Virus filtration is a key clearance unit operation in the manufacture of recombinant protein, monoclonal antibody, and plasma-derived biopharmaceuticals. Recently, a consensus has developed among filter manufacturers and end users about the desirability of a common nomenclature and a standardized test for classifying and identifying virus-retentive filters. The Parenteral Drug Association virus filter task force has chosen PR772 as the model bacteriophage to standardize nomenclature for large-pore-size virus-retentive filters (filters designed to retain viruses larger than 50 to 60 nm in size). Previously, the coliphage PR772 (Tectiviridae family) has been used in some filtration studies as a surrogate for mammalian viruses of around 50 to 60 nm. In this report, we describe specific properties of PR772 critical to the support of its use for the standardization of virus filters. The complete genomic sequence of virulent phage PR772 was determined. Its genome contains 14,946 bp with an overall G؉C content of 48.3 mol%, and 32 open reading frames of at least 40 codons. Comparison of the PR772 nucleotide sequence with the genome of Tectiviridae family prototype phage PRD1 revealed 97.2% identity at the DNA level. By dynamic light-scattering analysis, its hydrodynamic diameter was measured as 82 ؎ 6 nm, consistent with use in testing large-virus-retentive filters. Finally, dynamic light-scattering analysis of PR772 preparations purified on CsCl gradients showed that the phage preparations are largely monodispersed. In summary, PR772 appears to be an appropriate model bacteriophage for standardization of nomenclature for larger-pore-size virus-retentive filters.

Radioiron uptake from 59FeC13 by Streptococcus mutans OMZ176 was increased by anaerobiosis, sodium ascorbate, and phenazine methosulfate (PMS), although there was a 10-min lag before PMS stimulation was evident. The reductant ascorbate may have provided ferrous iron. The PMS was reduced by the cells, and the reduced PMS then may have generated ferrous iron for transport; reduced PMS also may have depleted dissolved oxygen. We conclude that S. mutans transports only ferrous iron, utilizing reductants furnished by glucose metabolism to reduce iron prior to its uptake.Streptococcus mutans is considered the major odontopathogen of human dental caries (11). However, the presence of S. mutans in the oral cavity does not imply disease in a simple cause-and-effect relationship; caries results from a complex interaction between host, microbial, and dietary factors (11). Some trace metals may shift this equilibrium to favor either the host or the microbe, and the presence and concentrations of these metals may be cariogenic or cariostatic (reviewed in reference 2). S. mutans has a superoxide dismutase that uses manganese as a cofactor but can substitute iron for manganese to produce active superoxide dismutase if manganese is absent (12, 13). Neither metal is required for anaerobic growth of S. mutans (13). Aerobic metabolic roles for iron in addition to its function as a cofactor for superoxide dismutase are suggested by experiments showing iron stimulation (nearly threefold) of S. mutans steady-state growth in manganese-containing medium (2). Iron-containing cytoplasmic fractions (other than superoxide dismutase) have been demonstrated in S. mutans (12). In other studies, iron was required for S. mutans growth (3), and an increase in colony size was obtained by adding iron to a solidified minimal medium (10). Lactoferrin (but not iron-saturated lactoferrin) increased the length of the S. mutans lag phase (5), suggesting that the organism is unable to obtain iron bound by lactoferrin.Transport of iron by S. mutans has not been extensively investigated. For efficient iron acquisition, many microorganisms produce siderophores that bind highly insoluble ferric iron, making it available for transport (14). Intracellular utilization of iron by S. mutans and the existence of specialized transport systems for iron in other organisms imply that S. mutans has membrane-associated iron uptake mechanisms. In the present studies, phenolate or hydroxamate siderophore production by S. mutans OMZ176 was not detected. The organism appeared to transport only reduced (ferrous) iron. Uptake of radioiron. For the radioiron uptake assays, the cells were grown aerobically without shaking in an atmosphere of 95% air-5% CO2 by the following procedure. A 0.1-ml amount of a 24-h culture of S. mutans OMZ176 in Todd-Hewitt broth (Difco Laboratories, Detroit, Mich.) was transferred to 10 ml of Chelex-100-treated FMC medium supplemented with magnesium, manganese, and iron. After 24 h of incubation, the entire 10-ml culture was transferred to 1 liter o...

Viral clearance studies are mandated prior to entering clinical trials and for commercial launch of biopharmaceuticals. These studies are a key component of risk mitigation to reduce the potential for iatrogenic transmission of pathogenic viruses. This paper reviews regulatory guidance and practical strategies for designing viral clearance studies. Essential elements of a developmental phase-appropriate viral clearance package are detailed. These include scale-down model qualification, virus spike experiments and validation (clearance evaluation) of manufacturing process steps. Heuristics and learnings from available data are shared. Developments in this area including generic validation strategies, multiviral spiking strategies and use of newer model viruses for nonconventional substrates are also described. This review provides a framework for a comprehensive viral validation package for regulatory submissions.

When cultured anaerobically in a chemically defined medium that was treated with Chelex-100 to lower its trace metal content, Streptococcus mutans OMZ176 had no apparent requirement for manganese or iron. Manganese or iron was necessary for aerobic cultivation in deep static cultures. During continuous aerobic cultivation in a stirred chemostat, iron did not support the growth rate achieved with manganese. Since the dissolved oxygen level in the chemostat cultures was higher than the final level in the static cultures, manganese may be required for growth at elevated oxygen levels. In medium supplemented with manganese, cells grown anaerobically contained a low level of superoxide dismutase (SOD) activity; aerobic cultivation increased SOD activity at least threefold. In iron-supplemented medium, cells grown anaerobically also had low SOD activity; aerobic incubation resulted in little increase in SOD activity. Polyacrylamide gel electrophoresis of the cell extracts revealed a major band and a minor band of SOD activity in the cells grown with manganese; however, cells grown with iron contained a single band of SOD activity with an Rf value similar to that of the major band found in cells grown with manganese. None of the SOD activity bands were abolished by the inclusion of 2 mM hydrogen peroxide in the SOD activity strain. S. mutans may not produce a separate iron-containing SOD but may insert either iron or manganese into an apo-SOD protein. Alternatively, iron may function in another activity (not SOD) that augments the defense against oxygen toxicity at low SOD levels.

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