Stability of therapeutic proteins in solution : The impact of excipients on chemical and physical degradation

Therapeutic proteins replicate the functions of naturally occurring proteins in the body, offering treatments for various diseases, including cancer.However, their efficacy, safety, and function can be compromised by chemical and physical degradation, such as deamidation and aggregation, which can occur during production, transportation, storage, or after administration.Therefore, stabilizing formulations are essential to maintain their native structure and prevent degradation.This thesis investigates how excipients can enhance the stability of therapeutic proteins in liquid formulations, with a focus on chemical stability.It also aims to develop an in vitro methodology for predicting the stability and behavior of therapeutic proteins in vivo.The study examines the effect of six excipients; glycerol, fructose, sucrose, melezitose, polysorbate 80, and n-Dodecyl--D-maltoside, on the chemical stability of the therapeutic affibody GA-Z.An accelerated stability study is used to assess the impact of these excipients.The results show that glycerol, fructose, sucrose, and melezitose enhance the chemical stability of GA-Z by reducing deamidation, isomerization, and hydrolysis.These excipients increase the chemical stability by stabilizing the folded state of the zdomain and inducing a small structural change, which lowers the conformational flexibility of the peptide backbone of GA-Z.In contrast, polysorbate 80 does not affect the stability or structure of GA-Z, while n-Dodecyl--D-maltoside induces significant changes in both stability and structure, possibly through micelle-protein complex formation.Additionally, a novel in vitro methodology combining Asymmetrical Flow Field-Flow Fractionation and Liquid Chromatography-Mass Spectrometry was developed, capable of predicting in vivo stability, analyzing serum albumin binding, detecting changes in protein size, and identifying aggregates.These findings provide valuable insights into the stabilization of therapeutic proteins in solution and present a useful method for predicting their stability and behavior in vivo.The results provided by this study will aid in the development of more effective protein formulations by enhancing stability, minimizing degradation, and advancing the development of safer and more efficient protein drugs.