**2. Biological drugs: critical aspects**

#### **2.1 Definition**

According to the definition of biologics provided by the European Medicines Agency (EMA), "a biological drug is one that contains one or more active substances derived from a biological source. Biologics are larger and more complex molecules than non-biologic ones. Only living organisms can reproduce this complexity" [4]. Most biologics in current clinical use are proteins. They can differ in size and structural complexity, from simple proteins, such as insulin or growth hormone, to more complex ones, such as coagulation factors or monoclonal antibodies [2, 3, 5].

Biologics, including "biotech" drugs, that is, those produced by biotechnological methods (including recombinant DNA technologies, controlled expression of genes encoding biologically active proteins in prokaryotes or eukaryotes, hybridoma-based methods, and monoclonal antibodies), consist of active substances obtained from living cells or organisms [6]. Biologics production is a complex process involving gene manipulation, fermentation, and purification steps. It requires a very high level of technical expertise, sensitivity, and control to ensure its safety and efficacy. Generally, the first step is modifying a cell or microorganism, considered to be the host, to introduce a genetic sequence coding the protein to be produced. Then the host is conserved, and a master cell bank is produced from a seed lot. They are picked up, cluttered, and grown in a bioreactor or fermenter. Finally, it is collected to purify the protein, which will be then stabilized and formulated for therapeutic use.

Any changes in these processes, such as differences in temperature or pH, or cell culture conditions, could cause a significant modification in the final product in terms of efficacy or safety [7]. Moreover, due to post-translational changes, such as glycosylation, oxidation, and deamination, the final product may differ slightly from batch-to-batch and even within the same batch, they may have an impact on the mechanism of action of the molecule.

Since an ineluctable and unpredictable variability characterizes all living organisms, even if minimal, what is obtained from a biotechnological process will have an "intrinsic degree of minimal variability." Therefore, unlike generics where an exact copy can be made, in the case of biologic production, it is said that "the process defines the product" [8].

#### **2.2 Immunogenicity**

Another aspect that differentiates biologics from "conventional drugs" is their immunogenic potential, that is, their ability to induce an immune response in the


#### **Table 1.**

*Differences between generics and biosimilars.*

body (**Table 1**). Immunogenicity can lead to the development of antidrug antibodies (ADAs). ADAs may be neutralizing antibodies (NA) that neutralize the activity of these therapeutic proteins, causing reduced efficacy [9].

In the case of vaccines, the ability to induce an immune response, immunogenicity, is the expected therapeutic effect.

Immunogenicity, being one of the significant concerns in relation to biologics, is assessed throughout their entire development and production process.

The ability of biologics to induce immune responses may depend on several factors: The particular properties of the biologic, the characteristics of the patient, the concomitant treatments, the routes and the features of administration, or, finally, any variations introduced in the manufacturing process [10].

It is known that in the 1990s, the replacement of serum albumin with stabilizing agents (polysorbate 80 and glycine) in epoetin alfa caused several cases of pure erythroid aplasia due to the development of antierythropoietin antibodies [11].

#### **2.3 Biosimilarity**

Biologics enjoy two protection mechanisms: Patent (usually lasting up to 20 years) and a period of data and market exclusivity (up to 11–12 years) [12].

Once this period of patent coverage and exclusivity is over, "biosimilars", nonidentical but similar copies of originator biologics, determined to be of equal quality, safety, and efficacy to the originators, can be produced [13, 14].

"Biosimilarity" is the regulatory term first used by the European Union (EU) and the EMA to denote the comparability between a biosimilar and its originator reference medicine [13].

The first commercially available biosimilar appeared in the EU in 2006, while the first approval of a biosimilar in the United States (US) was in 2015 [15].

Medicinal products produced by biotechnology differ from traditional pharmaceutical chemistry methods in many aspects, including molecular size, structural complexity, stability of the final product, and the possibility of different relevant coand post-translational modifications (e.g., of the glycosylation profile). Additionally, because of their production process, which involves the essential intervention of living systems (microorganisms or animal cells), biologics present numerous aspects of heterogeneity linked to the host cell used, the plasmids used to transfect the host cell, and, therefore, transfer the gene necessary to induce the expression of the desired protein, as well as the conditions of growth and fermentation and the different methods of purification. All these peculiarities are not immediately transferable from one laboratory to another and contribute to the uniqueness of the product [4]. In particular, changes in the glycosylation pattern, a process that naturally occurs during the formation of a protein, can affect the therapeutic effect of the drug as well as lead to pharmacokinetic and pharmacodynamic modifications, altering the final product [15].

Therefore, structural variability and nonexact identity are two problems already present in original biologics, between different batches of the same product or even between drugs of the same set, and not only linked to their copies, that is, the biosimilars. This is because the very concept of similarity and nonidentity, which underlies biologics and biosimilars, is due to the inherent inability to replicate biological molecules exactly [16].

However, the primary responsibility of regulatory authorities and manufacturers in this context is to avoid clinically significant structural differences, which could adversely affect the efficacy and safety of the proposed biosimilar. This is achieved by assessing and demonstrating a high degree of structural and functional similarity between the originator and the biosimilar through what is known as a "comparability exercise," via studies that are defined as "of comparability" or "comparative" [13].

Therefore, the registration process of a biosimilar is different from that of a nonbiological drug equivalent (for which only bioequivalence studies, showing pharmacokinetic parameters, are generally required) (**Table 1**).

The investigation of biosimilars starts with quality studies (biological and physicochemical) and then continues with comparison studies with the originator, initially nonclinical (comparative nonclinical studies), concerning toxicity, pharmacokinetics, and pharmacodynamics, and then clinical (comparative clinical studies), in which efficacy and safety are assessed. In addition, at least one clinical study of immunogenicity is required to compare this aspect between the biosimilar and the original biologic (**Figure 1**) [4, 17].

It is clear that since clinical efficacy studies have already been conducted for originators, the purpose of studies on biosimilars is not to establish clinical benefit, but to demonstrate clinical equivalence, that is, noninferiority, with the biologic originator, defined in terms of "similarity throughout."

### **2.4 Interchangeability and substitutability**

"Interchangeability" is generally defined as the medical practice of substituting one drug for another equivalent drug with the same clinical effect and the risk– benefit ratio [18]. It thus describes the process, following a clinical decision by the prescribing physician, of transition from the originator to the biosimilar or from the biosimilar to the originator or between two biosimilars [13]. Interchangeability can only be assessed after the biosimilar has received regulatory approval.

"Substitutability," on the other hand, is defined as the practice, not necessarily of exclusive medical pertinence, of replacing medicine with another, often

cheaper, that has the same qualitative and quantitative composition of active substances, the same pharmaceutical form, and route of administration and which is bioequivalent with the reference medicine based on bioavailability studies [13]. "Automatic substitution" (for equivalents) by pharmacists refers to the practice whereby the pharmacist has the faculty or is obliged by national or local regulations, to dispense an equivalent and interchangeable medicine in place of the prescribed medication, without consulting the prescribing physician. "Primary substitution" occurs when a new treatment is started with a biosimilar (or equivalent) rather than the original reference product, and "secondary substitution" occurs when the treatment of a patient, already receiving a biologic, is substituted with a biosimilar [4].

However, it should be specified that in the US, the concept of interchangeability corresponds to the European concept of substitutability or "switching," since in the US, when the biosimilar is designated for use interchangeably with the original biologic, the pharmacist can dispense and authorize automatic substitution. Specifically, the Food and Drug Administration (FDA) requires that the definition of interchangeability of a biosimilar with the reference product must be established by an internal committee (the Biologics Price Competition and Innovation Act) based on specific documentation. To receive a designation of interchangeability in the US, the manufacturer must demonstrate through ad hoc studies that (1) the biosimilar will produce the same clinical outcome as the reference product in a given patient, and (2) the risk in terms of safety or reduced efficacy of alternating or switching between the use of the originator and the biosimilar is no greater than the risk of using the originator without such "switches" (**Figure 2**).

Thus, for the FDA, once a single biosimilar is defined as interchangeable, the clinician's decision on the individual case is not required for its substitution [18].

Regarding the automatic substitutability of biosimilars, the EMA does not assume responsibility for interchangeability and refers this decision to the EU Member States; in fact, European legislation has given the competent national authorities of the various Member States decision-making legislative autonomy in this matter (**Table 1**). However, the EMA has clarified that the recommendations issued on the marketing of medicinal products do not include whether or not a biosimilar should be used interchangeably and that the decision on the prescriptive choice of the specific medicinal product to be used, reference rather than biosimilar, should be entrusted to qualified healthcare professionals [19]. Moreover, the EMA generally recommends continuity of treatment for any patient already on therapy; but also emphasizes that there is no reason not to prescribe biosimilars directly to naive patients, that is patients who have not been treated previously, especially about the cost savings that this entails [4].

In European countries, several national regulatory authorities support substitutability during initial treatment or with the consent of the prescribing physician, but it is not endorsed unequivocally and uniformly [20]. In other countries, interchangeability is treated even differently than in the EU and US [1, 21]. Certifying that the drug is interchangeable is very complex for regulators without sufficient supporting data. The substitutability of generic drugs with reference drugs is used because the two drugs are considered identical if they have been demonstrated bioequivalence, but, as biosimilars are not exact copies, the generic approach cannot be applied in the case of biosimilars, and the question of their interchangeability remains unclear and is still an open debate that essentially involves all regulatory agencies.

#### **Figure 2.**

*Interchangeability studies. FDA requires evidence of a single "switch" for approval of a non-interchangeable biosimilar, but will generally require data on multiple "switches" for the definition of interchangeability.*

Indeed, the main concern about interchangeability is that repeated switches between biosimilars and the reference biologic may increase immunogenicity, leading to adverse reactions, particularly therapeutic ineffectiveness.

#### **2.5 Extrapolation**

Extrapolation is a scientific rationale used to describe how the proposed biosimilar receives all of the approved indications from the originator while performing comparative clinical trials of only one or two signs [22]. This rationale is captured in confirmatory phase III clinical trials, although the results of each experimental phase affect the extrapolation of indications.

There are limitations if particular indications are still protected by patent.

This concept of transferability of safety and efficacy data from one indication to another is not always clear to prescribing clinicians. Still, the extrapolation of therapeutic indications is recognized by both the EMA and the FDA [23, 24], although there must be a valid scientific justification for it to be applicable.

It is up to the Committee for Medicinal Products for Human Use (CHMP) of the EMA in the EU and the FDA in the US to determine on a case-by-case basis whether multiple indications can be extrapolated based on sufficient scientific evidence [23, 24].

#### **2.6 Pharmacovigilance of biologics: Specific aspects**

For all drugs, and certainly also for biologics, which do not yet have an established history behind them, robust postmarketing surveillance is crucial for identifying and assessing adverse effects and any other issues under discussion, such as the rationalization of interchangeability itself. The current pharmacovigilance paradigm typical of the "small molecule drugs" is highly insufficient and unsuitable to monitor the safety of biologics and biosimilars due to the different manufacturing techniques and the typical complexity of biologics, the possible structural differences existing between biosimilars and their originators, the possibility of biologics to cause longterm or short-term immunological reactions. Biologics are considered a priority for pharmacovigilance activities and, for this reason, the Directive 2010/84/EU included them in the "List of medicines subject to additional monitoring," characterized by an inverted black triangle in the "summary of product characteristics" (SmPC) and package leaflet accompanied by a sentence encouraging healthcare professionals and patients to report any suspected adverse reaction (**Table 1**) [25]. The EMA adopted new recommendations for the pharmacovigilance of biosimilars in 2016, and it has a separate section for "biological medicinal products" [26]. On the other hand, the FDA includes the Center for Drug Evaluation and Research (CDER), which is responsible for the pharmacovigilance of biosimilars and has its own guidelines [27, 28]. In the EU, all marketing authorization applications for biologics, including biosimilars, are reviewed by the EMA through a centralized procedure; consequently, the resulting marketing authorization is valid in all EU Member States. For this procedure to be undertaken, it is first necessary that the reference product, to which the application for marketing authorization of a biosimilar product relates, is a medicinal product that has obtained a marketing authorization in the EU based on a complete registration dossier, by Article 8 of Directive 2001/83/EC (**Table 1**) [13]. Each company must submit a risk management plan (RMP) with the marketing authorization application. The EU-RMP must detail the risk management system, describing the safety profile of the medicine, also taking into account the known safety profile of the corresponding originator, and

outline how the manufacturer will continue to monitor the efficacy and safety of its product and the measures that the marketing authorization holders (MAHs) intend to introduce to prevent or minimize any risk during the use of the medicine. Every biosimilar on the market has an ongoing EU-RMP, with a summary published in the European public assessment report (EPAR) (**Table 1**). Finally, Directive 2010/84/ EU stipulates that marketing authorization may be conditional on post-authorization safety (PASS) and efficacy (PAES) studies. PASS studies aim to identify, characterize, and quantify a safety risk, confirm the safety profile of the drug, or even measure the effectiveness of risk management measures taken during the marketing of the drug (this includes, specifically, immunogenicity phenomena that represent a crucial safety issue for any biologics and are mandatorily managed in the EU-RMP). In contrast, PAES studies aim to assess and confirm efficacy in cases where there are uncertainties regarding some aspects of the effectiveness of medicine [4].

The nomenclature is also a particular aspect of pharmacovigilance of biologics. When only the international nonproprietary names (INN) are used to report biologics or biosimilars without a distinguishable identifier, it may be complex to attribute an adverse event to a specific product. Instead, each biosimilar should be easily differentiated from the reference product and other biosimilars to ensure the appropriate use, traceability, and accurate reporting of adverse drug reactions (ADR). Since 2006, the World Health Organization (WHO) has been looking for a name for biosimilars that is universal and more suitable than INN names. In the EU, using an INN is up to the manufacturer, and there is no specific legislation outlining how to name a biologic/ biosimilar. As required by European legislation, all authorized medicines must have a trading name, either a brand name or the name of the active substance, followed by a trademark or the company's name that holds the marketing authorization. Therefore, each biologic, including biosimilars, is identifiable by a unique name formally approved by the EMA as part of the authorization process. In the EU, the reporting of suspected ADRs requires the inclusion of the brand name of the biologic and its batch number, but it has been shown that only 5% of ADRs include both the brand name and the batch number [29]. The lack and omission of traceable information can delay identifying safety problems with a specific product [30, 31]. The FDA, in 2019, has recently adopted a new guideline for the nomenclature of biosimilars, whereby four lowercase letters must be added as a biologic qualifier to the INN in the case of biosimilars [32], for example, Filgrastim-sndz. This action could promote an accurate identification of biologics and facilitate pharmacovigilance, increasing patient and physician confidence in biologics and biosimilars by ensuring proper traceability [31].

Another pharmacovigilance issue specific to biologics is, as already mentioned, immunogenicity (see Paragraph 2.2). Intrinsic differences may cause different immunogenicity even within the same batch. The immune response can be humoral (producing ADA is neutralizing or non-neutralizing) or cellular. Anaphylaxis and hypersensitivity reactions are the two main safety issues due to immunological reactions to these drugs. Still, even cross-reactivity to endogenous proteins or lack of efficacy or alternated drug pharmacokinetics may occur [33].

Such immunogenic ADRs, and even more so those due to immunogenicity linked to the "switch" (between an originator and a biosimilar or vice versa or between a biosimilar and another biosimilar), are difficult to identify they may occur in a minimal number of patients. It is also essential to understand the time interval between the administration of biologics and the occurrence of adverse events because of the possibility of delayed immunogenic reactions, which create further serious difficulties in defining the causal relationship with the specific product. Full characterization of

immunogenicity cannot be established during approval studies but requires long-term studies and rigorous postmarketing surveillance.
