Exjobbspresentation: Monica Pernsved, MPBIO

Examinator: Mikael Molin
Handledare: Catharina Wising och Maria Lidqvist
Opponent: Elin Persson

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 in an attempt to increase antibody secretion from the cells further. Transient expressions 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 and Puromycin stable expression of 45 and 39 µg/ml were achieved for C192B and Ov185, respectively. The data indicates that a transient expression of at least 0.8 µg/ml is a prerequisite for success in the selection phase, and if this is not achieved the transfection should be repeated.
To select high producing clones from the cell pool achieved from the selection phase cloning by limiting dilution was performed on stably transfected pools. High producing C192B clones were obtained that produced up to 490 ug/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.