High Throughput Accurate Mass Screening of Monoclonal Antibodies

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

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

3 Analytical R&D 3

4 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

5 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

6 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

7 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

8 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

9 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

10 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 _BSA_INFUSION_3 28 (.519) M1 [Ev-72518,It25] (Gs,.75,1322:1984,1.,L5,R5); Cm (17:29) Desalted BSA 1 mg/ml _BSA_INFUSION_1 TOF MS ES TIC e6 TIC Deconvoluted Data Using MaxEnt1 TOF MS ES+ 6.66e Desalted BSA 1 mg/ml _BSA_INFUSION_3 28 (.519) Cm (17:29) MS Raw Data Sum ~2 scans Time TOF MS ES Cysteinylated BSA Δ= m/z mass 1

11 Intact Protein Analysis RP-HPLC-MS RP-HPLC of Intact Protein Purity and intact data Murine EGF Generic.1 TFA/ACN gradient E K _EGF_4 7.e :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 _EGF_ : TOF MS ES TIC 8.63e4 MS TIC 1.72 Sum across peak including potential impurities Time

12 Intact Protein Analysis RP-HPLC-MS RP-HPLC of Intact Protein Purity and intact data Murine EGF More than one isoform under main peak E K _EGF_4 614 (11.262) Cm (61:621-(633:648+58:6)) 1 Deconvoluted MS Data Theory = Experimental = Minor Component B =114 Truncated N-terminal ASN? A4 A4; A: ±.8 B: ±.6 1: TOF MS ES+ EGF Sequence NSDSECPLSHDGYCLHDG VCMYIEALDKYACNCVVGY IGERCQYRDLKWWELR A5 A B5 B B4 B B3 B A3 A m/z 12

13 Intact Protein Analysis RP-HPLC-MS RP-HPLC of Intact Protein Purity and intact data Murine EGF XICs demonstrate multiple isoforms are present E K _EGF_ :19:18 oxegf-n 12-Jun-28 1: TOF MS ES _EGF_4 1: TOF MS ES e _EGF_4 1: TOF MS ES e _EGF_4 1: TOF MS ES e oxegf EGF EGF-N Time 13

14 Intact Protein Analysis SEC-MS 3227_JB_2 AU 2.e+1 1.5e+1 1.e 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 Cytochrome C Aprotinin Insulin B LH-RH TRH Phenylalanine : Diode Array Range: 2.359e+1 Sample Prep Requirement Analyte Dependent MS Compatible Sample Load Requirement Throughput SEC SEC _JB_2 Sm (Mn, 2x5); Sm (Mn, 2x5) 1: TOF MS ES TIC 1.26e Time Sample Prep Requirement Analyte Dependent MS Compatible Sample Load Requirement Throughput 14

15 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 _BSA_JB_QTOF_ : TOF MS ES+ TIC 3.17e5 SEC BSA Non-Reduced Divert Sample Prep Requirement Analyte Dependent _BSA_JB_QTOF_2 1: TOF MS ES TIC 4.19e MS Compatible Sample Load Requirement Throughput BSA Reduced Time

16 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 _SO57_JB_QTOF_ mm.7 µg : Diode Array Range: 7.175e-3 1: TOF MS ES+ TIC 1.28e _SO57_JB_QTOF_1 1.e : Diode Array Range: 1.2e-2 Sample Prep Requirement Analyte Dependent MS Compatible Sample Load Requirement Throughput SEC AU 5.e _SO57_JB_QTOF_1 1: TOF MS ES TIC e mm 3.5 µg Time 16

17 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

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

19 Antibody Analysis HT Development SEC-MS Intact Protein Methodology Analysis of intact and reduced mab MS TIC Raw Specta Deconvoluted Data SO mg/ml Intact 7:33:2 22-Apr-29 SO mg/ml Intact 4229_SO57_2 1: TOF MS ES+ 4229_SO57_2 192 (3.559) Cm (185:29) 1: TOF MS ES TIC 4.27e Intact Antibody SO mg/ml Intact 4229_SO57_2 192 (3.559) M1 [Ev-17673,It13] (Gs,.75,26:4,1.,L33,R33); Cm (185:29) GF/GF GF/G1F 1: TOF MS ES GF/G2F G1F/G1F Time m/z mass SO mg/ml Reduced 8:7:4 4229_SO57_3 Heavy Chain Apr-29 1: TOF MS ES+ TIC 2.32e5 SO mg/ml Reduced 4229_SO57_3 27 (3.836) Cm (2: :189) SO mg/ml Reduced 1: TOF MS ES+ 4229_SO57_3 27 (3.836) M1 [Ev ,It32] (Gs,.75,131:2437,1.,L33,R33); Cm (2: :189) 1: TOF MS ES e GF Deglycosylated G1F G2F Time m/z mass

20 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

21 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

22 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

23 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: 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:

24 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

appropriate sequence and modifications Automated deconvolution

25 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

26 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 G1F Non-Glycosylated G2F G Man Model Antibody - Spent Media Control Modifiers Relative Abundance Average SD RSD GF G1F Non-Glycosylated G2F G Man

27 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 = Counts) A A T Average Mass Analyte: 21525_IgG2B_JB_QTOF_71.raw 1 Man5 A GF A A G1F A Min Intensity Threshold : counts A : IgG 2B HC N-Term Q No K : Clone 1 G2F Min Intensity Threshold : counts A : IgG 2B HC N-Term Q No K : (max = Counts) 75 5 A Clone 2 25 A A T Average Mass 27

28 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 Assay Replicate (1/2, 3/4, etc.) 28

29 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

30 HT Application Clone Screening Man5 Confirmation Confirm Man5 by glycopeptide analysis on the LTQ-FT Tryptic peptide EEQYNSTYR-Man5 ( ) RT: Relative Abundance GF XIC (+2) 1.9E6 Man5 XIC (+2) 4.7E5 (25 GF) Time (min) NL: 1.9E6 m/z= F: FTMS + p NSI Full ms [ ] MS 2179_IgG2B_JB_L TQFT_1 NL: 4.68E5 m/z= F: FTMS + p NSI Full ms [ ] MS 2179_IgG2B_JB_L TQFT_1 2179_IgG2B_JB_LTQFT_1 # RT: AV: 7 NL: 7.11E5 F: FTMS + p NSI Full ms [ ] Relative Abundance Man GF G1F Glycopeptides G2F m/z 3

31 HT Application Media Component Titration Component Titration Experiment Product Quality Impact Same Protein-A - SEC-MS - BPL workflow RMC.1.SOY Glycan Distribution Total Glycan 8H284B - 8H284B - 5 8H284B K48-9K48-5 9K E258-8E E L359-6L L L C23-9C23-5 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)

32 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

[ APPLICATION NOTE ] High Sensitivity Intact Monoclonal Antibody (mab) HRMS Quantification APPLICATION BENEFITS INTRODUCTION WATERS SOLUTIONS KEYWORDS

[ APPLICATION NOTE ] High Sensitivity Intact Monoclonal Antibody (mab) HRMS Quantification APPLICATION BENEFITS INTRODUCTION WATERS SOLUTIONS KEYWORDS Yun Wang Alelyunas, Henry Shion, Mark Wrona Waters Corporation, Milford, MA, USA APPLICATION BENEFITS mab LC-MS method which enables users to achieve highly sensitive bioanalysis of intact trastuzumab