title: Recombinant interferon-gamma secreted by Chinese hamster ovary-320 cells cultivated in suspension in protein-free media is protected against extracellular proteolysis by the expression of natural protease inhibitors and by the addition of plant protein hydrolysates to the culture medium.
abstract: Biosafety requirements increasingly restrict the cultivation of mammalian cells producing therapeutic glycoproteins to conditions that are devoid of any compound of animal origin. On cultivation in serum-free media, the proteases inhibitors, usually found in serum, cannot protect secreted recombinant proteins against unwanted endogenous proteolysis. Chinese hamster ovary (CHO) cells, secreting recombinant human interferon-gamma (CHO-320 cell line) and cultivated in suspension in an original protein-free medium, expressed at least two members of the matrix metalloproteinases (MMP), either at the cell surface (proMMP-14 and MMP-14) or secreted (proMMP-9). In addition, tissue- and urinary-type plasminogen activators were also secreted in such culture conditions. At the cell surface, dipeptidyl peptidase IV and tripeptidyl peptidase II (TPPII) activities were also detected, and their activities decreased during time course of batch cultures. The proteolytic activities of these proteins were counterbalanced by (1) their expression as zymogens (proMMP-9, proMMP-14), (2) the expression of their natural inhibitors, tissue inhibitors of metalloproteinases-1 and -2 and plasminogen activator inhibitor-1 (PAI-1), or (3) the addition of plant protein hydrolysates to the culture medium, acting as a nonspecific source of TPPII inhibitors. This study points out that, even in protein-free media, recombinant proteins secreted by CHO cells are actively protected against physiological and unwanted extracellular proteolysis either by endogenous or by exogenous inhibitors.
authors: Laboratoire de Biochimie cellulaire, Université catholique de Louvain, B-1348 Louvain-la-Neuve, Belgium.
authors: J
authors: J
authors: Mols
authors: C
authors: C
authors: Peeters-Joris
authors: R
authors: R
authors: Wattiez
authors: S N
authors: SN
authors: Agathos
authors: Y-J
authors: YJ
authors: Schneider
mesh_terms: D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003470:Culture Media; D006868:Hydrolysis; D007371:Interferon-gamma / Q000187:drug effects / Q000378:metabolism*; D007700:Kinetics; D010447:Peptide Hydrolases / Q000378:metabolism; D010940:Plant Proteins / Q000494:pharmacology*; D011480:Protease Inhibitors / Q000494:pharmacology*; D011994:Recombinant Proteins
chemical_list: D003470:Culture Media; D010940:Plant Proteins; D011480:Protease Inhibitors; D011994:Recombinant Proteins; D007371:Interferon-gamma; D010447:Peptide Hydrolases