fit the process chemist - almac · fit the process chemist ... transaminase + ketone aminoacid-dh/...

www.almacgroup.com

“Rational Design of Enzymes tofit the process chemist

timelines”

Dr. Derek Quinn / ALMAC Group

Organic Process Research &DevelopmentPasadena, 6-8th March 2017

Founded in 1968

Global Headquarters in Craigavon, Northern Ireland

Turnover >$500m

~ 4000 Personnel Globally

Unique ownership – charitable foundation

All Profit Re-invested

Arran Chemical Company - purchased 2015

ALMAC Group Overview

Advantages from using enzymes

Enzyme Hit Process Development options with economic assessment

Phaseappropriate

fit-for-purposeprocess

OrCommercialEconomic

Process withconstant pricing

assessmentEnzymeKits

Bioinformaticselection & insilico design

Enzymeengineering

1. ProcessOptimisation

2. SubstrateEngineering

3. Ultrasound& reactionengineering

4. Resinapplication forinhibition and

extraction

5. EnzymeImmobilisation

6. Enzymeengineering

Constant economic assessment

Enzyme process design

• Selectivity• Specificity• Stability• Activity

• Cost• Productivity• Quality• Sustainability

Product isolation, purification, removal of bioburden/catalyst

IRED no.

% HPLC peak area @ 260

nm

% HPLC peak area of

product enantiomersee

Product SM (S) (R)

3 9 91 54 46 8

5 2 98 0 100 100

8 77 23 89 11 78

9 2 98 13 87 74

12 3 97 100 0 100

16 3 97 77 23 54

20 100 0 100 0 100

26 1 99 89 11 78

27 31 69 92 8 84

28 8 92 0 100 100

SM

Product

• In silico selection of 50 IRED enzyme library• Gene synthesised, cloned and expressed• Screened against client substrate – 20% hit rate

IRED found by in-silico screening

• IRED-20 selected for development(100% ee and best activity in screening)

• Enzyme form defined: Spray-dried cells suitable

• Challenges encountered during PRD:• Product inhibition limits product titre 15g/L• Product highly water soluble difficult recovery

by extraction• Evolution???

• Solution implemented: Ion exchange column inrecirculation mode (54g/L) Solves inhibition problem in aqueous

reaction medium Enables efficient product recovery with

ammonia in methanol

IRED process and challenges

Enzyme evolution overview

• Classical mutagenesis techniquesslow, undirected, high screening burden

• Rational mutagenesis:Crystal structures/Modelling, computationally ,hungry.

• Random mutagenesis:Mutant generation simple, but very high screening burden

• Directed mutagenesis/combinatorial approach:Directed evolution, high screening burden reduced by recombination,very powerful, mimics nature

• Machine learning:Mathematical algorithms which predict mutations and learns fromboth positives and negatives

• Rational mutagenesis revisitied, computational power has increasedexponentially, new powerful algorithms can now exploit this.

• Awarding of Nobel prize in 2013 to Karplus, Levitt and Warshel forQM/MM

Rational Design / Modelling Methods

Classical mechanics Hybrid methods Quantum mechanics

• Newtonian mechanics andempirical

• Atom is the smallest unit• Force fields• Computationally fast• Less accurate

• QM/MM methods• EVB method

• Electronic structure• Based on approx. to the

time indep Schrödingereq

• HF, pos-HF, DFT andsemiempiricals

• Computationallyexpensive

• Size limitations

Molecular dynamics

E = Kr

r -req( )

2+

bonds

å Kq

q-qeq( )

2+

angl es

åV

n

21 +cos nj -g[ ]( ) +

Aij

Rij

12-

Bij

Rij

6

æ

èçç

ö

ø÷÷

i< j

atoms

åtors ions

å +qi qj

e Rij

æ

èçç

ö

ø÷÷

i< j

atoms

å [1]

Docking methods

Rational Design/Protein engineering

InitialStructuralanalysis

In silicopredictionof mutantEnzymes

Wet labvalidation

Docking/Mechanism

determination

Pointmutations

Typically 30-50rationally

designed mutantenzymesper round

Wet labvalidation

Rational Design

Hydrolase CarbonylReductase

AmidaseNitrileHydratase

Transaminase

How to make chiral amines

Chiral amine target

Transaminase+

ketone

Aminoacid-DH/Amine-DH

+a-ketoacid/ketone

Ketoreductase+

ketone

Amine oxidase+

racemate

Imine reductase+

imine

Hydrolase/Nitrilase+

ester/nitrile

Classical resolution,Auxilliary chemistry

Asymm. hydrogenation,Asymm. PTC

WT (S)-TAM Vibrio fluvialis

Bulky substrates – typically not wellaccepted by wildtype TAm enzymes

Rational engineering approach taken toopen up pocket and improve interactions

Substrate docked into active site andenergy minimisation carried out todetermine most likely pose

Non-catalytic poseDistance between the amino group of PMPand the substrate carbonyl is large (5.1 Å).

•Nucleophilic attack of PMP not possibledue to steric clash between the phenylgroup of substrate and PMP.

ACS Catalysis, DOI: 10.1021/acscatal.6b02380

TAm

Structural analysis

(S)-TAM Vibrio fluvialis

Am

ino

acid

sdis

trib

uti

on

Co-evolution network

Homologous Sequences analysis (~30000 seq.)


Best variant of (S)-TAMVibrio fluvialis

Catalytic pose

•Distance between the substrate carbonylcarbon and the PMP amino group (3.1 Å)quite close to the distance reported forthe PMP: acetophenone intermediate(2.65 Å) (Cassimjee et al. 2015).

•Strong T-shaped π-stacking and S/π interactions introduced.

•V153S mutation may increase the subunitinterface affinity. Calculated

ΔΔGV153S =-2.01 kcal/mol.

•After 18h the best variant retains 98 %and 90% of activity at 40°C and 50°C,respectively, while the other variantssuffer a significant decrease in activity at50°C


General Mechanism of Transaminase Enzymes

Complex mechanism: Planar quinonoid orientation and catalytic lysineresidue defines chirality of product

Best variant: Analysis of the quinonoid intermediate

• The spatial arrangement of the catalytic lysine with respect to the planar quinonoid commands theorientation of the amino group of the catalytic lysine, which undergoes nucleophilic attack towards thenitrogen binding carbon of the quinonoid intermediate.

• The spatial arrangement favours the formation of pro-(S) external aldimine and consequently theformation of the end S-amine product


TAM-WT

ConversionSubstrate: phenyl-acetophenone

Reaction conditions: 5 mM substrate, 1M IPA, 0.01 mM enzyme, 0.1 M K·phosphate buffer (pH 8.0), 10% DMSO (v/v), 40°C, 18 h. Allreactions were performed in triplicates.

TAM- best variantConversion: ND Conversion: 42.1%


• 113 rational designed variants, 2 in vitro screening rounds

• Best variant had > 1700 fold-improvement in the reaction rate

a Reaction conditions: 5 mM substrate, 1M IPA, 0.01 mM of purified enzyme, 0.1 M potassium phosphate buffer (pH 8.0), 10 %DMSO (v/v), 40 °C, 18 h. All reactions were performed in triplicates.b Kinetic parameters are the apparent rate constants calculated from the initial reaction rates at a fixed concentration ofamine donor (1 M).c Fold change with W57G/R415A as referenced Reaction rates are initial reaction rates. Reaction conditions: 5 mM substrate, 1 M IPA, 0.01 mM of purified enzyme, 0.1 MK·phosphate buffer (pH 8.0), 10 % DMSO (v/v), 40 °C. Reactions were performed in duplicates.e ND - not detectable. Reaction rate for WT was no more than 0.1 μM/h, considering the detection limit.


Rational Design



Transaminase

B/C:ter

300K341K

Molecular dynamics :B-factor calculated at two different temperatures

Amidase – Rational Design - Thermostability

Highly-flexible regions identified

Ni2

Ni1

Identify flexible regions: High B-factorMutate residues and test for reduction in B-Factor

SS-bond insertion criteria:

1 - Distance CB-CB 3Å-4.5Å2 – Distance C-SG 1.8 Å ; SG-SG ~2 Å3 – C-SG-SG-C dihedral angle ~90°4 - C-SG-SG angle ~103°

Amidase – Rational Design

Multimeric enzyme: 3 separately expressed proteinsStabilisation between protein interfacesStabilisation within subunits of proteinsS-S bonds, salt bridges, stabilising mutations

Amidase – Rational Design

SelectedMutants

Subst

rate

conc

ent

rati

on

(mM

)

• ~100 mutants generated in study for retention of activity at high ºC• Wild type and mutants were incubated at 70 ºC for 16h• Retention of activity measured (substrate depletion) at room temperature• Activity of mutants less than wild type, not surprising (less flexible enzyme)• Retention of mutant enzyme 4 greatly improved• >50 % of activity after 3 days, wild type activity absent

Rational Design



Transaminase

• Substrate inhibition: nM affinity• Molecular modelling, substrate docking, rational

mutant selection, gene synthesis, wet screening• Predicted affinity reduced to from nM to mM range

with 20 mutant enzymes

Wild type: 88.9 nM Variant: 12.7 mM

Addressing inhibition via CRED engineering

CRED-A231_F97A_L241A_W226A +NADPH docked

Carbonyl reductase - Rational evolution

Substrate

NADPH

• In silico analysis of WT CRED (Molecular Docking).

• 20 variants rationally designed.

• Best mutant increases conversion to >90% with aee of 98.7 % (S).

Result: Increased conversion of ketone 1 whilemaintaining the WT enzyme selectivity

1 2

Rational Design



Transaminase

Background:• Client wanted enzymatic process to replace chiral Simulated Moving Bed

Chromotography process for late phase and commercial API

Problems:• Expensive racemate – so yield is important• Two enzyme process Hydrolase 1 = resolution Hydrolase 2 = Desymmetrisation• Hydrolase-1 had low E-value of 13 (R)-diester yield < 30%• Hydrolase-2 had low stability high enzyme loading required with resulting

isolation problems impact on yield

Solutions:• Improve E-value of resolution step via substrate engineering• Improve stability of desymmetrization enzyme via homologues panel

“Genomes, Biocatalysts and Pharmaceutical Mater ials”

Humboldt University, 13 November 2015, [email protected]

Desymmetrization for 2nd generation process

Problems solved:• Hydrolase-1 has E-value of 60 with dipropyl ester (R)-diester now at 45+%.• Hydrolase-2 accepted (R)-propyl diester with unchanged selectivity.• Hydrolase-2* found via homologues search, from a thermophilic organism. At

maintained selectivity the process now runs at 50 deg C with low enzymeloading.

Thinking forward:• Solutions so far kept registration schedule on track but could be improved.

• Protein engineering of hydrolase-2* to open the way to3rd generation single enzyme process.

Desymmetrization for 2nd generation process

• single enzyme approach preferred• Substrate docking and rational

mutagenesis: 2 rounds completed• Greatly increased selectivity, catalytic

rate also increased• Mutations combined to hit target

E value of >100

W.T.

W.T.

Hydrolase engineering for 3rd generation process

E-value

E-value

Rational Design/Protein engineering


In silicopredictionof mutantEnzymes

Wet labvalidation

Docking/Mechanism

determination

Pointmutations

Typically 30-50rationally

designed mutantenzymesper round

Wet labvalidation


molecular docking /Molecular Dynamics

simulations

Co-evolutionanalysis

Rational design ofmutants

Bibliographic search /Homology Modeling /Structural analysis

Molecular docking /Molecular Dynamics

Final list ofmutants for in

vitro screening

1 week 1-2 weeks

2-3 weeks computational, 3-5 weeks mutant generation

Screening

2 weeks

Rational Design: Typical Timelines

• Rational design in a matter of weeks canproduce major improvements to solve arange of issues

• It can deliver in its own right and/or be aplatform to build upon

34

• Lignocellulose, cell algaeweakening

• Cell permeation• Biocatalysis

enhancement• Surface attrition• Surface activation• Improved heat/mass

transfer• Emulsification

• Lignocellulose, cell,algae weakening

• (Bio)polymerdegradation

• Crystallization• Cell permeation• Emulsification

IN THE CAVITYextremeconditions oncollapse 5000oCand 2000atmospheres

IN THE BULKMEDIAintense shearforces

UNSYMMETRICCOLLAPSEInrush of liquid fromone sideof the collapsingbubbleproduces powerfuljet of liquidtargeted at surface

Acknowledgement Prof. T Mason, Uni. of Coventry

CelbiusUltrasound

• Lignocelluloseweakening

• Cell permeation• Biocatalysis

enhancement• Surface attrition• Surface activation• Improved heat/mass

transfer

Video courtesyof University of Twente, Netherlands.and Shimadzu Europa GmbH,Duisburg,Germany

• Lignocellulose weakening• (Bio)polymer degradation• Crystallization• Cell permeation

IN THE CAVITYextremeconditions oncollapse 5000oCand 2000atmospheres

IN THE BULKMEDIAintense shearforces

UNSYMMETRICCOLLAPSEInrush of liquid fromone sideof the collapsingbubbleproduces powerfuljet of liquidtargeted at surface

Acknowledgement Prof. T Mason, Uni. of Coventry

CelbiusUltrasound

Process Operation

Reactor 50 -5000 L

Recirculation10 – 100L/min

50W - ~ 5 kWDepending onmain reactorvol. & powerreqd.

Temperature 0 to 70oC

Process intensification

• Process being developed formulti-tonne,

• Step 2 of a 5 step process• Process when scale-up had

mixing issues• Poor performance for reaction

time >70 hr• Options? Enzyme evolution? or

Application of US? or both?

0

20

40

60

80

100

120

0 10 20 30 40 50 60 70 80

Convers

ion

(%)

Time (hrs)

Ultrasound assisted biotransformation

Series1

Series2

Process intensification

Current technologyConfigured in reactor / recirculation systems

Flanged cylindricalpipe

Transducers bondedto deliver ultrasound

Medium out

Medium in

Pictures courtesy of Celbius

40

Development of methods including:- ID (FTIR, NMR, MS, XRPD)- Assay- Related substances- Chiral purity- Microbial and biomass, residual protein- Residual solvents, proteins, biomass- Particle size- Genotoxic impurities- Additional specification tests as required, developed inparallel and in conjunction with chemical development

AU

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

0.80

0.90

1.00

1.10

1.20

Min ute s

0.0 0 0.2 0 0 .4 0 0 .60 0.80 1.0 0 1.2 0 1 .4 0 1 .60 1.80 2.0 0 2.2 0 2 .4 0 2 .60 2 .80 3.0 0 3.2 0 3 .4 0 3 .60 3 .80 4.00 4.2 0 4 .4 0 4 .60 4 .80 5.00

Analytical Development

4/24/2017

Assay Range Comment

NanoOrange® 10 ng/mL to 10 μg/mLLow protein-to-protein signal variability

Detection not influenced by reducing agents or nucleic acids

BCA 0.5 μg/mL to 1.5 mg/mLSamples must be read within 10 minutes

Not compatible with reducing agents

Bradford 1 μg/mL to 1.5 mg/mL

Proteins precipitate over time

High protein-to-protein signal variability

Not compatible with detergents

Lowry 1 μg/mL to 1.5 mg/mLLengthy, multistep procedure

Not compatible with detergents, carbohydrates or reducing agents

Absorbance at 280 nm 50 μg/mL to 2 mg/mLHigh protein-to-protein signal variability

Detection influenced by nucleic acids and other residues

4/24/2017

Example 3:

API 3

Example 4:

API 4

Example 5:

API5

Example 6:

API 6

Enzymatic stepPre RSM, C-C bond

formation

Pre RSM, ketone

reduction

Post RSM,Alcohol

oxidation

Pre RSM Ketone

Reduction

Stage in synthesisSeveral steps before API

Several steps beforeAPI

Penultimate step Several steps before API

Enzyme type

Enzyme (Cells from E.

coli fermentation)

KRED (liquid

formulation from in E.

coli fermentation)

KRED (whole cell

formulation produced in

Gluconobacter oxydans)

KRED (Dry powder

produced from E. coli

fermentation)

Control strategyTest for total proteins,

DNA, endotoxins and

microbiological residues;

demonstrate fate and

purge; no enzyme

residue specifications for

API

Test for total

proteins; no enzyme

residue specifications

for API

Test for total proteins,

DNA, endotoxins and

microbiological

residues; demonstrate

fate and purge; no

enzyme residue

specifications for API

Test for total

proteins;

in-process testing;

demonstrate fate and

purge;

no enzyme residue

specifications for API

43

Where we are…..

The oldest Biocatalysis Shop in Ireland!

Dr. Derek Quinn

Biocatalysis: Biology Team Leader Leader

Email: [email protected]

ALMACDepartment of Biocatalysis & Isotope Chemistry

Seagoe Industrial EstateCraigavon, N. IrelandBT63 5QD UK

[email protected]

www.almacgroup.com

fit the process chemist - almac · fit the process chemist ... transaminase + ketone aminoacid-dh/...

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