Tips and Tricks for SFC

Method Development

Jeffrey Kiplinger, Paul Lefebvre, Mickey Rego

Averica

Massachusetts, USA

Waters User’s Meeting, Prague CZ, December 2015

The Good Enough Concept

• “Good enough” requires that the standard be

much lower than BEST, but offer valuable

compensations

– Flip™ camera

– MP3 music file compression

– Cloud-based applications

– Kindle books

– Micro-clinics

“Good Enough” Chromatography

• Not the same as “Fully Optimized Chromatography”

– Screen many conditions for optimal selectivity

– Do loading studies

– Appropriate for large-scale separations, esp. as an ongoing production

process

• Averica’s business:

– Contract chromatography services, small molecule pharmaceutical

sector, non-GMP, 5 mg to 500 g scale

– ~45% chiral separations, average sample size 5-20 grams

– Task: deliver scalable supply of high value compound fast

De-Risking Early Drug Development

Best Practice List for Pre-GLP Safety Assessment AveragePhys. Chem. prop prediction 94%

Genotox pilot (e.g. mini-Ames) 87%

Off-target assays (e.g. CEREP) 87%

hERG inhibition (e.g. auto patch clamp) 85%

Reactive metabolite detection 85%

1-2 week pilot tox in rodent 82%

Genotox prediction (e.g. DEREK) 79%

General tox & ADME prediction (e.g. MCASE, TOPKAT) 74%

Mouse micronucleus (gene tox) 72%

Ames (normal Ames) 68%

3-4 day mini-tox in rodent 68%

Safety pharmacology core battery 66%

Safety pharmacology telemetry 62%

1-2 week pilot tox in large species 60%hERG screening (e.g. Rb efflux) 57%Transporter binding/inhibition 57%

Percentage of programs requiring specific assay prior to full development- Source: Drug Safety Executive Council (DSEC) survey of 20 Directors of safety

assessment at major pharmaceutical companies, 2011

Principles

• Work with a small set of favored CSPs

• Move to prep columns rapidly

• Use “work arounds” that limit method

development effort

– Vary the co-solvent rather than the CSP

– Take a big valley

– Overlay peaks in stacking when possible

Reg

isPa

ck

AD IA

CC

A-E

X- b

12

Reg

isC

ell

OD

CC

O-E

X- b

14

OJ

CC

J

AS

Lux-

2

IC

Wh

elk-

O1

CC

4

CLA

-1

Sulfinpyrazone yes yes yes no no no no no no no no yes yes yes no

Chlorpheniramine yes yes no no no yes yes no no no yes yes yes yes no

Ibuprofen no no no no no no no yes yes no yes no yes yes no

Benzoflumethiazide yes yes no no no no no yes yes yes yes yes yes yes no

Tetramisole yes yes yes yes no no no yes yes yes yes yes yes yes yes

Sulpiride yes no yes no no no no no no yes no yes no no no

Sulconazole yes yes yes yes no no no yes yes yes no no no yes no

Octopamine no no no yes no yes no no no no no no no no no

Warfarin yes yes yes yes yes yes yes yes yes yes yes yes yes yes yes

Propranolol yes yes yes yes yes yes yes yes yes yes yes no no yes yes

Ornidazole no no no no no no no no no no no yes yes no yes

Acenaphthenol no no no no yes yes yes yes yes yes yes yes yes yes yes

trans-Stilbene oxide yes yes yes yes yes yes yes yes yes yes yes yes yes yes yes

2-methyl-2-propyl-sulfinamide no no no no no no no no no yes no no no no no

Ketoprofen no no no no no no no yes no no no yes yes no yes

Miconazole nitrate yes yes yes yes no no no yes no yes yes yes no yes no

Naphthol no no no yes yes yes yes yes yes yes no yes no no yes

(±)-Naringenin yes yes yes yes yes no yes no no no no no no no no

Terfenadine no no no yes yes yes no yes yes yes no yes yes no no

Bucetin no no yes no yes yes yes no no no yes no yes no yes

Cineole no no no no no no no no no no no no no no no

Practical Performance Characteristics

Capacity to load

• Overloading

decreases peak

capacity

• Measure peak

capacity with

high and low

standard

samples

Capacity to elute

• Compounds

resolved at lower

% co-solvent are

easier to recover

• Some columns

are more

retentive than

others

Capacity to resolve

• Percentage of test

compound set that

interact with CSP

• Rs = RT2 – RT1

Degree of resolution

Column Overall rank

AD 92.3

Whelk-O1 79.8

OD 78.8

RegisPack 77.8

CLA-1 76.6

IC 72.2

IA 70.8

Lux-2 Cel 69.1

CCA-EX-b12 69.0

AS 68.4

OJ 64.7

CC4 64.6

RegisCell 63.7

CCJ 60.8

CCO-EX-b14 58.1

Column Performance Ranking

• (%RCmax+%DRmax+%PCLmin)/3

• Simple, unweighted average of

performance criteria

• Lower ranked columns are more

“compound specific”

• Greater preference for immobilized

CSPs

Iterative Screening

• Initial screen 6 columns, 2-3 co-solvents

– Stop early if a separation looks promising

• Second set of columns only if no CSP interaction seen

• 3 minute 5-55% co-solvent gradient

• Pick a column, develop isocratic method

– Co-solvent , %, modifier +/-

• Transfer to prep column

• Reversal of elution order using simple alcohols

Co-solvent Mediates Selectivity

Co-solvent:

Blue = MeOH

Purple = 50:50 MeOH:iPrOH

Gold = iPrOH

Co-elution

E1

E2

E2

E1

AV-010163

(RegisPack)

Optimizing Prep Chromatography

• Limited density variation in operating region gives limited control

over solubility and k’

• Courtesy A. Tarafder

Critical point: Tc = 86oC, Pc

= 120 bar, rc = .48 g/mL

Operating

region

Isopycnic plot: CO2/MeOH = 80/20 (v/v)Generated by: REFPROP (NIST)

ABPR Backpressure Optimization

• ABPR can go ‘unstable’, esp. at high pressures, with negative impact

on separation

• Do we need this level of control?

Pressure Control – ABPR + BPR Cartridge

• Upchurch 1000 psi cartridge helps stabilize ABPR, but

changing pressure is slow

• Note variation with flow rate and solvent viscosity is minimal

Pressure Control Using Restrictor Tubing

30 cm 0.01”ID

20 cm 0.01”ID

10 cm 0.01”ID

• Selectable restrictor – Gilson valvemate + variable restrictor length

• In practice we use only one restrictor

Method Optimization on Prep Columns

• Vary co-solvent modifier concentration (if used)

• Adjust retention time using hexane or acetonitrile co-

solvent admixtures

• Elevated temperature (to 40oC) if retention is excessive

– Insulated column

• Set collection times carefully

– Valley timing to meet ee spec, not for recovery

– Stack design

• Larger campaigns are worth more effort

E1

E2

E1 E1 E1 E1

E2 E2 E2 E2

Racemic

compound

separation

F1 F2

“Valley”

Feedstock: Sample + Impurities

AV15166UPC2-110% 1:1 MeOH/IPA w/ 0.1% IPAm

Whelk-O (212)

Time-0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00

AU

0.0

1.0e-1

2.0e-1

3.0e-1

4.0e-1

5.0e-1

6.0e-1

7.0e-1

8.0e-1

9.0e-1

1.0

1.1

1.2

1.3

1.4

1.5

1.6

1.7

1.8

AV15166_09 Diode Array 230

Range: 1.995

1.15

0.49

1.59

1.41

2.251.84 1.98

72 % chemical purity

AV15166 UPLC-1 CSH C18 (223)

Time0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00

AU

-2.0e-1

0.0

2.0e-1

4.0e-1

6.0e-1

8.0e-1

1.0

1.2

1.4

1.6

1.8

2.0

2.2

2.4

2.6

AV15166_06 Sm (Mn, 1x2) 2: Diode Array 230

Range: 3.25 Area%

0.14

0.14

1.68

0.23

0.07

10.74

71.98

5.50

0.14

1.45

5.96

0.97

0.35

0.37

0.10

0.19

Area

115.28

115.44

1405.78

193.42

61.76

8993.10

60291.03

4604.30

118.81

1216.28

4993.35

809.82

289.52

307.64

81.79

158.41

Height

7438

6007

76457

13852

3918

631780

3085174

304178

9441

36956

363292

50488

17652

22121

6090

11208

Time

1.69

1.85

2.06

2.11

2.23

2.56

2.61

2.66

2.72

2.78

2.95

3.03

3.09

3.12

3.18

3.28

2.61

2.56

2.952.66

Gradient HPLC

Isocratic

SFC

Stacked Injection Design

• Estimated productivity 0.35 kkd, E1 >95% ee

• Requires knowing the “desired” peak

E1

E2

Advanced Strategies

• Resolve a key intermediate

• Evaluate different feedstocks

• Derivatize with removable

protecting groups

• Partially enrich one isomer

CN

N

CH3

O

BnOOC

CH3CH3

N

CH3

O

HOOC

CH3CH3

NH

NN

N

i) Bu3SnN

3, xylene, reflux

ii) HCl, MeOH

iii) NaF, aq. NaOHiv) filter, HCl

Key Intermediate

Valsartan (Diovan)

Chiralpak AD, Hex:IPA:TFA

Est. productivity 0.08 kkd

Proprietary Chiral SFC Method

Est. productivity 1.8 kkd

Feedstock Evaluation

• 150 grams active enantiomer needed

• By conventional chromatography: 8 FTE-wks to purify racemate

• SFC: Cleanup on 2-EP at 4.5 g/hour = 1 week to pure rac.

• Cost savings: client estimate $55K

silica

C18

• Case Study in purification strategy

Protection/Deprotection

NH2

OH

R1

R2

NH

OH

R1

R2

OCH3

CH3 CH3

O

Boc2O, NaHCO3

HCl

* *

Unprotected:

Inadequate

resolution, esp. on

loading

Chiral Chromatography of

Protected Compounds

FMOC: ChromegaChiral CCS,

Gradient, ACN: MeOH 3:1

BOC: ChiralPak AD-H,

Gradient, IPA

BOC-protected (Isocratic Analytical)

BOC-protected (Isocratic Preparative)

Est. Productivity 2.4 kkd

Selective Enantiomer Enrichment

• E1 production is more efficient than E2

enrichment

– Standard sample of active

– Pirkle columns, playing with selectivity, and CSP

changes can often reverse elution order

• Feedstock sources

– Synthetic: partial success of enantioselective process

– Chromatographic: overload and “polish”

Co-solvent Changes Selectivity

Co-solvent:

Blue = MeOH

Purple = 50:50 MeOH:iPrOH

Gold = iPrOH

Co-elution

E1

E2

E2

E1

AV-010163

(RegisPack)

Selective Enantiomer Enrichment

• E1 production is more efficient than E2

enrichment

– Standard sample of active

– Pirkle columns or phase changes can reverse elution

order

• Enriched feedstock sources

– Synthetic: partial success of enantioselective process

– Chromatographic: overload and “polish”

E1

E2

E1 E1 E1 E1

E2 E2 E2 E2

Racemic

compound

separation

F1 F2

“Valley”

Enriching E1 – Return on Effort

• Enriched

solutions of

warfarin (MeOH,

ChiralPak AD-H)

• Compare

feedstock

processing rate

to get to 95% ee

with production

rates of each

isomer

• Processing E1-enriched feedstock is 4.3x more efficient than

racemic, but enriched E2 is only 2.4x better

%E1 %E2 Processing

Rate (kkd)

Prod. Rate

E1 (kkd)

Prod. Rate

E2 (kkd)

10 90 1.20 0.13 1.07

25 75 0.93 0.23 0.70

50 50 0.85 0.42 0.43

75 25 1.16 0.86 0.30

90 10 2.00 1.80 0.20

Multi-step Separations

• Goal: separation of 10 mg of each target peak for full structure

elucidation

• Tools for separation effort: LCMS/UV(PDA), SFCMS, Prep SFC

50 min RP gradient:

impractical for

preparative HPLC

Fraction 11 – QC by HPLC

Typical “Fraction Tree”

F1

AV11083 Crude

F3 F4F2

F9 F10 F11 F5 F6 F8F7

F12 F13

F14 F15 F17F16

F19 F20 F21F18

F22 F23 F24 F25 F28F26 F29F27

F24-28

Method 1

Method 3

Method 4

Method 5

Method 6

Method 7 Method 7

F30 F31

Method 8

Method 7

F32F33

F34F35

Method 8

F39F36

F37F38F40

F41

Standard Fraction Manifold

UV

DetectorBPR

Collect valveF1

Waste

Fraction valve

F2

F3F4

Heat

Exchanger

MBPR and

manifold

Fraction bottles &

vent linesWaste

bottle

MBPR

waste

17 bar

Waters Prep-100

UV

DetectorBPR Heat

Exchanger

Tunable

Splitter

MS

Detector

Reverse Gradient

Solvent Pump

FC valve

Open bed

fraction

collector

GLS

MBPR

• Prep-100 GLS provides near-

perfect alignment of UV, MS, and

actual fraction traces

– Ebinger, Weller, Kiplinger, Lefebvre;

J. Amer. Assoc. Lab. Automation

2010

Fraction Collection Using GLS

F1

Waste

Fraction

valve

F2

F3

F4

UV

DetectorBPR

Heat

Exchanger

Collect valve

GLS

MBPR

• Reduced tubing length post-GLS

• MBPR set very low

• No need for reversed gradient pump

• Fraction valve is the fraction vessel

7 bar

Expanded Fraction Collection

Initial Cuts from Mother Liquor

F1

F4

Seven

initial

fractions

F1 Secondary Fractions

Six secondary

cuts

F4 Fractionation

AV15337 - UPLC

AV15337 HPLC

AV15337 - SFC

AV15337 Prep

AV15337 Prep Stack

AV15338 - UPLC

AV15338 HPLC

AV15338 - SFC

AV15338 Prep

AV15338 Prep Stack

Summary

• “Good enough” method development provides a speed

advantage that can compensate for sub-optimal

separation

• Work-arounds overcome sub-optimal method issues

• Drug Discovery working paradigm expects rapid

turnaround

• Problem-solving complex separations with good enough

approach, similarly, requires acceptance of sub-optimal

approach

Thanks

• Waters

• Abhijit Tarafder (Waters)

• Averica

– John Tipping

– Brittany Murphy

– Mickey Rego

– Keith Galyan

– Emily-Showell Rouse