Host cell residual DNA sample preparation program
May 21, 2021
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Recombinant protein drugs, antibody drugs, vaccines and other products in biological products are expressed and produced by continuous cell lines. Although they have undergone strict purification processes, there may still be residual host cell DNA fragments (rDNA) in the products.
These rDNA may affect the function of the key genes of the receptor, thereby bringing the risk of infectious or tumorigenicity.
Therefore, how to efficiently and stably obtain the trace rDNA in the sample is particularly important. At present, there are mainly the following methods:
| Methods | Advantage | Disadvantage |
| Phenol/Chloroform | High DNA purity and high content | Time-consuming, cumbersome steps, and toxic |
| Sodium iodide precipitation | easy to use | Time-consuming and unstable DNA content |
| Column method | Simple and high purity | Time-consuming and difficult to automate |
| Magnetic Bead Method | Simple, high purity, can be automated | - |
![NP-2032[1]](/Content/uploads/2021213197/20210522104030d04d958e72634ed5a62eff19fd61da6f.png "NP-2032[1]") |
This treatment scheme is based on the principle of the magnetic bead method and is used in the rDNA pretreatment system of biological products, which can realize an efficient, simple and automated pretreatment system.
The SCIENTZ automatic nucleic acid extraction instrument is equipped with a well-known brand of host cell residual DNA sample pretreatment kit (magnetic bead method), and the recovery rate of various samples is stable up to over 80%, which can stably and effectively control the host cell DNA residual content in various biological products
Experimental program
Experimental materials: automatic nucleic acid extraction instrument (NP-2032), fluorescent quantitative PCR instrument (LightCycler480), host cell residual DNA sample pretreatment kit (magnetic bead method), CAR/TCR gene copy number detection kit (multiplex PCR- Fluorescence probe method)
Experimental samples: retrovirus samples, CAR-T cell samples, negative control
Experimental steps:
For monitoring.
Experiment preparation-sample preparation-extraction preparation-program start-total residual content detection
Remarks: The instrument runs for about 30 minutes
Experimental results:
| Sample name | MeanCp | MeanConc | Amount recovered | Scalar | Recovery rate |
| Negative control | 36.43 | 0.00 | - | - | - |
| Negative control+(portable) | 18.67 | 86.62 | 8662.15 | 9707.85 | 89% |
| Negative control | 18.72 | 53.47 | 8020.723657 | 9868.821045 | 81% |
| Virus sample+ | 19.15 | 63.10 | 8194.27 | 9707.85 | 84% |
| Cell sample | 20.21 | 32.93 | 2811.241943 | 2740.516576 | 102% |
Experimental results:
Automated processing of different samples has stable and efficient results;
Batch processing automation efficiency is higher than manual extraction efficiency;
It meets the requirements of the new USP recovery rate; it can be a good assistant for the pre-processing of rDNA detection in biological products.
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