Detection of Residual Proteins UL5 and UL29 Using a Targeted Proteomics Approach in HSV529, a Replication-Deficient HSV-2 Vaccine Candidate.

HSV529 is a replication defective human herpes simplex virus (HSV)-2 viral vaccine candidate in clinical development. An engineered cell line is required to support production of HSV529 by transgenic expression of the HSV-1 transcription factors UL5 (HELI) and UL29 (DNBI). These 2 genes have been deleted from the vaccine candidate to ensure replication deficiency, and the transgene products are thus impurities that must be monitored in the final product. Multiple reaction monitoring (MRM) is a mass spectrometry (MS) workflow that can be used to quickly develop targeted protein detection and quantitation methods. An MRM method was developed for detection of the HSV-1 proteins UL5 and UL29 based on results from nano-liquid chromatography-MS/MS protein analysis of HSV529 material. Sensitivity, specificity, and linearity of response for the MRM workflow were established using high-flow ultra-performance liquid chromatography coupled to a tandem quadrupole mass analyzer. Results show that residual UL5 and UL29 proteins can be detected in the HSV529 candidate, and that MRM analysis provides the appropriate sensitivity and specificity required for quantitation. The transition from nano-flow to ultra-performance driven chromatography was found to improve method robustness without compromising the sensitivity of the assay.

Amatoxins (α- and ß-Amanitin) and phallotoxin (Phalloidin) analyses in urines using high-resolution accurate mass LC-MS technology.

Mycotoxin intoxications can result from the consumption of amatoxins like α- and ß-amanitin or of phallotoxin, present in several toxic mushrooms like Amanita phalloides. To identify and quantify amatoxins and phallotoidin in biological matrixes, we developed a method using liquid chromatography coupled with an ultra-high-resolution and accurate mass instrument (liquid chromatography-high-resolution-mass spectrometry, LC-HR-MS), Q Exactive™ (Thermo Fisher). The method includes a simple solid-phase extraction of urine samples spiked with flurazepam as internal standard (IS), using Bond Elut Agilent Certify cartridges (C18, 200 mg, 3 mL). LC separation was performed on a C18 Accucore column (100 × 2.1 mm, 2.6 µm) using a gradient of 10 mM ammonium acetate buffer containing 0.1% (v/v) formic acid and of acetonitrile with 0.1% (v/v) formic acid. Separation of analytes was obtained in 7 min, with respective retention times for α-amanitin, ß-amanitin, phalloidin and IS of 1.9, 1.7, 3.5 and 3.8 min, respectively. Quantitation on the LC-HR-MS system was performed by extracting the exact mass value of each protonated species using a 5-p.p.m. mass window, which was 919.3614, 920.3455, 789.3257 and 388.1586 for α-amanitin, ß-amanitin, phalloidin and IS, respectively. Calibration curves were obtained by spiking drug-free urine at 1-100 ng/mL. Mean correlation coefficients, r(2), were above 0.99 for each amatoxins and phalloidin. According to currently accepted validation procedures, the method was tested for selectivity, calibration, accuracy, matrix effect, precision and recovery. Authentic urine samples from 43 patients suffering from a suspected intoxication with mushrooms were analyzed by LC-HR-MS, and the results were compared with ELISA competitive immunoassay. The LC-HR-MS presented large benefits over immunoassay of being specific, faster and more sensitive, making it suitable for daily emergency toxicological analysis.