Allergen manufacturing and quality aspects for allergen immunotherapy in Europe and the United States: An analysis from the EAACI AIT Guidelines Project.

Adequate quality is essential for any medicinal product to be eligible for marketing. Quality includes verification of the identity, content and purity of a medicinal product in combination with a specified production process and its control. Allergen products derived from natural sources require particular considerations to ensure adequate quality. Here, we describe key aspects of the documentation on manufacturing and quality aspects for allergen immunotherapy products in the European Union and the United States. In some key parts, requirements in these areas are harmonized while other fields are regulated separately between both regions. Essential differences are found in the use of Reference Preparations, or the requirement to apply standardized assays for potency determination. As the types of products available are different in specific regions, regulatory guidance for such products may also be available in one specific region only, such as for allergoids in the European Union. Region-specific issues and priorities are a result of this. As allergen products derived from natural sources are inherently variable in their qualitative and quantitative composition, these products present special challenges to balance the variability and ensuring batch-to-batch consistency. Advancements in scientific knowledge on specific allergens and their role in allergic disease will consequentially find representation in future regulatory guidelines.

Challenges in the implementation of EAACI guidelines on allergen immunotherapy: A global perspective on the regulation of allergen products.

Regulatory approaches for allergen immunotherapy (AIT) products and the availability of high-quality AIT products are inherently linked to each other. While allergen products are available in many countries across the globe, their regulation is very heterogeneous. First, we describe the regulatory systems applicable for AIT products in the European Union (EU) and in the United States (US). For Europe, a depiction of the different types of relevant procedures, as well as the committees involved, is provided and the fundamental role of national agencies of the EU member states in this complex and unique network is highlighted. Furthermore, the regulatory agencies from Australia, Canada, Japan, Russia, and Switzerland provided information on the system implemented in their countries for the regulation of allergen products. While AIT products are commonly classified as biological medicinal products, they are made available by varying types of procedures, most commonly either by obtaining a marketing authorization or by being distributed as named patient products. Exemptions from marketing authorizations in exceptional cases, as well as import of allergen products from other countries, are additional tools applied by countries to ensure availability of needed AIT products. Several challenges for AIT products are apparent from this analysis and will require further consideration.

Accurate quantification of 5 German cockroach (GCr) allergens in complex extracts using multiple reaction monitoring mass spectrometry (MRM MS).

BACKGROUND: German cockroach (GCr) allergen extracts are complex and heterogeneous products, and methods to better assess their potency and composition are needed for adequate studies of their safety and efficacy. OBJECTIVE AND METHODS: The objective of this study was to develop an assay based on liquid chromatography and multiple reaction monitoring mass spectrometry (LC-MRM MS) for rapid, accurate, and reproducible quantification of 5 allergens (Bla g 1, Bla g 2, Bla g 3, Bla g 4, and Bla g 5) in crude GCr allergen extracts. RESULTS: We first established a comprehensive peptide library of allergens from various commercial extracts as well as recombinant allergens. Peptide mapping was performed using high-resolution MS, and the peptide library was then used to identify prototypic and quantotypic peptides to proceed with MRM method development. Assay development included a systematic optimization of digestion conditions (buffer, digestion time, and trypsin concentration), chromatographic separation, and MS parameters. Robustness and suitability were assessed following ICH (Q2 [R1]) guidelines. The method is precise (RSD < 10%), linear over a wide range (r > 0.99, 0.01-1384 fmol/µL), and sensitive (LLOD and LLOQ <1 fmol/µL). Having established the parameters for LC-MRM MS, we quantified allergens from various commercial GCr extracts and showed considerable variability that may impact clinical efficacy. CONCLUSIONS AND CLINICAL RELEVANCE: Our data demonstrate that the LC-MRM MS method is valuable for absolute quantification of allergens in GCr extracts and likely has broader applicability to other complex allergen extracts. Definitive quantification provides a new standard for labelling of allergen extracts, which will inform patient care, enable personalized therapy, and enhance the efficacy of immunotherapy for environmental and food allergies.

Defects of T-cell effector function and post-thymic maturation in X-linked hyper-IgM syndrome.

X-linked hyper-IgM syndrome (XHIM) results from mutations in the gene encoding for CD40 ligand (CD154). Patients with the syndrome suffer from infections with opportunistic pathogens such as Cryptosporidium and Pneumocystis carinii. In this study, we demonstrate that activated T cells from patients with XHIM produce markedly reduced levels of IFN-gamma, fail to induce antigen-presenting cells to synthesize IL-12, and induce greatly reduced levels of TNF-alpha. In addition, we show that the patients' circulating T lymphocytes of both the CD4(+) and CD8(+) subsets contain a markedly reduced antigen-primed population, as determined by CD45RO expression. Finally, we demonstrate that the defects in antigen priming are likely due to the lack of CD154 expression and insufficient costimulation of T cells by CD80/CD86 interactions. Taken together, this study offers a basis for the increased susceptibility of patients with XHIM to certain opportunistic infections.

Characterization of allergen extracts.

Allergen vaccines are complex extracts of natural products, and are used for the diagnosis and treatment of allergic diseases. In the U.S., 19 allergen extracts have been standardized. For these vaccines, the potency is estimated by the skin test responses of highly allergic individuals, and surrogate in vitro tests are established for lot release and quality control. The surrogate tests differ for different extracts. National reference standards to which manufactured lots are compared are maintained at FDA/CBER. Allergen standardization has facilitated the establishment of data-driven release limits.

Murine B-cell and T-cell epitopes of the allergen Hev b 5 from natural rubber latex.

Hev b 5, a proline-rich acidic protein with a predominantly random secondary structure, is a major allergen in natural rubber latex and a candidate for specific immunotherapy of latex allergy. As a first step in the identification of candidate peptides for immunotherapy, we have begun to identify the B-cell and T-cell epitopes of Hev b 5 in BALB/c mice. The mice were immunized with a Hev b 5 fusion protein. The B-cell epitopes were determined by the SPOTS method using overlapping octamers or by ELISA inhibition using a series of overlapping 20-mers. The T-cell epitopes were determined by the proliferation and cytokine release of splenocytes cultured in the presence of the 20-mers. Potential antibody binding regions included residues in regions 1-38, 55-74, 109-128 and 132-151. Examination of the binding sequences for common motifs suggested enhanced antibody binding to the KXEE or KEXE sequences, where X is empty, threonine or alanine. Splenocyte stimulation and cytokine release suggest T-cell epitopes with the regions 1-20, 37-56, 73-101 and 109-146. Since they may contain major T-cell epitopes but do not exhibit significant antibody binding, peptide regions 38-48 and 75-101 are candidates for specific immunotherapy to Hev b 5 in the BALB/c mouse model.

Recurrent anaphylaxis in menstruating women: treatment with a luteinizing hormone-releasing hormone agonist--a preliminary report.

Recurrent idiopathic anaphylaxis is an illness consisting of recurring anaphylactic or anaphylactoid attacks of unknown cause. A patient has been described whose attacks appeared to be associated with endogenous progesterone secretion and who was treated successfully with an analog of luteinizing hormone-releasing hormone (LHRH). This report summarizes the treatment of four additional women with recurrent anaphylaxis in a randomized, double-blind trial of an LHRH agonist and placebo. Two out of the four women experienced remission of their symptoms with the LHRH analog. The patients who responded to therapy had experienced systemic anaphylactoid reactions after provocation with an LHRH infusion and the intradermal injection of medroxyprogesterone; the nonresponders had no adverse reactions to either challenge. Ovarian suppression with LHRH agonist may benefit a subset of women with recurrent idiopathic anaphylaxis.

Biological potency of German cockroach allergen extracts determined in an inner city population.

BACKGROUND: Cockroach allergy is an important cause of inner city asthma. To perform valid studies on the diagnosis and treatment of cockroach allergy, biological potencies of test extracts need to be established, and a surrogate in vitro test for biological potency should be chosen. METHODS: Sixty-two cockroach-allergic adult subjects were recruited for quantitative skin testing with three commercial German cockroach extracts. The intradermal D50 values were determined using linear interpolation, and the biologic potencies were determined from D50 data. The extracts were also analysed for relative potency, using a competition ELISA, and for specific allergen content, using a two-site ELISA. RESULTS: Estimates of each extract's D50 were analysable in 48-55 subjects, with D50s between 10.3 and 11.8. All three extracts were bioequivalent using pre-set criteria. The biological potencies of the extracts were 1738-8570 bioequivalent allergy units (BAU)/mL (geometric mean=3300), and these relative potencies were similar to those estimated by competition ELISA and specific allergen content. IgE against cockroach allergens were detected in sera from 34 subjects with analysable D50s, and 17 subjects had IgE directed against specific cockroach allergens. Although the presence of anti-Bla g 5 correlated with the subjects' skin test responses for 2/3 extracts, no single allergen was immunodominant. Antibody responses among the subjects were heterogeneous. CONCLUSIONS: Although commercial cockroach extracts are relatively low in potency, immunotherapeutic doses should be achievable. Biological potency may be estimated using D50 testing, a combination of specific allergen determinations, or by an overall potency assay such as the competition ELISA. CAPSULE SUMMARY: The biological potency of three German cockroach allergen extracts, determined in an inner city population, was 1738-8570 BAU/mL. No one allergen was immunodominant, and surrogate in vitro testing methods were examined.

Competition enzyme-linked immunosorbant assay (ELISA) can be a sensitive method for the specific detection of small quantities of allergen in a complex mixture.

RATIONALE: The competition ELISA assay is used to determine the potency of US standardized allergen extracts. We have been concerned that the competition ELISA is not sensitive to changes in individual allergen levels. This study was designed to determine the sensitivity of the competition ELISA to detect the specific loss of Bla g 1 and Bla g 2 in cockroach extracts. METHODS: German cockroach extract E3Cg was made from defatted German cockroaches. New Zealand White rabbits were immunized with rBla g 1 or rBla g 2. Optimal dilutions of anti-Bla g 1 and anti-Bla g 2 sera were established by ELISA. E3Cg was selectively depleted of Bla g 1 or Bla g 2 by immunoabsorption with anti-Bla g 1 or anti-Bla g 2 attached to Protein G agarose beads. Competition ELISA using pooled human sera, or mixed anti-Bla g 1 and anti-Bla g 2 serum, was performed on the depleted extracts, and on depleted extracts reconstituted with rBla g 1 or rBla g 2. RESULTS: Unlike pooled human-allergic IgE sera, anti-Bla g 1 and anti-Bla g 2 IgG -- in dilutions as low as 10(-6), could be used in the competition ELISA to measure the loss of allergen in depleted E3Cg. As little as 0.001 microg/mL of added rBla g 1 and 0.1 microg/mL of added rBla g 2, could be detected. CONCLUSION: The competition ELISA can be highly sensitive to compositional differences in complex allergen mixtures, even when the specific detecting antibody is present in relatively small amounts.

Exploiting the avian immunoglobulin system to simplify the generation of recombinant antibodies to allergenic proteins.

BACKGROUND: Monoclonal antibodies are a valuable tool in the study of allergens, but the technology used in their generation can be slow and labour-intensive. Therefore, we have examined recombinant antibody development by phage-display against single allergens and protein mixtures. OBJECTIVE: We used the avian immunoglobulin system (generated from single V(H) and V(L) genes) to provide a rapid method for generating highly specific recombinant antibody fragments from a minimal number of animals. METHODS: A single-chain antibody fragment (scFv) library was generated from a single chicken immunized with model allergens. ScFvs were isolated by phage-display and their properties investigated by ELISA and Western blot. RESULTS: Mono-specific scFvs were generated against recombinant Fel d 1 and native Amb a 1. Pannings against yellow jacket venom extracts only yielded clones that reacted with multiple proteins in the venom extract. The scFvs from each panning type were effectively expressed in Escherichia coli and readily purified. Highly specific and sensitive recognition of Fel d 1 and Amb a 1 was demonstrated in ELISA, with scFvs displaying antibody-concentration-dependent absorbance curves down to picogram levels of antibody. The specificity of selected antibodies for their cognate antigen was further confirmed in Western blot analysis, with scFvs directed to either Fel d 1 or Amb a 1 showing no reactivity for the other antigens used in immunization. Anti-Amb a 1 scFvs also mapped Amb a 1-isoform location in Western blot of ragweed extracts separated by 2D SDS-PAGE. DNA sequence analysis of scFvs showed that multiple different clones had been generated against Fel d 1 and Amb a 1. Using two anti-Fel d 1 scFv for ELISA analysis of Fel d 1 content in crude cat pelt extracts, we could produce data which were highly similar (P=0.33 and 0.89 by paired t-test analysis) to those obtained using conventional assays (radial immunodiffusion). CONCLUSION: Phage-display technology may generate multiple allergen-specific recombinant antibody fragments from a single chicken, to allergens from mammalian, plant and insect sources. The resulting antibody fragments are of demonstrable use in allergen identification and quantification, in comparison with standard immunoassays.

Allergic reactions to natural rubber.

Immediate hypersensitivity reactions to natural rubber pose a significant risk to patients with spina bifida and urogenital abnormalities, health care workers, and rubber industry workers. Other patients, outside of these high risk groups, have experienced severe allergic reactions to natural rubber as well. Awareness of this life-threatening condition by health care providers is essential if reactions are to be prevented. History alone is inadequate to identify all patients at risk, and reliable testing materials are not yet approved or widely available. Nonrubber medical devices are readily available for most uses; however, the only rubber-free condoms currently on the market do not prevent the transmission of sexually transmitted disease. The identification and detection of rubber antigens, along with mandated labeling for rubber antigen content, will contribute to the care of this growing population of patients. Efforts by the rubber industry to decrease the antigen content of natural rubber products will decrease the risk of reaction among sensitized patients and will, very likely, decrease the rate of sensitization in the future.

Latex antigens.

Previous studies have demonstrated that some children with spina bifida have IgE to proteins in natural rubber. In this study we compare different sources of latex antigen and identify possible antigenic peptides by radioimmunoblotting technique. Sera were collected from 26 children with spina bifida, tested by RAST with ammoniated latex extract (AL), and frozen until use. Extracts were prepared from ammoniated and nonammoniated latex, and the proteins were separated by electrophoresis on a 15% sodium dodecyl suflate-polyacrylamide gel and transferred to polyvinylene difluoride (PVDF). Strips of PVDF were then incubated with individual sera and 125I-labeled rabbit antihuman IgE before development by autoradiography; 18/26 sera were AL RAST positive; 0/8 AL RAST-negative patients had any binding to the latex proteins on PVDF. Sera from all patients were tested in a RAST with a nonammoniated latex extract (NAL), and the results were comparable to the AL RAST. Liquid-phase AL and NAL were comparable in their ability to inhibit the binding of patient's IgE to solid-phase AL and NAL. Sera from 14 RAST-positive patients were tested by immunoblotting with separated, reduced, and nonreduced AL and NAL. All 14 sera demonstrated IgE binding to a 14 kd peptide, which was more pronounced in reduced NAL. These results suggest that the 14 kd peptide in NAL is a major antigen in rubber allergy but that AL is an acceptable antigen source for in vitro diagnostic studies.

Latex hypersensitivity in children.

Latex is a natural substance used in the manufacture of thousands of products. Although latex allergy is uncommon in the general population, health care workers and children with spina bifida appear to be at high risk for latex allergy. These patients may experience urticaria, rhinoconjunctivitis, bronchospasm, and anaphylaxis following contact with or inhalation of latex antigens. Several protein allergens in latex have been identified, some of which appear to cross-react clinically and immunochemically with fruit antigens. Good diagnostic tests are available for latex allergy, but strict avoidance is still the only available treatment.

Immunodetection of latex antigens.

BACKGROUND: Protein antigens in latex products can cause type I reactions. In the past antigens have been measured by protein assays, high-performance liquid chromatography, RAST inhibition, and skin tests. We examined the use of a mouse monoclonal antibody (CRI-C) to latex in the detection of latex antigens. METHODS: CRI-C was raised by standard techniques after immunization of a BALB/c mouse with ammoniated latex. Medical gloves were extracted and assayed with: (1) standard protein assays, (2) RAST inhibition assays with sera from health care workers allergic to latex and patients with spina bifida, (3) an ELISA with a biotinylated form of CRI-C (BiC). Reference proteins included bovine serum albumin for the protein assays and nonammoniated latex and affinity-purified C antigen for the immunoassays. RESULTS: Among the protein assays, the best correlation was between the Bradford and bicinchoninic acid assays. In absolute numbers the Bradford assay produced the lowest results, and OD280 the highest. The OD280, BCA, and Bradford methods "detected" protein in vinyl gloves. The results of RAST inhibition and BiC ELISA correlated with the protein assays. These immunoassays appeared to be more specific than the protein assays. CONCLUSIONS: The BiC ELISA is an easy and reproducible in vitro assay of relevant latex antigen. Clinical correlation will be required for validation.

Affinity purification of latex antigens.

Latex extracts are complex mixtures of antigenic peptides. We attempted to raise monoclonal antibodies to latex and to use these antibodies to purify latex antigens. A monoclonal antibody, CRI-C, was raised by standard techniques. Peptides of nonammoniated latex (NAL) and ammoniated latex were electrophoretically separated and transferred for immunoblots. CRI-C was covalently attached to an agarose column. NAL was passed over the column, and purified antigen was then eluted. The eluate was analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis and RAST inhibition with sera from health care workers and children with spina bifida. CRI-C recognized a single band in ammoniated latex immunoblots and several distinct bands in NAL. The affinity-purified antigen of CRI-C (C-Ag) had multiple bands of less than 20 kd and was 3.9 times more potent in RAST inhibition than NAL when sera from patients with spina bifida were used. However, when health care workers' sera were used, there was no significant difference in the inhibitory potency of NAL and C-Ag. CRI-C appears to recognize a distinct and important epitope in the IgE immune response to latex of patients with spina bifida.

The determination of equivalent doses of standardized allergen vaccines.

BACKGROUND: Standardized allergen vaccines are tested for potency by manufacturers by using assays proposed in their license applications and approved by the Center for Biologics Evaluation and Research, which reviews and verifies the test results before lot release. The current lot-release limits for mite and grass pollen allergen vaccines impose statistical equivalence to the national reference extract; thus the limits are primarily based on assay variability. OBJECTIVE: We sought to establish a clinical basis for lot-release limits for the relative potency of allergen vaccines and to evaluate alternative specifications. METHODS: We performed literature selection and review, linear and logistic regression analyses of selected studies, and analysis of lots submitted to the Food and Drug Administration for approval since 1995. RESULTS: Therapeutic equivalence is achieved over a 10-fold range of allergen concentration. Safety equivalence is more difficult to assign, but on the basis of injection data, a 4-fold increase in allergen concentration is associated with a 5% to 10% increase in adverse reaction rates. The SD in log relative potency for the submitted allergenic products was determined to be 0.090 for grasses and 0.061 for mites, compared with 0.079 for competition ELISA. CONCLUSIONS: Current lot-release limits are well within literature-based estimates of therapeutic, diagnostic, and safety equivalence ranges for the clinical use of allergen vaccines. In addition, the aggregate consistency of the submitted products is comparable with the precision of the assay that is used to assess the products. These results support expanded release limits for verification of relative potency, provided the submitted lots of material remain at their present level of consistency.

Characterization and comparison of commercially available German and American cockroach allergen extracts.

BACKGROUND: In this study we examine the variability among unstandardized cockroach allergen extracts. METHODS: We obtained 24 aqueous and glycerinated cockroach allergen extracts from nine manufacturers. We used previously characterized cockroach extracts, E2-Cg and E2-Ca, as references. The modified ninhydrin assay was used to determine protein concentration of each extract. Relative potencies of extracts were determined by competition ELISA, using a human allergic serum pool. Bla g 1 and Bla g 2 levels of glycerinated German cockroach extracts were determined by ELISA using monoclonal antibodies. Extracts were also analysed by SDS-PAGE. RESULTS: Commercial cockroach allergen extracts had highly variable protein contents that were lower than the protein contents of the references. Electrophoretic data confirmed the presence of a variable number and intensity of protein bands in extracts among manufacturers. The relative potencies of the commercial extracts were between 10 and 782 BAU/mL for German cockroach and 10-250 BAU/mL for American cockroach. The mean Bla g 1 content of the commercial extracts was significantly lower than that of the reference (P = 0.001). The mean Bla g 2 content of the commercial extracts was higher than that of the E2-Cg reference but the Bla g 2 levels were more variable compared to Bla g 1. In glycerinated German cockroach extracts, protein concentrations, relative potencies and specific allergen levels were significantly correlated (P < 0.001). CONCLUSION: Our tests indicate that commercially available cockroach allergen extracts are variable in protein content, electrophoretic banding patterns, relative potency and Bla g 2 levels. In glycerinated German cockroach extracts, protein concentrations, relative potencies and specific allergen levels were significantly correlated.