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Orbitrap mass spectrometry, a high- confidence screening tool in biopharmaceutical product development David Horn Thermo Fisher Scientific San Jose, CA.

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Presentation on theme: "Orbitrap mass spectrometry, a high- confidence screening tool in biopharmaceutical product development David Horn Thermo Fisher Scientific San Jose, CA."— Presentation transcript:

1 Orbitrap mass spectrometry, a high- confidence screening tool in biopharmaceutical product development David Horn Thermo Fisher Scientific San Jose, CA

2 2 Outline 1.Challenges in the characterization of biopharmaceuticals 2.Introduction to the new generation of Orbitrap TM mass spectrometers 3.Confident intact antibody analysis using the Q Exactive TM Hybrid Mass Spectrometer 4.Introducing Protein Deconvolution 1.0 software 5.Confident antibody analysis using the Orbitrap Elite TM Hybrid Mass Spectrometer 6.Summary and conclusions

3 3 Outline 1.Challenges in the characterization of biopharmaceuticals 2.Introduction to the new generation of Orbitrap TM mass spectrometers 3.Confident intact antibody analysis using the Q Exactive TM Hybrid Mass Spectrometer 4.Introducing Protein Deconvolution 1.0 software 5.High resolution analysis of antibody subunits using the Orbitrap Elite TM Hybrid Mass Spectrometer 6.Summary and conclusions

4 4 MAb Characterization and QA/QC is Challenging …unlike Aspirin This present some new analytical challenges... Aspirin (180 Da) MAb (150,000 Da)

5 5 Structure of an antibody Mass ~150 kDa 2 light chains (~20 kDa each) 2 heavy chains (~50 kDa each) 16 disulfide bonds Glycan heterogeneity Various degradation pathways: Deamidation Oxidation Disulfide scrambling Sequence truncation Deglycosylation Aggregation

6 6 The Challenge in Monoclonal Antibody Analytics Target Identification and Validation MAb Generation Drug Discovery Cell Line Development Clone Screening and Selection Preclinical Development Cell Culture and Purification Process Development Clinical Development Formulation Lot Release Testing Stability Studies Pre- Commercialization Product Improvements Patent Extensions Biobetters Post- Commercialization Product Titer Purity/Impurities Product ID - Intact mass - Sequence coverage Product Quality - Charge var. - Aggregates - Fragments - Modifications DMPK/Metabolite Glycans Increasing #s of MAb candidates in pipeline Advances in automation in cell culture & recovery dev., formulation screening QbD guidelines requiring enhanced MAb quality monitoring MAb Development Workflow MAb Analytics Trends that increase the number of MAb samples requiring analysis: Goal of analytical labs: speed, throughput and productivity Today’s focus

7 7 Thermo Scientific + Dionex: Combining Best-in-Class Technologies Best-in class consumables for protein science Unique ion-chromatography solutions Leading HR/AM mass spectrometers High resolution protein separation columns Bio-HPLC & UHPLC+ Platforms for Protein & MAb Characterization & QA/QC Leading chromatography & MS data systems Today’s Focus

8 8 Outline 1.Challenges in the characterization of biopharmaceuticals 2.Introduction to the new generation of Orbitrap TM mass spectrometers 3.Confident intact antibody analysis using the Q Exactive TM Hybrid Mass Spectrometer 4.Introducing Protein Deconvolution 1.0 software 5.Confident antibody analysis using the Orbitrap Elite TM Hybrid Mass Spectrometer 6.Summary and conclusions

9 9 A new season in life of Orbitrap mass spectrometry

10 10 The Orbitrap Mass Analyzer The first Orbitrap was introduced in 2005 The Orbitrap is a Fourier transform mass spectrometer Ions oscillate at a frequency proportional to m/z Image current detection produces a “transient” that is converted to a mass spectrum via a Fourier transform. High resolution, mass accuracy, and throughput z φ r

11 11 Q Exactive and Orbitrap Elite – What’s New? Q Exactive Quadrupole mass selection HCD MS/MS Advanced signal processing and electronics Improved ion optics High sensitivity and throughput! Orbitrap Elite New compact high field Orbitrap Velos Pro Ion Trap Advanced signal processing and electornics Improved vacuum Ultra high resolution and mass accuracy!

12 12 Advanced signal processing produces higher resolution for isotopically-resolved intact proteins Orbitrap Elite LTQ Orbitrap Velos 48+ charge state of yeast enolase (46.6 kDa) is baseline resolved Advanced signal provides higher resolving power. Higher resolution = improved mass accuracy = higher confidence.

13 13 Advanced Signal Processing Produces Higher Resolution for Unresolved Intact Proteins Q resolution Orbitrap resolution m/z Glycoforms Hardware and software improvements produce higher resolution peaks for IgG glycoforms m/z

14 14 Outline 1.Challenges in the characterization of biopharmaceuticals 2.Introduction to the new generation of Orbitrap TM mass spectrometers 3.Confident intact antibody analysis using the Q Exactive TM Hybrid Mass Spectrometer 4.Introducing Protein Deconvolution 1.0 software 5.High resolution analysis of antibody subunits using the Orbitrap Elite TM Hybrid Mass Spectrometer 6.Summary and conclusions

15 15 Q Exactive MS - High Performance for Intact Proteins HCD Cell MS/MS Orbitrap Mass Analyzer Resolution 140K Mass accuracy better than 2 ppm Advanced signal processing Ion Source Improved sensitivity Quadrupole Mass Filter m/z amu wide Precursor selection SIM scan High resolution, hardware improvements, and advanced signal processing all contribute to improve intact protein characterization

16 16 Intact MAb on a Q Exactive m/z m/z High S/N Well resolved glycoforms across full m/z range Clean baseline Smooth distribution of charge states

17 17 ReSpect TM Deconvolution of Q Exactive IgG Data 10 glycoforms identified, all within ~12 ppm of theoretical average mass (+/-2 Da mass accuracy) 2 deglycosylated forms detected with high mass accuracy at low relative abundance (both at ~2%) Deconvoluted spectrum produced by ReSpect is free of artifacts and the various forms of the protein are well resolved G1F (9 ppm) G0F (9 ppm) 2xMan5 (-9 ppm) G0+G0 (12 ppm) G0+G0F (6 ppm) G0F+G1F (0 ppm) G0F+G1F (-6 ppm) G0F+G2F (or 2 G1F) (-9 ppm) G1F+G2F (-12 ppm) G2F+G2F (12 ppm)

18 18 Detected Charge States for G0F/G1F glycoform ChargeMeasured m/zCalculated MassDelta Mass DaDelta Mass (ppm) The deconvoluted mass for each individual charge state are as accurate as the mass in the deconvoluted spectrum ( ) The calculated average mass is very consistent for all charge states

19 19 Mass Measurement Accuracy for 52+ charge state of IgG G0F/G1F Δ= 7 ppm G0F/G0F Δ= 0 ppm G0/G0F Δ= 12 ppm G1F/G1F or G0F/G2F Δ = 10 ppm G1F/G2F Δ = 12 ppm High mass measurement accuracy can be obtained from a single charge state of an intact antibody Actual raw data!

20 20 Mass and Abundance Reproducibility of IgG data on a Q Exactive For the same IgG sample as previously shown, 7 different LC/MS runs with various 10 minute LC gradients Two different Q Exactive instruments were used Data were acquired on 3 different days Deconvoluted mass spectra were produced using Protein Deconvolution 1.0 software Reproducibility in mass and relative abundance were determined for the 5 major glycoforms of IgG

21 21 Q Exactive produces reproducible mass measurement for IgG glycoforms IgG Glycoform Mass Measurement Accuracy (ppm) RAW fileQ ExactiveG0+G0FG0F+G0FG0F+G1FG0F+G2FG1F+G2F N/A /- 6.4 ppm mass tolerance across all measurements Mass measurement is highly reproducible, even across instruments

22 22 IgG glycoform relative abundances (%) RAW fileQ ExactiveG0+G0FG0F+G0FG0F+G1FG0F+G2FG1F+G2F CV3.4%1.6%N.A.3.9%14% (4.9%) Relative abundances are highly reproducible across runs Run 2 used a different gradient and thus there was an overlapping species that did not occur in the other runs. Q Exactive produces reproducible relative abundances for IgG glycoforms

23 23 What does low ppm mass measurement provide? At <10 ppm mass tolerance, a mass spectrometer can detect: The reduction of two disulfide bonds on a 150 kDa protein. High confidence that there are few to no modifications to the sequence. ppm errorMass error (Da)Detectable Modificcation Da1 disulfide bond Da2 or more disulfide bonds Da3 or more Disulfide bonds 203 Da3 Disulfide bonds Da5 Disulfide bonds 406 Da6 Disulfide bonds Da8 Disulfide bonds IgG, 150 kDa

24 24 Q Exactive Summary The Q Exactive produces very high mass accuracy for intact antibodies These masses and abundances can be very reproducibly measured This indicates that the Q Exactive will be excellent for high throughput confirmation of biopharmaceuticals

25 25 Outline 1.Challenges in the characterization of biopharmaceuticals 2.Introduction to the new generation of Orbitrap TM mass spectrometers 3.Confident intact antibody analysis using the Q Exactive TM Hybrid Mass Spectrometer 4.Introducing Protein Deconvolution 1.0 software 5.High resolution analysis of antibody subunits using the Orbitrap Elite TM Hybrid Mass Spectrometer 6.Summary and conclusions

26 26 Protein Deconvolution 1.0 Workflow software for intact protein mass determination Supports all Orbitrap mass spectrometers Includes 2 deconvolution algorithms: Xtract for isotopically resolved proteins ReSpect for isotopically unresolved proteins (e.g. IgG) Target release date: Early November For more information – create an account at the Thermo Proteomics Software Portal (http://portal.thermo-brims.com)http://portal.thermo-brims.com ReSpect is a trademark of Positive Probability, Ltd.

27 27 Protein Deconvolution 1.0 – A Workflow Design Select AlgorithmLoad File Create/Select Method

28 28 Protein Deconvolution Method Parameters Instrument Resolution (Detected Automatically) Estimated Target Mass The instrument resolution and the user-supplied protein target mass are used to calculate an accurate peak model for ReSpect deconvolution.

29 29 Protein Deconvolution – Chromatogram Tab Averaged spectrum created for deconvolution Select chromatogram

30 30 Protein Deconvolution - ReSpect Deconvolution Fast Data Processing: ~1-2 s Deconvoluted Spectrum Deconvolution Results

31 31 Protein Deconvolution - Report Comprehensive, Exportable Report

32 32 Outline 1.Challenges in the characterization of biopharmaceuticals 2.Introduction to the new generation in Orbitrap TM mass spectrometers 3.Confident intact antibody analysis using the Q Exactive TM Hybrid Mass Spectrometer 4.Introducing Protein Deconvolution 1.0 software 5.High resolution analysis of antibody subunits using the Orbitrap Elite TM Hybrid Mass Spectrometer 6.Summary and conclusions

33 33 Orbitrap Elite for High Performance Protein Characterization Multiple dissociation techniques provide several mechanisms for protein sequence characterization by top-down techniques New high field Orbitrap provides higher resolution and mass accuracy Electron transfer dissociation especially important for glycoproteins Electron Transfer Dissociation Compact high-field Orbitrap analyzer Advanced Signal Processing 240,000 resolution, low ppm mass accuracy Velos Pro Ion Trap Selectable m/z range CID, MS n HCD Cell High sensitivity ion optics

34 34 Orbitrap Elite: Compact High-Field Analyzer Compact Orbitrap unique to Orbitrap Elite Reduced Orbitrap size results in 2x improvement in resolution Compact Orbitrap + advanced signal processing = 4x resolution improvement in over previous generations of Orbitraps 240,000 maximum resolution, enabling routine isotopic resolution for proteins up to 66 kDa. 12 mm 20 mm 30 mm 10 mm Orbitrap EliteAll other Orbitraps

35 35 Isotopically Unresolved vs. Isotopically Resolved m/z m/z Orbitrap Elite: IgG light chain 17+ Resolution = 15,000 Isotopically unresolved (first beat only) Isotopically resolved (2 or more beats) Orbitrap Elite: IgG light chain 17+ Resolution = 240,000 Distance between peaks = ~1 Da/(charge state) Isotopic resolution provides improved mass accuracy and higher peak capacity

36 36 Isotopically-Resolved IgG Heavy Chain (~50 kDa) m/z of IgG heavy chain Acquired at 240,000 resolution on Orbitrap Elite Baseline resolved! Δm/z = Isotopic resolution for the heavy chain on an LC timescale

37 37 What does ppm mass measurement mean? ppm errorMass error (Da)Detectable Modifications Da Deamidation, Disulfide reduction Da Deamidation, disulfide reduction DaDisulfide reduction 201 DaDisulfide reduction Da Da Da--- At 5 ppm mass tolerance for a 50 kDa protein, we can detect: Deamidation of the IgG heavy chain The reduction of a single disulfide bond on a 50 kDa protein. Any amino acid substitution (except for Q->K) Heavy Chain, 50 kDa

38 38 Accurate MW Determination of reduced IgG light chain m/z Xtract deconvolution Measured mass = Target mass = Dalton Mass Deviation  2 S-S? How do we confirm this? 240,000 resolution Shiaw-Lin Wu, Barry Karger, Barnett Institute, Northeastern University IgG light chain 18+ charge state

39 39 “Top Down” vs. “Bottom Up” protein analysis Proteins are usually digested with a proteolytic enzyme and analyzed using peptide mass fingerprinting or data dependent MS/MS Peptide mass fingerprinting has some disadvantages, including introduction of artifacts into the sample and there is no guarantee of 100% sequence coverage An alternative strategy is to use a “top down” methodology, where the intact protein is isolated and fragmented in the mass spectrometer using either a data-dependent or targeted acquisition method High resolution mass spectrometry is a must References: Kelleher et al, “Top Down versus Bottom Up Protein Characterization by Tandem High Resolution Mass Spectrometry”, J. Am. Chem. Soc., 1999, 21, pp Bondarenko et al, “Mass Measurement and Top-Down HPLC/MS Analysis of Intact Monoclonal Antibodies on a Hybrid Linear Quadrupole Ion Trap-Orbitrap Mass Spectrometer”, J. Am. Soc. Mass Spectrom., 2009, 20, pp

40 40 Top-Down Analysis of mAb Light Chain – Electron Transfer Dissociation (ETD) m/z z= z= z= z= z= z=8 Xtract produces >200 mass values ProSightPC TM search results in unambiguous identification of the IgG light chain (E-value = 1.4e-16) Fragments detected between the two internal cysteines:

41 41 Top-Down Analysis of mAb Light Chain – HCD Xtract produces >500 mass values ProSightPC TM search results in unambiguous identification of the IgG light chain (E-value = 1.4e-26) Fragments are detected between the two internal cysteines and at the termini up to the first cysteine m/z

42 42 Top Down Fragment Map for IgG light chain identifies and localizes disulfide bonds 21 pairs of complementary fragments confirm 100% sequence coverage Combined ETD and HCD results produced fragments at 53 backbone cleavage sites, 13 more than HCD or ETD alone No fragments are identified between the disulfide-bound cysteines due to cyclization

43 43 Summary – Orbitrap Elite The Orbitrap Elite is well suited for both intact protein confirmation as well as top down protein characterization Top down protein characterization is an alternative to bottom-up peptide MS/MS for identification and confirmation of expected and unexpected changes to the target protein

44 44 Summary and Conclusions Orbitrap-based systems are excellent for biopharmaceutical characterization The Q Exactive and Orbitrap Elite are the best Orbitrap systems yet for intact biopharmaceutical analysis Protein Deconvolution 1.0 produces highly accurate confident intact protein masses and abundances ProSightPC is applicable to biopharmaceutical applications (not just top down proteomics) High confidence results allow scientists in biopharmaceutical labs to increase sample throughput by bypassing more time-consuming experiments

45 45 Thermo Scientific BioPharma Capabilities Unregulated Regulated ResearchDiscoveryDMPKQC/QA Sample Analysis Sample Preparation Data Interpretation The most complete portfolio of BioPharma solutions

46 46 Acknowledgements Marketing Andreas Hühmer Thomas Moehring Markus Kellmann Yi Zhang Zhiqi Hao Seema Sharma Rosa Viner Vlad Zabrouskov Amy Zumwalt Shannon Eliuk Reiko Kiyonami Justin Blethrow Julian Saba Scott Peterman Software R&D Steve Chaput Doug Miller Tom McClure Grace Li Paul Gazis Helen Tran Shijun Li Barbara Gibson Hardware R&D Alexander Makarov Jae Schwartz Martin Zeller

47 47 Thank you—Q&A Partners in driving value creation


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