The US Food and Drug Administration (FDA) hosted a workshop on February 22, 2024, to discuss the status of biomarkers in drug development for allergic asthma and food allergy. The workshop provided a forum for open discussion among regulators, academicians, National Institutes of Health staff and industry to inform stakeholders of the requirements for the FDA to adopt a biomarker as a surrogate end point for a clinical trial, and to inform FDA of the status of various biomarkers in development. The workshop was divided into 3 sessions: (1) FDA and European Union regulators discussing regulatory perspectives on use of biomarkers in drug development programs, (2) investigators discussing biomarkers for pediatric and adult asthma, and (3) investigators discussing biomarkers for food allergy. In this report, we review the information presented at the workshop and summarize the current status of potential biomarkers for these allergic diseases.

BACKGROUND:T-cell responses to the individual components of allergen extracts have not been fully elucidated in subcutaneous allergen immunotherapy (SCIT). Specifically, it is unknown whether T-cell responses to immunodominant allergens are more or less sensitive to modulation, and whether allergen abundance in the immunotherapy extract influences T-cell response modulation. OBJECTIVE:To fill these gaps, we evaluated CD4+ T-cell reactivity specific to each of the main cockroach allergens in the double-blinded, placebo controlled, multicenter CRITICAL (NCT03541187) SCIT trial. METHODS:Participants aged 8-17 years with mild-to-moderate, well-controlled asthma received 12 months' dosing with cockroach SCIT (n = 20) or placebo (n = 26). Peripheral blood mononuclear cells were isolated before and after 12 months of therapy. CD4+ T-cell responses at baseline and after treatment were assessed using overlapping peptide pools derived from 11 well-defined cockroach allergens and intracellular cytokine staining for IL-4, IFN-γ, and IL-10 production. T-cell responses were evaluated for magnitude, cytokine polarization, allergen immunodominance, and correlation with allergen content in the cockroach SCIT extract. RESULTS:SCIT modulation was more prominent in participants with the strongest and most TH2-polarized responses. Downmodulation was observed against Bla g 5 and Bla g 9, the most dominantly recognized allergens in the population study. Furthermore, effective modulation was observed independent of allergen content in the cockroach SCIT extract. CONCLUSION:Immunodominant responses are effectively modulated by SCIT, and this effect is independent of allergen abundance in the extract utilized for SCIT.

Introduction: German cockroach (GCr) aeroallergens are associated with allergic rhinitis and asthma. Vitellogenin (Vg) and vitellin (Vn) are abundant proteins in GCr blood and eggs (including egg cases), respectively, and are possible high molecular mass allergens. Prior efforts to purify Vg/Vn yielded amounts too small for subsequent studies. In this study, we report the affinity purification of Vg/Vn from whole-body defatted GCr powder and determination of the binding of Vg/Vn to anti-GCr IgE. Method: New Zealand white rabbits were immunized with pure Vg/Vn in Freund's adjuvant, and IgG was purified from the rabbit sera and conjugated to cyanogen bromide (CNBr)-activated Sepharose. Aqueous extracts from GCr powder were passed over the column. After extensive washing, putative Vg/Vn was eluted in low-pH buffer, neutralized, and analyzed by SDS-PAGE and liquid chromatography high-resolution mass spectrometry (LC-HRMS). IgE binding of Vg/Vn was evaluated by inhibition of IgE binding to GCr-ImmunoCAP(I6) in sera from 10 GCr-allergic individuals. In addition, Vg/Vn was biotinylated and bound to ImmunoCAP-streptavidin, and direct IgE antibody binding to the immobilized Vg/Vn was determined in sera from 26 GCr-allergic individuals. Results: Vg/Vn isolated by affinity chromatography was 91% pure by LC-HRMS; contaminants included Bla g 3 (0.9%), human keratin (6%), and rabbit IgG. Vg/Vn inhibited IgE binding to GCr-ImmunoCAP(I6) in 8 of 10 sera. In direct-binding experiments, 21/26 (80%) sera had anti-Vg/Vn IgE at >0.10 kU(A)/L, while 11/26 (42%) sera were >0.35 kU(A)/L. Conclusions: We affinity-purified Vg/Vn and demonstrated that Vg/Vn-specific IgE antibody is a major component of GCr-specific IgE.

Chapter 81 Quantitation and Standardization of Allergens Ronald L. Rabin, Ronald L. RabinSearch for more papers by this authorLynnsey Renn, Lynnsey RennSearch for more papers by this authorJay E. Slater, Jay E. SlaterSearch for more papers by this author Ronald L. Rabin, Ronald L. RabinSearch for more papers by this authorLynnsey Renn, Lynnsey RennSearch for more papers by this authorJay E. Slater, Jay E. SlaterSearch for more papers by this author Book Editor(s):Barbara Detrick, Barbara Detrick Johns Hopkins University, School of Medicine, Baltimore, MarylandSearch for more papers by this authorJohn L. Schmitz, John L. Schmitz University of North Carolina, School of Medicine, Chapel Hill, North CarolinaSearch for more papers by this authorRobert G. Hamilton, Robert G. Hamilton Johns Hopkins University, School of Medicine, Baltimore, MarylandSearch for more papers by this author First published: 27 January 2016 https://doi.org/10.1128/9781555818722.ch81 AboutPDFPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShareShare a linkShare onFacebookTwitterLinked InRedditWechat Abstract Allergic reactions and allergic diseases are the most common human disorders of immune regulation. Diseases may include localized responses in the skin and various portions of the airway, or systemic responses characterized by extensive skin involvement, severe airway compromise, or cardiovascular collapse. Mechanisms include mast cell or basophil activation by the cross-linking of allergen-specific homocytotropic IgE, cellular infiltration following mast cell or basophil mediator release, complement activation, the deposition of immune complexes in susceptible tissues, or the infiltration of activated T-lymphocytes. The degree of impairment from allergic disease varies widely, with most reactions posing minor inconvenience, but with rare episodes requiring intensive—and sometimes unsuccessful—interventions to prevent death. Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition RelatedInformation

The German cockroach (GCr) generates aeroallergens associated with allergic rhinitis and asthma. Vitellogenin(Vg)/vitellin(Vn), an abundant protein in GCr blood, eggs and egg cases, is a possible allergen with high molecular mass. Prior efforts to purify Vg/Vn yielded amounts too small for our studies. We report here an affinity purification of Vg/Vn from whole body defatted powder from adult GCr. We added protease inhibitors to all buffers containing GCr. Aqueous extracts from GCr powder (Greer) were prepared in ammonium bicarbonate buffer pH 8 (ABC) and paper-filtered. New Zealand white rabbits were sensitized with pure Vg in Freund's adjuvant (J Insect Physiol 45:431 (1999)). We purified IgG from anti-Vg rabbit serum using protein A Sepharose, and conjugated the rabbit IgG anti-Vg to cyanogen bromide-activated Sepharose. We passed GCr extract over the column, washed it extensively with neutral buffers and eluted bound material with glycine buffer pH 2.7. We neutralized, concentrated and evaluated the eluted material by SDS-PAGE and liquid chromatography high-resolution mass spectrometry (LC-HRMS). IgE binding of Vg/Vn was evaluated by inhibition of IgE binding to ImmunoCAP(i6) in sera from three GCr-allergic individuals. Vg/Vn isolated by affinity chromatography was 91% pure by LC-HRMS; contaminants included Bla g 3 (0.9%), human keratin (6%) and rabbit IgG. Vg/Vn inhibited IgE binding to ImmunoCAP(i6) in all 3 sera in a dose-dependent manner over a range of added vitellin/vitellogenin of 5-42.5 mcg, with maximal inhibition of 30-53%. We purified Vg/Vn and demonstrated that Vg/Vn-specific IgE is a major component of GCr-specific IgE in three GCr-allergic individuals.

Clinical studies demonstrate that efficacy and safety in allergen immunotherapy (AIT) are linked to a multiplicity of factors decisively influencing success or failure. In recent years, numerous trials were performed with correspondent study results published. Yet, the number of AIT products successfully obtaining licensure in the analogous time frame is comparably limited. Essential for licensure is that the AIT product investigated remains comparable in its qualitative and quantitative composition throughout the clinical development. Verification of efficacy is not solely demonstrated by a statistically significant difference between the test and control populations; it must also be shown to be clinically relevant. Choice of meaningful inclusion and end-point criteria is critical. Post hoc or subgroup analysis can be supportive but needs verification as predefined criteria in additional studies. Data analysis may be presented on varying analysis populations, while it should be based on the intention-to-treat population for regulatory review to allow objective assessment of the treatment effect on the overall study population. Apparently conflicting interpretations of clinical data between publications and regulatory review are frequently based on their inherently different objectives, with regulatory review taking into considerations the full data sets of all relevant clinical studies for the concerned AIT product to allow an informed decision on licensure.

Mouse allergy is an important cause of indoor asthma and allergic rhinoconjunctivitis. The major mouse allergen, Mus m 1, is a complex of homologous pheromone-binding lipocalins called major urinary proteins (MUPs). We analyzed the proteome of MUPs in mouse urine, commercial mouse epithelial extracts, and environmental samples using several approaches. These include as follows: two-dimensional electrophoresis and immunoblotting; liquid chromatography-high-resolution mass spectrometry (LC/HRMS); multiple reaction monitoring (MRM) mass spectrometry; and LC/HRMS analysis of glycans at the N-66 residue of MUP3. Albumin is predominant in the extracts, while MUPs are predominant in urine. LC/HRMS of 4 mouse allergen extracts revealed surprising heterogeneity. Of 22 known mouse MUPs, only 6 (MUP3, MUP4, MUP5, MUP13, MUP20, and MUP21) could be identified with MRM using unique peptides. Assessment of MUP content in urine, extracts, and dust samples showed good correlation between MRM and other methods working with different detection principles. All 6 identifiable MUPs were found in electrophoretically separated urine bands, but only MUP3 and MUP20 were above LOQ in unseparated mouse urine, and only MUP3, MUP4, and MUP20 were found in mouse epithelial extracts. Glycan heterogeneity was noted among 4 individual inbred mice: of 13 glycan structures detected, 8 were unique to one mouse, and only 2 glycan modifications were present in all 4 mice. Using mass spectrometry and MRM, mouse allergen extracts and urine samples are shown to be complex and heterogeneous. The efficacy and safety of commercial mouse allergen extracts will be improved with better controls of allergen content.

BACKGROUND:Mouse allergy is an important cause of indoor asthma and allergic rhinoconjunctivitis. The major mouse allergen, Mus m 1, is a complex of homologous pheromone-binding lipocalins called major urinary proteins (MUPs).METHODS:We analyzed the proteome of MUPs in mouse urine, commercial mouse epithelial extracts, and environmental samples using several approaches. These include as follows: two-dimensional electrophoresis and immunoblotting; liquid chromatography-high-resolution mass spectrometry (LC/HRMS); multiple reaction monitoring (MRM) mass spectrometry; and LC/HRMS analysis of glycans at the N-66 residue of MUP3.RESULTS:Albumin is predominant in the extracts, while MUPs are predominant in urine. LC/HRMS of 4 mouse allergen extracts revealed surprising heterogeneity. Of 22 known mouse MUPs, only 6 (MUP3, MUP4, MUP5, MUP13, MUP20, and MUP21) could be identified with MRM using unique peptides. Assessment of MUP content in urine, extracts, and dust samples showed good correlation between MRM and other methods working with different detection principles. All 6 identifiable MUPs were found in electrophoretically separated urine bands, but only MUP3 and MUP20 were above LOQ in unseparated mouse urine, and only MUP3, MUP4, and MUP20 were found in mouse epithelial extracts. Glycan heterogeneity was noted among 4 individual inbred mice: of 13 glycan structures detected, 8 were unique to one mouse, and only 2 glycan modifications were present in all 4 mice.CONCLUSIONS:Using mass spectrometry and MRM, mouse allergen extracts and urine samples are shown to be complex and heterogeneous. The efficacy and safety of commercial mouse allergen extracts will be improved with better controls of allergen content.

German cockroach (Blattella germanica) is a source of important urban indoor aeroallergens, associated with allergic rhinitis and asthma. Vitellogenin, a high molecular mass and abundant protein in B. germanica eggs and egg cases (oothecae), was purified and assessed for its allergenicity. To identify vitellogenin, extracts made from whole body acetone-defatted German cockroach were purified by ammonium sulfate precipitation followed by size exclusion chromatography, and extracts from cockroach egg cases were purified by size exclusion chromatography alone. SDS-PAGE (one- and two-dimensional) and bottom-up liquid chromatography high-resolution mass spectrometry (LC-HRMS) analyses were performed to determine the presence of vitellogenin. IgE-ELISA and immunoblots, using sera from individuals with confirmed allergy to German cockroach, were used to assess allergenicity. Vitellogenin was successfully identified in German cockroach whole body samples by LC-HRMS and was reactive to cockroach-allergic patient sera in ELISA and immunoblot experiments. Fragments of vitellogenin were identified in many of the bands and spots present on the SDS-PAGE gels, in addition to the 100 kDa band where one fragment had been previously identified. We partially purified vitellogenin from both whole bodies and egg cases. About one-third of patient sera had IgE binding to vitellogenin from egg case samples. Vitellogenin is an abundant German cockroach protein that is a candidate allergen. Further work is needed to determine the percentage of IgE that specifically binds vitellogenin in cockroach-allergic patients, and the importance of vitellogenin in the pathobiology of German cockroach allergy.

This chapter reviews the manufacturing procedures for food allergen extracts. The chapter also reviews applicable regulatory requirements, potential approaches to standardization, and clinical application of these products. Food allergen extracts are complex biological products used for the diagnosis, but not treatment, of food allergy. Preparation of a food allergen extract requires careful selection and identification of source materials, grinding, defatting, extraction conditions, clarification, sterilization, and product testing. Variation in any of these steps can affect the quality of the final product. Although extractions for all products licensed for use in the United States are performed using raw source materials, many foods are not consumed in their raw form. Thermal and nonthermal processing alters the allergenicity of many common food allergens. Processing prior to extraction may therefore change the allergenicity of the final product. The manufacture of food allergen extracts is a complex process with many considerations to maximize the quality of the final product. Allergen extracts for a select number of foods may be inconsistent between manufacturers or unreliable in a clinical setting, indicating a potential area for future improvement.

Allergenic source materials include pollen, molds, animal dander, and insects; food allergens from nuts, grains, and animals; venoms; and salivary proteins from insects and ticks. Clinical diagnostic tests have used heterogeneous extracts from allergen source materials for skin prick tests (SPTs). In vitro laboratory methods using immunoassays or microarrays can detect serum IgE directed against allergenic proteins where clinical testing may not be suitable. Clinicians rely primarily on licensed commercial extracts of allergens for SPTs. Manufacturers and regulatory agencies have standardized selected extracts for identity, composition, and potency. Allergen sources contain multiple proteins. The IgE antibody responses to these proteins vary between allergic subjects as does the quantity of specific IgE. Component-resolved molecular diagnostics can be used to improve the specificity of allergy testing and resolve clinical cross-reactivities that may affect treatment outcomes. This clinical commentary will review methods for the production, evaluation, and standardization of allergen extracts from the perspective of diagnostic testing that may be useful for allergists in practice. (C) 2020 American Academy of Allergy, Asthma & Immunology.

Allergen extracts are the most widely used allergy diagnostic and immunotherapeutic reagents worldwide. In this chapter, the authors discuss the regulation of these important products in the United States, the differences in regulation of standardized and nonstandardized products, and the additional potency determinations used for the labeling of standardized allergen extracts. In addition, the authors discuss the statistical basis on which bioequivalence may be established between allergen extracts and a reference standard, or a new product to an existing extract. The underlying considerations of allergen potency are specific to the allergen extracts themselves, and these differences are discussed in detail.

BACKGROUND:Allergen extracts are the primary tool for diagnosis and treatment of allergic diseases. In the United States, most allergen extracts are non-standardized. More sophisticated analytical approaches are needed to characterize these products and enable manufacturers and regulators to better determine potency.OBJECTIVE:To expand the multiple reaction monitoring (MRM) assay for an in-depth characterization of German cockroach (GCr; Blattella germanica) allergen extracts.METHODS:We applied advanced liquid chromatography (LC) and mass spectrometry (MS) techniques including MRM. The expanded LC/MRM-MS method was optimized to measure known GCr allergens and their isoforms/variants in commercial extracts and environmental samples. We performed isoform-specific allergen measurements in multiple extracts from four commercial sources and extracts prepared using environmental samples from urban homes. To investigate causes of heterogeneity, we examined over 30 extraction process variables.RESULTS:Evaluation of the commercial extracts confirmed the variability of production lots and commercial sources. Commonly used defatting and extraction protocols yielded extracts with comparable allergen profiles and content. However, the identity and quality of source materials was a major contributor to variability. In comparing commercial GCr extracts to environmental samples, relative quantities of Bla g 1, Bla g 2, Bla g 3, Bla g 4 and Bla g 11 were similar, while Bla g 5, Bla g 6, Bla g 7 and Bla g 8 were present in the environmental samples but largely absent for the commercial extracts.CONCLUSIONS AND CLINICAL RELEVANCE:LC/MRM-MS can be used to measure all known GCr allergens in commercial allergen extracts and environmental samples. Significant differences exist between allergen profiles of commercial extracts and the profiles of environmental samples from dwellings. This analytical platform can serve as a template to achieve better product characterization of similarly complex products.

We read with interest the CME review article “Understanding differences in allergen immunotherapy products and practices in North America and Europe,”1Mahler V. Esch R.E. Kleine-Tebbe J. Lavery W.J. Plunkett G. Vieths S. et al.Understanding differences in allergen immunotherapy products and practices in North America and Europe.J Allergy Clin Immunol. 2019; 143: 813-828Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar in which the authors compare the regulation of such products in Europe with their regulation in the United States. Unfortunately, the authors inaccurately describe the US regulatory framework for allergenic extracts. Here is a partial list of errors:1.The section titled “Regulation and Clinical Development of New Products, US Regulatory Standards” includes a discussion of the Food and Drug Administration's (FDA's) procedures for classification/reclassification of allergenic extracts into category I, II, IIIA, or IIIB. These procedures, formerly specified in regulations (21 CFR 601.25 and 601.26) and rendered obsolete in 2016, relate to allergenic extracts regulated by the National Institutes of Health before transfer to the FDA in 1972 and are irrelevant to regulation of new products. The authors also omit that, consequent to the most recent review of these extracts, the FDA revoked licenses for 15 extracts in 2013 because of safety concerns.2.The authors state, “There is no formal FDA guidance for clinical development of [AIT products]. Each product under development in the US is considered separately by the CBER.” The authors communicate that, lacking a formal guidance document, the FDA is unable to provide consistent, proactive advice to manufacturers. This is untrue. The FDA routinely provides pre- and post-licensure advice to manufacturers, consistent with applicable regulations and statutes. Furthermore, the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) documents described in Table E2 as European Union (EU) guidance documents are also considered as part of FDA's guidance regimen.3.Fig 1 contrasts a detailed EU clinical development framework with a superficial US framework that suggests, erroneously, that the FDA requires non-US phase II data for review of new products. This is untrue. Furthermore, the authors assert (without reference) that “much [in the US regulations] has been borrowed from EU precedents.” This is untrue as well. Although US and EU regulators appropriately learn from the other's experiences, their frameworks for pharmaceutical regulation evolved independently, the history of which is beyond the scope of this letter.4.The authors state, again without reference, that in the EU, “… the pharmaceutical company has to provide evidence for impeccable [emphasis added] quality, efficacy and safety” of an allergenic product. The obvious implication is that US standards for manufacturing allergenic extracts are inferior to the EU standards. This is untrue. In the United States—no less than in the EU—the standards for licensure include a demonstration of safety, purity, and potency. Furthermore, the word “impeccable” is an odd choice in this situation, which implies perfection rarely attainable in science or regulation.5.The authors state, “Only products with a positive benefit/risk ratio [can be licensed in the EU].” Obviously, this is also a requirement for US licensure. In conclusion, rather than presenting a disinterested comparison of EU and US regulatory practices, the authors have substituted opinion for fact in an article that is inconsistent with the standards of the Journal of Allergy and Clinical Immunology, and inappropriate as a CME review. Understanding differences in allergen immunotherapy products and practices in North America and EuropeJournal of Allergy and Clinical ImmunologyVol. 143Issue 3PreviewAllergen immunotherapy (AIT) is thought to be clinically effective and safe in treating allergic rhinitis, asthma, and stinging insect allergy in Europe and North America. However, there are intercontinental differences in AIT therapeutic products in terms of their application and regulation. In North America unmodified standardized and nonstandardized aqueous aeroallergen extracts are approved and used almost exclusively for subcutaneous immunotherapy, whereas more product options are available in Europe, including adsorbed allergens, chemically modified allergens, or both. Full-Text PDF ReplyJournal of Allergy and Clinical ImmunologyVol. 144Issue 4PreviewReviewing such a broad topic is formidable given many differences in extracts, manufacturing, clinical diagnoses, treatments, and regulation of new product development.1 In our attempt to present a concise review of differences between continents, we did not intend to minimize the role of the US Food and Drug Administration (FDA) in assuring quality, safety, effectiveness, and labeling of allergen products, as suggested in the correspondence by Rabin et al.2 Full-Text PDF

Allergenic extracts are complex biologics and good quality controls are needed to ensure manufacturing of products with consistent potency and quality. The existing approach uses customized antibodies to measure IgE-reactive proteins either individually or as a group. Although the approach is useful, it is not sufficient to provide a complete compositional picture of complex allergenic extracts. To develop a new and improved multiplex assay using a dynamic combination of high throughput LC/MS technology, multiple reaction monitoring (MRM) MS, and immunoassay for an in-depth allergenic protein identification, and simultaneous quantification, potency and stability measurements. The method development process involves immunoallergomics to establish a comprehensive profile of IgE-reactive proteins in extracts, and generate a peptide library of all allergens, isoforms, and variants. Prototypic and quantotypic peptides were selected for MRM method development for accurate quantification of all GCr allergens. ICH (Q2(R1)) guidelines were followed to assure assay robustness and suitability. Allergens from various commercial GCr extracts were quantified to show considerable variability between preparations. The LC-MRM MS method was also evaluated for as a stability assay of allergens subjected to various degradative conditions (temperature, pH, and enzymes). The MRM-based potency of selected allergens correlates well with ELISA-based measurements. Also, the LC-MRM was evaluated for its application towards measurement of GCr allergens in dust samples collected from various US households. The new assay is robust and valuable for potency measurement of GCr allergen extract and offers great promise in achieving full characterization.

Cockroach allergens can lead to serious allergy and asthma symptoms. Termites are evolutionarily related to cockroaches, cohabitate in human dwellings, and represent an increasing pest problem in the United States. The Formosan subterranean termite (Coptotermes formosanus) is one of the most common species in the southern United States. Several assays were used to determine if C. formosanus termite proteins cross-react with cockroach allergens. Expressed sequence tag and genomic sequencing results were searched for homology to cockroach allergens using BLAST 2.2.21 software. Whole termite extracts were analyzed by mass-spectrometry, immunoassay with IgG and scFv antibodies to cockroach allergens, and human IgE from serum samples of cockroach allergic patients. Expressed sequence tag and genomic sequencing results indicate greater than 60% similarity between predicted termite proteins and German and American cockroach allergens, including Bla g 2/Per a 2, Bla g 3/Per a 3, Bla g 5, Bla g 6/Per a 6, Bla g 7/Per a 7, Bla g 8, Per a 9, and Per a 10. Peptides from whole termite extract were matched to those of the tropomyosin (Bla g 7), arginine kinase (Per a 9), and myosin (Bla g 8) cockroach allergens by mass-spectrometry. Immunoblot and ELISA testing revealed cross-reaction between several proteins with IgG and IgE antibodies to cockroach allergens. Several termite proteins, including the hemocyanin and tropomyosin orthologs of Blag 3 and Bla g 7, were shown to crossreact with cockroach allergens. This work presents support for the hypothesis that termite proteins may act as allergens and the findings could be applied to future allergen characterization, epitope analysis, and clinical studies.

OBJECTIVE:To review the manufacturing procedures of food allergen extracts and applicable regulatory requirements from government agencies, potential approaches to standardization, and clinical application of these products. The effects of thermal processing on allergenicity of common food allergens are also considered. DATA SOURCES:A broad literature review was conducted on the natural history of food allergy, the manufacture of allergen extracts, and the allergenicity of heated food. Regulations, guidance documents, and pharmacopoeias related to food allergen extracts from the United States and Europe were also reviewed. STUDY SELECTIONS:Authoritative and peer-reviewed research articles relevant to the topic were chosen for review. Selected regulations and guidance documents are current and relevant to food allergen extracts. RESULTS:Preparation of a food allergen extract may require careful selection and identification of source materials, grinding, defatting, extraction, clarification, sterilization, and product testing. Although extractions for all products licensed in the United States are performed using raw source materials, many foods are not consumed in their raw form. Heating foods may change their allergenicity, and doing so before extraction may change their allergenicity and the composition of the final product. CONCLUSION:The manufacture of food allergen extracts requires many considerations to achieve the maximal quality of the final product. Allergen extracts for a select number of foods may be inconsistent between manufacturers or unreliable in a clinical setting, indicating a potential area for future improvement.