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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