Small changes in the manufacturing process can alter a product's efficacy and safety. According to the guidelines of the
EMA, extensive comparability testing will be required to demonstrate that the biosimilar has a comparable profile in terms
of quality, safety and efficacy as the reference product.
Various analytical assays are available to compare physicochemical and biological properties between production batches of
a biosimilar in comparison with a reference product. It is important to recognize the limits of existing assays so that the
results can be accurately interpreted for marketing authorization. It is also important to have careful interpretation of
results to ensure continued safety and efficacy in the target populations. Analytical assays, therefore, have an important
role in the decision-making process for marketing authorization of biosimilar products.
Demonstrating equivalence to the materials used in toxicology and early phase clinical trials are required if bridging studies
are to be avoided. Yet, demonstrating that two separate manufactured lots are identical is very difficult. The key is to achieve
a level of consistency that falls within a set of defined parameters based on testing and characterization.
Prior knowledge of the innovator product (i.e., biochemical, biological, and clinical data) is principally held by the regulatory
authorities based on historical filing of clinical and toxicology data. The ability to reduce development time for the sponsor
of the biosimilar is based on good regulatory interaction at the earliest stage of manufacture. This is important as such
additional studies (e.g., bridging studies) can significantly extend development timelines and the cost of biosimilar development.
Successful and effective comparability studies are thus key in the development of biosimilars.
As with small-molecule generic drugs, the structure of the biosimilar must be analyzed but unlike small molecules, it is not
so much a black-and-white question of proving that it is exactly the same but rather proving that it is sufficiently close
to resulting in no obvious or appreciable functional difference in its biological activity. Analytical characterization of
a biosimilar should include primary, secondary, tertiary, and quaternary structural assessment, biological activity and analysis
of product impurities. All of these components must be understood and further characterized during comparability studies of
the biosimilar with reference to the innovator drug. Molecular weight is assessed using one or more forms of mass spectrometry,
usually matrix-assisted laser desorption/ionization mass spectrometry (MALDI–MS), electrospray MS or liquid chromatography–mass
spectrometry (LC–MS). Techniques such as sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE), high-performance
liquid chromatography (HPLC), peptide mapping, glycosylation patterns, amino acid determination, and carbohydrate content
analysis are also used to carry out isoform and impurity studies.
There is no single analytical technique that will demonstrate all required criteria and characteristics of a biosimilar product
for the purposes of comparison; therefore, a wide variety of tools are needed for this purpose. Several examples of these
techniques are defined below:
- Mass spectrometry can be utilized to show any differences in molecular shift between the biosimilar and innovator product
- Capillary isoelectric focusing (cIEF) can be used to provide data for in-process samples
- Biacore analysis is used to assess receptor binding function
- Peptide mapping is utilized to differentiate enzymes or combinations of enzymes.
Biologically relevant tests must be used to measure the product's activity and can also be used to glean information about
higher order protein structure. In all instances, the results are compared with those of similar analyses for the reference
innovator product. It is, however, not expected that the quality attributes of the biosimilar and the reference product will
be completely identical. Minor structural differences between the two active substances may potentially be acceptable, but
must be justified, as must any variability in post-translational modifications and differences between the impurity profiles.
These differences will only be deemed acceptable if they are supported by the comparability exercise for quality attributes
in relation to safety and efficacy.