How Allergenic Extracts Are Made

Allergenic extracts are biological products used to diagnose and treat allergic patients. Knowledge about how allergenic extracts are manufactured is critical for the clinician to select appropriate extracts for the intended purpose.

Various steps are involved in the production of allergenic extracts. These steps include the proper selection and procurement of allergenic raw materials, the manufacturing processes utilized, the quality procedures in place to assure that the extracts have a consistent composition and potency, and the measures used to support particular expiration dates.

The allergens utilized to produce extracts can be classified as natural or recombinant. Natural allergens have been used for many years and will continue to be used for the time to come because of the varying limitations regarding the production and use of recombinant allergens.

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 allergenic extracts varies, depending on the product. Natural allergenic raw materials are obtained from the outdoor environment (i.e. pollen), from laboratory cultures (i.e mites and fungi), animal groomers (i.e. epithelia), and supermarkets (foods).

A series of articles, entitled “How Allergen Extracts Are Made – From Raw Materials to Final Extracts”, which address each type of product individually, was prepared to inform the clinician and other interested professionals about the topic.

Contact us to obtain copies of these articles.

Storage of Allergenic Raw Materials

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 (<0oC). 

Dust mite micro photograph

Relevance of Indoor Allergen Exposure

The prevalence of allergic diseases, including asthma and hay fever (allergic rhinoconjunctivitis), has increased in developed countries around the world during the past decades and continue to increase. According to the American Academy of Allergy Asthma and Immunology, allergic diseases currently affect more than 25% of individuals in the USA. This has impacted the global allergy market.

Many studies suggest that exposure to common indoor allergens is a relevant environmental factor partially responsible for the increase in prevalence of allergic diseases in developed countries. Individuals in these countries spend more than 90% of their time indoors, and therefore exposure to indoor allergens is prolonged. Genetically susceptible individuals exposed to indoor allergens, primarily during infancy, become sensitized to these allergens.

Allergen avoidance can prevent (to some extent) the development of sensitization to allergens and relieve allergic symptoms in allergen-sensitized individuals. Therefore, various medical and public-oriented professional organizations have emphasized the relevance of indoor allergen exposure and recommended several guidelines for affected subjects, encouraging the use of certain measures for optimal indoor allergen avoidance.

Several multi-center studies on asthma and allergens performed at the national level in homes of affected subjects (including the National Cooperative Inner-City Asthma Study and the National Survey of Lead and Allergens in Housing) have demonstrated the usefulness of allergen measurement in monitoring the efficacy of allergen avoidance interventions.

The high prevalence of allergies has contributed to the increased public concern about air quality in homes, schools, and the workplace during the past years. However, due to the growing demand for mold assessments, common allergens (other than fungi) are not typically addressed during the course of indoor air quality inspections, albeit these non-fungal allergens are often the cause of the allergy symptoms experienced by building occupants.

A number of allergens are present indoors. A dose-response relationship between exposure and sensitization has been described for dust mite, cockroach, cat, and dog allergens. Cat and dog allergens are contained in small particles (2-6 um in diameter), which  are passively transported to public buildings. Therefore, cat and dog allergens are ubiquitous and present in the majority of buildings, including those where these animals have never been housed, like new homes and schools, at levels that, in some cases, exceed proposed thresholds to cause sensitization.

Studies performed in several major inner cities of the USA have linked exposure to cockroach, rat, and mouse allergens with asthma, and indicate that these allergens account for 95% of the allergen load present in some homes.

The relevance of indoor allergen exposure during infancy to cause sensitization and development of asthma should be emphasized to the general public. Daycare centers and schools can be critical sites of exposure to indoor allergens, and it has been suggested that these buildings should be target locations for interventions. Epidemiologically, it can be more effective to target one single daycare center or school than multiple homes.

Production of Pollen Extracts: How Pollen Purity is Evaluated

After the pollen used for the production of allergenic extracts has been collected, their identity and purity must be evaluated. Microscopic analysis is performed for this purpose. 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.
For the purpose of microscopic pollen identity and purity evaluations, samples are stained with a solution and examined under various magnifications. Microscopic pollen analysis also can provide valuable information regarding pollen quality for the production of pollen 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 structure indicates that fungi have actively infested the pollen for a period. This pollen should be discarded for the production of pollen extracts.

Qualified individuals should perform the pollen identification and purity assessments in appropriate in-house laboratories at allergen manufacturing companies. However, programs to train individuals and certify such laboratories are currently lacking.
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 of pollen used to produce pollen extracts with other allergenic products will likely occur.

Pollen Collection to Produce Allergen Extracts

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 amount 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.

There are three main methods used to collect pollen from wind-pollinated plants; first, the water-set; second, the vacuum; and third, the cut/dry/sieve. Many pollen species can be collected using either method.

The three collection methods have advantages and disadvantages. The main advantage of the water-set method is that it results in very clean pollen. To the contrary, it is very labor intensive and often the resulting pollen is impacted by high moisture and subsequent microbial growth.

The main advantage of the vacuum and the cut/dry/sieve methods is that are relatively simple to perform. Their main caveat is that the resulting pollen often contains high levels of biological contaminants.

Following collection, pollen is dried to reduce moisture content and prevent microbial growth. Subsequently, the naturally present plant parts, fungal spores, insect fragments, and foreign pollen grains are removed. The two primary methods to clean pollen are mechanical gradation sieving and air classification, both described below.
Mechanical gradation sieving is the simplest and most widely used method because such sieving with various micron-sized meshes can remove biologic contaminants. The air classification method can separate particles that differ in both weight and size, resulting in very clean pollen for the production of pollen extracts. Though, air classification machines are expensive and often disrupt pollen, making its quality difficult to assess because some cytoplasmic material containing allergens can be lost during the cleaning process.

Proper understanding regarding pollen collection to produce allergen extracts is essential for allergen manufacturers and clinicians who use pollen allergen extracts.

Evaluation of pollen purity to prepare allergen extracts

Several factors are responsible for the composition, consistency, and stability of the allergen extracts used to diagnose and treat allergic diseases. The quality of the raw materials used to produce those extracts should be maximized. Proper evaluation of pollen purity to prepare allergen extracts is critical to assure the quality of the final product.

Pollen is a unique raw material because is exposed to the outdoor environment before and during collection. Therefore, pollen can be potentially impacted by 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.

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 variety of fungal spores, foreign pollen grains, and plant parts in pollen should be expected, and it simply indicates insufficient cleaning. This pollen can be further cleaned and used to produce allergen extracts.

To the contrary, the presence of large amounts of one single spore type, hyphae or sporulating structures indicates that fungi have actively colonized polled due to current or past excessive moisture. Thus, this pollen should be discarded.

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.  



Production of allergen 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 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 is also involved in the preparation and review of a series of articles, entitled “How Allergen Extracts Are Made”, which addresses each type of product individually. We have selected renowned scientists from different countries to contribute to the articles, which are being published sequentially in the Annals of Allergy and Immunology during 2017.

How Human Activities Impact Pollen

A large number of plants exist on Earth. Geographical location, soil composition, and climate are the main natural factors associated with plant distribution and botanical biodiversity. Speciation and hybridization are responsible for the existence of different plant varieties of the same species, which often produce pollen with allergenic capacities composition. Pollen is responsible for seasonal allergies worldwide.

The topic of how human activities impact pollen is complex. Urbanization alters the distribution of native plants by reducing their ecological niches. On the contrary, plants that produce highly allergenic pollen are often introduced in urban areas due to landscaping practices.

Invasive plants are also accidentally introduced in ecosystems, and they slowly replace native plants.  For example, short ragweed (Ambrosia artemisiifolia), a weed native from the USA that produces highly allergenic pollen, was introduced in Europe years ago, and now this pollen is responsible for seasonal allergies in many European countries.

“Global warming”, another phenomenon associated with human activities, also affects plant biodiversity. A global increase in temperature is altering the geographical distribution of many plant species and their pollination patterns.

In addition, to the effect of human activities on pollen previously mentioned, this phenomenon might also impact pollen directly. For example, many observations indicate that the increasing levels of air pollution associated with urbanization and extensive use of fossil fuels augment the allergenic potential of pollen derived from different plants. Other observations suggest that pollen exposed to high levels of air pollution have greater inflammatory properties than pollen present in non-polluted areas.

In summary, the effect of human activities on plant biodiversity, pollination patterns, and pollen allergenic potential has been documented. The potential impact of this phenomenon on human health is under investigation.

Allergists/immunologists should be aware of the potential impact of human activities on pollen, and that their patients could perhaps experience non-expected exacerbations of their allergy symptoms in the future. Companies that manufacture pollen allergen extracts to diagnose and treat allergic diseases should take the correct measures to assure that the appropriate pollen is collected for the intended purpose.

Factors Responsible for Allergies

According to the World Health Organization (WHO) and the World Allergy Organization (WAO), allergies or immediate-type hypersensitivity reactions, affect approximately 20-30% of the world population. Allergies are the most common chronic diseases in adults and children in developed countries, accounting for high levels of morbidity and social burden, as determined by various direct and indirect parameters. These parameters include self-reported symptoms, medication use, hospitalizations, emergency room admissions, and missed work or school days.

The prevalence and incidence of allergies has increased during the past decades and continue to increase in developed countries.  Several hypotheses, including the “Hygiene Hypothesis”, have been proposed to explain this phenomenon. This hypothesis postulates that the decreasing exposure to microbial products, resulting from civilization, plays a role in switching Th1 to the Th2 immune response. Th1 responses protect from developing allergies while Th2 responses promote them.

Many individuals in the world live in urban areas, which have grown during the past years. Developing countries are evolving, an over 90% of urban growth is occurring in those nations, adding an estimated 70 million new residents to urban areas each year. Therefore, an increase in the prevalence and incidence of allergies in developing countries should be expected over time.

Multiple factors responsible for allergies have been identified. The factors responsible for allergies are complex and result from a combination of both genetic and environmental components.

Allergies cluster in families. The risk of developing allergies in children is estimated to be around 30% if one parent has allergies and 70% if both parents suffer from them. A large number of genes have been associated with allergic asthma and particular allergic phenotypes.

The most important environmental factor responsible for allergies is exposure to allergens. Other environmental factors that can promote allergic sensitization and trigger symptoms, particularly asthma, include exposure to air pollutants.

Infants with particular genetic phenotypes predisposing to allergies initially develop food allergy, resulting in atopic dermatitis, which generally resolves with increasing age. It has been reported that approximately 40%-70% of those children are symptom-free when they reach an age of approximately 7 years.

In many cases, children with food allergies subsequently develop allergic rhinitis, resulting from exposure to aeroallergens. Allergic rhinitis often evolves to allergic asthma. This progression in the manifestation of allergic symptoms is known as “the allergic March”

Due to the complex interaction among the various factors responsible for allergies, a number of long-term birth cohort studies have been and are being conducted to ascertain the relevance of the combined factors responsible for allergic sensitization and its progression from childhood to young adulthood. These studies include the  International Study of Asthma and Allergies in Childhood (ISAAC), Mechanisms of the Development of Allergy (MeDALL), Developing a Child Cohort Research Strategy for Europe (CHICOS), Environmental Health Risks in European Birth Cohorts (ENRIECO), and Global Allergy and European Network (GA2LEN). These studies will identify the factors responsible for allergies.

 

 

 

 

Allergen Extracts on the Market

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.

Contact us for more information regarding the topic of this post.