Robinson, MJ; (2016) Investigating the relationship between forced degradation and long-term shelf life for biotherapeutics. Doctoral thesis , UCL (University College London).

Abstract

The expansion of the biologics market within the last decade means there is a greater need to optimise protein drug stability. In adopting a quality by design (QbD) approach, the biologics market is looking towards stability prediction models to facilitate process development. Two approaches are traditionally taken to enhance protein stability: protein engineering and formulation development. The thesis investigates the effectiveness and validity of combining a highthroughput scale-down technique with a design of experiments (DoE) approach to map excipient-mutant interactions, whilst also investigating predictability of longerterm isothermal degradation based on rapid measurements such as aggregation temperature (Tagg). Prediction of stability within aqueous and lyophilised formulations is the aim of this work. Two proteins are utilised as models: 1) granulocyte-colony stimulating factor (GCSF) – a well-characterised, globular protein to which the mutations will be applied – and 2) tissue plasminogen activator (t-PA), an example of a complex multi-domain protein. High-throughput thermostability screening identified a changing dependence of GCSF variants on pH and polysorbate-80. Orthogonal methods including HPLC SEC, circular dichroism and an activity assay verified these findings, proving these factors have long and short-term effects. Relationships were exhibited between the Tagg temperatures and the rates of decay of GCSF mutants within random formulations, demonstrating predictability despite excipient interactions. Strong correlations were also observed when GCSF mutants were freeze-dried within a uniform formulation. The t-PA studies highlighted the challenges currently faced with predicting stability of multi-domain proteins. Predictability using rapid measurements exhibited complicated trends, whilst DoE studies on t-PA singled out pH to be the most significant stability influencer. Studies proved that whilst the excipient arginine is critical for solubility, it has no significant effect on thermostability at the arginine and tPA concentrations studied here.

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