The EMA and US FDA guidelines on Biosimilars recommend the application of extensive state-of-the-art characterization assays. Studies including these protein characterization methods should include batches of both the biosimilar and reference medicinal product (RMP), in order to demonstrate that the quality of the biosimilar is comparable with that of the RMP.
The US FDA biosimilar scientific considerations guideline for industry states: “Sponsors should use appropriate analytical methodologies with adequate sensitivity and specificity for structural characterization of the proteins.” Generally, such protein characterization methods include comparisons of the following structural aspects:
The EMA and the FDA share a similar stance in terms of their expectations for early-stage biosimilar characterization and advise sponsors to submit analytical data from biosimilar testing early in product development.
During biosimilar product development, initial studies center around determining the primary amino acid sequence of the RMP at the protein level. This is an expectation of both the US FDA and the EMA and set out in their guidance documents. This analysis often occurs before any biosimilar product is manufactured, because the data ensures that transcription of the DNA sequence results in the production of a molecule with the correct amino acid sequence. State-of-the-art instrumentation such as modern mass spectrometers, chromatography systems, appropriate protein chemical techniques (including Edman N-terminal sequencing) and expertise in the area of structural characterization, are all used to generate the primary amino acid sequence of the RMP.
Once the manufacturing processes for the Biosimilar has been defined, high-end analytical techniques can be used to the most appropriate cell clone for the manufacture. For example, analysis of product intactness, peptide mapping based comparability assessments of the protein backbone and analysis of post-translational modifications (glycosylation in particular) and the aggregation state can help enormously in choosing the most appropriate clone for manufacturing the Biosimilar.
Buffer excipients can often interfere with biosimilar testing. Surfactants such as Tween can interfere with MS analysis and analysis of monosaccharides. It may be necessary to purify the active biopharmaceutical ingredient prior to analysis. A suitable purification method needs to be developed and qualified prior to use in characterization and comparability analysis.
Once a suitable Biosimilar batch is manufactured, the primary amino acid sequence of the Biosimilar should be determined and compared with the RMP sequence. This analysis is performed in the same way as it was for the RMP. Based on both the EMA and US FDA guidelines, the defined sequences should be identical if a claim of Biosimilarity is to be made.
As the development of the Biosimilar progresses and further batches of material are manufactured, data must be obtained to demonstrate that the Biosimilar is structurally comparable to the RMP.
The US FDA biosimilar scientific considerations guidance for industry states: “Sponsors should conduct extensive structural characterization of both the proposed product and the reference product in multiple representative lots to understand the lot-to-lot variability of both products in the manufacturing processes.”
The number of batches of each product that should be analyzed is product dependent but at least 10 RMP batches should be analyzed against 10 Biosimilar batches to provide a reflection of the variation of the RMP (i.e. different batches, expiry dates and manufacturing sites). RMP batches used as part of the comparability exercise and for assessment of the product structural profile should be commercially available and not reference standards of the material such as pharmacopoeial standards.
To satisfy these requirements, multiple batches of the RMP should be subjected to structural, physico-chemical and biophysical analyses alongside batches of Biosimilar.
|Structural characterization||Physicochemical analysis|
|Amino acid sequence||Molecular weight or size|
|Amino acid composition||Isoform pattern|
|Terminal amino acid sequence||Extinction coefficient (or molar absorptivity)|
|Peptide map||Electrophoretic patterns|
|Sulfydryl group(s) and disulfide bridges||Liquid Chromatographic patterns|
|Carbohydrate structure||Spectroscopic profiles|
Where possible, batches should be analyzed “as received”. Analysis of purified product should only be performed where significant interference from excipients is observed (as discussed above).
In summary, a battery of characterization methods is required to support the development of a Biosimilar product and to provide data showing comparability with the RMP.
This biosimilar testing should assess the structural and physico-chemical requirements stipulated within ICH topic Q6B and should: