Monitoring of the production of monoclonal antibodies by hybridomas. Part II: Characterization and purification of acid proteases present in cell culture supernatant.

Autor: van Erp R; Clinical Lab Systems Research Unit, Organon Teknika B.V., Boxtel, The Netherlands., Adorf M, van Sommeren AP, Gribnau TC
Jazyk: angličtina
Zdroj: Journal of biotechnology [J Biotechnol] 1991 Oct; Vol. 20 (3), pp. 249-61.
DOI: 10.1016/0168-1656(91)90298-a
Abstrakt: An acid proteolytic activity has been found in cell culture supernatants from long-term cultivations of hybridoma cells in hollow fibre bioreactors using serum free medium. The proteolytic activity has now been further characterized and the main results were: (1) the proteolytic activity showed a maximum around pH 3 and declined essentially to zero at pH 8; (2) the activity was specifically inhibited by pepstatin A; (3) the acid proteases consisted of two sets of closely spaced bands with apparent molecular weights of 40-45K and 90-105K, respectively; (4) the protease bands (40-45K and 90-105K) were reactive with anti-human cathepsin D; (5) the IEP values of the acid proteases ranged from pH 4.55-6.5. Furthermore, IgG incubation with the acid proteases isolated from hybridoma cells yielded fragments similar to those found in serum-free hollow fibre cell culture supernatants. These results indicated that the IgG fragments are the result of degradation by cathepsin D like proteases released after cell death or cell lysis.
Databáze: MEDLINE