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Biosimilar

Protein drugs including therapeutic antibodies and biosimilars are very complex biomolecules with thousands of atoms arranged into a defined but dynamic three-dimensional structure. The unique higher-order structure (HOS) is essential for them to function properly, and a misfolded structure can be ineffective and/or cause unpredictable toxicity to patients. Hence, to establish structural similarity between a biosimilar and the reference drug, not only the primary structure (amino acid sequence) needs to be characterized, but also the higher-order structure. Both US FDA and EMA require extensive structural characterization of biosimilar candidates. The importance of biosimilar HOS comparability assessment is described in detail in the US FDA biosimilar guidelines 2015 and also in the video:

“The three-dimensional conformation of a protein is an important factor in its biological function. Proteins generally exhibit complex three-dimensional conformations (tertiary structure and, in some cases, quaternary structure) because of their large size and the rotational characteristics of protein alpha carbons. The resulting flexibility enables dynamic, but subtle, changes in protein conformation over time, some of which may be required for functional activity. These rotations are often dependent on low-energy interactions, such as hydrogen bonds and van der Waals forces, which may be very sensitive to environmental conditions. Current analytical technology is capable of evaluating the three-dimensional structure of many proteins. Using multiple, relevant, state-of-the-art methods can help define tertiary protein structure and, to varying extents, quaternary structure and can add to the body of information supporting biosimilarity. At the same time, a protein’s three-dimensional conformation can often be difficult to define precisely using current physicochemical analytical technology. Any differences in higher order structure between a proposed product and a reference product should be evaluated in terms of a potential effect on protein function and stability. Thus, functional assays are also critical tools for evaluating the integrity of the higher order structures.”

Higher-Order Structure Characterization Methods

Traditional Spectroscopy Methods: Optical spectroscopy methods such as circular dichroism (CD), Fourier Transform Infrared (FTIR), fluorescence spectroscopy, and differential scanning calorimetry (DSC) have been utilized to study protein higher-order structure changes. The information obtainable from these techniques represents an average across the whole protein and across all of the protein conformers present in the sample, without localized and detailed structural information. These low resolution techniques can provide useful preliminary evaluation of the structrural comparability between a biosimilar and the innovator drug, but as the characterization of critical quality attributes have become more stringent as required by regulatory agencies, more sensitive and higher resolution structural analysis is needed to characterize the small and local conformational changes that may result in efficacy differences.

X-Ray Crystallography and NMR: Crystallography is regarded as the gold standard method for high-resolution HOS determination. If successful, it can solve protein structure at atomic resolution with high confidence. However, flexible regions on the protein can be missed, and this is why a number of regions are usually missing from the antibody crystal structures in Protein Data Bank. In addition, the challenge of antibody crystallization, the complex and time-consuming data analysis involved, and the high cost make X-ray crystallography impractical for routine testing of antibody drugs. In cases where crystal is not obtainable, smaller antibody segments such as the Fc (fragment crystallizable) may be analyzed. Crystallography cannot analyze dynamic conformations in solution, a condition where protein drugs are active. 2D NMR is able to provide solution-phase HOS data at amino acid resolution for smaller proteins, but whole antibodies are too large for this method.

Hydrogen Deuterium Exchange Mass Spectrometry (HDX-MS): This technique measures the solvent accessibility of hydrogens especially those in the protein backbone. The exchange rates are dependent on the local protein structure. At neutral pH and room temperature, HDX at unprotected backbone amides occurs within less than a second, while the exchange rate is reduced by up to 100 million times for sites that are hydrogen bonded and/or shielded from the solvent. Thus, this method is well suited for evaluating the higher-order structure similarity of biopharmaceuticals in their native solution. However, to obtain accurate and reliable results, this technique requires careful control of experimental elements such as temperature, pH, exchange time and back exchange. The traditional “bottom-up” HDX-MS approach provides valuable higher-order structural information at peptide level resolution, but with an incomplete coverage and high level of back exchange. NovoAb now has further improved the technology by using “top-down” and “middle-down” HDX-MS strategies as well as subzero temperature UPLC separation, which enhanced the spatial resolution up to single amino acid and extended the sequence coverage to 100% for monoclonal antibodies, and also significantly reduced back exchange down to a minimal level (ca. 2%). More information can be found here.