Biologics and Biosimilars Characterization

In contrast to small molecule drugs, which are chemically synthesized, biologics and biosimilars, also known as biotherapeutic drugs, are produced from living organisms such as bacteria, yeast and mammalian cells. Consequently, they are usually very large molecules (peptides, small proteins, antibodies, polysaccharides, etc.) that can exhibit post-translational modifications and certain degrees of structure variability.

Confirmation of the identity of the therapeutic drug through structural characterization, from the primary amino acid sequence to the higher order structure, together with impurity control is therefore critical to ensure efficacy and patient safety.

Bruker’s Mass Spectrometry Solutions go beyond traditional peptide mapping and intact mass analysis. These solutions combine ultimate performance, speed and versatility with powerful workflow-enabled software to provide biologics developers with ready access to orthogonal characterization approaches. These include monoclonal antibody (mAb) subunit analysis using middle up and middle down approaches for the unambiguous detection and characterization of sequence variants and modifications including deamidation, oxidation and N-terminal clipping: as well as higher order structure studies for the detailed characterization of disulfide bridges, conformational epitopes, structural changes upon perturbation to calculating accurate drug antibody ratios (DAR) in antibody drug conjugate (ADC) studies.

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 contain multiple disulfide bonds, analysis is challenging, often requiring several LC-MS runs with the tryptic digests from reduced and non-reduced protein and a manual comparison of these two analyses. 

In contrast to small molecule drugs, which are chemically synthesized, biologics and biosimilars, also known as biotherapeutic drugs, are produced from living organisms such as bacteria, yeast and mammalian cells. Consequently, they are usually very large molecules (peptides, small proteins, antibodies, polysaccharides, etc.) that can exhibit post-translational modifications and certain degrees of structure variability.

Confirmation of the identity of the therapeutic drug through structural characterization, from the primary amino acid sequence to the higher order structure, together with impurity control is therefore critical to ensure efficacy and patient safety.

Bruker’s Mass Spectrometry Solutions go beyond traditional peptide mapping and intact mass analysis. These solutions combine ultimate performance, speed and versatility with powerful workflow-enabled software to provide biologics developers with ready access to orthogonal characterization approaches. These include monoclonal antibody (mAb) subunit analysis using middle up and middle down approaches for the unambiguous detection and characterization of sequence variants and modifications - including deamidation, oxidation and N-terminal clipping; as well as higher order structure studies for the detailed characterization of disulfide bridges, conformational epitopes, and structural changes upon perturbation; and calculating accurate drug antibody ratios (DAR) in antibody drug conjugate (ADC) studies.

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 contain multiple disulfide bonds, analysis is challenging, 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 allows for intact protein analysis, enabling structural evaluation of therapeutic drugs without modification, in conditions that are physiologically relevant. The selectivity and quantitative nature of magnetic resonance means that potency determination, impurity profiling and degradation studies (eg polysorbates) can be performed directly enabling fast and easy testing without the need for response factor calculations or the method redevelopment activities required by traditional LC methods, thereby saving time and reducing costs. In addition, benchtop time domain NMR (TD-NMR) is becoming an important technique for the analysis of protein aggregates and 100% fill-check of vials and syringes.

Fourier Transform Infrared (FT-IR) spectroscopy can be used to analyze water-soluble and membrane proteins such as nuclear receptors, which are currently decisive targets in drug research and development, being associated with conditions such as Alzheimer's, and Parkinson's disease, diabetes and obesity. FT-IR is typically applied in protein secondary structure elucidation, detection of conformational changes and monitoring of protein dynamics like aggregation, precipitation and crystallization. Infrared protein analysis is a quick and relatively inexpensive technique used for formulation optimization, stability studies during drug development and QC of protein drug products.  

Surface Plasmon Resonance (SPR) is the gold standard methodology to characterize an interaction from a kinetic point of view. While solution-based approaches offer an equilibrium-based perspective on an interaction, the label-free and real-time analysis of SPR affords insight into the kinetics a key characteristic that enables more precise understanding of an interaction. Biologics demonstrating similar affinities using equilibrium-based methods like ELISA may have vastly different off-rates with subsequently different biological outcomes. SPR however enables the identification of biologics with the optimal half-life at your target protein.

SPR also allows researchers to identify antibodies that target different epitopes on a protein. Equally, conditional binding based on factors like pH, e.g. as in cancer tissue, or the effect of different buffers on the binding interaction can be investigated with ease. These approaches help to understand an interaction in a physiologically more translatable context.