Selective Stabilization and Destabilization of Protein Domains in Tissue-Type Plasminogen Activator Using Formulation Excipients

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 T m , and the Van't Hoff entropy for unfolding, ΔS vh , 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 (f 45 ) 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 T m (R 2 = 0.77), ΔS vh (R 2 = 0.71), but not T agg (R 2 = 0.01), we analysed how formulation excipients and pH Page 2 of 45 ACS Paragon Plus Environment Molecular Pharmaceuticswould be able to optimise T m and ΔS vh . Formulation excipient behaviours were found to group according to their combined impact on T m and ΔS vh . 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.

Fine‐tuning the activity and stability of an evolved enzyme active‐site through noncanonical amino‐acids

“…Previously, introduction of mutations that conferred rigidity into the cofactor-binding loop (e.g. H192P/ [42][43][44]. n = 3.…”

“…The fluorescence was measured as a function of temperature in the range of 30-90°C with steps of 1°C, equilibration time of 30 s at each temperature. Thermal denaturation curves were analysed by fitting the baseline and single transition to a two-state model [42][43][44].…”

“…The methodology of Analyses 1 and 2 was reapplied to the experimental thermostability data of the five variants ( Fig. 7) by similarly weighting a sum of twostate model functions for thermal denaturation [42][43][44] of each variant (Equation 4, Materials and Methods). In Analysis 2, the variants were globally fitted, with the stability parameters of 385Y and 385F shared, and the %(nc)AA-TK incorporated , %385F-TK misincorporated and %385Y-TK misincorporated weighting parameters fixed for each variant.…”

“…The thermal unfolding of all TK subspecies (ncAA biocat or AA biocat ) of variants 385pAMF (black), 385Y (red), 385F (blue), 385pCNF (green) and 385pNTF (magenta). The apparent thermal stability was determined by the change in the fluorescence emission ratio (350/330) as a function of time, and fitted to a two-state model of thermal denaturation, as described previously [42][43][44]. Error bars represent the standard error of the mean (SEM), n = 3. stable 385pNTF variant, 4.9°C higher than 385Y (3M), and remarkably, 2.4°C higher than wild-type transketolase (Table 3).…”

“…Summary of the thermal stability of TK biocat and TK incorporated of wild-type, 385pAMF, 385Y, 385F, 385pCNF and 385pNTF. Parameters were determined by fitting the change in the fluorescence emission ratio (350/330) as a function of temperature to a two-state model of thermal denaturation[42][43][44], using the methodology of Analyses 1 and 2. Associated errors are the fitting error for the thermal denaturation function AE 1.2 120.9 AE 33.2 67.7 AE 0.6 244.1 AE 2.…”