Meg Provenzano, Hayden Skalski, and Kaitlyn Vap - Veolia Water Technologies & Solutions, Sievers Instruments
Annex 1 and Cleaning Validation
Annex 1 was revised in August 2022 to reflect changes in pharmaceutical regulatory and manufacturing environments and address the need for enhanced process understanding as part of a contamination control strategy (CCS). Annex 1’s focus on contamination control strategies with respect to equipment revolves around cleaning programs. Analytical method selection for cleaning validation presents an opportunity to improve process understanding with the implementation of process analytical technologies (PAT) such as at-line or online monitoring of endotoxin, bioburden, total organic carbon (TOC), and conductivity.
Cleaning validation can be daunting, especially with high volume testing for various sources of contamination and the need to confidently make decisions that impact production times and product quality. However, with PAT for cleaning validation, process control and understanding are enhanced, real[1]time decisions can be made, and analytical testing becomes faster and simpler. Manufacturers can minimize hands-on time, decrease the amount of lab samples in the QC queue, and reduce the amount of training required to perform testing. An at-line or online approach allows for quicker results, increased uptime for equipment, and overall risk reduction with the ability to track and trend results throughout the process.
Analytical Methods for Cleaning Validation
Analytical methods for cleaning validation typically fall under two categories: spe[1]cific or non-specific methods, and should address various forms of contamination.
Specific vs non-specific methods:
- Non-specific methods are ideal for a robust contamination control strategy since they have the ability to monitor various sources of contamination, not just the most toxic or the most difficult to clean compounds. This provides a measure of absolute cleanliness.
- Specific methods use instruments such as HPLC, UV-Vis, or mass spectrometry. They quantify a specific contaminant or compound, such as an active pharmaceutical ingredient (API) or detergent, however they have limitations in regards to overall process understanding and control.
Types of contamination:
- Chemical contamination: TOC and conductivity testing are two non-specific analytical methods often used for cleaning validation and the detection of chemical contamination. These two methods in parallel provide process control and understanding for a cleaning program, as carbon and conductivity measurements can capture contaminants ranging from APIs to detergents and any potential excipients present. These two methods are also very versatile, which has made them compatible not only in the lab but also at-line and online, allowing for the real-time release of equipment back into production.
- Microbial contamination: Bacterial Endotoxin Testing (BET) and bioburden testing are also used in cleaning validation practices to detect microbial contamination or biofilm. Following proper sanitization steps in a cleaning cycle, it has been a growing practice in the industry to evaluate residue for both endotoxin and bioburden.
By implementing a combination of testing methods for bioburden, endotoxin, TOC, and conductivity, you can enhance your contamination control strategy and ultimately reduce equipment downtime.
At-line and Online Monitoring for Cleaning Validation
With the recent revisions to Annex 1 prompting action for continuous monitoring and improved process understanding, there is a drive to transition to PAT, including at-line and online monitoring for cleaning validation. Decentralizing testing from the QC lab can help provide a clear understanding of the manufacturing process.
At-line monitoring:
With at-line testing, you eliminate the transition time between the production floor to the QC laboratory for sample testing and instead leverage portable instruments for sampling at the site of the production equipment after a cleaning cycle. Eliminating the need for QC lab testing will remove delays associated with quality control workflow, ultimately reducing turnaround time for equipment to enter back into production and optimizing overall uptime.
TOC and conductivity testing performed at-line allows for both swab and rinse sampling. If methods for TOC have already been built in the lab for cleaning validation, they’re simple to transition from the lab to the production floor.
At-line testing for endotoxin and bioburden requires instruments that are easy to use and can provide more rapid results than traditional methods. Many traditional methods like plate counts for bioburden testing or 96-well plate assays for BET pose the risk of human error as you need to manually count colonies during the traditional evaluation of bioburden, and endotoxin testing requires hundreds of manual pipetting steps. These methods are limited by time, with bioburden often taking days or weeks to provide results.
With rapid microbial methods (RMMs) for bioburden, such as high throughput flow cytometry that can correlate back to plate counts, companies can make actionable decisions quickly, with results being obtained within an hour. However, only certain rapid bioburden testing methods can be correlated to plate counts, thus making the technology selection important to ensure streamlined adoption of these methods within a CCS
Centripetal microfluidic technology for the LAL assay is a newer, innovative way to test for bacterial endotoxins without the pain points of traditional LAL tests and provides a pathway to real-time release testing for BET. This technology used in the Sievers Eclipse BET Platform is less prone to errors and drastically reduces set up time, time to results, and hands-on time. It also eliminates the need for a highly trained microbiology analyst to perform the assay, making this test transferable to the production floor. Implementing centripetal microfluidic technology as part of a CCS for at-line testing in the manufacturing area or QC testing can detect endotoxins earlier in the process. This technology can also help mitigate time involved, risks to the business, and any potential risks to your process.
Online monitoring:
Online cleaning validation is achieved with TOC and conductivity and provides the ultimate ability to enhance process understanding and improve efficiency. TOC and conductivity are preferred over specific methods for online monitoring, as specific methods like HPLC, UV-Vis, or mass spectrometry for cleaning validation need to be performed in the lab due to the footprint of the instruments and lack of compatibility in an online setting.
Sievers TOC and conductivity analyzers can be integrated into a CIP or clean-in-place skid, allowing for online, real-time sampling of the final rinse. This method is supplemented with swab samples measured at-line or in the lab unless justified otherwise, but ultimately the ability to conditionally release in real time on the basis of TOC and conductivity rinse samples passing under the established cleaning validation limit allows companies to optimize uptime through the reduction of equipment downtime. This provides the potential to entirely eliminate the clean hold time equipment undergoes as production waits for passing rinse and swab results prior to being pushed back into production.
Conclusion
Risk mitigation can be achieved through the adoption of process analytical technologies for cleaning validation in alignment with Annex 1 guidelines. Rapid methods for TOC, conductivity, endotoxin, and bioburden can be used to ensure equipment cleanliness and improve process understanding and control. By deploying continuous or at-line monitoring systems for these contaminants, you can decrease the number of manual steps, detect contamination sooner, and avoid production delays by maximizing equipment uptime.
As noted in Annex 1, rapid or alternative methods and continuous monitoring systems “should be considered to increase the protection of the product from potential extraneous sources of endotoxin/ pyrogen, particulate and microbial contamination.” Implementation of rapid micro methods - such as microfluidic technology for endotoxin testing and flow cytometry for bioburden - in addition to at-line or online TOC and conductivity monitoring, will help lean out cleaning validation processes and reduce risks. These methods contribute to a contamination control strategy through the ability to make actionable decisions and better manage equipment by evaluating and managing potential risks to quality. With faster results and easier oversight of processes, delays to production or product release can be minimized, ensuring prompt delivery of safe, efficacious therapies to patients.
About the Authors
Meg Provenzano is the Product Manager for Sievers bio-detection instruments at Veolia Water Technologies & Solutions. She has over 10 years of experience in the bacterial endotoxin testing industry and has held several positions in Quality Control, Technical Support, and Product Management. She is customer-centric and enjoys hands-on problem solving, whether for technical issues, assay assistance, or software. Meg holds a B.S. in Marine Science and Biology from Coastal Carolina University where she focused on Bottlenose Dolphin population research.
Hayden Skalski is the Lead Life Sciences Product Application Specialist for the Sievers Instruments product line, specializing in bacterial endotoxins testing (BET). Hayden has over 8 years of experience in the pharmaceutical industry and Quality Control Microbiology and has presented on numerous topics surrounding endotoxin testing. Previously, Hayden held roles at Charles River Laboratories, Regeneron and Novartis, validating and executing method development protocols for endotoxin testing, providing customer support, troubleshooting and supporting high-volume product testing. Hayden has a B.S. from the University at Albany (SUNY) in Biology.
Kaitlyn Vap is the Life Sciences Lead Product Application Specialist responsible for supporting Sievers total organic carbon (TOC) customers at Veolia Water Technologies & Solutions. She works with companies in the pharmaceutical industry to implement Sievers analytical instruments for the validation of laboratory water and drug products in accordance with global pharmacopeia regulatory standards. As an application specialist, she also explores new and/or developing applications to streamline the process of water and drug product testing. Kaitlyn holds a B.S. in Chemical Engineering from the University of Wyoming.
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