New Challenges and Methods in Allergen Testing by romer-labs

Issue 7

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A Romer Labs® Publication

New Challenges and Methods in Allergen Testing Detecting Allergens in Processed Foods 10 Steps to Validating and Verifying a Cleaning Method

Contents

Challenges in Allergen Testing, Part Two: Detecting Allergens in Processed Foods

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Most of the food and drink we consume has been processed or modified in some way, potentially altering allergenic proteins. Adrian Rogers looks at incurred samples and how they might change how we test processed foods. By Adrian Rogers, Senior Research Scientist, Romer Labs®

Spot On is a quarterly publication of Romer Labs Division Holding GmbH, distributed free-of-charge. ISSN: 2414-2042

Editors: Joshua Davis, Cristian Ilea

Contributors: Chiara Palladino, Adrian Rogers, Paul Bagshaw Graphic: GraphX ERBER AG Research: Kurt Brunner

Publisher: Romer Labs Division Holding GmbH Erber Campus 1 3131 Getzersdorf, Austria Tel: +43 2782 803 0 www.romerlabs.com

10 Steps to Validating and Verifying Allergen Cleaning Procedures

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How do food producers know whether their cleaning regimen is working? Paul Bagshaw of Holchem Laboratories gives a detailed look into cleaning validation and verification. By Paul Bagshaw, Technical Services Manager, Holchem Laboratories Ltd.

All photos herein are the property of Romer Labs or used with license.

Spot On Issue 7

Editorial New Horizons in Allergen Testing As the prevalence of food allergies grows worldwide, so does the need for more and more people to observe a strictly allergen-free diet. Accurate food allergen detection enables correct food labeling, which keeps consumers safe and ensures compliance with food safety regulations. However, several difficult challenges come with the need to test for food allergens. Food producers routinely deal with a diverse variety of ingredients, which are subject to preparation and processing in several possible ways. Nonetheless, they still need to detect even the tiniest traces of allergens. Both the complexity of this topic and consumer demand for accurate food labeling require that food producers take a careful look into the steps of an allergen-testing program so that they can be sure to select the right detection method. In this issue of Spot On, we follow up on the discussion we began in the last Spot On devoted to challenges facing allergen testing with an investigation into one of the most difficult problems: how to detect allergens in processed foods. The article highlights the difficulties encountered when testing for allergens in highly processed foods, and sheds some light on recent advances in standardization and calibration materials in the food allergen analytical community. Our second article provides readers with a quick but detailed guide on how to validate and verify an allergen cleaning procedure in food processing facilities. Validation plays an essential role in avoiding allergen contamination of finished products and enhances the reliability of any allergen testing performed further down the production line. We hope you enjoy this issue of Spot On.

Chiara Palladino Product Manager Allergens

A R moam g ae zr i n L ea bosf® RPoumbel irc a L taibosn®

Challenges in Allergen Testing Detecting Allergens in Processed Foods P a r t

T w o

–

By Adrian Rogers - Senior Research Scientist, Romer Labs®

Processing foods can change the nature of the allergens hiding in them, making them harder to find.

Spot On Issue 7

Photo: shutterstock_ Rasica

Food producers are increasingly looking to food allergen analysis as a means of emphasizing greater transparency, traceability and integrity in the supply chain. While this growing awareness has extended to validation and verification of factory cleaning and investigation of recalls and incidents, producers are also investigating new ways to detect allergens in processed foods. Food allergen expert Adrian Rogers explores how incurred samples might show the way forward.

A Romer Labs® Publication

Quality control is an important factor when producing incurred reference materials. The allergen to be incorporated into the food matrix must be very well defined.

ost of the food and drink we consume has been processed or modified in some way. This processing brings about many benefits in terms of food safety, preservation and taste. However, processing changes the characteristics of the ingredients used to make the food; of particular interest are the changes that allergenic proteins can undergo. There are many kinds of changes relevant to allergen detection: allergenic proteins can be subjected to heat-accelerated chemical reactions including Maillard reactions and other protein-carbohydrate interactions, protein aggregation with loss of solubility, shear effects on protein structure, emulsion formation, pH effects, and water activity during food production. Recent studies have shown that processing allergens can alter their allergenicity, changing how an allergic individual may react to them. If immunoassay-based food allergen detection methods rely on the use of antibodies to detect allergens present in food, it follows that the ability to detect them may be affected by processing. Such processing effects must be taken into account when developing new analytical methods, either by improved extraction methods intended to increase the solubility of the aggregated proteins or by going back to basics and raising new sets of antibodies that specifically target processed allergens.

How do we create real-life samples with incurred controls? To evaluate these improved allergen detection methods, incurred sample controls are needed. These are food samples into which a known amount of the food allergen has been incorporated during processing, mimicking as closely as possible the actual conditions under which the sample matrix would normally be manufactured. Analyzing the real-life sample would give the most accurate representation of the recovery and response of a particular method for that particular matrix. Yet each food matrix and processing condition is different and it would not be practical to evaluate all possibilities with naturally incurred standards. However, evaluation of an ELISA with one or more typical combinations of food matrix and processing condition is a useful method in determining whether the ELISA can provide reliable results when applied to processed foods. Steve Taylor of the Food Allergy Research and Resource Program has described what should be considered when creating incurred reference materials for food allergen analysis. In essence, he suggests, the matrix of choice should be one in which the allergenic food residues would likely be found during typical pro-

cessing in an industrially produced food. The simplest way to do this would be to look around a supermarket and search for similar products with and without the specific allergenic component, for example, a cookie with and without milk. There are several things to consider when making incurred materials. Such materials are usually produced in a food processing pilot plant or test kitchen; the ability to clean the processing equipment thoroughly after each batch is an important consideration to assure that there is no batch-to-batch contamination. Quality control is an important factor when producing incurred reference materials. The allergen to be incorporated into the food matrix must be very well defined. Where official reference materials exist they are not usually affordable in the quantities needed to produce the incurred controls. Yet any official reference materials can be used for comparative purposes to define the nature of the material used. Furthermore, the homogeneity of the allergen added to the food matrix is critical for every step of the process. In order to ensure that the allergen is homogeneously incorporated into the incurred material, multiple analyses of the sub-sampled food at various stages of processing are required to make sure that uniform distribution of the allergen is achieved. Contamination between batches with increasing levels of allergen can be controlled by starting with the lowest concentration and steadily increasing the levels. Spot On Issue 7

Another important consideration is the stability of the incurred material. Any processing effects that the allergen undergoes can usually be determined quickly during analysis. But if the incurred material is to be stored for any length of time and used to generate real world standard curves for an analytical method, then the shelf life of the material must be ascertained. There is a possibility that further reactions between the proteins of the allergen and the components of the food matrix into which it is incorporated could occur during storage. Since the production of well-defined incurred controls is not easy, storage of these controls would al-

low their future use for evaluating improved analytical methods. Such incurred control materials may also be used to evaluate batch-to-batch variability for commercial ELISA kits.

MoniQA has developed

Early successes with incurred samples

closely as possible the

Very slowly, fully validated incurred reference materials for food allergen analysis are coming onto the market. The MoniQA Association has recently released a milk-incurred material that incorporates well-characterized dried skimmed milk powder into a gluten free cookie at two individual protein concentrations. This incurred reference material has been produced to mimic as closely as possible the processing through which an allergen-containing food goes during production. This is a distinct advantage as it more closely reflects the real-world foods that are routinely tested in analytical labs across the world and gives a good indication of the suitability of the analytical methods used to detect processed allergens. Advancing such research even further, work undertaken at the University of Manchester under the guidance of Professor Clare Mills has investigated how an allergen-incurred chocolate dessert material originally developed for the diagnosis of food allergies can be adapted for use as an analytical control material. Such research is encouraging and could play a vital role in expanding the comprehensiveness of allergen testing in processed foods. The use of a matrix and incurred food ingredients with demonstrable allergenic activity for analytical purposes will help ensure that efforts to standardize calibration materials and harmonize allergen-reporting units are meaningfully aligned with ongoing measures to protect allergic consumers from accidental exposure to problem foods.

an incurred reference material to mimic as processing through which an allergencontaining food goes during production.

References S. Taylor et al : Anal Bioanal Chem (2009) 395:83-92 Allergen immunoassays – considerations for use of naturally incurred standards. M. Abbott et al : Journal of AOAC International (2010) Vol 93, No 2 Validation Procedures for Quantitative Food Allergen ELISA Methods: Community Guidance and Best Practises. K. Verhoeckx et al : Food and Chemical Toxicology (2015) 80: 223-240 Food processing and allergenicity. M. Walker et al : Analyst (2016) 141: 24-35 Is food allergen analysis flawed? Health and supply chain A Romer Labs® Publication

risks and a proposed framework to address urgent analytical needs. Phil E. Johnson et al : Food Chemistry (2014) 148: 30-36 A Multi-laboratory evaluation of a clinicallyvalidated incurred quality control material for analysis of allergens in food. Nitride et al : Journal of AOAC International (2018) 101, No 1 Integrating Allergen Analysis Within a Risk Assessment Framework: Approaches to Development of Targeted Mass Spectrometry Methods for Allergen Detection and Quantification in the iFAAM Project.

How do food producers know whether their cleaning regimen is working? ELISA, lateral flow devices and ATP testing are methods that can help validate a cleaning program.

10 Steps to Validating and Verifying Allergen Cleaning Procedures About the author

Paul Bagshaw has worked at Holchem Laboratories Ltd. for nearly 10 years. His research interests include allergens, CIP and hygienic design. As Technical Services Manager, he provides technical support to Holchem’s sales team and customers.

By Paul Bagshaw - Technical Services Manager, Holchem Laboratories Ltd.

That cleaning is a vital component of any allergen management program is beyond dispute. But how do food producers know whether their cleaning regimen is working? Paul Bagshaw of Holchem Laboratories guides us through the ins and outs of cleaning validation and verification.

ood manufacturers and processors rely on a variety of differing policies and procedures to enforce allergen controls. These include personnel controls, such as a hand-washing procedure and the use of protective clothing and equipment (PPE), process controls such as segregated storage and color-coded equipment, production controls such as dedicated equipment and time segregation, and – most importantly – cleaning. Cleaning regimens are subject to rigorous valida-

tion. Global Food Safety Initiative (GFSI) retailer approval schemes such as the British Retail Consortium (BRC) state, “Where cleaning procedures are part of a defined prerequisite plan to control the risk of a specific hazard the cleaning and disinfection procedures and frequency shall be validated…” In simple terms, this means that the validation process should demonstrate that the cleaning procedure a site is using reduces the hazard – in this case, an allergen – to a level deemed to be acceptable. Spot On Issue 7

The validation guidelines at Holchem Laboratories Ltd. have been developed in accordance with the principles of the European Hygienic Engineering and Design Group (EHEDG) Cleaning Validation subgroup1 and Campden BRI2, together with in-house best practices. The overarching principle can be summed up in this way: validation should be carried out under worst-case scenarios. Here, we take a look at the steps involved in setting up a validation program and then verifying that program.

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Determine the objective of the cleaning The process begins by determining the objective of the cleaning: with allergens, the goal is to ensure the absence of detectable allergens in food products that undergo processing following the cleaning. The site should first define the products and process lines that the validation will cover as well as the type of cleaning to be validated (i.e. a product changeover cleaning or an end-of-production cleaning, open plant or closed plant (clean-in-place)). It has always been best practice to use the most sensitive detection technique available for detecting allergens in food products (traditionally based on ELISA). In addition, food-processing surfaces should be cleaned such that allergens cannot be detected using lateral flow devices (LFDs).

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Ensure organizational support A team approach will help ensure that the validation process is effective; the team should be multi-departmental (production, engineering, technical, hygiene, hazard specialist) and have the backing of senior management to ensure seamless collaboration.

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Know the hygienic design of your equipment A review of the hygienic design of the equipment is recommended. The primary purpose of this step is to determine the areas of the equipment that are most difficult to clean; this is helpful in determining the A Romer Labs® Publication

worst-case scenario. These areas will be assessed for cleanability during the validation process. As such, it may be necessary to strike a balance between the place that is hardest to clean (but may need specialist access equipment or engineering support to dismantle) and places that are hard to clean but are practicably accessible.

Validation should be carried out under worst-case scenarios.

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Review any current cleaning programs and applicable regulations If no cleaning program exists, then one is created at this stage. In practice, one often exists, meaning that sites should be recording that cleaning program, often by putting copies of CICs (cleaning instruction cards) into the validation pack. However, certain parameters of the clean are generally overlooked, such as the number of cleaning operatives and the cleaning window required. Certain parameters of the clean, such as those for chemical strengths and solution temperatures, are often formulated in terms of a range. In such cases, the validation should be undertaken in worst-case circumstances, i.e. at the lowest chemical strength or temperature in the range. Those responsible for a site should also take into consideration the implications concerning health and safety legislation when carrying out the clean. The site should already have undertaken COSSH assessments for the chemicals they intend to use on the validation, and should consider whether any risk assessments are required for any dismantling of equipment for cleaning. Chemical disinfectants should have the relevant efficacy data, including the European standards EN1276 and EN 13697, and comply with the requirements of the Biocidal Products Regulation (EU 528/2012).

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Determine the worst-case scenario for the soiling There are several reasons to choose a particular food product for the validation: it may have the strongest adhering soil, the highest level of allergens or the hardest allergen to remove. The processing that the food product undergoes will also have an impact on the removal of the soiling; this could include the longest processing time, the highest temperature or the

The cleaning has achieved its desired results when both the product and food contact surfaces are free from allergens.

period of time the equipment sits idle before cleaning. Whilst this determination of the worst-case soiling scenario will ensure a robust validation that stands up to scrutiny, an added benefit is that it increases efficiency by allowing food manufactures to carry out fewer validation processes. For example, if a food manufacturer has a number of allergens that are checked by the same cleaning and disinfection program, validating the program for the worst-case scenario for a single allergen (highest allergen presence, most difficult to clean soil) theoretically confers validation to cleaning programs for all allergens used.

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Sample, sample and sample again Sites should then determine the type of sampling to assess whether the objective of the cleaning and disinfection program has been met. The simplest form of assessment, the visual inspection, is often the most overlooked: are debris visible at the sample sites? Other options are direct and indirect sampling: direct sampling is likely to be via surface swabbing, whilst indirect sampling is commonly used only for CIP (cleaning in place) applications and is generally taken via rinse water samples. Finally, product sampling normally involves taking a sample of the first product off the line for testing after cleaning has been carried out. For CIP, it is considered good practice to take samples from the first, middle and last product from the line.

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Choose the right analytical test Now that we know how we are going to sample, we need to decide which analytical tests to use to determine whether the objective of the cleaning and disinfection program has been met. Such tests should be specific, sensitive, representative and reproducible. For allergens in the product itself, ELISA should be used wherever possible for validation. For surfaces, ELISA is also useful after validation to assess residues, though LFDs are the desired option, as they will be the method of choice for performing ongoing cleaning verification. If a lab-based ELISA test is undertaken for surface residues, LFDs should be run in parallel to establish any correlation between the two methods. For allergen testing, positive controls should be es-

tablished to ensure that the target allergen, in food products and on process surfaces, can be detected under the conditions of food manufacturing under test. This requires the food manufacturer to send samples of the product due to be run and swabs of the surfaces before the validation clean begins. This is helpful in accounting for the variations in allergen detection that the food matrix may cause. If, for example, the allergen is known to be a product ingredient but LFDs are unable to detect it, then an LFD is not a suitable verification method going forward. It is also good practice to determine if the cleaning or disinfectant residues present in the sample matrix have any effect on the sensitivity of the analytical detection technique.

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Carry out the validation Then, the actual validation process can go forward. It is generally accepted that it be repeated no fewer than 3 times. It is also good practice to carry out validation at varying times to account for different cleaning teams, seasonal variation in raw ingredients, variation in production pressures and other factors. Validations should be reviewed routinely, at least annually, or when any parameter changes, such as a product, machinery or a cleaning parameter.

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Interpret the results The cleaning has achieved its desired results when both the product and food contact surfaces are free from allergens. Conversely, the detection of allergens in the product as well as on food contact surfaces indicates that the cleaning has not achieved its desired results, meaning that the site must amend some aspect of the cleaning program to improve results before rerunning the validation. If a cleaning program can achieve surface cleanliness via LFD but not by ELISA (or other alternative, non-routine sensitive techniques), a risk assessment to ascertain whether the detectable allergen present on the surface is likely to be a significant risk to the subsequent batch of product should be undertaken. Within the risk assessment, two factors are important. Firstly, cross-contamination from the food contact surface to the food involves a transfer coefficient: not all allergens present on the surface will transfer to Spot On Issue 7

An approach

Table 1. Analytical methods for cleaning validation and verification. Method

Purpose

combining LFD and

LFD

Surface testing Ongoing verification

give some insight

ELISA

ATP testing

Protein swabs

Source: Romer Labs

Product testing Post-validation surface testing

ELISA methods can

Post-validation verification (ATP must be detectable on surfaces, even if allergens are not) Ongoing verification

into how successful a validation performs on surfaces and products,

the food. In practice, the transfer coefficient of the allergen to the foodstuff, and the area of the food contact surface touched by the portion size before it is packed will be unknown. Secondly, the nature of product and surface testing is different. Product testing involves macerating the product sample in a large volume of diluent, whilst the swab used in surface testing is recovered into a small quantity of diluent. Yet as the same volume of diluent is tested, in effect a lower detection sensitivity is recorded for product samples. In reality, therefore, a detection of allergen present on a food surface would likely result in an allergen level in the food product of approximately 100 times less.

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Verify with ATP or protein swabs After validation, food manufacturers require a method of verification. Cleaning verification is intended to demonstrate that on subsequent cleaning occasions, the cleaning and disinfection program has met its objectives. It may be possible to implement a verification routine via ATP measurements rather than allergen lateral flow strips. However, for this to be an acceptable method, ATP must be present on the surfaces when there is an absence of detectable allergen residues. If this is possible, ATP can be frequently measured (daily, for example), whilst allergen lateral flow strips could be used less frequently (weekly or monthly). The average results of the validation plus any ‘comfort factor’ should be set as target levels for ATP, which requires that ATP be used alongside other test methods during the validation. Similarly, protein swabs can serve to provide ongoing verification: as the vast majority of allergens are proteins, an absence of protein implies an absence of allergen. A Romer Labs® Publication

However, a presence of protein does not necessarily indicate a presence of allergen. If ATP or protein swabs are to be used, those operating the site must understand that they are not measuring the allergen itself but general hygiene indicators.

respectively.

Conclusion Cleaning and validation are complex processes, involving a variety of possible tools. Ultimately, a cleaning validation is a procedure that must be customized to the needs of a specific production environment. Detailed knowledge of the products and process lines and an understanding of current and previous cleaning programs and their efficacy should inform the decision about what constitutes the worst-case scenario. Only this way can a facility ensure that the validation is robust enough to meet both internal requirements and the standards of external accrediting bodies. An approach combining LFD and ELISA methods can give some insight into how successful a validation performs on surfaces and products, respectively. ATP and protein testing methods can help to verify the ongoing efficacy of a validation program.

References 1

EHEDG Guidance Document Cleaning Validation in the Food Industry - General Principles, Part 1, Available as a free download at https://www.ehedg. org/guidelines/

Arrowsmith H. and Brown H. (2009) Validation of cleaning to remove food allergens. Guideline No. 59, CampdenBRI, Chipping Campden, UK ISBN 978 0 907503 55 2

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