Establishment of bioanalytical workflows for the quality assessment of formulated proteins

Abstract

Proteins and peptides contribute to a significant class of therapeutic agents in the pharmaceutical market. Over the last decades, they gained growing interest due to their safety and specificity compared to small molecule drugs. Around 20% of the approved moieties by the Food and Drug Administration (FDA) in the last decade are of biological origin, such as antibodies, enzymes, peptides, and amino acids. One of the factors to be considered during the handling of proteins and peptides, especially on a large scale, is their stability. The sensitive nature of these biomolecules could lead to a loss of activity in cases of improper handling or storage. Therefore, biotechnologists focused on developing different formulation techniques to provide adequate protection and stabilization for the target protein or peptide. The design of a pharmaceutical formulation is a tailored mission according to the nature of the targeted protein, route of administration, and required mode of release. Furthermore, testing of the formulated protein is required to investigate the original characteristics of the protein and its stability after the formulation process. Thus, numerous analytical techniques and methods were developed and optimized for protein and peptide analysis. In the present thesis, the analysis of formulated proteins by employing different techniques and assessment of their stability was executed. <br /> The first aim of the present work was to develop bioanalytical workflows for the analysis of formulated proteins. Lysozyme was selected as a model protein in different formulations that were based on hot-melt extrusion as one of the appropriate methods for protein stabilization in the solid state. Analytical characterization of the lysozyme extrudates was performed to test identity, purity, and biological activity based on chromatographic, electrophoretic, and spectrofluorimetric methods. These validated methods were adapted into a workflow that was followed to test the stability of lysozyme hot-melt extrudates over six months of storage according to the guidelines of the International Council for Harmonization (ICH) Topic Q1A (R2). <br /> The second objective was focused on analysing the stability and structural integrity of the tested protein which is directly linked to its function. Thereby, the disulfide connectivity, one of the most important posttranslational modifications, in a protein or a peptide plays an essential role in maintaining the correct folding and, in turn, activity. In order to evaluate the protein’s folding state, validated methods are required, however, a lack of bioanalytical standards to test conformational isomerism based on disulfide bonds in a protein structure was obvious. Therefore, a series of standards were designed and validated according to the ICH M10 and Q14 guidelines and model peptides were generated to evaluate the developed bioanalytical standards. These standards were further applied for the elucidation of the connectivity in selected disulfide-rich peptides and proteins by application of optimized workflows and protocols using the developed standards. <br /> The objectives are extensively described and studied in the following chapters including the results (the respective manuscripts are enclosed in the appendix), which can be considered as a milestone in the development of bioanalytical methods for testing protein characteristics to evaluate their sequential, structural, and functional integrity in the pure and formulated state.