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 healthcare workers' exposure to cyclophosphamide, ifosfamide, methotrexate, and 5-fluorouracil by 24-h urine assay: A descriptive pilot study.

PURPOSE: The objective of this pilot study was to determine the frequency of urination and the concentration of four hazardous drugs (cyclophosphamide, ifosfamide, methotrexate, and fluorouracil) in workers' 24-h urine samples in relation to exposure to traces with hazardous drugs. METHODS: The study was conducted in three healthcare centers in the region of Montréal, Quebec, Canada. We recruited healthcare workers (nurses and pharmacy technicians) assigned to the hematology-oncology department. Each participant was asked to collect all urine voided during a 24-h period, to fill out an activity journal documenting tasks performed and to document the use of personal protective equipment. Samples were analyzed for cyclophosphamide, ifosfamide, methotrexate, and alpha-fluoro-beta-alanine (FBAL, the main urinary metabolite of 5-fluorouracil). Drugs were quantified by ultra-performance liquid chromatography-tandem mass spectrometry (positive electrospray MRM mode). RESULTS: Eighteen healthcare workers (10 nurses and 8 technicians) were recruited and provided consent to participate. Urine samples were obtained between 1 September and 30 September 2019. The number of urinations over the 24-h collection period ranged from 3 to 11 per participant. A total of 128 urine samples were analyzed for the 18 workers. All urine samples were negative for the four antineoplastics tested. CONCLUSION: No traces of cyclophosphamide, ifosfamide, methotrexate, or FBAL were found in the 24-h urine samples of 18 healthcare workers practicing in three healthcare facilities in Quebec. Although it was feasible to collect 24-h urine samples in this research project, it appears unrealistic to do so recurrently as part of a large-scale surveillance program.

The Experience of the ICNP Francophone Research and Development Centre of Canada with Validating the ICNP® French-Canadian Version.

The mission of the ICNP Francophone Research and Development Centre of Canada is to promote and support the use of the French ICNP® by French-speaking nursing students, health-care workers and health organizations. This paper presents the different steps performed to achieve validation of the ICNP's French-Canadian version, which is now available for its integration into the electronic health-care records.

Development of the Adolescent Cancer Suffering Scale.

BACKGROUND: While mortality due to pediatric cancer has decreased, suffering has increased due to complex and lengthy treatments. Cancer in adolescence has repercussions on personal and physical development. Although suffering can impede recovery, there is no validated scale in French or English to measure suffering in adolescents with cancer. OBJECTIVE: To develop an objective scale to measure suffering in adolescents with cancer. METHODS: A methodological design for instrument development was used. Following a MEDLINE search, semistructured interviews were conducted with adolescents 12 to 19 years of age who had undergone four to six weeks of cancer treatment, and with a multidisciplinary cohort of health care professionals. Adolescents with advanced terminal cancer or cognitive impairment were excluded. Enrollment proceeded from the hematology-oncology department/clinic in Montreal, Quebec, from December 2011 to March 2012. Content validity was assessed by five health care professionals and four adolescents with cancer. RESULTS: Interviews with 19 adolescents and 16 health care professionals identified six realms of suffering: physical, psychological, spiritual, social, cognitive and global. Through iterative feedback, the Adolescent Cancer Suffering Scale (ACSS) was developed, comprising 41 questions on a four-point Likert scale and one open-ended question. Content validity was 0.98, and inter-rater agreement among professionals was 88% for relevance and 86% for clarity. Adolescents considered the scale to be representative of their suffering. CONCLUSIONS: The ACSS is the first questionnaire to measure suffering in adolescents with cancer. In future research, the questionnaire should be validated extensively and interventions developed. Once validated, the ACSS will contribute to promote a holistic approach to health with appropriate intervention or referral.

Biochemical composition of haemagglutinin-based influenza virus-like particle vaccine produced by transient expression in tobacco plants.

Influenza virus-like particles (VLPs) are noninfectious particles resembling the influenza virus representing a promising vaccine alternative to inactivated influenza virions as antigens. Medicago inc. has developed a plant-based VLP manufacturing platform allowing the large-scale production of GMP-grade influenza VLPs. In this article, we report on the biochemical compositions of these plant-based influenza candidate vaccines, more particularly the characterization of the N-glycan profiles of the viral haemagglutinins H1 and H5 proteins as well as the tobacco-derived lipid content and residual impurities. Mass spectrometry analyses showed that all N-glycosylation sites of the extracellular domain of the recombinant haemagglutinins carry plant-specific complex-type N-glycans having core α(1,3)-fucose, core ß(1,2)-xylose epitopes and Lewis(a) extensions. Previous phases I and II clinical studies have demonstrated that no hypersensibility nor induction of IgG or IgE directed against these glycans was observed. In addition, this article showed that the plant-made influenza vaccines are highly pure VLPs preparations while detecting no protein contaminants coming either from Agrobacterium or from the enzymes used for the enzyme-assisted extraction process. In contrast, VLPs contain few host cell proteins and glucosylceramides associated with plant lipid rafts. Identification of such raft markers, together with the type of host cell impurity identified, confirmed that the mechanism of VLP formation in planta is similar to the natural process of influenza virus assembly in mammals.

Human antibody response to N-glycans present on plant-made influenza virus-like particle (VLP) vaccines.

BACKGROUND: Plant-made biotherapeutics are gathering momentum and some plant glycoproteins are allergens. Glycans with core ß1-2xylose and α1,3fucose motifs and antennae terminated by mannose residues (e.g.: MMXF) are found on several plant allergens and can cross-react with glyco-epitopes from other sources. To date, reactivity to these cross-reactive determinants has not been associated with clinical symptoms. OBJECTIVE: We produced VLP vaccines bearing the hemagglutinin(HA) of H5(A/Indonesia/5/05) or H1(A/California/07/09) influenza viruses by transfection of Nicotiana benthamiana. Subjects enrolled in Phase I/II trials were followed for evidence of allergy/hypersensitivity and development of antibodies against plant glyco-epitopes. METHODS: A total of 280/349 subjects received either one (H1) or 2 doses (H5) of vaccine (5-45 µg of HA/dose) intramuscularly including 40 with pre-existing plant allergies. Subjects were monitored for 6 months. IgG and IgE to plant glyco-epitopes were measured by ELISA using corn-/egg-derived avidin and bromelain as target antigens. RESULTS: No subject developed allergic/hypersensitivity symptoms. Some (34%) developed transient IgG and, in some cases IgE, to plant glyco-epitopes but no subject mounted an IgE response to the MMXF motif. Antibodies returned to baseline by 6 months in most subjects. CONCLUSION: VLP vaccines bearing influenza HA glycoproteins can elicit transient IgG and, in some cases, IgE responses that are not associated with either the development or worsening of allergic/hypersensitivity symptoms.

Impact and appreciation of two methods aiming at reducing hazardous drug environmental contamination: The centralization of the priming of IV tubing in the pharmacy and use of a closed-system transfer device.

OBJECTIVES: The main objective was to evaluate the impact of two methods aiming at reducing hazardous drug environmental contamination: the centralization of the priming of IV tubing in the pharmacy and the use of a closed-system transfer device. The secondary objective was to evaluate the satisfaction of pharmacy technicians using a survey. METHODS: Sites in the hematology-oncology satellite pharmacy and care unit were analyzed for the presence of cyclophosphamide, ifosfamide and methotrexate before and after the centralization of the priming of IV tubing in the pharmacy and before and after using a closed-system transfer device. The limits of detection for cyclophosphamide, ifosfamide and methotrexate were, respectively, of 0.0015 ng/cm(2), 0.0012 ng/cm(2) and 0.0060 ng/cm(2). The pharmacy technician satisfaction was evaluated using a questionnaire. RESULTS: A total of 225 samples was quantified. After the centralization of priming in the pharmacy, no significant difference was found in the proportion of positive samples for cyclophosphamide, ifosfamide and methotrexate. Traces of cyclophosphamide found on the floor in patient care areas was significantly reduced (median[min-max] 0.08[0.06-0.09]ng/cm(2) vs. 0.03[0.02-0.05], p < 0.0001). After using a closed-system transfer device, a significant difference was found for the proportion of cyclophosphamide positive samples (15/45(33%) vs. 0/45(0%), p < 0.0001), but no significant difference was found for ifosfamide (12/45(27%) vs. 5/45(11%), p = 0.059) and methotrexate (1/45(2%) vs. 2/45(4%), p = 0.557). Pharmacy technicians raised issues following the centralization of priming (e.g. workload) and the use of closed-system transfer devices (e.g. spills, particles, workload and handling difficulties). CONCLUSION: The centralization of the priming of IV tubing in the pharmacy reduced floor contamination in patient care areas without increasing surface contamination in the pharmacy. Closed-system transfer devices reduced contamination in pharmacy, but handling issues were raised by pharmacy technicians.

Differential proteomic screen to evidence proteins ubiquitinated upon mitotic exit in cell-free extract of Xenopus laevis embryos.

Post-translational modification of proteins via ubiquitination plays a crucial role in numerous vital functions of the cell. Polyubiquitination is one of the key regulatory processes involved in regulation of mitotic progression. Here we describe a differential proteomic screen dedicated to identification of novel proteins ubiquitinated upon mitotic exit in cell-free extract of Xenopus laevis embryo. Mutated recombinant His6-tagged ubiquitin (Ubi (K48R)) was added to mitotic extract from which we purified conjugated proteins, as well as associated proteins in nondenaturing conditions by cobalt affinity chromatography. Proteins eluted from Ubi (K48R) supplemented and control extracts were compared by LC-MS/MS analysis after monodimensional SDS-PAGE. A total of 144 proteins potentially ubiquitinated or associated with them were identified. Forty-one percent of these proteins were shown to be involved in ubiquitination and/or proteasomal degradation pathway confirming the specificity of the screen. Twelve proteins, among them ubiquitin itself, were shown to carry a "GG" or "LRGG" remnant tag indicating their direct ubiquitination. Interestingly, sequence analysis of ubiquitinated substrates carrying these tags indicated that in Xenopus cell-free embryo extract supplemented with Ubi (K48R) the majority of polyubiquitination occurred through lysine-11 specific ubiquitin chain polymerization. The potential interest in this atypical form of ubiquitination as well as usefulness of our method in analyzing atypical polyubiquitin species is discussed.

Comparative proteome analysis of human epithelial ovarian cancer.

BACKGROUND: Epithelial ovarian cancer is a devastating disease associated with low survival prognosis mainly because of the lack of early detection markers and the asymptomatic nature of the cancer until late stage. Using two complementary proteomics approaches, a differential protein expression profile was carried out between low and highly transformed epithelial ovarian cancer cell lines which realistically mimic the phenotypic changes observed during evolution of a tumour metastasis. This investigation was aimed at a better understanding of the molecular mechanisms underlying differentiation, proliferation and neoplastic progression of ovarian cancer. RESULTS: The quantitative profiling of epithelial ovarian cancer model cell lines TOV-81D and TOV-112D generated using iTRAQ analysis and two-dimensional electrophoresis coupled to liquid chromatography tandem mass spectrometry revealed some proteins with altered expression levels. Several of these proteins have been the object of interest in cancer research but others were unrecognized as differentially expressed in a context of ovarian cancer. Among these, series of proteins involved in transcriptional activity, cellular metabolism, cell adhesion or motility and cytoskeleton organization were identified, suggesting their possible role in the emergence of oncogenic pathways leading to aggressive cellular behavior. CONCLUSION: The differential protein expression profile generated by the two proteomics approaches combined to complementary characterizations studies will open the way to more exhaustive and systematic representation of the disease and will provide valuable information that may be helpful to uncover the molecular mechanisms related to epithelial ovarian cancer.