The time-honoured tradition of each higher-level regulatory authority publishing its own guideline on the same topic is alive and well. The detection of host cell impurities in bioengineered products is no exception: the European Medicines Agency (EMA) and the European Food Safety Authority (EFSA) have each published their own requirements.
Biotechnological processes are widely used to produce active pharmaceutical ingredients (APIs) for human and veterinary use. Human insulin, one of the first biotechnology products, has been produced biotechnologically in Europe for the treatment of diabetes mellitus since 1994. Many other recombinant proteins have since followed it onto the market. In 2022, around 59% of all medicines approved in Germany were biopharmaceuticals (as recently reported by the Verband Forschender Arzneimittelhersteller e.V. in the Annual Biotech Report 2023). For biopharmaceutical products intended for human and veterinary use, the European Medicines Agency (EMA) is the competent authority.
The European Food Safety Authority (EFSA) provides independent scientific advice on food-related risks. It is involved in the market authorisation of almost all food ingredients intended for use in the European Union. Among other things, EFSA also evaluates novel foods and novel food ingredients produced by cellular agriculture. In most of these production processes, biotechnology plays a fundamental role, whether or not genetic engineering is involved.
The product quality requirements, however, differ considerably. Many host organisms (e.g. Pichia pastoris) can be used both for biopharmaceutical production and for the production of novel food ingredients – yet a different authority is responsible for approving each product.
Validation of analytical methods against a recommended detection limit for host cell DNA impurities is a fundamental FDA requirement – but not an EMA requirement.
The EMA scientific guideline on "DNA and host cell protein impurities, routine testing versus validation studies" does not specify a limit of detection (LoD) for host cell genomic DNA impurities in the final biotechnological product.
The International Council for Harmonisation (ICH) provides several quality guidelines describing regulatory expectations for biopharmaceutical products. For example, process-related impurities from the host organism used for recombinant protein expression must be characterised in the final product. However, ICH Q6B (Specifications: Test Procedures and Acceptance Criteria for Biotechnological/Biological Products) does not define a detection threshold either. Essentially, it appears sufficient that the (unspecified) "level of DNA from the host cells can be detected by direct analysis on the product (such as hybridization techniques"; see section 6.2.1. of the EMA-adopted ICH Q6B guideline).
Everything appears to rest on a product-specific risk assessment: how residual host cell DNA in the final product is evaluated may depend on the type of host and its genomic structure, as well as on the type of recombinant DNA used for production.
The U.S. Food and Drug Administration (FDA) takes a different view in the "Guideline for Industry: Characterization and Qualification of Cell Substrates and Other Biological Materials Used in the Production of Viral Vaccines for Infectious Disease Indications." Section C.2. indicates that "you should limit residual DNA for continuous non-tumorigenic cells, such as low-passage Vero cells, to less than 10 ng/dose for parenteral inoculation." Vero cells are widely used for gene therapy production.
The World Health Organization (WHO) has also clearly set out its expectations in the TRS 987 Annex 4 "Guidelines on the quality, safety and efficacy of biotherapeutic protein products prepared by recombinant DNA technology". Section A.4.2.1. indicates that "in general, it has been possible to reduce rcDNA levels in rDNA-derived biotherapeutics to <10 ng per dose."
But what does this mean in practice? Let’s look at a recent example of an EMA-approved recombinant protein vaccine: AREXVY. The world’s first respiratory syncytial virus (RSV) vaccine is essentially a recombinant RSV-specific antigen, glycoprotein F. The FDA has approved AREXVY last year and provides more detailed dosing information in the package insert.
After reconstitution, each 0.5 mL dose is formulated to contain 120 micrograms of the recombinant RSVPreF3 antigen. Each dose must contain less than 10 ng of host cell rDNA, as required by the WHO and the FDA; the package insert, however, states < 0.80 ng per milligram of product. Extrapolated to one gram of final product, almost 800 ng of host cell rDNA per gram of product is still in line with WHO and FDA expectations.
Does analytical validation also play a role when submitting a dossier to EFSA for GMO-derived novel foods or novel food ingredients?
The European Food Safety Authority (EFSA) says YES, and published the "Guidance on the characterisation of microorganisms used as feed additives or as production organisms." EFSA Journal 2018;16(3):5206, 24 pp. https://doi.org/10.2903/j.efsa.2018.5206
Section 3.2. "Presence of DNA from the production strain" gives clear advice: "The presence of DNA from the production strain should be tested in the product by PCR [...] having detection threshold of 10 ng of DNA per gram or mL of product or lower." The challenge is to produce enough product material for spiking experiments and to start method validation, e.g. in accordance with the ICH Q2(R2) guideline. But at what point do you have enough product material (e.g. soy leghemoglobin) to start spiking controls as suggested in the EFSA guidance?
As an example of how insufficient or incomplete dossiers can cause extreme delays in processing an application, the 156th GMO Panel Plenary meeting on 15–16 March 2023 dealt with an application to place on the market soy leghemoglobin produced from genetically modified Komagataella phaffii (formerly known as Pichia pastoris; EFSA application No. EFSA-GMO-NL-2019-162).
According to the EFSA "Guidance on the risk assessment of genetically modified microorganisms and their products intended for food and feed use." EFSA Journal 2011; 9( 6):2193, 54 pp. https://doi:10.2903/j.efsa.2011.2193. the product could be classified as a category 2 "Complex product in which both GMMs and newly introduced genes are no longer present, e.g. cell extracts, most enzyme preparations". For this application, the clock remains stopped until EFSA receives, among other things, additional information on the "method used to estimate the presence of the recombinant DNA". This information was evidently missing from the dossier. Soy leghemoglobin approved by Food Standards Australia may contain less than 300 mg DNA per litre (the mass was not specified). Will this be the stumbling block for the EFSA dossier?
Please follow this link for further information: https://open.efsa.europa.eu/questions/EFSA-Q-2019-00651. Interestingly, the applicant submitted the dossier to the Norwegian Scientific Committee for Food and Environment (VKM).
As an example of how EFSA’s DNA detection requirements can indeed be met, EFSA published a "Scientific Opinion on the safety evaluation of the food enzyme phospholipase C from a genetically modified Komagataella phaffii (strain PRF). EFSA Journal 2019;17(4):5682, 12 pp. https://doi.org/10.2903/j.efsa.2019.5682.
Section 3.4. acknowledges that "no DNA was detected with primers that would amplify a 999-bp fragment specific for a region containing the alphaMF-PLC gene, with a limit of detection (LoD) of 1 ng spiked DNA/g food enzyme", which is ten times more sensitive than the guidance requires – and this was, of course, tested in the product by PCR.
Conclusion
For biopharmaceutical products (example: AREXVY), the limit for host cell DNA impurities is 10 ng DNA per administered dose, in line with WHO and FDA expectations. In fact, AREXVY may contain up to 800 ng per gram of product, which has been accepted for intramuscular administration in humans.
For biotechnology-derived novel food ingredients (example: soy leghemoglobin), the limit is 10 ng DNA per gram of product, in line with EFSA’s much stricter expectations – even though the product is intended for use only as a food ingredient.
In either case, the applicant should demonstrate that the analytical method delivers reliable and reproducible results. Even where the authority does not require formal method validation, it can make sense to follow the ICH Q2(R2)/Q14 guidelines.
Disclaimer:
The information in this article has been researched to the best of our knowledge and belief and was current at the time of publication. This article is for information purposes only and does not constitute scientific or regulatory advice. To ensure that the information and links provided here are up-to-date, please consult the relevant websites.
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Last update of this entry: Sunday 04 June 2023 by Christian Lange.
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