 Supercritical Fluid Chromatography Achiral Applications and Techniques Frank Riley on behalf of the Pfizer, Groton SFC Users Pfizer Global Research and Development Groton, CT 06340 M. T. Combs, M. Ashraf-Khorassani, L. T. Taylor Department of Chemistry, Virginia Tech, Blacksburg, VA 24061



Outline

• Introduction • Impurity Isolations – Structure Elucidation • Biocatalysis Reaction Monitoring • Carbohydrate Application – Simple Sugars • Peptide Separations – Protected and Unprotected • HydroOrganic Modified – Water Additive • Method Validation

 Why are we interested in supercritical fluids for chromatography? • • • • • • •

Fast Chromatography Rapid Method Development Scaleable Detector Friendly Unified Chromatography Cost effective Green Chemistry



Pfizer’s SFC Technology Development Initiatives 2010

• Align with Analytical R&D technology focus areas • Establish SFC platforms for routine chiral and achiral analytical testing in support of project progression • Capitalize on SFC’s enhanced speed, resolution and effectiveness • Collaborate with Internal and External resources on platform development/delivery for analytical and preparative applications



Pfizer’s SFC Technology Development Initiatives 2010 SSAT Pfizer Groton SFC Team

HPLC Method Dev’t initiatives

GC Expert Team

PARC

SFC platform development

2D LC-SFC Platform development SFC Chiral Screening/evaluation

Green Flash Reaction monitoring Technology collaborations

SFC Achiral screening/evaluation

Polytides

Validation: Method, equipment

Parallel screening

 Structure Elucidation – Paying The Bills • Structure Elucidation for impurities exceeding 0.2% area threshold for use in clinical application/ regulatory filing. • Project lab detects impurities, POI-1 at 0.25% area and POI-2 at 0.5% area during scale-up synthesis, previously un-detected in previous campaigns. • Attempts to degrade material, heat/solution, result in increased impurity levels, 0.5% and 1.2% area respectively • Validated method employs HCLO4 modifier – No MS clues 10.878

2.20 2.00 1.80

POI-1 Rt: 10.6 min

1.60 1.40

POI-2 Rt: 11.2 min

1.00 0.80

0.40 0.20

11.264 11.395

0.60

10.657

AU

1.20

0.00 1.00

2.00

3.00

4.00

5.00

6.00

7.00 8.00 Minutes

9.00

10.00

11.00

12.00

13.00

14.00

15.00

 • • • •

Structure Elucidation Step-1: Discard project lab method, Screen via. SFC. Step-2: Time based fractionation across gradient elution. Step-3: Re-inject fractions, validated method, targeting POI’s Step-4: Refine fractions based on Step-3, scale as appropriate

Scouting Fractions

F-1

F-2 F-3

F-4

Thar Investigator Princeton Diol 250x10mm, 5um Linear gradient 5-50% modifier (MeOH ) Flow Rate: 12.0mL/min Temp: 40C BP:120 bar



Structure Elucidation 10.878

2.20 2.00 1.80

POI-1 Rt: 10.6 min

1.60 1.40

AU

1.20

POI-2 Rt: 11.2 min

Project Lab

1.00 0.80

10.657

0.40

11.264 11.395

0.60

0.20 0.00 2.00

3.00

4.00

5.00

6.00

7.00 8.00 Minutes

9.00

10.00

11.00

0.50

F-4

13.00

14.00

15.00

0.12

10.671

0.40

F-3

0.10

0.35

0.08 AU

0.30 0.25 0.20

0.06 0.04

0.15

0.02 10.947

0.10 0.05

0.00

0.00

2.00 4.00

5.00

6.00

7.00 8.00 Minutes

9.00

10.00

11.00

12.00

13.00

14.00

4.00

6.00

15.00

8.00 Minutes

10.00

12.00

14.00

1.60

10.880

3.00

1.40 1.20

11.268

2.00

1.00

Spiked Impurities for verification

0.80 0.60 0.40

10.659

1.00

AU

AU

SFC Isolated Impurities

0.45

12.00

11.294

1.00

0.20 0.00 1.00

2.00

3.00

4.00

5.00

6.00

7.00 8.00 Minutes

9.00

10.00

11.00

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13.00

14.00

15.00



Structure Elucidation

• Project Time: • SFC Method Screen: 4-columns, 1-modifier, 36-mins • Scouting Fractions: Isolation (3-injs), re-analysis (4-fractions), 120-mins • Scale-up: 10-injections (focused isolates), fraction dry down, re-analysis 330-min • Spike fractions for verification, 15-min • Data Analysis: 30-min • Deliver Isolates, 0.4mg and 0.6mg respectively for MS and NMR • Total Project Time, Isolation perspective: 531-min (~9-hrs)



Structure Elucidation – Main Band Elimination

0.020

0.000 0.00

5.00

10.00

77

5 5

15.00

66

22 33

0.010

11

AU

0.030

CRD LC Method

4 4

0.040

Main Band

CP-945,598

• MBE: Extract major component  enrich impurities  LCNMR • LC-NMR is very difficult with < 1% level impurities • Minimal time investment

20.00

25.00

30.00

35.00

40.00

45.00

50.00

55.00

Minutes

Slide courtesy of T. Zelesky



SFC - MBE

0.14

5

7

CRD LC Method

0.04 0.02

4

6

0.06

1

AU

0.08

2 3

0.10

Main Band

0.12

0.00 0.00

5.00

10.00

peak

15.00

20.00

%Area

25.00 30.00 Minutes

peak

35.00

40.00

45.00

50.00

55.00

%Area

1

0.05

1.5

4

0.7

18.3

API

97.1

2.4

5

0.9

29.1

2

0.1

11.0

6

0.04

1.1

3 0.3 10.5 7 0.9 25.7 Significantly increase loadability (40X) of impurities Slide courtesy of T. Zelesky

Biocatalysis Application • Biocatalysis is the use of natural catalyst, such as protein enzymes, to perform chemical transformations on organic compounds. • Purpose of the enzyme is to selectively act on a single type of functional group. • Enzymes are chiral catalysts in which the substrate may be transformed into an optically active product. • Reaction proceeds under mild conditions, minimize problems of undesired side-reactions such as decomposition, isomerization, racemization and rearrangement. • Environmentally acceptable, being completely degraded in the environment.

Biocatalysis – Reaction Monitoring Work-Up Reaction mixture (containing substrate, whole cells, NADPH (reducing agent) and Isopropanol in phosphate buffer (pH 7.0, 100 mM) 100 ul Added to1900 ul of acetonitrile in 96-well plate to precipitate proteins Centrifuge the plate to remove precipitated proteins and biomass Supernatant (1ml)transferred to another 96-well plate SFC analysis

Biocatalysis – Reaction Monitoring SM, AD-H, ACN n-rileyf-MD_Cscreen_SM_CP81171_5

2: Diode Array Range: 2.662

1.52

Starting Material

2.2

2.0

O

1.8

1.6

Enzymatic Reduction

1.4

AU

1.2

1.0

S

8.0e-1

Thiolan

1.05

6.0e-1

4.0e-1

2.0e-1

0.0

0.27

-2.0e-1 0.50

1.00

1.50

2.00

2.50

3.00

3.50

4.00

4.50

5.00

5.50

6.00

6.50

7.00

7.50

8.00

8.50

Time 9.00

Lot 64504-10-1_racemate, AD_H, ACN 2.98

Product

1.4

2: Diode Array Range: 1.51

HO

1.3 1.2

S-alcohol

1.1

R-alcohol

1.0 1.02

9.0e-1

AU

Thar Method Station I – MassLynx 4.1 Chiralcel AD 250x4.6mm, 5um Isocratic: 85:15 CO2:CH3CN Flow Rate: 4.0mL/min Temp: 40C BP:120 bar Waters 2996 DAD Waters ZQ – peak confirmation Analysis Time: 3-min vs. 15-min LC

n-rileyf-MD_Cscreen_SM_CP81171_4

8.0e-1

S

7.0e-1 6.0e-1 5.0e-1 4.0e-1 2.62

3.0e-1 0.77

2.0e-1 1.0e-1

0.50

1.00

1.50

2.00

2.50

3.00

3.50

4.00

4.50

5.00

5.50

6.00

6.50

7.00

7.50

8.00

8.50

Time 9.00



Biocatalysis Impurity Isolation substrate [O] Impurity A isolation via SFC Reactant product oxidized product bug rxn

reaction

+

+

Impurity A

Impurity B

product/Impurity A sample via SFC Isolation Method

product/Impurity A sample via CRD LC Method 300

1.0

250

product

Imp. A

AU

0.5

Imp. A

200 150

product

100

0.0

50

-0.5

0

5.0

10.0

15.0

20.0

0

2

4

6

8

Minutes

Slide courtesy of T. Zelesky



Biocatalysis Impurity Isolation

600 550 500 450 400 350 300 250 200 150 100 50 0

5

10

15

20

25

• 1 hr. to dev. SFC method • Stacked injs. over 47 minutes • Fraction dry down = 1 hr. • 8 mg  NMR • Solvent Cost: < $2 • CRD sample  NMR sample = 3 hrs.!

30

35

40

45

2-ethylpyridine, 1 cm x 25 cm 10 mL/min 85/15, CO2/MeOH BP: 140 bar

Slide courtesy of T. Zelesky



Carbohydrate Application

• Carbohydrates are a very important class of naturally occurring chemicals that metabolize to water, carbon dioxide and heat/energy. • An organic compound with general formula Cm(H2O)n, that is, consisting only of carbon, hydrogen and oxygen, the last two in the 2:1 atom ratio.

• Evaluate the feasibility of using SFC for the separation.

Carbohydrate - Gluconolactone Gluconolactone is composed of multiple water-attracting hydroxyl groups, upon addition to water readily forms an equilibrium mixture of the lactone, aldehyde, gluconic acid O and furanose OH O

Aldehyde

O OH

O

O

OH OH

OH

O

HO

Gluconic Acid

HO

HO

OH

OH

OH

OH

OH

O

Gluconolactone

O

Furanose

OH HO OH

Carbohydrate - Gluconolactone • Globally protect the hydroxyl groups leaving the O-glycosidic site available for reaction. • Single method to detect conversion – speed. Glycosidic Reaction site

O O O OH O

Si

HO OH

Si

O O O

Globally protect hydroxyl groups

Si

O OH

Si

MW: 178 Log P: -2.38 (ACD) Very Polar

MW: 466 Log P: 4.18 (ACD) Very Non-polar

Carbohydrate - Gluconolactone 0.150

5.400

O

0.140 0.130

OH O

0.120 0.110 0.100

O

Volts

OH

Monitor Reaction

0.090 0.080

OH

0.070 0.060

7.378

0.030

5.647

0.040

5.107

2.667

0.050

0.020 0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

8.00

9.00

Minutes

1.00

O

1.817

0.80

O

Si

O

Si

O

0.60

O

Si

O

0.40 Si

2.129 2.346 2.731

Volts

Thar Method Station II – Empower Software Phenomenex Diol 250x4.6mm, 5um Linear gradient 5-50% modifier (MeOH) Flow Rate: 4.0mL/min Temp: 40C BP:120 bar Waters 2420 ELS detection (passive split) Waters ZQ – peak confirmation

0.20

0.00 0.00

1.00

2.00

3.00

4.00 5.00 Minutes

6.00

7.00

8.00

9.00



Polytide Application

• Historically RP/HPLC-MS is the most popular technique for the analysis of peptides when purification is necessary. • Complex peptide mixtures result in long analysis times or 2D-LC modes of operation. • Evaluate the feasibility of using SFC for the separation of Polytides. – Identify model peptide compounds for initial study – Screen multiple columns and modifiers – Understand separation mechanism

• Research initiative: Prof. Larry Taylor (VT).



Polytides - Protected Separation of Linear and End Capped Dodecapeptides with Identical Molecular Mass Exchange Single Amino Acid Sequence Ac-Gly-Phe-Leu-Gly-Leu-Ala-Leu-Gly-Gly-Leu-Lys-Lys-NH2 Ac-Gly-Gly-Leu-Gly-Leu-Ala-Leu-Gly-Phe-Leu-Lys-Lys-NH2 phenylalanine and glycine exchange Molecular Mass = 1214.5 Da ____________________________________________ Ac-Gly-Val-Leu-Gly-Leu-Ala-Leu-Gly-Gly-Leu-Lys-Lys-NH2 Ac-Gly-Gly-Leu-Gly-Leu-Ala-Leu-GlyVal-Leu-Lys-Lys-NH2 valine and glycine exchange Molecular Mass = 1166.4 Da

SFC of Protected Peptide Pairs That Differ in Amino Acid Sequence MeOH w/0.2% TFA:2-EP Polytide Mix_2

1: TOF MS ES+ 1166.4 1.43e3

9.14

100 %

9.43

0 2.00 Polytide Mix_1

4.00

6.00

8.00

10.00

12.00

14.00

9.419.53

%

100

16.00 1: TOF MS ES+ 1214.5 1.10e3

0

Time 2.00

4.00

6.00

8.00

10.00

12.00

14.00

Thar Method Station I – MassLynx 4.1 Princeton 2-EP 250x4.6mm, 5um Linear gradient 5-50% modifier (MeOH w/ 0.2% TFA) Flow Rate: 2.0mL/min Temp: 40C BP:100 bar Waters LCT (TOF)

16.00



Polytides – Un-Protected Separation of Linear and Un-Protected Dodecapeptides with Identical Molecular Mass Exchange Single Amino Acid Sequence Gly-Phe-Leu-Gly-Leu-Ala-Leu-Gly-Gly-Leu-Lys-Lys Gly-Gly-Leu-Gly-Leu-Ala-Leu-Gly-Phe-Leu-Lys-Lys phenylalanine and glycine exchange Molecular Mass = 1173.5 Da ____________________________________________ Gly-Val-Leu-Gly-Leu-Ala-Leu-Gly-Gly-Leu-Lys-Lys Gly-Gly-Leu-Gly-Leu-Ala-Leu-Gly-Val-Leu-Lys-Lys valine and glycine exchange Molecular Mass = 1125.4 Da

 Elution of a Single Un-Protected Peptide MeOH w/ 0.2% TFA

Work continues toward baseline resolution of the Un-Protected series with Aqueous modifier.

9.292

Single Peptide

9.905

320000

10.334

160000

11.418 11.674 11.996 12.469

10.744

Intensity

240000

90:10MeOH:Water w/ 0.2% TFA 1.00x106

0 0.00

1.50

3.00

4.50

6.00

7.50

9.00

10.50

12.00

13.50

Extracted: 1174.4

15.00

Single Peptide

6.852

80000

Minutes

GFLGLALGGLKK

Intensity

Thar Method Station II – Empower Software Princeton 2-EP 250x4.6mm, 5um Linear gradient 5-50% modifier Flow Rate: 2.0mL/min Temp: 40C BP:100 bar Waters ZQ

7.50x105

5.00x105

2.50x105

0.00 0.00

1.50

3.00

4.50

6.00

7.50

9.00

10.50

Minutes

GFLGLALGGLKK

12.00

13.50

15.00

 Peptide Application • Linear 12-mer peptides that differ only in amino acid sequence can be baseline separated. • Stationary phases containing nitrogenous bases were most successful, i.e. 2-Ethyl pyridine and Amino. • TFA appears to be the mobile phase additive of choice (suppress deprotonation of peptide carboxylic acid group, protonate the amino group)

• Water Addition: • Enhance solubility of hydrophilic compounds • Increase solvation of the stationary phase • Modify surface tension between the phases • Alter surface chemistry of the packed phase.



Peptide Application Continued Exploration

• Effects of/Impact of Separation: • Gradient Steepness • Temperature • Stationary/Mobile Phases • Additive Concentration • pH • Column Coupling – same/mixed phases • Continued Collaboration with Prof. Taylor



Separation of Nucleobases Facilitated with Water Additive

• During the past decade, the greatest success for improving SFC chromatographic peak shapes of polar solutes has been achieved using polar modifiers and even more polar additives with standard silica-based polar stationary phases. • Initial study concerned the chromatographic behavior of four water soluble nucleobases (thymine, uracil, adenine, and cytosine) utilizing polar stationary phases. • Incorporation of a fixed amount of water additive into the alcohol modifier yielded markedly improved chromatographic performance. • The high polarity of water and its ability to function as a hydrogen bond acceptor and hydrogen bond donor enhance its role as a neutral additive.

Thymine Uracil Adenine Cytosine

Thar Method Station – MassLynx Software Princeton 2-EP 250x4.6mm, 5um Gradient: Initial: 80:20; 6-min: 50:50; 8-min:50:50; 8.5-min: 80:20; 11-min 80:20 Flow Rate: 3.0mL/min Temp: 40C BP:200 bar Waters 2998 PDA

Thymine Uracil Adenine Cytosine

Thar Method Station – MassLynx Software Princeton 2-EP 250x4.6mm, 5um Gradient: Initial: 80:20; 6-min: 50:50; 8-min:50:50; 8.5-min: 80:20; 11-min 80:20 Flow Rate: 3.0mL/min Temp: 40C BP:200 bar Waters 2998 PDA

Thymine Uracil Adenine Cytosine



Implementation of SFC as an Analytical Tool in a GMP Regulated Environment: • Task: Evaluate platform feasibility for method validation, regulatory compliance and down-stream method transfer – currently targeting early development validation guideline • Challenge: – Take a single method developed in Discovery – Robust enough to pass through: • • • • • •

• • • •

Co-discovery Research Analytical Development Analytical Supply Chain Manufacturing QC release lab Meet regulatory scrutiny

Continuous Process Improvement Instrument Validation – Thar analytical – Completed Method Validation - Completed Stop reinventing the wheel at each stage of development



Method Validation: Criteria

•Regulatory Bodies – FDA and ICH • Validation of Analytical Procedures: Text and Methodology Q2(R1) • Specificity • Linearity • Range Green Chemistry 2009 • Accuracy L. Kalmbach • Precision • Repeatability • Intermediate Precision • Limit of Detection Show Stoppers • Limit of Quantitation



LOQ/LOD Acceptance Criteria

• LOQ • Determined using low level linearity values and should be at least 0.05% of nominal concentration • RSD ≤ 10% for 6 injections at 0.05% • S/N ≥ 30

• LOD • Determined using low level linearity values and should be at least 0.02% of nominal concentration • RSD ≤ 30% for 6 injections at 0.02% • S/N ≥ 10



Method Validation – LOQ/LOD Challenge

• Method Parameters – – – – – – – – – –

4ml/min, 20%MeOH 120 Bar 40 C 2-EP Column 250x4.6mm, 5u PDA detector (2998): Scan 210350nm, Extracted: 254nm Reference: 360-400nm Resolution: 1.2nm Sample Rate: 10 points/sec Filter Time: Normal Injection = 10uL

• Prepared Samples: 1.0mg/ml=100% 0.5ug/ml=0.05% - LOQ 0.2ug/ml=0.02% - LOD Test Mix: Flavone Carbamazepine Amcinonide Ketoprofen

Thar Analytical Method Station Empower Software 21CFR11 compliant

2.500

1.00

3.489

Wavelength Compensated 0.90

1.0 mg/mL 100% nominal 254nm

0.80

0.50

5.208

Volts

0.60

7.582

0.70

Flavone

0.40

Ketoprofen

0.30 0.20 0.10 0.00 6.00

0.005

0.003

5.019

0.002

0.001

0.000

0.000 0.00

LOD 0.2ug/mL 0.02% nominal 7.409

3.299

0.002

14.00

0.004

7.378

0.006

12.00

5.052

AU

0.008

10.00

3.319

0.010

8.00 Minutes

AU

0.012

0.004

4.00

LOQ 0.5ug/mL 0.05% nominal

2.331

0.014

2.00

2.347

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00 8.00 Minutes

9.00

10.00

s/n: Flavone: 123:1 Ketoprofen: 41:1 Target s/n >30:1

11.00

12.00

13.00

14.00

15.00

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00 8.00 Minutes

9.00

10.00

11.00

s/n: Flavone: 59:1 Ketoprofen: 18:1 Target s/n >10:1

12.00

13.00

14.00

15.00



SFC as an Analytical Tool in a GMP Regulated Environment:

• Detection limits and system reproducibility that once impeded SFC from entering the mainstream are achievable. • We have the capability to qualify instrumentation and validate analytical methods utilizing SFC. • SFC is the next building block in our “method development toolbox” at Pfizer. • SFC is perfect for chiral applications. We are currently pushing the technology for achiral and polytide applications.

SFC “Green” Flash Application • Complimentary Flash Technique • Rxn mixture clean-up, purification • Eliminate chlorinated/hazardous solvents • Reduced waste stream • Inexpensive, replaceable cartridges “LC-Flash”

“SFC-Flash”

Column Components SS-Tube, 30mmID

Modified Biotage 25M Packed cartridge

SS Dispersion Frit Fits inside of cartridge

Tapered Cap Sits inside of cartridge bevel

Column Components

SFC “Green” Flash Application Column Comparison/Test Conditions: Length: Particle size, Si: Column Vol: Flow Rate: BP: Temp: Inj. Volume: MP Comp: Sample:

GF-Column 25x210mm 40-63um 86 mL 69 mL/min 120bar 40C 585uL 90:10 CO2:MeOH 1,3-Dinitrobenzene

Sepax 21.2x250mm 40-60um 74 mL 50 mL/min 120bar 40C 500uL

Cost: Replaceable Bed:

$1500 (Reusable Hardware) $1350 $23 NA ($1350)

Initial Column Comparison 1,250

1,150

1,200

1,100

1,150

1,050

GF-Column Inj-1

1,100 1,050 1,000 950 900 850

950 900 850 800 750

800

700

750 700

650

650

600

600

550

550

500

400 350 300 250

SPW 0.20

100 50

STH 10.00

200

2.54min 341.2uV 1194.3uV 1.10

400 350 300 250 200 150 100

50

-50 0

-50 3

4

5

6

7

8

9

1

2

1,150 1,100 1,050

Overlay

1,000 950 900 850 800

Sepax

GF

750 700 650 600 550 500 450 400 350 300 250 200 150 100

50

STH 10.00

2

SPW 0.20

1

2.80min 378.2uV 1095.5uV 1.78

0

0

0

Rt: Area: Height: As:

450

STH 10.00

450

SPW 0.20

Rt: Area: Height: As:

500

150

Sepax Column Inj-1

1,000

0 -50 0

1

2

3

4

5

6

7

8

9

3

4

5

6

7

8

9

Initial Column Reproducibility 1,250 1,250

1,200

1,200

1,150

1,150

GF-Column Inj-1

1,100 1,050 1,000 950 900

GF-Column Inj-50

1,100 1,050 1,000 950 900 850

850

800

800

750 750

700

700

650

650

600

600

450 400 350 300 250

SPW 0.20

150 100

50

STH 10.00

200

2.54min 341.2uV 1194.3uV 1.10

500 450 400 350 300 250 200 150 100 50

0

0

-50

-50 2

3

4

5

6

7

8

9

0

1

2

3

1,250 1,200

Overlay Inj-1 and Inj-50

1,150 1,100 1,050 1,000 950 900 850 800 750 700 650 600 550 500 450 400

No indication of cartridge side-wall failure

350 300 250 200 150 100

50

STH 10.00

1

SPW 0.20

0

Rt: Area: Height: As:

550

STH 10.00

500

SPW 0.20

Rt: Area: Height: As:

550

0 -50 0

1

2

3

4

5

6

7

8

9

4

5

6

2.56min 339.7uV 1194.3uV 1.05 7

8

9

Green Flash – Next Steps • TLC to SFC correlation…….is it possible. • Additional phases applicable to SFC; 2-EP, Diol, Amino…other. • Can a vendor build a comparable, high pressure flash platform to compete in current process….cost. • User friendly, touch screen programming, look and feel of current platforms…. • Easy-load cartridge holder, high pressure rating… • End-User not concerned with “what” solvents are utilized, concerned with reliability, robustness, application….each and every time. • Potential: currently ~1-flash system/3-chemists within current Discovery organization…..



SFC conclusions Bottom line $$ : There are significant long term operational cost savings using a technology that performs strongly in Chiral/Achiral analytical and purification applications.

• Significant increase in production rate and throughput/instrument - Collection time is faster / stacked injs. - Evaporation time is faster / saves energy - cost savings in labor

• Significant solvent cost savings! - Less solvent consumed and less disposal - CO2 is inexpensive to purchase (~ 10 cents/L) - CO2 zero cost to dispose of - Alcohol modifiers $$