Extreme Point Sort Transformation Combined With a Long Short-Term Memory Network Algorithm for the Raman-Based Identification of Therapeutic Monoclonal Antibodies.

Therapeutic monoclonal antibodies (mAbs) are a new generation of protein-based medicines that are usually expensive and thus represent a target for counterfeiters. In the present study, a method based on Raman spectroscopy that combined extreme point sort transformation with a long short-term memory (LSTM) network algorithm was presented for the identification of therapeutic mAbs. A total of 15 therapeutic mAbs were used in this study. An in-house Raman spectrum dataset for model training was created with 1,350 spectra. The characteristic region of the Raman spectrum was reduced in dimension and then transformed through an extreme point sort transformation into a sequence array, which was fitted for the LSTM network. The characteristic array was extracted from the sequence array using a well-trained LSTM network and then compared with standard spectra for identification. To demonstrate whether the present algorithm was better, ThermoFisher OMNIC 8.3 software (Thermo Fisher Scientific Inc., U.S.) with two matching modes was selected for comparison. Finally, the present method was successfully applied to identify 30 samples, including 15 therapeutic mAbs and 15 other injections. The characteristic region was selected from 100 to 1800 cm-1 of the full spectrum. The optimized dimensional values were set from 35 to 53, and the threshold value range was from 0.97 to 0.99 for 15 therapeutic mAbs. The results of the robustness test indicated that the present method had good robustness against spectral peak drift, random noise and fluorescence interference from the measurement. The areas under the curve (AUC) values of the present method that were analysed on the full spectrum and analysed on the characteristic region by the OMNIC 8.3 software's built-in method were 1.000, 0.678, and 0.613, respectively. The similarity scores for 15 therapeutic mAbs using OMNIC 8.3 software in all groups compared with that of the relative present algorithm group had extremely remarkable differences (p < 0.001). The results suggested that the extreme point sort transformation combined with the LSTM network algorithm enabled the characteristic extraction of the therapeutic mAb Raman spectrum. The present method is a proposed solution to rapidly identify therapeutic mAbs.

Biosimilar or Not: Physicochemical and Biological Characterization of MabThera and Its Two Biosimilar Candidates.

The development of therapeutic biosimilar antibodies has become an important driving force of the modern biopharmaceutical industry. In this study, physiochemical characteristics (amino acid sequence, intact/subunit molecular weight, isoelectric point, post-translation modification, and disulfide linkage pattern), purity (charge variants, high and low molecular weight variants), antigen binding activity, Fc receptor binding affinity and Fc-effector function (CDC and ADCC) were analyzed by using an extensive set of state-of-the-art and orthogonal analytical technologies to provide a comprehensive characterization of the innovative product rituximab and two biosimilar candidates. The similarity study showed that biosimilar candidate 1 (BC1) and the reference product (RP) MabThera had an identical protein amino acid sequences and highly similar primary structures along with similar purity, heterogeneity profiles, antigen binding activity, Fc receptor binding affinity, and Fc-effector functions. Biosimilar candidate 2 (BC2), which had an amino acid replacement at a constant region, a different N-glycosylation profiling, and purity, was not analytically similar to RP. Although BC2 showed improvement such as an increased level of afucose, another IgG1 allotype, and similar biological activities, it was not recommended to be applied as a biosimilar compound in drug registration because the biosimilar manufacturer must first show that its primary structure was identical to that of RP. Our physicochemical characterizations and bioassay comparability study provided a deepened understanding of the structure-function relationship of quality attributes.

Ultrasound-assisted dispersive liquid-liquid microextraction followed by gas chromatography-mass spectrometry for determination of parabens in human breast tumor and peripheral adipose tissue.

An efficient method for the determination of eight parabens in human breast tumor (n = 102) and peripheral adipose tissue samples (n = 87) was successfully developed. After a precipitation procedure (acetonitrile-water-15% (w/v) zinc sulfate solution) was applied to finely-chopped tissue samples, ultrasound-assisted dispersive liquid-liquid microextraction was conducted on the sample solution. The extracted parabens were determined by gas chromatography-mass spectrometry. Significant factors influencing extraction efficiency were screened and optimized using Plackett-Burman design and central composite design. The optimized conditions were obtained at 93 µL 1­decanol, 500 µL methanol, and ultrasound irradiation power 156 W for 44 s. Under the optimum condition, the proposed method showed enrichment factors between 44.9 and 60.5. The detection limits for parabens were between 0.5 and 1.0 ng g-1. Calibration curves showed good linearity with coefficients of determination higher than 0.993. Relative standard deviations were lower than 11.8%. No significant matrix effects were observed as recoveries from spiked tissue samples ranged between 79.6% and 113.6%. The sample pretreatment procedure and analytical characteristics showed comparable or superior to those of the previously reported methods. Finally, the procedure was successfully employed in real samples and results indicated a wide existence of parabens in the tested tissues. At least one paraben was detected in 98.9% tissues and the levels ranged from 0.5 to 5931.1 ng g-1, depending on the types of analytes and tissues.

Application of dispersive liquid-liquid microextraction for the preconcentration of eight parabens in real samples and their determination by high-performance liquid chromatography.

A simple and sensitive method for the simultaneous determination of eight parabens in human plasma and urine samples was developed. The samples were preconcentrated using dispersive liquid-liquid microextraction based on the solidification of floating organic drops and determined by high-performance liquid chromatography with ultraviolet detection. The influence of variables affecting the extraction efficiency was investigated and optimized using Placket-Burman design and Box-Behnken design. The optimized values were: 58 µL of 1-decanol (as extraction solvent), 0.65 mL methanol (as disperser solvent), 1.5% w/v NaCl in 5.0 mL of sample solution, pH 10.6, and 4.0 min centrifugation at 4000 rpm. The extract was injected into the high-performance liquid chromatography system for analysis. Under the optimum conditions, the linear ranges for eight parabens in plasma and urine were 1.0-1000 ng/mL, with correlation coefficients above 0.994. The limit of detection was 0.2-0.4 and 0.1-0.4 ng/mL for plasma and urine samples, respectively. Relative recoveries were between 80.3 and 110.7%, while relative standard deviations were less than 5.4%. Finally, the method was applied to analyze the parabens in 98 patients of primary breast cancer. Results showed that parabens existed widely, at least one paraben detected in 96.9% (95/98) of plasma samples and 98.0% (96/98) of urine samples.

High-performance liquid chromatographic method for determination of leucovorin in plasma: validation and application to a pharmacokinetic study in healthy volunteers.

A sensitive and reliable high-performance liquid chromatographic (HPLC) method, using a solid-phase extraction (SPE), was developed and validated for determination of leucovorin (LV) in human plasma. Plasma sample was extracted by using a Sep-Pak cartridge which could be renewable. The sample was analyzed by HPLC with UV detection at 286 nm. The method was shown to perform selectively and sensitively for LV. The main metabolite of LV, 5-methyltetrahydrofolic acid, and endogenous substances in plasma did not show any interference in the analysis. The limit of detection was 10 ng/mL for LV in plasma and the linear range was 50-1500 ng/mL in plasma. The relative standard deviation (RSD) of intra-day and inter-day assays was 2.8-6.1% and 2.4-5.3%, respectively. The extraction recoveries of LV in plasma were over 90%. The method was proved to be applicable to the pharmacokinetic study of LV in healthy volunteers after a single oral administration (75 mg). The pharmacokinetic parameters and relative bioavailability were investigated for domestic LV tablet and capsule vs an imported tablet.