Biologics and Biosimilars

Analysis of Biotherapeutic Drugs

Biologics and biosimilars, also known as biotherapeutic drugs, are produced from living organisms such as bacteria, yeast and mammalian cells: this is in contrast to small molecule drugs, which are synthesised chemically. This means that on top of being very large molecules (peptides, small proteins, antibodies, polysaccharides etc.), they exhibit post-translational modifications and certain degrees of structure variability.

Confirmation of the identity of the therapeutic drug through structural characterisation, from the primary amino acid sequence to the higher order structure, together with impurity control are critical factors to ensure efficacy and patients safety.

Disulfide bond (DSB) analysis and hydrogen deuterium exchange (HDX) have emerged as technologies that can be employed to gain insights into protein structure. These insights are critical for determining structural similarity for biosimilars, or for monitoring protein stability during drug development. The tertiary structure of therapeutic proteins is key to their activity and stability. 

Research and development labs require technology capable of automated DSB analysis in biopharmaceuticals, based on a single digest of the unreduced protein and without prior knowledge of enzyme specificity or native DSBs. Due to the complexity of these proteins and the fact that they will contain multiple disulfide bonds, analysis is a challenge, often requiring several LC-MS runs with the tryptic digests from reduced and non-reduced protein and a manual comparison of these two analyses.

NMR is especially sensitive to changes to higher order structure at atomic resolution, making it ideally suited for similarity assessment of biologics and biosimilars. NMR also allows for intact protein analysis, enabling evaluation of the structure of therapeutic drugs without modification, in conditions that are physiologically relevant.

Fourier Transform Infrared (FT-IR) spectroscopy can be used to analyse water-soluble and membrane proteins such as nuclear receptors, which are currently a very important targets in drug research and development, being associated with conditions such as Alzheimer, Parkinson diseases, diabetes and obesity. Fast data acquisition and high sample throughput are some of the benefits of this technique. Infrared protein analysis is also relatively inexpensive and is a powerful technique used for formulation optimisation, stability studies during drug development and QC of protein drug products.