Drug manufacturers must demonstrate the comparability of their products after process and formulation changes to ensure similar quality, safety, and efficacy. Biosimilars also require evaluation of their equivalency to the innovators' products. By complementing traditional biochemical methodologies, biophysical characterization, using a variety of methodologies, can enhance product knowledge in terms of higher order structure, molecular size distribution, and the properties of aggregates. This article presents three case studies that show the advantages of applying state-of-the-art biophysical techniques in comparability assessments.
Changes in process, formulation, or a manufacturing site often are made in late-phase development or after commercialization of pharmaceuticals for various reasons, including meeting increased demand, improving a quality attribute, or reducing cost of goods. However, because of the complexity in structures and the structure–function relationship of biological therapeutics, such changes may lead to changes in molecular structures, which may adversely affect the quality, safety, or efficacy of the drug. For example, the structures may be changed in such a way that the molecules are more prone to aggregation. Large protein aggregates are considered to be potentially immunogenic.1 It is therefore essential to establish comparability in critical attributes between materials before and after production changes. The industry and regulatory authorities around the world have been discussing, adopting, and improving such practices. The FDA and EMA have published several guidance documents in recent years on comparability for biologics, including one for biosimilars.2–4 In-depth characterization of structure and conformation of biomolecules using physicochemical methodologies provides the primary indication for comparability, although ultimate affirmation of comparability in safety and efficacy can only be based on long-term clinical outcomes. Most physicochemical methodologies have limitations and caveats. Therefore, as stated in ICH Q5E, the industry should "apply more than one analytical procedure to evaluate the same quality attribute" to "maximize the potential for detecting relevant differences in the quality attributes of the product that might result from the proposed manufacturing process change."2
Currently established biophysical techniques enable in-depth characterization of biological molecules in higher order structure, molecular size and size distribution, intermolecular interactions, and conformational stability. For example, circular dichroism (CD) spectroscopy is widely used to evaluate secondary and tertiary structures. Tryptophan emission fluorescence spectroscopy also is very useful to probe changes in structure because of tryptophan's sensitivity to its local environment. Other spectroscopic tools used to analyze protein structures include Fourier transform infrared (FTIR), Raman, and nuclear magnetic resonance (NMR) spectroscopy. Applying a combination of these tools, which are based on different physical principles, maximizes the potential to detect structural changes. These methods also can be used to evaluate conformational stability along with other methods such as differential scanning calorimetry (DSC). Multiple techniques based on different separation mechanisms are available to analyze size distribution. For instance, size exclusion chromatography (SEC) and asymmetric flow field-flow fractionation (AF4 or aFFFF) use hydraulic pressure with and without a stationary phase to separate species of different hydrodynamic volume, while analytical ultracentrifugation sedimentation velocity (AUC–SV) separates species by centrifugal force in the solution phase. Dynamic light scattering (DLS), on the other hand, does not physically separate species, but mathematically resolves the size distribution according to their diffusion coefficients. A large variety of methodologies, including many spectroscopic, calorimetric, and sizing methods mentioned above, can be applied to evaluate intermolecular interactions. Also, biosensor-based techniques such as surface plasmon resonance (SPR), particularly the system offered by Biacore, and biolayer interferometry (BLI), are rapidly becoming key tools of in vitro functional characterization in the biotechnology industry.
(PHOTO COURTESY OF: VETTER PHARMA INTERNATIONAL GMBH)
This article presents three case studies, which show the advantages of applying biophysical techniques in product comparability assessments.