Robinson, MJ; Matejtschuk, P; Longstaff, C; Dalby, PA; (2019) Selective stabilisation and destabilisation of protein domains in tissue-type plasminogen activator using formulation excipients. Molecular Pharmaceutics , 16 (2) pp. 744-755. 10.1021/acs.molpharmaceut.8b01024.

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Abstract

Multi-domain biotherapeutic proteins present additional behavioural and analytical challenges for the optimisation of their kinetic stability by formulation. Tissue-type plasminogen activator (tPA) comprises six protein domains that exhibit a complex and pH-dependent thermal unfolding profile, due to partially independent domain unfolding. Here we have used tPA as a model for evaluating the relationships between various thermal unfolding and aggregation parameters in multi-domain proteins. We show that changes in the thermal unfolding profile of tPA were parameterised by the overall thermal mid-point transition temperature Tm, and the Van't Hoff entropy for unfolding, ΔSvh, which is a measure of unfolding cooperativity. The kinetics of degradation at 45 °C, leading to aggregation, were measured as rates of monomer and activity loss. These two rates were found to be coincident at all pH. Aggregation accelerated at pH 4 due to the early unfolding of the Serine Protease N-terminal domain (SP-N), whereas at pH 5-8, the fraction unfolded at 45 °C (f45) was <1%, resulting in a baseline rate of aggregation from the native ensemble. We used a Design of Experiments (DoE) approach to evaluate how formulation excipients impact and control the thermal unfolding profile for tPA, and found that the relative stability of each of the tPA domains was dependent on the formulation. Therefore, the optimisation of formulations for complex multi-domain proteins such as tPA may need to be multi-objective, with careful selection of the desired attributes that improve stability. As aggregation rates (ln v) correlated well to Tm (R2 = 0.77), ΔSvh (R2 = 0.71), but not Tagg (R2 = 0.01), we analysed how formulation excipients and pH would be able to optimise Tm and ΔSvh. Formulation excipient behaviours were found to group according to their combined impact on Tm and ΔSvh. The effects of each excipient were often selectively stabilising or destabilising to specific tPA domains, and changed the stability of particular domains relative to the others. The types of mechanism by which this could occur might involve specific interactions with the protein surface, or otherwise effects that are mediated via the solvent as a result of the different surface hydrophobicities and polarities of each domain.

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