High Throughput Accurate Mass Screening of Monoclonal Antibodies

High Throughput Accurate Mass Screening of Monoclonal Antibodies Jim Blasberg April 28, 211 sigma-aldrich.com

Life Science and High Technology Center Life Science and High Technology Center2 Sigma-Aldrich, St. Louis

Analytical R&D 3

Analytical R&D Objectives Serve as corporate center of excellence for mass spectrometry Provide analytical support to Research Biotech and SAFC product development teams Generate application data supporting marketing of new Biotech products and evaluation of new technologies for Business Development Provide high-end analytical characterization of proteins and antibodies Provide specialized testing in support of Quality Control Lead and/or support troubleshooting and problem-solving initiatives in support of high-value products 4

Generalized Protein/Glycoprotein Characterization Workflow Reduce & Separate SEC-MS Sialic Acid Analysis HPLC-FLD HPAEC, Colorimetry Bulk Sample Reduce Complexity Specific Structural Details Data Analysis Intact MW analysis with all PTM SDS-PAGE LC-MS Remove PTM Enzymatically Chemically Peptide/Glycopeptide Mapping LC-MS/MS Glycan Analysis MALDI/ESI HPAEC HPLC Correlate Data from All Methods 5

Why Study Intact Protein Mass? Intact mass analysis can detect altered chemical and physical properties related to protein function Detection of PTMs N/C-terminal variation Glycosylation state 5-9 of proteins are glycosylated Confirm primary sequence Evaluate stability Oxidation or degradation 6

Intact Protein Analysis General Considerations ESI We chose to focus on Electrospray Ionization or ESI as our technique of choice for intact protein analysis Infusion or RP-HPLC sample introduction 7

Intact Protein Analysis General Considerations Deconvolution How we get from multiply charged to zero-charge accurate mass Time of Flight (TOF) mass analyzer Waters LCT or QTof Premier Multiply charged ions centered around 1 m/z Good resolving power in this m/z range Deconvolute to zero charge A: The m/z values can be expressed as follows: m/z = (MW + nh+)/n B: With 2 adjacent m/z values we can determine charge state and solve for MW 8

Intact Protein Analysis General Considerations Sample Introduction - ESI Direct Infusion - MS Low throughput Good approach for small numbers of relatively pure samples Typically requires cleanup RP-HPLC - MS Moderate throughput Analyte dependent, MS compatible Good for protein purity and intact Sample Prep Requirement Analyte Dependent MS Compatible Sample Load Requirement Infusion RP-HPLC Throughput 9

Intact Protein Analysis Direct Infusion Desalting and Direct Infusion Request for intact mass of single protein sample UF solvent exchange/desalting Zero charge accuracy within.1 Desalted BSA 1 mg/ml 211421_BSA_INFUSION_3 28 (.519) M1 [Ev-72518,It25] (Gs,.75,1322:1984,1.,L5,R5); Cm (17:29) 66429 1 Desalted BSA 1 mg/ml 211421_BSA_INFUSION_1 TOF MS ES+ 1.15 1.28 1 TIC.85 1.6e6 TIC 66429 Deconvoluted Data Using MaxEnt1 TOF MS ES+ 6.66e3.2.4.6.8 1. 1.2 1.4 1.6 1.8 2. 2.2 2.4 2.6 2.8 Desalted BSA 1 mg/ml 211421_BSA_INFUSION_3 28 (.519) Cm (17:29) 151.485 1 1582.295 1384.5267 1444.7919 1444.7218 1384.6366 1444.8199 1582.3198 162.8247 1665.483 1665.7242 MS Raw Data Sum ~2 scans Time TOF MS ES+ 24 +32 66461 66548 Cysteinylated BSA Δ=119 66548 12 13 14 15 16 17 18 19 2 21 22 23 24 25 26 27 28 29 3 31 m/z 661 662 663 664 665 666 667 668 669 67 mass 1

Intact Protein Analysis RP-HPLC-MS RP-HPLC of Intact Protein Purity and intact data Murine EGF Generic.1 TFA/ACN gradient E1257 47K44 6128_EGF_4 7.e+1 11.17 19:19:18 12-Jun-28 2: Diode Array Range: 7.979e+1 6.e+1 AU 5.e+1 4.e+1 3.e+1 UV Trace 2.e+1 1.e+1. 6128_EGF_4 1.6 2. 4. 6. 8. 1. 12. 14. 16. 18. 2. 22. 24. 26. 28. 3. 1: TOF MS ES+ 11.26 TIC 8.63e4 MS TIC 1.72 Sum across peak including potential impurities 24.56 24.89.2 8.2 7.91 8.46 9.83 17.83 2.67 22.62 54 Time 2. 4. 6. 8. 1. 12. 14. 16. 18. 2. 22. 24. 26. 28. 3. 28.25 11

Intact Protein Analysis RP-HPLC-MS RP-HPLC of Intact Protein Purity and intact data Murine EGF More than one isoform under main peak E1257 47K44 6128_EGF_4 614 (11.262) Cm (61:621-(633:648+58:6)) 1 Deconvoluted MS Data Theory = 639.7 Experimental = 639.3 Minor Component B 639-5925=114 Truncated N-terminal ASN? A4 A4;151.6832 151.884 A: 639.32±.8 B: 5925.13±.6 1: TOF MS ES+ EGF Sequence NSDSECPLSHDGYCLHDG VCMYIEALDKYACNCVVGY IGERCQYRDLKWWELR A5 A5 128.7834 1511.348 17.53 B5 B5 1185.8752 1213.677 B4 B4 1482.2542 1511.7428 1513.5339 1516.4966 B3 B3 1976.13 A3 A3 214.394 2 4 6 8 1 12 14 16 18 2 22 24 26 28 m/z 12

Intact Protein Analysis RP-HPLC-MS RP-HPLC of Intact Protein Purity and intact data Murine EGF XICs demonstrate multiple isoforms are present E1257 47K44 6128_EGF_4 11.1 19:19:18 oxegf-n 12-Jun-28 1: TOF MS ES+ 1486.1 328 3 11.34 11.41 11.5 1.14 1.2 1.64 11.96 12.22 12.42 2 1.2 1.4 1.6 1.8 11. 11.2 11.4 11.6 11.8 12. 12.2 12.4 6128_EGF_4 1: TOF MS ES+ 11.4 1514.7 1.7e3 1 1.2 1.4 1.6 1.8 11. 11.2 11.4 11.6 11.8 12. 12.2 12.4 6128_EGF_4 1: TOF MS ES+ 11.26 151.8 7.43e3 1.2 1.4 1.6 1.8 11. 11.2 11.4 11.6 11.8 12. 12.2 12.4 6128_EGF_4 1: TOF MS ES+ 11.37 1482.2 1.89e3 11.32 oxegf EGF EGF-N 1 69 18 1.2 1.4 1.6 1.8 11. 11.2 11.4 11.6 11.8 12. 12.2 12.4 Time 13

Intact Protein Analysis SEC-MS 3227_JB_2 AU 2.e+1 1.5e+1 1.e+1 5.. SEC-MS of Intact Protein Traditionally not MS compatible Developed MS compatible MP system, 45 ACN/.1 TFA 3 3 x 7.8 mm columns in series Peak Analyte MW 1 BSA 66429 2 Cytochrome C 12327 3 Aprotinin 6511 4 Insulin B 3496 5 LH-RH 1183 6 TRH 362 7 Phenylalanine 165 1 28.16 2 29.84 3 31.71 4 5 38.3 6 41.56 7 5.58 2: Diode Array Range: 2.359e+1 Sample Prep Requirement Analyte Dependent MS Compatible Sample Load Requirement Throughput SEC SEC 5. 1. 15. 2. 25. 3. 35. 4. 45. 5. 55. 6. 65. 7. 3227_JB_2 Sm (Mn, 2x5); Sm (Mn, 2x5) 1: TOF MS ES+ 41.93 41.29 TIC 1.26e4 29.88 4 13. 23.19 28.14 33.73 38.13 43.57 47.15 Time 5. 1. 15. 2. 25. 3. 35. 4. 45. 5. 55. 6. 65. 7. 5.59 Sample Prep Requirement Analyte Dependent MS Compatible Sample Load Requirement Throughput 14

Intact Protein Analysis SEC-MS SEC-MS of Intact Proteins Modify for general use, single column Shorten run time Divert small MW after analyte(s) elute 8-minute run time, no re-equilibration required 211421_BSA_JB_QTOF_1 1 3.54 1: TOF MS ES+ TIC 3.17e5 SEC BSA Non-Reduced Divert Sample Prep Requirement Analyte Dependent.4.5 1. 1.5 2. 2.5 3. 3.5 4. 4.5 5. 5.5 6. 6.5 7. 7.5 8. 211421_BSA_JB_QTOF_2 1: TOF MS ES+ 3.41 TIC 4.19e5 5.54 6.13 MS Compatible Sample Load Requirement Throughput BSA Reduced Time.5 1. 1.5 2. 2.5 3. 3.5 4. 4.5 5. 5.5 6. 6.5 7. 7.5 8. 15

Intact Protein Analysis SEC-MS SEC-MS of Reduced Antibody Optimize for sample load, 2. and 4.6 mm id 29527_SO57_JB_QTOF_7 AU 5.e-3 2.5e-3. 1.75 2. 2.25 2.5 2.75 3. 3.25 3.5 3.75 4. 4.25 4.5 4.75 5. 5.25 5.5 5.75 29527_SO57_JB_QTOF_7 3.54 2. mm.7 µg 3.52 4.15 2: Diode Array Range: 7.175e-3 1: TOF MS ES+ TIC 1.28e4 4.14 4.17 4.25 1.65 1.76 2.8 1.98 2.48 2.28 2.32 4.43 2.97 3.26 2.71 2.86 4.69 4.97 5.77 4.86 5.19 5.45 5.67 57 1.75 2. 2.25 2.5 2.75 3. 3.25 3.5 3.75 4. 4.25 4.5 4.75 5. 5.25 5.5 5.75 29527_SO57_JB_QTOF_1 1.e-2 4. 2: Diode Array Range: 1.2e-2 Sample Prep Requirement Analyte Dependent MS Compatible Sample Load Requirement Throughput SEC AU 5.e-3 4.77 1.75 2. 2.25 2.5 2.75 3. 3.25 3.5 3.75 4. 4.25 4.5 4.75 5. 5.25 5.5 5.75 29527_SO57_JB_QTOF_1 1: TOF MS ES+ 3.95 TIC 4.6 1.29e4 53 1.65 1.95 2.13 2.17 2.28 2.58 2.8 4.6 mm 3.5 µg 3.6 3. 3.19 3.45 3.67 1.75 2. 2.25 2.5 2.75 3. 3.25 3.5 3.75 4. 4.25 4.5 4.75 5. 5.25 5.5 5.75 4.23 4.64 4.41 4.67 4.97 5.1 5.17 5.41 5.47 5.67 Time 16

Antibody Analysis HT Development SAFC Interests Glycoprofile and Protein Quality Media Development Raw Material Characterization Cell Line Engineering Ultimately the ability to tune glycoprofile Standard workflows not amenable to high throughput 17

Antibody Analysis HT Development Typical Mammalian Antibody Glycan Structures GF G1F G2F Galactose N-Acetylglucosamine Mannose Fucose 18

147574. 147691. 147929. Antibody Analysis HT Development SEC-MS Intact Protein Methodology Analysis of intact and reduced mab MS TIC Raw Specta Deconvoluted Data SO57 1127-62 4.3 mg/ml Intact 7:33:2 22-Apr-29 SO57 1127-62 4.3 mg/ml Intact 4229_SO57_2 1: TOF MS ES+ 4229_SO57_2 192 (3.559) Cm (185:29) 1: TOF MS ES+ 3.56 TIC 4.27e4 1 2876.686 2997.6689 49 2824.6846 359.9355 2574.74 Intact Antibody 49 3128.4614 245.3655 3267.7783 3342.3911 335.5981 SO57 1127-62 4.3 mg/ml Intact 4229_SO57_2 192 (3.559) M1 [Ev-17673,It13] (Gs,.75,26:4,1.,L33,R33); Cm (185:29) 14666. 1 GF/GF 146823. GF/G1F 1: TOF MS ES+ 627 627 14699. 147131. 147289. GF/G2F G1F/G1F 4.39.6 145761. 146238. 146513. 146462. 148543. 21.25.5.75 1. 1.25 1.5 1.75 2. 2.25 2.5 2.75 3. 3.25 3.5 3.75 4. 4.25 4.5 4.75 5. 5.25 Time 4 6 8 1 12 14 16 18 2 22 24 26 28 3 32 34 36 38 m/z 1458 146 1462 1464 1466 1468 147 1472 1474 1476 1478 148 1482 1484 mass SO57 1127-62 4.3 mg/ml Reduced 8:7:4 4229_SO57_3 Heavy Chain 3.84 4.45 22-Apr-29 1: TOF MS ES+ TIC 2.32e5 SO57 1127-62 4.3 mg/ml Reduced 4229_SO57_3 27 (3.836) Cm (2:218-162:189) 144.7675 1 1483.1755 1363.123 1528.118 1327.1686 1575.8411 1626.625 1293.1565 168.7927 SO57 1127-62 4.3 mg/ml Reduced 1: TOF MS ES+ 4229_SO57_3 27 (3.836) M1 [Ev-138428,It32] (Gs,.75,131:2437,1.,L33,R33); Cm (2:218-162:189) 1: TOF MS ES+ 961 5395. 1.85e5 1 961 185 GF 126.8181 123.119 12.8855 1738.6687 18.845 1867.4794 1939.2255 Deglycosylated G1F 5557. 1172.9645 G2F 48949. 5361. 5585. 4.25.5.75 1. 1.25 1.5 1.75 2. 2.25 2.5 2.75 3. 3.25 3.5 3.75 4. 4.25 4.5 4.75 5. 5.25 Time 4 6 8 1 12 14 16 18 2 22 24 26 28 3 32 34 36 38 m/z mass 488 49 492 494 496 498 5 52 54 56 58 51 19

Antibody Analysis HT Development Option 1 Direct from media Transfer clarified media to 96-well plate Reduce direct In-media Analyze by SEC-MS Process Data in BPL Fast and simple workflow Spiked pristine media ok Real samples precipitated and gave no signal in SEC-MS 2

Antibody Analysis HT Development Option 2 96 well UF membrane Transfer clarified media to a 96-well UF plate and wash to remove low MW Reconstitute and reduce Analyze by SEC-MS Process Data in BPL Two more steps than Option 1 Still fast and simple Spiked pristine media showed only polymer, likely Pluronic 21

Antibody Analysis HT Development Option 3 Protein-A purification Transfer Protein-A resin to a 96-well filter plate Wash Transfer clarified media to the 96-well filter plate Equilibrate/ Wash Process Data in BPL Analyze by SEC-MS Reduce Add elution buffer equilibrate and collect Several more steps than Option 1 or 2 A bit more involved but still reasonable time-wise, 2-3 hrs/plate To-date has been successful for numerous media samples of suitable titer 22

HT Application Clone Screening Purification Procedure 5-µL P3476 Protein A Agarose Fast Flow per well, wash w/eq buffer 75-µL clarified spent media containing target IgG preferably <1 µg/ml Equilibrate 1-min, wash 2x w/eq, add 1-µL ELUT buffer equilibrate 1-min Collect eluted antibody in a 96-well plate Add 5-µL 1M ABC and 1M DTT, incubate.5 hr at RT SEC-MS Procedure Waters Acquity UPLC Column: Toso Haas TSK Gel SW3XL, 3 x 2. mm, 4 µm MP:.1 TFA 45 CH 3 CN Flow Rate:.125 ml/min Inj. Vol.: up to 2 µl Run time: 8 minutes Flow Divert: 4.6-7.9 min MS: Waters QToF Premier (ESI+) Capillary: 3.2 kv Sample Cone: 4. V RF Lens: 6. V Extraction Cone: 3. V Desolvation: 24 C Source: 12 C Scan Range: 4-4 23

HT Application Clone Screening Purification Procedure Typical Plate Layout Real throughput 84 spent media samples complete overnight Data analysis day 2 Purified mab Reference Spent Media Control Clone Screening Assay Day 7 Biological duplicate samples Clone Screening Assay Day 1 Biological duplicate samples 24

HT Application BPL Output BiopharmLynx Data Processing Automated deconvolution of data Set up BPL method with appropriate sequence and modifications Automated deconvolution of 96 samples in <2 min Manual inspection of results with final summary in Excel 25

HT Application Reproducibility Protein-A + SEC-MS + Data Processing Holistic assay variability evaluation Coaching expectations Model Antibody - Reference Standard Modifiers Relative Abundance Average SD RSD GF 66.3 66 66.5 66.3.25.4 G1F 22.1 22 21.7 21.9.21.9 Non-Glycosylated 5.4 5.7 5.3 5.5.21 3.8 G2F 2.7 2.9 2.7 2.8.12 4.2 G 2.4 2.5 2.6 2.5.1 4. Man5 1.1 1 1.2 1.1.1 9.1 Model Antibody - Spent Media Control Modifiers Relative Abundance Average SD RSD GF 72.5 72.7 72.2 72.5.25.3 G1F 2 19.9 2 2..6.3 Non-Glycosylated 2.5 2.6 2.6 2.6.6 2.2 G2F 2.5 2.4 2.4 2.4.6 2.4 G 1.7 1.6 1.8 1.7.1 5.9 Man5.8.8 1.9.12 13.3 26

HT Application Clone Screening Clone Screen Data Processing Evaluate glycoprofile of various clones Biological replicates look good Discovered widely variable clonal Man5 levels Control: 21525_IgG2B_JB_QTOF_69.raw (max = 2255.1 Counts) 1 75 5 25 A 573.85 51171.47 595.17 A 51299.73 5561.22 51213. 51428.39 T 5374 56 58 51 Average Mass 512 514 51468 Analyte: 21525_IgG2B_JB_QTOF_71.raw 1 Man5 A 5619.7 GF A 5846.41 A 5846.64 G1F A 518.44 Min Intensity Threshold : counts A : IgG 2B HC N-Term Q No K : 5846.29 Clone 1 G2F Min Intensity Threshold : counts A : IgG 2B HC N-Term Q No K : 5846.29 (max = 532.6 Counts) 75 5 A 518.54 Clone 2 25 A 562.73 5947.2 A 5117.1 5788.49 5112.38 51271.93 5142.58 T 5374 56 58 51 512 514 51468 Average Mass 27

HT Application Clone Screening Man5 Content Export data from BPL to Excel Average biological replicates Normalize Man5 to GF and compare Man5 relative abundance Man5 Content 45 4 Average G Normalized 35 3 25 2 15 1 5 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 Assay Replicate (1/2, 3/4, etc.) 28

Generalized Protein/Glycoprotein Characterization Workflow Reduce & Separate SEC-MS Sialic Acid Analysis HPLC-FLD HPAEC, Colorimetry Bulk Sample Reduce Complexity Specific Structural Details Data Analysis Intact MW analysis with all PTM SDS-PAGE LC-MS Remove PTM Enzymatically Chemically Peptide/Glycopeptide Mapping LC-MS/MS Glycan Analysis MALDI/ESI HPAEC HPLC Correlate Data from All Methods 29

HT Application Clone Screening Man5 Confirmation Confirm Man5 by glycopeptide analysis on the LTQ-FT Tryptic peptide EEQYNSTYR-Man5 (245.9349) RT:. - 91.2 Relative Abundance 1 9 8 7 6 5 4 3 2 1 1 9 8 7 6 5 4 3 2 1 1.22 8.69 2.83 2.27 3.65 31.7 35.12 48.44 6.91 64.21 68.43 82.8 84.24 2.83 19.98 GF XIC (+2) 1.9E6 Man5 XIC (+2) 4.7E5 (25 GF) 38.9 84.32 21.49 45.66 37.19 48.44 65.8 67.87 75.78 83.69 1 2 3 4 5 6 7 8 9 Time (min) NL: 1.9E6 m/z= 1317.85-1318.2 F: FTMS + p NSI Full ms [35.-16.] MS 2179_IgG2B_JB_L TQFT_1 NL: 4.68E5 m/z= 123.8-124.11 F: FTMS + p NSI Full ms [35.-16.] MS 2179_IgG2B_JB_L TQFT_1 2179_IgG2B_JB_LTQFT_1 #2166-2228 RT: 2.64-21.21 AV: 7 NL: 7.11E5 F: FTMS + p NSI Full ms [35.-16.] 1 1318.3 Relative Abundance 95 9 85 8 75 7 65 6 55 5 45 4 35 3 25 2 15 Man5 123.98 GF G1F 1399.6 +2 Glycopeptides G2F 1 1216.49 1244.5 1358.4 148.9 5 1297.52 1346.54 1375.76 1176.44 1261.26 146.5 1439.32 1511.37 1533.1 1562.47 115 12 125 13 135 14 145 15 155 m/z 3

HT Application Media Component Titration Component Titration Experiment Product Quality Impact Same Protein-A - SEC-MS - BPL workflow RMC.1.SOY Glycan Distribution 6. 5. 4. 3. 2. 1.. 31 Total Glycan 8H284B - 8H284B - 5 8H284B - 15 9K48-9K48-5 9K48-15 8E258-8E258-5 8E258-15 6L359-6L359-5 6L359-1 6L359-15 9C23-9C23-5 9C23-15 Lot - g/l Soy Glycosylation GF N(1) Glycosylation G1F N(1) Glycosylation G2F N(1) Non-glycosylated Glycosylation G N(1) Glycosylation Man5 N(1)

Acknowledgements Sigma Analytical R&D Kevin Ray Ben Cutak Jim Walters Gordon Nicol Janet Irungu Mark Angeles SAFC Biosciences Nan Lin Joaquina Mascarenhas Andrew Christie Chas Hernandez Supelco Tracy Ascah 32