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Title:
ENZYMATIC PROCESS FOR THE PRODUCTION OF BETA-LACTAM ANTIBIOTICS IN THE PRESENCE OF PARTICULATE INOCULUM
Document Type and Number:
WIPO Patent Application WO/2017/186864
Kind Code:
A1
Abstract:
The invention provides an improved process for producing β-lactam antibiotics catalyzed by an enzyme, which is immobilized onto a carrier, wherein the resulting β-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.

Inventors:
ZEPECK FERDINAND (AT)
AGER CHRISTOPH (AT)
AUER ANDREAS (AT)
EBERL WALTER (AT)
Application Number:
PCT/EP2017/060094
Publication Date:
November 02, 2017
Filing Date:
April 27, 2017
Export Citation:
Click for automatic bibliography generation   Help
Assignee:
SANDOZ AG (CH)
International Classes:
C12P35/04; C12N11/00; C12P37/04
Domestic Patent References:
WO1998056486A11998-12-17
WO1993023164A11993-11-25
WO2012032040A12012-03-15
WO2004082661A12004-09-30
WO1997004086A11997-02-06
WO2004082661A12004-09-30
WO2010072765A22010-07-01
WO1996005318A11996-02-22
WO1998020120A11998-05-14
Other References:
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
BONOMI P ET AL., MOLECULES, vol. 18, 2013, pages 14349 - 14365, Retrieved from the Internet
Attorney, Agent or Firm:
GREINER, Elisabeth (DE)
Download PDF:
Claims:
Claims

1 . Process for the enzymatic production of a β-lactam antibiotic comprising

(a) providing a suspension comprising a compound having a β-lactam core structure, and an activated phenylglycine derivative in a reaction media,

(b) providing an enzyme immobilized onto a solid carrier,

(c) contacting said compound having a β-lactam core structure and said activated

phenylglycine derivative in the presence of said enzyme in a reaction vessel to obtain a β- lactam antibiotic product,

wherein a particulate inoculum is present in step (c). 2. The process according to claim 1 , wherein said compound having a β-lactam core structure is selected from 6-aminopenicillanic acid (6-APA) and 7-aminodesacetoxycephalosporanic acid (7- ADCA), and wherein said activated phenylglycine derivative is selected from phenylglycine methyl ester (PGM) and p-hydroxyphenylglycine methyl ester (HPGM).

3. The process according to claims 1 or 2, wherein the β-lactam antibiotic product is selected from the group of amoxicillin, ampicillin, cefadroxil and cephalexin, preferably, wherein the β-lactam antibiotic product is amoxicillin.

4. The process according to any one of the preceding claims, wherein the particulate inoculum has a mean particle size from about 0.1 to about 140 pm, or from about 1 to about 100 μπη, or from about 5 to about 90 pm, or from about 10 to about 80 μιη, or from about 10 to about 70 μιη, or from about 10 to about 60 pm, or from about 10 to about 50 μηπ, or from about 10 to about 40 pm, preferably from about 10 to about 80 μιτι, or from about 10 to about 70 pm, or from about 10 to about 60 μιη, or from about 10 to about 50 μιη, or from about 10 to about 40 μιτι, more preferably from about 10 to about 60 pm, or from about 10 to about 50 pm, or from about 10 to about 40 μιη, most preferably from about 10 to about 40 pm. 5. The process according to any of the preceding claims, wherein the particulate inoculum comprises at least 20% particles having a particle size of < 10 pm and between 10 and 20 μιτι, 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%, preferably 20% to 95%, or 20% to 90%, or 20% to 85%, or 20% to 80%, or 20% to 75%, or 20% to 70%, or 20% to 60%.

6. The process according to any of the preceding claims, wherein the ratio of particles having a particle size of < 10 pm and the particles having a particle size of between 1 0 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 . 7. The process according to any one of the preceding claims, wherein the particulate inoculum is present in step (c) in an amount of about 1 to 20 mol% based on the amount of said compound having a β-lactam core structure, or of about 5 to 15 mol%, or of about 8 to about 12 mol% .

8. The process according to any one of the preceding claims, wherein the particulate inoculum are particles corresponding to the β-lactam antibiotic product obtained in step (c), preferably, wherein the particulate inoculum is comprised of particles of amoxicillin if the β-lactam antibiotic product obtained in step (c) is amoxicillin, or of particles of ampicillin if the β-lactam antibiotic product obtained in step (c) is ampicillin, or of particles of cefadroxil if the β-lactam antibiotic product obtained in step (c) is cefadroxil, or of particles of cephalexin if the β-lactam antibiotic product obtained in step (c) is cephalexin.

9. The process according to any one of the preceding claims, wherein the particulate inoculum is added to the reaction vessel in the form of a suspension ("inoculum suspension"). 10. The process according to claim 9, wherein the inoculum suspension is produced by providing a suspension of said β-lactam antibiotic product in water, and reducing the size of the β-lactam antibiotic product particles in the suspension to a mean particle size from about 0.1 to about 140 μηπ, or from about 1 to about 100 pm, or from about 5 to about 90 m, or from about 10 to about 80 Mm, or from about 10 to about 70 pm, or from about 10 to about 60 Mm, or from about 10 to about 50 Mm, or from about 10 to about 40 m, preferably 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, more preferably from about 10 to about 60 pm, or from about 10 to about 50 Mm, or from about 10 to about 40 m, most preferably from about 10 to about 40 pm.

1 1 . The process of claim 9 or 10, wherein the inoculum suspension comprises at least 20% particles having a particle size of < 10 pm and between 10 and 20 Mm, 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%, preferably 20% to 95%, or 20% to 90%, or 20% to 85%, or 20% to 80%, or 20% to 75%, or 20% to 70%, or 20% to 60%. 12. The process of claim 9 or 1 1 , wherein 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.

13. The process according to claim 10 or 12, wherein the size of the particles is reduced by ultra- sonication, mixing or milling, preferably by milling or mixing, most preferably by milling.

14. The process according to any one of the preceding claims, further comprising a step (d), wherein the enzyme immobilized onto a solid carrier is recovered by separating it from the suspension containing the resulting β-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.

15. The process according to claim 14, wherein the separating step is performed via a sieve filtration.

16. The process according to any one of the preceding claims, wherein the reaction media is an aqueous media, preferably water.

17. The process according to any one of the preceding claims, wherein the enzyme is penicillin G acylase.

18. The process according to any one of the preceding claims, wherein the solid carrier is a polymeric resin, preferably polymethacrylate beads such as Relizyme® EP1 13/M beads.

19. β-Lactam antibiotic selected from amoxicillin, ampicillin, cefadroxil and cephalexin produced by the process according to any one of the preceding claims, preferably amoxicillin produced by the process according to any one of the preceding claims.

20. Use of amoxicillin according to claim 19 for producing amoxicillin trihydrate.

21 . Process for producing amoxicillin trihydrate using amoxicillin produced according to any one of claims 1 to 18.

22. Use of a particulate inoculum as defined in claims 4 to 7 for producing a β-lactam antibiotic

according to a process as defined in claims 1 to 18.

Description:
ENZYMATIC PROCESS FOR THE PRODUCTION OF BETA-LACTAM ANTIBIOTICS IN THE PRESENCE OF PARTICULATE INOCULUM

Field of the Invention

The invention relates to the field of heterogeneous catalysis. The invention provides an improved process for producing β-lactam antibiotics catalyzed by an enzyme, which is immobilized onto a carrier, wherein the resulting β-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. Background of the Invention

The enzymatic β-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 β-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, is typically immobilized onto a solid carrier, e.g., onto polymeric resin beads, and is also suspended in water so as to form a heterogeneous catalyst. The partly dissolved substrates and the respective activated amino acid derivatives are then converted to the desired β-lactam antibiotics in a reaction, which is catalyzed by said catalyst, e.g., by the penicillin G acylase immobilized onto a solid carrier.

The resulting β-lactam antibiotic products are poorly soluble in water and precipitate from the reaction media. However, it was observed that the conversion rate from the substrates and the activated amino acid derivatives to the desired β-lactam antibiotics decreases significantly over time, and in particular during the long-term or repeated use of the enzyme immobilized onto a solid carrier (heterogeneous catalyst) in consecutive cycles of the enzymatic reaction, which is particularly relevant for the large scale production of β-lactam antibiotics in an industrial setting. Consequently there is a need of process improvement.

It has now been surprisingly found that when producing β-lactam antibiotics with poor solubility in the reaction media, using an enzyme immobilized onto a carrier as a heterogeneous catalyst, the products tend to precipitate onto the surface of the heterogeneous catalyst, thus inhibiting the enzyme and resulting in low yields of the product. The present invention provides a process to avoiding and reversing precipitation of the product onto the heterogeneous catalysts to allow the reactions to be more efficient in terms of time and yield, thus saving significant costs. Brief Description of the Figures

Figure 1. EM images of the immobilized enzyme after the first reaction cycle according to Example 1 (approximately 120 min) wherein no inoculum was present during the reaction (Left image:

accelerating voltage = 4.64 kV, working distance = 1 1.5 mm, Signal A = SE1 ; right image: accelerating voltage = 4.64 kV, working distance = 12.0 mm, Signal A = SE1 ).

Figure 2. EM images of the immobilized enzyme after the second reaction cycle according to Example

1 (approximately 120 minutes) wherein no inoculum was present during the reaction (Left image: accelerating voltage = 4.64 kV, working distance = 12.0 mm, Signal A = SE1 ; right image: accelerating voltage = 4.64 kV, working distance = 1 1.0 mm, Signal A = SE1 ). Figure 3. EM images of the immobilized enzyme after the first reaction cycle according to Example 2 (approximately 120 min) wherein inoculum (9.0 g Amoxicillin * trihydrate crystals < 60μππ added) was present during the reaction (Left image: accelerating voltage = 4.64 kV, working distance = 1 1.5 mm, Signal A = SE1 ; right image: accelerating voltage = 4.64 kV, working distance = 1 1 .5 mm, Signal A = SE1 ). Figure 4. EM images of the immobilized enzyme after the second reaction cycle according to Example

2 (approximately 120 min) wherein inoculum (9.0 g Amoxicillin*trihydrate crystals < 60μιη added) was present during the reaction (Left image: accelerating voltage = 4.64 kV, working distance = 1 1 .5 mm, Signal A = SE1 ; right image: accelerating voltage = 4.64 kV, working distance = 1 1 .5 mm, Signal A = SE1 ). Figure 5. EM images of the immobilized enzyme after the third reaction cycle according to Example 2 (approximately 120 min) wherein inoculum (9.0 g Amoxicillin * trihydrate crystals < 60μππ added) was present during the reaction (Left image: accelerating voltage = 4.64 kV, working distance = 1 1.5 mm, Signal A = SE1 ; right image: accelerating voltage = 4.64 kV, working distance = 1 1 .5 mm, Signal A = SE1 ). Figure 6. EM images of the immobilized enzyme after the first reaction cycle according to Example 3 (approximately 120 min) wherein inoculum (9.0 g milled Amoxicillin * trihydrate added as an aqueous suspension) was present during the reaction (Left image: accelerating voltage = 4.64 kV, working distance = 12.0 mm, Signal A = SE1 ; right image: accelerating voltage = 4.64 kV, working distance = 12.0 mm, Signal A = SE1 ). Figure 7. EM images of the immobilized enzyme after the second reaction cycle according to Example

3 (approximately 120 min) wherein inoculum (75 g milled amoxicillin added as a suspension derived from the first reaction cycle) was present during the reaction (Left image: accelerating voltage = 4.64 kV, working distance = 12.5 mm, Signal A = SE1 ; right image: accelerating voltage = 4.64 kV, working distance = 12.0 mm, Signal A = SE1 ). Figure 8. EM images of the immobilized enzyme after the third reaction cycle according to Example 3 (approximately 120 min) wherein inoculum (75 g milled amoxicillin added as a suspension derived from the second reaction cycle) was present during the reaction (Left image: accelerating voltage = 4.64 kV, working distance = 12.5 mm, Signal A = SE1 ; right image: accelerating voltage = 4.64 kV, working distance = 12.0 mm, Signal A = SE1 ).

Figure 9. EM images of the immobilized enzyme after the fourth reaction cycle according to Example 3 (approximately 120 min) wherein inoculum (75 g milled amoxicillin added as a suspension derived from the third reaction cycle) was present during the reaction (Left image: accelerating voltage = 4.64 kV, working distance = 12.0 mm, Signal A = SE1 ; right image: accelerating voltage = 4.64 kV, working distance = 1 1.5 mm, Signal A = SE1 ).

Figure 10. EM images of the immobilized enzyme after the fifth reaction cycle according to Example 3 (approximately 120 min) wherein inoculum (75 g milled amoxicillin added as a suspension derived from the fourth reaction cycle) was present during the reaction (Left image: accelerating voltage = 4.64 kV, working distance = 1 1 .5 mm, Signal A = SE1 ; right image: accelerating voltage = 4.64 kV, working distance = 1 1.5 mm, Signal A = SE1 ).

Figure 11. EM images of the immobilized enzyme after the sixth reaction cycle according to Example 3 (approximately 120 min) wherein no inoculum was present during the reaction (Upper left image: accelerating voltage = 4.64 kV, working distance = 1 1.0 mm, Signal A = SE1 ; upper right image: accelerating voltage = 4.64 kV, working distance = 1 1.0 mm, Signal A = SE1 ; Lower left image:

accelerating voltage = 4.64 kV, working distance = 1 1.5 mm, Signal A = SE1 ; lower right image:

accelerating voltage = 4.64 kV, working distance = 1 1.0 mm, Signal A = SE1 ). Figure 12. EM images of the immobilized enzyme after the seventh reaction cycle according to

Example 3 (approximately 120 min) wherein inoculum (75 g milled amoxicillin added as a suspension derived from the fifth reaction cycle) was present during the reaction (Left image: accelerating voltage = 4.64 kV, working distance = 12.0 mm, Signal A = SE1 ; right image: accelerating voltage = 4.64 kV, working distance = 1 1.5 mm, Signal A = SE1 ). 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. (A) Solid inoculum (#02) and (B) suspension milled (#07) with Ultraturrax® T45. (C) Solid inoculum (#09) and (D) suspension milled (#14) with IKA Labor/Pilot 2000/04. Particle size distribution was determined by measuring the size of the particles on a microscopy picture and is depicted in size ranges < 10 μιη, 10-20 μιη, 21-30 μιη, 31 -40 μιη, 41 -50 μιη, 51 -60 μιτι, 61 -70 μιτι, 71 -80 μηι, 81 -90 μιτι, 91 -100 μιτι, >100 μιτι.

Figure 14. EM images of immobilized enzyme after use in batches #01 to 08 according to Example 4. The used inoculum was prepared with Amoxicillin suspension from previous batch and Ultraturrax® T45 (A: accelerating voltage = 5.57 kV, working distance = 12.5 mm, Signal A = SE1 ; B: accelerating voltage = 5.57 kV, working distance = 12.5 mm, Signal A = SE1 ). Description of the Invention

The present invention provides an improved process for the production of β-lactam antibiotics that have a poor solubility in the reaction media that proceeds via the use of a heterogeneous catalyst. In a preferred embodiment, 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 β-lactam antibiotics having a β-lactam core structure such as a 6-aminopenicillanic acid (6-APA) core structure, or a 7- aminodesacetoxycephalosporanic acid (7-ADCA) core structure. The β-lactam antibiotics having a 6- APA core structure are typically acylated with an amino acid side chain in position 6. β-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. The β- lactam antibiotics having a 7-ADCA core structure are typically acylated with an amino acid side chain in position 7. β-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.

While the process according to the invention is principally suited for the production of any β-lactam antibiotics having a poor solubility in the reaction media, the invention will be described in more detail in the following using amoxicillin as an example. Amoxicillin, and in particular, amoxicillin trihydrate, is a known substance, and processes for its production are disclosed in the state of the art. Traditionally, the industrial scale production of semi-synthetic β-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. Therefore, the synthesis of these β-lactam antibiotics catalyzed by enzymes constitutes a clear example of an enzymatic reaction of industrial importance and a more sustainable production process. The enzymatic synthesis of Amoxicillin is described, e.g., in WO 97/04086, WO 2004/082661 and WO 2010/072765. In a typical enzymatic procedure for producing amoxicillin, 6-APA is acylated with the aid of side chain p-hydroxyphenylglycine in the presence of an enzyme, e.g., penicillin G acylase, according to the reaction scheme below. 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). The resulting amoxicillin may then be subjected to further down-stream processing, e.g., to obtain the common end product amoxicillin trihydrate.

6-APA MW: 181 ,19 C 16 H 19 N 3 0 5 S

3a

MW: 216,26

The substrates 6-APA and p-hydroxyphenylglycine methyl ester (HPGM) are used as solids suspended in water. The enzyme, e.g., penicillin G acylase, is immobilized onto a solid carrier, e.g. polymeric resin beads, and is also suspended in water so as to form a heterogeneous catalyst. 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. It was observed that the conversion rate from 6-APA and HPGM to amoxicillin significantly decreased over time, i.e., during the repeated or long-term use of said enzyme immobilized onto the solid carrier in consecutive cycles of the enzymatic reaction. The present inventors found that when producing β- lactam antibiotics with poor solubility in the reaction media via a heterogeneous catalyst, the products precipitate onto the surface of the heterogeneous catalyst, thus resulting in low yields of the product. It has now been surprisingly found that the decrease of the conversion rate can be avoided by performing the reaction in the presence of a particulate inoculum.

According to this invention, a process for the enzymatic production of β-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 β-lactam antibiotics in this regard are, e.g., amoxicillin, ampicillin, cefadroxil or cephalexin. For example, 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 β-lactam antibiotic is amoxicillin.

In the process according to the invention, 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).

It has been found that after a saturation of the β-lactam antibiotic in the reaction mixture is reached (i.e., during step (c)), the β-lactam antibiotic product crystallizes and precipitates on the surface of the solid carrier, onto which the enzyme is immobilized. Thus, the surface of the solid carrier, onto which the enzyme is immobilized, is increasingly covered with crystals of the β-lactam antibiotic product. This leads to the blocking of this heterogeneous catalyst. It is believed that said blocking results in a reduced total enzymatic activity of the heterogeneous catalyst, and leads to a significant decrease of the rate of enzymatic conversion of the compound having a β-lactam core structure, e.g., 6-APA or 7- ADCA, and the activated phenylglycine derivative to the desired β-lactam antibiotic product over time. It has now been surprisingly found that the precipitation of the β-lactam antibiotic particles onto the surface of the solid carrier, onto which an enzyme is immobilized, can be avoided if a particulate inoculum with a defined particle size is present in the reaction vessel, when the reactants are contacted (i.e., during step (c)). The inoculum should be present in the reaction mixture before a saturation of the β-lactam antibiotic product obtained in step (c) is reached. In this case, the precipitation and/or crystallization of the β-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 β-lactam core structure and the activated phenylglycine derivative towards the desired β-lactam antibiotic product no longer decreases over time, but remains nearly constant over the whole process. This makes the enzymatic production process more robust during the reaction time, i.e., over multiple reaction cycles, e.g., over 10, 20, 50, 100, 200, 300, 400, 500 or even 1000 reaction cycles, as well as during long-term reactions, e.g., during continuous-flow reactions, and leads to high yields. Equally important is the fact that the enzyme immobilized onto a solid carrier thereby retains its activity and can be directly used in further reaction cycles. Thus, the time-consuming and laborious additional step of reactivating the heterogeneous catalyst by removing the β-lactam antibiotic product that adheres to the surface of the enzyme immobilized onto a solid carrier is avoided. This is particularly advantageous in terms of costs and efficiency of the overall work-flow, in particular in an industrial setting.

It has been further surprisingly found that 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 β-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. Consequently, it is important for the improved process according to the invention that a particulate inoculum is present in the reaction vessel during the contacting of said compound having a β-lactam core structure and said activated phenylglycine derivative in the presence of said enzyme, i.e., during said step (c).

In one embodiment, the process for the enzymatic production of a β-lactam antibiotic comprises the steps of (a) providing a suspension comprising a compound having a β-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 β-lactam core structure and said activated phenylglycine derivative in the presence of said enzyme in a reaction vessel to obtain a β-lactam antibiotic product. During the contacting of said compound having a β-lactam core structure with said activated phenylglycine derivative in the presence of said enzyme, i.e., during step (c), a particulate inoculum is present. In the process of the invention, the compound having a β-lactam core structure is selected from 6-APA and 7-ADCA, and is preferably 6-APA. Examples of side chains used in the synthesis of β-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). Preferably, the activated phenylglycine derivative is selected from phenylglycine methyl ester (PGM) and p-hydroxyphenylglycine methyl ester (HPGM), and is preferably HPGM . Consequently, the β-lactam antibiotic product is preferably selected from the group of amoxicillin, ampicillin, cefadroxil and cephalexin. Most preferably, the β-lactam antibiotic product is amoxicillin.

The reaction media may be any reaction media suitable for that purpose. In the production of β-lactam antibiotics according to the invention, the reaction media is typically an aqueous media, preferably water. If the β-lactam antibiotic product is amoxicillin, 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 β-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. in WO 96/05318, WO 98/20120 and WO 2010/072765. A preferred enzyme for producing the β-lactam antibiotics according to the process of the present invention is penicillin G acylase. For example, if the β-lactam antibiotic product is amoxicillin, the enzyme immobilized onto a solid carrier in step (b) is preferably penicillin G acylase.

In the process of the invention, 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. In preferred embodiments, 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. In highly preferred embodiments, the solid carrier is a polymeric resin, preferably polymethacrylate beads such as Relizyme® EP1 13/M beads. It is most preferred that the enzyme immobilized onto a solid carrier in step (b) is penicillin G acylase, and the solid carrier is polymethacrylate, e.g., penicillin G acylase immobilized on polymethacrylate beads known as Relizyme® EP1 13/M.

The term "particulate inoculum" as used herein 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). Other techniques for measuring particle size are the Laser Light Scattering Technique (0.02-2000 pm), the X-Ray Sedimentation Technique (0.1 um to 300 pm), the H.E.L. Limited Subsieve AutoSizer (SAS) (0.2 pm to 75 pm) or Light Obscuration (0.5 to 400 pm). In a preferred embodiment the particulate inoculum comprises a high percentage of particles having a particle size of < 10 pm and between 10 and 20 pm . Preferably, 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%.

In a preferred embodiment 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). Other techniques for measuring particle size are the Laser Light Scattering Technique (0.02-2000 pm), the X-Ray Sedimentation Technique (0.1 um to 300 pm), the H.E.L. Limited Subsieve AutoSizer (SAS) (0.2 pm to 75 pm) or Light Obscuration (0.5 to 400 pm). For example, particle size distribution (PSD) of the particulate inoculum can be estimated by measuring the size of the particles on a microscopy picture, counting and grouping the particles into different size ranges of < 10 pm, 10-20 pm , 21 -30 pm, 31 -40 pm, 41 -50 pm, 51 -60 pm, 61 -70 pm, 71 -80 pm, 81 -90 pm, 91 -100 pm, >100 pm . In a preferred embodiment, the solid inoculum particles are of the same nature as the β-lactam antibiotic product obtained in step (c). For example, in the process for the enzymatic production of amoxicillin according to the invention, the particulate inoculum is preferably comprised of particles of amoxicillin. Hence, the particulate inoculum is comprised of particles of ampicillin if the β-lactam antibiotic product obtained in step (c) is ampicillin, or is comprised of particles of cefadroxil if the β- lactam antibiotic product obtained in step (c) is cefadroxil, or is comprised of particles of cephalexin if the β-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.

During the contacting of said compound having a β-lactam core structure and said activated phenylglycine derivative in the presence of said enzyme in the reaction vessel to obtain a β-lactam antibiotic product, i.e., during step (c), the particulate inoculum is present in an amount of about 1 to 20 mol% based on the amount of said compound having a β-lactam core structure, or of about 5 to 15 mol%, or of about 8 to about 12 mol%. Preferably, 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 β-lactam core structure. If the β-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 0.1 to about 140 μιη, or from about 1 to about 100 μιη, or from about 5 to about 90 μιη, or from about 10 to about 80 μηι, or from about 10 to about 70 μιη, or from about 10 to about 60 μιη, or from about 10 to about 50 μιη, or from about 10 to about 40 μιη, 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 β-lactam core structure, i.e., 6-APA. In a particularly preferred embodiment, the β-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 μιη, and which are present in step (c) in an amount of about 8 to about 12 mol% based on said compound having a β-lactam core structure, i.e., 6-APA.

In one embodiment, 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 β-lactam antibiotic product in water, and reducing the size of the β-lactam antibiotic product particles in the suspension to a mean particle size from about 0.1 to about 140 μιη, or from about 1 to about 100 μιη, or from about 5 to about 90 μηι, or from about 10 to about 80 μιη, or from about 10 to about 70 μπη, or from about 10 to about 60 μηπ, or from about 10 to about 50 μιη, or from about 10 to about 40 μηι . The β-lactam antibiotic product source for preparing said suspension can be any solid β-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 β-lactam antibiotic product separated after step (c) in a previous reaction cycle.

In a preferred embodiment the inoculum added to the reaction vessel in the form of a suspension ("inoculum suspension") may comprise a high percentage of particles having a particle size of < 10 pm and between 10 and 20 pm. Preferably, 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%.

In a preferred embodiment 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 β-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. Alternatively, the particle size of the crystals of the β-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). For example, the inoculum suspension may be added to the reaction vessel before the other reactants of steps (a) and (b) are added to the vessel. However, 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). Vice versa, 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 β-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 β-lactam core structure and the activated phenylglycine derivative to the β-lactam antibiotic product, e.g., 6-APA and HPGM to amoxicillin, starts as soon as the reactants are brought into contact with the heterogeneous catalyst, i.e., already during the addition. Depending on the activity of the enzyme immobilized onto a solid carrier, the reaction is completed after about 60 - 180 minutes. The reaction suspension containing the β-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 β-lactam antibiotic product that adheres to its surface. The β-lactam antibiotic product that adheres to the surface of the heterogeneous catalyst, e.g., amoxicillin, can be removed from the surface of the heterogeneous catalyst by additional washing steps with alkaline aqueous solution at a pH of about 9.5, e.g., with aqueous solutions of NaOH, KOH, NH 3 or similar bases. Apart from the additional washing step necessary, which is time and cost consuming, this method bears the further disadvantage that the β-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.

Thus, 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 β-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. In the process according to the invention, the separating step is advantageously performed via a sieve filtration.

A part of the suspension containing the resulting β-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 β-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 β-lactam antibiotic according to a process of the invention.

The invention will now be illustrated by way of non-limiting examples. Examples

HPLC method

Gradient HPLC-System with UV-Detector

Column Bischoff; Nucleosil 100-53 C 18, 125 x 4,0, Bead Size: 5 pm

Eluent A Na-/K-phosphate buffer pH 3.11

Eluent B Acetonitrile Column oven temperature 30°C

Flow: 1.5 mL/min

Detection: 220 nm

Gradient

Preparation of an inoculum suspension

If no inoculum from the previous cycle or enzymatic conversion is available: About 9.0 g Amoxicillin trihydrate were suspended in 75 mL water and mixed (10 - 30 min) with a lab mixer (14000 rpm) for particle size reduction. Particle size: < 60 pm. If an inoculum from a previous reaction cycle or enzymatic conversion is available: about 75 g reaction mixture (amoxicillin suspension: 120 g/L; particle size: 0.1 -140 pm) from the previous cycle was mixed for 10 - 30 min. Particle size: < 60 pm.

Example 1 : Enzymatic synthesis of amoxicillin without particulate inoculum

First cycle (without inoculum):

23 g immobilized enzyme (Penicillin G acylase immobilized on Relizyme® EP1 13/M beads) was suspended in 100 ml. water, loaded into a reactor, and cooled to 10°C. 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). The pH was adjusted to 6.3 with 5% NaOH aqueous solution and maintained at 6.3 during the reaction with 5% 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.

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 .

Second cycle (without inoculum):

45 g 6-APA and 40 g HPGM (1 .04 mol equivalents) were suspended in 300 mL water and added to the reactor and the washed immobilized enzyme from the first cycle under stirring (50 rpm) at 10°C. pH was adjusted to 6.3 with 5% NaOH aqueous solution and maintained at 6.3 during the reaction with 5% NaOH aqueous solution. After 120 min the reaction mixture was separated from the immobilized enzyme over the bottom sieve (125 μπη) and the immobilized enzyme was washed two times with 130 mL water.

An EM image of immobilized enzyme after the second reaction cycle is depicted in Figure 2. The amount of amoxicillin crystals adhering to the surface of the immobilized enzyme was increased after the second reaction cycle compared to the first 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 . As can be seen from table 1 , the conversion of 6-APA after 120 min reaction time decreased significantly in the second reaction cycle.

Table 1 :

Example 2: Enzymatic synthesis of amoxicillin in the presence of particulate inoculum (three consecutive reaction cycles)

First 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 μηπ) 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 μιη) and the immobilized enzyme was washed two times with 130 mL water.

An EM image of the immobilized enzyme after the first reaction cycle is depicted in Figure 3. The amount of amoxicillin crystals adhering to the surface of the immobilized enzyme was significantly lower compared to example 1 , first reaction cycle. A sample of the separated reaction mixture was taken and analyzed by HPLC showing that conversion was achieved. The results are depicted in table 2. Indeed, the conversion rate was significantly higher than the conversion rate observed in example 1 , first reaction cycle.

Second and third cycle (particulate inoculum was present):

About 9.0 g Amoxicillin*trihydrate as fine needles (< 60 pm) were added and suspended with the washed immobilized enzyme from the first cycle in 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 5% NaOH aqueous solution and maintained at 6.3 during the reaction with 5% NaOH aqueous solution. After 120 min the reaction mixture was separated from the immobilized enzyme over the bottom sieve (125 μπτι) and the immobilized enzyme was washed two times with 130 mL water. A sample of the separated reaction mixture was taken and analyzed by HPLC showing that conversion is achieved. The results are depicted in table 2. For the third cycle the procedure of the second cycle was repeated.

EM images of the immobilized enzyme after the 2 nd and 3 rd reaction cycle are depicted in Figures 4 and 5, respectively. The amount of amoxicillin crystals adhering to the surface of the immobilized enzyme was significantly lower compared to the amount of crystals that adhered to the immobilized enzyme in example 1 , first reaction cycle. A sample of the separated reaction mixture was taken and analyzed by HPLC showing that conversion was achieved. The results are depicted in table 2. As can be seen from table 2, the conversion of 6-APA after 120 min reaction time in the 2 nd and 3 rd cycle remains stable compared to the 1 st cycle. The conversion rate was significantly higher than the conversion rate observed in example 1 , first reaction cycle, and was comparable to the conversion rate observed in the first reaction cycle of example 2. This result is in line with Figures 4 and 5, which show that the surface of the immobilized enzyme was not blocked with amoxicillin crystals, so that it was fully functional during all reaction cycles without further reactivation steps. Table 2:

Example 3: Enzymatic synthesis of amoxicillin with and without inoculum (7 consecutive reaction cycles)

Cycle no. 1 (using an inoculum suspension):

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 was suspended in water and milled to a particle size of < 60 pm. The milled Amoxicillin trihydrate suspension was added to the immobilized enzyme. pH was adjusted to 6.3 with 20% NaOH aqueous solution and maintained at 6.3 during the reaction with 20% 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 pm) and the immobilized enzyme was washed two times with 130 ml_ water.

An EM image of the immobilized enzyme after the 1 st reaction cycle is depicted in Figure 6. The amount of amoxicillin crystals adhering to the surface of the immobilized enzyme was significantly lower compared to example 1 , first reaction cycle. A sample of the separated reaction mixture was taken and analyzed by HPLC showing that conversion was achieved. The results are depicted in table 3.

Cycles no. 2 to 5 (using an inoculum suspension):

About 75 g 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. 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. 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. For the third to fifth cycle the procedure of the second cycle was repeated.

EM images of the immobilized enzyme after each of the reaction cycles 2-5 are depicted in Figures 7- 10. The amount of amoxicillin crystals adhering to the surface of the immobilized enzyme was significantly lower compared to example 1 , first reaction cycle. Samples of the separated reaction mixtures were taken and analyzed by HPLC showing that conversion was achieved. The results are depicted in table 3.

Cycle no. 6 (without inoculum):

For the sixth cycle no inoculum was used. 45 g 6-APA and 40 g HPGM (1 .04 mol equivalents) were suspended in 300 mL water and added to the reactor and the washed immobilized enzyme from the previous cycle 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 1 30 mL water. EM images of the immobilized enzyme after this reaction cycle 6 are depicted in Figure 1 1 . The adherence of amoxicillin crystals on the surface of the immobilized enzyme was comparable to example 1 , first reaction cycle. A sample of the separated reaction mixture was taken and analyzed by HPLC showing that conversion was achieved. The results are depicted in table 3.

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.

An EM image of the immobilized enzyme after reaction cycle 7 is depicted in Figure 12. The amount of amoxicillin crystals adhering to the surface of the immobilized enzyme was significantly lower compared to example 1 , first reaction cycle, and also compared to the previous reaction cycle 6, see Figure 1 1 . A sample of the separated reaction mixture was taken and analyzed by HPLC showing that conversion was achieved. The results are depicted in table 3. As can be seen from table 3, a constantly high conversion of 6-APA after 120 min reaction time was achieved in reaction cycles no. 1 to 5, wherein an inoculum was used. A significant decrease of the conversion of 6-APA after 120 min reaction time was, however, observed in reaction cycle 6, which was performed without the particulate inoculum . The conversion increased again in reaction cycle no. 7, when a particulate inoculum was present again. This is in line with the finding that the immobilized enzyme was not blocked with amoxicillin crystals as long as the inoculum was present during the conversion, so that it could be used without further reactivation steps, cf., Figures 6 to 10. However, in the reaction performed in the absence of particulate inoculum, surface of the immobilized enzyme became significantly covered with crystals, which had a negative impact on its activity, see Figure 1 1 , and table 3. As derivable from figure 12, the addition of the particulate inoculum to a reaction mixture with a blocked immobilized enzyme led to its full recovery, i.e., to the removal of the crystals from its surface, and the full catalytic reactivation.

Table 3:

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. Alternatively, 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) and 6-APA (3.6 kg, 16.6 mol) were suspended in 22 L water (cooled to 10°C), and after stirring (250 rpm) for 10 to 15 min the suspension was fed to the enzymatic reactor within 30 - 60 min. 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. At a 6-APA concentration < 3.5 g/kg (> 95.1 % 6-APA conversion) 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.

For the piloting campaign 16 consecutive reaction cycles were performed. Inoculum suspension was used in all 1 consecutive reaction cycles. Solid Amoxicillin as well as suspension from previous batch was used.

Inoculum samples of batches #02, #07, #09 and #14 were selected and subjected to particle size distribution (PSD) determination: (i) solid inoculum treated with Ultra Turrax® (#02), (ii) suspension from previous batch treated with Ultra Turrax® (#07), (iii) solid inoculum treated with colloid mill (#09), and (iv) suspension from previous batch treated with colloid mill (#14). PSD of amoxicillin suspensions used as inoculum was estimated by measuring the size of the particles on a microscopy picture. Subsequently, the particles were counted and grouped into different size ranges (< 10 μπη, 10-20 pm, 21 -30 pm, 31 -40 pm, 41 -50 μπτι, 51 -60 μπη, 61 -70 pm, 71 -80 μπη, 81 -90 μιη, 91-100 μπη, >100 μππ) to allow better comparability. The results of the PSD determination are shown in Figure 13 A-D. As can be seen from Figure 13, 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.

Table 4:

All batches showed good conversions and efficient separation of reaction suspension from immobilized enzyme. No major impact on reaction performance dependent on preparation and type of inoculum could be observed comparing solid inoculum and suspension from previous batch treated with homogenizer or colloid mill. The positive effect of inoculum was confirmed and as shown in EM images of Figure 14 no crystal growth on immobilized enzyme beads occurred.

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 μππ and between 10 and 20 μπη resulted always in satisfying results for 6- APA conversion as can be seen in table 4.