Viral shedding analysis for gene therapy products and mRNA vaccines

Shedding refers to the release of gene therapy products or oncolytic products from a patient through excreta (feces), secreta (urine, saliva, nasopharyngeal fluids, etc.), or through the skin (via pustules, sores, or wounds).

A shedding study aims to determine whether the product is shed and whether the shed product is infectious. Viral shedding assays present unique challenges. The analytes released are typically found in very low quantities, and the matrices involved in shedding assays often contain significant levels of PCR inhibitory agents and endogenous DNA.

Highly sensitive quantitative PCR (qPCR) or digital PCR (dPCR) are the preferred methods for detecting shed nucleic acids, including viral fragments, mRNA, and DNA/RNA vaccines in excreta and secreta.

Spiked-in samples should mimic the target particle as closely as possible during method development and validation. For example, if the virus is encapsulated in its infectious stage, the extraction process must effectively recover DNA or RNA from encapsulated viral particles.

Excreta and secreta contain high levels of PCR-inhibitory agents, which can impact assay performance. Therefore, the inhibitory effect must be minimized, well-characterized, and accounted for in the validated assay to ensure accurate and reliable detection.

Quantitative PCR (qPCR) is the standard method for shedding studies, with digital PCR (dPCR) now offering additional advantages. dPCR is less sensitive to PCR inhibition, requires minimal dilution, and enables absolute quantification without needing a standard curve.

We develop, optimize, qualify, validate, and run assays tailored to each viral shedding study, covering AAV, retroviral and lentiviral vectors, oncolytic viruses, and RNA and DNA vaccines. With over 20 years of experience in nucleic acid extraction and assay validation, we ensure reliable results by incorporating spike-in controls to assess recovery and efficiency. These spikes mimic the active drug product, whether encapsulated, in lipid nanoparticles, or as free nucleic acids.

Our validations align with context-of-use requirements and regulatory guidelines, including GLP and GCLP standards.

Ensuring compliance with Good Laboratory Practice (GLP) for viral shedding studies submitted to regulatory agencies guarantees data reliability, integrity, and traceability. As a GLP-accredited laboratory, we operate within a rigorous framework that includes sample management, controlled work environments, instrument maintenance, temperature regulation, staff training, and IT security. Additionally, our time-stamped sample handling safeguards the integrity of the samples and the resulting data.

For clinical shedding studies, Good Clinical Laboratory Practice (GCLP) bridges GLP principles with Good Clinical Practice (GCP) to ensure high-quality bioanalytical data in patient samples.

Meanwhile, our ISO/IEC 17025 accreditation signifies technical competence in laboratory testing, ensuring consistent, high-quality results. In viral shedding and biodistribution studies, ISO compliance enhances credibility, particularly for non-regulated studies and global collaborations, further supporting the development of gene therapies and advanced biologics.