Cell-based potency assays.
Potency is a critical quality attribute, and it is essential to prove the comparability of the biosimilar to that of the reference
product in a relevant biological system. A potency assay that measures biological activity is, therefore, required for both
lot release and stability testing. Biological potency assays can be in-vitro cell-based systems, in-vivo tests or enzymatic assays. Several different cell-based assays are available, including ligand binding assays, cell proliferation,
cytotoxicity and cell death studies, activation or inhibition signalling events such as cyclic adenosine monophosphate (cAMP),
measuring the cytopathic effect, and reporter gene assays. More than one bioassay may be required, depending on the biologic
product's mechanism of action or complexity.
Cell-based assays are increasingly being used to demonstrate the biological activity of a product because of their advantages
over in-vivo assays. Cell-based assays reduce animal usage while being both faster and cheaper, and they raise the regulatory standard
in terms of output. They also provide a demonstration of equivalence of biological function with the original reference product.
Potency assays typically measure the biological activity of the product over a range of concentrations, comparing it to that
of a well-characterized reference standard. The resulting dose–response curve may depict either the stimulation or the inhibition
of the biological response. This potency is typically expressed as either an EC50 value (half maximal effective concentration)
or an IC50 value (half maximal inhibitory concentration).
The inherent variability of biological assay systems and the resolution of such assays (a function of the dilution series,
for example) may result in differences in measured potency from one assay to the next. The potency of the biosimilar in the
test is thus expressed relative to that of the reference standard in the same assay to account for this. For example, it might
be described as, 92% as potent as the reference standard if its potency is a little lower, or perhaps 109% if it induces higher
Besides showing that the biosimilar induces a similar biological effect, the assay must be sufficiently sensitive to discriminate
small differences in biological activity and stability, with a quantitative readout over a range of treatment concentrations.
The cell line is the single most important factor in the development of most potency assays. Ideally, the cells will be of
a physiologically relevant origin, but they may also be genetically engineered. Either way, it is vital to use well-characterized
cells that respond predictably if the assay is going to be suitable for quality control use.
Once developed, potency assays need to be shown to be fit for the intended purpose, with experimental evidence of operation
within acceptable parameters. The stringency and extent of validation required depend on how far down the development process
the product is. For late-stage and commercial products, the assay must be well characterized with all specifications set and
justified, and full validation in accordance with ICH Q2(R1) is recommended (2). While this may take several months to complete,
it is required for product licensing, and such assays require ongoing maintenance to ensure robust performance, including
monitoring trending data and characterization of key reagents throughout the assay's lifespan. Any changes in reagents should
also be qualified, whether these are the internal reference standards or critical reagents such as growth factors, assay plates
or detection reagents.
Nonclinical animal studies.
Comparative in-vitro pharmacology and in-vivo studies comprising efficacy testing, pharmacokinetic assessment and toxicology studies, including toxicokinetics anti-drug
antibody and tolerance assessment, should be designed to maximise the information obtained in the comparisons between reference
and biosimilar products. The pharmacodynamics effect and activity relevant to the clinical application must be assessed, with
at least one repeat dose toxicity study. Toxicokinetic measurements will include antibody titres, cross reactivity and neutralizing
capacity. Normal safety pharmacology, reproductive toxicology, mutagenicity and carcinogenicity studies, however, are not
required for biosimilars. For biosimilars, the conclusion of nonimportant differences in pharmacological activity, pharmacokinetic
behavior or toxicological tolerance in comparison to the innovator drug are expected to be referenced in the regulatory filing.
Although full safety and efficacy studies in humans are not required for biosimilars in the EU, a degree of clinical investigation
is necessary. A Phase I safety study, usually in healthy volunteers, will have to be performed. Then, a Phase III comparative
study in patients to look at the relative effects of the biosimilar and the reference product must be performed, which should
include pharmacokinetic, pharmacodynamics and clinical efficacy assessments.
Even after approval, clinical safety and pharmacovigilance procedures must be put in place. One problem that can occur is
immunogenicity (i.e., patients developing an unwanted immune response to the product). While immunogenicity is rare in innovative
biological medical products, it can happen. The poster-child case was a packaging change for Eprex (erythropoietin) where
a substitute stopper interacted with the product formulation, causing an immunogenic response in patients. Biophysical comparison
of biosimilar and innovator drug showed that the two products were not structurally identical. Small differences were found
in the hydrodynamic structure, the degree of alpha helicity and the stability of these products (3). Thus, despite the rarity
of such occurrences, it remains important to test for immunogenicity using state-of-the-art methods.
There are a number of methods that may be selected to perform immunogenicity testing. A double antigen bridging assay has
been the preferred method because once it is optimized, it can be applied to immunogenicity testing in any host species. Alternate
methods also include application of enzyme-linked immunosorbent assay (ELISA) techniques, immunohistochemisry, electrochemiluminescence,
and also application of surface plasmon resonance. These techniques must be validated and be sufficiently sensitive to detect
low titre and low affinity antibodies. The latest draft guidance on biosimilars issued by FDA in February 2012, "Scientific
Considerations in Demonstrating Biosimilarity to a Reference Product" states that, at the very least, two separate immunogenicity
studies (using methods such as those previously described) should be conducted to compare a biosimilar to its reference product