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Log10 Reduction Factors in Viral Clearance Studies

by Horst Ruppach, PhD
Volume 12, Issue 4 (Winter 2013/2014)

Viral clearance studies are required for pharmaceuticals derived from human and/or animal sources such as recombinant proteins produced in eukaryotic cell lines, human blood products and vaccines, and even for some critical class III medical devices. It is mandatory to demonstrate that steps in the manufacturing process are capable of inactivating or removing potential viral contaminants. For this, a laboratory-scale (downscale) of the process step is developed and challenged with different model virus solutions. The viral concentrations are quantitatively determined in the feed material and the relevant product fraction. The ratio of both defines the reduction in virus and specifies the viral inactivation or viral removal capacity of the investigated process step.

In general, cell line-based infectivity assays like the plaque forming units (PFU) assay or the tissue culture infective dose (TCID50) assay are used to quantify the virus infectivity in the process solutions of a viral clearance study. In some cases, the quantitative polymerase chain reaction (qPCR) may be used. The viral titer derived from the viral infectivity assays is represented by log10/mL values. For instance, viral stock solutions used to challenge a process step typically have a viral titer of >107 log10 PFU/mL or TCID50/mL. The calculated ratio of the viral titer in the starting material and in the relevant product fraction defines the viral reduction, called log10 reduction factor (LRF), log10 reduction value(LRV), or sometimes simply log10 clearance. The mode of the LRF calculation is outlined in the relevant guidelines for viral clearance studies. If possible, the 95% confidence limit is calculated based on the 95% confidence limits of the single viral titers (the 95% confidence interval of the viral infectivity in: [A] the starting material; and [B] the final product fraction using the formula √ a2+b2 )...

Citation:
Ruppach H. Log10 Reduction Factors in Viral Clearance Studies. BioProcess J, 2014; 12(4): 24-30. http://dx.doi.org/10.12665/J124.Ruppach.

Posted online January 7, 2014.

 
Fetal Bovine Serum: The Impact of Geography

by William Siegel and Leland Foster, PhD
Volume 12, Issue 3 (Fall 2013)

Misunderstandings persist regarding geographic origin when sourcing fetal bovine serum (FBS), particularly as it affects quality and cost. This brief communication provides an overview of FBS and sourcing considerations, and direction to resources for further research on related questions. A key concept in evaluating quality in animal-derived raw material is that it is impossible to fundamentally improve the quality by means of any processing. Quality must begin at the source. The importance of geographic origin in suitability assessment is too often overlooked. Global geographic incidence of bovine disease or adventitious agents presents an opportunity for risk management by selecting material from geographic areas with the most limited disease/agent profiles...

Citation:
Siegel W, Foster L. Fetal Bovine Serum: The Impact of Geography. BioProcess J, 2013; 12(3): 28-30.
http://dx.doi.org/10.12665/J123.Siegel.

Posted online September 30, 2013.

 
Improved Production of Laccase and Peroxidase-Free Tyrosinase in Streptomyces antibioticus

by Krothapalli Raja Surya Sambasiva Rao, Niraj Kant Tripathy, Yadla Mahalaxmi, Reddy Shetty Prakasham, and Devarakonda Srinivasa Rao
Volume 12, Issue 3 (Fall 2013)

The impact of individual and interactive behaviors of various fermentation process parameters on laccase and peroxidase-free tyrosinase production were investigated by isolated Streptomyces antibioticus RSP-T1. Six key bioprocess factors (medium pH, rpm, incubation time, sodium chloride concentration, complex nitrogen [yeast extract + peptone], and carbon source [maltose]) were selected based on using a one variable at a time methodology. All selected parameters had an impact at individual and interactive levels on tyrosinase production. Only 25% of the improved tyrosinase production was attributed to the optimized fermentation parameters selected. Regarding the nutritional parameters, the complex nitrogen/carbon source concentration (maltose and yeast extract + peptone) was found to have the most significance impact on overall tyrosinase enzyme production. Physiological growth factors (pH, rpm, and incubation time) played key roles at an interactive level. A maximum yield of 12.60 IU/mL tyrosinase production was achieved with optimized medium, adjusted to 7.5 pH, consisting of 0.75% maltose (w/v), 0.2% yeast extract (w/v), 0.2% peptone (w/v), and 1.25% sodium chloride (w/v) at 160 rpm in 24 hours. This study identified the optimum medium component concentrations for improved tyrosinase production by S. antibioticus RSP-T1. This strain requires complex nitrogen sources (yeast extract and peptone) for increased product yield. Overall, a greater than 250% increase in tyrosinase production was achieved using this optimization approach, as compared to conventional methods...

Citation:
Rao KRSS, Tripathy NK, Mahalaxmi Y, Prakasham RS, Rao DS. Improved Production of Laccase and Peroxidase-Free Tyrosinase in Streptomyces antibioticus. BioProcess J, 2013; 12(3): 46-54.
http://dx.doi.org/10.12665/J123.RaoPrakasham.

Posted online September 30, 2013.

 
Expediting Clinical Batch Production of Viral-Vectored Vaccines and Gene Therapy Products

by Priyabrata Pattnaik, PhD and George Adams
Volume 12, Issue 3 (Fall 2013)

There are approximately 400 vaccines in commercial manufacture targeted at 24 infectious diseases. Of these vaccines, about 300 are viral-based. It is estimated that 1400 vaccines are in development, half of which are viral-based (~ 640 viral vaccines, ~ 200 viral vectors, ~ 60 virus-like particles). Another 240 gene therapy products are in development that utilize the same viral-based technology. A live vector vaccine is one that uses a weakened or harmless microorganism to transport antigenic viral pieces into a cell in order to stimulate an immune response. Vectored vaccines show promise in reliably inducing potent cell-mediated immunity, which is essential for complex diseases such as AIDS, malaria, and cancer...

Citation:
Pattnaik P, Adams G. Expediting Clinical Batch Production of Viral-Vectored Vaccines and Gene Therapy Products. BioProcess J, 2013; 12(3): 41-45.
http://dx.doi.org/10.12665/J123.Pattnaik.

Posted online September 30, 2013.

 
Purification of Recombinant Baculovirus by End-Point Dilution and gp64 Screening

by Brooks Hayes and Thera Mulvania, PhD
Volume 12, Issue 3 (Fall 2013)

The baculovirus expression vector system (BEVS) has emerged as a powerful tool for the production of recombinant proteins used as therapeutics, reagents, and diagnostics. In order to maximize the system’s efficiency and thereby reduce costs, optimizing production parameters is imperative. A critical factor in optimization is the production of a high-quality baculovirus stock with a high-titer, pure clonal population of recombinant virus that is stable over time. Baculovirus stocks may contain alternate varieties of infectious virus due to cross-contamination, outgrowth of non-recombinant virus, and excision of inserts attributable to some recombinant virus production technologies. Since the advent of the BEVS, the “gold standard” for production of pure baculovirus stocks has been plaque purification. Briefly, plaque purification involves infecting a monolayer of cells with dilutions of virus before applying an agarose overlay to the monolayer. After a 5–7 day incubation, isolated plaques can be picked, virus eluted from the agarose plug, and amplified. The drawbacks of plaque purification are: (1) it is time and labor-intensive; (2) the results hinge greatly on the health of the cells and the cell density at infection; (3) identification and picking of isolated plaques is challenging; and (4) the integrity of the procedure is easily compromised by virus diffusion and mass flow of virus-containing liquid beneath the agarose overlay...

Citation:
Hayes B, Mulvania T. Purification of Recombinant Baculovirus by End-Point Dilution and gp64 Screening. BioProcess J, 2013; 12(3): 20-27.
http://dx.doi.org/10.12665/J123.Hayes.

Posted online September 30, 2013.

 
Model and Method for Optimal Sizing of Serial Microfiltration Systems

by Sal Giglia and Greg Straeffer
Volume 12, Issue 3 (Fall 2013)

Classical filtration fouling models do not accurately predict fouling behaviors for sterilizing filtration applied to some bioprocess fluids, particularly when filters are operated in series. To overcome the limitations of these models, we extended a previously developed single-stage fouling model that combined blocking and adsorption mechanisms to dual filters operated in series. Our model demonstrates improvements in predicting bioprocess fluid filtration performance accuracy when applied to microfiltration membranes operated in series, from measured performance of each membrane filter operated individually. In addition, the model and developed method allows for rapid and efficient optimization of prefiltration to final filter area ratios. In redundant sterile filtration, two filters with the same pore size rating are operated in series to protect against an integrity failure of the primary sterilizing filter. Prefilters can also be used to protect final sterilizing-grade membrane filters from excessive fouling which in turn offer the benefits of reduced costs and filtration time...

Citation:
Giglia S, Straeffer G. Model and Method for Optimal Sizing of Serial Microfiltration Systems. BioProcess J, 2013; 12(3): 31-39.
http://dx.doi.org/10.12665/J123.Giglia.

Posted online September 30, 2013.

 
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