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C Ill El T H I Capillary Electrophoresis: Renaissance and New Applications Cap illary Elect roph oresi s: Renaissance and New Applications Gerard P. Rozing Agilent Techlihnologies, 76337 Waldbronn, Germany. ©Agilent Technologies Presentation at HPLC'2009 1 7/6/2009 Brief Retrospective of Capillary Electrophoresis ©Agilent Technologies Presentation at HPLC'2009 2 7/6/2009 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 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 market 1999 Introduction of commercial microchip electrophoresis instrumentation *Quoted in part from Compton & Brownlee, Biotechniques, 6, 432, 1986 ©Agilent Technologies Presentation at HPLC'2009 3 7/6/2009 Publications with Capillary Electrophoresis HPLC’2009 and/or Microfluidic in Title # of publications cited* 3000 *Source: ISI Web of Knowledge 2500 2000 1500 CE MF 1000 500 0 1989 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 ©Agilent Technologies Presentation at HPLC'2009 4 7/6/2009 HPLC’2009 Key Features of Capillary Electrophoresis • Ultra high efficiency separations (N ∝ 1/Di) • Ideal for separation of charged molecules (“when charged look for CE first”) • Inherently microscale separation X Mandates small samples X (Rel.) easy coupling MS • Several versatile separation modes in one box X Capillary Zone Electrophoresis X Capillary Gel Electrophoresis X Micellar Electrokinetic Chromatography X Capillary Isoelectric Focussing X Capillary Isotachophoresis X Capillary Electrochromatography • Regarded replacement technique for HPLC!!! ©Agilent Technologies Presentation at HPLC'2009 5 7/6/2009 Publications with Capillary Electrophoresis HPLC’2009 and/or Microfluidic in Title # of publications cited* 3000 2500 2000 1500 CE MF 1000 500 0 1989 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 Presentation at HPLC'2009 6 7/6/2009 HPLC’2009 Technology Adoption Life Cycle* *Bohlen, Iowa State University, 1957 ©Agilent Technologies Presentation at HPLC'2009 7 7/6/2009 HPLC’2009 The chasm adaption of TALC* ¾ Existing competitive technology ¾ Lack of confidence in new technology Visionaries Pragmatists ¾ Technology regarded incomplete ¾ Poor communication between visionaries and pragmatists *Discontinuous technology adoption model, G. Moore , Crossing the Chasm: Marketing and Selling High‐Tech Products to Mainstream Customers ©Agilent Technologies Presentation at HPLC'2009 8 7/6/2009 CE tried to cross the chasm*, hit some HPLC’2009 bowling pins but did not became a tornado** DNA sequencing “Bowling pins” like ion analysis, 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 ©Agilent Technologies Presentation at HPLC'2009 9 7/6/2009 Why did CE‐technology not turn into a HPLC’2009 tornado? • Other separation techniques (HPLC) remained dominant X Appearance of microfluidic CE devices on the market • High level of frustration X “Difficult” method. Low chance to get it right the first time. X Separation of analytes more difficult to predict and optimize than HPLC. X Sample matrix has large impact on the separation (invisible system peaks, sample zone properties modulate peak shape and separation). X Fused silica was not reproducible X DtDeparture of big players from the CE markktet after 8-10 years • Did not meet customer expectations and metrics full 100% X Sensitivity in UV-VIS detection (best case 1-10 ppm) X Quantitative reproducibility X Overall Robustness • Not enough “visionaries” – no critical mass among “pragmatists” X Initially not an approved official method (USP, EP, FDA) ©Agilent Technologies Presentation at HPLC'2009 10 7/6/2009 HPLC’2009 Market size and projections in 2006* *Global Assessment Report 2006 Strategic Directions Inc. ©Agilent Technologies Presentation at HPLC'2009 11 7/6/2009 Current Trends in Capillary Electrophoresis New “Bowling Pins”? ©Agilent Technologies Presentation at HPLC'2009 12 7/6/2009 HPLC’2009 CE New “Bowling Pins” • Biotechnology X “with over one third of all pipe-line products in active development are biopharmaceuticals”. Many of the leading pharmaceutical companies have entered the therapeutic MAb sector X Emerging Biogenerics requiring methodology for proof of equivalence and identity • Life Sci ence Research (esp. proteom ics ) X Replacing slab gel electrophoresis with CGE and/or MCE ©Agilent Technologies Presentation at HPLC'2009 13 7/6/2009 HPLC’2009 CE New “Bowling Pins” • CE-MS for X Metabolomics • Majority of metabolites are charged and highly hydrophilic solutes • Human Metabolome Technologies Solution X Biomar kers • Clinical proteomics (Mischak et al.) • Detection of chronic alcohol abuse (Thormann et al.) ©Agilent Technologies Presentation at HPLC'2009 14 7/6/2009 HPLC’2009 CE Î New Bowling Pins? • Food and Drug Safety and Authenticity X Counterfeited recombinant proteins Next slide ©Agilent Technologies Presentation at HPLC'2009 15 7/6/2009 HPLC’2009 Confessions of a professional cyclist* • Growth hormones Î counterfeit hGH** • Insuline • Gonadotropines • Corticosteroids • Anabolic steroids • Eryypthropoetine • Hemoglobine & Hemoglobine Oxygen Carriers*** • Plasma expanders • Blood transfusions *Source J‐L Veuthey, Université de Genève **Julie Schappler et al, poster # CPB66‐We ***Oral presentation Wednesday, Schappler et al. ©Agilent Technologies Presentation at HPLC'2009 16 7/6/2009 HPLC’2009 CE Î New Bowling Pins? • Food and Drug Safety and Authenticity X Counterfeit proteins (rhGH) X Heparin case ©Agilent Technologies Presentation at HPLC'2009 17 7/6/2009 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, 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 glycosaminoglycan family of carbohydrates (GAGs). It is a ppyolydisp erse oligomer, average MW 12‐15 kD. Ubiquituously found in mucosal tissue ©Agilent Technologies Presentation at HPLC'2009 18 7/6/2009 Determination of OSCS in Heparin HPLC’2009 Formulations* – Limit of Detection 75.0 DS Sodium 60. 0 Thiosulfate mAU OSCS 40.0 0. 15% OSCS/2.5% DS 0.1% OSCS/2% DS 20.0 0.075% OSCS/1.5% DS 0.05% OSCS/1.0% DS 0.04% OSCS/0.8% DS ‐505.0 0.00 1.25 2.50 3.75 5.00 6.25 7.50 8.75 9.96 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 mbar injection, –14 kV. 50 mg/mL Heparin. Detection 200:10 nm, BF 5 *Todd Wielgos et al., Journal of Pharmaceutical and Biomedical Analysis, 49(2), 2009, 319-326 ©Agilent Technologies 2/1/2009 ‐ 2/5/2009 Determination of OSCS in Heparin HPLC’2009 Formulations* – Repeatability 25.0 Sodium Thiosulfate 20.0 DS mAU 10. 0 OSCS 0.0 ‐5.0 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 *Todd Wielgos et al., Journal of Pharmaceutical and Biomedical Analysis, 49(2), 2009, 319-326 ©Agilent Technologies 2/1/2009 ‐ 2/5/2009 HPLC’2009 Recommendations by USP? • CE heparin method to be 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 to implement. No CE competence in labs WW. Redundancy of equipment not given ©Agilent Technologies 2/1/2009 ‐ 2/5/2009 HPLC’2009 CE Î New Bowling Pins? • Food and Drug Safety and Authenticity X Counterfeit proteins (rhGH) X Heparin case • “Traditional” market demands remain strong X Established methodology (Pharmacopeia) • Drug stability • Enantiomeric purity determination • Counterion determination • pKa determination • Longgp product life cy cle in p harmaceutical industr y X Forensic applications (illicit drugs, doping) ©Agilent Technologies Presentation at HPLC'2009 22 7/6/2009 HPLC’2009 Market size/trends in 2008* *Global Assessment Report 2008 Strategic Directions Inc. ©Agilent Technologies Presentation at HPLC'2009 23 7/6/2009 HPLC’2009 How to turn on the CE‐tornado? • Vendors must build confidence among “pragmatists” X Obvious long term commitment to CE analytical measurement technology X Thorough, coherent solutions from manufacturers X New hardware development (Agilent) ©Agilent Technologies Presentation at HPLC'2009 24 7/6/2009 Agilent 7100 Capillary
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