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Jorgen Nedergaard Larsen - One of the best experts on this subject based on the ideXlab platform.
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Standardization of Allergen Extracts
Allergy, 2019Co-Authors: Jorgen Nedergaard Larsen, Sten DreborgAbstract:Allergens are molecules with the capacity to elicit IgE responses in humans. When stimulated with Allergens, most allergic patients respond with production of IgE specific for several proteins/Allergens in the source material. The Standardization of Allergen extracts is essential in order to control variability and to achieve consistency and reproducibility in a clinical setting. Because the IgE binding capacity of an Allergen extract is related to the content of one or a few major Allergens, it is important that the Standardization procedure ensures consistency, not only in the overall IgE binding potency, but also in the content and ratio of individual major Allergens. Owing to the complexity of Allergen extracts, a key element in Standardization of Allergen extracts is the use of standards. This chapter describes the principles for Standardization of Allergen extracts to be used by research laboratories. Other chapters in this volume describe in vitro methods in detail.
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Absolute quantification of Allergens from complex mixtures: a new sensitive tool for Standardization of Allergen extracts for specific immunotherapy.
Journal of Proteome Research, 2011Co-Authors: Ulla Seppälä, Jorgen Nedergaard Larsen, Claire Dauly, Sarah Robinson, Martin Hornshaw, Henrik IpsenAbstract:Products for specific diagnosis and immunotherapy of IgE-mediated allergies are currently based on natural extracts. Quantification of major Allergen content is an important aspect of Standardization as important Allergens particularly impact vaccine potency. The aim of the study was to develop a mass spectrometry (MS) based assay for absolute quantification of Timothy (Phleum pratense) pollen Allergens Phl p 1 and Phl p 5 in P. pratense extract. High-resolution and accurate mass (HRAM) MS was selected for its ability to detect peptides with high selectivity and mass accuracy (
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absolute quantification of Allergens from complex mixtures a new sensitive tool for Standardization of Allergen extracts for specific immunotherapy
Journal of Proteome Research, 2011Co-Authors: Ulla Seppälä, Jorgen Nedergaard Larsen, Claire Dauly, Sarah Robinson, Martin Hornshaw, Henrik IpsenAbstract:Products for specific diagnosis and immunotherapy of IgE-mediated allergies are currently based on natural extracts. Quantification of major Allergen content is an important aspect of Standardization as important Allergens particularly impact vaccine potency. The aim of the study was to develop a mass spectrometry (MS) based assay for absolute quantification of Timothy (Phleum pratense) pollen Allergens Phl p 1 and Phl p 5 in P. pratense extract. High-resolution and accurate mass (HRAM) MS was selected for its ability to detect peptides with high selectivity and mass accuracy (<3 ppm). Isotope labeled heavy peptides were used for absolute quantification of specific isoAllergens of Phl p 1 and Phl p 5 at low femtomole level in P. pratense extract. Robustness and linearity of the method was demonstrated with intra day precision ≤5% (n = 3). Phl p 1b was shown to be 5 times less abundant than its variant Phl p 1a and Phl p 5b was shown to be 9 times more abundant than the Phl p 5a. The present study shows th...
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Standardization of Allergen-specific immunotherapy vaccines.
Immunology and Allergy Clinics of North America, 2006Co-Authors: Michael D. Spangfort, Jorgen Nedergaard LarsenAbstract:In the 1970s and 1980s, scientific methods were introduced in the Standardization of Allergen vaccines and, in combination with improved documentation of the clinical benefits obtained using standardized vaccines, specific allergy treatment as a scientifically based, reproducible, and safe treatment for allergic disease was established. This article describes important issues in the control of source materials and vaccine preparation as part of the European Standardization of Allergen vaccines, and also includes a discussion of vaccines that are based on recombinant Allergens, which may appear on the market in the near future.
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Recombinant Allergens/Allergen Standardization.
Current Allergy and Asthma Reports, 2001Co-Authors: H. Løwenstein, Jorgen Nedergaard LarsenAbstract:Recombinant Allergens are genetically engineered isoforms representing Allergen molecules from Allergen extracts. Immunologic responses of allergic patients toward Allergen extracts define the major Allergens. For the average allergic patient, the diagnostic sensitivity and treatment efficacy correlate with the concentration of major Allergen. Standardization of Allergen products (extracts or genetically engineered Allergens) can therefore advantageously be performed using a selected recombinant major Allergen as a standard. The Standardization will furthermore require reagents for which both monospecific, monoclonal, or preferably, recombinant antibodies can be used. Due to differences in the Allergenic activity of individual isoAllergens and the naturally occurring mixture of isoAllergens found in an Allergen extract, and due to additional contribution to the Allergenic activity from other molecules in the extract, a biologic potency assessment must always be performed as a supplement. This is also the case for a genetically engineered Allergen product.
Torben Sigsgaard - One of the best experts on this subject based on the ideXlab platform.
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Are Allergen batch differences and the use of double skin prick test important
BMC Pulmonary Medicine, 2015Co-Authors: Gert Thomsen, Lars Rauff Skadhauge, Tine Halsen Malling, David Lee Sherson, Øyvind Omland, Vivi Schlünssen, Torben SigsgaardAbstract:Background Skin prick tests (SPT) are widely used both in clinical diagnostics and in research. The Standardization of Allergen extracts is well documented to be crucial for the validity of SPT, whereas less emphasis has been placed on reproducibility and the SPT procedure itself. The objectives of this study are to clarify how the double skin prick test procedure influence the sensitivity and specificity of the test and to analyse the differences in weal size in skin prick tests between two batches of Allergen extracts from the same vendor.
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Are Allergen batch differences and the use of double skin prick test important?
BMC Pulmonary Medicine, 2015Co-Authors: Gert F Thomsen, Lars Rauff Skadhauge, Tine Halsen Malling, David Lee Sherson, Øyvind Omland, Vivi Schlünssen, Torben SigsgaardAbstract:Background Skin prick tests (SPT) are widely used both in clinical diagnostics and in research. The Standardization of Allergen extracts is well documented to be crucial for the validity of SPT, whereas less emphasis has been placed on reproducibility and the SPT procedure itself. The objectives of this study are to clarify how the double skin prick test procedure influence the sensitivity and specificity of the test and to analyse the differences in weal size in skin prick tests between two batches of Allergen extracts from the same vendor. Methods The association between rhinitis and SPT was assessed among 1135 persons from a general population sample. SPT was performed twice with 10 common aeroAllergens. In a subsample of 90 persons SPT was performed simultaneously with five of the Allergens using different batches. Results Thirty percent had at least one positive SPT. Among asthmatics this number was 62%. Only minor differences were seen between the sizes of two weals from the same batch. A second SPT with the same batch did not change the association between rhinitis and sensitization. When performing SPT with two different batches disagreement was observed in 2% (Birch) to 11% (Cat) of the subjects. Conclusions Performing SPT twice with the same Allergen batch does not enhance the validity of the test, and value of double testing can be questioned. Considerable differences in SPT response with different batches from the same manufacturer were observed. Thus inter batch differences in Allergen extracts might be a source of variability.
S Vieths - One of the best experts on this subject based on the ideXlab platform.
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Standardization of Allergen products 2 detailed characterization of gmp produced recombinant phl p 5 0109 as european pharmacopoeia reference standard
Allergy, 2016Co-Authors: Martin Himly, Angela Neubauer, K H Buchheit, Andreas Nandy, H Kahlert, M Thilker, Markus Steiner, Peter Briza, S Klysner, S ViethsAbstract:Background The Biological Standardization Programme of the European Directorate for Quality of Medicines and Healthcare (EDQM) aims at the establishment of well-characterized reference standards based on recombinant Allergens and validated assays for the quantification of major Allergen content. The objective of this study was to examine the detailed physicochemical and immunological characterization of recombinant Phl p 5.0109, the second available Allergen reference standard. Methods Recombinant Phl p 5.0109 PP5ar06007 was produced under GMP conditions and analyzed by an array of physicochemical and immunological methods for identity, quantity, homogeneity, and folding stability in bulk solution, as well as thermal denaturation, aggregation state, and biological activity when formulated for long-time storage. Results PP5ar06007 revealed as a highly homogeneous, monomeric, well-folded preparation of rPhl p 5.0109, as documented by mass spectrometry, SDS-PAGE, isoelectric focusing, size-exclusion chromatography with light scattering, circular dichroism, and infrared spectroscopy. Upon storage at +4°C, PP5ar06007 retained the monomeric state for at least 2 months. A protein quantity of 1.56 ± 0.03 mg/ml was determined by amino acid analysis in PP5ar06007, and its biological activity was shown to be comparable to natural Phl p 5 in terms of basophil activation and T-cell reactivity. Conclusions Recombinant Phl p 5.0109 PP5ar06007 was characterized extensively at the physicochemical and immunological level. It revealed to be a highly stable, monomeric, and immunologically equivalent of its natural counterpart. PP5ar06007 is now available as European Pharmacopoeia Allergen reference standard for grass pollen products.
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Standardization of Allergen products 1 detailed characterization of gmp produced recombinant bet v 1 0101 as biological reference preparation
Allergy, 2009Co-Authors: Martin Himly, E Nony, Henri Chabre, L Van Overtvelt, Angela Neubauer, K H Buchheit, S Vieths, P Moingeon, Fatima FerreiraAbstract:Background: Standardization of Allergen extracts requires the availability of well-characterized recombinant Allergens, which can be used as reference standards provided by the European regulatory authorities. The objective of this study was the detailed physicochemical and immunological characterization of rBet v 1.0101, which shall be used in a ring trial within the framework of the Biological Standardization Programme BSP090 of the European Directorate for Quality of Medicines and Healthcare. Methods: Recombinant Bet v 1.0101 Y0487 was produced under good manufacturing practice conditions and analysed by an array of physicochemical and immunological methods for identity, quantity, homogeneity, folding and denaturation, aggregation state and stability in solution, as well as biological activity. Results: Batch Y0487 was shown to contain monomeric and well-folded protein being identical with rBet v 1.0101, as determined by mass spectrometry. SDS-PAGE, isoelectric focusing, deamidation analysis and size-exclusion chromatography with light scattering revealed sample homogeneity of >99.9%. Upon storage at +4°C batch Y0487 retained the monomeric state up to 3 months. Protein quantification determined by amino acid analysis was found coinciding with half-maximal inhibition of serum IgE in ELISA. Biological activity of batch Y0487 was shown to be comparable to natural Bet v 1 by IgG and IgE immunoblotting, as well as basophil and T-cell activation. Conclusion: Recombinant Bet v 1.0101 Y0487 was characterized extensively by physicochemical and immunological methods. It was shown highly stable, monomeric and immunologically equivalent to its natural counterpart. Thus, it represents an appropriate candidate reference standard for Bet v 1.
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Development of a functional in vitro assay as a novel tool for the Standardization of Allergen extracts in the human system.
Allergy, 2005Co-Authors: Lothar Vogel, D. Lüttkopf, Lina Hatahet, D. Haustein, S ViethsAbstract:Background: Biochemical and immunochemical methods used for batch control of Allergen extracts rely on the binding of IgE molecules to Allergens. They do not measure the ability of a protein to induce type I allergic reactions. Therefore, a biological assay was established that is based on the cellular mechanisms of allergies in order to assess the cross-linking capacity of Allergens. Methods: Rat basophilic leukaemia cells were transfected with cDNA coding for the human high affinity IgE receptor chains. The surface expression of the IgE-binding α-chain was detected by FACS analysis and the functional integration of the ‘humanized’ receptors into the signal transduction cascade was addressed by intracellular calcium mobilization. Mediator release was measured in response to human IgE and a variety of cross-linking Allergen preparations. Results: Several clones were obtained that were able to bind Allergen-specific human IgE. The results of the biological assay were compared with those obtained by immunochemical methods. The biological assay was used to determine the potency of Allergen extracts, including highly diluted products that cannot be analysed by conventional methods. Conclusion: A stable ‘humanized’ basophil cell line was established that will be a useful tool for the Standardization and batch control of Allergen extracts. Because of its high sensitivity, it can also be used to detect minute quantities of potentially Allergenic proteins, e.g. in processed foods. In addition, the test may support the development of novel allergy vaccines, such as recombinant hypoAllergenic molecules.
Martin Himly - One of the best experts on this subject based on the ideXlab platform.
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Standardization of Allergen products 2 detailed characterization of gmp produced recombinant phl p 5 0109 as european pharmacopoeia reference standard
Allergy, 2016Co-Authors: Martin Himly, Angela Neubauer, K H Buchheit, Andreas Nandy, H Kahlert, M Thilker, Markus Steiner, Peter Briza, S Klysner, S ViethsAbstract:Background The Biological Standardization Programme of the European Directorate for Quality of Medicines and Healthcare (EDQM) aims at the establishment of well-characterized reference standards based on recombinant Allergens and validated assays for the quantification of major Allergen content. The objective of this study was to examine the detailed physicochemical and immunological characterization of recombinant Phl p 5.0109, the second available Allergen reference standard. Methods Recombinant Phl p 5.0109 PP5ar06007 was produced under GMP conditions and analyzed by an array of physicochemical and immunological methods for identity, quantity, homogeneity, and folding stability in bulk solution, as well as thermal denaturation, aggregation state, and biological activity when formulated for long-time storage. Results PP5ar06007 revealed as a highly homogeneous, monomeric, well-folded preparation of rPhl p 5.0109, as documented by mass spectrometry, SDS-PAGE, isoelectric focusing, size-exclusion chromatography with light scattering, circular dichroism, and infrared spectroscopy. Upon storage at +4°C, PP5ar06007 retained the monomeric state for at least 2 months. A protein quantity of 1.56 ± 0.03 mg/ml was determined by amino acid analysis in PP5ar06007, and its biological activity was shown to be comparable to natural Phl p 5 in terms of basophil activation and T-cell reactivity. Conclusions Recombinant Phl p 5.0109 PP5ar06007 was characterized extensively at the physicochemical and immunological level. It revealed to be a highly stable, monomeric, and immunologically equivalent of its natural counterpart. PP5ar06007 is now available as European Pharmacopoeia Allergen reference standard for grass pollen products.
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Standardization of Allergen products: 2. Detailed characterization of GMP-produced recombinant Phl p 5.0109 as European Pharmacopoeia reference standard.
Allergy, 2016Co-Authors: Martin Himly, Angela Neubauer, Andreas Nandy, H Kahlert, M Thilker, Markus Steiner, Peter Briza, S Klysner, R. Van Ree, K H BuchheitAbstract:Background The Biological Standardization Programme of the European Directorate for Quality of Medicines and Healthcare (EDQM) aims at the establishment of well-characterized reference standards based on recombinant Allergens and validated assays for the quantification of major Allergen content. The objective of this study was to examine the detailed physicochemical and immunological characterization of recombinant Phl p 5.0109, the second available Allergen reference standard. Methods Recombinant Phl p 5.0109 PP5ar06007 was produced under GMP conditions and analyzed by an array of physicochemical and immunological methods for identity, quantity, homogeneity, and folding stability in bulk solution, as well as thermal denaturation, aggregation state, and biological activity when formulated for long-time storage. Results PP5ar06007 revealed as a highly homogeneous, monomeric, well-folded preparation of rPhl p 5.0109, as documented by mass spectrometry, SDS-PAGE, isoelectric focusing, size-exclusion chromatography with light scattering, circular dichroism, and infrared spectroscopy. Upon storage at +4°C, PP5ar06007 retained the monomeric state for at least 2 months. A protein quantity of 1.56 ± 0.03 mg/ml was determined by amino acid analysis in PP5ar06007, and its biological activity was shown to be comparable to natural Phl p 5 in terms of basophil activation and T-cell reactivity. Conclusions Recombinant Phl p 5.0109 PP5ar06007 was characterized extensively at the physicochemical and immunological level. It revealed to be a highly stable, monomeric, and immunologically equivalent of its natural counterpart. PP5ar06007 is now available as European Pharmacopoeia Allergen reference standard for grass pollen products.
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Standardization of Allergen products 1 detailed characterization of gmp produced recombinant bet v 1 0101 as biological reference preparation
Allergy, 2009Co-Authors: Martin Himly, E Nony, Henri Chabre, L Van Overtvelt, Angela Neubauer, K H Buchheit, S Vieths, P Moingeon, Fatima FerreiraAbstract:Background: Standardization of Allergen extracts requires the availability of well-characterized recombinant Allergens, which can be used as reference standards provided by the European regulatory authorities. The objective of this study was the detailed physicochemical and immunological characterization of rBet v 1.0101, which shall be used in a ring trial within the framework of the Biological Standardization Programme BSP090 of the European Directorate for Quality of Medicines and Healthcare. Methods: Recombinant Bet v 1.0101 Y0487 was produced under good manufacturing practice conditions and analysed by an array of physicochemical and immunological methods for identity, quantity, homogeneity, folding and denaturation, aggregation state and stability in solution, as well as biological activity. Results: Batch Y0487 was shown to contain monomeric and well-folded protein being identical with rBet v 1.0101, as determined by mass spectrometry. SDS-PAGE, isoelectric focusing, deamidation analysis and size-exclusion chromatography with light scattering revealed sample homogeneity of >99.9%. Upon storage at +4°C batch Y0487 retained the monomeric state up to 3 months. Protein quantification determined by amino acid analysis was found coinciding with half-maximal inhibition of serum IgE in ELISA. Biological activity of batch Y0487 was shown to be comparable to natural Bet v 1 by IgG and IgE immunoblotting, as well as basophil and T-cell activation. Conclusion: Recombinant Bet v 1.0101 Y0487 was characterized extensively by physicochemical and immunological methods. It was shown highly stable, monomeric and immunologically equivalent to its natural counterpart. Thus, it represents an appropriate candidate reference standard for Bet v 1.
Pascal Demoly - One of the best experts on this subject based on the ideXlab platform.
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Real-life clinical practice and management of polysensitized patients with respiratory allergies: a large, global survey of clinicians prescribing Allergen immunotherapy
Expert Review of Clinical Immunology, 2017Co-Authors: Ulrich Wahn, Moises Calderon, Pascal DemolyAbstract:BACKGROUND: The allermix survey was conducted to gain insights into the most frequently diagnosed allergies worldwide, the use of Allergen immunotherapy (AIT) in clinical practice, and to identify current clinical approaches for the management of polyallergic patients. METHODS: The survey was conducted in 19 countries worldwide during 2016. Clinicians currently prescribing AIT completed an online questionnaire, designed to gather data on their daily clinical practice, most commonly diagnosed and treated allergies among their patients, awareness and adherence to recommendations and guidelines on AIT, the AIT format(s) that they choose to prescribe, and management of polyallergic patients. RESULTS: Respondents (N = 1029) considered a clinical demonstration of safety and effectiveness, and quality and Standardization of Allergen extract, as key factors influencing their choice of which AIT formulation to prescribe. 98% of respondents reported managing polyallergic patients; 58% were treated with single-Allergen AIT and 42% with multiple-Allergen AIT. 74% of respondents were aware of latest AIT practice guidelines, while 67% had an awareness of recent recommendations for pharmaceutical manufacturers. CONCLUSION: These findings highlight the need for high-quality AIT products that are supported by robust efficacy and safety data, and address the treatment habits of allergy specialists in their real-life clinical practice.
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Real-life clinical practice and management of polysensitized patients with respiratory allergies: a large, global survey of clinicians prescribing Allergen immunotherapy
Expert Review of Clinical Immunology, 2016Co-Authors: Ulrich Wahn, Moises A. Calderon, Pascal DemolyAbstract:ABSTRACTBackground: The allermix survey was conducted to gain insights into the most frequently diagnosed allergies worldwide, the use of Allergen immunotherapy (AIT) in clinical practice, and to identify current clinical approaches for the management of polyallergic patients.Methods: The survey was conducted in 19 countries worldwide during 2016. Clinicians currently prescribing AIT completed an online questionnaire, designed to gather data on their daily clinical practice, most commonly diagnosed and treated allergies among their patients, awareness and adherence to recommendations and guidelines on AIT, the AIT format(s) that they choose to prescribe, and management of polyallergic patients.Results: Respondents (N = 1029) considered a clinical demonstration of safety and effectiveness, and quality and Standardization of Allergen extract, as key factors influencing their choice of which AIT formulation to prescribe. 98% of respondents reported managing polyallergic patients; 58% were treated with single...
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Choosing the optimal dose in sublingual immunotherapy: Rationale for the 300 index of reactivity dose
Clinical and Translational Allergy, 2015Co-Authors: Pascal Demoly, Moises Calderon, Gianni Passalacqua, Tarik YalaouiAbstract:AbstractSublingual immunotherapy (SLIT) is an effective and well-tolerated method of treating allergic respiratory diseases associated with seasonal and perennial Allergens. In contrast to the subcutaneous route, SLIT requires a much greater amount of antigen to achieve a clinical effect. Many studies have shown that SLIT involves a dose–response relationship, and therefore it is important to use a proven clinically effective dose from the onset of treatment, because low doses are ineffective and very high doses may increase the risk of side effects. A well-defined Standardization of Allergen content is also crucial to ensure consistent quality, potency and appropriate immunomodulatory action of the SLIT product. Several methods of measuring antigenicity are used by manufacturers of SLIT products, including the index of reactivity (IR), standardized quality tablet unit, and bioequivalent allergy unit. A large body of evidence has established the 300 IR dose of SLIT as offering optimal efficacy and tolerability for allergic rhinitis due to grass and birch pollen and HDM, and HDM-induced moderate, persistent allergic asthma. The 300 IR dose also offers consistency of dosing across a variety of different Allergens, and is associated with higher rates of adherence and patient satisfaction. Studies in patients with grass pollen allergies showed that the 300 IR dose has a rapid onset of action, is effective in both adults and children in the short term and, when administered pre-coseasonally in the long term, and maintains the clinical benefit, even after cessation of treatment. In patients with HDM-associated AR and/or asthma, the 300 IR dose also demonstrated significant improvements in symptoms and quality of life, and significantly decreased use of symptomatic medication. The 300 IR dose is well tolerated, with adverse events generally being of mild or moderate severity, declining in frequency and severity over time and in the subsequent courses. We discuss herein the most important factors that affect the selection of the optimal dose of SLIT with natural Allergens, and review the rationale and evidence supporting the use of the 300 IR dose.