Characterization of the Higher-Order Structure of Biotherapeutics Biopharmaceutical Sciences Group Waters Corporation 2011 Waters Corporation 1
FDA desire for specific innovations in biotherapeutic analysis Steven Kozlowski, M.D. Director, Office of Biotechnology Products Office of Pharmaceutical Science Center for Drug Evaluation and Research Food and Drug Administration Three-dimensional Structure As described previously, proteins must be folded into a threedimensional structure to become functional (sometimes a threedimensional structure can be misfolded). The proteins within a biologic will have one major three-dimensional structure along with a distribution of other variants differing in three-dimensional structure. Our current ability to predict the potency of biologics would be enhanced if we had improved ability to measure and quantify the correct (major) three-dimensional structure, aberrant three-dimensional structures (misfolding), and the distribution of different threedimensional structures. TESTIMONY BEFORE THE SUBCOMMITTEE ON TECHNOLOGY AND INNOVATION COMMITTEE ON SCIENCE AND TECHNOLOGY U.S. HOUSE OF REPRESENTATIVES SEPTEMBER 24, 2009 2011 Waters Corporation 2
Answering Structural Questions with Advanced MS workflows 3+ 637 Å 2 3+ 6+ Drift 6+ 1231 Å 2 6+ Ion Mobility-TofMS Measure average cross-sectional area (Size/Shape) of proteins in gas-phase Detect multimeric and conformational variants for biotherapeutic ti proteins Resolve components at common m/z Hydrogen Deuterium Exchange MS D2O Measure incorporation of Deuterium on backbone amide groups Probe protein structure and dynamics Engen, J. R. (2009) Anal. Chem. 81(19), 7870 Assessment of structural differences 2011 Waters Corporation 3
Applying IMS and HDX to Biotherapeutic Development Questions Did my protein fold correctly? vs Can I identify sites of protein aggregation? Are there Conformational Changes after X? Formulations and Stability Testing? Did this Mutation affect protein structure? Where does my antibody bind its target? Where does the drug interact with the target protein?? Epitope mapping Do these processes make the same protein? Batch to Batch Site to Site Innovator vs. Biosimilar? Comparability 2011 Waters Corporation 4
Structural LC and LC-MS Low order to Higher order of protein structure Amino acid analysis Peptide mapping Sequence map Disulfide bonds PTM Intact protein Intact mass Top-Down sequence SEC, IEX Conformation HX MS Ion mobility 2011 Waters Corporation 5
Deuterium Exchange at Amide Hydrogen for LC/MS studies y g / R 1 Peptide bond NH + 3 C CO NH C CO NH C CO R 2 Peptide bond R 3 Back Exchange too fast for detection in seconds/ minutes H H H No exchange Amide H D D 2 O (solution) H s H s & D s at backbone amide positions at backbone amide positions - Measure mass difference to determine uptake Picture Credit: Engen, J. R. (2009) Anal. Chem. 81(19), 7870 2011 Waters Corporation 6
Waters HDX Workflow at the Intact Protein Level ive Deuterium Level (Da) Relat 140 120 100 80 60 40 20 Intact CaM, 10s, 1m, 5m, 10m, 60m Labeling 0 APO HOLO 0 1 10 100 1000 Time (min) in Logarithmic scale Protein in H 2 O, ph 7.40 at room temp Add 20 fold excess D 2 O Protein labeling occurs For various times Quench ph to 2.50, temp to 0 C OPTIONAL AUTOMATION Quenched protein is injected into HDX manager at ph 2.5 Inside HDX Manager at 0 C Global Analysis at Intact Level nanoacquity UPLC for fast desalting/protein separation Deuterium uptake determination ESI Q Tof MS 2011 Waters Corporation 7
Compare Different States over Time Normal Disease 2011 Waters Corporation 8
Compare Different States over Time Normal Disease Deut terium Uptake 180 160 140 120 100 80 0 50 100 150 Time (min) Normal Disease 2011 Waters Corporation 9
Compare Different States over Time Normal Disease Deut terium Uptake 180 160 140 120 100 80 0 50 100 150 Time (min) Normal Disease 2011 Waters Corporation 10
Compare Different States over Time Normal Disease Deut terium Uptake 180 160 140 120 100 80 0 50 100 150 Time (min) Normal Disease 2011 Waters Corporation 11
Waters HDX Workflow at the Peptide Level Relative Deuterium Level (D Da) peptide 128-140 12 APO HOLO 10 8 6 4 2 0 R0.10 010 100 1.00 10.0000 100.00 1000.00 Time (min) Undeuterated / H2O Deuterated / D2O Protein in H 2 O, ph 7.40 at room temp Add 20 fold excess D 2 O Protein labeling occurs For various times Quench ph to 2.50, temp to 0 C OPTIONAL AUTOMATION Inside HDX Manager at 0 C Quenched protein is injected into HDX manager at ph 2.5 Local Analysis at Peptide Level Online Pepsin Digestion ph 2.50 nanoacquity UPLC using 1.7 µm BEH130 C18 column for fast separation Peptide map PLGS and IDENTITY E for peptide ID ESI Q-Tof MS E Or ESI HDMS E Deuterium Uptake DynamX Processing Determination 2011 Waters Corporation 12
Measurement of average mass change over time Labeling time 240 m 60 m 100 % 100 0 3.00 4.00 5.00 6.00 7.00 8.00 % 0 3.00 4.00 5.00 6.00 7.00 8.00 100 Uptake of deuterium from 0 solution for different regions 100 vary according to time % % 0 10 m 1 m 100 % 0 300 3.00 400 4.00 500 5.00 600 6.00 700 7.00 800 8.00 100 % 0 3.00 4.00 5.00 6.00 7.00 8.00 100 % 0 100 % 0 Changes in mass (distribution) correlated to conformational changes 100 100 10 s % 0 3.00 4.00 5.00 6.00 7.00 8.00 % 0 0 s 100 % 0 Time 3.00 4.00 5.00 6.00 7.00 8.00 100 % 0 m/z 600 605 610 2011 Waters Corporation 13
Waters nanoacquity UPLC with HDX Technology gy NEW module for nanoacquity UPLC Module designed to maintain fluidics and columns at 0 degrees C Exclusively for use with nanoacquity and G2 mass spectrometers MS E enabled systems 2011 Waters Corporation 14
Waters nanoacquity UPLC with HDX Technology UPLC provides major advantages for HDX studies: Exceptional Reproducibility Minimize back-exchange by operating at 0 CC Rapid separations with high resolution to reduce time spent on column HPLC Example shown is a 52 kda protein digested with pepsin at physiological ph UPLC High-Speed and High-Resolution UPLC Separation at Zero Degrees Celsius; Anal. Chem. 2008, 80, 6815 6820; Thomas E. Wales et al; 10.1021/ac8008862 2011 Waters Corporation 15
Calmodulin (CaM) conformational change upon calcium binding Apo : calcium free Protein folds Holo : calcium bound Control peptide #43 (128-140AA) 10 sec. D 2 O labeling 33 % exchanged 6 % exchanged 0 10 20 30 40 50 60 70 80 90 100 % Undetermined Regions mapped onto 3D representation with colour code for percentage uptake Time course plots reveal which regions are more exposed to exchange Apo form reveals significant change in uptake compared to Holo form for region identified by AA residues 128-140140 (colour-coded coded on 3D view) Boxed region shows close-up of relative differences between Apo (unbound Ca) and Holo (bound Ca) 2011 Waters Corporation 16
Complex HDX Data can be Resolved by enabling Ion Mobility Iacob et al; Ion Mobility adds an additional dimension to mass spectrometric analysis of solutionphase hydrogen/ deuterium exchange; Rapid Commun. Mass Spectrom. 2008; 22: 2898 2904 2011 Waters Corporation 17
IMS separates co-eluting labeled peptides p UCA064_100901_077_bsa_2m 287 (3.504) Cm (278:306) 100 1: TOF MS ES+ 9.08e4 2 m Labeling No IMS separation % 0 m/z 709 710 711 712 713 714 715 716 717 718 719 Ion Mobility Enabled p p ( p) UCA064_100901_077_bsa_2m_rt_04_JA2 291 (3.567) Cm (278:294) 1: TOF MS ES+ 100 3.99e4 ASIQKFGERALKA 2+ % 2+ 1+ with IMS-enabled separation 0 m/z 709 710 711 712 713 714 715 716 717 718 719 UCA064_100901_077_bsa_2m_rt_04_JA 284 (3.472) Cm (278:294) 1: TOF MS ES+ 100 3.42e3 % 0 AVEGPKL 1+ m/z 709 710 711 712 713 714 715 716 717 718 719 2011 Waters Corporation 18
Waters HDX software: DynamX TM Automated uptake calculation Easy view Convenient interpretation 2011 Waters Corporation 19
DynamX: IMS Support 100 min Time Course Changes in Uptake 10 min Peptide Identified 1 min Interfering Peptide separated by Mobility 10 sec Ref 2011 Waters Corporation 20
HDX for Comparability: Experimental Difference Chart Is my protein different from a reference compound? Greater deuteration in APO Yes. Difference chart reveals locations of different deuteration. Four regions in Apo CaM displayed greater difference. APO HOLO No difference btw Apo & Holo 2011 Waters Corporation 21
HDX for Comparability: Experimental Butterfly Chart How different are the two protein states? Butterfly Chart reveals the exchange rate and deuteration incorporation in comparison for all peptides in all time points. APO HOLO 2011 Waters Corporation 22
Waters HDX software: Efficiency of data processing 100 kda protein typically generates ~200 reproducible peptic peptides Number of spectra to process in entire HDX study was calculated to be: 200 peptides x 6 labeling time points x 2-set comparison (bound vs. unbound) x 2 (N=2 duplicates) = 4800 spectra to determine deuterium uptake Manual Manual Calculation of deuterium uptake Semi- Automated Help via export to Excel Macro etc. Fully Automated DynamX Full Month 2 3 Weeks 1-4 Days data processing time 2011 Waters Corporation 23
nanoacquity UPLC System with HDX Automation Independent temperature zones for automated sampling processing HDX manager 2011 Waters Corporation 24
Ion Mobility and Higher Order Structure 2011 Waters Corporation 25
Synapt G2: A unique analytical platform for MS and HDMS (Ion Mobility MS) studies 2011 Waters Corporation 26
Engineering Ion Mobility within a QTof for protein structural analysis 2011 Waters Corporation 27
Obtaining protein cross-section values from calibrated IMS analyses Basic mobility equation is based on free drift (Inefficient) Traveling wave facilitates ion mobility and permits flexible tuning of mobility parameters IMS Calibration with known ion cross sections allows precise determinations for unknown protein ions An automated procedure has been developed to facilitate this calibrated measurement 2011 Waters Corporation 28
CALIBRATION: Ion Mobility drift (arrival) time for sperm whale myoglobin charge states p y g g Protein with Known Collision Cross Sectional Areas at various charge states plotted against Arrival time/ Mobility All published data from http://www.indiana.edu/~clemmer/ 2011 Waters Corporation 29
VALIDATION: Cross-sections for cytochrome c charge states vs. published data p 4000 Difference 2.0% Cross Sectio ons (Å 2 ) 3000 2000 Published Cross-sections Estimated Cross-sections 1000 6 8 10 12 14 16 18 Charge State Check Validity of CCS calcuation: Collisional Cross Sectional Areas derived from Mobility plotted against charge state 2011 Waters Corporation 30
Experimentally determined cross-sections for different charge states of human insulin analogs under near physiological conditions 2000 Insulin Cross Section vs. Charge State Humulin Humalog Cross Section (A 2 ) 1600 1200 800 Apidra Novolog Levemir Lantus EHSS theoretical Cross Section PA theoretical Cross Section 400 2 3 4 5 6 7 Charge State 2011 Waters Corporation 31
Two ways of viewing Synapt G2 HDMS data 2D DriftScope M/z Intensity Map Drift Time (ms) 2011 Waters Corporation 32
Two ways of viewing Synapt G2 HDMS data 3D Plot Angled 3D view 2D DriftScope M/z Intensity Map Drift Time (ms) 2011 Waters Corporation 33
Electrospray Mass Spectra of Human Insulin 4+ 3+/ 6+ 5+ 7+ 5+ 1162.339 2324.084 2011 Waters Corporation 34
IMS separation of monomeric insulin from dimeric insulin Intensity 3+ 637 Å 2 6+ 1231 Å 2 6+ 3+ Dift Drift Time (ms) IMS Separation of monomeric insulin from dimeric insulin 2011 Waters Corporation 35
Electrospray spectra of human insulin and insulin analogs from ph 7.2 solutions Lantus Levemir Novolog Apitra Humalog Humulin 2011 Waters Corporation 36
Analysis of human insulin analogs by Synapt G2 HDMS Humulin Humalog Apidra Lantus Novolog Levemir 2011 Waters Corporation 37
ESI spectra of INF α2b before and after high temperature stress Indistinguishable by MS alone INF α2b at ph 7.0 INF α2b at ph 7.0 Incubated at 55 o C for 20 min 2011 Waters Corporation 38
Probing conformation changes of Interferon under high temperature stress Mass Selected Arrival Time Distribution (5+) INFα2b after Incubated for 20 min INFα2b @@ ph ph 7.0 7.0 @ 55 o C SEC Dimer @ 70 C for 30 min Inten nsity Monomer Dimer 1533 Å 2 2015 Å2 Dimer IFN α2b Std Drift Time (ms) From A. Diress et al. J. Chromatogr. A 1217 (2010) 3297 3306 2011 Waters Corporation 39
Final Thoughts MS technologies have matured to the point where biotherapeutic MS characterization is routine for many companies: Peptide mapping, Intact protein, Glycan profiling Goals of routine analyses are to increase Quality and Productivity The challenge: biotherapeutic product and process understanding is greater than just primary structure Waters is actively innovating to provide tools that t better answer higher order structural and product quality questions. 2011 Waters Corporation 40
For more information Search www.waters.com for HDX nanoacquity UPLC System with HDX Technology o Video Improved Ion Mobility Separation of Protein Conformations in the Gas Phase with SYNAPT G2 HDMS Synapt G2-S HDMS o Videos 2011 Waters Corporation 41
Acknowledgments Richard Denny Hongwei Xie Weibin Chen Scott Berger Joomi Ahn Barry Dyson Keith Richardson 2011 Waters Corporation 42
2011 Waters Corporation 43