Hycells Biotechnology | A New Option for RCL Testing Platforms
Release Date:
2023-07-03 13:24
According to the recently issued “Technical Guidance Principles for Pharmaceutical Research and Evaluation of In Vitro Gene Modification Systems (Trial)”, RCL testing is critical as a safety assessment step throughout the research, development, and manufacturing of cell-based therapeutics.
Replicating viruses are associated with product safety; therefore, it is necessary to consult relevant research experience and literature to develop and establish sensitive detection methods. Common detection methods include: Instruction on cell culture method and direct qPCR method and so on. The test samples include the detection of replication-competent lentiviruses (RCL) harvested during the preparation of viral vectors: Based on the current state of research, it is recommended to use an indicator cell culture assay for RCL detection of lentiviral vectors. For HIV-derived lentiviral vectors, the positive control used for RCL detection may be an HIV virus that meets the assay requirements; if such a virus lacks auxiliary genes, the same preparation method should be employed, and the assay must be conducted and handled properly in a compliant environment. Regarding RCL detection parameters, it is generally believed that p24 protein, reverse transcriptase activity, psi-gag, and VSV_G sequences The detection of these markers can indicate the presence of RCL; appropriate assay parameters should be selected based on the specific characteristics of the viral vector and the research context.
I. Introduction to RCL Testing
In CAR-T cell manufacturing, most retroviruses and lentiviruses used for cellular gene therapy are engineered as replication-deficient viral vectors and are co-transfected using multiple plasmids to express the lentivirus or γ-retrovirus. During production, homologous recombination may occur between the deleted vector backbone and wild-type viral sequences, leading to the generation of replication-competent virus (RCL), which poses a serious risk to human health. Therefore, RCL detection is one of the most critical components of safety testing.
The FDA-recommended RCL assay involves culturing and amplifying any RCL that may be present in viral vectors using susceptible cells, followed by detection at the end of the culture period.
Amplification phase: Incubate the sample to be tested with HIV-1–susceptible cell lines that support robust viral replication (typically C8166), passage the cells at least five times, and culture for a minimum of three weeks.
Incubation period: Collect the culture supernatant after 3 weeks, inoculate it into C8166 cells, and culture for 7 days before assaying for RCL markers.
RCL induction period culture
RCL Standard Operating Procedure
Detection method:
A: The P24 ELISA method is suitable for detecting RCL formed by recombination between viral genomes during vector preparation.
B: PERT method: After RCL amplification, the PERT assay can also be used to measure reverse transcriptase activity. A drawback of this method is the presence of high background in certain cell types.
C: qPCR method: Real-time quantitative PCR (qPCR) is used to quantify the VSV-G gene sequence during the replication of pseudotyped VSV-G virus, or to detect the psi-gag sequence generated by recombination between the vector plasmid and the packaging plasmid.
II. Analysis of RCL Detection Methods
In its 2020 guidance on RCR/RCL testing, the U.S. FDA states that RCR/RCL assays encompass four methods: indicator cell culture, ELISA (for p24 protein detection), PCR/Q-PCR (using psi-gag or VSV-G–targeted polymerase chain reactions), and PERT (product-enhanced reverse transcription assay).
The indicator cell culture assay for replication-competent lentiviral RCL requires 28 days, making it time-consuming; in contrast, Q-PCR assays based on the VSV-G sequence and PCR assays based on the gag sequence offer advantages such as shorter turnaround times, higher sensitivity, and strong reproducibility. Currently, many companies submitting CAR-T product applications employ Q-PCR/PCR methods to directly quantify VSV-G or psi-gag sequences in the final product as a rapid release-testing approach.
Shanghai Hycells Biotechnology Co., Ltd. has leveraged market demand and its technological platform strengths to accumulate extensive development experience in methods such as ELISA for p24 protein detection, PCR targeting the Psi-gag sequence region, and VSV-G–based polymerase chain reaction. We welcome inquiries from all potential customers.
National toll-free consultation hotline: 021-36696819
1. ELISA method (p24 protein assay)
A 96-well plate pre-coated with HIV-1 core antibodies is incubated with the substrate TMB in the presence of peroxidase, resulting in a color change from blue to yellow upon acidification. The optical density (OD) is then measured at 450 nm using a microplate reader.
b) Quantitative analysis with high sensitivity
A standard curve is generated using reference standards, and the p24 protein concentration in the samples is calculated; RCL assays are also performed. The P24 ELISA method achieves a sensitivity of 3 pg/mL.
c) Suitable for multiple sample types
The P24 assay can detect a variety of sample types, including terminal cells, concentrated vectors, and serum or plasma. This platform employs stringent cutoff criteria to ensure the accuracy, validity, and reproducibility of test results.
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