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Heinz Goetz World Wide Product Manager Liquid Chromatography

Heinz Goetz World Wide Product Manager Liquid Chromatography. Protein Analysis by Aqueous Size Exclusion Chromatography (SEC) with the 1100 SEC System. SEC: What is it?. HPLC technique for analysis/characterization of macromolecules

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Heinz Goetz World Wide Product Manager Liquid Chromatography

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  1. Heinz GoetzWorld Wide Product ManagerLiquid Chromatography Protein Analysis by Aqueous Size Exclusion Chromatography(SEC) with the 1100 SEC System

  2. SEC: What is it? • HPLC technique for analysis/characterization of macromolecules • SEC: - w/ organic eluents (Gel Permeation Chromatogr.- GPC) - w/aqueous eluents (Gel Filtration Chromatogr. - GFC) • GPC: - typical eluents: tetrahydrofuran, toluene, dimethylacetamide … - for synthetic macromolecules(plastics) • GFC: - eluent: water, buffered water - biomacromolecules (proteins, poly- saccharides, DNA, RNA, water-soluble synthetic polymers • Tasks: - MW determination, mono-,di, ... purity analysis Agilent Restricted

  3. SEC: What is it? GPC (plastics analysis, organic eluents) SEC GFC (biomolecule analysis, aqueous eluents) Agilent Restricted

  4. SEC Separation mechanism Mobile phase Column packing A B C A Inject B B Pores C Elution volume C Polymer Analytes Agilent Restricted

  5. Mechanism of SEC Separation FLOW Small Change in elution volume ---->big change in molecular weight ---> excellent HPLC equipment required! 000855P1.PPT Agilent Restricted

  6. Sample chromatogram 30 20 10 0 Time (min.) 8 14 16 10 12 Steps in SEC Analysis Authoring Division Name File Name Security Notice (if required) • Determination of separation parameters (eluent, column(s), column temp. etc.) • Analysis of narrow distributed calibration standards • Set-up of calibration curve • Analysis of sample • Calculation of molecular weight data and molecular weight distribution Agilent Restricted

  7. SEC Applications with Proteins • Impurity testing * • Molecular weight characterization * • Expression and folding studies + , • Separation of Reaction Components and Products, (esp., antibodies, fragments, and conjugates) + • Purification + • Desalting and Exchange of Sample Buffer * SEC software (G2182AA) required, + recommended Agilent Restricted

  8. Life Science Applications with SEC and MW determination Agilent Restricted

  9. Recommended System • Isocratic pump w/ seal wash • Vacuum degasser • Thermostatted autosampler • Thermostatted column compartment • VWD, MWD or DAD • FLD (for highest sensitivity) • ChemStation w/ GPC-SEC data analysis software(G2182AA) RID not required! Agilent Restricted

  10. NEW! Agilent 1100 Series SEC System # 1 HPLC - worldwide HP 1100 HPLC Superior pump performance and thermostat stability for best retention time precision Peltier-column thermostat with temperature range up to 80°C Single valve injector for extended uptime, optional 4-40°C Choice of VWD, MWD and DAD for sensitive UV detection Sensitive fluorescence detector w/ “on the fly” spectra • Complete system solution for convenient operation • Hardware & software • Procedures for start up & system checkout • Columns Agilent Restricted

  11. Conditions for Protein Analyses • Columns: TSK Gel SW 2000xl (p/n 79911S3-597) and TSK Gel SW 3000xl (p/n 79912S3-597) in series • Mobile phases A: 0.10 M Na-Sulfate, 0.1 M Na-phosphat, pH 6.7 (A) B: 0.40 M NaCl, 10mM Na-phosphat, pH 7.4 (B) C: 0.15 M NaCl, 20mM Na-phosphat, 0.01% Tween 20, pH 6.8 (C) • Detection: DAD (278, 20/360,40, 214, 4/360,40) FLD (220/350) • Flow: 0.8 ml/min • Sample conc. < 0.1 % • Column Temp.: 25 and 42 C Autosampler thermostat: 4 C • Calibration standards: Proteins, supplier Sigma Chemicals Agilent Restricted

  12. Optimized SEC separation of Proteins and Peptides 555 OVA 45 K Lysozyme 14.3 K Tyrosin 180 Leucin-Enkephalin Cytochrome C 12.4 K Carbonic Anhydrase 29 K Insulin 5.8 K Thyroglobulin 660K Gamma-Globulin 150 K BSA 66 K Eluent: 0.4 M NaCl, 10mM Na-phosphat, pH 7.4Factor 1.5 - 2 in molecular weight needed for separation Agilent Restricted

  13. Calibration Curve Selected curve fit Deviation between data pointand curve Optimization of calibration standards and curve for each protein!!! Agilent Restricted

  14. Simultaneous Impurity Testing and Identiifcation: I. Impurity Testing Impurity Determination:standard HPLCreport 91.1 % Ovalbumin 8.9 % dimer Agilent Restricted

  15. Simultaneous Impurity Testing and Identification: II. Identif. by Molecular Weight Identification:SEC report Ovalbumin? Dimer ofOvalbumin? Mp = 42700 (OVA) and 84400(dimer)! Molecular weight data prove ovalbumin anddimer peak (A)!Reference molec. weight ovalbumin: 43000 Agilent Restricted

  16. Quality Control of Antibody w/ Fluorescence Det. Molecular weight Comparison:SEC: 149000Bioanalyzer: 157000 Antibody Native(SEC) vs. denaturingmethod High MWImpurity 0.2% Eluent Agilent Restricted

  17. QA/QC-Analysis of Erythropoietin (EPO) M = 55400 (ref. SEC ~ 55000)D = 1.007 Tyr(ISTD) EPO (B) 100% monomer - proof by DAD- and fluorescence signaland low polydispersity. Good Mp agreement! Agilent Restricted

  18. EPO Purity by Spectra Overlay No spectra difference between mono- and dimer! Agilent Restricted

  19. Fluorescence Spectra of EPO Excitation spectrum 278 nm 340 nm Emissiom spectrum Agilent Restricted

  20. Identification of Proteins in Human Plasma by Molecular Weight AlbuminMp = 65500 ImmumoglobulineMp = 717000 FLD DAD Improved separation required by further column TSK G2000SW Agilent Restricted

  21. Comparison of MW Results Agilent Restricted

  22. Features of Protein Analysis and Characterization by SEC • Not denaturing; collection of fractions; different detection principles • Eluent optimization for each protein required • Good accuracy - (typically < 2%)and precision (typically < 2% RSD) • Molecular weight and impurity testing simultaneously(peaks, polydispersity, spectra) in a single run • MW range: 100 - 10 MD • Sensitivity (~ 8 ng/µl (100 µl sample) - S/N = 3/1) • Resolution: ~ 100 %(factor 2 in MW difference) Workhorse ! Agilent Restricted

  23. Pharmacia/Upjohn, Italy Amgen, USA ---> 5 Systems in 2000 Roche Company, USA Novartis, Switzerland Bayer Pharma, Germany Fibrinogen, USA Hexal Company, Germany BioSyntech, Canada Angiotech Pharmaceutical, Canada Astra - Zeneca, Sweden Protein expression-, QA/QC- analysis Extract from > 300 customers (allindustries)! Company Confidential Pharma-/Lifescience Customers in SEC The MasterWorkhorse Agilent Restricted

  24. Alternative Techniques - SDS PAGE Agilent Restricted

  25. Alternative Techniques - CE -SDS Agilent Restricted

  26. Alternative Techniques - Bioanalyzer Agilent Restricted

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