1. Analytical Assessment

2. Product-Related Attributes

Figure 1. Inherent variations in biological products (FDA: https://www.fda.gov/drugs/biosimilars/curriculum-materials-health-care-degree-programs-biosimilars; accessed on 4 April 2022) ^[9].

• Peptide mapping (LC-MS), peptide mass fingerprinting (MALDI-MS), MALDI TOF, and MS amino acid sequencing are all examples of primary structure sequencing.
• Higher-order structures can be confirmed using thermodynamic DSC, NMR, SPR, ELISA, fluorescence, far and near UV CD, DSC, NMR, SPR, and ELISA. While process-related testing is straightforward and well-established, testing product-related attributes can be improved by testing the UV and fluorescence spectra under various stress conditions, temperature surfactants, electrolytes, and pH ^[10]. Newer and more sensitive methods are always needed.
• Cell-based assays, SPR, and ELISA, to test receptor binding.
• Forced degradation: degradation is forced to match intramolecular bond strength as a structural similarity measure.

3. Process-Related Attributes

• Protein content. Biological products label potency of 100 IU/mL for insulin in vials. Based on shared experience, the protein content cannot always be the same due to filling variability, concentration testing variability, and many other unpredictable factors. For this reason, most products are allowed an acceptable practical range of variability of ±5% ^[14]. However, this quality attribute is controversial, as the first FDA guideline required this attribute to be tested for equivalence. The 95% CI of the biosimilar product cannot go beyond 1.5*SD of the reference product in an equivalence test. This range was established entirely arbitrarily. If the SD of the reference product turns out to be small, all batches of the biosimilar product will fail despite being within the release specification of ±5%. This means that a biosimilar product might be acceptable for patients but not for approval by the FDA. This situation arose when the first biosimilar EP2006 required the testing of 50 lots to match the equivalence criteria of Amgen’s Neupogen, despite all lots meeting the release specifications ^[15]. We can use this as an example to remove the comparative testing of the protein content from side-by-side testing. However, if a biosimilar product has a higher variability, this must be confirmed with the variability in the reference product lots.
• Post-translation modifications, aggregates, and isomers should be tested in a range model, wherein 90% of the values of the biosimilar lots should fall within 3 × SD of the reference product to establish analytical similarity and the specification should include a range of no more than 3 × SD of the reference product.
• Bioassay limits are calculated as specified in the statistical analysis of biological assays and test results. They are typically expressed as an acceptable range for the estimated potency (e.g., 80–125 percent of the stated potency) and an acceptable range for the confidence limits of the estimated potency (e.g., 64–156 percent of the stated potency) ^[16].
• Impurities in biological products, also known as residuals, are of much greater importance than in chemical drugs. Impurities can be either process- or product-related. Process-related impurities are derived from the manufacturing process—for example, cell culture, downstream, or cell substrates. In contrast, product-related impurities are non-active molecular variants of the biologic and are formed during expression, manufacture, or storage. Understanding these impurities is essential to developing control strategies to reduce or remove them from the final product. The impurities caused by the upstream process may include cell culture reagents, antifoams, growth modifiers (insulin), antibiotics, protein a, solubilizers, residual solvents, chelating agents, extractable extracts, and leachable. The downstream-derived impurities may include detergent, protein a, process additives, chromatographic resins, extractable, and leachable. Cell-derived impurities include host cell DNA and host cell proteins. Product-related impurities include truncated forms such as fragments; modified forms such as disulfide, oxidation, deamidation, and glycosylation; and aggregates including multimers and subvisible particles. When present in a substantial quantity, these impurities may reduce the product’s potency and, worse, induce immunogenic responses or alter the product’s pharmacokinetics. While process-related impurities can be readily isolated, product-related impurities are often difficult to separate because of their close structural similarity to the active molecules. As a result, a biosimilar product must not have any unmatched impurity. There is also no analytical method or biological test that can ensure the safety of an unmatched impurity since any testing of immunogenicity in an animal species may not match the immune response in humans. In some cases, an unmatched impurity may be acceptable if the same regulatory agency has approved an identical structure or there is sufficient published proof of its safety. Since matched impurities can reduce efficacy if they are not as efficacious, a variation of 3% is generally allowed as a legacy attribute. Additionally, the 3% variation must not include more than 1% of any single impurity. However, these acceptance criteria can also be established by profiling the reference product.
• Particle size (subvisible), residual DNA, fill volume, and sterility standards are well defined in several official compendia, and these should be acceptable.
• Physical properties. If the formulation is the same, then the formulation’s physical properties, such as surfactants, osmolality, and pH, should fall within three standard deviations of that of the reference product. However, when the formulation is different, the release specifications will be based on testing multiple lots of biosimilar products. The BPCIA allows a biosimilar product to have a different formulation; however, using the same formulation as the reference product reduces the risk of higher immunogenicity, especially if the inactive component(s) are used in another biological product and have the same route of administration. This is in contrast to the WHO’s suggestion that “relevant differences in formulation (for example, use of excipients in the biosimilar that are not widely used in medicinal products)” can be tested using animal models ^[17], despite experience gained from the incidence of immunological reactions induced by erythropoietin formulations that used a different formulation ^[18]. No animal testing can establish the safety of inactive components when used in a biological drug formulation.