Pharma Manufacturing recently spoke with Jim Cannon, head of OEM and markets for Mettler Toledo Thornton, to get a better understanding of how this innovative bioburden testing technology is revolutionizing pharma manufacturing.
Q: What is bioburden and how is it consequential in pharma manufacturing?
A: Bioburden is a bit of an all-encompassing term. It refers to both the biofilm, which grows on the surfaces inside of pipes, manifolds, valves, and point of use, and to the planktonic, which are the floating organisms that come off the biofilm.
It is consequential because the gram-negative organisms can be pathogenic, and if they get into products such as API, IV solutions, or ophthalmic or nasal solutions, they can recover and contaminate them and cause serious detrimental health consequences for a patient.
And, unlike conductivity in total organic carbon (TOC), which could affect the quality or efficacy of a drug but are generally not considered fatal or associated with recalls, microbial contamination involving pathogenic organisms would have serious health consequences for anyone. So, the impact on pharma processing is enormous.
Q: Why are conventional methods for managing microbial contamination and pharma water falling short?
A: The traditional methods to maintain the microbial health of a water system are either sanitizing with chemicals, heat, or ozone, or keeping the water hot at all times. But if a system has not been sanitized properly, either because the duration or the heat or chemicals were not high enough, or you have areas where the organisms have been trapped behind a manifold, gasket, or dead leg, then the system is not up to speed.
The traditional monitoring method is to take a grab sample from a point of use and incubate those samples for five days to see if you’ve captured any organisms. At that point, you are trying to capture planktonic and enough viable organisms (meaning prepared to divide) out of a sample port, and then put them onto the right growth media and incubate it at the right temperature and the right length of time. There are a lot of “ifs” in this technique, which was first introduced in 1887.
The other challenge is controlling this and managing the microbial health of the water system. Because there are low salts in the water and no conductivity and organics of food source, these organisms protect themselves by slowing their metabolism and going into a state of almost hibernation or VBNC (viable but not culturable). That does not mean they will not culture, but they don’t culture in the normal five days in the normal nutrient media that are used. Some could take seven days or 21 days to multiply. And yet, at the same time, our pharma customers are monitoring conductivity in TOC real time but now we’re sending this water to production and they are hoping that in five days they get no microbial contamination showing up.
Q: How can implementing real time microbial monitoring help to improve process control?
A: By putting in an online system, they’ll still be doing their traditional plate count to meet compendial requirements. But by monitoring real time for their purified water or the water for injection (WFI), they are able to react to trends, because remember, when you take a grab sample, you are hoping that planktonic bacteria comes down that valve and is captured. Well, that’s pretty much of a big bet.
But by seeing this online, real time: Do I have a stable-state water system? Did my sanitization workproperly? Am I starting to get a trend where I could have an out-of-specification event? By seeing this trend, I can react immediately and not wait five days, because here’s the other challenge: If I culture a sample and in five days I show I have colonies, well, was that a sample error or was it a human contamination? So now if I go take another sample and wait another five days, that water has been used in API, ophthalmic, IV or other solutions and now I’m facing a possible recall.
Q: What makes the laser-induced fluorescence (LIF) technology used in the 7000RMS unique?
A: We are combining two known technologies. First of all, laser-induced fluorescence is the ability by using a laser of a specific wavelength (405 nm) to cause NADH and riboflavin metabolites in cells to get excited, and when they come back from the excitation state to a ground state, they emit a fluorescent light at a very specific emission spectrum which we detect.
In addition, we are using what is called Mie scattering. First introduced by a German physicist, the process says the angle of scatter off a particle (a bacteria is a physical particle) can also tell you the size of the particle.
We are combining the tech used in particle counters, common in the semiconductor industry, with fluorescence. Microbiologists have been able to make bacteria fluoresce forever. Bioluminescence, chemical luminescence, laser-induced fluorescence -— these are very well-known technologies. And these technologies, when combined, allow us to monitor in real time water flowing through our system.
Our system is connected to a pharma water system as an at-line measurement. In other words, it’s a sample stream just like your TOC instrumentation. We are flowing water at 30 ml/minute through our flow cell and we take 500,000 measurements per second. Then, we use algorithms to determine if it is an inert particle — particles from a gasket or a membrane, machining a new sample port orba filter — or if it is a microorganism. By doing this, we can determine whether the microbial health of the system is in a steady state.
We will also fluoresce bacteria that form spores when stressed, like bacillus subtilis. Even if you heated the water and think the bacteria should be dead, consider that bacteria have been found living in a volcanic vent at the bottom of the ocean and on plates outside the space station. Bacteria are ubiquitous — they’re everywhere — and you need to control this.
Given that the customer will see more data, it does not mean their system is out of control. It just means they have better, real-time data to put in place process controls to ensure they don’t have a microbial incident or OSS situation. So, by combining the laser-induced fluorescence and the Mie scattering, we are able to provide the customer a real-time data stream that shows the health of their water system.
Q: What are some other advantages of using the at-line analyzer?
A: One, you are bringing your microbial monitoring and bioburden detection up to the same analytical levels that our customers currently use for online conductivity, at-line TOC, and at-line ozone measurements. Those are compendial measurements, as is microbial. These are the tests required by the U.S. pharmacopoeia and others around the world. Every major pharmacopoeia requires conductivity, TOC, microbial, and endotoxin measurements of purified WFI. But until today, the microbial monitoring was done offline. You waited five days, seven days, 14 days, 21 days, but now at-line, real-time microbial monitoring is state of the art.
In addition, the FDA is pushing the process analytical technology (PAT) initiative it introduced a number of years ago. The EMA is pushing the same thing. PAT is the use of real-time analytical instrumentation so that you monitor and control the process real time and you can make changes and improvements real time.
Taking grab samples is the typical pharmaceutical QA/QC approach; you make a product, you take a sample, you test. But you cannot test quality into a product. You have to do it real time. The FDA is now taking the semiconductor approach, which relies heavily on real-time analytical instrumentation. PAT is part of their long-term plan and the FDA is urging the pharmacopeias to push more on endorsing real-time analytics instrumentation.
At a recent Parenteral Drug Association meeting, the FDA was very clear that the use of online microbial instrumentation as a process control tool was being strongly endorsed. They believe when used in conjunction with traditional methods for the time being (the grab sample plate-count method, the colony forming units), it gives you a better level of control. The FDA’s PAT initiative is being driven in the industry and you’re seeing it more in pharmacopeia regulations.
There are numerous major pharma companies that are early technology adopters, deploying online real-time microbial monitoring on their water systems to provide them better process control. And this is where the world is going. We are going to move away from a test from 1887 into real-time instrumentation.