Successfully reported this slideshow.
We use your LinkedIn profile and activity data to personalize ads and to show you more relevant ads. You can change your ad preferences anytime.

Native and IM-MS characterization of ADCs

21,203 views

Published on

Presentation by Dr. Sarah Cianférani-Sanglier, University of Strasbourg, Strasbourg, France. Talk given at Waters Antibody Drug Conjugates (ADC) 2014 Meeting, Nov. 20-21, Wilmslow UK.

Published in: Science
  • Be the first to comment

Native and IM-MS characterization of ADCs

  1. 1. Native and IM-MS characterization of ADCs Sarah CIANFERANI BioOrganic Mass Spectrometry Laboratory (LSMBO), Pluridisciplinary Hubert Curien Institute (IPHC) Analytical Sciences Department University of Strasbourg, CNRS UMR7178, 67087 Strasbourg - France sarah.cianferani@unistra.fr 1
  2. 2. mAbs as therapeutics  mAbs : the most important class of human therapeutics - more than 40 mAbs & derivatives currently approved (EMA/FDA) - used for multiple clinical indications (inflammation, autoimmunity, cancer, transplantation, etc.) - more than 400 mAbs & derivatives in pre-clinical development and clinical trial  mAbs as anti-cancer drugs in clinical studies (Jan 2012)* - 84 unmodified IgG (51%) - 25 ADC (Antibody Drug Conjugates) (15%) + 7 (dec 2012) - 10 bispecific (6%) - 17 engineered (10%) - 16 fragments (10%) *Reichert JM. and Dhimolea A., Drug Discovery Today 2012; Pauwels P. et al., mAbs 2012  mAbs are complex therapeutic proteins (150 kDa glycoproteins)  Next generation empowered mAbs : bispecific mAbs (bsAbs), antibody-drug-conjugates (ADCs) 4 umab) IgG4 (6F4-2) + GSH Bispecific mAb (bsAb) (natalizumab/6F4-2) + bsAb (2 different half mAbs) ADC (mAb + cytotoxic)
  3. 3. Native IM-MS Intact mAb analysis (150 kDa) Middle up mAb analysis (25-100 kDa) Bottom up mAb analysis (peptide mapping) (700-7000 Da) Top-down MS (FT-ICR + Orbital trap) LC-MS (nano)LC-MS/MS enzymatic digestion Higher Order Structure Aggregates mAb/Ag complexes (>150 kDa) Native MS Complexity Size MS toolbox for mAb characterization HDX-MS Crosslinking MS
  4. 4. 4 1991 Native MS 2006 Ion Mobility MS 2012 High resolution native MS Native MS: a brief history 2000 Automated nanoESI 2002 Haemocyanins 2.3 MDa Katta et al. JACS 1991 1991 Holo-myoglobine 17.5 kDa McKay et al. JACS 2006 2006 Ribosomes 2.3 MDa 2000 4000 6000 8000 10000 12000 14000 16000 18000 20000 22000 24000 m/z0 100 % x20x1010474.19 10236.41 10008.84 4422.33 4173.91 3949.99 9791.42 4696.49 14088.06 13870.68 10723.23 13659.90 13455.13 10982.64 13255.21 14313.58 16702.72 16496.93 16298.87 16101.28 15914.78 16912.72 17126.64 17349.11 19404.00 18990.11 18796.87 19615.76 19830.56 20053.19 21493.64 2000 4000 6000 8000 10000 12000 14000 16000 18000 20000 22000 24000 m/z0 100 % x20x1010474.19 10236.41 10008.84 4422.33 4173.91 3949.99 9791.42 4696.49 14088.06 13870.68 10723.23 13659.90 13455.13 10982.64 13255.21 14313.58 16702.72 16496.93 16298.87 16101.28 15914.78 16912.72 17126.64 17349.11 19404.00 18990.11 18796.87 19615.76 19830.56 20053.19 21493.64 Sanglier et al. JASMS 2002 2013 Bacteriophage HK97 capsid 18 MDa Increase in size, Number of partners And Heterogeneity Binding Conformation 1996 nanoESI Snijder et al. Angew. Chem 2013
  5. 5. Native MS analysis Native MS gives information on assemblies maintained by noncovalent interactions ESI-MS instrument 3000 3500 m/z Gas phase Solution Sample preparation step = desalting step Instrumental setting optimizations - Buffer exhange - Use of volatile aqueous buffer at neutral pH - Ammonium buffer (0-1M) with controled pH - Control of the energy communicated to the ions in the 1st pumping stage region of the instrument (Vc, Pi adjustment) Data interpretation - Binding stoichiometries - Binding specificity - Solution affinities - Dynamics of assembly/disassembly 5
  6. 6. Native MS workflow for mAb analysis Minimal and easy sample preparation Purified mAb, bsAb or ADC Deglycosylation Step (PNGase F, IgZero, etc.) Data Interpretation 144190 Mass 143700 144000 144300 144600 Mass 148200 PNGase-F 144190 143700 144000 144300 144600 Mass 146700 147200 147700 148200 147239 147077 147401 147563 PNGase-F G0F/G0F G1F/G0F G1F/G1F G2F/G0F G2F/G1F If necessary Buffer Exchange (spin columns, microconcentrators, SEC etc.) Native MS and IM-MS (with ESI, needles or Chip-based introduction) Synapt G2 HDMS (Waters) + nanoMate Triversa (Advion)
  7. 7. How do we jumped into the mAb field? 7 - mAb native MS - mAb/Ag binding stoichiometries - Native IM-MS  2008-2012: Reference Monoclonal Antibodies (mAb) - Native MS for monitoring of bsAb formation - IM-MS for bsAb - Real-time IM-MS to monitor bsAb formation  2013: towards bispecific mAbs (bsAbs) - Native MS mandatory for ADCs’ analysis - Native MS for drug load profiles, average DAR - IM-MS for ADC heterogeneity assessment - IM-MS for drug load profiles, average DAR  2014: immunoconjugate analysis (ADC)
  8. 8. 1. Native MS and IM-MS for reference mAb analysis 8 2. Native MS and IM-MS for bsAb formation 3. Native MS and IM-MS for ADC formation
  9. 9. Native MS analysis : benefits for intact mAb analysis  Denaturing Conditions mAb 2µM in H2O:ACN:FA (50:50:1) Trastuzumab Nap2 denat 2uM meth denatAb 120v ADC TfCE5 3325 3330 3335 3340 3345 3350 3355 3360 3365 3370 3375 3380 3385 3390 3395 3400 3405 3410 3415 3420 3425 3430 3435 3440 3445 3450 3455 3460 3465 3470 3475 3480 3485 3490 3495 3500 m/z0 100 % A: 147917.13±1.11 B: 148061.73±0.82 C: 148222.42±0.86 D: 148383.83±0.80 E: 148544.33±0.95 44+ 1 2 3 4 5 44+ 1: 147917.1 ± 1.1 Da: G0/G0F 2: 148061.7 ± 0.8 Da: (G0F)2 – error 30 ppm 3: 148222.4 ± 0.9 Da: G0F/G1F 4: 148383.8 ± 0.8 Da: (G2F)2 5: 148544.3 ± 1.0 Da: G1F/G2F M meth denatAb 120v ADC TfCE5 1800 2000 2200 2400 2600 2800 3000 3200 3400 3600 3800 4000 4200 4400 4600 4800 5000 5200 5400 5600 5800 m/z A: 147917.13±1.11 B: 148061.73±0.82 C: 148222.42±0.86 D: 148383.83±0.80 E: 148544.33±0.95 2000 3000 50004000 6000 7000 m/z m/z 2000 2500 3000 3500 4000 4500 5000 5500 6000 6500 7000 7500 8000 8500 9000 9500 9) Sm (Mn, 2x10.00); Cm (1:53) 1: TOF MS ES+ 179 6000 7000 m/z2000 3000 50004000 26+ Trastruzumab 5uMACNH4 150mM pH7.2 NAP2 viva2 FDEHighMass TDC 150v 6mbar 5860 5870 5880 5890 5900 5910 5920 5930 5940 5950 5960 5970 5980 5990 6000 6010 6020 6030 6040 6050 6060 6070 6080 6090 6100 6110 6120 6130 6140 6150 6160 6170 6180 6190 6200 6210 0 100 % 2 3 4 5  Native Conditions mAb 5µM in AcNH4 150mM pH7.5 26+  Data analysis is straightforward : - accurate mass measurement - Purity assessment - Homogeneity determination - Clarification/simplification of native mass spectra du to fewer charges (increased sensitivity) 2: 148071.7 ± 0.5 Da: (G0F)2 – error 100 ppm
  10. 10. Native MS analysis for aggregation studies Detection of oligomeric forms21-Jan-2014 m/z 3500 3750 4000 4250 4500 4750 5000 5250 5500 5750 6000 6250 6500 6750 7000 7250 7500 7750 8000 8250 8500 8750 % 0 100 m/z 3500 3750 4000 4250 4500 4750 5000 5250 5500 5750 6000 6250 6500 6750 7000 7250 7500 7750 8000 8250 8500 8750 % 0 100 O23060FDE 8 (0.553) Sm (Mn, 2x20.00); Cm (2:29) TOF MS ES+ 2.16e3 O9766FDE 1 (0.084) Sm (Mn, 2x20.00); Cm (1:74) TOF MS ES+ 2.65e3 21-Jan-2014 m/z 3500 3750 4000 4250 4500 4750 5000 5250 5500 5750 6000 6250 6500 6750 7000 7250 7500 7750 8000 8250 8500 8750 % 0 100 m/z 3500 3750 4000 4250 4500 4750 5000 5250 5500 5750 6000 6250 6500 6750 7000 7250 7500 7750 8000 8250 8500 8750 % 0 100 O23060FDE 8 (0.553) Sm (Mn, 2x20.00); Cm (2:29) TOF MS ES+ 2.16e3 O9766FDE 1 (0.084) Sm (Mn, 2x20.00); Cm (1:74) TOF MS ES+ 2.65e3 +25 +25 60005000 800070004000 m/z 60005000 800070004000 m/z +39 +39 mAb dimer: 7% mAb dimer : 3% Trastuzumab Batch # 1 Trastuzumab Batch # 2 Quantification based on peaks intensities; 22+ to 28+ for monomer; 36+ to 42+ for dimer ; MWdimer : 296526 ± 18Da Native MS for batch to batch comparisons Native MS allows assessment of higher order structure/oligomeric state
  11. 11. Native MS analysis of immune mAb/Ag complexes Direct determination of mAb/Ag binding stoichiometries m/z 5000 5200 5400 5600 5800 6000 6200 6400 6600 6800 7000 7200 7400 7600 7800 8000 8200 8400 8600 8800 9000 9200 9400 9600 9800 % 0 100 mass 130000 140000 150000 160000 170000 180000 190000 200000 210000 220000 230000 240000 250000 260000 % 0 100 147099  5 Da 23+ 6000 7000 8000 9000 m/z 0 % 100 kDa 150 200 250 0 % 100 mAb (5 µM) m/z 5000 5200 5400 5600 5800 6000 6200 6400 6600 6800 7000 7200 7400 7600 7800 8000 8200 8400 8600 8800 9000 9200 9400 9600 9800 % 0 100 mass 130000 140000 150000 160000 170000 180000 190000 200000 210000 220000 230000 240000 250000 260000 % 0 100 220878  15 Da 245485  15 Da +3 x 24.6 kDa +4 x 24.6 kDa 30+ 6000 7000 8000 9000 m/z 0 % 100 kDa 150 200 250 0 % 100 mAb (5 µM) + JAM-A (40 µM) m/z 5000 5200 5400 5600 5800 6000 6200 6400 6600 6800 7000 7200 7400 7600 7800 8000 8200 8400 8600 8800 9000 9200 9400 9600 9800 % 0 100 196368  8 Da 171783  15 Da mass 130000 140000 150000 160000 170000 180000 190000 200000 210000 220000 230000 240000 250000 260000 % 0 100 +2 x 24.7 kDa27+ 6000 7000 8000 9000 m/z 0 % 100 kDa 150 200 250 0 % 100 +1 x 24.7 kDa mAb (5 µM) + JAM-A (10 µM) Unexpected 1:4 mAb:Ag stoichiometry Atmanene C. et al. Anal Chem. 2009, 81(15):6364-73.
  12. 12. Native Ion Mobility MS (IM-MS) for conformational studies By IM-MS : simultaneous measurement of drift times and m/z ratios ESI source IM cell MS analyzer Detector Instrumental setting optimizations - Wave height and velocity - Pressure in the IM cell Data interpretation - Conformation of biomolecules - Conformational changes Trap T-Wave Transfer T-waveQuadrupole Ion Guide Pusher Detector Primary pumping Turbomolecular pump INTERFACE TOF ANALYZER Reflectron Source NanoESI Ar He ArN2 IMS Cell Vanne ArAr Drift time m/z 12
  13. 13. Native Ion Mobility MS (IM-MS) for conformational studies - Ions separation takes place according to ion mobility Ion Mobility cell 2+ 2+1+ Drift time + 1+ Ion separation according to ion mobility Size, shape Charge Compactness  Drift time  z  Drift time  2+ 1+ 2+ 1+ Drift time N2 z x y z x y z x y z x y x y zx y z L EtDz MMTkP T N e iongasb   112760 15.27316 3   Drift times can be related to collisional cross sections (CCS)  Information on ion gas phase conformation (2D representation of 3D structure) 13
  14. 14. Ion Mobility Mass Spectrometry (IM-MS) For mAb conformational characterization SM classique IM-MS SMSupra IM-MS Denaturing MS Native MS Native IM-MS Denaturing IM-MS Beck A, Sanglier-Cianférani S, Van Dorsselaer A. Anal Chem. 2012, 84(11):4637-46
  15. 15. IM-MS to probe mAb conformational heterogeneity
  16. 16. 16Pritchard C et al. Anal Chem. 2013, 85(15):7205-12 IM-MS for batch-to-batch conformational comparison
  17. 17. 1. Native MS and IM-MS for reference mAb analysis 17 2. Native MS and IM-MS for bsAb formation 3. Native MS and IM-MS for ADC formation
  18. 18. 18 Native MS analysis for analysis for bsAb formed by Fab Arm Exchange IgG4 (natalizumab) IgG4 (6F4-2) + GSH Bispecific mAb (bsAb) (natalizumab/6F4-2) +  2 mAb features are important for the exchange reaction of IgG4: - CH3 domain - Ser at position 228  In vivo, IgG4s can exchange half molecules by a dynamic process called Fab-arm exchange (FAE). In vitro, the FAE process can be mimicked by a reaction with glutathione (GSH)  Serine 228 in IgG4 introduces flexibility in the core hinge region. Besides the usual disulfide bonds connecting two heavy chains (covalent between HC), intrachain disulfide bonds may form instead (only non-covalent bonds between HC). covalent bonds between HC non-covalent bonds between HC
  19. 19. Native MS analysis of bsAB formed by FAE 19 AcNH4 150mM pH7.5 6mbar180v 2000 2500 3000 3500 4000 4500 5000 5500 6000 6500 7000 7500 8000 8500 9000 9500 m/z TOF MS ES+ 417 A: 148142.72±0.9 B: 148300.17±2.7 C: 148465.80±0.8 D: 148618.45±1.6 bispecif nataluzumab/6F4-2 mutant3 2uM 50v denat 500 750 1000 1250 1500 1750 2000 2250 2500 2750 3000 3250 3500 3750 4000 4250 4500 4750 5000 5250 5500 5750 6000 6250 0 100 % O14353FDE 26 (1.761) 30002000 500040001000 m/z +45 bispecif nataluzumab/6F4-2 mutant3 2uM 50v denat 3260 3265 3270 3275 3280 3285 3290 3295 3300 3305 3310 3315 3320 3325 3330 3335 3340 3345 m/z0 100 % O14353FDE 26 (1.761) TOF MS ES+ 3.17e5 A: 73728.33±0.19 B: 73890.42±0.24 C: 74068.96±1.71 D: 74407.87±0.35 E: 74569.96±0.26 F: 74731.84±0.32 G: 147947.25±4.86 H: 148139.83±0.37 I: 148301.66±0.36 J: 148462.72±0.34 K: 148621.50±0.58 L: 148773.14±2.01 M: 148928.86±2.08 +45 G0F/G0F G0F/G1F G1F/G1F G2F/G0F G2F/G1F 70005000 800070004000 m/z3000 bispecif nat/mut3 5uM AcNH4 150mM pH7.5 6mbar180v 6070 6080 6090 6100 6110 6120 6130 6140 6150 6160 6170 6180 6190 6200 6210 6220 6230 6240 6250 6260 6270 6280 6290 6300 6310 6320 6330 6340 6 0 100 % O14364FDE 26 (1.761) TO A: 148 B: 148 C: 148 D: 148 G0F/G0F G0F/G1F G1F/G1F G2F/G0F +24 +24 Hz6F4-2v3: 73729 ± 1 Da natalizumab: 74408 ± 1 Da Hz6F4-2 148302 ± 1 Da Intact Hz6F4-2v3 148300 ± 3 Da ∼17 % half-antibody a high proportion of S-S bond between half-mAbs recombine an almost exclusive homogeneous ion population is native MS  Native Conditions  Denatured conditions
  20. 20. Native MS to monitor FAE dynamics and bsAb formation 20 t = 0 t = 4 h t = 24h v 6mbar 0 5600 5700 5800 5900 6000 6100 6200 6300 6400 6500 6600 6700 6800 6900 m/z v 6mbar 0 5600 5700 5800 5900 6000 6100 6200 6300 6400 6500 6600 6700 6800 6900 m/z 6200 6400 660060005800 6800 m/z +23+24+25 Native MS can serve for online monitoring of FAE and bsAb formation 2D Graph 4 f = a*exp(-b*x) f = a*exp(-b*x) f = a*exp(-b*x) f=a*(1-exp(-b*x)) f = a*exp(-b*x) Time (min.) 0 200 400 600 800 1000 1200 1400 NormalizedIntensity(%) 0 10 20 30 40 50 Col 1 vs Col 2 x column 2 vs y column 2 Col 4 vs Col 5 Col 4 vs Col 5: 1080.0000 x column 5 vs y column 5 Col 7 vs Col 8 Col 7 vs Col 8: 120.0000 x column 6 vs y column 6 natalizumab bsAb 6F4-2 Apparent rate constant: 4.66 x 10-5 s-1* * compared to 7-10 x 10-5 s-1 as reported in Rispens, T. et al. J Am Chem Soc 2011, 133, 10302-11 Rose, R. J. et al. Structure 2011, 19, 1274-82. Detection of a 3rd population after 4h corresponding to bsAb Debaene F. et al. Anal Chem. 2013 85(20):9785-92.
  21. 21. tD (ms)22 242018 21.3 0.2 20.8 0.2 20.4 0.2 +24 t = 8h + GSH t = 8h - GSH control t = 0 + GSH • All 3 mAbs can be detected simultaneously in the IM cell • Native IM-MS can serve for online monitoring of FAE and bsAb formation natalizumab Bispecific mAb 6F4-2 B. O14611FDE.raw : 1 +23 +24 O14605FDE.raw : 1 Native IM-MS to monitor FAE dynamics and bsAb formation Debaene F. et al. Anal Chem. 2013 85(20):9785-92.
  22. 22. 1. Native MS and IM-MS for reference mAb analysis 22 2. Native MS and IM-MS for bsAb formation 3. Native MS and IM-MS for ADC formation
  23. 23. ADCs are next generation empowered therapeutic mAbs  ADCs are tripartite molecules, made by the chemical conjugation of cytotoxins to mAbs Cytotoxic drugLinker *Beck A. and Reichert JM., mAbs 2014 cell-killing capabilities + targeting delivery of the conjugated drug to tumor cells Highly cytotoxic drug  2 already approved ADCs’ on the market: - brentuximab vedotin (Adcetris®, Seattle Genetics) - ado-trastuzumab emtansine (Kadcyla®, Genentech) - > 30 in clinical trials*  3 main types of conjugation: - at cysteine sulfhydril groups after reduction of the interchain disulfide bonds (brentuximab vedotin) - at lysine side chains amine (ado-trastuzumab emtansine) - at engineered cysteine residues at specific sites without reduction (thiomabs) 23
  24. 24. ADCs’ analytical characterization is more challenging than unconjugated mAbs  Cysteine-conjugation generates heterogeneous population of drug-loads 24 S-S Reduction step Drug Conjugation step Covalent species Non-covalent species  Compared to “unconjugated” mAbs, ADCs have increased level of complexity: - formation of heterogeneous mixtures of species with DARs - heterogeneity coming from the presence of a mixture of covalent and non-covalently associated populations due to the presence of drugs at the interchain cysteine residues Need for translational complementary analytical techniques
  25. 25. Drug load profile Unconjugated D0 Average DAR Higher order structures Ion mobility MS? Native MS ? - Cristallography - HDX-MS - SEC-MS - Bottom up (nano)LC-MS/MS - Hydrophobic Interaction (HIC) - Ion Exchange (IEC) - Size exdclusion chromatography - Reversed phase (rpHPLC) - CE-SDS Minutes3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 D0 D2 D4 D6 D8 - Middle- and Top-down MS (MS/MS on intact ADCs ) The analytical toolbox for ADC analysis - Middle-up LC-MS    8 0 8 0 n n DAR DAR A nA DAR = 4.0 Site of conjugation 25
  26. 26. Our reference Cys-linked ADC: brentuximab vedotin mAb Brentuximab anti-CD30 (chIgG1) -Cys-SH (Hinge) Linker Peptide protease-clivable linker -Cit-Val- Drug antimicrotubule drug monomethylauristatin E  indicated for treatment of hematological malignancies (Hodgkin lymphoma, systemic anaplastic large cell lymphoma)
  27. 27. HIC chromatography : the gold standard for Cys-linked ADC Minutes3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 D4D2 D6 D0    8 0 8 0 n n DAR DAR A nA DAR = 4.0  3 main ADC quality attributes (QA) can be determined by HIC - average DAR - drug load distribution - Relative amount of unconjugated mAb (D0) D8
  28. 28. ec-2013Brentuximab IgZero Viva5 replicat 4 5uM AcNH4 150mM pH7.4 80v 6mbar m/z 0 1000 1500 2000 2500 3000 3500 4000 4500 5000 5500 6000 6500 7000 7500 6000 7000 m/z1000 3000 500040002000 37+ 15+ 37+  Full scan mass spectrum Native MS is mandatory for cysteine-linked ADC analysis - Very minor ion signals correspond to intact ADC - Non-covalent interactions are disrupted in denatured conditions 28 1008040 6020 120 140 Mass (kDa) 145,910 Da 123,500 Da 101,072 Da 75,586 Da 25,042 Da 53,177 Da  MaxEnt deconvoluted mass spectrum Denatured conditions IgGZero treated ADC @ 2 µM in H2O:ACN:FA (50:50:1)
  29. 29.  Full scan mass spectrum Native MS is mandatory for cysteine-linked ADC analysis 29 Native conditions IgGZero treated ADC @ 5 µM in 150 mM AcONH4 pH 7.5 ec-2013Brentuximab IgZero Viva5replicat 45uMAcNH4150mMpH7.480v6mbar m/z 00 1000 1500 2000 2500 3000 3500 4000 4500 5000 5500 6000 6500 7000 75006000 7000 m/z1000 3000 500040002000 Polymer contamination free LC + 1 payload Intact ADC signals Clarification/simplification of native mass spectra due to fewer charges (increased sensitivity)
  30. 30.  MaxEnt deconvoluted mass spectrum Native MS is mandatory for cysteine-linked ADC analysis 30 Native conditions IgGZero treated ADC @ 5 µM in 150 mM AcONH4 pH 7.5 mass 144000 145000 146000 147000 148000 149000 150000 151000 152000 153000 154000 155000 156000 157000 158000 159000 % 0 100 O19456FDE 24 (1.627) M1 [Ev-62181,It48] (Gs,10.000,5175:6905,10.00,L10,R10); Sm (Mn, 2x2.00); Sb (25,5.00 ); Cm (2:29) TOF MS ES+ 5.05e5 146 Mass (kDa) 150148 154152 158156 D4 D2 D6 D8 D0 MWexp (Da) mass (Da) Error (ppm) D0 145903 7.3 50.2 D2 148539 8.1 54.2 D4 151172 5.8 38.3 D6 153814 12.5 81.4 D8 156446 9.2 59.1 - ADC is almost detected as an intact molecule - Mass measurement affords drug binding stoichiometries to be assessed - Native MS maintains non-covalent interactions in the gas phase
  31. 31.  Native MS profile  HIC profile Semi-quantitative native MS analysis to assess average DAR - Native MS data are in good correlation with HIC data for all QA - Native MS affords in one single run 1) drug load profile determination and 2) average DAR calculation 31 = 4.0DAR Comparison between HIC and Native MS for brentuximab vedotin = 3.9DAR
  32. 32. Off-line HIC native MS coupling Native MS allows to confirm mass homogeneity of each HIC fraction : no mixtures of different drug binding stoichiometries are observed within one HIC fraction Minutes3.004.005.006.007.008.009.0010.0011.00 D0 1 3 D4 D2 2 D6 4 D8 5 2014-09-19 Bretux IgZero HIC Fraction1 3uM AcNH4 150mM pH7.4 6mbar 180v mass 140000 141000 142000 143000 144000 145000 146000 147000 148000 149000 150000 151000 152000 153000 154000 155000 156000 157000 158000 159000 % 0 100 O23505FDE 16 (1.090) M1 [Ev0,It27] (Gs,10.000,5261:7072,7.00,L30,R30); Sm (Mn, 2x5.00); Sb (2,40.00 ); Cm (1:29) TOF MS ES+ 4.22e5 A: 145939.97±0.85 145904 ± 2 Da D0 2014-09-23 Bretux IgZero HIC Fraction5 3uM AcNH4 150mM pH7.4 6mbar 80v mass 140000 141000 142000 143000 144000 145000 146000 147000 148000 149000 150000 151000 152000 153000 154000 155000 156000 157000 158000 159000 % 0 100 O23515FDE 11 (0.755) M1 [Ev-68567,It25] (Gs,10.000,5168:7016,7.00,L30,R30); Sm (Mn, 2x5.00); Sb (2,40.00 ); Cm (1:29) TOF MS ES+ 8.24e4 151173 ± 1 Da D4 2014-09-21 Bretux IgZero HIC Fraction3 0.5uM AcNH4 150mM pH7.4 6mbar 180v mass 140000 141000 142000 143000 144000 145000 146000 147000 148000 149000 150000 151000 152000 153000 154000 155000 156000 157000 158000 159000 % 0 100 O23509FDE 24 (1.627) M1 [Ev-81894,It25] (Gs,10.000,5383:7158,7.00,L30,R30); Sm (Mn, 2x5.00); Cm (1:29) TOF MS ES+ 3.27e5 A: 148558.11±0.99 148539 ± 1 Da D2 2014-09-25 Bretux IgZero HIC Fraction7 3uM AcNH4 150mM pH7.4 6mbar 180v mass 140000 141000 142000 143000 144000 145000 146000 147000 148000 149000 150000 151000 152000 153000 154000 155000 156000 157000 158000 159000 % 0 100 O23521FDE 6 (0.419) M1 [Ev0,It76] (Gs,10.000,5168:7016,7.00,L30,R30); Sm (Mn, 2x5.00); Cm (1:29) TOF MS ES+ 2.50e5 A: 156515.52±3.37 156457 ± 3 Da D8 150 155145 Mass (kDa) 1 3 2 5 2014-09-24 Bretux IgZero HIC Fraction6 3uM AcNH4 150mM pH7.4 6mbar 180v mass 140000 141000 142000 143000 144000 145000 146000 147000 148000 149000 150000 151000 152000 153000 154000 155000 156000 157000 158000 159000 % 0 100 O23518FDE 17 (1.157) M1 [Ev-97372,It21] (Gs,10.000,5168:7016,7.00,L30,R30); Sb (2,40.00 ); Cm (1:29) TOF MS ES+ 2.61e5 153815 ± 2 Da D6 4 32
  33. 33. Advantages of native MS compared to HIC 2 3 4 5 6 7 8 9 10 11 min. D0 D2? D4?D1? Hz1613F12 SG3249DAR2 IgZero 5uM AcNH4 150mM pH7.5 NAP2 6mbar 120v % 100 146 152 mass (kDa)145 150148147 151149 153 D0 D1 D3 D2 D4 = 2.0DAR  Native MS profile  HIC profile - Broad HIC peaks - Difficulty to unambiguously assess drug binding - No odd drug load expected = 1.8DAR - Native MS mass accuracues allows unambiguous drug binding stoichiometry assessment - odd drug load are clearly detected - No peak broadening as on HIC Native MS allowed to precise unclear HIC results 33 D1’?
  34. 34. Native IM-MS of intact cysteine-linked ADC Deglycosylated Parent mAb O25596FDE.raw : 1 24+ 12 16 20 22 tD (ms) 7500 7000 6000 5500 m/z 181410 6500 5000 23+ 21+ IgGZero ADC O23564FDE.raw : 1 12 16 20 22 tD (ms) 7500 7000 6000 5500 m/z 181410 6500 5000 Deglycosylated ADC SynaptG2, 3µM, AcNH4 150mM pH7,4 Vc=80v, Pi=6mbar, Bias25, WH=40V, WV=923m/s, He=130mL/min, N2= 45mL/min 34
  35. 35. Native IM-MS of intact cysteine-linked ADC Deglycosylated Parent mAb O25596FDE.raw : 1 24+ 12 16 20 22 tD (ms) 7500 7000 6000 5500 m/z 181410 6500 5000 23+ 21+ IgGZero ADC O23564FDE.raw : 1 12 16 20 22 tD (ms) 7500 7000 6000 5500 m/z 181410 6500 5000 Deglycosylated ADC 35 SynaptG2, 3µM, AcNH4 150mM pH7,4 Vc=80v, Pi=6mbar, Bias25, WH=40V, WV=923m/s, He=130mL/min, N2= 45mL/min
  36. 36. Native IM-MS of intact cysteine-linked ADC  ADC heterogeneity in drug binding is observed on native IM-MS plots  Each individual even-drug load can be separated in IM-MS  No positional isomers were detected (to low IM cell resolution on intact ADC) Deglycosylated Parent mAb Deglycosylated ADC O25596FDE.raw : 1 16 17 18 19 tD (ms) 6700 6600 6500 6400 m/z 23+ 22+ O23564FDE.raw : 1 16 17 18 19 tD (ms) 6700 6600 6500 6400 m/z DO 23+ D0 22+ D2 23+ D6 24+ D4 23+ D8 24+ 36 SynaptG2, 3µM, AcNH4 150mM pH7,4 Vc=80v, Pi=6mbar, Bias25, WH=40V, WV=923m/s, He=130mL/min, N2= 45mL/min
  37. 37. Native IM-MS of intact cysteine-linked ADC resolving power of IM cell tD/tD at FWHM : 16.4 ± 0.8 D0 14.2 D2 14.9 D4 15.6 D6 16.3 D8 17.0 13 14 15 16 17 18 19 tD (ms) 37  Each individual even-drug load can be separated in IM-MS Parent mAb (24+ charge state) ADC (24+ charge state) (brentuximab vedotin) - D0 D2 D4 D6 D8 tD (ms) 14.2 ± 0.0 14.2 ± 0.1 14.9 ± 0.1 15.6 ± 0.1 16.3 ± 0.1 17.0 ± 0.1 (tD) (ms) - - + 0.7 + 0.7 + 0.7 +0.7 CCS from IM-MS (nm²) 68.0 ± 0.0 68.1 ± 0.1 68.8 ± 0.1 69.5 ± 0.1 70.3 ± 0.1 71.1 ± 0.1 CCS (nm²) - - + 0.7 + 0.7 + 0.8 +0.8 Predicted* CCS (nm2) 68.2 68.2 69.0 69.8 70.6 71.4 Predicted CCS (nm²) - - + 0.8 + 0.8 + 0.8 +0.8 Drug binding induces constant and reproducible tD and CCS differences
  38. 38. Semi-quantitative analysis of native IM-MS  For each charge state, intensities of the extracted drift peaks were plotted across a series for each drug binding stoichiometry (D0, D2, D4, D6 and D8) and a Gaussian curve fitted.  To give a relative intensity of each conformer, the area underneath each curve was calculated as a percentage of the total observed signal, here (D0+D2+D4+D6+D8). = 3.7 ± 0.1DAR (triplicates) 38 NormalizedIntensity(%) Charge States 22 24 26 28 0 20 40 60 80 100 D0: 14% D2: 27% D4: 30% D8: 10% D6: 19%
  39. 39. Minutes3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 D0 D2 D4 D6 D8 Average DAR determination from semi quantitative native MS and IM-MS MaxEnt1 deconvolution Quantitation based on ion intensities n=8 D0 D2 D4 D6 D8 DAR = 3.9 ± 0.1DAR = 3.7 ± 0.1DAR = 4.0 Number of drug load Calculation based on area measured under fited gaussian of charge states extracted ions n =3 Native MS HIC Average DAR determination and drug-load profiles obtained from native MS or native IM-MS are in good agreement with HIC data mass 144000 145000 146000 147000 148000 149000 150000 151000 152000 153000 154000 155000 156000 157000 158000 159000 % 0 100 O19456FDE 24 (1.627) M1 [Ev-62181,It48] (Gs,10.000,5175:6905,10.00,L10,R10); Sm (Mn, 2x2.00); Sb (25,5.00 ); Cm (2:29) TOF MS ES+ 5.05e5 146 Mass (kDa)150148 154152 158156 D4 D2 D6 D8D0 Native IM-MS 39 Debaene F. et al. Anal Chem. 2014, 86(21):10674-83.
  40. 40. Take home message 40From May C. et al., Biochemical Pharmacology 2012. Native MS HDX-MS Ion Mobility -MS Native MS is a rapid, robust and reliable method for first- line semi-quantitative mAb characterization Native IM-MS for semi-quantitative global conformational characterization of mAbs HDX for structural mAb characterizartion (epitope mapping, comparability studies, etc.)
  41. 41. Special feature: Head comparison of trastuzumab and cetuximab with corresponding biosimilar and biobetter candidates Beck A, Debaene F, Diemer H, Wagner-Rousset E, Colas O, Van Dorsselaer A and Cianférani A, JMS; 2015 in press What’s coming next ? 41 originator  Trastuzumab  Trastuzumab-B originator biosimilar
  42. 42. Many Thanks for your attention ! The Antibody Physico-Chemistry group Elsa WAGNER-ROUSSET Olivier COLAS Nathalie CORVAÏA Amandine BŒUF (past) Daniel AYOUB (past) Alain BECK The native MS group of LSMBO Julien MARCOUX Guillaume TERRAL Johann STOJKO François DEBAENE (past) Cédric ATMANENE (past) Alain VAN DORSSELAER Sarah CIANFERANI 42

×