C illCapillary El t h iEl ectrophoresis: Renaissance and ... · X O th l ti i bi ti ith HPLCOrthogonal separation in combination with HPLC X More seamless integration with mass spectrometry

C ill El t h iCapillary Electrophoresis: Renaissance and New Applications

Gerard P. Rozingil h l iAgilent Technologies,

76337 Waldbronn, Germany.

©Agilent Technologies7/6/2009

1Presentation at HPLC'2009

Brief Retrospective of Capillary Electrophoresis

©Agilent Technologies 7/6/2009

Presentation at HPLC'2009 2

HPLC’2009

Key Dates of Capillary Electrophoresis*

< 1930’s exemplary results with tubular electrophoresis

1930’s Tiselius et al. moving boundary electrophoresis of proteins

1960’s Tiselius & Hjertén et al., initial Capillary Electrophoresis1960 s Tiselius & Hjertén et al., initial Capillary Electrophoresis

1970’s Everaerts et al., CZE, Isotachophoresis (ITF) in narrow polymer capillaries,

Pretorius et al., Capillary Electrochromatography (CEC)

1980’s Technology accelerated

Jorgenson et al, CZE in fused silica capillaries

Hjertén et al, SDS PAGE in capillaries, capillary Isoelectric Focussing (IEF)

Terabe et al., Micellar ElectroKinetic Chromatography

Knox et al, capillary electrochromatography

Karger, capillary gel electrophoresis

First introductions of commercial CE-instrumentation

1990’s Boisterous growth of applications publications and market1990’s Boisterous growth of applications, publications and market

1999 Introduction of commercial microchip electrophoresis instrumentation



*Quoted in part from Compton & Brownlee, Biotechniques, 6, 432, 1986

HPLC’2009Publications with Capillary Electrophoresis and/or Microfluidic in Title

3000

# of publications cited*

2500

3000

*Source: ISI Web of Knowledge

1500

2000

CE

1000

MF

0

500

1989 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 20081989 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008



HPLC’2009

Key Features of Capillary Electrophoresis

• Ultra high efficiency separations (N ∝ 1/Di)

• Ideal for separation of charged molecules Ideal for separation of charged molecules

(“when charged look for CE first”)

• Inherently microscale separation Mandates small samples

(Rel.) easy coupling MS

• Several versatile separation modes in one boxCapillary Zone Electrophoresis

Capillary Gel Electrophoresis

Micellar Electrokinetic Chromatography

Capillary Isoelectric Focussing

Capillary Isotachophoresis

Capillary Electrochromatography

• Regarded replacement technique for HPLC!!!©Agilent Technologies 7/6/2009


HPLC’2009Publications with Capillary Electrophoresis and/or Microfluidic in Title

3000

# of publications cited*

2500

3000

1500

2000

CE

1000

MF

0

500

1989 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 20081989 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008

*Source: ISI Web of Knowledge©Agilent Technologies 7/6/2009


HPLC’2009

Technology Adoption Life Cycle*



*Bohlen, Iowa State University, 1957

HPLC’2009

The chasm adaption of TALC*

Existing competitive technology

Visionaries PragmatistsLack of confidence in new technologyTechnology regarded incompletePoor communication between visionaries and pragmatists



*Discontinuous technology adoption model, G. Moore , Crossing the Chasm: Marketing and Selling High‐Tech Products to Mainstream Customers

HPLC’2009CE tried to cross the chasm*, hit some bowling pins but did not became a tornado**

“Bowling pins” like ion analysis, i i id

DNA sequencing

enantiomer separation, peptides, recombinant proteins, oligonucleotides, basic drugs

The early nineties



*G.A. Moore, Crossing the Chasm: Marketing and Selling High‐Tech Products to Mainstream Customers**Picture taken from website Chasminstitute, www.chasminstitute.com

HPLC’2009Why did CE‐technology not turn into a tornado?

• Other separation techniques (HPLC) remained dominant

Appearance of microfluidic CE devices on the market

• High level of frustration

“Difficult” method. Low chance to get it right the first time.

Separation of analytes more difficult to predict and optimize than HPLC.

Sample matrix has large impact on the separation (invisible system peaks, sample

zone properties modulate peak shape and separation).

Fused silica was not reproducible

D t f bi l f th CE k t ft 8 10 Departure of big players from the CE market after 8-10 years

• Did not meet customer expectations and metrics full 100%

Sensitivity in UV-VIS detection (best case 1-10 ppm)

Quantitative reproducibility

Overall Robustness

• Not enough “visionaries” – no critical mass among “pragmatists”

Initially not an approved official method (USP, EP, FDA)



HPLC’2009

Market size and projections in 2006*



*Global Assessment Report 2006 Strategic Directions Inc.

Current Trends in Capillary Electrophoresis

New “Bowling Pins”?



HPLC’2009

CE New “Bowling Pins”

• Biotechnology“with over one third of all pipe line products in active with over one third of all pipe-line products in active development are biopharmaceuticals”. Many of the leading pharmaceutical companies have entered the therapeutic MAbsector sector Emerging Biogenerics requiring methodology for proof of equivalence and identity

Lif S i R h ( t i )• Life Science Research (esp. proteomics)Replacing slab gel electrophoresis with CGE and/or MCE



HPLC’2009

CE New “Bowling Pins”

• CE-MS for MetabolomicsMetabolomics• Majority of metabolites are charged and highly hydrophilic solutes

• Human Metabolome Technologies Solution

Bi kBiomarkers• Clinical proteomics (Mischak et al.)

• Detection of chronic alcohol abuse (Thormann et al.)



HPLC’2009

CE New Bowling Pins?

• Food and Drug Safety and AuthenticityCounterfeited recombinant proteinsCounterfeited recombinant proteins

Next slide



Next slide

HPLC’2009

Confessions of a professional cyclist*

• Growth hormones counterfeit hGH**

• InsulineInsuline

• Gonadotropines

• CorticosteroidsCorticosteroids

• Anabolic steroids

• Erythropoetiney p

• Hemoglobine & Hemoglobine Oxygen Carriers***

• Plasma expandersp

• Blood transfusions

*Source J‐L Veuthey, Université de Genève



**Julie Schappler et al, poster # CPB66‐We ***Oral presentation Wednesday, Schappler et al.

HPLC’2009


• Food and Drug Safety and AuthenticityCounterfeit proteins (rhGH)Counterfeit proteins (rhGH)Heparin case



HPLC’2009

The Heparin Contamination Case

Heparin, a highly‐sulfated glycosaminoglycan, is widely used as an injectable anticoagulant, and has the highest negative charge density of any known biological molecule.

In March 2008, major recalls of heparin were announced by the FDA. According to the FDA contaminatedAccording to the FDA, contaminated heparin killed 81 people in the United States. The contaminant was identified as an "over‐sulphated" derivative of chondroitin sulfate, a popular shellfish‐derived supplement often used for arthritis.

Heparin is a member of the glycosaminoglycanfamily of carbohydrates (GAGs). It is a polydisperse oligomer, average MW 12‐15 kD.



p y p g gUbiquituously found in mucosal tissue

HPLC’2009Determination of OSCS in Heparin Formulations* – Limit of Detection

60 0

75.0DS

Sodium

40.0

60.0

mAU

0 15% OSCS/2 5% DS

OSCS

Thiosulfate

20.0 0.075% OSCS/1.5% DS

0.1% OSCS/2% DS

0.15% OSCS/2.5% DS

‐5 0

0.04% OSCS/0.8% DS

0.05% OSCS/1.0% DS

600 mM Li phosphate pH 2.5, 25 mm id x 21.5 cm effective x 30 cm total length, temperature 20°C, 1000 mbar injection, –14 kV. 50 mg/mL Heparin. Detection 200:10 nm, BF 5

0.00 1.25 2.50 3.75 5.00 6.25 7.50 8.75 9.96

‐5.0

minutes

©Agilent Technologies 2/1/2009 ‐ 2/5/2009

*Todd Wielgos et al., Journal of Pharmaceutical and Biomedical Analysis, 49(2), 2009, 319-326

HPLC’2009Determination of OSCS in Heparin Formulations* – Repeatability

20.0

25.0

DSSodium Thiosulfate

10 0

mAU

10.0

OSCS

0.0 2.0 4.0 6.0 8.0 10.0 12.0‐5.0

0.0

0.0 2.0 4.0 6.0 8.0 10.0 12.0

minutes

600 mM Li phosphate pH 2.5, 25 mm id x 21.5 cm effective x 30 cm total length, temperature 20°C, 1000 mbs injection, –14 kV. Wash 0.1 N NaOH. Detection 200:10 nm, BF 5, 12 injections 0.1% OSCS/2.0% DS in 50 mg/mL Heparin

©Agilent Technologies2/1/2009 ‐ 2/5/2009

*Todd Wielgos et al., Journal of Pharmaceutical and Biomedical Analysis, 49(2), 2009, 319-326

HPLC’2009

Recommendations by USP?

• CE heparin method to be replaced with AEX replaced with AEX chromatography in 2009

• LC method: 1 hr run time. Some samples require hours for heparin degradation

• USP says CE is too difficult CE• USP says CE is too difficult to implement. No competence in labs WW.

CERedundancy of equipment not given

©Agilent Technologies2/1/2009 ‐ 2/5/2009

HPLC’2009


• Food and Drug Safety and AuthenticityCounterfeit proteins (rhGH)Counterfeit proteins (rhGH)Heparin case

• “Traditional” market demands remain strongEstablished methodology (Pharmacopeia)• Drug stability

• Enantiomeric purity determination

• Counterion determination

• pKa determination

• Long product life cycle in pharmaceutical industryg p y p y

Forensic applications (illicit drugs, doping)



HPLC’2009

Market size/trends in 2008*



*Global Assessment Report 2008 Strategic Directions Inc.

HPLC’2009

How to turn on the CE‐tornado?

• Vendors must build confidence among “pragmatists”

Obvious long term commitment to CE analytical measurement technology

Thorough, coherent solutions from manufacturers

New hardware development (Agilent)



Agilent 7100 Capillary Electrophoresis System



HPLC’2009

How to turn the tornado on?

• Manufacturers & Suppliers building confidence among pragmatists

Obvious long term commitment to CE analytical measurement technology

Th h h t l ti f f t (“Kit ”)Thorough, coherent solutions from manufacturers (“Kits”)

New hardware development (Agilent)

• Complement and complementary technology

O th l ti i bi ti ith HPLCOrthogonal separation in combination with HPLC

More seamless integration with mass spectrometry

Multiple, sensitive detection methods (LIF, contactless conductivity detection, parallel UV detection)

Sharing and merging electrophoresis separation methods (slab gel off-gel capillary microchip)Sharing and merging electrophoresis separation methods (slab gel, off gel, capillary, microchip)

• Trained workforce

Academic education

Applying rigorous method protocols; “Good CE-Practice”Applying rigorous method protocols; Good CE Practice

Computer supported method development and optimization (e.g. Gas et al.)

• More “bowling pins”

Kinetic Capillary Electrophoresis (Sergey Krylov et al )Kinetic Capillary Electrophoresis (Sergey Krylov et al.)

Microbial Analysis (Kenndler et al, Armstrong et al)



HPLC’2009

Acknowledgement

Hans‐Peter Zimmermann, Stefan Falk‐Jordan, Tobias Preckeland other colleagues @ Agilent Technologies

Stacey Ma, Genentech, USAy , ,

Cari Sänger‐van de Grient, Pim Muyselaar, Solvay, Netherlands



Documents

C illCapillary El t h iEl ectrophoresis: Renaissance and ... · X O th l ti i bi ti ith HPLCOrthogonal separation in combination with HPLC X More seamless integration with mass spectrometry