FDA has not mandated the use of allergen residue testing to assess the effectiveness of preventive allergen controls such as cleaning of shared equipment. However, the prudent use of such methods is very useful in determining whether allergen cleaning procedures (SSOPs) are effective and consistent. Increasingly, food companies do use swab testing of equipment surfaces for SSOP validation.
The Food Safety Modernization Act (FSMA) stipulates that allergens are a potential hazard. FSMA requires preventive allergen control implementation in manufacturing facilities that handle allergens. The food industry makes extensive use of shared manufacturing equipment for multiple formulations, some containing allergenic foods or ingredients and others not. The cleaning of shared equipment is a critical preventive allergen control step. While FDA has not yet released its anticipated guidance on preventive allergen control, it has already begun to conduct FSMA inspections, and some of those inspections have included assessment of preventive allergen controls. Thus, food companies using allergenic foods or ingredients should develop an Allergen Control Plan, and effective and consistent procedures for the cleaning of shared equipment.
In the U.S., the major allergens are defined as milk, eggs, fish, crustacean shellfish (shrimp, crab, lobster), peanut, soybean, tree nuts (walnut, cashew, etc.), and wheat. Ingredients derived from the major allergenic foods are also considered allergens for labeling purposes, although the comparative allergen loads (the amount of protein from the allergenic source) are highly variable from non-detectable (e.g., butter oil, cold-pressed soybean oil) to low (e.g., lactose) to high (e.g., wheat flour, soybean flour, casein, whey protein concentrate). The effectiveness of allergen SSOPs is typically focused on the major allergenic foods and ingredients derived from them, especially ingredients with high allergen loads.
Allergen Swab Testing
Allergen swabs can be effective in assessing the cleanliness of equipment surfaces. The swabs can be tested directly using certain commercial kits such as Neogen Alert kits. More commonly, swab use is coupled with lateral flow devices (LFD), also known as strip tests. Many allergen-specific commercial companies offer LFDs, including Neogen, r-Biopharm, Romer Labs, and 3M. Commercial LFDs exist to detect residues of peanut, milk, egg, soy, gluten (wheat, rye, barley), various tree nuts, crustacean shellfish, and fish.
LFDs and swab tests are highly specific and based upon antibodies that bind to protein(s) from the allergenic food. These test methods are qualitative but capable of detecting very low residual levels of allergens on equipment surfaces. These methods can also be used for detection of residues in clean-in-place (CIP) final rinse water samples. Some companies use these qualitative methods on ingredients or processed food samples, but their use for such purposes is not recommended unless careful evaluations have been done to ensure that the food matrix does not interfere with the detection of residues using LFDs.
Because food allergens are proteins, allergen-specific swabs and LFDs are the most relevant approach to determine if allergen residues remain on equipment surfaces. However, other swab approaches are available, including general protein tests (e.g., 3M Clean-Trace) and ATP tests (e.g., Charm AllerGiene). General protein tests detect protein residues from any source, allergen or not. ATP is a molecule found in all biological organisms, so ATP testing will detect soil residues on equipment surfaces from many sources. In our experience, general protein and ATP swab methods are slightly less sensitive than allergen-specific LFD methods. Due to their specificity, allergen-specific swabs with LFDs are more suitable for validation of SSOP effectiveness.
Deciding on a Testing System
Careful thought should be given to selection of the optimal commercial kit. The following are some tips on choosing the correct test to help ensure allergen SSOPs are as effective as possible.
Choose the right swab. Swabs must remove protein residues that may adhere to equipment surfaces, but must also be adsorbent. Swabs must also release the proteins back into an extraction solution. The swabs provided with commercial kits, such as the environmental swabs from Neogen, outperform regular cotton swabs.
Sponges should be avoided for allergen testing, as they tend to hold on to proteins, failing to release them into extraction solutions. Furthermore, some sponges may contain microbial growth media made from allergenic foods such as milk and soy.
Choose the most appropriate test method. The choice of an LFD that fits your purpose is relatively straightforward: It must be able to detect the allergen residues in the product matrix of concern. Essentially, will the LFD detect residues on the equipment surface before cleaning?
Different commercial LFDs targeted at residues of the same allergenic food are not created equal. LFDs contain antibodies raised against the allergenic food or specific proteins from the allergenic food. Each commercial LFD kit has its own proprietary antibody/ies that may respond differently to the residues left on equipment surfaces. Food companies use a variety of ingredients derived from a particular allergenic food (e.g., milk-derived ingredients can include non-fat dry milk, caseinates, or whey derivatives). Don’t assume that a given LFD will detect all forms of milk equally well—some commercial milk LFDs do not detect whey or whey-based ingredients, for instance. The sensitivity levels of different commercial kits for the same analyte will also vary and be dependent on the nature of the ingredient derived from the allergenic source.
Processing conditions also affect a system’s ability to detect allergen residues on equipment surfaces. Heat processing causes protein aggregation, resulting in difficulty with removal of residues from the surface and challenges with solubilization. Fermentation can alter proteins through proteolysis, again affecting antibody recognition on the LFD.
False negative results due to overloading LFDs is a particularly important concern. Due to the “hook effect,” high levels of allergen residues can cause false negatives. The hook effect occurs when high levels of the allergen residue are present, which overwhelms the detector antibodies in the strip causing most (perhaps all) of the allergen-antibody complexes to miss binding to the allergen-specific antibodies bound at the test line in the strip—resulting in a false negative response. Some commercial LFDs have overload lines while others do not, and the level of allergen needed to generate a false negative due to overloading will be different for each commercial kit. With milk, overloading occurs at concentrations from 100 ppm up to >10,000 ppm (expressed as ppm NFDM), depending upon the type of commercial kit. Serial dilutions of the swab or CIP rinse water extraction solution can be used to avoid overloading.
Set an achievable cleaning goal. LFD swabs are used to validate the effectiveness of SSOPs for removal of detectable allergen residues from shared equipment surfaces (or CIP final rinse water). Each product and processing line should be evaluated separately. If formulations have similar physical attributes (e.g., dry powders), similar cleaning approaches can be used on multiple formulations. When formulations contain multiple allergens, the assessment can sometimes be limited to the allergen that is present in the highest allergen load.
A corporate target level for effective allergen cleaning should be set, such as no detection of allergen residues on swabs using a specific LFD with a particular sensitivity level (e.g., no detection of milk protein residues with an LFD having a detection limit of 5 ppm). The LFD sensitivity level will vary to some degree with the nature of the food matrix, but a general statement such as “no detection by swab with LFD” is usually sufficient.
Use the right technique. Swabbing of hard-to-clean spots on the processing lines (nooks and crannies) is important. Multiple swabs should be taken, especially in the initial stages of cleaning validation on a processing line to identify the spots that are hardest to clean. Those spots can then become the focus of subsequent cleaning validations and verifications. Allergen cleaning protocol effectiveness should be re-validated periodically or whenever anything changes (product formulation, equipment matrix, processing conditions, etc.). The frequency of re-validation is not fixed and is dependent upon the frequency of changeovers. When using the recommended environmental swabs, the swabbing technique can vary without much effect on the observations. Swabs and surfaces can be either wet or dry.
Interpret the results. LFDs offer qualitative results. Thus, results should be interpreted primarily as negative or positive. The goal for cleaning validation should be “negative by swab” after documenting that the chosen LFD is fit for purpose.
Commercial LFDs provide sensitivity limits in concentration terms, such as ppm, and relate to the allergen concentration in the swab extraction solution. This term has no bearing on the allergen concentration that might be found on a finished product that comes in contact with the equipment surface. Swab test results should instead be provided in terms of µg/cm2, but that presumes users will swab uniform areas of the equipment surface. Since irregular surface areas are swabbed, the most appropriate expression of results would be µg/swab. And since the degree of hazard to the finished product cannot be determined from a swab result, the goal should be “negative by swab” as noted above unless you are brave enough to test finished product (see below).
Know when to test finished product. The results of equipment surface swabs cannot easily be translated to finished food products. Swabs with LFDs offer qualitative results while finished product testing is usually quantitative. LFDs tend to be extremely sensitive; they can detect extremely small amounts of allergen on equipment surfaces. When the subsequent product is manufactured on the shared equipment, allergen residues will likely be transferred to that product especially on the initial amount of product made after changeover. However, depending upon the amount of residue remaining of the equipment surface and the volume of the next product that passes over that equipment surface, the residues may not always be detectable in that next product. The testing of finished product is the only way to determine if detectable residues are present.
For obvious reasons, food companies are reluctant to test finished food products for undeclared allergens because the presence of such residues means that the product cannot be sold. However, the ultimate validation of an allergen cleaning procedure involves ensuring that no detectable residues are present in the finished product. If a robust swabbing strategy has been used and no allergen residues have been detected by swab with LFD, then it is very unlikely that allergen residues will be detected in the finished product. In those circumstances, testing of the finished product does serve as the ultimate validation.
Dr. Taylor is the co-founder and co-director of the Food Allergy Research and Resource Program (FARRP) at the University of Nebraska, Lincoln. Reach him at email@example.com. Dr. Baumert is the co-director at FARRP. Reach him at firstname.lastname@example.org.