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WO2017186864A1 - Procédé enzymatique pour la production d'antibiotiques bêta-lactames en présence d'inoculum particulaire - Google Patents

Procédé enzymatique pour la production d'antibiotiques bêta-lactames en présence d'inoculum particulaire Download PDF

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Publication number
WO2017186864A1
WO2017186864A1 PCT/EP2017/060094 EP2017060094W WO2017186864A1 WO 2017186864 A1 WO2017186864 A1 WO 2017186864A1 EP 2017060094 W EP2017060094 W EP 2017060094W WO 2017186864 A1 WO2017186864 A1 WO 2017186864A1
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Prior art keywords
inoculum
amoxicillin
process according
particles
reaction
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Inventor
Ferdinand Zepeck
Christoph AGER
Andreas AUER
Walter EBERL
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Sandoz AG
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Sandoz AG
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
    • C12N11/082—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer obtained by reactions only involving carbon-to-carbon unsaturated bonds
    • C12N11/087—Acrylic polymers
    • 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
    • C12P37/00—Preparation of compounds having a 4-thia-1-azabicyclo [3.2.0] heptane ring system, e.g. penicillin
    • C12P37/04—Preparation of compounds having a 4-thia-1-azabicyclo [3.2.0] heptane ring system, e.g. penicillin by acylation of the substituent in the 6 position
    • 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 invention relates to the field of heterogeneous catalysis.
  • the invention provides an improved process for producing ⁇ -lactam antibiotics catalyzed by an enzyme, which is immobilized onto a carrier, wherein the resulting ⁇ -lactam antibiotics are poorly soluble in the reaction media.
  • the process according to the invention achieves particularly high yields and ensures that the enzyme immobilized onto a solid carrier retains its activity so that it remains stable for direct use in multiple further reaction cycles without the need for cost- and time-consuming reactivation.
  • the enzymatic ⁇ -lactam antibiotic synthesis is a suspension-to-suspension reaction.
  • the substrates 6- aminopenicillanic acid (6-APA) or 7-aminodesacetoxycephalosporanic acid (7-ADCA), as well as amino acid derivatives constituting the side chains of the ⁇ -lactam antibiotics, e.g., phenylglycine (PG) or p-hydroxyphenylglycine (HPG) are used as solids suspended in water.
  • the amino acid derivatives are typically used in an activated form, e.g., in the form of the corresponding ester or amide.
  • the enzyme e.g., penicillin G acylase
  • a solid carrier e.g., onto polymeric resin beads
  • the partly dissolved substrates and the respective activated amino acid derivatives are then converted to the desired ⁇ -lactam antibiotics in a reaction, which is catalyzed by said catalyst, e.g., by the penicillin G acylase immobilized onto a solid carrier.
  • Figure 13 Particle size distribution (PSD) of inoculum suspensions for enzymatic amoxicillin synthesis according to Example 4, wherein white squares ( ⁇ ) represent Amoxicillin suspension before and black squares ( ⁇ ) after size reduction.
  • PSD Particle size distribution
  • FIG. 14 EM images of immobilized enzyme after use in batches #01 to 08 according to Example 4.
  • the present invention provides an improved process for the production of ⁇ -lactam antibiotics that have a poor solubility in the reaction media that proceeds via the use of a heterogeneous catalyst.
  • it relates to an improved process for the production of amoxicillin via the use of an enzyme immobilized onto a solid carrier with an improved conversion rate from 6-APA and p- hydroxyphenylglycine methyl ester (HPGM) to amoxicillin.
  • the process according to the invention is principally suited for the production of ⁇ -lactam antibiotics having a ⁇ -lactam core structure such as a 6-aminopenicillanic acid (6-APA) core structure, or a 7- aminodesacetoxycephalosporanic acid (7-ADCA) core structure.
  • the ⁇ -lactam antibiotics having a 6- APA core structure are typically acylated with an amino acid side chain in position 6.
  • ⁇ -Lactam antibiotics having a 6-APA core structure are, for example, amoxicillin carrying a p- hydroxyphenylglycine (HPG) side chain, or ampicillin carrying a phenylglycine (PG) side chain.
  • ⁇ - lactam antibiotics having a 7-ADCA core structure are typically acylated with an amino acid side chain in position 7.
  • ⁇ -Lactam antibiotics having a 7-ADCA core structure are, for example, cefadroxil having a p-hydroxyphenylglycine (HPG) side chain, or cephalexin having a phenylglycine (PG) side chain.
  • amoxicillin and in particular, amoxicillin trihydrate, is a known substance, and processes for its production are disclosed in the state of the art.
  • the industrial scale production of semi-synthetic ⁇ -lactam derivatives such as amoxicillin, ampicillin, cefadroxil and cephalexin is performed by chemical methods under harsh conditions using reactive intermediates and organic solvents and processes that are environmentally unfriendly.
  • p-Hydroxyphenylglycine is preferably used in an activated form, such as an ester or amide form thereof, for example, in the form of a p-hydroxyphenylglycine methyl ester (HPGM).
  • HPGM p-hydroxyphenylglycine methyl ester
  • the resulting amoxicillin may then be subjected to further down-stream processing, e.g., to obtain the common end product amoxicillin trihydrate.
  • the substrates 6-APA and p-hydroxyphenylglycine methyl ester are used as solids suspended in water.
  • the enzyme e.g., penicillin G acylase
  • a solid carrier e.g. polymeric resin beads
  • the partly dissolved substrates 6-APA and HPGM are then converted to amoxicillin via the heterogeneous catalyst.
  • the resulting amoxicillin is poorly soluble in water and precipitates from the reaction media.
  • a process for the enzymatic production of ⁇ -lactam antibiotics with poor solubility in the reaction media is provided. If the reaction media is water, or an aqueous system, a solubility of about 0.1 to about 33 mg/mL in water is considered a "poor solubility".
  • Suitable ⁇ -lactam antibiotics in this regard are, e.g., amoxicillin, ampicillin, cefadroxil or cephalexin.
  • amoxicillin * trihydrate has a water solubility of about 4.5 mg/mL, ampicillin * trihydrate of about 9.0 mg/mL, cefadroxil of about 0.399 mg/mL, or cephalexin * monohydrate of about 17.2 mg/mL.
  • a preferred ⁇ -lactam antibiotic is amoxicillin.
  • a suspension of the reactants is provided (step (a)). Further provided is an enzyme immobilized onto a solid carrier (step (b)). The reactants are then contacted in the presence of said enzyme in a reaction vessel, e.g., in a bioreactor, (step c).
  • the inoculum should be present in the reaction mixture before a saturation of the ⁇ -lactam antibiotic product obtained in step (c) is reached.
  • the precipitation and/or crystallization of the ⁇ -lactam antibiotic product occurs on the surface of the particulate inoculum instead on the surface of the solid carrier, onto which the enzyme is immobilized. Consequently, the rate of conversion of the compound having a ⁇ -lactam core structure and the activated phenylglycine derivative towards the desired ⁇ -lactam antibiotic product no longer decreases over time, but remains nearly constant over the whole process.
  • the heterogeneous catalyst can also be reactivated if a particulate inoculum is added to the reaction vessel during said step (c). Even if the surface of the solid carrier, onto which the enzyme is immobilized, is covered to a significant extent with crystals of the ⁇ -lactam antibiotic product, these crystals are removed from the surface if a particulate inoculum is added to the reaction vessel during step (c). The blocking of the heterogeneous catalyst can thus be reversed.
  • a particulate inoculum is present in the reaction vessel during the contacting of said compound having a ⁇ -lactam core structure and said activated phenylglycine derivative in the presence of said enzyme, i.e., during said step (c).
  • the process for the enzymatic production of a ⁇ -lactam antibiotic comprises the steps of (a) providing a suspension comprising a compound having a ⁇ -lactam core structure and an activated phenylglycine derivative in a reaction media, (b) providing an enzyme immobilized onto a solid carrier, and (c) contacting said compound having a ⁇ -lactam core structure and said activated phenylglycine derivative in the presence of said enzyme in a reaction vessel to obtain a ⁇ -lactam antibiotic product.
  • the compound having a ⁇ -lactam core structure is selected from 6-APA and 7-ADCA, and is preferably 6-APA.
  • side chains used in the synthesis of ⁇ -lactam antibiotics are phenylacetyl side chains or activated derivatives of the phenlyacetyl side chains such as phenylglycine amides or esters therefrom, p-hydroxyphenylglycine amides or esters therefrom, dihydro-phenylglycine amides or esters therefrom, and the like containing a chiral alpha carbon due to the presence of an amino group (e.g., as in, for example, amoxicillin, ampicillin, cefadroxil, cephalexin).
  • an amino group e.g., as in, for example, amoxicillin, ampicillin, cefadroxil, cephalexin.
  • the activated phenylglycine derivative is selected from phenylglycine methyl ester (PGM) and p-hydroxyphenylglycine methyl ester (HPGM), and is preferably HPGM .
  • the ⁇ -lactam antibiotic product is preferably selected from the group of amoxicillin, ampicillin, cefadroxil and cephalexin. Most preferably, the ⁇ -lactam antibiotic product is amoxicillin.
  • the reaction media may be any reaction media suitable for that purpose.
  • the reaction media is typically an aqueous media, preferably water.
  • the suspension in step (a) is suspension of 6- APA and HPGM in an aqueous media, preferably water.
  • the enzyme used as a catalyst in the process according to the invention may be any enzyme suitable for catalyzing the enzymatic process for producing ⁇ -lactam antibiotics. Suitable enzymes are known in the art, and are, e.g., enzymes of the group of penicillin acylases, preferably penicillin G acylases including mutants thereof. Suitable enzymes and enzyme mutants are described e.g.
  • a preferred enzyme for producing the ⁇ -lactam antibiotics according to the process of the present invention is penicillin G acylase.
  • the enzyme immobilized onto a solid carrier in step (b) is preferably penicillin G acylase.
  • the enzyme is typically immobilized onto a solid carrier (heterogeneous catalyst).
  • the solid carrier may be any suitable carrier material known in the art.
  • the carrier material can include natural polysaccharide-based carriers, such as CNBr-activated agarose sepharose, epoxy activated agarose or sepharose, and ionic exchange CH-sepharose; hydrophobic adsorbents such as phenyl sepharose, synthetic organic carriers such as the epoxy carriers; the azalactone carrier, Emphaze; the ionic exchanger, Bio-Rex 70, the hydrophobic adsorbent, Amberlite XAD 4, XAD 8 and inorganic carriers, such as silanized CPG glass bead derivatives and silanized celite derivatives.
  • Suitable carriers for penicillin G acylase are further Eupergit C (PcA), polyacrylamide, gelatin-beads and agarose beads and acrylic carriers (https://ttnqmai. files. WordPress.com/2012/06/caocarrier- boundimmobilizedenzymes-principlesapolicationsdesiqn.pdf and Bonomi P, et al., 2013. Molecules. 78:14349-14365).
  • Other contemplated carriers are glyoxyl-agarose, amino-epoxy supports.
  • the solid carrier is a polymeric resin, in particular polymethacrylate beads known as Relizyme® EP1 13/M, Relizyme EP1 13/S, Sepabeads EC-EP/S, Sepabeads EC-EP/M, ECR8206M/5749 or ECR8206/5803.
  • the solid carrier is a polymeric resin, preferably polymethacrylate beads such as Relizyme® EP1 13/M beads.
  • the enzyme immobilized onto a solid carrier in step (b) is penicillin G acylase
  • the solid carrier is polymethacrylate, e.g., penicillin G acylase immobilized on polymethacrylate beads known as Relizyme® EP1 13/M.
  • particle inoculum refers to solid particles having a mean particle size from about 0.1 to about 140 pm, or from about 1 to about 100 Mm, or from about 5 to about 90 pm, or from about 10 to about 80 pm, or from about 10 to about 70 pm, or from about 10 to about 60 pm , or from about 10 to about 50 pm , or from about 10 to about 40 pm.
  • the mean particle size can be determined by methods known in the art, e.g., microscopic determination (particles of all kind; 0.5- 5000 pm (light microscope); 0.01 -10 pm (SEM/TEM); sieving (for dry powders/granulates; 100-10,000 pm (wire mesh/metal sieves), 5-100 pm (micro mesh); sedimentation techniques (gravitational settling) for dry powders/granulates; 2-200 pm; electrical Sensing Zone (e.g. Coulter) (for dry powders/granulates; 1 -1000 pm; and Phase Doppler Anemometry (PDA) (for dry powders/granulates; 1 -1000 pm).
  • microscopic determination particles of all kind; 0.5- 5000 pm (light microscope); 0.01 -10 pm (SEM/TEM); sieving (for dry powders/granulates; 100-10,000 pm (wire mesh/metal sieves), 5-100 pm (micro mesh); sedimentation techniques (gravitational settling) for dry powders/granulates; 2-200
  • the particulate inoculum comprises a high percentage of particles having a particle size of ⁇ 10 pm and between 10 and 20 pm .
  • the particulate inoculum comprises at least 20% particles having a particle size of ⁇ 10 pm and between 10 and 20 pm, or at least 30%, or at least 40%, or at least 45%, or at least 50%, or at least 55%, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or 100%, and more preferably, the particulate inoculum comprises 20% to 95% particles having a particle size of ⁇ 10 pm and between 1 0 and 20 pm , or 20% to 90%, or 20% to 85%, or 20% to 80%, or 20% to 75%, or 20% to 70%, or 20% to 60%.
  • the ratio of particles having a particle size of ⁇ 10 pm and the particles having a particle size of between 10 and 20 pm is from 1 : 10 to 10: 1 , or from 1 :9 to 9: 1 , or from 1 :8 to 8: 1 , or from 1 :7 to 7: 1 , or from 1 :6 to 6: 1 , or from 1 :5 to 5: 1 , or from 1 :4 to 4: 1 , or from 1 :3 to 3: 1 , or from 1 :2 to 2: 1 , or 1 : 1 .
  • the percentage of particles of a particular size can be determined by methods known in the art, e.g. microscopic determination (particles of all kind; 0.5-5000 pm (light microscope); 0.01 -10 pm (SEM/TEM); sieving (for dry powders/granulates; 100-10,000 pm (wire mesh/metal sieves), 5-100 pm (micro mesh); sedimentation techniques (gravitational settling) for dry powders/granulates; 2-200 pm; electrical Sensing Zone (e.g. Coulter) (for dry powders/granulates; 1 -1000 pm; and Phase Doppler Anemometry (PDA) (for dry powders/granulates; 1 -1000 pm).
  • microscopic determination particles of all kind; 0.5-5000 pm (light microscope); 0.01 -10 pm (SEM/TEM); sieving (for dry powders/granulates; 100-10,000 pm (wire mesh/metal sieves), 5-100 pm (micro mesh); sedimentation techniques (gravitational settling) for dry powders/granulates
  • PSD particle size distribution
  • the solid inoculum particles are of the same nature as the ⁇ -lactam antibiotic product obtained in step (c).
  • the particulate inoculum is preferably comprised of particles of amoxicillin.
  • the particulate inoculum is comprised of particles of ampicillin if the ⁇ -lactam antibiotic product obtained in step (c) is ampicillin, or is comprised of particles of cefadroxil if the ⁇ - lactam antibiotic product obtained in step (c) is cefadroxil, or is comprised of particles of cephalexin if the ⁇ -lactam antibiotic product obtained in step (c) is cephalexin.
  • the inoculum particles may be in any solid state form, e.g., crystalline particles, amorphous particles, or any mixed crystalline/amorphous form thereof. It is, however, preferred that the particles are in a crystalline state.
  • the particulate inoculum is present in an amount of about 1 to 20 mol% based on the amount of said compound having a ⁇ -lactam core structure, or of about 5 to 15 mol%, or of about 8 to about 12 mol%.
  • the particulate inoculum is present in an amount of about 5 to 15 mol%, and more preferably, in an amount of about 8 to about 12 mol% based on the amount of said compound having a ⁇ -lactam core structure.
  • the particulate inoculum are preferably particles of amoxicillin having a mean particle size from about 0.1 to about 140 ⁇ , or from about 1 to about 100 ⁇ , or from about 5 to about 90 ⁇ , or from about 10 to about 80 ⁇ , or from about 10 to about 70 ⁇ , or from about 10 to about 60 ⁇ , or from about 10 to about 50 ⁇ , or from about 10 to about 40 ⁇ , and which are present in step (c) in an amount of about 5 to 15 mol%, and preferably, in an amount of about 8 to about 12 mol% based on said compound having a ⁇ -lactam core structure, i.e., 6-APA.
  • the ⁇ -lactam antibiotic product produced by process according to the invention is amoxicillin
  • the particulate inoculum are preferably particles of amoxicillin having a mean particle size from about 10 to about 40 ⁇ , and which are present in step (c) in an amount of about 8 to about 12 mol% based on said compound having a ⁇ -lactam core structure, i.e., 6-APA.
  • the inoculum is added to the reaction vessel in the form of crystals having the particle size as defined above. In one embodiment, the inoculum is added to the reaction vessel in the form of a suspension ("inoculum suspension"). In this case, an inoculum suspension is prepared before the start of a reaction cycle.
  • the inoculum suspension may be prepared by providing a suspension of the desired ⁇ -lactam antibiotic product in water, and reducing the size of the ⁇ -lactam antibiotic product particles in the suspension to a mean particle size from about 0.1 to about 140 ⁇ , or from about 1 to about 100 ⁇ , or from about 5 to about 90 ⁇ , or from about 10 to about 80 ⁇ , or from about 10 to about 70 ⁇ , or from about 10 to about 60 ⁇ , or from about 10 to about 50 ⁇ , or from about 10 to about 40 ⁇ .
  • the ⁇ -lactam antibiotic product source for preparing said suspension can be any solid ⁇ -lactam antibiotic, e.g., amoxicillin, amoxicillin * trihydrate, ampicillin, cefadroxil or cephalexin, which is suspended in water.
  • Equally suited as a source for preparing said suspension is a suspension of the ⁇ -lactam antibiotic product separated after step (c) in a previous reaction cycle.
  • the inoculum added to the reaction vessel in the form of a suspension may comprise a high percentage of particles having a particle size of ⁇ 10 pm and between 10 and 20 pm.
  • the particulate inoculum may comprise at least 20% particles having a particle size of ⁇ 10 pm and between 10 and 20 pm, or at least 30%, or at least 40%, or at least 45%, or at least 50%, or at least 55%, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95% , or 100%, and more preferably, the particulate inoculum comprises 20% to 95% particles having a particle size of ⁇ 10 pm and between 10 and 20 Mm, or 20% to 90%, or 20% to 85%, or 20% to 80%, or 20% to 75%, or 20% to 70%, or 20% to 60%.
  • the ratio of particles having a particle size of ⁇ 10 pm and the particles having a particle size of between 10 and 20 pm in the inoculum suspension is from 1 : 10 to 10:1 , or from 1 :9 to 9:1 , or from 1 :8 to 8:1 , or from 1 :7 to 7:1 , or from 1 :6 to 6:1 , or from 1 :5 to 5: 1 , or from 1 :4 to 4:1 , or from 1 :3 to 3:1 , or from 1 :2 to 2: 1 , or 1 :1.
  • the reduction of the particle size of the ⁇ -lactam antibiotic product in the inoculum suspension may be effected by applying mechanical forces, e.g., mixing, milling or crushing with an appropriate equipment, but also by ultra-sonication, or another suitable method known in the art for that purpose.
  • the particle size of the crystals of the ⁇ -lactam antibiotic product in the inoculum suspension may be customized by crystallization methods known to the skilled in the art.
  • the order of adding the reactants as defined in steps (a) and (b) to the reaction vessel is not particularly critical, as long as the particulate inoculum is present during step (c).
  • the inoculum suspension may be added to the reaction vessel before the other reactants of steps (a) and (b) are added to the vessel.
  • the particulate inoculum may also be first mixed with the enzyme immobilized onto a solid carrier, or a suspension thereof, and contacting said mixture with the reactants as defined in step (a).
  • the particulate inoculum may also be first mixed with the reactants as defined in step (a), and contacting said mixture with the enzyme immobilized onto a solid carrier.
  • the suspension comprising the compound having a ⁇ -lactam core structure and an activated phenylglycine derivative are typically added as a mixed suspension into the reaction vessel; they may, however, also be added as individual suspensions.
  • the conversion of the compound having a ⁇ -lactam core structure and the activated phenylglycine derivative to the ⁇ -lactam antibiotic product starts as soon as the reactants are brought into contact with the heterogeneous catalyst, i.e., already during the addition.
  • the reaction is completed after about 60 - 180 minutes.
  • the reaction suspension containing the ⁇ -lactam antibiotic product may be discharged from the reactor, e.g., via a sieve on the bottom of the reaction vessel, whereas the enzyme immobilized onto a solid carrier remains on the bottom sieve and may be washed with water.
  • the enzyme immobilized onto a solid carrier is then ready for use in a further reaction cycle without any additional reactivation steps, i.e., without any extra steps of having to remove the ⁇ -lactam antibiotic product that adheres to its surface.
  • the ⁇ -lactam antibiotic product that adheres to the surface of the heterogeneous catalyst e.g., amoxicillin
  • this method bears the further disadvantage that the ⁇ -lactam antibiotic products such as amoxicillin are not stable under this conditions, and hence, a loss of product occurs during said reactivation step. This is avoided by the process according to the invention.
  • the process of the present invention may further comprise a step (d), wherein the enzyme immobilized onto a solid carrier is recovered by separating it from the suspension containing the resulting ⁇ -lactam antibiotic product after completion of the reaction, and optionally a step (e), wherein the enzyme immobilized onto a solid carrier is washed with water after the separation.
  • the separating step is advantageously performed via a sieve filtration.
  • a part of the suspension containing the resulting ⁇ -lactam antibiotic product that is separated from the enzyme immobilized onto a solid carrier in said step (d) may be used for producing a particulate inoculum, or for producing an inoculum suspension, respectively, that may be used in further reaction cycles.
  • the invention further relates to ⁇ -lactam antibiotic selected from amoxicillin, ampicillin, cefadroxil and cephalexin produced by the process according to the invention, preferably to amoxicillin produced by the process of the invention.
  • the invention also relates to the use of amoxicillin obtained according to the process of the invention for producing amoxicillin trihydrate.
  • the invention further relates to a process for producing amoxicillin trihydrate using amoxicillin obtained according the process of the invention.
  • the invention further relates to the use of a particulate inoculum as defined herein for producing a ⁇ -lactam antibiotic according to a process of the invention.
  • Example 1 Enzymatic synthesis of amoxicillin without particulate inoculum
  • FIG. 1 An EM image of the immobilized enzyme after the first reaction cycle is depicted in Figure 1. Adherence of amoxicillin crystals on the surface of the immobilized enzyme was observed. The immobilized enzyme was used without further removal of the adhering amoxicillin crystals for the next reaction cycle. A sample of the separated reaction mixture was taken and analyzed by HPLC showing that a conversion was achieved. The results are depicted in table 1 .
  • Example 2 Enzymatic synthesis of amoxicillin in the presence of particulate inoculum (three consecutive reaction cycles)
  • Second cycle (particulate inoculum was present): 36 g immobilized enzyme (Penicillin G acylase immobilized on Relizyme® EP1 13/M beads) was loaded into the reactor with the addition of 100 mL water and cooled to 10°C. About 9.0 g Amoxicillin*trihydrate as fine needles ( ⁇ 60 ⁇ ) were added and suspended with the immobilized enzyme. pH was adjusted to 6.3 with 5% NaOH aqueous solution and maintained at 6.3 during the reaction with 5% NaOH aqueous solution. 45 g 6-APA and 40 g HPGM (1 .04 mol equivalents) were suspended in 300 mL water and added to the reactor under stirring (50 rpm). After 120 min the reaction mixture was separated from the immobilized enzyme over the bottom sieve (125 ⁇ ) and the immobilized enzyme was washed two times with 130 mL water.
  • immobilized enzyme Penicillin G acylase immobilized on Relizyme® EP1 13/M beads
  • Second and third cycle (particulate inoculum was present):
  • Cycle no. 1 (using an inoculum suspension):
  • reaction mixture (amoxicillin suspension) obtained from the previous reaction cycle was milled to a particle size of ⁇ 60 pm and suspended with the washed immobilized enzyme from the first cycle in the reactor with 100 ml_ water.
  • 45 g 6-APA and 40 g HPGM (1 .04 mol equivalents) were suspended in 300 ml_ water and added to the reactor under stirring (50 rpm). pH was adjusted to 6.3 with 20% NaOH aqueous solution and maintained at 6.3 during the reaction with 20% NaOH aqueous solution.
  • the reaction mixture was separated from the immobilized enzyme over the bottom sieve (125 pm) and the immobilized enzyme was washed two times with 130 ml_ water.
  • a sample of the separated reaction mixture was taken and analyzed by H PLC showing that conversion is achieved. The results are depicted in table 3.
  • the procedure of the second cycle was repeated.
  • Cycle no. 7 (using an inoculum suspension): About 75 g reaction mixture (amoxicillin suspension) obtained from cycle no. 5 was milled to a mean particle size of ⁇ 60 pm and suspended with the washed immobilized enzyme from the sixth cycle in the reactor. 45 g 6-APA and 40 g HPGM (1 .04 mol equivalents) were suspended in 300 mL water and added to the reactor under stirring (50 rpm). pH was adjusted to 6.3 with 20% NaOH aqueous solution and maintained at 6.3 during the reaction with 20% NaOH aqueous solution. After 120 min the reaction mixture was separated from the immobilized enzyme over the bottom sieve (125 pm) and the immobilized enzyme was washed two times with 130 mL water.
  • Example 4 Enzymatic synthesis of amoxicillin in pilot-plant process using particulate inoculum
  • the enzymatic reactor was cooled to 10°C and charged with 3.3 kg immobilized enzyme (Penicillin G acylase immobilized on Relizyme® EP1 13/M beads).
  • the immobilized enzyme was washed 5 times with 40 L water.
  • 0.64 g Amoxicillin were suspended in 6 L water and crushed using Ultra Turrax® T45 Homogenizer or by milling for 10 minutes using colloid mill IKA MK Labor/Pilot 2000/04.
  • 4 kg of amoxicillin suspension from previous batch were crushed using Ultra Turrax or milled for 10 min and diluted with 2 L water.
  • the suspension was added to the reactor as inoculum, and the can was washed with 6 L water, and stirring was started (175 rpm).
  • HPGM 3.2 kg, 17.5 mol, 1 .06 equivalents related to 6-APA
  • 6-APA 3.6 kg, 16.6 mol
  • the pH was simultaneously adjusted to the value set at pH 6.3 with HCI (32%) and NaOH (20%), and the reaction mixture was pumped in circle over a loop. After transfer was completed the vessel was rinsed with 6 L water, and the wash water was added to the reactor.
  • pH-adjustment to pH 6,3 was continued during reaction-, discharging- and washing steps.
  • the conversion was monitored by HPLC as In-Process-Control.
  • the reaction was stopped by discharging the reactor over a bottom sieve (160 pm).
  • the reactor was washed twice with 10 L water, and the washing waters were added to the reaction mixture.
  • the enzyme reactor was then ready for the next cycle.
  • PSD particle size distribution
  • the particles were counted and grouped into different size ranges ( ⁇ 10 ⁇ , 10-20 pm, 21 -30 pm, 31 -40 pm, 41 -50 ⁇ , 51 -60 ⁇ , 61 -70 pm, 71 -80 ⁇ , 81 -90 ⁇ , 91-100 ⁇ , >100 ⁇ ) to allow better comparability.
  • the results of the PSD determination are shown in Figure 13 A-D.
  • the particulate inoculum of batches #02, 07, 09 and 14 comprises a significant percentage of particles ⁇ 10 pm and between 10 and 20 pm.
  • Indicators for the inoculum quality are the reaction course (conversion), and the separation of reaction mixture from immobilized enzyme, which can be assessed by amoxicillin concentration in wash waters.
  • Table 4 shows corresponding In-Process-Control results and concentration of amoxicillin in wash water from batches #02, 07, 09 and 14.
  • Comparison of Example 4 with Example 1 shows that the use of inoculum results in a high and constantly efficient conversion of 6-APA throughout multiple consecutive reaction cycles (table 4), while the conversion of 6-APA in Example 1 without inoculum as depicted in table 1 decreased significantly in the second reaction cycle.
  • Comparison of Example 4 with Example 3 shows that using in consecutive reaction cycles in one cycle no inoculum (cycle 6 of Example 3) gives immediate decrease of 6-APA conversion.
  • the broad variety of PSD of the used inoculum comprising a high percentage of particles ⁇ 10 ⁇ and between 10 and 20 ⁇ resulted always in satisfying results for 6- APA conversion as can be seen in table 4.

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Abstract

La présente invention concerne un procédé amélioré de production d'antibiotiques β-lactames catalysée par une enzyme, qui est immobilisée sur un support, les antibiotiques β-lactames résultants étant faiblement solubles dans le milieu de réaction. Le procédé selon l'invention permet d'obtenir des rendements particulièrement élevés et garantit que l'enzyme immobilisée sur un support solide conserve son activité de sorte qu'elle reste stable pour utilisation directe dans des cycles de réaction supplémentaires multiples sans nécessiter une réactivation coûteuse et longue.
PCT/EP2017/060094 2016-04-27 2017-04-27 Procédé enzymatique pour la production d'antibiotiques bêta-lactames en présence d'inoculum particulaire Ceased WO2017186864A1 (fr)

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Citations (8)

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Publication number Priority date Publication date Assignee Title
WO1993023164A1 (fr) * 1992-05-14 1993-11-25 Novo Nordisk A/S Procede de separation
WO1996005318A1 (fr) 1994-08-12 1996-02-22 Gist-Brocades B.V. Genes mutants d'acylases de la penicilline g
WO1997004086A1 (fr) 1995-07-18 1997-02-06 Gist-Brocades B.V. Penicilline g acylase immobilisee amelioree
WO1998020120A1 (fr) 1996-11-05 1998-05-14 Bristol-Myers Squibb Company Acylases mutantes de la penicilline g
WO1998056486A1 (fr) * 1997-06-10 1998-12-17 Dsm N.V. Procede de separation d'une substance solide
WO2004082661A1 (fr) 2003-03-21 2004-09-30 Dsm Ip Assets B.V. Poudre de trihydrate d'amoxicilline cristallin
WO2010072765A2 (fr) 2008-12-23 2010-07-01 Dsm Ip Assets B.V. Acylases de pénicilline g mutantes
WO2012032040A1 (fr) * 2010-09-07 2012-03-15 Dsm Sinochem Pharmaceuticals Netherlands B.V. Procédé de production de céphalosporines

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Publication number Priority date Publication date Assignee Title
WO1993023164A1 (fr) * 1992-05-14 1993-11-25 Novo Nordisk A/S Procede de separation
WO1996005318A1 (fr) 1994-08-12 1996-02-22 Gist-Brocades B.V. Genes mutants d'acylases de la penicilline g
WO1997004086A1 (fr) 1995-07-18 1997-02-06 Gist-Brocades B.V. Penicilline g acylase immobilisee amelioree
WO1998020120A1 (fr) 1996-11-05 1998-05-14 Bristol-Myers Squibb Company Acylases mutantes de la penicilline g
WO1998056486A1 (fr) * 1997-06-10 1998-12-17 Dsm N.V. Procede de separation d'une substance solide
WO2004082661A1 (fr) 2003-03-21 2004-09-30 Dsm Ip Assets B.V. Poudre de trihydrate d'amoxicilline cristallin
WO2010072765A2 (fr) 2008-12-23 2010-07-01 Dsm Ip Assets B.V. Acylases de pénicilline g mutantes
WO2012032040A1 (fr) * 2010-09-07 2012-03-15 Dsm Sinochem Pharmaceuticals Netherlands B.V. Procédé de production de céphalosporines

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BRUGGINK A ET AL: "Penicillin acylase in the industrial production of beta-lactam antibiotics", ORGANIC PROCESS RESEARCH AND DEVELOPMENT, CAMBRIDGE, GB, vol. 2, no. 2, March 1998 (1998-03-01), pages 128 - 133, XP002233341, DOI: 10.1021/OP9700643 *

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