Reverse-Phase Separation of Proteins, Peptide and Other Biomolecules. Bill Long Applications Engineer October 8, 2008

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1 Reverse-Phase Separation of Proteins, Peptide and Other Biomolecules Bill Long Applications Engineer October 8, 28

2 Where is Reversed Phase HPLC Useful in Bio Separations? Proteomics New Bio Pharmaceutical Development Analytical Development Stability Testing Quality Control Page 2

3 BioPharma Proteomics Peptide Mapping Bio-Reactor producing protein Purification Step 1 Purification Step 2 Final Product Pure Protein Mass Spectrometer + Software ID peptides and verify presence of protein and impurities 1-D and 2-D (Reverse Phase) CAP/NANO ZORBAX 3SB, Extend, Poroshell Trypsin enzyme digestion of proteins into peptides Page 3

4 BioPharma QA/QC and In-Process Control Bio-Reactor producing protein or antibody Purification Step 1 Purification Step 2 Final Product Pure LC/UV monitoring of purification process: Reverse Phase, Ion Exchange, SEC, Affinity ZORBAX 3SB, Extend, Poroshell Page 4

5 Agilent Reversed Phase Columns for Separations of Proteins and Peptides Requirements Wide pore - 3Å for unrestricted access to bonded phase LC/MS compatible bonded phases at low and high ph low bleed, high performance Multiple bonded phases for method optimization Many configurations for LC/MS compatibility, small sample sizes and 2-D HPLC for proteomics Columns available 3StableBond 3Extend Poroshell 3SB Configurations from nano to prep Page 5

6 ZORBAX 3SB and 3Extend-C18 Columns for the Analysis of Proteins and Peptides 3StableBond Four different bonded-phases, 3SB-C18, C8, CN, and C3 for selectivity optimization Extremely stable at low ph Use with TFA, Formate and Acetate mobile phases Stable at high temperature up to 8-9 C 3Extend-C18 Uses unique bidentate-c18 bonded phase for long lifetime at high ph (up to 11.5) Double endcapped Can also be used at low ph (2.) Ammonium hydroxide mobile phase good for high ph LC and LC/MS C18 Si O C18 Si O R R1 R1 R1 Si R R Si R R Si R Silica Support O OH O OH O Silica Support Page 6

7 Typical Conditions for Separations of Peptides and Proteins on 3SB Columns Column: 4.6 x 15 mm, 5 or 3.5 μm 3SB Mobile Phase: A: 95:5, H 2 O : ACN with.1% TFA B: 5:95, H 2 O : ACN with.85% TFA Flow Rate: 1 ml / min. Temp: 35-4 C Initial Gradient: - 6% B in 6 min. 1115P1.PPT Page 7

8 C18 and TFA/ACN Gradients Capable of High Resolution Separate complex peptide mixtures, even at low ph Page 8

9 What Options are Available if the Standard C18/TFA-ACN Gradient Is Not Sufficient? Page 9

10 Changes in α Can Be the Key to Improved Separation and Resolution R s = 4 N (α-1) α k' k'+1 Theoretical Plates Selectivity Retention Page 1 99P2.PPT

11 Resolution as a Function of Selectivity, Column Efficiency, or Retention Selectivity Impacts Resolution Most Change bonded phase Change mobile phase Resolution Increase N Increase Alpha Increase k' R s = N ½ /4 (α-1)/α k /(k +1) Plates: Alpha: k : Page 11

12 Comparison of Small Peptide Selectivity Differences on 3SB Bonded Phases 3SB-C18 3SB-C8 3SB-C3 Conditions: Columns: ZORBAX 3SB, 4.6 x 15 mm, 5 mm Mobile Phase: Gradient, - 26% B in 3min. A =.1% TFA in Water B =.1% TFA in Acetonitrile Temperature: 4 C Sample: 2 µg of each peptide Flow Rate: 1. ml / min. Detection: UV-21nm 3SB-CN Page 12

13 Comparison Separation of Large Polypeptides on 3SB Bonded Phases 3SB-C18 3SB-C8 3SB-C3 1 2, , Columns: ZORBAX StableBond 3SB 4.6 x 15 mm, 5 mm Mobile Phase: Linear Gradient, 25-7% B in 4 min A:.1% TFA in Water B:.9% TFA in 8% ACN/2% water Flow Rate: 1. ml/min Temperature:6 C Sample: 3 mg each protein 1. RNase 6. CDR 2. Insulin 7. Myoglobin 3. Cytochrome C 8. Carbonic 4. Lysozyme Anhydrase4 5. Parvalbumin 9. S-1β 1. S-1α 3SB-CN Retention Time (min) Four different 3SB bonded phases allow selectivity optimization of proteins. Page 13

14 Extended Column Lifetime of ZORBAX 3SB-C8 rhgh Tryptic Digest Column: ZORBAX 3SB-C8, 4.6 x 15mm Mobile Phase Gradient - 6% in 12 min.a=.1% TFA in Water, B=.86% TFA in ACN Temp.: 4 C Flow Rate: 1mL/min. Det. UV 21nm Sample: 5 µg of rhgh Tryptic Digest After 495 ml After 1368 ml Page 14

15 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 Parvalbumin Myoglobin RNase A Insulin Lysozyme Carbonic Anhydrase Calmodulin Columns: 4.6 x 15 mm Mobile Phase: 5-4% B in 2 min. A:.1% TFA / Water B:.1% TFA / ACN Flow Rate: 1 ml / min. 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 15

16 Optimize Recovery by Changing Temperature Example: ßAP Separation Column: ZORBAX 3SB-C18, 4.6 x 15 mm, 5 μm Mobile Phase: A-.1%TFA in water, B-.9%TFA in acetonitrile Gradient: 2-45% B in 35 min Flow Rate: 1 ml/min 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 (min.) Page 16

17 LC/MS Sensitivity vs. Mobile Phase Modifier TFA vs. Acetic Acid MSD1 TIC, MS 6 3x1 5% Acetic.1% TFA.5% TFA Column: ZORBAX 3SB-C3 2.1 x 15 mm, 5 μm Mobile Phase: Gradient: % B hold for 5 min. 4% B in 55 min. 4 1% B in 2 min A: 5% acetic acid B: Acetonitrile Flow Rate:.2 ml/min Instrument: Agilent 11 MSD Sample: GluC Digest of BSA.1% TFA A significant increase in sensitivity was observed using 5% acetic acid instead of TFA. Reducing TFA concentration to.1% improved sensitivity very little. min Page 17

18 Typical Conditions for Separations of Peptides and Proteins on 3Extend-C18 Columns at High ph Column: 15 mm Length, 5 or 3.5 mm 3 Extend-C18 Mobile Phase: A: 2 mm NH 4 OH in water B: 2 mm NH 4 OH in 8% ACN Flow Rate: 1 ml / min. Temp: 25-3 C Initial Gradient: 5-6% B in 3 min. At low ph use the same conditions as on the 3SB columns. Page 18

19 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 min. TFA Conditions, 8 C A-.1% TFA in water B-.85% TFA in 8%AcN 29-41%B in 3 min. 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 min. Aβ(1-38) Aβ(1-4) Aβ(1-42/3) Aβ(1-43) Aβ(1-38) Aβ(1-4) Aβ(1-42) Aβ(1-42) Aβ(1-43) Aβ(1-38) Aβ(1-4) Column: Flow Rate: Sample: ZORBAX 3Extend C x 15 mm, 5 μm.25 ml/min 5 µl sample (1 pmol each) 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 High ph and room temperature improve peak shape, recovery and change selectivity. Page 19

20 Use Extend-C18 for Different Selectivity at High and Low ph Comparison of TFA and NH 4 OH For Peptide RP-HPLC \ ESI-MS Analysis 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/min Temp: 25 C Gradient: 5-6% B in 2 min; LC/MS: Pos. Ion ESI- Vf 7V, Vcap 4.5 kv, N2-35 psi, 12L/min, 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 min Page 2

21 Peptide RP-HPLC/ESI-MS Using 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 μm Flow rate:.25ml/min Temp: 25 C Gradient: 5-6% B in 2 min LC/MS: Pos. Ion ESI- Vf 7V, Vcap 4.5 kv, N2-35 psi, 12L/min, 3 C Sample: 4µL (5 ng each peptide) 1.5E7 LLG LLL LLVF LLL-NH 2 1E7 LHL.5E min 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 21

22 Improved Resolution and Reduced MS Noise at High ph AI and AII elute together at lower ph AI, AII and AIII all resolved at high ph High efficiency and good recovery of hydrophobic peptides at high ph. Ideal for LC/MS with ammonium hydroxide-modified mobile phase Page 22

23 High Sensitivity, High Resolution and High Speed LC and LC/MS of Proteins and Peptides Higher speed separations of proteins and peptides are possible High throughput and high efficiency protein applications require reduced analysis times Shorter Columns with Smaller Particles Improve Speed for Gradient Separations Page 23

24 GRADIENT ELUTION Page 24

25 Why Gradients Are Preferred for Protein and Peptide Separations 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 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). φ Page 25

26 1% B t g = 5 Possible to Manipulate Terms to Increase Gradient Retention (k*) % B 1% B k* t g F t g = 1 S Δ%B V m % B 1% B 1/k* = gradient steepness = b % B t g = 2 1% B F = flow rate (ml/min.) t g = gradient time (min.) V m = column void volume (ml) ΔΦ = change in % B solvent S = constant t g = 4 % B Time (min)

27 Increase in t G Improves k* and Peak Capacity 4 3 Gradient time Peak Capacity min min 3 45 min min min % increase in peak capacity as tg extended min Page 27

28 Improving R s Using Short Column Length ( Vm ), Smaller Particle Size (N), Constant Time (t G ) 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 min. 1. ml / min. Ambient Sample: 1. Gly-Tyr 6. Leu-Enk 2. Val-Tyr-Val 7. Angiotensin II 3. [Gln 11 ] Amyloid-β- 8. Kinetensin Protein Fragm RNase 4. (TYR8) Bradykinin 1. Insulin (Eq.) 5. Met-Enk Page 28

29 Maintain Resolution and Reduce Time by Keeping t G / Vm Relationship Constant ZORBAX 3SB-C8 4.6 x 25 mm, 5 µm Rapid Resolution 4.6 x 15 mm, 3.5 µm Mobile Phase: A: 95:5, Water : ACN with.1% TFA B: 5:95, Water 3 : ACN with.85% TFA Rapid Resolution 4.6 x 5 mm, 3.5 µm 1-6% B in 5 min. 4 min % B in 3 min. 24 min. 1-6% B in 1 min. 9 min. 1. 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: Flow: 1. ml/min Detection: 215nm Sample: 1-1µg protein (1µL inj.) in 6M Guanidine HCL, ph Page 29

30 What Makes Very High Speed (HPLC Possible? Small particle sizes Short columns (<1mm) Low viscosity mobile phases Optimized HPLC minimal extra column volume, low volume flow cell, minimal delay volume for gradients Detector set to fast response time High diffusion coefficient of the sample in the mobile phase But large molecules have small diffusion coefficients!! Page 3

31 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) -- High back pressure and plugging Limit diffusion distance into a particle! Page 31

32 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 32

33 Poroshell is Used for Fast Separations at 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 Page 33

34 Flow Rates for Poroshell Columns Column Internal Diameter Porous Particle Flow Rate Range Poroshell Flow Rate Range 2.1 mm.1.3 ml/min.3 3 ml/min 1. mm 3 6 μl/min ml/min Very high flow rates can be used effectively with Poroshell columns Page 34

35 High Flow Rates with 2.1 mm ID Poroshell for High Resolution and Fast Separations Columns: Poroshell 3SB-C x 75 mm, 5 μm Mobile Phase:A:.1% TFA B:.7% TFA in ACN Gradient: 5 1% B in 1. min. Flow Rate: 3. ml/min. 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.Ovalbumin.5 1. Time (min) 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 35

36 Poroshell Has Different Selectivity from Totally Porous 3SB Different selectivity due to the different ratios of bonded phase on the surface. 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 min Flow Rate: 3 ml/min Piston Stroke: 2 µl Temp.: 7 C Det.: UV 215 nm , SB-C Poroshell 3SB-C18 min min Page 36

37 Effect of Increasing Flow Rate in Protein Analysis using Totally Porous Silica ml/min (15) -1%B /.67 min 2 ml/min -1%B in 1 min 1 ml/min -1%B in 2 min.5 ml/min (2.5) 1-1%B in 4 min Resolution Loss 2 3 Totally Porous 2.1x75mm, 5µm 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 4.5 min 3 min As flow rate increases, peak width increases and resolution is lost in protein analysis when using totally porous particles! min 1. Neurotensin 2. Rnase A min* 3. Lysozyme 4. Myoglobin min* min* Aligned Page 37

38 Effect of Increasing Flow Rate in Protein Analysis with Poroshell ml/min -1%B.67 min 2 ml/min -1%B 1 min 1 ml/min -1%B 2 min.5 ml/min -1%B 4 min Sustained Efficiency and Resolution 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 4 Poroshell 3SB-C18 2.1x75mm, 5µm.5 min 3 min As flow rate increases, peak width and resolution are maintained when using superficially porous particles! min 1. Neurotensin 2. Rnase min* A 3. Lysozyme 4. Myoglobin min* min* Aligned Page 38

39 Poroshell is Used for High Resolution High resolution and fast analysis High resolution of protein digests High resolution of protein impurities High resolution for LC/MS of proteins Page 39

40 High Resolution and Fast Analysis Separation of a Protein Digest on Poroshell 3SB-C ml/min -1%B 12 min.28 ml/min -1%B 12 min 6 min Poroshell 3SB-C18 2.1x75 mm, 5 µm BSA tryptic digest 15hrs., 7 pmol min Zorbax 3SB-C18 2.1x15 mm, 5 µm 5 6 min 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 min* Extremely fast (6 min), high, resolution of BSA tryptic digest, using Poroshell 3SB-C18. Under typical conditions, a comparable run takes 6min on a totally porous particle. Page 4

41 Fast Analysis of Insulin (5.7 kda) Impurities using Poroshell 3SB-C18 Column: Poroshell 3SB-C18, 2.1 x 75 mm, 5 μm Mobile Phase Gradient: 5-1% B in 5 min. A: water +.1% TFA B: ACN +..7% TFA Flow Rate: 2 ml/min Temperature: 7 C Sample: Bovine pancreas insulin 5 4 INSULIN - bovine pancreas 3 27 hrs. at 55 C min Untreated min Page 41

42 Ultra High Speed Resolution Optimization Using RP-HPLC on Poroshell 3SB-C18 Column: Poroshell 3SB-C18, 2.1 x 75 mm, 5 μm Mobile Phase Gradient: 5-1% B in tg min. A: water +.1% TFA B: ACN +..7% TFA Flow Rate: 2 ml/min Temperature: 7 C Sample: Carbonic Anhydrase 1 mg/ml Injection Volume: 2 μl t G = 1 min Carbonic Anhydrase (CA) Several Months at 4 C t G = 2 min t G = 4 min t G = 8 min High resolution is obtained in 2 or 3 minutes. min Page 42

43 Poroshell Bonded Phases Provide Selectivity Options to Enhance Resolution Poroshell 3SB- Poroshell SB-C18, 2.1 x 75 mm C x 75 mm Poroshell 3SB-C3 Poroshell SB-C3, 2.1 x x 75 mm Column: Agilent Poroshell (2.1 x 75 mm); Temp.: 7 C; Flow:.5 ml/min; Det: UV 215 nm Mobile Phase: A=.1% TFA/H 2 O, B=.7% TFA/ACN; Gradient: 5-1% B in 3. min Changing from SB-C18 to SB-C3, within the Poroshell family results in resolution of peaks 5 and 6, still in 3 minutes! , , Samples: 1. Angiotensin II 2. Neurotensin 3. RNase A 4. Insulin B Chain min 5. Insulin 6. Cytochrome C 7. Lysozyme 8. Myoglobin 9. Carbonic Anhydrase min Page 43

44 Ultra High Speed HPLC Peptide Maps of a Monoclonal Antibody on Several Zorbax Poroshell Phases Original method 12 min t G using a C x 25 mm 57 peaks detected Zorbax Poroshell 3SB-C18, 2.1 x 75mm, 5µ Zorbax Poroshell 3SB-C8, 2.1 x 75mm, 5µ Zorbax Poroshell 3SB-C3, 2.1 x 75mm, 5µ min min min Conditions: Mobile phase A =.1% TFA in water Mobile phase B =.1% TFA in ACN; Temperature: 7 C; Detection: VWD, 21nm; Injection: 1 µl Lys-C digest of Human Monoclonal Antibody; Flow: 1. ml/min; Gradient: min, % B; 5.5 min, 55% B; 5.6 min, 55% B; 7. min, % B 46 to 48 peaks, 1/2 analysis time Aligned Data courtesy of: Novartis Pharma, Biotechnology, Basel Dr. Kurt Forrer Patrik Roethlisberger For more details see EN Zorbax Poroshell technology facilitates ultra-fast HPLC analysis of peptides Page 44

45 High Flow Rates and High Sensitivity LC/MS Using 1. mm ID Poroshell Column: Poroshell 3SB-C18, 1. x 75 mm, 5 μm Mobile Phase Gradient: 2-1% B in 5.5 min. A: water +.1% formic acid B: ACN +.1% formic acid Flow Rate: 6 μl/min 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/min Drying gas temperature: 35 C Nebulizer: 45 psi, Fragmentor volatage: 14 V Scan: 6 25 Stepsize:.15 amu Peakwidth:.6 min 1E pmoles of protein on column These TIC s show good sensitivity with only.5 pmoles on column. min Page 45

46 Summary Variety of ZORBAX 3 Columns for RP HPLC Separation of Biomolecules 3SB for Low ph and High Temperature 3 Extend for ph 2 to 11.5 for Enhanced Resolution and Reduced LC/MS Noise 3 Poroshell for High Speed, High Resolution RP HPLC Variety of Bonded Phases, Particle Sizes and Column Sizes 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. All Columns Compatible with LC/MS Friendly Mobile Phases Page 46

47 Thank you for attending. To learn more about the sample simplification with the Multiple Affinity Removal System, visit Proteomics Solution Source at Page 47

48 Upcoming BioSeparation eseminars IDENTIFY - Identify, Characterize and Measure Bio-molecules in a Variety of Sample Sources Wednesday, October 22, 28 1:pm EDT To register, visit age/g.php?t=a&d= Page 48