The structural integrity of herpes simplex virus 2 (HSV-2) during freezing, thawing, and lyophilization has been studied using scanning and transmission electron microscopy. Viral particles should be thawed quickly from -80 to 37 degrees C to avoid artifacts of thawing. To avoid freezing damage, the virus should be rapidly frozen (>20 K s(-1)) rather than slowly frozen as occurs on the shelf of a lyophilizer (<1 K s(-1)). Fast freezing and thawing allows six cycles of freeze thaw with no loss of viral titer TCID50. Viral particles were characterized using immunogold labeling methods. Freshly thawed virus had 19 +/- 4 polyclonal immunogold particles virus(-1); virus stored at -80 degrees C for at least 4 months had 17 +/- 3 particles virus(-1); virus stored for 1 week at 4 degrees C had 8 +/- 4 particles virus(-1). By bulk lyophilization the number of particles was 4 +/- 4, but by fast freezing and lyophilization the number of gold particles improved to 12 +/- 5. The loss of viral membrane was directly observed, and the in vitro loss was demonstrated to occur through three possible pathways, including (i) simultaneous release of tegument and membrane, (ii) sequential release of membrane and then tegument, and (iii) release like by in vivo infection. The capsids were not further degraded as indicated by the lack of free DNA, which was only released by boiling the viral samples with 1% SDS, followed by a dilution to 0.001% w/v SDS for the real-time PCR reaction.
Lyophilization is the most popular method for achieving improved stability of labile biopharmaceuticals, but a significant fraction of product activity can be lost during processing due to stresses that occur in both the freezing and the drying stages. The effect of the freezing rate on the recovery of herpes simplex virus 2 (HSV-2) infectivity in the presence of varying concentrations of cryoprotectant excipients is reported here. The freezing conditions investigated were shelf cooling (223 K), quenching into slush nitrogen (SN2), and plunging into melting propane cooled in liquid nitrogen (LN2). The corresponding freezing rates were measured, and the ice crystal sizes formed within the samples were determined using scanning electron microscopy (SEM). The viral activity assay demonstrated the highest viral titer recovery for nitrogen cooling in the presence of low (0.25% w/v sucrose) excipient concentration. The loss of viral titer in the sample cooled by melting propane was consistently the highest among those results from the alternative cooling methods. However, this loss could be minimized by lyophilization at lower temperature and higher vacuum conditions. We suggest that this is due to a higher ratio of ice recrystallization for the sample cooled by melting propane during warming to the temperature at which freeze-drying was carried out, as smaller ice crystals readily enlarge during warming. Under the same freezing condition, a higher viral titer recovery was obtained with a formulation containing a higher concentration of sugar excipients. The reason was thought to be twofold. First, sugars stabilize membranes and proteins by hydrogen bonding to the polar residues of the biomolecules, working as a water substitute. Second, the concentrated sugar solution lowers the nucleation temperature of the water inside the virus membrane and prevents large ice crystal formation within both the virus and the external medium.
The presence of impurities in captured CO2 plays a vital role in the safe and effective CO2 transport and storage in the CCUS chain. Impurities can significantly increase the cost of processing, transport, and storage and moreover add additional challenges to the design, operation, health and safety and integrity aspects. The effects of various impurities on the aforementioned challenges have been addressed in this work. Despite the importance of this area, there are still some knowledge gaps in terms of assessing the impact of CO2 specification on CCUS design and operations. International standards address different elements of the CCS chain, but none cover the full chain or consider the full chain economics. There are also differences between industry and leading CO2 authorities regarding the potential issues and challenges of implementing those standards. This paper reviews available standards and references which provide specifications/limitations for impurities for the purpose of transport and storage. In this work, the modified cubic EoSs and GERG EoS have been used to predict the thermodynamic properties and tuned viscosity models have been used for the prediction of transport properties. The required specifications for the quality of CO2 streams have been investigated using the above methodology for fluid properties, followed by the use of commercial software packages for thermohydraulic analysis of CO2 pipelines. Additionally, the storage capacity and geochemistry of fluids under high-pressure and high-temperature (HPHT) storage conditions were investigated. The impact of impurities has been assessed based on various CO2 sources using commercial capturing technologies. The assessment considered the impact of impurities on thermodynamic, thermohydraulic, integrity and operation of CO2 transport, injection, and storage system. This would include the effects of various types of components and their typical concentrations, e.g., water content, non-condensable gases (N2, O2, CH4, Ar, H2and CO), toxic gases (H2S and SO2), and hydrocarbons, on the thermophysical properties including density, viscosity, phase envelope and hydraulic parameters. A comparison of modelling results against the available experimental data measured at elevated pressure and temperature conditions have also been presented. This paper has mainly focused on the lessons learned from past CO2 transport design and operational experiences in order to identify the areas where it could lead to an optimised system in terms of design, costs, and operation. Additionally, past experience in the design of CO2 pipelines and operation of CO2 injection has been used to identify opportunities where CO2 specifications and guidelines could potentially be modified in order to achieve an optimised and cost-effective CO2 transport and injection system. Keywords: CO2 Specification; CO2 Transport Pipelines; Design and Operation Challenges; CO2 impurities; CCUS;
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