Considerations for Biodistribution and Viral Shedding Assays in Regulated Bioanalysis

Viral vectors are often the delivery backbone of both in vivo and ex vivo gene therapies. Assays to track the biodistribution and shedding of these vectors are critical for ensuring their safety and efficacy. However, there is little formal regulatory guidance on the development and validation of these assays. 

In this webinar, we explore bioanalytical assays for viral vectors, with a focus on regulatory guidance, platform selection, and key considerations for method development, qualification, and validation of biodistribution and viral shedding assays. 

Gene and Cell Therapy Bioanalytical Strategy 

Various bioanalytical assays are needed to understand the pharmacokinetic (PK)/pharmacodynamic (PD) relationship for cell and gene therapies. For these advanced therapeutics, quantitative polymerase chain reaction (qPCR) and digital PCR (dPCR) are common molecular methods used for bioanalytical measurements. 

Biodistribution and Viral Shedding Assays 

Biodistribution assays assess the distribution and persistence of a cell or gene therapy product throughout the body. This includes measuring its distribution from the site of administration to both target and non-target tissues and biofluids, providing valuable PK information to evaluate the therapy’s safety and efficacy. 

Viral shedding assays measure the release of viral-based gene therapy products from the patient through excreta and secreta, such as feces, urine, saliva, nasopharyngeal fluids, and even skin. Shedding is an observed result of biodistribution and is measured to understand the risk of shedding virus into the environment and to other individuals.  

Regulatory Considerations 

Currently, there are no formal regulatory guidelines for the qualification and validation of qPCR and dPCR assays in cell and gene therapy bioanalysis. However, best practices and general considerations can be gleaned from key documents, including: 

• Recommendations for Method Development and Validation of qPCR and dPCR Assays in Support of Cell and Gene Therapy Drug Development, developed by an American Association of Pharmaceutical Scientists working group¹ 

• FDA Guidance on Long-Term Follow-Up After Administration of Human Gene Therapy Products.² 

• ICH S12 Draft Guidance on nonclinical biodistribution considerations for gene therapy products³ 

• FDA Guidance on Design and Analysis of Shedding Studies for Virus or Bacteria-Based Gene Therapy and Oncology Products.⁴ 

Given the lack of prescriptive guidance, implementing best practices in bioanalytical method development, based on scientific rigor, is crucial. 

Platform Selection 

Platform selection should be driven by the unique needs of the therapeutic and the study. Each platform has distinct advantages and limitations: 

• qPCR, the traditional platform, is widely used for its broad dynamic range, with an upper limit of quantitation that can reach up to 10⁸ copies per reaction. This platform is well-suited for biodistribution assays where very high copy levels of viral vectors are expected. 

• Digital PCR (dPCR), a newer platform, offers the advantage of high precision and absolute quantification without the need for standard curves. It can be useful for assays where sample matrices may contain PCR inhibitors, for example, shedding in feces or saliva, and where detection of lower levels of viral particles is needed.  

Method Development and Validation 

In validation, key parameters can include specificity, linearity, dynamic range, lower limit of quantitation (LLOQ), sensitivity/limit of detection (LOD), reproducibility(precision and accuracy), robustness, ruggedness, matrix interference, and stability. However, a fit-for-purpose approach should be utilized for development and validation of biodistribution and viral shedding assays based on the context of use and specific drug program. 

Conclusion 

Understanding the context of use is critical for the development and validation of biodistribution and viral shedding assays for bioanalysis of cell and gene therapies. For both assay types, the nature of the viral vector, the route of administration, and the expected concentrations of viral particles, among other considerations, all play a role in determining the most appropriate bioanalytical strategy, detection platform, and assay design.  

Regulatory considerations, while still evolving, provide general guidance on performance assessments. Given the absence of specific bioanalytical method validation guidelines, each assay must be developed and validated with careful attention to its intended purpose. 

For more information on developing and implementing a rigorous bioanalytical strategy for cell and gene therapies, speak to a scientist. 

References 

¹ Hays A, et al. Recommendations for Method Development and Validation of qPCR and dPCR Assays in Support of Cell and Gene Therapy Drug Development. AAPS J. 2024;26(1):24. 

² US Food and Drug Administration. Guidance for Industry: Long-Term Follow-Up After Administration of Human Gene Therapy Products, 2020. 

³ ICH Harmonized Guideline: Nonclinical distribution considerations for gene therapy products S12. Draft Guidance, 2021. 

⁴ US Food and Drug Administration. Guidance for Industry: Design and Analysis of Shedding Studies for Virus or Bacteria-Based Gene Therapy and Oncology Products, 2015.