Clinical development of biosimilars: an evolving landscape

Review

Mini Focus: Bioanalysis oF BiosiMilaRs

With the increasing access to quality healthcare and improved life-expectancy, the demand for life-saving, disease-modifying therapeutics is intensifying. With many developing nations joining the ranks of the Western world to improve healthcare access for their citizens, the societal demand for safe and effective therapeu-tics at manageable costs is expected to rise dra-matically in the next 10–20 years [101]. Despite the persistent market demand to make cop-ies of highly effective biological therapies, the complexity of therapeutic proteins produced by living cells had been a limitation to defining an abbreviated path to approval. In 2004, the EU proposed a regulatory pathway for bringing similar biological medicinal products to mar-ket and agreed upon a new legal provision for member states in November 2005 [102]. In 2006, the agency issued an overarching guidance on quality, nonclinical and clinical development [103,104]. Following that, the European Medicines Agency (EMA; London, UK) and the Scientific Committee for Medicinal Products for Human Use (CHMP) have published detailed product-specific guidance and guidance for biosimilar monoclonal antibody therapeutics as annex documents to the original legislation [105–114]. The WHO published a biosimilar guideline in 2009 [115], and the US FDA has codified bio-similar legislation in the Affordable Health Care Act under the Prescription Drug User Fee Act V [116–118]. Biosimilar guidance documents published by Canada [119], Japan [120], Brazil [121] and others share many common principles with

the EMA guidance. As of spring 2011, at least 14 biosimilar products, including two somatro-pins (Omnitrope® and Valtropin® [122–124]), five erythropoietins (Binocrit®, ABseamed®, Epoetin alfa HEXAL®, Silapo® and Retacrit® [125–128]) and seven granulocyte colony-stimulating factors (Filgrastim® Ratiopharm®, Ratiograstim®, Bio-grastim®, Tevagrastim®, Filgrastim HEXAL®, Zarzio® and Nivestim™ [129–132]) have been approved for marketing in the EU. Somatropin (human growth hormone), glucagon, hyaluroni-dase and recombinant salmon calcitonin have all been approved for marketing in the USA under the laws regulating biologics and, to date, there have been no biosimilar approvals in the USA.

Clinical development guidelinesAt a high level, there is general agreement between the EU and the FDA guidance on the foundational requirements for the biosimilar dossier. The FDA guidance document encour-ages biosimilar manufacturers to take a ‘stepwise approach’ and the expectations to demonstrate similarity appear consistent with therapeutic drug-development principles [1,2]. Rigorous com-parative analytical and bioactivity characteriza-tion forms the foundation of the biosimilarity assessment [3]. Such an assessment is dependent not only on a thorough understanding of the structure–activity relationship and critical product attributes, but also on the limitations of the characterization tools [4]. It is widely recognized that there are gaps in understand-ing of the structure–function relationship, as

Clinical development of biosimilars: an evolving landscape

Biosimilars, or similar biological medicinal products, can provide a meaningful option for patients and physicians provided they deliver the therapeutic value of a reference product at a more modest cost. Unlike generic small-molecule drugs that require primarily the demonstration of pharmaceutical equivalence, the complex nature of protein therapeutics warrants a rigorous evaluation of both pharmaceutical and therapeutic equivalence to the reference product in an abbreviated clinical program. Furthermore, the lack of comprehensive structure –activity relationship data increases the burden on appropriately designed human clinical studies with predefined acceptance criteria to demonstrate the absence of clinically meaningful differences between the biosimilar and reference product. Although a number of biosimilar proteins have been approved, especially in Europe, issues on substitutability, extrapolation to other disease indications, and selection of reference standards and comparators, remains to be standardized at a global level.

Meena SubramanyamTranslational Medicine, Biogen Idec, Cambridge, MA 02142, USA E-mail: [email protected]

575ISSN 1757-618010.4155/BIO.13.5 © 2013 Future Science Ltd Bioanalysis (2013) 5(5), 575–586

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well as the dose–response relationship, for some of the marketed protein products. As a result, the emphasis for similarity demonstration has shifted to the clinic to demonstrate the lack of clinically meaningful differences, especially when differences are observed in specific product quality attributes. With a focus on the totality of evidence, the guidance remains intentionally vague on defining specific acceptance criteria to demonstrate pharmaceutical and therapeutic equivalence. Given the complexity and hetero-geneity of biologics, the general expectation is that at least one comparative clinical study will be needed to establish therapeutic equivalence to the reference product.

Clinical developmentOvera l l, a s stated in the guidance, demonstration of comparability of the bio-similar protein to the reference product has to be accomplished in three critical areas during clinical development, namely PK/PD, efficacy and safety, including short- and long-term immunogenicity.

�� PK studiesA risk-based product development approach minimally entails a comparative PK study to demonstrate the predictable and quantifiable dose–response relationship of the biosimilar to the reference product. For demonstration of similarity in a dose–response relationship, a PK/PD equivalence study in healthy volun-teers with either a crossover design or a paral-lel design, based on the half-life of the thera-peutic protein, is recommended. Evaluation of typical parameters such as AUC, maximum observed plasma concentration (C

max) as well

as plasma half-life (T1/2

) is recommended. The single- or multi-dose PK study of limited dura-tion can be conducted as a standalone study or can be integrated into the first part of the pivotal biosimilar efficacy study, especially if it is designed to be a parallel ana lysis. Integrating PD evaluations as part of this study will help strengthen the similarity argument and may support a more targeted clinical-efficacy trial. One of the key considerations for the selection of the PD marker is the clinical relevance and meaningfulness, which may aid in predefining the acceptance range that will be used to dem-onstrate similarity to the reference product. For chemically synthesized therapeutics, typically an 80–125% acceptance range for compara-tive PK data is used [5]. The EMA and FDA

guidelines explicitly state that this range does not apply to complex biosimilar therapeutics, and provides the flexibility for the manufac-turers to specify and justify a product-specific range based on experiential knowledge. How-ever, it is important to recognize that the bio-similar manufacturer may not in general have an expansive manufacturing history for a given protein, nor adequate knowledge regarding the clinical impact of product attributes. Such information may not be readily available, even for the reference product. To minimize the uncertainty around therapeutic equivalence and in the absence of other supporting data, therefore, the biosimilar applicant should be expected to predefine a stringent upper and lower bioequivalence margin in order to remain within the therapeutic window of the reference product.

�� Bioanalytical considerationsAnother critical consideration for PK/PD studies is the development and validation of appropriate quantitative bioanalytic methods to determine the concentration of the biosimilar and reference product. It is recommended that samples from biosimilar- or reference product-administered subjects be evaluated in the same, or a very sim-ilar assay that utilizes similar technology and secondary reagents. It is important to demon-strate that the selected analytical method can quantify the biosimilar and reference product equivalently. This is readily accomplished by using the biosimilar or the reference product as assay calibrators to quantify the levels. Mini-mally, precision and accuracy, as well as the lack of interference from matrix components, in the detection of biosimilar and reference products should be demonstrated. The acceptance crite-ria for the various parameters should follow the principles outlined in the bioanalytical method validation guidelines [133]. For PK assays, the selection of the assay format, as well as the cap-ture reagent that determines the sensitivity and specificity of the assay, is very critical. In addi-tion, characterization of the assay calibrator, as well as establishing sample stability conditions are essential. With regard to PD assays, selection of the end point to demonstrate dose–response relationship and development of sensitive analytical methods for sample ana lysis are key activities. As with the PK data, it is important to gain agreement from regulatory agencies on the acceptance margins for the PD data in order to bolster the similarity demonstration.

Key Terms

Biosimilar: Similar biological medicinal products; refers to a new version of a marketed innovator product made in a living organism using recombinant DNA technology, by another manufacturer, following the expiration of the patent and exclusivity period for the innovator product.

Therapeutic equivalence: Drug that has a highly similar effect to another therapeutic in the same disease indication. Typically, demonstration of therapeutic equivalence requires bioequivalence and pharmaceutical equivalence assessments.

Bioanalytical method validation: Best practices used to develop and implement therapeutic-specific test methods to understand the PK, PD and immunogenicity of the therapeutic.

Pharmacovigilance: A systematic evaluation process established as part of a clinical trial to detect, assess and understand the number and type of adverse events that is associated with the administration of a therapeutic in man, short- and long-term. The pharmacovigilance plan also contains elements to actively monitor the occurrence of known safety events and in some instances may contain guidance for clinical management of events.

Review | Subramanyam

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Efficacy studiesDemonstrating comparable efficacy to the reference product at the marketed dose and route of administration in the intended disease popu-lation forms the second critical aspect of similar-ity demonstration. A double-blind, adequately powered, randomized, parallel-group efficacy study, employing an equivalence design in the most sensitive patient population, is required to perform a robust efficacy evaluation. The design of this clinical study will depend on the PK characteristics and pharmacological activity for each particular biologic. The upper and lower efficacy margins should be prespecified based on the largest difference that will have no clini-cal meaningfulness. Such an approach will help identify products with a dissimilar efficacy pro-file. When PD or surrogate markers are included in the study, the correlation of marker expression to clinical outcomes must be understood.

Immunogenicity assessmentImmunogenicity assessment, or the monitoring of anti-drug antibody (ADA) development, is an integral part of the third critical element, safety evaluation, in biosimilar development [6–8] and has been identified as an essential component of the clinical efficacy trial design. Development of ADAs is strongly influenced by drug-substance and drug-product characteristics, including process- and product-related impurities and formulation in addition to patient and disease characteristics [9,10]. Production and formula-tion of biosimilar products using a potentially alternate manufacturing process compared with the innovator product may result in subtle dif-ferences leading to differences in the immuno-genicity profile between the products. In general, the administration of therapeutic proteins has the potential to elicit product-specific ADAs, which in some instances have been shown to have clinical effects on pharmacology, PK or effi-cacy [11–15]. From a safety perspective, instances of ADA–drug immune complexes resulting in serum sickness, IgE isotype-mediated anaphy-lactic reactions [14], or neutralization of an endo-genous counterpart in rare instances, resulting in very serious consequences, are all reported in literature [16–19].

If the titer level and qualitative nature of ADAs developed against the biosimilar product differs from what is observed against the reference prod-uct, a differential impact on the pharmacology and PK, and thereby efficacy or safety of the product, may be observed in the same patient

population. During biosimilar development, therefore, similarity in the incidence, type of ADA (non-neutralizing, clearing and neutraliz-ing), titer of ADA and time to formation of ADA in patients administered the biosimilar product compared with the reference product should be demonstrated in at least one comparative clinical immunogenicity assessment. Although the FDA guidance suggests a one-sided trial to determine comparative immunogenicity, the sensitivity of this design is limited to determining higher immunogenicity incidence to the biosimilar. In contrast, a two-sided trial will also help deci-pher if the immunogenicity to the biosimilar is lower. Such a result may also be suggestive of differences between the biosimilar and refer-ence product. Immunogenicity data are essen-tial to interpret the totality of clinical similar-ity in PK and efficacy, as well as safety. Longer term pharmaco vigilance commitment will be required for biosimilars to gather safety data, especially for low-incidence, high-impact events. The experience with the erythropoietin reference product Eprex® highlighted the amount of time and data that is required to establish a firm link between changes to product formulation and the increase in the number of pure red cell aplasia (PRCA) cases. PRCA is a potentially fatal condi-tion in which ADAs crossreact with and neutral-ize endogenous erythropoietin. This abolishes erythropoiesis and results in severe anemia [16,17].

Immunogenicity challenges in biosimilar developmentThe link between ADA and patient safety events has already been experienced during the development of erythropoietin biosimilar products. Dosing had to be suspended in two clinical studies (“Treatment of anemia associated with chronic renal insufficiency in predialysis patients”, and “Study to evaluate the efficacy, safety and immunogenicity of subcutaneous HX575 in the treatment of anemia associated with chronic kidney disease” [134]) of the bio-similar product EPO HEXAL versus the ref-erence product Erypo® due to occurrence of PRCA. In one of the studies, a German patient developed PRCA and another patient from Rus-sia in the study was diagnosed with neutralizing antibodies against EPO, highlighting potential product differences [18,135]. Following dosing sus-pension, patients in the study had to be trans-ferred to alternative treatments at their treating physician’s option. PRCA was also reported in a hemodialysis patient with chronic kidney disease

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who was undergoing treatment with the biosimi-lar epoetin alfa product Wepox® (Wockhardt Ltd, Mumbai, India) [19]. These case studies serve to highlight the inadequacy of the cur-rent analytical and biochemical characteriza-tion studies of biosimilar drug substances and drug products to demonstrate rigorous similar-ity. In the case of the biosimilar epoetin zeta product Retacrit, a different immunogenicity profile compared with the reference epoetin alfa product was observed in a nonclinical study per-formed in dogs. However, the ADA was demon-strated to be non-neutralizing with no obvious clinical effect in both dogs and man. However, longer term immunogenicity data for humans are needed to confirm the absence of discernible ADA-mediated clinical effects of Retacrit [20,136]. The EMA rejection of marketing authorization for Marvel (Middlesex, UK) insulin [137] and Alpheon’s IFN-a biosimilar product [138] cited inadequacies in immunogenicity assessment procedure, besides other deficiencies. The early experience with biosimilar product development has shown that even when biosimilar products are deemed sufficiently similar in physicochemi-cal characteristics to the reference product, the risk of immunogenicity cannot be excluded with reasonable certainty.

�� Bioanalytical methods to assess immunogenicityIn principle, as part of the biosimilar development discussions with regulatory agencies, a risk-based immunogenicity evaluation strategy, as well as assay design and validation parameters, should be developed and presented [21–23]. The duration of the immunogenicity studies, and the periodic-ity and timing of sampling for testing of antibod-ies should also be justified. The tiered approach of screening, confirmation and characterization of ADA should be implemented with assays of adequate sensitivity as outlined in the immuno-genicity guidance [139]. International standards should be used whenever possible for method calibration. With regard to assay methodology, it is preferable to use a single-assay format to detect ADAs to biosimilar and reference product in order to demonstrate comparable sensitivity and drug tolerance. It is recommended that the neutralizing antibody assay format be reflective of the pharmacological mechanism of action of the product. Validation parameters outlined in regulatory guidelines should be executed using surrogate assay controls in order to show that the assay is capable of detecting antibodies to

both products. The same requirements also hold true for neutralizing antibody assays. In addition, immunogenicity data should be cor-related to clinical parameters such as PK, PD, efficacy and safety to assess impact. In view of the unpredictability of the onset and the qualita-tive nature of ADA that develop, post-marketing monitoring of immunogenicity at predetermined intervals will be required for at least 1 year for a biosimilar product. For high-risk products such as erythropoietin, a special vigilance plan of longer duration may be needed.

Biosimilar development: clinical experienceAn ana lysis of the clinical development approaches adopted by the biosimilar products approved for marketing by the CHMP reveals some deviations within the framework of the stated requirements. The following sections on granulocyte colony-stimulating factor (G-CSF), somatropins and epoetins provide an overview of the similarity requirements outlined in the EMA guidance for the various product classes and the data package developed by the biosimilar manufacturers for marketing authorization.

�� G-CSFRecombinant (r)G-CSF product Neupogen® (Amgen, CA, USA) produced in Escherichia coli contains a single, 174 amino acid poly peptide chain with two intra-molecular disulfide bonds, and one free cysteine at residue 17 [24]. It is used clinically for the reduction in duration of neutro-penia after cancer chemotherapy; for myelo-ablative therapy following bone marrow trans-plantation; in the mobilization of peripheral blood progenitor cells; and, for the treatment of severe congenital, cyclic or idiopathic neutro-penia, and persistent neutropenia in patients with advanced HIV infection [25,26]. A total of seven biosimilar rG-CSF products of N eupogen have been granted marketing authorization in Europe, and a few in other countries as well [27,28]. The explicit recommendations around allowable PD end points, the specific population to be studied, as well as a provision for treat-ment extrapolation to other indications with the same mechanism of action without additional clinical studies, appears to have accelerated the development of this biosimilar [106]. The Sandoz product is marketed as Zarzio and HEXAL and the other bio similar substance XM02 is mar-keted as B iograstim, Filgrastim Ratiopharm, Ratiograstim Nivestim and Tevagrastim. The

Key Term

Clinical trial: Set of controlled studies performed in humans to delineate the efficacy and safety of an administered therapeutic at a specified dose. Helps define the dose–efficacy and dose–safety relationship to calculate the therapeutic window.



development of Zarzio approved for treating neutropenia followed very closely the principles laid down in the EMA guidance document [106]. A total of four Phase I, randomized, double-blind, crossover, single- and multiple-dose PK studies were conducted in healthy volunteers via the subcutaneous (SC) and intravenous routes. The PD analysis monitored the absolute neu-trophil count (ANC); equivalency of CD34+ cell-count increase; mean ANC-time profiles; as well as dose-dependency and reversibility of effect. For demonstration of efficacy, neutrope-nic chemotherapy patients were enrolled in an open Phase III study (n = 170) evaluating Zarzio at 300 or 480 mg via the SC route. In addition, comprehensive safety assessments were con-ducted to demonstrate that the overall incidence of adverse events was within the range of the reference product. Immuno genicity assessment both in Phase I and III studies of Z arzio demon-strated that none of the 316 subjects developed anti-recombinant human (rh)G-CSF antibodies. The other approved biosimilar product Nivestim was studied in two randomized, single-center, Phase I trials in healthy volunteers to compare PK, PD and safety profiles of Nivestim with the reference product Neupogen [132,28]. In both studies, 90% confidence intervals for the pri-mary end points were within the predefined range (0.80 to 1.25%) to demonstrate bioequiva-lence. Nivestim was also shown to be well toler-ated, with no additional safety concerns com-pared with Neupogen. Efficacy and equivalence were demonstrated in a randomized, double-blind, multicenter, Phase III trial of 279 patients with breast cancer receiving myelosuppressive chemo therapy. The mean duration of severe neu-tropenia in cycle 1, the primary end point, was similar between Nivestim (1.6 days; n = 165) and Neupogen (1.3 days; n = 85), meeting predefined criteria for bioequivalence. Secondary end points supporting bioequivalence included the mean time to recovery of ANC and incidence of febrile neutropenia. Based on the strength of the clini-cal data generated, Nivestim was approved for all indications that Neupogen is approved for, and both Zarzio and Nivestim have ongoing pharmacovigilance monitoring.

�� SomatropinsSynthetic growth hormone (somatropin) is a 191 amino acid-containing single polypeptide chain protein. Similar to rhG-CSF, develop-ment and authorization of biosimilar somatro-pin appears to have proceeded without major

issues in Europe. For somatropin, the EMA guidance recommends a single-dose study in healthy volunteers using SC administration with endogenous growth hormone (GH) production suppression with a somatostatin analog to dem-onstrate similarity in PK/PD of the biosimilar to the reference product [107]. The guidance explicitly identifies IGF-1 and IGFBP-3 as the preferred PD markers for the activity of soma-tropin. However, due to the lack of a clear rela-tionship between serum IGF-1 levels and growth response, IGF-1 is not considered a suitable sur-rogate marker of the efficacy of a somatropin [29]. Clinical efficacy demonstration requires at least one comparative adequately powered, double-blind, randomized, parallel group clinical trial for a duration of 6–12 months with prespecified and justified comparability margins. Change in height velocity or (change in) height velocity standard deviation score from baseline to the prespecified end of the comparative phase of the trial is the recommended primary efficacy end point. Furthermore, the EMA guidance pro-vides a number of prespecified requirements to minimize height measurement errors and vari-ability between patient cohorts. As with other products, the requirement for a comprehensive pharmacovigilance plan containing comparative 12-month immunogenicity data of patients with sampling at 3-month intervals is mentioned.

For Omnitrope approval, five PK/PD s tudies, which included in total approximately 120 healthy volunteers, were conducted [123]. These studies included IGF-1 and IGFBP-3 as PD markers, but were not conducted as crossover comparative studies to the reference product Genotropin®. Omnitrope was also compared with the reference Genotropin in 89 prepuber-tal, GH-deficient treatment-naive children in an open-label 9-month trial for expanded use. Although the guidance allows the extrapolation of efficacy to other indications in which the refer-ence product is approved, it is unclear at this time whether the comparative efficacy claim will hold true if the treated population is not deficient in GH. Furthermore, in the trial, initially almost 60% of the children administered Omnitrope developed anti-host cell protein antibodies. The immunogenicity issue was resolved only when the manufacturer enhanced the Omnitrope purifica-tion process to decrease host cell protein burden. The requirement to monitor immunogenicity as well as risk of malignancies over long-term use is embedded in the Omnitrope pharmaco vigilance plan. Omnitrope has also been authorized for



marketing by the FDA under section 505(b)(2) of the FDC act in 2006 [140], well before the issuance of the biosimilar guidance. Since therapeutic equivalence demonstration was not needed for this approval, Omnitrope is not allowed to be substituted for other approved human GH products in the USA. Development of Valtropin® pretty much followed the same path using Humatrope® as the reference [124]. Besides GH-deficient children, Valtropin was also clinically evaluated in children with Turner syndrome prior to approval. The immunogenic-ity rate of 2% was highly similar to the reference product Humatrope.

�� EpoetinsEpoetins, which are erythropoiesis agents, are generally prescribed for renal anemia, chemotherapy-induced anemia and for predo-nation of blood prior to surgery for autologous transfusion [30–32]. There are a number of inno-vator epoetin products currently in the market including, Epogen® (Epoetin alfa) and Procrit® (Epoetin alfa), manufactured by Amgen and marketed in the USA; Eprex (Epoetin alfa), manufactured by Ortho Biologics LLC (PR, USA), and marketed by Johnson & Johnson (NJ, USA) outside the USA; NeoRecormon® (epoetin beta), manufactured by Roche (Basel, Switzerland) and marketed in Europe; Aranesp® (darbepoetin alfa), manufactured by Amgen and marketed in the USA, Europe, Canada and A ustralia, and MIRCERA® (methoxy polyethyl-ene glycol- epoetin beta), manufactured by Roche and available in Europe.

The approval of several epoetin biosimilar products has provoked intense debate about comparable product quality and the significance of the observed variation in glycosylation (man-nose levels) of the epoetin products [33–35]. Even though the biosimilar products share identical amino acid sequences to the reference product, there are inconsistencies in isoform composition and biological activity of biosimilar epoetin prod-ucts [36,37]. Despite these observed differences, interestingly, the products were not disqualified as biosimilars to the reference product.

From the clinical studies perspective, the com-parative PK, PD single- and multiple-dose intra-venous, and SC administration study in healthy volunteers, as well as the confirmative, double-blind, randomized, parallel group, multicenter Phase III efficacy and safety trial demonstrating therapeutic equivalence in mean absolute change in hemoglobin level in patients with renal anemia

on dialysis was demonstrated for the biosimilar products using Eprex (epoetin alfa) or NeoRecor-mon (epoetin beta) as the reference product [111]. Based on PRCA events observed with Eprex, the renal anemia studies using the SC route of admin-istration was waived for the early biosimilar epo-etins approved in 2007 [17,18]. Although another exploratory clinical study of the biosimilar epo-etin in the treatment of chemotherapy-associated anemia via the SC route was conducted, the trial size was deemed inadequate to establish compara-ble efficacy for the SC route of administration. In 2010, Hospira’s Retacrit became the first epoetin biosimilar to submit data from a rigorous Phase III clinical trial demonstrating comparable efficacy and safety to the reference product epoetin alfa when administered subcutaneously in patients with end-stage renal failure on chronic hemodi-alysis. The PRCA concern has made pharmaco-vigilance studies a central piece of erythropoietin biosimilar approvals, besides the requirement for a 300 patient immunogenicity assessment study for a minimum duration of 12 months. Monitoring an increase in PRCA incidence between biosimi-lar and reference products will minimally require tens of thousands of patients (incidence rates: 0.2 to 18 cases per 100,000 patient years between 2001 and 2003) [38–40]. In February 2012, the EMA published the first draft of the guidelines on good pharmacovigilance practice, including a module on management and reporting of adverse reactions to medicinal products [136]. The direc-tive 2010/84/EU article 102 of the medicinal products directive 2001/83/EU was amended to require the member states to record the name and batch number of any dispensed medicinal prod-uct in order to link a specific biological medicinal product to the adverse reaction. In addition, the directive requires doctors to maintain accurate prescription records to correlate adverse events to pharmacist-initiated product substitution. The article also requires pharmaceutical compa-nies to include warnings stating that changing to another biological medicinal product should be authorized by the prescribing physician for pharmacovigilance reasons.

Biosimilar recombinant soluble human insulin & IFN-a: not so quicklyThe rejection of the marketing authorization for biosimilar insulin and IFN-a products had very similar themes: differences in product quality, PK profile not equivalent to reference product and incomplete information on immunogenicity of biosimilar product.



Recombinant insulin is generally prescribed to diabetic patients to increase the uptake of g lucose by cells and reduce the concentration of glucose in the blood, thereby reducing long-term complications including damage to blood vessels, eyes, kidneys and nerves [41]. The rejec-tion of the marketing authorization application of a biosimilar insulin product, Marvel insulin (Humulin biosimilar) [42,137], by the EMA illus-trates the need for manufacturers to stringently address the criteria laid out in the guidance [108]. In this instance, the EMA cited several deficien-cies and inadequacies in the data package for the rejection: failure to perform a single-dose crossover, PK study via SC injection, as well as improper conduct of the PD euglycemic efficacy study in an unblinded manner in Type 1 diabetes patients [137]. In addition, even though the total AUC for glucose infusion rate for Marvel insulin was within 80–125% interval compared with the reference product Humulin S, the AUC 2 h following dosing was found to be significantly higher. The faster absorption and elimination of Marvel insulin compared with the reference product Humulin S resulted in a potential 45% greater glucose-lowering effect than Humulin S within the first hour after dosing, which was deemed highly clinically meaningful. Similarly, the AUC for the Marvel insulin long product was found to be 27% compared with Humulin I, but 23% higher for the mix product compared with Humulin M3, and the clearance of both these products could not be demonstrated within the study duration due to significant batch-to-batch variability. The CHMP also considered the immuno genicity assessments as well as the analytical methods used for the assessment to be inadequate. Even though the adverse event profile and immunogenicity rates in the first 24 weeks appeared to be similar in patients with Type 2 diabetes, there were significantly higher rates of adverse events and new antibody formation in patients with Type 1 diabetes. The CHMP also opined that immunogenicity data was not collected from treatment-naive patients and that the impact of antibodies on safety and efficacy was not presented. In addition, the pharmaco-vigilance system and the risk-management plan were not considered to address the requirements outlined in the EMA guidance. Based on the totality of evidence presented, the EMA rejected marketing authorization for Marvel insulin as a Humulin biosimilar. This case study exem-plifies not only the need to work closely with the agencies and seek appropriate consultation

throughout the development process, but also emphasizes the rigor that needs to be applied through the entire development process.

The biosimilar product Alpheon was being developed to treat adult patients with chronic (long-term) hepatitis C infection. Alpheon was expected to be used with the antiviral medicine, ribavirin. Besides physicochemical and biological similarity demonstration, the company studied the efficacy of Alpheon to reduce viral burden compared with the reference product Roferon-A in 455 patients with hepatitis C infection after 12 out of the 48 weeks of treatment and 6 months after stopping treatment.

The marketing authorization was rejected by the CHMP due to differences in the impurity profile between the biosimilar and reference product, lack of adequate stability data on the active substance, and inadequate validation of the fill/finish process [138]. Even though the num-ber of patients who responded to treatment was similar for the two products, more patients expe-rienced side effects and a return of disease after stopping treatment with Alpheon. In addition, the CHMP was also dissatisfied with the rigor in the immunogenicity assessment.

ConclusionSince the issuance of the earliest of the biosimilar guidance by the EMA there has been increas-ing urgency around the world to acknowledge the inevitability of biosimilar product launches. Many countries have developed and issued region-specific guidance to ensure patient safety while providing access to these life-saving medi-cines. The flexible regulatory framework has been built on the foundational belief that clini-cal meaningfulness and benefit–risk ratio are the key tenets for demonstration of similarity. The biosimilar development and marketing authori-zation experience to date illustrates the challenges in navigating this path. While several rG-CSF, epoetin and somatropin biosimilar products have gained marketing authorization, the EMA has rejected marketing authorization for biosimilar insulin and IFN-a and -b products due to defi-ciencies in study conduct, insufficient validation of assessment methods, different impurity profile to reference product, in addition to lack of com-parability in clinical data between the biosimilar and reference product [137,138]. A closer ana lysis of the EMA scientific assessment documents shows that data or study exceptions from the published guidance have been allowed in several cases of authorized biosimilar products based on the



totality of clinical data. The biosimilar filgrastim was given marketing authorization even though the total number of studies specified in the guid-ance was not conducted. Similarly, extrapolation of indications for somatropin and filgrastim was allowed, but not for epoetins based on benefit–risks. From a product characteristics perspective, Valtropin expressed from yeast was accepted as a biosimilar to Humatrope, a bacterial protein, and differences in isoform and glycosylation profile for erythropoietin biosimilars from the reference products was given less consideration based on insufficient information on clinical meaningfulness.

The CHMP and EMA have taken the lead, not only in providing a framework for biosimi-lar approvals, but also in providing marketing authorization for a number of protein biosimilars ranging from nonglycosylated to complex glyco-sylated proteins using an integrated step-wise approach. This early experience has set the stage for future biosimilar development. However, there are many challenging issues that remain to be addressed and standardized at a global level within the current framework: approach to estab-lishment of the upper and lower reference limits for demonstration of clinical similarity for the various product classes, number of product lots to be characterized and included in clinical trials, data required to bridge a foreign comparator to a US licensed product, clinical meaningfulness of difference in occurrence rates of adverse events, and immunogenicity rates, even if there is no immediately discernible clinical effect.

Future perspectiveTo date, biosimilar product approvals have shown the complexity associated with the pro-duction, characterization and evaluation of the similarity of these proteins. Even though the framework for biosimilar development has been broadly adopted by many countries, there are many open issues that require a thoughtful and deliberate approach. Expectations around the execution and follow-up period for pharmaco-vigilance studies, especially as it pertains to the development of a comprehensive understand-ing of the clinical impact of ADA, as well as the considerations around substitutability and extrapolation of indications, require further clarification. A cautious approach is warranted in authorizing extrapolation to additional indica-tions and to other populations, such as pediatric patients within the same indication, because of potential differences in expected toxicities and

the risk–benefit ratio, disease pathophysiology, and in comorbidities and concurrent therapies in the patient populations [141]. It will be prudent to restrict extrapolation considerations only to indications with shared molecular mechanism of action and well characterized and understood disease states.

Based on the therapeutic success achieved with targeted monoclonal antibody therapeu-tics, the future of the biosimilar landscape will likely include these structurally complex pro-teins. Based on the premise that pharmacologi-cally targeting a cell surface or soluble protein using mono clonal antibodies will ultimately result in similar therapeutic effects, the EMA issued a guidance for similar biological medici-nal products containing monoclonal antibodies in 2012 [114]. Due to the known limitations in the structure–activity relationship data and rec-ognizing the challenges associated with produc-tion, purification and charac terization of mono-clonal antibodies from different cell lines and manufacturing processes, the guidance places a lot of emphasis on the impact of manufactur-ing processes on product quality. The impact of qualitative and quantitative differences in product-related variants (post-translational modifications) on the biological function, and thereby the pharmacological activity of the prod-uct, has been identified as a critical parameter in the assessment. The speed of biosimilar product development is likely to trigger further advance-ments in analytical and product characterization tools in the future to enable better understand-ing of the contribution of product attributes to function and safety. Despite the justified need to enhance access and affordability of life-saving therapies, the development path for such prod-ucts cannot be overly abbreviated in the absence of convincing data on efficacy and long-term safety. A highly rigorous data-driven evaluation of the similarity of biosimilar to the reference product should continue to be advocated and maintained to avoid unanticipated risks in the future.

Financial & competing interests disclosureM Subramanyam is an employee of Biogen Idec and owns company stocks. The author has no other relevant a ffiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.



Executive summary

Background

�� Biosimilars, unlike generic small-molecule drugs, require a rigorous evaluation of both pharmaceutical and therapeutic equivalence to the reference product in an abbreviated clinical program.

�� The lack of adequate structure–activity relationship data for the majority of the protein reference products increases the burden on appropriately designed human clinical studies to demonstrate the absence of meaningful differences in efficacy and safety, including immunogenicity for the intended disease population.

�� Although a number of biosimilar therapeutic proteins have been approved in Europe and elsewhere, challenges on substitutability, extrapolation to other disease indications and use of reference standards sourced from other regions remains to be standardized at a global level.

Clinical study specifics

�� Equivalance of the biosimilar product to the reference product has to be demonstrated in three critical areas during clinical development of biosimilars, namely PK/PD, efficacy and safety, including short- and long-term immunogenicity.

�� For demonstration of similarity in a dose–response relationship, a PK/PD equivalence study in healthy volunteers with either a crossover design or parallel design based on the half-life of the therapeutic protein is recommended. Evaluation of typical parameters such as AUC, maximum observed plasma concentration (C

max) as well as plasma half-life (T

1/2), is recommended.

�� A double-blind, adequately powered, randomized, parallel-group efficacy study in the most sensitive patient population is required to demonstrate similarity in efficacy and safety, as well as to monitor immunogenicity. The design of this clinical study depends on the PK characteristics and pharmacological activity for each particular biologic. In addition, a comprehensive plan for long-term immunogenicity assessment and safety monitoring will need to developed and implemented.

Immunogenicity

�� Comprehensive monitoring (incidence, persistence, time to formation) of anti-drug antibodies to the biosimilar therapeutic in validated sensitive bioanalytical methods is a key requirement in biosimilar guidances issued to date.

�� The guidance generally recommend the collection of this information as part of the pivotal comparative efficacy study of prespecified duration, beyond which there is an expectation for data collection as part of the pharmacovigilance studies.

�� The experience with development of the various biosimilar proteins highlights the challenges in collecting the relevant information to assist with informed decision making with regard to immunogenicity and patient safety. Regulators have so far demonstrated considerable rigor in the ana lysis of immunogenicity data along with the totality of other clinical data in granting marketing authorization.

Future considerations

�� Biosimilar product approvals to date have shown the complexity associated with the production, characterization and clinical evaluation of the similarity of these proteins.

�� There are many open issues that require a thoughtful and deliberate approach, including the expectations around the execution and follow-up period for pharmacovigilance studies, substitutability and extrapolation of indications, and the sourcing of reference products from different jurisdictions.

�� Due to the known limitations in the structure–activity relationship data available for monoclonal antibody therapeutics, and potential impact of post-translational modifications and product and process variants on patient safety, a highly rigorous data-driven evaluation of the similarity of these complex biosimilar monoclonal antibody therapeutics to the reference product should be advocated.

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Clinical development of biosimilars: an evolving landscape