Testing of Residual Host Cell DNA: Importance in Quality Management of Biologicals

Mar 26, 2025 | Informative Articles

Testing of Residual Host Cell DNA: Importance in Quality Management of Biologicals
Photo: Isaac74 | Depositphotos.com

The testing of residual host cell DNA is a crucial aspect of quality management in the manufacturing of biologics. These tests are essential to ensure that the final products do not contain hazardous or unwanted DNA residues that could pose potential risks to patients. Below are key aspects of this testing process.

1. Background: Why is Residual Host Cell DNA Testing Critical for Biopharmaceuticals?

Residual DNA arises during the production of biopharmaceuticals when animal, bacterial, or human host cells are used to produce recombinant proteins, monoclonal antibodies, or vaccines. In these manufacturing processes, host cells are typically lysed to extract the desired therapeutic proteins. Despite extensive purification steps, small amounts of host cell DNA may remain in the final product. This residual DNA can be considered a contamination that may compromise both product quality and patient safety.

Doctor talking to patient Photo from freepik

2. Why is Residual DNA a Problem?

Residual DNA in biologics presents various risks:

• Immunogenic reactions:
Residual DNA may activate the patient's immune system, leading to inflammation, allergic reactions, or even more severe immune responses.
• Oncogenic potential:
Host cell DNA may contain oncogenes, which have the potential to cause cancer.
• Infectivity:
In rare cases, residual DNA may originate from viral particles or other infectious agents, potentially leading to disease transmission.

3. Regulatory Requirements for Residual Host Cell DNA Testing: Guidelines and Limits

Pharmaceutical Photo: Pexels.com | Thirdman

Major global regulatory authorities, such as the WHO, FDA, and the European Pharmacopoeia, have established strict limits on the amount of residual DNA in biologics to ensure patient safety.

• WHO:
Recommends that the residual DNA content in the final product should be less than 10 ng per dose.
• FDA:
Sets a limit of 10 ng per dose for biopharmaceuticals, with stricter requirements for vaccines (e.g., Hepatitis A: 100 pg per dose).
• European Pharmacopoeia:
Establishes a threshold of 10 ng per dose for most biopharmaceuticals, with even more stringent limits for specific vaccines.

These limits are considered safe and are intended to minimize the potential risks associated with residual DNA.

4. Best Methods for Detecting Residual Host Cell DNA: dPCR and Alternative Approaches

Various methods are available to quantify residual DNA in biopharmaceuticals. The most commonly used techniques include:

• Quantitative PCR (qPCR):
This method is highly precise and sensitive. It is often the preferred technique due to its high specificity and accuracy in determining the exact DNA concentration.
• Digital PCR (dPCR):
An advanced method that allows absolute quantification of residual DNA without relying on standard curves. dPCR provides extremely high accuracy and sensitivity, making it an excellent choice for residual DNA analysis.
• DNA probe hybridization:
Less commonly used but can be of interest for specific applications.
• Fluorescence staining and immunoassays:
Typically employed for specialized applications or as complementary techniques.

5. Importance of Fast and Accurate Testing

Since residual DNA in biologics can exist in minute amounts, a fast, precise, and highly sensitive testing method is required. The use of technologies such as digital PCR allows for real-time identification and quantification of DNA residues. These technologies not only provide high accuracy but are also faster than traditional methods like DNA probe hybridization, optimizing the entire quality control process and accelerating biologics manufacturing

6. Advantages of a Robust Residual DNA Testing Strategy

• Ensuring product quality:
Accurate determination of residual DNA levels ensures that biologics meet regulatory requirements and are safe for patients.
• Preventing recalls and regulatory issues:
By closely monitoring residual DNA levels, companies can ensure compliance with global regulations, reducing the risk of recalls or approval delays.
• Enhancing manufacturing processes:
Regular monitoring of residual DNA concentration helps manufacturers optimize their production processes and confirm the effectiveness of their purification procedures.

7. Conclusion

Residual DNA testing is an essential component of quality management in the production of biopharmaceuticals. It ensures that products comply with stringent regulatory requirements and guarantees patient safety. Techniques such as digital PCR enable the precise quantification of even the smallest amounts of residual DNA, making them a valuable tool in quality control for the biopharmaceutical industry.

Compliance with regulatory limits is not only a legal obligation but also a crucial step in establishing trust in biologics and maximizing their efficacy and safety.

To meet these requirements, Minerva Biolabs has developed the MiQuant® Residual DNA HEK-293 - dPCR Assay. This assay enables highly sensitive and robust detection of residual DNA from HEK-293 cells using digital PCR (dPCR).

MiQuant® Residual DNA HEK-293 - dPCR

The MiQuant® Residual DNA HEK-293 - dPCR assay targets short multi-copy sequences in the HEK-293 genome (repeat element, approximately 1 million copies per genome). The amplicon length is less than 200 base pairs, ensuring reliable amplification even for highly degraded DNA, in accordance with regulatory guidelines.

Key Features of the MiQuant® Assay:
• Platform open:
Compatible with all major commercial dPCR devices such as QIAcuity®, ddPCR™, and QuantStudio™ Absolute Q™.
• Efficient workflow:
Direct sample analysis without prior DNA extraction, simplifying and accelerating the process.
• Stability and handling:
Lyophilized kit components facilitate transport and storage.
• Absolute quantification:
A major advantage is absolute quantification of samples without the need for a standard curve.
• Regulatory compliance:
Meets the criteria set by EP, WHO, and FDA for residual DNA analysis.
• Validation report available:
The validation study was conducted using the QIAcuity® 26k nanoplate.

Validation Report Data:
• Limit of Detection (LOD):
The smallest concentration of HEK-293 DNA where at least 23 out of 24 replicates were considered positive (above LOB = 0.312 cp/μl) was 1.7 fg/μl per reaction.
• Limit of Quantification (LOQ):
A coefficient of variation ≤ 20% was obtained down to 5 fg/μl per reaction (15.72%).
• Precision:
Relative repeatability was below 18% between 200,000 fg and 200 fg target DNA and increased to 38.05% and 29.60% for 50 fg and 20 fg target DNA, respectively.

Relative run-to-run variation was below 5.4% for all tested DNA target quantities, except 20 fg (28.57%).

The validation report is available on the product website!

MiQuant® Residual DNA HEK-293 – dPCR

Overall, the MiQuant® Residual DNA HEK-293 - dPCR assay provides a reliable solution for quality control in biopharmaceutical production.

Process graphics

The MiQuant® Residual DNA HEK-293 - dPCR assay can be tested.
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