Allergen Standardization and Characterization - Josefina Zakzuk, MD, PhD; Jonathan Kilimajer, MD; Enrique Fernández-Caldas, PhD; Richard F. Lockey, MD (Updated 2019)

Posted: September 2009
Updated: January, 2019

Josefina Zakzuk, MD, PhD
Institute for Immunological Research, University of Cartagena

Jonathan Kilimajer, MD
Medical Department, Inmunotek SL, Spain
Enrique Fernández-Caldas Enrique Fernández-Caldas, PhD
Scientific Director, Inmunotek SK, Madrid, Spain
Clinical Professor of Medicine,
University of South Florida College of Medicine, Tampa, Florida

Richard F. Lockey Richard F. Lockey, MD
Professor of Medicine,
University of South Florida, College of Medicine, Tampa, Florida

Key words: allergens, allergen immunotherapy, vaccines, standardization, recombinant allergens.

Introduction
There is a worldwide increase in atopic diseases, which include allergic rhinoconjunctivitis, allergic asthma, atopic dermatitis and food allergies[1],[2]. Although the reasons for this increase are unclear, exposure to mite allergens and diesel exhaust particles is recognized as an important environmental risk factor in genetically predisposed individuals[3]. The diagnosis of allergic disease requires a detailed history, physical examination, and allergy testing, i.e., skin testing or in vitro determination of allergen-specific immunoglobulin E (IgE). Skin testing is performed by applying an allergen extract to the skin and then scratch or pricking it with an appropriate needle-like instrument. In sensitized individuals, it results in the formation of a raised wheal surrounded by an erythematous flair within 15 to 20 minutes, indicating a positive test reaction. In vitro specific IgE also supports the clinical diagnosis and helps to guide the allergist in the management of allergic diseases.

Once an individual is sensitized, symptomatic and long-term strategies, such as environmental control and immunomodulatory treatments using specific allergen immunotherapy (SIT) play an important role in treatment. SIT is the practice of administering gradually increasing doses of allergen vaccines to reduce allergic symptoms and the need for medications. It is currently classified as therapeutic vaccines with scientific support of its effectiveness to prevent and relieve allergic symptoms[4]. SIT is the only known treatment that affects the natural course of allergic diseases, especially when introduced early in life. This biological response modifier is capable of influencing allergen-driven immunological responses and restoring, to a certain degree, the Th1/Th2 balance in allergic subjects. B and T cells, blocking antibodies, IL-10 and other cytokines play an important role in the response to SIT[5]. Accuracy of SIT depend on the availability of well-characterized allergen vaccines. So too does effective skin tests and in vitro determinations; allergen standardization and characterization are paramount to achieve these goals.

Allergen-specific immunotherapy was first described by Noon in 1911[6]. From these early days, numerous attemps have been undertaken to establish a reliable, common method of allergen standardization[7]. Standardization of allergen vaccines/extracts used for therapy and diagnosis is necessary because their qualities may vary, depending on production and control methods of the manufactured batches[8].

Testing and Standardizing Allergen Products
Allergen products to diagnose and treat allergic diseases have been used for over 100 years. Allergen extracts are biological products that are administered to humans, and pets, to diagnose, prevent and treat allergic diseases. Allergen products are pharmaceutical preparations derived from extracts of naturally occurring source materials containing allergens, which are substances that cause, or provoke allergic (hypersensitivity) diseases.

Allergen standardization begins by the controlled selection af the source (raw) material which is going to be used in the preparation of  allergen extracts. Allergen source materials are obtained from validadated and audited suppliers and are obtained with a certificate of analysis confirming collection methods and source. Pollens from trees, weeds and grasses are collected under controlled conditions by certified suppliers; quality and purity must be documented. Source materials should be assigned an expiration date based on stability studies and receive a code to enable testing and tracking according to Good Manufacturing Practices (GMP) requirements. Pollen source materials are subjected to strong quality regulations and processes, and include purity, foreign contaminations, and stability data[9],[10]. Raw materials from mites and molds are grown in specialized facilities and are also subjected to regulatory controls[11],[12],[13]. In spite of recent progress, standardization of fungi allergenic extracts continuous to be an unmeet need. Great differences have been shown among unstandardized mold allergen extracts[14],[15]. Wurth et al reported that only one from the 4 comercially available Aspergillus extracts in USA contained detectable quantities of the major allergen Asp f 1, coinciding with results showing that this product was unique with potential to induce experimental anaphylaxis in sensitized mice[16].

Animal dander is collected from housed animals and is accompanied with a certificate from a veterinary doctor stating that the animals are healthy and free of infectious and contagious agents[17]. In most European countries, national regulations allow marketing of allergen products as "Named Patient Preparations" (NPPs), although in the last few years, registration procedures have been started. NPPs are industrially prepared allergen vaccines following the prescription issued by an allergy specialist. It is regulated mainly by the Guideline on Allergen Products: Production and Quality Issues[18] and the Monograph on Allergen Extracts of the European Pharmacopoeia[19]. European manufacturers of allergen extracts develop their own methods and in-house reference preparations for standardization. In the USA, the FDA Center for Biologics Evaluation and Research (CBER) reference preparations, standardized by approved methods, are used. The quality of allergen products is a key issue for both diagnosis and therapy, and the standardization of allergen extracts is of primary importance to improve their quality and offer physicians worldwide a reliable method to diagnose and treat allergic respiratory diseases. Effective diagnosis and treatment, using skin test reagents and SIT, requires the optimal amount of allergens for testing and the maintenance dose of vaccine for treatment. Skin test reactions should be large enough to suggest clinical sensitivity but not so large as to produce excessive discomfort or the risk for a serious systemic reaction.

The heterogeneity of allergen extracts makes it necessary to develop methodologies to assess their potency and ensure their consistency, stability and safety. Allergen products are legally considered medicines that require registration by government institutions[20], such as the Food and Drug Administration (FDA) in the United States and the Paul Ehrlich Institute in Germany, further increasing the need for standardization. Basic researchers, physicians, regulatory authorities and manufacturers have tried to define a common methodology to standardize allergen vaccines[21].

In Europe, manufacturers have implemented company-specific protocols to standardize the production and quality control of the allergen extracts and to compare production batches to assure batch-to-batch consistency[22]. All these production and quality control issues are controlled by strict protocols and adhered to Good Manufacturing Practices[23]. Thus, current allergen standardization requirements concentrate on the consistency of production and the safety and potency of allergen products. These protocols use in vivo and in vitro standardization techniques, a representative allergic patient population, and dose-response studies to assign biological activities. Dose-response studies are mandatory and are primarily based on skin testing (intradermal or prick tests) and on inhibition of allergen-specific IgEs compared to reference extracts known as in-house reference preparations (IHRPs). Furthermore, guidelines have been issued for the clinical development of products for specific immunotherapy for the treatment of allergic diseases. The IgE-binding potencies of the IHRPs are quantified by skin test reactivity (in vivo standardization) and by competitive IgE tests, such as RAST, ImmunoCAP, or ELISA inhibition assays (in vitro standardization). Although they may be similar, they are expressed in company-specific units. The quality of mite and pollen allergen extracts is better defined today than it was in the past, and the quality of food and epithelial allergen extracts has also improved[24],[25].

In the United States, allergen standardization is based on intradermal testing of allergic patients and the potencies of sequential batches are determined by appropriate surrogate in vitro assays, which are based on inhibition of binding of IgE from pooled allergic sera to solid phase reference allergen extracts, or measurement of specific allergen contents in the allergen vaccines8,20. Extracts in the United States are more homogenous with respect to total allergenic potency than the extracts produced in Europe, mainly because the FDA provides the same standardized reagent for internal use by all manufacturing companies. However, these standards are only limited to 19 sources, including the most clinically relevant in the country (hymenoptera venoms, cat hair and pelt, house dust mites, pollens from 8 grass species and short ragweed). These preparations are licensed by the Center for Biologics Evaluationand Research and their number has not raised since 10 years.  In case of non-standardized extracts, calculation of manteinance doses is more difficult  because weight by- volume (w/v) or protein nitrogen unit (PNU) labelling may not correlate with biological potency. The FDA organized an Steering Commitee to compile evidence about the safety and evidence of non-standardized allergenic extracts[26] finding that although most commercially available products were safe for clinical use, for about a half there was no evidence about their efficacy.  It is expected that the number of currently available extracts in the market will be drastically reduced.

In Japan, the Japanese Society of Allergology standardized Japanese cedar pollen allergen vaccines[27]. The in vivo allergenic potency of the reference extract was determined by intradermal testing and measurement of the content of the major allergen Cry j 1 was selected as the surrogate in vitro assay.  In Korea the allergen extracts manufactured  at the Research Center for the Standardization of Allergic Diseases, Department of Internal Medicine and Institute of Allergy, Yonsei University College of Medicine, Seoul, Korea, exhibit good allergen potency, in terms of in vitro (ELISA inhibition) and in vivo (intradermal skin test) tests, when compared with commercially available extracts, even detecting low levels of major allergens[28] .

The prevalence of food allergies is rising. The standard of care consists of food-allergen avoidance and treatment of allergen-induced systemic reactions with adrenaline. Thus, accurate diagnosis, prevention and treatment are pressing needs. Research is needed to enable an understanding of the mechanisms involved in food allergy at the cellular and molecular levels and to design prevention and intervention strategies[29] as well as food oral immunotherapy, in wich significant progress has been observed   in promoting immunomodulatory effects that lead to the clinical outcome of desensitization. Studies in the USA have revealed that the presence of food allergy in children may be of 6.7%[30].

 

Monoclonal Antibodies and Recombinant Allergens (RAs)
Allergenic extracts consist of complex mixtures of substances with a variation in allergenic activity and allergen composition. Patients are often not sensitized to all allergens in one extract and there may be great variability among individuals. Advances in the field of microarrays containing purified native and recombinant allergens (RAs) may be useful to identify more precise sensitization profiles and further personalization of  allergen composition in the vaccine [31],[32]. The characterization of major allergen components and the development of techniques to quantify them, such as ELISA systems based on monoclonal antibodies, have led more manufacturers to provide information on the major allergen content of their extracts, even though identification of major allergen content is not currently mandatory, except for a limited number of extracts, such as cat and ragweed. EMEA guidelines now recommend that allergen manufacturers state the content of representative major allergens in mass units for their allergen products using antibody-based techniques. However, this could not be mandatory due to the limited number of standards and immunoassays in comparison with the broad quantity of allergenic extracts. As noted above, differences in assays and methodologies for measuring the major allergens may preclude direct comparisons among products of different manufacturers.

Larenas-Linnemann and Cox reviewed the information obtained on unit definition and dosage of allergens from European manufacturers of allergen extracts used for sublingual immunotherapy (SLIT). They concluded that the monthly maintenance dose the manufacturers recommended for SLIT was 5-45 times higher than the recommended dose for subcutaneous immunotherapy. However, since each manufacturer in Europe uses its own IHRPs and its own units to express potencies, the comparison of different products from different companies at national and international levels is almost impossible. Even if the amount of major allergens is stated, differences in the quantification technique, the reference extracts and antibodies used can influence the outcome. Thus, for comparison of diagnostics and immunotherapeutics from different manufacturers, the same analytical methods and materials ideally should be used[33]. Another study quantified and compared the allergen content of different grass pollen preparations for skin prick testing (SPT) and SLIT used in Europe[34]. Protein concentrations of SPT solutions ranged from 15 to 427 µg/ml, and Phl p 5 concentrations, a major grass allergen, ranged from 0.15 to 18.3 µg/ml. Protein content of the maintenance doses of the immunotheurapeutics ranged from 5 to 153 µg and Phl p 5 content ranged from 0.2 to 21.6 µg. SDS-PAGE and immunoblots confirmed the differences in protein and allergen contents.

Since allergen extracts or vaccines may contain hundreds of allergenic determinants, and individuals can be sensitized to different combinations of these proteins, sensitization profiles in atopic populations are very heterogeneous. Therefore, before RAs are utilized in routine clinical practice, epidemiological studies using RAs to identify the immune response to specific allergens in the population at large and to clarify cross-reactivity are necessary. The number of cloned and purified allergens has increased substantially over the past two decades. The incorporation of Omic disciplines to Experimental Allergology has led to the identification of new allergens, some of them being important in terms of IgE binding frequency or allergenic potential[35],[36]. In this sense, the fact of reducing the allergenicity of one source to a few molecules for SIT is far from reality for some allergenic species such as house dust mites.

Recently, it has been demonstrated that Der p 23 is an new and clinically relevant house dust mite allergen. IT has sequence homology with peritrophins, which contains chitin-binding domains and is part of the peritrophic matrix lining the gut of arthropods. Recombinant Der p 23 reacted with IgE antibodies from 74% of D. pteronyssinus allergic patients (n = 347) at levels comparable to the two major HDM allergens, Der p 1 and Der p 2. The allergen is localized in the peritrophic matrix lining the midgut of D. pteronyssinus as well as on the surface of the fecal pellets.  The high allergenic activity of Der p 23, and its frequent recognition as a respiratory allergen, may be explained by the fact that it becomes airborne and respirable through its association with mite feces[37]. The availability of high quality recombinant allergens, is important for the follow-up of mite allergic patients, to establish clinical risk factors[38] and develop new vaccines[39]. The correct selection of candidate patients is also of landmark importance[40],[41].

Nowdays the use of RAs in diagnosis has made significnt progress, and when combined with precision medicine (PM) can change the scope of allergy diagnosis. One prototype PM method using recombinants components in the test for an in vitro IgE assay, ImmunoCAP®ISAC, was developed in the sixties and has attracted increasing interest. These microarrays of allergen components have significantly improved the way to describe the IgE binding profile of a patient. Lately a macroarray containing both “whole” allergens and molecular components has been developed, namely the Allergy Explorer (ALEX). This method allows the acquisition of an IgE biniding profile comprising 282 reagents (157 allergen extracts and 125 individual allergen components), resulting in the widest screening available of potential allergens [42].

To improve safety and efficacy of SIT, an alternative approach may be to use individual allergens for tailored immunotherapy after identifying the specific sensitization causes [43]. The possibility of obtaining allergens as recombinant products makes it possible to produce hundreds of allergens in large quantities and  quicker than the laborious purification  of natural extracts. Molecular cloning has provided an efficient way to obtain pure polypeptides. This has distinct advantages over using native sources which form complex mixtures and are often present in very small amounts. There has been considerable progress in understanding the molecular characteristics of allergens. Many allergens have been purified from aqueous extracts or produced as recombinant molecules. Allergen sequence information is available from different databases (www.allergen.org/www.allergome.org). These polypeptides enable the mapping of B- and T-cell epitopes and the identification of their binding sites. Sequence polymorphisms that influence antibody binding and T-cell recognition have also been established for several allergens, finding isoforms of clinical relevance in  restricted geographical areas[44],[45].

The expectation of using RAs for SIT is to overcome some of the pitfalls of using natural allergen products for immunotherapy. These approaches may enable physicians to administer only the clinically relevant allergen, thus avoiding exposure to unnecessary antigens. Moreover, RAs could also be rendered hypoallergenic using a variety of techniques. Several immunotherapy studies have been conducted with recombinant allergens with clinical benefits [46],[47],[48],[49]. More clinical trials are needed to compare the potential advantages and benefits of SIT with RAs versus current standard therapy with natural vaccines. This process will take years; in the meantime, optimizing SIT with naturally occurring vaccines is required to assure efficacy and safety. For sources with few important allergens, the use of single RAs for SIT is a more promising scenario.

The CREATE Project
A project financed by the European Union, "Development of Certified Reference Materials for Allergenic Products and Validation of Methods for their Quantification", (the CREATE Project), explored the idea of introducing standardized techniques to quantify major allergen content into standardized protocols. Mass units of major allergens would be used to quantify the active ingredients of the allergen while allowing comparison among manufacturers. The goals of the CREATE Project were to evaluate the potential of purified recombinant allergens as certified reference materials (CRMs) and to evaluate available ELISAs for the measurement of major allergens using the candidate CRMs as a standard. Eight major allergens originating from four of the most important inhalant allergen sources were selected: Bet v 1 from birch pollen, Phl p 1 and Phl p 5 from grass pollen, Ole e 1 from olive pollen and Der p 1 and 2 and Der f 1 and Der f 2 from house dust mites. Three were found to be suitable as biological reference materials; the rest, except rPhl p 1a, indicate potential for optimization, provided aspects of their protein expression processes are modified. As a result of this study, recombinant Bet v 1 and Phl p 5 were produced under "Good Manfacturing Practice" and evaluated by the European Directorate for the Quality of Medicines as biologic reference preparations to be included in the European Pharmacopoeia as international standards. Consequently, standardization of these allergen products will become global permitting comparisons among different manufacturing sources[50]. The project BSP090, sponsored by the Biological Standardisation Programme of the EDQM (European Directorate for the Quality of Medicines & HealthCare), was the continuation of the CREATE project and intended to establish biological reference preparations for Bet v 1 and Phl p 5 as well as to validate reference ELISA methods for their quantification. As a result of thiw work, the EMEA published that recombinant  Bet v 1 is intended for use as standard for calibration of secondary standards and/or in house reference preparations for determination of the Bet v 1 content in allergen extracts from birch (Betula verrucosa) pollen and recombinant Bet v 1 preparations by ELISA. A reference ELISA method for its quantification has also been selected after validation in multi-center studies[51], [42]. According to extensive physico-chemical analyses, recombinant Phl p 5.0109 is produced as a highly stable, monomeric, and immunologically equivalent to its natural counterpart and is also available as European Pharmacopoeia allergen reference standard for grass pollen products[52].

For allergen sources with many epidemiologically important allergens identified, such as HDM extracts, the development of many reference preparations for standardization is a real  need. The use of multiplex platforms for quantification may overcome the expensive and time consuming activity of measuring several allergens for standardization of only one source[53].

Characterization of allergoids
"Allergoid" is a term used to describe natural allergen products that have been modified with aldehydes (formaldehyde and glutaraldehyde) to decrease their allergenicity and potentially increase their safety. Allergoids are commercially available in Europe from the 1980s and have demonstrated successful clinical results[54].

Allergoids represent the fasted growing sector of the allergen immunotherapy market in Germany and Spain[55]. Numerous double-blind placebo-controlled studies using allergoids (modified allergens) have demonstrated efficacy[56] .

Chemical modifications of allergenic extracts aims to reduce its allergenicity in order to increase safety, albeit preserving its immunogeniticy.  In this sense, determination of total allergenic activity and measurement of allergen content is not applicable for standardization. Some quality control procedures are also unsuitable; for example, SDS-PAGE visualization of allergoids is completely different to the natural extract before polimerization due to coupling to high molecular weight compounds. As a consequence, particular requirements for quality control of these products are requested in the regulatory framework. According to the Guideline on Allergen Products: Production and Quality Issues, retention of immunogenicity needed to be demonstrated in vivo by means of animal models[57]. However, high heterogeneity has been observed in these results. Furthermore,  the evaluation of every batch of allergoid produced is conflictive with ethical principles in regard to animal testing. This gap on allergoid characterization may be overcome by the use of in vitro IgG inhibition ELISA, which is now mandatory for registration purposes in Europe. Other methods, such as peptide content analysis of allergen sequences by mass spectrometry, have been applied for allergoid characterization. Capillary liquid chromatography-tandem mass spectrometry based peptide mapping can be used to confirm the presence of allergens in allergoids of Betula alba. Peptide sequencing could even discriminate at the level of isoforms[58]. Size-exclusion chromatography can also be used to show consistency of the modification process, being applicable for batch release[59].

Other techniques which could be used for standardization
Beyond the use of monoclonal antibodies, other physico-chemical approaches have been explored for allergen standardization and offer new possibilities. Evaluation of mass spectrometry (MS) has been performed to determine its capacity to characterize the composition of allergen extracts. One advantage offered by the use of MS includes the measurement of several different allergenic components (allergens and isoforms) simultaneously rather than measuring individual allergens. This is advantageous while working with allergen preparations that contain a wide group of IgE binding proteins, such as mite extracts. Additionally, MS-based methods are available to discriminate between allergen isoforms, which is difficult to accomplish using immunologic based methods. This method could also be useful for the standardization of allergoids, since measuring major allergens is not possible in these preparations due to their chemical modification with aldehyde. One disadvantage of MS is that even though it may demonstrate the presence of allergen peptides inside the complex mixture, it is not a real quantitative method. Another issue is  the high costs of instruments and the requirement highly trained personnel.

Conclusions and Current challenges
Allergen standardization strategies should be uniform throughout the world. The different units, which vary among manufacturers as well as global regions, are confusing and unreliable with the potential to underestimate or overestimate the potency of allergen extracts. The variations can be attributed to the variability of the raw materials used, the production methods and the lack of consistent and reliable quantification of allergy content. Major allergen measurements are essential to overcome these problems. Some recombinant allergens are found to be suitable as certified reference materials and are currently released for allergen standardization. But, results from the CREATE and the BSP090 Project demonstrate that the process of obtaining recombinants and immunoassays which filfull requirements for standardization is slow. The limitations of recombinant allergens as CRM due to incorrect folding, aggregation, poor solubility and insufficient stability. However, evidence gathered in studies identifies the complexity of this approach. The European Medicines Agency (EMEA) recommends proving that each allergen extract contains the relevant allergens by antibody-based techniques or mass spectrometry. New techniques, such as nuclear magnetic resonance and small angle X ray scattering, are commonly applied to characterize allergenic molecules in the laboratory. They are slowly affecting the methods by which allergen extracts are standardized. This would be an important step forward towards a comprehensive characterization of allergen products. Future enhancements of SIT, using RAs, also require allergen standardization, which in turn, requires development of standardized methods to measure allergen content, homogeneity, folding, aggregation, solubility and stability of recombinant products. Allergen standardization will advance rapidly in the future, improving the effectiveness and safety of allergen vaccines. Allergen immunotherapy will remain an effective and safe treatment for allergic respiratory diseases.

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