Demonstrating Biosimilarity

Extensive comparability testing is required to ensure that biosimilars have comparable profiles to their reference products. (INGRAM PUBLISHING/THINKSTOCK IMAGES)Biosimilars are biologically derived therapeutics that are designed to be the equivalent, both functionally and structurally, as branded biologics. Because biologics are so sensitive to manufacturing changes, they can be particularly hard to copy. Unlike drugs made from small molecules, any change in the process or the products used to make them increases the possibility that they will be dissimilar from the originator product.

Biosimilars have been available in Europe since the 2006-approval of Omnitrope, Sandoz's version of Pfizer's growth-hormone product Genotropin (somatropin). Several versions of two other biologics, erythropoietin and filgrastim, are also now available from various companies. While none are yet approved in the United States, a legal framework towards biosimilar approval is now in place, and it is only a matter of time before the innovator companies face competition in this market.

The European Union (EU) authorities implemented their guidelines for biosimilars in 2004 as part of an amendment to the community code relating to medicinal products for human use. In Europe, however, the term "biosimilar" is not explicitly defined, and whether a product would be acceptable via the "similar biological medicinal product" approach is dependent on analytical procedures, the manufacturing processes used, and clinical and regulatory experience. The European Medicines Agency (EMA) also demands comparability studies to substantiate the similar nature of the two products in terms of quality, safety, and efficacy.

The clinical trials for biosimilars that have been demanded by the EMA ahead of approval have somewhat varied. Products have been approved despite differences in the glycosylation patterns and impurity profiles between the biosimilar and the innovator product. So far, all the biosimilars approved in Europe have been versions of naturally occurring hormones or cytokines, and draft guidelines for monoclonal-antibody products were published in 2011 (1).

FDA established the US framework in March 2010, and there are a number of key differences from the guidelines delineated in Europe. The US guidelines state that the clinically active ingredients in a biosimilar must be highly similar to the reference biologic, with no clinically meaningful differences in terms of safety, purity or potency. It must have the same mechanism of action, be administered in the same way via the same dosage form and have the same strength as the reference product.

With no biosimilars approved in the US to date, the guidelines have yet to be tested in practice for a marketed product. However, the experience gained in Europe during the past seven years may provide pointers. So what does the European experience tell us?

Safe substitution

To confirm the biosimilarity of a potential competitor biologic product to the original reference biologic, analytical studies must be carried out to show that it is indeed highly similar to the reference product. In general, extensive comparative physicochemical and functional studies are required.

Little is generally known about the originator company's manufacturing process as this is proprietary information. Variability in the exact nature of the biologic product is inevitable, thus, its intrinsic similarity to the reference product must be proven.

In addition, any impurities related either to the product itself or the process used to manufacture it must be identified, characterized, quantified, and compared to those of the reference product as must all the substances deliberately included within the product. The type, nature, and extent of any differences between the biosimilar and the reference must be clearly described and discussed in the application for approval.

A number of different factors should be considered when assessing if the two products are indeed highly similar. First, the expression system must be considered, including the identity of the cell line being transfected, the sequence of the transgene, any promotors or other control regions, and the overall genetic identity and stability of the cloned gene. Biosafety testing of the cellular substrates including the master cell bank (MCB) and cells at the limit of in-vitro cell age used for production must also be performed. The gene-copy number, and the gene-sequencing process, whether messenger ribonucleic acid (mRNA) or complementary deoxyribonucleic acid (cDNA) should also be determined in the cellular substrates. Fluorescence in-situ hybridization (FISH) analysis and restriction enzyme analysis should also be carried out. This comparison of the MCB and the cells at the limit of in-vitro cell age (considered to be the 'worst case scenario' in a bioproduction setting) enables the demonstration of the genetic stability characteristics of the MCB.

Next, the manufacturing process must be studied, including the cell line used, the glycosylation structure and heterogeneity it produces as well as both product expression levels and the expression of endogenous retroviral particles. Also, the viability and productivity of cells, product integrity, and degradation products and levels of host-cell protein and DNA must be assessed. The nature of the media is also important, such as whether or not it contains serum, is protein-free or chemically defined. Processing considerations, such as the bioreactor format and the downstream process, must also be considered, along with the removal of process- and product-related contaminants, virus inactivation and removal, product formulation, and product stability.

Finally, as with all medicinal products, the viral and microbial safety of the biosimilar must be considered. These are assured by three complementary approaches:

• Testing of starting materials for viral and microbial contaminants

• Testing process intermediates at appropriate stages in the manufacturing process for any contaminating viruses, mycoplasma, bacteria, and fungi

• Analytical characterization.

Comparability studies

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.

Chemical analysis. 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.

Biological analysis. 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.

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 activity.

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.

Clinical studies. 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 (4).

Conclusion

The EMA Committee for Medicinal Products for Human Use have issued a number of guidelines relevant to biosimilars that detail the requirements for market approval. The EMA guidelines cover a range of issues including manufacturing, measurement and comparability, chemical and biological analysis, and clinical trial requirements. In addition to the pharmaceutical, chemical, and biological data normally required for a generic-drug application, application for market approval of biosimilar products require additional toxicological and other nonclinical and clinical data.

The key is to demonstrate that the biosimilar product is similar to the reference product in terms of quality, safety, and efficacy. Products are dealt with on a case-by-case basis. The 2009 Biologics Price Competition and Innovation Act directed the US FDA to develop a regulatory framework in support of developing biosimilars and also defined the pathway to achieve drug approval. This pathway is based on a risk-based approach using what the agency has termed "totality of evidence." Working with a partner who has experience with the regulators for both innovator and biosimilar products will help to build this body of evidence.

Alison Armstrong is director of development services at BioReliance Ltd., Todd Campus, West of Scotland Science Park, Glasgow G20 0XA.

References

1. EMA, "Multidisciplinary: Biosimilar," www.ema.europa.eu/ema/index.jsp?curl=pages/regulation/general/general_content_000408.jsp, accessed July 22, 2013.

2. ICH, Q2(R1) Validation of Analytical Procedures, Step 4 version (1994).

3. S. Deechongkit et al., J Pharm. Sci. 95 (9) 1931–1943 (2006).

4. FDA, Guidance for Industry: Scientific Considerations in Demonstrating Biosimilarity to a Reference Product (Rockville, MD, Feb. 2012).