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Affinity Chromatography of Human Blood Coagulation Factor VIII on Monoliths with Peptides from a Combinatorial Library

K. Amatschek, R. Necina, R. Hahn, E. Schallaun, H. Schwinn, D. Josić, A. Jungbauer

Journal of Separation science, 23 (2000) 47-58

FVIII is a very complex molecule of great therapeutic significance. It is purified by a sequence of chromatographic steps including immunoaffinity chromatography. A peptide affinity chromatography method has been developed using peptides derived from a combinatorial library. Spot technology using cellulose sheets has been applied for this purpose. The dual positional scanning strategy was used for identification of the amino acids in random positions. Approximately 5000 possible candidates found in the first screening round were reduced to a panel of 36. Six candidates have been selected empirically. Five peptides seem to be directed against the light chain of FVIII, one peptide seems to be directed against the heavy chain. The peptides have been immobilized on conventional beaded material and CIM polymethacrylate monoliths. Much better performance with respect to capacity and selectivity has been observed with the monolithic material. Exposure of the ligand and its ensuing accessibility are responsible for these properties.

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