Microbiology Roundtable

In general, what are some current issues or problems pharm/biopharma companies are dealing with in regards to microbiology testing, analysis and identification?

Robert Lutskus, Global Product Delivery Manager – Informatics, Lonza Walkersville, Inc.: Current issues companies are dealing with include aging technology within their laboratories; managing and compiling data from individual or standalone systems into larger, enterprise systems and the level of IT support needed for laboratory-based environments. Aside from these issues, a number of major data integrity problems have been found by regulators recently during GMP inspections. As a result, auditors have been trained to perform more stringent inspections, which is a challenge pharma/biopharma companies have to address. Here is where having a quantitative endotoxin method offers a big advantage as it provides information about the manufacturing process in an electronic format. If software is compliant, it will automatically include an audit trail that will record many of the key issues that concern regulators looking for established data integrity rigor. To be fully secure, the data collected must be held in an encrypted database that cannot be altered unless it is recorded in the audit trail.

Sandra Gay, PhD, Global Pharma Marketing Manager, bioMerieux: The biggest challenge that pharma companies relay to us today is getting fast and accurate test results to ensure production is secure. Designing and maintaining a robust quality system both guarantees their products will be safe for the patient and protects their brands from damaging recalls.

Control of production has many components and starts with a clear risk assessment of your process, environment and water systems. Understanding your facility’s unique microflora and including them in your testing protocols and validations is critical and expected by auditors.

A real understanding of what microbiology is and the specificities of your situation will help you determine and implement the right method. Rapid Microbiological Methods (RMM) help ensure the quality control process by providing accurate results in days or even hours as opposed to weeks. Additionally, RMM will help you react quickly and not loose time on negative results, therefore enhancing your labs productivity (who is not supposed to do more with less?)

While there is no “perfect” method for every circumstance, it is essential to make sure the alternative method you choose is produced by a reliable, long-term partner, who understands microbiology and your specific needs and is very much up to date with the regulatory requirements for hot topics such as data integrity. You don’t want bits and pieces of solutions from here and there, but rather a more streamlined, global solution.

Mike Dingle, Field Application Specialist, Controlled Environments, TSI Incorporated: The current issue with pharmaceutical microbiology is that it isn’t current. Antiquated culture based methods are very much the norm and can be a significant hindrance to companies looking to modernize their manufacturing. The processes are very manual, making them labor intensive and at high risk for technician error/variability. When you include the fact that most take days to obtain results, it becomes readily apparent that these methods are not capable of providing the throughput and timeliness of results that is needed to meet the requirements of QbD, PAT, and continuous manufacturing initiatives.

I think pharma companies have a strong desire to move beyond traditional growth based methods and the regulatory agencies seem to be advocating for their implementation as well. The “how to make it happen” is what remains elusive in this equation. Despite a number of guidance documents that have come out, most still do not see a clear path to adoption and fear that satisfying inspectors will require an effort that is prohibitive. The good news is that there are a number of groups, across the industry, that are evaluating these technologies and as their results get published the path should be come clearer.

Erin Patton, Senior Product Specialist for Charles River Labs, Microbial Solutions Divison: Analysis of data from microbiology testing is always of concern, as it can be a challenge to ensure that data is not only secure, but also is easy to evaluate and accessible in a way that supports critical decision making about product quality, control of facility, and ultimately, patient safety. It’s common to encounter data management strategies that include manual transcription of data from one system to another and review of results individually, and not in the aggregate so that the “big picture” becomes clear. However, there are many solutions out there now, such as Charles River’s Cortex software for endotoxin data and Accugenix Tracking & Trending web tools for identification data, which eliminate tedious and risky transcription steps. In addition, such tools which centralize data for more powerful reporting allow quality personnel to easily see and act upon trends before problems have a chance to develop.

Gilberto Dalmaso, PhD, Senior Advisor and Pharma Customer Advisory Team Manager, Particle Measuring Systems: Current microbiology testing, analysis and identification issues impacting the pharmaceutical industry include method sensitivity, method validation and method quickness.

Claire Briglia, Technology Specialist, MilliporeSigma and Tim Cser, Technology Specialist, MilliporeSigma: Time to result, older methods and lack of personnel. The sheer volume of sampling required especially with EM versus labor costs, number of personnel available continue to be an issue. If testing is outsourced turnaround time becomes an issue with short shelf life of future biologics. The need for automated systems is becoming increasingly important, to align with the increasing volume of testing. The agility of regulatory changes to keep up with emerging methods and technology is often a bottleneck to new technology adoption. There are so many complex cell-based and personalized products coming into the market, that require specific applications and more advanced method development, a major hurdle for many labs. In addition, the cost of the test sample itself may be a challenge as more expensive personalized therapies are developed.

Dr. Michael J. Miller, President, Microbiology Consultants, LLC: The advancement of gene and cell therapies currently presents the microbiologist with a unique challenge for release testing. Many of these innovative products must be re-implanted or infused in patients in a timeframe that is significantly shorter than conventional pharmaceuticals (e.g., within days of manufacture). Additionally, small batch sizes compounds the issue of how much product is needed (or is available) for release testing because the more product that is sacrificed for this activity the less product is available for the patient. For example, a single batch of product may provide only a few IV bags or less than ten vials of product available for release testing and patient administration. Therefore, the development of alternative strategies for product release (especially those associated with sterility testing) is of the utmost importance.

Dr. Friedrich von Wintzingerode, Senior Manager Microbiology, Head of Endotoxin Task Force, Roche Diagnostics GmbH: There are currently two major fields of interest in regards to microbiology testing:

1. Microbial components/byproducts

• The impact of LER and other masking effects on LAL based endotoxin testing is still a hot topic. The issue of LER/masking makes industry microbiologists fully aware of the complexity of endotoxin structure and function.
• Microbial components like exotoxins, beta glucans, and non-endotoxin pyrogens like flagellin or lipopeptides potentially impact patient safety. Proteases and other enzymes can impact product quality. Biotech companies are challenged by these components because existing micro methods and concepts are largely based on intact and living microbial cells.

2. Testing for anaerobes

• Facultative anaerobes like Propionibacterium acnes can contaminate biotech products. However, current microbial control concepts are focused on aerobic bioburden. We need to better understand the risk associated with anaerobic bioburden and clarify whether additional tests are justified.

Looking at the latest in technology, can you tell us about some new technologies and/or processes that are available now that you are excited about? Do any of these new technologies have the potential to have a noticeable impact on pharma/ biopharma company’s microbiology testing and analyzing operations?

Lutskus: While not new to the market, MODA™ software can efficiently manage workflows for QC testing laboratories. Additionally, the ability to interface MODA™ software with rapid methods, such as the Rapid Micro Bio Growth Direct™ system, opens up a lot of exciting options for laboratories.

Gay: Coming from a Pasteurian company, I’m not only excited by all the powerful RMMs already on the market that allow you to detect contamination by a single microorganism, such as solid-phase cytometry and automated broth analysis with dual temperature mimicking the sterility testing; I’m also happy to see that even the 130-year-old Petri dish, can be taken to a whole new level without changing a company’s habits.

Automating the reading and incubation of your everyday plated media can actually transform a manual method into a paperless system. Having the plates read and photographed at regular intervals allows you to go back and review growth patterns of plates displaying questionable results to reveal behaviors like swarming. Solutions such as these will not only give you the final count but also alert you if your pre-established thresholds have been reached with full traceability; providing data integrity and 21CFR11 compliance to this basic, typically manual process.

Imagine you’re being audited and the inspectors asks what happened on a particular day - in a click you can show the results including a picture of the plate, even if the test happened years ago. I remember during a conference an FDA inspector telling the audience to at least take a picture of your plate with your phone. I think we can agree, that while taking a shot of your plate with a phone is better than nothing, having this performed automatically and systematically as part of your process is even better.

Dingle: There are a number of rapid methods out there that are pretty exciting, but at this point we’ve been hearing about them for years now. Laser induced fluorescence (such as TSI’s BioTrak® Real-Time Viable Particle Counter) is certainly exciting technology because of its ability to detect airborne viable contaminants in real-time with no manual interventions. This instrument was first introduced in 2012, so it’s hard to call it “new” anymore, but it certainly has matured. I think this holds true for many other rapid methods out there as well. What this means is that the specific applications that these methods can best serve have become clearer and they have been modified and improved to be used in these applications. This is making implementation less arduous and widespread adoption far easier to envision than it was just a few years ago. Adoption of these methods will have a noticeable impact by making microbiology testing and analysis more sensitive, accurate, and timely; all while reducing the risk to product caused by manual interventions and reducing or eliminating technician error.

Patton: Rapid microbiological methods (RMM), such as cartridge-based technology for endotoxin testing and amplified ATP technology for microbial detection are not as new as some might think, but are really exciting because of the impact they have on not only a company’s microbiology testing, but their entire operation. Rapid endotoxin detection cartridge technology allows for in-process control testing to be performed in just 15 minutes at the point of sample, removing the need for manufacturing processes to continue at risk while waiting on testing results from the centralized lab. Such a paradigm shift supports the pharmaceutical industry’s efforts to better adopt Quality by Design (QbD) principles. As environmental monitoring programs are key to QbD practices as well, taking advantage of newer applications of technologies, such as MALDI-TOF mass spectrometry for rapid microbial identification of EM isolates, is also impactful. Identification results that are faster and more accurate than those provided with older phenotypic methods allow for better control of a company’s EM program overall, contributing to increased control of facility and decreased risk. And lastly, RMMs like the Celsis microbial detection system offer positive impact to an organization by significantly shortening manufacturing cycles, finished product hold times, and enabling faster response in case of investigations.

Dalmaso: New technology with the potential for great impact on the pharmaceutical industry in relation to microbiology testing and analyzing operations include Laser Induced Fluorescence (LIF), flow cytometry, oxygen depletion, and advances in molecular biology techniques.

Briglia and Cser: I’m excited about the pace of technology. Super powerful computers make new technologies available faster. Sensors and the IOT should automate many of our tests that require manual interpretations. Real time laser flow testing and chip sensor driven molecular testing will change microbiology but balancing these methods versus traditional results will be the challenge. Flow cytometry also has significant potential, however statistically significant sample sizes remain a challenge in the regulated QC Microbiology sector. Nucleic acid technologies are extremely promising for specific organisms of interest and will hopefully be utilized more broadly fore sterility applications as better sample prep and contamination control technologies are developed to prevent false positives.

Miller: Rapid methods for sterility testing have been validated and implemented by a number of companies. These include the use of technologies that require a short enrichment phase (e.g., ATP bioluminescence) and those that do not (e.g., solid phase cytometry). One of the challenges the industry has is to determine the required time to result for a rapid sterility test and this will dictate whether a rapid scientific principle is appropriate for the firm’s user requirements. Obviously, the faster the sterility test can be performed the faster life-saving medicines can be delivered to the patient. Yet, there may be additional technology challenges that may have to be overcome. For example, some therapies may contain certain chemical components that may interfere with a method’s ability to detect microorganisms or may provide background noise or interference that could be interpreted as a positive response when there are no microorganisms present. These phenomena are more commonly referred to as “false negatives” and “false positives” respectively, and the validation of alternative methods for sterility testing must take into account the potential for these types of responses during method suitability studies.

Von Wintzengerode:

• Industry will take benefit from recombinant assays for endotoxin testing, which do not rely on natural resources.
• Automated test systems for endotoxins and bioburden have the potential to increase effectiveness of in-process testing.

In the past few years have regulatory expectations from the FDA and other global agencies resulted in more scrutiny placed on microbiology processes? Do inconsistent or different global regulations place a regulatory strain on pharmaceutical companies?

Lutskus: Yes, regulatory expectations from the FDA and other global agencies have resulted in more scrutiny being placed on microbiology processes. For example, in recent years there have been a number of focused audits involving data integrity and the trending of data. The volume of data generated in the lab is a particularly large target from a data integrity perspective. There have been numerous “guidance” documents for data integrity and there has been inconsistent guidance, specifically from the MHRA and the FDA. Additionally, the second person plate verification question has been a recent focus with no real global guidance. Companies are left to interpret this data and hope it is implemented satisfactorily. When it comes to biologics, the FDA has been cautious over the issue of LER in hold-time studies, despite the fact there is no data to suggest this poses a risk to patient safety. However, vaccine and biologic manufacturers have to reformulate their products to avoid LER-prone buffers.

Gay: The combined effect of the rise of developing countries in pharma manufacturing and the arrival of robust automated methods have indeed resulted in more expectations from the global regulatory agencies. Pharma companies all over the world need to show more accuracy, consistency and security for the patient. This is taking us in the right direction, of course, but it also means more pressure. Looking at the latest FDA warning letters, a large part of them point towards lack of data integrity and lack of global control. We strongly recommend to the pharma companies work with 21CFR11 systems and eliminate paper-based results when possible. Implementing rapid methods not only deliver accurate and timely results, but they automatically trace everything. Additionally, working with instruments where barcoding is employed down to the reagent level adds additional security. How else can you prove to your auditor that the testing went well and no step was forgotten or overlooked? Data integrity is at the heart of full control.

Dingle: There has always been scrutiny on microbiology, but as expectations increase, and they always do, the limitations of traditional microbiology are being more exposed. I think most companies would tell you that they cannot survive without high-speed internet, but the microbiological test methods that they are using are equivalent to a having a dial-up connection. The regulatory expectation is that every effort will be made to find issues and correct them before they become a problem, but when it takes days to get results and those results need to be manually compiled and analyzed, it is very difficult to proactively respond to issues. In fact, it may be difficult to even react to a problem in a timely manner. When you add to that the specter of “Data Integrity” that has companies doing things like having a second person count plates, it becomes easy to see why those relying on traditional microbiological methods struggle to meet the expectations of the regulatory agencies.

Patton: Recently, it has become more prevalent to hear accounts of FDA inspectors citing concerns regarding the data integrity of microbiology test results which rely on subjective readout and manual record-keeping of analysts. As data integrity is the requirement for complete, consistent, and accurate data, there has been increased expectation from global regulatory agencies that pharmaceutical companies make every effort to obtain accurate test results.

With regard to microbiology QC work, data is both quantitative and qualitative, and much of the qualitative data is generated through basic analyst observation and documentation, leaving room for error or differences in interpretation. As such, this expectation to ensure testing accuracy has resulted in agency recommendations for additional analysts’ participation in and review of subjective testing processes, in order to prevent failures in data integrity. Examples of tests impacted by this so-called ‘four-eyes principle” include endotoxin testing with gel clot technique, microbial identifications that rely on Gram staining steps, and traditional microbial limits and sterility testing.

With the ever-increasing availability of modern methodologies which rely on objective readouts, a developing expectation to improve accuracy by reducing subjectivity need not be a strain on the pharmaceutical industry, but rather an opportunity for improvement.

Dalmaso: No, global regulatory bodies are pushing the implementation of new and more sensitive technologies in microbiology. The problem is that pharma companies do not want to see more microorganisms in their processes. However, more sensitive technologies will ensure an increase to product quality.

Briglia and Cser: If you maintain a culture of quality, the differences usually aren’t too drastic. Data integrity is the current regulatory focus and hopefully data automation and IOT will help with the data stream. In reality microbiology testing regs aren’t that drastically different in most cases and all follow basic cGMP practices. I agree that data handling will continue to be an issue. Biologics with smaller and more frequent lots will only make this more critical.

Miller: I do not believe there are significantly different or inconsistent regulatory expectations in terms of microbiological processes. That being said, there is a global expectation that companies better understand their processes and adequately control these processes for potential contamination. Over the past few years the industry has experienced a number of recalls and serious patient clinical impacts as a direct result of microbial issues associated with poorly managed manufacturing facilities. For example, PharmaTech LLC recalled all of their liquid products due to at least 13 deaths and 58 cases associated with a multistate outbreak of Burkholdaria cepacia that was directly linked to contaminated water used to manufacture product (L. Ensor, FDA, PDA Europe Pharmaceutical Microbiology Conference, 2017; https:// www.fda.gov/Safety/Recalls/ucm515610.htm; https://www.fda.gov/Drugs/ DrugSafety/ucm511527.htm). Therefore, it is paramount that firms fully understand their process capabilities in preventing microbial contamination and to implement meaningful practices to meet this expectation.

Von Wintzengerode: In general FDA guidance regarding microbiology topics is well structured. There is no conflicting guidance between EMA, FDA and/or PMDA.

What advice would you give to a pharmaceutical company struggling with their microbiology processes/applications? Is there a top 10 list of items you would suggest to these companies to help them?

Lutskus: Our top 10 tips for companies struggling with their microbiology processes/applications are:

1. Evaluate your current systems and processes to identify bottlenecks.
2. Select an electronic system that will alleviate your identified pain points.
3. Efficiently implement the software so that returns can be seen immediately.
4. Re-examine processes with the new workflow to identify further savings.
5. Attend conferences and participate in roundtable discussions to better understand other companies’ issues, including how they identify these problems and implement solutions.
6. Work with vendors to understand the different solutions available and the challenges their customers report.
7. Consider implementing automation where possible to both reduce human error and improve efficiencies.
8. Commit to better training for your employees, such as encouraging them to watch webinars and use e-learning modules offered by many test suppliers.
9. For products where LAL is not the optimal endotoxin detection method, secure a secondary supplier or alternative methodology.
10. Implement more accurate and thorough data capture/analysis, helping to ensure rapid and easy traceability. Analysis software with trending capability means you can analyze data on demand and respond before out-of-specification errors occur.

Gay: The top list of items I would suggest to the pharma companies are:

• Chose a reliable partner to provide microbiology solutions. Make sure they can accompany you all the way from implementation to routine use, make sure they are microbiology experts and have the patient’s health at the heart of their preoccupations as well as your company’s long term relationship.
• Reduce the manual methods and implement automation for more standardized, accurate and traced results.
• Innovative and Rapid Microbiological Methods can bring high level added value for the industrial and financial performances of your manufacturing site: bring awareness and build business cases with your production team and you will become a performance leader in your organization.
• Ask your regulator! (We’ve all heard that one a lot in conferences.) It’s important to work hand-in-hand with your regulatory agency rather than waiting to the last minute and discovering your validation is not acceptable. And here again, your partner in microbiology should be able to accompany you to avoid such issues.
• Perform risk assessments.
• Walk away from non-21CFR11 systems.

Dingle:

• Get the right people, develop them, keep them, and make sure you have enough of them. If a pharma company is struggling with microbiology it is probably because they underestimated the importance of having enough educated, well trained, experienced microbiologists on staff.
• Microbiology cannot function on an island. Microbiology needs to understand the other functions and they need to understand microbiology or there will be issues, and they will probably happen repeatedly.
• Modernize, or at least get out of the last century, and the payoff can be huge. These endeavors seem scary and are out of the comfort zone of many microbiologists, but a number of the rapid methods and data handling solutions that are “cutting edge” have been around for years now and you don’t need to re-invent the wheel to implement.

Patton: There are many points that should be considered for a microbiology lab to perform optimally and avoid struggles:

• Always guard against testing interruptions by having secondary suppliers qualified.
• Recognize the importance of ongoing training for personnel. There are many conferences available to those in the micro QC industry, but other good sources include workshops, seminars, and webinars.
• Confirm that methods are validated appropriately in order to ensure optimal test performance and to avoid regulatory pitfalls.
• Consider automated testing technologies, which can offer a reduction in error rates and offer efficiencies to the lab.
• Beyond streamlining QC testing, consider the potential benefits that automated or rapid testing solutions can have on the speed, efficiency, and confidence in resolving out-of-specification events and investigations.
• When considering the financial case for implementation of improved technologies, be sure to take a broader view than just laboratory impact. In fact, there are multiple beneficiaries from implementing reliable rapid or automated technologies across an organization, such as manufacturing operations, Finance, and Quality Assurance groups.

Dalmaso: Environmental monitoring applications to aid struggling pharma-ceutical companies include water testing, bioburden testing and process gas testing.

Implement technology with the following requirements:

1. Sensitivity at single-cell level
2. Discrimination between viable and non-viable microorganisms
3. Able to detect viable but nonculturable microorganisms (VBNCs)
4. Rapid: as fast as possible (days vs. hours vs. minutes)
5. Qualitative and quantitative capabilities
6. Able to identify in case of contamination
7. Cheap
8. Easy to use and validate
9. Robust
10. Usable in the manufacturing environment

Briglia and Cser: Reach out to your vendors who help you with those processes/applications. The vendors work with a variety of manufacturers and can help you optimize and troubleshoot your problematic applications.

1. Reach out to your vendors. They should be the experts on their technology, the validation that goes into them, and method development support.
2. Take advantage of industry led resources (PDA Technical Reports, ISPE guidance, Microbiology Network PMFList)
3. Attend local PDA chapters to network with peers
4. Consult previously cited/top ten 483 lists to see what the FDA is focusing on
5. Purchase some PDA books like Environmental Monitoring or Method Development and Validation for the Pharmaceutical Microbiologist
6. Find a good consultant
7. Discuss with the regulatory agencies, they are a resource. Call the FDA, they don’t bite!

Miller: I have found many companies that have experienced significant microbiology issues did not have an appropriate contamination control program in place. In many instances these same companies lacked the necessary level of technical expertise to maintain their facilities in a microbiological state of control. Robust environmental, process and product monitoring programs should confirm the appropriateness of practices for the use of sanitizers, personnel and material flow, aseptic sampling, equipment and facilities preventive maintenance programs and other relevant contamination control activities. In the absence of a meaningful control program and in-house expertise, it is advisable to confer with a pharmaceutical microbiology consultant for expert guidance.

Von Wintzengerode:

• Build up internal micro expertise
• Keep in mind that all solutions are case specific
• Roll out micro training to production (e.g. basic hygiene)
• Conduct end to end Microbial Control Risk Assessments to identify gaps in your control system.
• Biotech DS manufacturing is NOT sterile, but they are targets for microbial contamination and requires strict microbial control

What do you see as the major industry critical issues over the next five years in regards to microbiology?

Lutskus: Over the next five years, top issues will range from managing laboratory data, including consolidating data from a variety of systems and ensuring data integrity is maintained, to the challenge of harmonization that arises with mergers and acquisitions. There will also be a focus on efficiency forcing laboratories to do more with less, such as evaluating data using risk-based methodologies, and adopting rapid methods and technologies.

Gay: We have seen pharma companies focusing strongly on bioproduction and cellular therapies in both mature and developing countries. Maintaining the same level of control whatever the country and situation is essential and a challenge that multinational or companies outsourcing part of their production face today. The methods they will chose have to be robust and standardized enough, with real global support, in order to maintain this control.

Specifically on bioproduction and the growth of biosimilars, the rapidity of in-process testing is an insurance for increasing competitiveness and proactivity.

Dingle: The most critical issue over the next five years is to gain the acceptance of modern microbiological methods. The pace of innovation is moving quickly and compendial methods often can’t keep up. To achieve the desired objectives of moving pharmaceutical manufacturing into the 21st century, the way most companies are doing microbiology today just won’t cut it. A prime example of this would be the completely automated, gloveless isolators that are being introduced. These offer unsurpassed contamination protection to the product, but the manual compendial microbiological monitoring methods are simply incompatible with this technology. Despite the rhetoric encouraging companies to move to these methods, many roadblocks still remain, with only “big pharma” having the resources available to even try. It is critical that a clearer path is made through collaboration between companies and with regulatory agencies to give companies of all sizes the ability to adopt these methods.

Dalmaso: Pharma industry microbiologists are not adequately prepared in Pharmaceutical Microbiology before entering the industry, and need a way to acquire the necessary skills to be successful. In Europe and the US, courses teaching pharmaceutical microbiology are largely unavailable. There should be more discussion between universities to introduce and promote pharmaceutical microbiology courses.

Briglia and Cser: Automating data records. The cloud, IOT, and rapid technologies should allow us to ensure data integrity is automated and 24 hour access to the data in whatever format we want. Integrating microbiology testing into the continuous processing/single-use systems. While the single-use systems are designed to reduce/eliminate contamination events, how are the QC labs going to be integrated into the increasingly automated processing. Slow adoption of new technologies such as molecular methods, data security of WiFi transfer, while keeping in mind that patient impact of future changes in methods and regulations will be a critical point. The industry cannot afford to get caught being too inward focused just to innovate. Operational efficiency will be a critical focus in the industry over the next 5 years. Some propose the integration of QC into manufacturing operations to streamline objectives and create a less siloed organization.

Miller: The industry requires an adequate level of pharmaceutical microbiology expertise to sustain a satisfactory level of microbiological control in manufacturing. Technologies to help achieve these goals are currently available but without the right microbiology capabilities and expertise to understand the science these same technologies will essentially be useless. Therefore, the industry must place a significantly greater emphasis on cultivating a strong microbiology culture (no pun intended!) that positively impacts the way we will manufacture safe and effective drugs in the years ahead.

Von Wintzengerode:'

• The impact of non-endotoxin pyrogens and microbial components on patient safety and product quality may lead to the expectation that Biotech Drug Substance manufacturing should be sterile.
• Data integrity (micro is manual!)