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

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

A different perspective regarding natural allergenic products 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 allergen extracts be assessed. This organization has not yet focused on assessing the quality of the products 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 products to prepare allergen extracts in the USA and Europe should be harmonized.

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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 contaminants. The potential impact of chemical contaminants in pollen is not discussed in this post.

Proper pollen collection is essential to produce allergen 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 man-made pollutants. However, this is an emerging strategy, which often does not allow for the collection of sufficient amounts of pollen necessary for the production of pollen allergen extracts. Many small family-owned pollen collection entities also obtain pollen for the production of pollen 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 associated with pollen used for the production of pollen extracts, including 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.

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 allergen 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 allergen 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 extracts.

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The image below describes our proposal to prepare a guidance document to assist the individuals and organizations involved in the evaluation of pollen purity to prepare allergen extracts. Please click the image to see it enlarged.  

A large number of allergen 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 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.

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Environmental factors are responsible for the deterioration of the 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). 

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 allergen extracts. 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.

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 allergen extracts list pollen, mites, fungi (molds), insects, epithelia (mammalian allergens), hymenoptera venoms, foods, and “others”. Therefore, the procurement of the raw materials to produce those allergen 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 allergen 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 allergen extracts as well as their stability and expiration dates.

Steps involved in the production of allergen extracts:

A number of steps are involved in the production of allergen extracts. These steps vary depending on the types of extracts and their intended purpose/s. For example, allergen 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 allergen 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, 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: 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. Allergen extracts are often lyophilized to preserve their integrity. Lyophilized extracts are common intermediate products used for further manufacturing.

Additional activities the Allergen Science & Consulting has been and is performing regarding the production of allergen 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 leaded 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.