Analysis of expression kinetics and activity of a new B-domain truncated and full-length FVIII protein in three different cell lines

Haack, A; Schmitt, Christoph; Poller, Wolfgang; Oldenburg, Johannes; Hanfland, P.; Brackmann, Hans‐Hermann; Schwaab, R.

doi:10.1007/s002770050486

Cited by 11 publications

(10 citation statements)

References 31 publications

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“…Haack et al [32] performed transient transfection of full-length and B-deleted rFVIII production in HEK293, COS, and CHO cells. The authors presented a kinetic expression profile but the levels reached were low (around 8-10 ng/mL).…”

Section: Discussionmentioning

confidence: 99%

Transient transfection of serum-free suspension HEK 293 cell culture for efficient production of human rFVIII

et al. 2011

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BackgroundHemophilia A is a bleeding disorder caused by deficiency in coagulation factor VIII. Recombinant factor VIII (rFVIII) is an alternative to plasma-derived FVIII for the treatment of hemophilia A. However, commercial manufacturing of rFVIII products is inefficient and costly and is associated to high prices and product shortage, even in economically privileged countries. This situation may be solved by adopting more efficient production methods. Here, we evaluated the potential of transient transfection in producing rFVIII in serum-free suspension HEK 293 cell cultures and investigated the effects of different DNA concentration (0.4, 0.6 and 0.8 μg/106 cells) and repeated transfections done at 34° and 37°C.ResultsWe observed a decrease in cell growth when high DNA concentrations were used, but no significant differences in transfection efficiency and in the biological activity of the rFVIII were noticed. The best condition for rFVIII production was obtained with repeated transfections at 34°C using 0.4 μg DNA/106 cells through which almost 50 IU of active rFVIII was produced six days post-transfection.ConclusionSerum-free suspension transient transfection is thus a viable option for high-yield-rFVIII production. Work is in progress to further optimize the process and validate its scalability.

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Section: Discussionmentioning

confidence: 99%

Transient transfection of serum-free suspension HEK 293 cell culture for efficient production of human rFVIII

et al. 2011

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“…An episomal expression system as efficient as the HEK293(EBNA) system has not been utilized for scaled-up transient production in CHO cells. Different host cell types can confer different post-translational modifications, which may significantly affect the properties of therapeutic recombinant proteins (Haack et al, 1999). The development of a stable cell line is costly and time-consuming.…”

Section: Discussionmentioning

confidence: 99%

Epi‐CHO, an episomal expression system for recombinant protein production in CHO cells

Kunaparaju

Liao

Sunstrom

2005

Biotech & Bioengineering

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This study describes the development of a transient expression system for CHO cells based on autonomous replication and retention of transfected plasmid DNA. A transient expression system that allows extrachromosomal amplification of plasmids permits more plasmid copies to persist in the transfected cell throughout the production phase leading to a significant increase in transgene expression. The expression system, named Epi-CHO comprises (1) a CHO-K1 cell line stably transfected with the Polyomavirus (Py) large T (LT) antigen gene (PyLT) and (2) a DNA expression vector, pPyEBV encoding the Py origin (PyOri) for autonomous plasmid amplification and encoding Epstein-Barr Virus (EBV) nuclear antigen-1 (EBNA-1) and OriP for plasmid retention. The CHO-K1 cell line expressing PyLT, named CHO-T was adapted to suspension growth in serum-free media to facilitate large-scale transient transfection and recombinant gene expression. Enhanced green fluorescent protein (EGFP) and human growth hormone (hGH) were used as reporter proteins to demonstrate transgene expression and productivity. Transfection of suspension-growing CHO-T cells with the vector pPyEBV encoding hGH resulted in a final concentration of 75 mg L(-1) of hGH in culture supernatants 11 days following transfection.

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“…However, HEK cells are not the optimal choice of host cell line for transient production of therapeutic proteins. For example, HEK293 cells are known to differ from CHO cells with respect to N ‐glycan processing (9), which may affect product bioactivity in vitro (10). Consequently, to rapidly produce protein product in the same cell type that would be employed for large‐scale production processes, there have been recent examples of optimized, scalable transient production systems employing PEI‐mediated transfection of nonengineered CHO cells (11, 12).…”

Section: Introductionmentioning

confidence: 99%

Control of Culture Environment for Improved Polyethylenimine-Mediated Transient Production of Recombinant Monoclonal Antibodies by CHO Cells

et al. 2006

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In this study we describe optimization of polyethylenimine (PEI)-mediated transient production of recombinant protein by CHO cells by facile manipulation of a chemically defined culture environment to limit accumulation of nonproductive cell biomass, increase the duration of recombinant protein production from transfected plasmid DNA, and increase cell-specific production. The optimal conditions for transient transfection of suspension-adapted CHO cells using branched, 25 kDa PEI as a gene delivery vehicle were experimentally determined by production of secreted alkaline phosphatase reporter in static cultures and recombinant IgG4 monoclonal antibody (Mab) production in agitated shake flask cultures to be a DNA concentration of 1.25 microg 10(6) cells(-1) mL(-1) at a PEI nitrogen:DNA phosphate ratio of 20:1. These conditions represented the optimal compromise between PEI cytotoxicity and product yield with most efficient recombinant DNA utilization. Separately, both addition of recombinant insulin-like growth factor (LR3-IGF) and a reduction in culture temperature to 32 degrees C were found to increase product titer 2- and 3-fold, respectively. However, mild hypothermia and LR3-IGF acted synergistically to increase product titer 11-fold. Although increased product titer in the presence of LR3-IGF alone was solely a consequence of increased culture duration, a reduction in culture temperature post-transfection increased both the integral of viable cell concentration (IVC) and cell-specific Mab production rate. For cultures maintained at 32 degrees C in the presence of LR3-IGF, IVC and qMab were increased 4- and 2.5-fold, respectively. To further increase product yield from transfected DNA, the duration of transgene expression in cell populations maintained at 32 degrees C in the presence of LR3-IGF was doubled by periodic resuspension of transfected cells in fresh media, leading to a 3-fold increase in accumulated Mab titer from approximately 13 to approximately 39 mg L(-1). Under these conditions, Mab glycosylation at Asn297 remained essentially constant and similar to that of the same Mab produced by stably transfected GS-CHO cells. From these data we suggest that the efficiency of transient production processes (protein output per rDNA input) can be significantly improved using a combination of mild hypothermia and growth factor(s) to yield an extended "activated hypothermic synthesis".

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Analysis of expression kinetics and activity of a new B-domain truncated and full-length FVIII protein in three different cell lines

Cited by 11 publications

References 31 publications

Transient transfection of serum-free suspension HEK 293 cell culture for efficient production of human rFVIII

Transient transfection of serum-free suspension HEK 293 cell culture for efficient production of human rFVIII

Epi‐CHO, an episomal expression system for recombinant protein production in CHO cells

Control of Culture Environment for Improved Polyethylenimine-Mediated Transient Production of Recombinant Monoclonal Antibodies by CHO Cells

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