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Site-specific and Quantitative N-glycan Heterogeneity Analysis of the Charge Isomers of an Anti-VEGF Recombinant Fusion Protein by High-resolution 2-dimensional Gel Electrophoresis and Mass Spectrometry.

Site-specific and Quantitative N-glycan Heterogeneity Analysis of the Charge Isomers of an Anti-VEGF Recombinant Fusion Protein by High-resolution 2-dimensional Gel Electrophoresis and Mass Spectrometry.
Posted by Troy

glycan modification request important concerns about quality control of biopharmaceutical production. Conbercept is a recombinant fusion protein glycosylation multi-drug approved for the treatment of age-related macular degeneration (AMD). With 14 N-glycosites in the molecule and 7 N-glycosites in monomer, charge separation and characterization conbercept isomer pose a big challenge because of the heterogeneity enormous.

Stability of batch-to-batch on the distribution charge isomers and the possible causal pattern requires the development of an effective analytical approach. Here, moving the pH gradient (IPG) -based approaches the two-dimensional gel electrophoresis (2-DE) was first optimized to achieve high resolution, high separation and preparation costs reproduced isomers. Then, combined with a quantitative analysis of heterogeneity strategy-specific N-glycan based diagnostic MS2 ion (peptide + GlcNAc, ion Y1) of glycopeptides, integrated approach to heterogeneities quantitation of specific N-glycan isomers established between charges.

Finally, the site-specific quantitation of N-glycoforms in each of the 2-DE resolved spots performed, and the results showed that sialylaion which tends to increase to a place situated in the area of ‚Äč‚Äčacid gel. This study provides an effective approach for separating isomer weight protein glycosylation drug charges, and quantitative exploring the site-specific N-glycans dynamics along with the different isomers charge.

Site-specific and Quantitative N-glycan Heterogeneity Analysis of the Charge Isomers of an Anti-VEGF Recombinant Fusion Protein by High-resolution 2-dimensional Gel Electrophoresis and Mass Spectrometry.
Site-specific and Quantitative N-glycan Heterogeneity Analysis of the Charge Isomers of an Anti-VEGF Recombinant Fusion Protein by High-resolution 2-dimensional Gel Electrophoresis and Mass Spectrometry.

PCR-Restriction Fragment Length Polymorphism and Pulsed-Field Gel Electrophoresis Characterization of Listeria monocytogenes isolates of Ready-to-Eat Foods, Food Processing Environmental and Clinical Samples in South Africa.

Listeria monocytogenes is a ubiquitous, intracellular foodborne pathogen responsible for invasive listeriosis. The ability of L. monocytogenes causes the disease has some correlation with serotype specific group of offspring, making identification of the lineage is important for epidemiological analysis. Development of methods for connecting strain typing for outbreak of listeriosis L. monocytogenes will help minimize the spread of disease. The purpose of this study was to design a PCR-restriction fragment length polymorphism (RFLP) method for distinguishing between groups of lineages of L. monocytogenes.

PCR-amplified gene fragment hly for 12 serotypes of L. monocytogenes were sequenced, aligned, and analyzed by Bioedit program, and single nucleotide polymorphisms (SNPs) in the gene region identified. Because of the difficulty in obtaining serotype reference strain 4AB, these serotypes not included in this study. We tested the specificity and accuracy of PCR-RFLP method in the reference strains of L. monocytogenes and validated the method with 172 L. monocytogenes strains recovered from humans, food, environmental and food processing in 2000 to 2002 and from 2008 to 2010 from regions in Africa South.

PCR-RFLP analysis applied in this study L. monocytogenes serotype were placed into one of three groups based on different lineages and SNP sequences each lineage group. SNP preserved in areas where RFLP analysis can be applied to the difference between L. monocytogenes groups.This genealogical studies were conducted to evaluate the implementation of the original agarose gel electrophoresis reported previously for analysis of various monoclonal and polyclonal antibodies.

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Experiments were conducted to test the electrophoresis system for the characterization of different monoclonal antibodies and serum of animals, analysis of antibodies expressed in cell culture and antibody stability evaluation.

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