“From Genomes to Biotransformation Scale Up” 05 November 2014 presentdted at RSC/SCI Symposium “Challenges in Catalysis for Pharmaceuticals and Fine Chemicals” by Dr. Stefan Mix . Dededcatedteaicated team of ch emi sts, m oecuaolecular boogstsabiologists an dad an aystsalysts . Extensive track record in use of enzymes to produce chiral intermediates / APIs / bulk chemicals
. Enzyme kits: Hydrolase, CRED, TAm, Nitrilase, P450s….. . Enzyme discovery and evolution . Scouting of biocatalytic synthesis routes . Enzyme screening and scale -up (internal and with partners) . Metabolite isolation, identification and synthesis
“From Genomes to Biotransformation Scale Up”, Burlington House, London, 05 November 2014 [email protected] Exploring manufacturing routes to chiral b uildi ng bloc ks an d APIs:
Relevance of chiral of chiral drugs drugs
Classical Resolution 1000 800
600 billion US$ All drugs 400 single enantiomer drugs 200
0 Chirality 1998 1999 2000 2010 Year Chemo / Organo Catalysis The trend for new chiral pharmaceutical reagents is Chiral Pool continuing. In 2000, 35% of intermediates were chiral and this number is expected to increase to 70% by 2010 Biocatalysis Woodley et al TRENDS in Biotechnology Vol.25 No.2, 2006
“From Genomes to Biotransformation Scale Up”, Burlington House, London, 05 November 2014 [email protected] Technology
Oxidise Esterify Epoxidise Reduce Reduction to -OH Form amine
Hydrolyse to amide Hydrolyse to acid Oxidise
Demethylate Hydroxylate
Hydroxylate Dihydroxylate Hydrolyse to acid Form an amide Esterify carboxyl Hydrolyse amide From ketone
“From Genomes to Biotransformation Scale Up”, Burlington House, London, 05 November 2014 [email protected] Biocatalytic Approaches to Statin Side Chains
OC(O)CH2OMe OC(O)CH2OMe OH O O O A EtO HO HO O OEt -Chymo- O OEt PLE O OEt trypsin 98.1% e.e.
KCN Nitrilase O OH OH B Cl NC CN NC COOH >95 % e.e.
O O O ADH OH O O OH OH O C Cl Cl R OR OC(CH3)3 OC(CH3)3 > 99.5% e.e.
O O ADH OH O HHDHO O HHDH OH O D Cl Cl NC OEt (S) OEt (S) OEt (R) OEt Schürmann et al. (2010) in “Green > 99% e.e. Chemistry in the Pharmaceutical Industry” OH (Eds. P.Dunn, A.Wells, O O DERA OH OH O M.T.Williams), Wiley-VCH O E Cl + Cl Cl OH 2eq. 96.6% d.e.
“From Genomes to Biotransformation Scale Up”, Burlington House, London, 05 November 2014 [email protected] Enzyme Platforms Product Classes Aldolases Alcohols, Diols, Amino alcohols Proteases Peptides, Amines, Carboxyesters Extensive portfolio Lipases and Esterases Alcohols, Esters, Carboxylic acids of off-the-shelf Ammonia lyases Amino acids enzymes: Hydantoinases, Carbamoylases, Racemases Amino acids • Made in-house Amidases Amino acids, Amides • From partners Acylases Amino acids, N-Acetyl-Amino acids Hy droxyn itr ile lyases ChdiCyanohydrins On-going enzyme Omega-Transaminases Amines discovery programs: • In-house Carbonyl Reductases Alcohols • With industry AA Dehydrogenases Amino acids partners Nitrilases Carboxylic acids, Nitriles • With academic Nitrile hydratases Amides, Nitriles partners Monooxygenases (P450 , Baeyer -Villiger) Alcohols, Sulfoxides Epoxide hydrolases Epoxides, Diols Haloalcohol dehalogenases Epoxides, Diols
“From Genomes to Biotransformation Scale Up”, Burlington House, London, 05 November 2014 [email protected] Biocatalyst libraries off-the-shelf: Speed matters!
mg-10g 100g-1Kg 1Kg-100Kg >100Kg
“Off-the-shelf” biocatalysts Process Dev Speed COST
“From Genomes to Biotransformation Scale Up”, Burlington House, London, 05 November 2014 [email protected] Recombinant enzymes – how they are made:
Homogenisation Freeze- or Spray- & Clarification drygying
Cell pasteLiquid CFE Solid CFE
• Whole cell biocatalysts are cheapest, but CFE often preferred. • Further protein purification is possible, but reduces fermentation yield and increases cost. • Scale up of fermentation, homogenisation and freeze/spray-drying needs PRD!
“From Genomes to Biotransformation Scale Up”, Burlington House, London, 05 November 2014 [email protected] Case Studies: Biotransformation route scouting and PRD at Almac, scale up at Almac / other CMO or client
“From Genomes to Biotransformation Scale Up”, Burlington House, London, 05 November 2014 [email protected] What do these molecules have in common?
They are important pharmaceutical intermediates. AikdiikihihiAsymmetric ketone reduction is key step in their synthesis. They have been made via bioreduction at Almac.
“From Genomes to Biotransformation Scale Up”, Burlington House, London, 05 November 2014 [email protected] Case study: (R)-3-Methoxydecanol
Application of CRED technology enabled large scale manufacture of chiral building block for late phase API. Success Criteria: Quality, Quantity, Speed, Cost
“From Genomes to Biotransformation Scale Up”, Burlington House, London, 05 November 2014 [email protected] Case study: (R)-3-Methoxydecanol Why was a new approach needed?
Noyori route too expensive, tedious impurity removal, MedChem route very inefficient , ee borderline – hard to scale, and could not Could not meet late phase deliver >99% ee requirements
“From Genomes to Biotransformation Scale Up”, Burlington House, London, 05 November 2014 [email protected] Case study: (R)-3-Methoxydecanol Proposed bioreduction route
Sample passes Further process Customer Analysis optimisation
PCDPoC Demons tra tion Process Development Tech Transfer & PdProduc tRlt Release 30 g sample sent to Customer kg delivery Scale-up From Almac
PoC to Commerical Supply of Building Block t = 0 t = Final Delivery of BB Project siting Customer Decision t = PoC Partner selection To initiate alternative route Completion investigation
“From Genomes to Biotransformation Scale Up”, Burlington House, London, 05 November 2014 [email protected] Recombinant CREDs – how they work, and how to work with them:
Screening phase: RECYCLE COFACTOR REDUCTION o Identify catalyst, o Identify co-factor, Gluconate o Identify recycling pH stat GDH O system. R' R'' Glucose NAD(P)+ ee is what matters! CRED OH NAD(P)H O R' R'' PRD phase – optimise: temperature, pH, CRED o OH co-solvent, enzyme form, catalyst loading, throughput. Cost is what matters!
“From Genomes to Biotransformation Scale Up”, Burlington House, London, 05 November 2014 [email protected] Case study: (R)-3-Methoxydecanol Key Step Bioreduction CRED A-161, GDH-102 glucose O O OH O NAD+
C7H15 OMe KH2PO4 buffer C7H15 OMe pH 7.0 5% v/v DMSO CRED Screening: • A161 identified as hit with >99.5% ee • Enzyme is NADH-dependent • GDH/glu cose u sed for co-factor rec ycle
Process Optimisation: • 5% DMSO co-solvent, pH 7.0, 30 deg C • 0.1% w/w of lyo cell free extract CRED/GDH sufficient • 180 g/L substrate >99.8% converted within 12 hours • Workup by extraction with MtBE; crude product telescoped into next step
“From Genomes to Biotransformation Scale Up”, Burlington House, London, 05 November 2014 [email protected] Case study: (R)-3-Methoxydecanol – impurity control
Control of Diol-impurity starts here. OH High reaction completion target C7H15 OH
of > 99.8% conversion. Diol-impurity
Control of Dec-3-enol impurity starts here.
High reaction completion targets. Choice of base for deprotonation. C7H15 OH Unstabilised THF used for methylation. Decenol-impurity Fractional distillation in both final steps.
Achieved: 99.3% ee; diol <0.2%; dec-2-enol <0.15% BHT and Me-BHT <0.2%, all other <0.1%.
“From Genomes to Biotransformation Scale Up”, Burlington House, London, 05 November 2014 [email protected] Case study: (R)-3-Methoxydecanol from POC sample to commercial output
5 Step Process Yields (%POC / %pilot batch / %final)
Step 1: 75% / 50% / 85% Step 2: 91% / 90% / 95% Step 3: 95% / 95% / 98% Step 4: 80% / 70% / 85% Step 5: 70% / 70% / 75%
Process modifications after pilot batch experience: Step 1: switched from Meldrum’ s acid to methyl potassium malonate SM Step 4: switched from n-BuLi to n-HexLi, relaxed specification Step 5: improved hydrolysis protocol
“From Genomes to Biotransformation Scale Up”, Burlington House, London, 05 November 2014 [email protected] Case study: (R)-3-Methoxydecanol Summary
• 5-Step process (()one biotransformation) • Asymmetric chemo-cat route not viable • Biocatalyst far cheaper and giving superior selectivity • Biocatalyst was off-the-shelf, facilitating rapid identification and PRD • Enzyme ori g ina lly foun dthd throug h genome m in ing, an dthd then expressed in E. coli • Improved route design and simplified purification strategy to deliver better quality at higher yield • POC sample followed by 25 kg pilot batch • Several full scale campaigns split between Almac and two partners with equivalent distillation capability
“From Genomes to Biotransformation Scale Up”, Burlington House, London, 05 November 2014 [email protected] What do these molecules have in common?
Me Et NH2 NH2 H2N CO2H OH
They are in demand as pharmaceutical intermediates. Almac chemists have worked on scaleable syntheses. They are very small molecules with even smaller substituent difference at the stereocentre = tricky to make. Chiral technology cost contribution is high due to low MW. We had some success,,q but further work is required.
“From Genomes to Biotransformation Scale Up”, Burlington House, London, 05 November 2014 [email protected] Mature enzyygpme technologies example: Applications of Hydrolases
Kinetic resolution Desymmetrization
: : O O O S + S S CO R 2 1 R2 R2 R2 CO2H CO2R1
R 2 R2 R2 + R CO R 3 2 1 R3 CO2H R3 CO2R1
+
CO2R CO H 2 CO2R
50% y ie ld 100% yield
“From Genomes to Biotransformation Scale Up”, Burlington House, London, 05 November 2014 [email protected] Case study: D-Isovaline
Background: • A number of synthetic methods are known for this type of structure, and a number of suppliers list it as stock item. • But price is very high (> $500/g) and quality insufficient. • Strecker-reaction provides easy access to racemate. • Classical resolution on basic amine intermediate un-successful after screening >30 commercial resolving agents. Is there a biocatalytic alternative to enable viable multi kg supply?
“From Genomes to Biotransformation Scale Up”, Burlington House, London, 05 November 2014 [email protected] Case study: D-Isovaline
Strecker + Resolution Approach: other substrates made and tried: NaCN, HOAc, Me Et NH4OAc, MeOH Me Et Me Et Me Et Me Et H N HN CN HN CO R O 2 CN H2N CO2R 2 PG PG
PG = Ac, Cbz PG = Ac, Cbz nitrilases: hydrolases: R=H,Me,Et,Pr unsuccessful low E-values Me Et O Me Et HN H N CO H NH 2 2 O Strecker + bioresolution approach envisaged to give shortest route and quickest success from cheap commercial starting material.
However, this did no t wor k due to low E-vald/lkflues and / or lack of reactivity.
10 different substrates screened against >200 enzymes – no luck.
“From Genomes to Biotransformation Scale Up”, Burlington House, London, 05 November 2014 [email protected] Case study: D-Isovaline hydrolase Me alkylation Me Et desymmetrisation Me Et
EtO2C CO2Et EtO2C CO2Et high ee HO2C CO2Et possible? commercially available and inexpensive Curtius
Me Et Me Et
H2N CO2H HN CO2Et xHCl Boc
Envisaged alternative approach with hydrolase mediated desymmetrisation as key step – this did work. Finally!
Screening of 180 off-the-shelf hydrolases gave a hit, BUT ee was only 85%, AND Curtius gave low yield.
“From Genomes to Biotransformation Scale Up”, Burlington House, London, 05 November 2014 [email protected] Case study: D-Isovaline AH-125 EtI, NaOEt buffer pH 7.5 Me EtOH Me Et cyclohexane Me Et
EtO2C CO2Et 90% EtO2C CO2Et 95% HO2C CO2Et 85% ee commercially available and 1) DPPA, 92% inexpensive 2) BnOH, CuCl PhCl
H2/Pd/C 1) NaOH/H2O Me Et EtOH/H2O/HCl Me Et 2) tBuNH2/MtBE Me Et
H2N CO2H 90% HN CO2H 82 % HN CO2Et 99.7% ee 99.4% ee xHCl Cbz tBuNH2 Cbz salt Enzyme step PRD was straightforward. 0.5% w/w recombinant enzyme were sufficient, used as cell free extract. Curtius problem was solved by switching to Cbz-product. ee-Upgrade was achieved via tBu-amine salt. Final step workup yielded highly pure product.
“From Genomes to Biotransformation Scale Up”, Burlington House, London, 05 November 2014 [email protected] Case study: D-Isovaline – Summary & Outlook AH-125 EtI, NaOEt buffer pH 7.5 Me EtOH Me Et cyclohexane Me Et
EtO2C CO2Et 90% EtO2C CO2Et 95% HO2C CO2Et 85% ee commercially available and 1) DPPA, 92% inexpensive 2) BnOH, CuCl PhCl
H2/Pd/C 1) NaOH/H2O Me Et EtOH/H2O/HCl Me Et 2) tBuNH2/MtBE Me Et
H2N CO2H 90% HN CO2H 82 % HN CO2Et 99.7% ee 99.4% ee x HCl Cbz tBuNH2 Cbz salt 10 kg of pilot batch material were made and delivered. Room for future improvement: Remove need for ee -upgrade by substrate engineering or protein engineering. Use cheaper enzyme preparation for larger scale. Alternative enzyme with reversed selectivity would enable replacing Curtius with Hofmann-rearrangement.
“From Genomes to Biotransformation Scale Up”, Burlington House, London, 05 November 2014 [email protected] What if no recombinant off-the- shelf biocatalyst is good enough?
• Option Zero: Substrate and reaction engineering
• Option A: Check for wild type microorganisms to do the job o Traditional approach – does traditional mean outdated? o Often - but not always! - narrow substrate scope and low productivity o Still preferred if complex multi-enzyme pathways are involved o Engineering of metabolic pathways possible via strain mutation
• Option B : Check for enzyme homologues in nature, clone and express (£) o Modern approach, enabled by advances in bioinformatics and genome mapping o Related: Meta-genomics approach o Can replace or merge with option C
• Option C: Start a directed evolution program (£££) o Modern approach, enabled by advances in gene synthesis, protein engineering and understanding of protein structure -activity-relationship o Impressive results but high up-front investment o Expanded off-the-shelf catalyst libraries as by-product useful to others
“From Genomes to Biotransformation Scale Up”, Burlington House, London, 05 November 2014 [email protected] Enzyme Evolution
Directed evolution in E.coli, Pichia and Aspergillus expression systems . Almac perform direc te d evoltilution by bthboth Parental gene random and site-specific mutagenesis. Mutagenesis X X X . Expertise in a number of different expression X X systems such as E.coli, Pichia and Expression Aspergillus.
. Bioinformatic expertise allowing saturation Screening mutagenesis
. Novel system that allows glycosylation of proteins in E.coli
FthFurther round s of evol ltiution
“From Genomes to Biotransformation Scale Up”, Burlington House, London, 05 November 2014 [email protected] Evolution Strategy
Literature and Directed Saturation bioinformatics evolution mutitagenesis
Selection of Base platform: Advanced Enzyme parent gene synthesis enzyme with superior activity and Modelling HTS assay selectivity and platform docking
“From Genomes to Biotransformation Scale Up”, Burlington House, London, 05 November 2014 [email protected] Biocatalyst improvement via hlhomologues Homologues can be identified via sequence and structural alignment.
These have a similar core motif, but are derived from a range of organisms.
Priority will be given to homologues with similar docking characteristics but originating from extremophile organisms.
Involves bioinformatic studies , gene synthesis, host transformation and reaction screening.
“From Genomes to Biotransformation Scale Up”, Burlington House, London, 05 November 2014 [email protected] Metagenomics
Metagenomics: application of genomics to uncultured microorganisms 1st STEP: Isolation of environmental DNA (eDNA)
• the ex situ method: cells are isolated and concentrated from environment prior to their lysis
• the in situ methods: cells are lysed directly within the soil material
• the enrichment method: microorganisms are enriched for a desired enzyme activity prior to the isolation of eDNA EtExtracti on of novel nat ural lbi bioca tltalys ts
“From Genomes to Biotransformation Scale Up”, Burlington House, London, 05 November 2014 [email protected] Metagenomics
Classical Almac & UCL Cloning eDNA into Isolation of DNA vectors
•Gene identification •2nd Generation by DNA sequencing sequencing (optional) •Bioinformatic •Expression cloning libraries into appropriate • Expression and vectors cloning
metagenome metagenome libraries libraries
Extraction of novel biocatalysts Handelsman et al. 1998 – CHEM BIOL . 5:R245-R249
“From Genomes to Biotransformation Scale Up”, Burlington House, London, 05 November 2014 [email protected] Metagenomics
• Novel salt mine enzyme discovery with QUB
• Grown under both aerobic and anaerobic conditions, range of temperatures
• Novel microbes identified
• Novel enzymes identified
• Salt mines in Northern Ireland
“From Genomes to Biotransformation Scale Up”, Burlington House, London, 05 November 2014 [email protected] How to use whole cell biocatalysts
Phase 1 : SiScreening SlSelec tion from pane l o f50f 50-100 organisms
Phase 2 : PRD Optimisation of growth + reaction conditions for maximised yield and productivity
Phase 3: Full scale manufacture via either of two modes:
Fermentation mode vs Re-suspension mode one-pot microbial growth off-line biomass growth, and desired reaction reaction with re-suspended cells
“From Genomes to Biotransformation Scale Up”, Burlington House, London, 05 November 2014 [email protected] A commercially viable biocatalyst is defined by…..
Performance Supply •Cost • Turnover • Security • Selectivity • Made at scale • Volume efficiency IP Issues • Right to use • License agreement • Simple business model
“From Genomes to Biotransformation Scale Up”, Burlington House, London, 05 November 2014 [email protected] Whereweare:Where we are:
“From Genomes to Biotransformation Scale Up”, Burlington House, London, 05 November 2014 [email protected] Whereweare:Where we are:
“From Genomes to Biotransformation Scale Up”, Burlington House, London, 05 November 2014 [email protected] Contacts
Dr. Tom Moody HdfBitliHead of Biocatalysis
Phone: +44 28 3836 5503 ALMAC Email: [email protected] Seagoe Industrial Estate Craigavon, N. Ireland Dr. Stefan Mix BT63 5QD UK Biocatalysis Team Leader www.almacgroup.com Phone: +44 28 3836 5917 Email: [email protected]
“From Genomes to Biotransformation Scale Up”, Burlington House, London, 05 November 2014 [email protected] Thank you for listening!
Thank you for listening!
“From Genomes to Biotransformation Scale Up”, Burlington House, London, 05 November 2014 [email protected]