supercritical fluid chromatography achiral applications ... · pdf filesupercritical fluid...

Supercritical Fluid ChromatographyAchiral Applications and Techniques

Frank Riley on behalf of the Pfizer, Groton SFC UsersPfizer Global Research and Development

Groton, CT 06340

M. T. Combs, M. Ashraf-Khorassani, L. T. TaylorDepartment 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

• 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

HPLCMethod Dev’t

initiativesGC

Expert Team

SFCTeam

PARC

SFC platform development

SFC Achiral screening/evaluation

SFC ChiralScreening/evaluation

2D LC-SFCPlatform development

Polytides

Green Flash

Reaction monitoring

Parallel screening

Pfizer’s SFC Technology Development Initiatives 2010

Validation:Method, equipment

Technologycollaborations

Structure Elucidation – Paying The Bills

10.6

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POI-1Rt: 10.6 min

POI-2Rt: 11.2 min

• 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 increasedimpurity levels, 0.5% and 1.2% area respectively

• Validated method employs HCLO4 modifier – No MS clues

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

Thar Investigator Princeton Diol 250x10mm, 5umLinear gradient 5-50% modifier (MeOH )Flow Rate: 12.0mL/minTemp: 40CBP:120 bar

F-1 F-2

F-3 F-4

Scouting Fractions

Structure Elucidation

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Project Lab

SFCIsolated

Impurities

Spiked Impuritiesfor verification

F-3F-4

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

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1 2 3M

a in

Ba n

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67

• MBE: Extract major component enrich impurities LC-NMR

• LC-NMR is very difficult with < 1% level impurities • Minimal time investment

Slide courtesy of T. Zelesky

CRD LC Method

SFC - MBE

peak %Area peak %Area1 0.05 1.5 4 0.7 18.3

API 97.1 2.4 5 0.9 29.12 0.1 11.0 6 0.04 1.13 0.3 10.5 7 0.9 25.7

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1

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Mai

n B

and

Significantly increase loadability (40X) of impuritiesSlide courtesy of T. Zelesky

CRD LC Method

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 MonitoringWork-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 plateto precipitate proteins

Centrifuge the plate to remove precipitated proteins and biomass

Supernatant (1ml)transferred to another96-well plate

SFC analysis

Biocatalysis – Reaction MonitoringSM, AD-H, ACN

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Starting Material

Product

S-alcohol R-alcohol

Enzymatic Reduction

Thar Method Station I – MassLynx 4.1Chiralcel AD 250x4.6mm, 5umIsocratic: 85:15 CO2:CH3CNFlow Rate: 4.0mL/minTemp: 40CBP:120 barWaters 2996 DADWaters ZQ – peak confirmation Analysis Time: 3-min vs. 15-min LC

O

S

HO

S

Thiolan

Impurity A isolation via SFC

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product/Impurity A sample via SFC Isolation Method

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Biocatalysis Impurity Isolation [O]

reactionbug rxn +

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Reactant product oxidized productsubstrate


Biocatalysis Impurity Isolation

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• 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.!

2-ethylpyridine, 1 cm x 25 cm 10 mL/min85/15, CO2/MeOHBP: 140 bar


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

OOH

OH

OHHO

O

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

OOH

OH

OHO

O

HOOH

OH

OHHO

O

O

O

OH

HO

OH

OH

Gluconolactone

Furanose

Gluconic Acid

Aldehyde

Carbohydrate - Gluconolactone

OOH

OH

OHHO

O

OO

O

OO

O

Si

Si

Si

Si

Globally protect hydroxyl groups

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

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

• Globally protect the hydroxyl groups leaving the O-glycosidic site available for reaction.

• Single method to detect conversion – speed.Glycosidic

Reaction site

Carbohydrate - Gluconolactone2.

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Si

Thar Method Station II – Empower SoftwarePhenomenex Diol 250x4.6mm, 5umLinear gradient 5-50% modifier (MeOH)Flow Rate: 4.0mL/minTemp: 40CBP:120 barWaters 2420 ELS detection (passive split)Waters ZQ – peak confirmation

Monitor Reaction

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 exchangeMolecular 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 exchangeMolecular Mass = 1166.4 Da

SFC of Protected Peptide Pairs That Differ in Amino Acid Sequence

Thar Method Station I – MassLynx 4.1Princeton 2-EP 250x4.6mm, 5umLinear gradient 5-50% modifier (MeOH w/ 0.2% TFA)Flow Rate: 2.0mL/minTemp: 40CBP:100 barWaters LCT (TOF)

MeOH w/0.2% TFA:2-EP

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1166.41.43e3

9.149.43

Polytide Mix_1 1: TOF MS ES+ 1214.51.10e3

9.539.41

Polytides – Un-Protected

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 exchangeMolecular 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 exchangeMolecular Mass = 1125.4 Da

Separation of Linear and Un-Protected Dodecapeptides with Identical Molecular Mass

Exchange Single Amino Acid Sequence

Elution of a Single Un-Protected Peptide

GFLGLALGGLKK

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Thar Method Station II – Empower SoftwarePrinceton 2-EP 250x4.6mm, 5umLinear gradient 5-50% modifierFlow Rate: 2.0mL/minTemp: 40CBP:100 barWaters ZQ GFLGLALGGLKK

Work continues toward baseline resolutionof the Un-Protected series with

Aqueous modifier.

Single Peptide

Single Peptide

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 ApplicationContinued 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.

Thar Method Station – MassLynx Software Princeton 2-EP 250x4.6mm, 5umGradient: Initial: 80:20; 6-min: 50:50; 8-min:50:50; 8.5-min: 80:20; 11-min 80:20Flow Rate: 3.0mL/minTemp: 40CBP:200 barWaters 2998 PDA

ThymineUracilAdenineCytosine

ThymineUracilAdenineCytosine

• 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

Implementation of SFC as an Analytical Tool in a GMP Regulated Environment:

Method Validation: Criteria•Regulatory Bodies – FDA and ICH

• Validation of Analytical Procedures: Text and Methodology Q2(R1)• Specificity• Linearity• Range• Accuracy• Precision

• Repeatability• Intermediate Precision

• Limit of Detection• Limit of Quantitation

Show Stoppers

Green Chemistry 2009L. Kalmbach

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 210-

350nm, 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% - LOQ0.2ug/ml=0.02% - LODTest Mix:FlavoneCarbamazepineAmcinonideKetoprofen

Thar Analytical Method StationEmpower Software21CFR11 compliant

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1.0 mg/mL100% nominal

254nm

LOQ0.5ug/mL

0.05% nominal

LOD0.2ug/mL

0.02% nominal

s/n: Flavone: 123:1Ketoprofen: 41:1

Target s/n >30:1

s/n: Flavone: 59:1Ketoprofen: 18:1

Target s/n >10:1

Flavone

Ketoprofen

Wavelength Compensated

• 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 as an Analytical Tool in a GMP Regulated Environment:

• Complimentary Flash Technique• Rxn mixture clean-up, purification• Eliminate chlorinated/hazardous

solvents• Reduced waste stream• Inexpensive, replaceable cartridges

SFC “Green” Flash Application

“LC-Flash”“SFC-Flash”

SS-Tube, 30mmID

ModifiedBiotage 25MPacked cartridge

SS Dispersion FritFits inside of cartridge

Tapered CapSits inside of cartridge bevel

Column Components

Column Components

SFC “Green” Flash Application

Column Comparison/Test Conditions:

GF-Column SepaxLength: 25x210mm 21.2x250mmParticle size, Si: 40-63um 40-60umColumn Vol: 86 mL 74 mLFlow Rate: 69 mL/min 50 mL/minBP: 120bar 120barTemp: 40C 40CInj. Volume: 585uL 500uLMP Comp: 90:10 CO2:MeOHSample: 1,3-Dinitrobenzene

Cost: $1500 (Reusable Hardware) $1350Replaceable Bed: $23 NA ($1350)

9876543210

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Initial Column Comparison

Rt: 2.54minArea: 341.2uVHeight: 1194.3uVAs: 1.10


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Initial Column Reproducibility

No indication of cartridge side-wall failure



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

• 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 $$ << $$ Acetonitrile/other organics

• In addition - scalable, reproducible, can use smaller particle size, higher efficiencies, etc.

Bottom line $$ : There are significant long term operational cost savings using a technology that performs strongly in

Chiral/Achiral analytical and purification applications.


General SFC Conclusions

• SFC has demonstrated superiority as an analytical/purification separations tool

• Chromatographic gains are realized as a result of the physical properties of supercritical CO2 !

- Low viscosity and high solute diffusivity result in faster analysis times and higher throughput w/out loss in efficiency

- Using supercritical CO2 is cost effective, safe, and inert!

• Recent advances in instrument robustness and UV detector sensitivity have made SFC a viable drug development chromatographic technique

• Innovation is key to the future of pSFC!

Acknowledgements

Pfizer - SSAT SFCTeam:Todd ZeleskyLynne KalmbachManisha PatelDuc VuongYun Huang

Collaboration/Guidance/Support Team:Todd ZeleskyJian WangManisha PatelLynne KalmbachBrian Marquez Steve BrownCarrie WagerMark DeludeJim BradowLaurence PhilippeYun HuangAnne AkinThar Technologies Prof. Larry TaylorProf. Tom ChesterDr. Terry BergerMany More……

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