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Background: Pegfilgrastim is a long-acting recombinant human granulocyte colony-stimulating factor biologic that is indicated to reduce the incidence of infections, manifested by febrile neutropenia, in patients receiving myelosuppressive anti-cancer drugs and to increase survival in patients acutely exposed to myelosuppressive doses of radiation. Due to the high cost of biologic therapy and the scarcity of biosimilar alternatives, there is an unmet medical need for targeted biologics., Objective: This comparative analytical investigation aimed to confirm the similarity of biosimilar Stimufend® (pegfilgrastim-fpgk) to reference product Neulasta® (pegfilgrastim)., Methods: The analysis was designed using state-of-the-art orthogonal techniques and side-by-side testing to compare the physicochemical and biological properties of these two products. The measured quality attributes included the primary structure and higher order structure of the molecule, purity/impurity profiles, product variants, process-related impurities, composition, content, and biological activity. The statistical analysis was based on risk ranking of the critical quality attributes (very low, low, moderate, high, very high), and scientific considerations in combination with the characteristics of the assay (sensitivity, selectivity, and variability). In addition, non-quantitative parameters were compared using a descriptive assessment of the product profile. Analytical similarity was concluded by quality attributes falling within the defined range of the originator product., Results: The results of this study confirm that Stimufend® is biosimilar to Neulasta® for all measured quality attributes. There are no clinically significant differences between Stimufend® and Neulasta®, which was confirmed by the marketing approval for Stimufend® by the Food and Drug Administration and the European Medicines Agency., Conclusion: The findings of this study provide robust evidence supporting the structural and functional biosimilarity between Stimufend® and Neulasta®., Competing Interests: The authors have read the journal’s policy and have the following competing interests: all authors are employees of Fresenius Kabi SwissBioSim. This does not alter our adherence to PLOS ONE policies on sharing data and materials. There are no patents, products in development or marketed products associated with this research to declare., (Copyright: © 2024 Sykes et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Glycan profile comparisons are one of the most tedious analytical exercises for establishing compliance with recombinant therapeutic protein batches. Based on its intensive research, the FDA has confirmed that lectin array binding with fluorescent monitoring is the fastest and most reliable method for profile comparisons. Using a database of over 150 biological products expressed in nine diverse mammalian cell systems, the FDA immobilized 74 lectins to study their binding using fluorescently labeled glycoproteins. The FDA identified nine distinct lectins from a custom-designed lectin microarray: rPhoSL, rOTH3, RCA120, rMan2, MAL_I, rPSL1a, PHAE, rMOA, and PHALs, which detect core fucose, terminal GlcNAc, terminal β-galactose, high mannose, α-2,3-linked sialic acids, α-2,6-linked sialic acids, bisecting GlcNAc, terminal α-galactose, and triantennary structures, respectively. This method can be used for screening and routine testing and to monitor batch-to-batch variability of therapeutic proteins, including establishing analytical similarity as a crucial part of biosimilar development.

Background: Pegfilgrastim-cbqv/CHS-1701 (UDENYCA ® ) (hereafter referred to as pegfilgrastim-cbqv) was approved in 2018 by the US Food and Drug Administration as a biosimilar for pegfilgrastim (Neulasta ® ) (hereafter referred to as pegfilgrastim). Both pegfilgrastim-cbqv and pegfilgrastim are conjugates of recombinant human granulocyte colony stimulating factor (r-metHuG-CSF) with a 20 kDa polyethylene glycol (PEG) indicated to decrease the incidence of infection, as manifested by febrile neutropenia, in patients receiving myelosuppressive anticancer drugs. The demonstration of analytical similarity for PEG-protein conjugates presents unique challenges since both the protein and PEG attributes must be characterized., Objective: The current study demonstrates the analytical similarity of pegfilgrastim-cbqv and the reference product, pegfilgrastim. In addition to the physicochemical and functional characterization of the protein, the study assessed attributes specific to PEGylation including PEG size and polydispersity, site of attachment, linker composition, and PEGylation process-related variants., Methods: The structural, functional, and stability attributes of pegfilgrastim-cbqv and pegfilgrastim were compared using state-of-the-art analytical methods. For the protein, the primary structure, disulfide structure, and secondary and tertiary structures were assessed using traditional protein characterization techniques such as mass spectrometry (MS), circular dichroism (CD), intrinsic fluorescence, and differential scanning calorimetry (DSC), as well as more advanced techniques such as two-dimensional (2D) nuclear magnetic resonance (NMR) and hydrogen deuterium exchange (HDX). For the PEG moiety, the site of attachment, occupancy, linker composition, size and polydispersity were compared using mass spectrometry (both intact and after endoprotease digestion), multiangle light scattering detection (MALS), and Edman degradation. Purity assessments included the assessment of both protein variants and PEGylation variants using chromatographic and electrophoretic analytical separation techniques. The functional similarity between pegfilgrastim-cbqv and pegfilgrastim was compared using both a cell-based bioassay and surface plasmon resonance (SPR). The degradation rates and stability profiles were compared under accelerated and stressed conditions., Results: Biosimilarity was demonstrated by a thorough assessment of physiochemical and functional attributes, as well as comparative stability, of pegfilgrastim-cbqv relative to pegfilgrastim. These studies demonstrated identical primary structure and disulfide structure, highly similar secondary and tertiary structure, as well as functional similarity. The impurity profile of pegfilgrastim-cbqv was comparable to that of pegfilgrastim with only minor differences in PEGylation variants and a slight offset in the PEG molar mass. These differences were not clinically relevant. The degradation profiles were qualitatively and quantitatively similar under accelerated and stress conditions., Conclusion: The structural, functional, and stability data demonstrate that pegfilgrastim-cbqv is highly similar to the reference product, pegfilgrastim., (© 2024. The Author(s).)

Background and Objective: ABP 654 is a proposed biosimilar to ustekinumab reference product (RP), a human immunoglobulin isotype class G subclass 1 kappa monoclonal antibody that acts as an antagonist of interleukin (IL)-23 and IL-12. Ustekinumab RP is indicated for the treatment of some forms of plaque psoriasis, active psoriatic arthritis, Crohn's disease, and ulcerative colitis. ABP 654 and ustekinumab RP utilize different expression systems, and the purpose of this study was to assess analytical similarity between ABP 654 and ustekinumab RP sourced from the United States (US) and the European Union (EU)., Methods: The analytical testing plan included general properties, primary structure, higher-order structure, product-related substances and impurities, particles and aggregates, biological activity, and thermal stability and degradation studies., Results: ABP 654 was found to be analytically similar to ustekinumab RP with respect to physicochemical and biological properties, including structure, function, purity, and potency., Conclusions: Based on a comprehensive similarity assessment, ABP 654 was found to be similar to ustekinumab RP, notwithstanding minor physicochemical differences that are not expected to have a clinically meaningful effect on safety or efficacy., (© 2023. The Author(s).)

Introduction: SB12 is a biosimilar to eculizumab reference product [Soliris TM (Soliris is a trademark of Alexion Pharmaceuticals, Inc.)] that acts as a C5 complement protein inhibitor. The infusion stability of in-use (diluted) SB12 outside the conditions stated in the reference product's label is unknown., Objective: The objective of this study was to assess the stability of SB12 after extended storage in conditions not claimed in the originator label., Methods: Infusion stability was assessed in SB12 samples (diluted in 0.9% NaCl, 0.45% NaCl, and 5% dextrose, final concentration of 5 mg/mL per clinical trial protocol and the reference product's label) kept at 5 ± 3 °C for up to 3 months, then 30 ± 2 °C/65 ± 5% relative humidity (RH) for 72 h. The product was stored in different containers [polyolefin (PO) bags, glass bottles and syringes], and the protocol followed International Conference on Harmonisation (ICH) and European Medicines Agency (EMA) requirements for stability evaluation of biological products. Stability was evaluated using complementary assays, including pH, protein concentration (A 280 ), purity (size exclusion-high-performance liquid chromatography, capillary electrophoresis-sodium dodecyl sulfate, and imaged capillary isoelectric focusing), biological activity (C5 binding and inhibition), and safety (subvisible particles)., Results: Except for charge variants in SB12 diluted in 5% dextrose, all results met the stability acceptance criteria. There were no major changes in terms of physicochemical stability, biological activity, and subvisible particles., Conclusions: The infusion stability of SB12 after extended storage (5 ± 3 °C for up to 3 months, then 30 ± 2 °C/65 ± 5% RH for 72 h) was demonstrated for longer periods and at higher temperatures than what is stated in the EU and US labels of the reference product. The physicochemical properties, biological activity, and subvisible particles of in-use SB12 diluted in 0.9% NaCl and 0.45% NaCl were maintained under the described conditions and for all tested containers. However, instability was observed for the diluted SB12 in 5% dextrose. These results may reduce the workload of clinical staff and minimize drug waste from treatment delays without any loss in product quality and biological activity., (© 2023. The Author(s).)

Background: Adalimumab-aqvh/CHS-1420 (YUSIMRY TM ) (hereafter referred to as adalimumab-aqvh) was recently approved by the US Food and Drug Administration as a biosimilar for adalimumab., Objective: The current study was conducted to investigate the analytical similarity of adalimumab-aqvh and the reference product, adalimumab., Methods: The structural, functional, and stability attributes of adalimumab-aqvh and adalimumab were compared using state-of-the-art assays. The primary structure, disulfide structure, glycan profile, secondary and tertiary structures, molar mass, size variants, free thiol, charge variants, hydrophobic variants, post-translational modifications, subvisible particles, host cell proteins, and protein concentration were investigated. The functional similarity between adalimumab-aqvh and adalimumab was demonstrated by comparing fragment antigen-binding (Fab)-associated and fragment crystallizable (Fc)-associated biological activities. The stability of adalimumab-aqvh and of adalimumab was compared through forced degradation., Results: The structural attributes of adalimumab-aqvh were identical to those of adalimumab or met the similarity criteria, with a few exceptions. Adalimumab-aqvh and adalimumab exhibited comparable stability profiles and functional activities. Any observed differences in the physiochemical attributes did not impact the conclusion of similarity because they did not influence any functional activities related to the adalimumab mechanism of action., Conclusion: The structural, functional, and stability data provide convincing evidence of biosimilarity between adalimumab-aqvh and the reference product, adalimumab., (© 2023. The Author(s).)

Therapeutical monoclonal antibodies are structurally and functionally complex, whereas the innovator's manufacturing processes are proprietary. With respect to the similarity assessment, a proposed biosimilar product needs to demonstrate a side-by-side comparison between the reference product (RP) and candidate product in terms of physicochemical properties and biological activities, as well as nonclinical and clinical outcomes. Here, a comprehensive analytical similarity assessment was performed for in-depth comparison of HLX04, China-sourced Avastin ® (CN-Avastin ® ), and Europe-sourced Avastin ® (EU-Avastin ® ) following a tier-based quality attribute (QA) evaluation. A series of orthogonal and state-of-the-art analytical techniques were developed for the assessment. Ten lots of HLX04 were compared with 29 lots bevacizumab RP. Referred to the characterization results, HLX04 is highly similar to the RPs with respect to physicochemical properties and biological functions. In addition, HLX04 was found with similar stability and degradation behaviors upon multiple stressed conditions to bevacizumab. Minor differences were observed in glycosylation, aggregates, FcγRIIIa(F), and FcγRIIIa(V) binding activities; nevertheless, they were evaluated and demonstrated not to impact clinical outcomes. According to the reported clinical results, the totality of evidence, including the pharmacokinetic, efficacy, safety, and immunogenicity, further shows that HLX04 is similar to CN-/EU-Avastin ® ., (© 2023. Springer-Verlag GmbH Germany, part of Springer Nature.)

Background: Post-translational modifications in bioprocessing and storage of recombinant mAbs are the main sources of charge variants. While the profile of these kinds of variants is considered an important attribute for the therapeutic mAbs, there is controversy about their direct role in safety and efficacy. In this study, the physicochemical and pharmacokinetic (PK) properties of the separated charge variants belonging to a trastuzumab potential biosimilar, were examined., Methods: The acidic peaks, basic peaks, and main variants of trastuzumab were separated and enriched by semi-preparative weak cation exchange. A panel of analytical techniques was utilized to characterize the physicochemical properties of these variants. The binding affinity to HER2 and FcγRs and the PK parameters were evaluated for each variant., Results: Based on the results, the charge variants of the proposed biosimilar had no significant influence on the examined efficacy and PK parameters., Conclusion: During the development and production of biosimilar monoclonal antibodies, evaluating the effect of their charge variants on efficacy and PK parameters is needed.

Background: PF-06439535 (bevacizumab-bvzr; Zirabev ® ) is a biosimilar of bevacizumab reference product (RP; Avastin ® ). This study describes the formulation development for PF-06439535., Methods: PF-06439535 was formulated in several buffers and stored for 12 weeks at 40 °C to determine the optimal buffer and pH under stressed conditions. Subsequently, PF-06439535 at 100 and 25 mg/mL was formulated in a succinate buffer with sucrose, edetate disodium dihydrate (EDTA), and polysorbate 80, and in the RP formulation. Samples were stored at - 40 °C to 40 °C for ≤ 22 weeks. The physicochemical and biological properties relevant to the safety, efficacy, quality, or manufacturability were investigated., Results: When stored at 40 °C for 13 days, PF-06439535 demonstrated optimal stability in histidine or succinate buffers and was more stable in the succinate formulation than the RP formulation, under both real-time and accelerated stability conditions. There were no significant changes in the quality attributes of 100 mg/mL PF-06439535 after storage at - 20 °C and - 40 °C for 22 weeks, and there were no changes in the quality attributes of 25 mg/mL PF-06439535 after storage at 5 °C (recommended storage temperature). Changes were observed at 25 °C for 22 weeks or at 40 °C for 8 weeks as expected. No new degraded species were observed in the biosimilar succinate formulation compared with the RP formulation., Conclusions: Results demonstrated that 20 mM succinate buffer (pH 5.5) is the PF-06439535 preferred formulation, and that sucrose is an effective cryoprotectant for processing and frozen storage, and an effective stabilizing excipient for 5 °C liquid storage of PF-06439535., (© 2023. The Author(s).)

Monoclonal antibodies (mAbs) represent a dynamic class of biopharmaceutical products, as evidenced by an increasing number of market authorizations for mAb innovator and biosimilar products. Stability studies are commonly performed during product development, for instance, to exclude unstable molecules, optimize the formulation or determine the storage limit. Such studies are time-consuming, especially for mAbs, because of their structural complexity which requires multiple analytical techniques to achieve a detailed characterization. We report the implementation of a novel methodology based on the accelerated stability assessment program (ASAP) in order to model the long-term stability of mAbs in relation to different structural aspects. Stability studies of innovator infliximab and two different biosimilars were performed using forced degradation conditions alongside in-use administration conditions in order to investigate their similarity regarding stability. Thus, characterization of post-translational modifications was achieved using liquid-chromatography-tandem mass spectrometry (LC-MS/MS) analysis, and the formation of aggregates and free chain fragments was characterized using size-exclusion chromatography-multi-angle light scattering (SEC-MALS-UV/RI) analysis. Consequently, ASAP models were investigated with regard to free chain fragmentation of mAbs concomitantly with N57 deamidation, located in the hypervariable region. Comparison of ASAP models and the long-term stability data from samples stored in intravenous bags demonstrated a relevant correlation, indicating the stability of the mAbs. The developed methodology highlighted the particularities of ASAP modeling for mAbs and demonstrated the possibility to independently consider the different types of degradation pathways in order to provide accurate and appropriate prediction of the long-term stability of this type of biomolecule., (© 2022. Springer-Verlag GmbH Germany, part of Springer Nature.)

Epidermal growth factor receptor (EGFR) is the primary target for the treatment of colorectal cancer, the third most diagnosed cancer worldwide. In recent years, regulatory changes have facilitated the approval of biosimilars aimed to bring more access to biologics to patients. However, it has also expended the requirements of non-clinical characterisation data using state-of-the-art and orthogonal methodologies to demonstrate similarity between proposed biologic and its reference medicinal product (RMP). The current study was aimed to develop a stable CHO-S cell line producing panitumumab biosimilar candidate, P-mAb, a fully human IgG2 anti-EGFR monoclonal antibody and assess its physicochemical and functional similarity with RMP, Vectibix. The single-cell clone from stably transfected CHO-S cell pools was used for the production of P-mAb. This was followed by purification and comparative physicochemical and biological characterisation of P-mAb and RMP using SDS-PAGE, LC/MS, MALDI, MS/MS, CD spectrometry, DSF, SAXS, ITF, MTT assay and binding affinity. SAXS and MST assays are being used for first time in biosimilarity analysis of therapeutic monoclonal antibody. The results of structural and functional analysis of anti-EGFR P-mAb, produced by stable CHO-S cell line revealed high similarity between P-mAb and RMP, vectibix, thus providing the scientific basis of its potential for therapeutic applications.

Omalizumab is an anti-IgE monoclonal antibody (mAb) approved for the treatment of moderate-to-severe asthma. Herein, we report physicochemical, biological, pharmacological, and toxicological characteristics of an Omalizumab biosimilar mAb named KA. We show that KA and its originator present only minimum differences. Their charge heterogeneity and primary, secondary structures are similar. The two molecules are comparable regarding in vitro activity, including molecular binding and cell-based inhibition. Pharmacological and toxicological properties were assessed using a mouse model of allergy and cynomolgus monkeys, and we determined that the efficacy, safety, and pharmacokinetic characteristics of KA are comparable to its originator. Our data, which demonstrated that KA has similar activity to the Omalizumab reference product in relevant preclinical models, calls for a clinical evaluation of its bio-similarity., Competing Interests: Declaration of Competing Interest The authors declare that they do not have any conflicts of interest regarding the publication of this paper., (Copyright © 2022. Published by Elsevier B.V.)

Due to the complex manufacturing process of therapeutic monoclonal antibodies, it is hardly possible to produce an identical copy of the original product (originator). Consequently, follow-on products (biosimilars) must demonstrate their efficacy being similar to the originator in terms of structure and function. During this process, a variety of analytical methods are required for this purpose. This study focuses on three particularly relevant analytical techniques: high-resolution mass spectrometry, fragment crystallisable (Fc) affinity chromatography, and two-dimensional peptide mapping. Each analytical method proved able to identify specific differences between originator and biosimilar. High-resolution mass spectrometry was used to characterize the glycan pattern. It was shown that a trastuzumab biosimilar did not have the G0:G0F sugar modification identified in the originator. The application of affinity chromatography to rituximab showed that originator and biosimilar interacted differently with the immobilized Fc receptor. Furthermore, 2D-HPLC peptide mapping demonstrated the influence of orthogonality of separation dimensions, leading to differentiation of a rituximab originator and biosimilar., (© 2022. Springer-Verlag GmbH Germany, part of Springer Nature.)

Aim: The purpose of this study was to evaluate the extended physicochemical and biological stability of Sandoz Rixathon®/Riximyo® (SDZ-RTX) after exposure to out-of-fridge (OOF) conditions., Materials and Methods: The impact of the short-term temperature excursion on stability parameters of SDZ-RTX was simulated by subsequently exposing the three batches of SDZ-RTX (100 and 500 mg) to OOF conditions, (I) 25 ± 2 °C/60 ± 5% relative humidity (RH) and (II) 30 ± 2 °C/65 ± 5% RH, for up to 21 days after more than the claimed 36-month shelf-life storage in long-term conditions (5 ± 3 °C). Analytical methods used included the cation exchange chromatography (CEX), size exclusion chromatography (SEC), and non-reducing capillary electrophoresis-sodium dodecyl sulfate (nrCE-SDS), as well as biological activity by complement-dependent cytotoxicity (CDC)-bioactivity as well as further methods, for example, related to identity and pharmacopoeia test methods., Results: No notable changes were observed across all batches with respect to identity (charge and primary structure), pharmaceutical tests (clarity, visible and subvisible particles analytics, container appearance, degree of coloration, pH, osmolality, extractable volume, and container closure integrity testing), protein content by UV and microbiological parameters (sterility and bacterial endotoxins) under both OOF conditions. Only minor changes were observed for parameters evaluated via SEC, CEX, and nrCE-SDS. For potency (CDC-bioactivity) only one of the batches showed a relevant change. Even for these stability-indicating test methods, all analyzed parameters complied with the shelf-life specifications., Conclusion: SDZ-RTX is safe for use even under worst-case conditions, for example, after subjecting it for up to 21 days at OOF conditions (25 ± 2 °C/60 ± 5% RH or 30 ± 2 °C/65 ± 5% RH) after the batches had reached an age that was already beyond the claimed shelf-life., (© 2022. The Author(s).)

Different manufacturing processes and storage conditions of biotherapeutics can lead to a significant variability in drug products arising from chemical and enzymatic post-translational modifications (PTMs), resulting in the co-existence of a plethora of proteoforms with different physicochemical properties. To unravel the heterogeneity of these proteoforms, novel approaches employing strong cation-exchange (SCX) high-performance liquid chromatography (HPLC) hyphenated to mass spectrometry (MS) using a pH gradient of volatile salts have been developed in recent years. Here, we apply an established SCX-HPLC-MS method to characterize and compare two rituximab-based biotherapeutics, the originator MabThera ® and its Indian copy product Reditux™. The study assessed molecular differences between the two drug products in terms of C-terminal lysine variants, glycosylation patterns, and other basic and acidic variants. Overall, MabThera ® and Reditux™ displayed differences at the molecular level. MabThera ® showed a higher degree of galactosylated and sialylated glycoforms, while Reditux™ showed increased levels of oligomannose and afucosylated glycoforms. Moreover, the two drug products showed differences in terms of basic variants such as C-terminal lysine and N-terminal truncation, present in Reditux™ but not in MabThera ® . This study demonstrates the capability of this fast SCX-HPLC-MS approach to compare different drug products and simultaneously assess some of their quality attributes.

Insulin glargine is a long-acting analogue of human insulin that has been used to manage hyperglycemia in patients with diabetes mellitus (DM) for nearly 20 years. Insulin glargine has a relatively constant concentration-time profile that mimics basal levels of insulin and allows for once-daily administration. MYL-1501D is a biosimilar insulin glargine designed to offer greater access of insulin glargine to patients, with comparable efficacy and safety to the marketed reference product. We conducted a comprehensive panel of studies based on a formal analysis of critical quality attributes to characterize the structural and functional properties of MYL-1501D and reference insulin glargine products available in the United States and European Union. MYL-1501D was comprehensively shown to have high similarity to the reference products in terms of protein structure, metabolic activity (both in vitro cell-based assays and in vivo rabbit bioassays), and in vitro cell-based assays for mitogenic activity. The structural analyses demonstrated that the primary protein sequence was identical, and secondary and tertiary structures are similar between the proposed biosimilar and the reference products. Insulin receptor binding affinity and phosphorylation studies also established analytical similarity. MYL-1501D demonstrated high similarity in different metabolic assays of glucose uptake, adipogenesis activity, and inhibition of stimulated lipolysis. Rabbit bioassay studies showed MYL-1501D and EU-approved insulin glargine are highly similar to US-licensed insulin glargine. These product quality studies show high similarity between MYL-1501D and licensed or approved insulin glargine products and suggest the potential of MYL-1501D as an alternative cost-effective treatment option for patients and clinicians., Competing Interests: P Goyal, B Vats, S Gouda, DJ Ranayhossaini, JP Smith, A Barve, and R Ullanat are paid employees of Viatris Inc and may hold stock in the company. HV Pai, P Kodali, N Vajpai, S Annegowda, K Mane, S Mohan, S Saxena, AB Veerabhadraia, M Palande, PS Nair, DC More, UR Karudumpa, K Jyothirmai, A Bhattacharya, F Almeida, SG Khyade, PR Moole, and R Melarkode are paid employees of Biocon Research Limited and may hold stock in the company. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

The present study investigates the impact of charge variants on bevacizumab's structure, stability, and biological activity. Five basic and one acidic charge variants were separated using semi-preparative cation exchange chromatography using linear pH gradient elution with purity > 85%. Based on the commercial biosimilar product's composition, two basic variants, one acidic and the main bevacizumab product, were chosen for further investigation. Intact mass analysis and tryptic peptide mapping established the basic variants' identity as those originating from an incomplete clipping of either one or both C-terminal lysine residues in the heavy chain of bevacizumab. Based on peptide mapping data, the acidic variant formation was attributed to deamidation of asparagine residue (N84), oxidation of M258, and preservation of C-terminal lysine residue, located on the heavy chain of bevacizumab. None of the observed charge heterogeneities in bevacizumab were due to differences in glycosylation among the variants. The basic (lysine) variants exhibited similar structural, functional, and stability profiles as the bevacizumab main product. But it was also noted that both the variants did not improve bevacizumab's therapeutic utility when pooled in different proportions with the main product. The acidic variant was found to have an equivalent secondary structure with subtle differences in the tertiary structure. The conformational difference also translated into a ~ 62% decrease in biological activity. Based on these data, it can be concluded that different charge variants behave differently with respect to their structure and bioactivity. Hence, biopharmaceutical manufacturers need to incorporate this understanding into their process and product development guidelines to maintain consistency in product quality.

Reliable and reproducible monitoring of the conformational state of therapeutic protein products remains an unmet technological need. This need is amplified by the increasing number of biosimilars entering the drug development pipeline as many branded biologics are reaching the end of their market exclusivity period. Availability of methods to better characterize protein conformation may improve detection of counterfit and unlicensed therapeutic proteins. In this study, we report the use of a set of modified DNA aptamers with enhanced chemical diversity to probe the conformational state of 12 recombinant human erythropoietin (rHuEPO) therapeutic protein products; one FDA-licensed rHuEPO originator biological product, three rHuEPO products that are approved for marketing in the US or EU as biosimilars, and eight rHuEPO products that are not approved for marketing in the US or EU. We show that several of these modified aptamers are able to distinguish rHuEPO reference products or approved biosimilars from non-licensed rHuEPO products on the basis of differences in binding kinetics and equilibrium affinity constants. These reagents exhibit sensitivity to the conformational integrity of various forms of rHuEPO and as such represent powerful, simple-to-use analytical tools to monitor the conformational integrity of therapeutic-proteins during manufacture and to screen for and identify both substandard and counterfeit products.

FKB327 was approved by the European Medicines Agency as a biosimilar to European-authorized adalimumab (Humira ® ; AbbVie Inc). Adalimumab is a monoclonal antibody, binding and inhibiting tumor necrosis factor (TNF)-α with use indicated for several immune-mediated, chronic, and inflammatory disorders. The approval is based on high similarity in the physicochemical properties between FKB327 and adalimumab. The objective of this study is to assess the biological similarity, with regard to Fab- and Fc-associated functions, and describe the relationship between physicochemical and biological characterization and functional activity. State-of-the-art orthogonal techniques were implemented to assess the structure and function of FKB327. Peptide mapping with liquid chromatography and mass spectrometry, capillary electrophoresis-sodium dodecyl sulfate, ultraviolet circular dichroism, size-exclusion high-performance liquid chromatography (HPLC), and cation exchange HPLC were the techniques used to assess structure. Functional activity was assessed with enzyme-linked immunosorbent assay, surface plasmon resonance, and cell-based assays. The polypeptide sequence of FKB327 was identical to that of adalimumab. FKB327 also was demonstrated to have a similar secondary and tertiary structure to adalimumab. Posttranslational heterogeneities, along with size and charge variants, were not clinically meaningful. FKB327 binds to TNF-α, FcγR, the neonatal Fc receptor, and C1q, and induces apoptosis, antibody-dependent cellular cytotoxicity, and complement-dependent cytotoxicity. The binding and activity of FKB327 were similar to that of adalimumab. FKB327 shares similar structure and activity with adalimumab. Based on characterization of physicochemical and biological properties, FKB327 is expected to have a similar safety, immunogenicity, and efficacy profile to adalimumab., (© 2020 The Authors. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd, British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics.)

Biosimilars are highly similar to, but not identical with, their originator products. As a result, structural differences between originators and biosimilars can be difficult to detect and characterize without the appropriate analytical tools. Therefore, we first focus on identifying initial structural differences between rituximab, bevacizumab, and trastuzumab originator and biosimilar pairs and later address how these differences change after applying thermal stress at 40 °C with orbital shaking for 4 weeks. Prior to incubation, we detected comparable secondary and tertiary structures for each pair and identified different levels of soluble aggregates, charge variants, and molecular weight variants due to differences in glycoforms and the number of C-terminal lysine groups. Over the course of incubation, we compared differences in charge variants and unfolding patterns. Taken together, our study provides a comparability exercise, providing information on the minor differences present between originator and biosimilar products and how those differences are impacted by stress.

The therapeutic antibodies and their by-products (antibody fragments, conjugated, etc.) establish one of the most dynamic biopharmaceutical market segments today. Due to their intrinsic properties of specificity towards their target, towards their flexible affinity and due to their stability, antibodies became therapeutic agents of the very first choice. One of the challenges of this sector is to create antibodies of very good quality, more and more quickly, while having less and less consequent development costs in fine., (© 2019 médecine/sciences – Inserm.)

Sandoz rituximab (SDZ-RTX; Rixathon ® ; GP2013), a rituximab biosimilar, was approved in June 2017 in Europe in all indications of reference rituximab. The stepwise SDZ-RTX development program generated extensive physicochemical, structural, functional, and biological data demonstrating a match with reference rituximab on all clinically relevant attributes. A focused clinical development program followed, in two indications selected for sensitivity to detect potential differences versus reference rituximab: rheumatoid arthritis (pivotal pharmacokinetics and efficacy evaluation) and follicular lymphoma (pivotal efficacy/safety evaluation). These trials demonstrated highly similar pharmacokinetics, pharmacodynamics, efficacy, safety, and immunogenicity profiles. The totality of evidence for biosimilarity for SDZ-RTX, combined with knowledge that B-cell depletion is common to each approved indication, allowed SDZ-RTX approval for use in all indications of reference rituximab.

Studies of breast cancer therapy have examined the improvement of bispecific trastuzumab/pertuzumab antibodies interacting simultaneously with two different epitopes of the human epidermal growth factor receptor 2 (HER2). Here, we describe the creation and production of plant-made bispecific antibodies based on trastuzumab and pertuzumab plant biosimilars (bi-TPB-PPB). Using surface plasmon resonance analysis of bi-TPB-PPB antibodies binding with the HER2 extracellular domain, we showed that the obtained Kd values were within the limits accepted for modified trastuzumab and pertuzumab. Despite the ability of bi-TPB-PPB antibodies to bind to Fcγ receptor IIIa and HER2 oncoprotein on the cell surface, a proliferation inhibition assay did not reveal any effect until α1,3-fucose and β1,2-xylose in the Asn297-linked glycan were removed. Another approach to activating bi-TPB-PPB may be associated with the use of disulfiram (DSF) a known aldehyde dehydrogenase 2 (ALDH2) inhibitor. We found that disulfiram is capable of killing breast cancer cells with simultaneous formaldehyde accumulation. Furthermore, we investigated the capacity of DSF to act as an adjuvant for bi-TPB-PPB antibodies. Although the content of ALDH2 mRNA was decreased after BT-474 cell treatment with antibodies, we only observed cell proliferation inhibiting activity of bi-TPB-PPB in the presence of disulfiram. We concluded that disulfiram can serve as a booster and adjuvant for anticancer immunotherapy.

Objective: PF-06438179/GP1111 (PF-SZ-IFX) is an infliximab biosimilar. We evaluated the extended in-use physicochemical and biological stability of PF-SZ-IFX upon preparation for intravenous infusion., Methods: Two batches of PF-SZ-IFX were reconstituted to a concentration of 10 mg/mL and subsequently diluted to 0.4 and 4.0 mg/mL, representing the clinically relevant range for intravenous infusion. Dilution was performed in polyethylene saline infusion bags, which are commonly used in clinical practice. To simulate product handling under worst-case conditions, reconstituted solutions were stored for up to 30 days at 5 ± 3 °C and up to 14 days at 25 ± 2 °C (60 ± 5% relative humidity); diluted solutions were stored for up to 30 days under the same sets of conditions. Physicochemical and biological stability were evaluated according to pH, osmolality, appearance, particulate content, protein concentration, proportions of molecular weight variants and charge variants and potency. Standard and state-of-the-art analytical techniques were employed, including imaged isoelectric focusing, size exclusion chromatography, reducing sodium dodecyl sulphate capillary electrophoresis and functional cell-based bioassay., Results: Across batches and concentrations of PF-SZ-IFX, all parameters resided within the predefined acceptance criteria, including pH, osmolality, particulate content, clarity, protein concentration, molecular weight variants, charge variants and potency, for up to 30 days under both storage conditions tested (up to 14 days for reconstituted samples stored at 25 ± 2 °C)., Conclusions: Physicochemical and biological analyses demonstrated that the infliximab biosimilar PF-SZ-IFX was not affected by extended storage of the diluted preparations used for intravenous infusion.

Development of bio-therapeutics has exhibited exponential growth in China over the past decade. However, no biosimilar drug has been approved in China (CN) due to the lack of a national biosimilar regulatory guidance. HLX01, a rituximab biosimilar developed in China under European Medicines Agency biosimilar guidelines and requirements, was the first such drug submitted for regulatory review in China, and it is expected to receive approval there as a biosimilar product. To demonstrate the analytical similarities of HLX01, CN-rituximab (sourced in China but manufactured in Europe) and EU-rituximab (sourced and manufactured in Europe), an extensive 3-way physicochemical and functional similarity assessment using a series of orthogonal and state-of-the-art techniques was conducted, following the similarity requirement guidelines recently published by China's Center for Drug Evaluation. The results of the similarity study showed an identical protein amino acid sequence and highly similar primary structures between HLX01 and the reference product (RP) MabThera®, along with high similarities in higher order structures, potency, integrity, purity and impurity profiles, biological and immunological binding functions, as well as degradation behaviors under stress conditions. In addition, HLX01 presented slightly lower aggregates and better photostability compared with the RP. Despite slight changes in relative abundance of glycan moieties and heavy chain C-terminal lysine modification, no differences in biological activities and immunological properties were observed between the RP and HLX01. In conclusion, HLX01 is highly similar to CN- and EU-sourced RP in terms of physicochemical properties and biological activities, suggesting similar product quality, efficacy, and safety. The regulatory requirements interpreted and applied towards the HLX01 marketing application sets a precedent for analytical similarity assessment of biosimilar products in China.

Background : The GRPR-antagonist 68 Ga-SB3 visualized prostate cancer lesions in animal models and in patients. Switching radiometal from 68 Ga to 111 In impaired tumor targeting in mice, but coinjection of the neprilysin (NEP)-inhibitor phosphoramidon (PA) stabilized 111 In-SB3 in circulation and remarkably increased tumor uptake. We herein report on the biological profile of 111 In-SB4: 111 In-[dAla 11 ]SB3. Methods : The biological responses of 111 In-SB3/SB4 were compared in PC-3 cells and animal models. Results : Gly 11 /dAla 11 -replacement deteriorated GRPR-affinity (SB4 IC 50 : 10.7 ± 0.9 nM vs. SB3 IC 50 : 4.6 ± 0.3 nM) and uptake in PC-3 cells ( 111 In-SB4: 1.3 ± 0.4% vs. 111 In-SB3 16.2 ± 0.8% at 1 h). 111 In-SB4 was more stable than 111 In-SB3, but PA-coinjection stabilized both radiotracers in peripheral mice blood. Unmodified 111 In-SB3 showed higher uptake in PC-3 xenografts (8.8 ± 3.0%ID/g) vs. 111 In-SB4 (3.1 ± 1.1%ID/g) at 4 h pi. PA-coinjection improved tumor uptake, with 111 In-SB3 still showing superior tumor targeting (38.3 ± 7.9%ID/g vs. 7.4 ± 0.3%ID/g for 111 In-SB4). Conclusions : Replacement of Gly 11 by dAla 11 improved in vivo stability, however, at the cost of GRPR-affinity and cell uptake, eventually translating into inferior tumor uptake of 111 In-SB4 vs. unmodified 111 In-SB3. On the other hand, in-situ NEP-inhibition turned out to be a more efficient and direct strategy to optimize the in vivo profile of 111 In-SB3, and potentially other peptide radiotracers.

The approval of biosimilars requires demonstration of biosimilarity, which rests on the base of thorough analytical characterization of the biosimilar product. In addition to demonstration of biosimilarity, the product related impurities need to be thoroughly characterized and controlled at minimal levels. Pegylation of peptides and proteins creates significant challenges for detailed structural characterization, such as PEG (Poly Ethylene Glycol) heterogeneity, site of addition and number of attached pegylated moieties. A combination of several methods including circular dichroism, FTIR (Fourier-transform Infrared Spectroscopy), fluorescence spectroscopy, DSC (Differential Scanning Calorimetry), 1D and 2D NMR (Nuclear Magnetic Resonance), Edman degradation and peptide mapping by LC-MS (Liquid Chromatography Mass Spectrometry) were used for characterization of N-terminally pegylated filgrastim. Product related impurities such as oxidized, reduced, deamidated, dipegylated variants and monopegylated positional isomers have been characterized in detail using various HPLC (High Performance Liquid Chromatography) based methods and LC-MS techniques. The functional characterization in terms of receptor binding and cell proliferation assay was conducted for the similarity assessment and the potential impact of the product variants on the in vitro biological activity has also been assessed. In summary, this study presents, for the first time, a detailed structural and molecular level characterization of a biosimilar pegfilgrastim providing a strong base for the demonstration of overall biosimilarity of the product with the innovator product., Competing Interests: All authors were employees of Intas Pharmaceutical Ltd at the time the experiments and data analysis was performed. This does not alter the author’s adherence to PLOS ONE policies on sharing data and materials.

Due to the increase in the number of infliximab products, the need for global harmonization of the bioactivity of this monoclonal antibody was recognized by the World Health Organization (WHO). In response, the National Institute for Biological Standards and Control (NIBSC) developed the first international standard (IS) for infliximab, which targets tumour necrosis factor (TNF). Each ampoule is assigned values of 500 IU of TNF neutralizing activity and 500 IU of binding activity. Two preparations of infliximab were formulated and lyophilized at NIBSC prior to evaluation in a collaborative study for their suitability to serve as an IS for the in vitro biological activity of infliximab. The study involved participants using in vitro cell-based bioassays (TNF neutralization, antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity) and binding assays. The results of this study showed that the candidate preparation, coded 16/170, is suitable as an IS for infliximab bioactivity. This infliximab IS from NIBSC, is intended to support in vitro bioassay calibration and validation by defining international units of bioactivity. The proposed unitages, however, are not intended to revise product labelling or dosing requirements, as any decisions regarding this relies solely with the regulatory authorities. Furthermore, the infliximab IS is not intended for determining the specific activity of products, nor to serve any regulatory role in defining biosimilarity. We briefly discuss the future use of WHO international standards in supporting the global harmonisation of biosimilar infliximab products.

Biosimilars are biologic products that are highly similar to a licensed reference product in terms of quality, safety, and efficacy. SB5 is a biosimilar of Humira® (adalimumab) developed by Samsung Bioepis. To demonstrate its biosimilarity in quality to Humira®, we performed a comprehensive characterization in terms of structure, physicochemical properties, and biological properties following the International Conference on Harmonization, US Food and Drug Administration, and European Medicines Agency guidelines. We analyzed all available batches of SB5 and more than 100 EU- and US-sourced lots of Humira® using state-of-the-art methods whenever possible, and compared the two sets of data. The structural properties comprised primary and higher-order structures and N-glycosylation. The physicochemical characteristics were categorized into liquid chromatographic patterns and electrophoretic pattern concerning size and charge heterogeneity. The biological properties were examined by in vitro functional assays. Overall, SB5 and Humira® were shown to be similar to each other in terms of quality attributes. For some of the quality attributes, minor differences were observed. However, the observed differences have been adequately addressed and demonstrated these do not translate into clinically meaningful differences in terms of safety, purity, and potency.

The successful development and regulatory approval of originator and biosimilar therapeutic proteins requires a systems approach to upstream and downstream processing as well as product characterization and quality control. Innovation in process design and control, product characterization strategies, and data integration represent an ecosystem whose concerted advancement may reduce time-to-market and further improve comparability and biosimilarity programs. The biopharmaceutical community has made great strides to this end, yet there currently exists no pre-competitive monoclonal antibody (mAb) expression platform for open innovation. Here, we describe the development and initial expression of an intended copy of the NISTmAb using three non-originator murine cell lines. It was found that, without optimization and in culture flasks, all three cell lines produce approximately 100 mg mAb per liter of culture. Sodium dodecyl sulfate polyacrylamide gel electrophoresis, size-exclusion chromatography, nuclear magnetic resonance spectroscopy, intact mass spectrometry, and surface plasmon resonance were used to demonstrate that the products of all three cell lines embody quality attributes with a sufficient degree of sameness to the NISTmAb Reference Material 8671 to warrant further bioreactor studies, process improvements and optimization. The implications of the work with regard to pre-competitive innovation to support process design and feedback control, comparability and biosimilarity assessments, and process analytical technologies are discussed.

Analytical similarity is the foundation of the totality-of-evidence-based approach for demonstrating that a proposed biosimilar is highly similar to a reference product. This review uses analytical similarity assessment of ABP 501, the first approved adalimumab biosimilar, as a case study to highlight considerations necessary to demonstrate analytical similarity. Similarity assessments start with risk-ranking the critical quality attributes based on scientific understanding of how they impact safety, efficacy, immunogenicity and/or pharmacokinetics/pharmacodynamics. Testing strategy and evaluation approaches are designed with the understanding of the analytical methods and their capabilities. Statistical considerations are used to establish objective assessment criteria. The result is a scientifically justified, objective analytical similarity assessment that demonstrates that a proposed biosimilar is structurally and functionally similar to a reference product.

The evaluation of analytical similarity has been a challenging issue for the biosimilar industry because the number of lots for reference and biosimilar products available at the time of development are limited, whilst measurable quality attributes of target molecule are numerous, which can lead to potential bias or false negative/positive conclusions regarding biosimilarity. Therefore, appropriate statistical analyses are highly desirable to achieve a high level of confidence in the similarity evaluation. A recent guideline for the risk-based statistical approaches recommended by the US Food and Drug Administration provides useful tools to systematically evaluate analytical similarity of biosimilar products compared with reference products. Here, we evaluated analytical similarity of CT-P6, a biosimilar product of trastuzumab, with the reference products (EU-Herceptin® or US-Herceptin®) following these statistical approaches. Various quality attributes of trastuzumab were first ranked based on the clinical impact of each attribute and subsequently adjusted to one of three tiers (Tier 1, Tier 2 and Tier 3) considering the characteristics of the assay, the level of attribute present and the feasibility of statistical analysis. Two biological activities with highest potential clinical impact were evaluated by an equivalent test (Tier 1), and other bioactivities and structural/physicochemical properties relevant to the clinical impact were evaluated by a quality range approach (Tier 2). The attributes with low risk ranking or qualitative assay were evaluated by visual comparison (Tier 3). Analytical similarity assessment analyzed by the three tiers clearly demonstrated that CT-P6 exhibits highly similar structural and physicochemical properties, as well as functional activities, compared with the reference products. There were small differences observed in a few quality attributes between CT-P6 and the reference products, but the differences were very minor, and unlikely to impact on clinical outcome. The recently reported equivalent clinical efficacy of CT-P6 with the reference product further supports that CT-P6 is highly similar compared with the reference product in the view of totality-of-evidence.

ABP 215 is a biosimilar product to bevacizumab. Bevacizumab acts by binding to vascular endothelial growth factor A, inhibiting endothelial cell proliferation and new blood vessel formation, thereby leading to tumor vasculature normalization. The ABP 215 analytical similarity assessment was designed to assess the structural and functional similarity of ABP 215 and bevacizumab sourced from both the United States (US) and the European Union (EU). Similarity assessment was also made between the US- and EU-sourced bevacizumab to assess the similarity between the two products. The physicochemical properties and structural similarity of ABP 215 and bevacizumab were characterized using sensitive state-of-the-art analytical techniques capable of detecting small differences in product attributes. ABP 215 has the same amino acid sequence and exhibits similar post-translational modification profiles compared to bevacizumab. The functional similarity assessment employed orthogonal assays designed to interrogate all expected biological activities, including those known to affect the mechanisms of action for ABP 215 and bevacizumab. More than 20 batches of bevacizumab (US) and bevacizumab (EU), and 13 batches of ABP 215 representing unique drug substance lots were assessed for similarity. The large dataset allows meaningful comparisons and garners confidence in the overall conclusion for the analytical similarity assessment of ABP 215 to both US- and EU-sourced bevacizumab. The structural and purity attributes, and biological properties of ABP 215 are demonstrated to be highly similar to those of bevacizumab.

There are numerous applications that use the structures of protein-ligand complexes from the PDB, such as 3D pharmacophore identification, virtual screening, and fragment-based drug design. The structures underlying these applications are potentially much more informative if they contain biologically relevant bound ligands, with high similarity to the cognate ligands. We present a study of ligand-enzyme complexes that compares the similarity of bound and cognate ligands, enabling the best matches to be identified. We calculate the molecular similarity scores using a method called PARITY (proportion of atoms residing in identical topology), which can conveniently be combined to give a similarity score for all cognate reactants or products in the reaction. Thus, we generate a rank-ordered list of related PDB structures, according to the biological similarity of the ligands bound in the structures., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

CT-P10 (Truxima™) was recently approved as the world's first rituximab biosimilar product in the European Union (EU) and South Korea. To demonstrate biosimilarity of CT-P10 with the reference medicinal product (RMP), extensive 3-way similarity assessment has been conducted between CT-P10, EU-Rituximab and US-Rituximab, focusing on the physicochemical and biological quality attributes. A multitude of state-of-the-art analyses revealed that CT-P10 has identical primary and higher order structures compared to the original product. Purity/impurity profiles of CT-P10 measured by the levels of aggregates, fragments, non-glycosylated form and process-related impurities were also found to be comparable with those of RMPs. In terms of the post-translational modification, CT-P10 contains slightly less N-terminal pyro-glutamate variant, which has been known not to affect product efficacy or safety. Oligosaccharide profiling has revealed that, although CT-P10 contains the same conserved glycan species and relative proportion with the RMPs, the content of total afucosylated glycan in CT-P10 was slightly higher than in EU- or US-Rituximab. Nevertheless, the effect of the observed level of afucosylation in CT-P10 drug product on Fc receptor binding affinity or antibody-dependent cell-mediated cytotoxicity was found to be negligible based on the spiking study with highly afucosylated sample. Arrays of biological assays representative of known and putative mechanisms of action for rituximab have shown that biological activities of CT-P10 are within the quality range of RMPs. Recent results of clinical studies have further confirmed that the CT-P10 exhibits equivalent clinical efficacy and safety profiles compared to EU- and US-Rituximab. The current 3-way similarity assessment together with clinical study results confidently demonstrate that CT-P10 is highly similar with EU- and US-Rituximab in terms of physicochemical properties, biological activities, efficacy, and safety for its final approval as a biosimilar product.

The NISTmAb Reference Material (RM) 8671 is intended to be an industry standard monoclonal antibody for pre-competitive harmonization of best practices and designing next generation characterization technologies for identity, quality, and stability testing. It must therefore embody the quality and characteristics of a typical biopharmaceutical product and be available long-term in a stable format with consistent product quality attributes. A stratified sampling and analysis plan using a series of qualified analytical and biophysical methods is described that assures RM 8671 meets these criteria. Results for the first three lots of RM 8671 highlight the consistency of material attributes with respect to size, charge, and identity. RM 8671 was verified to be homogeneous both within and between vialing lots, demonstrating the robustness of the lifecycle management plan. It was analyzed in concert with the in-house primary sample 8670 (PS 8670) to provide a historical link to this seminal material. RM 8671 was verified to be fit for its intended purpose as a technology innovation tool, external system suitability control, and cross-industry harmonization platform. Graphical abstract The NISTmAb Reference Material (RM) 8671 is intended to be an industry standard monoclonal antibody for pre-competitive harmonization of best practices and designing next generation characterization technologies for identity, quality, and stability testing.

The NISTmAb is a monoclonal antibody Reference Material from the National Institute of Standards and Technology; it is a class-representative IgG1κ intended serve as a pre-competitive platform for harmonization and technology development in the biopharmaceutical industry. The publication series of which this paper is a part describes NIST's overall control strategy to ensure NISTmAb quality and availability over its lifecycle. In this paper, the development and qualification of methods for monitoring NISTmAb charge heterogeneity are described. Capillary zone electrophoresis (CZE) and capillary isoelectric focusing (CIEF) assays were optimized and evaluated as candidate assays for NISTmAb quality control. CIEF was found to be suitable as a structural characterization assay yielding information on the apparent pI of the NISTmAb. CZE was found to be better suited for routine monitoring of NISTmAb charge heterogeneity and was qualified for this purpose. This paper is intended to provide relevant details of NIST's charge heterogeneity control strategy to facilitate implementation of the NISTmAb as a test molecule in the end user's laboratory. Graphical Abstract Representative capillary zone electropherogram of the NIST monoclonal antibody (NISTmAb). The NISTmAb is a publicly available research tool intended to facilitate advancement of biopharmaceutical analytics.

This industry update covers the period from 1 September through 30 September 2017, and is based on information sourced from company press releases, scientific literature, patents and various news websites. The month saw the US FDA approve three new molecular entities, Aliqopa (copanlisib dihydrochloride) (Bayer Healthcare); Solosec (secnidazole) (Symbiomix Therapeutics) and Verzenio (abemaciclib) (Eli Lilly and Co). Intarcia Therapeutics Inc. has its application for approval of a novel drug device combination of exenatide for the treatment of diabetes rejected by FDA but said that it will work to address the concerns and refile the application. The impact of biosimilars in the market is steadily increasing with seven biosimilars approved in the USA and Sandoz hoping to add to this with its announcement that FDA has accepted its Biologics License Application for a biosimilar version of Roche's Rituxan. Circassia announced positive top line results of a respiratory drug, Duaklir (for the treatment of chronic obstructive pulmonary disease) and Sarepta (for its new treatment for Duchenne muscular dystrophy). Axovant Sciences Ltd announced the failure if its drug Intepirdine in the treatment of Alzheimer's, adding to a growing list of drug failures in this area. There were a number of developments in the area of oncology with Bristol-Myers Squibb and Infinity Pharmaceuticals announcing an expansion of their collaboration looking at combination treatments, as well as Eli Lilly and Co's approval for Verzenio. Rani Therapeutics and Intra-Cellular Therapies announced successful funding rounds to support their drug programs. Allergan announced a novel licensing deal for its dry eye drug, Restasis, which it hopes would allow it to stave off patent challenges from several companies looking to develop generic versions of the drug. New research suggests that loss of sense of smell can be linked to an increased risk of developing Parkinson's disease.

Biosimilars are products that are similar in terms of quality, safety, and efficacy to an already licensed reference/ innovator product and are expected to offer improved affordability. The most significant source of reduction in the cost of development of a biosimilar is the reduced clinical examination that it is expected to undergo as compared to the innovator product. However, this clinical relief is predicated on the assumption that there is analytical similarity between the biosimilar and the innovator product. As a result, establishing analytical similarity is arguably the most important step towards successful development of a biosimilar. Here, we present results from an analytical similarity exercise that was performed with five biosimilars of rituximab (Ristova®, Roche), a chimeric mouse/ human monoclonal antibody biotherapeutic, that are available on the Indian market. The results show that, while the biosimilars exhibited similarity with respect to protein structure and function, there were significant differences with respect to size heterogeneity, charge heterogeneity and glycosylation pattern.

Patent expiration of first-generation biologics and the high cost of innovative biologics are 2 drivers for the development of biosimilar products. There are, however, technical challenges to the production of exact copies of such large molecules. In this study, we performed a head-to-head comparison between the originator anti-VEGF-A Fab product LUCENTIS® (ranibizumab) and an intended copy product using an integrated analytical approach. While no differences could be observed using size-exclusion chromatography, capillary electrophoresis-sodium dodecyl sulfate and potency assays, different acidic peaks were identified with cation ion exchange chromatography and capillary zone electrophoresis. Further investigation of the intact Fab, subunits and primary sequence with mass spectrometry demonstrated the presence of a modified light chain variant in the intended copy product batches. This variant was characterized with a mass increase of 27.01 Da compared to the originator sequence and its abundance was estimated in the range of 6-9% of the intended copy product light chain. MS/MS spectra interrogation confirmed that this modification relates to a serine to asparagine sequence variant found in the intended copy product light chain. We demonstrated that the integration of high-resolution and sensitive orthogonal technologies was beneficial to assess the similarity of an originator and an intended copy product.

Remsima™ (infliximab) is the first biosimilar monoclonal antibody (mAb) approved by the European Medical Agency and the US Food and Drug Administration. Remsima™ is highly similar to its reference product, Remicade®, with identical formulation components. The 2 products, however, are not identical; Remsima™ has higher levels of soluble aggregates, C-terminal lysine truncation, and fucosylated glycans. To understand if these attribute differences could be amplified during forced degradation, solutions and lyophilized powders of the 2 products were subjected to stress at elevated temperature (40-60°C) and humidity (dry-97% relative humidity). Stress-induced aggregation and degradation profiles were similar for the 2 products and resulted in loss of infliximab binding to tumor necrosis factor and FcγRIIIa. Appearances of protein aggregates and hydrolysis products were time- and humidity-dependent, with similar degradation rates observed for the reference and biosimilar products. Protein powder incubations at 40°C/97% relative humidity resulted in partial mAb unfolding and increased asparagine deamidation. Minor differences in heat capacity, fluorescence, levels of subvisible particulates, deamidation and protein fragments were observed in the 2 stressed products, but these differences were not statistically significant. The protein solution instability at 60°C, although quite significant, was also similar for both products. Despite the small initial analytical differences, Remicade® and Remsima™ displayed similar degradation mechanisms and kinetics. Thus, our results show that the 2 products are highly similar and infliximab's primary sequence largely defines their protein instabilities compared with the limited influence of small initial purity and glycosylation differences in the 2 products.

Ovaleap ® (XM17) is a recombinant human follicle-stimulating hormone to treat infertility by inducing ovulation or controlled ovarian stimulation for assisted reproductive technology (ART) procedures. Ovaleap ® (follitropin-α) was approved by the European Medicines Agency in 2013 as a biosimilar medicinal product to the reference medicine, Gonal-f ® . Information is often not easily accessible and/or publicly available regarding the rigorous manufacturing procedures for biosimilars. Objectives of the current analysis were to report on validation procedures for the Ovaleap ® manufacturing process, to compare the characteristics of Ovaleap ® versus Gonal-f ® , and to describe the performance and consistency of Ovaleap ® . Formal validation of the Ovaleap ® manufacturing process was performed at full commercial scale, consisting of several consecutive fermentation and purification runs. Comparison with Gonal-f ® involved numerous techniques to determine molecular structure, isoform distribution, biological activity, and product-related impurities. The stability of the multidose application system, targeted for long-term stability at ambient temperature, was assessed and demonstrated. All analyses showed the manufacturing process of Ovaleap ® to be robust and consistent. Ovaleap ® was found to have similar characteristics when compared with Gonal-f ® . This analysis supports the role of Ovaleap ® as a biosimilar to Gonal-f ® , thus providing patients and clinicians with another therapeutic option during ART procedures.

Monoclonal antibodies and other biologic drugs play important roles in the treatment of various hematological malignancies and solid tumors. However, such drugs are intrinsically more expensive to develop than small molecules and their clinical benefits are often accompanied by challenges relating to affordability and access. Patent expiry for 'originator' biologics is providing opportunities for a new generation of biosimilar drugs, potentially capable of relieving pressure on healthcare budgets. This article discusses key characteristics of biosimilars, distinguishes them from generics and noncomparable biologics and outlines the robust regulatory requirements that must be followed to establish biosimilarity with a reference product. The path to approval is discussed with reference to the rituximab biosimilar CT-P10, the first licensed monoclonal antibody biosimilar cancer therapeutic.

A biosimilar product needs to demonstrate biosimilarity to the originator reference product, and the quality profile of the latter should be monitored throughout the period of the biosimilar's development to match the quality attributes of the 2 products that relate to efficacy and safety. For the development of a biosimilar version of trastuzumab, the reference product, Herceptin®, was extensively characterized for the main physicochemical and biologic properties by standard or state-of-the-art analytical methods, using multiple lots expiring between March 2015 and December 2019. For lots with expiry dates up to July 2018, a high degree of consistency was observed for all the tested properties. However, among the lots expiring in August 2018 or later, a downward drift was observed in %afucose (G0+G1+G2). Furthermore, the upward drift of %high mannose (M5+M6) was observed in the lots with expiry dates from June 2019 to December 2019. As a result, the combination of %afucose and %high mannose showed 2 marked drifts in the lots with expiry dates from August 2018 to December 2019, which was supported by the similar trend of biologic data, such as FcγRIIIa binding and antibody-dependent cell-mediated cytotoxicity (ADCC) activity. Considering that ADCC is one of the clinically relevant mechanisms of action for trastuzumab, the levels of %afucose and %high mannose should be tightly monitored as critical quality attributes for biosimilar development of trastuzumab.

Protein aggregation is a common biological phenomenon which is responsible for degenerative diseases and is problematic in the pharmaceutical industry. According to the rules provided by regulatory agencies, industry is supposed to assess the product quality regarding the presence of subvisible particles. Also, they should evaluate the technologies that are used to measure these particles. Therefore, US FDA and industry have been looking for methods capable of accurately characterizing the protein products. Four sizing techniques reviewed here are good candidates to be used for characterization of protein and their aggregates: dynamic light scattering, size-exclusion chromatography, electron microscopy and Taylor dispersion analysis. The first three are more established techniques while the last one is a more recent and growing technique.