When cleaning samples fail to meet acceptance criteria without a clear root cause, it can be challenging to discern whether the failure is due to inadequate cleaning or poor sample handling. The time lost from repeating validation runs, investigating deviations, and performing re-cleaning/sampling/analysis, as a consequence of poor sample handling, cannot be recovered. Proper sample handling techniques are vital to any cleaning program. This memo will discuss common sample handling issues and recommendations to minimize their risk to your cleaning program.
Common sample handling issues
Incorrect selection or preparation of sample containers before sampling
Lack of clear ownership and communication of sampling plan and timelines from collection to analysis
Sample submission form and label are misaligned or incomplete
Inadequate qualification of operators on rinse, swab, and visual inspection
Incorrect selection or preparation of sample containers before sampling
Before analytical method validation, sample containers should be defined. Sample containers are selected based on the amount of sample needed for testing (and retesting) and the nature of the material sampled, such as temperature sensitivity, light sensitivity, or potential for sample interaction with the container. Using a non-validated container may have the following adverse impacts: yield inaccurate results, refusal by Quality Control (QC) to test the sample, or execution of an addendum method validation for the container used. The sampling procedure should define the validated container specifications. Also, the materials section in the validation protocol should include the validated container to ensure personnel know the proper containers to use during sampling. Personnel should be trained on the correct sample containers specified for each sample type.
Container preparation before sampling is an essential control for the accurate analysis of samples and the prevention of contamination. Upon receipt of the sampling containers, there should be controls in place for their acceptance and release, such as visual inspection for damage or contamination, a validated process for cleaning/sterilization, and verification of sterility status from the supplier. Before collecting a rinse sample, containers should be conditioned by triple rinsing the container and the inside of the lid with the final rinse sample solution being collected. Reused sample containers for product residue and cleaning agents must be cleaned and conditioned before sampling. For nonspecific methods such as conductivity and TOC rinse, it is vital to condition the container to reduce potential trace impurities that may interfere with the accuracy of results. For bioburden and endotoxin, it is recommended to use sterile single-use containers to reduce the risk of contamination.
Incorrect sample storage or exceeding sample stability
Storage conditions may vary depending on the solvent used during sampling and the intended analytical method (e.g., bioburden samples may be chilled but not frozen). The proper storage condition and stability timeframe are established during analytical method development and validation. Improper storage of samples can have irreversible effects due to damage to the sample, such as degradation or lysis. Similarly, exceeding the validated stability period may result in invalid samples, as QC will only test samples within validated parameters.
When handling samples, it is crucial to properly care for samples to prevent breakage, contamination, or impact on the physical stability of the sample. For example, viscous solvents may froth if inadvertently shaken, leading to difficulties when performing analytical methods. Training of sampling personnel should include proper storage conditions, handling techniques, and impact on samples if not correctly followed.
Lack of clear ownership and communication of sampling plan and timelines from collection to analysis
A well-thought-out and detailed sampling plan outlining roles and responsibilities should be established before cleaning validation. Incorrect execution of the sampling plan may lead to delays due to investigations. The sampling plan should clearly define details, such as the sampling locations, the number and type of samples, the quantity of material to be collected, and the associated acceptance criteria. At a minimum, the sampling submission form should contain:
The unique sample ID.
Sample description (e.g., "TOC swab sample from agitator shaft").
Sampling date/time.
Storage conditions.
Acceptance criteria.
Identity of the person who sampled.
Any other relevant information.
Sample labels should accurately reflect the information documented on the sample submission form.
The cleaning execution record (e.g., batch record or validation execution documentation) should document:
The unique sample ID.
A description of the sample, including location.
Sampling date/time.
Identity of the person who sampled.
Acceptance criteria.
Analytical results.
Furthermore, clear ownership and communication of the sampling plan, including the timeline for the end of cleaning, sample collection, and testing of samples, is necessary. If there is poor communication between departments or shifts, sampling errors can occur, or samples can exceed their validation. Maintaining and documenting a clear chain of custody for each person responsible for each step, from sample collection through reporting of analytical results, is vital. Failure to maintain the chain of custody is a data integrity issue that will call the sample results into question.
Using inappropriate or contaminated gloves
When performing rinse and then swab sampling, it is common for gloves to come into contact with the final rinse solution and become soiled. Sampling with soiled gloves can lead to sample contamination. It is imperative to consider the type of samples to be collected, the order of sample collection, and the potential risk of contamination of a sample from soiled gloves. For example, sterile gloves should be used when collecting bioburden and endotoxin samples. Similarly, gloves should be changed between collection of rinse and swab samples. It is essential to provide guidance and training on when it is appropriate to sanitize or replace gloves if they become contaminated during sampling.
Spraying/wiping down with alcohol before sampling
Alcohol or other solvents may be used to spray and wipe down containers, gloves, or areas to reduce the potential for contamination of samples. However, personnel should be made aware of the potential impact on samples when solvents, such as alcohol, are used before sampling. When performing microbial or TOC rinse samples, the sampling apparatus should be flushed, and containers should be conditioned with rinse water before collection.
Alcohol exposure can lead to cell membrane degradation of microbes, leading to inaccurate bioburden or endotoxin results. Post-sampling, microbial swab sample points should be wiped down with alcohol to remove trace residues of sampling materials. Due to this, different swab sampling locations must be used for product residues, which should be indicated in the sampling plan. Similarly, alcohol exposure can lead to increased organic carbon in TOC samples. A general recommendation is that alcohol should not be used in the area before and during sampling for nonspecific methods such as TOC.
Failure to use aseptic techniques during sampling
Aseptic techniques should be used when performing sampling in the field to decrease the risk of sample contamination. For example, personnel should change gloves if soiled, clean sample staging areas before use, and containers should remain closed until immediately before sample collection. When the total final rinse solution is collected, and sub-aliquots are taken for analysis, the sub-aliquot sampling tool (such as a mug or sterile beaker) must be cleaned before use. All personnel participating in sampling should be trained on and follow aseptic techniques in the field to protect the integrity of the samples and prevent contamination.
Collecting samples in the incorrect order
Order of sample collection should always be considered due to the potential for contamination during sample handling. When rinse, swab, and visual inspection are performed, rinse sampling should be performed first, followed by swabbing and visual inspection, which can occur concurrently. As gloves may be soiled during the collection of consecutive rinse samples, the collection order must be considered based on the analytical test. Similarly, when swab sampling is performed, equipment may become contaminated through swabbing. Therefore, microbial samples should be taken before product residue and cleaning agent samples when collecting rinse or swab samples. The sampling plan should specify the order of sample collection, which is based on the potential risk to the sample from contamination. Personnel should be trained on the sampling plan and the criticality of collecting samples in the correct order.
Under-filling of sample containers for TOC and conductivity
For TOC and conductivity samples, the underfilling of sample containers can lead to the rinse sample not being representative of the source. For example, if a TOC sample container is underfilled, atmospheric CO2 trapped in the container will dissolve into the sample, resulting in increased organic carbon. Similarly, underfilling of conductivity sample containers can introduce ions from the air trapped in the container that will dissolve into the sample, resulting in increased conductivity measurements. The amount of trapped air should be minimized by completely filling sample containers to prevent unintended impact of air interaction unless the impact on samples is accounted for during swab and rinse recovery studies. Personnel should be trained on the appropriate collection volume and the unintended consequences of underfilled sample containers.
Sample submission form and label are misaligned or incomplete
Missing or misaligned critical information between the sample labels and the submission form may lead to incorrect storage or failure to test samples within the validated stability timeframe. For example, if a sample label states to store the sample at room temperature and the submission form states to freeze the sample, QC sample submission may refuse to process the sample further until the misalignment is corrected, or the sample may be incorrectly stored, resulting in the inability to test or invalid results. Before submitting to QC, sampling personnel should verify that all critical information in the sample submission forms, and the labels have been documented and aligned.
Inadequate qualification of operators on rinse, swab, and visual inspection
Swab sampling qualifications should include demonstrating adequate recovery from soiled coupons with a known amount of material in triplicate, awareness of swab locations for all equipment types, and demonstration of swab technique in the field.
Visual inspection qualification should confirm the operators' ability to identify visible residues from coupons under conditions in alignment with the manufacturing environment (with consideration for viewing angle, distance, and lighting). In addition, personnel should be trained on surface anomalies, including stains, rouge, and scratches, and visual inspection should be demonstrated in the field.
Rinse sampling qualification should include training on aseptic techniques, proper conditioning of the sample containers, and proper fill volumes for each type of sample.
Records for personnel qualifications on swab, rinse, and visual inspection should be maintained and readily available.
Sample handling techniques are an essential aspect of any cleaning program. Designing robust sampling procedures and subsequent training and qualification processes that are periodically monitored for effectiveness are the most fundamental controls to ensure consistent and proper sampling. Sampling procedures and training programs should include, at a minimum:
Identification of appropriate container for each analytical test and how to properly prepare sample containers before sampling.
Sample storage and validated stability conditions.
Clear ownership of sampling and testing activities along with sampling plan. communication and chain of custody requirements.
Proper glove use to prevent contamination.
Potential impact to each analytical method due to solvent exposure, where appropriate.
Aseptic sampling techniques.
The correct order of sample collection.
Filling requirements for each analytical method.
Sample submission form and label completion and alignment requirements.
Qualification requirements for operators on rinse, swab, and visual inspection.
Incorporating these critical areas in your sampling procedures and training programs will minimize the risk of cleaning analytical failures due to noncompliant techniques.
References
ISPE. (2018). Baseline Pharmaceutical Engineering Guide (Third Edition): Sterile Product Manufacturing Facilities
PDA. (2012). Technical Report No. 29 (Revised 2012). Points to Consider for Cleaning Validation.
PDA. (2010). Technical Report No. 49. Points to Consider for Biotechnology Cleaning Validation.
PDA. (2015). Technical Report No. 72. Passive Thermal Protection Systems for Global Distribution: Qualification and Operational Guidance.
WHO. (2005). Annex 2 Good Manufacturing Practices for Biological Products.
WHO. (2003). Annex 4 Guidance on Good Manufacturing Practices: Inspection Report.
Contributors: Jenna Carlson, Alec Fufidio, Samson Goodrich, Joanna Joseph, and Harrison Sweeney