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EMA and EFSA: Expectations on Analytical GMO Detection Limits in Biotechnology Products

The old tradition of publishing a separate guideline covering the same topic is still maintained among the higher-level regulatory authorities. The detection of host cell impurities in bioengineered products is no exception, the European Medicines Agency (EMA) as well as the European Food Safety Authority (EFSA) have each published their own requirements on this topic.

Biotechnology processes have widely been applied for the production of active pharmaceutical ingredients (API) for animal and human use. As one of the first biotechnology products, human insulin has been biotechnologically produced for the treatment of diabetis mellitus since 1994 in Europe. Many more recombinant proteins have followed the path to the market. In 2022, about 59% of all pharmaceuticals approved for the market in Germany have been biopharmaceuticals (as recently been reported by the Verband Forschender Arzneimittelhersteller e.V. in the Annual Biotech Report 2023). For biopharmaceutical products intended for animal and human use, the European Medicines Agency (EMA) is the competent authority.

The European Food Safety Authority (EFSA) provides independent scientific advice on food-related risks. The authority is responsible for market authorisation for almost all nutrition ingredients intended for use in the European Union. Among others, the EFSA evaluates and approves also Novel Food and Novel Food ingredients produced in cellular agriculture production processes. In most of this productions, biotechnology plays a fundamental role, irrespective if genetic engineering is involved.

The requirements for product quality are quite different here. Many host cell organisms (e.g., like Pichia pastoris) may be applied for biopharmaceutical production as well as novel food ingredient production. However, the approval authority responsible for each product is different.


Analytical method validation with a recommended host cell DNA impurity detection limit is a fundamental FDA but not EMA requirement.

The EMA scientific guideline on "DNA and host cell protein impurities, routine testing versus validation studies" does not clearly indicate a detection limit (LoD) for host-cell genomic DNA impurities in the final biotechnological product.
The International Council for Harmonization (ICH) provides several quality guidelines describing the regulatory expectations for biopharmaceutical products. For example, process-related impurities from the host organism used for recombinant protein expression must be characterized in the final biopharmaceutical product. However, a pre-defined detection threshold can also not be found in the ICH Q6B Specifications: Test procedures and acceptance criteria for biotechnological/biological products. Basically, it seems to be sufficient that the (not further specified) "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).
It all seems to be based on a product-specific risk assessment, and evaluating residual host cell DNA impurities in the final product may depend on the host type and it´s genomic structure as well as the type of recombinant DNA used for biopharmaceutical production.

The U.S. Food and Drug Adminitration (FDA) provides a more 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 published 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 that mean in practice? Let´s take a look at a recent example for an EMA approved recombinant vaccine protein: AREXVY. The world´s first Respiratory Syncytial Virus (RSV) vaccine (basically) is a recombinant RSV-specific antigen glycoprotein F. The FDA has approved AREXVY already last year, and provides more detailled information on the dosing scheme in the package insert.
After reconstitution, each 0.5-mL dose is formulated to contain 120 microgram of the recombinant RSVPreF3 antigen. Each dose may contain less than 10 ng rDNA from the host cells, as required by the WHO and the FDA; However, the package insert indicates < 0.80 ng per milligram product. Extrapolated to one gram final product, almost 800 ng rDNA host cell DNA per gram of product is surely in line with the WHO and FDA expectations.


Does analytical validation also play a role in dossier submission to EFSA for GMO-derived Novel Food or its 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 a 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 sufficient product material for spiking experiments, and to initiate method validation e.g., according to the ICH Q2(R2) guideline. However, at which time do you have sufficient product material available (e.g., from soy leghemoglobin) to start with controls spiking as suggested in the EFSA guideline?

As a example on how insufficient and/or incomplete application dossiers can result in an extreme time delay in application processing, the 156th GMO Panel Plenary meeting on 15-16 March 2023 was dealing with a request for placing on the market of 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 the EFSA receives (among others) additional information on the "method used to estimate the presence of the recombinant DNA". This information was obviously not included in the dossier. Soy leghemoglobin approved by the Food Standards Australia agency may contain less than 300 mg DNA per liter (the mass was not specified). Will this be the stumbling stone 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 how the EFSA guideline requirements on DNA detection can indeed be met, the 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 10-fold more sensitive than required by the guideline. Certainly this has been tested in the product by PCR.



The host cell DNA impurity in biopharmaceutical products (Example: AREXVY) is 10 ng DNA per administered dose, as outlined by the WHO and FDA expectations. In fact, AREXVY may contain up to 800 ng per gram of product, which has been accepted for intramuscular administration to humans.

The host cell DNA impurity in biotechnology-derived Novel Food ingredients (Example: Soy leghemoglobin) is 10 ng DNA per gram of product, as outlined by the much strikter EFSA expectations. Even though the product is intended for use only as an nutrition add-on.

In either case, the applicant should demonstrate that the analytical method produces reliabe and reproducible results. Although a formal method validation is not always required by the authority, it can make sense to follow the ICH Q2(R2)/Q14 EWG guideline.


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 any scientific or regulatory advice. To ensure that the information and links provided here are up-to-date, please consult the relevant websites.

Do you have questions or comments? We are looking forward to receiving your note.
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Please send an e-mail to service [at] biosafety4u.berlin.


Last update of this entry: Sunday 04 June 2023 by Christian Lange.
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