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

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 Matthew Lauber, Ph.D. Senior Applications Chemist 2013 Waters Corporation 1

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

Charged Surface Hybrid 2013 Waters Corporation 3

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

The Peak Shape Problem 2013 Waters Corporation 5

Competitor s Industry Standard 5 µm m Porous Silica C18 3.0 0.1% TFA Ion Pairing 1 2 3 4 5 6 7 8 9 Strong 2.0 A214 0.02% TFA 0.08% FA 1 2 3 4 5 6 7 8 9 1.0 0.1% FA Weak 1 2 3 4 5 6 7 8 9 not detected 0.0 0 10 20 30 40 50 60 Time (min) Competitor s Industry Standard C18 2.1 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 UV @ 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

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 2 Si OEt EtO EtO OEt Tetraethoxysilane Bis(triethoxysilyl)ethane Anal. Chem. 2003, 75, 6781-6788 2013 Waters Corporation 7

Small Particle Size Mobile Phase Peptides 1500 Da Peptide 2 3.5 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 0 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 Velocity (mm/sec) 2013 Waters Corporation 8

Waters BEH130 C18 1.7 µm 3.0 Competitor s Industry Standard 5 µm C18 3 4 5 1 6 2 3 4 5 6 7 8 0.1% TFA 9 2.0 A214 3 4 1 5 6 2 3 4 5 6 7 8 9 0.02% TFA 0.08% FA 1.0 0.1% FA 1 2 3 4 5 6 3 4 5 6 7 8 9 0.0 0 30 10 40 20 30 40 50 60 Time (min) Time (min) Bridged Ethyl Hybrid BEH130 C18 Pore Size (Å) Ligand Waters ACQUITY UPLC BEH130 C18 2.1 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

A New Column Chemistry CSH130 C18 3.0 0.1% TFA 1 2 3 4 5 6 7 8 9 A214 2.0 1 2 3 4 5 6 7 8 9 0.02% TFA 0.08% FA 1.0 V o 1 4 5 6 7 8 0.1% FA 0.0 1 2 2 3 9 10 20 30 40 50 60 Time (min) Waters ACQUITY UPLC CSH130 C18 2.1 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

A New Column Chemistry CSH130 C18 Competitor s Industry Standard C18 Porous (300Å) 5 µm 2.1 x 250 mm Competitor s Superficially Porous Peptide C18 SPP (100Å) 1.7 µm 2.1 x 150 mm BEH130 C18 Porous (130Å) 1.7 µm 2.1 x 150 mm CSH130 C18 Porous (130Å) 1.7 µm 2.1 x 150 mm TFA UV absorbance (214 nm) Formic Acid 1 2 10 50 Time (min) 10 50 10 50 10 50 Time (min) Time (min) Time (min) 2013 Waters Corporation 11

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 720003929EN 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

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), 153-61. The best metric for determining the quality of gradient separations 1 100% 9 peaks could resolve ~300-400 Peak Height 50% 2.35σ w h 13.4% 4σ w 4σ 0% t gradient,4 1 2.35 4 t w h, 2013 Waters Corporation 13

Peak Capacity - FA vs TFA Competitor s Industry Standard Silica C18 5 µm 2.1 x 250 mm FA % TFA 0.00 % FA 0.10 0.05 0.05 TFA 0.10 0.00 2013 Waters Corporation 14

Peak Capacity - FA vs TFA BEH130 C18 1.7 µm 2.1 x 150 mm Competitor s Industry Standard Silica C18 5 µm 2.1 x 250 mm FA % TFA 0.00 % FA 0.10 0.05 0.05 TFA 0.10 0.00 2013 Waters Corporation 15

Peak Capacity - FA vs TFA Competitor s SPP Peptide C18 1.7 µm 2.1 x 150 mm BEH130 C18 1.7 µm 2.1 x 150 mm Competitor s Industry Standard Silica C18 5 µm 2.1 x 250 mm FA % TFA 0.00 % FA 0.10 0.05 0.05 TFA 0.10 0.00 2013 Waters Corporation 16

Peak Capacity - FA vs TFA CSH130 C18 1.7 µm 2.1 x 150 mm 90% Competitor s SPP Peptide C18 1.7 µm 2.1 x 150 mm BEH130 C18 1.7 µm 2.1 x 150 mm 20% Competitor s Industry Standard Silica C18 5 µm 2.1 x 250 mm FA % TFA 0.00 % FA 0.10 0.05 0.05 TFA 0.10 0.00 2013 Waters Corporation 17

MS Signal - FA vs TFA Peak Capacity MS Signal CSH130 C18 1.7 µm Competitor s SPP Peptide C18 1.7µm BEH130 C18 1.7 µm Competitor s Industry Standard Silica C18 5 µm FA % TFA 0.00 % FA 0.10 0.05 0.05 TFA 0.10 0.00 % TFA % FA FA 0.00 0.10 0.05 0.05 TFA 0.10 0.00 2013 Waters Corporation 18

Loadability Attribute how much analyte can be loaded before peak shape deteriorates 8 8 450 400 B Low Mass Load 0.6 µg of mixture Low Mass Load CSH130 CSH C18 C18 1.7 µm BEH C18 450 400 A Typical Mass Load 6 µg of mixture High Mass Load *Previously shown CSH C18 BEH C18 CSH130 C18 1.7 µm 350 BEH130 C18 1.7 µm 350 P c,4σ 300 P c,4σ 300 BEH130 C18 1.7 µm P c4σ 250 250 200 200 10 00 % TFA % FA 150 0.00 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09 TFA 0.10 0.10 FA Percent 0.05TFA 0.00 0.00 0.05 0.10 0.10 0.05 0.00 % TFA % FA % TFA % FA 150 0.00 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09 TFA0.10 0.10 FA Percent 0.05TFA 0.00 0.00 0.05 0.10 0.10 0.05 0.00 2013 Waters Corporation 19

CSH130 C18 and Separations without TFA 2013 Waters Corporation 20

LC-MS 2E+6 Enolase Tryptic Digest CSH130 C18 1.7 µm T6 0.1% FA Wh,avg= 4.0 s Intensity T38 T14 Pc,4σ = 532 T51 T23 1E+6 T35 T42 T45 T40 T27 T37 T10 0.1% TFA 0E+0 1 11 21 Waters ACQUITY UPLC CSH130 C18 2.1 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 2013 Waters Corporation 31 Time(min) 41 51 10x drop in sensitivity ACQUITY UPLC H-Class Bio Xevo G2 QTof 500 pmol MassPREP Enolase Digest p/n 186002337 21

CSH130 C18 vs Other Chemistries Intensity 2E+6 0.1% FA BEH130 C18 1.7 µm BEH C18 1.7 µm P = 399 Pc,4σ c,4σ = 399 1E+6 0E+0 Intensity 2E+6 0 10 20 30 40 50 60 Competitor s Superficially Porous Competitor A Peptide C18 1.7 µm Superficially Porous C18 1.7 µm = 405 405 PPc,4σ c,4σ = 1E+6 0E+0 0 10 20 Intensity 2E+6 30 40 50 60 Time(min) CSH130 C18 1.7 CSH C18 1.7 µmµm P = 532 Pc,4σ c,4σ = 532 1E+6 0E+0 0 10 20 30 40 50 60 Time(min) Improvement for both optical and MS detection 2013 Waters Corporation 22

LC-MS Retention and Selectivity 1E+6 BEH130 C18 T3 SVYDSR T12 ANIDVK T19 HLADSK T10 GVLHAVK T40 IATAIEK More positive charge T3 T12 T19 T10 T40 0E+0 5.5 6.5 7.5 8.5 9.5 10.5 11.5 12.5 1E+6 Competitor s Superficially Porous Peptide C18 Intensity T3 T12T10 T19 T40 0E+0 5.5 6.5 7.5 8.5 9.5 10.5 11.5 12.5 1E+6 CSH130 C18 T10 T19 T12 T3 T40 0E+0 5.5 6.5 7.5 8.5 9.5 10.5 11.5 12.5 Time(min) 2013 Waters Corporation 23

Large Peptides/Small Proteins 0.1% FA Peptide/Protein kda 1 Bradykinin 1.1 130 Å A 214 1.2 1 0.8 0.6 0.4 0.2 CSH130 C18 1.7 µm 1 2 0 1.2 10 15 20 25 30 35 40 45 50 BEH130 C18 1.7 µm Time (min) 1 3 4 5 6 2 Renin Substrate 1.8 3 Ubiquitin 8.6 4 Cytochrome C (Equine) 12.4 5 Insulin (Bovine) 5.7 6 Melittin 2.8 0.8 A 214 0.6 0.4 0.2 1 2 4 3 5 6 300 Å A 214 0 1.2 10 15 20 25 30 35 40 45 50 BEH300 C18 1.7 µm Time (min) 1 2.1 x 150 mm columns 2% ACN for 1 min, 0.8 then to 50% ACN over 60 min 5 0.3 ml/min 0.6 40 C 0.4 0.2 1 2 4 3 6 ACQUITY UPLC H-Class Bio UV @ 214 nm / Xevo G2 QTOF 1 µg each component 0 10 15 20 25 30 35 40 45 50 Time (min) 2013 Waters Corporation 24

Peptide Mapping of a Therapeutic mab 2013 Waters Corporation 25

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 antibody drug conjugate (ADC; Trastuzumab emantansine) o Phase III clinical trials completed High peak capacity at mass loads to detect trace modifications and thoroughly characterize o o o o o Disulfide linkages Deamidation Oxidation Glycosylation Conjugation in ADCs JCO 2010;28:2698-2704 2013 Waters Corporation 26

Non-Reduced Lys-C 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), 284-91. L: 1-42 43-45 46-103 104-107 108-126 127-145 146-149 150-169 170-183 184-188 189-190 191-207 208-214 Lys-C Cleavage (C-terminal Side of Lys) H: 1-30 31-43 44-65 66-76 77-124 125-136 137-150 151-213 214-216 217 218-221 222-225 226-251 252-277 278-291 292-320 321-323 324-325 326-329 330-337 338-341 342-343 344-363 364-373 374-395 396-412 413-417 418-442 443-449 H: L: 1-30 31-43 44-65 66-76 77-124 125-136 137-150 151-213 214-216 217 218-221 222-225 226-251 252-277 278-291 292-320 321-323 324-325 326-329 330-337 338-341 342-343 344-363 364-373 374-395 396-412 413-417 418-442 443-449 Disulfide Bond 1-42 43-45 46-103 104-107 108-126 127-145 146-149 150-169 170-183 184-188 189-190 191-207 208-214 Light Chain Heavy Chain 2013 Waters Corporation 27

Method Considerations ACQUITY UPLC BEH130 C18 1.7µm ACQUITY UPLC CSH130 C18 1.7µm 2.1 x 150 mm 400 0.02% TFA 0.08% FA Optimize peak capacity Small compromise to MS Sensitivity Elevated Temperature (60 C) Improved peak shape and recovery 5-11 kda species 350 CSH130 C18 2 5-11 kda Species BEH130 C18 4 40 C Peak Capacity 300 250 BEH130 C18 6 8 60 C 200 MS Signal 10 150 12 0.00 0.02 0.04 0.06 0.08 0.10 Percent TFA Other applications of elevated temp. Anal Chem 2011, 83 (15), 5912-9. Anal Biochem 2011, 411 (2), 284-91. MAbs 2010, 2 (4) J Biol Chem 2009, 284 (51), 35390-402. 2013 Waters Corporation 28

Non-Reduced Lys-C C Peptide Maps Trastuzumab 1.1 0.8 BEH130 C18 Peak Capacity = 207 97.1% Coverage 0.02% TFA 0.08% FA 5 A210 0.5 4 0.2 1 2 3-0.1 0 10 20 30 40 50 60 70 80 90 100 110 A210 1.1 0.8 0.5 CSH130 C18 Peak Capacity = 394 96.7% Coverage 4 5 wh (sec) Peak BEH130 C18 CSH130 C18 1 33.7 15.4 2 16.1 9.7 3 9.5 5.7 4 18.9 9.9 2 3 5 23.9 13.2 0.2 1-0.1 0 10 20 30 40 50 60 70 80 90 100 110 Time (min) 2013 Waters Corporation 29

LC-UV UV-MS with an MS-Compatible Mobile Phase CSH130 C18 Low TFA mobile phase 0.02% TFA 0.08% FA MS Optimized Peak Capacity + MS signal UV signal ACQUITY H-Class Bio CSH130 C18 1.7 µm UV Peak Area MS-98701 UV-96527 UV Detector ESI-MS Xevo G2 QTof 3+ 2+ m/z 200 400 600 800 1000 1200 1400 1600 1800 2013 Waters Corporation 30

Disulfide Characterization H:137-150 x H:151-213 L:1-42 x L:46-103 H:1-30 x H:77-124 H:226-251 x H:226-251 L:127-145 x L:191-207 0.7 H364-373 x H:418-442 0.9 A210 Non-Reduced L:208-214 x H:222-225 1.1 H:252-277 x H:324-325 CSH130 C18 - Optimized Gradient 0.5 0.3 0.1 Blank -0.1-1.1 0 10 Reduced 20 30 H:1-30 H:77-124 217 H:151-213 77-124 125-136 137-150 151-213 214-216 L:46-103 66-76 H:226-251 44-65 L:127-145 31-43 H:252-277 L:1-42 1-30 -0.9 218-221 222-225 226-251 252-277 278-291 292-320 321-323 324-325 326-329 330-337 338-341 342-343 344-363 364-373 374-395 396-412 413-417 418-442 443-449 H364-373 -0.7 46-103 104-107 108-126 127-145 146-149 150-169 170-183 184-188 189-190 191-207 208-214 H:418-442 43-45 L:191-207 1-42 H:137-150 -0.5 L:208-214 A210-0.3 40 222-225 50 226-25160 252-70 80 90 100 110 120 130 Time (min) 2013 Waters Corporation 31

Assaying Deamidation CSH130 C18 - Optimized Gradient 44 GLEWVARIYPTNGYTRYADSVK 65 Binding region from the heavy chain Prone to deamidation 0.5 A210 0.4 0.3 0.2 0.1 H:44-65 * * 92 % Asn 7 % isoasp 1 % Asp Unmodified Deamidated Intensity 3E+5 0E+0 853 2E+4 854 855 856 Mass shift +0.98 Da * 0E+0 853 5E+3 854 855 856 * 0 59 60 61 62 63 64 65 Time (min) 0E+0 853 854 855 856 m/z 2013 Waters Corporation 32

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

Peptide Separation Technology Columns 2013 Waters Corporation 34

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

UPLC and HPLC CSH130 C18 2.1 x150 mm 1.7 µm A 214 1.0 0.8 0.6 High peak capacity separations not limited to UPLC 0.1 % FA ~8000 psi 0.4 0.4 CSH130 C18 Peptide Separation Technology Columns A 214 1.0 0.8 0.6 0.1 % TFA 0.2 Available 0.0 Now: Upcoming: Method 10 20 30 40 50 Analytical Columns 1.0 Time (min) Nano (75, 150, 1.0 Time 300 (min) µm ID) Transfer 1.7 µm Prep Columns (5 µm) 2.5 µm XP Longer Run Time Lower Pressure 0.8 2.5 µm XP 0.6 3.5 µm A 214 0.4 0.2 0.0 14.5 24.5 34.5 44.5 54.5 64.5 74.5 Time (min) ~3000 psi A 214 0.2 0.0 10 20 30 40 50 0.8 0.6 0.4 0.2 0.0 13.5 23.5 33.5 43.5 53.5 63.5 73.5 Time (min) 2013 Waters Corporation 36

Quality Control CSH130 C18 0.1 % Formic Acid 2013 Waters Corporation 37

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 1.20 1.00 0.80 0.60 0.40 0.20 0.00 1.20 1.00 0.80 0.60 0.40 0.20 0.00 1.20 1.00 0.80 0.60 0.40 0.20 0.00 1.20 1.00 0.80 0.60 0.40 0.20 0.00 Batch 110 T1 T13-T14 T14 2.00 3.00 4.00 5.00 6.00 Minutes 7.00 8.00 9.00 Batch 108 2.00 4.00 6.00 Minutes 8.00 Batch 116 2.00 4.00 6.00 Minutes 8.00 Batch 102 T4 T9-T10 2.00 4.00 6.00 Minutes 8.00 *Data provided by S. McCall/P. Iraneta 2013 Waters Corporation 38 T10 T8 T15 T19C T19 T12-T13 T12

Analytical Standards and Reagents (ASR) MassPREP Peptide Mixture Digestion Standards Cytochrome C Digestion Standard Part Number: 186006371 Rapigest SF Amgen Digestion Protocol Anal Chem. 2009 81(4):1686-92 2013 Waters Corporation 39

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 720003929EN 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 720004568EN 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 720004571EN 2013 High Resolution Peptide Mapping Separations with MS-Friendly Mobile Phases and Charge Surface Modified C18 M.A. Lauber, S.M. Koza, S.A. McCall, B.A. Alden, P.C. Iraneta, and K.J. Fountain Manuscript in Preparation 2013 2013 Waters Corporation 40