The Stability of Monoclonal Antibodies

Heron, Andrew David (1999) The Stability of Monoclonal Antibodies. PhD thesis, University of Glasgow.

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Abstract

The Stability of Monoclonal Antibodies Monoclonal antibodies (mAbs) are antibodies which are homogeneous and are of constant specificity and affinity. These properties make them ideal targets for rational drug design as molecular biology techniques allows the therapy to be genetically designed to fit the disease or condition, but, the inherent instability and aggregation propensity of such protein drugs poses serious problems for the pharmaceutical industry. With these problems in mind, the stability of mAbs in solution has been extensively studied using a range of biophysical techniques. With a view to correlating thermal unfolding data with protein drug shelf-life, differential scanning calorimetry (DSC) was used to study the thermal unfolding of three mAbs (Campath-IH, 4162W94 and 1209W95). It was found that each of the antibodies unfolds irreversibly and, by complementing DSC results with results from circular dichroism (CD) and intrinsic tryptophan fluorescence spectroscopy, we show all three mAbs unfold by firstly undergoing a small change in tertiary structure which is then followed by, at a higher temperature, major changes in both tertiary and secondary structure after which all structure is lost. Kinetic analysis of these irreversible processes over a range of pH and DSC scan rates resulted in the calculation of the activation energy (EA) and rate constants of unfolding. Extrapolation of these rate constants to physiological conditions afforded an estimate of drug shelf-life which we have shown is relevant only when thermal unfolding is the only reaction resulting in protein denaturation. The effect of various additives on the thermal unfolding of the antibodies has also been studied using both DSC and fluorescence spectroscopy, in particular fluorescence quenching techniques and the use of the extrinsic fluorescent probe ANS (8-anilino-1-naphthalene sulfonic acid). These studies revealed that mannitol exerts a strong stabilising influence i.e. increases the mean unfolding temperature (Tm) whereas cyclodextrins decrease the Tm and are therefore considered destabilising additives. Cyclodextrins also prevent gross thermal aggregation of the mAbs possibly as a result of binding to exposed hydrophobic amino acid residues and masking hydrophobic patches which are indicated in aggregate formation.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: Alan Cooper
Keywords: Biophysics, Pharmacology
Date of Award: 1999
Depositing User: Enlighten Team
Unique ID: glathesis:1999-76343
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 19 Nov 2019 15:23
Last Modified: 19 Nov 2019 15:23
URI: http://theses.gla.ac.uk/id/eprint/76343

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