Reversible inhibition of enzymes is caused by association and dissociation between enzymes and inhibitors. Therefore, reversible inhibitors can be trapped and extracted using enzyme-inhibitor interaction. The purpose of this study was to establish a method in which the reversible inhibitors retaining the original inhibitory activities are extracted from a single drop of biological sample using the enzyme-inhibitor interaction on the surface of a membrane. A membrane-immobilized carboxypeptidase Y (CPY) was produced after the biotinylated CPY was bound to the avidin separated by nondenaturing electrophoresis, transferred to a polyvinylidene fluoride and stained by Ponceau S. Ovomucoid, possessing reversible CPY inhibitory activity, was trapped and extracted from a single drop of egg white and isolated using the membrane-immobilized CPY. The isolated ovomucoid using this membrane-immobilized CPY possessed a feature that more than 85 % of the relative carboxylesterase activity was suppressed. The results indicate that ovomucoid retaining enzyme inhibitory activities can be isolated from a single drop of egg white sample using enzyme-immobilized membrane.
Liquid chromatography/mass spectrometry (LC/MS) method is becoming an important approach for therapeutic antibody assays as an alternative to the ligand-binding assay (LBA) method. The LC/MS method has some advantages over the LBA method, such as a wider dynamic range and short developing time. However, the development of the LC/MS method is often challenging because of complicated sample preparation processes involving affinity purification, denaturation, reduction and methylation, enzymatic digestion, and peptide purification. In addition, it is difficult to select a sensitive and specific surrogate peptide that allows the determination of the lower limit of quantitation of the analytical target. Another issue remains in the bioanalytical method validation (BMV) of the LC/MS method for large molecules. The BMV guideline on the LC/MS method for small molecules and that on the LBA method are helpful while developing a bioanalytical method for large molecules using LC/MS; however, these guidelines lack inherent characteristics related to bioanalysis of large molecules by the LC/MS method. In this review, we describe points to be considered regarding selection of surrogate peptides and optimization of the sample preparation processes in the LC/MS method for therapeutic antibody assays. Furthermore, we propose criteria for BMV of the LC/MS method. We expect that this review will aid in the development of sensitive, specific, and robust bioanalytical LC/MS methods for therapeutic antibodies.
In this study, the quantitative determination of gaseous biogenic volatile organic compounds (BVOCs) including monoterpenes and sesquiterpenes was accomplished using a solid-phase extraction-type collection device. The collection device was fabricated by packing styrene-divinylbenzene polymer particles into a specially designed glass cartridge. The retention performance of the collection device for BVOCs was quantitatively evaluated at 35°C with different volumes of air samples. The device showed good retention performance for the investigated BVOCs, that is, no breakthrough occurred for monoterpenes up to an air sampling volume of 150 L or for sesquiterpenes up to a sampling volume of 30,000 L. The elution performance was evaluated by passing organic solvents into the collection device, and an excellent elution recovery was obtained with 10 mL dichloromethane. Finally, the analytical method using the collection device was applied to determine the gaseous monoterpenes and sesquiterpenes from a grated carrot and the air inside a wooden house, and the results demonstrated the applicability of the method for the quantitative determination of BVOCs in several sample matrices.
We optimized several analytical conditions for more sensitive and precise HT-RPLC analysis of the therapeutic monoclonal antibody (mAb), bevacizumab. Specifically, we (1) optimized the sample preparation process to reduce adsorption and aggregation of bevacizumab, (2) introduced a sample concentration process using a centrifugal ultrafiltration unit to increase detection sensitivity, and (3) used another therapeutic mAb as an internal standard to improve analytical precision. The optimized method for bevacizumab analysis was shown to have low detection and quantification limits of 0.010 and 0.032 µg/mL, respectively, good correlation coefficients (r2 > 0.9997), and good intra- and inter-day precisions within < 12.0 %. This study provides an important methodology for the intact bioanalysis of therapeutic mAbs, not merely their LC measurement.