Increased Protein/Peptide Mass Transfer Rates for Improved Resolution and Faster LC

Transcription

1 Increased Protein/Peptide Mass Transfer Rates for Improved Resolution and Faster LC

2 Applications for Fast HPLC of Large Molecules and Proteins Proteomics studies LC/MS studies Combinatorial chemistry analyses Rapid monitoring for process control 2D-methods Page 2

3 First, A Few Basics on Bio Molecule Separations Factors That Optimize Bio Separations Sample Solubility Appropriate Pore Size Bonded Phase Low ph Mobile Phase High ph Mobile Phase Temperature Gradient Page 3

4 Optimize Solubility Selected Sample Solvents and Application for Protein and Peptides Solvent Application Comments.5-5% TFA in Water General Effective solubilization of many samples 6 M Guanidine, buffered General Very good for many proteins and at ph % Acetic Acid or Formic Acid;.1-.5M Perchloric Acid Peptides 6 M Urea/ 5% Acetic aid Hydrophobic Peptides, Proteins Water-Miscible Organic Solvents: Acetonitrile, Methanol, THF, Dioxane, DMSO; +/- TFA; +/- Water Hydrophobic Peptides, Polypeptides peptides Frequently used to extract peptides from tissues, precipitating many proteins and cellular debris Useful for membrane proteins, fragments, aggregating systems Limit injection volume to avoid problems; add water, as possible, to improve volume tolerance; acidify with TFA as required Page 4

5 Improved Peak Shape for Large Molecules in Solution Columns: 4.6 x 15 mm, 5 mm Mobile Phase: 6% MeOH: 4%.1% TFA Flow Rate:.75 ml/ Temperature: RT Detection: UV 282 nm Sample: Tylosin (MW 916) O SB-C3 (8Å) HO O OCH OCH 3 3 O H 3 C CH 2 CH 2 CH 3 CH 3 O H 3 C O CH 3 CHO CH 2 H HO 3 C O O OH N CH 3 O CH 3 O 3SB-C3 (3Å) HO CH3 OH CH 3 Pw 1/2 =.442 Pw 1/2 = Time () 5 1 Time () The size of a molecule in solution deteres which pore size column is best. The narrower peak width indicates unrestricted access to the pores. Page 5

6 Wide Pore (3Å) Columns are Needed for Proteins and Polypeptides Effect of Pore Size and Molecular Size on Peak Width, Gradient Separations SB-C18 (3Å) SB-C18 (8Å).14 PW 1/ Leu Enkephalin M.W. = 556 Angiotensin II M.W. = 146 Insulin B M.W. = 3,496 Cyt C M.W. = 12,327 RNase M.W. = 13,684 Lysozyme M.W. = 13,9 Proper pore size selection results in sharper peaks for large molecules Page 6

7 Recovery of Polypeptides from ZORBAX 3SB Columns Relative Recovery to 3SB-C18 11% 1% 9% 8% 7% 6% 5% 3SB-C8 3SB-C3 3SB-CN Parvalbu Myoglobin RNase A Insulin Lysozyme Carbonic Anhydrase Calmodulin Columns: 4.6 x 15 mm Mobile Phase: 5-4% B in 2. A:.1% TFA / Water B:.1% TFA / ACN Flow Rate: 1 ml /. Temperature: 6 C Sample: 4 µg each protein 25 µl injection Recovery may vary depending on bonded phase. All 3SB bonded phase generally provide good recovery. Page 7

8 Comparison Separation of Large Polypeptides on 3SB Bonded Phases 3SB-C18 1 2, SB-C8 3SB-C , Columns: ZORBAX StableBond 3SB 4.6 x 15 mm, 5 mm Mobile Phase: Linear Gradient, 25-7% B in 4 A:.1% TFA in Water B:.9% TFA in 8% ACN/2% water Flow Rate: 1. ml/ Temperature: 6 C Sample: 3 mg each protein 1. RNase 6. CDR 2. Insulin 7. Myoglobin 3. Cytochrome C 8. Carbonic Anhydrase 4. Lysozyme 9. S-1β 5. Parvalbu 1. S-1α 3SB-CN Ret ent ion Time () Four different 3SB bonded phases allow selectivity optimization of proteins. Page 8

9 Effect of Bonded-Phase Ligand on Recovery of a Synthetic Lipopeptide 3SB-CN Recovery 59% 3SB-C8 3SB-C18 Recovery 75% Recovery 89% Conditions: Column: ZORBAX 3SB, 4.6 x 15 mm, 5 mm Mobile Phase: A.1% TFA/water, B.1% TFA/can Gradient: 1-9 % B in 4 Flow Rate: 1 ml/ Temperature: 6 C, Sample: 15 µl ( 15 µg ) of peptide in.1% TFA/DMSO Retention Time, (.) Recovery on each bonded phase is not always predictable so evaluate different bonded phases. Page 9

10 ZORBAX 3SB HPLC Column Developed Expressly for Bio Separations Page 1

11 ZORBAX 3SB Designed for Long Life in Low ph Bio Separations rhgh Tryptic Digest Column: ZORBAX 3SB-C8, 4.6 x 15mm; Mobile Phase Gradient - 6% in 12.A=.1% TFA in Water, B=.86% TFA in ACN Temp.: 4 C Flow Rate: 1mL/. Det. UV 21nm Sample: 5 µg of rhgh Tryptic Digest After 495 ml After 1368 ml Reference 3 336P1.PPT Page 11

12 High ph Can be Used for Separating Hydrophobic or Other Low-Solubility Peptides Comparison of Aß Peptide RP-HPLC Separations at Low and High ph TFA Conditions, 25 C A-.1% TFA in water B-.85% TFA in 8%AcN 33-45%B in 3. Aβ(1-38) Aβ(1-4) Aβ(1-42/3) Column: Flow Rate: Sample: ZORBAX 3Extend C x 15 mm, 5 mm.25 ml/ 5 µl sample (1 pmol each) TFA Conditions, 8 C A-.1% TFA in water B-.85% TFA in 8%AcN 29-41%B in 3. Absorbance (21 nm) NH 4 OH Conditions, 25 C A- 2 mm NH 4 OH in water B- 2 mm NH 4 OH in 8%AcN 26-38%B in 3. Aβ(1-43) Aβ(1-38) Aβ(1-4) Aβ(1-42) Aβ(1-42) Aβ(1-43) Aβ(1-38) Aβ(1-4) Amyloid ß Sequences: Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys Leu 17 Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile Gly Leu Met Val Gly Gly 38 Val Val 4 Ile Ala 42 Thr 43 -COOH Reference 1 High ph and room temperature improve peak shape, recovery and change selectivity. Page 12

13 ph Can Change Selectivity in Bio Separations Comparison of TFA and NH 4 OH For Peptide RP-HPLC \ ESI-MS TIC (15-15 m/z) 3E7 2.5E7 2E7 1.5E7 1E7.5E7 3E7 2.5E7 2E7 1.5E7 1E7.1% TFA 2mM NH 4 OH LLG LHL LLL-NH 2 LLG LLL LHL LLVF LLL LLL-NH 2 LLVF Column: ZORBAX Extend C18, 2.1 x 15 mm Flow rate:.25ml/ Temp: 25 C Gradient: 5-6% B in 2 ; LC/MS: Pos. Ion ESI- Vf 7V, Vcap 4.5 kv, N2-35 psi, 12L/, 3 C 4 µl (5 ng each peptide); TFA Conditions: A-.1% TFA in water B-.85% TFA in 8% AcN NH 4 OH Conditions: A- 2 mm NH 4 OH in water B- 2 mm NH 4 OH in 8% AcN.5E Page 13

14 Peptide RP-HPLC/ESI-MS NH 4 OH Mobile Phase Yields Positive and Negative Ion Spectra 3E7 Positive Ions LLL LLL-NH 2 TIC (15-15 m/z) 2.5E7 2E7 1.5E7 1E7.5E7 3E7 2.5E7 2E7 Negative Ions LLG LHL LLVF Column: ZORBAX Extend C x 15 mm, 5 mm Flow rate:.25ml/ Temp: 25 C Gradient: 5-6% B in 2 LC/MS: Pos. Ion ESI- Vf 7V, Vcap 4.5 kv, N2-35 psi, 12L/, 3 C Sample: 4µL (5 ng each peptide) 1.5E7 LLG LLL LLVF LLL-NH 2 1E7 LHL.5E The Extend-C18 column is ideal for LC/MS of proteins and peptides at high ph Both positive and negative ion MS are possible with NH 4 OH mobile phase. Page 14

15 LC/MS at Low ph on Extend-C18 Column: ZORBAX Extend-C18, 2.1 x 15 mm, 5 mm Flow Rate:.2 ml/ Temperature: 35 C Mobile Phase Gradient: 15-5% B in 15. A:.1% TFA in water B:.85% TFA in 8% ACN LC/MS: Pos. Ion ESI Vf 7V, Vcap 4.5 kv, N 2-35 psi, 12L/., 325 C Sample: Angiotensin I, II, III 2.5 ml (5 pmol each) TIC (2-15 m/z) 5.E6 4.E6 3.E6 2.E6 1.E6 AII+ AIII AI Max: m/z Acidic conditions can not resolve all three Angiotensins. Page 15

16 LC/MS at High ph on Extend-C18 Column: ZORBAX Extend-C18, 2.1 x 15 mm, 5 mm Flow Rate:.2 ml/ Temperature: 35 C Mobile Phase Gradient: 15-5% B in 15. A: 1 mm NH 4 OH in water B: 1 mm NH 4 OH in 8% ACN LC/MS: Pos. Ion ESI Vf 7V, Vcap 4.5 kv, N 2-35 psi, 12L/., 325 C Sample: Angiotensin I, II, III, 2.5 ml (5 pmol each) 5.E7 Signalx1 TIC (2-15 m/z) 4.E7 3.E7 2.E AII AI AIII Max: E m/z At high ph all 3 Angiotensins are resolved and the mass spectrum shows improved spectral clarity. Page 16

17 Optimize Recovery by Changing Temperature ßAP Peptide Separation Column: ZORBAX 3SB-C18, 4.6 x 15 mm, 5 mm Mobile Phase: A-.1%TFA in water, B-.9%TFA in acetonitrile Gradient: 2-45% B in 35 Flow Rate: 1 ml/ Sample: 1 µl injection of 5 µg peptide in 6M Urea/5% HOAc ßAP(1-38) ßAP(1-43) * Recovery <1% 25 C Absorbance (21 nm) 4 C 6 C 8 C Recovery >7% Time (.) Page 17

18 Protein and Peptide Retention by RP-HPLC High Sensitivity to Changes in % Organic: 1 Leucine Enkephalin s = 11 Lysozyme s = 4 1 log k«k Benzene s = 2.7 log k = log k -Sφ 1 φ = Water/Organic φ Large proteins like Lysozyme (14, MW) are more sensitive to changes in mobile phase conc of organic modifier (15X) than small molecules like benzene (78 MW) and 4X more sensitive than leucine enkephalin (6 MW). Page 18

19 Smaller Particles Improve Mass Transfer Effect on Column Dispersion Plate Height H H = A + B/u + C u Large Particle Small Particle Sum Curve: van-deemter Resistance to Mass Transfer H Longitudinal diffusion Eddy Diffusion u opt Linear Velocity u The smaller the plate height, the higher the plate number and the greater the chromatographic resolution Page 19

20 Improved Efficiency Over Wide Flow Range with Smaller Particles HETP (cm ) μm 3.5μm 1.8μm Column: ZORBAX Eclipse XDB- C18 Dimensions: 4.6 x 5/3mm Eluent: 85:15 ACN:Water Flow Rates:.5 5. ml/ Temp: 2 C Sample: 1.μL Octanophenone in Eluent H = A + B/u + Cu Volum etric Flow Rate (m L/m in) Smaller particle sizes yield flatter curves, ima shift to higher flow rates Page 2

21 1% B t g = 5 Changing Gradient Time to Affect Retention (k*) and Resolution % B 1% B k* t g F t g = 1 S Δ%B V m % B % B t g = 2 1% B DF = change in volume fraction of B solvent S = constant F = flow rate (ml/.) t g = gradient time (.) V m = column void volume (ml) 1% B % B Time () t g = 4 S 4 5 for small molecules 1 < S < 1 for peptides and proteins 995P1.PPT Title ofpresentation AgilentRestricted

22 Optimize Resolution: Column Length, Particle Size and Gradient Time ZORBAX 3SB-C8 4.6 x 25 mm,5 µm Rapid Resolution 4.6 x 15 m m,3.5 µm Mobile Phase: A: 95:5, Water : ACN with.1% TFA B: 5:95, 28 Water 3: ACN with.85% TFA % B in % B in Met-enkephalin 2. Leu-enkephalin 3. Angiotensin II 4. Neurotensin 5. RNase 6. Insulin (Bov) 7. Lysozyme 8. Calmodulin 9. Myoglobin 1. Carbonic Anhydrase Conditions: Rapid Resolution 4.6 x 5 m m,3.5 µm 1-6% B in 1. Flow: 1. ml/ Detection: 215nm Sample: 1-1µg protein (1µL inj.) in 6M Guanidine HCL, ph Page 22

23 Improving Resolution Using Short Column Length ( Vm ) and Particle Size (N) 3SB-C8, 4.6 x 15 mm, 5µm 3SB-C8, 4.6 x 5 mm, 3.5µm Mobile Phase: Flow Rate: Temperature: A: 95% Water : 5 % ACN,.1% TFA B: 5% Water : 95% ACN,.85% TFA Gradient: 1-6% B in 3. Sample: 1. Gly-Tyr 2. Val-Tyr-Val 3. [Gln 11 ] Amyloid-β- 6. Leu-Enk 7. Angiotensin II 8. Kinetensin 1. ml /. Protein Fragm RNase Ambient 4. (TYR8) Bradykinin 1. Insulin (Eq.) 5. Met-Enk 997P1.PPT Page 23

24 Gradient Resolution and Shorter Columns Faster Analyses for Complex Samples: HSA Tryptic Digest 2.1x15mm, 5μm P/N gradient.2 ml/ 12 peaks 6 s x5mm, 1.8μm P/N gradient.5ml/ 125 peaks 1 s! x5mm, 1.8μm P/N gradient.5ml/ 156 peaks! 25 s Conditions:Mobile Phase A:W aterw/.1% TFA,B:ACN w/.1% TFA, Gradient2% B to 5% B, Tem p:5 C UV 214 nm Page 24

25 Particle Size - More Peak Capacity with 1.8 um RRHT Columns - Peptide Map of BSA Conditions: Columns: as listed, Mobile Phase: A:.1% TFA in Water B:.8% TFA in ACN Gradient: 5% B to 6%B in 25. Temperature: 8 C Sample: BSA tryptic digest Column used SB-C18, 2.1x15mm, 1.8µ Peak Capacity 673 Starting Pressure 38 bar 35% More peak capacity, more resolution SB-C18, 2.1x15mm, 3.5µ Peak Capacity 52 Starting Pressure 15 bar Less peak capacity, less resolution 2-2 Smaller particle size = sharper peaks = greater peak capacity Page 25

26 What Makes High Speed HPLC Possible? Small particle sizes (< 5 mm) Short columns (1 cm or less) Low viscosity mobile phases Optimized HPLC imal extra column volume, low volume flow cell, imal delay volume for gradients Detector set to fast response time High diffusion coefficient of the sample in the mobile phase Rapid diffusion = less band broadening = higher linear velocities with efficient peaks But large molecules have small diffusion coefficients!! Page 26

27 Slower Diffusion of Large Molecules Limits Speed and Resolution So, decrease the diffusion time for macromolecules! Increase the Diffusion Rate Elevate operating temperature -- Need stable bonded phase Decrease solvent viscosity -- Helps but changes elution Decrease the Diffusion Distance Develop very small particles (<2-um) Limit diffusion distance into a particle! Porous She l Solid Core Page 27

28 What is Poroshell 3SB? A solid silica core with a superficially porous surface of smaller silica sol particles derivatized with bonded phase. Poroshell 3SB superficially-porous particles, reduce diffusion distance for proteins for fast HPLC. A RP-HPLC phase such as C18, C8 or C3 are bound to sol particles in the porous shell. Scale: = 1 µm Scanning Electron Micrograph of Poroshell Particles Page 28

29 Comparison of Diffusion Distance Totally porous silica vs. superficially porous silica 5 µm Totally Porous Particle 5 µm Superficially Porous Particle 2.5 µm Required diffusion distance for a macromolecule reduced 1 fold!.25 µm Page 29

30 Maintain Efficiency at High Velocity with Poroshell 3SB-C18 Van Deemter Plot for Urease (83,D) and Insulin (5,7D) Column: Poroshell 3SB-C18, 5 μm Mobile phase: Urease, 44.7% ACN / 55.2%.1% TFA, Insulin1, 32.6% ACN / 67.3%.1% TFA Insulin 2, 28% ACN / 72%.1% TFA Detection:UV 214 nm Samples: in 6 M guanidine, ph 6.8 phosphate buffer Temperature: 6 C.2 Data fitted to Knox equation Urease; MW=83, (sub-unit); column: 3 x 1 mm.15 Plate Height, cm.1 Insulin1; MW=5,7 3 x 1 mm column.5 Insulin2; MW=5,7 2.1 x 75 mm column Mobile Phase Velocity, cm/sec Slow increase in plate height with velocity means high flow rates can be used for fast separation with modest loss in resolution Steeper plot for urease reflects slower diffusion of much larger protein Page 3

31 How Do We Use Poroshell Columns? Faster separations High flow rates High flow rates for fast analysis with high resolution in comparison to totally porous silica Polypeptides Large proteins High recovery of large proteins Elevated temperature High resolution High resolution of protein digests High resolution of protein impurities High resolution for LC/MS of proteins Change selectivity Different bonded phases Comparison to totally porous 3SB materials Page 31

32 High Flow Rates with 2.1 mm ID Poroshell for High Resolution and Fast Separations Columns: Poroshell 3SB-C x 75 mm, 5 mm Mobile Phase:A:.1% TFA B:.7% TFA in ACN Gradient: 5 1% B in 1.. Flow Rate: 3. ml/. Temperature: 7 C Pressure: 25 bar Detection: UV 215 nm Sample: 1. Angiotensin II 2. Neurotensin 3. Rnase 4. Insulin 5. Lysozyme 6. Myoglobin 7.Carbonic Anhydrase 8.Ovalbu.5 1. Time () Only Poroshell can provide high efficiency at higher flow rates for extremely rapid separations of proteins and peptides. This is due to more rapid mass transfer of the superficially porous particle Page 32

33 Flow Rates for Poroshell Columns Column Internal Diameter Porous Particle Flow Rate Range Poroshell Flow Rate Range 2.1 mm.1.3 ml/.3 3 ml/ 1. mm 3 6 ml/ ml/ Very high flow rates can be used effectively with Poroshell columns Page 33

34 Poroshell for Fast Analysis of High MW Proteins - ß-amylase (2kdal) Agilent 11 WPS with AutoBypass; Piston Stroke: 2µL, Column: as shown Gradient: as shown Mobile Phase: A= 95% H 2 O, 5% ACN with.1%tfa; B= 5% H 2 O, 95% ACN, with.7%tfa Flow Rate: as shown Temp.: 7 C, Detection.: UV 215 nm (Poroshell chromatogram - expanded) ß-am ylase F =2 ml/, 5-1 %B / 1 Poroshell chromatogram expanded F =1 ml/, 5-1 %B / 2 12 ß-am ylase ß-am ylase 12 Poroshell 3SB-C18 2.1x75 mm, 5 µm 1\DATA\PORSHL3\PrSHL1.d\ßamy-ovr.win * Zorbax 3SB-C18 4.6x15 mm, 5 µ* Poroshell technology facilitates ultra-fast HPLC analysis of proteins Note the similar separation of or impurities in the 2 and 2 separations. Page 34

35 Poroshell Has Different Selectivity from Totally Porous 3SB Agilent 11 WPS with AutoBypass Column: 3SB-C18, 2.1 x 75 mm, 5 mm Mobile Phase: A= 95% H 2 O, 5% ACN with.1%tfa B= 5% H 2 O, 95% ACN, with.7%tfa Gradient: 5 1%B in.67 Flow Rate: 3 ml/ Piston Stroke: 2 µl Temp.: 7 C Det.: UV 215 nm 8 3SB-C18 1, Poroshell3SB-C Poroshell and 3SB-C18 can have different selectivity due to the different ratios of bonded phase on the surface. Page 35

36 Effect of Increasing Flow Rate in Protein Analysis with use of Totally Porous Silica Lysozyme ml/ -1%B in.67 2 ml/ -1%B in ml/ -1%B in 2 Totally Porous 2.1x75mm, 5µm.5 ml/ -1%B in Agilent 11 WPS with AutoBypass; Piston Stroke: 2µL, Temp.: 7 C, Det.: 215 nm Mobile Phase: A= 95% H 2 O, 5% AcN with.1%tfa; B= 5% H2O, 95% AcN, with.7%tfa 1. Neurotensin 2. Rnase A 4. Myoglobin 5 Increased flow rate decreases run time, but, decreases resolution in protein analysis when using totally porous particles! Page 36

37 Effect of Increasing Flow Rate in Protein Analysis with Poroshell Rnase A Sustained Efficiency and Resolution 3 ml/ -1%B ml/ -1%B ml/ -1%B ml/ -1%B 4 Agilent 11 WPS with AutoBypass; Piston Stroke: 2µL, Temp.: 7 C, Det.: 215 nm Mobile Phase: A= 95% H 2 O, 5% ACN with.1%tfa; B= 5% H 2 O, 95% ACN, with.7%tfa Poroshell 3SB-C18 2.1x75mm, 5µm 1. Neurotensin 3. Lysozyme 4. Myoglobin 5 As flow rate increases, protein/peptide analysis time is dramatically reduced when using superficially porous particles! Page 37

38 Poroshell for Fast Analysis of High-Molecular Weight Proteins ß-amylase (2kdal) ß-amylase A=.1% TFA,H 2 O;B=.7% TFA,ACN *DAD1 A, Sig=215,16 Ref=31, ß-amylase *DAD1 A, Sig=215,16 Ref=31, ß-am ylase F =2 ml/, 5-1 %B / 1 Poroshell chromatogram (Poroshell chromatogram - expanded) expanded A=.1% TFA,H 2 O;B=.7% TFA,ACN 12 ß-am ylase ß-am ylase ß-amylase Poroshell 3SB-C18 2.1x75 mm, 5 µm Temp: 7ºC * F =1 ml/, 5-1 %B / 2 1\DATA\PORSHL3\PrSHL1.d\ßamy-ovr.win ZORBAX 3SB-C18 * 4.6x15 mm, 5 µm Temp: 7ºC Poroshell technology facilitates ultra-fast HPLC analysis of proteins Note the similar separation of or impurities in the 2 and 2 separations. Page 38

39 Good Peak Shape and Recovery with Poroshell Analysis of High-Molecular Weight Proteins - Thyroglobulin 66 kda DAD1 A, Sig=215,16 Ref=31,1 (PORSHL3\PRSHL1.D) Thyroglobulin subunits F=2, 5-1%B/1, 7 C A=.1%TFA, H2O; B=.7%TFA, ACN *DAD1 A, Sig=215,16 Ref=31,1 (PORSHL3\PRSHL1.D) (Poroshell chromatogram - expanded) Thyroglobulin subunits *DAD1 A, Sig=215,16 Ref=31,1 (PORSHL3\PRSHL42.D) Thyroglobulin subunits F Poroshell =2 ml/, 3SB-C18, 2.1x75mm, 5-15µm%B / 1 expanded F =1 ml/, 5-1 %B / 2 Zorbax 3SB-C18, 4.6x15mm, 5µm F=1, 5-1%B/2, 7 C 12 Thyroglobulin subunits Thyroglobulin subunits Thyroglobulin subunits Poroshell 3SB-C18 2.1x75 mm, 5 µm 5 1\DATA\PORSHL3\PrSHL42.d\Thyr-ovr.win Zorbax 3SB-C * 12 Poroshell technology facilitates ultra-fast HPLC analysis of very large protein subunits. Note the improved peak shape and recovery on Poroshell, in <2. 2 * 4.6x15 mm, 5 µm Page 39

40 High Resolution and Fast Analysis Separation of a Protein Digest on Poroshell 3SB-C18 1 ml/ -1%B ml/ -1%B 12 6 Poroshell 3SB-C18 2.1x75 mm, 5 µm BSA tryptic digest 15hrs., 7 pmol Zorbax 3SB-C18 2.1x15 mm, 5 µm Agilent 11 WPS with AutoBypass; Piston Stroke: 2µL, Temp.: 7 C, Det.: 215 nm Mobile Phase: A= 95% H 2 O, 5% AcN with.1%tfa; B= 5% H2O, 95% AcN, with.7%tfa * Aligned Extremely fast (6 ), high, resolution of BSA tryptic digest, using Poroshell 3SB-C18. Under typical conditions, a comparable run takes 6 on a totally porous particle. Page 4

41 UltraHigh Speed HPLC Peptide Maps ofa Monoclonal Antibody on SeveralZorbax PoroshellPhases Originalmethod 12 t G using a C x 25 mm 57 peaks detected Zorbax Poroshell3SB-C18,2.1 x 75m m,5µ Zorbax Poroshell3SB-C8,2.1 x 75m m,5µ Zorbax Poroshell3SB-C3,2.1 x 75m m,5µ Conditions:Mobile phase A =.1% TFA in water Mobile phase B =.1% TFA in ACN;Tem perature: 7 C;Detection:VW D,21nm ;Injection:1 µllys-c digestofhum an M onoclonalantibody;flow :1. ml/m in;gradient:,% B;5.5,55% B;5.6,55% B;7.,% B Aligned Zorbax Poroshelltechnology facilitates ultra-fasthplc analysis ofpeptides 46 to 48 peaks, 1/2 analysis tim e Data courtesy of: Novartis Pharma, Biotechnology,Basel Dr.KurtForrer PatrikRoethlisberger Formore details see EN Page 41

42 LC/MS of Proteins with Poroshell Formic acid Provides High Sensitivity Column: Poroshell 3SB-C18, 2.1 x 75 mm, 5 mm Mobile Phase Gradient: 2-1% B in 5.5. A: water +.1% TFA or FA B: ACN +.1% FA or TFA Flow Rate: 5 ml/ Temperature: 6 C Injector: 1 ml Sample: 1 pmol of BSA Electrospray ionization: positive ion Vcap:6V Drying Gas: 12L/ 35ºC Nebulizer: 45 psi Scan: 6-25 amu Step size:.15 amu Peak width: Abundance 25 2 TIC Formic Acid m/z TFA m/z Page 42

43 High Flow Rates and High Sensitivity LC/MS Using 1. mm ID Poroshell Column: Poroshell 3SB-C18, 1. x 75 mm, 5 mm Mobile Phase Gradient: 2-1% B in 5.5. A: water +.1% formic acid B: ACN +.1% formic acid Flow Rate: 6 ml/ Temperature: 8 C Injection volume: 1 ml Sample: insulin, lysozyme, cytochrome C, myoglobin, BSA, carbonic anhydrase LC/MS: Pos. Ion ESI, Vcap 6 V, Drying gas flow: 12 l/ Drying gas temperature: 35 C Nebulizer: 45 psi, Fragmentor volatage: 14 V Scan: 6 25 Stepsize:.15 amu Peakwidth:.6 1E pmoles of protein on column These TIC s show good sensitivity with only.5 pmoles on column. Page 43

44 Fast Analysis of Insulin (5.7 kda) Impurities using Poroshell 3SB-C18 Column: Poroshell 3SB-C18, 2.1 x 75 mm, 5 mm Mobile Phase Gradient: 5-1% B in 5. A: water +.1% TFA B: ACN +..7% TFA Flow Rate: 2 ml/ Temperature: 7 C Sample: Bovine pancreas insulin INSULIN - bovine pancreas 27 hrs. at 55 C Untreated Page 44

45 Increasing Gradient Times on Poroshell Rapid Analysis of Fresh Carbonic Anhydrase Column: Poroshell 3SB-C18, 2.1 x 75 mm, 5 mm Mobile Phase Gradient: 5-1% B in tg. A: water +.1% TFA B: ACN +..7% TFA Flow Rate: 2 ml/ Temperature: 7 C Sample: Carbonic Anhydrase CA CA CA t G = Impurities t G = CA t G = 4 t G = 16 t G = 8 Poroshell technology is compatible with a wide range of gradients that result in a corresponding range of resolution and run times. Note the consistent separation of or impurities even in the 1 run at top. CA 8 Page 45

46 Rapid Analysis of Carbonic Anhydrase Aging RP-HPLC on Poroshell 3SB-C18 3 Carbonic Anhydrase (CA) F= 2m L/m in, 5-1% B/4, 7 C A=.1% TFA in H2O,B=.7% TFA in ACN Poroshell 3SB-C18 (2.1x75mm) Flow=2mL/, 5-1%B/4, 7 C hrs. Incubation at 55 C 5 hrs.incubation at55 C CA hr.incubation at55 C 1 hrs. Incubation at 55 C CA Poroshell 3SB-C18 2.1x75 mm, 5 µm CA 3 2 FRESH FRESH Impurities Rapid HPLC analysis of carbonic anhydrase and its aging products is achieved in 4 utes. Page 46

47 Fast Separation of Aged Carbonic Anhydrase RP-HPLC on Poroshell 3SB-C18 Colum n: Poroshell3SB-C18,2.1 x 75 mm,5 μm Mobile Phase Gradient: 5-1% B in tg. A:water+.1% TFA B:ACN +..7% TFA Flow Rate:2 ml/ Tem perature:7 C Sam ple:carbonic Anhydrase 1 mg/ml Injection Volum e:2 μl 1 t G = 1 Carbonic Anhydrase (CA) SeveralMonths at4 C t G = t G = t G = Extremely fast resolution of carbonic anhydrase aging products using Poroshell under four different gradient times. High resolution is obtained in 2 or 3. Page 47

48 More Poroshell Bonded Phases Provide Selectivity Options to Enhance Resolution 7 5, 6 Poroshell3SB-C Poroshell SB-C18, 2.1 x 75 mm 2.1 x 75 mm , Poroshell3SB-C3 Poroshell SB-C3, 2.1 x x 75 mm Samples: 1. Angiotensin II 2. Neurotensin 3. RNase A 4. Insulin B Chain 5. Insulin 6. Cytochrome C 7. Lysozyme 8. Myoglobin 9. Carbonic Anhydrase Column: Agilent Poroshell (2.1 x 75 mm); Temp.: 7 C; Flow:.5 ml/; Det: UV 215 nm Mobile Phase: A=.1% TFA/H 2 O, B=.7% TFA/ACN; Gradient: 5-1% B in 3. Changing from SB-C18 to SB-C3, within the Poroshell family results in resolution of peaks 5 and 6, still in 3! Page 48

49 Summary Fundamentals of Bio Separations apply whether using conventional columns or columns that optimize mass transfer These fundamentals can be used to optimize bio separations 1.8 µm particles can be used for much faster gradients peptide maps Poroshell columns have greatly reduced surface area and markedly reduce resistance to large molecule mass transfer This allows for fast analysis of proteins including very large proteins with high recovery. Optimized bonded phases for low and high ph offer more options for RPLC and Mass Spec assays Page 49

50 Column Description Monolith stationary phase. Page 5

51 Column Description Currently there are 4 Bio-Monolith Products: Bio-Monolith QA (strong anion-exchanger) Bio-Monolith DEAE (weak anion-exchanger) Bio-Monolith SO3 (strong cation-exchanger) Bio-Monolith Protein A (immunoaffinity) Further products to expand the product portfolio (CM: weak cation-exchanger; Protein G (?), Specialty columns (pdna analytics, virus analytics). Page 51

52 Basic characteristics Bio-Monolith Columns: Data Sheets Catalog number Column chemistry Matrix Ligand density Matrix dimensions Dynamic binding capacity Maximum loading capacity Working flow rates Maximum allowed pressure over the column QA: Strong anion exchanger Poly(glycidyl methacrylate ethylene dimethacrylate) 2. ±.2 mmol/g Diameter: 5.2 mm; Length: 4.95 mm; Bed volume:.1 ml > 25 mg BSA/ml 1.2 to 1.5 mg.2 to 2 ml/ (max. 3 ml/) 15 bar Temperature stability 4 to 4 C Recommended ph 2 to 13 working 1 to 14 CIP Page 52

53 Column Length Bio-Monolith Columns are characterized by a high resolving power at extremely short column lengths only 5 milimeter. 1 ml/ = 45 cm/h = 36 CV/ (res. time:,1 s) Page 53

54 Different Loadings on Bio-Monolith QA 1uL 5uL 1uL Gradient Page 54

55 Consecutive injections Bio Monolith QA Analytical Column Page 55

56 Dynamic Binding Capacity for DNA Dynamic Capacities 16, 14, 1% 5% 1% 12, DBC mg DNA 1, 8, 6, 4, 1% 5% 1% 2,, 1% 5% 1% QFF porous particles Q nano - membrane QA - monolith Note 5 times higher dynamic binding capacity than particle based resin while operating at 4-fold higher flow rate! Courtesy of Pete Gagnon, for details visit validated.com Page 56

57 Bio Monoliths Monolithic supports are highly porous rigid polymers with: High porosity (over 6 %) Flow-through channels ( pores ) having large diameter (1.5 µm) Uniform channel connectivity in 3D (homogeneous structure) Pore diameter [nm] Page 57