Allergenic Materials to Produce Extracts – Natural or Recombinant Allergens

Allergenic materials are used to prepare allergen extracts to diagnose and treat allergic patients. Natural allergenic materials have been used for this purpose for many years.

It is often difficult to obtain enough sufficient quantities of natural allergenic materials. In addition, the safety of those allergenic materials is questioned, particularly in Europe, where medical regulatory agencies have proposed guidelines to monitor the quality of these products.

A different perspective regarding natural allergenic materials exists in the United States of America (USA). Currently, the Food and Drug Administration in the USA only demands that the quality and safety of the final allergenic extracts be assessed. This organization has not yet focused on assessing the quality of the allergenic materials used to prepare the associated extracts.

Recombinant allergens are alternatives to circumvent the caveats of using natural allergenic materials. While the science of obtaining recombinant allergens is rapidly evolving, natural products most likely will continue to be used for many years to come because of the varying limitations regarding the production and use of recombinant allergens. For example, it is currently not possible to obtain recombinant forms of all clinically relevant allergens. In addition, the safety and efficacy of such allergens need to be properly documented through appropriate clinical trials, which takes time to complete and is extremely expensive.

We are in a transition period. Meanwhile, the perspectives regarding the use of natural allergenic materials to prepare allergenic extracts in the USA and Europe should be harmonized.

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Fungal Allergenic Extracts

A large number of fungal allergenic extracts for diagnosis and treatment of allergic diseases are available on the market. This can be confusing for the allergist/immunologist to properly select the most clinically relevant fungal allergenic extracts.

While the proper selection of the appropriate fungal allergenic extracts derived from particular genera and/or species is critical, other factors regarding the materials used to obtain those fungal allergenic extracts also play a pivotal role responsible for the quality and consistency of fungal allergenic extracts. It is essential for the allergist/immunologist to properly understand those factors because they are ultimately responsible for a large variation in allergen content and potency among fungal allergenic extracts of the same species produced by different manufacturers.

Fungi have a high level of genetic adaptability to the environment. They often mutate as a result of external stimuli. Those mutations and the direct effect of environmental conditions are associated with selective allergen production and the potential secretion of secondary metabolites, particularly mycotoxins and polysaccharides. Therefore, when fungi are cultured to manufacture fungal allergenic extracts, those factors must be controlled.

For the purpose of manufacturing fungal allergenic extracts, the fungal strains used in cultures should be obtained from accredited sources such as the American Type Culture Collection or the Centraalbureau voon Schimmelcultures. The selected fungal strains must be grown under very strict conditions in compliance with specific regulations and general manufacturing guidelines.

The environmental factors responsible for fungal allergen production are many. The most relevant are the specific media formulations and culture conditions used to grow fungi. They are considered intellectual property that belongs to the companies that produce fungal allergenic extracts.

While materials derived from animals or plants are generally used to prepare laboratory culture media, they can potentially be allergenic and/or contain pathogenic microorganisms. Therefore, the media used to grow fungi must be carefully formulated to assure its safety.

The common culture conditions are static or under agitation. These conditions are responsible for the amount of oxygen available in the cultures, which affects the types and levels of fungal allergens and secondary metabolites produced, including mycotoxins.

Once fungal cultures achieve a particular level of maturity, they are harvested and inactivated to assure that no live fungi are present in the materials. Proof of inactivation is required before further processing can be initiated.

The materials used to produce fungal allergenic extracts vary, depending on suppliers. Mycelia, culture filtrates, or both can be used to prepare fungal allergenic extracts.

Fungal allergenic extracts should be obtained from one single reliable supplier. The allergist/immunologist should be aware of the fact that extracts derived from the same fungal genera and species produced by different manufactures are not equivalent in terms of allergen content and potency.

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Dog Allergenic Extracts

Dog allergenic extracts are used to diagnose and treat allergic diseases. Mammalian allergens are present in a variety of sources, and different raw materials are used to produce dog allergenic extracts extracts. Those materials include dog dander, hair, epithelium, pelt, skin scrapings, and hides. However, dog dander is the most commonly used product.

Because most individuals sensitized to cats react to Fel d 1, standardized allergenic extracts to cat allergens are available, based on particular concentrations of this allergen. On the contrary, a distinctive major dog allergen recognized by the specific IgE of most dog-sensitized individuals has not been identified. Therefore, a large variety of heterogeneous products are on the market.

Several dog allergens, including Can f 1, Can f 2, and Can f 3 have been traditionally considered relevant to particular individuals sensitized to dogs, with Can f 2 (lipocalin) and Can f 3 (albumin) being responsible for cross-reactivity among mammals. More recently, other dog allergens (Can f 4, Can f 5, and Can f 6) have been identified and proven clinically relevant to particular individuals. These allergens also are responsible for cross-reactivity.

It is important to note that Can f 5 is mainly secreted by non-neutered adult male dogs. Because female and neutered dogs produce lower quantities of this allergen, it has been suggested that they could be appropriate as household pets for individuals allergic to dogs. Therefore, different types of dog allergenic extracts could be needed to properly diagnose the allergic patient.

The fact that major clinically relevant dog allergens that affect most sensitized individuals have not been identified to date, combined with the heterogeneous composition of dog raw materials is responsible for the presence of a number of products on the market with differential diagnostic value. The currently available allergenic preparations may not be suitable for immunotherapy.

The intrinsic nature of sensitization to dog allergens is unique, and consistent diagnostic tools are needed. The production of individual dog allergens is an emerging technology with applications for diagnosis and treatment to target specific patient populations.

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Pollen Allergenic Extracts

Pollen is a unique raw material because it is exposed to the outdoor environment before and during collection. Therefore, pollen can be potentially impacted by moisture and airborne components present outdoors. They include both ubiquitous biological and chemical environmental pollutants. The potential impact of chemical contaminants in pollen is not discussed here.

Proper pollen collection is essential to produce allergenic extracts. Pollen collection is tedious and requires a high level of specialization. A few large pollen collection entities own land to cultivate the desired plants for pollen collection under conditions that minimize exposure to environmental pollutants. However, this is an emerging strategy, which often does not allow for the collection of sufficient amounts of pollen necessary to produce pollen allergenic extracts. Many small family-owned pollen collection entities also obtain pollen to produce pollen allergenic extracts.

After the pollen used for the production of allergenic extracts has been collected, it is dried to prevent potential microbial growth. Subsequently, their identity and purity are evaluated. Qualified individuals should perform the pollen identification and purity assessments in appropriate laboratories.

It is essential to perform all activities to produce pollen allergenic extracts, including pollen identification and purity assessments, in dedicated areas of allergen manufacturing companies where other types of raw materials are not present. Otherwise, cross-contamination with other allergenic products might occur.

Pollen microscopic analysis is performed to verify pollen identify and assess its purity. Currently, pollen purity assessments include counting and identifying biological components contaminating the pollen. However, this method of evaluating pollen purity does not consider either the potential clinical relevance of specific contaminants or their relative volume compared to that of the pollen in the final sample. While the potential clinical relevance of many pollen contaminants is unknown, volumetric counting instead of particle counting could provide a better estimation of the amount of foreign biological materials contained in pollen. However, standardized and approved methods to perform this evaluation are lacking.

Microscopic pollen analysis also can provide valuable information regarding pollen quality for the production of pollen extracts. The biological components that typically contaminate pollen include plant parts, foreign pollen, fungal structures, and other biological agents.

The pollen collected for the production of allergenic extracts must meet particular purity specifications. Specifications for the maximum allowed concentrations of biological contaminants in pollen are typically internally proposed by allergen manufacturing companies based on the likelihood of achieving a particular purity level after cleaning pollen. These specifications are not based on any potential health effects upon exposure to the contaminants because those effects are unknown.

The presence of biological contaminants in pollen is impossible to avoid, difficult to evaluate, and the interpretation of the associated purity data is very subjective. However, if the evaluation of pollen purity to prepare allergenic extracts is properly performed and interpreted, it can benefit the allergen manufacturing companies that produce such extracts. For example, the presence of a large number of plant parts and a variety of miscellaneous fungal spores generally indicates that the pollen is not sufficiently clean, and that additional activities to remove biological contaminants are necessary. To the contrary, the presence of large amounts of one single spore type, hyphae, and/or sporulating structures indicates that fungi have actively infested the pollen. This pollen should be discarded for the production of pollen allergenic extracts.

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Allergenic Extracts on the Market

A large number of allergenic extracts for diagnosis and/or treatment of allergies are available on the market. However, the safety and efficacy of many of them are controversial.

In 2004, the Food and Drug Administration (FDA) Center for Biologics Evaluations and Research (CBER), which regulates allergenic products, created a committee to review scientific data about the safety and efficacy of non-standardized allergen extracts. Such extracts were classified into five categories according to the level of scientific information to justify their use. For example, for pollen, Categories 1, 4, and 5 include 451, 250, and 7 species, respectively.

CBER proposed to remove all products classified in categories 4 and 5 due to safety concerns (Docket #FDA-2011-N-599). While this activity is taking place slowly due to the associated regulatory implications, the number of pollen allergenic extracts likely will be reduced to 451 species, all classified in Category 1. Allergen redundancy and/or cross-reactivity considerations should also indicate which pollen genera and/or species are clinically relevant.

Because of the increasing regulatory requirements regarding allergenic raw materials used in Europe, Lorentz et. al.,in 2009, proposed the concept of “homologous groups” to classify allergen sources. This concept is based on similar biochemical composition and homology/cross-reactivity of allergens or allergen sources. European regulatory agencies adopted this concept, and now require that allergen manufacturing companies obtain quality data for representative allergen sources classified in each homologous group, seven of which represent pollen species.

European regulatory agencies also have proposed norms to reduce the number of allergenic preparations available on the market. These approximations are analogous in concept to that proposed by CBER in the USA, but are generally more rigorous, and the number of allergen extracts permitted are fewer than those currently available in the USA.

France has stricter regulations regarding allergenic products, and therefore allergen manufacturers must also comply with the demands of the French medical authorities [Agence Française de Sécutité Sanitaire del Produits de Santé (AFSSAPS)]. The AFSSAPS groups aeroallergens in four classes, based on the scientific information currently available, but in an opposite ranking scale as adopted by CBER. Pollen classified in categories 1, 2, 3, and 4 include 24, 2, 7, and 10 species, respectively.

Storage of Allergenic Raw Materials

Environmental factors are responsible for the deterioration of the allergenic raw materials used to manufacture allergenic extracts. Among these factors, moisture content and storage temperature of the materials play a combined role, responsible for the allergenic stability of raw materials. The proper storage of allergenic raw materials is critical to guarantee a particular shelf-life.

We performed a study to examine the stability of several allergenic raw materials over several years. The materials, stored under various moisture and temperature conditions, were tested at different time-intervals. The results of this study indicate that for maximum shelf-life, raw materials should be stored under conditions of low moisture (<8%) and temperature (<0^oC).

Production of Allergenic Extracts

Introduction:

Allergen extracts are biological products used to diagnose and treat allergic diseases. Allergy diagnosis is performed by a careful review of the patient’s medical history, often followed by in vivo and/or in vitro testing, which involve the use of allergen extracts. Allergen avoidance should be the first measure to prevent allergies. However, this approximation is not always possible to implement, and it is not always effective.

Immunotherapy can modulate the immune system and prevent allergies. Medication, is the third alternative to manage allergies because is expensive and does not prevent the immune reaction responsible for allergies. However, medication can relieve allergy symptoms.

Various steps are involved in the production of allergenic extracts for diagnosis and treatment. These steps include, but are not limited to, the proper selection and procurement of allergenic raw materials, the manufacturing procedures utilized, the quality measures in place to assure that the extracts have a consistent allergen composition and potency, and the measures in use to guarantee a particular shelf-life and expiration date.

The allergens utilized in allergen extracts can be classified as natural or recombinant. The associated materials to produce them are manufactured differently.

Both natural or recombinant allergens can be used to diagnose and treat allergic diseases. Natural allergens have been used for many years in allergen extracts and will continue to be used for the time to come because of the varying limitations regarding the production and use of recombinant allergens. For example, it is currently not possible to obtain recombinant forms of all clinically relevant allergens. In addition, the safety and efficacy of such allergens need to be properly documented through appropriate clinical trials, which takes time and is extremely expensive.

Most catalogs of allergenic extracts list pollen, mites, fungi (molds), insects, epithelia (mammalian allergens), hymenoptera venoms, foods, and “others”. Therefore, the procurement of the allergenic raw materials to produce those allergenic extracts varies, depending on the product.

Natural allergenic raw materials are diverse in origin and have different characteristics. They are obtained from the outdoor environment (i.e. pollen), from laboratory cultures (i.e mites and fungi), animal groomers (i.e. epithelia), or supermarkets (foods).

This is the first post of a series associated with the topic of the production of allergenic extracts. In this post, we provide a brief description of the manufacturing steps associated with the manufacturing of extracts. Subsequently, we will address the complex topics of the natural allergenic materials used in the production of allergenic extracts as well as their stability and expiration dates.

Steps involved in the production of allergenic extracts:

Many steps are involved in the production of allergenic extracts. These steps vary depending on the types of extracts and their intended purpose/s. For example, allergenic extracts for in vitro diagnosis are not subjected to the same rigorous quality measures as those for in vivo use. However, a general, overview of the steps involved in the production of allergenic extracts is provided below.

Step #1: Once raw materials are received at allergen manufacturing companies, batch records are initiated, and the materials are placed on quarantine. Subsequently, the identity and purity of the raw materials are determined by macroscopic and/or microscopic inspection or by chemical/immunochemical testing.

Step #2: Moisture content in the materials is measured. Subsequently, the materials should be dried, if necessary, to prevent potential microbial growth and product bio-deterioration. Then, raw materials are released and stored until further processing. Ideally, allergenic raw materials should be stored frozen to preserve their stability and shelf-life over a given period, as indicated by an expiration date.

Allergen manufacturing companies should have real-time stability protocols in place for different raw material types, according to accepted guidelines. The information derived from the completion of those studies would likely identify selective expiration dates for different raw materials, as expected due to the intrinsic nature of their allergens.

Step #3: Allergenic raw materials are processed to various levels before extraction. The materials are typically washed and defatted with a solvent. Subsequently, they are sieved, milled, and/or powdered, as appropriate.

After raw materials are processed, they are known as “source materials”, which are the products that will be extracted to produce intermediate and/or final extracts. Source materials should also be placed on stability programs.

Step #4: After source materials are extracted, the resulting extracts are clarified, dialyzed, sterile-filtered, mixed with a stabilizing agent (i.e., glycerin), and stored. Allergenic extracts are often lyophilized to preserve their integrity. Lyophilized allergenic extracts are common intermediate products used for further manufacturing.

Additional activities the Allergen Science & Consulting has been and is performing regarding the production of allergenic extracts:

Because pollen is a challenging allergenic material, we have published a paper that exclusively addresses the production of pollen extracts. Please contact us if you would like to receive a copy of this article.

Allergen Science & Consulting led the preparation and review of a series of articles, entitled “How Allergen Extracts Are Made”, which addresses each type of product individually. We selected renowned scientists from different countries to contribute to the articles, which were published in the Annals of Allergy and Immunology. Please contact us if you have any inquiries about their content.