• Share
  • Email
  • Embed
  • Like
  • Save
  • Private Content
Purification process speed-and efficiency-Hayward-etal
 

Purification process speed-and efficiency-Hayward-etal

on

  • 841 views

 

Statistics

Views

Total Views
841
Views on SlideShare
789
Embed Views
52

Actions

Likes
0
Downloads
25
Comments
1

2 Embeds 52

http://www.linkedin.com 51
https://www.linkedin.com 1

Accessibility

Upload Details

Uploaded via as Adobe PDF

Usage Rights

© All Rights Reserved

Report content

Flagged as inappropriate Flag as inappropriate
Flag as inappropriate

Select your reason for flagging this presentation as inappropriate.

Cancel

11 of 1 previous next

  • Full Name Full Name Comment goes here.
    Are you sure you want to
    Your message goes here
    Processing…
  • Holistic approach to making compound purification fast and efficient
    Are you sure you want to
    Your message goes here
    Processing…
Post Comment
Edit your comment

    Purification process speed-and efficiency-Hayward-etal Purification process speed-and efficiency-Hayward-etal Presentation Transcript

    • Utilizing LC & SFC separations with UV, ELSD & MS detection for purification in drug discovery: Driving toward capacity, quality, efficiency, and rapid turnaround Xu Zhang, David P. Budac, Qing Ping Han, and Mark J. Hayward Lundbeck Research USA Paramus, NJ
    • The need for process efficiency and quality Med chemists, on average, spend ≥50% of their time on purification, even when state of the art tools are provided. 20 medicinal chemists means ≥10 FTE effort in purification. If 1-2 experts could do all the purification, a huge human efficiency increase in medicinal chemistry can be realized. Time is of the essence in drug discovery (LO cycle time). So, turnaround time must be fast (high need: hand crafted cmpds). Losing compounds costs a lot of Med chemist time. Thus, quality (minimized losses) has crucial impact on efficiency. Med Chem compounds cost ≈2k$ ea. (total FTE cost / average # of compounds). You can’t afford not to make every effort not to lose them (Aim for highest success rate or at least 6 sigma). Don’t be penny-wise (on instrument, solvent, or salary) and then pound foolish on the big $ = compounds. Quality and speed are crucial in every respect! 1Lundbeck Research USA Chemistry – Analysis and Purification
    • Purification – needs and goals Early Drug Discovery Full coverage (Med Chem definition) Hit to Lead Parallel Synthesis – 40+ mg, approximately 20-100 compound/batch, every few days Lead Optimization – hand crafted for in vivo – 100 - 500 mg, some requiring ultra high purity, approximately 20+ compound/batch daily Development Candidate Candidates – Up to 50 g with at least 10 g at ultra high purity, 2 day turnaround on 20 g, 20 - 25 compounds/yr Variety of sample quantities – 10s of mg to 10s of grams Variety of sample qualities Sample purities range from 5 to 95% prior to purification. Impurities may or may not be baseline resolved according to OA-LCMS (levers needed).* Dissolution remains an ongoing challenge.* Above needs define scale or capacity! Success requires following capabilities: 100 mg per injection 20 – 120 compounds/day *Experts are crucial ≥120 injections & collections/day in addressing these ≥0.5 g/hr in full gradient mode challenges 2Lundbeck Research USA Chemistry – Analysis and Purification
    • Given the needs and goals there are some significant challenges• Operational philosophy must be simple and streamlined yet diverse enough to cover most chemical space and keep quality high – Integrate Med Chem, analysis, purification, drying, and Compound Management into the way of working – Trusted partnership throughout the process is crucial for efficiency• Mass per injection is 5 fold higher than norm (LC/MS based) – Adapt off the shelf components to increase capacity• Cycle times are 3 fold faster than norm (LC/MS based) – Apply fast LC techniques• FTE load must be low to achieve desired gains in efficiency – Automate everywhere possible for tasks and transparency of data• Med Chemists already have their own instrumentation. Why should they come to you? – Must be able to do it much better / faster than Med Chemists – Must gain the trust of the Med Chemists 3Lundbeck Research USA Chemistry – Analysis and Purification
    • Purification Operational Philosophy Tried and true technique: get a quality separation at analytical scale, then scale up (50 fold in volume). Required to achieve high success rate. Nevertheless, must be able to adapt at prep scale 2 x 2 x 2 matrix of gradient fast analytical LC methods adapted to the preparative scale (needs more levers): 2 columns – C18 and C8. 2 gradients – C18 gradients favor moderate LogP to polar compounds and C8 moderate to high LogP 2 pHs – 4 and 6.5 (extremes rarely needed). Crucial efficiency component: align with OA-LC/MS and achieve high Med Chemist competence (simplicity) Routine 100 mg per injection & Gaussian peaks. Success rate as close to 100% as possible (measure and collect waste, if needed). Automated with optimized conditions built into predictable, calibrated methods. 4Lundbeck Research USA Chemistry – Analysis and Purification
    • Operational Philosophy: Why use analytical data and scale up?In many cases,only data from the HNJ_19813-127-002_03 18:38:1305-Jun-2006 2487 TUVactual sample 100 Waste stream 3.38 An2 1.99e6allows accurate after fractionthreshold collector when 1.86 collection works 2.16prediction due to % perfectly 4.18 4.31earlier eluting 0.58 1.47 2.64 2.95peaks and 0baseline rise from 0.50 HNJ_19813-127-002_03 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 2: Diode Arraylow level 1.0e-1 Collected peak 3.38 229.9 254 Range: 1.007e-1impurities. 8.0e-2 Stream prior 6.0e-2 to fraction 1.85Losing collector AU 238.9 2.47 4.0e-2 2.15 228.9compounds is not 227.9 4.18 4.32 2.0e-2 2.93 228.9 228.9a viable option for 209.9achieving 0.0 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 Time 5.00increased speed. Background levels at ~15% peak height (UV & MS) 5Lundbeck Research USA Chemistry – Analysis and Purification
    • 8 LC/MS method choices: 2 x 2 x 2 polarity matrix (3 binary choices = simplicity) Neutral-pH~7 Most likely: pH choice mod LogP & polarity,(Ionization/Polarity) neutral pH (for CNS cmpds) Acid-pH~4 C8 Low polarity Chemical space: & high LogP More typical pH dimension (polarity) is trulyColumn choice orthogonal to column/ gradient (Polarity) (see Thu talk) Column/gradient choices allow Highly polar access to extremes & optimization (H2O soluble) in mid regionnot as commonsome intermediates C18 Low Organic Moderate Organic High Organic C18 only C8 & C18 C8 only Gradient choice (Polarity) 6 Lundbeck Research USA Chemistry – Analysis and Purification
    • 2x2x2 approach: Optimize pH & polaritySimple technique to achieve optimal separation:•Send molecules through column un-ionized (adjust pH of sample and mobile phase).•Select column/gradient combo in order to achieve near 1.0 minute retention time. Goal: sharp peaks in the middle 60% of the chromatogram (0.4min < RT < 1.6min) Broad Peak(s) Solution: Elutes too early 1. Change pH, then Elutes too late Solutions: re-evaluate RT / polarity Solutions: 1. C8 C18 Column 1. C18 C8 Column 2. Less organic gradient 2. More organic gradient 3. Change pH 3. Change pH 7Lundbeck Research USA Chemistry – Analysis and Purification
    • Operational philosophy: Med Chemistsperform the pre-analysis used for purification • Why make Med Chemists do all that work? – Our Med Chemists perform OA-LC/MS 11 times per day (avg) • Following reaction progress (>90%) and final products (<10%) – They want to do it. They already do it = efficiency • How will Med Chemists know which method to use? – We train them – Med Chemists can become highly competent in the use of OA- LC/MSs choosing column, gradient, & pH (2 x 2 x 2) – Binary choices often sufficient to allow expert results without being expert • Med Chemist entry point for purification – OA-LC/MS data where one can say “I want that peak or those peaks in bottle(s)” and structure of compound – Highly interactive process with purification expert – Trusted partnership throughout the process is crucial for efficiency – Making excellent progress toward same approach with SFC 8Lundbeck Research USA Chemistry – Analysis and Purification
    • Challenges of 100 mg/injection Compared to analytical scale, injection mass increased 105 fold but mobile phase volume for separation can increase only about 50 fold Maintaining speed and resolution requires compensation in the chromatographic system. Injection process must be adapted for high load. Adsorption and buffering capacity must be adjusted for high load. Collection volume and separation time can limit number of compounds collected High separation efficiency (analytical like) must be routine to keep collection volumes reasonable. High separation efficiency (fast analytical like) must be routine to keep cycle times reasonable. Additional processes must be automated Adapted injection process must be automated. Extra adsorption / buffering capacity must be on-line. Automatic column switching & regeneration. Solvent and waste handling must be streamlined 9Lundbeck Research USA Chemistry – Analysis and Purification
    • Challenges of apparent mass-overload Ionized Un-ionized form form 100 mgDoxylamine(pKa 8.7): 25-100 mg 50 mginjection. NMobile phasetemperature: 25 mg O45°C. ° NBuffer: 0.2%formate.Mobile phasepH = 6.5 Conventional Wisdom: Higher mass loading kills chromatographic performance! Thus one injects lower mass (10-20 mg: common LC/MS scale). 10Lundbeck Research USA Chemistry – Analysis and Purification
    • Achieve 100 mg/injection Injection via “at column dilution” Deliver fully-dissolved sample to column – improve mass and volume loading Completely separate sub-system (pump, valves), enabling sample-dependent injection method selection from software High separation capacity Larger diameter column (30 mm) provides enough stationary phase surface area to retain compounds Higher temperature improves adsorption kinetics Higher buffer concentration enhances buffer capacity Completely separate buffer mixing sub-system enables buffer capacity selection within the separation methods software High separation efficiency Higher temperature allows for lower back pressure and faster, higher velocity separations Use all other known techniques, i.e. minimize extra-column volume, small particles (3 µm) On-line back-flush maintains column condition (>>2000 injections) and eliminates delay time for column re-equilibrium 11Lundbeck Research USA Chemistry – Analysis and Purification
    • RP-LC Purification System Schematic All components under full software control (MassLynx V4.1) [except automatic heaters] Back-flush heater SunFire C18 regenerating pump XBridge Back-flush solvent selections Column C18(ACN, 5% acetic acid and DMF) Makeup pump water bath Inertsil C8 Photo diode Inertsil splitter C18 array 6-pos. column selectors choose up to 6 column Dilution solvent selections (ACN, chemistries 50/50 ACN water mixture, etc..) MS ELSD heater At column dilution pump Fraction Injection collector port Waste UV heater Concentrated buffers at 1-4 M: NH4COOH, NH4COOCH3, CH3COOH, NH3, H2O etc… Waste level sensor and auto switcher Concentrated buffer pump mixer MilliQ Gradient water B A purification and auto- Binary pump delivery system Waste barrels Degassers AcetonitrileLundbeck Research USA Chemistry – Analysis and Purification 12
    • NP-SFC Purification System Schematic Most components under full software control (MassLynx V4.1) Back –flush/regeneration heater AD P50 CO2 + P50 Modifier (awaiting software) OD Columns 5-6: 515 515 Ethyl pyridine – 5 AS Variety of columns - 6 heater OJ Splitter heater 6-pos. column selectors Dilution solvent selections Photo diode choose up to 6 column (alcohols, 50/50 alcohols and CO2 Array-2996 chemistries mixture, etc..) MS-ZQ ELSD-2420 heater At column dilution pump (Thar analytical FDM) Fraction Injection collector port SIII CO2 vent Waste UV (2487) G Make up PR 40 PSI Diethylamine, triethylamine, isopropylamine, L ammonium formate, formic acid etc in alcohols pump S heater Concentrated buffer Waste level sensor pump (515) and auto switcher mixer (front panel control) P-200 P-50 CO2 modifier G700 with Bulk Tank Degassers Alcohols Waste barrels Much of our SFC design philosophy comes from our established approach toward RP-LC/MS based purification 13Lundbeck Research USA Chemistry – Analysis and Purification
    • LC System Photo: overall picture 14Lundbeck Research USA Chemistry – Analysis and Purification
    • LC System Photo: pumps (5) picture 15Lundbeck Research USA Chemistry – Analysis and Purification
    • LC System Photo: injector/collector (combo), columns, UV detectors (2) picture 16Lundbeck Research USA Chemistry – Analysis and Purification
    • LC System Photo: columns & detectors [MS, ELSD & UV (2)] picture 17Lundbeck Research USA Chemistry – Analysis and Purification
    • LC/MS & SFC/MS System Photos: note on vacuum pump ergonomics There are noise abatement solutions that work. (required in a Danish lab) 18Lundbeck Research USA Chemistry – Analysis and Purification
    • SFC Instrument photo (viewed from right) 19Lundbeck Research USA Chemistry – Analysis and Purification
    • Instrument photo (viewed from front) 20Lundbeck Research USA Chemistry – Analysis and Purification
    • “At Column Dilution” Approach for Sample Injection Nature of injection instantaneous >10x dilution! Choice of dilution solvent can have big impact on keeping samples in solution as injection mass increases and can help chromatographic performance Goal: Deliver sample to stationary phase as individual molecules in solution (best way: separate pump) Flow parameters are important Flow rate must be sufficiently high to deliver sample without introducing band-broadening (7.5 mL/min into 100 mL/min). Diverting at column dilution solvent after injection process can be helpful to eliminate the effects of injection solvent on the separation (divert at 0.3- 0.5 min). Dilution solvent composition is an untapped resource for scaling up injection mass 100% B not always universal best choice 50/50 clearly much better in about half the cases Binary choice (50%/50% A/B & 100%B) covers small scale (100mg inj) Further refinement of %B and buffering can be worthwhile for larger scales where ≥200mg injections are desired (it’s all about solubility) 21Lundbeck Research USA Chemistry – Analysis and Purification
    • At Column Dilution: RP-LC Flow Rate & Divert Elevated flow rate toavoid wasting time and 8 mL/min 2 mL/minminimize band-broadeningbefore column. Target: injector sweeptime 15 s max, 8 s typical. 1 mL/min Elevated flow rate alsohelps prevent sample loss. Diverting “at columndilution” flow also can 50:50:H2O/ACN 1% acetic Acidimprove separation byallowing dissimilarinjection and separation Nsolvents. N ACN Improved solubility bylowering pH eliminatesinjection precipitation Example: 200 mg imipramine 22Lundbeck Research USA Chemistry – Analysis and Purification
    • At column dilution: all the same applies to SFC Example: challenging chiral resolution 100 mg injection (all aspects mirror RP-LC, i.e. goal is solubility) 60742-048-010-boc-t79 2: Diode Array 2.24 250 Range: 1.379 1.25 100% B injection 1.0 7.5e-1 AU 2.67 Classic sign of precipitation 5.0e-1 (in addition to pressure spike): 2.5e-1 Second peak for same analyte 0.0 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 60742-048-010-boc-t94 2: Diode Array 2.54 250 1.25 Range: 1.315 1.0 50/50 CO2/MeOH injection 7.5e-1 (best choice in about half of cases) AU 5.0e-1 3.62 2.5e-1 0.0 Time 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 Already purified by RP-LC: we know that only 2 enantiomers present 23Lundbeck Research USA Chemistry – Analysis and Purification
    • Separation capacity and efficiency (LC & SFC) Column particle size and diameter: Smaller particles (3-5 µm) enhance surface area, adsorption capacity, separation efficiency, and speed. We have tested 3 µm particles at prep scale and they work as well as they do in analytical scale However, we have not found suppliers that pack 3 cm columns with 3 µm particles consistently (but we would like to) Thus, we use 5 µm particles for all prep scale work 30 mm diameter consistently provides enough capacity for 100 mg injections under “infinite diameter” conditions (1 mL injections typical, 2 mL max). Column length: Column length is an expensive and slow way to gain resolution Column cost approximately proportional to length Separation time approximately proportional to length Stationary phase (SFC) and eluent choices (RP-LC & SFC) are the most time effective way to achieve resolution We find 50 mm length optimum for RP-LC Adjusting eluent conditions can be done much faster to achieve resolution at this length (total time starts to increase at shorter lengths) More in buffering section We find 100 mm length optimum for SFC We haven’t found shorter chiral columns to be available (but we would like to) 24 Lundbeck Research USA Chemistry – Analysis and Purification
    • Separation capacity and efficiency Temperature: A crucial parameter that affects adsorption / desorption rate, and thus must be properly controlled: Four independent heaters used: Mobile phase heater (up to 400 Watts applied, J-KEM Sci.) Column heater (water bath kept at temp of mobile phase) Dilution solvent heater (up to 20 Watts applied, Sererity). Back-flush solvent heater (up to 80 Watts applied, J-KEM Sci) Benefits of temperature control: Improved peak shapes due to faster adsorption kinetics fronting = missed adsorption opportunities and tailing = delayed desorption Significant selectivity changes also possible for SFC. Maintain highly concentrated samples in solution. This can be especially crucial during the injection process. Reduces back pressure allows higher flow rates and faster runs combination of solvent choice / temp = speed for RP-LC! 25Lundbeck Research USA Chemistry – Analysis and Purification
    • RP-LC Temperature Effect on Peak Shape Reserpine 55oC More heat! 45oC o O = more mass transfer 35 C 25oC N N = less fronting H O O O O O O O O 0.5 26Lundbeck Research USA Chemistry – Analysis and Purification
    • RP-LC: Temperature Effect on Speed• To achieve resolution, one generally must have retention – No retention = no separation • elutes in void volume – Must sweep multiple column volumes (k’) • k’ must be greater than 2 • 5 < k’ < 15 very often optimal & ∆%B / k’ should be <5 (LSS)• To get resolution fast one must sweep column volumes quickly (k’/min ≥ 3 whereas typically k’/min ≤ 1 prep scale) – Maximize/increase velocity (flow) while maintaining mod ∆%B / k’ – Tune temperature to match velocity – Make good choices (required to achieve first 2): • Column type – polymer vs. silica/BEH • Mode of operation – isocratic (static) vs. gradient (linear sweep from A to B) • Solvent (B) – acetonitrile (ACN) vs. methanol (MeOH) 27Lundbeck Research USA Chemistry – Analysis and Purification
    • RP-LC: Velocity vs. temperature for: elution mode and column choice Optimal Velocity vs. Temperature 160 Fit gradient ACN - silica and BEH Observed gradient ACN - silica and BEH BEH or 135 Isocratic ACN silica - Guiochon etal polymer Separation Temperature (C) only (particle Iso (& grad) MeOH silica - Guiochon etal 110 stability) Isocratic (& grad) ACN polymer - Carr etal 85 MeOH Gradient ACN Limited stability for silica 60 High Velocity BEH or silica with Range where both reduced H2O 35 analyte and silica content only That’s stability are well (not polymer) Why established 10 ACN! 0 5 10 15 20 25 30 Optim um Eluent Velocity (m m /s) 28Lundbeck Research USA Chemistry – Analysis and Purification
    • RP-LC: Scientific case for gradient ACN operation• Temperature offers much greater ability than other techniques to achieve higher velocities• Gradients with acetonitrile are truly fast! – Optimum velocity with gradient ACN is much more responsive to temperature elevation than any other mode of operation – Isocratic operation is at least 3 fold slower than gradient (ACN) – Other solvents (alcohols) behave like isocratic operation even in gradient mode (still >3 fold slower) – ACN gradients appear to be uniquely crucial to achieving high productivity! 29 Lundbeck Research USA Chemistry – Analysis and Purification
    • RP-LC: Acetonitrile (ACN) usage – Why?• We use gradient ACN for purification Let’s compare methanol use with ACN – One third velocity or 3 fold more time (35 vs. 100mL/min @50°C) – Half the solubility (loading) or double the number of injections – Total 6 fold loss in productivity! (add instruments and FTEs!) – Double injections = double solvent = 12k$/mo (vs. 20k$/mo for ACN) (also doubles waste volume) – Lower organic strength = lower column lifetime (more column cost) & less reliability – Overnight runs = lower reliability – Lower reliability = lower quality & lost compounds – Must also change OA-LC/MS to MeOH (Med Chem disruptive)• Conclusion: the expense of 6 fold loss in productivity and lower quality would seem to thoroughly outweigh the potential 8k$/mo in savings on solvent 30Lundbeck Research USA Chemistry – Analysis and Purification
    • SFC: Effect of separation temperature on carbamazpine peaks carbamazepine-4 oC 40 oC oC 2: Diode Array 30 2.25 50 230 Range: 5.515 5.0 4.0 3.0 AU 2.0 1.0 Time 2.10 2.15 2.20 2.25 2.30 2.35 2.40 2.45 2.50 Peak shape doesn’t change much with increasing temperature compared with RP HPLC condition (note opposite effect on retention). However, temperature can still be very helpful with selectivity (see next). 31Lundbeck Research USA Chemistry – Analysis and Purification
    • SFC: Temperature tuning the separation ATemperature candramaticallyenhance Bselectivity,sometimes inunexpected ways C D °Mixture of endo/exo isomers/enantiomers at (A) 30, (B) 40, (C) 50, and (D) 60°C 32Lundbeck Research USA Chemistry – Analysis and Purification
    • RP-LC Mobile Phase Buffer Capacity Background: Goal: send desired compound through column un-ionized. Buffer must be more concentrated than analytical scale because sample is more concentrated. Don’t over rely on aqueous pKa and Henderson–Hasselbach equation to know ionization state – Use peak shape! (see talk on Thu) KEY MESSAGE: must view un-ionized state loosely, as pKa shift or anion complex with buffer can be sufficiently un-ionized for good separation peak shape for basic drugs where pH < pKa (pKa - pH = 2-4 is OK!). i.e. more buffer goes a long way toward reducing analyte charge. Practical Approach: Mix buffer on-line like “at column dilution:” Flow rate proportional to buffer concentration. Valve makes it easy to have 6 buffers on-line & method selectable. Target high pH and buffer concentration for high pKa compounds: pH 4 and 6.5 can cover a very full range of drug-like compounds. Lower buffer concentration (0.2%) usually works well for bases when pH > pKa. Also, more quickly removed during drying process. Higher buffer concentration crucial for high mass loading. Higher buffer concentration (1%) at pH 7.5 usually works well with high loading of stronger base intermediates when pH ≤ pKa. 33Lundbeck Research USA Chemistry – Analysis and Purification
    • RP-LC Mobile Phase Buffer Capacity Effect on peak shape & peak capacityDoxylamine [formate] = 260 mM (1%)pKa = 8.7100 mginjection.Mobile phasepH = 7.5 [formate] = 130 mM (0.5%)Mobile phasetemperature45°C ° N O [formate] = 52 mM (0.2%)Buffer needsto reach 10x Npeakconcentrationto correctpeak shape 34Lundbeck Research USA Chemistry – Analysis and Purification
    • RP-LC Mobile Phase Buffer Capacity Direct effect on loading and relative to pH shift Amitriptyline @ pH = 7.5 (pKa = 9.2) Buffer capacity (buffer concentration) Peak width @ 0.7 base (min) 100 mg has a large impact on 0.4 50 mg column loading (peak 25 mg width). 0.1 0.0 0.5 1.0 Buffer concentration (%) 100 mg Amitriptyline (pKa = 9.2) 1.0 The effect on loading Peak width @ base (min) 0.7 pH 6.5 from buffer pH 7.5 concentration can be 0.4 considerably larger 0.1 than that of the pH 0.0 0.5 Buffer concentration (%) 1.0 effect. 35Lundbeck Research USA Chemistry – Analysis and Purification
    • RP-LC peak shape / loading with pH: 3 distinct states 3.15 6.0e-1 AU 4.0e-1 pH = 7.5 1 st pH range with distinct chromatographic behavior: 2.0e-1 Basic compound exists as free base despite buffer 0.0 Time 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 pH = 6.5 Small 3.12 6.0e-1 transition pH being at or below the aqueous pKa. Buffer 4.0e-1 from a big concentration does not affect chromatographic AU 2.0e-1 change in pH. behavior (linear Langmuir behavior). Acetonitrile 0.0 0.50 1.00 1.50 2.00 2.50 3.00 3.50 Time 4.00 likely mitigates protonation. Peak shape and loading still suggest linear Langmuir behavior. 3.07 6.0e-1 pH = 5.5 4.0e-1 AU 3 rd pH range with distinct chromatographic behavior: 2.0e-1 0.0 0.50 1.00 1.50 2.00 2.50 3.00 3.50 Time 4.00 Distinctive peak shape, loading pattern and analyte state. Retention increases with loading (anti- 6.0e-1 pH = 5.0 Langmuir behavior). Analyte is likely to be at least 4.0e-1 2.78 AU 2.0e-1 partially protonated, but buffer anions may form 0.0 Time neutrally charged complex when present in sufficient concentration. Rapid equilibrium seems to 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 6.0e-1 pH = 4.5 4.0e-1 2.20 result in behavior like non-ionized complex (not AU 2.0e-1 protonated base) even at the effective pKa ≈ 4.5 in 0.0 0.50 1.00 1.50 2.00 2.50 3.00 3.50 Time 4.00 eluent. 6.0e-1 pH = 4.0 Big transition 4.0e-1 1.53 from a small 2 nd pH range with distinct chromatographic behavior: AU q e F u n r l t y o p i v d s i t l c u v y e x m s a 2.0e-1 change in pH. Basic compound primarily exists as protonated 0.0 Time base, buffer anions provide charge shielding (nonlinear Langmuir behavior). 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 6.0e-1 pH = 3.5 1.12 4.0e-1 AU 2.0e-1 0.0 Time 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 Noscapine at 25 and 100 mg loading at 20% ACN 60% ACN different pH conditions. (Aqueous pKa = 7.8)Lundbeck Research USA Chemistry – Analysis and Purification 36
    • Another practical aspect of getting the right buffer concentration FEB2007_317 2: Diode Array 0.55 Un-retained desired compound 254 Range: 5.028e-1 4.0e-1 3.0e-1 AU 2.0e-1 1.53 1.0e-1 2.12 (a) 1#1,1:10 1.78 1 0.0 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 FEB2007_321 2: Diode Array 1.52 254 Range: 5.28e-1 4.0e-1 3.0e-1 AU 2.0e-1 1.22 1.0e-1 1.82 2.17 (b) 0.98 1#1,2:2 1 0.0 Time 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 Chromatograms of a compound synthesized in-house. (a) 60 mg loading with 48 mM of formate (b) 80 mg loading with 96 mM of formate. 37Lundbeck Research USA Chemistry – Analysis and Purification
    • Another practical aspect of getting the right buffer concentration (selectivity) 1.2 1.0 8.0e-1 2.38 6.0e-1 4.0e-1 1.53 2.72 3.62 2.0e-1 0.75 (a) 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 2.23 1.2 1.0 8.0e-1 Impurity resolved 6.0e-1 4.0e-1 1.57 2.75 3.63 2.0e-1 0.87 2.43 (b) Time 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 Chromatograms of a compound synthesized in-house. (a) 40 mg loading with 48 mM of formate (b) 55 mg loading with 96 mM of formate. Mobile phase pH: 6.5 38Lundbeck Research USA Chemistry – Analysis and Purification
    • While we don’t understand the chemistry as well as we do RP-LC (yet), buffering also helps SFC 1 0 0 m g , 5 % to 2 0 % M E O H + 0 .2 % D E A , 1 0 0 G /M IN , b p 1 2 0 , s p 3 0 0 ,4 0 o C im p r a m in e - 8 2 : D io d e A r r a y 2 .9 1 320 R a n g e : 3 .7 3 6 e -1 No additive 2 .7 5 e -1 2 .5 e -1 Imipramine 2 .2 5 e -1 2 .0 e -1 0.2% DEA 100 mg injection 1 .7 5 e -1 A U 1 .5 e -1 1 .2 5 e -1 1 .0 e -1 7 .5 e -2 5 .0 e -2 2 .5 e -2 0 .0 T im e 1 .6 0 1 .8 0 2 .0 0 2 .2 0 2 .4 0 2 .6 0 2 .8 0 3 .0 0 3 .2 0 3 .4 0 3 .6 0 3 .8 0 4 .0 0 Buffering can help a lot with peak shape under high loading conditions SFC peak shape becomes much better with adding 0.2%DEA in MeOH. 39Lundbeck Research USA Chemistry – Analysis and Purification
    • Other Automation Features: Column Back-flush Regeneration – a simple but crucial component for success Without back-flushing, columns show increased peak width in as few as 50 injections Benefits of back-flushing column Prolongs lifetime of columns; >4000 injections (> 400 g) without loss of performance (increased peak width). Prevents carryover and pressure gain. Much more consistent performance. Allows for re-equilibration of column prior to starting next cycle, i.e. no time lost. Back-flushing technique Gradient back-flush repeated 3 times over duration of run Flow rate of 20 mL/min sufficient (1/5th of prep flow) Acidic buffer (5% acetic acid in water removes bases well) and organic (ACN) removes lipophilic compounds. Resolution difference between columns (in pair) may be ideal way to evaluate condition (significant difference = dead column) DMF is a quick way to dislodge nitrogen containing tarLundbeck Research USA Chemistry – Analysis and Purification 40
    • Other Automation Features (LC & SFC): ELSD collected mass estimation• ELSD Characteristics Mass based detection (not concentration) Fairly analyte independent +/- 20% accuracy readily achievable Automated inclusion in FractionLynx reportLundbeck Research USA Chemistry – Analysis and Purification 41
    • Other Automation Features (LC & SFC): Immediate access to data by Med Chemists• FractionLynx (FL) Reports captured by NuGenesis SDMS Our pipeline the the Med Chemist ELN Automated by printing FL browser reports to SDMS. Report includes waste UV chromatogram to show compound was collected (not lost). This (and ELSD mass) builds data driven trust with Med Chemists Convincing nature of data presentation minimizes need for post purification QC for ordinary compounds (95% purity threshold cmpds). Lundbeck Research USA Chemistry – Analysis and Purification 42
    • RP-LC Other Automation Features: Water plumbed directly to point source • Benefits of making buffers on-line Greater selection (6) Far less labor: people handle only small volumes of concentrated buffer Software select buffer concentration Achieves best water quality direct from Millipore Gradient Water circulates in ceiling & loop is tapped at point of useLundbeck Research USA Chemistry – Analysis and Purification 43
    • SFC has analogous set up of “A” solvent CO2 source photosLundbeck Research USA Chemistry – Analysis and Purification 44
    • LC - Other Automation Features: Waste collected in drums w/ automatic switching• Waste set up: 30 gal. drum (110 liter) Keeping drums in ventilated cabinet achieves best safety & aesthetics. Simple industrial level sensor detects full & switches to stand-by drum. Simple industrial level sensor detects full & switches on blue light to indicate need to replace drum. Entire waste handling process can be maintained by anyone. SFC process is same but waste package volume reduced to 20 liters and is easily placed under lab bench (smaller sensor used) Lundbeck Research USA Chemistry – Analysis and Purification 45
    • LC - Other Automation Features: Waste collected in drums w/ automatic switching• Waste set up: 110 liter (30 gal) drum is best balance between capacity & move-ability (55 gal. drums would work in same set up). 30 gal. capacity allows a full (24 hr) day of operation before drum must be replaced (2nd drum full). Thus, waste management workflow is decoupled from LC/MS workflow. Use of DOT approved containers & labels allowed us to shift drum removal & replacement to night time cleaning staff. Lundbeck Research USA Chemistry – Analysis and Purification 46
    • LC & SFC drying fractions • Med Chemists perform drying and downstream aspects through delivery to compound management • Med Chemists and Analytical own the process • We have used lean 6σ approach to streamline our way of working • We have found a simple, time efficient way to remove buffers and water without extra heating • We have 3 collection packages and workflows depending on mass purified that rapidly move compounds to transfer to compound management (CM) • Automated SD file generation for registrationLundbeck Research USA Chemistry – Analysis and Purification 47
    • RP-LC: Drying fractions - volatile buffer removal • Can be washed, but that is a manual approach = laborious • Can use more heat and time (roto-vap, Biotage V-10, or Genevac), but that may not be good for compound and we want faster not slower (8-12 hr Genevac) • Alternative: dry down to viscous goo at 35°C achieving approximately 95% volume reduction (2-3 hr in Genevac), then re-dissolve in pure acetonitrile – High organic content drives off buffer and water first • Dry again (4-5 hr in Genevac) – Extra step for Genevac but less total time – Result: no formate, acetate, or water (by NMR) and 1-2% residual acetonitrile – Easily achieved / automated with V-10 using acetonitrile as wash solvent or by adding pure acetonitrile tube at end of batch *volatile buffers are easily removed with a single pass dry for SFC fractions because there is no waterLundbeck Research USA Chemistry – Analysis and Purification 48
    • LC & SFC collection packaging: operating with downstream process in mind• Libraries (≤50 mg): collect directly into pre-tared, bar- coded 16 x 100 mm tubes accepted by CM in Genevac racks (Genevac to dry = done)• Singletons (≤2 g approx): collect into EPA tubes w/ one tube for each 100-300 mg injection V-10 xfer & dry to done into one or two pre-tared, bar-coded 4 mL tubes accepted by CM• Multi-gram: (≥10 g) collect into 500 mL jars rotovapLundbeck Research USA Chemistry – Analysis and Purification 49
    • LC & SFC drying tools: 3 needed• Genevac for libraries – 100% next day• Biotage V-10 for singletons (≤1 g) – Like Genevac with automatic pipetting and serial work flow – Acetonitrile tube added to drive off buffer / H2O at low temp (35°C) – Multiple tubes transferred to one or two 4 mL pre- tared CM tube – >75% same day• Roto-vap for multi-gram – Mostly next dayLundbeck Research USA Chemistry – Analysis and Purification 50
    • RP-LC purification example I: Crude Synthesized Product Analytical result submitted samplePurification performed Prep LCusing a neutral pH method purificationand C18 columnTemperatures: 40 o C Analytical result purified sample 51Lundbeck Research USA Chemistry – Analysis and Purification
    • RP-LC purification example II: Closely Eluting Species with High Background 11:47:0623-Aug-2006 MAJG_42700-018-001_03 2487 TUV 2.88 An2 100 2.28e6 2.45 Waste streamPurities of after fraction>95% are % collector 3.84 2.27routinelyachieved 0 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 MAJG_42700-018-001_03 Collected peak 2: Diode Arrayfor samples 2.88 250.9 254 Range: 7.557e-1such as 6.0e-1 Stream prior to fractionthese. 4.0e-1 AU collector 2.68 2.0e-1 2.48 209.9 209.9 0.0 Time 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 Background levels at >20% peak height (UV & MS) 52Lundbeck Research USA Chemistry – Analysis and Purification
    • LC purification example III: 200 mg Injection of Development Candidate Full Scale Same Data 2% Scale Concentrations of fractions 10-20mg/mL. On cooling crystals fall out of solution.Typical injection for purification of gram quantities of material. In this case 8grams were purified in 4 hours for ultra high purity (no visible impurities fortoxicology study). Level of recovery was >90%. 53Lundbeck Research USA Chemistry – Analysis and Purification
    • SFC example 1: Chiral purification comparison of enantiomeric mixture by prep SFC/MS and NP-LC (in house compound)NP-HPLC: cycle time 23min SFC/MS: cycle time 5 min 40 mg/injection 60 mg/injection. IA column - 2x25 cm, 5 um AD-H column - 3x15 cm, 5umUV detection – must fish out UV & MS detection – one peak relevant tubes desired, one tube collection Resulting ee is 90% Resulting ee is 100% 2.00 1.80 1.60 1.40 1.20 1.00AU 0.80 0.60 0.40 0.20 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 Comparison: SFC is 7 fold more productive, far less laborious, and delivers higher quality! 54 Lundbeck Research USA Chemistry – Analysis and Purification
    • SFC example 2: Chiral resolution of flurbiprofen (well known chiral example, comparison of isocratic with gradient) 100 m g, 5 to 30 % M E O H , 100 G /M IN , bp120 sp 280,40oC flurbiprofen-t7 2: D iode A rray 2.71 3.36 250 R ange: 5.515 4.0 AU 2.0 0.0 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 flurbiprofen-t6 2: D iode A rray 2.76 3.46 250 R ange: 5.512 4.0 AU 2.0 0.0 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 flurbiprofen-t5 2: D iode A rray 1.97 2.57 250 R ange: 5.341 4.0 AU 2.0 0.0 T im e 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 Top and middle: 100 mg and 50 mg/injection with 5 to 40% MeOH in 5 min gradient; Bottom: 50mg/injection with 15% MeOH isocratic 100g/min and 30x150 mm AD-H column, BP 120 bar Gradient often gives better separation / loading and does not cost time! Message: USE GRADIENT! 55Lundbeck Research USA Chemistry – Analysis and Purification
    • SFC example 3: Purification comparison of reaction mixture by prep LC/MS and SFC/MS (in house compound) Prep LC/MS: desired Prep SFC/MS fully separates product and starting desired product (m/z = 364) material are partially from starting material (m/z = co-eluted 288) m/z = 364 m/z = 364 m/z = 288 m/z = 288 We couldn’t find LC separation. SFC/MS was straight forward. 56Lundbeck Research USA Chemistry – Analysis and Purification
    • SFC example 4: Purification of achiral product isomers by prep SFC/MS with chiral column (in house compound) AD-H, 3x15cm, 30:70 IPA/CO2, 100g/min, 280 nmA mix of isomers (meta/para 55:45) separated by SFC – 250 mg loading Complete co-elution with RP HPLC 15:44:12 04-Mar-2010 AF28962-1c Sm (Mn, 3x4) 2: Diode Array 230 Range: 5.078e-1 Time Height Area Area% 3.24 503336 72270.71 55.27 4.5e-1 6.49 204468 58490.75 44.73 4.0e-1 3.5e-1 3.0e-1 AU 2.5e-1 2.0e-1 1.5e-1 1.0e-1 5.0e-2 0.0 Time 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 We couldn’t find RP-LC separation. SFC/MS was straight forward. 57Lundbeck Research USA Chemistry – Analysis and Purification
    • RP-LC summary Achieved analytical quality for 100+ mg injections Good RT / threshold correlation with OA-LC/MS. Gaussian peaks routinely. High throughput 1-3 g/hr full gradient routine purification rate OR >20 g day (single cmpd). High velocity separations – 4 mm/s (UPLC = 5 mm/s). 5 min run time, k’ = 20 separations, 6 min cycle time (early terminate library gradients). Column switching eliminates need for column wash or equilibration time. Theoretical >200 compounds purified / day (actual peak demand 400 / week). Versatile Range of buffers and columns selected (C8 and C18) cover a wide range of compound purification applications. Equipped with additional column selection to allow purification at high pH, and if desired, HILIC or reverse phase chiral purification. High Reliability/Success Rate Back-flushing prolongs column life (>4000 injections per or >400 grams purified on each column). 2 full systems means “always on.” (2 work flows, no down time). >99.99% success rates with >99% same day turnaround. Quality = Human Efficiency = Saves You $ One expert purifies > 95% of all compounds (>150g / month). >10 to 1 increase in human efficiency (Med Chemist time reduction). Even-though all out quality would seem to cost ≈20% more, it saves a lot of Med Chem effort worth many, many fold more. 58Lundbeck Research USA Chemistry – Analysis and Purification
    • SFC summary and overall thoughts We are doing essentially the same thing with SFC that we do with RP-LC (100 mL/min, 3 cm columns, most hardware same) and we achieve essentially the same results in the same time 1-3 g/hr full gradient routine purification rate Reliability and success rates are the same Same day turnaround on achiral separations However, there are some exceptions Chiral column / solvent screening takes longer (turnaround 1-3 days chiral) Sometimes chiral strategy requires resolution of intermediates We don’t understand buffering as well, so we can’t yet exercise the same degree of control on resolution using this lever RP-LC came first, so we tend to go there first Med Chemists just starting to learn OA-SFC/MS It is important not to be religious about one technique Many want to force fit into one or the other there is no such thing as a universal technique Instead, one should play to the strengths of multiple techniques RP-LC is best suited for separating based on the sum of the lipophilic parts of molecules NP-SFC is best suited for separating based on the specific polar functional groups and shapes of the molecules in specific regions There is considerable overlap between RP-LC & NP-SFC in the kinds of molecules that can be separated Use this to enhance capacity! 59Lundbeck Research USA Chemistry – Analysis and Purification
    • RP-LC Purification ParametersFlow Rates: 100 mL/min total with 1- 5 mL/min buffer and 7.5 mL/min fromthe dilution pump (for the first 0.3-0.5 minutes if dissimilar to eluent).Temperatures: 45-55°C for mobile phase, columns (water bath), and back-flushing. 45-75°C for the dilution heater.Work horse columns: C18 Inertsil ODS-3, 30x50mm, 3 µm particles. C8 Inertsil C8-3, 30x50mm, 3 µm particles.Tubing ID: 0.03” prior to column, 0.02” after column.Splitter: 1/10000 split with 1 mL/min MeOH / 0.1% formic acid as makeupsolvent.Mobile Phase: (A) Water purified by Millipore Milli-Q Gradient system (B) ACN UV grade from B&J (important).Buffers: Neutral, 0.2-1.0% ammonium formate (high purity); Acidic, 0.2-1.0 % acetic acid in ACN/water (high purity).Dilution Solvents: Varies but predominantly 1:1 ACN/water or 100% ACN.Back-flush Solvent: 3 gradient sweeps (A) 5% acetic 1% ACN buffer (B) ACN (sometimes DMF plug).Pumps / Injector / Detectors / Collectors / Software: WatersHeaters: J-KEM Scientific, LAUDA and Selerity Technologies 60 Lundbeck Research USA Chemistry – Analysis and Purification
    • Highlight of new application in SFC: Open Access (OA) SFC/UV/ELSD/MS• To gain efficiency, complementary capabilities, and greater capacity, we have deployed OA-SFC/UV/ELSD/MS – True orthogonal separation option for Med Chem support (TLC with awesome detectors) – Still has broad overlap with RP-LC/UV/ELSD/MS for Med Chem support, thereby providing added capacity for routine reaction monitoring – Also opens up chiral method development and ee measurement to “everyone” – 3 achiral column choices & 7 for chiral (6 modifier / buffer options)• Using new detector interfacing techniques and recent software releases, SFC/UV/ELSD/MS is ready for prime time in providing immediate gratification in the above applications 61Lundbeck Research USA Chemistry – Analysis and Purification
    • Orthogonal SFC separations can be highly complementary to the frequently used RP-LC60744-024-003 1: Scan ES+ • Truly orthogonal 100 0.61 251.0 MH=251 LC/MS 251 2.67e6 SFC approach % can separate 0 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 starting material60744-024-003 1: Scan ES+ 100 0.69 213 and products 213.0 MH=213 9.67e6 that RP-LC can’t % 0 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 Time 2.00 • These SFC methods also 60744-024-003, diol 60744-024-003b 1: Scan ES+ are aligned with 2.21 SFC/MS 251 6.89e7 preparative MH=251 SFC/MS scale methods % allowing 0 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 immediate 60744-024-003b 0.82 1: Scan ES+ 213 purification 6.13e7 • MS used in this MH=213 application due % 0 Time to lack of 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 chromaphore Normal phase separation gives TLC- like outcome for polar intermediates 62 Lundbeck Research USA Chemistry – Analysis and Purification
    • OA-SFC/UV/ELSD/MS can provide similar information as OA-LC/UV/ELSD/MS 0.65 UV • Chromatograms 0.41 LC/MS showing starting % 28 1.24 material and 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 product (reaction 0.69 ELSD 1.66 1.76 1.85 1.93 2.00 2.03 progress) 0.39 % 0.07 0.11 0.20 0.33 0.44 0.57 0.66 • Essentially same 92 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 Time data with either approach except reverse elution 1.0 order (TLC-like) SFC/MS UVAU 5.0e-1 • Note the 0.0 0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 improved quality of ELSD with 200.000 ELSD SFC!LSU 0.000 Time 0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 63 Lundbeck Research USA Chemistry – Analysis and Purification
    • Chiral screening of many methods on a singlesample login (MassLynx / OpenLynx SCN 798) Method set for achiral analysis First set of methods for chiral column screening Second set of methods for chiral column screening if first set doesn’t work New software makes method screening easy! 64Lundbeck Research USA Chemistry – Analysis and Purification
    • Screening chiral conditions for preparative method development (4 x 3)chiral-sample1-9 3: Waters 2998 chiral-sample1-5 3: Waters 2998 1.48 Range: 7.103e+1 4.37 4.50 Range: 7.051e+1 5.0e+1 MeOH AD-H 5.0e+1 EtOH AD-H 0.0 0.0 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00chiral-sample1-10 3: Waters 2998 chiral-sample1-6 3: Waters 2998 2.54 2.67 Range: 5.639e+1 3.55 Range: 8.666e+1 5.0e+1 0.63 OD-H 5.0e+1 OD-H 0.0 0.0 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00chiral-sample1-11 3: Waters 2998 3.00 3.17 Range: 6.07e+1 chiral-sample1-7 3: Waters 2998 5.0e+1 AS-H 5.0e+1 2.53 2.66 Range: 5.994e+1 AS-H 0.0 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 0.0chiral-sample1-12 3: Waters 2998 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 3.41 Range: 6.864e+1 chiral-sample1-8 3: Waters 2998 5.0e+1 OJ-H 3.48 5.0e+1 3.12 3.20 Range: 5.463e+1 0.0 0.66 OJ-H Time 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 0.0 Timechiral-sample1-1 3: Waters 2998 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 3.72 IPA 4.29 Range: 5.865e+1 5.0e+1 IPA AD-H AD-H Screening 4 columns and 3 solvent 0.0chiral-sample1-2 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 3: Waters 2998 gradients showed AD-H with IPA gives a 4.35 4.46 useful separation Range: 6.471e+1 5.0e+1 OD-H 0.0chiral-sample1-3 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 3: Waters 2998 Scaled preparative version of same 4.0e+1 3.62 3.87 AS-H AS-H Range: 4.232e+1 method was immediately used to resolve 2.0e+1 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 10g on same daychiral-sample1-4 3: Waters 2998 5.0e+1 1.03 3.86 OJ-H Range: 7.964e+1 OA-SFC/UV/ELSD/MS is a viable OJ-H 0.0 1.00 2.00 3.00 4.00 5.00 6.00 7.00 Time 8.00 screening approach for preparative work Lundbeck Research USA Chemistry – Analysis and Purification 65