Patent ID: 9612247
Date: 2017-04-04
CPC Classifications: C07K,G01N

Claim:
1. A method of determining the optimal relative positioning of an inner antibody variable domain and an outer antibody variable domain in a multispecific binding protein, the method comprising: a) providing a first antibody variable domain and a second antibody variable domain; b) contacting the first antibody variable domain and the second antibody variable domain with a first predetermined concentration of a denaturing agent selected from the group consisting of a chaotropic agent and a detergent, wherein the denaturing agent does not cause complete denaturation of the first antibody variable domain and the second antibody variable domain; c) contacting the first antibody variable domain and the second antibody variable domain with a first detectable sulfhydryl-reactive alkylating reagent under reducing conditions such that all reducible sulfhydryl groups in the first antibody variable domain and the second antibody variable domain are alkylated; d) contacting the first antibody variable domain and the second antibody variable domain with a second predetermined concentration of the denaturing agent that is greater than the first predetermined concentration and causes complete denaturation of the first antibody variable domain and the second antibody variable domain; e) contacting the first antibody variable domain and the second antibody variable domain with a second detectable sulfhydryl-reactive alkylating reagent that is distinguishable from the first detectable sulfhydryl-reactive alkylating reagent under reducing conditions such that all reducible sulfhydryl groups in the first antibody variable domain and the second antibody variable domain are alkylated; f) measuring the relative amount of the first and second detectable sulfhydryl-reactive alkylating reagents incorporated into the first antibody variable domain and the second antibody variable domain relative to a suitable control, wherein the relative amount of incorporation of the first sulfhydryl-reactive alkylating reagent inversely correlates with the stability of the first antibody variable domain and the second antibody variable domain; g) determining whether the first antibody variable domain is more or less stable than the second antibody variable domain, thereby identifying a less stable antibody variable domain and a more stable antibody variable domain; and h) identifying the less stable antibody variable domain as being suitable as an inner variable domain in a multispecific binding protein and the more stable antibody variable domain as being suitable as an outer variable domain in the multispecific binding protein, thereby determining the optimal relative positioning of the first and the second antibody variable domain in the multispecific binding protein wherein the chaotropic agent is selected from the group consisting of guanidine hydrochloride, guanidine thiocyanate, urea, thiourea, sodium trichloroacetate, trifluoroethanol (TFE), butanol, ethanol, lithium perchlorate, lithium acetate, magnesium chloride, phenol, and propanol and wherein the detergent is selected from the group consisting of Octyl-b-Dglucopyranoside, Decyl-b-D-1-thioglucopyranoside, Octyl-b-Dthioglucopyranoside, Digitonin, Dimethyldecylphosphine, oxide (APO-10), Dodecyldimethylphosphine, oxide (APO-12), octylphenoxypolyethoxyethanol, N-octyl glucoside, N-Octanoyl-N-methylglucamine (MEGA-8), N-Nonanoyl-N-methylglucamine (MEGA-9), N-Decanoyl-N-methylglucamine (MEGA-10), 4-Nonylphenyl-polyethylene glycol, Polyoxyethylene-polyoxypropylene block copolymer, Saponin, Polyethylene glycol tert-octylphenyl ether, polysorbate 20, polysorbate 40, polysorbate 80, Sodium cholate, Sodium deoxycholate, Sodium glycocholate, Sodium taurocholate, Sodium taurodeoxycholate, N-Lauroylsarcosine, Lithium dodecyl sulfate, Sodium dodecyl sulfate (SDS), Hexadecyltrimethyl ammonium bromide (CTAB), Trimethyl(tetradecyl)ammonium bromide (TTAB), ASB-14 (amidosulfobetaine-14), ASB-16 (amidosulfobetaine-16), C7BzO, CHAPS, CHAPSO, N,N-Dimethyl-N-dodecylglycine betaine, 3-(N,N-Dimethyloctylammonio)propanesulfonate inner salt (SB3-8), 3-(Decyldimethylammonio)propanesulfonate inner salt (SB3-10), 3-(Dodecyldimethylammonio)propanesulfonate inner salt (SB3-12), 3-(N,N-Dimethylmyristylammonio)propanesulfonate (SB3-14), 3-(N,N-Dimethylpalmitylammonio)propanesulfonate (SB3-16), 3-(N,N-Dimethyloctadecylammonio)propanesulfonate (SB3-18), 3-(1-Pyridinio)-1-propanesulfonate (NDSB 201), and 3-(Benzyldimethylammonio)propanesulfonate (NDSB 256).