RESUMO
We have compared various ion exchangers for monoclonal antibody (MAb) purification using different starting materials such as ascitic fluid and cell culture supernatant. Twelve cation and anion exchange resins were tested so far. Purification of MAbs with regard to the starting material is described. In well-defined conditions of adsorption (20 mM MES buffer, pH 6.50), one purification step based on cation-exchange chromatography is generally sufficient to achieve at least 90% purity of the MAb, even when produced by animal cell culture. Cation-exchange supports exhibit higher capacity for MAbs compared to anion exchangers. Among the cation exchangers tested, we have selected the cross-linked matrix S Sepharose FF for its large specificity and capacity for MAbs. Considering these key parameters and also the good mechanical resistance of the S Sepharose FF, we describe how, by varying the flow rate, sample concentration, and size of the column, the productivity may be improved in a monoclonal antibody purification process. Finally, a general 'gram scale' purification protocol of MAbs produced by animal cell cultures is proposed. This protocol, based on economical adsorption conditions and three steps of elution (100 mM, 200 mM and 1 M NaCl), allows the recovery of highly purified MAbs.
Assuntos
Anticorpos Monoclonais/isolamento & purificação , Resinas de Troca Iônica , Animais , Biotecnologia , Soluções Tampão , Células Cultivadas , Cromatografia por Troca Iônica/métodos , Meios de Cultura , Estudos de Avaliação como Assunto , Hibridomas/imunologia , Concentração de Íons de Hidrogênio , Camundongos , Muromonab-CD3/isolamento & purificação , Ribonucleoproteína Nuclear Pequena U1/imunologiaRESUMO
Various serum-free media for use in hybridoma cell cultures are reviewed and their protein composition analysed. The chromatographic behaviour of the serum-free components was analysed on ion exchange, hydrophobic and sizing columns in order to facilitate the design of a purification scheme for monoclonal antibodies produced from various cell cultures.