Purification and quantitation of tumor necrosis factor receptor immunoadhesin using a combination of immunoaffinity and reversed-phase chromatography
| Autor: | Martin Vanderlaan, Andrew J. S. Jones, John E. Battersby |
|---|---|
| Rok vydání: | 1999 |
| Předmět: |
Chromatography
Elution medicine.drug_class Chemistry Coefficient of variation General Chemistry Reversed-phase chromatography Monoclonal antibody High-performance liquid chromatography Sensitivity and Specificity Chromatography Affinity Receptors Tumor Necrosis Factor Etanercept Electrophoresis Macaca fascicularis Pharmacokinetics Affinity chromatography Immunoglobulin G medicine Animals Humans Electrophoresis Polyacrylamide Gel Spectrophotometry Ultraviolet Chromatography High Pressure Liquid |
| Zdroj: | Journal of chromatography. B, Biomedical sciences and applications. 728(1) |
| ISSN: | 1387-2273 |
| Popis: | The development of an automated, dual column assay to quantitate and recover the glycoprotein, tumor necrosis factor receptor immunoadhesin (TNFr-IgG) from monkey plasma, human serum, cell culture fluid and buffer samples is described. A combination of immunoaffinity and reversed-phase chromatographies are used. The targeted protein was captured using an anti-TNFr-1 monoclonal antibody immobilized on POROS resin. After non-specific adsorption had been reduced, the affinity column was placed in-line with a reversed-phase column and eluted with dilute acid. The reversed-phase column was subsequently eluted with an acetonitrile gradient and the TNFr-IgG collected and quantitated by comparison with peak areas of similarly treated standards. Detection was performed by measurement of absorbance at 214 nm. The dynamic range is from 0.5-15 microg total sample. Samples were quantitated and recovered from monkey and human pharmacokinetics samples, as well as from cell culture fluid and buffers. The lowest concentrations assayed were 100 ng ml(-1). Quantitation is reproducible, with a coefficient of variation of 2%. The procedure was used to develop a pharmacokinetic profile for the clearance of TNFr-IgG in humans and cynomolgus monkeys. Sufficient material was recovered such that the glycoforms could be identified. Additionally it has been used for process monitoring. The results compared favorably with data generated by ELISA. Optimization of the method and results are presented. |
| Databáze: | OpenAIRE |
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