Regulatory Verification by Health Canada of Content in Recombinant Human Insulin, Human Insulin Analog, and Porcine Insulin Drug Products in the Canadian Market Using Validated Pharmacopoeial Methods Over Nonvalidated Approaches.

BACKGROUND: For diabetes mellitus treatment plans, the consistency and quality of insulin drug products are crucial for patient well-being. Because biologic drugs, such as insulin, are complex heterogeneous products, the methods for drug product evaluation should be carefully validated for use. As such, these criteria are rigorously evaluated and monitored by national authorities. Consequently, reports that describe significantly lower insulin content than their label claims are a concern. This issue was raised by a past publication analyzing insulin drug products available in Canada, and, as a result, consumers and major patient organizations have requested clarification. METHODS: To address these concerns, this study independently analyzed insulin drug products purchased from local Canadian pharmacies-including human insulin, insulin analogs, and porcine insulin-by compendial and noncompendial reversed-phase high-performance liquid chromatography (RP-HPLC) methods. RESULTS: We demonstrated the importance of using methods fit for purpose when assessing insulin quality. In a preliminary screen, the expected insulin peak was seen in all products except two insulin analogs-insulin detemir and insulin degludec. Further investigation showed that this was not caused by low insulin content but insufficient solvent conditions, which demonstrated the necessity for methods to be adequately validated for product-specific use. When drug products were appropriately assessed for content using the validated type-specific compendial RP-HPLC methods for insulin quantitation, values agreed with the label claim content. CONCLUSIONS: Because insulin drug products are used daily by over a million Canadians, it is important that researchers and journals present data using methods fit for purpose and that readers evaluate such reports critically.

Proteome characterization of the culture supernatant of Mycobacterium bovis in different growth stages.

This study aimed to identify proteins secreted by Mycobacterium bovis into culture medium at different stages of bacterial growth. A field strain of M. bovis was grown in Middlebrook 7H9 media and culture supernatant was collected at three-time points representing three different phases of growth (early exponential, late exponential, and stationary phases). Supernatants were double filtered, digested by trypsin and analyzed by LC-MS/MS. The study found 15, 21, and 16 proteins in early, mid and late growth phases, respectively. In total, 22 proteins were identified, 18 of which were reported or predicted to have a cell wall or extracellular localization. To our knowledge, this is the first study to identify proteins secreted into the culture medium by a field strain of M. bovis in three different stages of growth. The dataset generated here provides candidate proteins with the potential for the development of serological diagnostic reagents or vaccine for bovine tuberculosis. Data are available via ProteomeXchange with identifier PXD017817.

Dysregulation of Ephrin receptor and PPAR signaling pathways in neural progenitor cells infected by Zika virus.

Zika virus (ZIKV) infection is a serious public threat with cases reported in about 70 countries and territories. One of the most serious consequences of ZIKV infection is congenital microcephaly in babies. Congenital microcephaly has been suggested to result from infection of neural progenitor cells (NPCs) in the developing fetal brain. However, the molecular and cellular mechanisms underlying microcephaly development remains to be fully elucidated. In this study, we employed quantitative proteomics to determine protein expression profile that occur during viral replication in NPCs. Bioinformatics analysis of the protein expression changes resulted in the identification of a wide range of cell signaling pathways. Specifically, pathways involved in neurogenesis and embryonic development were markedly altered, along with those associated with cell cycle, apoptosis, lipid metabolism and oxidative stress. Notably, the differential regulation of Ephrin Receptor and PPAR signaling pathways, as revealed by quantitative proteomics and validated by qPCR array, underscores the need to explore these pathways in disease development. Collectively, these results indicate that ZIKV-induced pathogenesis involves complex virus-host reactions; the findings reported here could help shed light on the mechanisms underlying ZIKV-induced microcephaly and ZIKV replication in NPCs.

Cellular immune responses of older adults to four influenza vaccines: Results of a randomized, controlled comparison.

Cellular immunity is important for protection against the serious complications of influenza in older adults. As it is unclear if newer influenza vaccines elicit greater cellular responses than standard vaccines, we compared responses to 2 standard and 2 newer licensed trivalent inactivated vaccines (TIVs) in a randomized trial in older adults. Non-frail adults ≥ 65 y old were randomly assigned to receive standard subunit, MF59-adjuvanted subunit, standard split-virus or intradermal split-virus TIV. Peripheral blood mononuclear cells (PBMC) harvested pre- and 3-weeks post-vaccination were stimulated with live A/H3N2 virus. PBMC supernatants were tested for interleukin 10 (IL-10) and interferon gamma (IFN-γ), and lysates for granzyme B (GrB). Flow cytometry identified CD4+ and CD8+ T- cells expressing intracellular IL-2, IL-10, IFN-γ, GrB, or perforin. Differences following immunization were assessed for paired subject samples and among vaccines. 120 seniors participated, 29-31 per group, which were well matched demographically. Virus-stimulated PBMCs were GrB-rich before and after vaccination, with minimal increases evident. Immunization did not increase secretion of IFN-γ or IL-10. However, cytolytic effector T-cells (CD8+GrB+perforin+) increased significantly in percentage post-vaccination in all groups, to similar mean values across groups. CD4+GrB+perforin+ T-cells also increased significantly after each vaccine, to similar mean values among vaccines. Vaccination did not increase the low baseline percentages of CD4+ or CD8+ T-cells expressing IFN-γ, IL-2 or IL-10 . In conclusion, participants had pre-existing cellular immunity to H3N2 virus. All 4 vaccines boosted cellular responses to a similar but limited extent, particularly cytolytic effector CD8+ T-cells associated with clinical protection against influenza.

Investigation of the differences in thermal stability of two recombinant human serum albumins with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine liposomes by UV circular dichroism spectropolarimetry.

Previous studies have demonstrated that liposome-protein interactions can result in changes to the thermal stability of the protein. We utilized far-UV circular dichroism spectropolarimetry and fluorescence spectroscopy to investigate the interaction of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) liposomes with two recombinant human serum albumins (rHSA). We demonstrate that rHSA expressed in Oryza sativa (OsrHSA) has improved secondary structure thermal stability compared to rHSA expressed in Pichia pastoris (PprHSA). A similar stability profile was observed when comparing bovine serum albumin (BSA) and defatted bovine serum albumin (dfBSA), suggesting the presence of fatty acids may be responsible for the improved stability of OsrHSA. Addition of DPPC liposomes reduced the thermal stability of both OsrHSA and BSA, but not of PprHSA or dfBSA. DPPC liposomes may disrupt stabilizing native fatty acids on OsrHSA and BSA.

Quantification of protein isoforms in mesenchymal stem cells by reductive dimethylation of lysines in intact proteins.

Mass spectrometry (MS)-based quantification of highly homologous proteins in complex samples has proven difficult due to subtle sequence variations and the wide dynamic range of protein isoforms present. Herein, we report the use of reductive dimethylation on intact proteins to quantitatively compare protein isoform expression in the nucleus and cytoplasm of mesenchymal stem cells (MSC) and normal stroma. By coupling fixed-charge MS/MS scanning, high-resolution UPLC FT-MS data-dependent acquisition and MASCOT-based data mining, hydrogen/deuterium-labeled dimethyl-lysine peptides were simultaneously captured allowing the accurate comparison of 123 protein isoforms in parallel LC MS/MS runs. Thirty-four isoforms were identified that had expression levels specific to MSC. Where possible, proteomic analyses were verified by Western blotting and were demonstrated to be divergent from the level of gene transcription detected for certain proteins. Our analysis provides a protein isoform signature specific to MSC and demonstrates the suitability of dimethyl-lysine labeling on intact proteins for quantifying highly homologous proteins on a proteome-wide scale.

A novel surface autolysin of Listeria monocytogenes serotype 4b, IspC, contains a 23-residue N-terminal signal peptide being processed in E. coli.

The 86-kDa protein IspC of 774 amino acids in Listeria monocytogenes serotype 4b has been recently identified as the target of humoral immune response to listerial infection and as a novel surface autolysin. A signal peptide is predicted at the N-terminal end of IspC, but no biochemical data has been shown to confirm the presence of the cleavage site of a signal peptidase. To address this and prepare sufficient amount of the protein for biochemical and structural characterization, we present a strategy for efficient expression and purification of IspC and analyze the purified protein by N-terminal sequencing and mass spectrometry. Expression of IspC in Escherichia coli using a pET30a-based expression construct was efficiently improved by incubating the culture at 37 degrees C for 2h followed by 4 degrees C for 16-18 h. The recombinant product rIspC remained as a soluble form in the cellular extract and was purified to electrophorectic homogeneity by the combination of metal chelate affinity chromatography with cation-exchange chromatography. The IspC was shown to contain a 23-residue N-terminal signal peptide being processed between Thr 23 and Thr 24 in E. coli, resulting in an 84-kDa mature protein. The highly purified form of rIspC from this study, exhibiting both peptidoglycan hydrolase activity and immunogenicity as previously reported, would facilitate further biochemical, structural, and functional studies of this autolysin.