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Studies to Prevent Degradation of Recombinant Fc-Fusion Protein Expressed in Mammalian Cell Line and Protein Characterization.

ABSTRACT: Clipping of recombinant proteins is a major issue in animal cell cultures. A recombinant Fc-fusion protein, VEGFR1(D1-D3)-Fc expressed in CHOK1SV GS-KO cells was observed to be undergoing clippings in lab scale cultures. Partial cleaving of expressed protein initiated early on in cell culture and was observed to increase over time in culture and also on storage. In this study, a few parameters were explored in a bid to inhibit clipping in the fusion protein The effects of culture temperature, duration of culture, the addition of an anti-clumping agent, ferric citrate and use of protease inhibitor cocktail on inhibition of proteolysis of the Fc fusion were studied. Lowering of culture temperature from 37 to 30 °C alone appears to be the best solution for reducing protein degradation from the quality, cost and regulatory points of view. The obtained Fc protein was characterized and found to be in its stable folded state, exhibiting a high affinity for its ligand and also biological and functional activities.

SUBMITTER: Chakrabarti S

PROVIDER: S-EPMC4926446 | biostudies-literature | 2016 Jun

REPOSITORIES: biostudies-literature

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Studies to Prevent Degradation of Recombinant Fc-Fusion Protein Expressed in Mammalian Cell Line and Protein Characterization.

Chakrabarti Sanjukta S Barrow Colin J CJ Kanwar Rupinder K RK Ramana Venkata V Kanwar Jagat R JR

International journal of molecular sciences 20160609 6

Clipping of recombinant proteins is a major issue in animal cell cultures. A recombinant Fc-fusion protein, VEGFR1(D1-D3)-Fc expressed in CHOK1SV GS-KO cells was observed to be undergoing clippings in lab scale cultures. Partial cleaving of expressed protein initiated early on in cell culture and was observed to increase over time in culture and also on storage. In this study, a few parameters were explored in a bid to inhibit clipping in the fusion protein The effects of culture temperature, du ...[more]

PMID: 27294920

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Project description:BackgroundAlpha-1 antitrypsin (AAT) is a major serine protease inhibitor. AAT deficiency (AATD) is a genetic disorder characterized by early-onset severe emphysema. In well-selected AATD patients, therapy with plasma-derived AAT (pAAT), "augmentation therapy", provides modest clinical improvement but is perceived as cumbersome with weekly intravenous infusions. Using mouse models of emphysema, we compared the effects of a recombinant AAT-IgG1 Fc-fusion protein (AAT-Fc), which is expected to have a longer half-life following infusion, to those of pAAT.MethodsIn an elastase model of emphysema, mice received a single intratracheal instillation of porcine pancreatic elastase (PPE) or human leucocyte elastase (hLE). AAT-Fc, pAAT, or vehicle was administered intraperitoneally 1 day prior to or 3 weeks following elastase instillation. Lung function and histology assessments were performed at 7 and 32 days after elastase instillation. In a cigarette smoke (CS) model of emphysema, mice were exposed to CS daily, 5 days a week, for 6 months and AAT-Fc, pAAT, or vehicle were administered every 10 days during the last 3 months of CS exposure. Assessments were performed 3 days after the last CS exposure. Immune responses to lung elastin peptide (EP) and the effects of AAT-Fc or pAAT treatment on dendritic cell (DC) function were determined ex vivo.ResultsBoth elastase instillation and CS exposure triggered emphysema-like alveolar enlargement, increased lung compliance, and increased markers of inflammation compared to controls. Administration of AAT-Fc either prior to or following elastase instillation or during CS exposure provided greater protection than pAAT against alveolar enlargement, lung dysfunction, and airway inflammation. When challenged ex vivo with EP, spleen mononuclear cells from elastase-exposed mice exhibited dose-dependent production of IFNγ and IL-17, suggesting immune reactivity. In co-culture experiments with splenic CD4+ T cells isolated from elastase-exposed mice, AAT-Fc treatment prior to EP-priming of bone marrow-derived dendritic cells inhibited the production of IFNγ and IL-17.ConclusionsCompared to pAAT, AAT-Fc more effectively prevented or attenuated elastase- and CS-induced models of emphysema. These effects were associated with immunomodulatory effects on DC activity. AAT-Fc may provide a therapeutic option to individuals with AATD- and CS-induced emphysema.

Dataset Information

Studies to Prevent Degradation of Recombinant Fc-Fusion Protein Expressed in Mammalian Cell Line and Protein Characterization.

Publications

Studies to Prevent Degradation of Recombinant Fc-Fusion Protein Expressed in Mammalian Cell Line and Protein Characterization.

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