|Year : 2021 | Volume
| Issue : 1 | Page : 7-16
Biosimilars: An Update
Saravanan Bhojaraj1, Thirumoorthy Durai Ananda Kumar2, Abhinav Raj Ghosh1, BS Sushmitha1, Srinivasan Ramamurthy3, Thirunavukkarasu Velusamy4, Thiyagarajan Ramesh5, MK Jayanthi6, Musthafa Mohamed Essa7, Saravana Babu Chidambaram1, M. Walid Qoronfleh8
1 Department of Pharmacology, JSS College of Pharmacy, JSS Academy of Higher Education & Research, Mysuru-570015, Karnataka, India
2 Department of Pharmaceutical Chemistry, JSS College of Pharmacy, JSS Academy of Higher Education & Research, Mysuru-570015, Karnataka, India
3 College of Pharmacy & Health Sciences, University of Science and Technology of Fujairah, Fujairah, UAE
4 Department of Biotechnology, School of Biotechnology and Genetic Engineering, Bharathiar University, Coimbatore 641046, Tamil Nadu, India
5 Department of Basic Medical Sciences, College of Medicine, Prince Sattam Bin Abdulaziz University, Al-Kharj-11942, Kingdom of Saudi Arabia
6 Department of Pharmacology, JSS Medical College, JSS Academy of Higher Education & Research, Mysuru-570015, Karnataka, India
7 Department of Food Science and Nutrition, CAMS, Sultan Qaboos University, Muscat, Oman
8 Research and Policy Department, World Innovation Summit for Health (WISH), Foundation, P.O. Box 5825, Doha, Qatar
|Date of Submission||07-May-2020|
|Date of Decision||06-Oct-2020|
|Date of Acceptance||20-Oct-2020|
|Date of Web Publication||12-Feb-2021|
PhD, MBA M. Walid Qoronfleh
Research and Policy Department, World Innovation Summit for Health (WISH), Qatar Foundation, P.O. Box 5825, Doha
PhD, FASc(AW) Saravana Babu Chidambaram
Associate Professor, Department of Pharmacology, JSS College of Pharmacy, Mysuru, Karnataka 570015.
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Biologics are medicines primarily derived from living systems and produced through recombinant DNA (rDNA) and monoclonal technologies. Generic version of biologics with improved efficacy and safety is called biosimilar. Patent and copyright expiration of biological products permits the entry of biosimilars. Synthesis of biosimilars involves two main processes, such as monoclonal antibodies and rDNA technology, and characterized by various methods such as posttranslational modification, mass spectrometry, peptide mapping, three-dimensional (high-order) structure, X-ray crystallography, ion mobility spectrometry, and hydrogen deuterium exchange mass spectrometry. Though both generic and biosimilar products follow the same regulatory approval, the requirements are not the same due to the variability in composition and instability. Hence, it is essential to develop pharmacokinetic and pharmacodynamic data to support the efficacy and safety data on biosimilars. This review summarizes the recent updates on biosimilars, synthesis, characterization, and current market status. Brief information on the role of biosimilars in multiple sclerosis is also provided in the review.
Keywords: Biologics, biosimilars, monoclonal antibodies, multiple sclerosis, patent dance, recombinant DNA
|How to cite this article:|
Bhojaraj S, Ananda Kumar TD, Ghosh AR, Sushmitha B, Ramamurthy S, Velusamy T, Ramesh T, Jayanthi M, Essa MM, Chidambaram SB, Qoronfleh MW. Biosimilars: An Update. Int J Nutr Pharmacol Neurol Dis 2021;11:7-16
|How to cite this URL:|
Bhojaraj S, Ananda Kumar TD, Ghosh AR, Sushmitha B, Ramamurthy S, Velusamy T, Ramesh T, Jayanthi M, Essa MM, Chidambaram SB, Qoronfleh MW. Biosimilars: An Update. Int J Nutr Pharmacol Neurol Dis [serial online] 2021 [cited 2021 May 8];11:7-16. Available from: https://www.ijnpnd.com/text.asp?2021/11/1/7/309286
| Introduction|| |
Biologics are the complex large molecular weight compounds (4000–140,000 Da) used in the treatment of life-threatening ailments such as cancer, neurodegenerative diseases, immunological disorders, and so on. Biologics are different from conventional medicines, which are prepared from pure chemical compounds. Biologics are made from living sources like animals and microorganisms such as bacteria or yeast using recombinant DNA (rDNA) and cutting-edge cell culture techniques. Development of high-throughput techniques such as protein engineering and rDNA has revolutionized biologic production., Human insulin was the first biologics approved in the year 1982 and thereon many in the same class entered the market with appreciable therapeutic outcomes. Between 1995 and 2007, The United States Food and Drug Administration (USFDA) has approved 136 and European Union (EU) permitted 105 biological products. At present, 907 medicines and vaccines of biologics are developed by the US pharmaceutical companies. The turnover of biologics in the year 2008 was 30%, and in the year 2015 it constituted around 50% of the total pharmaceuticals. However, the manufacturing process for biologics is cumbersome and expensive. Average cost of a biologic is approximately 22 folds higher than the conventional medications. Many of the biologics are under patent and when they come off patent, it generates opportunities for generic versions. Owing to complicated manufacturing process and higher cost of biologics, the development of the cost-effective product called “Biosimilar” received a substantial attention, which is kind of a generic small molecule. Biosimilars are biologic origins that are structurally and functionally more similar to an approved biologic (reference product). Biosimilars may have a different structure from the reference product, but the active substances are same with almost no difference in safety and efficacy. Biosimilars have the same amino acid sequences, dose, and route of administration as those of a reference biological product. Biosimilars are cheaper by 20 to 30% as compared to reference product. Zarxio (filgrastim) is the first biosimilar of Neupogen approved by USFDA in 2015. More than 50 biosimilar products are currently under development. Biosimilar products approved by USFDA till date are listed in [Table 1].
Development of Biosimilars
Production of biosimilars involves several stages during development., It starts with an understanding and characterization of the reference product in terms of molecular structure and determines the posttranslational modifications (PTM) over a specific period of time or between batches. Structural and functional elements necessary for the clinical action should be within the acceptable limits as per the regulatory requirements. Second stage involves developing a suitable approach to manufacture the biosimilar molecule by various methods, and each step is optimized to ensure that the final product has similar molecular and functional properties as the reference product. Next, the preclinical properties such as toxicity, tolerability, and nonhuman pharmacology are compared with suitable in vitro and in vivo models. The clinical studies are then initiated with pharmacokinetics (PK) in Phase I trial, which usually involves testing on healthy volunteers to determine bioequivalence. The confirmatory studies are successively performed upon the completion of preliminary trials to identify the possible relevant differences between the biosimilar and the reference product, if any exist. Biosimilar products are similar and has no clinical difference from an existing USFDA approved reference product. These evidences obtained during the study may be represented during the justification of its potential for the indications claimed for reference product.
Biopharmaceuticals are frequently known to increase the concern over safety issues, such as immunotoxicity, which is one of the major reasons for the loss of efficacy and/or side effects. Immunosuppression, immunostimulation, hypersensitivity, and autoimmunity are the four different aspects of immunotoxicology. Clinical trial data indicate frequent incidences of adverse reactions such as immunosuppression, immunostimulation, and hypersensitivity (immunogenicity) with monoclonal antibodies (mAbs). From the immune safety point of view, mAbs intended to target a specific component of the immune system needs careful and adequate assessment procedures. Nonclinical immunotoxicity studies are insufficient to answer all safety concerns about the potential immunotoxicity of mAbs, and it is uncommon to predict the immunotoxicity in clinical studies with validated end points.,
Nocebo effects are the negative expectancies of a patient about the treatment that alter the physiological and neurobiological functions but are not the known pharmacological actions of therapy. Nocebo affects adherence to the medication, including biosimilars. For example, nocebo effects of infliximab and etanercept originator biologics have been identified in clinical biosimilar trials. Women, psychiatric patients, and patients with aggressive behavior (personality traits) are more susceptible to nocebo effects. This negative perception along with impact on adverse events leads to nonadherence to biosimilar therapy. Nocebo effects also pose a great difficulty on biosimilars administration in neurological disorders. Nocebo effects as a cause of discontinuation of few biosimilars is given in [Table 2].
Synthesis of Biosimilars
rDNA technology is a complex process used in medical research that involves injecting DNA fragments from a source with a suitable sequence of genes into a vector. Manipulation in the genome of an organism is achieved either by the introduction of one or more new genes and regulatory elements or by reducing or blocking the expression of endogenous genes by recombining genes and elements. Enzymatic cleavage is employed to obtain different DNA fragments using restriction endonuclease for particular target DNA sequence followed by DNA ligase action to connect the fragments to repair the desired gene in the vector. The vector is then injected into a host organism, which is grown to generate multiple copies of the incorporated DNA fragment in culture, and finally the selected and harvested clones contain the related DNA fragment. The products developed through rDNA technology exhibit better therapeutic activity and considered as the first generation of biopharmaceuticals., There are certain limitations associated with biotechnology-derived products since they are restricted to a few therapeutic proteins, for example, recombinant human proteins such as insulin, somatotropin, and so on, [Figure 1].
|Figure 1 Production of rDNA technology. First, the DNA of interest is identified and isolated. The identified DNA is inserted into a suitable host vector and transferred into the host cell. Cells are then grown in culture media and expanded, followed by production in bioreactors. Then, they are purified and characterized in the form of bulk drugs. rDNA, recombinant DNA|
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Hybridoma technology was first discovered by Köhler and Milstein in 1975 for predominantly production of mAbs. Hybridoma generation includes immunizing a certain species against a particular epitope of an antigen and collecting the B-lymphocytes from the host. B-lymphocytes are then fused with immortal cell lines of myeloma. These hybridoma cells are cultivated in vitro in suitable media where only the hybridomas survive because they have inherited immortality from the myeloma cells and the B-lymphocyte selective resistance. The initial hybridoma culture comprises a mixture of antibodies derived from several different primary B-lymphocyte clones, each of which secretes its own antibody into the culture medium. The cell culture medium is then screened for the individual antibody activities obtained from several hundreds of wells. Recently, mAbs have been identified as the benchmark for development of biosimilars. Development of biosimilar using mAbs is comparatively less cumbersome than biologic production, but due to their complexity in terms of structure and functions, it mandates intense focus on nonclinical and clinical studies, including immunogenicity potential [Figure 2].
|Figure 2 Production of monoclonal antibody. mAbs are produced by initially immunizing the mice and isolating the splenocytes. These when combined with myeloma cells by means of polyethylene glycol form hybridoma cells. Hybridoma cells undergo screening process followed by validation and successive characterization of optimal clones. They are further subjected to scale-up process and expansion for large-scale manufacturing. mAbs, monoclonal antibody|
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Characterization of Biosimilars
A biosimilar requires comparison with reference product and its market entry requires dedicated pharmacovigilance plan. Three properties of therapeutic proteins including biosimilars are very important for the expected efficacy and safety profile. They are PTMs, three-dimensional (3D) structures, and protein aggregations.
The biosynthetic pathway for the production of biological therapeutics including biosimilars through high-throughput techniques such as rDNA technology and protein engineering is very complex. Even small changes in the amino acid composition leads to the structural perturbation. PTM is the major cause for the disturbance in the high-order protein structure known as misfolding. Different types of PTMs include glycosylation, phosphorylation, lipidation, oxidation, disulfide bond formation, sulphation, and deamidation. These changes occur both at in vitro and in vivo stages. This is the major reason for the failure of peptides to function in their respective targets. PTM influences the immunogenicity of biological therapeutics and it is a serious concern. Hence, PTM characterization is very essential. Mass spectrometry based methods and peptide mapping are the major characterization tools available for PTM studies.
MS is a valuable tool for the investigation of the primary structure of peptides (amino acid sequence). The soft ionization techniques, namely, electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI), are the more suitable ionization techniques for protein analysis. MALDI-MS is suitable for the simple proteins and ESI-MS is for the complex proteins.
Glycosylation process depends on cell lines, expression hosts, and protocols. Batch-to-batch variation can be determined through glycosylation studies. The amino acid changes can be inferred from chromatographic patterns. Reverse phase chromatography, hydrophobic interaction chromatography, ion exchange chromatography, size exclusion chromatography (SEC), and hydrophilic interaction liquid chromatography are the most suitable chromatographic methods. The investigation on biosimilar of tissue-type plasminogen demonstrates ∼2.5-fold higher rate of glycosylation. Difference in the fucosylation site on the recombinant and human-derived protein is detected through LC-MS analysis. Aspartic acid readily isomerizes into isoaspartic acid (isoAsp), which triggers immunogenicity issues. MS coupled with electron transfer dissociation and electron capture dissociation techniques are more suitable for the detection of isoAsp.
Peptide mapping evaluates the identity, stability, and possible mutations in the biological products. This technique utilizes the trypsin digested peptides (endoproteinase digestion, proteolysis) or chemically modified peptides for the analysis. The peptide fragments are characterized through mass spectrometric analysis. The mass spectral ions are compared with database peptides for the conclusions. This investigation provides details about the peptide primary structure, disulfide nature, PTMs, and peptide impurities.,
Three-dimensional (high-order) structure
The impact of PTM on the integrity of 3D structure protein requires rigorous characterization. Hence, the 3D structural characterization (high order) of proteins is more important. The difference in the high order of protein results in altered biological functions, including immunological reactions. 3D structural (high-order) difference also provides the rationalization for the lack of comparability between biosimilar and reference product. The biophysical techniques, namely, X-ray crystallography, nuclear magnetic resonance (NMR), circular dichroism, isothermal calorimetry, SEC, MS, (hydrogen deuterium exchange mass spectrometry [HDX-MS)] method, and ion mobility spectrometry (IMS) are other major methods for the protein structure determination. But for various reasons, X-ray crystallography and NMR methods are more preferred.
- X-ray crystallography: It is practically difficult with molecules that resist crystallization.
- NMR: Protein dynamics in solution is an important attribute. The high-order protein structures are maintained by collective forces and are evaluated by NMR.
- HDX-MS method: This method has more potential in the determination of small changes in the high-order proteins, more of recombinant immunoglobin G1 mAbs.
- IMS: This method provides the conformation on the protein.
Protein aggregate is a major concern in biosimilar production. These aggregates of protein can be reversible or irreversible, which may cause immunological and toxicological reactions. This demands the detection of aggregation site in the protein. SEC, Raman spectrometry, and Fourier-transform infrared spectroscopy are useful in the detection of protein aggregation.
Current Status of Biosimilars in Clinical Trials
Clinical data is essential in the case of biosimilars and these products undergo three main phases, namely:
- Phase 1: Safety studies carried out on healthy volunteers
- Phase 2: Efficacy testing performed on specific subjects
- Phase 3: Comparative studies done on a large number of patients
The mechanisms of biosimilar products are determined from the identical primary structures of the reference products. Similarly, the efficacy and safety are determined in comparison with reference as well as other such analytical studies, which are considered relevant by clinicians., Several adverse effects are reduced by the development of structurally complex heterogeneous biologics such as fusion proteins and mAbs. According to European Crohn’s and Colitis Organization, “for cost savings, switching from an proven biologic to a biosimilar is likely to be as inappropriate and ineffective as switching between the existing biologics That act on the same target, except when there is lack of response”. Switching study provides useful data on safety and efficacy. Significant clinical success has been observed with biosimilar drugs, especially in the treatment of chronic inflammatory disorders [Figure 3].
|Figure 3 Method for process and clinical development. Clinical development is initiated by preliminary point of care (POC) toxicity trial along with investigational new drug toxicity trial, followed by Phases 1, 2, and 3 of clinical trials. Finally, biologics license application review is performed. Process development takes place simultaneously with preliminary studies of POC toxicity being performed and pre-IND process development of Phase 1 trial. This is then followed by pre-phase 2 and pre-phase 3 of the process development. The preapproval validation is carried out along with the biologics license application. IND, Investigational New Drug|
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Regulatory Requirements for Biosimilars
Globally, regulatory authorities are working to develop a common mechanism for the approval of biosimilars. European Medicines Agency was the first major regulatory body that developed main framework for approving biosimilars. Biosimilars approved in EU were later approved in Australia, Japan, and Latin America for therapeutic use. All the applications for the approval of a biosimilar undergo a rigorous evaluation to ensure efficacy, safety, and quality. The applicant should submit reliable and robust data on the safety and efficacy, including clinical trial data on disease indications for approval. Hence, the regulatory approval for the biosimilar is much difficult than small drug molecules. The qualification of a biosimilar involves the following steps.
Step 1: comparative quality studies
It involves testing of quality and heterogeneity of biosimilar. This includes both analytical and pharmacological studies.
- Internal comparison: Comparability studies for biosimilar products at pre- and post-process stages.
- External comparison: These studies prove that biosimilar is similar or highly similar to the biologics.
Step 2: comparative nonclinical studies
This step focuses on the evaluation of nonclinical pharmacodynamic (PD) aspects along with toxicity profiling of biosimilar products.
Step 3: comparative clinical studies
This phase evaluates the PK, PD, and safety aspects along with immunogenicity profile in patients. Immunogenicity studies in humans are mandatory for approval.
Step 4: risk management plans
Long-term efficacy and safety aspects along with registry are covered in this phase.
- Pharmacovigilance: It refers to postmarketing drug safety monitoring, which provides additional confirmation on the efficacy and safety.
Under the Biologic Price Competition and Innovation Act (BPCIA), an interchangeable biosimilar means a biosimilar product that produces equivalent clinical benefits as the reference product in any particular case. According to the USFDA, an interchangeable product can replace the reference product without the prescriber’s involvement. The USFDA approval requirements ensure the safety and efficacy of an interchangeable product, just as the approved reference product., Purple book provides the type of data and information needed to meet the standard for interchangeability.,
Upon submission of biosimilar application to the licensing regulatory body (e.g., USFDA), the process for settling any patent disputes is generally referred as “patent dance”. Critical phases of this procedure include the following: the biosimilar applicant should provide the reference product sponsor (RPS) with confidential access to the application and related production details within 20 days of the USFDA having approved the abbreviated application. Within 60 days, RPS should provide the list of valid and off patent for biosimilar license. The applicant also should provide patent list and response regarding license within 60 days. RPS should provide basis for infringement and response to the applicant within the same 60-day period. The parties then have up to 15 days to negotiate a list of patents, if any, that should be subject to a patent infringement action. Then, within next 30 days, RPS must bring an infringement action for each patent on the negotiated list, if the parties reach the agreement. If the parties fail to reach an agreement, the biosimilar applicant will inform the RPS on the number of patents to be provided in a second list and should immediately exchange a list of patents subject to infringement litigation within 5 days of the notice. RPS will bring an infringement action on all the patents on the simultaneously exchanged lists within 30 days of this exchange. The right to request a preliminary injunction is integrated into the law, as the BPCIA allows the biosimilar applicant to notify the RPS at least 180 days before the first commercial marketing [Figure 4].
|Figure 4 Patent dance. The diagram is the pictorial representation that involves timing, sequence requirements, and several rounds of information exchange between the reference product sponsor and the biosimilar applicant|
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Biosimilars for Multiple Sclerosis
Multiple sclerosis (MS) is the progressive autoimmune disorder of the nervous system that causes inflammation in the myelin sheath of neurons and impairs electrical conductivity or blocks nerve signals. More than 2.1 million people are affected globally with MS. Primary progressive MS, secondary progressive MS, relapsing-remitting MS (RRMS), and progressive relapsing MS are the four major types of MS. Increased expression of inflammatory proteins such as interleukin-17 (IL-17) and interferon-gamma (IFNγ) is recorded in MS. The affordability of the currently available biologics for the treatment of MS is a major concern.
IFN-β1a inhibits the proliferation of leukocytes and antigen presentation, cytokines production, and T-cell migration across the blood-brain barrier in MS. Several studies have shown that IFN-β1a reduces the formation of new lesions and controls brain atrophy., Hence, biosimilar products for IFN-β are under development for MS. CinnoVex is a biosimilar form of intramuscular IFN-β1a, manufactured by Cinna Gen Co. of Iran. Avonex, Betaseron, and Rebif are the three IFN-β used in the treatment of MS. A clinical trial (NCT04115488) is in progress to access the safety and efficacy of a biosimilar Natalizumab PB006 in RRMS. Our group is currently focusing on the development of a biosimilar for MS.
| Conclusion|| |
Biosimilars are products of biological origin with almost the same or equivalent safety and efficacy of a generic biologics, but are cost-effective. Biosimilars undergo nonclinical and clinical testing as that of reference products and interchangeable product can replace the reference product without the prescriber’s involvement. Development of rDNA and monoclonal antibodies technologies, robust methods on the characterization, safety, and efficacy including immunogenicity evaluation of biosimilars helps in clinical acceptance in a larger mean. Since biosimilars usually originate with reference biologics as the base molecule, there exists the controversy between the reference product sponsor and the new biosimilar applicant for licensing (patent dance), wherein the disputes are normally sorted by the associated legal process of the concerned country. Biosimilars are now widely accepted to treat various ailments such as cancer, autoimmune disorders including MS, and so on. Owing to the safety, specific action, and cost-effectiveness, undoubtedly it can be foreseen that biosimilars will replace many of the small molecules and impart greater impact on both patient benefits and health care sectors.
| Acknowledgement|| |
Authors sincerely acknowledge the management and authorities of JSS Academy of Higher Education & Research, Mysuru, for providing the required infrastructure for this review preparation and also for the full financial support to meet manuscript processing fee. The language and technical editing support provided by The Editing Refinery, MD, USA is highly acknowledged.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2]