Purification of proteins from cell culture supernatants.
Desired proteins excreted by animal cells usually reach rather low concentrations in the culture supernatant and have to be purified from an excess of serum proteins which are added to the animal cell culture to maintain its viability and/or productivity. Defined media offer among others the advantage of an easier purification procedure. In addition lysis of cells may contribute also to the complexity of the protein mixture encountered. If fetal calf serum or new born calf serum is used in cultivation, bovine serum albumin and globulins will be the most abundant protein in the supernatant. The interaction of albumin especially with hydrophobic proteins represents significant problems for the effective purification of minor constituent. Isolation of a desired protein follows the general scheme: concentration: by precipitation, ultrafiltration, batch adsorption or partition in aqueous phase system; enrichment: by chromatography or partition; high resolution purification: by chromatography and/or immuno adsorption; final concentration and finishing: pyrogen removal, sterilization, formulation. Biochemical engineering aspects of proteins purification are discussed using human interferon-beta produced in a recombinant mouse cell line as an example. The process developed encloses extraction of interferon-beta from the production medium in aqueous two-phase systems and subsequent recovery of interferon-beta from the top phase by high pressure liquid chromatography.
D000418:Albumins / Q000032:analysis; D000818:Animals; D001709:Biotechnology / Q000379:methods*; D002478:Cells, Cultured / Q000032:analysis*; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003470:Culture Media / Q000032:analysis; D005347:Fibroblasts; D006801:Humans; D007370:Interferon Type I / Q000302:isolation & purification; D011506:Proteins / Q000302:isolation & purification*; D012995:Solubility
D000418:Albumins; D003470:Culture Media; D007370:Interferon Type I; D011506:Proteins