Detection and Characterization of In Vitro Payload-Containing Catabolites of Noncleavable Antibody-Drug Conjugates by High-Resolution Mass Spectrometry and Multiple Data Mining Tools.

The formation and accumulation of payload-containing catabolites (PCCs) from a noncleavable antibody-drug conjugate (ADC) in targeted and normal tissues are directly associated with the therapeutic effect and toxicity of the ADC, respectively. Understanding the PCC formation is important for supporting the payload design and facilitating preclinical evaluation of ADCs. However, detection and identification of PCCs of a noncleavable ADC are challenging due to their low concentrations and unknown structures. The main objective of this study was to develop and apply a generic liquid chromatography-high-resolution mass spectrometry (LC-HRMS) method for profiling PCCs in vitro. Noncleavable ADCs, ado-trastuzumab emtansine (T-DM1) and ADC-1, were incubated in liver lysosomes, liver S9, and/or cancer cells followed by data acquisition using LC-HRMS. Profiling PCCs mainly relied on processing LC-HRMS datasets using untargeted precise and thorough background subtraction (PATBS) processing and targeted product ion filtering (PIF). As a result, 12 PCCs of T-DM1 were detected and structurally characterized in human liver lysosomal incubation, a majority of which consisted of 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (MCC)-DM1 and a few amino acids. Additionally, the incubation of ADC-1 in human, rat, and monkey liver S9 and cancer cells generated one major and three very minor PCCs, verifying the payload design. The results demonstrate that PATBS enabled the comprehensive profiling of PCCs regardless of their molecular weights, charge states, and fragmentations. As a complementary tool, PIF detected specific PCCs with superior sensitivity. The combination of the in vitro metabolism systems and the LC-HRMS method is a useful approach to profiling in vitro PCCs of noncleavable ADCs in support of drug discovery programs. SIGNIFICANCE STATEMENT: Profiling in vitro payload-containing catabolites (PCCs) of a noncleavable antibody-drug conjugate (ADC) is important for optimization of the payload design and preclinical evaluation of ADC. However, currently used analytical approaches often fail to quickly provide reliable PCC profiling results. The work introduces a new liquid chromatography high resolution mass spectrometry method for comprehensive and rapid detection and characterization of PCCs released from a noncleavable ADC in liver lysosomes and S9 incubations.

GACN: Generative Adversarial Classified Network for Balancing Plant Disease Dataset and Plant Disease Recognition.

Plant diseases are a critical threat to the agricultural sector. Therefore, accurate plant disease classification is important. In recent years, some researchers have used synthetic images of GAN to enhance plant disease recognition accuracy. In this paper, we propose a generative adversarial classified network (GACN) to further improve plant disease recognition accuracy. The GACN comprises a generator, discriminator, and classifier. The proposed model can not only enhance convolutional neural network performance by generating synthetic images to balance plant disease datasets but the GACN classifier can also be directly applied to plant disease recognition tasks. Experimental results on the PlantVillage and AI Challenger 2018 datasets show that the contribution of the proposed method to improve the discriminability of the convolution neural network is greater than that of the label-conditional methods of CGAN, ACGAN, BAGAN, and MFC-GAN. The accuracy of the trained classifier for plant disease recognition is also better than that of the plant disease recognition models studied on public plant disease datasets. In addition, we conducted several experiments to observe the effects of different numbers and resolutions of synthetic images on the discriminability of convolutional neural network.

Effect of fast freeze-thaw cycles on mechanical properties of ordinary-air-entrained concrete.

Freezing-thawing resistance is a very significant characteristic for concrete in severe environment (such as cold region with the lowest temperature below 0°C). In this study, ordinary-air-entrained (O-A-E) concrete was produced in a laboratory environment; the compressive strength, cubic compressive strength of C50, C40, C30, C25, and C20 ordinary-air-entrained concrete, tensile strength, and cleavage strength of C30 ordinary-air-entrained concrete were measured after fast freeze-thaw cycles. The effects of fast freeze-thaw cycles on the mechanical properties (compressive strength and cleavage strength) of ordinary-air-entrained concrete materials are investigated on the basis of the experimental results. And the concise mathematical formula between mechanical behavior and number of fast freeze-thaw cycles was established. The experiment results can be used as a reference in design, maintenance, and life prediction of ordinary-air-entrained concrete structure (such as dam, offshore platform, etc.) in cold regions.

Enantioselective separation of mirtazapine and its metabolites by capillary electrophoresis with acetonitrile field-amplified sample stacking and its application.

A simple, rapid and sensitive chiral capillary zone electrophoresis coupled with acetonitrile-field-amplified sample stacking method was developed that allows the simultaneous enantioselective separation of the mirtazapine, N-demethylmirtazapine, 8-hydroxymirtazapine and mirtazapine-N-oxide. The separation was achieved on an uncoated 40.2 cm×75 µM fused silica capillary with an applied voltage of 16 kV. The electrophoretic analyses were carried out in 6.25 mM borate-25 mM phosphate solution at pH 2.8 containing 5.5 mg/mL carboxymethyl-ß-cyclodextrin. The detection wavelength was 200 nm. Under these optimized conditions, satisfactory chiral separations of four pair enantiomers were achieved in less than 7 min in vitro. After one step clean-up liquid-liquid extraction using 96-well format, sample was introduced capillary zone electrophoresis with acetonitrile-field-amplified sample stacking to enhance the sensitivity of enantiomers. The method was validated with respect to specificity, linearity, lower limit of quantitation, accuracy, precision, extraction recovery and stability. The lower limit of quantification was 0.5 ng/mL with linear response over the 0.5-50 ng/mL concentration range for each mirtazapine, N-demethylmirtazapine and 8-hydroxymirtazapine enantiomer. The developed and validated method has been successfully applied to the enantioselective pharmacokinetic studies in 12 healthy volunteers after oral administration of rac- mirtazapine.