Utilizing novel technology for the analysis of therapeutic antibodies and host cell protein contamination

Transcription

1 Utilizing novel technology for the analysis of therapeutic antibodies and host cell protein contamination Kelli Jonakin, Ph.D. Senior Field Application Scientist, Seattle, WA Eric Johansen, Ph.D. Senior Application Scientist, Biologics, Redwood City, CA

2 Outline Introduction MS Antibody characterization strategies. Intact protein analysis. Peptide mapping and quantitation. Protein sequence/modifications (oxidation, deamidation, etc). Sequence Variant Analysis MS XIC of All Quantitative Workflow Simple, Sensitive and Powerful Technique for Host Cell Protein Detection MSMS of ALL Fragment Ions of Every Peptide: SWATH AB SCIEX

3 Therapeutic Immunoglobulin Molecules Reported IgG1 Structure and Heterogeneity Changes in Sequence: Sequence Variant Analysis Cysteinylation (+119 Da) Thioether (-32 Da) CDR Intact IgG1: MW ~150 kda Homodimer H2L2 (symmetrical) aa 32 Cys - 16 disulfide bridges 4 interchain + 12 intrachain N-term blocking (Gln/Glu PyroGlu: - 17/-18 Da) VH VL IgG1 = H2L2 ( kda) CH1 CH1 CL CL Glycosylation Heterogeneity (Fab & Fc: +1445, 1607, 1769 Da ) Oxidation (Met, Trp: +16 Da) Deamidation (Asn: +1 Da) Succinimide (Asn/Asp Succ: -17 Da) Glycation (Lys-Glc: +162 Da) C H 2 C H 3 C H 2 C H 3 Host Cell Protein Analysis Other Proteins Other Proteins C-term clipping (-Lys: -128 Da) C-term amidation (OH NH2: -1 Da) AB SCIEX

4 Intact mab Analysis

5 Why Perform Intact Protein Analysis? Biological therapeutics are produced in cells. Cells can alter the glycosylation patterns and PTMs over time. Protein cleavage or truncation can also occur. Rapid method of global observation of changes to product from lot-to-lot. Intact protein analysis provides: Mass of complete protein product i.e. clipped or truncated? Glycosylation heterogeneity Drug-Antibody-Ratio of antibody-drug conjugates Reproducible, reliable, trustworthy data is the most important factor. DAR and Relative abundance of each glycoform must be determined reliably AB SCIEX

6 Excellent Sensitivity for Antibody Analysis Intact Molecule Weight Determination Anti Actin IgG (Sigma) 10µg mab on column Expanded mass region to view heterogeneity +40 (~150kDa) µg mab on column ~33.3 pmol 1µg mab on column ~6.6 pmol Note the Linear Response to the increasing mab Concentration: 1ug:50 counts, 5ug:250 counts, 10ug:500counts AB SCIEX

7 BioPharma View Deconvolution TOFMS Deconvoluted AB SCIEX

8 Qualitative and Quantitative Peptide Map Analyses

9 Why Perform Peptide Map Analysis? Biological therapeutics are produced in cells. Cells can alter sequence and PTMs in response to changing environmental conditions, through natural processes of change, or mistakes. It s important to determine critical quality attributes: properties that are necessary for safety and efficacy of the drug, and to track these attributes through scale-up, purification, and formulation changes. Peptide mapping analysis provides: Product sequence Post-Translational Modifications Glycosylation Heterogeneity Low-Level Sequence Variants. Adding simultaneous quantitation of each peptide from a protein product and its modified forms gives you an additional dimension of information: allows you to quantitatively monitor changes over time AB SCIEX

10 Quant-Qual Peptide Mapping MS XIC of All peptides ID OF ALL PEPTIDES and MODIFIED PEPTIDES TripleTOF IDA RUN MS XIC of ALL Batch Comparison Sample A Sample B AB SCIEX

11 Peptide Mapping Experimental details Denaturation > Reduction > Alkylation > Trypsin digestion TripleTOF 5600 system with the Turbo V Source. UHPLC system Injection: 1 ug on column TripleTOF TripleTOF Parameters: One 0.25 second TOF-MS scan second MS/MS scans Total Cycle Time 1.3 seconds. Time Solvent A Solvent B AB SCIEX

12 Sample Acquisition on TripleTOF System Antibody Digest: 45 min chromatography, 20 x 50msec msms/second 2 ul injection (1.0 ug total) Full scan MS TIC blue MS/MS TIC pink MS/MS spectra AB SCIEX

13 Sample Acquisition on TripleTOF System Antibody Digest: 45 min chromatography, 20 x 50msec msms/second Selected Start Mass/Charge: Da Selected End Mass/Charge: Da Selected Points:27791 to Min. Intensity: Max. Intensity: 8.410e5 Sum Intensity: 1.152e7 Sum Intensity Above 50%: 1.007e6 2 ul injection (1.0 ug total) Full scan MS TIC blue MS/MS TIC pink Peak Mass/Charge: Da Peak Width at 50%: Da Points Across Peak at 50%: 4 Peak Width at Base: Da Points Across Peak at Base: 18 Peak Area: Resolution: 36,023 MS/MS spectra 23,591 Resolution 36,661 Resolution 36,225 Resolution 36,844 Resolution AB SCIEX

14 ProteinPilot Software Version 4.5 (Beta) Smart data processing for protein characterization Automatic extensive identification with the Paragon search algorithm Search for Hundreds of Biological Modifications All Sequence Variants Unexpected Cleavages Without the Large Number of False Positives from Error Tolerant Searching AB SCIEX

15 The Problem with Trying to ID More Variations Sequence Database Match Approach (Mascot, Sequest, etc) MH How Many Peptides in Database within 10 ppm of ~20 Score each based on MSMS ions in Spectrum vs 20x Theoretical PEPTIDER Good at ID of peptides In database. Adding mods and amino acid substitutions increases size of database. Error Tolerant Search to find more Mods AB SCIEX

16 The Problem with Error Tolerant Searching How to Avoid the Combinatorial Explosion? (SIGNAL) (NOISE) New right answers increasing search space New wrong answers increasing search space Variations allowed Variations allowed As search space increases, additional right answers become harder to find while the cost in incorrect peptide hypotheses considered explodes. The signal to noise drops rapidly as we consider more variations. How can we change this situation? AB SCIEX

17 Limited de novo Sequencing Generate Taglets The Paragon Algorithm is a tag based database search algorithm. A large number of short sequence tags Taglets are called for an MS/MS spectrum. They are rated with a chance the Taglet is correct. S L L L L D S L G D AB SCIEX

18 Varying Search Space on a Continuum Taglets for Sequence Temperature Value (STV) AB SCIEX

19 The Paragon Algorithm Key Idea Database sequence regions with: hotter STV searched more extensively cooler STV are searched less extensively Assumption: it is worth more effort to find a MW match for sequence regions with more tag evidence indicating the right answer may be found there. Thus, allow more mods, soften digestion rules and allow bigger MW deltas as STV increases. Mol. Cell. Proteomics, 6: , (2007) AB SCIEX

20 Protein Pilot Search Results: Antibody Digest Color Peptide Confidence Gray No match Red > 0 and < 50 Yellow >= 50 and < 95 Green >= AB SCIEX

21 Quant-Qual Peptide Mapping MS XIC of All peptides ID OF ALL PEPTIDES and MODIFIED PEPTIDES TripleTOF IDA RUN MS XIC of ALL Batch Comparison Lot 1 Lot AB SCIEX

22 Peptide Mapping TripleTOF 5600 System High Speed MS/MS Acquisition TIC XIC AB SCIEX

23 Zoom in TIC & XIC windows MS XIC data gives higher specificity vs. TIC (similar to UV trace) TIC XIC AB SCIEX

24 Peptide modification Deamidation HT07 vs. Deamidated HT07 Unmodified Pept (large peak) HT min Modified Small Pept Deamidated HT07 HT07, +3 Precursor Ion HT07 MS/MS HT07 y11 +2 ion 1 Da Shift Dea-HT07, +3 Precursor Ion Dea-HT07 MS/MS 1 Da Shift Dea-HT07 y11 +2 ion AB SCIEX

25 Relative quantitation of individual peptides Automatable Reports. Quant + Qual = Fingerprint of the current state of your biologic product AB SCIEX

26 Recap: Advanced Characterization Peptide Mapping & Quantitation Workflow 1. Data Acquisition 2. Peptide Mapping 3. MS Peak Extraction, Relative Quant of all Peptides, and Report Generation TOF MS and MS/MS acquired at high speed TripleTOF 5600 System Datafiles Protein Database Search Results Obtained XIC peak integration across samples Automated Peptide Map Quantitative Report AB SCIEX

27 Batch Comparison: Scale-Up/Process Development/Formulation Studies can be Automated Data Acquisition TOF MS and MS/MS acquired at high speed BioPharmaBeta 2 is the latest preview release of our powerful new solution for automation of core biologics development workflows AB SCIEX

28 Characterize Standard for Peptide Mapping workflow 1. Load the file 2. Process 3. Review results AB SCIEX

29 BioPharma Beta 2 Quant Peptide Map Quantitative Batch Comparison TIC & XIC Matched peptide list MS MS/MS AB SCIEX

30 MSMS of All with SWATH Data-Independent Analysis for Host Cell Protein Detection

31 Why Perform Host cell Protein (HCP) analysis? Biological therapeutics are produced in cells. Any of the cell s proteins that copurify with the drug may provoke an immunogenic response in the patient. The current standard is an ELISA assay to detect HCPs. Polyclonal serum is raised against the host cell proteome. Usually in Rabbit. The ELISA only works if both Rabbit and Human react to the antigen: If Rabbit does not produce an immunogenic response but Human does: A deadly and costly problem could result. Mass spectrometry is being adopted as the new standard of unbiased HCP assessment AB SCIEX

32 MS/MS ALL with SWATH Acquisition What is it? MS/MS ALL A data-independent workflow enabled by TripleTOF system technology that acquires high resolution quantifiable MS/MS data for all detectable analytes in a complex sample, in a single run How does it work? SWATH Acquisition Uses wide isolation windows stepped across a mass range, collecting high resolution composite MS/MS spectra in a chromatographic time scale What does this enable? Data processing post-acquisition: generate fragment ion XICs at high resolution for quantitation with confirmation of identity Quantitation and confirmation of everything in the sample Digital record of everything in your sample Single method for acquiring all your data AB SCIEX

33 Targeted Quantitative Workflows Top Two Techniques MRM Workflow SWATH Acquisition High Res XICs AB SCIEX

34 Powerful MS System for Biologics Analysis Accurate-mass, high-resolution MS system for characterization and quantification of biologics High speed MS/MS acquisition for comprehensive identification and quantification for peptide mapping and SVA experiments Sequence-tag based searching in Protein Pilot can pinpoint sequence variants with high confidence in less time. SWATH Technology for Host Cell Protein Detection Easy transition from qualitative workflows to targeted quantitative workflows AB SCIEX

35 AB SCIEX Solutions in Biologics Preclinical Basic Research Development Clinical Development Commercialized Product Basic and Advanced Characterization Targeted Quantitation TripleTOF TripleTOF 4600 LINAC Unifying link QTRAP 4500 QTRAP 6500 w/selexion TM Eksigent ekspert 425 System AB SCIEX

36 Thank You for Your Attention

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