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Executive summary
The Therapeutic Goods Administration (TGA) has conducted a comprehensive evaluation of the residual DNA and endotoxin levels in the two mRNA vaccines supplied in Australia - Comirnaty (Pfizer) and Spikevax (Moderna). This report provides scientific evidence confirming that residual DNA and endotoxin levels meet internationally agreed limits.
The TGA's batch release process for COVID-19 vaccines, which involves thorough review and independent laboratory testing, ensures the quality and safety of vaccines before they reach the public. As of 24 October 2024, 212 batches of Comirnaty and Spikevax have been released by the TGA Laboratories. The report includes the results of independent testing by the TGA Laboratories for residual DNA and endotoxins.
Key findings include:
- Residual DNA Testing: The TGA Laboratories tested 28 batches of mRNA vaccines and confirmed that all batches complied with the World Health Organization recommended limit of up to 10 ng per dose. This confirmed the manufacturing process removes almost all DNA starting material and the vaccines are within the safe DNA residual limit. The quantitative PCR (qPCR) method was validated to ensure accuracy and reliability.
- Endotoxin Testing: Every batch of both mRNA vaccines released in Australia was tested for bacterial endotoxins using a validated Limulus amebocyte lysate (LAL) assay. Endotoxin was tested to a level of 5 Endotoxin Units (EU) per mL and all batches were below this level.
- The report concludes that the current mRNA manufacturing processes, as evaluated and approved by the TGA, effectively control levels of residual DNA and bacterial endotoxins to a safe level.
Background
The World Health Organization (WHO) considers batch release as a critical process in ensuring the quality and safety of vaccines[1]. This process involves the evaluation of each individual batch (or lot) of a licensed vaccine before it is released onto the market. This process is typically carried out by National Control Laboratories (NCLs). The evaluation includes a thorough review of the manufacturer’s production data and quality control test results. According to the WHO guidelines, testing is an additional component.
The purpose of batch release is to confirm each batch meets the approved release specifications before it reaches the public. In Australia, batch release of all vaccines is conducted by the TGA Laboratories, which is Australia’s National Control Laboratory for therapeutic goods[2]. The TGA has approved two mRNA vaccines for Severe Acute Respiratory Syndrome Coronavirus 2, (SARS-CoV-2) in Australia. These two vaccines, Comirnaty (Pfizer) and Spikevax (Moderna), constitute the majority of SARS-CoV-2 virus vaccines currently supplied in Australia. Testing was established for these products to ensure critical quality attributes were independently verified by the TGA.
As of 24 October 2024, 212 batches of Comirnaty (154 batches) and Spikevax (58 batches) have been batch released in Australia by the TGA Laboratories. Summary results for all batches are published on the TGA website.
This report contains the more detailed results of the TGA Laboratories independent testing of the two mRNA vaccines for two manufacturing impurities.
- Residual DNA testing may be conducted by the manufacturer prior to final formulation. The TGA Laboratories has reviewed residual DNA levels in the manufacturer’s batch release information or through the evaluation process to ensure it complies with approved specifications. To date, the TGA has also tested 28 batches to confirm the accuracy of the batch data submitted by the manufacturers.
- Endotoxin testing has been included in the batch release testing of all batches of both mRNA vaccines released to date.
Residual DNA
Monitoring for Residual DNA content
Residual DNA may be an impurity in biological medicines that could be present in finished products. It was first encountered as an impurity originating from mammalian cell culture lines used to produce recombinant proteins. It is made up of DNA fragments originating from the host cell used to produce biotechnology medicines or, in the case of mRNA products, the template plasmid used to make the mRNA. Manufacturing controls, such as breakdown through enzymatic DNA digestion and then purification, have been developed and optimised by manufacturers to remove DNA from these preparations. This is evaluated by the TGA prior to marketing authorisation. Safe residual DNA limits, of up to 10 ng per dose, were established for these products to ensure extremely high safety margins[3][4].
Testing for residual DNA may be conducted by the manufacturer at various stages in the manufacture of vaccines and other biological medicines. Acceptable results for these tests provide an assurance that the manufacturing process removes almost all DNA starting material, such that residual levels are below those internationally accepted by the scientific community.
Following the establishment of a manufacturing process which consistently produces comparable commercial batches, with residual DNA levels demonstrated to be below international standard levels, the requirement for residual DNA testing may be safely removed from the release testing process. Consideration of removal of the residual DNA testing should be in accordance with the principles of risk-based regulation. Testing should only be removed if the regulatory authority has assessed the data submitted and there is appropriate justification provided.
Although the test for residual DNA might not be included in the batch release process, routine monitoring may still occur. The TGA Laboratories have independently tested this manufacturing impurity as an additional check. This testing is intended to monitor the effectiveness of the purification process to remove residual DNA.
Residual DNA testing
The residual DNA measurements performed by the TGA Laboratories conform to the standard test procedures for residual DNA quantitation available in the European Pharmacopoeia monograph 2.6.35, Quantification and Characterisation of Residual Host-Cell DNA. The test method included in this monograph is quantitative PCR (qPCR). In this test, vaccine samples are compared with standard curves of reference material (plasmid DNA) prepared in a series of known concentrations. The amplification of test samples is compared with that of the standard curve materials to quantitate the amount of residual DNA present in the vaccine, as shown in figures 1 and 2 below. Use of a standard curve and comparison with a measurement from a test sample is a standard method for quantifying substances. The amount of amplification is monitored by a fluorescent reporter, which may either be a dye-labelled DNA probe specific for the gene sequence of interest, or a double stranded DNA-binding fluorescent dye. In the latter case, any background fluorescence arising from RNA is a constant amount that is subtracted from fluorescence measurements in the later cycles during which the exponential amplification curve is observed. Background fluorescence is measured separately for each reaction over the cycles between the green and red markers in the plot below. The cycle at which reaction fluorescence exceeds a certain threshold (where the amplification plots cross the horizontal, maroon line) is the sample threshold cycle (CT) value, and is proportional to the amount of qPCR target DNA initially present in the reaction.
