Ultra scale-down analysis and bioprocessing of fusion proteins.
Doctoral thesis, UCL (University College London).
Cancer, Alzheimer’s and Parkinson’s are a few of the very complicated diseases facing our ageing population. While small chemically derived pharmaceuticals have a more major role in curing these illnesses, it is hoped that a range of new larger biopharmaceuticals, namely antibody based therapies, could be effective due to their ability to target sites for treatment. This next generation of complex proteins, such as fusions of macromolecules and antibodies are likely to be sensitive to the engineering environment. Hence new insight is needed on where in a bioprocess this damage may occur and how this can be avoided. This thesis focuses on the stability, in a bioprocess engineering environment, of antibody fusion proteins used to treat colorectal cancer with a novel drug delivery system called Antibody Directed Enzyme Prodrug Therapy (ADEPT). An ultra scale-down shear device was used to mimic bioprocess conditions using millilitre quantities of process solution. This allowed protein characterisation and identified new engineering parameters which resulted in the reduction of yield impurities. The amount of intact fusion protein was measured over time during a constant shear field. It was found that the presence of an air/liquid interface exacerbated the rate of fusion protein degradation. First order rate constants were used to characterise the fusion protein degradation for defined bioprocess environments. The use of a surface active agent was shown to protect the protein even at high shear rates and air/liquid interfaces. However, when this surface active agent was added to the large scale production, no discernable effects on the yield were observed. Nevertheless the results showed that the surfactant did improve the proportion of intact protein giving a less heterogeneous product after FPLC purification. The research presented in this thesis shows that detailed characterisation in a ultra scale-down shear device of millilitre quantities of dilute protein solutions can be used to identify problems, such as increased impurities levels in the final product generated during the large scale production.
|Title:||Ultra scale-down analysis and bioprocessing of fusion proteins|
|Additional information:||Permission for digitisation not received|
|UCL classification:||UCL > School of BEAMS > Faculty of Engineering Science > Biochemical Engineering|
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