Reversed New Presentation 1-25-06

Simple Synthesis to Purification Techniques Jeff Horsman

Biotage AB

Corporate Headquarters Uppsala, Sweden

Biosystems

Discovery Chemistry

Genetic Analysis

Microwave Synthesis Uppsala

Uppsala, Sweden

Charlottesville, USA

Flash Purification Charlottesville

Outline • Modern Purification Techniques – Using pre-packed cartridge technologies – Using automated Flash purification systems

• Combining These Synthesis and Purification Techniques – Using Samplets and sample introduction modules (SIM’s) – Synthesis combined with purification from mgs to kgs – Produce bulk intermediates quickly and cost effectively

• Modern Synthesis Techniques – Microwave Assisted Organic Synthesis – Parallel Organic Synthesis

Modern Purification Techniques • Modern Purification Techniques – What is Flash chromatography? – Optimizing purifications

– Using pre-packed cartridge technologies – Reduce purification time and costs – Convenient, simple sample loading

– Using automated Flash purification systems – Quickly optimize purification protocols – Simple scale-up from small scale cartridges

What is Flash Chromatography? •TLC

Flow

1.0 0.9 0.8 0.7 Rf 0.6 0.5 0.4 0.3 0.2 0.1 0rigin

------10.00 5.00 3.33 CV 2.50 2.00 1.67 1.43 1.25 1.11 1.00

Flow

Flash

TLC

–Used to define flash purification conditions; –Isocratic; –CV = 1/Rf –Gradient; –CV vs Rf is dependent on elution conditions. –Biotage TLC to gradient

Influencing Purification Goals • TLC – Surface chemistry – Resolution (DCV) – Elution solvents

• Sample solvent • Impurities – Excess starting materials – Synthetic by-products

• Elution conditions – Isocratic – Gradient

Optimizing Purification Use TLC to determine: • Optimal solvent conditions – Solvent selectivity – Solvent strength

• Sample load factors – Resolution (∆CV) – The ∆CV / ∆Rf relationship – Sample mass effects

• Sample load – Discovery – scale – Development – scale – Use Biotage loading chart

Solvent Selectivity Solvent Diethyl Ether Methanol Ethanol 2-Propanol Tetrahydrofuran Acetone Ethyl Acetate Acetonitrile Dichloromethane Toluene Chloroform Hexane

Selectivity Group I Solvent Front II II ? II A B III C ? VIa VIa VIb V Origin VII Hexane/EtOAc VIII 2:1 ----

(From L.R. Snyder, J. Chromatogr., 92, 223 (1974)).

Solvent Front

C B ?

? A

Origin

100% CH2Cl2

Solvent Strength Solvent

Solvent Strength

Methanol Ethanol 2-Propanol Acetonitrile Ethyl Acetate Tetrahydrofuran Acetone Dichloromethane Chloroform Diethyl Ether Toluene Hexane

.95 .88 .82 .65 .58 .57 .56 .42 .40 .38 .29 .01 Calculated Solvent Strength

Solvent Front

Origin

Hexane/EtOAc 1:1 0.30

Solvent Front

Origin

Hexane/CH2Cl2 1:2 0.28

A B

Origin

A B

Origin

A B

Origin

∆CV vs. ∆Rf

1 .9 .8 .7 .6 .5 .4 .3 .2 .1 0

1 .9 .8 .7 .6 .5 .4 .3 .2 .1 0

1 .9 .8 .7 .6 .5 .4 .3 .2 .1 0

R f A = .80 Rf B = .67 ∆Rf = .13 ∆CV = 0.08 B

RfA= .47 RfB = .34 ∆Rf = .13 ∆CV = 0.8

RfA = .32 RfB = .18 ∆Rf = .14 ∆CV = 2.4 B

B

0 1 10

A

A

A

2

3

4

5

6

7

8

9

0 1 10

2

3

4

5

6

7

8

Column Volumes

9

0 1 10

2

3

4

5

6

• No ∆Rf change with lowering of Rf • Increasing ∆CV with decreasing Rf • Predict maximum sample loading better with ∆ CV than ∆Rf

7

8

9

The Rf - CV Relationship Rf

Optimal Range

0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.60 0.70 0.80 0.90 1.00

CV ∆Rf

0.05 0.05 0.05 0.05

10.0 6.7 5.0 4.0 3.3 2.8 2.5 2.2 2.0 1.6 1.4 1.25 1.11 1.0

∆CV

1.7 1.0 0.7 0.5

• Lower Rf values mean larger CV and ∆CV values • Equal changes in Rf (∆Rf) do not translate to equal changes in CV (∆CV) • Optimal Rf range(0.15 – 0.35) – For compound of interest with isocratic elution – Maximum resolution – Maximum loading capacity – Minimal solvent consumption

Determining Loading Capacity • Compound resolution key to good loading capacity • TLC data measured in Rf (retention factor) − ∆Rf not a useful term

• Rf values are inversely proportional to FLASH column volumes (CV)

Rf = 1/CV or CV = 1/Rf • Resolution (∆CV) determines load for any size cartridge: ∆CV = CV1 - CV2 • FLASH separations and loading capacity governed by ∆CV, not ∆Rf

Sample Load Factors • Resolution (∆CV) – Larger ∆CV = larger loads

• Mass ratios – Beware of overload – Total loadable mass based on amount of crude, not amount of product

• Required purity – Higher purity requirements = lower loads, lower yields

• Required yield – Higher yield requirements = lower purity

• Cartridge size – Larger cartridges = larger loads

Why use Column Volumes • Easy scale-up – Optimization work on TLC – Quick and cost effective – Test many solvent systems at the same time – Direct scale up to any cartridge size

– Direct relationship between cartridge sizes – Using CV is independent of flowrate – Scale up based on flowrate requires column diameter ratio calculations

Optimizing Gradient Purification • Always TLC Sample – Measure Rf – Try for compound of interest Rf = 0.4 – Set gradient conditions – Initial = ¼ polar solvent concentration from TLC – Final = Twice polar solvent concentration from TLC – Segment 1 = 1 CV @ initial conditions – Segment 2 = 10 CV, Initial to Final conditions – Segment 3 = 2 CV @ final conditions – Segment 4 = 3CV Final conditions to 100% polar solvent (may not be required)

• Difficult samples are no problem • Set load based on Biotage ∆CV/cartridge chart • Use flow rate = cartridge diameter

TLC to Gradient Example • TLC 80:20 Hexane:Ethylacetate (20% EtOAc) – Segment 1, – Segment 2, 10CV’s – Segment 3, – Segment 4, over 3CV’s

Initial Segment 5% EtOAc Increase from 5% to 40% EtOAc over Hold for 2CV’s at 40% EtOAc If required increase to 100% EtOAc

• Above example is for initial work – If the same or similar sample is run again vary slightly based on earlier separation – Remove segment 3 or 4 if not required – Use 8CV’s instead of 10 for main gradient

Biotage Pre-packed Cartridges •Broad range of cartridge sizes – 4 grams to 40 kilograms

•Pre-packed, so you don’t have to!! – Proven proprietry packing methods

• purification scales mgs to kgs

Fully Scaleable FLASH Solutions

Discovery

Development

Production

Fully Automated Production

Sp1, automated

Flash 75 & 150

Flash 400

MP- 8000

Pressure system

10g – 250g

Kg Quantities

Kg Quantities

5mg – 10g

FLASH+ cartridges and modules • • • • •

• • • •

KP-SIL, KP-NH, KP-C18 Others on request Recessed inlet ISO 9002 certified automated manufacturing Complies with FDA 21 CFR 177.1520 extractable requirements Wide variety of sizes Stable to >100 psi Disposable Axial compression

FLASH 12+

FLASH 25+

FLASH 40+

Sample Loading Techniques • 5 Sample loading methods are commonly used – Samplets – The preferred method enhances separation

– Direct liquid injection – Ideally sample is soluble in low polarity solvents

– ZIF-SIM – Sample only soluble in high polarity solvents

– Dry-load heads – A cross between Samplets and ZIF-SIM’s

– Pour liquid directly on cartridge – Least recommended

Why use Samplets Conditions Cartridge: Eluent: Flow: Load:

FLASH 40+S 8:2 Hexane/EtOAc 25 mL/min 500 mg (in 1-mL acetone)

Direct injection Samplet Mixed fractions

• Samplet use increases separation performance – More compound retention – Tighter elution bands – Increased resolution – Less mixed fractions – Improved compound purity – Increased product yield

• Limitations 0

5

10 Minutes

15

20

Samplet Capacities Max.Volume 1 mL

– Sample is not soluble in suitable volumes, see table

Size 12+

Mass 0.5 g

Max.Mass 500 mg

25+

2.5 g

5 mL

2500 mg

40+

5.0 g

10 mL

5000 mg

When to use Samplets •

High capacity sample concentrator

•

Ideal for

•

•

– Typical load is 1/2 g sample per gram of silica – Designed for multiple samples preparation – Soluble samples – Insoluble samples – Chromatographically strong solvents

Samplet Family

– Dissolve sample but interfere with separation

Convenient, easy to use

– Eliminates rotary evaporation – Directly load crude reaction mixtures – Easy and safe disposal after use

Inserted into top of FLASH+ cartridge – Requires no additional hardware or connections

FLASH 40+ FLASH 25+ FLASH 12+

Direct Liquid Injection • First check suitability of dissolution solvent – Run TLC in dissolution solvent – Spot should not move more than Rf 0.05

• Easy load technique – Use barrel as funnel – Remove plunger – Attach barrel to 3-way valve

• However – Often requires sample filtration – Solvent strength impacts chromatography – Uses top of chromatographic bed to adsorb sample, diminishing the effective column length – Reduced purification performance

ZIF-SIM Loads Conditions Cartridge: Eluent: Flow: Injection:

FLASH 40+S Hexane/EtOAc 8:2 25 mL/min A. ZIF-SIM 60 1:1 B. ZIF-SIM 60 1:2 C. ZIF-SIM 60 1:10 D. Samplet

• ZIF-SIM vs Samplet – Samplet

– 1-mL (500 mg) added to FLASH 40+ Samplet, dried – Inserted in FLASH 40+ cartridge (or into ZIF-SIM)

– 3 solid samples prepared

– 1-mL sample (500 mg/mL) per g silica, dried – 1-mL sample per 2 g silica, dried – 1-mL sample per 10 g silica, dried

Absorbance

A

– Packed into separate ZIF-SIM modules

B

• Performance difference

– Samplet (D) retains and resolves components completely – Sample/ silica ratio important for optimal ZIF-SIM performance

C

– 1:1 (A) too heavy (overload) – 1:10 (C) too light (excessive bandspreading)

D 0

5

10 Minutes

15

20

Dry-load Heads • Ideally sample loads – 0.3 to 0.5 the mass of the Samplet silica – Large dissolution volumes – Should be free flowing powder

• Performance – Between Samplets and ZIF-SIM’s

Direct Pour-onto Cartridge • Least preferred method – Stand cartridge vertical – Ensures even sample load

• Reduces usable cartridge length • Sample load issues – Uneven load due to “percolation” effects

All Purifications Fully Scaleable • Flash 400 System • Complete skid mounted system meets – XP Class I Division 1 & 2 explosion-proof & CE ATex – USA/Canada ASME – CE/EU Pressure Directive – Japan certification pending

• Designed & documented for cGMP

Even Pre-absorbed Sample Loading • SIM 10,000 • For Low Solubility and or Highly Fouling Compounds • Skid Mounted Sample Injection Module • Radial Compression • Disposable User Packed Cartridges

SP1 Automated Flash Purification

SP1/SP4 Overview Automated, single or sequential cartridge flash system with touch screen control and state-of-the-art functionality – Binary Gradient Pumping System from four solvents – UV Sample Detection – Automated Fraction Collection – Built in computer – Intuitive Touch Screen Control – Automatic gradient creation based on TLC – Pre-packed Columns…

SP1/SP4 Extended Bed • NEW Expanded fraction capacity – Collect up to 5.8 L with at least 24 fractions for each four-column run. – Upgrade any existing SP System to the new EXP functionality, independent of its configuration.

Compact footprint

•48 x 58 x 61cm (19 x 23 x 24”) •Fits into any standard fumehood •Built-in PC •Touch screen control •Same footprint for SP4

Sample loss/contamination control Automatic pause High pressure Loss of detector Fraction collector arm misalignment

Three built-in fluid-line flushes UV-zero flush Line flush Detector flush

Auto-extend At the end of run if remaining UV activity is detected

All New Software • Robustness and flexibility • Change in software architecture • Common Biotage platform

New Software

Wizard

Run

Status

Results

TLC to gradient

Rf 0.57 Rf 0.36 Rf 0.21 Rf 0.12

Case Study Nitro-organics • Base Separation on TLC • Compounds in mix

Rf approx. 0.6 (0.57 actual) Rf approx. 0.35 (0.36 actual) Rf approx. 0.2 (0.21 actual) Rf approx. 0.1 (0.12 actual)

– – – –

Napthalene 1-nitroaniline 2-nitroaninline 3-nitroaniline

• Initially use Sp TLC gradient – Optimize if required – Use less solvent – Less time – Higher loads

12+M Sp1 Chromatograms

Recommended load 200mgs

Increase load by 50%, increase polar solvent to 100%

Optimize by changing gradient to 50% polar solvent over 10CV

• Choosing Recommended conditions – Simply using Rf and TLC solvent strengths – Excellent separation 1st time – Simple optimization for scale-up – Just enter new cartridge size! – Sp1 automatically changes solvent volumes!

Sp1 Scale-up Chromatograms • Scale-up factors work 1.2g on 25+M

– 25+M scale increase x 4 – 40+M scale increase x 10 – 75L scale increase x 100

• Separations collected using slope collection algorithm 8.7g on 40+M

25g on 75L

For True Scale-up The Advancer and Flash400 system • • • • • • • • •

50-600 ml working volume Patent pending ”instant” adiabatic cooling Mechanical stirrer Best-in-class safety Multiple vessel entry points Easy-to-use software 60-250°C operating temperature 1-22 Bar operating pressure range 1200 W power supply

•

Complete skid mounted system meets – – – –

•

XP Class I Division 1 & 2 explosion-proof & CE ATex USA/Canada ASME CE/EU Pressure Directive Japan certification pending

Designed & documented for cGMP

More Traditional • Optimize chemistry – 2410 lab system

• Check chemistry at prep scale – 3400 prep system

• Fully validate at process scale – 4100 process system

• Purify using Biotage Flash • Scale-up to Flash 400

Modern Synthesis Techniques • Modern Synthesis Techniques – Microwave Assisted Organic Synthesis – Reducing synthesis reaction times from days to minutes – Optimizing reaction schemes in hours not weeks – Produce bulk intermediates quickly and at high purity

– Parallel Organic Synthesis – Argonaut synthesis units – Offer easy scale-up from mgs to kgs

Why does microwave heating speed up reactions? Arrhenius Equation:

K = A e–∆G/RT

For every 10 oC temperature increase, the reaction rate doubles This leads to an exponential time savings as the temperature is raised: ∆=

10oC 60oC 100oC

~ ½ the reaction time ~ 1/60 the reaction time ~ 1/1000 the reaction time

Advantages Hydantoin Synthesis

• Shorter reaction times  Rapid testing of creative ideas  Increased productivity

HN O

H N

COOEt NH

O Ba(OH)2 / DMF

N

O

 Higher yields, improved purity • Expanded reaction diversity

Microwave Heating: 4 min; 90% yield Conventional Heating: 48 h; 54% yield

 Some difficult reactions made possible

Epoxide Ring Opening OH

• Less reagents and solvents • Less reactive reagents • Reproducible and Scalable results

O

NH4OH NH2

Microwave Heating: 7 min; 93% yield Conventional Heating: 10 days; 13% yield

Initiator Product Line • A small and upgradeable microwave synthesis platform • The system is controlled via a touch screen • Can run volumes of 0.2-20 mL (with EXP) either manually or automated

UPGRADEABLE A modular system with several upgrade paths  the expanded volume range (EXP, 0.2-20 ml)  automation (Robot Eight or Robot Sixty)

Initiator EXP Initiator Eight EXP

Initiator

Initiator Eight

Initiator Sixty

Initiator Sixty EXP

Selecting a starting point (Temp/time)

If the reaction was run conventionally

o

Using the 10 Rule to determine a starting point K = A e–∆G/RT

Prepare Three Vials and Bracket the new determined Temperature & Time

o

140 C 5 min

o

160 C 5 min

First Choice

o

180 C 5 min

First Step in the Synthesis of Oxaprozin (DAYPROTM)

O O

OH

+

STEP 1 DMAP

PhMe

Coherent SynthesisTM 5 min (96%)

* Dr Vita Ozola; Ilga Mutule (1) Breviglieri, G. et al. US patent US 6,096,896, 2000

O

O OH

*

150 oC;

O

O O

Step 1

O

Conventional Synthesis1 Reflux;

4h

First Step in the Synthesis of Oxaprozin Optimization Studies – varying concentration

O

O OH

+

O 20 mol% DMAP

O O

O

O

O

"Solvent" (2.5 mL)

OH

1 Benzoin

2 Succinic anhydride

Entry

1 (mmole)

2 (mmole)

Solvent

T (min)

t (oC)

HPLC % yield

1

0.1

0.12

DMF

30

110

12

2

0.1

0.12

PhMe

30

80

4

3

0.1

0.12

PhCF3

30

110

27

4

1.0

1.2

PhMe

30

110

66

5

1.0

1.2

PhMe

30

150

82

6

6.0

12.0

PhMe

5

150

96

Can reaction conditions developed using “small scale” microwave heating be directly SCALED-UP?

YES! Conventionally & (reverse Arrhenius rule)

0.2-5 ml

20 ml

300 ml

mg – g

1 – 10 g

10 g – 1 kg

Advantage Series Synthesizers • Advantage Series – 2410 Small scale optimization – Parallel synthesis – Allows rapid screening of reaction conditions – Direct scale-up parameters

– 3400 Prep scale automated workstation – Validation of 2410 results – Calorimetric determinations

– 4100 Process scale-up reactor – Validation of 3400 results – Direct scale-up to standard reactors

Advantage 2410 • Utilize the Advantage Series 2410 for screening reactions – Four reactors – 5 to 30 mL working volume – Individual temperature control – -10 to +150 oC

– Reflux – Distillation – Powerful magnetic stirring – Mimics overhead stirrers

– Inert atmosphere

Advantage 3400 • Four Independent reactors – Individually jacketed – 125 or 250mL working volume – Standard glass joints

• Mechanical stirring • Individual temperature control – -20 to +180C

• Two feed pumps – Feed rates based on temperature The selected process would be optimized on the lab scale 2410, then characterized and validated on the Advantage Series 3400 Process Chemistry Workstation (AS 3400)

• Recipe or on-the-fly operation • Automated report generation • Data logging 21CFR part 11 compliant

Advantage 4100 Series • Reactions then run in the Advantage Series 4100 Process Scale-Up Reactor at the 2L scale • Collect heat flow data: examples – reduction step: 470 kJ/mol – methanol quench step: 160 kJ/mol – condensation step: 425 kJ/mol

• Data agrees with 3400 results • Product isolated at high yield & purity at 10x scale