The Havoc of Non-Existent Biofilms

A possibility of biofilm can derail bioburden investigations. Is biofilm really as common on our equipment as we think?

Biofilm is a nightmare. Microbes latch to your equipment and become near impossible to remove. They can hide indefinitely, showing intermittent signs when they happen to flake off and pop up in random test samples. Any organism is welcome to the party, so the different organisms you recover can throw off your investigation. My colleague Jordi Iglesias gave a great webinar on using rapid Endotoxin tests to monitor for biofilm.

But this isn’t a story about biofilms. This is the story about how the possibility of biofilm weighs heavy on any contamination investigation. They can drive you crazy questioning your process. Is your cleaning strong enough to remove the film? What if just a little bit remains and slowly grows back? Can pieces float away to colonize spots all over our system? I’ve ran through those questions too many times for some critical manufacturing equipment. Luckily, I never¹ ran into a biofilm as a root cause.

Biofilm? A case study

I recently presented a microbial contamination case study at the 2022 Informa Compounding Pharmacy Compliance Event. I discussed the manufacturing process pictured below. My former company received frozen raw material, funneled it into thawing vessels, then pumped it to a pooling tank. We tested the pooled material. It was filled with Bacillus , most prominently Bacillus cereus .

After months of investigating, we determined the CIP cycles were missing a 2-meter section of product contact piping, allowing Bacillus to proliferate in our growth supporting material between runs. The section is circled with the dotted red line here:

The important question for this post: How did our thoughts about biofilm evolve during the investigation? The equipment was cleaned after each run. The investigation team collected rinse samples after cleaning to see if Bacillus was still present in the system. Those samples always looked great because the rinse cycles didn’t contact that 2-meter section.

Our team struggled to find the Bacillus source. Since Bacillus forms spores and biofilms, we thought low counts were hiding in a biofilm and resisting our cleaning. During the investigation, we had a contract cleaning service fill the line with peracetic acid for 24 hours to kill off the spores. Batches made after this sporicidal treatment looked great – further solidifying our assumption of a biofilm resistant to our routine cleaning cycle.

It’s important to note: the sporicidal cleaning worked because the contractor manually opened all valves, not because of the extra sanitizing action of the peracetic acid. This cleaning solution hit all product contact surfaces while the routine cleanings were stuck with the pre-programmed valve cycles. When we simply corrected the valve programming for the routine cleaning cycle, we never saw Bacillus again.

Biofilm proliferation . . . by word-of-mouth

We discovered the cleaning cycle deficiency with an investigative rinse sample that mimicked the product pathway. It seems simple now, but we easily could have completed this investigation without that sample.

Had we not collected that sample, the investigation would have incorrectly attributed the root cause to biofilm with spore-forming organisms. Corrective Action: We continue using the contracted cleaning service before each run. Since that process manually opened all valves, it would have worked. It cost $100k and delayed manufacturing a full day between lots. We thought we were lucky the product had great margins!

With this outcome, dozens of industry professionals would have their own story to tell about biofilm wreaking havoc. They’d tell the wrong story.

Around 20 individuals, including engineers, validations groups, quality personnel, and microbiologists, were intimately involved in this investigation. Most were at the manager, director, or global/corporate level. They all reported to their managers on the investigation findings. If a suspected biofilm showed up in their future careers, they’d have this event to refer to. Maybe they jump to a biofilm conclusion and don’t find a much simpler root cause. They’d relay the tale about biofilm’s resistance to routine cleanings, further solidifying its’ reputation in the industry. They could even suggest a similar expensive cleaning. For our site, that would have led to catastrophic long-term cleaning costs.

Can you see how biofilm is dangerous in the way it impacts our investigations? It doesn’t even need to exist at a site to cause problems. With my experience working in similar teams, I’m convinced bio burden problems in the industry have been attributed to biofilms before a true root cause was detected. Don’t let that happen to you!

1. Please reach out to me about my one “exception” and other nuances!