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Effect of Arginine Glutamate on Protein Aggregation in Biopharmaceutical Formulation
[Thesis]. Manchester, UK: The University of Manchester; 2016.
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Abstract
Monoclonal antibodies (mAbs) represent one of the fastest growing classes of therapeutic proteins. This success is due to a number of attractive properties such as high binding affinity, specificity, low immunogenicity and high aqueous solubility. Despite this, mAbs can suffer from undesirable physical instabilities, especially reversible self-association (RSA), which can lead to aggregation and phase separation. One aspect of formulation is therefore to find solution conditions which minimise mAb aggregation propensity during storage at high concentrations. Hence, the buffer, excipient and pH must be carefully considered to obtain the optimal formulation. Currently, if a platform formulation process is non-ideal for a particular candidate mAb, then an alternative strategy is to utilise high-throughput screening to measure various physical parameters indicative of physical stability. Arginine (in the form of hydrochloride salt Arg路HCl) is often used in formulations exhibiting high RSA and a propensity for aggregation. The interaction of Arg with the protein surface is complex and dependent on both the salt form and concentration. Here the focus was on the glutamate salt of arginine (Arg路Glu), to quantify its effect on mAb conformational and colloidal stability under different pH conditions. Arg路Glu was able to decrease the propensity of the mAbs to aggregate, particularly at pH values closer to their pI.The work also included the use of in vitro cell culture models to examine cell viability in the presence of the various arginine salts over a range of osmolalities. Whilst Arg路Glu is composed of two naturally occurring amino acids and both of which are considered non-toxic individually, the effect of the increased concentrations of their combination, on cells has not been explored previously. In vitro cell lines were chosen to represent the subcutaneous tissue, the effect of Arg路Glu on cell viability was compared against NaCl, Arg路HCl and sodium glutamate (NaGlu). The work concluded there was no additional toxicity associated with the presence of Arg路Glu in the cell culture models studied, therefore Arg路Glu has the potential as an excipient as it reduces aggregation and is nontoxic.Another aspect of the work was to assess the use of solution NMR spectroscopy as an orthogonal technique in mAb formulation characterisation. 1H NMR spectroscopy was used to measure a number of experimental parameters for high concentration mAb solution. The work proposed that 1H NMR spectroscopy can serve as a valuable orthogonal method for mAb characterization and formulation.