Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D501/00—Heterocyclic compounds containing 5-thia-1-azabicyclo [4.2.0] octane ring systems, i.e. compounds containing a ring system of the formula:, e.g. cephalosporins; Such ring systems being further condensed, e.g. 2,3-condensed with an oxygen-, nitrogen- or sulfur-containing hetero ring
  • C07D501/14—Compounds having a nitrogen atom directly attached in position 7
  • C07D501/16—Compounds having a nitrogen atom directly attached in position 7 with a double bond between positions 2 and 3
  • C07D501/20—7-Acylaminocephalosporanic or substituted 7-acylaminocephalosporanic acids in which the acyl radicals are derived from carboxylic acids
  • C07D501/24—7-Acylaminocephalosporanic or substituted 7-acylaminocephalosporanic acids in which the acyl radicals are derived from carboxylic acids with hydrocarbon radicals, substituted by hetero atoms or hetero rings, attached in position 3
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/54—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame
  • A61K31/542—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame ortho- or peri-condensed with heterocyclic ring systems
  • A61K31/545—Compounds containing 5-thia-1-azabicyclo [4.2.0] octane ring systems, i.e. compounds containing a ring system of the formula:, e.g. cephalosporins, cefaclor, or cephalexine
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P35/00—Preparation of compounds having a 5-thia-1-azabicyclo [4.2.0] octane ring system, e.g. cephalosporin
  • C12P35/04—Preparation of compounds having a 5-thia-1-azabicyclo [4.2.0] octane ring system, e.g. cephalosporin by acylation of the substituent in the 7 position

Definitions

• the present invention relates to a crystalline form of an intermediate for cefoperazone and to a process for the preparation thereof by enzymatic condensation of a 3'-thiosubstituted ⁇ -lactam nucleus with a phenylglycine derivative.
• Enzymatic production of semi-synthetic ⁇ -lactam antibiotics by acylation of the parent amino ⁇ -lactam moiety with a side chain acid derivative has been widely described (e.g. DE 2163792, DE 2621618, EP 339751 , EP 473008, EP 1394262, NL 1010506, WO 1992/01061 , WO 1993/12250, WO 1996/02663, WO 1996/05318, WO 1996/23796, WO 1997/04086, WO 1998/56946, WO 1999/20786, WO 2005/00367, WO 2006/069984, WO 2008/1 10527 and US 3,816,253).
• the enzymes used in the art are in most cases penicillin acylases obtained from Escherichia coli and are immobilized on various types of water-insoluble materials (e.g. WO 1997/04086).
• cephalosporins have been developed with the objective to change, preferably improve, antibacterial properties. Virtually all of these have been, and still are, chemically prepared from fermentatively obtainable cephalosporin C by introduction of alternate groups at the C- 3' position and exchange of the aminoadipyl side chain for other side chains.
• nucleus is defined as the ⁇ -lactam moiety of a 3'-thiosubstituted cephalosporin (i.e. a compound of formula (2)) and is 7-amino-3-(1 ,2,3-triazol-4(5)-ylthiomethyl)-3-cephem-4-carboxylic acid.
• side chain is defined as the moiety which, in the semi-synthetic ⁇ -lactam compound, is attached to the 7-amino position of the nucleus as defined herein, for instance D-4-hydroxyphenylglycine.
• free side chain is defined as the acid form of the side chain, for instance D-4-hydroxyphenylglycine.
• side chain ester is the ester form of the free side chain whereby the carboxyl group of the free side chain is esterified with an alcohol to give an ester, for instance D-4-hydroxyphenylglycine methyl ester or ethyl ester.
• the side chain ester may be in the form of the free base or as a salt, for instance as the HCI-salt and the side chain ester may be in a solid form or dissolved in a suitable solvent.
• the nucleus is a compound of general formula (2) referred to as 7-amino-3-(1 - methyl-1 H-tetrazol-5-ylthiomethyl)-3-cephem-4-carboxylic acid (7-TMCA).
• the phenylglycine derivative or the like preferably is an ester such as D-4-hydroxyphenylglycine ethylester, D-4-hydroxyphenylglycine methylester.
• the phenylglycine derivative or the like is an amide such as D-4-hydroxyphenylglycine amide.
• the phenylglycine derivative or the like is D-4-hydroxyphenylglycine amide, D-4-hydroxyphenylglycine ethylester or D-4-hydroxyphenylglycine methylester since the 3'-thiosubstituted cephalosporins thus obtained can serve as suitable intermediates in the preparation of cephalosporins with antibacterial properties (for example cefoperazone and cefpiramide).
• the most preferred phenylglycine derivative is D-4-hydroxyphenylglycine methylester.
• the phenylglycine derivative or the like has the following properties:
• an ee preferably equal to or greater than 90%, more preferably equal to or greater than 95%, preferably equal to or greater than 96%, preferably equal to or greater than 97%, preferably equal to or greater than 98% and most preferably equal to or greater than 99%;
• a salt content preferably of 20 mole% or less, more preferably of 10 mole% or less, more preferably of 5 mole% or less, more preferably of 2 mole% or less, most preferably of 1 mole% or less, expressed as moles of salt relative to moles of ester.
• the enzyme used for the enzymatic condensation conveniently is an enzyme suitable for recognizing as substrate amides or esters of a-amino acids such as dihydrophenylglycine, 4-hydroxyphenylglycine and phenylglycine such as penicillin acylases and a-amino acid ester hydrolases.
• a-amino acids such as dihydrophenylglycine, 4-hydroxyphenylglycine and phenylglycine
• penicillin acylases and a-amino acid ester hydrolases In a preferred embodiment it was found that mutant penicillin G acylases as described in WO 2010/072765 are well-suited for the synthesis of 3'-substituted cephalosporins since relatively low amounts of unwanted thiols are generated.
• the enzyme is immobilized in order to facilitate separation from the reaction medium and recovery for repeated use. Immobilization can be carried out using a multitude of carrier materials such as silica or gelatin-based carriers
• the reaction may be carried out at a wide temperature range, i.e. -20°C to 40°C.
• the temperature range is from -5°C to 20°C as the balance between reaction speed, degradation rate and optimal conversion is best tuned within this range.
• the most optimal temperature range, where high conversions are obtained in combination with low product degradation, was found to be from 0°C to 10°C.
• the pH at which the reaction is carried out is from 5.0 to 10.0.
• the pH range is from 8.0 to 9.5 as the balance between reaction speed, degradation rate and optimal conversion is best tuned within this range.
• the most optimal pH range, where high conversions are obtained in combination with low product degradation, was found to be from 8.5 to 9.0.
• This approach facilitates rapid dissolution of starting material on the one hand while limiting the formation of side products, such as free side chain and thiols on the other hand. It was found that highest conversions are obtained when the starting nucleus is dissolved in the reaction mixture.
• Suitable bases are ammonia, aqueous potassium hydroxide and aqueous sodium hydroxide. It was found that aqueous sodium hydroxide gives superior results compared to ammonia in terms of co-formation of undesired thiols. In contrast to ammonia, alkaline earth hydroxides such as aqueous lithium hydroxide, potassium hydroxide or sodium hydroxide surprisingly reduced the co-formation of undesired thiols to almost neglectable. Preferably the concentration of alkaline earth hydroxide is from 1 M to 10M.
• a more preferable concentration range of alkaline earth hydroxide is from 2M to 8M, most preferably from 3M to 6M.
• Addition of the side chain amide or ester can be performed by addition of said side chain amide or ester as a solid or dissolved. When dissolved this is preferably in water wherein the resultant solution is brought at low pH with an acid such as, for example, hydrochloric acid or sulfuric acid.
• a side chain ester is dissolved and the resulting solution is added to the reaction mixture during a certain time interval.
• the side chain ester is added as described in WO 2008/1 10527 and WO 2008/1 10529. Said time interval may be from 10 to 300 min, preferably from 30 to 200 min, most preferably from 60 to 180 min.
• the 3'-thiosubstituted cephalosporin of formula (1 ) is recovered using known methods, usually following lowering the pH to a value between 1 .5 and 6.5.
• a reactor that is equipped with a sieve in the bottom compartment and an outlet at the bottom may be used. The contents of the reactor may then be discharged through the sieve, preferably using upwards stirring.
• the resulting 3'-thiosubstituted cephalosporin suspension, free of immobilized enzyme, may then be filtered or centrifuged.
• the isolation of the product (1 ) includes removal of immobilized enzyme by filtration, precipitation of the product by lowering the pH with aqueous sulfuric acid, preferably to a value of 2.5 to 6, removal of about 80% water by filtration, dilution of the resulting suspension with methanol and filtration to dryness.
• a further washing step with methanol may be applied.
• product (1 ) can be isolated as a stable, manageable solid in 94% yield, and with high quality.
• Preferred solvents are alcohols and ketones such as acetone, ethanol or methanol.
• the organic solvent can be added to the slurry before filtration, but in this case the impurities present in the mixture (such as D-4-hydroxyphenylglycine methylester, D-4-hydroxyphenylglycine and/or 7-TMCA) lose solubility and may contaminate the final product. Consequently, it is preferred to add the alcohol or ketone after 50-90% of the liquid has been removed from the slurry by filtration.
• the 3'-thiosubstituted cephalosporin obtained after enzymatic coupling is derivatized in a subsequent reaction such as outlined below, the 3'-thiosubstituted cephalosporin is not isolated and/or not crystallized.
• the product (1 ) obtained by the process of the invention described above is further reacted to give a desired pharmaceutical product of general formula (3).
• R-i is a radical chosen from th e l ist con si sti n g of 4-ethyl-2,3-dioxo-1 - piperazinecarbonyl, 4-hydroxy-6-methyl-nicotinyl (or the corresponding keto-form 6-methyl-4-oxo-1 ,4-dihydropyridine-3-carbonyl), 1 /-/-imidazole-4-carbonyl-5-carboxylic acid or derivatives such as amides, ethers and esters thereof.
• R 5 is 4-ethyl- 2,3-dioxo-1 -piperazinecarbonyl or 4-hydroxy-6-methyl-nicotinyl.
• Such further reaction preferably comprises derivatization of the amino group of the side chain, for example by reaction with an acid halide in water/ethyl acetate with potassium carbonate, such as described in DE 2600880.
• the product may be converted into a pharmaceutically acceptable salt, preferably the sodium salt, for example by reaction with an inorganic sodium salt such as sodium hydrogencarbonate.
• the product of the reaction (1 ) is isolated in a crystalline form which is novel as a result of the hitherto unprecedented enzymatic reaction conditions and downstream processing steps.
• this crystalline form is highly stable and pure thereby making it an excellent starting material for the high yield, high purity production of both cefoperazone and cefpiramide.
• the XRD spectrum of the crystalline form of the invention shows major peaks at 2 ⁇ values of 13.9 ⁇ 0.3, 15.6 ⁇ 0.3, 19.1 ⁇ 0.3, 19.9 ⁇ 0.3, 22.4 ⁇ 0.3, 23.2 ⁇ 0.3, 24.8 ⁇ 0.3, 28.1 ⁇ 0.3, 29.0 ⁇ 0.3, 32.2 ⁇ 0.3, 33.9 ⁇ 0.3, 38.6 ⁇ 0.3 and 48.8 ⁇ 0.3.
• the intensity of any of said major peaks is more than 10% of the intensity of the most intense of said major peaks.
• the product obtained by the processes of the invention is used for the manufacture of a medicament with antibacterial properties.
• the medicament thus obtained has the advantage of being produced in high purity and with low environmental burden as compared to their congeners being synthesized chemically.
• Figure 1 is the X-ray powder diffraction pattern recorded from the compound of formula (1 ) after storage under ambient conditions for 2.5 h.
• X-axis 2-theta value (deg).
• Y-axis intensity (cps). The following distinct peaks can be discerned:
• Figure 2 is the X-ray powder diffraction pattern recorded from the compound of formula (1) prior to storage under ambient conditions for 2.5 h.
• X-axis 2-theta value (deg).
• Y-axis intensity (cps). The discernible peaks are the same as in Figure 1, however there is also a broad maximum at 2 ⁇ ⁇ 27.
• mutant penicillin G acylases may be carried out as described in WO 1996/005318 and WO 2003/055998.
• genes encoding mutant penicillin G acylases may be obtained by gene synthesis.
• Production of the mutant penicillin G acylase was achieved by cloning the genes encoding mutant penicillin G acylases into an appropriate expression vector, transforming a suitable host such as Escherichia coli with said vector and culturing the transformed host under conditions suitable for the production of the mutant penicillin G acylases and recovery and purification of the mutants was carried out as described in WO 2010/072765.
• Penicillin G acylase AA (the Escherichia coli wild type penicillin G acylase with mutations B:F24A and B:V148L) and penicillin G acylase mutant 1 (the Escherichia coli wild type penicillin G acylase with mutations V1 1A, A:S3L, A:V192E, B:F24A, B:V148L and B:F460L) as disclosed in Example 1 of WO 2010/072765 were immobilized according to the method disclosed in EP 839192 and EP 222462.
• the measurement is performed using 718 STAT Titrino from Metroh m .
• the pH electrode is from Metrohm, series number 6.0234.1 10. It contains 3M KCI.
• the pH meter calibration is performed at 20°C at pH 4 and pH 7 using standard solutions from Merck, using the calibration program present in the instrument.
• Example 1 a 7-Amino-3-(1 -methyl-1 /-/-tetrazol-5-ylthiomethyl)-3-cephem-4-carboxylic acid (7-TMCA; 5 g) was added to distilled water (38 g) and cooled to 3°C. The mixture was stirred at 400 rpm and the pH was brought to 9.0 with aqueous NaOH (5M) whereafter the remainder of the reaction was carried out at pH 8.8. After 60-80 min, the suspension was filtered.
• 7-TMCA 7-Amino-3-(1 -methyl-1 /-/-tetrazol-5-ylthiomethyl)-3-cephem-4-carboxylic acid
• the filtrate containing 4.1 g of 7-TMCA, was place back in the reactor and immobilized penicillin G acylase mutant 1 (3.5 g, see Materials and Methods) was added and to the resulting mixture a solution of D-4-hydroxyphenylglycine methylester (HPGM) was dropped at speed of 7 mL/h by a syringe pump (dosing time 2 h).
• HPGM D-4-hydroxyphenylglycine methylester
• This solution was prepared by dissolving HPGM (4.0 g, 1 .7 equiv.) in water (6.4 g) and H 2 S0 4 25% (4.25 g in water).
• the enzymatic reaction was followed by analytical HPLC (see Materials and Methods) and stopped at the end of HPGM addition, by enzyme filtration.
• the conversion was 98% (w.r.t. 7-TMCA).
• the mixture contained 1 .1 % (w/w) D-4-hydroxyphenylglycine (HPG), 0.1 % (w/w) 7-TMCA, 1 .0% (w/w) HPGM, 7.9% (w/w) of the title compound and 0.07% (w/w) 5-mercapto-1 - methyltetrazole.
• HPPG D-4-hydroxyphenylglycine
• the enzyme was removed by filtration on glass filter no. 1 to give filtrate 65 g at pH 8.8. Under vigorous stirring at 3°C a 25% aqueous solution of H 2 S0 4 (2.70 g) was added in 10 min to give a pH of 5.7. The obtained suspension was stirred for 10 min at 3°C and then filtered under vacuum on filter glass no. 3. When 60% of the volume was removed by filtration (40 g mother liquor recovered), MeOH (14 g) was added to the suspension remaining on the filter and the solvent mixture was completely removed by filtration. The final solid was washed with MeOH (15 g). The title product was recovered with a yield of 90% based on the starting material 7-TMCA.
• Example 1 b as Example 1 a with the following differences: the reaction was titrated using 1 M sodium hydroxide and the down stream processing performed without MeOH. The conversion was 93% (w.r.t. 7-TMCA). The mixture contained 0.75% (w/w) HPG, 0.3% (w/w) 7-TMCA, 0.2% (w/w) HPGM, 6.6% (w/w) of the title compound and 0.13% (w/w) 5-mercapto-1 -methyltetrazole. In the HPLC chromatogram two minor not identified impurities were visible with ⁇ 1 % H PLC area percentage. At the end of the enzymatic reaction, the enzyme was removed by filtration on glass filter no. 1 to give 81 g of filtrate at pH 8.6.
• Example 1 c as Example 1 b with the following differences: HPGM added as solid during the reaction and the reaction was performed at pH 8.7.
• the mixture contained 0.43% (w/w) HPG, 1 .6% (w/w) 7-TMCA, 0.12% (w/w) HPGM, 6.1 % (w/w) of the title compound and 0.12% (w/w) 5-mercapto-1 -methyltetrazole.
• Example 1 d as Example 1 c with the following difference: the reaction was titrated using ammonia 25% in water.
• the conversion was 63% (w.r.t. 7-TMCA).
• the mixture contained 0.34% (w/w) HPG, 3.04% (w/w) 7-TMCA, 0.14% (w/w) HPGM, 6.1 % (w/w) of the title compound and 0.23% (w/w) 5-mercapto-1 -methyltetrazole.
• Example 1e as described in the Example 1 a with the following differences: the reaction titrated using ammonia 25% in water at pH 8.8 and the down stream processing was carried out without MeOH.
• the conversion was 96% (w.r.t. 7-TMCA).
• the mixture contained 0.88% (w/w) HPG, 0.26% (w/w) 7-TMCA, 0.16% (w/w) H PGM, 7.14% (w/w) of the title compound and 0.9% (w/w) 5-mercapto-1 -methyltetrazole.
• HPLC chromatogram two minor not identified impurities were visible with ⁇ 1 % HPLC area percentage.
• the enzyme was removed by filtration on glass filter no. 1 to give 65 g of filtrate at pH 8.8.
• Example 1f as Example 1 e with the following differences: reaction performed at pH 7.2 and 18°C and filtration applied in the down stream processing at the same pH. After 3 h the conversion was 81 % (w.r.t. 7-TMCA). The mixture contained 1 .05% (w/w) HPG, 0.78% (w/w) 7-TMCA, 0.02% (w/w) HPGM, 6.4% (w/w) of the title compound and no 5-mercapto-1 -methyltetrazole. In the HPLC chromatogram two minor not identified impurities were visible with ⁇ 1 % HPLC area percentage. At the end of the enzymatic reaction, the enzyme was removed by filtration on glass filter no. 1 to give a solid presenting 95% area percentage of desired product and 5% area percentage of starting material 7-TMCA. The NMR analysis of this sample indicated a purity of the product of 83.7%, with 6.8% of 7-TMCA present.
• Example 1 g as Example 1 d with the following difference: HPGM was added as solid at the beginning of the enzymatic reaction, that was carried out at 10°C and pH from 8.2 to 7.7.
• the conversion was 88% (w.r.t. 7-TMCA).
• the mixture contained 0.77% (w/w) HPG, 0.66% (w/w) 7-TMCA, 0.76% (w/w) HPGM, 10.77% (w/w) of the title compound and not quantified amount of (w/w) 5-mercapto-1 -methyltetrazole.
• the S/H ratio was 9.4.
• Example 1 h as Example 1 g, using immobilized penicillin G acylase AA instead of immobilized penicillin G acylase mutant 1 .
• Example 1 i as Example 1 g with the following difference: HPGM added as solution during the reaction.
• the conversion was 95% (w.r.t. 7-TMCA).
• the mixture contained 0.89% (w/w) HPG, 0.35% (w/w) 7-TMCA, 0.1 6% (w/w) H PGM , 1 0.43% (w/w) of the title compound and not quantified amount of (w/w) 5-mercapto-1 -methyltetrazole.
• the S/H ratio was 4.1 .
• Example 1j as Example 1 a with the following differences: reaction was titrated using NaOH 10 M and product was isolated from pH 2.72.
• the conversion was 98% (w.r.t. 7-TMCA).
• the mixture contained 1 .12% (w/w) HPG, 0.12% (w/w) 7-TMCA, 1 .18% (w/w) HPGM, 8.9% (w/w) of the title compound and 0.04% (w/w) 5-mercapto-1 -methyltetrazole.
• the enzyme was removed by filtration on glass filter no.1 to give 59 g of filtrate at pH 8.8. Under vigorous stirring at 3°C a 25% aqueous solution of H 2 S0 4 was added in 5 min to give a pH of 2.72.
• the resulting suspension was diluted with same volume of MeOH and then the product was recovered by filtration. Based on HPLC analysis, the mixture contained 3.8% HPG, 2.9% HPGM, 0.51 % 7-TMCA and 92.7% of the title compound (data based on HPLC area percentage).
• Example 1 k as Example 1j, adding acetone during the down stream process instead of MeOH.
• the enzyme was removed by filtration on glass filter no. 1 to give 59 g of filtrate at pH 8.8. Under vigorous stirring at 3°C a 25% aqueous solution of H 2 S0 4 was added in 5 min to give a pH of 2.72. The resulting suspension was diluted with same volume of acetone and then the product was recovered by filtration. Based on HPLC analysis, the mixture contained 4.3% HPG, 2.3% HPGM, 0.51 % 7-TMCA, 0.5% 5-mercapto-1 -methyltetrazole and 92.1 % of the title compound (data based on HPLC area percentage).
• Example 11 as Example 1j with the following differences: reaction performed at pH 8.5 and MeOH added at the beginning of the down stream processing. The conversion was 97% (w.r.t. 7-TMCA). The mixture contained 1 .44% (w/w) HPG, 0.1 1 % (w/w) 7-TMCA, 0.85% (w/w) HPGM, 8.2% (w/w) of the title compound and 0.1 1 % (w/w) 5-mercapto-1 -methyltetrazole. In the HPLC chromatogram two minor not identified impurities were visible with ⁇ 1 % HPLC area percentage. At the end of the enzymatic reaction, the enzyme was removed by filtration on glass filter no.1 to give 63 g of filtrate at pH 8.5.
• Example 1 m as Example 11 with the following differences: during the down stream processing the compound was precipitated at pH 4.8 and acetone was used during the filtration instead of MeOH.
• the conversion was 94% (w.r.t. 7-TMCA).
• the mixture contained 0.64% (w/w) HPG, 0.36% (w/w) 7-TMCA, 0.64% (w/w) H PGM, 8.37% (w/w) of the title compound and 0.03% (w/w) 5-mercapto-1 -methyltetrazole.
• HPLC chromatogram two minor not identified impurities were visible with ⁇ 1 % HPLC area percentage.
• the enzyme was removed by filtration on glass filter no.1 to give 59 g of filtrate at pH 8.6.
• 7-TMCA underwent chemical degradation, that occurred also in the absence of enzyme, it was faster at basic pH and at high concentration of 7-TMCA.
• the degradation product was the major side product formed during the enzymatic reaction and was identified by mass analysis to be 5-mercapto-1 -methyltetrazole.
• the chemical degradation occurred using aqueous ammonia (25%) for the titration but was neglectable when aqueous NaOH (1 or 5 or 10M) was applied.
• the amount of 5-mercapto-1 -methyltetrazole was not detectable during the dissolution step using aqueous NaOH 5M and 0.05% (w/w) and at the end of the enzymatic reaction; 0.01 % (w/w) and 0.24% (w/w) during the dissolution step and at the end of the enzymatic reaction respectively using aqueous ammonia (25%) under the same reaction conditions, or greater under different reaction conditions.

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