Development and optimization of stable CHO cell lines for expression of Recombinant antibodies for use in the diagnosis of cancer
Publicerad
Författare
Typ
Examensarbete för masterexamen
Master's Thesis
Master's Thesis
Program
Modellbyggare
Tidskriftstitel
ISSN
Volymtitel
Utgivare
Sammanfattning
This master's thesis project aimed to investigate and evaluate the development of stable CHO
cell lines for the expression of recombinant antibodies (rAbs), for use in the diagnosis of
cancer and thereby increase the know-how of this technique at Fujirebio Diagnostics AB
(FDAB). Three antibodies targeting cancer biomarkers; C192, Ov185, and SCC140 were
selected for recombinant expression in an attempt to increase yield and to get more stable
productivity, to meet the increased demand at the market. Biomarkers produced from cancer
cells can be detected by antibodies in in vitro diagnostics.
In this project, the suspension cell line CHO-S adapted to high density serum-free culture was
used in combination with the pCHO1.0 vector, constructed for the expression of two-subunit
proteins, such as IgG. An alternative signal sequence (B), originating from human albumin
was evaluated in comparison with the original signal sequence to increase antibody secretion
from the cells further. Transient expressions with concentrations of 0.8-1.5 μg/ml were
achieved 48 h post-transfection for pCHO 1.0 construct containing C192 with alternative
signal sequence (C192B), Ov185 and Ov185 with alternative signal sequence (Ov185B).
After selection with Methotrexate (MTX) and Puromycin stable expression with
concentrations of 45 and 39 μg/ml were achieved for C192B and Ov185, respectively. The
data indicate that a transient expression with concentrations of at least 0.8 μg/ml is a
prerequisite for success in the selection phase, and if this is not achieved, that the transfection
should be repeated.
To select high producing clones from the cell pool achieved, in the selection phase cloning by
limiting dilution was performed on stably transfected pools. High producing C192B clones
were obtained that produced up to 490 μg/ml in productivity assessment, corresponding to at
least 10 times increase in expression as compared to the original hybridoma. Recombinant
C192B was purified and characterised regarding immunoactivity, with high purity and with
immunoactivity in comparison to hybridoma produced C192.
In this project two antibodies were successfully expressed in recombinant form and the
technique can now be considered established at FDAB. Next step will be to scale up for
manufacturing of rAbs at FDAB.