Microbial QC Testing in the Manufacture of Biopharmaceuticals

by | Nov 18, 2021 | Biologics Manufacturing

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Contamination control of bioprocessing cell lines is essential. The same environments that encourage desirable microbiological growth also encourage growth of undesirable contaminants including yeasts, molds, mycoplasma, bacteria, and viruses.

Bioprocessing Overview

Bioprocessing can be broadly defined as any process using live cells or even cellular components to manufacture end products for various industries. These products might include new vaccines, antibiotics, biopharmaceuticals, and renewable biofuels. The industry began to emerge in the 1940s with the introduction of the novel polio vaccine and is currently undergoing an explosive growth in new fields, such as the production of stem cell and precision genetic therapies.

The biomanufacturing industry is highly regulated to ensure drug safety and quality; however incidents such as the Genzyme Incident still occur. An entire manufacturing plant in Massachusetts was shut down for several months due to a viral contamination introduced through a cell culture nutrient.

A recent study reviewed 20 incidents of viral contamination of gene cell therapy cell lines and the results indicated that significant delays in production were attributed to delays in getting correct quality control data.

Bioprocessing workflow
Figure 1. Bioprocessing Workflow

Typical Bioprocessing Workflow

A typical bioprocessing workflow consists of three stages (Figure 1). In the upstream flow, formulation and hydration of the cell line leads to culture in a bioreactor. Once sufficient growth has occurred the specific cells are harvested, and sent downstream, where they are separated and purified. Afterwards the appropriate sterile containers are filled for bulk storage and eventually filled in the final customer specific container.

Microbial Contaminants in Biopharmaceutical Manufacturing

Microbial contamination sources have numerous entry paths and introduce extensive product variability due to degradation of product as well as changes in purity levels. These entry points are the key locations where contamination control or quality control should simultaneously occur.  Many of these contaminant sources are human in origin.

Typical contaminants include bacteria (such as Escherichia coli, Staphylococcus spp., and Streptococcus spp.), viruses, and mycoplasma. Each of these contaminants presents a detection and ultimately, a mitigation strategy. Products with short viability and shelf lives are most severely affected. Cell and gene therapies may only have usable lives of hours to a few days, and prolonged product release cycle time can increase holding/inventory costs and limit manufacturing throughput.

Traditional growth-based microbial testing methods are labor intensive and time consuming. Microbial culturing requires considerable time to allow sufficient growth of bacteria. A huge lag time exists between sampling and reporting of results. Among the worst cases, culture-based detection of contamination can take up to two weeks.

Traditional microbial tests typically require more than a dozen manual steps before results are available. Microbial growth is limited by the growth medium used and incubation conditions, thus impacting testing sensitivity, accuracy, and reproducibility. Even more rapid methods such as electrophoresis and flow cytometry require incubation, detection, and complex cell counting algorithms.

Viruses are also a significant concern to maintaining contamination free bioprocessing. Different strains of viruses have very different structures which make them harder to detect with conventional culture methods. This has serious consequences to manufacturing and even more problematic, consumers if not identified in time. A recent study reported examples of viral contaminations between 1985 – 2020 in protein and vaccine manufacturing cell lines. These included herpes, parainfluenza, and human adenovirus. The use of qPCR is increasing in popularity as a rapid screening tool to detect predefined targets but typically requires sending samples out to reference labs.

Mycoplasmas are a class of cell wall-less bacteria and are serious contaminants in bioprocessing. The most common cases of mycoplasma contamination have involved the species such as M. orale, M. hyorhinis, and A. laidlawii.

Like bacteria, the presence of mycoplasma is typically tested for by cell culture. However, cell culture requires up to 35 days for this class of bacteria, which can cause even greater loss of product in manufacturing than bacterial contamination.

Commercial PCR is available to detect certain species of mycoplasma. However due to the broad range of mycoplasma species, multiplexed detection of conserved sequences is highly desirable to capture as wide a range as possible.

Bioprocessing Quality Control Methods

Quality Control Using Conventional Cell Culture

The most common form of regulated quality control (QC) for bioprocessing is culturing cells to quantitatively determine microbial contents and limits, and to positively identify a specific microorganism. Bioburdens of contaminants are typically calculated using cell culture methods. There are numerous drawbacks to cell culture techniques, mainly the length of time to enrich and grow sufficient cell colonies for positive identification. A lengthy time to answer delays product release.

Cell culture techniques require that samples be prepared on site with extensive, specialized facilities and trained personnel or are sent to an off-site reference lab which can incur additional time penalties as well as high cost. These QC methods are also highly regulated and require extensive time and costs to validate and verify a new testing methodology.

Extensive resources are required to make changes to QC tests, and these tests are not easily customizable. Inventory requirements can be substantial to maintain a full microbiology lab that must accommodate increasingly specialized testing.

Rapid Testing by Real-time PCR

Introduction of new rapid testing methods is increasing popular in bioprocessing. These methods significantly reduce the time to result compared to conventional techniques. Screening for the presence or absence of targeted species allows biopharmaceutical manufacturers a faster way of making decisions, and provides for rapid product release, improved process control, and lowered inventory at all processing stages. Traditional test methods can then be used to confirm the prescreening results.

Real-time PCR (qPCR) is recognized as one of several rapid microbial detection technologies due to the availability of improved automation, ease of use, and high sensitivity and specificity (Table 1). Further, regulatory agencies and corporate initiatives encourage the use of rapid methods for faster decision making and to prevent loss of product. Table 1 shows examples of emerging rapid and novel techniques for microbial detection.

novel and rapid microbiological technologies for microbial detection
Table 1. Novel and rapid microbiological technologies for microbial detection.


Controlling microbial contamination is critical to ensuring drug safety and quality. Bioprocessed products, particularly injectable biopharmaceuticals require the utmost sterility as microbial contamination can lead to serious issues with product yield, safety, and efficacy resulting in loss of time and money as well as patient morbidity and worst-case mortality.

Routine microbial contamination monitoring of in-process samples is essential. However, conventional QC relies on cultured microbial growth, with a time-to-result measured in days or weeks. This not only slows production but allows microbial contaminants time to spread and establish themselves before action is taken. There is a need for more rapid detection methods for microbial contamination monitoring.

<a href="https://lexagene.com/author/dianestewart/" target="_self">Dr Diane Stewart</a>

Dr Diane Stewart

Diane Stewart has a PhD in chemistry from Brown University, and has had a lengthy career as scientist and manager for numerous scientific instrumentation products including molecular diagnostics instrumentation, nanotechnology, and ion microscopy. Currently she is the product manager for the MiQLab point-of-care system at LexaGene. She has extensive experience in federal grant writing, negotiating and administration. In addition to program and product management, she is skilled with in-house legal duties have included drafting invention disclosures, tracking patent office actions, patent searching, patent landscape analysis, leading intellectual property review boards, and negotiating legal documentation for collaborations.

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