Biosimilars: Key regulatory considerations and similarity assessment tools

Abstract A biosimilar drug is defined in the US Food and Drug Administration (FDA) guidance document as a biopharmaceutical that is highly similar to an already licensed biologic product (referred to as the reference product) notwithstanding minor differences in clinically inactive components and for which there are no clinically meaningful differences in purity, potency, and safety between the two products. The development of biosimilars is a challenging, multistep process. Typically, the assessment of similarity involves comprehensive structural and functional characterization throughout the development of the biosimilar in an iterative manner and, if required by the local regulatory authority, an in vivo nonclinical evaluation, all conducted with direct comparison to the reference product. In addition, comparative clinical pharmacology studies are conducted with the reference product. The approval of biosimilars is highly regulated although varied across the globe in terms of nomenclature and the precise criteria for demonstrating similarity. Despite varied regulatory requirements, differences between the proposed biosimilar and the reference product must be supported by strong scientific evidence that these differences are not clinically meaningful. This review discusses the challenges faced by pharmaceutical companies in the development of biosimilars.

small-molecule drugs or as stand-alone medications. Patents for some currently licensed biologic products have already expired or will expire in the coming years. Moreover, 7 of the top 10 pharmaceutical products sales in 2016 were biologic drugs (EvaluatePharma, 2017). As such, the concept of developing biologic products that are "biosimilar" (highly similar) to the approved biologic products has aroused great interest worldwide, across government bodies as well as in scientific and medical communities (Li et al., 2015). Biosimilars may offer increased treatment options for patients and physicians, and may optimize efficiencies across healthcare systems. Therefore, biosimilars have the potential to provide lower cost alternatives and offer greater access to biologics, and thereby allow increased use of biologic therapies (Baer, Maini, & Jacobs, 2014).
In broad terms, a biosimilar is highly similar to a reference product in terms of structure and function (WHO, 2016). The development of biosimilars is associated with numerous challenges, including the proprietary nature of the production processes of the reference product (the approved product) and the complexity of biologic molecules (Bandyopadhyay, 2013). By definition, and in contrast with small-molecule generic products, it is impossible to manufacture identical copies of biologic products (WHO, 2016). However, information from European Public Assessment Reports, US Summary Basis of Approval, published literature, and requests via the Freedom of Information Act (EMA, 2017;FDA, 2015a) can be harnessed to obtain methodology and results from key studies pertaining to the reference product. Together with the biosimilar developer's knowledge of analytical characterization and manufacturing, these data act as the basis for a strategy for biosimilar development.
Additional information on the reference product is acquired through extensive structural and functional characterization encompassing many product quality attributes, including primary sequence, higher order protein structure, post-translational modifications, protein aggregation and product-related impurities, and biological activities (Tsuruta, Lopes dos Santos, & Moro, 2015). Through a process of reverse engineering, a manufacturing process is developed that results in a biologic product that is highly similar to the reference product (Tsuruta et al., 2015). Because the development of a biosimilar likely begins several years after the development of the reference product, the relevant technologies may have evolved in the intervening years. The regulatory expectation is that the biosimilar manufacturer applies contemporary technologies in their product development, and must also adhere to current industry standards and regulatory expectations, which may also have evolved from the time that the reference product was developed and approved.
The approval of biosimilars is a highly regulated and detailed process. The European Medicines Agency (EMA) and the United States (US) Food and Drug Administration (FDA) guidance documents stipulate that a biosimilar manufacturer must perform a series of extensive similarity assessments in order to demonstrate biosimilarity to the reference product, and to ultimately gain regulatory approval or licensure (EMA, 2015;FDA, 2015b) (Table 1). The World Health Organization (WHO) has also published general guiding principles for the development of biosimilars, with the aim of providing a coherent approach for national regulatory guidelines (WHO, 2016).
Globally, regulatory expectations for the development and approval of biosimilars are not completely harmonized. Regional-and countryspecific biosimilar pathway legislation and guidance are at different stages of development and implementation. As a result, there is no global harmonization on certain aspects of biosimilar development, including the selection of the reference product, nomenclature, and the design of analytical, non-clinical, or clinical comparative studies. Indeed, global agreement on the regulatory requirements will optimize the development and manufacturing of biosimilars worldwide.
The purpose of this review is to discuss some of the challenges of biosimilar development.

| REGULATORY REQUIREMENTS FOR BIOSIMILARS: RIGOROUS, COMPREHENSIVE, AND EVOLVING
Demonstrating biosimilarity requires rigorous evaluation of the proposed biosimilar including side-by-side comparison with the reference product. During the development of the reference product, the developer must conduct extensive preclinical studies and large clinical trials in all indications for which approval will be sought. However, for a biosimilar developer, the comparative analytical characterization and the demonstrated similarity between a proposed biosimilar and the reference product reduces the requirement for large clinical trials in all the indications approved for the reference product (WHO, 2016).
The biosimilars approval pathway was pioneered in the European Union (EU), which has established regulatory architecture, with 11 product classes (under 31 different trade names) currently authorized by the EMA (EMA, 2017). The FDA has also developed extensive guidance on the regulatory requirements for the evaluation of similarity and granted approval of five biosimilars to date (Table 2). A biological medicinal product that contains a version of the active substance of an already authorized product (reference medicinal product) in the EEA FDA (FDA, 2015b) A biological product that is highly similar to a US-licensed reference product notwithstanding minor differences in clinically inactive components, and for which there are no clinically meaningful differences between the biological product and the reference product in terms of safety, purity, and potency of the product WHO (WHO, 2016) A biotherapeutic product that is similar in terms of quality, safety, and efficacy to an already licensed reference product

| Evidence requirements for the approval of biologics and biosimilars: A different way of thinking
The development pathway of an originator biologic requires extensive clinical evaluations, with the ultimate aim of establishing superiority (vs. placebo or comparator agents) in terms of efficacy and an adequate safety profile. In contrast, the pathway for biosimilar development is to demonstrate similarity to the reference product with respect to quality, safety, and efficacy using a stepwise approach that includes analytical, nonclinical, and clinical studies, rather than establish de novo safety and efficacy (FDA, 2015b) ( Figure 2). Based on this model, biosimilarity is evaluated using a scientifically tailored approach, with approval based on the "totality of the evidence," including analytical, (structural and functional), animal toxicity, pharmacokinetic (PK), pharmacodynamic (PD), immunogenicity, and clinical safety and effectiveness (FDA, 2015b). Individual regulatory agencies across the globe determine biosimilarity by assessing all of the available data provided by the biosimilar developer.
As such, a biosimilar may be deemed similar to a reference product even if there are minor analytical differences between the two, provided that sufficient scientific data and appropriate justification are supplied to show that these differences are not clinically meaningful (FDA, 2015b). Throughout the development of biosimilars, the nature and potential impact of residual uncertainty are evaluated and addressed at each stage and, in certain cases, may warrant the need for additional studies (FDA, 2015b).

| MANUFACTURING OF BIOSIMILARS: A CHALLENGING PROCESS
Due to their size and complexity, as well as differences in host cell lines and biological expression systems, the manufacture of biologics, including biosimilars, is challenging (Schiestl, Zabransky, & Sorgel, 2017). Control of biological expression systems is complex, and even small changes in bioreactor parameters may influence the clinical performance of a potential biosimilar. Factors such as pH, temperature, oxygen, light, forces experienced during cell culture, purification, formulation, and storage can also influence the quality of potential biosimilars throughout the manufacturing process (Mellstedt & Ludwig, 2008). In addition, the structure, biological activity and intrinsic stability can be influenced by events that occur throughout the manufacture of biosimilars. As such, caution must be exercised throughout the entire manufacturing process to avoid structural changes. Moreover, developers maintain strict control over the quality of incoming raw materials and extensively characterize their manufacturing processes to maintain batch-to-batch variability within an acceptable range (Kresse, 2009;Mellstedt & Ludwig, 2008). Biosimilar developers use the same manufacturing principles, basic processes, and current good manufacturing practices (cGMP) as those of the originator biologic (FDA, 2015b). The manufacture of biosimilars is a multistep process, beginning with the selection of an appropriate host cell line and transfecting the host with DNA that encodes the protein sequence of the reference product. Routine production of biologic products and biosimilars include fermentation, purification, formulation, fill, and finish, followed by analytical testing of the product. For biosimilars, determining the correct protein sequence of the reference product, and thus correctly encoding the DNA to be transfected into the host cell is a challenge because, although literature and patent information contain protein sequence details, this information can often be misleading or incomplete (Nowicki, 2007). Therefore, the biosimilar developer must confirm the amino acid sequence of the reference product prior to constructing the DNA sequence. The optimum host cell line for production is subsequently identified, based on product quality, cell growth, and protein expression characteristics. After transfecting a host cell, the specific clone is chosen principally based on the desired/critical product attributes of the protein produced. This is an iterative process to identify not only an appropriate clone, but also the production conditions that will deliver target product quality attributes similar to those of the reference product (Lee, Litten, & Grampp, 2012).
The purification process of biologic products and biosimilars involves chromatographic and filtration steps. This too is based on the biosimilar developer's manufacturing knowledge and is an iterative process to select the purification process that will deliver target product quality attributes similar to those of the reference product and meet current product safety standards and expectations.
Differences between the proposed biosimilar and the reference product may arise due to differences in host cell line, growth media, culture conditions such as temperature, pH, and agitation rate, as well as differences in the purification process (Mellstedt & Ludwig, 2008).
However, the biosimilar developer must demonstrate similarity of target product quality attributes with the highest scrutiny on those that impact the mechanism of action of the biotherapeutic. The characterization of multiple lots of the reference product at the outset and, thereafter at regular intervals, enables the design of an appropriate and robust manufacturing process, which will consistently produce a highly similar product. Manufacturing consistency ensures a product that meets approved specifications over the life cycle of a product (FDA, 2015b).
During development, manufacturing processes are designed, developed, and understood through the science-and risk-based approaches of quality by design. Quality by design is defined as a "systematic approach to development that begins with predefined objectives and emphasizes product and process understanding and process control, based on sound science and quality risk management (FDA, 2012)." It is particularly important that the product quality attributes of the reference product, which are pertinent to the intended clinical profile (known as critical quality attributes [CQAs]), are within an appropriate range, limit, or distribution for the proposed biosimilar.
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CQAs are product-specific. The primary amino acid sequence must be identical to the reference product as the sequence is critical in determining the structure and biological activity of a biologic product.
Other CQAs likely include aggregate levels, bioactivity, charge heterogeneity, and the glycosylation profile, particularly for monoclonal antibodies (mAbs), which may be part of the mechanism of action of the biologic (ICH, 2009;Tsuruta et al., 2015). While biosimilars guidelines allow for the use of different cell lines for the biosimilar than that used by the originator, particular attention should be paid to quality attributes that may be impacted by the cell line chosen, such as glycosylation (EMA, 2015;FDA, 2015b).
Once the cell culture conditions and purification process are optimized, the production process is scaled up to the proposed commercial scale and refined to maximize product yield, while maintaining product quality attribute ranges of the proposed biosimilar. The biosimilar manufacturer must consider potential lotto-lot variability in quality attributes and carefully examine changes across and within multiple production runs (Tsuruta et al., 2015). The Although there are no specific types of analyses or assays for evaluating all biologics, including potential biosimilars, the selection of analyses is influenced by the properties of the reference product. As such, similarity is determined on a case-by-case basis and the exact requirements can vary across regulatory agencies (Markenson, Alvarez, Jacobs, & Kirchhoff, 2017). The aim of an analytical similarity assessment is to investigate structural and functional elements such as primary structure, glycosylation, post-translational modifications, purity, charge heterogeneity and higher order structure, as well as bioactivity features that may impact the clinical properties of the proposed biosimilar. For example, structural and functional characterization of a proposed biosimilar to Rituxan (rituximab) was conducted using multiple state-of-the-art analytical tools (Visser et al., 2013) ( Table 3).
A variety of techniques may be used to assess analytical similarity, and a few examples are outlined below. However, it is important to note that other techniques may be appropriate for the biosimilar in development, and future advances must also be considered.
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| 2701 each peak. AUC-SV, an orthogonal technique to SE-HPLC, is used to assess the level of aggregate species, and to detect large aggregates that cannot enter a chromatography column, and may otherwise be undetected. In addition, CGE is used to assess low molecular mass or fragment species.
Charge heterogeneity is determined using isoelectric focusing, IEC, and isoelectric capillary electrophoresis (iCE). These techniques are useful in separating protein products into acidic, main, and basic species. Isoelectric focusing is the classic gel technique that is stained for detection, IEC uses cation or anion exchange chromatography columns to separate intact proteins, and iCE uses a capillary to separate denatured proteins. IEC and iCE typically use a UV detector to detect eluting protein, which allows for integration and percentage area of each peak to be calculated (Jung et al., 2014). For mAb biosimilars, charge heterogeneity is assessed using IEC to separate intact proteins that are detected by UV (Visser et al., 2013). The use of a preparative IEC column allows for the isolation of purified peaks, followed by identification of the species eluting under each peak, and analysis of both reduced and non-reduced sample preparations (Jung et al., 2014).
The higher order structure of the proposed biosimilar and the reference product is determined using X-ray crystallography, which provides high resolution information on the protein fragments (Fab and Fc). Secondary structure is assessed using far-UV circular dichroism and Fourier transform infrared spectroscopy, and the tertiary structure is evaluated using near-UV circular dichroism and fluorescence spectroscopy. Thermal stability is determined using differential scanning calorimetry, which monitors the specific unfolding of the protein (Visser et al., 2013).
Additionally, techniques such as hydrogen deuterium exchange mass spectrometry (HDX-MS) and proton nuclear magnetic resonance spectroscopy ( 1 H-NMR) can be used to characterize higher order structure (Wang and Li, 2014). HDX-MS is used to evaluate localized small differences in structure, based on the observed ion exchange rates (Engen, 2009;Houde, Berkowitz, & Engen, 2011). 1 H-NMR is used to determine the environment of the protein protons as a comparative tool. However, the ability of these assessment techniques to identify meaningful differences remains undetermined.
In addition to structural characterization of biosimilars, key functional tests are required to assess biologic potency and activity, such as target and receptor binding, complement binding assays, cellmediated toxicity, and cytotoxicity, where appropriate. The functional evaluation of potential biosimilars is based on the mechanism(s) of action of the reference product as reported in the scientific literature (FDA, 2015b). Unlike the structural similarity assessment described above, which is accomplished using a common strategy for biosimilars, the functional similarity assessment is unique for each biosimilar.
The potency of the proposed biosimilar and the reference product is determined using cell-based assays, which are used to examine the ability of the antigen to bind to its target and neutralize its biologic activity. Binding of the potential biosimilar to the target antigen is evaluated using target antigen binding assays, such as enzyme-linked immunosorbent assay, surface plasmon resonance, or flow cytometry (Visser et al., 2013). These techniques are also used to assess the binding of the potential biosimilar to pertinent receptors. Table 4 provides an example of the functional assessments of a proposed biosimilar to Remicade (infliximab) and highlights the diverse nature of the functional assessment of a proposed biosimilar (Jung et al., 2014).

| US FDA guidance on the statistical aspects of establishing analytical similarity
A three-tier approach to the statistical evaluation of analytical similarity between the proposed biosimilar and the reference product has been outlined by the FDA (Chow, Song, & Bai, 2016;FDA, 2015b).
The CQAs are identified and divided into three tiers based on a risk assessment of their impact on biological activity, PK/PD, safety, and immunogenicity.
CQAs that have a high impact on biological activity, safety, or immunogenicity with results from the analytical testing amenable to statistical evaluation are assigned to a tier 1 analysis, which involves an equivalency analysis between the proposed biosimilar and the US reference product. CQAs assessed to have moderate impact on these risks and with results from the analytical analysis amenable to statistical analysis are assigned to a tier 2 analysis. This involves a quality range analysis between the proposed biosimilar and the US reference product. CQAs that have low or no impact on these risks or have results that are not amenable to statistical analysis, regardless of risk ranking, are assigned to a tier 3 analysis. This involves raw data or graphical presentation of results between the proposed biosimilar and the US reference product.

| THE ROLE OF COMPARATIVE IN VIVO NONCLINICAL STUDIES
Nonclinical and clinical studies form the backbone of the efficacy and safety dossier of the reference product, and indeed for all new drug applications. In contrast, regulatory agencies recognize that  Hence, because of species differences and the small sample size, these studies are considered less informative than larger, statistically powered clinical trials (EMA, 2015;Van Meer et al., 2015). In addition, safety (including immunogenicity) in nonclinical species, even nonhuman primates, is not considered predictive of the potential for immunogenicity in humans (Ponce et al., 2009). In light of these limitations and concerns, fit-for-purpose comparative nonclinical in vivo evaluations may be needed to address specific residual analytical uncertainty or to meet regulatory requirements in some jurisdictions.
In certain circumstances, such as identification of impurities or bridge manufacturing scale-up, nonclinical studies of the biosimilar only (and not the reference product) may be appropriate.

| ESTABLISHING CLINICAL EFFICACY AND SAFETY SIMILARITY
The clinical assessment of similarity of a proposed biosimilar to the reference product involves comparative PK/PD, immunogenicity, and efficacy and safety studies. Establishing PK and PD similarity is a key part of the development of biosimilars, as it is not possible to accurately determine the PK and PD profiles based solely on nonclinical studies (FDA, 2015b). Furthermore, data from analytical and comparative PK studies can be used as a "bridge" that permits use of a single (US or EU) reference product in larger, comparative clinical efficacy studies.
Comparative clinical efficacy and safety trials are conducted, including immunogenicity assessments and, in some cases, confirmatory PK/PD studies to demonstrate clinical similarity of the proposed biosimilar to the reference product. The aim of clinical comparative studies is not to re-establish efficacy and safety, but to identify any clinically meaningful differences between the proposed biosimilar and the reference product, and to resolve residual uncertainty.

| THE ROLE OF COMPARATIVE IMMUNOGENICITY ASSESSMENT
Treatment with biologic products, including biosimilars, may provoke an immunogenic response, which could potentially alter the PK, efficacy, and safety properties of these agents (Bendtzen, 2012;Pendley, Schantz, & Wagner, 2003). Several factors have the potential to trigger an immunogenic response, including structural differences, aggregates, heterologous protein/amino acid mismatch, host cell proteins or other impurities, and varying glycosylation patterns (Liu, Zou, Sadhu, Shen, & Nock, 2015). Furthermore, concomitantly administered immunosuppressive therapy and chemotherapy can influence immunogenicity (Buttel et al., 2011). Therefore, careful examination of the formation of anti-drug antibodies in patients treated with biologics is critical throughout development and during post-marketing surveillance of biosimilars.

| THE SCIENTIFIC PRINCIPLES OF EXTRAPOLATION ACROSS INDICATIONS
Extrapolation is a scientific and regulatory term that describes the approval of a biosimilar for use in an indication held by the reference product, which is not directly studied in a comparative clinical trial with a biosimilar. Extrapolation is based on establishing a similar mechanism of action for the biosimilar in various disease indications (FDA, 2015b).
Extrapolation of clinical data can reduce or eliminate the need for studies in multiple indications, and therefore, may increase access to biosimilars sooner. Although the decision to extrapolate data from one indication to another is made on a case-by-case basis, with strong scientific justification, based on the totality of evidence, the concepts are supported by the EMA and the US FDA regulatory guidelines (EMA, 2015;FDA, 2015b clinical studies are a blunt instrument in the development of biosimilars, and that analytical evaluation is a far more sensitive tool in assessing similarity.

ACKNOWLEDGMENTS
This review was sponsored by Pfizer Inc. Writing support was provided by Neel Misra, MSc, of Engage Scientific Solutions and was funded by Pfizer.

CONFLICTS OF INTEREST
CFK, SA, HDC, AMR, and XZMW are full-time employees of Pfizer Inc.
AB was a full-time employee of Pfizer Inc at the time this manuscript was initiated and has now retired.