Purification and quantitation of tumor necrosis factor receptor immunoadhesin using a combination of immunoaffinity and reversed-phase chromatography |
| |
| Affiliation: | 1. Department of Biology, University of Pennsylvania, Philadelphia, PA 19104, USA;2. Department of Cell and Developmental Biology, University of Pennsylvania, Perelman School of Medicine, Philadelphia, PA 19104, USA;1. Fraunhofer Institute for Structural Durability and System Reliability (LBF), Division Plastics, Group Material Analytics, Schlossgarten Str. 6, 64289 Darmstadt, Germany;2. SABIC, Analytical Technology, Plasticslaan 1, 4612 PX Bergen op Zoom, The Netherlands;3. SABIC,1600 Industrial Blvd., Sugar Land, TX 77478, USA;1. Laboratory for Human Disease Models RIKEN Center for Integrative Medical Sciences, Yokohama, Japan;2. Department of Pediatrics and Developmental Biology, Tokyo Medical and Dental University, Tokyo, Japan;3. Laboratory for Integrative Genomics, RIKEN Center for Integrative Medical Sciences, Yokohama, Japan;4. Division of Leukemia and Lymphoma, Children’s Cancer Center, National Center for Child Health and Development, Tokyo, Japan;5. Department of Pediatrics, Ehime University Graduate School of Medicine, Ehime, Japan;6. Department of Hematology/Oncology, Saitama Children’s Medical Center, Saitama, Japan;7. The Jackson Laboratory, Bar Harbor, ME;8. Kazusa DNA Research Institute, Kisarazu, Chiba, Japan |
| |
| Abstract: | 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 μg 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. |
| |
| Keywords: | |
| 本文献已被 ScienceDirect 等数据库收录！ |
|
