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The maytansinoid antibody-drug conjugates (ADCs) in clinical development for cancer therapy each contain a derivative of the microtubule-targeting agent, maytansine, covalently attached to the antibody via an engineered linker. A sample of any of these conjugates contains molecules with different numbers of maytansinoid molecules, or "drug" loads, the relative abundance of which can be determined by mass spectrometry. We examined the accuracy of the Poisson distribution and the binomial distribution in predicting the relative abundance of species with different drug loads for three antibody-maytansinoid conjugates with different antibodies and linker-maytansinoid pairings. We used variance, calculated from the experimental mass distribution data, as the parameter to determine the optimal value n of the binomial distribution number of trials. The accuracy of the Poisson distribution in predicting distribution of the species abundance in these conjugates varied among the conjugates. In contrast, the accuracy of the binomial distribution was similar for all three conjugates and comparable to the best accuracy of the Poisson distribution, as supported by a paired t-test.

A reversed-phase high-performance liquid chromatography (RP-HPLC) method was developed for detection and quantification of free maytansinoid drug in disulfide-linked conjugates between monoclonal antibodies and the maytansinoid drug DM1 (MAb-DM1). Mobile phases and gradient conditions were optimized for separation of several DM1-related free drug species from MAb-DM1 conjugates. The selectivity, linearity, and reproducibility of the method are reported. Reduction of the disulfide-linked DM1 followed by RP-HPLC allowed estimation of purity of MAb-linked DM1 as well as recovery of L-DM1. The method was also used to estimate drug per MAb ratios, which were consistent with those determined by UV spectroscopy.

Summary The rate of hydrolysis of suboptimal concentrations of N-benzoyl- L ¯ ¯ -phenylanalyl- L ¯ ¯ -valyl- L ¯ ¯ -arginine- p- nitroanilide hydrochloride (S-2160) by activated bovine plasma protein C is greatly enhanced by the presence of monovalent cations. Of the cations tested, at their maximal effective concentrations, the order of the ability to enhance the S-2160 amidase activity of activated protein C is Cs+ > Rb+ > K+ > NH4+ > Na+ > Li+. Progressive substitution of methyl groups for hydrogen in NH4+ leads to a concomitant decrease in the ability of these compounds to enhance the amidase activity of activated protein C. Divalent cations, at low concentrations, weakly enhance the amidase activity of activated protein C and show a decreased tendency in this regard at high concentrations. The above observations also apply to hydrolysis of the substrate α -N-tosyl- L ¯ ¯ -arginine methyl ester.

The binding of calcium to human plasma fibronectin has been measured by equilibrium dialysis at 25 degrees in 0.1 M NaCl 50mM Tris HCL, pH 7.4. Curve fitting of the binding data indicates that fibronectin has two strong calcium binding sites per chain (Mr 220,000), KD = 1.3 mM and approximately 12 weak sites, KD = 2.3 mM. No significant displacement of bound calcium by magnesium was observed at magnesium concentrations up to 1 mM. Calcium binding to a pair of tryptic fragments of fibronectin (Mr congruent to 160,000 and 180,000) that bind to gelatin has also been investigated. These fragments have a single class of calcium binding sites, with 2.2 sites per chain, KD = 1.1 mM. Negligible calcium binding to tryptic fragments derived from other regions of the fibronectin molecule was observed.

The binding isotherms of Ca 2+ and Sr 2+ to human blood coagulation Factor IX have been obtained at 25 °C and pH 7.4. In the case of both cations, a Scatchard plot of the data reveals that a single class of binding sites exist. For Ca 2+ , a total of 16.0 ± 1.0 sites, of K D 7.3 ± 0.2 × 10 −4 m , are present on human Factor IX. Similar analysis of the Sr 2+ data indicates that Factor IX contains 11.0 ± 1.0 binding sites, with a K D of 1.9 ± 0.1 × 10 −3 m . Both Sr 2+ and Mn 2+ effectively displace Ca 2+ from human Factor IX; whereas Mg 2+ is considerably less potent in this regard. Conversely, Ca 2+ is capable of nearly complete displacement of Sr 2+ from its binding sites on human Factor IX. The activation of human Factor IX, by human Factor XIa, shows a complex dependence on the Ca 2+ concentration. Sr 2+ can substitute for Ca 2+ in this activation process. Mn 2+ cannot, in itself, substitute for Ca 2+ in activation of Factor IX, but does significantly enhance the activation of Factor IX by Factor XIa at suboptimal levels of Ca 2+ . The rate of activation of human Factor IX by the coagulant protein of Russell's viper venom also shows a dependence on the presence of divalent cations. Here, however, a rigid specificity is not noted, since Ca 2+ , Sr 2+ , and Mn 2+ all allow activation to proceed equally well.