Isolation/Purification. Drug Development. Drug Discovery

Transcription

1 Isolation/Purification Drug Development Drug Discovery

2 XTerra Columns The Most Successful Column in ver 0 Years of Stationary Phase Technology The universal acceptance by pharmaceutical scientists in drug discovery, method development and isolation and purification has enabled XTerra columns to become the fastest selling and most successful column product in the world of chromatography. The reason: the enabling science of Hybrid Particle Technology. Drug discovery scientists benefit from the DMS injection-resistance of the rugged XTerra particle. Method development scientists are able to operate at whatever ph is necessary for optimal selectivity due to XTerra columns wide ph range. Isolation and purification scientists can load up to 60x more material per injection because of the high mass loading capacity of XTerra columns.

3 Hybrid Particle Technology XTerra Columns For over 0 years, scientists have been forced to work within certain boundaries when performing HPLC separations. Restrictions on speed, resolution, ph, temperature, and loading capacity were imposed upon the chromatographer by limitations of the stationary phase material. The patented* Hybrid Particle Technology of XTerra columns allows chromatographers to break these boundaries and realize the full potential of their analytical and preparative separations. In Hybrid Particle Technology, one out of every three silanols is replaced with a methyl group during synthesis. This hydrophobicity is distributed throughout the entire structure of the particle backbone. The result is a rugged hybrid (inorganic/organic) particle that can be operated at high speeds, high temperatures, and high ph. The presence of % fewer residual silanols (after endcapping and bonding) also means that XTerra columns give exceptionally sharp, high-efficiency peaks for basic compounds. *US Patent No. 6,686,05 B Traditional versus XTerra Manufacturing Process Traditional lica Manufacturing Process XTerra Manufacturing Process: Much More Than a Surface Modification H H H H H H H H H H H H H H H H H Bonded and Endcapped lica Particle Lower surface coverage Poor peak shape for bases ph range -8 HC H CH H HC H H HC CH H CH H HC H H H HC CH HC CH H CH H CH HC CH Bonded and Endcapped XTerra Particle Highest, most homogenous coverage / less silanols Superior peak shape ph range - HC Et Et Et Et Et Et Et Et Et Et Et Et Et Et CH Et Et Et Et Et Et Et Et Et + Et HC Et Et Unbonded lica Particle Polyethoxysilane (PES) Tetraethoxysilane (TES) Unbonded XTerra Particle Methylpolyethoxysilane (MPES) Tetraethoxysilane (TES) Methyltriethoxysilane (MTES)

4 The Efficiency of lica with Stability of Polymers The vast majority of reversed-phase HPLC separations take place on silica-based stationary phases. lica has long enjoyed such attributes as high efficiency and mechanical strength. However, silica suffers from poor peak shape for bases and a limited ph range. ne way that chromatographers have attempted to overcome these limitations is by turning to polymer-based stationary phases. Polymers, however, have not enjoyed the acceptance of silica due to poor efficiency, low mechanical strength, and unpredictable peak elution order when transferring methods from polymeric to silicabased columns. Hybrid Particle Technology overcomes these limitations and combines the best attributes of both these materials while overcoming each material s weaknesses. The result is a rugged material that has high mechanical strength, high efficiency, excellent peak shape for bases, and easy scale-up from analytical to preparative chromatography. Pharmaceutical scientists in the areas of drug discovery, drug development, and drug isolation and purification have enthusiastically embraced the Hybrid Particle Technology of XTerra columns. This acceptance has resulted in XTerra columns becoming the fastest selling family of HPLC columns in the history of Waters Corporation. In addition, R&D Magazine awarded Waters a 000 R&D 00 Award for the XTerra family of HPLC columns. XTerra Combines the Best Properties of lica and Polymers Inorganic (lica) rganic (Polymer) rganic vs. Inorganic HPLC packings Advantages Mechanically strong High efficiency Predictable retention Wide ph range No ionic interactions Chemically stable Hybrid Particle Technology Disadvantages Limited ph range Tailing peaks for bases Chemically unstable Mechanically weak Low efficiency Unpredictable retention lica Separations at Polymer ph XTerra RP 8 :.6 x 50 mm, 5 µm, ph 0.7 Polymer Column:. x 50 mm, 0 µm, ph 0.7. Analytes:. Codeine. Thebaine 5. Reserpine. Yohimbine. Cocaine 6. Methadone 0.5 Mobile Phase A: 0 mm NH H, ph 0.7 Mobile Phase B: ACN Flow Rate: ml/min AU 0.8 AU Gradient: Time Profile (min) %A %B Injection Volume: 5 µl Temperature: Ambient Detection: 0 nm Instrument: Waters 690, 996 PDA

5 XTerra Chemistries XTerra MS XTerra MS C 8 and MS C 8 columns were designed to be compatible with Mass Spectrometry applications and provide sharp peaks, good sensitivity, and large peak capacities. These chemistries exhibit peak shape usually only achievable with columns employing embedded polar group technology (see XTerra RP columns). The tri-functional bonding chemistries of XTerra MS columns deliver the longest lifetimes over the widest ph range (ph -) and provide maximum throughput with excellent resolution and ultra-low bleed. XTerra RP XTerra RP 8 and RP 8 columns combine Hybrid Particle Technology with Shield Technology* by incorporating an embedded polar group to deliver the best possible peak shape. These general purpose columns allow fast method development by offering unique selectivity. Like all XTerra columns, the shielded XTerra RP columns exhibit excellent water wettability, even in 00% aqueous mobile phases. The combination of a greatly widened ph range for selectivity choices and the additional selectivity options of two ligands are often the only tools necessary for successful method development. XTerra Phenyl XTerra Columns XTerra phenyl columns offer complementary selectivity as compared to straight-chain alkyl stationary phases, especially for compounds with aromatic rings. The combination of Hybrid Particle Technology and the XTerra phenyl chemistry results in a stationary phase with high ph stability, optimal surface coverage, excellent peak shape for all compounds and highly reproducible retention times. In addition, the difunctional bonding chemistry of the short chain phenyl ligand imparts improved low ph hydrolytic stability. This unique combination of substrate and ligand adds a new dimension in selectivity for separations of complex mixtures. XTerra MS XTerra RP XTerra Phenyl Particle/Ligand Structure MS C 8 MS C 8 MS C 8 RP 8 MS C 8 RP 8 CH RP 8 RP 8 CH Polar Group CH CH Phenyl Polar Group CH Polar Group CH CH Polar Group CH CH CH CH CH Comments Sharpest peaks Trifunctional bonding Longest lifetimes ph - Ideally suited for Mass Spec Best peak shape for bases Shield technology ph - Ideally suited for UV, fluorescence, electrochemical, and RI detectors Complimentery selectivity Difunctional bonding Excellent peak shape for all compounds ph - Ideally suited for Mass Spec Effect of XTerra Stationary Phase on Selectivity Analytes:. Quercetin. Isorhamnetin. Kaempferol XTerra MS C 8 XTerra RP XTerra Phenyl Available XTerra Columns and Chemistries Packing Particle Shape Particle ze(s) (µm) Pore ze (Å) Carbon Load (%) End-capped XTerra MS C 8 Spherical.5,.5, 5, yes XTerra MS C 8 Spherical.5,.5, 5, yes XTerra RP 8 Spherical.5, 5, yes XTerra RP 8 Spherical.5, 5, yes XTerra Phenyl Spherical.5, yes Temperature: Detection: XTerra columns are available in over 600 configurations, ranging from capillary to prep. XTerra stationary phases come in five different chemistries, each with a range of particle sizes appropriate for most applications. Columns:.6 x 50 mm, 5 µm Mobile Phase: 5% 0.% H P, ph.0 8% MeH (v/v) Flow Rate:.0 ml/min *US Patent No. 5,7,755 5 C 70 nm Waters 690, 996 PDA

6 Drug Discovery Ruggedness, Efficiency and Speed The Hybrid Particle Technology of XTerra columns results in an extremely rugged and efficient particle that is resistant to the DMS injections commonly performed in the drug discovery laboratory. These small XTerra particles are then packed into short columns which allow for faster gradients and shortened analysis times. The benefits for drug discovery scientists include high throughput, minimized sample backups and faster library screening. 6

7 Drug Discovery Speed with Resolution Speed, resolution, capacity these attributes are crucial to the success of the drug discovery scientist. With automated synthesizers capable of producing over a thousand potential lead compounds per day, sample backups are common in the chromatography laboratory. Eliminate sample backups while characterizing, analyzing and purifying combinatorial libraries faster than ever before by running ultrafast gradients on XTerra MS columns. Shorter Columns, Higher Flow Rates, and Higher Peak Capacities To maximize sample throughput, analytical run times must be shortened while still maintaining high peak capacity. The combination of short XTerra columns containing.5 µm particles, together with high flow rates and fast gradients creates a breakthrough in speed, resolution and capacity without compromise. In addition, XTerra columns can be operated at temperatures up to 80 C, thereby lowering system backpressure. Increasing Flow Rates and Higher Peak Capacity Smaller Particles and Shorter Columns Result in Faster Runs Short Gradient (t g ) with Higher Peak Capacity Van Deemter Curves for XTerra MS C 8 Columns Peak Capacity Plate Height (µm) Smaller Plate Height=Higher Efficiency=Increased Peak Capacity 5 µm Flow rate tg Flow rate tg Flow rate.6 x 50 mm 5 µm.6 x 0 mm.5 µm.6 x 0 mm.5 µm tg Smaller Particles Provide Higher Efficiency at Faster Flow Rates.5 µm Linear Velocity (mm/sec) As particle size decreases, improved peak capacity and resolution is obtained at high speed. These parameters can be successfully optimized to obtain faster separations while retaining maximum resolution. Faster flow rates and shorter columns allow for shorter gradient run times without compromising peak capacities. For mass spectrometry compatible flow rates, these same advantages are realized for smaller diameter columns as well.

8 Columns Designed with Mass Spectrometry (MS) in Mind Automated LC/UV/MS is the technique of choice for the characterization of combinatorial libraries produced by high output automated synthesizers. XTerra MS columns were created for LC/MS with ultra-low MS bleed from ph -. XTerra columns provide the speed and resolution necessary to fully utilize the selectivity, sensitivity and structural characterization capabilities of today s powerful Mass Spectrometers. High Sensitivity LC/MS Analyses In order to achieve the high sensitivities and low limits of detection required for drug metabolism studies, a total analysis solution is required. Waters provides this complete answer by combining asis sample preparation products, XTerra columns, Alliance LC systems, and Micromass mass spectrometers to produce the best possible combination of speed, resolution and sensitivity for your LC/MS analyses. LC/MS Separation at Low, Medium, and High ph Low Level Drug Determination by SPE/LC/MS Analytes: Conc. (µg/ml DMS) : Lidocaine 0.9 : Prednisolone.70 : Naproxen 0.06 : Amitriptyline 0.6 5: Ibuprofen TIC ES ph= ph=5 0 0 ph= Gradient (tg) = min TIC ES+ ph.7 Column: XTerra MS C 8. x 0 mm,.5 µm Mobile Phase A: 0.% HCH in % ACN (v/v) Mobile Phase B: 0.% HCH in 00% ACN Mobile Phase A: 0 mm CH CNH in % ACN (v/v) Mobile Phase B: 0 mm CH CNH in ACN Mobile Phase A: 0.% NH H in % ACN (v/v) Mobile Phase B: 0.% NH H in ACN Gradient: Time Profile (min) %A %B Flow Rate:.5 ml/min Splitter ca. 80 µl/min (split 0:) Injection Volume: 5 µl Temperature: MS Conditions Ion Source: Source Temperature: Desolvation Temperature: Instrument: Ambient Electrospray positive Electrospray negative Using a single XTerra column, both resolution and MS signal can be optimized for acids, bases and neutrals while maintaining unsurpassed peak shape and peak capacity. ph 5 ph C 50 C Alliance HT 795 w/ ZQ Mass Detector CID Mass Spectra Daughters of 5ES+ cid 5.99e8 S (.005) H H H C N N N S CH H C Ranitidine CHN Relative Abundance (%) % 0 00 % 0 00 % Plasma spike.0 ng/ml ranitidine 6 Sm (Mn, x ) Internal Standard spike.0 ng/ml m/z MRM of channels ES+ 5> e MRM of channels ES+ 7>0 9.5e5 Column: XTerra MS C 8. x 0 mm,.5 µm Mobile Phase: 0% 0. M HCNH 70% ACN (v/v) Flow Rate: 00 µl/min Injection Volume: 5 µl Temperature: Ambient MS Conditions Ion Source: Electrospray positive Source Temperature: 50 C Desolvation Temperature: 50 C Instrument: Alliance HT 795 w/ ZQ Mass Detector Using an asis MCX extraction method with a 0 mg 96-well extraction plate, EDTA rat plasma was extracted and analyzed for the histamine H-receptor antagonist ranitidine using an XTerra MS C 8 column on an Alliance LC/MS System. 8

9 Intelligent Speed (IS ) Columns for Fast Generic Gradients Direct Scale-up Capability Drug Discovery pen access walk-up LC systems run fast, generic gradients and are often the starting point for testing samples that contain a wide range of chemical compounds. XTerra IS columns are ideally suited for these high throughput systems. XTerra IS columns combine a rugged and efficient particle with advanced column hardware and packing techniques to provide faster run times, increased sample throughput and shorter product development time. The fast and efficient chromatography realized with XTerra columns can be directly and easily scaled to preparative applications. This allows new drug candidates to be taken directly from the lead generation to the lead optimization stage of drug discovery. The ability to operate at high ph provides excellent peak shapes for bases and increased mass loading (up to 60x) as compared to conventional silica-based materials. Complex Test Mix Run on XTerra MS C 8 IS Column Accurate Discovery Scale-up AU Analytes:. 8-Bromoguanosine. Acetanilide. Triamcinolone. Hydrocortisone 5. -Amino-7-chloro-5-oxo-5H []benzopyrano [,-b] pyridine- -carbonitrile 6. 6 a-methyl-7a-hydroxyprogesterone 7. -Aminofluoranthene 8. -Bromofluorene 9. Perylene 0. Naphtho(,-a)pyrene V 0 5 Column: XTerra MS C 8,. x 0 mm IS TM,.5 µm Mobile Phase A: 0.% HCH Mobile Phase B: 0.% HCH in ACN Flow Rate: 0.6 ml/min Gradient: Time Profile (min) %A %B Injection Volume: 5 µl Sample Concentration: 0 µg/ml Temperature: 0 C Detection: 5 nm Instrument: Alliance 795 with 996 PDA % - 00 % - Mobile Phase A: 0 mm NH HC, ph 0 Mobile Phase B: ACN/00 mm NH HC, ph 0 (90/0) Sample Concentration: 5 mg/ml each in DMS Temperature: Ambient Detection: 0 nm Instrument: Waters AutoPurification System DMS DMS Analytes:. Ethyl--hydroxybenzoate. Diphenhydramine. Dibenzofuran. N-phenylcarbazole Column: XTerra MS C 8.6 x 50 mm, 5 µm Mass Load:.5 mg Column: Mass Load: Flow Rate:.05 ml/min Gradient: Time Profile (min) %A %B Injection Volume: 59 µl Flow Rate: 8.0 ml/min Gradient: Time Profile (min) %A %B XTerra MS C 8 Prep BD 9 x 50 mm, 5 µm mg Injection Volume: 000 µl A wide range of compounds was baseline separated in less than.5 minutes. This separation is representative of LC column testing in major pharmaceutical laboratories around the world. With XTerra columns, redevelopment time is minimized and separations can easily be scaled up from analytical to prep.

10 Method Development Harness the Power of ph XTerra columns allow the method development scientist to operate under whatever conditions are necessary for optimal selectivity. With a ph operating range that is twice as wide as silica, XTerra columns enable the development scientist to harness one of the most powerful tools for optimizing retention times during method development ph. This results in faster and easier development of more robust and reproducible chromatographic separations. 0

11 Method Development ptimize Selectivity for Ionizable Compounds ne of the greatest challenges facing the method development scientist is creating a robust separation in the most time efficient manner possible. Tools available to the separations scientist that influence elution order (selectivity) include solvent type, ph column chemistry and/or temperature. The unique Hybrid Particle Technology of XTerra columns allows the greatest flexibility for selecting whatever chromatographic conditions are necessary for optimal selectivity and separation. Develop Robust Separations for Basic Compounds at High ph ne of the most powerful tools for manipulating the selectivity for ionizable compounds is ph. Acidic compounds have increased retention at ph values below their pk a and basic compounds exhibit longer retention at ph values above their pk a. nce many pharmaceutically active compounds are ionizable, a column with a wider usable ph range allows for greater retention time manipulation during method development. XTerra columns have a usable ph range (ph -) that is twice as wide as silica based columns, thereby making method development twice as easy and twice as fast. Maximize Selectivity with the Wider ph Range Increase Retention of Basic Compounds at Higher ph Capacity Factor (k) Neutral (N) 0 9 Acid (A) Base (B) ph B A N ph 0 A,B N ph 5 0 B N A ph 0 0 Capacity Factor (k) Good peak shape Poor retention CH N CH N CH H Increasing retention Good peak shape Need critical control of ph for chromatographic reproducibility Amitriptyline Nortriptyline Best retention Good column lifetimes Good peak shape Improved chromatographic reproducibility ph Nortriptyline { } Amitriptyline Nortriptyline Amitriptyline 0 0 Co-elution at ph Separation of very similar bases at ph 0 The wider usable ph range of XTerra columns is a powerful tool for changing the selectivities of ionizable analytes. For the first time, XTerra columns allow the development scientist to utilize the full power of ph as a method development tool.

12 Use ph to Improve Peak Shape and Increase Retention The quality of peak shapes for basic analytes is dependent upon the concentration, type and activity of residual silanols that are present after bonding. It is difficult to reduce the number of silanols on silica by more than 50%, even after ligand bonding and endcapping. For XTerra columns, one third of the surface silanol groups are replaced with methyl groups prior to ligand bonding and endcapping, resulting in the highest, most homogeneous coverage of any reversed phase material. This % reduction in silanol concentration delivers the sharpest and most symmetrical peaks for basic analytes regardless of mobile phase. Superior Peak Shape for Any Compound in Any Mobile Phase The exceptional peak shape achieved on XTerra columns extends across a ph range that was not previously achievable with silica-based columns. With a single XTerra column, superior peak shapes can be realized for acidic, neutral and basic compounds. The result is a more robust separation for compounds in their neutral form acidic compounds below their pk a and basic compounds above their pk a since ph control becomes less critical. Excellent Resolution and Peak Shape at High ph Improved Peak Shape for XTerra at Any ph Column: XTerra RP 8,.6 x 50 mm,.5 µm Mobile Phase A: H Mobile Phase B: MeH Mobile Phase C: 00 mm NH HC, ph 9.0 Flow rate:.0 ml/min Gradient: Time Profile (min) %A %B %C Injection Volume: 0 µl Temperature: 0 C Detection: 5 nm Instrument: Alliance 695, 996 PDA Analytes:. Trimethoprim. Nordoxepin. Nortriptyline. Doxepin 5. Imipramine 6. Amitriptyline 7. Trimipramine Acetonitrile (ACN) Methanol (MeH) USP Tailing Factor USP Tailing Factor Salicyclic acid Salicyclic acid 0 mm HCNH 5% MeH ph.0 Nicotine p-toluamide Salicyclic acid 0 mm HCNH 5% MeH ph.0 Nicotine p-toluamide Salicyclic acid 0 mm NH HC 5% ACN ph 7.0 Nicotine 0 mm NH HC 7% MeH ph 7.0 Nicotine p-toluamide p-toluamide Salicyclic acid Salicyclic acid 0 mm NH HC 5% ACN ph 0.0 Nicotine 0 mm NH HC 5% MeH ph 0.0 Nicotine ph.0 ph 7.0 ph 0.0 p-toluamide p-toluamide Acidic Compound Neutral Compound Basic Compound The wider ph range of XTerra columns allows greater selectivity choices while improving peak shape and resolution. Regardless of mobile phase composition or analyte, XTerra columns provide the most flexibility in developing fast and robust separations.

13 Use Column Chemistries to Change Selectivity Streamlined Method Development Method Development Another powerful method development tool is column stationary phase chemistry. The choice of XTerra columns, available in five different ligands, can have a marked effect on selectivity. These complementary column chemistries are part of an overall method development routine that also includes ph, organic modifier and temperature. These chemistries, along with a wider ph range, superior peak shape and excellent lot-to-lot reproducibility make XTerra the only columns necessary to develop robust HPLC methods. XTerra columns are part of a streamlined method development routine that includes: two ph levels (e.g., ph.0 and ph 0.0), three XTerra columns (e.g., XTerra MS C 8, RP 8, and Phenyl), and two organic modifiers (ACN and MeH). Using a generic gradient and MS for rapid peak elution and identification, experiments (xx) provide the exploratory work necessary to find the most promising separation. After this initial exploratory work, fine-tuning can be performed using solvent selectivity, ph or temperature. Effect of XTerra Column Chemistry on Selectivity and Retention Easy and Fast Method Development XTerra Phenyl C H C H CH CH 5 6 Columns:.6 x 50 mm, 5 µm Mobile Phase A: Mobile Phase B: Mobile Phase C: H MeH 50mM HCH, ph.5 MeH, ACN, MeH, ACN, low ph low ph high ph high ph XTerra RP 8 XTerra RP 8 XTerra RP 8 XTerra RP 8 XTerra RP 8 MeH, ACN, MeH, ACN, low ph low ph high ph high ph XTerra MS C 8 XTerra RP 8 CH CH Gradient: Time Profile (min) %A %B %C Flow Rate:.0 ml/min Injection Volume: 5 µl Temperature: Detection: Instrument: Analytes: 0 C 5nm Waters 695, 996 PDA. Suprofen. Tolmetin. Naproxen. Fenoprofen 5. Ibuprofen 6. Diclofenac XTerra MS C 8 XTerra MS C 8 XTerra MS C 8 XTerra MS C 8 XTerra MS C 8 MeH, ACN, MeH, ACN, low ph low ph high ph high ph XTerra Phenyl XTerra Phenyl XTerra Phenyl XTerra Phenyl XTerra Phenyl MeH, ACN, MeH, ACN, low ph low ph high ph high ph This twelve experiment gradient method development routine can easily be automated and is often all that is necessary to develop separations of complex samples The column stationary phase chemistries of XTerra columns are key components in an overall method development plan.

14 Develop Shorter and Faster Methods Using Intelligent Speed (IS ) Columns Because pharmaceutical companies are being pressed to bring products to market sooner, method development scientists are being asked to develop shorter and faster separations. The challenge is to do this without sacrificing analyte resolution or method robustness. Waters has made this possible by producing XTerra IS columns which combine state-of-the-art column packing techniques and hardware to produce efficient, stable and reproducible 0 mm length analytical columns. Excellent Batch-to-Batch Reproducibility Waters set the standard for reproducibility with the Symmetry family of columns in 99. This high level of quality continues with XTerra columns. As with all modern Waters stationary phases, reproducibility, excellent peak shape and long column lifetimes are assured since all Waters columns are manufactured in cgmp, IS 900 certified facilities. The method development scientist can be confident in, not only the results but also in the ability to transfer the method to QC. Develop Faster Methods Reproducible and Predictable Results Year After Year 0.00 Sample Preparation: SPE: asis HLB µelution Plate Condition: 00 µl MeH Equilibrate: 00 µl H Load: 50 µl Spiked rat plasma with % H P, diluted with : H Wash: 00 µl 5% MeH in H Elute: 50 µl MeH Dilute: 00 µl H MRM of 6 Channels ES+ TIC.86e6 MRM 95 > 8.6e5 MRM 67 > 90..8e5 MRM 78 > e6 MRM 60 > 8 6.6e5 MRM 85. > 65..8e6 MRM 78 >..8e6 LC Column: XTerra MS C 8,. x 0 mm IS,.5 µm Mobile Phase A: 0 mm NH HC, ph 0 Mobile Phase B: MeH Flow Rate: 0. ml/min Gradient: Time Profile (min) %A %B Injection Volume: 0 µl Sample concentration: 5 µg/ml Temperature: Ambient Detection: MS Instrument: Waters 777 Sample Manager, Waters 55 Binary HPLC Pump and Waters Micromass Quattro Ultima MS MS Quattro Ultima ES+ MRM Cone (V) 5.0 Capillary (kv):.5 Source ( C): 50 Desolvation ( C): 00 Cone gas flow (L/hr): 50 Desolvation gas flow (L/hr): 550 LM resolution &:.5 HM resolution &:.5 Ion Energy : 0. Ion Energy : 0.8 Multiplier (V): 650 Analytes:. Caffeine. Practolol. N-acetyl procainamide. Propranolol 5. Methoxyverapamil 6. Amitriptyline %RSD verlay of QC batch test chromatograms taken from five batches of XTerra RP 8, 5µm stationary phase material. XTerra columns produce reproducible and predictable results year after year. Year

15 Method Development Rugged Particles Mean Longer Column Lifetime In addition to a wider ph range and superior peak shape, another benefit of Hybrid Particle Technology for method development is extended column lifetime. This is due to the ruggedness of the XTerra particle. The molecular structure of the XTerra particle imparts greater resistance to dissolution at high ph as compared to traditional silica-based particles. XTerra columns exhibit the ph resistance of a polymer and the performance of silica. Longer Column Lifetimes at High ph The Hybrid Particle Technology of XTerra columns provides longer column lifetimes across a much wider ph range than silica. Even the newer silica-based columns that are reported to have longer lifetimes when running at high ph, require several different column chemistries to span the ph range of one XTerra column. When running separations, using XTerra columns over extended periods of time at high ph, no loss in retention time or deterioration of peak shapes is observed. Improved Robustness of the XTerra Particle Bonded lica Particle Complete dissolution of silica Catastrophic column failure Short lifetimes Bonded XTerra Particle Slow rate of surface dissolution Incorporated methyl groups uncovered slow rates of dissolution Longer column lifetimes USP Tailing Factor Day Day 5. Methamphetamine... Propranolol... Nortriptyline.. Column: XTerra RP 8.6 x 50 mm, 5 µm with.9 x 0 mm XTerra RP 8 guard column Mobile Phase: 50% 50 mm Pyrrolidine, ph.5 50% ACN (v/v) (H) (H) (H) (H) (H) Flow Rate:.0 ml/minute Surface Surface CH H CH H CH H CH H CH CH Injection Volume: µl Temperature: 0 C Core H H H H H H H H H H H H H H H H Core H H CH CH CH Detection: Instrument: 5 nm Waters 695, 996 PDA Initial Injection H - H - Surface H - H - H - H - H - H H H H H Surface CH H CH H CH CH Core Core CH CH CH After 5 Days At high ph, the hydroxyl anion in the mobile phase attacks the silica surface, thereby causing dissolution of the silica. This is accelerated as more silanol sites become exposed. In the XTerra particle, the presence of the methyl siloxane bond throughout the particle results in increased resistance to dissolution in high ph mobile phases. 5 0 XTerra columns exhibit superior stability at high ph, separating basic compounds with no deterioration of plate count or peak symmetry.

16 Isolation/Purification Bridging the Performance Gap from Analytical to Prep Introducing the new XTerra Prep BD columns from Waters that deliver analytical performance in preparative dimensions with maximum column stability the result of patent-pending ptimum packed Bed Density (BD ) design. Combining BD design with the unique chromatographic properties of XTerra Hybrid Particle Technology provides a range of high performance preparative columns with unprecedented flexibility, both through the range of analytes basic, acidic and polar and the wide operating range of mobile phases necessary for successful, predictable preparative scale-up. 6

17 Isolation/Purification Introducing ptimum Bed Density (BD ) Design The purification process invariably requires HPLC injections of large volumes of sparingly soluble material dissolved in strong sample solvents. This combination has been recognized to be one of the primary contributors to early preparative column failure. The challenge for the column supplier is to reproducibly manufacture preparative columns with the same performance and stability as the equivalent analytical column. The proprietary ptimum Bed Density BD procedure yields preparative columns with the same packed bed densities as their analytical counterpart resulting in durable bed stability, better efficiency and higher loadability. Increase Mass Capacity and Resolution for Drug Development Interaction between basic analytes and ionizable silanols on silica surfaces results in poor peak shape. Therefore, separation of basic compounds is often performed at low ph to suppress silanol ionization, thereby reduce peak tailing. However, in this mode, basic analytes are protonated and are not retained well by reversed-phase sorbents. However, XTerra Prep columns with Hybrid Particle Technology unlike traditional silica columns can be used routinely at high ph so that basic compounds can be separated in their neutral form. This results in a tremendous improvement, not only in peak shape, but also resolution and loadability. Moving from an isocratic to a gradient separation mode further increases capacity. Consistency of column lifetime is a key consideration not only for high throughput applications, but also in the planning of the annual budget requirements of a purification laboratory. Consistent Column-to-Column Performance Low vs High ph Loading 0 % 0 0 % - Day Column Day 0 Column Injection Injection Before and After QC Test Comparisons on XTerra Prep BD Columns Columns: Mobile Phase A: Mobile Phase B: Flow Rate: in Column New Columns Post 000 Injections % Change Plates USP Tail Plates USP Tail Eficiency,78., ,0.05, ,9.0, ,09.0, Average,86.06, Four XTerra Prep MS C 8 BD 9 x 50 mm, 5 µm.0% HCH.0% HCH in ACN 8.0 ml/min Gradient: Time Profile (min) %A %B Analytes: Tylosin Sulfathiazole Ketoprofen Injection Volume: 00 µl DMS Temperature: Ambient Detection: 5 nm Instrument: Waters Purification Factory 00 % % % - ph. Load 6. mg ph 0 Load 6. mg ph 0 Load 80 mg Column: XTerra Prep MS C 8 BD 9 x 00 mm, 5 µm Mobile Phase A: 0.% TFA ph. Mobile Phase B: 0.% TFA in ACN Mobile Phase A: 0 mm NH HC ph 0 Mobile Phase B: 00 mm NH C /ACN (0/90) Gradient: Time Profile Analytes: (min) %A %B. Miconazole Econazole Injection Volume: 000 µl ph. 000 µl ph 0.0 Temperature: Ambient Detection: 70 nm Instrument: Waters Autopurification System XTerra Prep BD exhibit unmatched column-to-column stability XTerra columns provide superior stability at high ph, separating basic compounds with enhanced resolution and loadability.

18 Accurate Scalability to Larger Column Dimensions The key to successful purification is obtaining a good analytical separation and the ability to scale-up directly without modifying the mobile or stationary phase. The isolation and purification of critical compound pairs is a major challenge to purification scientists. ften a successful analytical separation will not scale up directly due to the reduced performance of the preparative column. The BD design ensures equivalent chromatographic performance from analytical to preparative dimensions, eliminating the need for any subsequent method redevelopment. Predictable sample loadability allows efficient purification of the required mass for preclinical drug metabolism studies. Scalability for Drug Discovery / Lead ptimization Analytes:. xacillin. Cloxacillin. Dicloxacillin Pictured here is the Waters FractionLynx MS with a 767 Sample Manager for inject/collect/re-inject, 55 Binary Gradient Module for high pressure mixing, Column Fluidics rganizer for software-controlled valving, fluidics, and column management, 996 PDA and ZQ Mass Detector. Column: XTerra MS C 8 Column: XTerra Prep MS C 8 BD Column: XTerra Prep MS C 8 BD.6 x 50 mm, 5 µm 9 x 50 mm, 5 µm 0 x 50 mm, 5 µm Load: mg Load: 5 mg Load: 8 mg Excellent scalability of acidic compounds at low ph across three dimensions. Mobile Phase A: Mobile Phase B: Flow Rate: % HCH in H % HCH in ACN.8 ml/min (.6 x 50 mm).0 ml/min (9 x 50 mm)* 77.0 ml/min (0 x 50 mm)** Gradient: Time (min) %A %B Curve Temperature: Ambient Detection: Instrument: 70 nm Waters Autopurification System Gradient Adjustment: *Add 0.5 min to each time point on gradient table. **Add 0.5 min to each time point on gradient table. 8

19 Screen and Purify Libraries in Half the Time Isolation/Purification XTerra Prep columns deliver the speed you need for high throughput lead generation speed that enables rapid analysis and purification of thousands of samples. Now you can screen and purify your libraries twice as fast, compared to traditional silica-based columns. XTerra packings, engineered specifically for preparative chromatography, allow you to operate routinely in a wider ph range (-). Choosing an optimum ph enables maximum column loading (low ph for acidic compounds or high ph for basic compounds). Hybrid Particle Technology combined with ptimum Bed Density always assures the best peak shapes and longest column lifetimes. Peak widths (volumes) halved Concentration of fractions doubled Sensitivity increased ptimization of High Throughput Applications Analytes: Conc. (mg/ml DMS) : Diphenhydramine 0 Column: XTerra Prep MS C 8 BD 9 x 50 mm, 5 µm Column: XTerra Prep MS C 8 BD 9 x 0 mm, 5 µm Column: XTerra Prep MS C 8 BD 9 x 0 mm, 5 µm : xybutynin 0 : Terfenadine 0 Mobile Phase A: 0mM NH HC, ph 0 Mobile Phase B: ACN Flow Rate: 0 ml/min Mobile Phase A: 0mM NH HC, ph 0 Mobile Phase B: ACN Flow Rate: 0 ml/min Mobile Phase A: 0mM NH HC, ph 0.0 Mobile Phase B: ACN Flow Rate: 0 ml/min Gradient: Time Profile (min) %A %B Gradient: Time Profile (min) %A %B Gradient: Time Profile (min) %A %B Injection Volume: 500 µl Injection Volume: 500 µl Injection Volume: 500 µl Instrument: FractionLynx MS Instrument: FractionLynx MS Instrument: FractionLynx MS Fast throughput purification at high ph for basic compounds shows excellent peak shape and resolution at high loading conditions. The larger capacity obtained at high ph enables the use of shorter columns to double purification throughput without compromising purity. Higher throughput can be achieved by increasing the flow rate to take advantage of the reduced backpressure of this shorter column.

20 Life Sciences The New Standard in Synthetic ligonucleotide Analysis and Purification Use of XTerra hybrid-particle technology provides significant advantages over current methods used to analyze or purify synthetic oligonucleotides. Compared to gel electrophoresis or other HPLC techniques, the XTerra reversed-phase, ion-pairing HPLC method yields superior component resolution and product recovery for either analytical or semi-preparative applications. XTerra hybrid particles are also available in Waters new NanoEase 75, 00, and 50 µm nano columns and 00 µm capillary columns for LC/MS applications. 0

21 Life Sciences HPLC Purification of Synthetic ligonucleotides The successful purification of a long-chain synthetic oligonucleotide product containing substantial amounts of failure sequences can be challenging. A novel method has been developed for the purification of such sequences using reversedphase high performance liquid chromatography (RP-HPLC) with an ion-pairing (IP) mobile phase. Up to 0.5 µmole of synthetic DMT-off oligonucleotide can be purified in a single injection with this method on a.6 x 50 mm XTerra column. In addition, the isolated oligonucleotide requires no post-purification desalting or detritylation preparation prior to use. HPLC Analysis of Synthetic ligonucleotides The high coupling efficiency of DNA/RNA synthesis methods can produce high quality product provided that the automated synthesizers are properly functioning. While sub-optimally functioning systems can yield acceptable material for some applications (e.g., PCR primers), only high purity oligonucleotides (e.g., >98% pure) can be used for some diagnostic and therapeutic applications. Use of XTerra hybrid-particles contained in Waters Intelligent Speed (IS ) columns is well suited for the fast, IP-RP- HPC analysis of synthetic oligonucleotides and RNAi. High Mass Load Purification for 5 nt ligonucleotide Separation of Synthetic ligonucleotides on XTerra Intelligent Speed (IS ) Columns AU Start Collection 60 nm 90 nm Stop Collection An example of a 5 nt oligonucleotide purified using an XTerra column. The dashed lines indicate the start and stop of the collection. The chromatogram shows monitoring at two separate wavelengths. 60 nm is the typical wavelength used to detect DNA. nce the detector is saturated with 0.5 mmole mass load, the less sensitive 90 nm detection wavelength was used for visual confirmation of the oligonucleotide separation from n- failure products. AU Conditions Column: XTerra MS C 8,.5 µm.6 x 50 mm Mobile Phase A: 5% ACN in 0.M TEAA, ph 7.0 Mobile Phase B: 5% ACN in 0.M TEAA, ph 7.0 Gradient: Time Profile (min) %A %B Flow Rate: Temperature: Detection: HPLC System: 97% Pure.0 ml/min 60 C 60 and 90 nm Waters Alliance System with Waters 695 PDA UV 5 nm N- 0mer G N- N-9 N- N- 0mer C N- 0mer A N- 0mer T 0 Separation was performed on a.6 x 0 mm XTerra MS C 8,.5 µm IS column. Mobile phases were comprised of,,,,,-hexafluoroisopropanol (HFIP) and triethylamine (TEA) with a concave methanol (MeH) gradient. nce XTerra columns are packed with hybrid particles, they can be operated at the elevated ph and temperature levels needed for DNA separation (ph 7 9; 60 C). The short column length (0 mm) allows for separation times under 5 minutes. Figure shows the IP-RP-HPLC separation of four different homooligonucleotides (dg 0, dc 0, da 0, and dt 0 ). Baseline separation of closely eluting impurities (i.e. N,,, etc.) from the target oligonucleotide was routinely achieved. Column: Mobile phase A: Mobile phase B: Buffer preparation: Flow rate: Gradient: Injection volume: Temperature: Detection: HPLC System: XTerra MS C 8,.5 µm,.6 x 0 mm IS Column 5 % MeH in TEA/HFIP (6. mm/ 00 mm), ph % MeH in TEA/HFIP (6. mm/ 00 mm), ph 7.9 Dissolve.5 ml of HFIP in ~ 950 ml of water. While mixing vigorously, add. ml of TEA. Adjust final volume to L with water. The ph of the solution should be about ml/minute 0 60 % B in.5 min.; gradient profile (concave) µl (dissolved in mobile phase A). 00 pmole total mass load 60 C Waters 87 Dual Wavelength Absorbance Detector; 5 nm Waters Alliance HT Separations Module with Waters 87 Dual Wavelength Absorbance Detector

22 XTerra Capillary and Narrowbore Columns XTerra Analytical Columns ID Length Particle ze Type MS C 8 MS C 8 RP 8 RP 8 Phenyl 75 µm 50 mm.5 µm Capillary µm 00 mm.5 µm Capillary µm 50 mm.5 µm Capillary µm 50 mm.5 µm Capillary µm 00 mm.5 µm Capillary µm 50 mm.5 µm Capillary µm 50 mm.5 µm Capillary µm 00 mm.5 µm Capillary µm 50 mm.5 µm Capillary µm 50 mm.5 µm Capillary µm 00 mm.5 µm Capillary µm 50 mm.5 µm Capillary µm 50 mm 5 µm Capillary µm 00 mm 5 µm Capillary µm 50 mm 5 µm Capillary mm 50 mm.5 µm Column mm 50 mm.5 µm Column mm 00 mm.5 µm Column mm 50 mm.5 µm Column mm 0 mm.5 µm Guard mm 0 mm 5 µm Guard mm 5 mm.5 µm Direct Connect Column NEW. mm 5 mm.5 µm Direct Connect Column NEW. mm 5 mm 5 µm Direct Connect Column NEW. mm 5 mm 0 µm Direct Connect Column NEW. mm 0 mm.5 µm IS Column NEW. mm 0 mm.5 µm IS Column mm 0 mm.5 µm Guard NEW. mm 0 mm 5 µm IS Column mm 0 mm 5 µm Guard mm 0 mm.5 µm Column mm 0 mm.5 µm Column mm 50 mm.5 µm Column mm 50 mm.5 µm Column mm 50 mm.5 µm Cartridge mm 50 mm 5 µm Column mm 50 mm 5 µm Cartridge mm 00 mm.5 µm Column mm 00 mm.5 µm Cartridge mm 00 mm 5 µm Column mm 00 mm 5 µm Cartridge mm 50 mm.5 µm Column mm 50 mm.5 µm Cartridge mm 50 mm 5 µm Column mm 50 mm 5 µm Cartridge mm 50 mm 5 µm Column mm 50 mm 5 µm Cartridge ID Length Particle ze Type MS C 8 MS C 8 RP 8 RP 8 Phenyl NEW.0 mm 0 mm.5 µm IS Column mm 0 mm.5 µm Cartridge NEW.0 mm 0 mm.5 µm IS Column mm 0 mm.5 µm Guard NEW.0 mm 0 mm 5 µm IS Column mm 0 mm 5 µm Guard mm 0 mm.5 µm Column mm 0 mm.5 µm Column mm 50 mm.5 µm Column mm 50 mm.5 µm Column mm 50 mm.5 µm Cartridge mm 50 mm 5 µm Column mm 50 mm 5 µm Cartridge mm 00 mm.5 µm Column mm 00 mm.5 µm Cartridge mm 00 mm 5 µm Column mm 00 mm 5 µm Cartridge mm 50 mm.5 µm Column mm 50 mm.5 µm Cartridge mm 50 mm 5 µm Column mm 50 mm 5 µm Cartridge mm 50 mm 5 µm Column mm 50 mm 5 µm Cartridge NEW.9 mm 0 mm.5 µm IS Column NEW.9 mm 0 mm.5 µm IS Column mm 0 mm.5 µm Guard NEW.9 mm 0 mm 5 µm IS Column mm 0 mm 5 µm Guard mm 50 mm.5 µm Cartridge mm 50 mm 5 µm Cartridge mm 00 mm.5 µm Column mm 00 mm.5 µm Cartridge mm 00 mm 5 µm Column mm 50 mm.5 µm Column mm 50 mm 5 µm Column mm 50 mm 5 µm Cartridge NEW.6 mm 0 mm.5 µm Guard NEW.6 mm 0 mm 5 µm Guard NEW.6 mm 0 mm.5 µm IS Column mm 0 mm.5 µm Cartridge NEW.6 mm 0 mm.5 µm IS Column NEW.6 mm 0 mm 5 µm IS Column mm 0 mm.5 µm Column mm 0 mm.5 µm Column mm 0 mm 5 µm Column

23 XTerra Prep Columns ID Length Particle ze Type MS C 8 MS C 8 RP 8 RP 8 Phenyl ID Length Particle ze Type MS C 8 MS C 8 RP 8 RP 8.6 mm 50 mm.5 µm Column mm 50 mm.5 µm Column mm 50 mm.5 µm Cartridge mm 50 mm 5 µm Column mm 50 mm 5 µm Cartridge mm 00 mm.5 µm Column mm 00 mm.5 µm Cartridge mm 00 mm 5 µm Column mm 50 mm.5 µm Column mm 50 mm.5 µm Cartridge mm 50 mm 5 µm Column mm 50 mm 5 µm Cartridge mm 50 mm.5 µm Column mm 50 mm 5 µm Column mm 50 mm 5 µm Cartridge XTerra ligonucleotide Purification Columns ID (mm) Length (mm) Particle ze Type MS C 8.6 mm 50 mm.5 µm Column mm 50 mm.5 µm Column XTerra Method Validation Kits ID (mm) Length (mm) Particle ze Type MS C 8 MS C 8 RP 8 RP 8 Phenyl. mm 00 mm.5 µm Col MVK Custom Custom Custom. mm 50 mm 5 µm Col MVK Custom Custom Custom Custom.9 mm 50 mm 5 µm Col MVK Custom Custom Custom.6 mm 00 mm.5 µm Col MVK Custom Custom Custom.6 mm 50 mm.5 µm Col MVK Custom Custom mm 50 mm 5 µm Col MVK Custom Custom mm 50 mm 5 µm Col MVK Custom All other Method Validation kits are custom made on request 7.8 mm 0 mm 5 µm Cartridge mm 0 mm 0 µm Cartridge mm 50 mm 5 µm Column mm 00 mm 5 µm Column mm 50 mm 5 µm Column mm 50 mm 0 µm Column mm 00 mm 0 µm Column mm 0 mm 5 µm Cartridge mm 0 mm 0 µm Cartridge mm 0 mm 5 µm Column mm 50 mm 5 µm Column mm 00 mm 5 µm Column mm 50 mm 5 µm Column mm 50 mm 0 µm Column mm 50 mm 0 µm Column mm 00 mm 0 µm Column mm 0 mm 5 µm Cartridge mm 0 mm 0 µm Cartridge mm 0 mm 5 µm BD Column mm 50 mm 5 µm BD Column NEW 9 mm 50 mm 0 µm BD Column mm 00 mm 5 µm BD Column mm 50 mm 5 µm BD Column mm 50 mm 0 µm BD Column mm 50 mm 0 µm BD Column mm 00 mm 0 µm BD Column mm 50 mm 5 µm BD Column mm 75 mm 5 µm BD Column mm 00 mm 5 µm BD Column mm 50 mm 0 µm BD Column mm 50 mm 0 µm BD Column mm 00 mm 0 µm BD Column mm 50 mm 5µ m BD Column mm 50 mm 0 µm BD Column mm 00 mm 5 µm BD Column mm 50 mm 0 µm Column mm 50 mm 0 µm Column mm 00 mm 0 µm Column Needs Guard Holder Part No WAT Needs Cartridge Column Holder Part No Needs Cartridge Column End Connector Kit Part No Needs Part No WAT0690 (Universal, use with standard columns) or WAT06905 (Integrated into cartridge column) Needs End Connector Kit Part No WAT0755 Needs 7.8 x 0 mm Cartridge Holder Part No Needs 0 x 0 mm Cartridge Holder Part No Needs 9 x 0 mm Cartridge Holder Part No

24 Sales ffices AUSTRIA AND EXPRT (CENTRAL SUTH EASTERN EURPE, CIS AND MIDDLE EAST) () AUSTRALIA (6) BELGIUM () DENMARK (5) FINLAND (58) FRANCE () IRELAND (5) ITALY (9) 0 7. JAPAN (8) 7 79 NRWAY (7) PUERT RIC (787) SINGAPRE (65) TAIWAN (886) UNITED KINGDM () ALL THER CUNTRIES: WATERS CRPRATIN U.S.A / BRAZIL (55) GERMANY (9) KREA (8) SPAIN () Waters Corporation. CANADA HNG KNG MEXIC (555) SWEDEN (6) All rights reserved. Waters, XTerra, asis, BD, IS, Alliance, Autopurification, NanoEase, Micromass, FractionLynx, ZQ, and Quattro Ultima are trademarks of Waters Corporation. BioCAP is a trademark of Bio-Rad Laboratories. CHINA INDIA AND INDIA SUBCNTINENT (9) THE NETHERLANDS (0) SWITZERLAND () EN 07/0