Cultivation of Chinese hamster ovary (CHO) cells in protein-free media supplemented with plant peptones is sufficient to prevent proteolysis of secreted recombinant glycoproteins
(2004)
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- Chinese hamster ovary (CHO) cells are widely utilized to produce complex recombinant (glyco)proteins due to their ability to perform efficiently post-translational modifications and to grow in suspension at very high densities. For therapeutic purposes, biosafety has to be ensured at each step of the protein purification, but also during the cultivation of the cells. Consequently, serum and animal components are progressively banned from new culture medium formulations and now protein-free (PF) media are available for the cultivation of CHO cells. Unfortunately, a decrease in cell density and productivity is often associated with the shift from serum-containing media to PF media as well as an increase in the risk of proteolysis due to the lack of serum protease inhibitors. We have used CHO-320 cells that are secreting recombinant human interferon-ã (IFN-ã), as a cellular model to study cell growth, glycoprotein secretion, glycosylation heterogeneities, as well as the extent of proteolysis in such media. After a direct adaptation from adhesion in serum to suspension in a homemade PF medium supplemented with different plant protein hydrolysates (peptones), CHO-320 cells grew faster than in serum-containing medium. IFN-ã production was similar from a quantitative viewpoint, but most the IFN-ã was bi-glycosylated whereas in serumcontaining medium, the non-, mono-, and bi-glycosylated glycoforms were found in equal proportions. Further supplementation of this PF medium with some plant peptones increased the IFN-ã secretion by up to 60% and cell density by up to 30% upon cultivation in suspension. The cultivation of CHO-320 cells inside microcarriers in PF media with plant peptones was also investigated. It appears that in such culture conditions, plant peptones were only able to increase the IFN-ã secretion while the cell density remains apparently unaffected. Depending on the type of plant peptones utilized, IFN-ã underwent proteolysis in the culture medium. Some extracellular proteases expressed by CHO cells were identified and their relationships to this proteolytic event was investigated. CHO cells express members of the matrix metalloproteinases (MMP) and of serine proteases, either at the cell surface i.e. (pro)MMP-14, dipeptidyl peptidase IV (DPPIV) and tripeptidylpeptidase II (TPPII) or released them in the extracellular medium i.e. proMMP-9, urinary-type (uPA) and tissue-type plasminogen activators (tPA). In addition and probably more importantly, the presence of their natural inhibitors was also detected i.e. TIMP-1 and -2 as well as plasminogen activator inhibitor type 1 (PAI-1). It has also been shown that peptones could act as a source of exogenous inhibitors of tripeptidylpeptidase II (TPPII). Our results indicate that the proteolytic activity of these proteases was tightly regulated at a posttranslational level by (i) their expression as inactive precursors (proMMP-9 and proMMP-14), (ii) by the presence of some endogenous inhibitors and (iii) by the presence of some peptides in plant peptones acting as exogenous TPPII inhibitors. Nevertheless, when some plant peptones were added to the nutritive medium, IFN-ã proteolysis was observed. We showed that this proteolytic event was related to a cysteine protease already present as residual enzymatic contamination of the peptones and not to extracellular proteases naturally expressed by CHO cells. Finally, at a molecular level, we have some evidence that plant peptones could act as a nutritional source. Indeed, plant peptones could behave as competitive substrates of peptide transporters and/or TPPII. Consequently, di- and or tripeptides present in the peptones can directly be absorbed by peptide transporters, whereas some larger peptides in the peptones could be cleaved into tripeptides by TPPII and afterwards be internalised. Nevertheless, the effects of the peptones cannot be reproduced by amino acids supplementation and some of their properties have to be interpreted as an indication of the presence of bioactive substances. To conclude, plant peptones are highly beneficial supplements for the cultivation of CHO cells in protein-free media. Unfortunately, their undefined nature represents an important limitation, which can have adverse effects and, therefore, the biologically active components present in the peptones should be identified to substitute peptones from the formulation of culture media as well as the transduction cascades should be dissected at the molecular level.
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Mols, J. (2004). Cultivation of Chinese hamster ovary (CHO) cells in protein-free media supplemented with plant peptones is sufficient to prevent proteolysis of secreted recombinant glycoproteins. https://hdl.handle.net/2078.5/98432