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Use of Solid Core Chromatography for the Analysis of Pharmaceutical Compounds
The document discusses the advantages of solid core chromatography in the pharmaceutical industry, highlighting its ability to provide greater efficiency, improved separation, and lower operational pressures. It covers the features and benefits of solid core materials, optimization strategies, and their effectiveness compared to traditional methods. Additionally, the document explores the stability of these columns under varying pH and temperature conditions, emphasizing their practicality for high-throughput analytical workflows.
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Use of Solid Core Chromatography for the Analysis of Pharmaceutical Compounds
- 1. Use of SolidCore Chromatography for the Analysis of Pharmaceutical Compounds Dafydd Milton Product Manager, LC & LC/MS columns Tony Edge R&D Principal March 2014
- 2. Introduction • The PharmaceuticalProcess • Mapping out different sectors Wh t t f h ll f d• What types of challenges are faced • Understanding the drivers in the pharmaceutical industry • Solid Core Chromatography • Understanding the benefits of the technology • Bar for bar greater efficiency• Bar for bar greater efficiency • How to optimise the separation • Understanding chemistry • Optimization of the morphology • Coupling Solid Core Chromatography to Pharmaceutical Analysis• Coupling Solid Core Chromatography to Pharmaceutical Analysis • Design of new workflows • Improving column lifetimes 2 • Improving assay robustness
- 3. The Pharmaceutical Process– Beginning 3
- 4. The Pharmaceutical Process– Further Development 4
- 5. Solid Core Material– Features and Benefits • Features • More uniform particle sizing• More uniform particle sizing • Better packing of particles • Reduced pore depth • Reduced mass transfer effects in mobile phase • BenefitsBenefits • More Efficient Chromatography • Allows the use of low pressure systems • Competitive Edge • Bar for bar gives better separations than porous materialsg p p 5
- 6. Liquid Chromatography ParticleDesign 2.6 µm 80 Å Solid Core Particles 80 Å 2.6 µm 150 Å Conventional Fully Porous Non-Porous Solid Core 4 µm 80 Å 1.x µm 80 Å Reduce Size to improve kinetics at expense of operating pressure Low sample capacity Very high pressure Small particle kinetics Reasonable pressure Very High Sample Capacity Lower Efficiency Low Sample Capacity Very High Efficiency High Sample Capacity High Efficiency 6 operating pressurey y g y g y
- 7. Pressure Comparison 900 1000 BAP 2 00 2 11 600 700 800 900 600 bar limit pp ddL 3 0 23 0 400 500 600 ressure(bar) HPLC pressure limit 100 200 300 Pr u = 8 7mm/s 0 0 200 400 600 800 1000 Flow rate (µL/min) Accucore RP MS 2 6µm <2µm 3µm 5µm u = 8.7mm/s Accucore RP-MS 2.6µm <2µm 3µm 5µm Columns: 100 x 2.1 mm Mobile phase: H2O / ACN (1:1) Temperature: 30 °C Wide flow rate range with P < 600 bar 7 Temperature: 30 °C
- 8. The Theory …the van Deemter equation CC B AHETP uCuC u AHETP sm 8
- 9. A – TermEddy Diffusion or Multiple Paths Packing Efficiency D90/10~1.5 Porous Silica Packing Efficiency D90/10~1.1Accucore 9
- 10. B – TermLongitudinal Diffusion Longitudinal diffusion: (HETP = H + H + H )Longitudinal diffusion: (HETP = HA + HB + Hc) Th l t t ti i l t th d f b d l tThe solute concentration is lower at the edges of a band; solute diffuses to the edges. Time High concentration C t ti t ilib iHigh concentration. Concentration at equilibrium • The B-term depends on; 10 • Void volume of the column
- 11. C – TermResistance To Mass Transfer • The C term depends on; Diff i diff i th i th ili• Differences in diffusion path in the silica pores • Differences in the radial diffusion path in the liquid • Size of molecule 11 • Significant for large molecules but not for small molecules
- 12. Efficiency Comparison –Van Deemter 20.0tical 10 0 15.0 Theoret 5.0 10.0 uivalent Plates 0.0 0 0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0 10 0 eightEq 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 He Linear velocity of mobile phase (mm/s) Accucore RP-MS 2.6µm 5µm 3µm <2µm Columns: 100 x 2.1 mm Mobile phase: H2O / ACN (1:1) Temperature: 30 °C Detection: UV at 254 nm Flow rate range: 0.1 to 1.0 mL/min Highest efficiency and lowest rate of efficiency loss with flow rate for solid core 12 Flow rate range: 0.1 to 1.0 mL/min Analyte: o-xylene rate for solid core
- 13. Van Deemter –Limitations • Classical interpretation of how a column is performing • 3 parameters • A – eddy diffusiony • B – longitudinal diffusion • C – Resistance to mass transfer • Optimization of these parameter will give the best peak shape/efficiency • However it does not take into account; • Analysis time P t i ti t• Pressure restrictions on a system 13
- 14. Kinetic Plots • Allowsfor fairer comparisons of analytical systems • Van Deemter just compares pure separation ability• Van Deemter just compares pure separation ability • Incorporates time of analysisp y • Analysts want FASTER chromatography • Van Deemter plots do not specify the time of analysis • Incorporates pressure limitations of systems • Van Deemter does not account for a pressure limitationp on system • Based on three very simple classical equations• Based on three very simple classical equations 14
- 15. Kinetic Plots –Retention Time 1000010000 1000 (s)ofpeak 100 iontime Accucore allows optimisation of retention times Retenti Solid core produces sharper peaks in less time 10 1,000.00 10,000.00 100,000.00 Efficiency p 15 Accucore RP-MS 2.6µm 5µm 3µm <2µm Efficiency
- 16. Impedance • Devised byKnox and Bristow in 1977 Defines the resistance a compound has to moving• Defines the resistance a compound has to moving down a column relative to the performance of that column • Allows for pressure to be incorporated Often plotted with a reverse axis• Often plotted with a reverse axis • Mimics van Deemter plot • Minimum value optimum conditions 2 Pt E p • Often plotted as a dimensional form 2 N E • t/N2 • t0 or tr both used 16
- 17. 100,000 Kinetic Plots –Impedance 100,000 2 0 N Pt E ce 2 N 10,000 mpedanIm S lid i lSolid core requires less pressure to obtain sub 2 µm efficiencies 1,000 100.001,000.0010,000.00100,000.001,000,000.00 Accucore RP-MS 2.6µm 5µm 3µm <2µm Efficiency 17 µ µ µ µ Efficiency
- 18. Resolution Equation '11 50 k '1 1 4 1 5.0 k k NRs Efficiency Particle size / packing Selectivity (Chemistry) Retention FactorParticle size / packing (Chemistry) Factor (Surface area) 18
- 19. The Impact ofSelectivity on Resolution Efficiency SelectivityRetentionEfficiency SelectivityRetention 2 5 3.0 2 5 3.0 N R= k’ k’+1 -1 4 N R= k’ k’+1 k’ k’+1 -1 -1 4 2.0 2.5 (R) 2.0 2.5 (R) k +1 4 k2 k +1k +1 4 k’22 1.5 N solution( 1.5 N solution( = k2 k’1 = k2 1 = 2 1 0.5 1.0 k Res 0.5 1.0 k ResSelectivity () has the greatest impact on 1.00 1.05 1.10 1.15 1.20 1.25 0.0 N 1.00 1.05 1.10 1.15 1.20 1.25 0.0 N improving resolution. 0 5000 10000 15000 20000 25000 0 5 10 15 20 25 N k 0 5000 10000 15000 20000 25000 0 5 10 15 20 25 N k S 19 Stationary phase, mobile phase, temperature
- 20. Stationary Phase Characterization •Hydrophobic retention (HR) Hydrophobic Interactions y p ( ) • k’ of neutral compound • Hydrophobic selectivity (HS) • α two neutral compounds that have different log P • Steric Selectivity (SS) • α sterically different moleculesα sterically different molecules • Hydrogen bonding capacity (HBC)y g g y ( ) • α molecule that hydrogen bonds and a reference • Good measure of degree of endcapping 20 • Gives indication of available surface area
- 21. Stationary Phase Characterization •Activity towards bases (BA) Interactions with Bases and Chelators • Activity towards bases (BA) • k’, tailing factor (tf) of strong base • Indicator of free silanols • Activity towards chelators (C) • k’, tailing factor (tf) of chelator • Indicator of silica metal content 21
- 22. Stationary Phase Characterization Interactionswith Acids and Ion Exchanges • Activity towards acids (AI) • k’, tf acid • Indicator of interactions with acidic compounds• Indicator of interactions with acidic compounds • Ion Exchange Capacity (IEX pH 7.6)g p y ( p ) • α base / reference compound • Indicator of total silanol activity • All silanols above pKa I E h C it (IEX H 2 7)• Ion Exchange Capacity (IEX pH 2.7) • α base / reference compound • Indicator of acidic silanol (SiO-) activity 22 • Indicator of acidic silanol (SiO ) activity
- 23. Column Characterization (Visualization) HR/10 HSAI Accucore C18 HR /10 HSAI Accucore RP-MS SSIEX (2.7) SSIEX (2.7) HBC IEX (7.6)BA C HBC IEX (7.6)BA C HR /10 HSAI Accucore PFP HR /10 HSAI Accucore Phenyl-Hexyl HS SSIEX (2.7) AI HS SSIEX (2.7) AI HBC IEX (7.6)BA C HBC IEX (7.6)BA C 23
- 24. Widest Range ofSolid Core Selectivity Options 500 mAU 1,2,3 curcuminoids 2 00 2.50 HR /10 HSAI Accucore RP-MS Solid Core C18 0.50 1.00 1.50 2.00 HS SSIEX (2.7) AI Accucore C18 Accucore 150-C18 Accucore C8 Accucore 150-C4 Accucore Polar Premium 1 0.00 HBCC Accucore aQ Accucore Polar Premium Accucore Phenyl-Hexyl Accucore PFP 2 3 Polar Premium shows different selectivity and separates the peaks IEX (7.6)BA Accucore Phenyl-X Accucore C30 0.0 1.0 2.0 3.0 0 Minutes 24
- 25. Accucore Columns –Selectivity Choices Columns: Thermo Scientific™ Accucore™ C30 Column Accucore C18 Column Ki t C181 Different selectivity for K2 isomers 350 mAU Accucore C18 Kinetex C18 Dimensions: 2.6 µm, 100 x 3.0 mm Mobile Phases: Methanol:buffer, 98:2 Buffer = 2 mM ammonium acetate 1 250 300 2+2’ Flow: 650 µL/min Temperature: 20 ºC Injection: 5 µL Detector: UV 250 nm 2+2’ Ki t C18 200 250 Detector: UV 250 nm Peaks: 1. Vitamin K2, 50 µg/mL 2. Vitamin K1, 50 µg/mL Other peaks formed by UV irradiation 1 C30 shows better separation for K1 Kinetex C18 100 150 2 2’ separation for K1 isomers Accucore C30 50 0 Vitamin K2 Vitamin K10.00 1.25 2.50 3.75 5.00 6.25 8.00 -25 min 25
- 26. Very Fast Separationswith Superb Resolution • Separation of atorvastatin i t di t (ATC AT1 Fully porous C18 5 μm, 250 x 4.6 mm intermediates (ATC-AT1 with ATC-AT1-Difluro) • All customer requirements 60 min• All customer requirements were met • Reduction of run time from 60 min to 60 min 8 min • Resolution improved using the Accucore PFP column Accucore PFP 2.6 μm, 150 x 3.0 mm 8 min • Mobile phase constituents kept similar • HPLC compatible method (270 bar) 26
- 27. Available Databases forColumn Characterisation http://www.usp.org/app/USPNF/columnsDB.html 27
- 28. Some Basic ColumnRequirements • Column Ruggedness • Stable under isocratic conditions • Stable under gradient conditions • Stable at low pH • Stable at high pHg p • Stable at elevated temperatures 28
- 29. Solid Core ColumnStability – Ruggedness Accucore RP-MS 2.6 µm 100 x 2.1 mm ID Mobile Phase: 60/40 ACN/H2O Flow Rate: 400 µL/min Accucore RP-MS 2.6 µm 100 x 2.1 mm ID Mobile Phase A: Water (0.05% TFA) Mobile Phase B: Acetonitrile (0.05% TFA) W h H O (0 05% TFA)Injection Volume: 1 µL Column Temp: 30 °C Wash: H2O (0.05% TFA) Injection Volume: 1 µL Column Temperature: 30 °C Efficiency (o-Xylene) Asymmetry (o-Xylene)Asymmetry (o Xylene) 4 000 + isocratic test 6 000 f t di t4,000 + isocratic test injections with no decrease in performance 6,000 + fast gradient injections with no change in retention 29
- 30. Solid Core ColumnStability – Low pH 40 Column Stability at pH < 2 pH 1.8 (0.1% TFA) 30 35 30,000 l 20 25 tionFactor Acetaminophen p-HBA o-HBA column volumes (5.5 days) 10 15 Retent Amitriptyline Nortriptyline DIPP DNPP (5 5 y ) 5 0 0 5000 10000 15000 20000 25000 30000 35000 Column Volumes Solid core columns are stable at low pH 30 Solid core columns are stable at low pH
- 31. Solid Core ColumnStability – High pH pH 10.5 (0.1% ammonia) 30,000 column r column volumes (5.5 days) ntionFactorReten S lid l t bl t hi h H 31 Solid core columns are stable at high pH
- 32. Solid Core ColumnStability – Elevated Temperature 8 9 Column Stability at 70°C Mobile phase: MeOH/H2O (65:35) Flow rate: 0.4 mL/min 6 7 8 r Column temperature: 70 °C Column: Accucore C18 50 x 2.1 mm Run time: 5 min 4 5 6 entionFactor Phenol Butylbenzene Run time: 5 min 2 3 Rete o-Terphenyl Pentylbenzene 0 1 0 2000 4000 6000 8000 10000 9,000 column volumes (400 i j ti ) 0 2000 4000 6000 8000 10000 Column Volumes (400 injections) Solid core columns are stable at high temperature 32 Solid core columns are stable at high temperature
- 33. Work Flow Solutions– Generic Methods • Used where sample throughput is critical • Compound management • Discovery DMPK • Ability to run at high flow rates without compromising chromatographyAbility to run at high flow rates without compromising chromatography • Require robust methods • Assays cannot afford to fall over • Many samples means long column lifetime • For bioanalytical samples need columns that are robust with plasma extractsFor bioanalytical samples need columns that are robust with plasma extracts • Require orthogonal chemistries • Reversed phase / HILIC etc. 33
- 34. Faster than 5and 3 µm Fully porous 5 µm, 150 x 4.6 mm Rs = 2.64 5 µL injection ∆P = 59 bar Gradient and flow rate: • Fully porous 5 μm 150 x 4.6 mm 35–60 %B in 10.0 min 1000 µL/min solvent used 17 mL1µL injection ∆P 23 b 1000 µL/min solvent used 17 mL •Fully porous 5 μm, 100 x 2.1 mm 35–60 %B in 6.7 min 210 µL/min solvent used 2.4 mL F ll 3 100 2 1 Fully porous 5 µm, 100 x 2.1 mm Rs = 1.64 1µL injection ∆P = 23 bar • Fully porous 3 μm, 100 x 2.1 mm 35–60%B in 4.0 min 350 µL/min solvent used 2.4 mL • Accucore RP-MS 2.6 μm, 100 x 2.1 mm Fully porous 3 µm, 100 x 2.1 mm Rs =1.96 1µL injection ∆P = 97 bar 35–60 %B in 3.5 min 400 µL/min solvent used 2.4 mL ACCUCORE 2.6 µm, 100 x 2.1 mm Rs = 2.50 1µL injection ∆P = 218 bar Reduced analysis time and solvent costs Minutes 0 1 2 3 4 5 6 7 8 9 10 -100 100 x 2.1 mm 34 Reduced analysis time and solvent costs
- 35. Shorter Columns –Faster Separations ACCUCORE 2.6 µm, 100 x 2.1 mm Gradient and flow rate: • Accucore RP-MS 2.6 μm, 100 x 2.1 mm Rs = 2.50 35 –60%B in 3.5 min 400 µL/min • Accucore RP-MS 2.6μm, 50 x 2.1 mm 35–60%B in 1.8 min mAU 400 µL/min Rs = 1.51 ACCUCORE 2.6 µm, 50 x 2.1 mm Double productivity with 50 mm column Minutes 0.0 1.0 2.0 3.0 4.0 35 Double productivity with 50 mm column
- 36. Example of aHILIC Separation Column: Accucore HILIC 2.6 μm, 150 x 3.0 mm Flow: 0.5 mL/min B k 290 b Separation of decitabine and α anomer impurity Backpressure: 290 bar Temperature: 40 °C Injection: 5 µL and α-anomer impurityj µ Detection: UV @ 244 nm Mobile phase: 5% A (20 mM ammonium acetate)ammonium acetate) 95% B (acetonitrile) 36
- 37. Regulatory DMPK –Bioanalysis • Methods can be optimized • Possibilities to optimize stationary phase chemistries• Possibilities to optimize stationary phase chemistries • Analysis times still important for PK studies • Methods will tend to form final clinical method • Sensitivity can be an issue due to efficacious nature of drug • Injections with plasma• Injections with plasma • Columns must not block • Can result in peak splitting • Can result in columns overpressurising • Can result in retention time shift • Metabolism studies • Need columns with high resolution 37 • Ideally limited sample prep, so columns stable with diluted urine
- 38. Lower Pressure thanSub 2m • Flow rate: 500 μL/min • Mobile phase: A Water; B Acetonitrile Sub 2 µm, 100 x 2.1 mm • Mobile phase: A–Water; B–Acetonitrile Accucore RP-MS 2.6 µm, 100 x 2.1 mm Accucore RP-MS 2.6 µm, 100 x 2.1 mm Sub 2 µm, 100 x 2.1mm Maximum pressure (bar) 171 338 Minutes 0.0 0.5 1.0 1.5 2.0 2.5 Equivalent performance, lower pressure 38 (50% lower)
- 39. Accucore with TLX– Method Conditions Sample Preparation • Acetonitrile with internal standard added to spike plasma sample • 2 (600 μL) parts acetonitrile : 1 part spiked plasma (300 μL) Precipitated sol tion mi ed on orte mi er Autosampler Method LC Method • Precipitated solution mixed on vortex mixer • Sample centrifuged and supernatant transferred to 2 mL vial • 10 μL injected onto system p • Wash Solvent A – 20% acetonitrile + 0.1% Formic Acid + 80% water • Wash Solvent B – 45%IPA + 45% acetonitrile + 10% Acetone + 0.1% Formic Acid • Mobile Phase A – 0.1% Formic Acid in water • Mobile Phase B – 0.1% Formic Acid in acetonitrile • Columns: Cyclone 50 x 0.5 mm (TFC), Accucore C18 50 x 2.1 mm (Analytical) • 100 μL sample loop, 100 μL syringe 39
- 40. Accucore with TLX– Retention Stability 4.4000 Rosuvastatin Retention 4.1000 4.2000 4.3000 3.9000 4.0000 Minutes 3 6000 3.7000 3.8000 3.5000 3.6000 0 500 1000 1500 2000 2500 Injection ~2,400 injections on Accucore column with TLX system – no change in retention 40 y g
- 41. Accucore with TLX– Backpressure Stability 140 Backpressure Plots 100 120 60 80 Bar Linear (Loading Pump Pressure at t=0) Linear (Eluting Pump Pressure at t=0) 20 40 0 0 500 1000 1500 2000 2500 Injection ~ 2,400 injections on Accucore column with TLX system – no increase in t=0 backpressure 41 y p
- 42. Accucore with TLX–PressureTraces 300 Backpressure Traces 250 150 200 Bar 100 Injection 2 Elute Injection 500 Elute Injection 2395 Elute 50 0 50 100 150 200 250 300 350 Seconds ~ 2,400 injections on Accucore column with TLX system – backpressure traces 42 y p
- 43. Greater Peak Capacitythan 5 or 3m 220 240 5 µm,100 x 2.1 mm Gradient: 65–95%B in 2.1 min, 95% B for 0.4 min 3 100 2 1 Flow rate: 400 μL/min 3 m, 100 x 2.1 mm 120 140 160 180 capacity ACCUCORE 2.6 µm, 100 x 2.1 mm 40 60 80 100 120 malisedpeakc 0 0 0 5 1 0 1 5 2 0 2 5 3 0 0 20 40 Accucore 2.6µm 3µm 5µm Norm Minutes 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Higher peak capacity – more peaks can be separated per injection 43 g p p y p p p j
- 44. More Sensitive thanFully Porous 5 and 3m Gradient and flow rate: • 5 μm, 100 x 2.1 mm 35–60 %B in 6 7 min S/N = 169 5m, 100 x 2.1 mm 35–60 %B in 6.7 min 210 μL/min • 3μm, 100 x 2.1 mm 35–60 %B in 4.0 min 350 μL/min S/N = 368 350 μL/min •Accucore RP-MS 2.6μm, 100 x 2.1 mm 35–60 %B in 3.5 min 400 μL/min mAU S/N = 399 3m, 100 x 2.1 mm S/N = 399 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 ACCUCORE 2.6m, 100x2.1mm Higher S/N ratios – detection and quantification of low level impurities Minutes 44 of low level impurities
- 45. Production - QualityControl Workflows • Robust methods • Loading capacity can be an issue as looking for impurities• Loading capacity can be an issue as looking for impurities • Selectivity important • Resolution also important • Need to be able to transfer the methods to CRO’s • Need to be aware of differences caused by different instrumentation• Need to be aware of differences caused by different instrumentation 45
- 46. Loading Capacity Columns: • AccucoreRP-MS 2.6 μm,100 x 2.1 mm • <2 μm,100 x 2.1 mm2,500,000 1 2 Effect of Loading - Accucore R² = 0.9998 R² = 0.9993 2,000,000 0.8 1 1.2 dValue A R² = 0.9721 1,000,000 1,500,000 eakarea 0.2 0.4 0.6 Normalise As N Tr As N Tr500,000 Pe 0 0 5 10 15 20 25 Load on Column (µg) 0 0 0.5 1 1.5 2 2.5 Load on column (µg) <2µm Accucore 2 6μm Competitor No loss in performance with 2 μg loaded on a <2µm Accucore 2.6μm Competitor 46 2.1 mm ID Solid core column
- 47. Method Transfer andOptimisation 5 μm, 150 x 4.6 mm Method Transfer Calculator: www.thermoscientific.com/crcRs = 2.64 5 µL injection Gradient and flow rate: • 5 μm, 150 x 4.6 mm 35 60 %B in 10 0 minRs = 2 50 35–60 %B in 10.0 min 1000 μL/min • Accucore RP-MS 2.6 μm, 100 x 2.1 mm 35–60 %B in 3.5 min Rs = 2.50 400 μL/min • Accucore RP-MS 2.6 μm, 50 x 2.1 mm 35–60 %B in 1.8 minutes 400 μL/min ACCUCORE 2.6 μm, 100 x 2.1 mm Rs = 1.51 1µL injection 400 μL/min ACCUCORE 2.6 μm, 50 2 1 1µL injection Minutes 0 1 2 3 4 5 6 7 8 9 10 0 50 x 2.1 mm Scalable from fully porous 5 μm columns µ j 47 Scalable from fully porous 5 μm columns
- 48. System Considerations • Column:Accucore RP-MS 2.6 μm, 100 x 2.1 mm • Gradient: 65–95 % B in 2.1 min Dwell volume: 100 µL 95 % B for 0.4 min • Flow rate: 400 µL/min Accela 1250 Dwell volume: 800 L Surveyor Accela Surveyor Agilent 800 µL Minutes 0.00 1.00 2.00 3.00 4.00 Accela 1250 Surveyor Agilent 1100 Run time (min) 2.5 3.0 3.5 Dwell volume: 1000 µL min0 0 5 1 1 5 2 2 5 3 3 5 Agilent 1100 Average PW (1/2 Height) 0.02 0.02 0.04 min0 0.5 1 1.5 2 2.5 3 3.5 Solid core can deliver performance on a b f diff t t 48 number of different systems
- 49. System Considerations • Minimisevolume dispersion Always Optimize System Configuration • Tubing–short L, narrow ID • Low injection volume • Low volume flow cell• Low volume flow cell • Optimise detector sampling rate Need enough points to define peak (minimum of 10, >20 for quantitation) 5 pts Fast scanning MS • Low dwell volume pump for fast 45 pts 9 pts Low dwell volume pump for fast gradients 49
- 50. Analyzing Biomolecules • Moveto produce bigger molecules • Difficult to copy• Difficult to copy • Greater success rates • Chromatography requirements • Need less retentive phase • Need wide pores to cope with larger molecules• Need wide pores to cope with larger molecules 50
- 51. Peptides – Resolution& Peak Shape RT: 0 11 15 03RT: 0.11 - 15.03 1.12 8.74 3.76 7.09 13.468.91 11 20 Accucore 150-C18 11.20 13.63 1.01 C ti l S lid C C18 1.01 8.52 6.983.60 13.13 15.0310.84 Conventional Solid Core C18 100000 10.20< 2 µm Wide Pore Fully Porous C18 0 50000 uAU 8.491.59 14.4912.035.25 14.31 y 2 4 6 8 10 12 14 Ti ( i ) -50000 51 Time (min)
- 52. Proteins – ExcellentResolution vs < 2 µm Wide Pore • Sharper and higher peaks 80000 100000 Accucore 150-C4 Backpressure: 185 bar higher peaks than < 2 µm Wide Pore Fully Porous C4 40000 60000 uAU Porous C4 • Better resolution and sensitivity 100000 0 20000 • Significantly lower backpressure60000 80000 100000 < 2 µm Wide Pore Fully Porous C4 Backpressure: 320 bar p 40000 60000 0 1 2 3 4 5 6 7 8 9 10 Ti ( i ) 0 20000 52 Time (min)
- 53. Conclusions • The PharmaceuticalProcess • Mapping out different sectors Wh t t f h ll f d• What types of challenges are faced • Understanding the drivers in the pharmaceutical industry • Solid Core Chromatography • Understanding the benefits of the technology • Bar for bar greater efficiency• Bar for bar greater efficiency • How to optimize the separation • Understanding chemistry • Optimization of the morphology • Coupling Solid Core Chromatography to Pharmaceutical Analysis• Coupling Solid Core Chromatography to Pharmaceutical Analysis • Design of new workflows • Improving column lifetimes 53 • Improving assay robustness