Biophysical Characterization for Product Comparability - Spectroscopic methods such as circular dichroism can detect subtle differences in higher order structure before and after changes in process
Biophysical Characterization for Product Comparability
Spectroscopic methods such as circular dichroism can detect subtle differences in higher order structure before and after changes in process and formulation.
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.
(PHOTO COURTESY OF: VETTER PHARMA INTERNATIONAL GMBH)
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.
This article presents three case studies, which show the advantages of applying biophysical techniques in product comparability
assessments.
Qin Zou, PhD, is a senior principal scientist in analytical research and development, BioTherapeutics Pharmaceutical Sciences, Pfizer Inc.
Articles by Qin Zou, PhD
