Furthermore, it is a regulatory requirement. We have designed and implemented a software system, named PhyloID, that can be used to detect putative adventitious agents in biological samples characterized by next-generation sequencing. Adventitious virus detection. Sterility Testing. Detection of adventitious agents Identify and quantify adventitious agents present in your sample The safety of vaccines, monoclonal antibodies, cell and gene therapy products and other biologically-derived products is strictly regulated and required for a product release. Cell passages followed by direct immunofluorescence were carried out to check viability of the mentioned adventitious agents. Our HTS method is a general-purpose non-specific method that can be used to detect both known and unexpected viruses in multiple types of samples . In the detection of adventitious agents in bioprocessing, it is necessary to detect the lowest abundance of virus or bacterial cells, often contained in a large volume of the original sample. Outline of Talk. Real-time detection methods could be translated into gene therapy platforms. Sterility Testing. Novel biosensors present avenues for real-time adventitious agent detection. CO1 Barcode Assay. Testing of adventitious agents can be done using various methods such as in-vivo tests, in-vitro tests, nucleic acid based methods, etc. According to the developers, their anomaly detection model can be used to detect the presence of adventitious microbial contamination in cell cultures within a few minutes, overcoming one of the key challenges with cell therapy manufacture - the long (multiple day) incubation periods required for sterility testing. Adventitious Agents / Virology Testing. Pre-studies to determine potential sample cytotoxicity. Virus Bank Characterization. These pathogens can enter the manufacturing process at multiple stages and impact yield and safety. Raw Material Testing. Consult our experts. Group: CAMP. IRG_CAMP_2021_020. Senior/Research Engineer - Adventitious Agent Detection by CRISPR. Supaporn Phumiamorn 11 March 2016 34 Sample selection -Cellular genome (DNA) -Transcriptome (RNA) -Virus particle Amplification schemes -Family-specific primers -Degenerate primers, etc. Detection of adventitious agents in three indicator cell lines. High-throughput sequencing (HTS) is an approach for such testing; however, an optimized testing method is one with a sample-processing pipeline that can help detect any viral adventitious agent. Traditional methods for adventitious agent testing are well established and typically rely on biological amplification and detection, which can take several weeks. Rapid Detection of Adventitious Agents . Adventitious agent testing in biomanufacturing requires assays of broad detection capability to screen for as many infectious agents as possible. Electron Microscopy Services - Adventitious Agents Electron microscopy services provide high-resolution images of viral or biological specimens at an ultrastructural level to detect the presence, absence, and quantitation of microbial or adventitious agents in biological fluids or in cell substrates. Ideally, products could be screened throughout the manufacturing and production process, enabling real-time or rapid detection of any potential contamination in order to take action as soon as possible. With a high depth of coverage, the sensitivity is sufficient to detect very low levels of DNA contamination. Such methods have already been successfully applied to detection of adventitious agents in vaccines [1], cell lines [6,7], serum [8,9] and bioreactors [10] and multiple laboratory and informatics . The following sections provide a more in-depth overview of NGS and specifically its use for adventitious . Adventitious agents include numerous lifeforms such as bacteria, fungi, viruses, mycoplasma, and others that are inadvertently introduced into biological systems. Molecular analyses indicated that frequencies of . NGS-Based Detection of Adventitious Agents Experimental set-up We explored the utility of next-generation sequencing using a model for: 1. Both random PCR combined with MPS and microarray tests have the advantage to detect a much wider range of potential extraneous agents than the currently mandated methods like cell cultures or agent-specific PCR . Oncogenicity & Tumorigenicity Testing . Risk evaluation may support potential regulatory actions by the national regulatory authority (NRA) and/or national control laboratory (NCL), the relevant inspectorate (for example, the Good Manufacturing Practice The adventitious agent tests required today detect contaminating adventitious agents in biopharmaceutical products throughout the pre-clinical, clinical and licensing stages of the product lifecycle. Bioinformatics will not be covered. The methods used have ensured that very few products have reached the market with viral contaminants, and in the cases where they have, the contaminants have not posed a risk to human health. They present significant problems to the stability of cell cultures and the sterility of manufacturing products. They present significant problems to the stability of cell cultures and the sterility of manufacturing products. Blind study to detect any DNA or RNA viruses present 11 The presence of neutralizing antibodies against pestiviruses was determined. According to the developers, their anomaly detection model can be used to detect the presence of adventitious microbial contamination in cell cultures within a few minutes, overcoming one of the key challenges with cell therapy manufacture - the long (multiple day) incubation periods required for sterility testing. A highly specific assay such as the ViralSEQ Vesivirus Assay, is needed to detect viruses that are common in biopharmaceutical plants that standard culture tests are unable to detect. 2:00 Next-Generation Sequencing Detection of Adventitious Agents Brenda Richards, Staff Scientist, Functional Genomics, Genzyme, a Sanofi Company Next-generation sequencing (NGS) is an emerging technology for pathogen detection in biopharmaceutical manufacturing allowing for the identification of both known and novel microorganisms. The Accukit Adventitious Agents (AA20) assay and standard system is an NGS-library prep workflow for 20 common adventitious agents found in Chinese hamster ovary cell bioproduction processes. Adventitious Agents. Posted on 11 November 2021. Our laboratory has consistently found that the source of bovine viral diarrhea contamination of CCLs has been the use of contaminated fetal bovine cell culture enrichment serum. Based on the results, the adventitious virus test using HTS technology is capable of detecting adventitious viruses at least at 10 4 genome copies per mL in the viral vaccine crude harvest matrix.. Several alternative and rapid microbiological methods (ARMMs) are available for detecting and enumerating microorganisms and adventitious agents during testing, and regulatory authorities are encouraging industry to . Design a testing plan around your needs. This paper serves to summarize current testing approaches and challenges, along with opportunities for the use of these new technologies. NGS-based detection of adventitious agents can fulfill a critical unmet need in the detection of known and novel microbes in biopharmaceutical manufacturing. Additional wor k is needed to determine if these technologies could be suitable screening tool s for adventitious agents . Adventitious Agents Detection Test Bioburden Testing Cell Line Authentication and Characterization Tests Endotoxin Tests Residual Host Contaminant Detection Tests Sterility Testing Other Tests . A similar sample is prepared for in vivo viral adventitious agent testing. The first is the assess - ment of cytopathic effect (CPE) on the detector cell. The detection of virus in biologic drug manufacturing facilities and the resulting drug preparations is of critical importance. The detection of adventitious agents, including viruses, is an important component of biopharmaceutical production. detection technologies Transcript pdf available: www.fda.gov May, 2013: WHO Cell Substrate Study Group (Beijing) discussed DNA-based detection of adventitious agents in cell substrates Nov. 2013: PDA/FDA Advanced Technologies for Virus Detection in the Evaluation of Biologicals: Applications and Challenges. The routine testing method for detecting of viral adventitious agents is based on the observation of cytopathic effects (CPEs), hemadsorption of erythrocytes, or immunofluorescence on the indicator cells. This will also include a user requirements specification to facilitate the . Design a testing plan around your needs. detect possible contaminating viruses. Adventitious Agent Detection . NGS testing for adventitious viruses can offer faster turnaround time and broad detection of adventitious agents, including known and novel viruses. This time-consuming approach is not compatible with novel treatment modalities such as modified T-cell therapy which requires a rapid time-to-result, nor with antibody manufacturing processes which are undergoing intensification in . Adventitious agent testing in biomanufacturing requires assays of broad detection capability in order to screen for as many infectious agents as possible. Back to Job Listing. Viral Detection Technologies Must Continue to Evolve Viral Detection Technologies Must Continue to Evolve Challener, Cynthia A. Charles A. SWOFFORD, Massachusetts Institute of Technology, U.S.A. 9:45 am NGS Implementation Strategy for Adventitious Virus Detection in an Adenovirus Vaccine Aurora SIGNORAZZI, Janssen Vaccines & Prevention B.V., Netherlands 10:10 am Break 10:35 am Next Generation Sequencing to Unravel a Positive Signal in QC Investigation: Pitfalls and Learnings . Thus, next-generation sequencing-based detection of adventitious agents is a valuable approach that can fill a critical unmet need in the detection of known and novel microorganisms in biopharmaceutical manufacturing. followed by mass spectrometric analysis of the amplicon(s), nanotechnology, consisting of direct detection of nucleic acids using labelled probes, massively . Alternative adventitious agent detection methods in biopharmaceuticals: A proposal for a structured best practice approach for their evaluation, validation, and implementation. Viral detection methods currently considered as industry standard generally fall into two categories: broad spectrum screening assays such as cell culture (in vitro) and animal (in vivo) based testing and transmission electron microscopy (TEM); Based on test types market is segmented into bioburden tests, adventitious agent detection tests, residual host contamination detection tests, cell line authentication and characterization tests, sterility tests, endotoxin tests and other test types. Mycoplasma Detection. LAY ABSTRACT: Because biological products are manufactured in cells, the living environment must be kept sterile. The current gold standard for general infectious adventitious virus screening is the in vitro assay in which test articles are cultured onto a panel of different cell lines and observed for cytopathic effect (CPE). The current gold standard for general infectious adventitious virus screening is the in vitro assay in which test articles are cultured onto a pa The scope of safety tests might be however not sufficient in some cases. Mycoplasma Detection. The Accukit contains all the necessary reagents for library preparation of cell lysate samples to target a panel of adventitious agents (Table 1) to achieve a sensitivity of 10 copies of target per ml . Lay abstract: Because biological products are manufactured in cells, the living environment must be kept sterile. Reads from AGM WGS and transcriptome systematically produce false . The global adventitious agent testing market is estimated to expand . Virus Titre Determination. adventitious agent or novel endogenous agent is detected in an already licensed or registered vaccine. The manufacture of biopharmaceutical agents such as therapeutic monoclonal antibodies, vaccines and cell-based therapies like CAR-T cells require the highest quality control against the contamination of adventitious agents. Based on these positive experiences, I am confident that the framework document for alternative adventitious agent tests will be just as helpful for colleagues." Looking ahead, a case study is planned to apply this framework to alternative mycoplasma detection methods. This contamination event illustrates the gaps within the existing adventitious agent strategy and the potential use of new broader molecular detection methods. In this review, detection methods for bacteria, viruses, and mycoplasma are comprehensively addressed. A number of methods can be used to detect endogenous and adventitious viruses. Patient viral safety is the first and most pressing concern, but also the economics of production management. Adventitious agent testing in biomanufacturing requires assays of broad detection capability to screen for as many infectious agents as possible. Cell Line Authentication. Adventitious agent testing in biomanufacturing requires assays of broad detection capability in order to screen for as many infectious agents as possible. Adventitious Agents / Virology Testing. Adventitious agents present significant complications to biopharmaceutical manufacturing. 1. Adventitious agents include numerous lifeforms such as bacteria, fungi, viruses, mycoplasma, and others that are inadvertently introduced into biological systems. By phyre Detection methods . Approaches for the detection of adventitious agents . Mycoplasma Detection. In addition, growing animal and microbe-derived biologics will further increase the necessity of . Consult our experts. Adventitious agent testing requires the detection of both the vaccine virus and any adventitious viruses, with the adventitious viruses potentially present at much lower concentrations. The current sample preparation workflow is not adequate to handle this challenge, therefore limiting the lower limit of detection regardless of the . The workshop will cover all aspects of the sequencing process from upstream sample processing, extraction, cDNA synthesis, library preparation, and sequencing. 3.1.2 Working Cell Bank Each WCB as a starting cell substrate for drug production should be tested for adventitious virus either by direct testing or by analysis of cells at the limit of in vitro cell age, initiated from the WCB. Also, molecular detection of mycoplasma and pestiviruses was performed on the FBS samples. FDA's Approach to Adventitious-Agent Testing of Cell Substrates and Viral Vaccines: Traditional and Novel Methods Keith Peden Division of Viral Products OVRR, CBER, FDA May 7, 2010. Sartorius offers a wide range of in vitro and in vivo assays for detection of adventitious agents and supports manufacturers in selecting the optimal combination of tests for their safety strategy: In vitro testing. The second is the presence of erythrocyte hemagglutination activity in the conditioned medium removed from the cells on days 14 and 28. The strength of metagenomics is that this method does not target any specific virus/pathogen, but has a general capacity to detect a wide range of infectious agents, including unknown . Thus, next-generation sequencing-based detection of adventitious agents is a valuable approach that can fill a critical unmet need in the detection of known and novel microorganisms in biopharmaceutical manufacturing. SMART CAMP (Critical Analytics for Manufacturing Personalized-Medicine) is a new interdisciplinary research programme in Singapore (CREATE international research campus and innovation hub) and at the Massachusetts Institute of . At MDS we test for adventitious agents by detection of cytopathic effects on indicator cell lines such as MRC-5 cells, Vero cells, and a number of other different cell lines, per guidance outlined by the regulatory agencies (US Food and Drug Administration (FDA PTC 1993 and others) and the International Conference on Harmonisation (ICH Q5A). Adventitious Agents . Therefore, detection of adventitious agents during the manufacturing of biotechnological products is essential for the protection of the patient. In this case, however, the test sample is thawed and injected without clarification into the . Various reviews of HTS for adventitious agent detection have shown that there is a need for unbiased extraction methods, relevant controls, the use of spike recovery experiments, and quality control measures during library preparation 7,14-17. A wide range of in vitro and in vivo assays for detection of adventitious agents. In general, the AAT involves three orthogonal measures for detecting adventitious agents. 2015-07-01 00:00:00 Advances in adventitious agent detection methodology are bringing benefits, but more work needs to be done.The raw materials used in the manufacture of biologic drugs come from different sources: animals (although use of such materials is . PhyloID is run in two steps, each being a self-contained automated process amenable to GMP validation. adventitious agents in each lot/batch of material in adherence with current Good Manufacturing Practices (cGMP). MPS/NGS offers the ability to detect all existing nucleotides, RNA, and DNA from adventitious virus contamination, thus fine-tuning the degree of sensitivity by increasing or decreasing the depth of coverage. Adventitious agent detections are important due to regulatory affair and versatility. Current methods for detection of adventitious agents are expensive, lack . Adventitious agent detection testing is required during cell cultures due to elevated threat of contamination by viruses, endotoxins and mycoplasma among others. Low-speed centrifugation will remove larger cellular debris and yield a supernatant that may be inoculated directly onto detector cells in cell-based viral infectivity assays. In the case of LVVs, this process is sometimes complicated by the fact that . However, this assay is inherently subjective due to the nature of visual observation of cell morphology and labor and time intensive When appropriate non-endogenous virus tests have been performed on the Adventitious virus detection is an integral part of safety testing of biopharmaceutical products. The adventitious agents most frequently detected in CCL's have been bovine viral diarrhea virus and mycoplasma. An advantage of NGS sequencing is that detection does not require prior knowledge of potential adventitious viral agents, meaning it is capable of detecting a broad range of viral contaminations of both novel and unknown viral sequences. Detection methodologies -Mass spectrometry -Microarray -High-throughput sequencing Strategy for Adventitious Agent Detection (Arifa S. Khan, CBER, USA 2005 . Adenovirus-based vaccine stock a. systems that can be used for the detection of adventitious agents have been developed. A facility closure for bioremediation of viral contaminants represents an . Risk of viral contamination poses a huge threat for human use of biological products. Bacteria and other contaminants can be easily removed by 0.1 m or 0.2 m sterilizing grade membranes, however small viruses are not removed by these filters. Endotoxin test type segment held largest market share in 2018. sterility test type segment will witness highest growth rate over the forecast . The Adventitious Agent Detection as a part of HIVE is designed to facilitate and standardize the vaccine data review process. These include but may not be limited to: oligonucleotide arrays to detect viral genomes or mRNA, PCR (degenerate, multiplex, etc.) Manufacturers have to demonstrate the ability of the manufacturing process to produce a biopharmaceutical product that is . Adventitious virus testing - Detection, removal, and inactivation of virus contamination in biopharmaceuticals and biological materials are key steps that help ensure the quality of biotherapeutics. Biopharmaceutical manufacturers must select and design the most appropriate virus detection test methods for use across a manufacturing process, from initial cell lines up to the final-product stage, including raw materials and bulk drug-substance harvest. 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