Method validation and assessment of the biodistribution and shedding for adenovirus vector-based vaccine using qPCR and dPCR
Assessment of the biodistribution and shedding is required for the development of viral vector-based vaccines. Vector copy number is commonly quantified using digital PCR (dPCR) or quantitative PCR (qPCR). However, the regulatory guidelines for bioanalytical PCR assays have not been released at pres...
Saved in:
| Main Authors: | , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Elsevier
2025-09-01
|
| Series: | Molecular Therapy: Methods & Clinical Development |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2329050125001445 |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| _version_ | 1849233559351459840 |
|---|---|
| author | Yoichi Tanaka Sayaka Hamano Akiko Ishii-Watabe Yoshiro Saito Ruri Kikura-Hanajiri |
| author_facet | Yoichi Tanaka Sayaka Hamano Akiko Ishii-Watabe Yoshiro Saito Ruri Kikura-Hanajiri |
| author_sort | Yoichi Tanaka |
| collection | DOAJ |
| description | Assessment of the biodistribution and shedding is required for the development of viral vector-based vaccines. Vector copy number is commonly quantified using digital PCR (dPCR) or quantitative PCR (qPCR). However, the regulatory guidelines for bioanalytical PCR assays have not been released at present. To consider the future setting of acceptance criteria for method validation in a guideline, we aimed to develop and validate a method for quantifying a model adenoviral (Ad) vector vaccine (Ad-hCovHKU1S-2A-GFP) using dPCR and qPCR in biological matrices and assessed its biodistribution in mice. Primers and probes were designed for the region between the sequences of the Ad and HKU1S. Method development and validation were performed using dPCR and qPCR with 1 μg of mouse genomic DNA (gDNA)/reaction. The validation parameters and their acceptance criteria were pre-defined as possible values referred from the literature. Lower limits of quantitation were set as 12 copies and 48 copies/reaction for dPCR and qPCR, respectively. Both dPCR and qPCR met the pre-defined acceptance criteria for intra- and inter-run accuracy and precision. Cross-validation showed similar quantitative results using both dPCR and qPCR. The pre-defined acceptance criteria on dPCR and qPCR may be applicable to the biodistribution and shedding of viral vector-based vaccines. |
| format | Article |
| id | doaj-art-99d374cc85b04905a7b1a6b0a24d1fe2 |
| institution | Kabale University |
| issn | 2329-0501 |
| language | English |
| publishDate | 2025-09-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Molecular Therapy: Methods & Clinical Development |
| spelling | doaj-art-99d374cc85b04905a7b1a6b0a24d1fe22025-08-20T05:07:02ZengElsevierMolecular Therapy: Methods & Clinical Development2329-05012025-09-0133310154910.1016/j.omtm.2025.101549Method validation and assessment of the biodistribution and shedding for adenovirus vector-based vaccine using qPCR and dPCRYoichi Tanaka0Sayaka Hamano1Akiko Ishii-Watabe2Yoshiro Saito3Ruri Kikura-Hanajiri4Division of Medicinal Safety Science, National Institute of Health Sciences, Kanagawa 210-9501, Japan; Corresponding author: Yoichi Tanaka, Division of Medicinal Safety Science, National Institute of Health Sciences, Kanagawa, Japan.Division of Medicinal Safety Science, National Institute of Health Sciences, Kanagawa 210-9501, JapanDivision of Biological Chemistry and Biologicals, National Institute of Health Sciences, Kanagawa 210-9501, JapanNational Institute of Health Sciences, Kanagawa 210-9501, JapanDivision of Medicinal Safety Science, National Institute of Health Sciences, Kanagawa 210-9501, JapanAssessment of the biodistribution and shedding is required for the development of viral vector-based vaccines. Vector copy number is commonly quantified using digital PCR (dPCR) or quantitative PCR (qPCR). However, the regulatory guidelines for bioanalytical PCR assays have not been released at present. To consider the future setting of acceptance criteria for method validation in a guideline, we aimed to develop and validate a method for quantifying a model adenoviral (Ad) vector vaccine (Ad-hCovHKU1S-2A-GFP) using dPCR and qPCR in biological matrices and assessed its biodistribution in mice. Primers and probes were designed for the region between the sequences of the Ad and HKU1S. Method development and validation were performed using dPCR and qPCR with 1 μg of mouse genomic DNA (gDNA)/reaction. The validation parameters and their acceptance criteria were pre-defined as possible values referred from the literature. Lower limits of quantitation were set as 12 copies and 48 copies/reaction for dPCR and qPCR, respectively. Both dPCR and qPCR met the pre-defined acceptance criteria for intra- and inter-run accuracy and precision. Cross-validation showed similar quantitative results using both dPCR and qPCR. The pre-defined acceptance criteria on dPCR and qPCR may be applicable to the biodistribution and shedding of viral vector-based vaccines.http://www.sciencedirect.com/science/article/pii/S2329050125001445adenovirus vectorbiological sampledigital PCRquantitative PCRvalidationbiodistribution |
| spellingShingle | Yoichi Tanaka Sayaka Hamano Akiko Ishii-Watabe Yoshiro Saito Ruri Kikura-Hanajiri Method validation and assessment of the biodistribution and shedding for adenovirus vector-based vaccine using qPCR and dPCR Molecular Therapy: Methods & Clinical Development adenovirus vector biological sample digital PCR quantitative PCR validation biodistribution |
| title | Method validation and assessment of the biodistribution and shedding for adenovirus vector-based vaccine using qPCR and dPCR |
| title_full | Method validation and assessment of the biodistribution and shedding for adenovirus vector-based vaccine using qPCR and dPCR |
| title_fullStr | Method validation and assessment of the biodistribution and shedding for adenovirus vector-based vaccine using qPCR and dPCR |
| title_full_unstemmed | Method validation and assessment of the biodistribution and shedding for adenovirus vector-based vaccine using qPCR and dPCR |
| title_short | Method validation and assessment of the biodistribution and shedding for adenovirus vector-based vaccine using qPCR and dPCR |
| title_sort | method validation and assessment of the biodistribution and shedding for adenovirus vector based vaccine using qpcr and dpcr |
| topic | adenovirus vector biological sample digital PCR quantitative PCR validation biodistribution |
| url | http://www.sciencedirect.com/science/article/pii/S2329050125001445 |
| work_keys_str_mv | AT yoichitanaka methodvalidationandassessmentofthebiodistributionandsheddingforadenovirusvectorbasedvaccineusingqpcranddpcr AT sayakahamano methodvalidationandassessmentofthebiodistributionandsheddingforadenovirusvectorbasedvaccineusingqpcranddpcr AT akikoishiiwatabe methodvalidationandassessmentofthebiodistributionandsheddingforadenovirusvectorbasedvaccineusingqpcranddpcr AT yoshirosaito methodvalidationandassessmentofthebiodistributionandsheddingforadenovirusvectorbasedvaccineusingqpcranddpcr AT rurikikurahanajiri methodvalidationandassessmentofthebiodistributionandsheddingforadenovirusvectorbasedvaccineusingqpcranddpcr |