Cumulative viral titer reduction demonstrated by sequential challenge of a tangential flow membrane filtration system and a direct flow pleated filter cartridge.

The efficiency of sequential incorporation of two approaches to virus removal, i.e., tangential flow ultrafiltration using the Omega 300K VR Maximate System, and, direct flow microfiltration using the Ultipor VF grade DV50 virus removal filter, was evaluated using bacteriophage phi 6 in Dulbecco's modified Eagle medium (MEM) as the carrier fluid. A combined log titer reduction value of > 10 was demonstrated, with each step, individually, providing > 4.5 log reduction. Significant improvement in filter life was also demonstrated when the combined system was used. The data suggest that this system may have applications in the manufacture of biologicals and biopharmaceuticals, where there is a requirement for multiple robust methods of virus removal to ensure freedom from endogenous or adventitious viral contamination.

Sequence and phylogenetic analysis of the Rhizobium leguminosarum biovar trifolii pyrE gene, overproduction, purification and characterization of orotate phosphoribosyltransferase.

The pyrE gene of Rhizobium leguminosarum biovar trifolii (Rl) was subcloned and its sequence is presented. The nucleotide sequence analysis suggests that this gene is not regulated by transcriptional attenuation as seen for the pyrE and pyrB genes of Escherichia coli (Ec) and Salmonella typhimurium. The Rl pyrE gene was subcloned into Ec AT2538 pyrE60 where the Rl pyrE gene product, orotate phosphoribosyltransferase (OPRTase), was overproduced. Using Ec AT2538 pyrE60 overproducing Rl OPRTase, the enzyme was purified to homogeneity utilizing ammonium sulfate fractionation and affinity chromatography with an orotate monophosphate agarose matrix. The electrophoretically pure OPRTase was characterized and found to be a 24.7 +/- 0.3-kDa protein with a K(m) of 27.6 micromol l(-1). The deduced amino acid sequence for OPRTase was compared with OPRTases from other organisms and found to be most similar to that of Bacillus subtilis (Bs). The Rl OPRTase exhibits 37% identity and 46% similarity to the Bs OPRTase.

Preparation and evaluation of bacterial stocks for filter validation.

Bacterial cell stock paste of Brevundimonas diminuta (American Type Culture Collection # 19146) used for filter validation was evaluated by standard microbiological procedures to determine culture purity, monodispersion, cell size, and strain identity. Tests performed included direct microscopic count, standard plate count, gram stain, streak plate, sizing by microfiltration, scanning electron microscopy, and biochemical identification. Results indicated the bacterial stocks were of consistent quality in terms of bacteriological purity, cell size, viability, and concentration. Sizing by microfiltration confirmed consistent monodispersion and cell retention characteristics for each lot tested. Use of the commercially available RapID NF Plus system confirmed each lot to be B. diminuta as indicated by biochemical profile and identification database. These tests represent parameters necessary for qualification of bacterial stocks to be used for filter validation.