Charged Surface Hybrid C18 for High Resolution LC and LC/MS Peptide Separations

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1 Charged Surface Hybrid C18 for High Resolution LC and LC/MS Peptide Separations Higher Quality Peptide Separations Using Hybrid Particle-Based Reversed-Phase Columns and CSH Technology 2013 Waters Corporation 1

2 Reversed Phase Peptide Separations Peptide separations critically important peptide mapping, bottom-up proteomics reversed phase challenges remain LC performance MS performance TFA Ionic analytes peptides Secondary interactions - Poor peak shape Overloading at very low loads (<< neutrals) MS signal suppressing ion pairing agents needed TFA Peptide Still needed: high resolution, high sensitivity peptide separations regardless of eluent additive i.e. formic acid (FA) separations for LC-MS 2013 Waters Corporation 2

3 Charged Surface Hybrid 2013 Waters Corporation 3

4 Agenda The Peak Shape Problem Column Chemistries CSH Technology Peak Capacity CSH130 C18 and separations without TFA LC-MS of protein digests Small protein separations Peptide Mapping a Therapeutic mab CSH130 C18 for LC-UV-MS Disulfides and Deamidation Peptide Separation Technology (PST) Columns Quality control - QC tested with digests Analytical Standards and Reagents (ASR) 2013 Waters Corporation 4

5 The Peak Shape Problem 2013 Waters Corporation 5

6 Competitor s Industry Standard 5 µm Porous Silica C18 Competitor s Industry Standard C x 250 mm, Porous 5 µm, 300Å ACQUITY UPLC H-Class Bio 2% ACN for 1 min, then to 50% ACN over 60 min 0.3 ml/min 40 C 214 nm / Xevo G2 QTOF 5.6 µg MassPREP Peptide Mixture MassPREP Peptide Mixture Peptide Sequence 1 RASG-1 RGDSPASSKP 2 Angiotensin 1-7 DRVYIHP 3 Bradykinin RPPGFSPFR 4 Angiotensin II DRVYIHPF 5 Angiotensin I DRVYIHPFHL 6 Renin Substrate DRVYIHPFHLLVYS 7 Enolase T35 WLTGPQLADLYHSLMK 8 Enolase T37 YPIVSIEDPFAEDDWEAWSHFFK 9 Melittin GIGAVLKVLTTGLPALISWIKRKRQQ 2013 Waters Corporation 6

7 Ethylene Bridged Hybrid - BEH Technology U.S. Patent No. 6,686,035 B2 and others patent pending Bridged Ethanes In Silica Matrix Organo Silica Hybrid Particles ph stability Reduced ionic interactions Basis of Peptide Separation Technology EtO CH 2 CH 2 OEt OEt O Si Si Si O O Si O O Si O O Si O EtO OEt OEt Polyethoxysilane Et Et n EtO EtO OEt 4 EtO CH 2 Si + Si EtO OEt CH Si OEt 2 EtO EtO OEt Tetraethoxysilane Bis(triethoxysilyl)ethane 2013 Waters Corporation Anal. Chem. 2003, 75,

8 Small Particle Size Mobile Phase Peptides 1500 Da Peptide µm Porous Particle Diffusion-related band broadening Adsorption Equilibria H (mm) 1 Diffusion distances decrease Reduced Eddy diffusion Improved mass transfer kinetics 1.7 µm Column efficiency Narrower peaks 40 µl/min 2.1 mm ID 400 µl/min Velocity (mm/sec) 2013 Waters Corporation 8

9 Waters BEH130 C µm Competitor s Industry Standard 5 µm C Bridged Ethyl Hybrid BEH130 C18 Pore Size (Å) Time (min) Ligand Waters ACQUITY UPLC BEH130 C x 150 mm, Porous 1.7 µm, 130Å EtO CH 2 CH 2 OEt O Si Si EtO Si O O O Si OEt O Si Si OEt O OEt O O Peptide Sequence 1 RASG-1 RGDSPASSKP 2 Angiotensin 1-7 DRVYIHP 3 Bradykinin RPPGFSPFR 4 Angiotensin II DRVYIHPF 5 Angiotensin I DRVYIHPFHL 6 Renin Substrate DRVYIHPFHLLVYS 7 Enolase T35 WLTGPQLADLYHSLMK 8 Enolase T37 YPIVSIEDPFAEDDWEAWSHFFK 9 Melittin GIGAVLKVLTTGLPALISWIKRKRQQ 2013 Waters Corporation 9

10 A New Column Chemistry CSH130 C18 Waters ACQUITY UPLC CSH130 C x 150 mm, Porous 1.7 µm, 130Å Peptide Sequence 1 RASG-1 RGDSPASSKP 2 Angiotensin 1-7 DRVYIHP 3 Bradykinin RPPGFSPFR 4 Angiotensin II DRVYIHPF 5 Angiotensin I DRVYIHPFHL 6 Renin Substrate DRVYIHPFHLLVYS 7 Enolase T35 WLTGPQLADLYHSLMK 8 Enolase T37 YPIVSIEDPFAEDDWEAWSHFFK 9 Melittin GIGAVLKVLTTGLPALISWIKRKRQQ 2013 Waters Corporation 10

11 A New Column Chemistry CSH130 C Waters Corporation 11

12 Charged Surface Hybrid (CSH) Technology patent pending Charged Surface Hybrid (CSH) Technology and Its Use in Liquid Chromatography. P.C. Iraneta, K.D. Wyndham, D.R. McCabe, and T.H. Walter Waters White Paper EN 2011 Expands upon the robust BEH particle technology CSH130 C18 = BEH130 base particle + low level of basic moieties + trifunctional C18/end cap Acidic ph Positive Surface Charge Peptide 2013 Waters Corporation 12

13 Peak Capacity Peak Capacity = The number of peaks that can be separated within a retention window Neue, U. D., J Chromatogr A 2005, 1079 (1-2), The best metric for determining the quality of gradient separations 100% 9 peaks could resolve ~ Peak Height 50% 2.35σ w h 13.4% 4σ w 4σ 0% t gradient 2013 Waters Corporation 13

14 Peak Capacity - FA vs TFA P c,4 4σ Competitor s Industry Standard C18 5 µm 2.1 x 250 mm % TFA , , ,10 % FA 0.10 Percent 0.05TFA Waters Corporation 14

15 Peak Capacity - FA vs TFA BEH130 C µm 2.1 x 150 mm P c,4 4σ Competitor s Industry Standard C18 5 µm 2.1 x 250 mm % TFA , , ,10 % FA 0.10 Percent 0.05TFA Waters Corporation 15

16 Peak Capacity - FA vs TFA Competitor s SPP Peptide C µm 2.1 x 150 mm BEH130 C µm 2.1 x 150 mm P c,4 4σ Competitor s Industry Standard C18 5 µm 2.1 x 250 mm % TFA , , ,10 % FA 0.10 Percent 0.05TFA Waters Corporation 16

17 Peak Capacity - FA vs TFA 370 CSH130 C µm 2.1 x 150 mm % Competitor s SPP Peptide C µm 2.1 x 150 mm BEH130 C µm 2.1 x 150 mm 20% P c,4 4σ Competitor s Industry Standard C18 5 µm 2.1 x 250 mm % TFA , , ,10 % FA 0.10 Percent 0.05TFA Waters Corporation 17

18 MS Signal - FA vs TFA Peak Capacity MS Signal 370 CSH130 C µm Competitor s SPP Peptide C18 1.7µm P c,4σ BEH130 C µm Competitor s Industry Standard C18 5 µm Fold Decrease in MS Pea ak Area % TFA , , ,10 % FA 0.10 Percent 0.05TFA % TFA , , ,10 % FA 0.10 Percent 0.05TFA Waters Corporation 18

19 Loadability Attribute how much analyte can be loaded before peak shape deteriorates B Low Mass Load 0.6 µg of mixture Low Mass Load CSH130 CSH C18 C µm BEH C A Typical Mass Load 6 µg High of Mass mixture Load *Previously shown CSH C18 BEH C18 CSH130 C µm 350 BEH130 C µm 350 P c,4σ 300 P c,4σ 300 BEH130 C µm P c,4σ Percent 0.05 TFA 0.00 % TFA % FA Percent 0.05TFA Waters Corporation 19

20 CSH130 C18 and Separations without TFA 2013 Waters Corporation 20

21 LC-MS 0.1% TFA Waters ACQUITY UPLC CSH130 C x 150 mm, 1.7 µm, 130Å 2% ACN for 1 min, then to 50% ACN over 60 min 0.3 ml/min 40 C ACQUITY UPLC H-Class Bio Xevo G2 QTof 500 pmol MassPREP Enolase Digest p/n x drop in sensitivity 2013 Waters Corporation 21

22 CSH130 C18 vs Other Chemistries 0.1% FA Improvement for both optical and MS detection 2013 Waters Corporation 22

23 LC-MS Retention and Selectivity 2013 Waters Corporation 23

24 Large Peptides/Small Proteins 0.1% FA Peptide/Protein kda 1 Bradykinin Renin Substrate Ubiquitin Cytochrome C (Equine) Insulin (Bovine) Melittin Å 300 Å 2.1 x 150 mm columns 2% ACN for 1 min, then to 50% ACN over 60 min 0.3 ml/min 40 C ACQUITY UPLC H-Class Bio 214 nm / Xevo G2 QTOF 1 µg each component 2013 Waters Corporation 24

25 Peptide Mapping of a Therapeutic mab 2013 Waters Corporation 25

26 Peptide Mapping a Therapeutic mab Trastuzumab (Herceptin; Genentech) Breast Cancer, Anti-HER2 One of the highest grossing therapeutic mabs (~5 billion $/yr) Biosimilars Basis for a new ADC (Trastuzumab emantansine) o Phase III clinical trials completed High peak capacity at mass loads to detect trace modifications and thoroughly characterize o Disulfide linkages o Deamidation o Oxidation o Glycosylation o Conjugation in ADCs JCO 2010;28: Waters Corporation 26

27 Non-Reduced Lys-C Peptide Mapping Non-Reduced Lys-C Digests Minimal complexity + disulfides preserved 27 different linear peptides 8 different disulfide linked peptides 150 to 11,000 Da A recent Amgen protocol: Anal Biochem 2011, 411 (2), L: Lys-C Cleavage (C-terminal Side of Lys) H: H: L: Disulfide Bond Light Chain Heavy Chain 2013 Waters Corporation 27

28 Method Considerations ACQUITY UPLC BEH130 C18 1.7µm ACQUITY UPLC CSH130 C18 1.7µm 2.1 x 150 mm Peak Capacity % TFA 0.08% FA Optimize peak capacity Small compromise to MS Sensitivity Percent TFA CSH130 C18 BEH130 C18 MS Signal A Elevated Temperature (60 C) Improved peak shape and recovery 5-11 kda species 5-11 kda Species BEH130 C Time (min) Anal Chem 2011, 83 (15), Anal Biochem 2011, 411 (2), MAbs 2010, 2 (4) J Biol Chem 2009, 284 (51), C 60 C 2013 Waters Corporation 28

29 Non-Reduced Lys-C Peptide Maps Trastuzumab 2013 Waters Corporation 29

30 LC-UV-MS with an MS-Compatible Mobile Phase CSH130 C18 Low TFA mobile phase Optimized Peak Capacity + MS signal UV signal ACQUITY H-Class Bio CSH130 C µm UV Detector ESI-MS Xevo G2 QTof m/z Waters Corporation 30

31 Disulfide Characterization H: x H: L:1-42 x L: H:1-30 x H: H: x H: L: x L: H x H: A210 Non-Reduced L: x H: H: x H: CSH130 C18 - Optimized Gradient Blank -0.1 A H: H: L: H: H: L: Reduced 217 H: L: H H: H: L: L: Time (min) 2013 Waters Corporation 31

32 Assaying Deamidation CSH130 C18 - Optimized Gradient 44 GLEWVARIYPTNGYTRYADSVK 65 Binding region from the heavy chain Prone to deamidation 0.5 A H:44-65 * * 92 % Asn 7 % isoasp 1 % Asp Unmodified Deamidated Intensity 3E+5 0E E Mass shift Da * 0E E * Time (min) 0E m/z 2013 Waters Corporation 32

33 Conclusions CSH130 C18 Peptide Separations Improved loadability and greater peak capacity vs. other C18 columns Excellent peak shape with both TFA and FA mobile phases (highly compatible with MS) 130Å pore size optimal for species up to 8-10 kda Unique selectivity (basic residues) o Less retentive Exceptional chemistry for peptide separations Peptide mapping... proteomics and peptide isolation 2013 Waters Corporation 33

34 Peptide Separation Technology Columns 2013 Waters Corporation 34

35 New Addition to the Suite of Waters Peptide Separation Technology Peptide Separation Technology Peptide C18 Columns QC Tested with Digests BEH Technology BEH130 C18 and BEH300 C18 Industry Leading Performance for Most Applications Two Pore Sizes Particle Sizes: 1.7 µm, 3.5 µm, 5 µm Analytical, Nano and Prep Columns Now even more tools in the toolbox CSH Technology CSH130 C18 Best columns for formic acid separations Unique selectivity 2013 Waters Corporation 35

36 UPLC and HPLC CSH130 C x150 mm 1.7 µm A High peak capacity separations not limited to UPLC 0.1 % FA ~8000 psi CSH130 C18 Peptide Separation Technology Columns A % TFA Method Transfer 2.5 µmxp Longer Run Time Lower Pressure 0.2 Available 0.0 Now: Upcoming: Analytical Columns 1.0 Time (min) Nano (75, 150, 1.0 Time 300 (min) µm ID) 1.7 µm Prep Columns (5 µm) µm XP µm A Time (min) ~3000 psi A Time (min) 2013 Waters Corporation 36

37 Quality Control CSH130 C % Formic Acid 2013 Waters Corporation 37

38 Quality Control CSH130 C18 Batch-to-Batch Reproducibility Cytochrome C Digest, 0.1% Formic Acid Each new column will perform comparably to one previously used AU AU AU AU Batch 110 T1 T13-T14 T Minutes Batch 108 T4 T9-T Waters Corporation 38 T10 T8 T Minutes 8.00 Batch Minutes 8.00 Batch 102 T19C T19 T12-T13 T Minutes 8.00 *Data provided by S. McCall/P. Iraneta

39 Analytical Standards and Reagents (ASR) MassPREP Peptide Mixture Digestion Standards Cytochrome C Digestion Standard Part Number: Rapigest SF Amgen Digestion Protocol Anal Chem (4): Waters Corporation 39

40 Useful Literature Charged Surface Hybrid (CSH) Technology and Its Use in Liquid Chromatography P.C. Iraneta, K.D. Wyndham, D.R. McCabe, and T.H. Walter Waters White Paper EN 2011 Increasing Peak Capacity in Reversed Phase Peptide Separations with Charged Surface Hybrid (CSH) C18 Columns M.A. Lauber, S.M. Koza, K.J. Fountain Waters Application Note EN 2013 Peptide Mapping and Small Protein Separations with Charged Surface Hybrid (CSH) C18 and TFA-Free Mobile Phases M.A. Lauber, S.M. Koza, K.J. Fountain Waters Application Note EN 2013 High Resolution Peptide Separations with a Charged Surface C18 Stationary Phase M.A. Lauber, S.M. Koza, S.A. McCall, B.A. Alden, P.C. Iraneta, and K.J. Fountain Manuscript in Preparation Waters Corporation 40