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Characterization of proteins and their interactions is fundamental to the understanding of biochemical processes since most biomolecular processes involve the molecular recognition and ligand binding by proteins. Protein based drugs represent special challenges in terms of drug development and manufacturing. The structural integrity is sensitive to the conditions encountered during formulation, production and storage. The therapeutic efficacy is closely related to the conformational structure; thus development of protein drugs requires a detailed knowledge of the physical and chemical stability. In this context, it would be beneficial if biophysical and functional characterization could be combined into one method.
Pharmacopoeial dissolution methods are the routine test of in-vitro product performance, but whilst they measure product consistency they provide little detailed information about dissolution mechanism. Drug product dissolution is complex, and involves several more fundamental processes such as: hydration, disintegration, erosion, particle de-agglomeration, and dissolution of the drug substance. Thus what is called drug product “dissolution” is actually a combination of several processes. In this poster we present work on the development and application of UV area imaging as a tool to investigate the dissolution of the drug substance. Theories covering the dissolution of chemical substances (e.g. starting from Noyes & Whitney) stress the importance of the concentration gradient between the solid/solution interface and the bulk solution. UV imaging at the micron scale allows this concentration gradient to be investigated.
The ActiPix™ D100 is a novel analytical instrument using UV area imaging and Taylor dispersion analysis (TDA)1,2 for determining diffusion coefficients and hydrodynamic radii of proteins in solution. The detector monitors broadening of a band of a therapeutic protein or small molecule solution injected into a stream of buffer solution and driven by a syringe pump through a fused-silica capillary. The band is imaged at two windows, the first on entry to and the second on exit from a loop in the capillary. The hydrodynamic radius follows from the measured differences between peak times (first moments) and variances (second moments) at the two windows3. Preliminary experimental work has focused on comparison of this technique with that of dynamic light scattering (DLS) which is routinely used for particle size characterization in solution. Preliminary results indicate good correlation between the two techniques for measuring protein (BSA, Ovalbumin, Lysozyme and IgG) and small molecule (caffeine) hydrodynamic radii. Initial Actipix measurements for protein solutions show hydrodynamic radii results in good correlation with literature values. Repeatability and precision were also tested using model protein and small molecule solutions. The ability to determine protein aggregate levels is being explored.
In this poster, Paraytec introduce the principles, operation and key benefits of using surface dissolution imaging to the formulations community for rapid drug development. Model compounds are used to demonstrate practicle application of this revolutionary technique.
Characterization of proteins and their interactions is fundamental to the understanding of biochemical processes since most biomolecular processes involve the molecular recognition and ligand binding by proteins. Protein based drugs represent special challenges in terms of drug development and manufacturing. The structural integrity is sensitive to the conditions encountered during formulation, production and storage. The therapeutic efficacy is closely related to the conformational structure; thus development of protein drugs requires a detailed knowledge of the physical and chemical stability. In this context, it would be beneficial if biophysical and functional characterization could be combined into one method.
The Bioconversion-Chemistry-Engineering Interface Programme (BiCE) is a multidisciplinary collaboration between three academic Departments at UCL focusing on the integration of biocatalysis and chemistry with engineering. One of the outputs of the BiCE programme are families of evolved enzymes with improved conversion rates and substrate ranges [1]. In parallel we are looking at automated techniques for the high-throughput analysis of these enzymes under process conditions in particular in microwell [2] and microfluidic [3] formats.
Use of the ActiPix™ UV area imaging detector with multiple windows on a single looped capillary provides new insights with species of pharmaceutical & biopharmaceutical interest. Two key applications are demonstrated: Determination of hydrodynamic radius from imaging pressure driven flow of analyte bands at two windows. Results are shown for proteins and small molecules injected as single species and in mixtures, and for a mixture of proteins with simultaneous electrophoretic separation. Determining substrate specificity of a biocatalyst towards a mixture of UV active compounds using a continuous engagement electrophoretically mediated microanalysis (EMMA) method.
An A0 poster describing the benifits of the ActiPix™ D100 for determining hydrodynamic radius
Presented at PittCon 2007, Chicago.
Professor David Goodall presented "A miniature UV imaging detector based upon active pixel technology: applications with single and multiplexed capillary separations" at the recent Chromotographic Society Golden Jubilee Triad Symposia held at GlaxoSmithKline in Stevenage on 21-22 November 2006.
"Miniature Capillary UV Detector Based on Active Pixel Sensor Imaging: Development and Applications" Presented at HPLC2006, the 30th International Symposium on High Performance Liquid Phase Separations and Related Techniques, 17 - 23 June 2006, San Francisco.
"A New Active Pixel Based Miniature In-Line UV Detector: Technology and Applications" Poster presented at HPLC2006, the 30th International Symposium on High Performance Liquid Phase Separations and Related Techniques, 17 - 23 June 2006, San Francisco.
"Active Pixel Miniature UV Detector: High Resolution Imaging for Low Volume In line Applications" Poster presented at PAT 2006, the Application of Process Analytical Technology in the Biotechnology Industry, 23-24 June 2006, San Francisco.
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