Improve Efficiency of Variant Screening for Biological Drugs. Guillaume Tremintin, Market Area Manager, Biopharma Bruker Daltonics, Fremont, CA

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1 Improve Efficiency of Variant Screening for Biological Drugs Guillaume Tremintin, Market Area Manager, Biopharma Bruker Daltonics, Fremont, CA

2 2 Biological Products Characterization Detailed physicochemical characterization early in the development helps understanding the possible heterogeneities and support the specification setting and process control strategy. Cell Line Selection Purification Formulation Characterization Clinical Development Commercialization

3 3 Biological Products Characterization In early stages large numbers of alternative clones/culture conditions preclude analysis strategies requiring excessive sample prep and data handling Top-down and middle-down mass spectrometry workflows can be automated Minimizes user intervention Mitigates risks of artifacts creation during sample prep Manageable data complexity Ensures process consistency

4 Topics Automation of MALDI workflows Intact mass Termini verification Peptide map and glycopeptides identification Automation of ESI workflows Intact mass Sub-units analysis 4

5 5 Poll Question 1 Are you currently using mass spectrometry to assist your clone selection process? ESI-qTOF Other ESI-MS MALDI-TOF N/A

6 6 Poll Question 2 Which attributes do you monitor by MS during clone selection? Identity (intact mass) Primary sequence (peptide map, PMF) Glycoprofile N/A

7 Top-Down Mass Spectrometry Tools Solutions to Match the Analytical Question Ultimate Resolving Power Versatility and throughput solarix FT MALDI/ESI maxis 4G ESI TOF < 10,000,000 res < 500 ppb ma Resolved intact IgG Flexibility and outstanding Data Quality < 60,000 full sensitivity < 600 ppb ma Resolved IgG subunit < 40,000 res < 1ppm ma Resolved IgG subunit ultraflextreme MALDI TOF/TOF m/z Workhorse & PTM analysis < 20,000 res < 50 ppm ma Resolved myoglobin amazon speed ESI Trap with ETD 7

8 Rapid Characterization with MALDI-TOF MALDI provides: Robustness & Ease of use Versatility (Biotyper/Imaging/Oligonucleotides) High resolution intact protein data Rapid answers to challenging characterization tasks Automated acquisition & processing with visual reporting Characterization Task MALDI Acquisition Speed Product confirmation (intact mass) Sequence coverage Identification of unexpected PTMs & AA substitutions Automated N & C termini sequencing Automated glycopeptides detection & identification Seconds Seconds Seconds Minute Minutes 8

9 Intens. [a.u.] Intact Protein Workflow Product Identity Confirmation Intact Myoglobin (17 kda) x m/z Resolved isotopes for confident monoisotopic mass determination 5 second acquisition of ESI Quality Data Approach can also readily highlight any potential contaminants present High quality data achievable on a much simpler instrument than thought Ideal for a QC or late development laboratory 9

10 MALDI Top Down Sequencing (TDS) with N&C Terminal Confirmation N T E R M S E Q U E N C E C T E R M N N T N T E N T E R MALDI Intact Sequencing M R M E R M T E R M 30 seconds! N T E R M N T E R M S C T E R M E C T E R M N T E R M S E C E C T E R M Confirm N & C terminal sequences Identify truncations/terminal PTMs Generate sequence information without proteolytic digestion In less than 1 minute; no other technique generates this much information in this short a time! 10

11 TDS of IgG1 HC On ultraflextreme Both Termini Confirmed PyroGlu K??? IgG1 (human) heavy chain MALDI matrix: 1,5-DAN 11

12 T 3 Sequencing Comprehensive Termini Sequence pyro-glu-qvqlqqsgaelasvkls Confirm N&C terminal sequence with T 3 sequencing Bruker patent 12

13 Intact Antibody QC by TDS Workflow N&C Terminal Sequencing: Reduced LC & HC HC Intact Analysis Glycosylation heterogeneity 1 minute Analysis with automated QC reporting LC Top Down Sequencing 140 residues identified N & C termini sequenced N-term pyro-glu detected Data with resolved isotopes Additional confirmation by patented T3 sequencing 13

14 GlycoQuest TM Advanced Glycopeptide Identification Complement the peptide analysis tools already available in Proteinscape Compatible with all instruments data Automated detection & identification of glycopeptides & glycans from complex samples Opens glycosylation analysis to the non-expert S X X N X X X X X X R 14

15 MSMS Spectrum of N-glycosylated Peptide from IgG Abs. Int. * 1000 b Q F N S T b-17 S T Y R b-18 E E Q F T y R Y T S y-17 R Y T S N F Q Peptide sequence : EEQF N STYR Peptide moiety b-18 7 Glycan moiety y-17 4 y-17 6 b-18 2 b-18 4 b+18 6 R a 2 y-17 3 b-18 6 y-17 1 b-18 3 b-17 6 y-17 9 y 1 b 3 y-17 5 b-17 7 y 9 b-17 5 b+18 9 y-17 2 b-18 1 y 2 y 4 b-17 9 b 6 y-17 7 y 3 Q b 2 b 4 b 5 b 7 b m/z 15

16 Glycopeptide Analysis Workflow LC-MALDI Non-Glycosylated Peptides Identification Proteins identified Example: LC-MALDI Analysis of a tryptic digest of a Murine mab (MOPC21, Sigma). Peptides identified Database search (Mascot) of LC-MS/MS run Identified HC peptides Glycosylation site Glycopeptide fractions 16

17 Glycopeptide Analysis Workflow LC-MALDI Find the Glycopeptides Spectra Example: LC-MALDI Analysis of a tryptic digest of a Murine mab (MOPC21, Sigma). Glycopeptide classification Glycopeptide spectra detected Peptide mass determined MSMS spectrum of Compound 757 Compound 757 highlighted 17

18 Glycopeptide Analysis Workflow LC-MALDI Peptide ID + Glycan ID => Glycopeptide ID Glycans identified Example: LC-MALDI Analysis of a tryptic digest of a Murine mab (MOPC21, Sigma). Glycan Search (Glycoquest) on the basis of classification result Glycan fragments detected Glycan identified for Compound 757 Compound 757 highlighted MSMS spectrum of Compound

19 Results: GlycoQuest Automated Report Generation GlycoQuest automatically generates PDF reports Glycan fragments in MSMS spectra are automatically annotated giving additional confidence that correct identification has been made Confidence at a glance view the structure with confirmation 19

20 Top-Down with UHR-ESI q-tof High resolution with no sensitivity compromise Characterize & detect impurities simultaneously High mass accuracy and isotopic fidelity Confident mass assignment for proteins or protein fragments compact R = 23,000 MA = 1 ppm Intact, intact subunit & digest accurate mass Rapid QC / clone analysis / stability tests Peptide map / Primary sequence verification Glycoprofiling 100% sequence coverage Quantitation of PTMs / AA substitutions impact R = 40,000 MA = 0.8 ppm maxis 4G R = 60,000 MA = 0.6 ppm 20

21 BioPharma Compass Routine Analysis Automation Sample acquisition (LC & MS) Sample processing Peptide Mapping Sequence coverage Map with multiple enzymes Reference comparison Report generation Released glycan screening Relative quantitation Peptide BPC Annotation Library Match or mass search Subunit Analysis Theoretical or reference Comparison Intact Mass Analysis Glycoprofiling Comparison with reference 21

22 Submit a Batch of Samples Samples are submitted as a file (Excel spreadsheet) 22

23 Submit a Batch of Samples Submission of the sample list in spread sheet format Alternative Sample Submission via Excel Spreadsheet with Defined Columns 23

24 Start Batch Processing from Acquisition PC 24

25 QC Result Table QC criteria (No. of compounds, No. of unmatched reference peaks, average mass deviation) are defined during processing of analyses QC values displayed in the table are calculated using workflow parameters 25

26 QC Result View 26

27 Batch Report 27

28 Intact Protein Analysis Workflow Human IgG (150kDa) Glycoforms Intact (150 kda) raw data Close up of raw data showing resolved glycoforms Intact analysis = Minimum sample processing = Minimum artefacts High quality data before deconvolution = Certainty 28

29 Intact Protein Analysis Workflow Automated Comparison with a Reference Resolved glycoforms (150 kda) Relative quantitation of glycoforms in comparison to reference standard Intact Mass Da mass ppm Relative Quant (S / R) Glyco Glyco Glyco Glyco Glyco Glyco Glyco Key for product development and quality Evaluate different production/feed systems Evaluate batch to batch consistency 29

30 BioPharma Compass 1.1 Intact Protein Workflow Biopharma Compass software allows for fully automated analysis and data processing, summarizing results in a simple format for high-throughput screening, that can be tailored specifically for various types of analysis. In this particular workflow, Biopharma Compass was able to provide a very simple method used for comparing measured intact mass to the theoretical mass. Establishing Biopharma Compass methods for our routine analysis has resulted in a much higher throughput workflow, along with a simplified user intervention and generation of consistent and reliable data processing. 30

31 Intact Subunit Analysis Workflow LC-MS and MALDI mab sub units analysis LC Fd Fc/2 1. Endoglycosidase F2 2.IdeS cleavage 3.Full TCEP reduction FabRICATOR (immunoglobulin degrading enzyme) cleaves the HC of mabs at the conserved Gly-Gly motif in the hinge region Highly specific Reduction of the fragments yields a pool of 6 fragments of 3 types: Light chain Fc/2: ~CH2+CH3 Fd: ~CDR+CH1 31

32 Intact Subunit Analysis Workflow Excellent S/N Isotopic resolution Isotopic fidelity Sub ppm mass accuracy Identification Certainty Rapid amino acid sequence confirmation in a small data set Definitive, state-of-the-art structural characterization Rapid comparison of top clones, drug substance batches, and commercial product lots 32

33 Intact Subunit Analysis Workflow Clone Comparison At Subunit Level Isotopic resolution Entire isotopic envelope shifts by Da Confirmed by peptide mapping Clone B 33

34 Intact Subunit Analysis Workflow Panitumumab Sub Units Dionex ProSwift RP-4H column 1x50 mm A : 0.1% HCOOH, B : 60 %ACN, 40% iospropanol, 0.1% HCOOH 0.3 ml/min 15% to 45% B in 30 min 4.2 µg mab (Ides treated) LC Fd pe Fc/2-K+ Fc/2-K + + Ox 34

35 Automated Antibody Verification for Lead Discovery Besides time reduction based on parallel sample preparation and shortening acquisition times, we are able to get a result overview within seconds. Analysis and report time for each sample have been reduced by about 90%. We are now able to offer a Quality Control (QC) workflow for our early hit finding process within Global Biologics. 35

36 36 Poll Question 3 Would better automation of the data acquisition and processing be a factor in introducing MS assays earlier in the clone selection process? Yes No

37 Conclusions An array of mass spectrometry tools are available for rapid topdown characterization of biological products MALDI is well suited for high throughput identity assays and termini confirmation. It is also useful to extend peptide maps usefulness by providing high confidence automated glycopeptide identification ESI UHR-qTOF resolution and high isotopic fidelity combined with automation software enable fast and accurate determination of the intact mass of biological products and process related impurities 37

38 Acknowledgements Robert L. Dufield, Heather S. DeGruttola, Matthew S. Thompson, Keith A. Johnson, Melissa D. Zolodz, Paul W. Brown, Jason D. Batchelor, Justin B. Sperry, James A. Carroll, Lisa A. Marzilli, Jason C. Rouse, Pfizer Andover & St Louis, USA Ryan Hylands, Malcolm J. Saxton, Joanne Waters, Novozymes, Nottingham, United Kingdom Alain Beck, Elsa Wagner, Daniel Ayoub, Pierre Fabre, Saint-Julien-en-Genevois, France Simone Greven, Birge Dübel, Heiner Apeler, Bayer, Wuppertal, Germany Anja Resemann, Andrea Schneider, Arndt Asperger, Ulrike Schweiger-Hufnagel, Wolfgang Jabs, Lars Vorweg, Christian Albers, Carsten Baessman, Detlev Suckau, Waltraud Evers, Markus Meyer, Bruker Daltonics, Bremen, Germany Jeremy Wolff, Mike Easterling, Bruker Daltonics, Billerica, MA 38

39 Innovation with Integrity 5/3/2013 Corporation. All rights reserved. 39 Copyright 2011 Bruker Corporation. All rights reserved.