1. Introducing Milligram Screening Reactions into the Kilogram World of Chemical Development
Simon Yates, AstraZeneca.
FreeSlate European User Meeting 24th September 2013

2. Our Journey so far…

3. What is Chemical Development?
We produce a scalable process, not just a compound
From a few grams in the lab to multi-kilo plant campaigns
Emphasis on SELECT criteria
Safety
Economics
Legal
Environment
Control
Throughput
Some images to describe:
Process takes research compounds to commercial scale
Emphasis on SELECT
Not traditionally the place HT experimentation / equipment aimed at
Most of reactions on >500mg (1mL) going up to 200g (3L) (maybe in 2007?) – singular.
Definitely not 96 reactions at once, and not on 10mg scale. Which is what we now do!
Picture :
Volumetric with sold in going to massive reactor – image of what’s the problem
Had to learn a lot along the way

5. Scavenging Challenge? Transition Metals are toxic!
In number of metal catalysed reactions in Med Chem
The time to develop process for early compounds
Conventional work up takes time to develop
Development time by using scavengers
100’s
Of scavengers and carbons to choose from
1st real driver to do this
Use of metal catalysed reactions shown to be increasing in Med Chem
Needed to reduce time to early key manufactures.
Use of functionalised silica and polymers to grab catalyst residues out of solution – save time on extractive wu that can be done later
Loads to choose from
Screening was the way forward
A key was to have it inerted though – changing oxidation state changing behaviour of scavenger – need to be like manufacture. Failures in the past.
Purchased CM2 in a glovebox
Time, effort and missed opportunity by automated screening
Reproducibility by screening under inert conditions

6. What are scavengers?

7. But what kit? Integrity 10 10 reactions at a time Minimum volume ~2mL
Typical reactions run on >500mg in small automated equipment or Jacketed Vessels

8. But what kit? Inerted Glovebox 20mL reaction tubes 100mg reactions
24 wells
MT Flexiweigh
Very manual
Lots of programming
18months earlier we had established catalyst screening at Avlon
In a glovebox
With Mettler Toledo equipment – 24 wells, ~100mg per reaction, very step wise approach
We didn’t have the space and we wanted to be more automated
We did take a lot of learning from them though, especially about gloveboxes and workflows!
2x MT Mini Mapper

9. Wanted to…. In number of reactions we could run (up from 24)
Reaction scale
Manual intervention, to make this as routine as possible
Ease of programming and data analysis
‘Future proof’ our investment
1st real driver to do this
Use of metal catalysed reactions shown to be increasing in Med Chem
Needed to reduce time to early key manufactures.
Use of functionalised silica and polymers to grab catalyst residues out of solution – save time on extractive wu that can be done later
Loads to choose from
Screening was the way forward
A key was to have it inerted though – changing oxidation state changing behaviour of scavenger – need to be like manufacture. Failures in the past.
Purchased CM2 in a glovebox

10. Purchased a Symyx CM2 Solid Dispensing 10G needle (5mL syringe)
Vial gripper
16G to 20G needle (with N2 pressure)
- 1mL & 500uL syringes attached to 10 off deck solvents
9 heat-stir bays
Better picture with it all set up.
Point out what happens and at what point by talking though the picture.
48s in duplicate
Filter – didn’t work now centrifuge off deck
Bespoke filter equipment
- Symyx filter too small

11. Scavenging Workflow ~40 different scavengers 96 x 1mL vials
Seal, heat, stir 16 hours
Stock of reaction solution
Use robot where needed, use manual pipetting where needed.
Case study on JH stuff
Dispense IS, metal and ligand as stocks – multidisp. from 1 asp.
Vac off
Add in solid Ar-BOR
Stock solution of AR-X as it was large crystals
Manual addition of bases (aq)
Sampling manually.

12. Scavenging Workflow Backing Solvent Air Gap Overshoot ICP sample
RAS
Centrifuge for 5 min
HPLC sample
ICP = Inductively Coupled Plasma

13. Scavenging today
We have run 20+ screens and saved projects time and money
Numerous examples of where scavengers used in all scales of manufacture
Pd is most common metal scavenged, now have a generic plate of 22 scavengers.
Improvements in whole workflow reduced time from 5 working days to 2 days.

15. Cross Coupling
R1-M
R2-X
R1-R2

16. Background Original plan was to run X-Coupling
Timing forced by closure of our existing facility
In number of reactions we could run cover more experimental space, quicker.
Reaction scale
Apply our previous learning
Always had plans to do x-coupling
AZ strategy forced our hand in 2009 with closure of Avlon
Great opportunity though
Move to a greater number of reactions on smaller scale (24 to 96)
With time I had become much more efficient in writing AS protocols
Assessed numerous ways of working
List out
Came to 96 well and a 24 well DoE.

17. Optimisation of continuous parameters
What we developed
Pre-screening
~10 reactions
Optimisation of continuous parameters
DoE (24-plate)
96-well plate screening
96-well plate screening
Optimisation of continuous parameters
DoE (24-plate)
Always had plans to do x-coupling
AZ strategy forced our hand in 2009 with closure of Avlon
Great opportunity though
Move to a greater number of reactions on smaller scale (24 to 96)
With time I had become much more efficient in writing AS protocols
Assessed numerous ways of working
List out
Came to 96 well and a 24 well DoE.
Discovery
Life Cycle Management
Development

18. Generic Reaction Plates
Developed ‘Generic plates’ for 5 different reaction types
Based on literature and in house expertise
Use robot where needed, use manual pipetting where needed.
Case study on JH stuff
Dispense IS, metal and ligand as stocks – multidisp. from 1 asp.
Vac off
Add in solid Ar-BOR
Stock solution of AR-X as it was large crystals
Manual addition of bases (aq)
Sampling manually.

19. Generic plate formation
Manual Pipette
-or - Automated dispensing
Evaporate off carrier solvent
Stock solutions of - Ligands
- Metals
- Internal std
and store
Use robot where needed, use manual pipetting where needed.
Case study on JH stuff
Dispense IS, metal and ligand as stocks – multidisp. from 1 asp.
Vac off
Add in solid Ar-BOR
Stock solution of AR-X as it was large crystals
Manual addition of bases (aq)
Sampling manually.

20. Running a project… Add in reaction solvent Add in solid(s) Reactants
Define:
Bases and Solvents
Use robot where needed, use manual pipetting where needed.
Case study on JH stuff
Dispense IS, metal and ligand as stocks – multidisp. from 1 asp.
Vac off
Add in solid Ar-BOR
Stock solution of AR-X as it was large crystals
Manual addition of bases (aq)
Sampling manually.

21. Running a project… Quickly add in Aq. Base ‘Start of reaction’
2 x Manual Sample Prep
~2hrs and 20hrs
Seal up
Heat and Stir
Use robot where needed, use manual pipetting where needed.
Case study on JH stuff
Dispense IS, metal and ligand as stocks – multidisp. from 1 asp.
Vac off
Add in solid Ar-BOR
Stock solution of AR-X as it was large crystals
Manual addition of bases (aq)
Sampling manually.

22. Future improvements Reactions run on 200-400uL scale at 10C below bp
Solvent loss / corrosion of sealing material
Fully closed plate
Can’t sample by CM2
1st sample will always be manual
2nd sample could be automated – middle of night.
Need a pierceable, but re-sealable, membrane
Any ideas welcome!!

23. 24 well - DoE type Expanding on 96 well hit(s) B A Discrete variables
Continuous variables
Use robot where needed, use manual pipetting where needed.
Case study on JH stuff
Dispense IS, metal and ligand as stocks – multidisp. from 1 asp.
Vac off
Add in solid Ar-BOR
Stock solution of AR-X as it was large crystals
Manual addition of bases (aq)
Sampling manually.
Material consumption
Profile reactions
Statistical Analysis

24. Catalyst Plate Reaction Plate 24 well – DoE type Weigh in
Ligands Metal
Cap, heat and stir, 60min.
Catalyst Plate
24 x 4mL vials
Add reaction solvent
Weigh in
Reactants, Bases
Internal Standard
Reaction Plate
24 x 4mL vials
Add reaction solvent/liquid reagents
Use robot where needed, use manual pipetting where needed.
Case study on JH stuff
Dispense IS, metal and ligand as stocks – multidisp. from 1 asp.
Vac off
Add in solid Ar-BOR
Stock solution of AR-X as it was large crystals
Manual addition of bases (aq)
Sampling manually.

25. Reaction Plate 24 well – DoE type
Transfer from Catalyst plate to reaction plate
Reaction Plate
Automatically sample
4 times over 16 hours
Run LC-Mass Spec on samples
Use robot where needed, use manual pipetting where needed.
Case study on JH stuff
Dispense IS, metal and ligand as stocks – multidisp. from 1 asp.
Vac off
Add in solid Ar-BOR
Stock solution of AR-X as it was large crystals
Manual addition of bases (aq)
Sampling manually.
Heat and Stir

26. Future improvements 1 Temperature (plate) per run Lose a key factor
Multiple Plates / run
 sampled into 1 HPLC plate

27. Future improvements
Sampling always run at the end, not good for fast reactions
Allow sampling as part of ‘dispense’
And/or move vials to heat zones to ‘start’ reactions (Josh Denette and Kristin Price at Pfizer)

28. Data Handling
MDB
Use robot where needed, use manual pipetting where needed.
Case study on JH stuff
Dispense IS, metal and ligand as stocks – multidisp. from 1 asp.
Vac off
Add in solid Ar-BOR
Stock solution of AR-X as it was large crystals
Manual addition of bases (aq)
Sampling manually.
RAS

30. Asymmetric Hydrogenation
Successfully built capability in Sodertalje, Sweden
site end of 2012
Opportunity to purchase new equipment
CM3 (in glovebox) and SPR
Off deck hydrogenation – make use of CM3 during long reactions
Knowledge of LEA / CM2
Commissioning - NOW
CM2 / CM3 used as 1 resource for X-Coupling and Asymm. Hydrog.

31. Conclusion We have come along way since 2007
Scavenger was the warm up act
Scavenging and X-Coupling saved millions of dollars already
Success led the way to keep Asymm. Hydrog. in house
Asymm. Hydrog. will have similar if not bigger impact
Continue to develop workflows
Keep control of the data
96 x 2 x 5 x 50 = 48K data points / year

32. Conclusion
User community carry on sharing and learning
What’s good for me, could be good for you too.

33. Acknowledgments AstraZeneca John Leonard, Gair Ford, Barney Squires
Phil Hogan, Keith Mulholland, Andy Campbell
Rachel Munday, Kevin Leslie
Sarah Thompson, Andy Poulton
Ex AstraZeneca
Paul Murray and co. (catsci.com)
Per Ryberg and co. (SP Technical Research Institute of Sweden)
Symyx/FreeSlate Steve Yemm, Zack Hogan, Colin Masui, Anny Tangkilisan, Rob Rosen, Eric Carlson
Peter Huefner, Grant Gavranovic, Justin Fisher, Jonathan Harris
Peter Gravil, Jos De Keijzer, Rick Sidler, Tony Mani, Guillaume Magan, Ludovic Edvard

34. Went to a lot of meeting like this one and talk to a lot of experienced people to understand what had been done and what we could hope to achieve.
Freeslate, San Francisco, 2013

35. Confidentiality Notice
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36. What it looks like under the hood
Why its important
Numerous solvents at numerous temps. Different viscosities.
What's under your control in AS
Air gaps and overshoots
Where to dispense to (Zmax)
Protocol

37. Time Out.… Accurate Sampling
Accuracy and Precision of sampling critical for ICP analysis
Narrower needle
No bubbles in your backing solvent
Use as small an airgap (20uL)
Use a suitably large overshoot
If sampling hot, aspirate an airgap before coming out of the vessel
Dispense below solvent level if possible.
Best syringe speeds (parameters) – varies for different solvents.
Use DoE to define
Why its important
Numerous solvents at numerous temps. Different viscosities.
What's under your control in AS
Air gaps and overshoots
Where to dispense to (Zmax)
Protocol

38. What are these syringe parameters?
Speeds for 500uL syringe
Time
Speed
Start Speed
Top Speed
Cut Off Speed
Slope
Option 25
420 1
900
125 2 3 4 5 75
462.5
4210 6 7 8 9
8000 10 11 12 13 14 15 16
Why its important
Numerous solvents at numerous temps. Different viscosities.
What's under your control in AS
Air gaps and overshoots
Where to dispense to (Zmax)
Protocol

39. Randomised Speed Options, in triplicate
Gravimetric checking
Randomised Speed Options, in triplicate 1 2 3 4 5 6 7 8 9 10 11 12 A 13 15 14 16 B C D
Tare all 48 vials
For each well
Aspirate 100uL Toluene, plus 30uL overshoot from a 20mL vial
Dispense into the 1mL vial using given parameters
Immediately reweigh vial
Calculate the volume dispensed by change in mass
Why its important
Numerous solvents at numerous temps. Different viscosities.
What's under your control in AS
Air gaps and overshoots
Where to dispense to (Zmax)
Protocol

40. The result… For 100uL of toluene, at 25C Why its important
Start Speed
Top Speed
Cut Off Speed
Slope
Option 25
125
8000 10
Why its important
Numerous solvents at numerous temps. Different viscosities.
What's under your control in AS
Air gaps and overshoots
Where to dispense to (Zmax)
Protocol

41. The result… For 100uL of toluene, at 25C 12 different solvents
Start Speed
Top Speed
Cut Off Speed
Slope
Option 25
125
8000 10
Why its important
Numerous solvents at numerous temps. Different viscosities.
What's under your control in AS
Air gaps and overshoots
Where to dispense to (Zmax)
Protocol
12 different solvents
A range of speed options shown to be best
2-MeTHF – option 3
THF – option 16