Cell surface staining of recombinant factor VIII is reduced in apoptosis resistant BHK-21 cells

Ishaque, Adiba; Thrift, John; Murphy, John E.; Konstantinov, Konstantin

doi:10.1016/j.jbiotec.2008.07.1856

Cited by 5 publications

(4 citation statements)

References 20 publications

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“…A recent study showed that an apoptosis resistant cell line exhibited less membrane bound rFVIII and further, it was shown that the annexin V based apoptosis assay was impeded by masking of PS by the rFVIII (Ishaque et al, 2008). In support of these data we found that annexin V was able to reduce the membrane bound fraction by 70% in a radio labelled rFVIII competition assay (Fig.…”

Section: Discussionsupporting

confidence: 82%

Optimisation of the Factor VIII yield in mammalian cell cultures by reducing the membrane bound fraction

Kolind

Nørby

Berchtold

et al. 2011

Journal of Biotechnology

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Section: Discussionsupporting

confidence: 82%

Optimisation of the Factor VIII yield in mammalian cell cultures by reducing the membrane bound fraction

Kolind

Nørby

Berchtold

et al. 2011

Journal of Biotechnology

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“…About 2000 events were collected and analyzed using CytoSoft TM Software (Guava Technologies). Additional details are also given in Ishaque et al, 2007Ishaque et al, , 2008.…”

Section: Apoptosis Evaluationmentioning

confidence: 99%

Characterization of cell‐banking parameters for the cryopreservation of mammalian cell lines in 100‐mL cryobags

Heidemann

Lünse

Tran

et al. 2010

Biotechnology Progress

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This article describes a cell banking process for rBHK cell lines in 100-mL cryobags. As the use of larger volume cell banks requires greater cell numbers and longer preparation time, extensive characterization of key process parameters beyond the conventional ranges was performed to support a cGMP banking process. All experiments were conducted using two recombinant BHK21 cell lines, one of them cotransfected with Hsp70. The results show that the entire cell banking process for these BHK cell lines can be performed at room temperature. A DMSO exposure time up to 5 h either directly in a bioreactor or in shaker flasks did not result in any significant negative effect after cell thaw, when the cryocontainers were frozen immediately after filling. Extensive characterization did not indicate any significant apoptotic effects after thaw. However, the Hsp70 cotransfected cell line did show a slightly better protection from potential cryopreservation-induced apoptosis. Surprisingly, it was found that cells transferred into cryobags showed a low recovery rate after thaw if the incubation time exceeded 1.5 h before freezing. Additional experiments confirmed that the DMSO exposure time inside the cryocontainer in contrast to the DMSO exposure in a reactor or shaker flasks is much more critical. The cryobag cell banking process should therefore be performed within a 1(1/2)-2 h window; a banking process for vials should not exceed 2(1/2) h.

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“…This prompted the authors to speculate that secreted rFVIII, by virtue of its phosphatidylserine (PS) binding properties, would be able to shield PS binding sites and thereby block Annexin V binding. It was subsequently shown by FACS analysis, that non-engineered host cells indeed bound more rFVIII than engineered cells (Ishaque et al, 2008), indicating a higher level of apoptosis in the host cells and a resulting increase in PS exposure.…”

Section: Introductionmentioning

confidence: 99%

The B-domain of Factor VIII reduces cell membrane attachment to host cells under serum free conditions

Kolind

Nørby

Flintegaard

et al. 2010

Journal of Biotechnology

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Cell surface staining of recombinant factor VIII is reduced in apoptosis resistant BHK-21 cells

Cited by 5 publications

References 20 publications

Optimisation of the Factor VIII yield in mammalian cell cultures by reducing the membrane bound fraction

Optimisation of the Factor VIII yield in mammalian cell cultures by reducing the membrane bound fraction

Characterization of cell‐banking parameters for the cryopreservation of mammalian cell lines in 100‐mL cryobags

The B-domain of Factor VIII reduces cell membrane attachment to host cells under serum free conditions

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