ABSTRACT
Scale-down models (SDM) are pivotal tools for process understanding and improvement to accelerate the development of vaccines from laboratory research to global commercialization. In this study, a 3 L SDM representing a 50 L scale Vero cell culture process of a live-attenuated virus vaccine using microcarriers was developed and qualified based on the constant impeller power per volume principle. Both multivariate data analysis (MVDA) and the traditional univariate data analysis showed comparable and equivalent cell growth, metabolic activity, and product quality results across scales. Computational fluid dynamics simulation further confirmed similar hydrodynamic stress between the two scales.
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
RotaTeq is a pentavalent rotavirus vaccine that contains five human-bovine reassortant strains (designated G1, G2, G3, G4, and P1) on the backbone of the naturally attenuated tissue culture-adapted parental bovine rotavirus (BRV) strain WC3. The viral genomes of each of the reassortant strains were completely sequenced and compared pairwise and phylogenetically among each other and to human rotavirus (HRV) and BRV reference strains. Reassortants G1, G2, G3, and G4 contained the VP7 gene from their corresponding HRV parent strains, while reassortants G1 and G2 also contained the VP3 gene (genotype M1) from the HRV parent strain. The P1 reassortant contained the VP4 gene from the HRV parent strain and all the other gene segments from the BRV WC3 strain. The human VP7s had a high level of overall amino acid identity (G1: 95-99%, G2: 94-99% G3: 96-100%, G4: 93-99%) when compared to those of representative rotavirus strains of their corresponding G serotypes. The VP4 of the P1 reassortant had a high identity (92-97%) with those of serotype P1A[8] HRV reference strains, while the BRV VP7 showed identities ranging from 91% to 94% to those of serotype G6 HRV strains. Sequence analyses of the BRV or HRV genes confirmed that the fundamental structure of the proteins in the vaccine was similar to those of the HRV and BRV references strains. Sequences analyses showed that RotaTeq exhibited a high degree of genetic stability as no mutations were identified in the material of each reassortant, which undergoes two rounds of replication cycles in cell culture during the manufacturing process, when compared to the final material used to fill the dosing tubes. The infectivity of each of the reassortant strains of RotaTeq, like HRV strains, did not require the presence of sialic acid residues on the cell surface. The molecular and biologic characterization of RotaTeq adds to the significant body of clinical data supporting the consistent efficacy, immunogenicity, and safety of RotaTeq.
Subject(s)
Reassortant Viruses/genetics , Rotavirus Vaccines , Rotavirus/genetics , Amino Acid Sequence , Animals , Cattle , Cluster Analysis , Genomic Instability , Humans , Molecular Sequence Data , Phylogeny , RNA, Viral/genetics , Sequence Alignment , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Vaccines, Attenuated , Viral Proteins/geneticsABSTRACT
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.
