Hong Weon Lee

Carbon nanotube-assisted enhancement of surface plasmon resonance signal.

We describe a method of amplifying the biosensing signal in surface plasmon resonance (SPR)-based immunoassays using an antibody-carbon nanotube (CNT) conjugate. As a model system, human erythropoietin (EPO) and human granulocyte macrophage colony-stimulating factor (GM-CSF) were detected by sandwich-type immunoassays using an SPR biosensor. For the amplification of the SPR signal, the CNT was conjugated with a polyclonal antibody, and then the conjugates were reacted with antibodies coupled with the target proteins. This amplification strategy increases the dynamic range of the immunoassays and enhances the detection sensitivity. The SPR immunoassays, combined with the CNT-assisted signal amplification method, provided a wide dynamic range over four orders of magnitude for both EPO and GM-CSF (0.1-1,000 ng/ml). The CNT amplification method is expected to realize the detection of picogram levels and a wide dynamic detection range of multiple proteins, enabling it to offer a robust analysis tool for the development of biopharmaceutical production.

New cell line development for antibody-producing Chinese hamster ovary cells using split green fluorescent protein.

BackgroundThe establishment of high producer is an important issue in Chinese hamster ovary (CHO) cell culture considering increased heterogeneity by the random integration of a transfected foreign gene and the altered position of the integrated gene. Fluorescence-activated cell sorting (FACS)-based cell line development is an efficient strategy for the selection of CHO cells in high therapeutic protein production.ResultsAn internal ribosome entry site (IRES) was introduced for using two green fluorescence protein (GFP) fragments as a reporter to both antibody chains, the heavy chain and the light chain. The cells co-transfected with two GFP fragments showed the emission of green fluorescence by the reconstitution of split GFP. The FACS-sorted pool with GFP expression had a higher specific antibody productivity (qAb) than that of the unsorted pool. The qAb was highly correlated with the fluorescence intensity with a high correlation coefficient, evidenced from the analysis of median GFP and qAb in individual selected clones.ConclusionsThis study proved that the fragment complementation for split GFP could be an efficient indication for antibody production on the basis of high correlation of qAb with reconstitution of GFP. Taken together, we developed an efficient FACS-based screening method for high antibody-producing CHO cells with the benefits of the split GFP system.

Effect of constitutively active Ras overexpression on cell growth in recombinant Chinese hamster ovary cells.

Constitutively active Ras (CA‐Ras) is known to enhance cell growth through the induction of various signaling cascades including the phosphoinositide 3‐kinase (PI3K)/Akt and mitogen‐activated protein kinase (MAPK)/ERK signaling pathways, although the cellular response is highly dependent on the cell type. To evaluate the effect of CA‐Ras overexpression on cell growth in recombinant Chinese hamster ovary (rCHO) cells, an erythropoietin (EPO)‐producing rCHO cell line with regulated CA‐Ras overexpression (EPO‐off‐CA‐Ras) was established using the Tet‐off system. The CA‐Ras expression level in EPO‐off‐CA‐Ras cells was tightly regulated by doxycycline addition. Although CA‐Ras overexpression slightly increased the viable cell concentration during the late exponential phase, it did not increase the maximum viable cell concentration or specific growth rate to a significant degree. Unexpectedly, CA‐Ras overexpression in rCHO cells led only to the enhancement in the activation of the MAPK/ERK signaling pathway and not the PI3K/Akt signaling pathway. Taken together, CA‐Ras overexpression in rCHO cells did not significantly affect cell growth; it also had no critical impact on viable cell concentration or EPO production, possibly due to a failure to activate the PI3K/Akt signaling pathway.