High Resolution and Fast LC and LC/MS of Proteins and Peptides with Poroshell Columns

Transcription

1 High Resolution and Fast LC and LC/MS of Proteins and Peptides with Poroshell Columns

2 The Path to Ultra-Fast HPLC Separations Superficially Porous Particles - 4µ 5 µm particles 15 mm length High Temp with Steric-Protection 25 mm length Non-Porous Particles Micro-Bore 1 µm particles 3.5 µm particles 15 mm length Superficially Porous Particles - 5µ Narrow-Bore Capillary Columns High Flow Rates Well-Plate Samplers 1.8 µm particles 2/6/24 Small-Column HPLC Technology Overlapped Injections Nano HPLC

3 What Makes High Speed HPLC Possible? Small particle sizes (< 5 µm) Short columns (1 cm or less) Low viscosity mobile phases Optimized HPLC imal extra column volume, low volume flow cell Detector set to fast response time High diffusion coefficient of the sample molecules in the mobile phase = rapid diffusion = less band broadening = higher linear velocities with efficient peaks But large molecules have small diffusion coefficients 2/6/24

4 Slower Diffusion of Large Molecules Broadens Peaks at High Flow Rates Decrease the diffusion time for macromolecules! How? Increase the Diffusion Rate Elevate operating temperature Decrease solvent viscosity Decrease the Diffusion Distance Develop very small particles Limit diffusion distance into a particle Zorbax Poroshell Approach 2/6/24

5 Comparison of diffusion distance Totally porous silica versus superficially porous silica 5 µm Totally Porous Particle 5 µm Superficially Porous Particle 2.5 µm Maximum diffusion depth for a macromolecule.25 µm 2/6/24

6 What are Zorbax Poroshell 3SB and 3Extend? A solid silica core with a superficially porous surface of smaller silica sol particles derivatized with a sterically-protected bonded phase Scale: Schematic of Zorbax Poroshell 3 Superficially-Porous Particle. A RP-HPLC phase such as C18, C8, C3 or Bidentate-C18 are bound to sol particles in the porous shell. 2/6/24 = 1 µm Scanning Electron Micrograph of Poroshell Particles

7 Poroshell is Part of the ZORBAX StableBond Family of Sterically-Protected Silica Phases HYDROLYTICALLY UNSTABLE CONVENTIONAL, SILOXANE ATTACKED BY HYDRONIUM ION HYDROLYTICALLY STABLE STERICALLY PROTECTED, SILOXANE RESISTS ATTACK BY HYDRONIUM ION 2/6/24 Advantage of Monolayer Bonding: Single Step, Reproducible Reaction

8 ZORBAX Extend-C18 - A Bidentate C18 Bonded Phase designed for Use at High ρh Now available as Zorbax Poroshell 3Extend-C18 C18 C18 O Si Si O Silica Support 2/6/24

9 ZORBAX StableBond-C18 shows exceptional stability at low ph and high temperatures (ph.8, 9 C) Purge Solvent: 5% methanol/water with 1.% TFA Solute: Toluene Kirkland, J.J. and J.W. Henderson, Journal of Chromatographic Science, 32 (1994) /6/24

10 Plate Heights, cm ZORBAX Extend-C18 is Very Stable at High ph Aging of Extend-C18 column in NH 4 OH at ph k Values Chloro-1-nitrobenzene Trimiprae Column: ZORBAX Extend-C x 15 mm, 5 µm Mobile phase:8% Methanol 2% 2 mm NH 4 OH, ph 1.5 Flow Rate: 1.5 ml/; Aging at 24 C, tests at 4 C Column Volumes of Mobile Phase 1-Chloro-1-nitrobenzene Trimiprae Consistent retention and plate heights over time indicate stable column at high ph. Long column lifetime achieved. 2/6/24

11 Gradient Equation for Fast HPLC of Proteins k* = t G F S Φ V m Because: S is a constant for a separation system. Assug: Φ S and Φ Φ is set for maximum resolution. Φ drop out of this equation. t G F k* = ~ V G V m V m t G X F = V G or gradient volume. k* (relative retention) is dependent upon the ratio of V G over V m. 1. If one keeps the same ratio of V G /V m, then k* and the peak elution pattern will stay the same. 2. Flow rates of 1 to 3 ml/ on a 2.1 mm i.d. Poroshell column are the equivalent of 5 to 15 ml/ on a 4.6 mm I.d. column! 3. When F is large, t G should be reduced proportionally; therefore, the very short run times! 4. In comparison of larger columns to the Poroshell configuration, t G and F are also adjusted. 994P2.PPT 2/6/24

12 How Do We Use Poroshell Columns? Faster separations High flow rates for fast analysis with high resolution in comparison to totally porous silica Polypeptides Large proteins Impurities High recovery of large proteins Elevated temperature Change selectivity Different bonded phases - Comparison to totally porous 3SB materials High resolution of proteins, heterogeneous proteins, and digests on different bonded phases LC/MS Factors affecting LC/MS of proteins High resolution LC/MS of proteins Different bonded phases comparisons to each other Peptide mapping at high and low ph 2/6/24

13 Ultra-Fast Analysis of High-Molecular Weight Protein and Impurities -- ß-Amylase (2KDal) Comparison of Zorbax Poroshell and Conventional Porous Particle F =2 ml/, 5-1 %B / 1 Zorbax Poroshell 3SB-C18 2.1x75 mm, 5 µm * 5 4 Above Poroshell chromatogram 3 2 expanded ß-amylase 5 Zorbax 3SB-C18, 4.6x15mm, 5µm 4 F F=1, =15-1%B/2 ml/,, C %B / Impurities * Zorbax 3SB-C18 4.6x15 mm, 5 µm 2 * Poroshell technology facilitates ultra-fast HPLC analysis of proteins Note the similar separation of or impurities in the 2 and 2 separations. 2/6/24

14 Poroshell Has Different Selectivity from Totally Porous 3SB , SB-C18 Agilent 11 WPS with AutoBypass Column: 3SB-C18, 2.1 x 75 mm, 5 µm 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 Poroshell 3SB-C Poroshell and 3SB-C18 can have different selectivity due to the different ratios of bonded phase on the surface. 2/6/24

15 Temperature sharpens peaks and lowers back pressure in ultra-fast protein analysis 16 bar 7 C Sterically protected C18 Superficially Porous Particle 2.1 x 75mm, 5µm 165 bar 6 C Agilent 11 WPS with AutoBypass Thermostatted Column Compartment 182 bar 5 C Mobile Phase: A= 95% H 2 O, 5% AcN with.1%tfa B= 5% H2O, 95% AcN, with.7%tfa 195 bar 4 C Piston Stroke: 2µL Temp.: 7 C, Det.: 215 nm 25 bar 3 C Instrument-Column Synergy in Optimizing Reversed Phase-HPLC Analysis of Proteins and Peptides, ISPPP 2, Ricker, R.D., and B. E. Boyes, Agilent Technologies, Inc. Use of elevated temperature with temperature-resistant bonded phases, reduces viscosity and back pressure, sharpens peaks, increases sample solubility, and reduces retention times. 2/6/24

16 Improved Peak Shape of IgG at Elevated Temperature Intact IgG (15 ug) at.5 ml/ o C 8 o C Zorbax Poroshell 3SB-C18, 5µ, 2.1 x 75 mm 75mm, Flow =.5mL/ 15µg intact IgG at 214 nm 1 Data courtesy of: Genentech. Inc Analytical Chemistry, Baojen Shyong, Galahad DePeralta, and Victor Ling 5 2/6/ Time (.) Immunoglobulins have retention characteristics similar to very hydrophobic peptides peaks are narrower at elevated temperatures and on shorter chain length columns

17 Effect of Increasing Flow Rate in Protein Analysis with use of Totally Porous Silica ml/ -1%B in ml/ -1%B in ml/ -1%B in 1 Totally Porous 2.1x75mm, 5µm 1. Neurotensin 2. Rnase A 3. Lysozyme 4. Myoglobin ml/ -1%B in Time () Efficiency and resolution are lost at high flow rates on a totally porous 3Å RP column 2/6/24

18 High Efficiency at High Flow Rates for Ultra-Fast Protein Analyses with Poroshell.5 ml/ 5 1%B in ml/ 5 1%B in 2 2 ml/ 5 1%B in 1 3 ml/ 5 1%B in.67 4 ml/ 5 1%B in.5 Agilent 11 DAD Agilent 11 WPS with ADVR Column: Poroshell 3SB-C x 75 mm, 5 mm Mobile Phase: A: 95% H 2 O, 5% ACN with.1% TFA B: 5% H 2 O, 5% ACN with.1% TFA Temperature:7 C Detector: UV 215 nm Sample: 1. Neurotensin3. Lysozyme 2. RNaseA 4. Myoglobin Resolution is maintained at high flow rates with Poroshell, allowing for efficient peaks and rapid analysis times. 2/6/24

19 Fast, High Resolution Separation of Peptides and Proteins With Poroshell 3SB-C18... In Seconds 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 2/6/24

20 Break Number 1 For Questions and Answers Press *1 on Your Phone to Ask a Question 2/6/24

21 How Do We Use Poroshell Columns? Faster separations High flow rates for fast analysis with high resolution in comparison to totally porous silica Polypeptides Large proteins Impurities High recovery of large proteins Elevated temperature Change selectivity LC/MS Different bonded phases - Comparison to totally porous 3SB materials High resolution of proteins, heterogeneous proteins, and digests on different bonded phases Factors affecting LC/MS of proteins High resolution LC/MS of proteins Different bonded phases comparisons to each other Peptide mapping at high and low ph 2/6/24

22 Separation of Seven Monoclonal Antibodies on Zorbax Poroshell 3SB-C8 2 5 Antibody Type: 1 = IgG1 Column: Zorbax Poroshell 3SB-C8, 2.1x75mm, 5u Flow: 1. ml/ Detection: 21nm Mobile phase A: H2-ACN (9:1) + 3 ml/l of MW 3 PEG Mobile phase B: H2O-ACN (1:9) +3 ml/l of MW 3 PEG Gradient:, 19% B; 12 41% B; 12.1, 19% B; 14, 19% B Temperature: 7 C = IgG1 3 = IgG1 4 = IgG4 5 = IgG1 6 = IgG1 7 = IgG1 5 Data courtesy of: Novartis Pharma, Biotechnology, Basel Dr. Kurt Forrer and Patrik Roethlisberger Zorbax Poroshell 3SB-C8 shows different selectivity for several pairs of monoclonal antibodies. 2/6/24

23 Separation of Seven Monoclonal Antibodies on Zorbax Poroshell 3SB-C3 Column: Zorbax Poroshell 3SB-C3, 2.1x75mm, 5u Flow: 1. ml/ Detection: 21nm Mobile phase A: H2-ACN (9:1) + 3ml/L of MW 3 PEG Mobile phase B: H2O-ACN (1:9) + 3ml/L of MW 3 PEG Gradient:, 19% B; 12 41% B; 12.1, 19% B; 14, 19% B Temperature: 7 C Antibody Type: 1 = IgG1 2 = IgG1 3 = IgG1 4 = IgG4 5 = IgG1 6 = IgG1 7 = IgG1 4 Data courtesy of: Novartis Pharma, Biotechnology, Basel Dr. Kurt Forrer and Patrik Roethlisberger Zorbax Poroshell 3SB-C3 shows different selectivity for several pairs of monoclonal antibodies. 2/6/24

24 Monoclonal IgG1 Chains on ZORBAX Poroshell 3SB-C8 Glycosylated Heavy Chains? Light chains Treated with: DTT 6 Column: Zorbax Poroshell 3SB-C8, 2.1x75mm, 5u 4 Flow: 1. ml/ 2 Detection: 21nm Mobile phase A: H2-ACN (9:1) +3 ml/l of MW 3 PEG Mobile phase B: H2O-ACN (1:9) + 3 ml/l of MW 3 PEG Gradient:, 25% B; 1 4% B; 1.1, 25% B; 12, 25% B Temperature: 7 C Treated with: DTT Peptide-N-glycosidase F Data courtesy of: Novartis Pharma, Biotechnology, Basel Dr. Kurt Forrer and Patrik Roethlisberger Treated with: DTT Peptide-N-glycosidase F Carboxypeptidase-B It is known that the heavy chain is glycosylated. What is not yet known for certain is whether one or both peaks above are glycosylated initially and what the difference is between the two peaks after the glycosylation is removed. Current thought is that they are likely to be conformers but there is no real proof of this yet. 2/6/24

25 Monoclonal Antibody Chains on ZORBAX Poroshell 3SB-C8 Heavy Chains Column: Zorbax Poroshell 3SB-C8, 2.1x75mm, 5um Flow: 1. ml/ Detection: 21nm Mobile phase A: H2-ACN (9:1) + 3 ml/l of MW 3 PEG Mobile phase B: H2O-ACN (1:9) + 3 ml/l of MW 3 PEG Gradient:, 2% B; 1 5% B; 1.1, 2% B; 14, 2% B Temperature: 7 C Light Chains differing by 2 ao acids IgG1(Antibody 7) treated with: DTT Peptide-N-glycosidase F Carboxypeptidase F 1 Data courtesy of: Novartis Pharma, Biotechnology, Basel Dr. Kurt Forrer and Patrik Roethlisberger /6/24

26 Separation of Large and Glycosylated Proteins on ZORBAX Poroshell 3SB-C18, C8, and C3 Column: Zorbax Poroshell 3SB-C18, C8 or C3, 1. x 75mm, 5µ Flow:.454ml/ Mobile phase A:.1% TFA in H 2 O Mobile phase B:.7% TFA in ACN Gradient: 5% B > 1% B in 1 Detector: DAD, 212nm, 1.7 µl flow cell <.1 peak width Temperature: 7 C Bypass mode, Binary Pump, no mixer Zorbax Poroshell 3SB-C18, 1.x75mm, 5µ mouse monoclonal IgG MW = 15KDa. Zorbax Poroshell 3SB-C8, 1.x75mm, 5µ human α 2 -Macroglobulin MW = 72KDa. human IgM MW = 95KDa * Column: Zorbax 3SB-C18, 1.x5mm, 3.5µ Flow:.71ml/ Mobile phase A:.1% TFA in H 2 O Mobile phase B:.7% TFA in ACN Gradient: 5%B > 1%B in 5 Detector: DAD, 212nm, 1.7µl flow cell <.1 peak width Temperature: 7 C Bypass mode, Binary Pump, no mixer Zorbax Poroshell 3SB-C3, 1.x75mm, 5µ Zorbax 3SB-C18,1.x5mm, 3.5µ human Glycophorin MW ~ 5KDa. 6% carbohydrate * 5X Faster * 3 Aligned Peak widths, resolution, and analysis times are superior on Poroshell columns 2/6/24

27 Separation of Large and Glycosylated Proteins on ZORBAX Poroshell 3SB-C18, C8, and C3 Column: Zorbax Poroshell 3SB-C18, C8 or C3, 1.x75mm, 5µ and Zorbax 3SB-C18, 1.x5mm, 3.5µ Flow:.454ml/ Mobile phase A:.1% TFA in H 2 O Mobile phase B:.7% TFA in ACN Gradient: 5% B > 1% B in 1 Detector: DAD, 212nm, 1.7 µl flow cell <.1 peak width Temperature: 7 C Bypass mode, Binary Pump, no mixer Zorbax 3SB-C18,1.x5mm, 3.5µ Zorbax Poroshell 3SB-C18, 1.x75mm, 5µ Zorbax Poroshell 3SB-C8, 1.x75mm, 5µ human IgM MW = 95KDa. human Glycophorin MW ~ 5KDa. 6% carbohydrate Zorbax Poroshell 3SB-C3, 1.x75mm, 5µ rabbit monoclonal IgG MW = 15KDa. human α 2 -Macroglobulin MW = 72KDa. Zorbax Poroshell C8 and C3 offer advantages over Zorbax 3SB-C18 for large and heavily glycosylated proteins Zorbax Poroshell C18 offers advantages for all but the most heavily glycosylated proteins /6/24

28 Chromatography of Thyroglobulin - a Doubly Heterogeneous Protein on ZORBAX Poroshell C3, C8, and C18 Column: as shown Flow:.454mL/ Mobile phase A:.1% TFA in H 2 O Mobile phase B:.7% TFA in ACN Gradient: 5% B > 1% B in 1 Detector: DAD, 212nm, 1.7 µl flow cell <.1 peak width Temperature: 7 C Bypass mode, Binary Pump, no mixer Peakwidth at Half Height (.) Symmetry ZORBAX Poroshell 3SB-C3, 1 x 75mm, 5µm ZORBAX Poroshell 3SB-C18, 1 x 75mm, 5µm ZORBAX Poroshell 3SB-C8, 1 x 75mm, 5µm ZORBAX 3SB-C18, 1 x 5mm, 3.5µm Bovine Thyroglobulin MW 67 KDa. Glycosylated and Partially Iodinated /6/ Best peak width and symmetry on Zorbax Poroshell 3SB-C3

29 High Speed HPLC Peptide Maps of a Monoclonal Antibody on Several ZORBAX Poroshell Phases Competitor C18, 4.6 x 25mm, 5µm ZORBAX Poroshell 3SB-C18, 2.1 x 75mm, 5µm ZORBAX Poroshell 3SB-C8, 2.1 x 75mm, 5µm ZORBAX Poroshell 3SB-C3, 2.1 x 75mm, 5µm 8 16 * * Conditions: Mobile phase A =.1% TFA in water Mobile phase B =.1% TFA in ACN; Gradient: 1, % B; 1, % B; 11, 5% b; 115, 7% B;12, 7% B; 125, % B; 135, % B Temperature: ambient; Detection: VWD, 21nm; Injection: 5 µl Lys-C digest of Human Monoclonal Antibody; Flow:.3 ml/; Conditions: Mobile phase A =.1% TFA in water Mobile phase B =.1% TFA in ACN; Gradient:, % B; 2, 5% B; 2.5, 1% B; 21.5, 1% B Temperature: 7 C; Detection: VWD, 21nm; Injection: 1 µl Lys-C digest of Human Monoclonal Antibody; Flow: 1. ml/ Aligned * 2/6/24

30 Ultra High Speed HPLC Peptide Maps of a Monoclonal Antibody on Several ZORBAX Poroshell Phases ZORBAX Poroshell 3SB-C18, 2.1 x 75mm, 5µm ZORBAX Poroshell 3SB-C8, 2.1 x 75mm, 5µm ZORBAX Poroshell 3SB-C3, 2.1 x 75mm, 5µm Conditions: Mobile phase A =.1% TFA in water Mobile phase B =.1% TFA in ACN; Gradient:, % B; 5.5, 55% B; 5.6, 55% B; 7., % B Temperature: 7 C; Detection: VWD, 21nm; Injection: 1 µl Lys-C digest of Human Monoclonal Antibody; Flow: 1. ml/; Aligned ZORBAX Poroshell technology facilitates ultra-fast HPLC analysis of peptides 2/6/24

31 Peaks detected in peptide maps of a Lys-C digest of a monoclonal antibody Column # of Peaks # of Peaks # of Peaks recognized recognized recognized (12. run) (2.5. runs) (5.6. runs) Competitor V C18 57 Poroshell-C Poroshell-C Poroshell-C /6/24

32 Separation of a Tryptic Digest on Poroshell Capillary HPLC Column Column : ZORBAX Poroshell 3SB-C18, 15 x.5 mm, 5µ Solvent :.1% TFA in water/.1% TFA in acetonitrile, gradient 5-65% in 15 Flow : 2 µl/ Detection : 214 nm Temp. : 25 C Sample :.1 µl, 1.5 pmol, digest of horse skeletal myoglobin System : Agilent 11 Series Capillary LC System /6/24

33 More Poroshell Bonded Phases Provide Selectivity Options to Enhance Resolution Conditions: columns as listed Mobile Phase: A.1% TFA/H 2 O, B.7% TFA/MeCN; Gradient: 5-1% B in 3. Temperature: 7 C; Flow rate:.5 ml/; Detector: UV 215 nm; 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 Poroshell 3SB-C18, 2.1 x 75 mm, 5 µm 7 1 5, Poroshell 3SB-C3, 2.1 x 75 mm, 5 µm Poroshell 3SB-C3 provides complete resolution of Insulin and Cytochrome C, where as the 3SB-C18 does not. 2/6/24

34 Break Number 2 For Questions and Answers Press *1 on Your Phone to Ask a Question 2/6/24

35 How Do We Use Poroshell Columns? Faster separations High flow rates for fast analysis with high resolution in comparison to totally porous silica Polypeptides Large proteins Impurities High recovery of large proteins Elevated temperature Change selectivity LC/MS Different bonded phases - Comparison to totally porous 3SB materials High resolution of proteins, heterogeneous proteins, and digests on different bonded phases Factors affecting LC/MS of proteins High resolution LC/MS of proteins Different bonded phases comparisons to each other Peptide mapping at high and low ph 2/6/24

36 Poroshell Columns for LC/MS of Proteins High resolution separations of proteins Formic or acetic acid containing mobile phases for better LC/MS sensitivity Optimize and balance LC and MS parameters for high sensitivity MS Parameters Flow rate Scan speed Peak width Step size LC Parameters Flow Rate Resolution Peak width Excellent elution and recovery of proteins improves LC/MS 2/6/24

37 Effect of LC/MS Acid Modifier on Peak Width using Poroshell Column: Poroshell 3SB-C18, 2.1 x 75 mm, 5 µm Mobile Phase Gradient: 2-1% B in 5.5. A: water +.1% TFA or FA B: ACN +.1% FA or TFA Flow Rate: 5 µl/ Temperature: 6 C Injector: 1 µl DAD: 22/4 nm DAD 22, 4 nm Norm. 2 Insulin Norm. 35 Cytochrome C Norm. BSA FA = formic acid TFA = trifluroacetic acid pw = pw = pw = FA pw =.31 TFA /6/24 5 FA TFA pw = FA pw =.59 TFA Formic acid gives high efficiency with only slightly larger peak widths on Poroshell

38 /6/ TFA Abundance TIC FA Effect of Modifier on MS Response: 1 pmol BSA m/z m/z

39 Good Sensitivity with Formic Acid Mass Spectra of Several Proteins pmol Insulin MW pmol Myoglobin MW pmol Carbonic anhydrase MW m/z m/z m/z Column: Poroshell 3SB-C18, 2.1 x 75 mm, 5 µm Mobile Phase Gradient: 2-1% B in 5.5. A: water +.1% formic acid B: ACN +.1% formic acid Flow Rate: 5 µl/ Temperature: 6 C Injector: 1 µl DAD: 22/4 nm 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 Peakwidth:.6 Both small and large proteins generate clear mass spectra using Poroshell with formic acid. 2/6/24

40 2/6/ m/z m/z pmol BSA 1 pmol phosphorylase B Impact of Protein Heterogeneity on Mass Spectra

41 Optimize LC/MS Separation: Effect of Flow Rate Column: Poroshell 3SB-C18, 1. x 75 mm, 5 µm Mobile Phase Gradient: 2-1% B in 5.5. A: water +.1% formic acid B: ACN +.1% formic acid Flow Rate: as shown Temperature: 8 C Injection volume: 1 µl 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 1E8 1E8 3 µl/ µl/ 1E8 1E8 1E µl/ µl/ µl/ The flow rate is more critical to the chromatographic separation than the MS. 2/6/24

42 Effect of Flow Rate on Mass Spectra 8 Max: Max: m/z m/z Max: e+6 m/z 8 Max: m/z m/z 2/6/ Max: Flow rate changes have almost no impact on the mass spectra From 3 6 µl/ µl/ 4 µl/ 5 µl/ 6 µl/ 7 µl/

43 Impact of Scan Speed on Sensitivity Column: Poroshell 3SB-C18, 2.1 x 75 mm, 5 µm Mobile Phase Gradient: 2-1% B in 5.5. A: water +.1% TFA or FA B: ACN +.1% FA or TFA Flow Rate: 5 µl/ Temperature: 6 C Injector: 1 µl Electrospray ionization: positive ion Vcap:6V Drying Gas: 12L/ 35ºC Nebulizer: 45 psi Scan: 6-25 amu Step size:.15 amu Peak width:.6 Abundance 1E8 Scan 6-25 Stepsize.15 Peakwidth.6 (.65 sec/cycle) 8 6 Peakwidth.5 (.34 sec/cycle with scan speed override turned on) 4 2 2/6/ A slower scan speed as defined by MS settings, allows for better peak definition for better sensitivity

44 Effect of MS Settings on TIC Signal 1.2E8 1E Pw=.1 Stepsize =.15 Loose chromatographic resolution 2 1.2E8 1E Pw=.6 Stepsize =.15 Best compromise of speed, chromatographic behavior and MS results E8 1E Pw=.4 Stepsize =.15 Scanspeed override ON Sacrifices MS signal (ion transmission) E8 1E Pw=.4 Stepsize =.25 Better chromatographic resolution but loss of MS signal /6/24

45 Effect of MS Settings on Spectral Quality Max: m/z Max: m/z 12 Max: m/z /6/24 m/z Max: Pw=.1 Stepsize =.15 Lose chromatographic resolution Pw=.6 Stepsize =.15 Best compromise of speed, chromatographic behavior and MS results Pw=.4 Stepsize =.15 Scanspeed override ON Sacrifices MS signal (ion transmission) Pw=.4 Stepsize =.25 Better chromatographic resolution but loss of MS signal

46 High Flow Rates and High Sensitivity LC/MS Using 1. mm ID Zorbax Poroshell 1E Column: Zorbax Poroshell 3SB-C18, 1. x 75 mm, 5 mm pmoles of protein on column 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, LC/MS: Pos. Ion ESI Vcap 6 V Drying gas flow: 12 L/ Drying gas temperature: 35 C Nebulizer: 45 psi Fragmentor voltage: 14 V Scan: 6 25 Stepsize:.15 amu Peakwidth:.6 Sample: Mixture of insulin, lysozyme, cytochrome C, myoglobin, BSA, carbonic anhydrase These TIC s show good sensitivity with only.5 pmoles on column. 2/6/24

47 Selectivity, Resolution Options with Multiple Poroshell Bonded Phases Poroshell 3SB-C3 Poroshell 3SB-C8 Poroshell 3SB-C18 * Column: Poroshell 3SB, 2.1 x 75 mm, 5 µm Mobile Phase Gradient: 2-1% B in 5.5. Mobile Phase: A: water +.1% Formic Acid B: ACN +.1% Formic acid Flow Rate: 5 µl/ Temperature: 6 C Injection: 1 ml Sample: Phosphorylase B, BSA, Ovalbu, Carbonic anhydrase, Soyben trypsin inhibitor, alpha-lactalbu and sucrose Detection: Electrospary ionization: positive ion Vcap: 6V Drying Gas: 12L/ 35ºC Nebulizer: 45 psi Scan: 6-25 amu Step size:.15 amu Peak width: /6/24

48 Pharmacia pi 3-1 Standard run on Poroshell 3SB-C Contents of sample: amyloglucosidase 65 kd soybean trypsin inhibitor 21.5 kd beta-lactroglobulin A 18.3 kd bovine carbonic anhydrase B 29 kd human carbonic anhydrase B 28.8 kd horse myoglobin kd lentil lectin 23.3 kd trypsinogen 24 kd sucrose methyl red Myoglobin expected MW measured MW Human carbonic anhydrase expected MW measured MW Bovine carbonic anhydrase expected MW measured MW Trypsinogen expected MW measured MW /6/24

49 Comparison of Speed in Peptide Mapping by LC-MS using ZORBAX Extend-C18 and Poroshell Extend-C MSD1 TIC, API-ES, Pos, Scan, Frag: Var EIC=583 unassigned EIC=48 (mw 48.2) Fragment 4 (43-45) FDK EIC=636 (mw 127.7) Fragment 3 (32-42) LFTGHPETLEK EIC=661 (mw 661.3) Fragment 8 (57-62) ASEDLK EIC=748 (mw 747.4) Fragment 18 ( ) ALELFR EIC=84 (mw 166.1) Fragment 2 (17-31) VEADIAGHGQEVLIR 4 gradient 4 gradient ZORBAX Extend-C18 (2.1 x 15mm, 3.5µm) Flow=.2mL/, Gradient 1%B in 4 Mobile Phase A= 1mM NH 4 OH, H 2 O; B= 1mM NH 4 OH, MeOH Agilent 11 LC-MSD, Scan 2-15m/z, Drying Gas= 1L/; 35psig; Temp= 35 C POS ESI mode, 45V, Fragmentor Ramp 5m/z to 15m/z Sample: myoglobin tryptic digest 5 pmol in 5µL ZORBAX Poroshell Extend-C18 (2.1 x 75mm, 5µm) Flow= 1.mL/, Gradient 1%B in 4 Mobile Phase A= 1mM NH 4 OH, H 2 O; B= 1mM NH 4 OH, MeOH Agilent 11 LC-MSD, Scan 2-15m/z, Drying Gas= 13L/; 6psig; Temp= 35 C POS ESI mode, 45V, Fragmentor Ramp 5m/z to 15m/z Sample: myoglobin tryptic digest 5 pmol in 5µL 2/6/24

50 Comparison of Peptide Mapping by LC-MS using Poroshell 3Extend-C18 and Poroshell 3SB-C18 at high and low ph 1 15 MSD1 TIC, API-ES, Pos, Scan, Frag: Var EIC=48 (mw 48.2) Fragment 4 (43-45) FDK Extend-C18 SB-C18 Ext 4 SB 15 EIC=636 (mw 127.7) Fragment 3 (32-42) LFTGHPETLEK Ext SB EIC=661 (mw 661.3) Fragment 8 (57-62) ASEDLK EIC=748 (mw 747.4) Fragment 18 ( ) ALELFR Separation EIC=84 (mw 166.1) Fragment 2 (17-31) VEADIAGHGQEVLIR Co-elution Ext SB Ext SB Ext SB ZORBAX Poroshell 3Extend-C18 (2.1 x 75mm, 5µm) Flow= 1.mL/, Gradient 1%B in 4 Mobile Phase A= 1mM NH 4 OH, H 2 O; B= 1mM NH 4 OH, MeOH Agilent 11 LC-MSD, Scan 2-15m/z Drying Gas= 13L/; 6psig; Temp= 35 C, POS ESI mode, 45V Fragmentor Ramp 5m/z to 15m/z,.52 sec/cycle Sample: myoglobin tryptic digest: 5 pmol in 5µL ZORBAX Poroshell 3SB-C18 (2.1 x 75mm, 5µm) Flow= 1.mL/, Gradient 1%B in 4 Mobile Phase A=.1% Formic Acid, H 2 O; B=.1% Formic Acid, MeOH Agilent 11 LC-MSD, Scan 2-15m/z Drying Gas= 13L/; 6psig; Temp= 35 C, POS ESI mode, 45V Fragmentor Ramp 5m/z to 15m/z,.52sec/cycle Sample: myoglobin tryptic digest: 5 pmol in 5µL 2/6/24

51 Summary Poroshell columns have a porous shell and solid core to reduce the diffusion distance and time for proteins. This allows for fast analysis of proteins including very large proteins with high recovery. It also allows for high efficiency and good resolution of protein impurities. Use of sterically protected 3SB and 3 Extend phases further allows these columns to be operated at very high temperature and low or high ph. These columns are ideal for LC/MS of proteins and peptides because they give high resolution of complex samples. 2/6/24

52 Bibliography [1]. J.J. Kirkland, F.A. Truszkowski, C. H. Dilks Jr., G.S. Engel, Superficially Porous Silica Microspheres for Fast High-Performance Liquid Chromatography of Macromolecules, J. of Chromatography A, 89 (2), 3-13 [2]. R.D. Ricker, B.J. Permar, and B.A. Bidlingmeyer, HPLC Analysis of Proteins in the Fast-Lane, PittCon22, March 18-21, 22 New Orleans [3]. W. Chen and R. Ricker, The Ideal Reversed-Phase HPLC Phases for High Throughput and Ultra-Fast Separations at Low ph, PittCon22, March 17-22, 22 New Orleans [4] Cliff Woodward, Ron Majors, Bernard J. Permar, and Robert D. Ricker, The Use of a Family of Superficially Porous Columns compatible with Standard and Capillary HPLCs to achieve fast separations of very Large, Heterogeneous Proteins, ISPPP 23, Orlando Acknowledgements 1) Genentech. Inc, Analytical Chemistry (Baojen Shyong, Galahad DePeralta, and Victor Ling) for the use of their data on the effects of temperature on antibody separations 2) Novartis Pharma, Biotechnology, Basel (Dr. Kurt Forrer and Patrik Roethlisberger) for their data and discussions on the analysis of monoclonal antibodies and their fragments. 2/6/24

53 HPLC Column Technical Support (phone: US & Canada) * (phone) * * Select option 4, then option (fax) 2/6/24

54 Q & A for breaks 1 & 2 Break 1 Q1: When you show the speed capabilities of Poroshell you have an implied assumption that everything in the LC is optimized for it.what happens if the system is set up for standard columns? A1: What happens is not necessarily pretty. You will see less of an advantage for high speed columns. To optimize means to install narrow bore tubing throughout, eliate as much extra column volume as possible, choose the best flow cell for your column diameter, and optimize the speed of data collection of your detectors. Once this is done, the advantages of Poroshell are obvious. Q2: When you show data for column lifetime, you do it in terms of column volumes and run standards at regular intervals. Why don t you do it in terms of number of injections? A2: With the number of columns to be checked and samples to be run, it simply takes too long; so, we run it the other way. The examples shown are really only for comparing one column to another not for running against an absolute standard. Every manufacturer wants to know how well they do relative to their competition. This helps improve products. Break 2 Q1: I noticed that PEG 3 was included in the mobile phases during the monoclonal antibody chromatograms. Why? A1: It turns out that antibodies are very sticky on reversed phase columns and always leave some molecules bound to the column. After several runs an increase in backpressure becomes very noticeable. If PEG 3 is included in the mobile phase, this buildup does not happen. I should note, however, that you must not use PEG 3 when running into your LC/MS; you will see PEG forever as a background contaant. The buildup can be washed off by running at 1% ACN until the pressure returns to initial conditions. Q2: C3 seems to be better than C18 on the biggest molecules. I do not understand why. A2: C3 was better on the very heterogeneous large molecules studied. We think that this is because C3 has the lowest retention; i.e., it has fewer ways to interact with a large complicated molecule and thus will show less broadening due to interaction than the long chain, C18. It is not absolutely certain that it will be best on all large molecules. 2/6/24