Home / Our services / Biologics Testing / Biosafety testing / Microbiology testing

Microbiology testing

To ensure the safety of a drug product for patients’ health, production must meet the standard of Good Manufacturing Practices through environmental controls and adequate bioprocess designs with the goal of preserving product attributes. In addition, release of products involves the performance of tests – microbiology testing – to demonstrate the absence of microbiological infection.

Microbiology testing encompasses a variety of tests which aim to ensure the absence of microbial contaminants in the final Drug Product.

A basis of all biosafety testing programs, it is required by international medicine agencies at critical stages of biomanufacturing and carried out on starting material (cell line, media, etc.) that may carry contaminants.

Under the umbrella of microbiology testing, the detection of the following contaminants is covered:

Bacterial and fungal contaminants (Sterility tests, Bioburden)
Endotoxins (Pyrogenic substances)
Mycoplasma
Mycobacteria and other specific contaminants.

Clean Cells offers a full range of cGMP-qualified microbiology tests applicable for all biologics including rapid microbial detection techniques more fitted to time sensitive products such as cell-based therapies.

Mycoplasma:
let’s detect the invisible threat

Clean Cells was founded in 2000 on the idea that there is no way to be successful with biologics if they are contaminated by mycoplasma.

Mycoplasma at a glance

Mycoplasma are small bacteria, without cell walls, belonging to the Mollicutes family. They are insidious contaminants that are often present in cell culture but are not visible under microscope. They are true parasites which means they use the organism of hosts for their own needs. These microorganisms interfere with many biological activities including metabolism and functionality of eukaryotic cells. It is therefore necessary to regularly test for any mycoplasma contamination during Research & Development phases.

Moreover, they may infect humans, animals and plants. They are involved in many diseases such as respiratory or genital infections. It is therefore crucial to test for this infection all along the culture process of a drug product that will be injected in patients.

As mentioned, mycoplasma detection is a crucial part of microbiology testing and is mandatory. Due to their characteristics, mycoplasma are only detectable by specific assays which have to be compliant with EP 2.6.7 and USP 63. These specific assays are implemented for many years at Clean Cells and can be performed either in non GMP or GMP grades.

Mycoplasma testing

Classical compendial culture methods:

Direct culture in microaerophilic conditions for 28-day incubation period

Requires specific culture medium (liquid and solid) allowing the growth of different mycoplasma species (at least 2 strains: ie M.pneumoniae and M.orale)

Requires microaerophilic conditions: nitrogen containing 5-10 percent of carbon dioxide.

Requires important amount of samples : around 35 ml

28 day duration with subcultures

cc_photos9dp_200514_01_bacterio_9depack_DSCF0393-635x635px

Indirect culture using amplification on permissive cell lines with endpoint fluorescence

Cultivation in Vero cells for 7 days and staining with Hoechst (fluorescent intercaling DNA agent)

Requires 2 to 4 ml and the use of two different mycoplasma strains (M. Hyorhinis and M. orale)

State-of-the-art NAT based, for turnover and material amount reductions:

In-house PCR

This assay targeting specific DNA sequences was developed and is now available at Clean Cells for around 24 years. Our in-house kit is fully validated with all Mycoplasma strains – including M.pneumoniae and M.orale – required to fulfil the more recent recommendations of EP2.6.7 and in line with EP2.6.21 chapter and ICHQ2R2 guideline.

Our kit has demonstrated its ability to detect Mycoplasma contamination in hundreds of samples and matrices. Equipment and software are qualified and compliant to 21CFR part 11 chapter ensuring a suitable data traceability.

Small amounts of material (1ml) are required and experimental phase is reduced to 24 to 48 hours.

Biofire mycoplasma PCR test dedicated to short-life products

It may be crucial for cell therapy products to be released very quickly and close to the patient bed. To achieve this goal, Biofire test was developed by Biomérieux for Mycoplasma detection and presents the advantage of being very fast : only a couple of hours.

We have implemented and validated this assay with all required strains for a suitable comparison to classical methods. Thanks to its experience, Clean Cells is able to perform the matrix validation which is mandatory before any routine use.

Sterility and bioburden testing:
key go-no go checkpoints for treatment

Microbiology testing ensures that biologics are free of bacterial and fungal agents. A basis of all biosafety testing programs to ensure the safety of patients, it is required by international medicine agencies at critical stages of biomanufacturing and carried out on starting material (cell line, media, etc.) that may carry contaminants.

Probably the most common test of microbiology, a sterility test is mandatory for the release of any drug product. Thus, products must be demonstrated as sterile to meet specifications.

Clean Cells has developed a panel of methods and approaches to assess the microbiological status of a drug product and its intermediates of production. Clean Cells’ offer covers a full range of cGMP-qualified microbiology tests applicable for all biologics including rapid microbial detection techniques more fitted to time sensitive products such as cell based therapies.

Sterility and bioburden assays

Sterility test is based on the use of two nutritive broth media which allow the growth of anaerobic, aerobic bacteria and fungi.

Depending of the material to be tested for its absence of microbial contaminants, it may consist to different approaches such as inoculation or filtration, and final read-out can be either manual or automatic reading but in any case will use the property of the culture media to allow the growth of such contaminants.

As part of microbiology testing, Clean Cells offers R&D-grade or GMP-certified tests including:

Compendial assays compliant with international pharmacopoeia (EP2.6.1, USP71, JP4.06)
Rapid microbial detection with BACT/ALERT 3D system (applicable for cell based therapies -EP2.6.27)
Steritest® System for filtration-based sterility

Besides the sterility test, one finds bioburden testings:

Bioburden assay is based on the same principle but the aim is to enumerate bacteria and fungi that may be present in the products. Products will be filtered through a specific membrane which will be then dropped on solid nutritive culture media which allow the growth of present bacterial or fungal contaminants. Any colony forming units (cfu) will be then counted to determine the load of contamination in the product.

This assay is classically required for non-sterile products but can also be applied on intermediate stages of production to determine the number of microorganisms present in the product.

Clean Cells offer:

Compendial assays compliant with international pharmacopoeia (EP2.6.12, EP5.1.4)
R&D-grade or GMP-certified

As a complementary test if sterility test or bioburden test appear to reveal a contamination, Clean Cells offer a service of identification.

Endotoxins:

Among the tests to detect bacterial contaminants in a drug product that will be injected into patients, sterility test and endotoxin quantification are probably the most classical ones.

As defined by the National Institute of health (NIH), endotoxin refers to lipopolysaccharide (LPS) that constitutes the outer leaflet of the outer membrane of most Gram-negative bacteria such as E. Coli. The presence of LPS may reflect either a current contamination of the product or an infection that occurred during the production process although the bacteria is not alive anymore. Indeed, these endotoxins are toxins which can persist without viable microorganisms.

A key future of these toxins is that they may lead to fever (pyrogenic substances) and sepsis shock if they are injected into humans !

Consequently, endotoxin testing and quantification is a prerequisite to injected biopharmaceuticals intravenously and are expressed in IU (international units). The acceptable level of endotoxins in a drug product will be determined according to the posology of treatment taking into account the weight of the patient and the duration of treatment ie IU/Kg of patient/ 24 hours.

Hence a very precise quantification is necessary.

Endotoxin testing

The test for bacterial endotoxins (BET) is used to detect or quantify endotoxins from gram-negative bacteria using amoebocyte lysate from the horseshoe crab (Limulus polyphemus or Tachypleus tridentatus).

At Clean Cells, endotoxin quantification is based on a chromogenic kinetic method. To ensure the accuracy and the reliability of the test, a validation of the absence of interfering effects on the measurement due to the matrix is performed in presence of a known amount of endotoxins (Spike)

Rich of more than 20 years of experience performing this assay on many types of samples and formulation buffers, Clean Cells offers

Golden standard LAL detection assay
Fully GMP compliant 21 CFR part 11 compliant
Compliant with EP 2.6.14 and USP85

Antibodies and recombinant proteins-635x635px

Mycobacteria

Mycobacteria belong to a specific category of bacteria: acid-alcohol resistant bacteria.

According to WHO: Human tuberculosis is still the greatest single cause of human morbidity and mortality in many developing countries. Mycobacterium tuberculosis (Kock bacillus) is the most frequent cause of human tuberculosis.

As a consequence, a detection of the potential presence of Mycobacteria spp is therefore required for the biosafety release of cell substrates used for the production of vaccines for human use (see EP chapter 5.2.3) and human vaccines (see chapter 2.6.16).

Our mycobacteria detection test panel includes:

Classical compendial culture method, according to to EP chapter 2.6.2:

Requires specific culture medium (liquid and solid) allowing the growth of Mycobacteria species (at least one strain)

Requires a 56-day culture period at 37°C.

State-of-the-art NAT based, for turnover reduction:

In-house PCR

This assay was developed at Clean Cells and is available at Clean Cells in non GMP and GMP format. Our detection test is fully validated to fulfil the regulatory expectations, including EP chapter 2.6.21 and ICHQ2R2 guideline.

Equipment and software are qualified and compliant to 21 CFR part 11 chapter ensuring a suitable data traceability.

Leaflets

Leaflet

Mycoplasma Leaflet

Download via media library

Get in touch with our team to know more about this test or for any of your microbiology testing needs.

"*" indicates required fields