Characterization of Antibody Drug Conjugates by Liquid Chromatography Mass Spectrometry

Transcription

1 Characterization of Antibody Drug Conjugates by Liquid Chromatography Mass Spectrometry Olga Friese 1, Jacquelynn Smith 1, Paul Brown 1, James Carroll 1, and Jason Rouse 2 Mass Spectrometry and Biophysical Characterization (MSBC) Pfizer Biotherapeutics PharmSci 1 St. Louis, MO and 2 Andover, MA 3 November 214

2 Overview Introduction of ADCs Structure and Role Complexities of ADC Construction Heightened Characterization of ADCs Top-down Strategy Intact Mass Analysis and Challenges Subunit Domain Analysis and Challenges Peptide Mapping: Reduced and Non-reduced Summary 2

3 Antibody Drug Conjugates ( ADCs ) Come of Age In past 5 years, more IND submissions for ADCs than in the previous 15 years Naked antibodies against only 8 distinct tumor targets currently marketed mechanism of action insufficient (ADCC & CDC) Cytotoxic payload of ADCs producing compelling efficacy data (Mylotarg, Adcetris & Kadcyla) Shapiro, M.A. et al. American Laboratory. August 27, 212. Goals Improve efficacy Improve selectivity Decrease toxicity Improve therapeutic index 3

4 Key Components of ADC: An Intricate Morphology Target Selective expression in disease Abundant expression Internalization upon binding Drug Highly potent Non-immunogenic Dormant during circulation in blood Validated mechanism of action (microtubule inhibition, DNA damage) Antibody Highly selective recognition Targets antigen found only on target cells Minimal non-specific binding ADC Linker Not altering mab characteristics Stable in plasma Labile upon internalization to release drug 4

5 Bioconjugation Chemistries: Different Level of ADC Complexity Lysine conjugation Cysteine conjugation 25/22 Lys in IgG1/IgG4 constant regions 8 Lys in κ constant region Site-specific conjugation 4 IgG1 interchain disulfide bonds 8 potential Cys conjugation sites Advantages of site specific conjugation Controlled drug loading, eliminate mixtures Improvement in pharmaceutical properties Pharmacokinetics Safety Stability Simplify analytics and development Junutula, J. R et al, Nat Biotechnol, 28, 26, Strop, P, et al Chem Biol, 213, 2,

6 Top-down Characterization Strategy for ADC by Mass Spectrometry SEC/UV/MS Intact Mass Reduction - or - IdeS digestion UHPLC/UV/MS Subunit Mapping Lys-C proteolysis RP-HPLC/UV/MS Intact mass: untreated and de-n-glycosylated Confirmation of sequence fidelity and extent of conjugation via average mass measurement of intact and de-n-glycosylated molecule Two-part subunit domain assay using reduction Confirmation of integrity and extent of conjugation via average mass measurements of LC and HC domains (triple mutants, conjugation near IdeS cleavage site) Three-part subunit domain assay using IdeS enzyme Confirmation of integrity and extent of conjugation at anticipated and unanticipated sites via monoisotopic mass measurements of scfc, LC, and Fd subunit domains Proteolytic mapping using enzymatic digestion Complexity of Mixture, Resolution of Modifications Confirmation of sequence coverage, PTM, and drug site occupancy via reduced/alkylated peptide mapping; Confirmation of disulfide connectivity via non-reduced peptide mapping (cysteine chemistry specific); Low Peptide Mapping Heightened characterization data is needed for: Reference material characterization (IND filing); Large scale process development; Lot-to-lot comparability. High 6

7 Role of Intact Mass Analysis in Characterization of ADC Intact mass analysis is used to confirm: Intended ADC primary structure with anticipated conjugation Characterization of bulk material Drug to antibody ratio (DAR) determination May or may not be accurate with LC/MS ionization efficiency might be impacted by number of drugs Drug loading distribution - fraction of antibodies containing zero, one, two,, n drugs May or may not be accurate with LC/MS ionization efficiency might be impacted by number of drugs Drug distribution of ADC by HIC Characterization of fractionated material SEC/UV/MS Intact Mass 7

8 Challenges in LC/MS Analysis of ADC: Compromise between Large and Small Molecule Analysis High LC column temperature High cone voltage High ion source temperature LC/MS technology for Small Molecules (linker payloads) Ambient column temperature Low cone voltage Low ion source temperature 8

9 Challenges Encountered During Intact Mass Analysis of ADC Using mab LC/MS Methods ADCs (Calicheamicin based ADCs) with acid labile linker payloads are unstable in the presence of strong acid modifiers (TFA) in mobile phases; Utilized.1% formic acid in water as mobile phase A and no acid in ACN ADCs with partially reduced disulfides (Cys-based ADCs) do not remain intact under denaturing conditions of osec/ms analysis: Utilized native (non-denaturing) SEC/MS using ammonium acetate as a mobile phase; Increased in-source pressure and collision cell voltage for collisional stabilization (cooling) of the ions ADCs with surface exposed linker payloads (site-specific ADCs) are susceptible to in-source fragmentation under ESI conditions (high cone voltages and/or source temperatures): Utilized lower cone voltages 9

10 Structure of ADC with Acid Labile Linker Calicheamicin AcBut Linker Acid labile DMH Linker Acid labile linker poses a challenge to many analytical techniques 1

11 % % Mass Spectrum of Intact ADC with Acid Labile Linker: Impact of Mobile Phase ph (+/-TFA) %TFA +.1% FA +3 Linker GF/ GF Linker GF/GF +4 Linker GF/GF Linker GF/ GF +3 Calich GF/ GF Calich GF/ GF Calich GF/ GF Calich GF/ GF In the presence of strong acid (TFA) acid labile linker in calicheamicin payload is cleaved leaving only a 24 Da linker In the absence of strong acid (TFA) acid labile linker in calicheamicin payload is not cleaved leaving intact calicheamicin attached to the mab mass 11

12 Possible Structures of ADC with Conventional Cysteine Conjugation Chemistry mab2 mab2 L1 + HHL1 L1 + HHL3 L1 + HHL3 2*L1 + HH2 2*HL2 Partial Reduction Conjugation 2*L1 + HH4 2*L1 + HH4 L1 + H3 + HL2 Partial reduction followed by conjugation leads to multiple ADC isoforms, in some case no interchain disulfide bonds 2*L1 + 2*H3 Sun, et al., Bioconjugate Chem. 25, 16, (Examples of potential ADC isoforms) 12

13 Zero-charged Deconvoluted Mass Spectra of Cys- Conjugated ADC under Denaturing osec/ms % % UV 214nm Mobile Phase.1% TFA, 4% AcN Under denaturing conditions of LC/MS only covalent subunits of ADC are observed GC3 ADC 1 HL + 2drugs GC3 ADC 1 L+1drug Mass Spectra mass H+3drugs H+1drug HH+2drugs HHL+1drug mass

14 Zero-charged Deconvoluted Mass Spectra of Cys- Conjugated ADC under Native SEC/MS % % 1 Mobile Phase Ammonium acetate ph 7. No acid; Intact No organic; (+ L/P) GF/GF (+2 L/P) GF/GF UV 214nm GF/GF (+4 L/P) GF/G1F G1F/G1F Intact Intact ADC molecule with, 2, 4, 6, and 8 L/P is observed under nondenaturing SEC/MS conditions (+6 L/P) GF/GF GF/G1F G1F/G1F (+8 L/P) GF/GF mass (+4 L/P) de-n-glycosylated (+2 L/P) (+6 L/P) (+ L/P) (+8 L/P) mass

15 Structure of ADC with the Heavy Chain C-terminal Conjugation Site O specific acyl donor + H 3 N + NH 2 glutamine tag (LLQGA) Transglutaminase nonspecific acyl acceptor payload TG O N H ADC Transglutaminase (TG) from Streptoverticillium mobaraense catalyzes the formation of covalent bond between glutamine side chain and a primary amine Surface accessible glutamine tag LLQGA at the C-terminus of the heavy chain results in 2 drugs conjugated to the mab AGQLL LLQGA AGQLL LLQGA mab ADC Strop, P, et al Chem Biol, 213, 2,

16 Mass Spectra of ADC with the Surface Exposed Linker Payload (TG Chemistry): In-source Fragmentations Xevo QTOF CV 4V * * mab(2) Optimization of ESI source parameters to reduced in-source fragmentation: cone voltage; source temperature; source pressure (backing pressure); flow rate CV 3V * In-source fragmentation is the most sensitive to cone voltages; In-source fragmentation is reduced by lowering cone voltage, adduct formation is increased; * in source fragmentation 16

17 Role of Subunit Domain Analysis in Characterization of ADC Subunit domain analysis is used to confirm: Intended ADC primary structure with anticipated conjugation Quick and reliable sequence verification via accurate mass with 1% coverage Localize major/minor/trace modifications to subunits/domains; Rapid scfc N-glycan profiling 3 min 37 C ph 6.6 Guanidine DTT 9 min 37 C 17

18 Three-Part Subunit Domain Mapping of Cysteine-Conjugated ADC: Confirmation of Conjugation at Intended Sites Intens. [mau] 4 scfc() LC() Fd () Notch 3 DSI Ref Material _25.d UV 215nm Profiles 3 2 mab Ides Enzyme -S-S - Reduction Hinge Region Sequences of IgGs 1 Intens. [mau] scfc Ox ADC scfc agly Time [min] scfc() scfc Ox scfc agly LC() IdeS IdeS LC(1) Fd Ox Fd () Fd - water Fd (1) Fd (1)- water Notch 3 DS _248.d Fd (2b) Fd (2c) Fd (2a) Fd (3) Number in ( ) indicates number of drugs attached Time [min] 1% sequence coverage for mab and ADC Complete, definitive assessment of intended ADC covalent structure (integrity) Quantitative assessment of the extent of conjugation for each subunit/domain 18

19 Mass Spectra of scfc and LC in Three-Part Subunit Domain Mapping of Cysteine Conjugated ADC scfc Intens. x GF 1+ ' G1F 1+ ' MS, min, 1%=2427, Deconvoluted (MaxEnt) scfc with no drug conjugated This is expected since this subunit does not contain any interchain Cys.5. Man5 1+ ' ' m/z G2F G2F + NeuAc LC Intens. [%] 1 8 LC() 1+ ' Da Notch-3 ADC Reduced CN_1356.d: +MS, min, 1%=1874, Deconvoluted (MaxEnt) [%] LC(1) Notch-3 ADC Reduced CN_1356.d: +MS, min, 1%=1171, Deconvoluted (MaxEnt) 1+ ' LC with one drug conjugated This is expected since this subunit contains 1 interchain Cys m/z 19

20 Mass Spectra of Fd in Three-Part Subunit Mapping of Cysteine Conjugated ADC Intens. [%] 4 2 [%] 8 6 Fd () 1+ ' Da Fd (1) 1+ ' Notch-3 ADC 3-part_1359.d: +MS, min, 1%=724, Deconvoluted (MaxEnt) Notch-3 ADC 3-part_1359.d: +MS, min, 1%=764, Deconvoluted (MaxEnt) 4 2 [%] [%] Da Notch-3 ADC 3-part_1359.d: +MS, min, 1%=816, Deconvoluted (MaxEnt) 1+ ' ' Fd (2) 1341 Da Fd (3) Notch-3 ADC 3-part_1359.d: +MS, min, 1%=769, Deconvoluted (MaxEnt) 1+ ' m/z Fd with one, two and three drugs conjugated This is expected since this subunit contains 3 interchain Cys 2

21 Challenges Encountered During RP-HPLC/MS Analysis of ADCs with Highly Hydrophobic Subunits Trop 2 ADC RN927 1mm_1877.d Waters C4 BEH Column LC() scfc(1) Trop2 TS1 ADC_1646.d: Base Peak UV Chromatogram, 215 nm Fd domain did not elute from C4 column due to its hydrophobic nature Fd () Trop2 TS1 32ADC_1646.d: Base Peak 34 UV Chromatogram, nm 38 Time [min] Agilent C3 Column LC() scfc(1) Fd () C3 column separation resulted in a partial elution of Fd domain Trop2 RN927 DS 7mgml_2392.d: Base Peak UV Chromatogram, 215 nm LC() Separation Method Technology C2 (MEB2) Column Trop2 RN927 DS 7mgml_2392.d: Base Peak UV Chromatogram, 215 nm scfc(1) Fd () Low carbon content C2 column resulted in a complete elution of Fd domain, however peak shape is broad and shelf life of column is very short Time [min] Note: separation conditions are different between experiments 21

22 LC Ox LC + 1 Linker LC + 1 Linker HC + 1 Linker HC + 2 Linker HC + 2 Linker HC + 3 Linker HC + 3 Linker HC + 3 Linker HC + 3 Linker HC + 4 Linker HC(D/P ) LC Ox, Deamidation HC (D/P 1 274) Two-Part Subunit Mapping of Lysine-Conjugated ADC: Confirmation of Conjugation at Intended Sites Intens. [mau] mab HC SCRx4 Red DSI _223.d 1 LC 75 -S-S- Reduction 5 25 [mau] ADC Three-part subunit domain data is very complex; IdeS digestion is incomplete due to the nearby conjugation at the IdeS cleavage site; Two-part subunit assay is preferred. It provides good resolution of HC with various number of linkers N-acetyl epsilon calicheamicin LC SCRx4 Red DS _227.d Time [min] HC + linker(s) 22

23 uau HK12 +1 drug1 LK15+1 drug HK13HK14+1drug HK13HK14+1drug HK3 # HK27# HK27# HK15ox LK7 HK1^ HK13 HK4 * uau LK5succ * HK13HK14+2drugs HK3, HK31 C- term ext HK27 HK15, HK28 HK17HK18 GF HK2 HK13HK14 LK5 HK7HK8 uau HK9, LK4, HK21 HK19 HK12 HK23 LK12 HK1HK11, HK11 LK15 HK29 LK6 LK1 LK9 HK3, Hk31 Pam HK31 HK22 LK14 HK24HK25, HK25 HK6 LK1^ HK26 LK11, HK5 HK16 Reduced and Alkylated Lys-C Mapping of ADC: Monitoring Sites of Conjugation LK15(1) HK12(1) HK13HK14(1, or,1 or 2) All linker payload-conjugated species are as expected; They correspond to addition of linker payload to Cys involved in the interchain disulfide bonds; No unexpected linker payload conjugation sites are observed; Due to hydrophobic nature of linker payload they elute late in the peptide map RT: Time (min) min LK8 HK1-85 min Time (min) LK1 LK2 NL: 7.51E5 nm= PDA 21348OV F5 mab NL: 6.74E5 nm= PDA 21348ovf 6 ADC NL: 7.51E5 nm= PDA 21348OV F5 mab NL: 6.74E5 nm= PDA 21348ovf 6 ADC 23

24 R/A Lys-C Mapping of Cysteine Conjugated ADC at High ph Leads to Hydrolysis of Linker Payload RT: min HK13HK Da +1 drug UV Profiles HK13HK drug ph 8.2 ph 7.5 NL: 1.59E5 Channel A UV 21342O VFJNS2_ NL: 1.82E5 Channel A UV 21348o vf6 Maleimide conjugated ADCs undergo hydrolysis at high ph 8 75 O 7 S N Payload 65 O Time (min) HK13HK Da+ 1 drug HK13HK drug m/z Mass Spectra NL: 4.1E ovf6_xtract#285 4 RT: AV: 1 T: FTMS + p ESI Full ms [ ] S NL: 5.85E ovf6_xtract#289 6 RT: 148. AV: 1 T: FTMS + p ESI Full ms [ ] CO 2 H H N O +H 2 O Payload ph optimal for enzymatic digestions of mabs are not always suitable for ADCs, especially for those with hydrolysable linker payload 24

25 Characterization Strategy of Alkylated Non-Reduced Peptide Maps of Cysteine-Conjugated ADC Alkylate Cys to prevent SH mispairing during proteolytic digestion; Look for expected disulfide linked peptides predicted for IgG1 connectivity; Look for interchain cysteine-containing peptides with drug payload attachment; Look for potential mispaired peptides for interchain cysteine-containing peptides with drug; Look for levels of free cysteines at every cysteine-containing peptide mass shifted due to alkylation; 25

26 Disulfide Linked Peptides in Non-Reduced Tryptic Map of Cysteine Conjugated ADC Intrachain Disulfide Linked Peptides Interchain Disulfide Linked Peptides trypsin Light Chain LT2-ss-LT7 LT11-ss-LT18 LT2-drug HT19-drug LT2-ss-HT19* Hinge region HT2-(2x)ss-HT2* IgG1 ADC Heavy Chain HT2-ss-HT1 HT14-ss-HT15 HT22-ss-HT28 HT36-ss-HT41 *Observed only if partially reduced HT2-drug HT2-drug-ss- HT2 HT2-drug-ss- HT2-drug* HT2-2xdrugs or 26

27 Non-Reduced Peptide Mapping of Cysteine-Conjugated ADC: EMC for Disulfide and Drug Linked Peptides RT: x x1 6 HT22-ss-HT HT36-ss-HT41 HT2-ss-HT LT11-ss-LT18 LT2-ss-LT HT19-drug HT14-ss-HT HT2-(2x)ss-HT LT2-drug Disulfide-linked peptides All interchain disulfide-linked peptides observed except LT2-ss-HT19; No HT2-drug is observed; No HT2-drug-ss-HT2 is observed; No HT2-drug-ss-HT2-drug is observed: Drug-conjugated peptides LT - light chain tryptic peptide HT - heavy chain tryptic peptide HT2-2xdrug Time (min) 27

28 Non-Reduced Peptide Mapping of Cysteine Conjugated ADC: EMC for Mispaired Peptides RT: Mispaired hinge region HT2-drug peptide ~ 4% by ion intensities HT2-drug-ss-LT HT2-drug-ss-LT HT2-drug-ss-HT HT2-drug-ss-HT19 6x Time (min) 28

29 Heightened Characterization of ADCs by MS is Driven by Conjugation Chemistry Methods Conventional Chemistry Site-Specific Chemistry Native SEC/MS Lysine Cysteine Glutamine Cysteine Organic SEC/MS Two-Part Subunit Domain Mapping (LC and HC) Three-Part Subunit Domain Mapping (scfc, LC, and Fd ) Reduced/Alkylated Peptide Mapping Non- Reduced/Alkylated Peptide Mapping - Only if linker payload is acid sensitive 29

30 Summary Mass Spectrometry is a powerful tool for heightened characterization of ADCs A tiered top-down analysis approach utilizing the latest technologies (UHR MS) can provide rapid, definitive product quality information at each stage of development Intact mass - Confirms sequence fidelity and extent of conjugation Subunit domain assay - Confirms integrity and extent of conjugation at the scfc, LC, and Fd subunit domain level Proteolytic mapping using enzymatic digestion - Confirms sequence coverage, PTM, and drug site occupancy at the peptide level Improvements in productivity of ADC s characterization can be realized when several new technologies merge IdeS or other proteolytic enzymes, UHPLC, and UHR MS have revolutionized ADCs characterization, and coming next is automated data analysis with report generation 3

31 Acknowledgement Projects Team members Jeff Borgmeyer - analytical leader Heyi Li analytical leader Jason Starkey analytical leader Qingping(Jim) Jiang Lawrence Chen Debra Meyer Scott Sprague Bill Romanow Jennyfer Smith Libbey Yates Tom Schomogy Margaret Ruesch VP of ARD Steve Max project leader Mass Spectrometry group members Jacquelynn Smith Paul Brown Justin Sperry Kathleen Cornelius Matthew Thompson James Carroll group leader Jason Rouse Sr. director Oncology RU project team members Rinat team members 31