ABSTRACT
Cell culture derived rotavirus preparations contain a mixture of double-layered particles (DLPs) and triple-layered particles (TLPs). Characterization of rotavirus vaccine products is important to demonstrate a consistent manufacturing process. A capillary zone electrophoresis (CZE) method was developed to separate and quantitate rotavirus DLPs and TLPs in cell lysate samples and CsCl-purified vaccine preparations of each of the five reassortant rotavirus vaccine strains (G1, G2, G3, G4 and P1) contained in the pentavalent rotavirus vaccine, RotaTeq. The CZE electropherograms showed that migration of DLPs and TLPs from both CsCl-purified and cell lysates resulted in a separation distance of approximately 3 min between the two rotavirus particle types. The identification of the peak(s) containing TLPs was confirmed for both CsCl-purified and cell lysate samples by treatment of the samples with 50mM EDTA, which converted TLPs to DLPs. The migration pattern of the DLPs was consistent (23-24 min) among all reassortant strains tested, whether the DLPs were CsCl-purified or from cell lysates. However, the migration pattern of the TLP electropherograms of the reassortant rotavirus strains in cell lysates differed from those of the CsCl-purified reassortant rotavirus strains. In the cell lysate samples, the TLPs of the G1 and G2 reassortant rotavirus strains migrated slower that the corresponding TLPs from the CsCl-purified samples, while the migration time of the TLPs of the G3, G4 and P1 reassortants strains from the cell lysate and CsCl-purified samples appeared similar. Also, the TLPs from the CsCl-purified samples appeared as a defined single peak, while most of the TLPs from the cell lysate samples appeared as a broad peak or as multiple peaks. All the migration patterns were reproducible and consistent. Taking into account reproducibility, objective quantitation, and minimal sample manipulation as well as volume, CZE allowed consistent and quantitative characterization of rotavirus vaccine preparations, which is required for evaluation of vaccine products, including process validation.
Subject(s)
Electrophoresis, Capillary/methods , Rotavirus Vaccines , Rotavirus/isolation & purification , Viral Load , Animals , Centrifugation, Density Gradient , Cesium , Chlorides , Chlorocebus aethiops , Culture Media , Humans , Reassortant Viruses/isolation & purification , Reproducibility of Results , Vero CellsABSTRACT
A sensitive and reproducible method to determine the in vitro infectious potency of a pentavalent reassortant rotavirus vaccine (RotaTeq) has been developed as an alternative to classical potency assays. Potency was determined based on cell-based viral replication followed by quantitative reverse-transcription polymerase chain reaction (RT-QPCR) analysis. In the assay, confluent Vero cell monolayers in 96-well plates were inoculated with serial dilutions of test samples, a pentavalent reassortant rotavirus reference standard and assay controls, followed by incubation for 24h. The cells were lysed with a Triton X-100 solution and the lysates assayed by RT-QPCR to quantitate viral nucleic acid produced during replication. The RT-QPCR utilizes primer/probe sets specific to each virus reassortant and the potencies of each sample were determined relative to the reference standard. This assay, hereafter referred to as the Multivalent QPCR-Based Potency Assay (M-QPA), permits the specific quantitation of each individual reassortant virus in the presence of the other four reassortant viruses. In addition, the assay was demonstrated to be concordant with a traditional method (plaque assay) for the quantitation of infectious virus particles. It is anticipated that assays of this type will become a valuable tool in the assignment of potency values and in the monitoring of stability of live virus vaccines.
