Application note nucleic acid extraction highperformance. In this paper we describe an optimized assay based on realtime polymerase chain reaction pcr for the quantification of residual per. Host residual dna hrdna is an impurity and needs to be monitored in the purified drug to ensure purity and safety. Realtime polymerase chain reaction pcr has become a popular. There are some similarities and some differences between pcr and dna replication. A key finding of the present work was the observation that dna is carried into nonphagocytic host cells via omvs and that this dna was detectable by pcr in the nuclear fraction of cells. Residual host cell dna analysis tools cygnus technologies. The current method for residual dna quantification in raav was adapted from protein programs and required sample digestion by proteinase prior to qpcr analysis. Pcr is an in vitro process occurring in a test tube that imitates the in vivo process occurring in a living cell of dna replication.
Realtime polymerase chain reaction pcr has become a popular technology for dna quantitation and monitoring of process impurities associated with biomanufacturing. Basic steps of gene cloning 4 when the host cell divides, copies of the recombinant dna molecule are passed to the. Picogreen and dna threshold being two of the historical means of determining dna levels. Quantitation of host cell dna contaminate in pharmaceutical. In order to reduce the risk of introducing contaminations and to increase the throughput, the assay was designed to require minimum sample handling. Us5393657a detection of residual host cell dna by pcr. They demonstrated that inactivated viruses do not attach to cell monolayers and can be easily removed by rinsing the monolayer.
In the case of host cell proteins, immunological assays e. In the very earliest days of the polymerase chain reaction amplifications were carried out using water baths and lab timers and the best available dna polymerases of the time, klenow or t4 dna polymerase. Among impurities to be eliminated during the downstream purification process, two components which are of major interest for safety and tolerance reasons are residual host cell dna and residual host cell proteins hcp. Jul 07, 2015 introducing of recombinant dna rdna into host cell. Mar 14, 2020 timely detection of hcc recurrence and its clonality is required to implement salvage therapies appropriately. The vector acts as a vehicle that transports the gene into a host cell usually a bacterium, although other types of living cell can be used. The restriction part of the system is an enzyme that recognizes a speci. Among these methods, qpcr is considered to be the most practical for residual dna quantitation due to their sensitivity, accuracy, precision, and timesaving features. Available in tube or 96well format for dna extraction step. Analysis of residual dna and rna of host cells within biopharmaceuticals and other biotechnological products. To facilitate screening of large numbers of transformants, set up pcr reactions using freshly grown cells as a source of chromosomal dna template.
That can be achieved, for example, by a random priming procedure. Typical qpcr protocols involve dna extraction steps complicating sample handling. In this article we will discuss about the gene isolation and cloning of dna. Here, we describe and demonstrate an in vitro procedure, ligationduringamplification lda, for selective amplification of closed circular dna using sequencespecific primers. Regulatory guidelines that cover the development of biopharmaceutical products require testing of host cell deoxyribonucleic acid dna impurities. Usp reference standards for measurement of residual host. Residual host cell dna is a process impurity in recombinant biotherapeutic products. Accuseq realtime pcr software 1 license 4443421 service. Sep 10, 2019 methylation marks are cell, tissue, and organ type specific. In theory, only one single template strand is needed to generate millions of new dna molecules. Pcr was invented in 1983 by the american biochemist kary mullis.
Jan 04, 2020 a fragment of dna, containing the gene to be cloned, is inserted into a suitable vector, to produce a recombinant dna molecule. Digiusto 1laboratory for cell and gene medicine, department of pediatrics, stanford university school of medicine, palo alto, ca 94304, usa. Introducing of recombinant dna into host cell biology go easy. Effects of ph, conductivity, host cell protein, and dna. Goren,1,2 rea globus,1 shahar molshanskimor,1 and udi qimron1,3, 1department of clinical microbiology and immunology, sackler school of medicine, tel aviv university, tel aviv 69978, israel 2these authors contributed equally 3lead contact. Today however the most common means of detecting and quantitating host cell dna is by quantitative pcr qpcr technology.
Molecular cell technology extending the host range of bacteriophage particles for dna transduction ido yosef,1,2 moran g. The life technologies resdnaseq host cell residual dna quantitation systems measure levels of residual dna from common host cell lines used in. This kit can be used with dna from different prokaryotic and eukaryotic species. Nuanualsuwan and cliver studied interference with virus attachment to cell monolayers as a way of assessing viral inactivation by uv, hypochlorite, and heat. These are several methods to introduce recombinant vectors and these are dependent on several factors such as the vector type and host cell. A digestionfree method for quantification of residual host cell.
Residual hostcell dna in biopharmaceutical products. Droplet digital pcr ddpcr supermixes biorads ddpcr supermix for residual dna quantification is a readytouse 2x supermix optimized to deliver maximum pcr efficiency, specificity, and sensitivity for direct quantification of residual host cell dna hcd with the qx100, qx200, or qx200 autodg droplet digital pcr system. Doublestranded host cell dna consists of two complementary strands of dna that are held together by hydrogen bonding. Pdf quantification of residual host cell dna in adenoviral.
Quantitative detection of residual porcine host cell dna by. Pbmc peripheral blood mononuclear cell pcr sscp polymerase chain reaction singlestrand conformation polymorphism pd pharmacodynamics pk pharmacokinetics ppnd prepostnatal development rcdna residual cellular dna rdnaderived prepared by recombinant dna technology rflp restriction fragment length polymorphism. Direct realtime quantitative pcr for measurement of host. Extending the host range of bacteriophage particles for dna. Direct realtime quantitative pcr for measurement of hostcell. Levels of host cell dna needs to be monitored during process development and validation. Specific detection of residual cho host cell dna by real. Dna replication in vivo pcr in vitro what does it accomplish. Detecting residual cho host cell dna using the agilent mx3005p qpcr system authors charmian cher, ph. Pdf dna isolating and amplifying by pcr and determining.
Cho cell dna standard recovered from the m4 and m5 matrices after extraction using the prepseq kit and kingfisher flex system was quantified using the standard curve in figure 2. Aim of practical series was to isolate dna from buccal cheek cells of btn 322 male population and used it as template dna in polymerase chain reaction pcr of the yap element on the ychromosome and analysis the amplified dna by isolating it on. Usp reference standards for residual dna testing of. Detecting residual cho host cell dna using the agilent. One critical challenge associated with host cell dna impurity testing is that recombinant proteins e. The removal or reduction of host cell dna is, therefore, mandatory for biopharmaceuticals, requiring accurate and sensitive tests to quantify such impurities and to meet guidelines established by regulatory agencies. The use of biologically derived drugs, in which host cell lines are used to express biological product, has increased dramatically in recent years. The products still contains fragments of dna from the host cells even after. Potency estimation of live virus vaccines using infectivity pcr. Design and validation of a new method to detect and quantify. Data are collected throughout the process to monitor the increase in pcr product formation, enabling quantitative determination of the starting amounts of dna in a sample. There is a need for standardised procedures and physical reference standards that can be used by the manufacturers. Pbmc peripheral blood mononuclear cell pcr sscp polymerase chain reactionsinglestrand conformation polymorphism pd pharmacodynamics pk pharmacokinetics ppnd prepostnatal development rcdna residual cellular dna rdnaderived prepared by recombinant dna technology rflp restriction fragment length polymorphism. Among these methods, qpcr is considered to be the most practical for residual dna quantitation.
Goren,1,2 rea globus,1 shahar molshanskimor,1 and udi qimron1,3, 1department of clinical microbiology and immunology, sackler school of medicine, tel aviv university, tel aviv 69978, israel 2these authors contributed equally. Among impurities to be eliminated during the downstream process, residual host cell dna is a major interest for safety. Pdf development and validation of quantitative realtime. Prior to pcr, it would have been impossible to do forensic or genetic studies with this small amount of dna.
This kit contains reagents for dna extraction as well as calibrated dna concentrate and primers for dna amplification. The recombinant vector carrying foreign dna needs to be transferred into the suitable host cell. For reasons of safety, residual hostcell dna must be eliminated during processing. Specific detection of residual cho host cell dna by realtime pcr. It is fundamental to much of genetic testing including analysis of. A digestionfree method for quantification of residual host. Amplification of closed circular dna in vitro nucleic. Clinical dna publications and presentations 2015 presentationsja buck, pm vallone, and j harenza investigation of reference sequence and variant caller effects on single nucleotide polymorphism snp calls made across six bk virus genotypes. M5 matrices were spiked with cho cell dna standard to obtain final pcr concentrations of 100, 10, and 1 pgwell. The hallmark of a great system is not that it makes one aspect better, but that it makes everything betterin this case, highly efficient process characterization. The formation of new combinations of genetic material by the insertion of nucleic acid produced outside the cell into a virus, bacterial plasmid or any other vector system to allow its incorporation into a host. The present invention relates to a method for quantifying residual host cell genomic dna in recombinant protein biologics using quantitative real time pcr. Thus, spiking studies were used to demonstrate and understand.
The host cell derived impurities pose safety concerns and the regulatory agencies have defined acceptable levels of such impurities in the protein drug 3. The resdnaseq quantitative dna kits use taqman quantitative pcr to perform rapid, specific quantitation of subpicogram levels of residual hostcell dna. For sensitive realtime pcr detection of residual host cell dna and an internal control using sequencespecific probes. The quan titative polymerase chain reaction qpcr is widely. Dna cloning dna cloning allows a copy of any specific part of a dna or rna sequence to be. The realtime pcr assay may thus be a promising new tool for the quantitative detection and clearance validation of residual e. A cellfree dna metagenomic sequencing assay that integrates. Jun 14, 2019 it is well documented that illegitimate dna, including genomic dna of the host cells, could become encapsidated. Residual host cell dna in recombinant pharmaceuticals has been identified as a potential risk factor that must be quantitated carefully both during the manufacturing process and in the final product. Determination of viral attachment to the host cell by pcr.
Three methods dna hybridization assay, threshold assay, and quantitative pcr have been recommended by regulatory agencies for residual host cell dna quantitation 14, 15. For extraction information, see the prepseq residual dna sample preparation kit user guide pub. Dna replication starts when dna helicase unravels the doublehelix structure to expose singlestranded dna. Currently, realtime quantitative pcr qpcr based methods are widely employed for quantification of hrdna, however, digital pcr technology promises higher assay sensitivity and precision. Demands of the guidelines suggest that those probes be manufactured independent of the dna sequence. Dna, generated from junctions of hepatitis b virus hbv integration in the hcc chromosome, as a circulating biomarker for this clinical setting. Host cell dna amplification kits cygnus technologies host cell dna amplification kits are used to measure the level of host cell dna contamination in products manufactured by recombinant expression in cho or e. Gene cloning requirements, principle, steps, applications. Highperformance extraction and quantitation of hostcell residual. Development and validation of quantitative realtime pcr for. The cleavage is on both strands of the dna so that a doublestranded break is made. The first residual dna assays were based on dna hybridization, wherein a dna probe created from host cell dna detects and quantifies the amount of complementary dna present in the product under assay. Development and validation of quantitative realtime pcr.
This would typically involve the demonstration of clearance of intentionally added host cell dna, of the size distribution expected in the drug manufacturing process, at greater than the expected concentration of residual dna to steps in the process and quantifying the reduction in dna. Bacterial membrane vesicles transport their dna cargo into. Development and validation of quantitative realtime pcr for the. Proposes a new hostcell dna extraction based on 96deepwell plates to. Application of pcrbased methods to assess the infectivity. Until now, closed circular dna, the replicatively competent form of many dna molecules, could only be amplified in appropriate host cells. Three methodsdna hybridization assay, threshold assay, and quantitative pcrhave been recommended by regulatory agencies for residual host cell dna quantitation 14, 15. Polymerase chain reaction pcr is a method used widely in molecular biology to make millions to billions of copies of a specific dna sample rapidly, allowing scientists to take a very small sample of dna and amplify it to a large enough amount to study in detail. Guidelines on the quality, safety, and efficacy of. The next step in a gene cloning experiment is to introduce the recombinant molecule into living cells usually bacteria, which will then grow and divide to produce clones brown, 1990. Extending the host range of bacteriophage particles. This has created a need to quantify residual host cell dna during manufacturing and in final products.
Original article detection of replication competent lentivirus using a qpcr assay for vsvg lindsey m. During the essential dna denaturation step, 94 o c or 95 o. Pcr primers, a few issues surrounding dna polymerases should be presented. Quantification of residual host cell dna by realtime quantitative pcr download pdf info. Pcr using components of the resdnaseq quantitative. The basic principle of a hybridization assay is binding of dna probes to immobilized and denatured host cell dna.
Usp biologics tm residual host cell dna must be minimized as much as possible during production of therapeutic proteins. Due to the theoretical potential for the transfer of oncogenes from the host cells, regulatory agencies have set allowable limits between 100 pgdose and 10 ngdose depending on the cell line used as well as the mode and frequency of dosing. Detection of replication competent lentivirus using a qpcr. Dna cloning dna cloning is a technique for reproducing dna fragments. A droplet digital pcr method for cho host residual dna. Development and validation of quantitative realtime pcr for the detection of residual cho host cell dna and optimization of sample pretreatment method in biopharmaceutical products. The method comprises using oligonucleotides with sequences directed to those of repetitive host cell line dna sequences, as primers in a pcr reaction. Currently, realtime quantitative pcr qpcr based methods are widely employed for quantification of hrdna, however, digital pcr technology. Nov, 2014 based on the quantification of genomic dna purified from the sample by quantitative realtime pcr step b of the method of the invention, the quantity of residual genomic dna from the eukaryotic host cell in the recombinant protein product can be easily determined step c by comparing the signal from the test sample with a standard curve. Samples with very high concentrations of drug substance can be tested with minimal dilution, effectively. Accuracy and sensitivity of residual dna detection by qpcr is not. Like other drug substances manufactured in cell lines, raav vectors are commonly contaminated with host cell dna, and the levels must be carefully monitored. Pdf development and validation of quantitative realtime pcr.
Quantitative detection of residual porcine host cell dna. To assay hostcell dna content in biopharmaceutical products derived from porcine sources, this study applies the quantitative realtime polymerase chain reaction qpcr method. Residual host cell dna contamination can be introduced during production of biotherapeutics. Relative standard deviation introduction many therapeutic protein including monoclonal antibody mab drugs are manufactured in cho cells 1,2. A range of different chemistries can be used to detect hostcell dna when using qpcr, including the commonly used sybr green i dye molecular probes and sequencespecific reporters such as hybridization and 5.
Quantification of residual host cell dna in adenoviral. Dna from any foreign dna that enters their cytoplasm. There are a number of methods which can be used to measure host cell dna. We describe a pcr method to quantitate contaminating levels of host cell dna in clinical plasmid dna preparations intended for human gene therapy. Here, we describe a direct qpcr approach without dna extraction. A method for detecting the presence of small mounts of contaminant dna, present in an amount of at least about 0. The alphalisa host cell residual dna detection kit is designed for detection and quantitation of dna contaminants in cell culture supernatants using a homogeneous nowash steps, no separation steps assay. The present invention relates to the field of analytical molecular biology and specifically to the field of dna quantitation.
While several methods can be applied for host cell dna detection, pcr is one of the most robust used by industry today. The chinese hamster ovary cho cells are the preferred host for manufacturing therapeutic biomolecules in the biopharmaceutical industry. Detection and quantitation of residual host cell dna. In particular, the present invention relates to quantitation of residual host cell dna in recombinant protein biologics using quantitative real time pcr. Realtime quantitative pcr qpcr is important for quantification of residual host cell dna resdna in therapeutic protein preparations. The resdnaseq quantitative dna kits use taqman quantitative pcr to perform rapid, specific quantitation of subpicogram levels of residual host cell dna.
To assay host cell dna content in biopharmaceutical products derived from porcine sources, this study applies the quantitative realtime polymerase chain reaction q pcr method. Host cell residual dna alphalisa detection kit, 100 assay. Cell replication the process of dna synthesis and replication in a cell involves dna helicase, dna polymerase, dna template, and deoxynucleotides. The applied biosystems resdnaseq host cell residual dna quantitation systems measure levels of residual dna from common host cell lines used in the production of biopharmaceutical products.
A range of different chemistries can be used to detect host cell dna when using. Development and validation of quantitative realtime pcr for the detection of residual cho host cell dna and optimization of sample. For reasons of safety, residual host cell dna must be eliminated during processing. Request pdf specific detection of residual cho host cell dna by realtime pcr chinese hamster ovary cells have been widely used to manufacture recombinant proteins for human therapeutic use.
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