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The nature of alpha-D-mannose-natural aldohexose sugar, C-2 glucose epimer, whose intended use is for preventing urinary tract infections-in the interaction with E. coli is addressed in order to drive the issue of its regulatory classification as a medicinal product or medical device. PRISMA systematic review approach was applied; Delphi Panel method was used to target consensus on statements retrieved from evidence. Based on regulatory definitions and research evidence, the mechanism of D-mannose does not involve a metabolic or immunological action while there is uncertainty regarding the pharmacological action. Specific interaction between the product and the bacteria within the body occurs, but its nature is inert: it does not induce a direct response activating or inhibiting body processes. Moreover, the action of D-mannose takes place, even if inside the bladder, outside the epithelium on bacteria that have not yet invaded the urothelial tissue. Therefore, its mechanism of action is not directed to host structures but to structures (bacteria) external to the host's tissues. On the basis of current regulation, the uncertainty as regard a pharmacological action of alpha-D-mannose makes possible its medical device classification: new regulations and legal judgments can add further considerations. From a pharmacological perspective, research is driven versus synthetic mannosides: no further considerations are expected on alpha-D-mannose., (© 2023. The Author(s).)

The primary purpose of this study was to assess the translatability of preclinical to early clinical tolerable and pharmacologically active dose ranges for central nervous system (CNS) active drugs. As a part of this, IBs were reviewed on reporting quality. Investigator's Brochures (IBs) of studies performed at the Centre for Human Drug Research (CHDR) reporting statistically significant results of CNS activity related to the drug's mechanism of action were included. The quality of IBs was assessed based on the presence of a rationale for the chosen animal model, completeness of pharmacokinetic (PK) results in reporting and internal validity information of the preclinical evidence. The IB-derisk tool was used to generate preclinical and early clinical data overviews data. For each compound, the overlap between pharmacologically active dose ranges and well-tolerated levels was calculated for three pharmacokinetic (PK) parameters: human equivalent dose (HED), maximum plasma concentration (C max ) and area under the curve (AUC). Twenty-five IBs were included. In general, the quality of reporting in IBs was assessed as poor. About a third of studies did not explore the entire concentration-effect curve (pre)clinically. Single dose tolerability ranges were most accurately predicted by C max . Human equivalent dose and AUC were the best predictors of pharmacologically active ranges. Tolerable and pharmacologically active dose ranges in healthy volunteers can be reasonably well predicted from preclinical data with the IB-derisk tool. The translatability of preclinical studies can be improved by applying a higher reporting standard in IBs including comparable PK measurements across all preclinical and clinical studies., (© 2023. The Author(s).)

National and international laws and regulations exist to protect animals used for scientific purposes in translational and applied research, which includes drug development. However, multiple animal models are available for each disease. We evaluated the argumentation behind the selection of a specific animal model using thematic content analysis in project applications issued in 2017-2019 in the Netherlands. In total, 125 animal models for translational and applied research from 110 project applications were assessed. Explanations to select a specific model included: the model’s availability (79%); the availability of expertise (62%); and the model showing similar disease pathology/symptoms (59%) to humans. Therefore, current selection of a specific animal model seems to be based on tradition rather than its potential predictive value for clinical outcome. The applicants’ explanations for the implementation of the 3R prin­ciples (replacement, reduction and refinement) as to the animal model were unspecific. Replacement was achieved by using data from prior in vitro studies, reduction by optimal experimental design and statistics, and refinement by reducing discomfort. Additionally, due to the stated need for a test model with high complexity (47%) and intactness (30%), the full replacement of animal models with alternative (non-live animal) approaches was thought unachievable. Without a clear, systematic and transparent justification for the selection of a specific animal model, the likelihood of poorly trans­latable research remains. It is not only up to the researcher to demonstrate this, as ethical committees and funding bodies can provide positive stimuli to drive this change.

The current pharmaceutical system is not sustainable; new drugs have limited impact on public health, they are (too) expensive and innovation has stalled because of overregulation of the industry. We suggest reviving compounding by pharmacists as the pharmaceutical model for the future allowing for innovation. Bedside production of biologics by magistral compounding under direct control by a pharmacist is considered key to the introduction of personalized medicines. Although specific regulations are lacking, appropriate quality testing will be essential for magistral preparations to be accepted by patients and doctors. In classical drug development a myriad of different, often redundant, techniques are being used for protein characterization. In most cases, the specification of these test are set based on history, the cut-off of the test, specifications of comparable products rather than on clinically set limits. Here, we propose a quality testing approach for two hypothetical biopharmaceuticals, a monoclonal antibody and an enzyme replacement therapy, based on common sense and a risk analysis., (Copyright © 2020 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

Previous qualitative research has suggested there are only minor differences between the db/db mouse and the Zucker Diabetic Fatty (ZDF) rat, both animal models of type 2 diabetes. However, it is not known whether these models are also comparable regarding drug response in quantitative terms (effect size). To investigate the extent of these differences, we conducted a systematic review and meta-analysis of approved drugs in these models. We searched on PubMed and Embase on July 3, 2019 for studies including either model, a monotherapy arm with an EMA/FDA approved drug for the treatment of type 2 diabetes, HbA1c assessment and a control group. Studies aimed at diabetes prevention or with surgical interventions were excluded. We calculated the Standardised Mean Difference (SMD) to compare effect sizes (HbA1c reduction) per drug and drug class across models. We included a risk of bias assessment for all included publications. A total of 121 publications met our inclusion criteria. For drugs with more than two comparisons, both models predicted the direction of the effect regarding HbA1c levels. There were no differences between the db/db mouse and ZDF rat, except for exenatide (P = 0.02) and GLP-1 agonists (P = 0.03) in which a larger effect size was calculated in the ZDF rat. Our results indicate the differences between the db/db mouse and ZDF rat are not relevant for preliminary efficacy testing. This methodology can be used to further differentiate between animal models used for the same indication, facilitating the selection of models more likely to predict human response., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Objective: The high cost of orphan drugs limits their access by many patients, especially in low- and middle-income countries. Many orphan drugs are off-patent without alternative generic or biosimilar versions available. Production of these drugs at the point-of-care, when feasible, could be a cost-effective alternative., Methods: The financial feasibility of this approach was estimated by setting up a small-scale production of recombinant human acid alpha-glucosidase (rhGAA). The commercial version of rhGAA is Myozyme™, and Lumizyme™ in the United States, which is used to treat Pompe disease. The rhGAA was produced in CHO-K1 mammalian cells and purified using multiple purification steps to obtain a protein profile comparable to Myozyme™., Results: The established small-scale production of rhGAA was used to obtain a realistic cost estimation for the magistral production of this biological drug. The treatment cost of rhGAA using bedside production was estimated at $3,484/gram, which is 71% lower than the commercial price of Myozyme ™., Conclusion: This study shows that bedside production might be a cost-effective approach to increase the access of patients to particular life-saving drugs., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Since the introduction of the first biosimilar the discussion about their interchangeability has persisted. The body of evidence gathered for biosimilars provides reassurance that they are approved based on a rigorous comparability exercise and do not show clinically meaningful differences to their reference products. There are no data suggesting that the risk of switching to a biosimilar in terms of increased immunogenicity is greater than switching between two batches of any biologic. The key concern around switching biosimilars is the nocebo effect, which reinforces the need for physician involvement when switching. Whereas this might argue against automatic substitution of biosimilars, it is not a biosimilars-specific concern. To increase physician confidence in biosimilars, regulators should acknowledge that biosimilars are interchangeable., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Clinical trial failures (>99%) in Alzheimer's disease are in stark contrast to positive efficacy data in animals. We evaluated the correlation between animal and clinical efficacy outcomes (cognition) in Alzheimer's disease using data from registered drugs as well as interventions tested in phase II or III clinical trials for Alzheimer's disease. We identified 20 interventions, which were tested in 208 animal studies in 63 different animal models. Clinical outcome was correlated with animal results in 58% of cases. But, individual animal models showed divergent results across interventions, individual interventions showed divergent results across animal models, and animal model outcomes were determined with 16 different methods. This result is unsurprising due to poor external validity (what do we model) of the animal models. Although the animal models all share Alzheimer's disease symptoms, none represents the whole syndrome. Investigators did not motivate why one model was chosen over another, and did not consider the ways the disease phenomena were generated (spontaneous, (experimentally) induced or by genetic modification), or the species characteristics, which determine the outcomes. The explanation for the lack of correlation between animal and human outcomes can be manifold: the pathogenesis of Alzheimer's disease is not reflected in the animal model or the outcomes are not comparable. Our conclusion is that currently no animal models exist which are predictive for the efficacy of interventions for Alzheimer's disease., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Niemann-Pick disease type B is a hereditary rare condition caused by deficiency of the acid sphingomyelinase (ASM) that is needed for lysosomal hydrolysis of sphingomyelin to ceramide and phosphocholine. This deficiency leads to a massive accumulation of sphingomyelin in cells throughout the body, predominantly in the liver, spleen and lungs. Currently, there is no effective treatment available. Olipudase alfa (recombinant human acid sphingomyelinase; rhASM) is an investigational drug that has shown promising results. However, dose-dependent toxicity was observed in mice upon the intravenous administration of rhASM, potentially due to the systemic release of ceramide upon the extracellular degradation of sphingomyelin by rhASM. Using a nanocarrier to deliver the rhASM to cells could improve the therapeutic window by shielding the rhASM to prevent the off-target degradation of sphingomyelin. For this aim, we recombinantly expressed hASM in human cells and loaded it into different liposomal formulations at a drug-to-lipid ratio of 4% (w/w). Among four formulations, the liposomal rhASM formulation with the composition DPPC:DOPS:BMP:CHOL:DiD (59:20:10:10:1 mol%) was selected because of its superiority concerning the encapsulation efficiency of rhASM (21%) and cellular uptake by fibroblasts and macrophages. The selected liposomal rhASM formulation significantly reduced the accumulated lyso-sphingomyelin in NPD-B fibroblasts by 71%, part of this effect was stimulated by the used lipids, compared to 55% when using the free rhASM enzyme. More importantly, the undesired extracellular degradation of sphingomyelin was reduced when using the selected liposomal rhASM by 61% relative to the free rhASM. The presented in vitro data indicate that the liposomal rhASM is effective and may provide a safer intervention than free rhASM., (Copyright © 2019. Published by Elsevier B.V.)

The development of hybridoma technology for producing monoclonal antibodies (mAbs) by Kohler and Milstein (1975) counts as one of the major medical breakthroughs, opening up endless possibilities for research, diagnosis and for treatment of a whole variety of diseases. Therapeutic mAbs were introduced three decades ago. The first generation of therapeutic mAbs of murine origin showed high immunogenicity, which limited efficacy and was associated with severe infusion reactions. Subsequently chimeric, humanized, and fully human antibodies were introduced as therapeutics, these mAbs were considerably less immunogenic. Unexpectedly humanized mAbs generally show similar immunogenicity as chimeric antibodies; based on sequence homology chimeric mAbs are sometimes more "human" than humanized mAbs. With the introduction of the regulatory concept of similar biological medicines (biosimilars) a key concern is the similarity in terms of immunogenicity of these biosimilars with their originators. This review focuses briefly on the mechanisms of induction of immunogenicity by biopharmaceuticals, mAbs in particular, in relation to the target of the immune system.

Purpose: Filgrastim, a recombinant human granulocyte-colony stimulating factor, is widely used to treat congenital and acquired neutropenia. Following patent expiration of the innovator filgrastim product, biosimilar filgrastim products have been approved in the EU and shown to be comparable with the innovator with respect to quality, safety and efficacy. In less regulated markets, copy filgrastim products are available but data about their quality are scarce. In the present study, we provide a head-to-head comparative study on the quality of biosimilar and copy filgrastim products., Methods: Innovator filgrastim product, Neupogen®, two EU-licensed biosimilars, Zarzio® and Tevagrastim®, and two copy filgrastim products, Biocilin® and PDgrastim®, were subjected to peptide mapping, circular dichroism spectroscopy, fluorescence spectroscopy, sodium dodecyl sulfate polyacrylamide gel electrophoresis, high performance size-exclusion chromatography, reversed-phase ultra-performance liquid chromatography, endotoxin test, flow imaging microscopy and in vitro potency assay., Results: Zarzio® and Tevagrastim® have comparable quality to Neupogen®, while Biocilin® showed a significantly lower and PDgrastim® a higher specific activity. Moreover, PDgrastim® showed a higher level of impurities and a lower thermo stability than the other products., Conclusions: Except for the deviating specific activities of the two copy filgrastim products, we found no substantial differences in product quality between the filgrastim products studied.

The aim of the study was to examine the reproducibility of a rat model to assess the preclinical similarity in safety profiles and tissue accumulation of iron products. Accordingly, the effect of several doses of intravenously administered Venofer® and of Ferrlecit® on blood parameters, and on kidney and particularly liver toxicity were examined in non-anemic Sprague Dawley rats. The different analysis showed neither a clear treatment nor a dose effect after multiple injections. The parameters measured in this rat strain showed some iron induced adverse effects, but these could not be correlated to treatment specific differences. The findings presented in this paper indicate the difficulty to define a useful preclinical model to evaluate iron-based nano-colloidal preparations., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Polyphosphate is an inorganic polymer that can potentiate several interactions in the blood coagulation system. Blood platelets contain polyphosphate, and the secretion of platelet-derived polyphosphate has been associated with increased thrombus formation and activation of coagulation factor XII. However, the small polymer size of secreted platelet polyphosphate limits its capacity to activate factor XII in vitro. Thus, the mechanism by which platelet polyphosphate contributes to thrombus formation remains unclear. Using live-cell imaging, confocal and electron microscopy, we show that activated platelets retain polyphosphate on their cell surface. The apparent polymer size of membrane-associated polyphosphate largely exceeds that of secreted polyphosphate. Ultracentrifugation fractionation experiments revealed that membrane-associated platelet polyphosphate is condensed into insoluble spherical nanoparticles with divalent metal ions. In contrast to soluble polyphosphate, membrane-associated polyphosphate nanoparticles potently activate factor XII. Our findings identify membrane-associated polyphosphate in a nanoparticle state on the surface of activated platelets. We propose that these polyphosphate nanoparticles mechanistically link the procoagulant activity of platelets with the activation of coagulation factor XII., (© 2017 by The American Society of Hematology.)

Approximately a dozen of intravenous iron nanomedicines gained marketing authorization in the last two decades. These products are generally considered as safe, but have been associated with an increased risk for hypersensitivity-like reactions of which the underlying mechanisms are unknown. We hypothesized that iron nanomedicines can trigger the innate immune system. We hereto investigated the physico-chemical properties of ferric gluconate, iron sucrose, ferric carboxymaltose and iron isomaltoside 1000 and comparatively studied their interaction with Toll-like receptors, the complement system and peripheral blood mononuclear cells. Two out of four formulations appeared as aggregates by Scanning Transmission Electron Microscopy analysis and were actively taken up by HEK293T- and peripheral blood mononuclear cells in a cholesterol-dependent manner. These formulations triggered in vitro activation of intracellular Toll-like receptors 3, -7 and -9 in a dose- and serum-dependent manner. In parallel experiments, we determined that these compounds activated the complement system. Finally, we found that uptake of aggregation-prone iron nanomedicines by peripheral blood mononuclear cells in whole blood induced production of the proinflammatory cytokine IL-1β, but not IL-6., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Many biopharmaceutical products exhibit extensive structural micro-heterogeneity due to an array of co-occurring post-translational modifications. These modifications often effect the functionality of the product and therefore need to be characterized in detail. Here, we present an integrative approach, combining two advanced mass spectrometry-based methods, high-resolution native mass spectrometry and middle-down proteomics, to analyse this micro-heterogeneity. Taking human erythropoietin and the human plasma properdin as model systems, we demonstrate that this strategy bridges the gap between peptide- and protein-based mass spectrometry platforms, providing the most complete profiling of glycoproteins. Integration of the two methods enabled the discovery of three undescribed C-glycosylation sites on properdin, and revealed in addition unexpected heterogeneity in occupancies of C-mannosylation. Furthermore, using various sources of erythropoietin we define and demonstrate the usage of a biosimilarity score to quantitatively assess structural similarity, which would also be beneficial for profiling other therapeutic proteins and even plasma protein biomarkers.

Biosimilars are considered to be one of the solutions to combat the substantially increasing costs of cancer treatment, and its imminent introduction is expected to expand affordability worldwide. However, biosimilar monoclonal antibodies provide many challenges compared with first-generation biosimilars, growth factors, and hormones, because they have shown only a modest clinical effect, and are often used in combination with other more toxic therapies, making it difficult to design studies that allow appropriate efficacy and safety assessments compared with the original products. The value of comparative clinical trials for showing clinical equivalence of biosimilars that demonstrate a high degree of similarity in physical, chemical, structural, and biological characteristics with the original product is increasingly being questioned, and advances in analytical methods that provide robust non-clinical data might reduce the need for extensive clinical comparisons. In this Series paper, the third of three papers on drug safety in oncology, we review the safety and efficacy of biosimilars in oncology, assessing biosimilar monoclonal antibodies in relation to first-generation biosimilars, the issues surrounding interchangeability and extrapolation of biosimilars to other disease and patient indications, and reassessing the safety approval pathway in light of 10 years worth of biosimilar experience., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Anemia resulting from iron deficiency is one of the most prevalent diseases in the world. As iron has important roles in several biological processes such as oxygen transport, DNA synthesis and cell growth, there is a high need for iron therapies that result in high iron bioavailability with minimal toxic effects to treat patients suffering from anemia. This study aims to develop a novel oral iron-complex formulation based on hemin-loaded polymeric micelles composed of the biodegradable and thermosensitive polymer methoxy-poly(ethylene glycol)-b-poly[N-(2-hydroxypropyl)methacrylamide-dilactate], abbreviated as mPEG-b-p(HPMAm-Lac 2 ). Hemin-loaded micelles were prepared by addition of hemin dissolved in DMSO:DMF (1:9, one volume) to an aqueous polymer solution (nine volumes) of mPEG-b-p(HPMAm-Lac 2 ) followed by rapidly heating the mixture at 50°C to form hemin-loaded micelles that remain intact at room and physiological temperature. The highest loading capacity for hemin in mPEG-b-p(HPMAm-Lac 2 ) micelles was 3.9%. The average particle diameter of the hemin-micelles ranged from 75 to 140nm, depending on the concentration of hemin solution that was used to prepare the micelles. The hemin-loaded micelles were stable at pH 2 for at least 3 h which covers the residence time of the formulation in the stomach after oral administration and up to 17 h at pH 7.4 which is sufficient time for uptake of the micelles by the enterocytes. Importantly, incubation of Caco-2 cells with hemin-micelles for 24 h at 37°C resulted in ferritin levels of 2500ng/mg protein which is about 10-fold higher than levels observed in cells incubated with iron sulfate under the same conditions. The hemin formulation also demonstrated superior cell viability compared to iron sulfate with and without ascorbic acid. The study presented here demonstrates the development of a promising novel iron complex for oral delivery., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Determining to what extent biophysical characteristics of aggregates affect immunogenicity of therapeutic interferon beta-1b. Three recombinant human interferon beta-1b (rhIFNβ-1b) samples with different levels of aggregates generated by copper oxidation, thermal stress, or left untreated, as well as Avonex(®) drug substance and Betaferon(®) drug product, were injected intraperitoneally in nontransgenic and interferon beta transgenic FVB/N mice 5 times per week for 3 weeks. Antibodies against interferon beta were measured using enzyme-linked immunosorbent assay. UV and fluorescence spectroscopy, dynamic light scattering, size exclusion chromatography, reversed-phase high-performance liquid chromatography (RP-HPLC), fluid imaging microscopy, and resonant mass measurement, as well as sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting, were used to characterize and quantitate aggregates in the 3 rhIFNβ preparations, to correlate biophysical characteristics with immunogenicity. In immune-tolerant interferon beta transgenic FVB/N mice, Betaferon drug product showed the highest immunogenicity, while Avonex drug substance showed the lowest level of immunogenicity. Of the 3 forms of rhIFNβ-1b, copper-oxidized rhIFNβ-1b showed lower immunogenicity than thermally stressed rhIFNβ-1b, despite containing larger aggregates. Both copper-oxidized rhIFNβ-1b and thermally stressed rhIFNβ-1b exhibited changes in protein structure as shown using fluorescence spectroscopy and RP-HPLC. Nontransgenic, nonimmune-tolerant FVB/N mice generated high antibody titers against all interferon beta samples tested. The level of immunogenicity and the breaking of tolerance in FVB/N transgenic mice are not only related to the level of aggregation but also depend on the size and structure of the aggregates.

Human serum albumin (HSA)-free formulation of Escherichia coli-derived human interferon beta (IFNβ-1b) with a high percentage of monomeric protein and low immunogenicity is developed and characterized in the current study. UV spectroscopy, fluorescence spectroscopy, dynamic light scattering, sodium dodecyl sulfate polyacrylamide gel electrophoresis, Western blotting, Micro-Flow Imaging, resonant mass measurement, size exclusion, and reversed-phase high performance liquid chromatographies were applied to assess the effect of excipients on the stability of IFNβ-1b to establish a HSA-free formulation. The antiviral activity of IFNβ-1b was evaluated using human lung carcinoma cell line. Immune tolerant mice to hIFNβ were used to assess the immunogenicity of the HSA-free formulated IFNβ-1b in comparison to Betaferon drug product and Avonex drug substance as standards through IgG titering of plasma. HSA-free formulated IFNβ-1b, including 200 mM L-arginine, 200 mM trehalose, and 0.1% n-dodecyl β-D-maltoside in 10 mM sodium acetate buffer, pH 7.4, showed the highest biological activity. The stability of IFNβ-1b in the HSA-free formulation was monitored for 3 weeks at 4°C and 37°C with relative humidity of 10% and 75%, respectively. Protein aggregation and immunogenicity in transgenic mice were decreased in the HSA-free formulated IFNβ-1b compared to Betaferon. The stability, biological activity, and immunogenicity of the HSA-free formulation and Betaferon were evaluated. Incubation of formulations at 4°C and 37°C for 3 weeks showed that the HSA-free formulated IFNβ-1b was more stable and less immunogenic in transgenic FVB/N mice. Low immunogenicity and the absence of HSA, which reduces the potential risk of viral infection (eg, HIV and HCV), are promising for clinical studies.

Comprehensive physicochemical characterization and biological assays are essential parts in assessing quality attributes of biologicals. Here, we compared the quality of different marketed recombinant human erythropoietin (epoetin) products: originators, Eprex and NeoRecormon as well as 2 biosimilars, Retacrit and Binocrit. In addition, assessment of batch-to-batch variability was included by collecting 2 or more batches of each product. Common assays which included sodium dodecyl sulfate-polyacrylamide gel electrophoresis, high-performance size-exclusion chromatography, asymmetrical flow field-flow fractionation, capillary zone electrophoresis, and potency testing were used. Of the tested products and among batches of single products, variations in epoetin content, isoform profiles, and potency were found. Ultimately, this study demonstrated the high quality of epoetin products with some degree of variation among products and batches, confirming the "similar but not identical" paradigm of biologicals., (Copyright © 2016. Published by Elsevier Inc.)

Protein glycosylation is among the most common and well-defined post-translational modifications due to its vital role in protein function. Monitoring variation in glycosylation is necessary for producing more effective therapeutic proteins. Glycans attached to glycoproteins interact highly specific with lectins, natural carbohydrate-binding proteins, which property is used in the current label-free methodology. We have established a lectin microarray for label-free detection of lectin-carbohydrate interactions allowing us to study protein glycosylation directly on unmodified glycoproteins. The method enables simultaneous measurement of up to 96 lectin-carbohydrate interactions on a multiplex surface plasmon resonance imaging platform within 20 min. Specificity determination of lectins succeeded by analysis of neoglycoproteins and enzymatically remodeled glycoproteins to verify carbohydrate binding. We demonstrated the possibilities for glycosylation fingerprinting by comparing different Erythropoietin sources without the need for any sample pretreatment and we were able to accurately quantify relative sialylation levels of Erythropoietin.

The European Union (EU) was the first region to establish a regulatory framework for biosimilars, in which animal studies are required to confirm similarity to a reference product. However, animal studies described in European public assessment reports (EPARs) or marketing authorization applications (MAAs) did not identify clinically or toxicologically relevant differences despite differences in quality, suggesting that animal studies lack the sensitivity to confirm biosimilarity. Scientific advice provided learning opportunities to evolve existing guidance. Altogether, the data support a step-wise approach to develop biosimilars that focuses on quality and clinical efficacy of biosimilar. This approach might be more effective and does not necessarily require animal studies, which is also reflected in new EU draft guidance., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Immunological processes leading to formation of antidrug antibodies (Abs) against recombinant human proteins remain poorly understood. Animal and clinical studies revealed that immunogenicity shares both T-cell-dependent (requirement of CD4(+) T cells, isotype switching) and T-cell-independent (involvement of Marginal Zone B cells, apparent lack of memory) characteristics. We used immune tolerant mice to study the mechanism underlying immunogenicity in more detail. We found that CD4(+) T cells were crucial at early stages of Ab responses against rhIFNβ. In addition, we found a similar number of germinal centers (GCs) in spleen after rhIFNβ treatment as after treatment with a foreign protein. However, neither Ab titers nor the number of GCs was increased by adsorption of rhIFNβ on aluminum hydroxide. Therefore, we tested the effect of several immune adjuvants in a follow-up study. We found that only conjugation of rhIFNβ to a carrier protein (cholera toxin subunit B) was effective in boosting Ab titers. However, these conjugates failed to trigger rhIFNβ specific memory formation. Our findings show that early events of the immunogenicity reaction to self-proteins are CD4(+) T-cell dependent. Nevertheless, despite those similarities, immunogenicity of human proteins is clearly not a classical CD4(+) T-cell-dependent response., (© 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.)

Introduction: Biosimilars are biologic products that receive authorization based on an abbreviated regulatory application containing comparative quality and nonclinical and clinical data that demonstrate similarity to a licensed biologic product. Extrapolation of safety and efficacy has emerged as an important way to simplify biosimilar development. Regulatory authorities have generally reached the consensus that extrapolation of similarity from one indication to other approved indications of the reference product can be permitted if it is scientifically justified., Areas Covered: Recently, the first biosimilar, biosimilar infliximab (Remsima/Inflectra) to the innovator monoclonal antibody infliximab (Remicade), was approved in the European Union, Canada and South Korea; the USA subsequently approved its first biosimilar, a less complex molecule (filgrastim-sndz). Based on two clinical trials of biosimilar infliximab in patients with rheumatoid arthritis and ankylosing spondylitis, the European Medicines Agency allowed extrapolation to all eight approved indications for innovator infliximab, whereas Health Canada did not permit extrapolation to the indications for ulcerative colitis and Crohn's disease. These differing decisions on extrapolation of indications for biosimilar infliximab highlight important unanswered regulatory and scientific questions. Here, we propose substantive scientific considerations for indication extrapolation., Expert Opinion: The preclinical and clinical criteria that are currently required to merit indication extrapolation have not been rigorously evaluated.

Interferon beta may induce antibodies in multiple sclerosis patients and the incidence of immunogenicity depends on the type of product. These antibodies can reduce the efficacy of interferon beta. Two transgenic immune tolerant mouse models for human interferon beta (hIFNβ) (C57Bl/6, and C57Bl/6×FVB/N F1 hybrid mice) have been developed previously for studying immunogenicity. These models, however, may not be used for every interferon beta product because of the lack of immunogenicity in the wildtype genetic background. We therefore developed a modified transgenic mouse model by backcrossing the F1 hybrid C57Bl/6×FVB/N transgenic mice with wildtype FVB/N for 10 generations. These F10 offspring (referred to hear as FVB/N) have a genetic background consisting of mostly FVB/N (99.9%) and very little C57Bl/6 (0.1%), and are expected to have the more sensitive antibody producing phenotype of the parental FVB/N strain. The newly generated "FVB/N" strain was assessed for antibody formation against different rhIFNβ formulations compared to the C57Bl/6, and C57Bl/6×FVB/N transgenic mouse models. The new FVB/N transgenic mouse model was more sensitive for all tested rhIFNβ products, and the difference in antibody titers between the transgenic and non-transgenic mice of the FVB/N strain was much bigger compared to the antibody levels of the C57Bl/6, and C57Bl/6×FVB/N strains., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Conjugation of polyethylene glycol (PEG) to therapeutics has proven to be an effective approach to increase the serum half-life. However, the increased use of PEGylated therapeutics has resulted in unexpected immune-mediated side-effects. There are claims that these are caused by anti-PEG antibodies inducing rapid clearance. These claims are however hampered by the lack of standardized and well-validated antibody assays. PEGylation has also been associated with the activation of the complement system causing severe hypersensitivity reactions. Here, we critically review the clinical and analytical tools used. In addition, we propose an explanation of the immune-mediated side-effects of PEGylated products based on the haptogenic properties of PEG, responsible for complement activation and the induction of anti-PEG antibodies., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Understanding the mechanism of aggregation of a therapeutic protein would not only ease the manufacturing processing but could also lead to a more stable finished product. Aggregation of recombinant interferon (IFNβ-1b) was studied by heating, oxidizing, or seeding of unformulated monomeric solution. The formation of aggregates was monitored by dynamic light scattering (DLS) and UV spectroscopy. The autocatalytic monomer loss model was used to fit the data on aggregation rates. The influence of pre-nucleation on aggregation step was demonstrated by inducing the liquid samples containing a monomer form of folded IFNβ-1b by heat and also an oxidizing agent. Results tend to suggest that the nucleus includes a single protein molecule which has been probably deformed. Seeding tests showed that aggregation of IFNβ-1b was probably initiated when 1.0% (w/w) of monomers converted to nucleus form. Chemiluminescence spectroscopy analysis of the sample indicated the generation of 3.0 μM of hydrogen peroxide (H2O2) during nucleation stage of IFNβ-1b aggregation. Arginine with a concentration of 200 mM was sufficient to suppress aggregation of IFNβ-1b by decreasing the rate of pre-nucleation step. We proposed the formation of pre-nucleus structures prior to nucleation as the mechanism of aggregation of IFNβ-1b. Furthermore, we have showed the positive anti-aggregation effect of arginine on pre-nucleation step.

Drug development has become the exclusive activity of large pharmaceutical companies. However, the output of new drugs has been decreasing for the past decade and the prices of new drugs have risen steadily, leading to access problems for many patients. By analyzing the history of drug development and the pharmaceutical industry, we identified the main factors causing this system failure. Although many solutions have been suggested to fix the drug development system, we believe that a combination of reforms of the regulatory and patent systems is necessary to make drug development sustainable. These reforms must be combined with a larger, open-access role for public research institutes in the discovery, clinical evaluation and safety evaluation of new drugs., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Aggregation often occurs during manufacturing and storage of protein drugs. Detergents such as sodium dodecyl sulfate are commonly used to prevent aggregation but need to be eliminated before final formulation for safety reasons. We studied the ability of dodecylmaltoside (DDM), a nontoxic alkyl saccharide surfactant, to reduce aggregation and increase the stability of interferon beta-1b (IFN)-β-1b. An increase of 8°C in the Tm of IFN-β-1b was observed when 0.1% of DDM was present in the protein solution. The absorption of DDM on hydrophobic surfaces of IFN-β-1b enables the surface to become hydrophilic and non-ionic, and increases the stability of the protein. 0.1% DDM also results in a 62% increase in helical and a 25% decrease in β-sheet structures. 0.1% DDM not only suppresses aggregate formation but also improves IFN-β-1b solubilization. Furthermore, we have showed the protective effect of DDM on the anti-viral activity of IFN-β-1b in solution.

Mouse models are commonly used to assess the immunogenicity of therapeutic proteins and to investigate the immunological processes leading to antidrug antibodies. The aim of this work was to develop a transgenic (TG) Balb/c mouse model for evaluating the immunogenicity of recombinant human insulin (insulin) formulations. Validation of the model was performed by measuring the antibody response against plain and particulate insulin in TG and nontransgenic (NTG) mice. Intraperitoneal administration of insulin (20 μg/dose, 12 doses over a period of 4 weeks) did not break the immune tolerance of the TG mice, whereas it did elicit antibodies in NTG mice. The immune tolerance of TG mice could be circumvented, albeit at low titers, by administering insulin covalently bound to 50-nm polystyrene nanoparticles. The TG mouse model was employed to compare the immunogenicity of oxidized aggregated insulin, oxidized nonaggregated insulin, and three commercially available formulations of insulin variants (i.e., Levemir®, Insulatard®, and Actrapid®). Oxidized insulin, aggregated or nonaggregated, was moderately immunogenic in TG mice (50% and 33% responders, respectively), whereas the immunogenicity of the commercial formulations was low. This model can be used to compare the immunogenicity of insulin formulations and to study immune mechanisms of antibody formation against insulin., (© 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.)

Purpose: The high prevalence of pure red cell aplasia in Thailand has been associated with the sharp increase in number of recombinant human erythropoietin (rhEPO) copy products, based on a classical generic regulatory pathway, which have entered the market. This study aims to assess the quality of rhEPO copy products being used in Thailand., Methods: Twelve rhEPO copy products were purchased from pharmacies in Thailand, shipped under controlled cold chain conditions to the Netherlands and characterized using (1) high performance size-exclusion chromatography, (2) asymmetrical flow field-flow fractionation, (3) sodium dodecyl sulfate polyacrylamide gel electrophoresis in combination with (4) Western blotting and additionally tested for (5) host cell protein impurities as well as (6) endotoxin contamination., Results: Some of the tested rhEPO copy products showed high aggregate levels and contained a substantial amount of protein fragments. Also, one of rhEPO copy products had a high endotoxin level, exceeding the FDA limit., Conclusions: Our observations show that some of the tested copy products on the Thai market differ significantly from the originator rhEPO product, Epogen®. This comparison study supports a link between the quality attributes of copy rhEPO products and their immunogenicity.

Protein aggregates are a major risk factor for immunogenicity. Until now most studies on aggregate-driven immunogenicity have focused on linking physicochemical features of the aggregates to the formation of anti-drug antibodies. Lacking is however, basic knowledge on the effect of aggregation on the biodistribution and clearance of therapeutic proteins in vivo. The aim of current study was to get insight into the effect of aggregation on biodistribution in mice using different routes of administration. Fluorescently labeled stressed and unstressed mouse serum albumin was injected via different routes in mice and detected via in vivo fluorescence imaging up to 48 hrs post-injection. We found that biodistribution of stressed MSA significantly differed from its unstressed counterpart. Subcutaneous and intramuscular administration resulted in accumulation of protein at the site of injection, from which clearance of stressed MSA was considerably slower than clearance of unstressed MSA. Upon intravenous and intraperitoneal injection of stressed MSA, fluorescent "hotspots" were observed in the spleens, livers and lungs. Further and more detailed examination of biodistribution after intraperitoneal injection showed higher fluorescence in most of tested organs suggesting more efficient diffusion and/or lymphatic uptake from peritoneum of unstressed MSA than the stressed formulation.

The aim of this critical review is to reach a global consensus regarding the introduction of follow-on versions of nonbiological complex drugs (NBCD). A nonbiological complex drug is a medicinal product, not being a biological medicine, where the active substance is not a homo-molecular structure, but consists of different (closely related and often nanoparticulate) structures that cannot be isolated and fully quantitated, characterized and/or described by state of the art physicochemical analytical means and where the clinical meaning of the differences is not known. The composition, quality and in vivo performance of NBCD are highly dependent on manufacturing processes of both the active ingredient as well as in most cases the formulation. The challenges posed by the development of follow-on versions of NBCD are illustrated in this paper by discussing the 'families' of liposomes, iron-carbohydrate ('iron-sugar') drugs and glatiramoids. It is proposed that the same principles for the marketing authorization of copies of NBCD as for biosimilars be used: the need for animal and/or clinical data and the need to show similarity in quality, safety and efficacy. The regulatory approach of NBCD will have to take into consideration the specific characteristics of the drugs, their formulation and manufacturing process and the resulting critical attributes to achieve their desired quality, safety and efficacy. As with the biosimilars, for the NBCD product, family-specific methods should be evaluated and applied where scientifically proven, including sophisticated quality methods, pharmacodynamic markers and animal models. Concerning substitution and interchangeability of NBCD, it is also advisable to take biosimilars as an example, i.e. (1) substitution without the involvement of a healthcare professional should be discouraged to ensure traceability of the treatment of individual patients, (2) keep an individual patient on a specific treatment if the patient is doing well and only switch if unavoidable and (3) monitor the safety and efficacy of the new product if switching occurs.

In the last decade, discussions on the development of the regulatory framework of generic versions of complex drugs such as biologicals and non-biological complex drugs have attracted broad attention. The terminology used is far from harmonized and can lead to multiple interpretations of legal texts, reflection papers, and guidance documents regarding market introduction as well as reimbursement. This article describes the meaning of relevant terms in different global regions (Europe, USA, WHO) and offers a proposal for a globally accepted terminology regarding (non-) biological complex drugs.

The availability of biosimilar insulins can potentially lead to lower insulin costs and increased access for patients with diabetes, worldwide. However, clinicians and regulatory agencies have raised several concerns regarding the safety and efficacy of these new medications. The European regulatory agencies have established guidelines for market approval of biosimilar insulins; however, many issues remain unresolved. Moreover, although the FDA has developed preliminary pathways for biosimilar protein development and is prepared to review each application on a case-by-case basis, insulins do not fall under this pathway at this time. The development of effective postmarketing surveillance protocols, determination of product interchangeability, and product identification/labeling remain key concerns. Numerous issues surround the development and commercialization of biosimilar insulins; thus, it is important that all stakeholders fully understand the complexity of these issues and how they can potentially affect patient care. Bridging the educational gap among clinicians and regulatory agencies will be challenging but necessary for ensuring patient safety., (© 2014 Diabetes Technology Society.)

The immunogenicity of biopharmaceuticals used in clinical practice remains an unsolved challenge in drug development. Non-human primates (NHPs) are often the only relevant animal model for the development of monoclonal antibodies (mAbs), but the immune response of NHPs to therapeutic mAbs is not considered to be predictive of the response in humans because of species differences. In this study, we accessed the drug registration files of all mAbs registered in the European Union to establish the relative immunogenicity of mAbs in NHPs and humans. The incidence of formation of antidrug-antibodies in NHPs and patients was comparable in only 59% of the cases. In addition, the type of antidrug-antibody response was different in NHP and humans in 59% of the cases. Humanization did not necessarily reduce immunogenicity in humans. Immunogenicity interfered with the safety assessment during non-clinical drug development when clearing or neutralizing antibodies were formed. While important to interpret the study results, immunogenicity reduced the quality of NHP data in safety assessment. These findings confirm that the ability to compare relative immunogenicity of mAbs in NHPs and humans is low. Furthermore, immunogenicity limits the value of informative NHP studies.

Biosimilars have been available on the European market since 2006 and experience with their use is increasing. The next wave of biopharmaceuticals that are about to lose patent protection consists of more-complicated products, including many monoclonal antibodies. Guidance has been released on the particulars of a biosimilarity exercise involving these products. Considerable challenges exist to establish biosimilarity for anticancer products. An especially challenging product is bevacizumab (Avastin(®)). On the basis of data available for the innovator product (bevacizumab) we will discuss strengths and weaknesses of preclinical and clinical models and explore the application of novel endpoints to the biosimilar comparability exercise., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

An increasing number of pegylated therapeutic proteins and drug targeting compounds are being introduced in the clinic. Pegylation is intended to increase circulation time and to reduce an immunogenic response. Recently however a number of publications have appeared claiming that the polyethylene glycol (PEG) moiety of these products in itself may be immunogenic and that the induced anti-PEG antibodies are linked to enhanced blood clearance and reduced efficacy of the products. A critical review of the literature shows that most, if not all assays for anti-PEG antibodies are flawed and lack specificity. Also the biological effects induced by anti-PEG antibodies lack the characteristics of a bona fide antibody reaction. Standardization of the anti-PEG assays and the development of reference sera are urgently needed.

Purpose: Interferon beta is commonly used as therapeutic in the first line of therapy for multiple sclerosis. However, depending on the product, it induces an antibody response in up to 60% of patients. This study evaluated the impact of therapy related factors like dose, route of administration and administration frequency on the immunogenicity of one of the originator interferon beta drugs (Betaferon®) in an immune tolerant transgenic mouse model., Methods: Immune tolerant transgenic mice received injections with Betaferon® via different routes, doses and injection frequencies. Anti-drug antibody (ADA) production was measured by ELISA to assess immunogenicity., Results: A single injection of Betaferon® was found to be sufficient for the induction of ADAs. The antibody titer was enhanced with increasing dose and treatment frequency. Among the tested administration routes, the intravenous route was the most immunogenic one, which is in contradiction with one of the dogma in immunogenicity research according to which subcutaneous administration is the most immunogenic route. Intramuscular, intraperitoneal and subcutaneous injections resulted in comparable immunogenicity., Conclusion: This study shows that treatment related factors affect significantly immunogenicity of Betaseron® and therefore substantiate the need for further studies on these factors in patients.

Background: The nature of adverse drug reactions observed post-authorization for biopharmaceuticals differs from that observed for chemically synthesized, small molecules (SMs). However, it remains unclear how much of the observed differences can be attributed to differences in authorized indications of the two product groups., Objective: To investigate if the nature of adverse drug reactions identified post-authorization for biopharmaceuticals differs from those of SMs within the same anatomical therapeutic chemical (ATC) group., Methods: We analyzed safety issues included in post-authorization, changes to the Summary of Product Characteristics of centrally approved products in the European Union classified in the ATC main group of 'antineoplastic and immunomodulating agents'. Generics and biosimilars were excluded. All issues identified during 2004-2011 were analyzed for differences in nature and timing between biopharmaceutical and SM products, at different ATC levels., Results: A total of 747 adverse drug reactions were identified; 361 for biopharmaceuticals and 386 for SMs. Within the sub group of immunosuppressants, neoplasms (20 % vs 2 %, p < 0.01) and infections and infestations (22 % vs 9 %, p < 0.01) occurred significantly more frequent for biopharmaceuticals. Adverse drug reactions of SMs were more often renal and urinary disorders (7 % vs 0 %, p < 0.01), blood and lymphatic system disorders (10 % vs 3 %, p = 0.04), and vascular disorders (7 % vs 1 %, p = 0.02). In the subgroup of antineoplastics, immune system disorders occurred more frequently for biopharmaceuticals, (6 % vs 1 %, p = 0.04). With the exception of immune system disorders and renal disorders, the overall differences between biopharmaceuticals and SMs were mostly caused by products authorized as immunosuppressants. For the subset of products authorized after 2004, the median time to the first safety issue was 18 months (95 % CI 12.4-21.5) for biopharmaceuticals and 17 months (95 % CI 12.5-21.5) for SMs and did not differ significantly within subgroups., Conclusion: Even within a group of medicinal products approved in the same indication, differences were observed in the nature of adverse drug reactions between biopharmaceuticals and SMs. The considerable differences in the nature of adverse drug reactions between biopharmaceuticals and SMs were not associated with differences in the timing of regulatory actions.

Purpose: To describe and assess the outcomes of Periodic Safety Update Report (PSUR) evaluations of biopharmaceuticals., Methods: A cross-sectional analysis was performed of follow-up requirements of PSURs submitted for centrally approved biopharmaceuticals in the European Union between 1 July 2008 and 30 June 2010. A follow-up analysis on a subset of products that submitted multiple PSURs within the study period was also performed., Results: The cross-sectional analysis included 70 PSURs. Potential safety concerns occurred in 57 (83 %) of all PSURs, and 26 (37 %) concluded a need to change the Summary of Product Characteristics (SPC). In comparison to newer products, products authorized for more than 10 years contained significantly fewer potential safety concerns (60 vs. 92 %; p < 0.01) and required fewer SPC changes (15 vs. 46 %; p = 0.03). For 45 products, multiple PSURs were submitted that could be included in a follow-up analysis. For this subset of products, of the 106 newly identified safety potential safety issues, 7 (7%) resulted in requirements for label changes in the following PSUR., Conclusions: PSURs facilitate communication between regulators and marketing authorization holders. Potential safety concerns occur for the majority of biopharmaceuticals and throughout their lifecycle, but for established products PSUR evaluations rarely lead to regulatory actions.

Purpose: The immunological processes underlying immunogenicity of recombinant human therapeutics are poorly understood. Using an immune tolerant mouse model we previously demonstrated that aggregates are a major trigger of the antidrug antibody (ADA) response against recombinant human interferon beta (rhIFNβ) products including Betaferon®, and that immunological memory seems to be lacking after a rechallenge with non-aggregated rhIFNβ. The apparent absence of immunological memory indicates a CD4+ T-cell independent (Tind) immune response underlying ADA formation against Betaferon®. This hypothesis was tested., Methods: Using the immune tolerant mouse model we first validated that rechallenge with highly aggregated rhIFNβ (Betaferon®) does not lead to a subsequent fast increase in ADA titers, suggesting a lack of immunological memory. Next we assessed whether Betaferon® could act as Tind antigen by inactivation of marginal zone (MZ) B-cells during treatment. MZ B-cells are major effector cells involved in a Tind immune response. In a following experiment we depleted the mice from CD4+ T-cells to test their involvement in the ADA response against Betaferon®., Results: Inactivation of MZ B-cells at the start of Betaferon® treatment drastically lowered ADA levels, suggesting a Tind immune response. However, persistent depletion of CD4+ T-cells before and during Betaferon® treatment abolished the ADA response in almost all mice., Conclusion: The immune response against rhIFNβ in immune tolerant mice is neither a T-cell independent nor a classical T-cell dependent immune response. Further studies are needed to confirm absence of immunological memory (cells).

The value of animal studies to assess drug safety is unclear because many such studies are biased and have methodological shortcomings. We studied whether post-marketing serious adverse reactions to small molecule drugs could have been detected on the basis of animal study data included in drug registration files. Of 93 serious adverse reactions related to 43 small molecule drugs, only 19% were identified in animal studies as a true positive outcome, which suggests that data from animal studies are of limited value to pharmacovigilance activities. Our study shows that drug registration files can be used to study the predictive value of animal studies and that the value of animal studies in all stages of the drug development should be investigated in a collaborative endeavour between regulatory authorities, industry, and academia., (Copyright © 2012 Elsevier Inc. All rights reserved.)