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  1. Biologics Research Projects

Improving Safety and Potency Testing of Allergen Extracts

Principal Investigator: Jay E. Slater, MD
Office / Division / Lab: OVRR / DBPAP / LI


General Overview

Allergen extracts are used in the US to diagnose and treat allergic diseases. Skin tests containing these extracts determine the causes of allergies. "Allergy shots" containing them are used to reduce or eliminate allergic symptoms. As with all drugs and biologics, good quality controls are needed to ensure that allergen extracts are safe and effective. The goal of our research is to provide new tools and data that allow allergen extract manufacturers to maintain and improve the quality of these important products.

Our work has two major areas of focus. First, we are attempting to improve the methods by which FDA and manufacturers measure the potency, or strength, of an allergen extract. Existing methods either measure a single, dominant protein in the extract, or determine the potency by measuring several undefined proteins as one group. In contrast, we are developing a method that will measure many specific proteins simultaneously. In this way, we can measure not only the overall potency but also the levels of individual proteins.

In our second project we are investigating the presence of endotoxin in certain allergen extracts. Endotoxins are ubiquitous in our environment, and are even present in some biological products; but their presence in allergen extracts has not been studied. We are using sophisticated laboratory techniques to identify the sources of endotoxins and the factors that affect endotoxin levels in these extracts.


Scientific Overview

The existing competition ELISA is a sensitive and specific method of determining overall potency, but its performance characteristics for each component allergen is uncertain. When an individual allergen is removed from the extract, the polyvalent sera cannot reliably discern its absence (Soldatova LN, et al. J Allergy Clin Immunol 2000;105:482-488; Dobrovolskaia E, et al. Clin Exp Allergy 2006; 36:525-530). Until each relevant allergen in a mixture is identified, regulators must ask manufacturers to measure overall potency. Once important allergens are determined, their measurement requires a change in technology that can be time-consuming and difficult.

The purpose of this study is to construct an assay that will have the strengths of both the RID and the competition ELISA, and that will allow manufacturers and regulators to determine the amounts of specific allergens in a mixture, as well as its overall allergenicity. Multiplex assays have been used with success for a variety of assays, such as detecting allergen-specific IgE and the amount of allergen in the environment. Following this approach, we have designed a multiplex allergen extract potency assay (MAEPA). In work so far, the assay has been optimized for cat hair and short ragweed pollen allergen extracts. We are currently developing the assay for German cockroach allergens, as well as to detect allergens in Neurospora crassa, Agarigus bisporus, and Nicotiana benthamiana/excelsiana.

We are also working to identify endotoxins in allergen extracts using bacterial genotyping by specific PCR and sequencing, and specific LPS-typing.


Publications

J Allergy Clin Immunol 2019 Oct;144(4):1140
Regulation of allergen immunotherapy products in Europe and the United States.
Rabin RL, Bridgewater J, Slater JE

Allergy 2018 Apr;73(4):816-26
Allergen manufacturing and quality aspects for allergen immunotherapy in Europe and the United States: an analysis from the EAACI AIT Guidelines Project.
Bonertz A, Roberts G, Slater JE, Bridgewater J, Rabin RL, Hoefnagel M, Timon M, Pini C, Pfaar O, Sheikh A, Ryan D, Akdis C, Goldstein J, Poulsen LK, van Ree R, Rhyner C, Barber D, Palomares O, Pawankar R, Hamerlijnk D, Klimek L, Agache I, Angier E, Casale T, Fernandez-Rivas M, Halken S, Jutel M, Lau S, Pajno G, Sturm G, Varga EM, van Wijk RG, Bonini S, Muraro A, Vieths S

Allergy 2018 Jan;73(1):64-76
Challenges in the implementation of EAACI guidelines on allergen immunotherapy: a global perspective on the regulation of allergen products.
Bonertz A, Roberts G, Hoefnagel M, Timon M, Slater J, Rabin R, Bridgewater J, Pini C, Pfaar O, Akdis C, Goldstein J, Poulsen LK, van Ree R, Rhyner C, Barber D, Palomares O, Sheikh A, Pawankar R, Hamerlijnk D, Klimek L, Agache I, Angier E, Casale T, Fernandez-Rivas M, Halken S, Jutel M, Lau S, Pajno G, Sturm G, Maria Varga E, van Wijk RG, Bonini S, Muraro A, Vieths S

Clin Exp Allergy 2017 Dec;47(12):1661-70
Accurate quantification of 5 German cockroach (GCr) allergens in complex extracts using multiple reaction monitoring mass spectrometry (MRM MS).
Mindaye ST, Spiric J, David NA, Rabin RL, Slater JE

PLoS One 2017 Aug 2;12(8):e0182260
Cross-reaction between Formosan termite (Coptotermes formosanus) proteins and cockroach allergens.
Mattison CP, Khurana T, Tarver MR, Florane CB, Grimm CC, Pakala SB, Cottone CB, Riegel C, Bren-Mattison Y, Slater JE

Ann Allergy Asthma Immunol 2017 Aug;119(2):101-7
Food allergen extracts to diagnose food-induced allergic diseases: how they are made.
David NA, Penumarti A, Burks AW, Slater JE

Ann Allergy Asthma Immunol 2016 May;116(5):440-446.e2
The NPC2 protein: a novel dog allergen.
Khurana T, Newman-Lindsay S, Young PR, Slater JE

PLoS One 2015 Oct 7;10(10):e0140225
Multiplex assay for protein profiling and potency measurement of German cockroach allergen extracts.
Khurana T, Dobrovolskaia E, Shartouny JR, Slater JE

Clin Exp Allergy 2005 Aug;35(8):1040-8
Exploiting the avian immunoglobulin system to simplify the generation of recombinant antibodies to allergenic proteins.
Finlay WJ, Devore NC, Dobrovolskaia EN, Gam A, Goodyear CS, Slater JE

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