Multiplexed total antibody and antibody-conjugated drug quantification assay

1 . A method of quantifying both an antibody-conjugated drug concentration and a total antibody concentration of an antibody-drug conjugate (ADC) in a single analysis sample, the method comprising:

(a) contacting the ADC comprising an antibody, a drug moiety, and a linker moiety that attaches the antibody to the drug moiety, wherein the linker moiety of the ADC is a disulfide linker, and which is bound to an affinity capture media selected from the group consisting of: (1) bead- or resin-supported Protein A/G, and (2) target antigen-paramagnetic bead capture media, with (i) a reductant that reduces the antibody portion of the ADC to form a denatured antibody portion of the ADC, wherein the reductant is selected from the group consisting of dithiolthreitol (DTT), 2-mercaptoethanol, and tris(2-carboxyethyl)phosphine (TCEP), whereby the drug moiety is separated from the denatured antibody portion of the ADC; (ii) at least one denaturant selected from the group consisting of formamide, dimethylformamide, acetonitrile, SDS, urea, an acid labile surfactant, a decyl furanyl sulfonic acid salt, and guanidine HCl; and (iii) at least one chemical selected from the group consisting of methanol, ethanol, HCl, ammonium bicarbonate, Tris buffer, HEPES, ammonium acetate, and acetonitrile; whereby the antibody portion of the ADC is denatured;

(b) digesting the denatured antibody portion of the ADC bound to the affinity capture media to form a digested ADC peptide mixture from the single analysis sample with a proteolytic enzyme selected from the group consisting of trypsin, chymotrypsin, papain, pepsin, LysN, LysC, AspN, GluC, ArgC, and PNGaseF; and

(c) eluting from the affinity capture media and analyzing the digested ADC peptide mixture by LC-MS/MS to detect at least one antibody signature peptide and the drug moiety in the single analysis sample.

2 . The method of claim 1 , wherein the ADC is suspended in a matrix selected from the group consisting of a buffer, whole blood, serum, plasma, cerebrospinal fluid, saliva, urine, lymph, bile, feces, sweat, vitreous, tears, and tissue, prior to the contacting step.

3 . The method of claim 1 , wherein the ADC is enriched by a technique selected from the group consisting of size exclusion chromatography, dialysis, selective precipitation, differential centrifugation, filtration, gel electrophoresis, liquid chromatography, reversed-phase chromatography, immunoprecipitation, spin columns including protein A and protein G, and desalting, prior to the contacting step.

4 . The method of claim 1 , further comprising washing ADC bound to the affinity capture media to reduce non-antibody proteins in contact with the ADC.

5 . The method of claim 1 , wherein the affinity capture media is bead- or resin-supported Protein A/G.

6 . The method of claim 5 , wherein the bead- or resin-supported Protein A/G is Protein A magnetic beads.

7 . The method of claim 1 , further comprising dephosphorylating the ADC bound to the affinity capture media.

8 . The method of claim 1 , wherein the reductant is tris(2-carboxyethyl)phosphine (TCEP).

9 . The method of claim 1 , wherein the denaturant is an acid labile surfactant.

10 . The method of claim 1 , wherein the total antibody concentration of the ADC is calculated from the analysis of the digested ADC peptide mixture.

11 . The method of claim 1 , wherein the antibody-conjugated drug concentration of the ADC is calculated from the analysis of the digested ADC peptide mixture.

12 . The method of claim 1 , wherein the average drug-to-antibody ratio (DAR) of the ADC is calculated from the analysis of the digested ADC peptide mixture.

13 . The method of claim 1 , wherein the drug moiety is selected from the group consisting of a maytansinoid, dolastatin, auristatin, calicheamicin, pyrrolobenzodiazepine (PBD), PNU-159682, anthracycline, duocarmycin, vinca alkaloid, taxane, trichothecene, CC1065, duocarmycin, camptothecin, and elinafide.

14 . The method of claim 1 , wherein the antibody portion of the ADC is an antibody fragment.

15 . The method of claim 1 , wherein the antibody portion of the ADC is a human or humanized antibody.

16 . The method of claim 1 , wherein the antibody portion of the ADC is an antibody which binds to one or more tumor-associated antigens or cell-surface receptors selected from the group consisting of (1)-(53):

(1) BMPRIB (bone morphogenetic protein receptor-type IB);

(2) E16 (LAT1, SLC7A5);

(3) STEAP1 (six transmembrane epithelial antigen of prostate);

(4) MUC16 (0772P, CA125);

(5) MPF (MPF, MSLN, SMR, megakaryocyte potentiating factor, mesothelin);

(6) Napi2b (NAPI-3B, NPTIIb, SLC34A2, solute carrier family 34 (sodium phosphate), member 2, type II sodium-dependent phosphate transporter 3b);

(7) Sema 5b (FLJ10372, KIAA1445, Mm.42015, SEMA5B, SEMAG, Semaphorin 5b Hlog, sema domain, seven thrombospondin repeats (type 1 and type 1-like), transmembrane domain (TM) and short cytoplasmic domain, (semaphorin) 5 B);

(8) PSCA hlg (2700050C12Rik, C530008O16Rik, RIKEN cDNA 2700050C12, RIKEN cDNA 2700050C12 gene);

(9) ETBR (Endothelin type B receptor);

(10) MSG783 (RNF124, hypothetical protein FLJ20315);

(11) STEAP2 (HGNC_8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, prostate cancer associated gene 1, prostate cancer associated protein 1, six transmembrane epithelial antigen of prostate 2, six transmembrane prostate protein);

(12) TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptor potential cation channel, subfamily M, member 4);

(13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, teratocarcinoma-derived growth factor);

(14) CD21 (CR2 (Complement receptor 2) or C3DR (C3d/Epstein Barr virus receptor) or Hs 73792);

(15) CD79b (CD79B, CD79β, IGb (immunoglobulin-associated beta), B29);

(16) FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain containing phosphatase anchor protein 1a), SPAP1B, SPAP1C);

(17) HER2;

(18) NCA;

(19) MDP;

(20) IL20Rα;

(21) Brevican;

(22) EphB2R;

(23) ASLG659;

(24) PSCA;

(25) GEDA;

(26) BAFF-R (B cell-activating factor receptor, BLyS receptor 3, BR3);

(27) CD22 (B-cell receptor CD22-B isoform);

(28) CD79a (CD79A, CD79a, immunoglobulin-associated alpha);

(29) CXCR5 (Burkitt's lymphoma receptor 1);

(30) HLA-DOB (Beta subunit of MHC class II molecule (Ia antigen));

(31) P2X5 (Purinergic receptor P2X ligand-gated ion channel 5);

(32) CD72 (B-cell differentiation antigen CD72, Lyb-2);

(33) LY64 (Lymphocyte antigen 64 (RP105), type I membrane protein of the leucine rich repeat (LRR) family);

(34) FcRH1 (Fc receptor-like protein 1);

(35) FcRH5 (IRTA2, Immunoglobulin superfamily receptor translocation associated 2);

(36) TENB2 (putative transmembrane proteoglycan);

(37) PMEL17 (silver homolog; SILV; D12S53E; PMEL17; SI; SIL);

(38) TMEFF1 (transmembrane protein with EGF-like and two follistatin-like domains 1; Tomoregulin-1);

(39) GDNF-Ral (GDNF family receptor alpha 1; GFRA1; GDNFR; GDNFRA; RETL1; TRNR1; RET1L; GDNFR-alpha1; GFR-ALPHA-1);

(40) Ly6E (lymphocyte antigen 6 complex, locus E; Ly67, RIG-E,SCA-2,TSA-1);

(41) TMEM46 (shisa homolog 2 ( Xenopus laevis ); SHISA2);

(42) Ly6G6D (lymphocyte antigen 6 complex, locus G6D; Ly6-D, MEGT1);

(43) LGR5 (leucine-rich repeat-containing G protein-coupled receptor 5; GPR49, GPR67);

(44) RET (ret proto-oncogene; MEN2A; HSCR1; MEN2B; MTC1; PTC; CDHF12; Hs.168114; RET51; RET-ELE1);

(45) LY6K (lymphocyte antigen 6 complex, locus K; LY6K; HSJ001348; FLJ35226);

(46) GPR19 (G protein-coupled receptor 19; Mm.4787);

(47) GPR54 (KISS1 receptor; KISS1R; GPR54; HOT7T175; AXOR12);

(48) ASPHD1 (aspartate beta-hydroxylase domain containing 1; LOC253982);

(49) Tyrosinase (TYR; OCA1A; OCALA; tyrosinase; SHEP3);

(50) TMEM118 (ring finger protein, transmembrane 2; RNFT2; FLJ14627);

(51) GPR172A (G protein-coupled receptor 172A; GPCR41; FLJ11856; D15Ertd747e);

(52) CD33; and

(53) CLL-1.