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Rapid recombinant protein production from pools of transposon-generated CHO cells.

Matasci M, Bachmann V, Baldi L, Hacker DL, De Jesus M, Wurm FM - BMC Proc (2011)

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Affiliation: Laboratory of Cellular Biotechnology, Faculty of Life Sciences, Ecole Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland.

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Transient gene expression (TGE) is the most commonly used technology for the rapid production of moderate quantities of recombinant proteins for preclinical studies or for analytical assay development... Expression from stably integrated transgene is highly dependent upon the chromatin structure surrounding the site of integration... As a consequence stable cell populations generated by conventional transfection techniques generally show low productivity and reduced expression stability... The clones showed levels of TNFR:Fc expression up to 360 mg/L with an overall mean of 96 +/- 54 mg/L... PB-transposition resulted in a high percentage (more than 98%) of TNFR:Fc expressing clones (data not shown)... Analyses on the stability of transgene expression over time were conducted using a bicistronic PB-donor plasmid allowing co-expression of TNFR:Fc and the enhanced green fluorescent protein (eGFP)... The stability of transgene expression was further confirmed by TNFR:Fc productivity studies performed in 50-ml cultures at different time points post-transfection... At one month post-transfection, the five pools showed comparable growth and production characteristics, reaching TNFR:Fc titers in the range of 350-500 mg/L in 14-day batch cultures... Similar results were obtained when productivity was tested 2 – 3 months post-transfection (Table 1)... The batch bioprocess was finally started at 12 d post-transfection using orbitally shaken TubeSpin Bioreactor 600 tubes... Using stable cell pools expressing either an IgG antibody (Fig. 1B) or two TNFR:Fc variants (Fig. 1C, D), we produced 500-750 mg of recombinant protein within a month after transfection... Our results demonstrated an improved level and stability of transgene expression in transposed pools, indicating usefulness of PB transposed cell pools as a valuable alternative to TGE for the rapid production of recombinant proteins.

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(A) Schematic representation of the protocol for the rapid production of recombinant proteins from pools of transposed cells. This protocol was successfully used to produce a recombinant monoclonal antibody (B) and two variants of TNFR:Fc (C and D). For each bioprocess shown, the percentage of viable cells (dotted lines) the viable cell density (dashed lines), and the recombinant protein titer (solid lines) were measured at the times indicated
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Figure 1: (A) Schematic representation of the protocol for the rapid production of recombinant proteins from pools of transposed cells. This protocol was successfully used to produce a recombinant monoclonal antibody (B) and two variants of TNFR:Fc (C and D). For each bioprocess shown, the percentage of viable cells (dotted lines) the viable cell density (dashed lines), and the recombinant protein titer (solid lines) were measured at the times indicated

Mentions: We developed a protocol for protein production from transposed cell pools at the 0.5-L scale (Figure 1A). Starting at 2 d post-transfection cells were subjected to 10 days of puromycin selection during which cells were expanded from TubeSpin® Bioreactor 50 tubes into orbitally shaken 250-mL cylindrical bottles. The batch bioprocess was finally started at 12 d post-transfection using orbitally shaken TubeSpin® Bioreactor 600 tubes. Using stable cell pools expressing either an IgG antibody (Fig. 1B) or two TNFR:Fc variants (Fig. 1C, D), we produced 500-750 mg of recombinant protein within a month after transfection.


Rapid recombinant protein production from pools of transposon-generated CHO cells.

Matasci M, Bachmann V, Baldi L, Hacker DL, De Jesus M, Wurm FM - BMC Proc (2011)

(A) Schematic representation of the protocol for the rapid production of recombinant proteins from pools of transposed cells. This protocol was successfully used to produce a recombinant monoclonal antibody (B) and two variants of TNFR:Fc (C and D). For each bioprocess shown, the percentage of viable cells (dotted lines) the viable cell density (dashed lines), and the recombinant protein titer (solid lines) were measured at the times indicated
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3285008&req=5

Figure 1: (A) Schematic representation of the protocol for the rapid production of recombinant proteins from pools of transposed cells. This protocol was successfully used to produce a recombinant monoclonal antibody (B) and two variants of TNFR:Fc (C and D). For each bioprocess shown, the percentage of viable cells (dotted lines) the viable cell density (dashed lines), and the recombinant protein titer (solid lines) were measured at the times indicated
Mentions: We developed a protocol for protein production from transposed cell pools at the 0.5-L scale (Figure 1A). Starting at 2 d post-transfection cells were subjected to 10 days of puromycin selection during which cells were expanded from TubeSpin® Bioreactor 50 tubes into orbitally shaken 250-mL cylindrical bottles. The batch bioprocess was finally started at 12 d post-transfection using orbitally shaken TubeSpin® Bioreactor 600 tubes. Using stable cell pools expressing either an IgG antibody (Fig. 1B) or two TNFR:Fc variants (Fig. 1C, D), we produced 500-750 mg of recombinant protein within a month after transfection.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Cellular Biotechnology, Faculty of Life Sciences, Ecole Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland.

AUTOMATICALLY GENERATED EXCERPT
Please rate it.

Transient gene expression (TGE) is the most commonly used technology for the rapid production of moderate quantities of recombinant proteins for preclinical studies or for analytical assay development... Expression from stably integrated transgene is highly dependent upon the chromatin structure surrounding the site of integration... As a consequence stable cell populations generated by conventional transfection techniques generally show low productivity and reduced expression stability... The clones showed levels of TNFR:Fc expression up to 360 mg/L with an overall mean of 96 +/- 54 mg/L... PB-transposition resulted in a high percentage (more than 98%) of TNFR:Fc expressing clones (data not shown)... Analyses on the stability of transgene expression over time were conducted using a bicistronic PB-donor plasmid allowing co-expression of TNFR:Fc and the enhanced green fluorescent protein (eGFP)... The stability of transgene expression was further confirmed by TNFR:Fc productivity studies performed in 50-ml cultures at different time points post-transfection... At one month post-transfection, the five pools showed comparable growth and production characteristics, reaching TNFR:Fc titers in the range of 350-500 mg/L in 14-day batch cultures... Similar results were obtained when productivity was tested 2 – 3 months post-transfection (Table 1)... The batch bioprocess was finally started at 12 d post-transfection using orbitally shaken TubeSpin Bioreactor 600 tubes... Using stable cell pools expressing either an IgG antibody (Fig. 1B) or two TNFR:Fc variants (Fig. 1C, D), we produced 500-750 mg of recombinant protein within a month after transfection... Our results demonstrated an improved level and stability of transgene expression in transposed pools, indicating usefulness of PB transposed cell pools as a valuable alternative to TGE for the rapid production of recombinant proteins.

No MeSH data available.