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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 MF, Guedon E, Yen FT, Ogier V, Goergen JL - BMC Proc (2011)

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratoire Réactions et Génie des Procédés UPR-CNRS 3349, ENSAIA-INPL, Nancy-Université, 54505 Vandoeuvre-lès-Nancy, France ; Lipidomix (EA4422), ENSAIA-INPL, Nancy-Université, 54505 Vandoeuvre-lès-Nancy, France ; Genclis SAS, 54505 Vandoeuvre-lès-Nancy, France.

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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... 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... EDTA (ethylenediaminetetraacetic acid) = inhibitor of metalloprotease activities Complete, Mini, EDTA-free (Roche) = serine and cysteine proteases inhibitor cocktail PMSF (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... Among the 5 caseinase activities detected when CHO cell cultures were performed with serum (Table 1), only the protease activities present all over the process could be potentially involved in the IFN-γ proteolysis (220, 90 and 85 kDa)... EDTA inhibited all the gelatinase activities, identifying these enzymes as metalloproteases, whereas PMSF and Complete inhibitor Cocktail inhibited all the caseinase activities, classifying these enzymes as serine proteases (data not shown)... Compositions of both BDM and RPMI with serum were compared and 3 components which are present in BDM but completely absent in RPMI were identified... Furthermore, when CHO cell cultures were performed in BDM medium without iron citrate during the first 30 hours of the culture, IFN-γ proteolysis was detected (data not shown)... 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.

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Western blot analysis of excreted IFN-γ produced by CHO cells cultivated in various media; RPMI serum, BDM and RPMI serum supplemented with iron citrate
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Figure 1: Western blot analysis of excreted IFN-γ produced by CHO cells cultivated in various media; RPMI serum, BDM and RPMI serum supplemented with iron citrate

Mentions: 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).


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 MF, Guedon E, Yen FT, Ogier V, Goergen JL - BMC Proc (2011)

Western blot analysis of excreted IFN-γ produced by CHO cells cultivated in various media; RPMI serum, BDM and RPMI serum supplemented with iron citrate
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3285009&req=5

Figure 1: Western blot analysis of excreted IFN-γ produced by CHO cells cultivated in various media; RPMI serum, BDM and RPMI serum supplemented with iron citrate
Mentions: 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).

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratoire Réactions et Génie des Procédés UPR-CNRS 3349, ENSAIA-INPL, Nancy-Université, 54505 Vandoeuvre-lès-Nancy, France ; Lipidomix (EA4422), ENSAIA-INPL, Nancy-Université, 54505 Vandoeuvre-lès-Nancy, France ; Genclis SAS, 54505 Vandoeuvre-lès-Nancy, France.

AUTOMATICALLY GENERATED EXCERPT
Please rate it.

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... 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... EDTA (ethylenediaminetetraacetic acid) = inhibitor of metalloprotease activities Complete, Mini, EDTA-free (Roche) = serine and cysteine proteases inhibitor cocktail PMSF (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... Among the 5 caseinase activities detected when CHO cell cultures were performed with serum (Table 1), only the protease activities present all over the process could be potentially involved in the IFN-γ proteolysis (220, 90 and 85 kDa)... EDTA inhibited all the gelatinase activities, identifying these enzymes as metalloproteases, whereas PMSF and Complete inhibitor Cocktail inhibited all the caseinase activities, classifying these enzymes as serine proteases (data not shown)... Compositions of both BDM and RPMI with serum were compared and 3 components which are present in BDM but completely absent in RPMI were identified... Furthermore, when CHO cell cultures were performed in BDM medium without iron citrate during the first 30 hours of the culture, IFN-γ proteolysis was detected (data not shown)... 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.

No MeSH data available.