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the ancient pond a frog jumps in sound of water —Matsuo Bashō (1686) Bioanalysis of Antibody-Drug Conjugates (ADCs): an overview of current approaches and case studies illustrating the challenges presented by hybrid biotherapeutic molecules Rand Jenkins 7th JBF Symposium Tokyo Japan, 09 March 2016 Overview 3 1 Introduction to ADCs 2 BA complexity & current approaches 3 LC-MS/MS assay options 4 Need for characterization—LC-HRAMS 5 BA practicality challenges 6 Final thoughts Biotherapeutics innovation peptides mAbs bispecifics (bsAbs, DART®s, BiTE®s) ADCs prodrug/carrier conjugates (albumin, PEG) fusion proteins messenger RNA therapeutics™ oligonucleotides (ssDNA, siRNA, mRNA) CAR-Ts 4 ADCs Monoclonal antibody drugs 6 Monoclonal antibody drugs in cancer therapy ● Effective for blood cancers − ● Limited effectiveness for solid tumors − − 7 e.g. Rituximab for B-cell non-Hodgkin lymphoma e.g. Herceptin for HER2+ breast cancers (20-25%) Usually require co-therapy with standard cytotoxic “chemo” drugs such as paclitaxel or doxorubicin Creating a “magic bullet” Small Cytotoxic “Drug” 8 + Monoclonal Antibody (mAb) Antibody-Drug Conjugate 9 Antibody-Drug Conjugate Like a mAb drug ● Seeks out & binds to specific tumor cells ● Long half life ADC ADC advantages ● Non-toxic in circulation ● Delivers highly toxic payload to tumor cells ● Toxin released inside to “safely” kill cells 10 ↖ ↑ linker drug “payload” drug-antibody ratio (DAR) = 6 ADC technology – how they function 11 FDA approved ADC products NEWS RELEASE: Aug. 19, 2011 (Seattle Genetics) FDA approves ADC to treat two types of lymphoma “The U.S. Food and Drug Administration today approved ADCETRIS (brentuximab vedotin) to treat Hodgkin lymphoma (HL) and a rare lymphoma known as systemic anaplastic large cell lymphoma (ALCL)” http://www.fda.gov/NewsEvents/Newsroom/PressAnnouncementsucm268781.htm NEWS RELEASE: Feb. 22, 2013 (Genentech/Roche) FDA approves new treatment for late-stage breast cancer “The U.S. Food and Drug Administration today approved Kadcyla (ado-trastuzumab emtansine), a new therapy for patients with HER2-positive, late-stage (metastatic) breast cancer” http://www.fda.gov/NewsEvents/Newsroom/PressAnnouncementsucm340704.htm 12 Kadcyla® ADCETRIS® S. Kaur, Land O'Lakes, ADC Bioanalysis, 07.18.13 newer engineered designs ADC diversity linkers & payloads Examples of ADC’s and Linkers and Payloads ADCETRIS® Monomethyl auristatin E (MMAE) Adcetris Brentuximab vedotin (SGN-35) Seattle Genetics Payload - Monomethyl auristatin E (MMAE) Cleavable payload Kadcyla Trastuzumab emtansine, adotrastuzumab emtansine (T-DM1) Genentech Payload – Maytansinoid (DM) Non-cleavable payload Maytansinoid (DM) Kadcyla® Inotuzumab ozogamicin (INO, CMC-544) and Mylotarg Pfizer Payload - N-Ac- Calicheamicin DMH Cleavable payload N-Ac- Calicheamicin DMH 2013 NBC meeting, ADC Bioanalysis Workshop, 18-May-2013 / LCMS Assays for payloads and intact ADC’s / Leo Kirkovsky (Pfizer) , [email protected] 4 Other payloads… Pyrrolobenzodiazapine (PBD) dimers Tubulysin analogues 16 New ADC conjugation idea: GlycoConnect™ Remon van Geel et al, Chemoenzymatic Conjugation of Toxic Payloads to the Globally Conserved N-Glycan of Native mAbs Provides Homogeneous and Highly Efficacious Antibody−Drug Conjugates, Bioconjugate Chem. 2015, in press 17 New ADC conjugation idea: Fleximer polymers 18 ADC complexity What should be measured? S. Kaur, Land O'Lakes, ADC Bioanalysis, 07.18.13 Heterogeneity challenge for bioanalysis Pharmacokinetic Assay Strategy: T-DM1 ® example) (Kadcyla T-DM1: Trastuzumab“active” with DM1 conjugated (1-8; DAR: 3.5) Conjugated ADCs 12 12 -- serum Total Trastuzumab: unconjugated Trastuzumab plus conjugated T-DM1 -- serum Total mAb (with or without drugs) DM1 with TCEP reduction of disulfide – plasma “Free” drug (and otherbonds forms) *X * DM1 dimer DM1 Other DM1-S-S-X * DM1-albumin Linker-containing catabolites: MCC-DM1, Lysine-MCC-DM1 21 * DM1 disulfide bound to antibody Standard ADC bioanalytical assays Assay Technology Total Antibody, DAR ≥0 Assess general mAb PK behavior—efficacy LBA (ELISA) Conjugated Antibody, DAR ≥1 Measure total ADC molecules—efficacy LBA (ELISA) Anti-Therapeutic Antibody (ATA) Detect immune response—efficacy & safety LBA (ELISA) and Cell-based NAb Unconjugated Drug Detect “free” drug—safety LC-MS/MS Pharmacokinetic data (Kadcyla® example) Phase I Study Trastuzumab-DM1 in Patients With Breast Cancer T-DM1 Total Trastuzumab DM1 Mean Concentration (ng/ml) 1,000,000 100,000 10,000 1,000 100 10 1 0.1 0 100 200 300 400 500 600 Time (hours) Fig 3. Mean levels of trastuzumab-DM1 (T-DM1), total trastuzumab, and DM1 over time are shown for patients after the first dose of T-DM1 administered at the maximum-tolerated does of 3.6 mg/kg. Standard deviation is shown in error bars. Adverse Events v3 (NCI CTCAE v3), except for cardiac troponin I elevation 23 and segmental wall-motion abnormalities (not described in CTCAE v3). in Table 1, received a cumula doses ranging from 0.3 to 4 treated at each dose level and d shown in Figure 2. ← Total ThreemAb patients were treate 0.6-, 1.2-, and 2.4-mg/kg coho served drug-related grade ! 2 e ←enrolled Conjugated in the 4.8-mg/kg coh ADCpromp 4“active” thrombocytopenia, dose level—3.6 mg/kg (as schema). After six patients wer was declared ←mg/kg “Free” drugthe MTD, of 15 patients. Study treatment was ongo 13, 2009. Fifteen patients withd sive disease, two because of th cause of AEs (grade 4 thrombo grade 3 pulmonary hypertens grade 3 muscular weakness in ered related to study treatment; metastatic disease in the brain] LBA Assays for ADCs are More Complex than LBA for mAb Therapeutics mAb Assay S. Kaur, AAPS, San DIego, 11.04.14 11 © 2014, Genentech LBA Assays for ADCs are More Complex than LBA for mAb Therapeutics mAb Assay Total Antibody Assay S. Kaur, AAPS, San DIego, 11.04.14 Conjugated Antibody Assay 11 © 2014, Genentech Characterize Ligand Binding Assays with Individual DARs Identified in Plasma to Evaluate Assay performance Anti-STEAP1 ADC Purified DAR Assay 0 1 2 4 6 Total Antibody ELISA (% recovery)1 88 78 86 92 92 Conjugated-Antibody ELISA (% recovery) 1 1 Based • • NA 11 102 99 64 specificity issue on expected nominal concentration of individual DARs spiked into serum Used individual DAR controls from plasma to test with ELISA ligand binding reagents No single anti-Drug mAb reagent in conjugate Ab assay appropriate for all DARs S. Kaur, AAPS, San DIego, 11.04.14 12 © 2014, Genentech ADC bioanalysis using LBAs Advantages • Traditional approach – the gold standard • High throughput and low cost per sample Challenges • MD expensive and time-consuming • Potential interference from soluble target • Reagents may show variable response to DARs 27 ADC bioanalysis LC-MS/MS alternatives Alternative ADC bioanalytical assays Assay Technology Total Antibody, DAR ≥0 LBA (ELISA) * Conjugated Antibody, DAR ≥1 Measure of conjugated antibodies only LBA (ELISA) * Antibody-conjugated Drug, DAR ≥1 Measure of conjugated “active” drug load Affinity capture LC-MS/MS (requires cleavable linkers) Total Antibody, DAR ≥0 Most convenient for non-clinical Affinity capture LC-MS/MS ** ** Develop Appropriate PK Assays: Molar Concentration Hybrid Binding LC-MS/MS & LBA Conjugate Assay Formats Conjugated Conjugated Antibody AntibodyAssay Assay Detection reagent (Biotin antigen/SAHRP or Biotin antiCDR/SA-HRP) ADC (carrying 1 or more drugs) Total-Antibody Due to DAR 0 Conjugated-Antibody Free Drug Capture reagent (anti-drug mAb) Molar Concentration Time AntibodyConjugated Drug Assay Total-Antibody Due to DAR Changes Antibody-Conjugated Drug Free Drug LC-MS/MS detection of released drug Linker cleavage/ ADC digestion step ADC and Endogenous IgG Capture by Protein A (resin) Time 13 S. Kaur, AAPS, San DIego, 11.04.14 © 2014, Genentech Ab-conjugated drug AC-LC-MS/MS assay Inter-assay accuracy & precision Total mAb universal AC-LC-MS/MS assay (non-clinical applications) 1. Generic affinity capture ● ● Anti-human Fc Protein A or G 2. “On-bead” processing ● LC-MS/MS Typical chemistry steps: - Denaturation - Reduction - Alkylation - Trypsin digestion Peptides Trypsin Anti-hFc antibody 3. LC-MS/MS Detection ● Human IgG peptides 32 Magnetic bead mAb target LC–MS/MS assays. sensitivity; identify the most promising light Unique human choices The most critical performancemAb metric forpeptide an IS and heavy chain candidate peptides based upon is how reliably the ISapplications) tracks the analyte through- their individual performance; and combine the (non-clinical out bioanalytical extraction and LC–MS/MS most promising light and heavy chain candidates Table 1. Amino acid sequences of universal peptide candidates. Peptide Sequence Human heavy chain subclass TVAAPSVFIFPPSDEQLK SGTASVVCLLNNFYPR‡ VDNALQSGNSQESVTEQDSK DSTYSLSSTLTLSK NA NA NA NA TPEVTCVVVDVSHEDPEVK‡ FNWYVDGVEVHNAK VVSVLTVLHQDWLNGK§ GFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSK IgG1 IgG1 IgG1, IgG4 IgG1, IgG2, IgG4 IgG1 Light chain candidates† TVAA→ SGTA→ LC-1 LC-2 LC-3 LC-4 Heavy chain candidates HC-1 HC-2 HC-3 HC-4 HC-5 VVSV→ TTPP→ All light chain peptide candidates are in the constant region of the -class. Peptide quantified as the S-carboxymethylcysteine derivative. § Peptide HC-3 was previously identified as a universal peptide [7]. † ‡ Michael T Furlong, Song Zhao, William Mylott, Rand Jenkins, Mian Gao, Vendana Hegde, James Tamura, Adrienne Tymiak & Mohammed Jemal, Bioanalysis 2013, 5(11), 1363−1376. future science group 33 www.future-science.com Total mAb Method Performance Evaluation (MPE): calibration standards and batch acceptance QCs Run ID 1RBFE2-A 2RBFE2-A Theoretical Concentration Mean %C.V. CAL 1 CAL 2 CAL 3 CAL 4 CAL 5 CAL 6 CAL 7 CAL 8 QC 1 (µg/mL) (µg/mL) (µg/mL) (µg/mL) (µg/mL) (µg/mL) (µg/mL) (µg/mL) (µg/mL) (µg/mL) (µg/mL) 0.859 3.05 11.4 41.7 48.7 0.148 1.46 35.2 0.0509 0.105 0.203 0.835 2.98 11.4 39.0 50.7 0.144 1.39 34.6 0.0538 0.096 0.217 0.802 3.02 12.5 42.7 49.8 0.151 1.49 35.9 0.0473 0.0971 0.194 0.805 3.01 11.6 37.5 48.5 0.140 1.51 36.8 0.0500 0.100 0.200 0.0503 0.0982 0.201 0.800 0.825 3.00 3.01 12.0 11.7 40.0 40.2 50.0 49.4 0.150 0.146 1.50 1.46 37.5 35.6 3.25 3.13 0.892 0.437 4.28 -2.27 5.99 0.548 2.06 -1.15 3.33 -2.98 3.48 -2.40 2.62 -5.04 4.78 -1.82 5.87 0.545 Acceptance criteria: ±25% for the LLOQ and ±20% for all other levels 34 QC 3 0.0492 0.0946 0.191 5.51 0.579 % Bias QC 2 references “Dual universal peptide approach to bioanalysis of human monoclonal antibody protein drug candidates in animal studies” Michael T Furlong, Song Zhao, William Mylott, Rand Jenkins, Mian Gao, Vendana Hegde, James Tamura, Adrienne Tymiak & Mohammed Jemal, Bioanalysis 2013, 5(11), 1363−1376. “A general LC-MS/MS method approach using a common whole molecule SIL-IS and a common immuno-capture for sample clean up and enrichment that is applicable to various mAbs in different matrixes” Li, H.; Ortiz, R; Tran, L; Hall, M; Spahr,C; Walker, K; Laudemann, J; Miller, S; Salimi-Moosavi, H; and Lee, J.W., Anal. Chem. 2012, 84, 1267−1273. 35 ADC bioanalysis using LC-MS/MS Advantages • Standard approach for small molecules • “Direct” detection of molecules or fragments • High sensitivity and selectivity/specificity • MD relatively rapid; universal methods possible • Data complimentary to LBAs Challenges • Complex and expensive instrumentation • Lower throughput and higher costs per sample 36 through falling snow a spring breeze blows —Kobayashi Issa (1818) ADC biotransformation characterization needed ADCs Can Undergo Biotransformations In Vivo: Analytes & Assay Standard Curve May Differ Analysis of small molecule drug transformations in vivo is well established – Absorption, Distribution, Metabolism and Excretion (ADME) by LCMS/MS. (E.g., cytochrome P450 induction/inhibition, metabolic profiling, plasma protein binding and P-gp transporters) Analysis of protein or ADC transformations in vivo is challenging Metabolites/ catabolites Adducts Intact antibodies Reference Standard Complexes with Antigen and other antibodies S. Kaur, AAPS, San DIego, 11.04.14 Small catabolites/metabolites E.g., Drug, linker, linker-drug Metabolites/catabolites 4 © 2014, Genentech ADC characterization needed Because: • Drug-antibody (DAR) distribution in vivo is dynamic • Drug payload can prematurely deconjugate • Higher drug-loaded species undergo faster clearance • Standard bioanalytical assays don’t detect post-dose changes in DAR distribution • PK-PD modeling may be incorrect • Catabolism/metabolism-related changes may impact efficacy and/or safety 40 LC-MS ADC characterization Assay Technology Drug or drug-linker metabolites Which species exist and are important? LC-MS/MS Catabolites (ADC or mAb) Which species exist and are important? Affinity capture LC-HRAMS DAR Distribution Assess ADC stability (in vitro / in vivo) Affinity capture LC-HRAMS or HIC Critical reagent DAR specificity Assess LBA reagents Affinity capture LC-HRAMS or HIC Major focus on DAR distribution To: • Evaluate ADC reference and dosing material • Assess stability-related changes in vitro • Assess biotransformation-related changes in vivo • Evaluate critical reagent specificity (for LBAs) Results are generally qualitative or semi-quantitative 42 ADC characterization Drug-Antibody Ratio (DAR) Affinity Capture-LC-HRAMS workflow courtesy of Keyang Xu, Genentech Affinity Capture LC-HRAMS allows direct measurement of intact ADC changes ABIO 10293 of Pages 11, Model 5G YABIO No. 10293 January 2011 No. of Pages 11, M 25 January 2011 Characterization of intact antibody–drug conjugates / K. Xu et al. / Anal. Biochem. xxx (2011) 4xxx–xxx A Characterization of intact antibody–drug conjugates / K. Xu et al. / Anal. Biochem. xxx (2011) xxx–xxx 3 A B TIC! 7.0e4 6.0e4 Intensity (cps) Biotinylated target antigen, e.g., ECD Wash 5.0e4 4.0e4 3.0e4 2.0e4 1.0e4 Elute Streptavidin Coated Paramagnetic Bead Magnet Plasma with ADCs Biotin-Target Antigen 0 Incubate @ 20-25 oC Inject onto LC-MS ADCs B Intensity (counts) mAb 20.0 16.0 14.0 8 10 Time (min) × × + × + + × × + + + × × ** × + + 6.0 2000 2200 70 Magnet separation 60 + + 2400 Deconv MS! × + + + × +: DAR 0 ×: DAR 1 *: DAR 2 × × 8.0 14 × × 10.0 12 × × × C Deglycosylation, Wash 6 12.0 2.0 C 4 Raw MS! 18.0 4.0 D 2 × + * * * * * * 2600 2800 m/z (amu) + × + × × * * + + * * + +× * * * 3000 3200 148 149 × 3400 DAR 1 : streptavidin coated paramagnetic bead, : biotinylated capture probe, : antibody or antibody drug conjugate (ADC) : proteins, peptides and other species in plasma Intensity (cps) 50 40 30 DAR 0 20 DAR 2 10 143 144 145 146 147 Schematic of the affinity capture LC–MS method to analyze ADCs in plasma or serum. (A) Biotinylated target antigen (e.g., ECD) is immobilized on paramagnetic Mass (kDa) tavidin beads by incubating at room temperature. Excess reagents are washed away. (B) The modified beads are then incubated with plasma/serum containing ADCs at Fig.2. Measurement of intact with different temperature to enable the capture of ADCs by target antigen. The inset depicts the immunoaffinity interactions between target antigen and ADCs the antibodies. (C)drug-to-antibody ratios in plasma by affinity capture LC–MS. The results show unbiased capture of anti-M standards (DAR DAR 1, and 2) spiked into rat plasma at a fixed composition. (A) Representative TIC of the TDC mixture. A retention time window (hig ycosylation is subsequently performed. Following extensive washing to remove unbound and nonspecifically bound proteins, beads are0,isolated by DAR magnetization. (D) selected to extract the mass spectrum of the TDCs. (B) Corresponding extracted mass spectrum showing characteristic charge envelopes for individual TDC ADCs are then eluted from the beads and injected onto the capillary LC–MS system for analysis. 44 Xu K, Liu L, Saad O, et al, Anal Biochem, 412, 56–66 (2011). Deconvoluted mass spectrum from panel B showing relative abundances of TDC species. Characterization of intact antibody–drug conjugates / K. Xu et al. / Anal. Biochem. xxx (2011) xxx–xxx Intensity (cps) A Intensity (cps) 144 145 35 30 ∆ = 1203 Da 20 15 10 Intensity (cps) 44% DAR1 * 145 146 28 24 20 16 12 ∆ = 1209 Da 17% DAR0 144 16 12 8 4 143 140 48% DAR1 145 144 145 * 146 253 146 148 147 ^ 149 150 151 94% DAR2 140 100 80 6% DAR1 60 40 143 144 145 146 Buffer_96h Buffer_96h * 148 147 Mass (kDa) 149 150 151 149 150 151 Rat_96h Rat_96h 149 150 Drug release in a plasma stability study of anti-MUC16 TDC in vitro. An aliquot of 100 μg/ml anti-MUC16 TDC (DAR 2) was incubated in plasma from different species and buffer control (PBS with 0.5% BSA) at 37° C for up to 96 h.! 151 44% DAR2 ∆ = 1206 Da Human_96h Human_96h * 147 252 liquid chromatography–mass spectrometry, Anal. Biochem. (2011), doi:10.1016/j.ab.2011.01.004 ^ 148 ∆ = 1207 Da 13% DAR0 151 ∆ = 1210 Da 147 * 251 Monkey_96h 35% DAR2 43% DAR1 150 Human_96h Fig.4. Drug release in a plasma stability study of anti-MUC16 TDC in vitro. An aliquot of 100 lg/ml anti-MUC16 TDC (DAR 2) was incubated in plasma from different spec and buffer control (PBS with 0.5% BSA) at 37 !C for up to 96 h. Samples were collected at different time points and analyzed by affinity capture LC–MS. Representati deconvoluted mass spectra are shown here for the anti-MUC16 TDC in cynomolgus monkey plasma at 0 h (A), cynomolgus monkey plasma at 96 h (B), rat plasma at 96 h ( human plasma at 96 h (D), and buffer control at 96 h (E). Minor components due to incomplete deglycosylation were observed and labeled as ‘‘^’’. Peaks labeled as ‘‘⁄’’ are y Da to be identified structurally and not included in calculations. Percentage DAR values shown refer to relative ratios. ^ * 146 * 250 Monkey_96h Please cite this article in press as: K. Xu et al., Characterization of intact antibody–drug conjugates from plasma/serum in vivo by affinity capture capillar 148 147 * 28 24 20 149 40% DAR2 * * 8 143 Intensity (cps) 148 147 * 144 ∆ = 1206 Da 249 20 ∆ = 1209 16% DAR0 4 E * 25 143 D 146 44% DAR2 ∆ = 1207 Da 120 Monkey_ 0h Monkey_0h 7% DAR1 5 C E 120 143 B 93% DAR2 40 24 20 generate mixtures of known compositions for16 affinity capture and 13% 12 subsequent LC–MS analysis. Fig. 2A shows a representative TIC of DAR0 the TDC mixture from affinity capture LC–MS. 8The retention time window (!9.0–10.3 min), representing the LC elution of the TDCs, 4 was selected and the corresponding mass spectrum was extracted 143 144 145 160 80 43% DAR1 28 in vitro ADC plasma stability Intensity (cps) he method was extensively characterized using anti-MUC andards (DAR 0, DAR 1, and DAR 2) to mimic situations occur in vivo. MUC16 TDC standards were spiked into rat plasma at varyntrations (with a total TDC concentration of 30 lg/ml) to Intensity (cps) D ary 2011 ^ 148 94% 149 150 151 Xu K et al, Anal. Biochem, 412, 56–66 (2011) A DAR2 100 in vivo ADC characterization 90 Intensity (cps) 80 70 day post-dose 11day post-dose 60 50 40 ∆ = 1199 Da 30 DAR1 20 10 143 B 144 145 147 148 150 149 151 DAR2 50 45 DAR1 40 Intensity (cps) 146 35 ∆ = 1201 Da 30 days post-dose 7 7days post-dose 25 20 ∆ = 1200 Da 15 10 DAR0 5 143 Intensity (cps) C 13 12 11 10 9 8 7 6 5 4 3 2 1 143 144 145 146 147 148 149 150 151 DAR1 ∆ = 1213 Da Fig.6. Drug release observed in a multipledose toxicokinetic study of anti-MUC16 TDC in vivo. Anti-MUC16 TDC (DAR 2) was administered to cynomolgus monkeys intravenously once every 3 weeks for a total of four doses. Three dose groups at 6, 10, and 20 mg/kg were evaluated. Deconvoluted mass spectra of the TDC species of a representative animal receiving the first dose of 6 mg/kg antiMUC16 TDC show the drug release from DAR 2 to form DAR 1 and DAR 0 with time: (A) 1 day postdose; (B) 7 days postdose; (C) 21 days postdose. ∆ = 1207 Da DAR0 DAR2 days post-dose 21 21days post-dose Xu K et al, Anal. Biochem, 412, 56–66 (2011) 144 145 146 148 147 Mass (kDa) 149 150 151 observed in a multiple-dose toxicokinetic study of anti-MUC16 TDC in vivo. Anti-MUC16 TDC (DAR 2) was administered to cynomolgus monkeys every 3 weeks for a total of four doses. Three dose groups at 6, 10, and 20 mg/kg were evaluated. Deconvoluted mass spectra of the TDC species of a al receiving the first dose of 6 mg/kg anti-MUC16 TDC show the drug release from DAR 2 to form DAR 1 and DAR 0 with time: (A) 1 day postdose; (B) Conjugation site influences linker stability and therapeutic activity of ADCs Shen B-Q et al. Nature Biotechnology, 30(2), 184–191 (2012) in vivo ADC (reduced interchain S-S) characterization Figure 7. Summary of mass shifts and peak asymmetry observed in cysteine-linked ADCs in vivo. Detection of odd loaded species at masses higher than expected was attributed to modification at the deconjugated drug-linker sites, specifically cysteinylation, and observed asymmetry in the deconvoluted data over time is hypothesized to be a consequence of increased sample heterogeneity due to in vivo modifications, including hydrolysis. Hengel SM et al. Anal. Chem. 2014, 86, 3420–3425. 49 Non-cleavable ADC with site-specific catabolism Stability of Non-Cleavable Site-Specific ADCs Fig 1. Stability studies of site-specific non-cleavable ADCs. a) Positions of conjugation sites on an antibody. b) Structure of the PEG6-C2-MMAD noncleavable payload conjugated to the glutamine tag on the antibody, and its cleavage product. The glutamine residue is shown in blue. c) Structure of the PEG6-C2-Aur3377 non-cleavable payload conjugated to the glutamine tag shown in blue. doi:10.1371/journal.pone.0132282.g001 (PEG6-C2-MMAD), a potent analogue of the antimitotic agent Dolastatin 10 [22], attached to specific sites across the anti-M1S1 C16 antibody (Fig 1a and 1b). To evaluate linker-payload Magdalena Dorywalska et al, Site-Dependent Degradation of a Non-Cleavable Auristatin-Based Linker-Payload in Rodent stability, we performed comparative in vitro plasma stability assays by incubating conjugates in Plasma and Its Effect on ADC Efficacy, PLOS ONE | DOI:10.1371/journal.pone.0132282 July 10, 2015 mouse plasma, purifying them and comparing their drug-antibody ratio (DAR) to that of untreated conjugates. Unlike the cleavable Aminocaproyl-valine-citrulline-p-aminobenzylcarbamate (C6-VC-PABC) linker [21], the non-cleavable PEG6-C2-MMAD-derived conjugates remained stable across most of the sites tested over a 4.5 day incubation period in mouse Non-cleavable ADC with site-specific catabolism Stability of Non-Cleavable Site-Specific ADCs Fig 2. Mass spectrometric analysis of non-cleavable conjugates Fig 2. Mass spectrometric analysis of non-cleavable conjugates. The Fig labels represent experimentally observed masses for conjugates before (upper panel) and after (lower panel) in vivo exposure. a) Intact mass deconvolution of C16 Site A-PEG6-C2-MMAD conjugate. The metabolic products of the DAR 2 species show a mass loss from either 1 x 186 Da (one payload) or 2 x 186 Da (both payloads). b) Intact mass of C16 Site I-PEG6-C2-MMAD conjugate. The metabolic product shows a 186 Da loss from one of the conjugated payloads. c) Intact mass of C16 Site A-PEG6-C2-Aur3377 conjugate. The in vivo exposed Magdalena Dorywalska et compared al, Site-Dependent Degradation conjugate shows no mass shift to the untreated compound. of a Non-Cleavable Auristatin-Based Linker-Payload in Rodent Plasma and Its Effect on ADC Efficacy, PLOS ONE | DOI:10.1371/journal.pone.0132282 July 10, 2015 doi:10.1371/journal.pone.0132282.g002 50 A comparative in vivo efficacy study confirmed that the position-dependent C-terminal residue clipping in certain PEG6-C2-MMAD conjugates results in a reduced potency in the BxPC3 mouse xenograft tumor model. Following a single intravenous dose of 10 mg/kg, the Engineered ADC (DAR4) in vitro plasma stability Control t=0h DAR 4 37 C t=72h DAR 3 DAR 2 37 C t=7d 51 LC-MS ADC characterization and beyond Assay Technology Drug or drug-linker metabolites Which species exist and are important? LC-MS/MS Catabolites (ADC or mAb) Which species exist and are important? Affinity capture LC-HRAMS DAR Distribution Assess ADC stability (in vitro / in vivo) Affinity capture LC-HRAMS or HIC Critical reagent DAR specificity Assess LBA reagents Affinity capture LC-HRAMS or HIC Intact ADC quantification Useful to measure individual DARs or catabolites? Affinity capture LC-HRAMS Intact ADC quantification PPD-ThermoFisher Scientific collaboration 53 Raw HR mass spectrum 3066.42 100 2948.52 90 Relative Abundance 80 Engineered DAR4 ADC (mcMMAF conjugate) 3194.13 70 2839.33 60 2737.99 50 40 30 2643.61 3332.96 2555.51 20 10 0 54 2500 2600 2700 2800 2900 3000 3100 3200 3300 3400 3500 3600 m/z LC-HRAMS quantitation data processing Potential options using full spectrum (FS) data acquisition + XICs and traditional peak integration - select m/z charge state(s), individual or summed - use narrow data extraction window (e.g. 5 mDa) - integrate XIC peak area + Deconvoluted composite spectrum - average/sum FS across entire chromatographic peak - peak area = total “zero-charge” state response + Deconvoluted individual FS - generate “zero-charge” state XIC - integrate XIC peak area 55 Intact analysis 100 10.53 TIC Relative Abundance Relative Abundance 100 3066.42 80 60 40 9.92 20 0 6 8 10 12 40 Relative Abundance 3332.96 2500 2800 3000 3200 3400 3600 m/z 2937.40 100 SILu™Lite (IS) 80 2719.89 60 3192.73 2532.25 20 0 2643.61 20 Time (min) 40 2839.33 60 0 14 ADC 80 2500 2700 2900 3100 m/z 3300 3500 Sample: 50 µg/mL ADC standard Method: • 30 µL mouse EDTA plasma • Add 2 µg/mL mAb IS • AC with anti-hFc agarose beads • No deglycosylation • Elute and analyze Summed XICs approach (with mAb IS) Calibration Curve (XICs: m/z 2839, 2892, 2948, 3006, 3066) Analyte/IS Area Ratio 120 100 y = 1.1969x - 0.805 r=0.997 80 60 40 20 0 0 20 40 60 ADC Conc. (ug/mL) 57 80 100 A&P - Summed XICs approach (with mAb IS) QCs QC 1-1 QC 1-2 QC 1-3 QC 1-4 QC 1-5 QC 2-1 QC 2-2 QC 2-3 QC 2-4 QC 2-5 QC 3-1 QC 3-2 QC 3-3 QC 3-4 QC 3-5 58 Analyte/IS Cal. Conc. (ug/mL) Nominal Conc. (ug/mL) Accuracy 3.00 2.79 2.49 2.51 2.56 17.3 15.2 14.9 17.8 16.0 108 106 108 102 102 1.83 1.66 1.41 1.42 1.47 13.8 12.0 11.8 14.2 12.7 89.3 88.2 89.9 84.3 84.6 1.6 1.6 1.6 1.6 1.6 12.5 12.5 12.5 12.5 12.5 100 100 100 100 100 115 104 88.1 89.0 91.9 110 96.3 94.1 114 101 89.3 88.2 89.9 84.3 84.6 CV% 11.7 8.35 3.01 Deconvoluted mass spectrum Engineered DAR4 ADC (mcMMAF conjugate) glycoforms 59 Deconvoluted MS approach (no IS) Calibration curve (DAR 4 mass 153430, 2nd glycoform) Deconvolved Signal 2.50E+08 y = 2197502x - 123876 r=0.997 2.00E+08 1.50E+08 1.00E+08 5.00E+07 0.00E+00 0 20 40 60 ADC Conc. (μg/mL) 60 80 100 A&P - Deconvoluted MS approach (no IS) QCs QC 1-1 QC 1-2 QC 1-3 QC 1-4 QC 1-5 QC 2-1 QC 2-2 QC 2-3 QC 2-4 QC 2-5 QC 3-1 QC 3-2 QC 3-3 QC 3-4 QC 3-5 61 Intensity 3040465 3171365 3384557 2532637 3566412 28494807 26480701 25990923 28256266 27660784 201954923 202279068 200604530 210591547 206835150 Cal. Conc. (ug/mL) 1.44 1.50 1.60 1.21 1.68 13.0 12.1 11.9 12.9 12.6 92.0 92.1 91.3 95.9 94.2 Nominal Conc. (ug/mL) 1.60 1.60 1.60 1.60 1.60 12.5 12.5 12.5 12.5 12.5 100 100 100 100 100 Accuracy 90.0 93.7 99.8 75.6 105.0 104.2 96.9 95.1 103.3 101.1 92.0 92.1 91.3 95.9 94.2 CV% 12.1 3.99 2.03 ADC bioanalysis other challenges Practicality issues for ADC bioanalysis Resource and time intensive • Several different assays needed for each study • Parallel sample analyses desirable, requiring multiple technologies, staff and instruments Preclinical/early development • Several species to evaluate • Multiple ADC constructs to compare • Restricted sample volumes 63 A work in progress... Final thoughts • Keeping up with biotherapeutics innovation is a challenge for analytics (CMC) and bioanalysis • Answering critical drug development questions requires both LBA and LC-MS assay technologies • Inherent ADC heterogeneity and dynamic changes in vivo require in-depth characterization • MS-based methods, including AC-LC-HRAMS, are emerging as increasingly versatile tools • Regulators are aware and likely to increase their expectations 65 Acknowledgements Acknowledgements PPD® Laboratories • Dongliang Zhan • Diego Cortes • William Mylott • Pat Bennett • • • • • Kwasi Antwi Jonathan Josephs Urban A. Kiernan Keeley Murphy Eric E. Niederkofler Genentech • Keyang Xu • Surinder Kaur Seattle Genetics • Shawna Hengel • Steve Alley Pfizer • Leo Kirkovsky Also • Jun Hosogi (JBF) • Shinobu Kudoh (JBF) …and many others 67 A story... 1.3 billion light years from Earth Two black holes merging into one Simulating eXtreme Spacetimes project (http://www.black-holes.org) Event GW150914 C. Henze/NASA Ames Research Center The “sound” of two black holes merging 14 September 2015 at 09:50:45 UTC Top panels show the measured signals in the LIGO detectors. Bottom panels show the expected signal based on numerical simulations. B. P. Abbott et al. (LIGO Scientific Collaboration and Virgo Collaboration), “Observation of Gravitational Waves from a Binary Black Hole Merger,” Phys. Rev. Lett. 116, 061102 (2016) Advanced LIGO Hanford, Washington, USA Laser Interferometer Gravitational-wave Observatory (LIGO) LIGO can detect relative length variations of order 10−21, or one ten-thousandth the diameter of a single proton Observatories and LIGO Scientific Collaboration (LSC) Kamioka Gravitational Wave Detector (KAGRA), Japan Important players in the LIGO project, from left to right: Kip Thorne of the California Institute of Technology, France A. Córdova of the National Science Foundation, Rainer Weiss of the Massachusetts Institute of Technology, David Reitze of Caltech and Gabriela González of Louisiana State University. Credit: Lexey Swall for The New York Times Thank you