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The Development and Scale-up of a Biocatalytic Process to Form a Chiral Sulfoxide William Robert Fraser Goundry, Bradley Adams, Helen Benson, Julie Demeritt, Steven Mckown, Keith R. Mulholland, Amy Robertson, Paul S Siedlecki, Paula M. Tomlin, and Kevin Vare Org. Process Res. Dev., Just Accepted Manuscript • DOI: 10.1021/acs.oprd.6b00391 • Publication Date (Web): 04 Jan 2017 Downloaded from http://pubs.acs.org on January 5, 2017

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Organic Process Research & Development

The Development and Scale-up of a Biocatalytic Process to Form a Chiral Sulfoxide William R. F. Goundry,*ǂ Bradley Adams, ǂ Helen Benson, ǂ Julie Demeritt, †ǁ Steven McKown, ǂ Keith Mulholland, ǂ Amy Robertson, ǂ Paul Siedlecki, ǂ Paula Tomlin, ǂ and Kevin Vareǂ ǂ

The Departments of Pharmaceutical Sciences and Pharmaceutical Technology and Development, AstraZeneca, Silk Road Business Park, Macclesfield, United Kingdom, Cheshire, SK10 2NA

ACS Paragon Plus Environment

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TABLE OF CONTENTS GRAPHIC

BVMO KRED NADPH triethanolamine water, 27oC

74% yield on a 60kg scale


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KEYWORDS Biocatalysis, sulfoxide, Baeyer-Villiger monooxygenase

ABSTRACT

A Baeyer-Villiger monooxygenase enzyme has been used to manufacture a chiral sulfoxide drug intermediate on a kilogram scale. This paper describes the evolution of the biocatalytic manufacturing process from the initial enzyme screen, development of a kilo lab process, to further optimization for plant scale manufacture. Efficient gas-liquid mass transfer of oxygen is key to obtaining a high yield.

INTRODUCTION Since the discovery of naturally occurring chiral sulfoxides1 and sulfoximines2, most notably methionine sulfoxide 1 and methionine sulfoximine 2 (Figure 1), interest in the use of a chiral sulfur motifs in pharmaceutical molecules has steadily increased. Many sulfoxides and sulfoximines have a high biological activity across a broad range of indications. Representative examples are: anti-ulcer agent esomeprazole 3; 3 potassium channel activator aprikalim 4;4 immuno suppressor oxisurane 5;5 the platelet adhesion inhibitor OPC-29030 6;6 antipsychotic agent ZD3638 7;7 anticancer drug buthionine sulfoximine 8;8 and anticancer drug AZD6738 9 (Figure 2).9 Figure 1. methionine sulfoxide 1 and methionine sulfoximine 2.


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1

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2

Figure 2. Examples of biologically active molecules containing a sulfoxide or sulfoximine: esomeprazole 3; aprikalim 4; oxisurane 5; OPC-29030 6; ZD3638 7; buthionine sulfoximine 8; and AZD6738 9.

4 6

3

5

7

8

9

Due to the importance of chiral sulfoxides in medicinal chemistry, as well as their use as chiral auxiliaries in asymmetric synthesis, strategies for their preparation in high chiral purity continue


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to evolve.10-12 Two main approaches are currently used: transition metal catalysis and biocatalysis. Transition metal catalysis is commonly achieved using the “Katsuki-Sharpless reagent” ((Ti(OPri)4:L-diethyl tartrate = 1:1) with tert-butyl hydroperoxide (TBHP) under conditions discovered by Kagan and Modena.13,14 Variants of these conditions have been used for the manufacture of esomeprazole 3 and ZD3638 7.7,15 Other metal approaches include Nakajima’s vanadium chiral Schiff bases with TBHP,16 Bach’s manganese salen system17 and more recently iron-based systems.18 Biocatalytic methods employed can be divided into whole-cell systems and the use of isolated enzymes; in both cases enzymes with a natural ability to oxidise sulfur have been exploited. Whole cell preparations have been used for the synthesis of both enantiomers of 5.19 The advent of modern biotechnology techniques has seen a preference for the use of isolated enzymes, which can be modified by directed evolution for both selectivity and stability. Enzymes used include: Horseradish Peroxidase;20 chloroperoxidase;21 cytochrome P450;22 Baeyer-Villiger monooxygenases (BVMO)23 and dioxygenases.24

AZD6738 9 is the lead candidate for an oncology project, undergoing development for the treatment of colon and hematological cancers.9 The molecule contains a chiral sulfoximine, which is derived from a chiral sulfoxide 10 (Scheme 1).25 For early pre-nomination deliveries, the route was the unselective m-CPBA oxidation of sulfide 11; due to the presence of the chiral morpholine the material is diastereomeric allowing easy chromatographic separation of undesired 12 to afford a maximum 50% yield of 10 (Scheme 1). For the delivery of batches destined for toxicological studies and the first clinical trials, an efficient method of introducing the chiral sulfoxide moiety was desired in order to reduce the manufacturing time and omit chromatography.


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Scheme 1. The original medicinal chemistry approach to the sulfoxide.

i) m -CPBA ii) Chromatography 11

10

12

50%

50%

Several reactions were tried using Kagan type conditions; however, for this substrate, the reaction was low yielding and degradation was observed. The biocatalytic chiral oxidation of the sulfide 11 to selectively give the (R,R)-diastereomer 10 was recognised by the discovery team as a key improvement for the project. This option was explored by partnering with Codexis to access their BVMO technology; Codexis have evolved a library of stable isolated enzymes with diverse specificity. An initial screen was performed using 129 BVMO enzymes in aqueous buffer, with iso-propanol (IPA) added to aid substrate solubility; 87 enzymes gave >90% conversion to sulfoxide of which eight gave >90% (by peak area) of desired (R,R)-diastereomer, 10. The catalytic cycle uses a BVMO in tandem with nicotinamide adenine dinucleotide phosphate (NADPH), which is oxidised to NADP+. NADPH is then regenerated through reduction of NADP+ by an additional ketoreductase (KRED) enzyme, which in turn oxidises the co-solvent IPA to acetone (Scheme 2). Following these initial hits, the project transferred from discovery to AstraZeneca’s Process Development department for scale-up and kilo delivery. Scheme 2. Overview of the biocatalytic process for BVMO reaction


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BVMO 11

10

NADPH

NADP+

KRED

REACTION DEVELOPMENT The enzyme process was rapidly assessed to provide confidence the process would work on scale; this information was required before the significant spend on enzyme purchase was committed. A design of experiment (DOE) was used to investigate: substrate concentration; enzyme concentration; IPA charge; enzyme type; buffer type (Table 1). A scale-up fact sheet for the enzymes was provided by Codexis, which was a useful guide for the setting of the levels in the DOE. After stirring for 24 hours, the reactions were extracted into ethyl acetate to aid analysis by HPLC. The design showed a lower substrate loading and higher enzyme loading was preferred whilst the amount of IPA was unimportant. There was little to choose between the enzymes but BVMO-P1/D08 (D08) was marginally better. Triethanolamine (TEA) as the buffer gave better conversions, probably due to increased organic solubility. Table 1. Initial DOE to assess the biocatalytic conditions.◊

Exp Starting

BVMO BVMO KRED NADP Buffer

Total IPA

11

10


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amount 11 (g/L)

Type♯

charge (g/L)

1

100

D08

2

1

0.5

TEA

2.5

5

16.3

81.4

2

60

D08

20

1

0.5

TEA

2.5

10

42.6

15.4

3

100

C11

20

1

0.5

TEA

2.5

5

34.7

62.6

4

100

C11

2

1

0.5

TEA

2.5

10

4.5

91.3

5

20

C12

2

1

0.5

TEA

2.5

5

8.2

11.3

6

20

C12

11

1

0.5

TEA

2.5

10

17.4

1.9

7

100

D08

2

1

0.5

Phosphate

2.5

10

6.8

89.2

8

20

D08

11

1

0.5

Phosphate

2.5

5

15.0

4.2

9

20

C11

2

1

0.5

Phosphate

2.5

10

2.6

16.9

10

60

C11

20

1

0.5

Phosphate

2.5

5

20.8

37.7

11

100

C12

2

1

0.5

Phosphate

2.5

5

5

91.1

12

100

C12

20

1

0.5

Phosphate

2.5

10

18.8

78.8

13

60

C12

11

1

0.5

Phosphate

2.5

7.5

17.5

41.1

14

60

C12

11

1

0.5

Phosphate

2.5

7.5

18.4

40.3

15

60

C12

11

1

0.5

Phosphate

2.5

7.5

20.0

38.6

No

(g/L)

(g/L)

Type

vol. (mL)

(vol %)

(g/L)

(g/L)

♯ Codexis panel screen reference for BVMO enzymes. ◊ All reactions were at 25˚C,stirred at 800rpm and used KRED-P1-H10. Each reaction was adjusted to pH 9.0, by titration with dilute HCl(aq), prior to enzyme addition. Two further DOE were used to improve the reaction concentration and enzyme loading. A solution conversion of 94% was achieved with a substrate concentration of 40g/L and an enzyme loading of 24% wt/wt. Once these conditions had been checked in a relatively small scale-up reaction, the purchase of the enzyme for manufacture was sanctioned at risk. Development work was restricted due to the availability of the enzyme (manufacture required a custom order) with all early work being performed in tubes with stirrer bars. Upon scaling to


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100mL jacketed vessels with overhead stirring two interlinked problems were noticed that were expected to cause an issue on scale up: i.

Variable reaction times between one and three days.

ii.

The loss of solvent over time.

It was conjectured the variable reaction times were resulting from slow air flow into the vessel and hence slow oxygen uptake. When a reaction in a jacketed vessel was left open only through the condenser, the reaction stalled. With three charge port stoppers removed, the reaction progressed but the IPA and acetone (a reaction byproduct) slowly evaporated. IPA is required by the KRED for the enzymatic cycle to be complete and also enables dissolution of the substrate in the aqueous solvent mixture. As IPA evaporated the rate of turnover could be reduced, whilst conversely the evaporation of acetone could increase turnover. Mass transfer of oxygen was noted as a potential issue for the kilo manufacture but was not explored further at this stage. Attention then turned to the work-up. Although the enzyme purchased from Codexis delivered a good diastereomeric ratio (d.r.), it had not been specifically evolved for our substrate, merely it was the best catalogue enzyme available. The resulting high enzyme loading of 24% wt/wt represented a significant challenge in the removal of the biological material. The desired product crystallised directly from the buffer but in poor yield. Extraction from the reaction system into organic solvent also proved problematic due to the high solubility of sulfoxide 10 in the buffer. Several methods of denaturing (precipitating) the enzyme were investigated, including combinations of extremes of heat and pH with large volumes of organic solvent (Table 2). Table 2. Work-up conditions investigated during initial development.


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Conditions

Observation/Result

pH 1

Denatured enzyme, however some product crystallised as HCl Salt. Product still in the aqueous layer.

pH 14

Possibly denatured the enzyme but no precipitate observed.

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Dilute with EtOAc and Product and enzyme in the aqueous layer. then aqueous washes Remove IPA with Small amounts of product crystallised straight from buffer. distillation then extract

Conditions above pH 14 did not adequately denature the enzymes (assessed as the appearance of a precipitate), whilst low pH resulted in sulfoxide 10 forming salts and crystallising out with the denatured enzyme. The approach selected for campaign 2 was to add 50 relative volumes of acetonitrile followed by filtration of the denatured enzyme. Filtration times were slow but acceptable; the addition of filter aid (e.g. Celite) resulted in inoperable filtrations. The mixed organic solvents could then be removed by distillation and sulfoxide 10 extracted into dichloromethane (DCM) ready for solvent swap into a crystallisation solvent. Whilst this process resulted in a low throughput and high solvent burden the process was deemed fit for purpose for campaign 2. A solubility screen was performed which highlighted the desired (R,R)-diastereomer 10 was many times less soluble than the (S,R)-diastereomer 12 in several solvents (Figure 3). A crystallisation from 2-butanone which selectively crystallised (R,R)-diastereomer 10 was developed; typical isolated yields were 70%. Despite the addition of heptane anti-solvent, the high solubility of (S,R)-diastereomer 12 appeared to prevent the full crystallisation of 10; further crystallisation did not occur once a the ratio of 10:12 reached 1:1 in the solution.


10

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Figure 3. Solubility of 10 and 12 in a range of solvents; bars reaching 100 mg/ml indicate >100mg/ml solubility (limit of the screening method).

100.00 90.00 80.00 70.00 60.00 50.00 (R,R)-diastereomer 10 40.00

(S,R)-diastereomer 12

30.00 20.00 10.00 0.00

KILO MANUFACTURE Pharmaceutical project teams are sometimes reticent to use enzymes with concerns in four key areas: the enzyme source, quality and specification; processing issues including poor filtrations and foaming; enzyme residues in API and strategies for managing potential impurities; the toxicity of the enzyme itself.26 These issues were discussed and logged during the project Good Manufacturing Practice (GMP) risk assessment. The enzymes were accepted from Codexis on the basis of a certificate of analysis combined with a use test. AZD6738 has an oral formulation and any protein or peptide fragments should be broken down by normal digestion pathways;


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however this should not be used as a basis of safety. A purge factor approach - analogous to that used for potential mutagenic impurities - was applied to the enzymes, predicting all biological material would be fully rejected downstream prior to isolation of AZD6738.27 This reaction requires oxygen for the oxidation to occur. The team recognised at an early stage this would require working under at least an atmosphere of air and engaged with the process safety group to understand the associated hazards; for a standard manufacture the basis of safety is to work under a blanket of nitrogen to remove the risk of fire. The reaction solvent system of 10% IPA in triethanolamine buffer (water, triethanolamine and HCl) was predicted not to be a flammable mixture at the reaction temperature, and the reaction could be run as an open system. Since the enzymatic cycle produces acetone the altered solvent composition as the reaction progressed was also evaluated. After an overnight hold, the first 100L batch in the kilo lab failed its success criteria with only 10% conversion vs the expected >95%. Suspecting a lack of oxygen in the vessel, the main charge port was left open to ensure flow of air; it was calculated the reaction required approximately 450L of air to reach completion. The reaction slowly progressed over the next 24 hours stopping at only 75% completion; as a pragmatic necessity IPA was regularly charged to offset evaporation. An additional charge of BVMO, KRED and NADP resulted in no further conversion. The balance of the material in the batch was sulfide 11. Although no lab reactions had been worked-up with this much remaining starting material is was predicted not to be a problem. To date a crystalline form of sulfide 11 has not been identified (it is charged to this stage as a solution in IPA) and hence it was expected to be highly soluble. The batch was worked-up and fortuitously the remaining sulfide 11 was fully rejected to the mother liquors which were retained for reprocessing.


12

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Clearly the level of dissolved oxygen in the reaction solution is key to how this reaction progresses; the reaction was considered to be first order with respect to oxygen concentration, although no detailed enzyme kinetic studies were performed. Gas solubility increases with decreasing temperature, but the system is constrained by the operating temperature of the BVMO. The use of an oxygen atmosphere to improve the performance of a BVMO has been reported by Wilson,28 however we did not investigate this approach as the Kilo lab and plant equipment available was incompatible with a pure oxygen atmosphere. Hilker described the use of a bubble column reactor to maximize oxygen mass transfer in a BVMO process,29 however this equipment was also not available and so not investigated. Instead, to improve conversion the second kilo batch was sparged with a stream of air, whilst simultaneously monitoring the level of IPA in the reaction system as greater losses were expected. Batch two was simply sparged using a standard polypropylene tube to bubble compressed air into the reaction vessel. The level of IPA was determined by a 1H NMR strength measurement and topped up as required. Under these conditions the reaction passed its success criteria with 98% conversion in 21 hours. Repeated additions of IPA were required and sub-surface sparging resulted in large amounts of foaming in the vessel. For batch one the filtration of the denatured enzyme went well, however during the subsequent extraction of the product into DCM, large amounts of interfacial material was seen coupled with flocculation. Believing this to be caused by tiny protein fragments passing the initial filter, subsequent batches were filtered through a 20µm filter which resolved the issue. Solvent swap into 2-butanone and the subsequent crystallisation occurred as expected with no issues. For the next campaign, an improved method of sparging the reaction was investigated. The optimum position to sparge the reaction was predicted to be just above the vortex about 1/3 of


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the way along the radius of the agitator. This was trialed on a 1L and 3L scale but in practice the position of the sparging tube was orders of magnitude less important than having effective stirring. During the first batch of this second kilo manufacture, the reaction was progressing reasonably slowly. When the agitation was increased from 250rpm to 350rpm, the reaction was complete after the usual timeframe of 17 hours. All the other batches were operated with the agitation at the maximum possible 500rpm, going to completion in around 6 hours. This highlighted the importance of agitation coupled with a low vessel fill volume. As the subsequent work up required the addition of a significant volume of solvent, the reaction volume occupies at most 1/3 of the vessel volume. Vigorous agitation leads to generation of a vortex deep enough to reach the base of the agitator shaft where the blades whip the headspace air into the stirred liquid. However, the overall process was still long due to the work-up, with difficult separations to perform. The yield for the last few batches was around 80% and this was assumed to be due to the operatives being better able to judge the separations. A repeat third kilo manufacture was required for early clinical supply; some minor improvements to the process were developed during the accommodation of the manufacture. The process had originally required purified water for the reaction, however the borehole water used routinely by the facility was sufficient. Through experience, the method and rate of air introduction was optimized to minimize solvent loss; no additional IPA charges were required. For the fourth and final kilo lab campaign, additional process development was undertaken. The team evaluated the kilo lab data and predicted that the improved mass transfer set up may allow the enzyme loading to be reduced. The original enzyme loading had been set by DOE in reaction


14

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tubes with reaction times of one to two days; now with air piped into the kilo vessel and with highly efficient agitation, the reaction was complete in six hours. During this manufacture, the amount of BVMO was incrementally reduced to 5% wt/wt BVMO, with minimal impact on product quality or diastereomeric ratio. This resulted in a significant cost saving for the project. In all batches, the reactions appeared to be first order until they neared completion, as demonstrated by the almost linear relationships in Figure 4. This is consistent with mass transfer being the limiting factor until most of the starting material is consumed. Batch C615/10 demonstrated that agitation still affects the reaction rate at 5% wt/wt enzyme loading. Overall, in the kilo lab, 29 batches were delivered (Table 3); all batches were on a ~1kg scale in 100L vessel. Figure 4. Area counts of sulfoxide 10Δ vs reaction time, showing how reaction rate varied with BVMO enzyme loading in the 4th kilo lab campaign. Each batch used the same vessel set-up and purging method.


15


1400 1200

Area counts

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1000 800 600 400 200 0 0

100

200

300

400

500

600

700

800

900

Reaction time (minutes) C615/3 - 20%w/w BVMO (500 rpm)

C615/6 - 12%w/w BVMO (500 rpm)

C615/8 - 6%w/w BVMO (500 rpm)

C615/9 - 5%w/w BVMO (500 rpm)

C615/10 - 5%w/w BVMO (300 rpm) C615/10 - 5%w/w BVMO (500 rpm)**

C615/10 - 5%w/w BVMO (400 rpm)

∆

Each HPLC sample was prepared to the same concentration by diluting 75 µL of the reaction mixture up to 25 mL. An internal standard was not used in these GMP batches. Therefore, the area counts results will contain some errors due to variations in batch volumes and sampling preparations. **Batch C615/10 was held after 420 minutes for approximately 18 hours with no oxygen sparging or agitation. The batch was sampled before and after this hold. Some product formed during this hold but at a significantly slower rate.

Table 3. A summary of the kilo lab manufactures. Manufacturing Campaign

Number of Agitation BVMO loading batches (RPM) (%wt/wt)

Average isolated yield (%)

1st

2

250

24

78†

2nd

8

500

24

77

3rd

9

500

24

71‡

4th

10

500

5-24

77


16

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†Includes reprocessing of the ﬁrst batch. ‡Lower average yield due to a single batch yield of 28% due to a mischarge.

SECOND GENERATION DEVELOPMENT Following the observations of the kilo lab that only 5% wt/wt BVMO was sufficient, the process underwent a second phase of development. The 5% enzyme loading process was scaled down to a lab 250mL jacketed vessel, ensuring the correct agitation type, agitation speed, vessel geometry and vessel fill were used. The DynoChem® gas-liquid vortexing “Drawdown” utility was used to calculate the mass transfer coefficient that was achieved for the optimum reaction set-up in the kilo lab. The agitation speed of the scale-down lab vessel was then set to replicate this mass transfer coefficient. Analysis during the kilo campaigns had highlighted two low level impurities; based on mass spectroscopy measurements these were believe to be sulfone (13) and N-oxide (14) (Figure 5). Clearly, these were forming from over oxidation of the desired product, possibly due to background hydrogen peroxide generation under the reaction conditions. If present, hydrogen peroxide could also be attacking the BVMO enzyme and slowing down the reactions. It was proposed that the introduction of an additional enzyme (catalase) would breakdown any hydrogen peroxide produced and remove the issue of over oxidation. Two reactions were compared in the lab with 5% wt/wt BVMO with or without 2.5% wt/wt catalase to see if the addition of catalase offered any advantages. The levels of the sulfone 13 (0.3%) and N-oxide 14 (3.4%) did not differ between the two reactions; hydrogen peroxide presence was discounted.


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Figure 5. The structures of the main impurities in the reaction. O

O –

N O

O S

–

N N

13

+ O N

Cl

O + S

N N

Cl

14

Now with a lower bio-burden, simpler approaches to the work-up were developed to reduce the high maximum volume and increase manufacturing throughput. The reaction mass was reduced to 1/3 of the volume to remove the organic co-solvents and majority of the water. This required vacuum conditions to maintain

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