Bioburden detection is one of the quality control measures required to ensure drug safety – and a possible bottleneck for product release.
Most of the challenges of drug development are ticked off the list once the product has been launched. Others stay relevant throughout the product’s lifetime – for example, bioburden detection. Remarkably, bioburden detection is not only required for the final product but also for raw materials and process intermediates.
Over the course of the production run, delays related to bioburden detection add up, creating a bottleneck both for patients waiting for their therapy and the pharma company to deliver to the market.
Bioburden can be defined as the number of microorganisms (bacteria, yeast, and mold) on a surface or in a solution. To ensure a safe product, the detection of bioburden is a mandatory, tightly regulated quality control step in pharmaceutical manufacturing. Membrane filtration has evolved as a preferred method because it is robust, economical, and easy to use.
As a first step, drug solutions are filtered to retain the microorganisms without affecting the drug. The choice of filter materials and pore size can already have a considerable impact both on efficient collection of the bacteria to obtain a representative sample, the removal of growth inhibitors, and the quality of the resulting product.
Which bugs to look for?
Bacteria immobilized on the filter are placed on a petri dish containing growth media adjusted to the expected microorganism species. These are often suggested by the regulatory bodies.
“The relevant chapters of the European and US Pharmacopeia list microorganisms that are to be used for method and culture validation, representing gram-positive and gram-negative bacteria, yeast, and mold,” Stéphanie Huck, WEU Application Services Scientist and Industrial Microbiology Scientific Liaison at Merck* told me in an interview.
“However, these are not necessarily the microorganisms that reflect the situation at the customer’s site, so it is often seen as a plus by the regulatory body to look for and validate against in-house strains that are more likely to be found in the product,“ she added.
Qualitative analyses using commonly available phenotypic or genotypic identification methods, including PCR or mass spectrometry, help identify suitable species and strains.
Waiting… and waiting… for colonies to grow
Bioburden detection is applied both in sterile and non-sterile manufacturing processes. Some products (e.g. injectables) must be sterile. For other, non-sterile pharmaceutical products and medical devices, certain thresholds exist that must not be exceeded. The actual values vary and depend on the product and its intended use, and the microorganisms in question.
Once colonies appear, they are counted manually and evaluated against the allowed thresholds. Only after passing bioburden quality control, products are released as an end product, or for sterile filling. In the meantime – usually, for three to seven days – production lines standstill, and products sit in the warehouse.
Speeding up bioburden detection and product release
Merck has developed the Milliflex® system to speed up bioburden detection. The Milliflex Oasis® pump and membrane filtration device ensure an efficient collection of microorganisms and removal of growth inhibitors. Larger sample volumes can be processed through the Milliflex Oasis® funnel.
After filtration and incubation, a reagent substrate provided in the Milliflex® Quantum Universal kit is applied to the membrane. If viable microorganisms are present, cellular enzymes cleave the substrate to release a fluorescent dye that accumulates inside the cells, amplifying the signal.
Inside the Milliflex® Quantum reader, the cells are exposed to the excitation wavelength of the dye and can be counted well before colonies become visible to the naked eye. The result is a process similar to the standard method but is shortened to half or even one-third of the time.
In contrast to many other rapid bioburden test systems, the Milliflex® Quantum system is non-destructive, so microorganisms stay viable. If unknown types or unexpectedly high levels of contamination occur, the filter can be placed on fresh agar media for reincubation.
The resulting cultured material is suitable for any standard identification tests, including biochemical, morphological, or nucleic acid analyses. Microbial ID data help to identify the root cause of the contamination and to set up a corrective/preventive action (CAPA) plan.
Switch to speed – or speedy from scratch
However, as in any highly regulated environment, establishing a new method is not always straightforward. Merck provides support with setup and regulatory documentation, on-site training of lab personnel, consulting, and help with result interpretation.
“For customers starting from scratch, or for new samples with difficult properties, we optimize filtration conditions in our labs as part of our service offering,” Huck explains. “Sometimes a different filter does the trick, for example switching from cellulose to PVDF materials. Sometimes you need to dilute the sample or use additives to reduce the viscosity. There is no one size fits all, but we usually find a solution.”
Bioburden detection is here to stay
Drug safety will remain an essential topic for years to come, and microorganisms are unlikely to disappear. So bioburden detection is here to stay, fostering trends to faster methods with higher throughput – on behalf of both patients and industry needs.
Interested to learn more about our pharmaceutical analysis & QC portfolio? More information here.
*The life science business of Merck operates as MilliporeSigma in the U.S and Canada.
Images via Shutterstock.com and Merck
Author: Dr. Ute Boronowsky, Freelance science writer and member of Science Inbound