Glutathione EP Impurity D

With advanced genetic engineering technologies and better understanding of disease biology, antibody-based therapeutics are emerging as promising new generation biopharmaceuticals. These novel antibody formats are carefully designed to possess desired features such as enhanced selectivity. However, their high level of structural complexity with multiple components often leads to long development and complex multi-step manufacturing processes, through which a variety of potential small molecule impurities can be introduced. In this work, an in-process assay was developed in which mixed-mode chromatography coupled with charged aerosol detection was utilized for multiplexed detection of nine reagents commonly used in development and manufacturing of antibody-based therapeutics: isopropyl β-d-1-thiogalactopyranoside, methionine sulfoximine, ampicillin, guanidine, dehydroascorbic acid, glutathione, tris(2-carboxyethyl)phosphine, N-acetyl cysteine, and arginine. This method utilized a mixed-mode column with ion-exchange properties operated in the hydrophilic interaction chromatography mode. Various parameters were systematically optimized and under optimal conditions, the method demonstrated excellent specificity, sensitivity, linearity, precision, accuracy, and was successfully applied to determine residual impurities in multiple samples from antibody-derived molecules.

Background:DNA methylation alterations have emerged as hallmarks of cancer and have been proposed as screening, prognostic, and predictive biomarkers. Traditional approaches for methylation analysis have relied on bisulfite conversion of DNA, which can damage DNA and is not suitable for targeted gene analysis in low-input samples. Here, we have adapted methyl-CpG-binding domain protein 2 (MBD2)-based DNA enrichment for use on a semi-automated exclusion-based sample preparation (ESP) platform for robust and scalable enrichment of methylated DNA from low-input samples, called SEEMLIS.Results:We show that combining methylation-sensitive enzyme digestion with ESP-based MBD2 enrichment allows for single gene analysis with high sensitivity for GSTP1 in highly impure, heterogenous samples. We also show that ESP-based MBD2 enrichment coupled with targeted pre-amplification allows for analysis of multiple genes with sensitivities approaching the single cell level in pure samples for GSTP1 and RASSF1 and sensitivity down to 14 cells for these genes in highly impure samples. Finally, we demonstrate the potential clinical utility of SEEMLIS by successful detection of methylated gene signatures in circulating tumor cells (CTCs) from patients with prostate cancer with varying CTC number and sample purity.Conclusions:SEEMLIS is a robust assay for targeted DNA methylation analysis in low-input samples, with flexibility at multiple steps. We demonstrate the feasibility of this assay to analyze DNA methylation in prostate cancer cells using CTCs from patients with prostate cancer as a real-world example of a low-input analyte of clinical importance. In summary, this novel assay provides a platform for determining methylation signatures in rare cell populations with broad implications for research as well as clinical applications.

Amino acids playing important roles in metabolic processes are often included in dietary supplements whose use has largely expanded over the last 20 years not only in patients with particular deficiencies, but also in athletes and even common people that want to enrich their regular daily diet. In the present study, a bare silica Kinetex core-shell 2.6μm HILIC column was used for separation of some important hydrophilic amino acids and amino acids-like molecules i.e., aspartic acid, creatine, carnitine, arginine and the tripeptide glutathione (GSH), by optimizing the chromatographic conditions for their determination in complex alimentary supplements. The contribution of partition, adsorption and ion exchange on the retention mechanism was studied by varying parameters such as water content and the counter-ion concentration in the mobile phase. Optimum conditions employed a Phenomenex Kinetex core-shell 2.6μm HILIC (100×4.6mm i.d.) column and a mobile phase of acetonitrile/potassium phosphate buffer (12.5mM; pH=2.8) 85:15, v/v, at the flow rate of 1.4mL/min, using UV detection at 200nm. A reference HPLC method for the selective determination of GSH by using 1,4-naphthoquinone as derivatization reagent was also introduced for comparative purposes. The developed HILIC method was validated and applied to the analysis of the considered compounds in dietary supplements. Interestingly, in some of the real samples, oxidized glutathione which is an inactive impurity of GSH, was found at the level of about 20%. The proposed study confirms the importance of simple analytical methods for a rigorous quality control of dietary supplements containing unstable active ingredients.