Issue 7

A Romer Labs®
Publication

New Challenges
and Methods in
Allergen Testing
Photo: shutterstock_pipicato

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
4-7
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
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10 Steps to Validating and Verifying

www.romerlabs.com

©Copyright 2018, Romer Labs®

All rights reserved. No part of this Allergen Cleaning Procedures
publication may be reproduced in any
material form for commercial purposes How do food producers know whether their cleaning regimen is working? Paul Bagshaw
without the written permission of the of Holchem Laboratories gives a detailed look into cleaning validation and verification.
copyright holder.
All photos herein are the property of By Paul Bagshaw, Technical Services Manager, Holchem Laboratories Ltd.
Romer Labs or used with license.
Romer Labs is part of ERBER Group

2 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® 3
Challenges in Allergen Testing
P a r tDetecting Allergens
T w o –

in Processed Foods
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.

4 Spot On Issue 7
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.

Photo: shutterstock_ Rasica

A Romer Labs® Publication 5

 M
Quality control is ost of the food and drink we consume has
been processed or modified in some way.
an important factor This processing brings about many ben-
efits in terms of food safety, preservation
when producing
and taste. However, processing changes the characteris-
incurred reference tics of the ingredients used to make the food; of partic-
ular interest are the changes that allergenic proteins can
materials. The undergo. There are many kinds of changes relevant to
allergen detection: allergenic proteins can be subjected
allergen to be
to heat-accelerated chemical reactions including Mail-
incorporated into lard reactions and other protein-carbohydrate interac-
tions, protein aggregation with loss of solubility, shear
the food matrix must effects on protein structure, emulsion formation, pH
be very well defined. effects, and water activity during food production. Re-
cent studies have shown that processing allergens can
alter their allergenicity, changing how an allergic indi-
vidual may react to them. If immunoassay-based food
allergen detection methods rely on the use of antibod-
ies 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 cessing in an industrially produced food. The simplest
basics and raising new sets of antibodies that specifical- way to do this would be to look around a supermarket
ly target processed allergens. and search for similar products with and without the
specific allergenic component, for example, a cook-
How do we create real-life samples ie with and without milk. There are several things to
with incurred controls? consider when making incurred materials. Such ma-
To evaluate these improved allergen detection meth- terials are usually produced in a food processing pilot
ods, incurred sample controls are needed. These are plant or test kitchen; the ability to clean the processing
food samples into which a known amount of the food equipment thoroughly after each batch is an important
allergen has been incorporated during processing, consideration to assure that there is no batch-to-batch
mimicking as closely as possible the actual conditions contamination.
under which the sample matrix would normally be Quality control is an important factor when produc-
manufactured. Analyzing the real-life sample would ing incurred reference materials. The allergen to be
give the most accurate representation of the recovery incorporated into the food matrix must be very well
and response of a particular method for that particular defined. Where official reference materials exist they
matrix. Yet each food matrix and processing condition are not usually affordable in the quantities needed to
is different and it would not be practical to evaluate all produce the incurred controls. Yet any official reference
possibilities with naturally incurred standards. How- materials can be used for comparative purposes to de-
ever, evaluation of an ELISA with one or more typical fine the nature of the material used. Furthermore, the
combinations of food matrix and processing condition homogeneity of the allergen added to the food matrix
is a useful method in determining whether the ELISA is critical for every step of the process. In order to en-
can provide reliable results when applied to processed sure that the allergen is homogeneously incorporated
foods. into the incurred material, multiple analyses of the
Steve Taylor of the Food Allergy Research and Re- sub-sampled food at various stages of processing are
source Program has described what should be con- required to make sure that uniform distribution of the
sidered when creating incurred reference materials allergen is achieved. Contamination between batches
for food allergen analysis. In essence, he suggests, the with increasing levels of allergen can be controlled by
matrix of choice should be one in which the allergenic starting with the lowest concentration and steadily in-
food residues would likely be found during typical pro- creasing the levels.

6 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 pro-
teins 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-
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 7
low their future use for evaluating improved analytical MoniQA has developed
methods. Such incurred control materials may also be
used to evaluate batch-to-batch variability for commer- an incurred reference
cial ELISA kits.
material to mimic as
Early successes with incurred samples closely as possible the
Very slowly, fully validated incurred reference ma-
terials for food allergen analysis are coming onto the processing through
market. The MoniQA Association has recently released which an allergen-
a milk-incurred material that incorporates well-charac-
terized dried skimmed milk powder into a gluten free containing food goes
cookie at two individual protein concentrations. This
incurred reference material has been produced to mim-
during production.
ic 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 analyt-
ical 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 fur-
ther, work undertaken at the University of Manchester
under the guidance of Professor Clare Mills has inves-
tigated 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 in-
curred food ingredients with demonstrable allergenic
activity for analytical purposes will help ensure that
efforts to standardize calibration materials and harmo-
nize allergen-reporting units are meaningfully aligned
with ongoing measures to protect allergic consumers
from accidental exposure to problem foods.
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 clinically-
validated 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 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.

F
Paul Bagshaw has
worked at Holchem ood manufacturers and processors rely on a tion. Global Food Safety Initiative (GFSI) retailer ap-
Laboratories Ltd. for variety of differing policies and procedures proval schemes such as the British Retail Consortium
nearly 10 years. His
to enforce allergen controls. These include (BRC) state, “Where cleaning procedures are part of a
research interests
include allergens, CIP personnel controls, such as a hand-washing defined prerequisite plan to control the risk of a specif-
and hygienic design. procedure and the use of protective clothing ic hazard the cleaning and disinfection procedures and
As Technical Services and equipment (PPE), process controls such as segre- frequency shall be validated…” In simple terms, this
Manager, he provides gated storage and color-coded equipment, production means that the validation process should demonstrate
technical support to controls such as dedicated equipment and time segre- that the cleaning procedure a site is using reduces the
Holchem’s sales team
gation, and – most importantly – cleaning. hazard – in this case, an allergen – to a level deemed
and customers.
Cleaning regimens are subject to rigorous valida- to be acceptable.

8 Spot On Issue 7
 The validation guidelines at Holchem Laboratories
Ltd. have been developed in accordance with the prin-
ciples of the European Hygienic Engineering and De-
sign Group (EHEDG) Cleaning Validation subgroup1
and Campden BRI2, together with in-house best prac-
tices. 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.
 and applicable regulations
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).
 for cleaning. Chemical disinfectants should have the
Ensure organizational support
A team approach will help ensure that the validation
process is effective; the team should be multi-depart-
mental (production, engineering, technical, hygiene,
hazard specialist) and have the backing of senior man-
agement to ensure seamless collaboration.
 There are several reasons to choose a particular food
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 9
worst-case scenario. These areas will be assessed for Validation should
cleanability during the validation process. As such, it
may be necessary to strike a balance between the place be carried out
that is hardest to clean (but may need specialist ac-
under worst-case
cess equipment or engineering support to dismantle)
and places that are hard to clean but are practicably scenarios.
accessible.

Review any current cleaning programs
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 pa-
rameters 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 tem-
peratures, 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 valida-
tion, and should consider whether any risk assess-
ments are required for any dismantling of equipment
relevant efficacy data, including the European stan-
dards EN1276 and EN 13697, and comply with the re-
quirements of the Biocidal Products Regulation (EU
528/2012).

Determine the worst-case scenario
for the soiling
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 lon-
gest processing time, the highest temperature or the
The cleaning has period of time the equipment sits idle before cleaning.
Whilst this determination of the worst-case soiling
achieved its desired scenario will ensure a robust validation that stands up
to scrutiny, an added benefit is that it increases effi-
results when both
ciency by allowing food manufactures to carry out few-
the product and food er validation processes. For example, if a food manu-
facturer has a number of allergens that are checked by
contact surfaces are the same cleaning and disinfection program, validat-
ing the program for the worst-case scenario for a single
free from allergens.
allergen (highest allergen presence, most difficult to
clean soil) theoretically confers validation to cleaning
programs for all allergens used.
 tection technique.
Sample, sample and sample again
Sites should then determine the type of sampling to
assess whether the objective of the cleaning and dis-
infection program has been met. The simplest form
of assessment, the visual inspection, is often the most
overlooked: are debris visible at the sample sites? Oth-
er options are direct and indirect sampling: direct sam-
pling is likely to be via surface swabbing, whilst indi-
rect 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.
 The cleaning has achieved its desired results when
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 deter-
mine whether the objective of the cleaning and disin-
fection 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-
10 Spot On Issue 7
tablished to ensure that the target allergen, in food
products and on process surfaces, can be detected un-
der 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 surfac-
es 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 de-

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 valida-
tion 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, ma-
chinery or a cleaning parameter.

Interpret the results
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 in-
dicates that the cleaning has not achieved its desired
results, meaning that the site must amend some as-
pect of the cleaning program to improve results be-
fore 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 like-
ly to be a significant risk to the subsequent batch of
product should be undertaken.
Within the risk assessment, two factors are import-
ant. Firstly, cross-contamination from the food con-
tact surface to the food involves a transfer coefficient:
not all allergens present on the surface will transfer to
 Table 1. Analytical methods for cleaning validation and verification.
Method Purpose
ELISA Product testing
Post-validation surface testing
LFD Surface testing
Ongoing verification
ATP testing Post-validation verification (ATP must be detectable on surfaces, even if allergens are not)
Protein swabs Ongoing verification
Source: Romer Labs
the food. In practice, the transfer coefficient of the al-
lergen 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 dilu-
ent 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.
 inform the decision about what constitutes the
Verify with ATP or protein swabs
After validation, food manufacturers require a
method of verification. Cleaning verification is in-
tended 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 aver-
age 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 11
An approach
combining LFD and
ELISA methods can
give some insight
into how successful a
validation performs
on surfaces
and products,
However, a presence of protein does not necessarily
respectively.
indicate a presence of allergen. If ATP or protein swabs
are to be used, those operating the site must under-
stand that they are not measuring the allergen itself
but general hygiene indicators.
Conclusion
Cleaning and validation are complex processes, in-
volving a variety of possible tools. Ultimately, a
cleaning validation is a procedure that must be cus-
tomized to the needs of a specific production envi-
ronment. Detailed knowledge of the products and
process lines and an understanding of current and
previous cleaning programs and their efficacy should
worst-case scenario. Only this way can a facility en-
sure that the validation is robust enough to meet
both internal requirements and the standards of ex-
ternal 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/
2
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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