Changes in residual DNA detection standards in biological products
At the end of March 2015, a product numbered 410001 was added to the National Standard Drug Substance List of the China Food and Drug Control Research Institute website: CHO Host Cell DNA Residue Detection Kit (PCR-Fluorescence Probe Method). This kit, jointly developed by the Chinese Inspectorate and the Chinese Academy of Sciences, is synchronized with the current United States Pharmacopoeia (USP) recommended method, but is different from the exogenous DNA residue assay in the 2010 edition of the Pharmacopoeia Appendix. It may affect the upstream and downstream enterprises of domestic biological products research and development and production.
The residual DNA of host cells in biological products has potential tumorigenic and infectious risks, so the drug regulatory authorities in various countries have strict requirements on the limits of DNA impurities. The US Pharmacopoeia introduces three common techniques in General Chapter <1130>, but will add a new chapter (General Chapter <30>) to the new edition (USP38–NF33) issued in 2015 to further standardize methods and reference materials for residual DNA detection. . Unlike the section above 1000, the USP No. 1000 section specifies the inspection techniques, system suitability standards, and reference materials. The new USP will only recommend the qPCR method as the standard method for host residual DNA in biological products. The technical advantages of the qPCR method are high sequence specificity, high sensitivity, good reproducibility, and few human interference factors, which can provide a reliable detection method for the biopharmaceutical industry in process research and quality control of finished products.
China has long been restricting the amount of residual DNA in biological products with reference to WHO, FDA and EU standards. From the "Quality Control Points for Human Recombinant DNA Products" promulgated by the Ministry of Health to the "Chinese Biological Products Regulations" issued in recent years, strict requirements have been imposed on DNA content, and some standards are higher than international standards. The 2010 edition of the Chinese Pharmacopoeia appendix includes the DAN probe hybridization method and the fluorescent dye method. Both of these methods have technical defects, and it is difficult to achieve the sensitivity of the impurity limit detection, which has been abandoned by the European and American Pharmacopoeia. At present, there are still many domestic companies using these two methods to detect residual DNA, which makes it difficult to achieve the world-class level of production technology and product quality. According to the development trend of residual DNA detection technology, Xiaobian expects that the qPCR method will appear in the 2015 edition of the Pharmacopoeia or Supplement. In the production and R&D, the company uses the kits provided in the Catalogue of Standards in the Central Inspection Institute, which can greatly reduce the time-consuming, labor-intensive and costly methodological investigation. Only a small number of method-adaptive experiments can be carried out in the production process and The optimization of the quality control system plays a practical role, while meeting the requirements of the relevant FDA standards. At the same time, the kit joint research and development unit Huzhou Nutrition Center of the Chinese Academy of Sciences provides free technical advice and training to help enterprises solve various technical problems in the application.
Biological products can be used to treat and prevent diseases, and it is related to the safety of medication for patients and healthy people, and the quality of products must be guaranteed. China's drug regulatory authorities have very strict standards for the determination of residual DNA in biological products, but there are certain lags in the revision of the pharmacopoeia. There is still a gap between the testing technology in the appendix and the advanced countries. Enterprises should have certain forward-looking in research and development and production. Otherwise, efforts to improve the process, improve quality, and ensure safety will be greatly reduced.
Why test residual DNA?
Biologics are the fastest growing segment of the pharmaceutical industry. In 2014, 7 of the top 10 best-selling drugs in the world were biologics. These sales of blockbusters are clinically effective, but the cost of research and development is high, and production and quality control requirements are very strict. Most biological preparations do not enter the body directly through the gastrointestinal tract, so in addition to biological activity, the regulatory authorities have strict requirements on the limits of impurities in medicines. Among them, host cell residual DNA has been the focus of drug regulatory agencies at home and abroad because of its special potential safety risks. The US Pharmacopoeia has organized a panel discussion since 2011 to revise the method for detecting residual DNA in biological products, and announced at the 2014 Prescription/Non-Prescription Stakeholder Forum Meeting #5 that a new chapter will be added to the 2015 revision of the Pharmacopoeia ( General Chapter <30>) to standardize test methods and reference materials. Why did the panel of the US Pharmacopoeia spend a few years discussing a method for detecting trace components (<100 pg/dose)? Also add special chapters to standardize? To answer these questions, you must first understand its source and potential harm. Products such as recombinant protein drugs, antibody drugs, vaccines and the like in biological products are produced by serial passage of animal cell lines. Although a strict purification process is carried out, DNA fragments of host cells may remain in the product. The fragment size and composition of these residual DNA are variable, and the potential risks are uncertain, which may lead to contagious, or tumorigenic, or increased immunogenicity, or mutagenicity. For example, residual DNA may carry HIV or Ras oncogenes; the LINE-1 sequence distributed in the genome of mammalian cells may play a retrotransposon insertion into the chromosome, and this insertion may affect the function of key genes, such as activating cancer. Genes or inhibition of tumor suppressor genes; in addition, because microbial-derived genomic DNA is rich in CpG and unmethylated sequences, the risk of immunogenicity of recombinant protein drugs in vivo is increased. The current research results show that the tumorigenicity of residual DNA is lower than the infectious risk, but considering that the tumorigenicity experiment is an animal experiment, the infectious experiment is done at the cellular level, and the risk of both aspects should not be taken lightly. . It is well known that foreign proteins may cause severe immune responses, but little research has been done on residual DNA-induced immune responses. High-dose nucleic acid samples, such as DNA vaccines or CpG oligonucleotides in adjuvants, have been reported in a number of preclinical and clinical studies to induce immune responses and to induce production of DNA antibodies.
Host residual DNA in biological products is not only an impurity in production, but also has certain safety hazards. Therefore, WHO and national drug registration regulators generally only allow 100pg/dose of residual DNA in biologics, and encourage the minimization of residual DNA and fragment length (less than the length of a functional gene, according to current evidence, approximately 200bp). Depending on the source and process of the impurities, a maximum of 10 ng/dose is allowed under special circumstances.
The pros and cons of various residual nucleic acid detection techniques
As mentioned earlier, the 2015 edition of the United States Pharmacopoeia will add a chapter to normalize residual DNA testing. How is it different from existing methods? Why did you only identify one method in the end? Let's take a look at the current US Pharmacopoeia's overall requirements for residual DNA testing [General Chapter <1130> NUCLEIC ACID-BASED TECHNIQUES-APPROACHES FOR DETECTING TRACE NUCLEIC ACIDS (RESIDUAL DNA TESTING)]. “ Because residual DNA involves key issues such as potential sources (infectious viral DNA) and regulatory protocols, the drug regulatory agency recommends that a DNA residue detection method for the product be established, whether or not the routine testing of the finished product involves DNA residue detection or process development. DNA clearance, residual DNA specifications, and quantitative analysis monitoring procedures have been established. Methods of analysis include hybridization, DNA-binding protein-based immunochromatography (threshold method), quantitative PCR (Q-PCR), or other DNA. Amplification method. The sensitivity of the ideal quantitative detection method should be able to detect residual DNA of about 10pg / dose. Hybridization, threshold and quantitative PCR methods are classical methods because sensitivity can meet the detection requirements." The contents of the Pharmacopoeia (USP) <1130> are described separately.
Hybridization-based : In this method, a DNA probe is designed based on the host DNA sequence to determine the amount of paired DNA in the product. The double-stranded DNA is denatured into a single strand and then immobilized on a nylon membrane or a nitrocellulose membrane. After the DNA probe is randomly incorporated into the label, it hybridizes to the sample host DNA immobilized on the membrane and appears in the corresponding position of the film or imager. spot. For fluorescently labeled probes, the optical density results of the spots can be quantified in the instrument. The spot optical density corresponds to the number of probes bound to the target DNA, and the amount of residual DNA is estimated. The residual DNA in the sample can be detected semi-quantitatively by visual inspection. The instrument reading can draw a standard curve corresponding to the spot optical density, and the corresponding detection result is more accurate.
DNA binding protein immunogen threshold method (DNA-BINDING PROTEIN-BASED) : This method uses the DNA antibodies and DNA binding protein, detected in four steps. In the first step, the DNA is denatured into single-stranded DNA by heating, and the denatured DNA is mixed with the avidin-conjugated DNA-binding protein and the urease-conjugated DNA monoclonal antibody, and the single-stranded DNA in the liquid phase, The DNA binding protein and the DNA antibody together form a sequence non-specific complex. In the second step, the sample mixture is fixed to the membrane by a biotin-labeled membrane, avidin-biotin binding, to wash away non-specific adsorption. In the third step, the membrane is placed in a test instrument to react with the urea solution, and the reaction product ammonia changes the pH of the solution and is recorded by the instrument. This change in pH is directly related to the number of DNA in the sample. In the fourth step, the instrument software automatically analyzes the raw data to determine the amount of residual DNA in the sample.
QUANTITATION PCR-BASED : The qPCR method has been applied to some areas of biopharmaceuticals (copy number detection and virus detection) for its fast, high-throughput characteristics. This technique is able to determine the exact number of target DNA sequences in various samples. The design of DNA probes is critical, and this DNA probe contains one dye molecule at one end and a quencher molecule at the other end. When a specially designed DNA primer directs the DNA polymerase to replicate along the template sequence to synthesize another corresponding sequence, the DNA polymerase cleaves the probe dye end bound to the target DNA, and the dye signal released into the reaction solution is measured by the instrument. After dozens of cycles of DNA amplification, the fluorescence signal is mapped to the starting DNA template, and the corresponding standard curve can accurately calculate the amount of residual DNA in the sample.
The US Pharmacopoeia appendix further evaluated the application of the three methods. Hybridization method can detect target DNA in a sequence-specific manner, but 32 P-labeled probes are not widely used because of short half-life, radiation, and the like. Fluorescently labeled probes If the instrument is used to read the signal, the hybridization method theoretically achieves the sensitivity required for quantitative detection, but the detection time takes 48 hours. Because the threshold method is a non-specific sequence immunodetection technique using DNA antibodies, it cannot specifically recognize the host residual DNA sequence, and is easily contaminated by the environment and the operator's DNA, resulting in a high reading value. The qPCR method has sequence specificity, sensitivity, accuracy, precision, and high-throughput screening, but it is not easy to develop a qualified q-PCR reagent to detect host residual DNA. Some people may ask whether the DNA residue of any species should be defined in the final product to ensure safety, so is the threshold method the most reasonable analysis method? It is also necessary to emphasize the purpose of host residual DNA detection: 1. Confirm that the purification process is reasonable and can effectively remove the host DNA residue; 2. Confirm that the impurity content in the product meets the standard requirements. Non-specific DNA detection results cannot distinguish between DNA contamination caused by contamination in production, contamination detection, or process defects, and cannot provide effective information for the solution. In the strict production system, residual DNA detection is to solve the problem of process rationality, and any external pollution problems are solved by SOP or GMP management system. Finally, the sensitivity of the classical residual DNA detection method is different. The detection limits of qPCR, DNA binding, and hybridization methods reach the levels of <1, 3, and 6 pg/sample, respectively (the sensitivity of qPCR is up to 10 fg/reaction), but technically. Restriction requires that the DNA fragments to be tested are not less than 50, 150, and 600 bp, respectively, for hybridization, q-PCR, and threshold detection, while fragments within the DNA limits acceptable by WHO and FDA are <200 bp. It can be seen that among the three methods, the applicability and technical indicators of the qPCR method are the best. From the principle of qPCR technology, the Taqman method is superior to the SYBR Green method in which fluorescent dyes are randomly incorporated. As previously described in the USP revision, after several years of systematic research and application feedback on the three detection methods, the US Pharmacopoeia will only recommend qPCR as the standard method for DNA detection of biological residues in the new edition of the Pharmacopoeia.
Standards and Techniques for Detection of Residual DNA in Biological Products in China
China has long been restricting the amount of residual DNA in biological products with reference to WHO, FDA and EU standards. Yeast, E. coli expressed biological products are limited to no more than 10 ng / dose, CHO and vero cells expressed EPO, rabies vaccine, hepatitis B vaccine, etc. no more than 100 or 10 pg / dose. The 2010 edition of the Chinese Pharmacopoeia appendix includes DNA probe hybridization and fluorescent dye methods as residual DNA detection methods. The DNA hybridization method represented by the digoxigenin-labeled dot blot hybridization method has been complicated, time-consuming, and only semi-quantitative, and has been eliminated in the 2014 US Pharmacopoeia revised consultation draft (PF40(2)). . The fluorescent dye method represented by PicoGreen is based on the fact that the fluorescent dye molecule directly binds to double-stranded DNA and is excited by a microplate reader with fluorescence detection function to generate a fluorescent signal because single-stranded DNA cannot be detected and has no sequence specificity. And the reason for low sensitivity (>300pg), etc., has long been abandoned by the European and American Pharmacopoeia.
The DNA probe hybridization method and the fluorescent dye method were included in the consultation draft of the 2010 edition of the Pharmacopoeia and the 2015 edition of the Pharmacopoeia (Appendix IX B Exogenous DNA Residues), and the USP included the hybridization method in 2009. , threshold method and qPCR method, by the end of 2015 USP will only include high sensitivity, high specificity qPCR method as the recommended method for residual DNA detection. It can be seen that the qPCR method will become an internationally recognized method for detecting residual DNA, and it may also be the main revision of the next edition of the Pharmacopoeia in China. Domestic bioproduct research and development and production enterprises, as well as upstream and downstream enterprises producing purified fillers, establish a host residual DNA detection system based on qPCR method as soon as possible, which will help improve the process, improve product quality, and achieve biopharmaceutical standards with developed countries in Europe and America. Connected.
Technical performance of kits in the National Standards Library
In order to improve the technical level of detection of host residual DNA in biological products by domestic enterprises and regulatory authorities, and to achieve integration with international standards, the Chinese Inspectorate, the Chinese Academy of Sciences, the production and R&D enterprises have developed the first domestically based qPCR technology with independent intellectual property rights. CHO host cell DNA residue detection kit. In addition to routine methodological studies and stability studies, R&D has also carried out research on DNA fragmentation, species specificity, versatility of different instruments, high protein, low pH, high salt, and performance comparison of similar foreign products. In the experiment, the applicability of different sample matrices to many domestic manufacturers and R&D enterprises was investigated. The kits, which are calibrated and verified by the Chinese National Standards CHO cell DNA, provide a complete set of reagents from sample extraction and purification to PCR detection with a detection limit of 10fg/rxn and resistance to human, rat, mouse and other genomes. The internal standard sample recovery rate is 70~130% (the new USP standard will be adjusted to 50~150%), which can provide flexible and accurate DNA limit detection for CHO cells expressing different varieties and different doses of biological agents. The kit has a 2-year shelf life and is provided by the joint research and development unit Huzhou Nutrition Center of the Chinese Academy of Sciences to provide free technical consultation, operational training and method validation services to help enterprises master the technology as soon as possible. The joint research and development project of the Chinese Academy of Sciences and the Huzhou Nutrition Center of the Chinese Academy of Sciences will launch E.coli, Yeast and vero cell residual DNA testing kits to provide technical support for the development of the domestic biopharmaceutical industry and the development of national standards for regulatory authorities.
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