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Fig. 1 (abstract O-002). | BMC Proceedings

Fig. 1 (abstract O-002).

From: Abstracts from the 25th European Society for Animal Cell Technology Meeting: Cell Technologies for Innovative Therapies

Fig. 1 (abstract O-002).

Matriptase knock-out in CHO cells prevents clipping of recombinant proteins. a Serine protease inhibitors protect model mAb from proteolytic degradation in CHO-K1 cell derived conditioned medium. The model mAb was incubated in conditioned medium for 0h or 48h at 37°C, subsequently samples were analyzed by western blot. Broad spectrum serine protease inhibitors (Aprotinin, Leupetin) were added during incubation. Aprotinin and Leupetin are inhibiting proteolytic degradation. The intact mAb (upper band) and the clipped mAb (lower band) are indicated by arrows. b Gene expression profiling of CHO-K1 versus CHO-A by NGS. Shown is the gene expression profile of “secreted/shedded members of the S1A trypsin–like serine protease family” for CHO-K1 and CHO-A cell lines using next generation sequencing. The gene expression analysis highlights that five proteases were more than 1.5 fold higher expressed in CHO-K1 cells (labelled with a red asterix). The y-axis shows the transcript abundance as RPKM (Reads Per Kilobase of exon model per Million mapped reads). c siRNA knock-down identifies matriptase as major clipping protease and CHO matriptase KO clone shows no detectable clipping activity. Upper figure: siRNAs directed against the five protease genes and scrambled (scr.) siRNA were transfected and conditioned medium was collected three days after transfection. The model mAb was incubated in fresh medium as control (first lane) and conditioned medium from the siRNA transfected cells. Samples were analyzed by western blot. Only siRNA targeting matriptase (ST14) showed reduced proteolytic degradation. The intact mAb (upper band) and the clipped mAb (lower band) are indicated by arrows. Lower figure: The model mAb was incubated for 48h in conditioned medium collected from wt CHO-K1 as well as the matriptase knockout clone. Samples were analyzed by western blot. The intact mAb (upper band) and the clipped mAb (lower band) are indicated by arrows. No proteolytic degradation could be detected in the samples originating from the matriptase KO clone. d Cell growth, viability and productivity in AMBR (fed batch with temperature shift). Cell growth, viability and volumetric productivity profiles of wt CHO-K1 (red circles, N=2) and matriptase KO clone (blue squares, N=1) cultivated in 15-mL AMBR. No significant differences were seen between WT and matriptase KO clone regarding cell growth and viability. Comparable or slightly higher productivity was detected for the matriptase KO clone compared to the WT. e Significant reduced proteolytic clipping applying matriptase KO clone. The model mAb was stable expressed in CHO-K1 (WT) as well as the CHO-K1 matriptase KO clone. Samples were analyzed by western blot. The intact mAb (upper band) and the clipped mAb (lower band) are indicated by arrows. Significant reduced proteolytic degradation could be detected in the samples originating from the matriptase KO clone (3 samples each is shown for wt and KO cells)

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