Biosafety

Sterility can be defined as the freedom from the presence of viable microorganisms.

Sterility testing of sterile pharmaceuticals is an important part of GMP microbiology, and is used to ensure that pharmaceutical and biopharmaceutical therapeutics are actually sterile and safe for human use.

Classic sterility tests may not be suitable for all products due to 14 days of incubation time.

Rapid methods are more suitable for cell-based products (e.g. ex vivo genetically modified cells administered fresh or with limited hold time between final formulation and patient administration) considering the advantage of shortening the release timelines for patients with no alternative therapeutic solutions.

Testing performed:

• Classic sterility test (EP 2.6.1/USP <71>)
• Rapid sterility test (EP 2.6.27)

Parenteral administered pharmaceutical products must be free of pyrogenic (fever-inducing) contamination as these substances may induce life-threatening systemic response of the patient’s innate immune system. A sterile drug product could be pyrogenic therefore dedicate testing is needed to evaluate this endpoint.

The most common in vitro tests for detection of pyrogenicity is known as Bacterial endotoxin Test (BET) / Limulus Amebocyte Lysate Assay (LAL) since endotoxin, in particular from gram gram-negative bacteria, are the most common cause of toxic reactions resulting from contamination with pyrogens.

However Non-Endotoxin Pyrogens (NEPs) are undetectable by the bacterial endotoxin test, and there is therefore a risk of overlooking a NEP contamination.

In recent years an alternative in vitro pyrogen test, the Monocyte Activation Test (MAT), has been developed to detect and quantify endotoxin and NEP contaminations.

Various combinations of proteins and/or formulation components that compose finished sterile biological drug products can produce low endotoxin recovery (LER), generating the failure to detect spiked endotoxin in some finished sterile biological drug products using BET. Parenteral Drug Association (PDA) issued Technical Report n. 82 about LER presenting guidelines for developing LER hold-time study design and providing strategies for mitigation of LER. LER studies have been requested from 2013 by FDA at BLA (Biologics License Application) submission.

Testing performed:

• Bacterial Endotoxin Test (2.6.14/USP <85>/JP); Method D (EP): Chromogenic kinetic (quantitative method)
• Method A (EP): Gel-clot method (limit test)Low Endotoxin Recovery test
• Monocyte Activation Test EP 2.6.30, Method A (quantitative method)

Mycoplasma species are bacteria that can infect cell cultures.

Mycoplasma contamination of cell lines used to produce biopharmaceutical products can disrupt cellular growth and metabolism and lead to changes in gene expression, which results in decreased product quantity and quality posing a potential threat to patient.
For these reasons, worldwide regulatory agencies require that biotechnological products produced in cell substrates be tested to ensure the absence of mycoplasma contamination.

The conventional mycoplasma tests, according to EP 2.6.7 and USP <63>, are culture method and the indicator cell culture method. Based on different products, one single method or both are necessary.

Currently several nucleic acid amplification technique (NAT) based methods are available to reduce the turnaround time drastically, especially for biopharmaceutical products that have short half-lives.

NAT may be used for detection of mycoplasmas by amplification of nucleic acids extracted from a test sample with specific primers that reveal the presence of the target nucleic acid. NAT indicate the presence of a particular nucleic acid sequence and not necessarily the presence of viable mycoplasmas.

Testing performed:

• Culture method
• Indicator cell culture method
• NAT method: Real Time PCR
• NAT method: PCR on agarose gel

Adventitious agents are defined by the World Health Organization (WHO) as microorganisms that may have been unintentionally introduced into the manufacturing process of a biological medicinal product. Among wide range of adventitious agents, viruses are one the most common source of contamination.

Adventitious virus assays are performed as part of raw materials testing, cell-line characterization, and lot-release testing of biologicals such as monoclonal antibodies, gene therapy vectors, recombinant proteins, and vaccines.

Different tests can be performed to detect adventitious viruses based on the specific purpose of the assay and the technique that can be applied.

Mainly adventitious viruses can be detected through:

• PCR technique, both qualitative and quantitative
• cell-based assays.

Testing performed:

• Quali/Quantitative PCR Testing to detect adventitious and endogenous viruses
  • human viruses
  • respiratory viruses
  • porcine viruses
  • bovine viruses
  • other specific target PCRs.
• Retrovirus testing assays
• Cell-based assays: cytopathic effect, Hemagglutination assay (HA-test), Hemadsorption assay (HAD-test), Plaque assay, Immuno-fluorescence
• Staining Assay (IFA)

Microbial identification is a characterization of a certain microorganism through an appropriate test method able to return the name of the analyzed species.

It is an essential tool for pharmaceutical companies that need to identify the sources and characteristics of microbial contaminations in products, environment, water, etc.

DNA sequencing is considered the state-of-the-art identification technique that provides accurate results and which is/had rapidly substituting biochemical methods in the pharmaceutical industry, driven also by the increasing requirements of pharmaceutical companies and competent authorities.

Microbial identification cover bacteria, yeasts and fungi.

Testing performed:

• Bacteria Identification (Comparative near-full gene sequencing analysis of 16S rDNA, MicroSeq 500 16S rDNA PCR and sequencing kit)
• Fungi and yeasts identification (MicroSeq D2LSU PCR and sequencing kit)

Sequencing of DNA and RNA by Sanger method in order to evaluate genetic stability of samples, with the possibility to start from PCR products, sample DNA or MCB/WCB

Residual DNA, or Host Cell DNA, can be a contaminant present in biologicals that are co-purified with drug substances.Residual DNA testing ensures drug’s final dosage form meets the requirements as established by regulatory agencies.

Testing performed:

• Detection of genomic DNA from multiple species (E. coli, Mouse, CHO cells, Yeast (Pichia), Canine and Vero cells)
• Detection of human genomic DNA

EP 2.7.5. Assay of heparin

Microbial enumeration tests (EP 2.6.12)

Test for specified micro-organisms (EP 2.6.13)