Functional analysis of the herpes simplex virus UL42 protein

The herpes simplex virus UL42 gene encodes a multifunctional polypeptide (UL42) that is essential for virus DNA replication. To further understand the relationship between the structure of UL42 and the role that it plays during virus replication, we analyzed an extensive set of mutant UL42 proteins for the ability to perform the three major biochemical functions ascribed to the protein:binding to DNA, stably associating with the virus DNA polymerase (Pol), and acting to increase the length of DNA chains synthesized by Pol. Selected mutants were also assayed for their ability to complement the replication of a UL42 null virus. The results indicated that the N-terminal 340 amino acids of UL42 were sufficient for all three biochemical activities and could also support virus replication. Progressive C-terminal truncation resulted in the loss of detectable DNA-binding activity before Pol binding, while several mutations near the N terminus of the polypeptide resulted in an altered interaction with DNA but had no apparent affect on Pol binding. More dramatically, an insertion mutation at residue 160 destroyed the ability to bind Pol but had no effect on DNA binding. This altered polypeptide also failed to increase the length of DNA product synthesized by Pol, and the mutant gene could not complement the growth of a UL42 null virus, indicating that the specific interaction between Pol and UL42 is necessary for full Pol function and for virus replication. This study confirms the validity of the Pol-UL42 interaction as a target for the design of novel therapeutic agents.     

Manufacturing Vaccines: An Illustration of Using PAT Tools for Controlling the Cultivation of Bordetella pertussis

An illustration of the operational consistency of the upstream part of a biopharmaceutical process is given. For this purpose four batch cultivations of Bordetella pertussis have been executed under identical conditions. The batches have been monitored by means of two fundamentally different process sensors. First, common single channel measurements such as temperature, pH, dissolved oxygen (DO), and flow rates are used and second, the multichannel measurements from the NIR (Near Infrared) analyzer. Because of the fundamental differences between the two types of measurements, two models have been developed to evaluate the operational consistency. The last sensor studied is a typical representative of process analyzers which are described in the PAT (Process Analytical Technology) guidance document issued in 2004 by the American Food and Drug Administration (FDA). Data from both sensors have been evaluated by a multivariate tool for data acquisition. This resulted in two different performance models. Again this approach is characteristic for the implementation of PAT for the manufacture of biopharmaceuticals.

With both performance models, we were able to explore the operational consistency of the batches. In addition, the performance models were also able to detect a deviating batch. Further, it was shown that both sensor types gave partly overlapping information since a deviation in the batch profiles of the logged process variables was accompanied by a deviation in the spectral batch profiles.

The performance models are valuable tools in developing advanced monitoring and control systems for biopharmaceutical processes. Using such models, advanced knowledge based systems can be developed to detect abnormal situations in an early stage and remove the cause.

The procedure of data processing described in this article is relatively new in the biopharmaceutical industry. The NIR analyzer and both performance models presented in this article are clear ingredients for better process understanding and process control, as intended in the FDA's PAT Initiative. This initiative is part of the FDA's strategy of cGMP (current good manufacturing practice) for the 21st century and aims at introducing innovations in both the manufacturing of biopharmaceuticals and the development of new biopharmaceuticals.

This study shows the feasibility of two typical PAT tools for controlling the manufacturing of biopharmaceuticals. To the best of our knowledge such feasibility study is not documented up to now in the scientific literature.     

Metal requirements of low-carbon power generation

Today, almost 70% of the electricity is produced from fossil fuels and power generation accounts for over 40% of global CO₂ emissions. If the targets to reduce climate change are to be met, substantial reductions in emissions are necessary. Compared to other sectors emission reductions in the power sector are relatively easy to achieve because it consists mainly of point-sources. Carbon Capture and Storage (CCS) and the use of low-carbon alternative energy sources are the two categories of options to reduce CO₂ emissions. However, for both options additional infrastructure and equipment is needed. This article compares CO₂ emissions and metal requirements of different low-carbon power generation technologies on the basis of Life Cycle Assessment. We analyze the most critical output (CO₂) and the most critical input (metals) in the same methodological framework. CO₂ emissions and metal requirements are compared with annual global emissions and annual production for different metals. It was found that all technologies are very effective in reducing CO₂ emissions. However, CCS and especially non-fossil technologies are substantially more metal intensive than existing power generation. A transition to a low-carbon based power generation would require a substantial upscaling of current mining of several metals.