2013-05-13 1 New Developments in Capillary Electrophoresis with focus on Bioanalysis Lecture 10 Christian Nilsson Today • CE in drug development CE in drug industry • Liquid Chromatography dominant • The field of LC more mature • Limited by availability of experienced CE users Preclinical stage • Find new compounds for group of compounds (i.e. lead compounds) with desired therapeutic effect • Generally small amounts of each compound • Many compounds • Combinatorial approch of synthesis Passive adsorption across biological membranes • Dissociation coefficients (pKa) • Partitioning behavior (e.g. octanol-water partioning) • Solubility • Membrane permeability Determination of pKa by CE • An alternative to potentiometry or UV spectrometry • CE – Smaller amount of samples – Less sensitive to contaminations – Automated – Chromophore necessary for UV detection 2013-05-13 2 Determination of pKa by CE • Measuring the ionic effective mobility as a function of the pH Example of 2-aminopyridine (pKa 6.7) A equilibrium equation is fitted to the data points Determination of pKa by CE Determination of pKa by CE Determination of pKa by CE • High throughput measurements • Commercially available 96 capillary instruments • Commercially available kits • Pressure-assisted CE to shorten the run times Multiplexed CE-UV Drug Discovery Today 2004, 9, 1072-1080 Low Molecular weight drug candidates of possess aromatic rings or conjugated double bond structures, which make UV detection useful Multiplexed CE-UV • Compatible with 96 well plates (8*12) at inlet side • 96 capillaries side by side at detection window • Can use vacuum to introduce different buffer solutions in different capillaries • An alternative is to use multichannel microfluidic electrophoresis devices 2013-05-13 3 Multiplexed CE-UV Determination of pKa by CE-MS • Pressure-assisted CE • A series of 10 volatile buffers covering pH 2.5- 10.5 • High throughput screening • Higher sensitivity than CE-UV • Can be used for non-UV-absorbing compounds Rapid Communications in Mass Spectrometry 2003, 17, 2639-2648 Determination of pKa by CE-MS • Simultaneous measurement of more than 50 compounds in less than 150 min Rapid Communications in Mass Spectrometry 2003, 17, 2639-2648 Determination of distribution coefficients • Methods based on: – Micellar Electrokinetic Chromatography (MEKC) – Microemulsion Electrokintetic Chromatography (MEEKC) • Separation based on hydrophobicity • Compared to standard compounds of known hydrophobicity • Separation of uncharged compounds – High pH for weak bases – Low pH for weak acids Microemulsion Electrokinetic Chromatography • Use of a microemulsion as electrolyte – Oil-in-water or water-in-oil microemulsion – Stable clear emulsion – Water, hydrocarbon and surfactant, co-surfactant Electrophoresis 2013, 34, 159-177 Microemulsion Electrokinetic Chromatography 2013-05-13 4 Comparsion between MEEKC and MEKC • Enhanced solubilization capacity and separation of MEEKC • Analytes can partition more effectively into the microemusion droplets compared to the more rigid micelles – Higher rate of mass transfer give more efficient separation Analysis of pharmaceutical counterions High throughput screening and lab on chip • HTS to identify chemical hits against a therapeutic target to detect lead candidates • Tradtional HTS method use radioactive or fluorescent labeling – Label may affect the result High throughput screening by CE • Screening for enzyme inhibitor • Enzymes common therapeutic drug targets • L-glutamic dehydrogenase (GLDH) used as model enzyme • Inline enzyme bioreactor in capillary column • GLDH immobilized on gold nanoparticles • The gold nanoparticles was absorbed on the inner wall of the capillary at the inlet Analytical Biochemistry 2011, 411, 88-93 High throughput screening by CE • Method used to screen plant extracts Analytical Biochemistry 2011, 411, 88-93 High throughput screening by CE Analytical Biochemistry 2011, 411, 88-93 The capillary surface is pretreated with a polyelectrolyte The enzyme is absorbed based on ionic binding 2013-05-13 5 High throughput screening by CE • Enzyme loading will be much greater if meditated by gold nanoparticles compared to in free solution – Enzyme enriched on GNPs that possess a high surface-to-volume ratio • Enzyme-Nanoparticle conjugates much more stable than enzyme-polyelectrolyte conjugates – Thiol groups present in proteins bind to the gold nanoparticles strongly Analytical Biochemistry 2011, 411, 88-93 Nanoparticle enhancement • Examples of other application – Immobilization of trypsin on nanoparticles to enhance the trypsin activity – Immobilization of cyclodextrin on nanoparticles to enhance chiral separation High throughput screening by CE NAD+ NADH Quantification of NAD+ at 254 nm Conversion of glutamate to α-ketoglutarate High throughput screening by CE Perphenazine (a known GLDH inhibitor Blank Other methods for high throughput screening • Immobilization of enzymes on silica particles that were packed into LC columns • Enzyme bioreactors based on entrapment of enzymes in sol-gel-derived monoliths in CE • Immobilized enzymes on magnetic nanoparticles Enzyme purification and characterization Produced to recover NADH During hard work / Shortage of oxygen 2013-05-13 6 Enzyme purification and characterization • Genetic modification to make purification easier • Examples: – Modification to make target enzyme more resistant to heat – Attachment of Histidin tag to allow IMAC Example of enzyme purification • Production of enzyme in E.coli. Using a plasmid containing target enzyme (genetic modified) • Destroy the cell membrane – Sonication or lyzosyme • Heat treatment • IMAC purification IMAC • Immobilized metal ion affinity chromatography • Metals such as Cupper and Nickel have affinity for histidine • Imidazole for elution • Phosphorylated peptides or proteins can be used purified by e.g. Zink Affinity Chromatography • Lectins for purification of glycoproteins • Antibodies • Aptamers Enzyme purification and characterization Spectrophotometry to determine LDH amount indirectly by measuring NAD+ concentrations CE Enzyme Assays • Pre-capillary enzyme assay – Initiation of reaction – Termination of reaction – Analysis of product(s) and or reactant(s) by CE • In-capillary enzyme assay – The enzyme, reactant and product have different mobility – Low consumption of reactants, enzymes and cofactors 2013-05-13 7 CE Enzyme Assays