Applying UHPLC-HRAM MS/MS Method to Assess Host Cell Protein Clearance during the Purification Process Development of Therapeutic mAbs.

Host cell proteins (HCPs) are one of the process-related impurities that need to be well characterized and controlled throughout biomanufacturing processes to assure the quality, safety, and efficacy of monoclonal antibodies (mAbs) and other protein-based biopharmaceuticals. Although ELISA remains the gold standard method for quantification of total HCPs, it lacks the specificity and coverage to identify and quantify individual HCPs. As a complementary method to ELISA, the LC-MS/MS method has emerged as a powerful tool to identify and profile individual HCPs during the downstream purification process. In this study, we developed a sensitive, robust, and reproducible analytical flow ultra-high-pressure LC (UHPLC)-high-resolution accurate mass (HRAM) data-dependent MS/MS method for HCP identification and monitoring using an Orbitrap Ascend BioPharma Tribrid mass spectrometer. As a case study, the developed method was applied to an in-house trastuzumab product to assess HCP clearance efficiency of the newly introduced POROS™ Caprylate Mixed-Mode Cation Exchange Chromatography resin (POROS Caprylate mixed-mode resin) by monitoring individual HCP changes between the trastuzumab sample collected from the Protein A pool (purified by Protein A chromatography) and polish pool (purified by Protein A first and then further purified by POROS Caprylate mixed-mode resin). The new method successfully identified the total number of individual HCPs in both samples and quantified the abundance changes in the remaining HCPs in the polish purification sample.

A Suitable HPLC-MS/MS Methodology for the Detection of Oxytetracycline, Enrofloxacin, and Sulfachloropyridazine Residues in Lettuce Plants.

Oxytetracycline (OTC), enrofloxacin (EFX), and sulfachloropyridazine (SCP) are critically important antimicrobials (AMs) in both human and veterinary medicine, where they are widely used in farm animals. Lettuce has become a matrix of choice for studying the presence of residues of these AMs in plants, as the concentrations of residues detected in lettuce can range from ng to mg. While several analytical methodologies have been developed for the purpose of detecting AMs in lettuce, these currently do not detect both the parent compound and its active metabolites or epimers, such as in the case of ciprofloxacin (CFX) and 4-epi-oxitetracycline (4-epi-OTC), which also pose a risk to public health and the environment due to their AM activity. In light of this situation, this work proposes an analytical method that was developed specifically to allow for the detection of OTC, 4-epi-OTC, EFX, CFX, and SCP in a lettuce matrix. This method uses acetonitrile, methanol, 0.5% formic acid, and McIlvaine-EDTA buffer as extraction solvents, and dispersive solid-phase extraction (dSPE) for the clean-up. The analytes were detected using a liquid chromatography technique coupled to mass spectrometry (HPLC-MS/MS). Parameters such as the specificity, linearity, recovery, precision, limit of detection, and limit (LOD) of quantification (LOQ) were calculated according to the recommendations established in the European Union decision 2021/808/EC and VICH GL2: Validation of analytical procedures. The LOQ for the analytes OTC, 4-epi-OTC, CFX, and SCP was 1 µg·kg-1, whereas for EFX, it was 5 µg·kg-1 dry weight. All calibration curves showed a coefficient of determination (R2) of >0.99. The recovery levels ranged from 93.0 to 110.5% and the precision met the acceptance criteria, with a coefficient of variation of ≤14.02%. Therefore, this methodology allows for the precise and reliable detection and quantification of these analytes. The analysis of commercial samples confirmed the suitability of this method.

Wheat germ, a byproduct of the wheat milling industry, as a beneficial source of anti-aging polyamines: A quantitative comparison of various forms.

Polyamines have received a lot of attention since the 1990s because of their anti-aging, anti-chronic disease, and proliferative effects. Wheat germ was reported as one of the natural sources of high polyamine, especially spermidine. The current study used three types of wheat germ: group A was industrially separated germ from whole grain, group B was the commercially available germinated wheat germ, and group C was manually separated wheat germ from germinated grain. The polyamine content of putrescine, spermidine, and spermine has been determined using a simplified isocratic LC-MS/MS method. An optimized extraction procedure was performed on all seven samples for obtaining a polyamine-enriched extract. The three dominant carbomylated polyamines were identified by analyzing the extracted samples in order to determine their relative abundance. Wheat germ powders contain the highest amount of polyamines (220-337 µg/g) of which spermidine is one of the most important. Germinated wheat grains, on the other hand, contain the least amount of this polyamine. The commercially available separated wheat germs are suggested as a good nutrition source of these polyamines.

Development of an HPLC-MS/MS Method for the Determination of Silybin in Human Plasma, Urine and Breast Tissue.

Silybin is a flavonolignan extracted from Silybum marianum with chemopreventive activity against various cancers, including breast. This study was designed to develop an HPLC-MS/MS method for the determination of silybin in human plasma, urine and breast tissue in early breast cancer patients undergoing Siliphos® supplementation, an oral silybin-phosphatidylcholine complex. The determination of silybin was carried out by liquid-liquid extraction with methyl-tert-butyl ether (MTBE); total silybin concentration was determined by treating the samples with ß-glucuronidase, while for the determination of free silybin, the hydrolytic step was omitted. Naringenin and naproxen were selected as internal standards. The detection of the analyte was carried out by mass spectrometry and by chromatography. The HPLC-MS/MS method was evaluated in terms of selectivity, linearity, limit of quantification, precision and accuracy, and carryover. The method proved to be selective, linear, precise and accurate for the determination of silybin. To the best of our knowledge, this presents the first analytical method with the capacity to quantify the major bioactive components of milk thistle in three different biological matrices with a lower limit of quantification of 0.5 ng/mL for plasma. Silybin phosphatidylcholine, taken orally, can deliver high blood concentrations of silybin, which selectively accumulates in breast tumor tissue.

Metabolomic Profile of Primary Turkey and Rat Hepatocytes and Two Cell Lines after Chloramphenicol Exposure.

The purpose of this study was to assess the formation of chloramphenicol metabolites in primary turkey and rat hepatocyte cultures and human hepatoma (HepG2) cells and nonhepatic, Balb/c 3T3 fibroblasts. Additionally, the cytotoxicity of the drug was assessed through three biochemical endpoints: mitochondrial and lysosomal activity and cellular membrane integrity after 24 and 48 h exposure. The two metabolites of the drug, chloramphenicol glucuronide and nitroso-chloramphenicol, were detected to the greatest extent in both primary hepatocyte cultures by liquid chromatography-tandem mass spectrometry. Toxic nitroso-chloramphenicol was the main metabolite in the primary turkey hepatocyte cultures, but it was not in the primary rat hepatocyte cultures. The most affected endpoint in turkey and rat hepatocyte cultures was the disintegration of the cellular membrane, but in the cell lines, mitochondrial and lysosomal activities underwent the greatest change. The primary hepatocyte cultures represent valuable tools with which to study the species differences in the biotransformation and toxicity of drugs. To the best of our knowledge, this is the first report of differences in chloramphenicol metabolism in primary turkey and rat hepatocyte cultures.

Green Analysis: High Throughput Analysis of Emerging Pollutants in Plant Sap by Freeze-Thaw-Centrifugal Membrane Filtration Sample Preparation-HPLC-MS/MS Analysis.

Emerging and fugitive contaminants (EFCs) released to our biosphere have caused a legacy and continuing threat to human and ecological health, contaminating air, water, and soil. Polluted media are closely linked to food security through plants, especially agricultural crops. However, measuring EFCs in plant tissues remains difficult, and high-throughput screening is a greater challenge. A novel rapid freeze-thaw/centrifugation extraction followed by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) analysis was developed for high-throughput quantification of 11 EFCs with diverse chemical properties, including estriol, codeine, oxazepam, 2,4-dinitrotoluene, 1,3,5-trinitroperhydro-1,3,5-triazine, bisphenol A, triclosan, caffeine, carbamazepine, lincomycin, and DEET, in three representative crops, corn, tomato, and wheat. The internal aqueous solution, i.e., sap, is liberated via a freeze/thaw cycle, and separated from macromolecules utilizing molecular weight cutoff membrane centrifugal filtration. Detection limits ranged from 0.01 µg L-1 to 2.0 µg L-1. Recoveries of spiked analytes in three species ranged from 83.7% to 109%. Developed methods can rapidly screen EFCs in agriculture crops and can assess pollutant distribution at contaminated sites and gain insight on EFCs transport in plants to assess transmembrane migration in vascular organisms. The findings contribute significantly to environmental research, food security, and human health, as it assesses the first step of potential entry into the food chain, that being transmembrane migration and plant uptake, the primary barrier between polluted waters or soils and our food.

Using a single tablet daily to treat latent tuberculosis infection in Brazil: bioequivalence of two different isoniazid formulations (300 mg and 100 mg) demonstrated by a sensitive and rapid high-performance liquid chromatography-tandem mass spectrometry method in a randomised, crossover study

The recommended treatment for latent tuberculosis (TB) infection in adults is a daily dose of isoniazid (INH) 300 mg for six months. In Brazil, INH was formulated as 100 mg tablets. The treatment duration and the high pill burden compromised patient adherence to the treatment. The Brazilian National Programme for Tuberculosis requested a new 300 mg INH formulation. The aim of our study was to compare the bioavailability of the new INH 300 mg formulation and three 100 mg tablets of the reference formulation. We conducted a randomised, single dose, open label, two-phase crossover bioequivalence study in 28 healthy human volunteers. The 90% confidence interval for the INH maximum concentration of drug observed in plasma and area under the plasma concentration vs. time curve from time zero to the last measurable concentration “time t” was 89.61-115.92 and 94.82-119.44, respectively. The main limitation of our study was that neither adherence nor the safety profile of multiple doses was evaluated. To determine the level of INH in human plasma, we developed and validated a sensitive, simple and rapid high-performance liquid chromatography-tandem mass spectrometry method. Our results showed that the new formulation was bioequivalent to the 100 mg reference product. This finding supports the use of a single 300 mg tablet daily strategy to treat latent TB. This new formulation may increase patients’ adherence to the treatment and quality of life.