Overview
Host cell residual DNA is a process-related impurity involved in the production of biologics, which not only reduces the effectiveness of biologics, but also may pose safety concerns such as infectiousness or tumorigenicity. Therefore, regulatory agencies in various countries have imposed limits on the amount of residual DNA in biologics.
Current WHO and FDA guidelines recommend residual DNA in finished products of no more than 10 ng/dose, and FDA also states that residual DNA in host cell DNA of biologics should be no more than 100 pg/dose. The General Principles of the European Pharmacopoeia stipulate that most of the residual DNA limits of biological products should be no more than 10 ng/dose, but the residual DNA limits of individual vaccines are more stringent, e.g., the residual DNA in the inactivated vaccine against hepatitis A should not be more than 100 pg/dose, and that the residual DNA in the vaccine against hepatitis B should not be more than 10 pg/dose. The 2020 edition of the Chinese Pharmacopoeia, Part III, stipulates that the DNA residue in biological preparations produced on a cellular matrix should not exceed 100 pg/dose, and the DNA residue in vaccines produced on a bacterial or fungal matrix should not exceed 10 ng/dose.
In addition, for the methods of exogenous DNA residue determination, national pharmacopoeias also give guidance recommendations. The 2017 edition of USP40-NF35 General Provision 1130 of the U.S. Pharmacopoeia describes 3 methods for the determination of exogenous DNA residues, which are DNA probe hybridization, threshold method and real-time quantitative PCR method. The European Pharmacopoeia proposes real-time quantitative PCR and immunoenzymatic methods, which are 2 sensitive analytical methods for quantifying residual DNA in host cells. The Chinese Pharmacopoeia 2020 version of the three general rules 3407 also stipulates that the host cell DNA residue detection methods are DNA probe hybridization, fluorescence staining and quantitative PCR.
Among them, qPCR method has very high sensitivity, sequence specificity and accuracy, which can provide a reliable detection means for the biopharmaceutical industry in process research and quality control of finished products, and has now become the preferred detection method for each biological product manufacturer.
Yeasen Biotechnology Residual DNA Detection Kits
Based on the principle of fluorescent probe qPCR, Yeasen has developed a series of rapid and specialized host cell residual DNA detection kits, including CHO, HEK293, E.coli, Vero, Human, MDCK, Hansenula polymorpha, Pichia pastoris and so on. These kits provide specialized and rapid detection of DNA residues in intermediate, semi-finished and finished products during the development and production of biologics such as antibody drugs, cell and gene therapy products, recombinant protein drugs and vaccines.
Feature
High sensitivity: developed based on fluorescent probe qPCR method with LLOD as low as 0.05fg/µL;
High accuracy: sample spiking recoveries in the range of 70%~130%;
High specificity: no cross-reactivity with irrelevant DNA, reducing false positives in detection;
Compliance with regulations: fully validated in accordance with Chp, USP, ICHQ2(R1) and other requirements, performance in accordance with Chinese and foreign regulatory standards;
Cooperate with auditing: product production conforms to ISO13485 quality system standards, with perfect audit documents.
Guarantee quality: the raw materials of the kits are all independently developed, and the qPCR Mix and other enzyme products are produced in an ultra-clean enzyme factory.
Application
Antibody DrugsHost cell residual impurity detectionVaccinesHost cell residual impurity detection
Recombinant Protein drugsHost cell residual impurity detection
Specification
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Testing and validation program |
Reference standards or requirements |
Note |
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1. Kit Standards (Reference Standards) |
Benchmarking to national standards or Prepared standards |
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2. Linear Range |
Scope of the standard curve |
Refer to the manual or actual situation (eg.30fg/μL~300pg/μL） |
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R2 |
≥0.98 (P.s.Yeasen Kits≥0.99） |
Requirements for HCD in Chinese Pharmacopoeia 2020 Edition 3407 General Provisions |
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Slope |
-3.1~-3.8 (P.s.Yeasen Kits -3.1~-3.6） |
Requirements for HCD in Chinese Pharmacopoeia 2020 Edition 3407 General Provisions |
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Amplification efficiency |
90%~110%（（P.s.Corresponding slope -3.1~-3.6, Requirements within the industry） |
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3. Accuracy |
Deviation from national standard DNA |
＜15% |
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Sample spiking recovery rate |
50%~150%（Requirements within the industry 70~130%） |
Requirements for HCD in Chinese Pharmacopoeia 2020 Edition 3407 General Provisions |
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4. Precision |
Repeatable |
CV＜15% |
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Intermediate precision |
CV＜15% |
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5. Speciality |
No interference with exogenous DNA |
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6. Limit of quantification |
fg/μL level |
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7. Stability |
Repeated freezing and thawing |
10 times |
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Acceleration Stability |
2~8℃ 30 days, 37℃ 14 days |
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Validity period |
-20°C storage for 2 years |
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Figures
High sensitivity: LLOQ as low as 0.3 fg/µL, LLOD as low as 0.05 fg/µL.
1.The linear range of CHO Host Cell Residual DNA Detection Kit (3G) was 3fg/μL~300pg/μL, R2=1, the amplification efficiency was 99.29%, and the CV of the detection value of each concentration was <15%.
Figure 1. CHO DNA (3G) calibration graph (left) and amplification curve linear mapping (right)
2.Detect CHO DNA (3G) at the lowest concentration point of the standardized song at 3fg/uL and below concentrations, 10 replicates per concentration. The results showed that at concentrations of 0.3 fg/uL and above, the CV was <20%, i.e., the limit of quantification of the CHO Host Cell DNA Residue Assay Kit (3G) was 0.3 fg/uL.
Figure 3. 0.05fg/μL CHO DNA (3G) qPCR assay results
High specificity: no cross-reactivity with other host cell DNA.
Assessing the interference of genomic DNA of mouse species with high affinity to CHO DNA (3G) as well as genomic DNA of cells commonly used in the production of biologics to the CHO DNA detection reagent, the amplification curves of the interference group and the control group overlapped and no interference was seen (the following graphs show, in order, the interference data of Mouse, HEK293, and E.coli genomic DNA to the CHO DNA detection reagent).
Figure. 4. Results of interference experiments with CHO DNA (3G) kit
Product Information
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Categorization |
Item No |
Product Name |
Specification |
|
Sample Pre-treatment |
18461ES |
25T/100T |
|
|
18467ES |
MolPure® Mag48 Sample Preparation Kit FN |
3×16T/6×16T |
|
|
Nucleic Acid Extraction Instrument |
80511ES |
48-Channel Automated Nucleic Acid Extractor |
48 Fluxes |
|
Residual DNA Detection |
41307ES |
50T/100T |
|
|
41308ES |
50T/100T |
||
|
41310ES |
SV40LTA&E1A Residue DNA Detection Kit |
50T/100T |
|
|
41317ES |
Hansenula polymorpha Host Cell DNA Residue Detection Kit |
50T/100T |
|
|
41319ES |
MDCK Host Cell DNA Residue Detection Kit |
50T/100T |
|
|
41323ES |
Plasmid DNA Residue Detection Kit |
50T/100T |
|
|
41324ES |
S. cerevisiae Host Cell DNA Residue Detection Kit |
50T/100T |
|
|
41325ES |
Human Host Cell DNA Residue Detection Kit |
50T/100T |
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|
41328ES |
Pichia pastoris Host Cell DNA Residue Detection Kit |
50T/100T |
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41330ES |
Sf9 and Baculovirus DNA Residue Detection Kit |
50T/100T |
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|
41331ES |
50T/100T |
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|
41332ES |
50T/100T |