An isotope dilution mass spectrometry assay to track Norovirus-like particles in vaccine process intermediates by quantifying capsid protein VP1.

The coronavirus disease (COVID-19) pandemic shows the rapid pace at which vaccine development can occur which highlights the need for more fast and efficient analytical methodologies to track and characterize candidate vaccines during manufacturing and purification processes. The candidate vaccine in this work comprises plant-derived Norovirus-like particles (NVLPs) which are structures that mimic the virus but lack any infectious genetic material. Presented here is a liquid chromatography-tandem mass spectrometry (LC-MS/MS) methodology for the quantification of viral protein VP1, the main component of the NVLPs in this study. It combines isotope dilution mass spectrometry (IDMS) with multiple reaction monitoring (MRM) to quantify targeted peptides in process intermediates. Multiple MRM transitions (precursor/product ion pairs) for VP1 peptides were tested with varying MS source conditions and collision energies. Final parameter selection for quantification includes three peptides with two MRM transitions each offering maximum detection sensitivity under optimized MS conditions. For quantification, a known concentration of the isotopically labeled version of the peptides to be quantified was added into working standard solutions to serve as an internal standard (IS); calibration curves were generated for concentration of native peptide vs. the peak area ratio of native-to-isotope labeled peptide. VP1 peptides in samples were quantified with labeled versions of the peptides added at the same level as that of the standards. Peptides were quantified with limit of detection (LOD) as low as 1.0 fmol µL-1 and limit of quantitation (LOQ) as low as 2.5 fmol µL-1. NVLP preparations spiked with known quantities of either native peptides or drug substance (DS) comprising assembled NVLPs produced recoveries indicative of minimal matrix effects. Overall, we report a fast, specific, selective, and sensitive LC-MS/MS strategy to track NVLPs through the purification steps of the DS of a Norovirus candidate vaccine. To the best of our knowledge, this is the first application of an IDMS method to track virus-like particles (VLPs) produced in plants as well as measurements performed with VP1, a Norovirus capsid protein.

Quantification of SARS-CoV-2 spike protein expression from mRNA vaccines using isotope dilution mass spectrometry.

The advent of mRNA vaccine technology has been vital in rapidly creating and manufacturing COVID-19 vaccines at an industrial scale. To continue to accelerate this leading vaccine technology, an accurate method is needed to quantify antigens produced by the transfection of cells with a mRNA vaccine product. This will allow monitoring of protein expression during mRNA vaccine development and provide information on how changes to vaccine components affects the expression of the desired antigen. Developing novel approaches that allow for high-throughput screening of vaccines to detect changes in antigen production in cell culture prior to in vivo studies could aid vaccine development. We have developed and optimized an isotope dilution mass spectrometry method to detect and quantify the spike protein expressed after transfection of baby hamster kidney cells with expired COVID-19 mRNA vaccines. Five peptides of the spike protein are simultaneously quantified and provide assurance that protein digestion in the region of the target peptides is complete since results between the five peptides had a relative standard deviation of less than 15 %. In addition, two housekeeping proteins, actin and GAPDH, are quantified in the same analytical run to account for any variation in cell growth within the experiment. IDMS allows a precise and accurate means to quantify protein expression by mammalian cells transfected with an mRNA vaccine.

Liquid Chromatography-Mass Spectrometry-Based Qualitative Profiling of mRNA Therapeutic Reagents Using Stable Isotope-Labeled Standards Followed by the Automatic Quantitation Software Ariadne.

mRNA-based medicines are a promising modality for preventing virus-caused illnesses, including COVID-19, and treating various types of cancer and genetic diseases. To develop such medicines, methods to characterize long mRNA molecules are needed for quality control and metabolic analysis. Here, we developed an analytical platform based on isotope-dilution liquid chromatography-mass spectrometry (LC-MS) that quantitatively characterizes long, modified mRNAs by comparing them to a stable isotope-labeled reference with an identical sequence to that of the target medicine. This platform also includes database searching using the mass spectra as a query, which allowed us to confirm the primary structures of 200 to 4300 nt mRNAs including chemical modifications, with sequence coverage at 100%, to detect/identify defects in the sequences, and to define the efficiencies of the 5'-capping and integrity of the polyadenylated tail. Our findings indicated that this platform should be valuable for quantitatively characterizing mRNA vaccines and other mRNA medicines.

Accurate quantification of SARS-CoV-2 RNA by isotope dilution mass spectrometry and providing a correction of reverse transcription efficiency in droplet digital PCR.

The novel coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected more than 505 million confirmed cases, including over 6 million deaths. Reference materials (RMs) of SARS-CoV-2 RNA played a crucial role in performance evaluation and quality control of testing laboratories. As the potential primary characterization method of RMs, reverse transcription digital PCR (RT-dPCR) measures the copy number of RNA, but the accuracy of reverse transcription (RT) efficiency has yet to be confirmed. This study established a method of enzymatic digestion followed by isotope dilution mass spectrometry (IDMS), which does not require an RT reaction, to quantify in vitro-transcribed SARS-CoV-2 RNA. RNA was digested to nucleotide monophosphate (NMP) within 15 min and analyzed by IDMS within 5 min. The consistency among the results of four different NMPs demonstrated the reliability of the proposed method. Compared to IDMS, the quantitative result of RT-dPCR turned out to be about 10% lower, possibly attributed to the incompleteness of the reverse transcription process. Therefore, the proposed approach could be valuable and reliable for quantifying RNA molecules and evaluating the RT efficiency of RT-based methods.

A simple and efficient derivatization strategy combined with switchable solvent liquid-liquid microextraction hydroxychloroquine methyl acetate-d3 -based quadruple isotope dilution gas chromatography mass spectrometry for the determination of hydroxychloroquine sulfate in biological fluids.

RATIONALE: A derivatization switchable solvent liquid-liquid microextraction quadruple isotope dilution gas chromatography mass spectrometry (D-SS-LLME-ID4 -GC/MS) method is presented for the determination of hydroxychloroquine sulfate in human biofluids. METHODS: While mixing type/period and concentration of NaOH were optimized via a univariate optimization approach, a multivariate optimization approach was used to determine optimum values for relatively more important parameters such as volumes of derivatization agent (acetic anhydride), NaOH and switchable solvent. RESULTS: Under the optimum experimental conditions, limit of detection and limit of quantification were calculated as 0.03 and 0.09 mg/kg (mass based), respectively. An isotopically labelled material (hydroxychloroquine methyl acetate-d3 ) was firstly synthesized to be used in ID4 experiments which give highly accurate and precise recovery results. After the application of D-SS-LLME-ID4 , superior percent recovery results were recorded as 99.9 ± 1.6-101.3 ± 1.2 for human serum, 99.9 ± 1.7-99.8 ± 1.8 for urine and 99.6 ± 1.5-101.0 ± 1.1 for saliva samples. CONCLUSIONS: The developed D-SS-LLME-ID4 -GC/MS method compensates the complicated matrix effects of human biofluids and provides highly accurate quantification of an analyte with precise results.

Quantification of SARS-CoV-2 spike and nucleocapsid proteins using isotope dilution tandem mass spectrometry.

The emergence and subsequent global outbreak of the novel coronavirus SARS-CoV-2 prompted our laboratory to launch efforts to develop methods for SARS-CoV-2 antigen detection and quantification. We present an isotope dilution mass spectrometry method (IDMS) for rapid and accurate quantification of the primary antigens, spike and nucleocapsid proteins. This IDMS method utilizes liquid chromatography-tandem mass spectrometry (LC-MS/MS) to analyze sample tryptic digests for detection and quantification of selected conserved peptides of SARS-CoV-2 spike and nucleocapsid proteins. The IDMS method has the necessary attributes to be successfully utilized for accurate quantification in SARS-CoV-2 protein-based vaccines and as targets of rapid diagnostic tests. Absolute quantification was achieved by quantifying and averaging 5 peptides for spike protein (3 peptides in the S1 subunit and 2 peptides in the S2 subunit) and 4 peptides for nucleocapsid protein. The overall relative standard deviation of the method was 3.67% for spike protein and 5.11% for nucleocapsid protein. IDMS offers speed (5 h total analysis time), sensitivity (LOQ; 10 fmol/µL) and precision for quantification of SARS-CoV-2 spike and nucleocapsid proteins.

Simultaneous quantification of seven repurposed COVID-19 drugs remdesivir (plus metabolite GS-441524), chloroquine, hydroxychloroquine, lopinavir, ritonavir, favipiravir and azithromycin by a two-dimensional isotope dilution LC-MS/MS method in human serum.

BACKGROUND: The present COVID-19 pandemic has prompted worldwide repurposing of drugs. The aim of the present work was to develop and validate a two-dimensional isotope-dilution liquid chromatrography tandem mass spectrometry (ID-LC-MS/MS) method for accurate quantification of remdesivir and its active metabolite GS-441524, chloroquine, hydroxychloroquine, lopinavir, ritonavir, favipiravir and azithromycin in serum; drugs that have gained attention for repurposing in the treatment of COVID-19. METHODS: Following protein precipitation, samples were separated with a two-dimensional ultra-high performance liquid chromatography (2D-UHPLC) setup, consisting of an online solid phase extraction (SPE) coupled to an analytical column. For quantification, stable isotope-labelled analogues were used as internal standards for all analytes. The method was validated on the basis of the European Medicines Agency bioanalytical method validation protocol. RESULTS: Detuning of lopinavir and ritonavir allowed simultaneous quantification of all analytes with different concentration ranges and sensitivity with a uniform injection volume of 5 µL. The method provided robust validation results with inaccuracy and imprecision values of ≤ 9.59 % and ≤ 11.1 % for all quality controls. CONCLUSION: The presented method is suitable for accurate and simultaneous quantification of remdesivir, its metabolite GS-441525, chloroquine, hydroxychloroquine, lopinavir, ritonavir, favipiravir and azithromycin in human serum. The quantitative assay may be an efficient tool for the therapeutic drug monitoring of these potential drug candidates in COVID-19 patients in order to increase treatment efficacy and safety.