Allergen stabilities and compatibilities in immunotherapy mixtures that contain cat, dog, dust mite, and cockroach extracts

Allergen stabilities and compatibilities in immunotherapy mixtures that contain cat, dog, dust mite, and cockroach extracts

Ann Allergy Asthma Immunol 115 (2015) 496e502 Contents lists available at ScienceDirect Allergen stabilities and compatibilities in immunotherapy mi...

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Ann Allergy Asthma Immunol 115 (2015) 496e502

Contents lists available at ScienceDirect

Allergen stabilities and compatibilities in immunotherapy mixtures that contain cat, dog, dust mite, and cockroach extracts Thomas J. Grier, PhD *; Dawn M. Hall, BS *; Elizabeth A. Duncan, BS *; and Satyen M. Gada, MD y * Research y

and Development Laboratory, Greer Laboratories Inc, Lenoir, North Carolina Walter Reed National Military Medical Center, Bethesda, Maryland



Article history: Received for publication August 18, 2015. Received in revised form October 1, 2015. Accepted for publication October 2, 2015.


Background: Indoor allergen mixtures that contain cat, dog, dust mite, and cockroach extracts are commonly used in allergy clinics for subcutaneous immunotherapy, but product-specific stabilities and mixing compatibilities in these complex patient formulas have not been determined. Objectives: To assess the recoveries of cat, dog epithelia, dog dander, dust mite Dermatophagoides farinae, and cockroach mix allergen activities in 5 component mixtures and 1:10 (vol/vol) dilutions stored for up to 12 months. Methods: Concentrated stock mixtures, 10-fold dilutions of these mixtures in human serum albuminesaline diluent, and analogous single-extract controls were analyzed for major allergen concentrations (cat Fel d 1, dog dander Can f 1) and multiallergen IgE-binding potencies (dog epithelia, D farinae, cockroach mix) after storage for 3, 6, 9, and 12 months at 2 C to 8 C. Results: The selected immunoassays were specific for individual target extracts in the 5-component mixtures and exhibited analytical sensitivities sufficient for evaluation of both the concentrated and diluted indoor allergen formulas. All control samples except diluted cockroach extract had near-complete stabilities during refrigerated storage. Mixtures that contained cat, dog epithelia, dog dander, and D farinae extracts exhibited favorable mixing compatibilities in 1:1 (vol/vol) concentrates (47.5% glycerin) and 1:10 (vol/vol) dilutions (4.75% glycerin), relative to corresponding control sample reactivities. Cockroach allergens in both 1:1 (vol/vol) and 1:10 (vol/vol) concentrations were stabilized significantly by mixing with the other 4 indoor allergen extracts. Conclusion: Extracts in mixtures that contained 5 common sources of indoor allergens possess favorable stabilities and mixing compatibilities and support the practice of combining these products in the same patient treatment formulations for subcutaneous immunotherapy. Published by Elsevier Inc. on behalf of the American College of Allergy, Asthma & Immunology.

Introduction Subcutaneous immunotherapy (SCIT) using specific combinations and concentrations of allergenic extracts is an effective treatment regimen for individuals with allergic rhinitis, conjunctivitis, and/or asthma.1 Efficient preparation of extract vaccines optimized for treatment of seasonal and perennial allergen sensitivities is challenged by the diverse nature of these customized mixtures and interactive effects that can directly affect their stability, efficacy, or safety in SCIT formulations.1e4 Reprints: Thomas J. Grier, PhD, Greer Laboratories, Inc, PO Box 800, Lenoir, NC 28645; E-mail: [email protected] Disclosures: Drs Grier, Ms Hall, and Ms Duncan are employed by Greer Laboratories Inc, who provided financial support for this study. The views expressed by Dr Gada do not reflect the official policy of the US Department of Army, US Department of Navy, US Department of Defense, or the US government. Funding Sources: This study was funded by Greer Laboratories, Inc.

The design of SCIT formulations can be affected by the stabilities and mixing compatibilities of candidate extracts. Allergenic proteins in multiextract vaccines are susceptible to potential physical and biochemical interactions with companion extract components (such as proteolytic enzyme degradation) that can compromise their IgE-binding properties and recognition by sensitive patients.5e7 These changes could result from compositional effects (product concentrations, glycerin content, activities and specificities of proteolytic enzymes, diluent properties), environmental changes (storage times, storage and normal-use temperature ranges, volumes in stored vials), or combinations of these and other factors. Laboratory investigations of allergen compatibility in SCIT vaccines have focused on quantitative assessments of individual extracts or prominent allergenic proteins in 2-component mixtures vs single-extract controls.8e18 In most prior studies, significant reductions in the potencies or concentrations of 1081-1206/Published by Elsevier Inc. on behalf of the American College of Allergy, Asthma & Immunology.

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target allergens were reported in extract mixtures that contained high levels of endogenous protease activities (fungi, insect) combined with others that possessed low or undetectable hydrolase levels (pollen, dust mites, animal epithelia or dander). On the basis of these results, separation of low-protease products from protease-rich extracts in patient treatment vials has been recommended in SCIT practice parameter guidelines published between 2003 and 2011.3,19,20 The diminished immunochemical stabilities of several fungal and insect allergens in mixtures that contain only high-protease extracts suggest that further segregation of these products into different treatment vials may also be beneficial.8,9 Owing to the limited stability data available for multiextract formulas representative of patient-specific SCIT vaccines administered throughout North America (mean of 8e10 products per treatment vial), along with cost and patient adherence concerns, many clinics continue to include extracts from the low-protease and high-protease groups in the same vaccine preparations.21e23 Evaluations of the compatibilities of these complex mixtures are desirable but challenging and are often restricted by interferences in the recognition or binding of these samples by antibody probe reagents (matrix effects) or changes in their dose-response characteristics that affect the accuracy and precision of the resulting activity measurements.24 Independent potency assessments of more than 2 extracts in the same immunotherapy mixtures have not been reported to date in any published study. SCIT regimens with indoor allergen mixtures may be indicated for sensitive patients whose chronic symptoms or exposures cannot be controlled adequately by avoidance measures and pharmacologic treatments.3,25,26 One author (S.M.G.) has encountered numerous patients presenting with sensitivities to 5 major sources of indoor allergens (cat, dog epithelia, dog hair or dander, dust mites, cockroach) that were viable candidates for immunotherapy with these products. Because indoor allergens fall into both the low-protease (animals, dust mites) and high-protease (certain fungi, insects) categories, combining these extracts into a single patient treatment vial could pose a potential risk to the stabilities of these component in maintenance vial preparations and an even greater risk in diluted (build-up) vials that contain considerably lower protein and stabilizing glycerin concentrations.3,19,20,27 As noted previously, although some stability data have been reported for 2-part mixtures of indoor allergens, extract recoveries in formulations that contain most or all of these products have not yet been evaluated.5 The present study was designed to address several of the information gaps and deficiencies described above; specifically, concurrent quantitation of the immunochemical potencies of 5 different indoor allergen extracts (standardized cat hair and dust mite Dermatophagoides farinae; nonstandardized dog epithelia, dog hair or dander, and American and German cockroach mix) in the same mixtures at both maintenance (1:1 [vol/vol]) and buildup (1:10 [vol/vol]) vial strengths during storage for up to 12 months at 2 C to 8 C. Multiple extract characterization methods were examined to determine the most appropriate procedures for these analyses. The antibody or serum-based reagents, allergen standards, and incubation conditions that yielded assay results specific for each individual analyte (component extract) in the 5-product mixtures were selected for the experiments reported herein. The test methods included in this study recognized individual major allergenic proteins (radial immunodiffusion assays specific for cat hair allergen Fel d 1, double-bind enzyme-linked immunosorbent assays [ELISAs] specific for dog hair or dander allergen Can f 1) or multiple allergens (IgE inhibitions ELISAs for dust mite D farinae, dog epithelia, or cockroach mix) in each target extract.


Methods Allergenic Extracts Extracts and diluents were purchased by the US Army Centralized Allergen Extract Laboratory from Greer Laboratories (Lenoir, North Carolina; 1:10 [wt/vol] aqueous dog epithelia, 10,000 BAU/ mL of glycerinated cat hair, 10,000 AU/mL of glycerinated dust mite [D farinae], 1:10 [wt/vol] aqueous dog epithelia, 1:20 [wt/vol] glycerinated American and German cockroach mix, 50% glycerinsaline diluent, human serum albumin [HSA]esaline diluent), or Hollister-Stier Laboratories (Spokane, Washington; 1:100 [wt/vol] glycerinated acetone-precipitated dog hair or dander). Products were stored at 2 C to 8 C (extracts) or 20 C to 25 C (diluents), as recommended by the manufacturers. Extract Mixtures and Controls A concentrated extract mixture (labeled as 1:1 [vol/vol], 47.5% glycerin, 10-mL total volume) was prepared at the US Army Centralized Allergen Extract Laboratory by combining aqueous dog epithelia (0.5 mL) with glycerinated cat (4 mL), dust mite D farinae (2 mL), cockroach mix (1 mL), dog dander (2 mL), and glycerin-saline diluent (0.5 mL) at final concentrations consistent with current SCIT practice parameter recommendations.3,4 Single-extract control solutions were prepared in a similar manner using the same volume of each extract concentrate cited above and glycerin-saline diluent. Diluted extract mixtures and controls (labeled as 1:10 [vol/vol], 4.75% glycerin) were prepared by combining 1.0 mL of each 1:1 (vol/vol) sample with 9.0 mL of HSA-saline diluent. Samples were shipped to Greer Laboratories overnight on cold packs, stored at 2 C to 8 C, and analyzed after 3, 6, and 9 months (1:10 [vol/vol]) or 3, 6, 9, and 12 months (1:1 [vol/vol]). Freshly prepared control solutions identical in composition to the stored controls were also evaluated to provide estimates of both relative and absolute allergen recoveries in the test samples. All samples were prepared and tested before the expiration date of each extract and diluent component. Assay Reagents Freeze-dried human serum pools that contained allergenspecific IgE to dust mite D farinae (lot ZE-P3), dog epithelia (lot ZE-P2), or cockroach (lot ZE-P7) were prepared at Greer Laboratories in 1994 using plasma obtained from commercial vendors (PlasmaLab International, Everett, Washington; Roche Biomedical Laboratories, Burlington, North Carolina). The number of samples from distinct donors in these pools ranged from 4 (lot ZE-P2) to 23 (lot ZE-P7). Clinical data (skin test reactivities, histories) were available for some but not all donors. All samples tested negative for antibodies to hepatitis B and human immunodeficiency virus antigens. In addition to the target allergens, other specificities found in some pools included cat, fungi (Alternaria, Aspergillus), and pollens (ragweeds, grasses, English plantain). Mouse antieCan f 1 capture antibody, rabbit antieCan f 1 probe antibody, and primary Can f 1 antigen standard were obtained from Indoor Biotechnologies (Charlottesville, Virginia). Sheep antiserum (lot S9-Cat) and antigen standard (lot C13-Cat) for the major cat allergen Fel d 1 were obtained from US Food and Drug Administration (FDA; Rockville, Maryland). Analytical Methods The quantitative methods selected for in vitro analyses of target extract compositions or major allergen concentrations in the test mixtures and controls are given in Table 1. Human IgE inhibition ELISAs for dust mite D farinae, dog epithelia, and cockroach allergen potencies were performed as described previously


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Table 1 Analytical methods for target extracts in indoor allergen mixtures and controls Analyte

Assay method


Cat hair Dog hair or dander Dog epithelia Dust mite Dermatophagoides farina Cockroach mix (American and German)

Radial immunodiffusion Double-bind (sandwich) ELISA IgE ELISA inhibition IgE ELISA inhibition IgE ELISA inhibition

Major cat allergen Fel d 1 Major dog allergen Can f 1 Multiple dog allergens, including prominent allergen Can f 3 (albumin) Multiple dust mite allergens Multiple allergens from both cockroach species

Abbreviation: ELISA, enzyme-linked immunosorbent assay.

using IgE-positive serum pools and dilutions of each specific glycerinated extract as coating and reference reagents.10,13 These methods were very similar in configuration and performance to those used regularly for lot release and stability testing of standardized dust mite and grass pollen extracts by all licensed US allergen manufacturers.24 Microtiter plates (Immulon 4; Thermo Electron Corp, Milford, Massachusetts) were coated with saturating levels of each target extract in carbonate buffer (pH 9.6) for 15 to 20 hours at 2 C to 8 C. After they were washed with phosphate-buffered saline that contained 0.05% polysorbate 20 (Tween 20), plates were incubated with the human serum pools specified above for each product (1:20 [vol/vol] final dilutions) that contained serial 3-fold dilutions of extract mixtures (test samples) or corresponding single-extract controls (reference samples) for 4 to 6 hours at 20 C to 25 C. Bound IgE was then determined by successive incubations with biotinylated antihuman IgE (KPL, Gaithersburg, Maryland), streptavidinealkaline phosphatase conjugate (Jackson Immunoresearch Laboratories, West Grove, Pennsylvania), and para-nitrophenyl phosphate substrate (Amresco, Solon, Ohio). Absorbance values were obtained at 405 nm in a calibrated microplate reader (Molecular Devices, Sunnyvale, California). Assay validities were confirmed by statistical analyses (paired t tests for parallelism) of the dose-response curves for reference and test sample pairs, and the resulting relative potencies (test-reference ratios) were determined by parallel line bioassay. Can f 1especific double-bind ELISA analyses were conducted as detailed previously.10 Microtiter plates (Nunc Maxi-Sorp, Nalge Nunc International, Rochester, New York) were incubated stepwise with antieCan f 1 capture antibody, Can f 1 antigen standard, antieCan f 1 probe antibody, and detection reagents (conjugate, substrate) using conditions comparable to those recommended by Indoor Biotechnologies. Standard curves were constructed using absorbance changes during specified periods (reaction rates) and log concentrations of the Can f 1 standards, with the range of standard dilutions exhibiting the highest correlation coefficients determined by linear regression. Mean test sample results were calculated using all absorbance values falling within the dose-response range of the selected standards. Fel d 1especific radial immunodiffusion assays were conducted using 1% agar gels that contained sheep antieFel d 1 cast onto GelBond support films (Cambrex BioScience, Rockland, Maine) and Fel d 1 allergen standards ranging from 5 to 20 mg/mL.24,28 Statistical Analyses Allergen recoveries for extract mixtures (relative to singleextract controls) and controls (relative to freshly prepared solutions of identical composition) were analyzed by 2-sample t tests assuming equal variances and means, with significance achieved for data comparisons yielding 2-tailed (2-sided) P < .05. Excluding preparation of the allergic human serum pools, human subject participation was not required to perform these experiments. This study was approved as nonhuman use research by the Walter Reed National Military Medical Center Institutional Review Board.

Results Single-Extract Controls The stabilities of the 1:1 (vol/vol) and 1:10 (vol/vol) control samples during storage for up to 12 months at 2 C to 8 C are shown in Figure 1. All 1:1 (vol/vol) controls had near-complete recoveries of extract potencies or specific allergen concentrations after storage for up to 9 months at 2 C to 8 C. The cockroach control at 12 months produced virtually no reactivity, possibly a consequence of very low residual sample volumes in this vial at the 12-month time point (high surface-volume ratios during storage between 9 and 12 months). Among the 1:1 (vol/vol) controls, only the cockroach control was limited in volume at the final time point because of the need to repeat several cockroach inhibition ELISAs at earlier test intervals to achieve valid dose-response curves and relative potency values. The 1:10 (vol/vol) control samples diluted with the HSA-saline diluent (5% glycerin) also exhibited favorable recoveries in most cases, with significant reductions (compared with 1:1 [vol/vol] controls in 50% glycerin) observed only with cockroach controls (P ¼ .002), consistent with the reduced stabilities reported for whole-body cockroach extracts at lower (0%e10%) glycerin concentrations.8,9 The control activities illustrated in Figure 1 were sufficient in quantitative terms to provide meaningful assessments of target extract compatibilities in the indoor allergen mixtures. Extract Mixtures The mixing compatibilities of multiallergen extracts (dust mite D farinae, dog epithelia, cockroach mix) or prominent allergenic proteins (dog hair or dander Can f 1, cat hair Fel d 1) in the indoor allergen mixtures are summarized in Figure 2. Allergen recoveries were favorable and consistent for dust mite D farinae, dog epithelia, Can f 1, and Fel d 1 in both the 1:1 (vol/vol) and 1:10 (vol/vol) mixtures during storage for up to 9 months at 2 C to 8 C. All activities in 1:1 (vol/vol) mixture except cockroach were also found to be stable after 12 months at these temperatures. With the exception of the 12-month time point, cockroach extract potencies in the 2 mixtures were significantly higher than corresponding control sample values (P < .001 to .005), presumably due to stabilization by proteins or nonallergenic constituents in the companion products. It is also possible that cross-reactive allergens, such as tropomyosin in dust mite extract, could have contributed to the observed increases in cockroach allergen potencies during refrigerated storage.29 When examined directly using freshly prepared extract controls at the same concentrations as those included in the study mixtures, dust mite extract alone produced very low relative potencies (<20% of cockroach reference levels) under analogous assay conditions (data not shown). In addition to the characterization methods described above, dog hair or dander extract activities in mixtures and controls were examined by an IgE inhibition ELISA. Contrary to the results obtained with the other selected methods, the 1:1 (vol/vol) and 1:10 (vol/vol) mixtures yielded consistently higher responses (6-fold to 12-fold increases) relative to those of the corresponding dog hair or

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Figure 1. Recoveries of allergen activities in single-extract controls after storage for up to 12 months at 2 C to 8 C. Results for dust mite Dermatophagoides farinae (IgE enzyme linked immunosorbent assay [ELISA] inhibition), dog epithelia (IgE ELISA inhibition), cockroach mix (IgE ELISA inhibition), dog hair or dander (Can f 1 ELISA), and cat hair (Fel d 1 radial immunodiffusion [RID]) in 1:1 (vol/vol) (left column) and 1:10 (vol/vol) (right column) control samples were expressed relative to the same activities determined for freshly prepared target extract control samples at each time point. Values greater than 70% and below 50% are shaded in green and red, respectively.

dander controls, the latter of which were within the expected equivalence ranges for this method (data not shown). Similar increases in allergen activity were not observed when these same mixtures were assayed specifically for Can f 1 (Fig 2). Crossreactions with cat hair and dog epithelia extracts in the indoor allergen mixtures accounted for most of the relative potency increases observed in the dog hair or dander inhibition ELISAs. As expected, the relatively low concentrations of dog serum albumin present in the dog hair or dander extract did not produce any appreciable interference of dog epithelia potency determinations (Fig 2). Discussion The diagnosis and management of IgE-mediated sensitivities to indoor allergens remain a formidable challenge for clinicians across North America and the world. Interventions aimed at reducing the levels of provocative allergens in indoor environments, such as homes, schools, and workplace settings, can be effective but must

be performed on a regular basis and are challenged by differences in the physical locations, size distributions, and buoyancy characteristics of airborne particles from dust mite, animal (pet, rodent), insect, and other sources.30e33 Immunochemical measurements of environmental (air, dust) samples in these settings can help to identify the particular allergens and sources present and estimate their mean and local (sample site specific) concentrations for comparisons with established threshold levels.26,34 For many patients whose indoor allergy symptoms cannot be controlled satisfactorily by avoidance measures or pharmacotherapy, immunotherapy is a viable option or complementary intervention that, unlike the other approaches, can potentially modify the underlying cause of allergic reactions by induction of immune tolerance.1,35 Treatment regimens with standardized (cat hair, dust mites) and nonstandardized (animal epithelia or danders, whole-body insects) extracts have been described, including recommendations for maintenance dose targets and mixing compatibilities in patient vaccines that contain these products.3e8,29,36,37 Following the current guidelines, patients


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Figure 2. Recoveries of allergen activities in 5-extract mixtures after storage for up to 12 months at 2 C to 8 C. Percent recoveries for dust mite Dermatophagoides farinae (IgE enzyme-linked immunosorbent assay [ELISA] inhibition), dog epithelia (IgE ELISA inhibition), cockroach mix (IgE ELISA inhibition), dog hair or dander (Can f 1 ELISA), and cat hair (Fel d 1 radial immunodiffusion [RID]) in 1:1 (vol/vol) (left column) and 1:10 (vol/vol) (right column) mix samples were expressed relative to the same activities determined for stored target extract control samples at the same time point. Values greater than 150%, between 70% and 140%, and between 50% and 75% are shaded in purple, green, and yellow, respectively.

presenting with sensitivities to multiple indoor allergens could require at least 2 injections per visit, with the insect and dust mite or animal extracts formulated in separate vaccines to maintain their potencies throughout the 6- to 12-month refrigerated storage period for these vials. On the basis of concerns related to insufficient stability data for allergen extracts commonly used in SCIT prescriptions, increased costs of vaccine preparations, and reduced patient adherence with additional injections, many allergy clinics continue to combine low-protease and high-protease products with potential or established incompatibility into the same patient treatment formulation.21e23 One report that examined nearly 115,000 SCIT prescriptions written within a large military health care system between 1990 and 2010 found a decreasing trend toward mixing pollen and mold extracts, but toward the end of this assessment period (2007e2010), 72% of single-injection and 30% of total mold prescriptions still contained pollen extracts.21

Several published studies have examined the stabilities of lowprotease indoor allergen extracts in 2-component mixtures with cockroach preparations.5 In independent assessments that used different assay methods and probe reagents for target extracts, cat hair allergens were found to be very stable when combined with American cockroach or German cockroach and stored for up to 12 months at 2 C to 8 C, but dust mite D farinae allergens exhibited partial or inconsistent potency reductions in analogous mixtures with these cockroach extracts.13,16 In a subsequent study, dog epithelia and dog hair or dander allergens exhibited susceptibilities to cockroach extract similar to those reported for dust mites and were most pronounced in test mixtures that contained low to moderate glycerin levels (10%e25% final concentrations).10 The data cited above support the separation of cockroach from cat, dog, or dust mite extracts in maintenance-strength patient treatment vials with reduced glycerin content. Patient treatment formulations that contain higher numbers or percentages of

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extracts typically possess elevated total protein concentrations and often increased glycerin levels, which could help protect specific allergenic components from the actions of proteolytic enzymes. On the other hand, the stabilities of these hydrolases could be enhanced to promote further degradation of important macromolecular constituents during refrigerated storage.2,3,5,18,20,38,39 The structural integrity of allergens in the dilute solutions administered during dose escalation steps can also be affected by the resulting concentration changes and the stabilizing properties of the diluting fluids selected for these vaccines.16,27 The present study was conducted to examine the stabilities of prominent (major) allergen concentrations and total extract IgEbinding potencies in SCIT mixtures and dilutions that contain a diverse group of 5 common indoor allergens that are representative of custom vaccine formulations for certain patients in one author’s clinical practice (military personnel stationed at bases across the United States and abroad). Because of the compositional complexity of these mixtures relative to those evaluated in earlier studies, only those data derived from laboratory methods with high specificities for target extracts, sensitivities sufficient for quantitation of mixtures and controls at 1:1 (vol/vol) maintenance and 1:10 (vol/vol) buildup strengths, and parallel dose-response curves yielding favorable dilution recoveries or intermediate precision for all samples were reported. As noted earlier, the selected assays were specific for major allergens (cat hair Fel d 1, dog hair or dander Can f 1) or multiple IgE-binding extract components (dust mite D farinae, dog epithelia, cockroach mix) in the test mixtures and controls. All single-extract control samples except cockroach at 1:10 (vol/ vol) had IgE-binding potencies or specific allergen concentrations closely comparable to those determined for freshly prepared solutions of identical composition during storage for up to 12 months at 2 C to 8 C (Fig 1). The stability of cockroach extract controls at 1:1 (vol/vol) (47.5% glycerin) and the instability of these solutions at 1:10 (vol/vol) (4.75% glycerin) were consistent with recent data from this laboratory, which revealed that recoveries of cockroach extract potencies at low (10%) glycerin concentrations were reduced significantly compared with control samples at moderate (25%) or high (50%) glycerin levels.8,9 These data provide further support for the effectiveness of glycerin as a stabilizer of immunotherapy vaccines that contain protease-rich extracts.3,5,27 The inclusion of 0.03% human serum albumin-saline diluent in the 1:10 (vol/vol) cockroach control did not appear to provide adequate protection from the actions of endogenous cockroach proteases, as noted previously.27 High recoveries of IgE-binding potencies for dust mite D farinae and dog epithelia and of major allergen concentrations for dog hair or dander Can f 1 and cat hair Fel d 1 were determined in the 5component mixtures at both 1:1 (vol/vol) and 1:10 (vol/vol) (Figure 2). As expected, the minor compositional changes observed previously for dust mite (D farinae, Dermatophagoides pteronyssinus) and dog (hair, dander, epithelia) allergens in mixtures with cockroach extracts at relatively low (10%e25%) glycerin concentrations were not detected in the 1:1 (vol/vol) mixture at 47.5% glycerin.10,13 The stability of these allergens in the 1:10 (vol/ vol) mixture could be affected by the residual glycerin concentration (4.75%), the presence of additional protein-based extracts or human serum albumin diluent, or combinations of these and other factors. The dust mite analyses were performed with a human serum pool (Greer Laboratories lot ZE-P3) that did not detect potency differences in the prior study with 2-component dust mite-cockroach mixtures at 10% glycerin.13 Unfortunately, the human serum pool that detected such changes (FDA lot S5-Dpf) was no longer available at the time of the current study. As a result, the recoveries of dust mite D farinae allergens in the 5-component mixtures should be interpreted with caution, and additional


assessments of these mixtures with other mite-positive sera are warranted. Cockroach extracts in the 5-component mixtures had significant increases in IgE-binding potency relative to cockroach control samples analyzed side by side at each time point (Fig 2). The consistency of the activities and percent recoveries for both the 1:1 (vol/vol) and 1:10 (vol/vol) mixtures at multiple intervals could be interpreted as an interference or cross-reaction with companion extract constituents or stabilization of labile cockroach allergens by the same or different components. The most likely source of a cross-reactive allergen is tropomyosin from dust mite D farinae (Der f 10), which has been reported to share structural epitopes with homologous 33-kDa allergens from American cockroach (Per a 7) and German cockroach (Bla g 7).29 In the cockroach potency assay, very low relative potencies (<20% of cockroach extract reactivities) were found with a freshly prepared dust mite D farinae extract control solution under conditions analogous to those used with the stored test mixtures and controls (data not shown). These contributions are not sufficient to explain the observed increases in cockroach activity. However, it is possible that these increases could result from enhanced exposure of tropomyosin (or other) epitopes in dust mite D farinae extracts by the actions of endogenous cockroach proteases in the stored test mixtures. When compared with initial (time zero) values or those expressed by freshly diluted control samples, the cockroach extract relative potency values illustrated in Figure 2 ranged from 1.6 to 2.0 in the 1:1 (vol/vol) mixture and from 1.0 to 1.3 in the 1:10 (vol/vol) mixture. These values fall within the equivalence ranges for standardized extracts tested during formal stability studies (mandated and monitored by the FDA) and are well within those suggested for maintenance of both efficacy and safety in SCIT vaccines.40,41 There are limitations to the present investigation. The experimental data were obtained using commercial extracts procured primarily from one licensed manufacturer (Greer Laboratories), and these products can vary somewhat in compositional profiles (standardized cat and dust mite D farinae) or in both qualitative and quantitative characteristics (nonstandardized dog and cockroach) from one manufacturer to another.3,24,42e47 Similarly, a relatively small number of serum samples or pools were available for use in the selected assays. In vivo analyses (skin testing) and Tcell assays that measured the activities of allergen fragments that retain considerable antibody binding avidities are also needed to further characterize the potential consequences of extract changes detected by the in vitro methods.48,49 To date, no study has evaluated the clinical outcomes of enzymatic degradation in extract mixtures. In summary, SCIT mixtures and dilutions that contain cat, dog epithelia, dog hair or dander, dust mite, and cockroach extracts exhibited favorable recoveries of immunochemical activities for all products during refrigerated storage for up to 12 months, supporting their use in individuals with diverse indoor allergen sensitivities whose symptoms cannot be controlled sufficiently by avoidance measures or pharmacotherapy. To the authors’ knowledge, this is the first peer-reviewed extract stability or compatibility publication to report selective and concurrent quantitation of allergen activities (potencies, concentrations) in more than 2 extracts from the same test samples. One other study published as a meeting abstract in 2007 described extract recoveries in mixtures that contained 6 to 10 products.50 The 2007 study included products from a different US extract manufacturer (ALK-Abello) than the present investigation (Greer Laboratories, Hollister-Stier Laboratories), and a panel of proprietary, noncommercial, double-bind ELISAs specific for prominent individual allergens in a variety of target extracts. Continued development and critical comparisons of diverse in vitro and in vivo methods for extract characterization are essential to expanding our understanding of allergen stabilities and


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compatibilities in product mixtures formulated for patient-specific SCIT regimens.5,24,27,50,51 Acknowledgments We thank Drs Silvia Huebner and Terrance Coyne for their encouragement and support of this project and appreciate the critical reviews and helpful suggestions to manuscript content provided by Bonnie Grier, Susan Kosisky, and Dr Jack Kelly. References [1] Malling H-J, Bousquet J. Subcutaneous immunotherapy for allergic rhinoconjunctivitis, allergic asthma, and prevention of allergic diseases. In: Lockey RF, Ledford DK, eds. Allergens and Allergen Immunotherapy. 4th ed. New York, NY: Informa Healthcare; 2008:343e358. [2] Cox L, Esch RE, Corbett M, Hankin C, Nelson M, Plunkett G. Allergen immunotherapy practice in the United States: guidelines, measures, and outcomes. Ann Allergy Asthma Immunol. 2011;107:289e299. [3] Cox L, Nelson H, Lockey R, et al. Allergen immunotherapy: a practice parameter third update. J Allergy Clin Immunol. 2011;127(1 suppl):S1eS55. [4] Grier TJ. How’s my dosing? a one-step, math-free guide for comparing your clinic’s maintenance immunotherapy doses to current practice parameter recommendations. Ann Allergy Asthma Immunol. 2012;108:201e205. [5] Esch RE, Grier TJ. Allergen compatibilities in extract mixtures. Immunol Allergy Clin N Am. 2011;31:227e239. [6] Esch RE. Allergen immunotherapy: what can and cannot be mixed. J Allergy Clin Immunol. 2008;122:659e660. [7] Nelson HS. Specific immunotherapy with allergen mixes: what is the evidence? Curr Opin Allergy Clin Immunol. 2009;9:549e553. [8] Grier TJ, LeFevre DM, Duncan EA, Coyne TC. Mixing compatibilities of Aspergillus and American cockroach allergens with other high-protease fungal and insect extracts. Ann Allergy Asthma Immunol. 2015;114:233e239. [9] Grier TJ, LeFevre DM, Duncan EA, Esch RE, Coyne TC. Allergen stabilities and compatibilities in mixtures of high-protease fungal and insect extracts. Ann Allergy Asthma Immunol. 2012;108:439e447. [10] Grier TJ, LeFevre DM, Duncan EA, Esch RE. Stability and mixing compatibility of dog epithelia and dog dander allergens. Ann Allergy Asthma Immunol. 2009; 103:411e417. [11] Rans TS, Hrabek TM, Whisman BA, et al. Compatibility of imported fire ant whole body extract with cat, ragweed, Dermatophagoides pteronyssinus, and timothy grass allergens. Ann Allergy Asthma Immunol. 2009;102:57e61. [12] Iraneta SG, Acosta DM, Duran R, et al. MALDI-TOF MS analysis of labile Lolium perenne major allergens in mixes. Clin Exp Allergy. 2008;38:1391e1399. [13] Grier TJ, LeFevre DM, Duncan EA, Esch RE. Stability of standardized grass, dust mite, cat, and short ragweed allergens after mixing with mold or cockroach extracts. Ann Allergy Asthma Immunol. 2007;99:151e160. [14] Meier EA, Whisman BA, Rathkopf MM. Effect of imported fire ant extract on the degradation of mountain cedar pollen allergen. Ann Allergy Asthma Immunol. 2006;96:30e32. [15] Hoff M, Krail M, Kastner M, et al. Fusarium culmorum causes strong degradation of pollen allergens in extract mixtures. J Allergy Clin Immunol. 2002; 109:96e101. [16] Nelson HS, Ikle D, Buchmeier A. Studies on allergen extract stability: the effects of dilution and mixing. J Allergy Clin Immunol. 1996;98:382e388. [17] Kordash TR, Amend MJ, Williamson SL, et al. Effect of mixing allergenic extracts containing Helminthosporium, D. farinae, and cockroach with perennial ryegrass. Ann Allergy. 1993;71:240e246. [18] Esch RE. Role of proteases on the stability of allergenic extracts. Arb Paul Ehrlich Inst Bundesamt Sera Impfstoffe Frankf A M. 1992;85:171e179. [19] Cox LS, Li JT, Nelson H, Lockey R. Allergen immunotherapy: a practice parameter second update. J Allergy Clin Immunol. 2007;120:S25eS83. [20] Li JT, Lockey RF, Bernstein IL, Portnoy JM, Nicklas RA. Allergen immunotherapy: a practice parameter. Ann Allergy Asthma Immunol. 2003;90:1e40. [21] Gada S, Haymore B, McCoy L, Kosisky S, Nelson M. Frequency of mold and pollen mixing in allergen immunotherapy prescriptions within a large health case system, 1990-2010. J Allergy Clin Immunol. 2012;129:1151e1153. [22] Esch RE. Formulation of therapeutic allergen mixtures: problems associated with the number, proportion, and enzymatic activities of allergens. Arb Paul Ehrlich Inst Bundesamt Sera Impfstoffe Frankf A M. 2000;93:57e61. [23] Esch RE. Specific immunotherapy in the U.S.A: general concepts and recent initiatives. Arb Paul Ehrlich Inst Bundesamt Sera Impfstoffe Frankf A M. 2003;94: 17e22.

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