Volume 5 Supplement 8
Characterization of metalloprotease and serine protease activities in batch CHO cell cultures: control of human recombinant IFN-γ proteolysis by addition of iron citrate
© Clincke et al; licensee BioMed Central Ltd. 2011
Published: 22 November 2011
During the production of any recombinant proteins, an evaluation of the product quality is crucial. Proteolytic events may occur during the process and could influence the product quality. Indeed, proteolysis is an unpredictable process and relatively little is know regarding the proteolytic enzymes produced and released by mammalian cells. In fact, proteases originating from the host cell line cannot be avoided in cell culture. Due to regulatory and safety prospects, the addition of serum, fetuin or albumin that usually limit protease activities, is not desirable. Thus, in serum-free cultures of mammalian cells, control of protease activity constitutes a major challenge.
In the present work, the presence of proteases and their effect on quality of IFN-γ produced by a recombinant CHO cell line cultivated in a stirred-tank bioreactor were studied. Whereas the quality of IFN-γ remained constant during the CHO cell cultures performed in BDM medium, IFN-γ proteolysis was observed when cultures were carried out in RPMI medium with serum [1, 2].
Materials and methods
IFN-γ producing CHO cell lines (CHO 320: dhfr+, α2,6 ST-) were grown in RPMI supplemented with 5% serum and in BDM medium . Whereas RPMI is a classical medium containing serum, BDM is a chemically defined medium without any proteins or serum addition, but supplemented with 0.1% pluronic F-68, 750 µM ethanolamine and 500 µM iron citrate.
Batch cultures were performed in stirred-tank bioreactor (Inceltech, SGI). Dissolved oxygen concentration was set at 50% of air saturation. Agitation rate used was 50 rpm; pH and temperature were set at 7.2 and 37°C respectively.
Glycosylation macroheterogeneity of IFN-γ was characterized by Western Blot (Amersham Biosciences).
Gelatinase and caseinase activities were performed using zymography. Cell-free culture supernatants were concentrated 2-fold on a 10-kDa cutoff filter. Then, the concentrated samples were instantly mixed 3:1 with nonreducing electrophoresis sample buffer (4.8 mL H2O; 1.2 mL Tris-HCl 0.5 M pH 6.8; 2 mL SDS 10%; 1 mL glycerol; 0.5 mL bromophenol blue) and loaded on the zymogram gels. The SDS-PAGE gels (10% acrylamide) contained either 0.05% caseine or 0.1% gelatin. The gels were run at 25 mA for 1h. To remove SDS, the gels were soaked twice for 30 min in 2% Triton X-100 on a shaker, then washed in distilled H2O followed by an 24h incubation in developing buffer (0.5 M Tris, pH 7.4, 1 µM Zn2+ and 5 mM Ca2+). Inhibitor supplementations were also performed using EDTAa, PMSFb and Complete inhibitor Cocktailc. Visualization of protease activity was carried out by incubation for around 3h in a Coomassie blue solution.
aEDTA (ethylenediaminetetraacetic acid) = inhibitor of metalloprotease activities
bComplete, Mini, EDTA-free (Roche) = serine and cysteine proteases inhibitor cocktail
cPMSF (phenylmethylsulfonyl fluoride) = inhibitor of serine protease activities
CHO cell cultures producing human recombinant IFN-γ were cultivated in stirred-tank bioreactor in both RPMI supplemented with 5% serum and BDM media. In both media, three major molecular weight variants (2N, 1N, 0N) were detected during the process with a majority of IFN-γ doubly-glycosylated (2N) whatever the medium used. However, using the RPMI medium with serum, IFN-γ proteolysis was observed during the whole culture (Figure 1).
Protease activities detected by zymography during CHO cell cultures performed in RPMI medium with serum and BDM medium
Molecular Weight (kDa)
RPMI medium with serum
220, 140, 90, 85 and 40 kDa
90 and 85 kDa
95, 90 and 65 kDa
Using zymogram analysis, gelatinase and caseinase activities in CHO batch cultures performed with or without serum were detected, and belong most likely to the metalloprotease and serine protease families. When cultures were carried out in RPMI with serum, a degradation of recombinant IFN-γ was observed, while no IFN-γ proteolysis was detected in culture performed with BDM medium. Furthermore, our results showed that despite the medium used (RPMI, BDM, with or without serum), addition of iron minimized IFN-γ proteolysis, probably due to the inhibition of a 90 kDa metalloprotease activity. Thus, we demonstrated that the addition of iron citrate can be advantageously considered for industrial processes to prevent the proteolysis of a recombinant protein, in particular if one or several metalloproteases are present in the culture.
This work was financed by the Agence Nationale pour la Recherche Technique (ANRT) and Genclis SAS (Vandoeuvre-lès-Nancy, France).
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