Dimethylacetamide → 2,2'-Dipyridyl disulfide

According to the general two-step method, 10.4 mg (0.071 μmol, 1.174 ml) of naked antibody hu2H11 with an initial concentration of 8.86 mg/ml are treated with 7 eq. of the N-hydroxy-succinimidyl ester of 4-(2-pyridyldithio)butanoic acid (0.16 mg, 0.496 μmol) dissolved in 34.2 μl of DMA such that the final antibody concentration is 8 mg/ml in the mixture. After purification, 2.2 ml of modified antibody hu2H11 at a concentration of 4.28 mg/ml (9.42 mg, 91%) are Obtained with, on average, 4.68 pyridyldisulfide molecules per antibody. 1.68 ml (7.2 mg, 0.049 μmol) of modified antibody hu2H11 are treated with 1.03 mg of (E)-(3S,10R,16S)-10-(3-chloro-4-methoxy-benzyl)-3-isobutyl-16-[(S)-1-((2R,3R)-3-{4-[4-(4-mercapto-4-methylpentanoyl)piperazin-1-ylmethyl]phenyl}oxiranyl)ethyl]-6,6-dimethyl-1,4-dioxa-8,11-diazacyclohexadec-13-ene-2,5,9,12-tetraone (compound Ex. 1, 1.148 μmol) dissolved in 101.2 μl of DMA. After purification on Superdex in the presence of 10% NMP and concentration on Amicon Ultra-15, the final change of buffer is performed in an aqueous pH 6.5 buffer containing 0.01 M of phosphate and 0.14 M of NaCl. 1.5 ml of conjugate Ex. 9 are thus obtained at a concentration of 1.1 mg/ml with, on average, 3 cryptophycin derivatives per antibody (HRMS) and a monomer purity of 99.9%.

1. customAfter purification, 2.2 ml of modified antibody hu2H11 at a concentration of 4.28 mg/ml (9.42 mg, 91%)

ORD record

ord-d0e212f018d446bbb806bab67be2734b

Source dataset

uspto-grants-2015_02

DOI

10.6084/m9.figshare.5104873.v1

Patent number

US08952147B2