Separation of rotavirus double-layered particles and triple-layered particles by capillary zone electrophoresis.

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.

Citrate-mediated disaggregation of rotavirus particles in RotaTeq vaccine.

For the routine manufacture of live virus vaccines, virus is diluted into a formulation buffer to stabilize it for long-term storage, and to facilitate vaccine administration. The characteristics of this buffer are dependent on the storage temperature of the vaccine, as well as the desired characteristics of the product. The formulation buffer for RotaTeq, Merck's live, pentavalent, oral rotavirus vaccine to prevent rotavirus gastroenteritis was developed as a fully liquid solution that requires no pre-feeding prior to administration, and is stable for 24 months at refrigerated temperatures. In studying the effects of the formulation buffer on the live virus contained within RotaTeq, we observed that the formulation buffer also directly impacts the state of rotavirus aggregation. This observation, termed "the matrix effect," was first noted as an approximately 50% increase in measured in vitro infectivity, following dilution of the virus into the buffer. Subsequent experiments confirmed that citrate in the formulation buffer facilitates the disaggregation of viral particles, likely through a carboxylic-acid mediated interaction. For vaccine manufacture, bulk virus is titered and subsequently diluted to a target concentration for dosing. Aggregation of the virus and subsequent inaccurate measurement of the amount of virus contained in either the bulk sample or in the final dosing container could lead to an inability to accurately predict final vaccine concentrations. Thus, discerning the nature and extent of the matrix effect was key principally for providing an accurate prediction of final virus concentration upon dilution, to ensure a robust manufacturing process. In addition, understanding potential contributions of the formulation buffer to clinical efficacy of the vaccine was critical. Clinical data have confirmed that the citrate-mediated disaggregation had no measurable impact on vaccine safety, immunogenicity, or efficacy.