Detecting residual cho host cell dna using the agilent mx3005p qpcr system authors charmian cher, ph. Prepare standard dilutions as follows in 1x alphalisa dna detection buffer mix solution gently with pipette, excessive pipetting andor vortexing could break down dna, change tip between each standard dilution. Residual host cell dna and protein quantities contained in final. In the following experiments, adherent cells were stained with dapi for host cell nuclei and parasites dna detection and markers commonly used for macrophage classification the integrin. Lance ultra dna contam hu kittrf31ctrf31mperkinelmer. Host cell proteins hcps constitute a major part of processrelated impurities during biologics production. Corporate template for external use thermo fisher scientific. The accurate detection, identification and quantification of hcps during biotherapeutic development is thus critical in ensuring patient safety, and is a regulatory requirement. Host cell dna levels are also a contaminant, and regulatory bodies such as the world health organization, the eu and the fda have set maximum levels that they consider safe in products. Application note nucleic acid extraction highperformance. First and foremost, the composition is closely associated with the host expression. Pdf residual dna analysis in biologics development. Drug characterisation to ich q6b requires the accurate detection and quantitation of residual host cell dna.
Among these methods, qpcr is considered to be the most practical for residual dna quantitation. The pcr amplification kits contain dna extraction reagents, dna calibrators, and validated specific primers that are compatible with your pcr system. Depending on the microscope configuration and the equipped filters, different markers can be analyzed in combination. Specific detection of residual cho host cell dna by real. Cho host cell dna detection kit in tubes d550t 761. The average genome size of an aav2 is 5000 base, therefore. Specific detection of residual cho host cell dna by realtime. Cygnus host cell dna detection kits utilize picogreen dna binding dye and include dna extraction reagents and cellline specific dna calibrators. To quantify host cell dna, quantitative realtime pcr offers superior sensitivity with a limit of quantification in the subpicogram range. Manual alphalisa host cell residual dna detection kit al331. Residual hostcell dna in biopharmaceutical products. Probes are labeled with radioactive tags or fluorescent dyes and bind to complementary targets during hybridization. Another major advantage of our approach being based on the host response to the infection, in comparison to tests based on the detection of the infectious organisms, for example via the t2 biosystems system t2 biosystems, 2020, which are based on particular dna sequences of a limited set pathogenic strains, is that our assay can be used to. Existing pcr systems cover a wide variety of host cell types like e.
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. The amount of residual hcps in drug product is generally considered a critical quality attribute cqa, due to their potential to affect product safety and efficacy. Building on its expertise in host cell protein analysis, cygnus technologies developed a broad range of. Host cell proteins hcps are unique to their respective hosts types and strains used for biologics production. Detection plate configuration and assay settings for detection using 96 biosensor mode.
Rapid functionalisation and detection of viruses via a. C6 vero cell hela mdck cap host cell dna detection cho e. The removal of host cell impurities is a critical step in the purification of biopharmaceutical products. The presence of residual dna carried by biological products in the body may lead to an. An emerging approach for parallel quantification of. To ensure product quality, the amount of residual dna in a drugs final dosage form. To test this possibility, raav9 samples produced in hela cells with or without spike of 1,000 pgml hela dna were either di gested according to figure 1a or. Using the threshold total dna assay molecular devices it is possible to perform the quantitation of residual dna with high precision and sensitivity up to 4 pgml. Development and validation of quantitative realtime pcr for the. Host cell residual dna contamination lance ultra trfret detection kit, 500 assay points false the lance ultra residual dna contamination detection kit is designed for detection and quantitation of dna contaminants in buffered solution and cell culture media using a homogeneous trfret nowash steps, no separation steps assay. Pdf development and validation of quantitative realtime pcr. A major challenge is the accurate and sensitive quantitation of host cell dna impurities in both the purification process and in drug substance samples.
The composition and abundance of hcps present in various steps of manufacturing processes and in the. Host cell dna and other dna species is a common processrelated impurity encountered when manufacturing viral vectors for gene therapy. The nv1pcr protocol may offer a rapid screening tool to detect samples contaminated with host cell dna. Design and validation of a new method to detect and. The challenges of hcp detection include 1 low levels of residual hcps present in large excess of product protein, 2 the assay must measure a large number of different protein analytes, and 3 the population of hcp species may change during process. Evaluation of hostbased molecular markers for the early. Sep 17, 2018 presence of host cell protein hcp contaminants in the final product, even at very low levels, may elicit unpredictable and serious immunogenic response in humans. Three methodsdna hybridization assay, threshold assay, and quantitative pcrhave been recommended by regulatory agencies for residual host cell dna quantitation 14, 15. Therefore, quantification of residual host cell or process related dna in purified product is a prerequisite for product release of biopharmaceuticals. Test samples were prepared by spiking cho cell dna standard. Viruses must first get into the cell before viral replication can occur. Rapid functionalisation and detection of viruses via a novel. The qrtpcr technique is highly sensitive and specific.
Host cell protein testing by elisas and the use of orthogonal. Highperformance extraction and quantitation of hostcell residual. Upon internalization of dnaharboring evs by human monocytes, p. Dna was first extracted from whole blood with a blood dna extraction kit, amplified via rpa, and applied to paper for snipr detection figure 6e. Due to safety issues, the maximum amount of dna allowed in a final dose administrated to humans are set to 10 ng per dose 1. Proper performance of the whole analytical procedure, including sample preparation, dna extraction and pcr analysis is thoroughly. 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. Guidelines for effective duplex detection of residual host cell dna and the. Analysis of residual dna and rna of host cells within biopharmaceuticals and other biotechnological products. Viral replication is the formation of biological viruses during the infection process in the target host cells. The individual requirements of these assays will depend of the particular stage in the drug lifecycle, although high sensitivity is needed to meet the guidelines of the world health organization1,2.
Detecting and quantifying residual host cell dna is an essential impurity test for all biopharmaceutical products. Omitting digestion resulted in underdetection of residual hela. Detecting residual cho host cell dna using the agilent. Highly sensitive and accurate methods for detection and quantitation of low level dna are needed to meet this requirement. Development and validation of quantitative realtime pcr for. Signal detection is achieved with autoradiography or by phosphor or fluorescenceimaging systems, and the signal detected is proportional to the amount of dna.
Improved enzymatic hostcell dna removal in aav manufacturing. Net rfu rfu viral sample rfu 0 ngml standard titer. A hostproduced quorumsensing autoinducer controls a. One critical challenge associated with hostcell dna impurity testing is that. Therefore, it is a regulatory requirement to monitor the removal of.
A digestionfree method for quantification of residual host cell. Host cell protein analysis in biopharma technology networks. Overcoming challenges of host cell dna removal in vaccine. Our results suggest that dna damage mediated by gp62 and qs communication mediated by dpo, vqma phage, and gp62 constitute two distinct triggers that promote phage vp882induced host cell lysis. Residual host cell dna analysis tools cygnus technologies. This primer set could detect hamster dna to 30 fg level, this level of sensitivity is within the limits recommended by the fda and eu as specified in their respective documents. Gp62 is a phage antiterminator that regulates an operon involved in lysis. Detection and quantitation of residual host cell dna. Sgs host cell impurity testing is a vital service for quality and safety assurance in your products. Critical issues for biopharmaceutical development protein quantification workshop, waters. A reliable and sensitive qpcr method for detecting the residual dna of host cells is required for biological products expressed from cho cell.