Thank you for joining us! The Webinar will begin shortly Peptide Mapping 101: Essential Tools for Effective Development and Characterization Part 1:Introduction to Peptide Mapping Stephan M. Koza, Ph. D. Principal Applications Chemist Waters Technologies Corporation 2013 Waters Corporation 1
Friendly Reminders We will have LIVE Technical Support available to address your questions. Please use text chat functionality to submit questions during the Webinar. Upon conclusion, follow up information will be available: http://www.waters.com/aug20 Recorded version of today s presentation Copies of today s slides Product specific discount offers Product specific information Categorized reference materials 2013 Waters Corporation 2
Agenda What is Peptide Mapping and Why Do It? Protein Digestion Peptide Separations 2013 Waters Corporation 3
What is Peptide Mapping? For biotherapeutic proteins and peptides peptide mapping is: The chemical or enzymatic treatment of a protein to produce peptide fragments Separation and identification of these fragments in a reproducible manner In-depth analysis that can identify minor and even isobaric differences in protein primary structure such as errors in the transcription of complementary DNA, point mutations., and PTMs (CQAs) Due to the complexity and inherent variability of the method peptide mapping is generally a comparative procedure where the peptide map of the test sample is compared to that of a reference substance prepared in a side-by-side experiment. 2013 Waters Corporation 4
Uses of Peptide Mapping Proteomics Studies Protein Biopharmaceutical Analysis Structural characterization o Pattern conforms to primary structure o Used with MS for primary structure determination o Non-Reduced Mapping for Disulfide Bond Assignment Protein modification o Identify post-translational modifications Glycosylation, substitution, truncation o Determine product related impurities: deamidation, oxidation, etc. o Characterization of variants observed in other methods (IEX, SEC) Protein identity o Confirm presence of signature peptides o Product integrity lot-to-lot analysis 2013 Waters Corporation 5
Biopharmaceutical Classes That Use Peptide Mapping Methods Peptides/Proteins derived through recombinant DNA-based processes Insulin Diabetes Erythropoietin Cancer Monoclonal antibodies derived by recombinant DNA processes, and their derivatives o Herceptin Cancer Protein Conjugates ADC PEGylated proteins Synthetic peptides Oligonucleotides/siRNA Vaccines Gene therapy Cells/Stem cells 2013 Waters Corporation 6
Why Do We Develop Peptide Maps for Biotherapeutic Proteins? Guidance for Industry Q6B Specifications: Test Procedures and Acceptance Criteria for Biotechnological/Biological Products 1. Structural Characterization and Confirmation (6.1.1) d. Peptide map Selective fragmentation of the product into discrete peptides is performed using suitable enzymes or chemicals.peptide mapping of the drug substance or drug product using an appropriately validated procedure is a method that is frequently used to confirm desired product structure for lot release purposes. 2013 Waters Corporation 7
Agenda What is Peptide Mapping and Why Do It? Protein Digestion Peptide Separations On to Part II 2013 Waters Corporation 8
Enzymes and Chemistries for Protein Digestion EUROPEAN PHARMACOPOEIA 5.0, 2.2.55. PEPTIDE MAPPING 2013 Waters Corporation 9
Enzymes and Chemistries for Protein Digestion EUROPEAN PHARMACOPOEIA 5.0, 2.2.55. PEPTIDE MAPPING Trypsin, Lys-C,and Asp-N are most commonly used and can provide high fidelity digestions for reproducible peptide maps. 2013 Waters Corporation 10
In silico Digestion Tools for Selecting an Enzyme (or Chemical):MassLynx Protein/Peptide Editor 2013 Waters Corporation 11
In silico Digestion Tools for Selecting an Enzyme (or Chemical) Trypsin results in 2 amino acids and 1 di-peptide, Lys-C might be a better choice as it generates 3 manageable peptides Further digestion would be needed to assign disulfide bonds in this example 2013 Waters Corporation 12
Flow Chart of Peptide Mapping Protein (e.g. antibody) Denaturation, Disulfide Reduction/Alkylation, Buffer Exchange Enzymatic Digestion (e.g. Trypsin) Peptide Map Analysis UPLC/ UV UPLC/ MS UPLC/ MS/MS UPLC/ MS E 2013 Waters Corporation 13
What s RapiGest SF Anionic detergent that improves solubility and digestion of many proteins for improved enzymatic digests. Unlike conventional denaturants, RapiGest SF does not inhibit enzyme activities so it can reduce digestion times and reduces the amount of enzyme used. It does not cause protein modifications (e.g., urea causing carbamylation) unlike some other protein denaturants. It s an acid labile surfactant whose degradation products do not interfere with LC/MS or MALDI MS analysis. 2013 Waters Corporation 14
Reproducible Peptide Mapping Pitfalls of Peptide Mapping that can affect robustness, reproducibility and accuracy: Sample preparation o Incomplete digestion o Non-reproducible digestion conditions o Non-specific cleavages (over-digestion) o Enzyme lot-to-lot variability (activity units or mass?) Non-reproducible chromatography It is critical that SOPs be written clearly and transferred precisely in order for peptide maps to be reproducible between different labs or even analysts Preparing a blank digest is always recommended for troubleshooting purposes 2013 Waters Corporation 15
Agenda What is Peptide Mapping and Why Do It? Protein Digestion Peptide Separations On to Part II 2013 Waters Corporation 16
Peptide Separations Column Selection Ethylene Bridged Hybrid (BEH) Particle Technology UPLC vs HPLC Charged Surface Hybrid Technology Fine Tuning Your Separation 2013 Waters Corporation 17
Ethylene Bridged Hybrid - BEH Technology U.S. Patent No. 6,686,035 B2 and others patent pending Bridged Ethanes In Silica Matrix Organo Silica Hybrid Particles ph stability Reduced ionic interactions Basis of Peptide Separation Technology EtO CH 2 CH 2 OEt OEt O Si Si Si O O Si O O Si O O Si O EtO OEt OEt Polyethoxysilane Et Et n EtO EtO OEt 4 EtO CH 2 Si + Si EtO OEt CH Si OEt 2 EtO EtO OEt Tetraethoxysilane Bis(triethoxysilyl)ethane 2013 Waters Corporation Anal. Chem. 2003, 75, 6781-6788 18
Small Particle Size Mobile Phase Peptides 1500 Da Peptide 2 3.5 µm Porous Particle Diffusion-related band broadening Adsorption Equilibria H (mm) 1 Diffusion distances decrease Reduced Eddy diffusion, A-Term Improved mass transfer kinetics, C-Term 1.7 µm Column efficiency Narrower peaks 40 µl/min 2.1 mm ID 400 µl/min 0 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 Velocity (mm/sec) 2013 Waters Corporation 19
Why UPLC for peptide mapping More resolution even using a shorter gradient 7.0e-2 6.0e-2 HPLC 2.1 x 300 mm, 3.5 µ 90 min 5.0e-2 AU 4.0e-2 3.0e-2 2.0e-2 1.0e-2 Time 30.00 35.00 40.00 45.00 50.00 55.00 60.00 65.00 70.00 75.00 80.00 85.00 90.00 1.0e-1 9.0e-2 UPLC 2.1 x 150 mm, 1.7 µ 55 min 8.0e-2 7.0e-2 AU 6.0e-2 5.0e-2 4.0e-2 3.0e-2 2.0e-2 Time 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00 42.00 44.00 46.00 48.00 50.00 52.00 54.00 56.00 2013 Waters Corporation 20
Charged Surface Hybrid (CSH) Technology patent pending Charged Surface Hybrid (CSH) Technology and Its Use in Liquid Chromatography. P.C. Iraneta, K.D. Wyndham, D.R. McCabe, and T.H. Walter Waters White Paper 720003929EN 2011 Expands upon the robust BEH particle technology CSH130 C18 = BEH130 base particle + low level of basic moieties + trifunctional C18/end cap Acidic ph Positive Surface Charge Peptide 2013 Waters Corporation 21
Peak Capacity Peak Capacity = The number of peaks that can be separated within a retention window Neue, U. D., J Chromatogr A 2005, 1079 (1-2), 153-61. The best metric for determining the quality of gradient separations 100% 9 peaks could resolve ~300-400 Peak Height 50% 2.35σ w h 13.4% 4σ w 4σ 0% t gradient 2013 Waters Corporation 22
A Novel Column Chemistry: CSH130 C18 (0.1% TFA) BEH130 C18 Competitor s Industry Standard C18 Porous (130Å) 1.7 µm 2.1 x 150 mm UV absorbance (214 nm) Porous (300Å) 5 µm 2.1 x 250 mm 10 10 50 10 Time (min) 20 30 Time (min) 40 50 50 CSH130 C18 Competitor s Superficially Porous Peptide C18 Porous (130Å) 1.7 µm 2.1 x 150 mm SPP (100Å) 1.7 µm 2.1 x 150 mm 1 10 50 Time (min) 2013 Waters Corporation 2 50 10 Time (min) 23
Peak Capacity - FA vs TFA 370 320 270 P c,4σ 220 170 120 Competitor s Industry Standard Silica C18 5 µm 2.1 x 250 mm 70 20 0.00 0.05 0.10 FA TFA Percent TFA % TFA 0.00 0.05 0.10 % FA 0.10 0.05 0.00 2013 Waters Corporation 24
Peak Capacity - FA vs TFA 370 320 270 BEH130 C18 1.7 µm 2.1 x 150 mm P c,4σ 220 170 120 Competitor s Industry Standard Silica C18 5 µm 2.1 x 250 mm 70 20 0.00 0.05 0.10 FA TFA Percent TFA % TFA 0.00 0.05 0.10 % FA 0.10 0.05 0.00 2013 Waters Corporation 25
Peak Capacity - FA vs TFA 370 320 270 Competitor s SPP Peptide C18 1.7 µm 2.1 x 150 mm BEH130 C18 1.7 µm 2.1 x 150 mm P c,4σ 220 170 120 Competitor s Industry Standard Silica C18 5 µm 2.1 x 250 mm 70 20 0.00 0.05 0.10 FA TFA Percent TFA % TFA 0.00 0.05 0.10 % FA 0.10 0.05 0.00 2013 Waters Corporation 26
Peak Capacity - FA vs TFA 370 CSH130 C18 1.7 µm 2.1 x 150 mm 320 270 90% Competitor s SPP Peptide C18 1.7 µm 2.1 x 150 mm BEH130 C18 1.7 µm 2.1 x 150 mm 20% P c,4σ 220 170 120 Competitor s Industry Standard Silica C18 5 µm 2.1 x 250 mm 70 20 0.00 0.05 0.10 FA TFA Percent TFA % TFA 0.00 0.05 0.10 % FA 0.10 0.05 0.00 2013 Waters Corporation 27
Loadability Attribute how much analyte can be loaded before peak shape deteriorates CSH C18 BEH C18 450 400 B Low Mass Load 0.6 µg 0.6 of Low µg mixture Mass of Load mixture (Equivalent to ~ 4.5 µg of a mab) CSH C18 CSH130 BEH C18 1.7 µm 450 400 A Typical Mass Load 6 µg 6 µg of mixture High of Mass mixture Load (Equivalent to ~ 45 µg of a mab) CSH C18 BEH C18 CSH130 C18 1.7 µm 350 BEH130 C18 1.7 µm 350 P c,4σ 300 P c,4σ 300 BEH130 C18 1.7 µm P c,4σ 250 250 200 200 *Previously shown 0.10 0.00 % TFA % FA 150 0.00 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09 TFA 0.10 0.10 FA Percent 0.05 TFA 0.00 0.00 0.05 0.10 0.10 0.05 0.00 150 % TFA 0.00 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09 TFA0.10 % FA 0.10 FA Percent 0.05TFA 0.00 % TFA 0.00 0.05 0.10 % FA 0.10 0.05 0.00 2013 Waters Corporation 28
Which Column do I choose CSH130 C18 or BEH130 C18? Intensity 2E+6 1E+6 0.1% FA BEH130 C18 C18 1.7 1.7 µm µm P c,4σ = P c,4σ 399= 399 0E+0 0 10 20 30 40 50 60 Intensity 2E+6 1E+6 0 10 20 30 40 50 60 Time(min) CSH130 C18 C18 1.7 1.7 µm µm P c,4σ P= c,4σ 532 = 532 0E+0 0 10 20 30 40 50 60 Time(min) 2013 Waters Corporation 29
LC-MS Retention and Selectivity BEH130 C18 1E+6 T3 SVYDSR T5 GVFR T12 ANIDVK T19 HLADSK More positive T5/ T10 GVLHAVK T12 T19 charge T3 T10 T40 IATAIEK 0E+0 5.5 6.5 7.5 8.5 9.5 10.5 11.5 12.5 Time(min) T40 1E+6 CSH130 C18 T5/ T19 T12 T10 T3 T40 0E+0 5.5 6.5 7.5 8.5 9.5 10.5 11.5 12.5 13.5 Time(min) 2013 Waters Corporation 30
UPLC and HPLC CSH130 C18 2.1 x150 mm 1.7 µm A 214 1.0 0.8 0.6 0.4 High peak capacity separations not limited to UPLC 0.1 % FA ~8000 psi A 214 1.0 0.8 0.6 0.4 0.1 % TFA 0.2 0.2 Method Transfer 0.0 10 20 30 40 50 1.0 Time (min) 0.0 10 20 30 40 50 1.0 Time (min) 0.8 0.8 2.5 µm XP A 214 0.6 0.4 ~3000 psi A 214 0.6 0.4 Longer Run Time Lower Pressure 0.2 0.0 14.5 24.5 34.5 44.5 54.5 64.5 74.5 Time (min) 0.2 0.0 13.5 23.5 33.5 43.5 53.5 63.5 73.5 Time (min) 2013 Waters Corporation 31
UPLC and HPLC CSH130 C18 2.1 x150 mm 1.7 µm A 214 1.0 0.8 0.6 High peak capacity separations not limited to UPLC 0.1 % FA ~8000 psi 0.4 0.4 CSH130 C18 Peptide Separation Technology Columns A 214 1.0 0.8 0.6 0.1 % TFA Method Transfer 2.5 µm XP Longer Run Time Lower Pressure 0.2 Available 0.0 Now: Analytical Columns 1.0 Time (min) 1.7 µm 0.8 2.5 µm XP 0.6 3.5 µm Prep 0.4 Columns (5 µm) A 214 0.2 10 20 30 40 50 0.0 14.5 24.5 34.5 44.5 54.5 64.5 74.5 Time (min) ~3000 psi 0.2 Upcoming: 0.0 Nano (75, 150, 300 µm ID) A 214 1.0 10 20 30 40 50 Time (min) 0.8 0.6 0.4 0.2 0.0 13.5 23.5 33.5 43.5 53.5 63.5 73.5 Time (min) 2013 Waters Corporation 32
Three Outstanding Peptide Separation Technology Columns Peptide/Protein kda 1 Bradykinin 1.1 130 Å A 214 1.2 1 0.8 0.6 0.4 0.2 CSH130 C18 1.7 µm 1 2 0 1.2 10 15 20 25 30 35 40 45 50 BEH130 C18 1.7 µm Time (min) 1 3 4 5 6 2 Renin Substrate 1.8 3 Ubiquitin 8.6 4 5 Cytochrome C (Equine) Insulin (Bovine) 12.4 5.7 6 Melittin 2.8 0.8 A 214 0.6 0.4 0.2 1 2 4 3 5 6 300 Å A 214 0 1.2 10 15 20 25 30 35 40 45 50 BEH300 C18 1.7 µm Time (min) 1 2.1 x 150 mm columns 2% ACN for 1 min, 0.8 then to 50% ACN over 60 min 5 0.3 ml/min 0.6 40 C 0.4 0.2 1 2 4 3 6 ACQUITY UPLC H-Class Bio UV @ 214 nm / Xevo G2 QTOF 1 µg each component 0 10 15 20 25 30 35 40 45 50 Time (min) 2013 Waters Corporation 33
New Addition to the Suite of Waters Peptide Separation Technology Peptide Separation Technology Peptide C18 Columns QC Tested with Digests BEH Technology BEH130 C18 and BEH300 C18 Outstanding Performance for Most Applications Two Pore Sizes Particle Sizes: 1.7 µm, 3.5 µm, 5 µm Analytical, Nano and Prep Columns Now even more tools in the toolbox CSH Technology CSH130 C18 Highest peak capacities in TFA and FA mobile phases. Unique selectivity Particle Sizes: 1.7 µm, 2.5 XP, 3.5 µm, 5 µm Analytical and Prep Columns (Nano in development) 2013 Waters Corporation 34
All Waters Peptide SeparationColumns are Quality Control Tested with Tryptic Digest of Cytochrome c CSH130 C18 0.1 % Formic Acid 2013 Waters Corporation 35
Fine Tuning Your Separation Parameters that Influence Selectivity Ion Pairing Reagent (TFA, HFBA, etc.) and Concentration Organic Eluent (MeCN, MeOH, IPA) Column Temperature Gradient Slope/Column Length Peak Tracking Ideally using LC-MS can expedite separation optimization Make several incremental changes Peak areas and A280/A214 UV absorbance ratios 2013 Waters Corporation 36
Method Optimization: Gradient Slope Rate of Change 0.75%/ col. vol. % * * * 1 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 % * * Rate of Change 1.5%/ col. vol. * 1 Time 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 2013 Waters Corporation 37
Why Does This Switch in Elution Order Occur? Log k Elution at Lower % MeCN w/ Shallower Gradient Elution at Higher % MeCN w/ Steeper Gradient % MeCN Adapted from: Spicer, V., Grigoryan, M., Gotfrid, A., Standing, K. G., & Krokhin, O. V. (2010). Predicting retention time shifts associated with variation of the gradient slope in peptide RP-HPLC. Analytical chemistry, 82(23), 9678-9685. 2013 Waters Corporation 38
Gradient Slope and Segmented Gradients Changes in gradient slope should occur in regions of separation where there are no peaks of interest Potential selectivity differences should be tracked Approach could also be used to generate a focused gradient if only specific peptides are of interest 2013 Waters Corporation 39
CSH130 C18 Useful Current Literature/Resources http://pubs.acs.org/doi/abs/10.1021/ac401481z Previously recorded webinar available: http://www.waters.com/waters/promotiondetail.htm?id=134727909 Recent Application Notes Increasing Peak Capacity in Reversed Phase Peptide Separations with Charged Surface Hybrid (CSH) C18 Columns M.A. Lauber, S.M. Koza, K.J. Fountain Waters Application Note 720004568EN 2013 Peptide Mapping and Small Protein Separations with Charged Surface Hybrid (CSH) C18 and TFA-Free Mobile Phases M.A. Lauber, S.M. Koza, K.J. Fountain Waters Application Note 720004571EN 2013 High Mass Loading of Peptides with Hybrid Particle C18 Columns and Acetic Acid Mobile Phases M.A. Lauber, S.M. Koza, K.J. Fountain Waters Application Note 720004674EN 2013 2013 Waters Corporation 40
End of Part 1 2013 Waters Corporation 41
Part 2 - Gaining Efficiency: Instrumentation and Informatics Platforms for Peptide Mapping Asish Chakraborty, Ph.D Asish_Chakraborty@waters.com 2013 Waters Corporation 42
A History of Relieving the Pressure on Analysts Sample Generation Sample Preparation Acquisition Data Analysis Report Generation Chemistries Sample Generation Instrumentation and Automation Data Analysis Informatics Report Generation Sample Generation Report Generation Sample Generation Now It Becomes Routine 2013 Waters Corporation 43
Performance and Usability through Engineered Simplicity Automatically ensuring the system is ready to run 2013 Waters Corporation 44
Automating batch processing, annotatation, and comparison tools in BiopharmaLynx TM increases productivity First shown at WCBP 2007 Meeting 2013 Waters Corporation 45
Biopharmaceutical Platform Solution with UNIFI 1.7 An analytical system for biotherapeutic analysis integrating UPLC/UV and UPLC/MS Intact Protein Mass Peptide Mapping Biopharmaceutical Platform Solution DDA (Peptide & Glycan) Released Glycan GU + Mass Xevo G2-S QTof ACQUITY UPLC H-Class & H-class BIO Bioseparations Size Exclusion (UV) Intact Protein: Peptide Mapping: Released Glycan: Bioseparations: TUV, MS TUV, MSE, MS/MS FLR (+MS, NIBRT Library), MS/MS TUV, FLR Workstation or Workgroup (Compliance) 2013 Waters Corporation 46
Deploy high resolution analytics across a biotherapeutic organization Few compliance issues GxP Labs Regulatory Compliance Discovery Development Production QC/QA Post-Approval Characterization Monitoring Release 2011 Waters Corporation 47
The Biopharmaceutical Workgroup Office PC Office PC Intact Mass, Peptide Mapping Lab PC UPLC-TUV- Xevo G2-S Released Glycan Analysis Lab PC UPLC-FLR- Xevo G2-S Office PC Lab Network Device (LND) LND LABORATORY NETWORK Lab PC LND Lab PC LND UPLC-TUV UPLC-TUV UPLC-FLR UPLC-FLR Data Processing & Database Storage Server Bioseparations 2013 Waters Corporation 48
UNIFI Meets the Biopharmaceutical industry s Global reach A Scientific management system for the global nature of the Biopharmaceutical business 2013 Waters Corporation 49
Peptide Mapping in UNIFI TM Advanced Reporting Capabilities in a GxP-ready Environment 2013 Waters Corporation 50
Experimental Setup for Peptide Mapping LC/MS E Reduced Peptide Map Reduction & Alkylation Therapeutic Proteins Denature & Alkylate LC/MS E Non-Reduced Peptide Map Trypsin Digest UNIFI Scientific Information System UPLC BEH300 C18, 1.7 µm, 2.1 x 100 mm 2011 Waters Corporation 51
UPLC/MS E Comprehensively Analyzes Complex Samples UPLC/MS E is a simple method of unbiased data acquisition that comprehensively analyzes all components in a single analysis. 2011 Waters Corporation 52
Surveying Chromatography and Complexity in Peptide Mapping Data Chromatogram with Peak Assignments 2013 Waters Corporation 3D Chromatogram 53
Peptide Mapping Data Assignments Annotated Chromatograms Fragment ions Spectrum 2013 Waters Corporation 54
Peptide Mapping Data Data Table (linked to Coverage Map) Assignments Coverage Map Fragment ions Spectra 2013 Waters Corporation 55
Access to both raw and processed data 2013 Waters Corporation 56
Case Study 1 2011 Waters Corporation 57
Experimental Setup for Peptide Mapping LC/MS E Reduced Peptide Map Reduction & Alkylation Therapeutic Proteins Denature & Alkylate LC/MS E Non-Reduced Peptide Map Trypsin Digest UNIFI Scientific Information System UPLC BEH300 C18, 1.7 µm, 2.1 x 100 mm 2011 Waters Corporation 58
Equivalent protein coverage was obtained for innovator and biosimilar Innovator Innovator HC Biosimilar Biosimilar HC Innovator LC Biosimilar LC BEH, C18, 1.7 µm, 130, 2.1x 100 mm, Gradient 1 to 35% ACN, 0.05%TFA, 60 min 2011 Waters Corporation 59
Asp Isomerization of Peptide T24 (FNWYVDGVEVHNAK) XIC Iso ASP Innovator Biosimilar Isomerization: Asp to iso-asp (no mass difference). isoasp is not a natural amino acid and can potentially be immunogenic. 2013 Waters Corporation 60
Oxidation of HC Peptide T42 Biosimilar Batch I % Oxidation Innovator Batch I Sample Injections 2013 Waters Corporation 61
Peptide Maps Report: Unifi enables researchers to focus on critical attributes of a molecule Analysis Information LC Coverage HC Coverage % Oxidation Biosimilar Batch I Innovator Batch I 2011 Waters Corporation 62
Experimental setup for disulfide bond mapping LC/MS E Reduced Peptide Map Reduction & Alkylation Therapeutic Proteins Denature & Alkylate LC/MS E Non-Reduced Peptide Map Trypsin Digest UNIFI Scientific Information System UPLC BEH300 C18, 1.7 µm, 2.1 x 150 mm 2011 Waters Corporation 63
Expected disulfide bonds in IgG1 Antibody Trypsin Digest Heavy chain IgG1 mab contains 16 S-S bonds (12 intra, and 4 inter) Light chain S - S Light Chain (1) S - S VH C H 1 S - S CHO Heavy Chain (2) S - S S-S S-S S-S -S-S- S-S CH2 CH3 S S S S S S S S S-S S- S S- S Humanized IgG K K C L S- S CHO S- S V L Light Chain (4) Heavy Chain (3) Digestion Enzyme: Trypsin Symmetry of IgG1 molecule provides redundancy in mass-based search 8 unique S-S bonded peptides LC: 2 Intra, HC: 4 Intra, HC-HC(Hinge): 1 inter HC-LC:1 inter 2013 Waters Corporation 64
Nonreduced peptide mapping enabled ID of all canonical S-S S peptides A simple filter to only display disulfide containing peptides Disulfide Containing Peptides 2:T21-3:T21 Additional studies show there are no scrambled disulfide presence 2:T21-3:T21 MS E Fragment Ions 2:T21-3:T21 2:T21-3:T21 2:T21-3:T21 UNIFI enables researchers to focus on critical attributes of a molecule 2013 Waters Corporation 65
Disulfide Bonds Report: Unifi enables researchers to focus on critical attributes of a molecule Component Plot for S-S peptides Analysis Information KK Disulfide containing peptides identified in both innovator and biosimilar mab samples Component Summary 2011 Waters Corporation 66
Case Study 2 2013 Waters Corporation 67
Automated Processing and Reporting with UNIFI : Intact Protein Analysis INTACT PROTEIN ANALYSIS MaxEnt1 deconvoluted mass spectra in compare mode G0F/G0F G0F/G1F G1F/G1F G0F/G2F Innovator G0F/G0F G0F/G1F G1F/G1F G0F/G2F Innovator G1F/G2F G1F/G2F G0/G0F G2F/G2F G0/G0F G2F/G2F Biosimilar 1 Biosimilar 2 m = 56 Da Biosimilar 1 glycoforms broadly match the innovator Biosimilar 2 glycoforms have a systematic mass shift of 56 Da compared to innovator mab Discrepancy needs to be explained UNIFI workflow automatically acquires, processes and reports the intact mass Deconvolution with MaxEnt Reporting with Flexible templates Ivleva et al Poster - ASMS 2012; 2011 Waters Corporation http://www.waters.com/webassets/cms/library/docs/2012asms_ivleva_rituximab.pdf 68
Reduced Protein Analysis LCs identical Biosimilar 2 LC w/ PyQ HC Reduction + Biosimilar 1 Mass Analysis of the Light Chain Light Chain Innovator MaxEnt1 deconvoluted mass spectra in compare mode LC Light Chain Innovator Innovator Biosimilar 1 Biosimilar 2 Automated Processing and Reporting Light chain masses are identical for Innovator, Biosimilar 1 and Biosimilar 2 2011 Waters Corporation 69
Reduced Protein Analysis of Heavy Chain G0F G1F MS Response Biosimilar 1 G0 G0 G0F+K Innovator G2F G1F+K 7.00E+06 6.00E+06 5.00E+06 4.00E+06 3.00E+06 Innovator Quantification of C-terminal Lys Variation G0 G0F G0F+K G1F Biosimilar 1 Innovator G2F G1F+K Summary plots Based on UNIFI results 2.00E+06 1.00E+06 0.00E+00 MS Response 1.20E+07 1.00E+07 8.00E+06 6.00E+06 Biosimilar 2 1 2 3 4 5 6 7 8 9 Distribution of G0 Glycoform Biosimilar 2 G0 G1F Biosimilar 2 4.00E+06 2.00E+06 Innovator Biosimilar 1 G0F G0F m = 28 Da 0.00E+00 Automated Processing and Reporting 1 2 3 4 5 6 7 8 9 Detailed Information automatically reported in UNIFI Multiple aspects available from the dataset Response for each batch of each protein measured and compared 2011 Waters Corporation 70
LC/MS E Tryptic peptide mapping to locate the sequence variance Tryptic Digest comparison between Innovator and Biosimilar 2 does not show sequence differences in Light Chain Light Chain - Innovator Compare mode Biosimilar 2 Coverage Map Automated Reporting 2013 Waters Corporation 71
LC/MS E Tryptic peptide mapping to locate the sequence variance Compare mode Heavy Chain - Innovator HC - Biosimilar 2 Biosimilar Coverage Map: shows section where no sequence match is made 2013 Waters Corporation 72
LC/MS E Tryptic peptide mapping to locate the sequence variance Compare mode Heavy Chain - Innovator Biosimilar 2 Coverage Map Alternative Enzyme to Trypsin needed to ascertain if there is a different sequence here in Biosimilar 2 2013 Waters Corporation 73
LC/MS E Chymotryptic peptide mapping analysis of Biosimilar 2 Innovator Innovator Biosimilar 2 BPI Peptide Map Biosimilar 2 m = 28 Da HC Coverage Map Chymotryptic Digest used to reveal differences Peptides are highlighted in the coverage map as each is selected by the user Amino Acid Substitution can be identified 2011 Waters Corporation 74
UNIFI Peptide Map Workflow: MS E data confirming the sequence Variant K 218 R 218 Automated fragment information from MS E data MS E Spectrum of chymotryptic digest confirms amino acid substitution K for R at position 218 2011 Waters Corporation 75
Summary of the Structural Analysis of Rituximab by LC/MS Approach The differences between Innovator and Biosimilars Rituximab candidates are: Biosimilar 1 vs. Innovator o Same AA Sequences o Higher percentage of C- terminal variation o Increased G0 glycoform o Different percentage of pyroglutamation at the N- termini of both LC and HC Biosimilar 2 vs. Innovator o Sequence Variant in HC, K 218 > R 218 o Lower percentage of C-terminal Lys variation o Much higher percentage of G0 o Different percentage of pyroglutamation at the N-termini of both LC and HC 2011 Waters Corporation 76
Summary Intact Mass Analysis Reduced Peptide Mapping Glycan Analysis Reduced LC Mass Analysis Non-Reduced Peptide Mapping Aggregate Analysis Reduced HC Mass Analysis Charge variant Analysis 2013 Waters Corporation 77
2013 Waters Biopharmaceutical Application Notebook www.waters.com/ biopharmbook 2013 Waters Corporation 78
Thank You! Questions? Landing Page http://www.waters.com/aug20 Promotional Discount Offers on Peptide Separation Columns PDF Slide Deck Full Webinar Recording of Today s Session Compilation of Literature, White Papers, Brochures General Questions email: mychemrep@waters.com 2013 Waters Corporation 79