Huub Schellekens

Netherlands Institute for Space Research, Utrecht, Utrecht, Utrecht, Netherlands

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Publications (122)697.96 Total impact

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    ABSTRACT: 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.
    Journal of immunological methods. 12/2014; 415:17-23.
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    ABSTRACT: 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 authorisation 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.
    Drug Discovery Today. 11/2014;
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    ABSTRACT: 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 J Pharm Sci
    Journal of Pharmaceutical Sciences 09/2014; · 3.13 Impact Factor
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    ABSTRACT: 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.
    Drug Discovery Today 09/2014; · 6.55 Impact Factor
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    ABSTRACT: 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.
    AAPS PharmSciTech 08/2014; · 1.58 Impact Factor
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    ABSTRACT: 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.
    Journal of Interferon & Cytokine Research 06/2014; 34(11):894-901. · 3.30 Impact Factor
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    ABSTRACT: 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 J Pharm Sci
    Journal of Pharmaceutical Sciences 03/2014; · 3.13 Impact Factor
  • Ellen H.M. Moors, Adam F. Cohen, Huub Schellekens
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    ABSTRACT: 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.
    Drug Discovery Today 02/2014; · 6.55 Impact Factor
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    ABSTRACT: 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.
    PLoS ONE 01/2014; 9(1):e85281. · 3.53 Impact Factor
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    Jacques Rottembourg, Huub Schellekens
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    ABSTRACT: When the patent of a classical small molecule drug expires, generics can be marketed if their therapeutic equivalence to the original drug has been established. 1,2 Conventional generics for an orally administered drug are considered to be therapeutically equivalent to a reference once pharmaceutical equivalence (that is, identical active substances) and bioequivalence (that is, comparable pharmacokinetics essentially on healthy volunteers) have been established in a crossover study, and do not require formal clinical efficacy and safety studies. The therapeutic equivalence allows therapeutic interchangeability. 3 The acceptance intervals to show that bioequivalence for the logarithm transformed are under the curve and C max ratios lie within an acceptance range of 0.80–1.25 for the 90% confidence intervals. In some cases, the acceptance interval needs to be tightened, and in other cases a wider acceptance may be acceptable. The classical generic approach based on showing pharmaceutical equivalence and bioequivalence has been the basis of the introduction of many safe and effective alternatives to innovative medicines. However, this approach has so far only been applied to products that can be fully characterised. For more complex molecules that are difficult to characterise, such as proteins, the demonstration of bioequivalence requires an alternative approach. Therapeutic proteins are the most widely used class of drugs for which the classical generic paradigm cannot be used. 4,5 The molecular mass of most of the therapeutic proteins varies from 5 to 150kDa, which is 25–1000-times greater than the average small drug molecule. Nearly all therapeutic proteins are produced by living cells and these cells in general make mixtures of different proteins, for example, by variation in the process of post-translation modification, such as glycosylation. There are many tools to analyse the structure and in vitro activity of therapeutic proteins (biologics). However, because of their complexity there is no combination of techniques that can completely describe
    Hosp Nephrol Europ. 01/2014; 2012(3):37-40.
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    ABSTRACT: 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. 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. 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. 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.
    Pharmaceutical Research 11/2013; · 4.74 Impact Factor
  • Nature Biotechnology 10/2013; 31(10):882-883. · 32.44 Impact Factor
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    ABSTRACT: 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 AAPS Journal 09/2013; · 4.39 Impact Factor
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    ABSTRACT: 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.
    The AAPS Journal 09/2013; · 4.39 Impact Factor
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    ABSTRACT: Advanced therapy medicinal products (ATMPs) are the cutting edge of drug innovation. ATMPs have different challenges than other drug classes. To accommodate these challenges and facilitate science-driven development, flexibility in the requirements to demonstrate the safety and efficacy of this rapidly evolving drug class is necessary. To create flexibility, the European Union introduced the risk-based approach. This approach provides the possibility of omitting guideline-based studies based on risk analyses. To gain insight into the effect of the risk-based approach on the non-clinical development of ATMPs, two questions are addressed in this paper. Firstly, "Do companies use a risk-based approach for the non-clinical development of ATMPs?" and, secondly, "Does the Committee for Medicinal Products for Human Use(CHMP) of the European Medicines Agency (EMA) accept non-clinical development programs based on the risk-based approach?". Scientific advice letters formulated by the CHMP were analyzed. The risk-based approach was used to justify deviations from the guidelines in the majority (75%) of the cases. The CHMP accepted 40% of the proposals to omit studies and stated that additional data was necessary to make an informed decision for 35% of the proposals. This indicates that the risk-based approach facilitates the science-driven development of ATMPs.
    Regulatory Toxicology and Pharmacology 08/2013; · 2.13 Impact Factor
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    ABSTRACT: 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.
    mAbs 06/2013; 5(5). · 5.28 Impact Factor
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    ABSTRACT: 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 are posed by anticancer products and there is ongoing controversy regarding which basis to establish biosimilarity for such products. An especially challenging product is bevacizumab (Avastin®). Based on 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.
    Drug discovery today 05/2013; · 6.63 Impact Factor
  • Huub Schellekens, Wim E Hennink, Vera Brinks
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    ABSTRACT: 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.
    Pharmaceutical Research 05/2013; · 4.74 Impact Factor
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    ABSTRACT: 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.
    BioDrugs 02/2013; · 2.12 Impact Factor
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    ABSTRACT: 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.
    European Journal of Clinical Pharmacology 02/2013; 69(2):217-226. · 2.74 Impact Factor

Publication Stats

3k Citations
697.96 Total Impact Points


  • 2004–2014
    • Netherlands Institute for Space Research, Utrecht
      Utrecht, Utrecht, Netherlands
  • 2002–2014
    • Universiteit Utrecht
      • • Department of Pharmaceutical Sciences
      • • Faculty of Science
      • • Division of Pharmacology
      • • Division of Innovation Studies
      • • Central Laboratory Animals Institute (CLAI)
      Utrecht, Utrecht, Netherlands
  • 2012
    • Erasmus Universiteit Rotterdam
      • Institute for Medical Technology Assessment (iMTA)
      Rotterdam, South Holland, Netherlands
  • 2008–2012
    • Leiden University
      • Leiden Amsterdam Center for Drug Research
      Leiden, South Holland, Netherlands
    • Technische Universität Braunschweig
      • Institut für Technische Chemie
      Braunschweig, Lower Saxony, Germany
    • Azienda Ospedaliero Universitaria San Luigi Gonzaga
      Orbassano, Piedmont, Italy
  • 2011
    • Ecole normale supérieure de Cachan
      Cachon, Île-de-France, France
  • 2010
    • University of Delaware
      Delaware, United States
  • 2006
    • Centre Hospitalier Universitaire de Nancy
      Nancy, Lorraine, France
  • 2005
    • Heinrich-Heine-Universität Düsseldorf
      • Neurologische Klinik
      Düsseldorf, North Rhine-Westphalia, Germany
    • Université Victor Segalen Bordeaux 2
      Burdeos, Aquitaine, France