fungi and mold. These proteases are relatively inexpensive and commercially available. Examples of preferred serine protease for use in this invention are derived from bacterial organism Bacillus subtilis. A preferred subtilisin of present invention is subtilisin Carlsberg and subtilisin BPN. After the resolution, N-acyl-D-methionine ester is extracted with organic solvents such as chloroform, dichloromethane, ethyl acetate, and toluene. The preferable solvent is chloroform and dichloromethane; chloroform is most preferred solvent. Distillation of solvent gave thick mass of N-acyl-D-methionine ester. Acidic hydrolysis of this mass by mineral acid followed by neutralization to pH 5.8 by concentrated base offered D-methionine. The acids that could be used are hydrochloric, sulfuric acid in 1-1 ON concentration, and the bases are sodium, potassium hydroxides in 5% -60% w/w concentration. The most preferred acid is hydrochloric acid at 2N concentration and most preferred base is 40% sodium hydroxide. The crude D-mefhionine thus obtained by filtration of reaction mass is chemically pure (-98%) with >99.8%ee optical purity. However this material could not pass for tests such as clarity of solution, chlorides, sulfates, heavy metal and loss on ignition tests. This material is purified by recrystallization in suitable solvent in presence of suitable adsorbent. The solvents are methanol, ethanol, isopropyl alcohol, water; either as single solvents or as mixture and the adsorbents are activated charcoal, silica gel, talc, bentonite. The most preferred solvent is water and the most preferred adsorbent is activated charcoal. Preferred temperature to dissolve the material is 80°C and cooling temperature is 0°C. The dilution of water to material is 3-20 times and concentration is 1-6 times. However, the preferred dilution is 7-8 times and concentration is 2 times. Dissolved material is filtered by nutsch followed by candle filter with 5-50 micron pore size diaphragm. The synthetic process for the present invention is given in Scheme 1 Example 1 Part A: Production of N-Acetyl-DL-methionine methyl ester Into a 100 L glass reactor 60 L methanol is charged, 3 kg of sodium hydroxide added and stirred to dissolve. 10 Kg of DL-methionine is added to above solution while maintaining reaction mass at 30±5°C, then 10 Kg of acetic anhydride is added. Reaction mixture is stirred till a clear solution obtained, then 3 L of sulfuric acid is added arid the reaction mass maintained at 30±5°C for 24±2h. Precipitated sodium sulfate is filtered and methanol removed from the reaction mass at a temperature not exceeding 40°C. To the reaction mass about 20 L of water is added and pH of aqueous mass was brought to 8.0 by adding solid sodium bicarbonate. Precipitated N-acetyl-DL-methionine methyl ester was filtered in a centrifuge. Yield, 10.6 Kg (76.4%). Mp.76-78, assay >98% (alkalimetry). Part B: Production of Crude D-Methionine Into a 100 L glass reactor fitted with on-line pH indicator, 25 L of water and 10 Kg of N-acetyl-DL-methionine methyl ester are added; pH adjusted to 7.6 by adding solid sodium bicarbonate. 200 g of subtilisin Carlsberg is added, then pH starts decreasing due to hydrolysis. By adding solid sodium bicarbonate pH is maintained at 7.6+0.2, The hydrolysis is complete in about one hour as indicated by constant value of pH. Further another lot of 50 g enzyme is added and reaction mixture maintained for additional 2-3 hours. Finally pH of the reaction mass is raised 8.0, and 500 g of activated carbon was added and stirred for 0.5 hours, filtered in nutsch filter with hyflow bed. The filtrate is extracted with chloroform (20+10+10 L), and distilled to get residue at 40±5°C (about 5.6 Kg). To the residue of 10 L, 2N hydrochloric acid is added and heated to 100°C and maintained at this temperature for 8h. Cooled to 10°C, 40% sodium hydroxide solution is added till the reaction mixture shows pH 5.8. Then the reaction mixture is further cooled to 5°C, filtered in a nutsch filter and dried at 60+5°C. 2.9 Kgs (80.1%). Assay: 98.3%, (Non- Aqueous), Chiral purity: 99.8% (HPLC using chiral columns), Chlorides: 0.39%, Heavy metals: >10 ppm, Sulfates: 300 ppm, Ash: 0.71%; 1.0 % solution in water and 2.0% in 6N hydrochloric acid is opalescent. Part C: Purification of Crude D-Methionine to Pharmaceutical Grade D-Methionine Into to 100 L glass reactor, 70 L of water is added and heated to 60±5°C, 7 Kg of crude D-methionine is added and temperature of reaction raised to 80±5°C. After reaction mixture becoming a clears solution, 500 g of activated charcoal is added and stirred for one hour and filtered through a candle filter filled with hyflow, attached with five micron filter. Filtrate was concentrated to 20% of its volume, at temperature not exceeding 50±5°C, and then cooled to 2±2°C; separated white shining amorphous crystals were centrifuged. 6.2 Kg (88.6%) Assay: 99.7%, (Non- Aqueous), Chiral purity: 99.8% (HPLC using chiral columns), Chlorides: lOppm, Heavy metals: Example 2 To a solution of DL-methionine (10g) in absolute alcohol (60 mL), sodium hydroxide (3g) is added and stirred to dissolve. Acetic anhydride (9.7g) is added slowly and stirred for 2.5 h at room temperature; sulfuric acid (3 mL) is added and stirred for 48h at room temperature. Precipitated sodium sulfated is filtered and solvent is removed at 40°C. Residue is dissolved in chloroform (100 mL) and washed with water (25mL x3), after drying with anhydrous sodium sulfate, solvent is removed to obtain brownish yellow thick liquid. Into a three necked 250 mL round bottom flask fitted with pH indicator electrode, the above crude N-acetyl-DL- methionine ethyl ester, water (50 mL) is added and pH adjusted to 7.6 by adding solid sodium bicarbonate. Subtilisin Carlsberg (0.8g) is added, then pH starts decreasing due to hydrolysis, by adding solid sodium bicarbonate pH is maintained at 7.6+0.2. The hydrolysis is complete in about one hour as indicated by constant value of pH. Further, another lot of enzyme (0.2 g) is added and reaction mixture maintained for additional 2-3 hours. Finally pH of the reaction mass is raised 8.0, and activated carbon (2g) is added and stirred for 0.5 hours and filtered. The filtrate is extracted with chloroform (50 ml x 3), and distilled to get residue at 40°C. To the residue 2N hydrochloric acid (30 mL) is added and heated to 100°C and maintained at this temperature for 8h. Cooled to 10°C, 40% sodium hydroxide solution is added till the reaction mixture shows pH 5.8. Then the reaction mixture is further cooled to 5°C, filtered and dried at 60±5°C to get crude D-methionine. This is dissolved in water (20 mL) by heating to 80°C, activated charcoal (0.2g) is added and stirred for 20 min, filtered, concentrated, cooled to 5°C and precipitated solid filtered and dried. Yield 2.4g (Over all yield, 48.0%) Assay: 99.3%, (Non- Aqueous), Chiral purity: 99.8% (HPLC using chiral columns), Chlorides: lOppm, Heavy metals: Example 3 Into a three necked 250 mL round bottom flask fitted with pH indicator electrode, N-acetyl-DL- methionine methyl ester (10 g), water (50 mL) is added and pH adjusted to 7.6 by adding solid sodium bicarbonate. Alpha chymotrypsin (80 mg) is added, then pH starts decreasing due to hydrolysis, by adding solid sodium bicarbonate pH is maintained at 7.6±0.2. The hydrolysis is complete in about one hour as indicated by constant value of pH. Further, another lot of enzyme (20 mg) is added and reaction mixture maintained for additional 2-3 hours. Finally pH of the reaction mass is raised 8.0, and activated carbon (2g) is added and stirred for 0.5 hours and filtered. The filtrate is extracted with chloroform (50 ml x 3), and distilled to get residue at 40°C. To the residue 2N hydrochloric acid (30 mL) is added and heated to 100°C and maintained at this temperature for 8h. Cooled to 10°C, 40% sodium hydroxide solution is added till the reaction mixture shows pH 5.8. Then the reaction mixture is further cooled to 5°C, filtered and dried at 60±5°C to give crude D-methionine, which is dissolved in water (20 mL) by heating to 80°C; activated charcoal (0.2g) is added and stirred for 20 min, filtered, concentrated, cooled to 5°C, precipitated solid filtered and dried. Yield 2.3g (63.9%). Assay: 99.8%, (Non- Aqueous), Chiral purity: 99.8% (HPLC using chiral columns), Chlorides: lOppm, Heavy metals: Example 4 Into a three necked 250 mL round bottom flask fitted with pH indicator electrode, N-acetyl-DL- methionine methyl ester (lOg), water (50 mL) is added and pH adjusted to 7.6 by adding solid sodium bicarbonate. Subtilisin Carlsberg (0.8g) is added, then pH starts decreasing due to hydrolysis, by adding solid sodium bicarbonate pH is maintained at 7.6±0.2. The hydrolysis is complete in about one hour as indicated by constant value of pH. Further, another lot of enzyme (0.2 g) is added and reaction mixture maintained for additional 2-3 hours. Finally pH of the reaction mass is raised 8.0, and activated carbon (2g) is added and stirred for 0.5 hours and filtered. The filtrate is extracted with chloroform (50 nil x 3), and distilled to get residue at 40°C. To the residue, 2N hydrochloric acid (30 mL) is added and heated to 100°C and maintained at this temperature for 8h. Cooled to 10°C, 40% sodium hydroxide solution is added till the reaction mixture shows pH 5.8. Then the reaction mixture is further cooled to 5°C, filtered and dried at 60±5°C to give crude D-methionine. This is dissolved in water (20 mL) by heating to 80°C, bentonite (0.2g) is added and stirred for 20 min, filtered and concentrated, cooled to 5°C, precipitated solid filtered and dried. Yield 2.1g (58.3%). Assay: 99.6%, (Non- Aqueous), Chiral purity: 99.8% (HPLC using chiral columns), Chlorides: 15ppm, Heavy metals: Advantages of Present Method 1. D-Methionine produced by this process is of pharmaceutical grade with very high optical purity (>99.8% ee) 2. The process uses cheaply and abundantly available commercially raw materials, DL-methionine, acetic anhydride, methanol and enzymes (subtilisin Carlsberg) etc. 3. The process is highly environment friendly. 4. In this process the enzymatic resolution is carried out in aqueous solution, hence monitoring of rater of hydrolysis and the final work up is easy. 5. The process poses no problems in up scaling and is industrially viable as a manufacturing process. We Claim 1. An improved process for preparation of enantiomerically pure (99.8%) pharmaceutical grade D-methionine (>99.8%ee). Which comprises a. Synthesis of N-acyl-DL-methionine ester by treating DL-methionine with an acylating agent and alcohol and isolating N-acyl-DL-methionine ester by known methods b. resolving N-acyl-DL-methionine ester to N-acyl-D-methionine ester using proteases c. hydrolyzing N-acyl-DL-methionine ester using acid, to give crude D- methionine d. purifying of crude D-methionine pharmaceutical grade enantiomerically pure (99.8%ee) D-methionine. 2. An improved process as claimed in 1, wherein acyl group in the acylating agent contain lto 3 carbon atoms and ester group contain from 1 to 3 carbon atoms. 3. An improved process as claimed in 1 & 2, wherein N-acyl-DL-methionine comprises N-acetyl-DL-methionine methyl ester 4. An improved process as claimed in claims 1 to 3, wherein N-acetyl-DL-methionine methyl ester is isolated from reaction mixture by neutralizing excess acid with potassium bicarbonate or sodium bicarbonate at 0-20°C. 5. An improved process as claimed in claims lto 4 wherein N-acyl-DL-methionine ester is subjected to enzymatic hydrolysis in aqueous-organic solvents. 6. An improved process as claimed in claims 1 to 5 wherein the organic solvents employed are selected from acetone, methanol, ethanol, isopropyl alcohol, acetonitrile. 7. An improved process as claimed in claims 1 to 6 wherein enzyme selected from subtilisin Carlgberg, subtilisin BPN and chymotrypsin are employed as the proteases. 8. An improved process as claimed in claims 1 to 6, wherein the purification of crude D-methionine obtained after hydrolysis of N-acyl-D-methionine ester is effected by dissolving in a solvent and treating with adsorbents. 9. An improved process as claimed in claims 8 wherein in solvents employed is selected from water, methanol, ethanol, isopropyl alcohol. 10. An improved process as claimed in claims 8&9 wherein adsorbents use are selected from activated carbon, silica gel, talc or bentonite. 11. An improved process for the preparation of enantiomerically pure (99.S%ee) pharmaceutical grade D-methionine substantially as herein described with reference to the examples. Dated this 2nd Day of August, 2004

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