Monitoring of the production of monoclonal antibodies by hybridomas. Part II: Characterization and purification of acid proteases present in cell culture supernatant.
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.
Clinical Lab Systems Research Unit, Organon Teknika B.V., Boxtel, The Netherlands.
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van Sommeren
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Gribnau
D000371:Agglutination; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000276:immunology; D016282:Aspartic Acid Endopeptidases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D001709:Biotechnology / Q000295:instrumentation / Q000379:methods*; D006063:Chorionic Gonadotropin / Q000276:immunology; D002845:Chromatography / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D006825:Hybridomas / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D015151:Immunoblotting; D007128:Immunoglobulin Fragments / Q000276:immunology / Q000302:isolation & purification; D007074:Immunoglobulin G / Q000276:immunology / Q000378:metabolism; D007525:Isoelectric Focusing
D000911:Antibodies, Monoclonal; D006063:Chorionic Gonadotropin; D007128:Immunoglobulin Fragments; D007074:Immunoglobulin G; D016282:Aspartic Acid Endopeptidases