1. INTRODUCTION:

The use of monoclonal antibodies (mAbs) has revolutionized the treatment of several diseases, including cancer, rheumatoid arthritis, and other immune-mediated disorders, and provided both clinicians and patients with a great set of previously unattainable therapeutic options¹. mAbs significantly advanced cancer treatment due to their high target selectivity as they are designed to target specific molecular targets involved in the development and pathogenesis of carcinogenesis and consequently have fewer side effects than their traditional chemotherapeutic counterparts².

Bevacizumab is a humanized monoclonal antibody that inhibits tumor cells' neovascularization and, consequently, their angiogenic potential through binding to all the different types of vascular and soluble isoforms of vascular endothelial growth factor (VEGF)³. As angiogenesis is a prerequisite to tumor invasion and metastasis, the Inhibition of VEGF and its interaction with VEGF overexpressed receptors on cancer cells blocks new blood vessel formation and normalizes the tumor vasculature and microenvironment, which consequently facilitates the delivery of cytotoxic chemotherapeutic agents effectively⁴. Bevacizumab has demonstrated significant clinical efficacy against several types of solid tumors with elevated intratumoral VEGF levels, such as metastatic colorectal cancer, non-small cell lung cancer, metastatic renal cell carcinoma, and glioblastoma multiforme⁵. The use of Bevacizumab and, specifically in combination with other chemotherapeutic agents, has proved to provide positive clinical benefits to patients suffering from these diseases in all oncological parameters, such as progression-free survival (PFS), overall survival (OS) in addition to overall and partial response rates, (OR) and (PR), respectively⁶.

As a monoclonal antibody, Bevacizumab is considered a biological agent belonging to the class of immune biotherapeutics⁷. This class of molecules is highly complex in structure and requires specialized manufacturing processes as they are produced within living systems, unlike their conventional small molecule drug counterparts⁸. This complexity is evident in the overall three-dimensional structure of these glycoproteins. It is also responsible for their activity and pharmacodynamics through their interaction with their targeted receptor in a highly topology-dependent process⁹. Biologics are produced in living systems and their manufacturing is highly complex, requiring several quality-driven process controls and parameters to ensure that these agents are produced with high quality¹⁰. Bevacizumab (Avastin®) is produced by upstream hybridoma technology through the humanization of its parent murine antibody (muMAb VEGF) A.4.6.1, which was achieved through site-directed mutagenesis¹¹. The manufacturing process employs recombinant CHO cell lines produced by suspension cell culture and fed-batch mode of upstream processing¹². The originator bevacizumab (Avastin®) lost its patency in 2019, allowing several pharmaceutical companies to pursue the development and regulatory approval of bevacizumab copies or what is known as "Biosimilars"¹³. However, unlike traditional small-molecule generics, the regulatory pathway for the approval of mAb biosimilars requires that the manufacturer prove that the biosimilar has achieved satisfactory evidence to prove that its molecule is pharmaceutically and biologically equivalent to the originator mAb¹⁴. This poses serious technical challenges and limitations on the manufacturer to provide in order to provide this evidence by employing standard analytical and characterization techniques due to the nature and complexity of the mAbs, in addition to the limited analytical technology available to provide extensive evidence of total comparability between the "reference" biologic and it's biosimilar¹⁵. Producing copies of reference biologicals with inferior quality and lack of similarity creates what are known as "biomimics" or “intended copies”¹⁶. These agents could expose the patient population to serious safety risks due to unexpected immunogenicity or lack of efficacy that could arise in these products. Accordingly, the issue of compromising patients' safety through the use of intended copies, in addition to the need for a dedicated regulatory registration pathway for biosimilars, allowed both the European Medicines Agency (EMA) and the US Food and Drug Administration (FDA) to develop dedicated registration tracks for biosimilars registration and approval¹⁷. This pathway paved the way for the approval of the first biosimilar by the European Medicines Agency (EMA) (Epoitein alpha) in 2007 and the first by the FDA in 2015 for (filgrastim)^18,19. The guidelines of the FDA and EMA require that the regulatory evidence used to demostrate that a biological product is similar to the reference biologic must first be based on a stepwise comparison exercise²⁰. This includes a comprehensive biological and functional characterization, as well as in vivo and clinical studies and in a flipped approach when compared with the originator development program (Figure. 1). The data required from the clinical and non-clinical studies are mainly dependent on the results of the first step in the comparability exercise, which involves performing a comprehensive biological and physicochemical analysis of the biosimilar with its reference originator^21,22. This step is regarded as the start of the development program for any biosimilar. Accordingly, any biosimilar development program must start with complete comprehensive head-to-head physiochemical and in vitro functional comparability exercises to advance to the program's later non-clinical and clinical stages²³.

Figure 1: Regulatory pathways for the development program of both originator drugs and biosimilars. The biosimilar development program depends on a stepwise” totality of evidence” approach.

This review aims to guide and assist pharmaceutical personnel working on the development and commercialization of Bevacizumab biosimilars in addition to regulatory officers worldwide responsible for of the regulatory review of bevacizumab application dossiers. The review will detail the minimal technical requirements required to perform a full comparability exercise between any Bevacizumab biosimilar and its reference product. It will build on the EMA and U.S. FDA guidelines and the current literature to provide a comprehensive analysis of the various aspects of the bevacizumab comparability exercise and ensure that the product is similar to the reference biologic. The technical information and data used in this review article depend on several studies and regulatory guidelines extracted from various databases, such as Pubmed, Google Scholar, and Elsevier. The authors have also relied on the different protocols of the U.S. Food and Drug Administration and the European Medicines Agency. The keywords used for the search criteria included: "Bevacizumab", "Biosimilars", "Primary structure", "higher-order structure", "Post-translational modifications", "In vivo study", "Clinical study", "regulatory approval," and "functional comparability". The data and articles covered the years starting from 2010 until 2022.

2. NON-CLINICAL DATA:

2.1 Physicochemical Comparability:

The first step in establishing a biosimilar's similarity to Bevacizumab is to perform state-of-the-art head-to-head comparative physicochemical analytical techniques between the originator and the reference product²⁴. These orthogonal analytical techniques are aimed at elucidating the primary, secondary, and higher-order structures, as well as the purity/impurity, charged variants, and glycan structures of both reference bevacizumab and its biosimilar²⁵. This step is pivotal in performing Bevacizumab's biosimilarity exercise as slight differences in the three-dimensional structure and topology of mAbs can drastically affect the immunogenicity, pharmacodynamics, and activity of the biosimilar. Table 1. details the significant physicochemical properties and the various analytical techniques, including their acceptable limits that are expected to be performed to confirm the biosimilarity of Bevacizumab and detect any potential variables that could affect its biological activity. The properties include head-to-head comparison covering the following: primary structure and posttranslational modifications, secondary structure, higher order structure, purity and related species variants, glycan profile, and hydrophobic variants^26-30. The analytical techniques that should be employed to investigate each of the previously mentioned properties should be highly sensitive and capable of validating the structural similarity between the reference bevacizumab and its biosimilar³¹. Some of these techniques are detailed in Table 1. and include Liquid Chromatography Electrospray Ionization Tandem Mass Spectrometric techniques (LC-ESI-MS) for determining the primary structure, including the amino acid sequence, peptide mapping, and posttranslational modifications^32-34. Far and near U.V. analysis is employed to determine secondary structural content, while Fourier-transform infrared spectroscopy (FTIR) is usually employed to determine the amidation profile of both mAbs^35,36. The purity and size variants are determined through the use of high-performance size exclusion chromatography (HP-SEC) combined with reducing and non-reducing SDS-PAGE^37,38. Other analytical techniques such as Hydrophilic interaction liquid chromatography (HIILC) and reverse phase liquid chromatography (RP-HPLC) can be used for the determination of the purity in addition to the related size and charge variants of both mAbs^39,40.

2.2 Functional Comparability:

After completing the structural and physicochemical comparability exercise, the manufacturer of Bevacizumab's biosimilars can advance to the non-clinical functional and biological comparability assays. These assays are designed to determine if both MAbs are similar in regards to their ability to bind their target receptors/ligands and proper activation of the immune system⁴¹. The in vitro functional similarity results for Bevacizumab should investigate the comparability of the biosimilar and its reference product in three main domains, and these include the following: Fab-mediated activities, Fc-mediated Characterization, and Fc/Fb activation of both the Antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC)⁴². Fab mediated activities include assays specifically designed to assess similarity in VEGF binding, Inhibition of VEGF-A and its isoforms, Inhibition of proliferation in human umbilical vein endothelial cells (HUVEC), and Inhibition of receptor tyrosine kinases (RTK)^42-44. The Fc-mediated characterization should investigate Bevacizumab's biosimilarity with its reference product in binding FcγRIa, FcγRIIa, FcγRIIb, FcγRIIIa, FcRn, as well as C1q binding⁴⁵. Finally, both agents should display similarity in their in their inability to activate both ADCC and CDC in specifically designed assays, as VEGF is a soluble target⁴². Table 2. lists the major in vitro assays required for the functional similarity between Bevacizumab and its biosimilars.

Table 2: Summary of the biological (Fab & Fc mediated) quality parameters and analytical techniques required for the functional comparability exercise for Bevacizumab biosimilars.

Quality attribute	Technical parameter	Methods	Approved limits
Functional activity (Fab-mediated)	Soluble VEGF Binding	ELISA	Similar binding activity
	Inhibition of VEGF isoforms induced proliferation of HUVEC cells	Cell based assay	Similar potency
	VEGF binding kinetics	SPR assay	Similar affinity
Biological activity (Fc-mediated)	(FcRIIIa, FcRIAa, FcRIIb, FcRIIIb) binding affinity and kinetics	SPR	Similar affinity
	FcRn binding affinity and kinetics	SPR
	C1q binding	C1q binding ELISA	Similar affinity
	ADCC	Cellular assays	Lack of activity
	CDC	Cellular Assays	Lack of activity

2.3 In vivo Comparability:

According to both the EMA and U.S. guidelines regarding biosimilars, most mAb biosimilars can be exempted from performing in vivo animal studies due to the extensive data generated from both physicochemical and functional comparability exercises. However, in the case of Bevacizumab, additional animal studies are required to support the effectiveness of the biosimilar. These studies are designed to mimic the various pathological features of the solid tumors with significant elevation in VEGF production in addition to the neovascularization events accolated with angiogenesis and carcinogenesis⁴⁶. Both the reference Bevacizumab and its biosimilar should be comparable in their ability to cause tumor growth inhibition, reduce microvessel density and decrease vascular permeability⁴⁷. This is usually performed using mice xenograft tumor models as secondary pharmacodynamic studies are not required for Bevacizumab biosimilars. In addition to the in vivo animal efficacy studies, toxicological in vivo data are also required to support the safety of Bevacizumab's biosimilar before it can be submitted to human clinical trials⁴⁸. These studies usually involve performing single and repeated dose toxicity assessments on rabbits or monkeys with a focus on the effect of both the reference agent and its biosimilar in regard to animal mortality and clinical signs such as body weight, food consumption, physiologic measurements (heart rate and body temperature), ophthalmic or electrocardiogram examinations, hematology, serum chemistry, coagulation, urinalysis, or on macroscopic necropsy observations⁴⁹. These studies can also be used to generate toxicokinetic data derived from single-dose animal studies (half-life, Clearance rat, volume of distribution, Cmax, AUC) and to support the development of a drug's pharmacokinetic (PK) profile⁵⁰. However, any PK conclusions should mainly rely on the pharmacokinetic studies performed in both Phase I and 3 of human clinical trials. Table 3. Lists the major non-clinical studies required to perform the non-clinical similarity exercise for Bevacizumab and its biosimilar.

3. CLINICAL DATA:

3.1 Pharmacokinetic Comparability:

The first phase of the clinical comparability program for Bevacizumab biosimilars should involve a Phase I PK study to determine the safety and immunogenic properties of both the reference product and its biosimilar and the pharmacokinetic profile of the biosimilar⁵¹. This study should be conducted on healthy volunteers, and the design should follow standard parameters and assess the bioequivalence of both the reference and its biosimilar in regard to the area under the curve (AUC) and the maximum observed concentration (Cmax) using a single-blind or a double-blind crossover design with a statistically convenient sample size⁵². The products would be considered bioequivalent if the 90% confidence interval for the ratio of geometric means of the biosimilar-to-reference was within the predefined acceptance interval of 80% to 120% for the primary endpoints of the study⁵³. Additionally, the Phase I PK study on healthy volunteers could be used for the extrapolation of critical secondary endpoints such as treatment-emergent adverse events (TEAEs) and the immunogenic profile of the subjects by detecting the emergence of antibody-drug antibodies (ADAs) and the neutralizing antibodies (Nabs) over different time intervals post bevacizumab treatment⁵⁴.

3.2 Phase III Clinical Study:

According to the EMA and FDA guidelines and as displayed in Figure. 1, the stepwise approach for the development of a comparability exercise for Bevacizumab biosimilar should expand on the initial bioequivalence and immunogenicity results obtained from the Phase I clinical trials that were performed on healthy volunteers and perform a comparability exercise between the biosimilar and the reference product in a Phase III clinical trial designed to compare the efficacy and safety of both agents within the clinical setting⁵⁵. This study should complete the comparability exercise and provide the evidence needed to judge if Bevacizumab's biosimilar is comparable to that of the reference agent and provide the totality of evidence needed to make that judgment. The Phase III clinical trial is considered the most pivotal step in the comparability exercise as it will test both products head-to-head with patients in a clinical setting⁵⁶. A comparative Phase III study should be conducted to compare the safety and efficacy of Bevacizumab to its reference product and should not be conducted to demonstrate the superiority of the new drug over the existing one. Instead, it should be conducted to demonstrate that the biosimilar does not have significant differences in its clinical characteristics from the originator, Bevacizumab⁵⁷.

This study should be designed carefully in regards to the chosen patient population, sample size, primary and secondary endpoints for the chosen indication, in addition to the proper determination of the acceptable limits of comparability for each clinical parameter⁵⁸. For Bevacizumab patients, the most sensitive patient population and the main adopted indication for all approved biosimilars would be to perform a comparative Phase III multicenter, double-blind parallel randomized trial in patients with advanced untreated non-squamous non-small cell lung cancer (nsNSCLC) in order to evaluate the efficacy and safety of the biosimilar represented by the different A.E.s the patients could experience during treatment in addition to the immunogenicity of the biosimilar and its reference products, the most sensitive patient population mainly depends on indication in which the originator is approved for first-line use⁵⁹. The primary comparable endpoint would usually include the overall response rate (ORR) between the two patient subgroups included in the trial, in addition to having a group of secondary endpoints focusing on the comparable safety and immunogenicity of the two products. The trial could also include other secondary oncological efficacy parameters such as disease progression survival (PFS), disease control rate (DCR), duration of response (DOR), and overall survival (OS) rate⁶⁰. Finally, the phase III clinical trial should have an adequate sample size to display the non-inferiority of the biosimilar to its reference product. The sample size is one of the major parameters within a clinical comparability exercise. It is dependent on different statistical variables, including a non-inferiority design of clinical margin not exceeding 10%, a power of 80%, and a dropout rate of 10%⁶¹. Accordingly, a typical sample size for a Bevacizumab biosimilar in (nsNSCLC) should range between 500 and 706 patients for for phase III, randomized, parallel, active-reference controlled design⁶². Figure 2. summarizes the different technical, non-clinical, and clinical requirements for performing a comparability exercise for Bevacizumab biosimilars. It should be noted that the Phase III clinical trial is one of the significant determinants of the authenticity of the comparability exercise as several biological intended copies fail to meet the minimal requirements needed for clinical comparability at this stage.

Figure 2: The multiple technical, non-clinical, and clinical stages for performing a comparability exercise for Bevacizumab biosimilars.

4. JUSTIFICATION OF EXTRAPOLATION OF INDICATIONS:

The regulatory pathway for bevacizumab biosimilars does not require the manufacturer to conduct a clinical trial for each approved indication for the reference originator product. Accordingly, the totality of the data generated during the comparability exercise and development program of the biosimilar can be utilized to extrapolate the biosimilars to other indications and is considered justifiable if the biosimilar is targeting the same elements contributing to the pathogenesis of other indications⁶³. In addition, extrapolation of indications is considered essential in reducing the time and cost of a biosimilar development program, as performing clinical trials for each approved indication of the biosimilar would be financially challenging for the biosimilar manufacturer and would defy the purpose of the regulatory pathway for biosimilars registration and approval⁶⁴. Accordingly, the regulatory approval of a Bevacizumab biosimilar in (nsNSCLC) would allow its extrapolation for the treatment of other indications approved for the originator product and these include metastatic colorectal cancer, renal cell carcinoma, cervical cancer, ovarian cancer, and glioblastoma.

5. AUTOMATIC SUBSTITUTION AND INTERCHANGEABILITY OF BEVACIZUMAB BIOSIMILARS:

The concept of interchangeable medications refers to the practice of switching the treatment of a patient from one type of biological agent to its biosimilar to ensure that the biosimilar will have the same clinical outcome and therapeutic effect as the originator reference medication⁶⁵. Once the interchangeability of a biological agent has been confirmed, this could result in the clinical practice of what is known as "auto substitution," which allows the pharmacist to substitute the biological treatment by replacing one biological agent with its biosimilar without the approval of the prescribing physician⁶⁶. The debate about the interchangeability of biosimilars has generated a great debate among regulators and specialists⁶⁷. The U.S. Food and Drug Administration (FDA) allows the use of biological interchangeability if the manufacturer can demonstrate that the biosimilar can meet the same clinical results as the reference biological agent in all approved indications⁶⁸. If a biosimilar should be given to a patient more than once, the manufacturer must provide adequate data to show that switching from the originator biologic to its biosimilar does not pose a safety hazard to the patient^69,70. Such technical requirements are significantly more challenging in terms of cost and time for the manufacturer to provide to regulatory bodies for the approval for a biosimilar. As a result, if a manufacturer seeks "interchangeability" status for its biosimilar, they need to provide a substantial amount of technical and clinical evidence of biosimilarity of its product. The U.S. FDA also states that if a biosimilar gets the status of "interchangeability," every state has the option of allowing the automatic substitution of the new product⁷¹. In September 2022, the EMA decided to approve interchangeability for all its approved biosimilars in all of the European Union (E.U.). As stated by the EMA, this decision is based on the clinical experience gained within the E.U. with the therapeutic administration of biosimilars in millions of patients without raising any major clinical concerns regarding the biosimilar's efficacy, safety, and immunogenicity⁷². However, automatic substitution at the pharmacy level remains within the jurisdiction of each E.U. member state. This decision would allow all biosimilars of Bevacizumab to be interchangeable within the E.U. and would, reduce the treatment costs of several diseases significantly. Accordingly, this would release additional funds that could be employed to enhance the healthcare outcomes for several diseases within the EU and improve the overall health of the population. Within the US, there are currently no Bevacizumab interchangeable biosimilars with Cyltzeo®, a biosimilar of adalimumab being the only interchangeable Mab biosimilar in the US⁷³.

6. CONCLUSIONS:

The originator Bevacizumab (Avastin®) has achieved total sales of 2.8 billion dollars in 2021 placing the medication among the top selling 15 mAbs globally⁷⁴. Currently, there are 3 Bevacizumab biosimilars approved in by the U.S. FDA and eight biosimilars authorized within the European Union by EMA^75,76. Due to the efficacy and safety profile of Bevacizumab in the treatment of several oncological disorders, in addition to its significant sales potential, it is expected that more additional Bevacizumab biosimilars will continue to be developed globally in the future. This will expose the different national regulatory bodies to a significant number of Bevacizumab biosimilars, some of whom will be inferior to the originator Bevacizumab, thus risking the overall safety and therapeutic outcomes of the patient population exposed to these medications. In this review article, we have listed the technical and clinical requirements needed for any Bevacizumab manufacturer to acquire regulatory approval for a biosimilar of Bevacizumab according to EMA and US FDA guidelines. This review will aid manufacturers pursuing Bevacizumab biosimilar development in designing the appropriate measure needed to launch their biosimilar. More importantly, it will also provide regulatory personnel in all national regulatory agencies the needed guidance to make informed decisions regarding approving any Bevacizumab biosimilar submitted to their agencies. Although the guidelines presented in this review do not meet the specific requirements of a particular agency, they should still meet the minimal threshold that all regulatory bodies have regarding the approval of biosimilars.

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Received on 05.10.2022 Modified on 30.11.2022

Research J. Pharm. and Tech 2023; 16(7):3499-3506.

DOI: 10.52711/0974-360X.2023.00578

Quality attribute	Technical parameter	Methods	Approved limits
Primary Structure	Primary sequence	LC-ESI-MS	Identical amino acid sequence
	Primary sequence	Peptide mapping	Identical amino acid sequence
	Intact molecular mass	LC-ESI-MS	Highly similar
	Reduced molecular mass	LC-ESI-MS	Highly similar
	Isoelectric point	cIEF	Similar isoelectric point
	N and C Terminal Variants	LC-ESI-MS	Identical HC and LC
	Qualitative Peptide Mapping	RP-UHPLC-UV	Highly similar visually
	Partial N- terminal subunits sequence	EDMAN degradation	Identical
	Post-Translational modifications including sequence variants	LC-ESI-MS	Highly similar variants and glycol variants
Higher order structure	Secondary structural content	Far UV CD Spectroscopy	Highly similar
	Secondary structure	FTIR Spectroscopy	Similar location of Amide I and Amide II bands analysis
	Tertiary structure	Near UV CD Spectroscopy	UV spectra similar and superimposable
	Intrinsic Fluorescence	Spectrofluorimetry	Highly similar
	Disulfide Bridge mapping	LC-ESI-MS	Cysteine content similar
	Free Thiol Estimation	Free cysteine analysis	Highly similar
	Thermal Denaturation	DSC	Highly similar
	Tertiary structure	Intrinsic Fluorescence	Highly similar spectra
Physico-chemical properties	Purity and related size species variants	HP-SEC	Highly similar
		Reducing and non-reducing SDS PAGE	Highly similar
		Reducing and non-reducing CE-SDS	Highly similar
		HP-IEC	Highly similar
		IEF	Highly similar
		cIEF	Highly similar
	Hydrodynamic Size	DLS	Similar
	Hydrophobic Variants	RP-HPLC	Similar
	Glycan Profile	HILIC of 2 AB labelled Glycans	Similar
Strength	Protein Concentration	OD₂₈₀ method	Highly similar