Browsing by Author "Berger, Verena"

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    Cell line development for and characterization of mono- and bivalent antibody derivatives
    (2013) Berger, Verena; Kontermann, Roland (Prof. Dr.)
    Immune mediated inflammatory diseases (IMDM) can affect a variety of tissues and organs causing a broad spectrum of symptoms. Yet, rheumatoid arthritis, multiple sclerosis, psoriasis or inflammatory bowel disease share mechanistic similarities. A major factor in the development is the imbalance of tumor necrosis factor (TNF) regulation. With a prevalence of 5-7 % in the western society, a great interest exists in the development of an effective therapy. Current treatment involves primarily corticosteroids and non-biologic disease-modifying antirheumatic drugs (DMARDs) like methotrexate (MTX). The fast progression made in the development of biopharmaceutics, especially of antibody based therapeutics allowed for a more specific treatment. TNF-related diseases are tried to tackle by intervention in the TNF signaling. To date, five such therapeutics are approved by the US Food and Drug Administration (FDA). They are aiming at the neutralization of TNF or lymphotoxin alpha (LTα), thereby reducing exaggerated TNF signaling and curtailing inflammation. However, neutralization of TNF facilitates opportunistic infections and several warnings have been issued by the FDA. In 1990, an antibody directed specifically against TNFR1 was discovered, when mice were immunized with human TNFR1. A humanized variant was generated in 2008, paving the way for the use of this antibody as a therapeutic in humans. The present study describes the development of chinese hamster ovary (CHO) cell lines expressing three different variants of the humanized TNFR1 specific antibody IZI06.1, a full length antibody and two human serum albumin fusion proteins (scFv-HSA and Fab-HSA). Cell lines were generated by site directed integration of the GOI into the CHO host genome in order to investigate the influence of the individual construct on growth performance and producibility. Cell lines expressing the IgG1 and the scFv-HSA perfomed similarly, showing productivities of 5-10 pg/c/d. Both proteins assembled properly and could be purified to satisfactory purity. These constructs were found to be applicable for the generation of a regulatory compliant cell line. The Fab HSA revealed multiple issues in terms of productivity and purification and was therefore excluded from further investigation. The monovalent fusion protein scFv HSA and full length antibody (termed ATROSAB) were investigated in more detail to determine the impact of variables like composition, size and valency on their biochemical and functional properties. Reduced size of the fusion protein translated into a reduced plasma half-life compared to the antibody. But although being also more susceptible to thermal degradation, comparison with the respective scFv showed that the scFv-HSA clearly benefits from fusion to HSA in terms of stability. It could be further demonstrated that both proteins specifically bind TNFR1, but not TNFR2 and that both proteins are generally able to inhibit TNF-induced TNFR1 signaling. However, potency of the fusion protein was demonstrated to be significantly reduced, most probably due to its monovalency. Without further improvement of the scFv HSA, the full length IgG remains first choice for the development of a prospective, highly specific therapeutic with indication for TNF mediated inflammatory diseases. These results are in accordance with bio-functional data generated earlier for the three constructs that also indicated a better affinity of the full length IgG to hTNFR1 compared to the scFv HSA and upon which it was decided to generate a full regulatory compliant cell line in CHO for the IgG1 (ATROSAB). A set of cell lines could be developed, that meet all requirements for a prospective use in an industrial, GMP regulated environment. This includes next to a high productivity and favorable growth characteristics the absence of adventitious agents. The cell lines were generated by Limiting Dilution, clonality was verified by microscopy. More than 400 clones were screened from which high producers were identified and cell banked. The most promising clone K20-3 achieved a productivity of 10 pg/c/d. In order to further enhance productivity without employing destabilizing gene amplification, this clone was re-transfected with the slightly modified expression vector for the IgG1. A more than 3-fold increase in productivity could be achieved with clone K35-1. However, quality analysis of ATROSAB produced by the re transfected clone revealed a greater heterogeneity and also bio functionality was affected negatively. Therefore it was decided for the time being to proceed with original, not re-transfected clone in order to set up a GMP production process. Yet, further analysis of the re-transfected clones may be worthwhile regarding their increased productivity. Adapted process and handling protocols probably can overcome the encountered limitations in product quality.