Orthogonal Analytical Methods Research Articles

Understanding in-vivo refolding of antibody fragments (Fab): Biosimilar Ranibizumab a case study

In-vitro protein refolding is a major bottleneck step in the large-scale manufacturing of antibody fragments expressed in a microbial host system. The formation of an inter-chain disulfide bond is a key rate-limiting step during in-vitro refolding of antibody fragment therapeutics. In this investigation, we report the use of two redox mutant strains of E. coli viz., SHuffle® T7 (DE3) and SHuffle® T7 Express (DE3) possessing an oxidizing cytoplasm for the soluble expression of a refolded biosimilar antibody fragment. The effect of various critical process parameters on antibody fragment refolding yield was studied using a Design of Experiment (DoE) approach. The optimized upstream processing led to the expression of 167 ± 2.53 mg/L and 95 ± 1.44 mg/L of soluble refolded biosimilar Ranibizumab using the SHuffle® T7 (DE3) and SHuffle® T7 Express (DE3) strains, respectively, at the bioreactor scale. Physicochemical characteristics of the in-vivo refolded antibody fragment were studied and compared with an innovator molecule using various orthogonal analytical methods. A biological activity study of in-vivo refolded Ranibizumab using HUVEC cell-based bioassay proved that cell proliferation inhibition is comparable to the innovator Ranibizumab. The proposed strategy offers a time and cost-effective manufacturing platform for antibody fragments.

Studying Macromolecular Interactions of Cellular Machines by the Combined Use of Analytical Ultracentrifugation, Light Scattering, and Fluorescence Spectroscopy Methods.

Cellular machines formed by the interaction and assembly of macromolecules are essential in many processes of the living cell. These assemblies involve homo- and hetero-associations, including protein-protein, protein-DNA, protein-RNA, and protein-polysaccharide associations, most of which are reversible. This chapter describes the use of analytical ultracentrifugation, light scattering, and fluorescence-based methods, well-established biophysical techniques, to characterize interactions leading to the formation of macromolecular complexes and their modulation in response to specific or unspecific factors. We also illustrate, with several examples taken from studies on bacterial processes, the advantages of the combined use of subsets of these techniques as orthogonal analytical methods to analyze protein oligomerization and polymerization, interactions with ligands, hetero-associations involving membrane proteins, and protein-nucleic acid complexes.

Orthogonal Analytical Methods to Elucidate How Low Levels of Residual Crystallinity in Posaconazole Amorphous Solid Dispersions Impact Phase Behavior during Release

Orthogonal Analytical Methods to Elucidate How Low Levels of Residual Crystallinity in Posaconazole Amorphous Solid Dispersions Impact Phase Behavior during Release

Liquid chromatography coupled with tandem mass spectrometry for simultaneous quantification of valproate, valproate-glucuronide and lamotrigine in various biological matrices of rat.

Valproate and lamotrigine are commonly used as antiepileptic drugs even in pregnant and breastfeeding women. The extent and effects of drug exposure on the developing brain of the offspring are not well understood. Animal models can be utilised to investigate the transfer of substances into fetal brain with the ultimate aim of providing insights to aid clinical decisions. In the present study, an LC-MS/MS method was developed and validated for quantification of valproate (VPA), valproate-glucuronide (VPA-Gluc, a major metabolite of valproate) and lamotrigine (LTG) in rat blood plasma, cerebrospinal fluid and brain tissue. A 10 μl sample was spiked with stable isotope-labelled internal standards and extracted by methanol. An Agilent RRHD Eclipse Plus C18 column (2.1 × 100 mm, 1.8 μm) was used. The MS/MS transitions were 143.1016-143.1016 (VPA), 319.1392-143.0978 (VPA-Gluc) and 256.0157-210.9826 (LTG). The linear ranges of VPA, VPA-Gluc and LTG were 30-250, 10-140 and 0.3-1 μg/ml, respectively. The intra- and inter-day accuracy and precision, carryover, sensitivity and recovery were evaluated according to the US Food and Drug Administration guidance for bioanalytical method validation. Finally, the validated method was applied to a set of experimental animal samples and produced results highly comparable with those from an orthogonal analytical method.

Combined physicochemical and functional assessment of pertuzumab integrity supports extended in-use stability.

Pertuzumab (Perjeta®) is a monoclonal antibody approved for the treatment of HER2-positive breast cancer. Before treatment, the concentrate must be diluted to obtain the ready-to-use infusion solution. Data on the storage stabilities of these preparations arelacking but important for all healthcare professionals in the area of outpatient chemotherapy. The aim of this study was to investigate the storage stability of the ready-to-use infusion bags and the concentrates from once-opened vials over a period of up to 42 days. For a comprehensive and unambiguous assessment of pertuzumab's integrity, a panel of orthogonal analytical methods was employed, including a newly established mass spectrometry-based peptide mapping procedure along with a reporter gene assay for monitoring cellular bioactivity. The herein presented data showed that the ready-to-use infusion solutions stored at 4 ± 2°C and at 20 ± 3°C without light protection, as well as the undiluted Perjeta®concentrates stored at 4 ± 2°C,were physicochemically stable and biologically active for 28 days. These results might eventually allow for infusion preparations in advance, thus improving the quality of patient care as well as the economic usage of pertuzumab.

Equivalence study of extractables from single-use biopharmaceutical manufacturing equipment after X-ray or gamma irradiation

Single-use (SU) devices and assemblies used as manufacturing equipment in the biopharmaceutical industry require comprehensive qualifications. These qualifications include the assessment of compounds released from SU devices in contact with the process fluids, and how these leachable compounds potentially influence process performance, drug product quality, and patient safety. SU suppliers need to provide comprehensive qualification data for several parameters, for both new products and product changes, such as changes in the sterilization process applied to the SU device. The introduction of X-ray irradiation as an alternative to the currently used and established gamma irradiation of SU devices represents a situation where robust data is required to demonstrate equivalency between these two radiation technologies. Here, we present the results of a comprehensive extractables study for three SU components, bags, tubing, and sterilizing grade filters, evaluated after X-ray and gamma-ray irradiation. The selected study conditions were set up to allow a direct comparison of the results from the two sterilization methods, and to allow conclusions to be made on the impact of irradiation type on the polymers and their additives. Orthogonal analytical methods are applied to identify and quantify all organic compounds present. The data package provided here supports risk assessments for application of irradiated SU equipment in biopharmaceutical manufacturing. The formation of reaction products and the fundamental chemical pathways are discussed and found to be independent of the irradiation type. The results demonstrate the equivalency of both irradiation methods for extractables from plastic components used in pharmaceutical and biopharmaceutical manufacturing.

Metabolite variation and discrimination of five licorice (Glycyrrhiza) species: HPTLC and NMR explorations

The roots and rhizomes of several Glycyrrhiza species are widely used as sweetening and flavoring agents in food, as well as important ingredients in formulations of traditional medicines. Five Glycyrrhiza species, G. uralensis, G. glabra, G. inflata, G. echinata, and G. lepidota, often share the name "licorice roots" in the botanicals’ marketplace. Unfortunately, misidentification/mislabeling is very common due to their similarities in morpho-anatomical features. Significant metabolite alterations among the different Glycyrrhiza species and their hybrids have been reported, suggesting that the biological activities could vary with the licorice roots or products derived from different species. Development of simple, effective methods for species identification and differentiation is of key importance. In this study, 78 licorice samples were investigated using HPTLC and NMR as analytical tools. Significant metabolite variations were observed between the five species. The species-specific fingerprint patterns for the five Glycyrrhiza species were determined with HPTLC and NMR; then applied to the sample identification and discrimination. The results obtained from these two orthogonal analytical methods agreed with each other. Furthermore, the NMR signals and the species-specific constituents that made significant contributions to the differentiation of the five Glycyrrhiza species were confirmed based on the multivariate analysis of the NMR spectral data. Using the established OPLS-DA models, the classification of hybrids was evaluated and confirmed. The developed methods, particularly the HPTLC method with its simplicity and low cost, could be used as a rapid and reliable approach for the authentication of licorice species and quality control of licorice raw material and products.

Phase separation in surfactant-containing amorphous solid dispersions: Orthogonal analytical methods to probe the effects of surfactants on morphology and phase composition

Amorphous–amorphous phase separation (AAPS) is an important phase transition process for amorphous solid dispersion (ASD) performance both in terms of drug release as well as physical and chemical stability during storage. Addition of surfactants to ASD systems can impact both of these processes. One possible mechanism through which surfactants affect ASD performance is via their impact on AAPS. Unfortunately, despite their increasing usage in ASD formulations, the effect of surfactant on AAPS is still poorly understood, and there are limited analytical techniques that provide microstructural and composition information about phase separated ASDs. In this study, the impact of four surfactants (sodium dodecyl sulfate, Tween 80, Span 20 and Span 85) on water-induced phase separation in ASDs formulated with ritonavir and polyvinylpyrrolidone/vinyl acetate (PVPVA) was investigated using a variety of orthogonal analytical methods. Transparent films of ASDs with different compositions were prepared by spin coating. Fluorescence confocal microscopy in combination with an in situ humidity chamber was used to monitor the kinetics and morphology of phase separation following exposure to high relative humidity. Optical photothermal IR analysis of phase separated films enabled characterization of domain composition and surfactant distribution. Liquid-liquid phase separation concentration, zeta potential and solution nuclear magnetic resonance spectroscopy measurements enabled interpretation of interaction with and partition of surfactants into the drug-rich phase. It was found that phase separation kinetics and morphology were notably changed by the surfactants. Further, the surfactants showed different affinities for the drug-rich versus the aqueous/polymer-rich phases. The employed analytical techniques were found to be complementary in providing insight into surfactant location in phase separated systems. This study highlights the complexity of phase separation, especially in the presence of surfactants, and provides a foundation to understand the impact of AAPS on ASD performance.

Comprehensive Analytical Characterization of the Proposed Biosimilar Trastuzumab

Abstract: Background: Biosimilars to monoclonal antibodies were developed to increase the accessibility of this drug and reduce the costs of this therapy without compromising on its quality, safety, and efficacy. The objective of this research is to describe the orthogonal analytical methods used for the physicochemical and biological characterization of the biosimilar proposed to trastuzumab, which is the basis of the biosimilar development program. Methods: The orthogonal analytical methods for characterization of the proposed biosimilar to trastuzumab were segregated based on the quality attributes of the product, such as the primary structure was evaluated using peptide mapping and high-end mass spectrometry, secondary and tertiary structure was evaluated using circular dichroism, fluorescence, and absorbance spectroscopy, identity and size heterogeneity were evaluated using Western blotting, size exclusion chromatography and capillary electrophoresis, charge heterogeneity, glycan heterogeneity and hydrophobic heterogeneity were evaluated using by weak cation exchange liquid chromatography, hydrophilic interaction chromatography and reverse phase liquid chromatography, antigen binding and Fc gamma receptor binding was evaluated using surface plasmon resonance, flow cytometry and cell-based enzyme linked immunosorbent assay, antiproliferation and antibody dependent cellular cytotoxicity were evaluated using in vitro cell-based bioassay. Results and Conclusion: The results of a comprehensive analytical characterization of the proposed biosimilar to the innovator trastuzumab confirm that the physicochemical and biological properties of the proposed biosimilar were comparable to those of the innovator molecule trastuzumab. Key words: Therapeutic Monoclonal Antibody, Biosimilar, Critical Quality Attributes, Charge Variants, Aggregates, Fragments, Post-translational modifications, N-Linked Glycan and Antibody dependent cellular cytotoxicity.

Revisiting Reduction of CO2 to Oxalate with First-Row Transition Metals: Irreproducibility, Ambiguous Analysis, and Conflicting Reactivity.

Construction of higher C≥2 compounds from CO2 constitutes an attractive transformation inspired by nature’s strategy to build carbohydrates. However, controlled C–C bond formation from carbon dioxide using environmentally benign reductants remains a major challenge. In this respect, reductive dimerization of CO2 to oxalate represents an important model reaction enabling investigations on the mechanism of this simplest CO2 coupling reaction. Herein, we present common pitfalls encountered in CO2 reduction, especially its reductive coupling, based on established protocols for the conversion of CO2 into oxalate. Moreover, we provide an example to systematically assess these reactions. Based on our work, we highlight the importance of utilizing suitable orthogonal analytical methods and raise awareness of oxidative reactions that can likewise result in the formation of oxalate without incorporation of CO2. These results allow for the determination of key parameters, which can be used for tailoring of prospective catalytic systems and will promote the advancement of the entire field.

Analytical Techniques for Phytocannabinoid Profiling of Cannabis and Cannabis-Based Products-A Comprehensive Review.

Cannabis is gaining increasing attention due to the high pharmacological potential and updated legislation authorizing multiple uses. The development of time- and cost-efficient analytical methods is of crucial importance for phytocannabinoid profiling. This review aims to capture the versatility of analytical methods for phytocannabinoid profiling of cannabis and cannabis-based products in the past four decades (1980–2021). The thorough overview of more than 220 scientific papers reporting different analytical techniques for phytocannabinoid profiling points out their respective advantages and drawbacks in terms of their complexity, duration, selectivity, sensitivity and robustness for their specific application, along with the most widely used sample preparation strategies. In particular, chromatographic and spectroscopic methods, are presented and discussed. Acquired knowledge of phytocannabinoid profile became extremely relevant and further enhanced chemotaxonomic classification, cultivation set-ups examination, association of medical and adverse health effects with potency and/or interplay of certain phytocannabinoids and other active constituents, quality control (QC), and stability studies, as well as development and harmonization of global quality standards. Further improvement in phytocannabinoid profiling should be focused on untargeted analysis using orthogonal analytical methods, which, joined with cheminformatics approaches for compound identification and MSLs, would lead to the identification of a multitude of new phytocannabinoids.

Site-Specific Conjugation Quantitation of a Cysteine-Conjugated Antibody-Drug Conjugate Using Stable Isotope Labeling Peptide Mapping LC-MS/MS Analysis.

Drug-load (DL) characterization of antibody-drug conjugates (ADCs) is an important analytical task due to its designation as a critical quality attribute (CQA) affecting potency and stability. Intact and subunit liquid chromatography-mass spectrometry (LC-MS) analyses can determine global drug-to-antibody ratios (DARs) that correlate well with other orthogonal analytical methods; however, peptide mapping liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis has struggled to provide complementary site-specific quantitation of drug conjugation sites. The peptide mapping method described herein utilizes stable isotope labeling to accurately quantitate the site-specific conjugation levels of a cysteine-conjugated ADC to provide "bottom-up" DAR characterization in parallel with protein sequence and post-translational modification (PTM) characterization in one multi-attribute analytical method (MAM).

Development of a process for large scale production of PfRH5 in E. coli expression system

The Plasmodium falciparum reticulocyte binding protein homologue 5 (PfRH5) has recently shown great promise to be developed as a vaccine candidate to prevent blood-stage malaria. However, because of its molecular complexity, most previous efforts were focused on expressing PfRH5 in its native and soluble form. Here, we describe the E. coli expression of full-length PfRH5 as inclusion bodies (IBs), followed by its high cell density fermentation at 1, 5 and 30 L scale. Denatured full-length PfRH5 was purified using a two-step chromatography process before being refolded using design of experiments (DoE). Refolded PfRH5 was further purified using size exclusion chromatography (SEC), recovering high purity antigen with an overall yield of 102 mg/L from fermentation cell harvest. Purified PfRH5 was further characterized using orthogonal analytical methods, and a short-term stability study revealed −80 °C as an optimum storage temperature. Moreover, refolded, and purified PfRH5, when formulated with adjuvant Glucopyranosyl A lipid stable emulsion (GLA-SE), elicited high antibody titers in BALB/c mice, proving its potential to neutralize the blood-stage malarial parasite. Here, we establish an E. coli-based process platform for the large-scale cGMP production of full-length PfRH5, enabling global malaria vaccine development efforts.

Forced degradation of cell-based medicinal products guided by flow imaging microscopy: Explorative studies with Jurkat cells

Cell-based medicinal products (CBMPs) offer ground-breaking opportunities to treat diseases with limited or no therapeutic options. However, the intrinsic complexity of CBMPs results in great challenges with respect to analytical characterization and stability assessment. In our study, we submitted Jurkat cell suspensions to forced degradation studies mimicking conditions to which CBMPs might be exposed from procurement of cells to administration of the product. Flow imaging microscopy assisted by machine learning was applied for determination of cell viability and concentration, and quantification of debris particles. Additionally, orthogonal cell characterization techniques were used. Thawing of cells at 5 °C was detrimental to cell viability and resulted in high numbers of debris particles, in contrast to thawing at 37 °C or 20 °C which resulted in better stability. After freezing of cell suspensions at −18 °C in presence of dimethyl sulfoxide (DMSO), a DMSO concentration of 2.5% (v/v) showed low stabilizing properties, whereas 5% or 10% was protective. Horizontal shaking of cell suspensions did not affect cell viability, but led to a reduction in cell concentration. Fetal bovine serum (10% [v/v]) protected the cells during shaking. In conclusion, forced degradation studies with application of orthogonal analytical characterization methods allow for CBMP stability assessment and formulation screening.

A Comprehensive Workflow for the Analysis of Bio-Macromolecular Supplements: Case Study of 20 Whey Protein Products

The presence of bio-macromolecules as major ingredients is a primary factor in marketing many biologically derived macromolecular supplements. Workflows for analyzing these supplements for quality assurance, adulteration, and other supply-chain difficulties must include a qualitative assessment of small-molecule and macromolecular components; however, no such integrated protocol has been reported for these bio-macromolecular supplements. Twenty whey protein supplements were analyzed using an integrated workflow to identify protein content, protein adulteration, inorganic elemental content, and macromolecular and small-molecule profiles. Orthogonal analytical methods were employed, including NMR profiling, LC-DAD-QToF analysis of small-molecule components, ICP-MS analysis of inorganic elements, determination of total protein content by a Bradford assay, SDS-PAGE protein profiling, and bottom-up shotgun proteomic analysis using LC-MS-MS. All 20 supplements showed a reduced protein content compared to the claimed content but no evidence of adulteration with protein from an unclaimed source. Many supplements included unlabeled small-molecule additives (but nontoxic) and significant deviations in metal content, highlighting the importance of both macromolecular and small-molecule analysis in the comprehensive profiling of macromolecular supplements. An orthogonal, integrated workflow allowed the detection of crucial product characteristics that would have remained unidentified using traditional workflows involving either analysis of small-molecule nutritional supplements or protein analysis.

Part 2: Physicochemical characterization of bevacizumab in 2 mg/mL antibody solutions as used in human i.v. administration: Comparison of originator with a biosimilar candidate

The physicochemical properties of Avastin® manufactured in the USA (Originator USA) and in Europe (Originator EU) and ABX-BEV, a bevacizumab biosimilar drug product candidate produced by Apobiologix Inc., were characterized at a clinically relevant concentration of 2 mg/mL following dilution of the 25 mg/mL drug products with 0.9% NaCl. Measurements using 14 orthogonal analytical methods performed within 5 h after dilution showed good similarity of the three antibodies as regards secondary structure, conformation, aggregation properties, subvisible and visible particles. No significant protein aggregation was observed within 5 h at 24 °C in the 2 mg/mL bevacizumab solutions. The same solutions that were measured within 5 h after dilution were analyzed again after 24 h overnight refrigeration at 2–8 °C: all 2 mg/mL bevacizumab solutions showed an increase in the number and sizes of aggregates, especially the particles larger than 10 μm and 25 μm. The data show the very good similarity in the physicochemical properties of ABX-BEV with Originators USA and EU at a clinically relevant concentration.

Cyanobacterial neurotoxin BMAA and brain pathology in stranded dolphins.

Dolphin stranding events occur frequently in Florida and Massachusetts. Dolphins are an excellent sentinel species for toxin exposures in the marine environment. In this report we examine whether cyanobacterial neurotoxin, β-methylamino-L-alanine (BMAA), is present in stranded dolphins. BMAA has been shown to bioaccumulate in the marine food web, including in the muscles and fins of sharks. Dietary exposure to BMAA is associated with the occurrence of neurofibrillary tangles and β-amyloid plaques in nonhuman primates. The findings of protein-bound BMAA in brain tissues from patients with Alzheimer’s disease has advanced the hypothesis that BMAA may be linked to dementia. Since dolphins are apex predators and consume prey containing high amounts of BMAA, we examined necropsy specimens to determine if dietary and environmental exposures may result in the accumulation of BMAA in the brains of dolphins. To test this hypothesis, we measured BMAA in a series of brains collected from dolphins stranded in Florida and Massachusetts using two orthogonal analytical methods: 1) high performance liquid chromatography, and 2) ultra-performance liquid chromatography with tandem mass spectrometry. We detected high levels of BMAA (20–748 μg/g) in the brains of 13 of 14 dolphins. To correlate neuropathological changes with toxin exposure, gross and microscopic examinations were performed on cortical brain regions responsible for acoustico-motor navigation. We observed increased numbers of β-amyloid+ plaques and dystrophic neurites in the auditory cortex compared to the visual cortex and brainstem. The presence of BMAA and neuropathological changes in the stranded dolphin brain may help to further our understanding of cyanotoxin exposure and its potential impact on human health.

Variance Between Different Light Obscuration and Flow Imaging Microscopy Instruments and the Impact of Instrument Calibration

Subvisible particles (SVPs) are an obligatory critical quality attribute of the product, and yet, they are found in all biopharmaceutical products intended for infusion or injection. Light obscuration (LO) is the primary pharmacopeial method used to quantify SVPs. However, the method may not be equally sensitive toward all particles that can possibly occur. Calibration of LO instruments is usually performed using polystyrene beads suspended in water. In this study, the dependence of the sizing accuracy of LO analysis was evaluated by using a calibration suspension of lower refractive index beads made of silica suspended in sucrose solution. It was demonstrated that the sizing accuracy was strongly dependent on the reference material’s properties used for calibration. It was also demonstrated that flow imaging microscopy suffered from the same artifact, albeit to a smaller extent. We further tested different LO sensors and instruments. Interestingly, our results show that the sizing accuracy varied from instrument to instrument, strongly depending on the properties of the sensor.To summarize, sizing and counting accuracies were dependent on the material used for calibration and its optical properties as well as the calibration curve, the sensor, and the instrument supplier. Closer match of optical properties between calibration system and test system seems to improve the sensitivity of the measurement. The results of this study raise the following major practical implications: (1) LO and flow imaging microscopy are not truly orthogonal analytical methods, (2) while matching optimal properties of material used for calibration and test items increased sensitivity, this is of poor practical applicability given that analytes contain multiple particles, and (3) setting product-specific limits for SVPs require special considerations with regard to the data sets used.

Chemical Structure and Composition of Major Glycans Covalently Linked to Therapeutic Monoclonal Antibodies by Middle-Down Nuclear Magnetic Resonance.

Glycosylation of monoclonal antibodies (mAbs) is a critical quality attribute that can impact mAb drug efficacy and safety. The mAb glycans are inherently heterogeneous in chemical structure and composition of monosaccharides. The established fluorescence or mass-spectrometry (MS) detection methods for glycosylation evaluation may require multiple steps of glycan cleavage or extensive digestion of the mAb, chemical labeling of the glycans, column separation and report the chemical identity of glycans indirectly through retention time and molecular weight values. In demonstrating chemical structure similarity and comparability among mAb drugs, orthogonal analytical methods for measuring glycan chemistry are needed to ensure the quality of drug products. Here, a "middle-down" NMR method is developed as a proof-of-concept approach to measure the domain-specific glycosylation of marketed mAb drugs without cleavage of the glycan moieties. Complete glycan 1H/13C chemical shift assignments were obtained at 13C natural abundance from commercial standard glycans that allowed unambiguous determination of the chemical structure, glycosidic linkage position, and anomeric configuration of each monosaccharide in the major N-glycan scaffolds found in mAb molecules. The analysis of glycan anomeric peaks in two-dimensional (2D) 1H-13C NMR spectra yielded metrics for clinically important mAb quality attributes (i.e., galactosylation (Gal%) and fucosylation (Fuc%)), consistent with literature results using a standard glycan-mapping method. Therefore, the middle-down NMR method provided a facile orthogonal measurement for mAb glycosylation characterization with improved chemical information content on glycan structure determination and quantification, compared to standard approaches.

The End of Phase 3 Clinical Trials in Biosimilars Development?

Most patients still have limited or no access to life-changing therapeutic proteins in the treatment of their cancer or autoimmune disorders. The current clinical development model of biosimilars is expensive, and in most cases, large, phase 3 trials do not provide meaningful information on the clinical equivalence of biosimilars and reference compounds. At the same time, the development of state-of-the-art orthogonal analytical methods has enabled a better understanding of the structure and structure-function relationship of biotherapeutics. Hence, we suggest here that a solid chemistry, manufacturing, and controls (CMC) package and meaningful phase 1 studies will leave limited uncertainty on biosimilarity, which can be addressed-if needed-by post-approval, long-term follow-up studies (post-approval studies, pharmacovigilance, real world evidence data and registries, and possibly new post-approval models to be developed). We believe that this new approach may be more appropriate than 600- to 1000-patient, phase 3 trials in assessing biosimilarity and therapeutic equivalence, under the condition that the administered biosimilar given to individual patients can beclearly identified. Obviously, there will probably never be a "one size fits all" development model, and an individualized, risk-based approach to biosimilar development will always have to be considered and discussed early with regulators.