Rapid automation of a cell-based assay using a modular approach: case study of a flow-based Varicella Zoster Virus infectivity assay.

Vaccine manufacturing requires constant analytical monitoring to ensure reliable quality and a consistent safety profile of the final product. Concentration and bioactivity of active components of the vaccine are key attributes routinely evaluated throughout the manufacturing cycle and for product release and dosage. In the case of live attenuated virus vaccines, bioactivity is traditionally measured in vitro by infection of susceptible cells with the vaccine followed by quantification of virus replication, cytopathology or expression of viral markers. These assays are typically multi-day procedures that require trained technicians and constant attention. Considering the need for high volumes of testing, automation and streamlining of these assays is highly desirable. In this study, the automation and streamlining of a complex infectivity assay for Varicella Zoster Virus (VZV) containing test articles is presented. The automation procedure was completed using existing liquid handling infrastructure in a modular fashion, limiting custom-designed elements to a minimum to facilitate transposition. In addition, cellular senescence data provided an optimal population doubling range for long term, reliable assay operation at high throughput. The results presented in this study demonstrate a successful automation paradigm resulting in an eightfold increase in throughput while maintaining assay performance characteristics comparable to the original assay.

Serglycin proteoglycan expression and synthesis in embryonic stem cells.

The serglycin proteoglycan is expressed in most hematopoietic cells and is packaged into secretory vesicles for constitutive or regulated secretion. We have now shown serglycin mRNA expression in undifferentiated murine embryonic stem (ES) cells and in embryoid bodies, and synthesis and secretion in undifferentiated ES cells. Serglycin was localized to ES cell cytoplasm by immunostaining. Serglycin mRNA is expressed in tal-1((-/-)) ES cells and embryoid bodies; tal-1((-/-)) mice cannot produce hematopoietic cells. Thus, ES serglycin expression is probably not associated with hematopoiesis. Serglycin expression was increased by treatment of ES cells with retinoic acid (RA) and dibutyryl cAMP (dbcAMP). The serglycin core protein obtained from control ES culture medium after chondroitinase digestion appears as a doublet. Only the lower Mr band is present in serglycin secreted from RA-treated and the higher Mr band in RA+dbcAMP-treated cells, suggesting that core protein structure is affected by differentiation.