A Series of Collaborations Between Various Pharmaceutical Companies and Regulatory Authorities Concerning the Analysis of Biomolecules Using Capillary Electrophoresis

An international project team (including members from US, Canada and UK) has been formed from a number of interested biopharmaceutical companies and regulatory authorities to conduct a cross-organisation collaboration exercise. The results from this exercise demonstrate the robustness of CE-SDS across eight different organisations that used instruments of the same equipment model, the same reagents, and the same methodology. Data generated from the analysis of a series of molecular weight markers showed very good precision with regards to relative migration time (RMT) both within and between organisations. The apparent molecular weight of bovine serum albumin (BSA) was measured with good precision to within approximately 2% RSD across the participants. A representative IgG sample showed similar results with regards to relative migration time of its 3 main components, IgG Light Chain, IgG Non-glycosylated Heavy Chain, and IgG Heavy Chain. Fractional peak area for each peak also showed good agreement, with less than 9% RSD for all peaks. This exercise will facilitate both increased regulatory and industrial opinion of CE for biopharmaceutical analysis.CE in the Biotechnology & Pharmaceutical Industries: 7th Symposium on the Practical Applications for the Analysis of Proteins, Nucleotides and Small Molecules, Montreal, Canada, August 12–16, 2005     

A Series of Collaborations between Various Pharmaceutical Companies and Regulatory Authorities Concerning the Analysis of Biomolecules Using Capillary Electrophoresis: Additional Instruments/Buffer

An international project team (including members from US, Canada and UK) was formed from a number of interested biopharmaceutical companies and regulatory authorities to conduct a cross-organisation collaboration exercise. The results of the first comparison with eight different organisations that used instruments of the same equipment model, the same reagents, and the same methodology has been reported previously [1]. This report represents the addition of other instruments using a different run buffer. The relative migration times were different, as expected, prohibiting a direct comparison between companies. The within-organisation variability was low for both relative migration time (<0.34% RSD% for all companies save one) and the peak area (<5% RSD% for all companies save one) when measuring the purity of a representative IgG sample. The apparent molecular weight of bovine serum albumin was measured with good precision (less than 10% RSD% across all companies) to the theoretical value when all data is utilized (67.5 kDa compared to 66.4 kDa). For a representative IgG sample, the three main components, IgG Light Chain, IgG Non-glycosylated Heavy Chain, and IgG Heavy Chain, could not be separated, specifically the IgG Non-glycosylated Heavy Chain and IgG Heavy Chain. When the IgG Non-glycosylated Heavy Chain and IgG Heavy Chain were combined for all organisations, the fractional peak area for the IgG Light Chain and IgG Non-glycosylated Heavy Chain + IgG Heavy Chain peak also showed excellent agreement, with less than 7.5 and 3.5% RSD%, respectively. The value of this exercise is in demonstrating the reliability of CE for the determination of apparent size of biopharmaceutical proteins. This underpins the appropriate use of such CE data in support of regulatory submissions.     

Chemical capacitance of nanoporous-nanocrystalline TiO2 in a room temperature ionic liquid

The electrochemical behaviour of nanoporous TiO(2) in a room temperature ionic liquid (RTIL), 1-ethyl-3-methylimidazolium bis((trifluoromethyl)sulfonyl)amide (EMITFSI), was investigated by cyclic voltammetry (CV) and impedance spectroscopy. Exponentially rising currents in voltammetry were attributed to the charging/discharging of electrons in the TiO(2) film and a charge transfer mechanism. The main features of the voltammetry and impedance followed the same trends in the ionic liquid as in other organic solvents and also in aqueous electrolytes. In the presence of lithium ions, the onset potential of the charge accumulation increased due to the change of the initial position of the TiO(2) conduction band. The results show that substitution of organic solvents contained in solar cells, supercapacitors or other electrochemical devices is in general feasible, though requires some adjustment in the electrolyte composition for optimal performance.     

Rapid and selective characterization of influenza virus constituents in monovalent and multivalent preparations using non-porous reversed-phase high performance liquid chromatography columns

The characterization of influenza vaccine composition has been approached through a novel methodology suitable for routine analysis. It is based on a two-stage process involving an initial sample processing step followed by analysis by reversed-phase HPLC with UV detection. The sample processing involves an initial concentration step carried out in the presence of a combination of detergents and organic solvents to enhance solubilization and ultimately to provide adequate detection. Conditions that provided fast, reproducible and selective separations of vaccine constituents were investigated by reversed-phase HPLC. The use of non-porous silica stationary phases was found to minimize carry-over and non-specific adsorption observed with conventional columns. An evaluation of separation parameters, including mobile phase composition and column temperature, allowed optimization of the selectivity of the method. The optimized method was suitable for the characterization of processed monovalent preparations (containing influenza virus constituents from a single strain). In addition, it allowed the simultaneous detection of the three influenza subtypes in trivalent vaccines in a single analysis. Several influenza constituents were detected including nucleoprotein, the highly hydrophobic matrix protein and the primary surface antigen, haemagglutinin (HA).     

Comparative metabolomic study of transgenic versus conventional soybean using capillary electrophoresis-time-of-flight mass spectrometry

In this work, capillary electrophoresis-time-of-flight mass spectrometry (CE-TOF-MS) is proposed to identify and quantify the main metabolites found in transgenic soybean and its corresponding non-transgenic parental line both grown under identical conditions. The procedure includes optimization of metabolites extraction, separation by CE, on-line electrospray-TOF-MS analysis and data evaluation. A large number of extraction procedures and background electrolytes are tested in order to obtain a highly reproducible and sensitive analytical methodology. Using this approach, a large number of metabolites were tentatively identified based on the high mass accuracy provided by TOF-MS analyzer, together with the isotopic pattern and expected electrophoretic mobility of these compounds. In general, the same metabolites and in similar amounts were found in the conventional and transgenic variety. However, significant differences were also observed in some specific cases when the conventional variety was compared with its corresponding transgenic line. The selection of these metabolites as possible biomarkers of transgenic soybean is discussed, although a larger number of samples need to be analyzed in order to validate this point. It is concluded that metabolomic procedures based on CE-MS can open new perspectives in the study of transgenic foods in order to corroborate (or not) the equivalence with their conventional counterparts.