Search

CN-122012678-A - CHO cell gene copy number detection method

CN122012678ACN 122012678 ACN122012678 ACN 122012678ACN-122012678-A

Abstract

The invention provides a detection method of the gene copy number of CHO cells, which comprises the steps of carrying out pretreatment on genome DNA extracted from recombinant cells, wherein the pretreatment comprises thermal denaturation treatment or enzyme digestion treatment, carrying out qPCR detection on the genome DNA after pretreatment, and measuring the copy number of target genes, wherein the recombinant cells contain anti-repressor elements. The method improves the accuracy, stability and sensitivity of detecting the gene copy number of the CHO cells for production, and particularly provides a detection method for detecting the copy number of the target nucleic acid with high-structure and low abundance in genome. Provides more reliable evaluation basis for the construction and screening of cell lines, the production of medical proteins and the quality control in the drug research and development process.

Inventors

  • ZHANG MENG
  • CHEN JI
  • ZHANG CHENGDAN
  • YANG JIAXIN
  • GAO JIAN
  • LI HONGCHENG

Assignees

  • 长春金赛药业有限责任公司

Dates

Publication Date
20260512
Application Date
20260114

Claims (10)

  1. 1. A method for detecting the copy number of a gene of interest in a recombinant cell, comprising: Pretreating genomic DNA extracted from recombinant cells, wherein the pretreatment comprises heat denaturation treatment or enzyme digestion treatment; qPCR detection is carried out on the pretreated genome DNA, and the copy number of the target gene is determined; Wherein the recombinant cell contains an anti-repressor element.
  2. 2. The method of claim 1, wherein the anti-repression subclass element is positioned near the target gene and is capable of causing the genomic DNA to form a supercoiled structure, thereby inhibiting qPCR signaling of the unpretreated genomic DNA.
  3. 3. The method for detecting the copy number of a gene of interest in a recombinant cell according to claim 1, wherein the recombinant cell is a recombinant mammalian cell, preferably a Chinese Hamster Ovary (CHO) cell.
  4. 4. A method for detecting the copy number of a gene of interest in a recombinant cell according to claim 1, wherein the temperature of the thermal denaturation treatment is 80 to 100 ℃, preferably 85 to 99 ℃, further preferably 95 to 98 ℃, and/or the time of the thermal denaturation treatment is 5 to 30 minutes, preferably 5 to 20 minutes, further preferably 5 to 10 minutes; Preferably, the genomic DNA is cooled after the thermal denaturation treatment, preferably to 0-10 ℃, further preferably in an ice bath or 4 ℃; or the temperature of the enzyme digestion treatment is 37 ℃ for 5-60 minutes, preferably 5-15 minutes, and more preferably 10 minutes; Preferably, the cleavage treatment cleaves genomic DNA by using a non-specific endonuclease to remove supercoiled structure, or the thermal denaturation treatment denatures genomic DNA by heating to remove supercoiled structure, and the pretreatment does not destroy the target gene sequence; Preferably, the cleavage treatment uses a tool enzyme that uses nonspecific endonuclease activity for random DNA fragmentation.
  5. 5. The method of claim 1, wherein the anti-repression subclass element is a UCOE element.
  6. 6. The method for detecting copy number of target gene in recombinant cells according to claim 1, wherein the digestion treatment is performed by using a digestion system comprising 10X FRAGMENTASE REACTION BUFFER V < 2 >, DSDNA FRAGMENTASE, genomic DNA and water; preferably, the enzyme is inactivated by heating or purifying after the enzyme digestion treatment; Preferably, the qPCR assay uses the TaqMan probe method; Preferably, the qPCR reaction procedure: initial denaturation at 95 ℃ for 30s; The reaction was cycled (40 times) at 95 ℃,5s, 60 ℃ and 35s. Preferably, the pretreatment is performed prior to formulation of the qPCR reaction system, preferably after dilution of the extracted genomic DNA.
  7. 7. The method for detecting the copy number of a target gene in a recombinant cell according to claim 1, wherein the determination of the copy number of the target gene comprises relative quantification or absolute quantification, preferably by calculating the copy number ratio of the target gene to the reference gene by a standard curve method; Preferably, the method further comprises providing a control cell line group without anti-repression subclass elements, performing the same qPCR assay to verify that the pretreatment does not disrupt the gene sequence of interest; preferably, the method is used to assess the genetic stability of cell lines, preferably detected at the Main Cell Bank (MCB), working Cell Bank (WCB) and end-of-production cell (EOPC) stages of CHO cells; preferably, the method is applied to high throughput screening of recombinant cell lines, preferably CHO cell lines containing UCOE elements; preferably, the method is used for evaluating genetic stability of recombinant protein drug development.
  8. 8. A kit for determining the copy number of an exogenous gene in a recombinant cell containing an anti-repressor element comprises a genomic DNA extraction reagent, a reagent or buffer for thermal denaturation or cleavage treatment, qPCR primers and probes specifically designed for the target gene; Optionally, internal reference gene primers and probes are included.
  9. 9. Kit according to claim 8, wherein the kit further comprises a non-specific endonuclease and a corresponding reaction buffer, preferably DSDNA FRAGMENTASE and 10 x Reaction Buffer v2 thereof; alternatively, the kit is suitable for exogenous gene copy number detection and genetic stability assessment of CHO cell lines containing UCOE elements.
  10. 10. Use of the method of any one of claims 1-7, the kit of claim 8 or 9 in the assessment of genetic stability of recombinant cells; Preferably, the use comprises accurately determining the exogenous gene copy number in CHO cells containing the UCOE element; Preferably, the detection is performed at the Main Cell Bank (MCB), working Cell Bank (WCB) and production end cell (EOPC) stages of CHO cells; Preferably, the use comprises high throughput screening of recombinant cell lines, preferably CHO cell lines containing UCOE elements; Preferably, the use comprises genetic stability assessment for recombinant protein drug development.

Description

CHO cell gene copy number detection method Technical Field The invention relates to the technical field of gene detection, in particular to a detection method for genetic stability of a CHO cell strain, and more particularly relates to detection of CHO cell gene copy number by utilizing a genome subjected to enzyme digestion or thermal denaturation pretreatment, and interference or inhibition of a supercoiled structure of the genome on qPCR (Real-time Fluorescent Quantitative Polymerase Chain Reaction, real-time fluorescence quantitative polymerase chain reaction) detection is relieved. Background CHO cells (CHINESE HAMSTER Ovary, chinese hamster Ovary cells) have significant advantages in the production of biotherapeutic recombinant proteins, including adaptation to serum-free suspension culture, ease of genetic manipulation, high-yielding monoclonal screening capacity, and post-translational modification functions similar to human origin, while being less sensitive to human viruses. However, the cells also present risks of genetic instability, such as gene rearrangement, mutation, epigenetic silencing, and multicopy expression pressure, among others. Therefore, regulatory authorities place clear demands on their genetic stability to ensure consistency and safety of drug production. UCOE (Ubiquitous Chromatin Opening Element, ubiquitin-like chromatin opening element) is an unmethylated CpG enrichment sequence derived from a housekeeping gene promoter region, and can effectively inhibit DNA methylation, inhibitory histone modification and other epigenetic silencing mechanisms by regulating and controlling a chromatin structure, and is matched with the upstream of a gene expression frame, so that long-term, stable and high-level expression （Neville, Jonathan J et al. "Ubiquitous Chromatin-opening Elements (UCOEs): Applications in biomanufacturing and gene therapy." Biotechnology advances vol. 35,5 (2017): 557-564.）. of a transgene in mammalian cells can be realized, the UCOE element can remarkably enhance the expression stability and yield of recombinant proteins in the application of biopharmaceuticals, accelerate the development of high-yield cell strains, reduce the production variability and the gene silencing risk in long-term culture, and further improve the efficiency and economy of upstream biological manufacturing processes. In CHO cell line construction, accurate quantification of UCOE vector copy number is particularly critical for screening "low copy-high expression" type clones. Exogenous gene copy number refers to the number of genes of interest that are integrated into the host cell genome by genetic engineering means. The insertion of UCOE into a transgenic vector creates a localized, open topological domain near the integration site, characterized by a higher level of negative supercoiled, which further facilitates sustained, high level expression of the transgene downstream thereof, but it is noted that DNA in supercoiled form has significantly lower amplification efficiency in qPCR than relaxed form （Chen, Jinsong et al. "DNA supercoiling suppresses real-time PCR: a new approach to the quantification of mitochondrial DNA damage and repair." Nucleic acids research vol. 35,4 (2007): 1377-88.）, and thus the unique chromatin open structure of UCOE may have a significant impact on qPCR-based absolute quantitative detection values. The anti-repression sub-element enhances chromatin openness and forms exogenous gene region structural modification, the pre-denaturation step of qPCR is insufficient to untwist a high-stability supercoiled structure, and the primer binding efficiency is obviously reduced, so that the copy number of transgenes in a genome cannot be truly reflected, and the judgment of the genetic stability of a cell strain or a cell bank can be influenced. The mesogenic regulations or guidelines provide for genetic stability (CHO recombinant expression) and for cell lines containing DNA expression constructs, the identity of these constructs should be determined between MCB (MASTER CELL Bank ), WCB (Working Cell Bank, working cell Bank) and EOPC (End-of-Production Cells), including the copy number of inserted foreign genes, the site of insertion of chromosomes, the sequence of the gene of interest, etc. The copy number of the exogenous gene plays a key bridge role between genetic engineering, cell phenotype and the quality of the final product, and is a scientific basis for guaranteeing the high efficiency, stability, safety and compliance production of therapeutic proteins. The screening of high-yield cell strains can be effectively guided by accurately measuring the copy number of the exogenous gene, the genetic stability of the cell lines is evaluated, and the consistency of the product quality is ensured so as to meet the declaration requirement of a regulatory agency. In the construction of CHO cell lines and the reporting process of related biological medicines, the detection of