Purifying by preparative HPLCHow can we improve the recovery of our sample

Colinda van Tilburg

| Recovery of prep LC(MS)1 11-5-2017

Group Leader Analytical Chemistry

Oss, 11th May 2017

Content

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• Mercachem

• Analytical department

• Purification and Recovery

• Recovery problems

• Results

• Conclusion

Introduction to Mercachem

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An innovative chemistry service provider in the field of Discovery

Chemistry, Parallel Chemistry, Medicinal Chemistry, and Process

Research

Founded in 1997

160 employees (45% Ph.D. and 55% M.Sc./B.Sc.)

Headquarters in Nijmegen, NL

GMP facility in Prague, CZ

Modern research facilities

Independent, privately held

Customers and Markets

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Introduction to Mercachem

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Parallel Chemistry

Medicinal Chemistry

Process Research

Discovery Chemistry

Innovation in Chemistry- Development novel chemistry- Synthesis of novel compounds- Complex chemistry

Conservation of knowhow- Electronic Lab Notebook- Knowledge portal

Scaffold and library design- Challenging compounds- Cheminformatics support

High level of automation- Parallel synthesis- Parallel work-up & purification- Workflow „de-bottle-necking“

Early scale-up- Route scouting, Catalyst Screening- Tox-batches (non-GMP)

GMP-production- API for Phase 1/Phase 2 Clinical- Analytical method development- Stability studies, Impurity research

Target FamiliesKinases, Neuro-Kinases, HDACsHKMTs, HKDMs, PPIs

Jump-start projectsCDK-8, CK-1d, IL-17, HKMT, DOT1L

Clinical candidates delivered

Analytical department

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Open access Equipment:

• NMR (2)

• LC-MS (7)

• LC (6) - reversed phase and chiral

• GC(MS) (4)

• UPC2

• Prep LC

• Reveleris (NP and RP) (13)

• Prep Reveleris (2)

Other analytical tools:

2-Dimensional LC, FTIR, UV, Optical Rotation,

Melting Point, DSC

Analytical assays:

Solubility, Lipophilicity, Stability, Organic Volatile

Impurities (OVI), Permeability

Preparative LC-MS:

5 prepLCMS and 1 prepLC, 1 prep100 (SFC)

Purification

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Revelaris

Prep LC(MS)AcidicBasicSFC

Reveleris NPReveleris RPReveleris Prep – acidic

– basic

WatersAgilent

RecoveryRecovery of ~750 single samples on prep LCMS (over the last 2 year)

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1 project

n=754

Purification with prepLC(MS)

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Collection

Waste

Possible loss of product

Chromatography1. Good shape of the column2. Optimization to focus gradient

Detector and fraction collector1. Delay time2. Threshold3. Fraction switching

Injector1. Injection valve2. Sample - SW/ACD/Normal

Recovery

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Separation

1. Good shape of the column - QC standard

2. Optimization to focus gradient

PDA

TIC

PDA

PDA

Recovery

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1. Delay time

Detector and Fraction collector

2. Threshold

3. Fraction switchingLost(%): fraction switchingPrep Revelaris 0 %LCMS -Waters 1-3 %

PDA

TIC

PDA

Recovery

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Injector

2. Sample - SW/ACD/Normal

1. Injection valve

Sandwich ACD: at-column dilution Normal

Flow through needle Partial loop

Solvent - Air - Sample - Air - Solvent Sample

Recovery analysis

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Method

Injection : 50 mg/ml sample; injection volume 1000µlChromatography: Column: Waters XSelect CSH (C18, 150x19mm, 10µm); flow: 24 ml/min Eluent A: 99% ACN + 1% 10mM ammonium bicarbonateEluent B: 10mM ammonium bicarbonate in water pH=9.5Collection: Mass/UV basedPooling: Reveleris: pooling in round bottom flask, freeze drying

LCMS: calibrated tubes, genevac drying

Weigh-methodCollection

Waste

Gradient:

Recovery

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HASP – reaction mixture

1 2 3 4 5 6 7 8 9 10 11 12 13 14Retention time (min)

0.0E+00

5.0E+07

1.0E+08

1.5E+08

1 2 3 4 5 6 7 8 9 10 11 12 13 14Retention time (min)

0.0E+00

5.0E+07

1.0E+08

1.5E+08

2.0E+08

2.5E+08

Pentoxyfiline – pure compound

Sample

Recovery

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Results

injection Pentoxyfiline HASPPrep Reveleris Normal 96 xLCMS -Agilent SW 97-98 75LCMS - Waters SW 89-92 63LCMS - Waters ACD 86-90 62LCMS - Waters ACD+SW 90-92 x

13%!

13%!

Recovery

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Overview

Chromatography parameters- Separation should be appropriate- Choose right sample method- Threshold right

Apparatus parameters- Delay time right ~ 1%- Choose right fraction volume, reduce your fractions ~1-3% - Choose (if possible) a FTN injector or mix sample method

(ACD+SW) ~2-4%

4-8% profit

RecoveryRecovery of ~750 single samples on prep LCMS (over the last 2 year)

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n=754

Recovery errors

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Probably main fault: injector of the Waters Prep

Nee

dle

Syringe

Sam

ple

Loop

Injector

Injector

Loop

Injector

Loop

Conclusion

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• Chromatography and solubility are the main reason to choose for either of the preparative LCMS systems

• Reveleris and LCMS (Agilent) gave high recovery of ~97%

• LCMS (Waters) gave recovery between 86-92% depending on injection method

• The injector is probably the main reason why the recovery is low, if the solubility and chromatography are good.

Mercachem team

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Analytical team: Bram van Oss, Patrick Strijbos, Jan Sadownik, Eef van de Elzen, Vivian Moorman and Colinda van Tilburg

Questions

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PurityPurity of ~750 single samples

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At-Column DilutionConventional separation ACD separation

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