How powerful is this series of HCD kits?

Although the downstream purification process can remove residual impurities such as HCD, there may still be some HCD residues, and these residual exogenous DNA are easy to cause potential immunogenicity and safety issues, so HCD detection is one of the important quality detection indicators in the biologics production process.

The importance of HCD testing

Residual host cell DNA can have a variety of consequences when it enters the body:

• Carcinogenicity:It is easy to introduce dominant oncogenes, which makes some cells differentiate into tumor cells. At the same time, residual host cell nucleic acids may cause cell proliferation to go out of control in vivo and turn into tumor cells, such as Ad5E1A, SV40.
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• Infectious:Infectious viral genes may be present in the DNA of the host cell, which are amplified through replication and transcription and produce infectious virions, such as HIV-1
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• Increased immune-genetic:Host cell genomic DNA is rich in CpG, which may be mediated by Toll-like receptors (TLRs) to elicit immune responses.
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• Potential recombination risk:LTRs in the host cell genome can be transferred to the vicinity of cellular proto-oncogenes, so that these proto-oncogenes are activated by LTR, transforming normal cells into cancer cells.
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Methods for the detection of HCD

The Chinese Pharmacopoeia 2020 Edition, General Chapter 3407 includes three methods for the determination of exogenous DNA residues: DNA probe hybridization, fluorescent staining, and quantitative PCR.

DNA probe hybridization

The exogenous DNA in the test sample was heated, denatured, and then adsorbed on a solid-phase membrane, and hybridized and refolded with specifically labeled single-stranded DNA to double-stranded DNA. After comparison with a known amount of positive DNA control, the amount of DNA is deeply reflected in color development, and the amount of exogenous DNA residue in the test sample can be determined.

Characteristics of the method:

• When the DNA content of the sample is less than 10pg, the chemicals in the sample can have a significant impact on the test results.
• The test results are unstable, and the residual DNA content of real host cells is very different, and the detection time is relatively long.
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Fluorescent staining method

A fluorescent signal is generated by application of a double-stranded DNA fluorescent dye that binds to double-stranded DNA to form a complex and is detected at a wavelength of 520 nm using a microplate reader, with fluorescence intensity proportional to the DNA concentration over a range of DNA concentrations. Based on the fluorescence intensity of the standard and test sample, the amount of residual DNA in the test sample is calculated.

Characteristics of the method:

• Fluorescence signals are susceptible to interference, resulting in poor specificity.
• DNase contamination of experimental consumables and the environment must be avoided.
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Quantitative PCR

After the reaction starts, the host cell DNA is denatured and melted into a single strand, the probe is preferentially annealed with the template strand, and the primer is then annealed to the template for strand extension, and the amplification enzyme exerts 5′-3′ exonuclease activity during the extension process, and the probe will be excised from the 5′ end one base by one when encountering the probe, and the fluorophore will be separated from the quencher to form a free fluorescent signal, which will be detected by the fluorescence detection system. A DNA standard of known concentration was used to construct a standard curve and the amount of exogenous DNA residue in the test sample was determined.

Quantitative PCR is the only standard method for the determination of exogenous DNA residues in biological products recommended by the new USP in the Chinese Pharmacopoeia, and the only method recommended in the new USP for the detection of host residual DNA in biological products.

Characteristics of the method:

• Primer and probe-specific binding of the target region to ensure the accuracy and specificity of the detection results.
• Fluorescent probes have higher sensitivity for detection and accurate quantification of trace amounts of residual DNA.
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Host cell residual DNA standards

Features:

Hanhai New Enzyme has upgraded its host residual DNA detection products, covering multiple cell types such as CHO, E. coli, Vero, HEK293, Pichia pastoris, and mycoplasma. The primer and probe sequences of the kit are from the pharmacopoeia, so you don’t need to do a full set of validation experiments, just need to confirm the method. Developed based on a quantitative PCR method, LODs can reach 0.003 pg/μL with no cross-reactivity with other DNA, reducing false positives in the assay reaction. Strict production management system, batch-to-batch difference controllable CV<10%. Provide a complete set of solutions for manual extraction and automatic instrument extraction</b40>, which can quickly complete the detection within 2 hours and ensure the accuracy of quantitative detection.

Performance data

The standard products are calibrated by the national standard products

The standards in the CHO HCD kit were calibrated using the national CHO DNA standard, and the standard from different manufacturers was detected with the same kit, and the deviation of the measured value of the new enzyme standard was less than 5%.

The primer probe sequences for HCDs of CHO, Vero, E. coli, and yeast are from pharmacopoeia

Sensitivity, specificity, repeatability, precision

The test results of real samples are consistent

Simple and convenient

The finished product of the extraction reagent is assembled and used directly, without the need to prepare reagents separately, and the whole set is stored stably at room temperature. Fully automated instrument extraction with pre-packaged kits for flexible on-demand access.

Product Information

References

[1] Technical Guidelines for Pharmaceutical Research and Evaluation of In Vivo Gene Therapy Products (Trial), Center for Drug Evaluation, National Medical Products Administration, May 2022

[2] Chinese Pharmacopoeia Commission, Pharmacopoeia of the People’s Republic of China (2020 edition).

[3] Yan Luyao, Zhang Jiayou, Yang Xiaoming. Research Progress on the Detection of Residual DNA from Host Cells in Biological Products[J]. Int J Biologics, 2021, 44(3): 170-174