Microbiological Contaminants
Meeting your Microbiology Testing Needs
During the production of biopharmaceuticals, contaminating microorganisms can enter the process at many stages. Once introduced, bacteria and fungi can replicate exponentially, severely compromising the final product. In addition, the use of mammalian cell lines for production introduces the potential of mycoplasma and mycobacterium contamination.
The presence of microorganisms in vaccines and other biopharmaceuticals can, over time, both affect the product and pose a major risk to patient health. As such, regulatory bodies, including the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA), have stringent tests in place to ensure contamination has not occurred. This is determined through microbiology testing of representative samples of a batch to identify the presence of any viable microorganisms in the material. Proof that your product is free from microbial contaminants is part of the documentation required for the release of biopharmaceuticals.
Sartorius provides a comprehensive package of microbiology biosafety assays to cover your microbiological contamination testing requirements including:
- Sterility by direct inoculation
- Microbial enumeration of non-sterile products by plate method
- Mycoplasma culture assay
- Mycoplasma by PCR
- Detection of Mycobacterium spp.
Detection of endotoxin using the Pyros® Kinetix® Flex
Our experts design a microbiology testing plan around your unique drug development or commercial product needs
Ready-to-use, quality-assured microbial test methods, with qualification or validation of your product matrix in the assay, reduces testing timelines
PCR-based methods accelerate manufacturing; compendial testing is carried out in parallel to meet your drug development or commercial lot release needs
Create Your Microbiology Testing Strategy
Sampled from | Sterility Testing | Microbial Enumeration Testing | Mycoplasma Testing (Compendial) | Mycoplasma Testing by PCR | Mycobacterium Testing | Endotoxin Testing |
|---|---|---|---|---|---|---|
Research Cell Bank | X | - | - | X | X* | - |
Master Cell Bank | X | - | X | - | X* | - |
Working Cell Bank | X | - | X | - | X* | - |
End of Production Cell Bank | X | - | X | - | X* | - |
Master Virus Seed Stocks | X | - | X | (X) | X* | - |
Working Virus Seed Stocks | X | - | X | (X) | X* | - |
Unpurified Bulk Harvest | - | X | X | (X) | X* | - |
Drug Substance | X | - | - | - | - | - |
Drug Product | X | - | - | - | - | X |
X* Mycobacterium testing only required for human or primate cell lines and products derived from human or primate cell lines.
(X) Other risk-based approaches to mycoplasma testing may be employed when there is an inability to neutralize the sample virus. This should be discussed with your regulator.
Sterility Testing
Sterility testing must be performed and demonstrated throughout the production process of a biologic or viral vaccine.
Sartorius offers sterility tests by direct inoculation and by membrane filtration to GMP regulations and in compliance with the European Pharmacopoeia (EP), Japanese Pharmacopoeia (JP) and the Unites States Pharmacopoeia (USP) standards.
Prior to testing, the sample matrix should be qualified in the assay to examine any inhibitory effects; this is achieved by spiking the test material with representative test organisms at low concentrations and observing acceptable growth.
The following organisms are suitable as model organisms:
Aerobic bacteria
- Staphylococcus aureus
- Bacillus subtilis
- Pseudomonas aeruginosa
Anaerobic bacterium
- Clostridium sporogenes
Fungi
- Candida albicans
- Aspergillus brasiliensis
In the direct sterility test, the sample is inoculated into two different growth media: fluid thioglycolate and soybean casein digest. For the membrane filtration test, the sample is filtered through 0.45 µM filters which are then inoculated into Trypticase soya broth (TSB) and fluid thioglycolate medium. In both methods, the test is incubated for 14 days. Sartorius can report the results of the assay with a certificate of analysis within 5 days of the final observations.
It should be noted that these sterility tests are not applicable for articles that are not intended to be sterile; a bioburden test should be used in this case.
Sartorius can advise on the appropriate testing for your material, as well as sample number and volumes.
Microbial Enumeration
The purpose of a bioburden test is to enumerate mesophilic bacteria and fungi that may grow under aerobic conditions. The test is designed primarily to determine whether or not a substance or preparation complies with an established specification for microbiological quality. It is similar to sterility testing but performed where the sample is not required to be sterile, as it will undergo further processing. Therefore, this test is generally used for raw materials and in-process samples, such as bulk harvests, where the customer has set acceptable limits of bioburden for their internal processes. (Sterility testing can also be performed on bulk harvest samples that are expected to be sterile. It is at the developer’s discretion whether to use a bioburden or sterility test.)
Two methods are available for the bioburden test — plate or membrane filtration. Both are acceptable to regulators and may be used at the developer’s discretion. The plate method is more typical for processing a large sample volume. If certain inhibitory effects are observed, such as from antibiotics, then membrane filtration may be used.
In the plate-count method, the test material is diluted 10-fold in buffered water before predefined volumes are spread onto 2x TSA plates and 2x SDA plates. These are incubated, and the average CFU/mL is recorded.
Sartorius can advise on the appropriate tests and testing methods for your material.
Mycoplasma Testing
Mycoplasma detection and removal is critical for biologics produced for clinical studies and licensed products for human use. The testing is complicated, as the family of microbes has many different and often fastidious requirements for growth. Sartorius offers mycoplasma testing services that comply with cGMP standards and to the current International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH), European Pharmacopoeia (EP) and United States Pharmacopoeia (USP) guidelines.
There are two traditional components to mycoplasma testing described in the EP — the agar and broth method and the in vitro indicator cell culture method. The sample matrix to be tested should be qualified in the assay prior to testing; qualification involves spiking the test material with test organisms at low concentrations and demonstrating detection.
The following organisms are suitable as test organisms:
- A. laidlawii
- M. gallisepticum
- M. fermentans
- M. hyorhinis
- M. orale
- M. pneumoniae
Agar and Broth
For the agar and broth method, the sample is inoculated into different liquid and solid media types, which are subsequently incubated in different environmental conditions (aerobic and anaerobic). During the incubation period, samples from the inoculated broth are also taken and inoculated onto the agar growth media. Detection of colonies indicates a positive test; the entire test lasts for 28 days.
In vitro Indicator Cell Culture
The in vitro indicator cell culture method requires that the sample be inoculated onto a suitable cell substrate (Mouse 3T3 or Vero cells), cultured and stained with a fluorescent dye that binds to DNA. The cultures are observed by light microscopy to identify fluorescent spots, or granules, surrounding the nucleus of the indicator cells, which indicates the presence of mycoplasmas. Sartorius is able to report the results of the assay with a certificate of analysis within 5 days of the final observations.
Rapid Mycoplasma Polymerase Chain Reaction (PCR)
In addition to the agar and broth methods, the European Pharmacopoeia now accepts a PCR-based mycoplasma test, following appropriate validation. The EP joins the US Food and Drug Administration (FDA), which has accepted the use of PCR-based mycoplasma detection for release of a biopharmaceutical product since 2012. With this test, manufacturers can replace the existing methods, which can last up to 28 days, with a rapid technique. Mycoplasma PCR testing can be performed at Sartorius and a result reported in 48 hours from receipt of sample.
Mycobacterium Testing
This assay is performed when testing human or primate cell lines, which are susceptible to Mycobacterium spp.
The sample is inoculated onto two types of selective agar media and one type of broth medium. The media is cultured for 56 days at 36 to 38oC and are observed for the presence of mycobacteria.
In order to qualify the test, a spiked control is included, where the test material is challenged with the positive control Mycobacterium tuberculosis to test for inhibition of mycobacteria growth.
This method complies with European Pharmacopoeia section 2.6.2.
Endotoxin Testing
Endotoxin testing is typically performed on final drug product only. Endotoxins are part of the outer membrane of the cell wall of gram-negative bacteria, which can potentially contaminate cell cultures during production of biopharmaceuticals or contaminate final therapeutic products.
The turbidimetric test is performed by adding limulus amebocyte lysate (LAL) — an aqueous extract of blood cells (amoebocytes) from the Atlantic horseshoe crab — to the liquid sample or standard endotoxin dilution. Then the turbidity of the reaction mixture is measured. At higher endotoxin concentrations, clotting enzyme is activated more rapidly, so the sample becomes more turbid. As a kinetic assay, the time for each sample to reach a particular optical density (OD) is the critical measure.
The Pyros® Kinetix® Flex and Pyros® eXpress software have been validated to perform turbidimetric kinetic analysis. Sample onset times are referenced against a standard curve to quantify sample endotoxin concentration.
The kinetic endotoxin assay performed at Sartorius follows harmonized USP <85> Bacterial Endotoxins and EP 2.6.14 Bacterial Endotoxins.
As per regulations, sample matrix qualification for inhibition and enhancement is required prior to testing a new sample matrix.
