Recombinant human interferon-β1 (rhIFN-β1) glycoprotein is used as a therapeutic agent for a variety of diseases, such as multiple sclerosis and hepatitis. In the present work, different strategies were applied to produce rhIFN-β1a in mammalian cell cultures. Transfected population of CHO-K1, CHO dhfr-, BHK and HEK cells were compared for their ability to produce rhIFN-β1a, and clones of the most promising cell line (CHO-K1) were isolated by the limit dilution method. Likewise, different culture conditions were assayed by changing the amounts of fetal calf serum, sodium butyrate and/or ZnSO4, to improve cell productivity. The presence of each additive increased the rhIFN-β1a yield ranging from 2 to 8 times, depending on the tested cell clone, but when these components were simultaneously added to the medium, the rhIFN-β1a concentration in the supernatants was even greater.

Two receptor-binding sites for hGM-CSF are described. Competitive binding ELISA using four monoclonal antibodies (MAbs) showed different epitope recognitions. The antibody combining sites were mapped using sets of overlapping peptides and hexapeptide libraries prepared by the SPOT synthesis technique. We identified the conformationally dependent epitopes A18E21R23R24F119 and R23E21N17W13 bound by MAb CC5B5 and the nonlinear epitope P118F119W13E14 bound by MAb M1B8. The epitopes recognized by these two MAbs are very closely located on the native protein surface. The peptide L61YKQGKLRGSLTK72 was recognized by MAb M7E10 and the peptide A1PAR4, representing the N-terminal sequence of the protein, was bound by the nonneutralizing MAb CC1H7. Inhibition assays of the GM-CSF biological activity demonstrated that MAb M1B8, CC5B5 and M7E10 bind to domains which are responsible for the interaction of the cytokine with the GM-CSF receptor.

Two groups of isoforms of rhEPO, at a concentration of 300 µg/ml, were tested as putative inhibitors of the lectinic hemagglutination reaction in order to obtain affinity ligand(s) for hormone purification: groups I (pI: 3.80; 3.89; 3.95; 4.07, 4.15 and 4.26) and groups II (pI: 4.15, 4.26; 4.38; 4.51; 4.72 and 4.93) Crude extracts from the vegetable materials Abrus precatorious (Abrin), Artocarpus incisa (Frutalin), Artocarpus integrifolia (Jacalin), Canavalia ensiformes (ConA), Canavalia brasiliensis (Conbr), Cratylia floribunda, Dioclea altissima (DAL), Dioclea grandiflora (DGL), Erythrina vellutina (EVL), Erythrina cristagalli, Lutaelburgia auriculata (lectin not fully characterized yet), Lycopersicum esculentum (LEA), Phaseolus vulgaris (PHA), Ricinus communis (Ricin) and Triticum vulgaris (WGA) were used. Only some of the galactose-specific lectins and the GlcNAc-specific lectins showed rapid full inhibition of the hemagglutination reaction for the less acidic isoforms and the total isoforms of rhEPO, respectively. On this basis, the selected lectins were purified by affinity chromatoghraphy and covalently coupled to cyanogen bromide activated Sepharose® (Amersham-Pharmacia). CHO.K1 cell culture supernatant containing rhEPO was loaded onto the lectin resins and the recoveries were calculated by using specific elutions.