RESUMO
A comparative study was carried out to investigate the influence of different mouse antibody subclasses on the chromatographic behaviour on thiophilic supports. Cell-free supernatants from different mouse-mouse hybridoma cultures in a standard medium were purified on thiophilic agarose and Fractogel EMD TA. The adsorption capacities and purification factors were monitored under optimised adsorption conditions. The different isotypes did not differ significantly regarding capacity of the thiophilic matrix, but the purity of the eluted antibody fractions was significantly lower for the IgG2a subclass compared to all other murine antibodies. A significant copurification of proteins from cell culture supernatant with antibodies of the IgG2a subclass indicated a restriction in the universal nature of thiophilic interaction.
Assuntos
Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos , Animais , Anticorpos Monoclonais/classificação , Especificidade de Anticorpos/efeitos dos fármacos , Extratos Celulares , Sistema Livre de Células , Hibridomas , Camundongos , Fenolsulfonaftaleína/farmacologiaRESUMO
The application of thiophilic membranes for the purification of monoclonal antibodies from hybridoma culture media was studied. Affinity filtrations were performed with membrane stacks and also in a cross flow module with a spiral filtration channel. Purification factors up to five and concentration factors of about eight could be achieved. The flux behaviour was analysed and interpreted according to existing models of filtration. The results were confirmed by scanning electron microscopy. The binding capacity of the membranes differed considerably with the mode of operation. The main component responsible for membrane fouling was identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis and amino acid sequence analysis as bovine serum albumin or its fragments.