• Korean Journal of Microbiology
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• v.41 no.3
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• pp.216-224
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• 2005

Chromatography has now been used successfully to provide the requisite purity for human plasma-derived biop-harmaceuticals such as coagulation factors and immunoglobulins. Recently, increasing attention has been focused on establishing efficient cleaning procedures to prevent potential contamination by microorganisms as well as carry-over contamination from batch to batch. The purpose of present study was to develop a cleaning validation system for the assurance of virus removal and/or inactivation during chromatography process. In order to establish an assay system for the validation of virus clearance during chromatography cleaning process, a quantitative real-time PCR method for porcine parvovirus(PPV) was developed, since PPV, a model virus for human parvovirus B19, has a high resistance to a range of physico-chemical treatment. Specific primers for amplification of PPV DNA was selected, and PPV DNA was quantified by use of SYBR Green I. The sensitivity of the assay was calculated to be 1.5 $TCID_{50}/ml$. The established real-time PCR assay was successfully applied to the validation of PPV removal and cleaning during SP-Sepharose cation chromatography for thrombin purification and Q-Sepharose anion chromatography for factor VIII purification. The comparative results obtained by real-time PCR assay and infectivity titrations suggested that the real-time PCR assay could be a useful method for chromatography cleaning validation and that it could have an additive effect on the interpretation and evaluation of virus clearance during the virus removal process.

• Journal of The Korean Society of Agricultural Engineers
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• v.56 no.1
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• pp.81-88
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• 2014

Accurate wind data is essential for predicting airborne spread of virus. OpenFOAM was used for computational fluid dynamics (CFD) simulation procedure which is under GNU GPL (General Public License). Using complex terrain, DEM (Digital Elevation Map) that was prepared from GIS information covering a research site is converted to a three dimensional surface mesh that is composed by quad and full hexahedral space meshes. Around this surface mesh, an extended computational domain volume was designed. Atmospheric flow boundary conditions were used at inlet and roughness height and was considered at terrain by using rough wall function. Two different wind conditions that was relatively stable during certain periods were compared in 3 different locations for validating the accuracy of the CFD computed solution. The result shows about 10 % of difference between the calculated result and measured data. This procedure can simulate a prediction of time-series data for airborne virus spread that can be used to make a web-based forecasting system of airborne virus spread.

• Microbiology and Biotechnology Letters
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• v.38 no.2
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• pp.168-176
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• 2010

Acellular dermal matrix (ADM), produced by decellularization from human cadaveric skin, has been used for various biomedical applications. A manufacturing process for ADM ($SureDerm^{TM}$) using tri-n-butyl phospahate (TnBP) and deoxycholic acids as the decellularization solution has been developed. The manufacturing process for $SureDerm^{TM}$ has 70% ethanol treatment and ethylene oxide gas sterilization for inactivating infectious microorganisms. The purpose of this study was to examine the efficacy of the 70% ethanol treatment, decellularization process using 0.1% TnBP and 2% deoxycholic acids, and EO gas sterilization process in the inactivation of viruses. A variety of experimental model viruses for human pathogens, including the human immunodeficiency virus type 1 (HIV-1), bovine herpes virus (BHV), bovine viral diarrhoea virus (BVDV), hepatitis A virus (HAV), and porcine parvovirus (PPV) were all selected for this study. Enveloped viruses such as HIV-1, BHV, and BVDV were effectively inactivated to undetectable levels by 70% ethanol treatment. However HAV and PPV showed high resistance to 70% ethanol treatment with the log reduction factors of 1.85 and 1.15, respectively. HIV-1, BHV, and BVDV were effectively inactivated to undetectable levels by decellularization process. All the viruses tested were completely inactivated to undetectable levels by EO gas treatment. The cumulative log reduction factors of HIV-1, BHV, BVDV, HAV, and PPV were $\geq12.71$, $\geq18.08$, $\geq14.92$, $\geq6.57$, and $\geq7.18$, respectively. These results indicate that the production process for $SureDerm^{TM}$ has a sufficient virus-reducing capacity to achieve a high margin of the virus safety.

• Korean Journal of Microbiology
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• v.48 no.4
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• pp.314-318
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• 2012

Most types of collagen used for biomedical applications, such as cell therapy and tissue engineering, are derived from animal tissues. Therefore, special precautions must be taken during the production of these proteins in order to assure against the possibility of the products transmitting infectious diseases to the recipients. The ability to remove and/or inactivate known and potential viral contaminants during the manufacturing process is an ever-increasingly important parameter in assessing the safety of biomedical products. The purpose of this study was to evaluate the efficacies of the 70% ethanol treatment and pepsin treatment at pH 2.0 for the inactivation of bovine viruses during the manufacture of collagen type I from bovine hides. A variety of experimental model viruses for bovine viruses including bovine herpes virus (BHV), bovine viral diarrhea virus (BVDV), bovine parainfluenza 3 virus (BPIV-3), and bovine parvovirus (BPV), were chosen for the evaluation of viral inactivation efficacy. BHV, BVDV, BPIV-3, and BPV were effectively inactivated to undetectable levels within 1 h of 70% ethanol treatment for 24 h, with log reduction factors of ${\geq}5.58$, ${\geq}5.32$, ${\geq}5.11$, and ${\geq}3.42$, respectively. BHV, BVDV, BPIV-3, and BPV were also effectively inactivated to undetectable levels within 5 days of pepsin treatment for 14 days, with the log reduction factors of ${\geq}7.08$, ${\geq}6.60$, ${\geq}5.60$, and ${\geq}3.59$, respectively. The cumulative virus reduction factors of BHV, BVDV, BPIV-3, and BPV were ${\geq}12.66$, ${\geq}11.92$, ${\geq}10.71$, and ${\geq}7.01$. These results indicate that the production process for collagen type I from bovine hides has a sufficient virus-reducing capacity to achieve a high margin of virus safety.

• Korean Journal of Microbiology
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• v.44 no.3
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• pp.228-236
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• 2008

Validation of viral safety is essential in ensuring the safety of mammalian cell culture-derived biopharmaceuticals, because numerous adventitious viruses have been contaminated during the manufacture of the products. Mammalian cells are highly susceptible to Reovirus type 3 (Reo-3), and there are several reports of Reo-3 contamination during the manufacture of biopharmaceuticals. In order to establish the validation system for the Reo-3 safety, a real-time RT-PCR method was developed for quantitative detection of Reo-3 in cell lines, raw materials, manufacturing processes, and final products as well as Reo-3 clearance validation. Specific primers for amplification of Reo-3 RNA was selected, and Reo-3 RNA was quantified by use of SYBR Green I. The sensitivity of the assay was calculated to be $3.2{\times}10^0\;TCID_{50}/ml$. The real-time RT-PCR method was proven to be reproducible and very specific to Reo-3. The established real-time RT-PCR assay was successfully applied to the validation of Chinese hamster ovary (CHO) cell artificially infected with Reo-3. Reo-3 RNA could be quantified in CHO cell as well as culture supernatant. When the real-time RT-PCR assay was applied to the validation of virus removal during a virus filtration process, the result was similar to that of virus infectivity assay. Therefore, it was concluded that this rapid, specific, sensitive, and robust assay could replace infectivity assay for detection and clearance validation of Reo-3.

• Journal of Microbiology
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• v.44 no.1
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• pp.72-76
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• 2006

Plasma-derived products are produced from plasma via fractionation and chromatography techniques, but can also be produced by other methods. In the performance of nucleic acid amplification tests (NAT) with plasma-derived products, it is necessary to include an internal control for the monitoring of all procedures. In order to avoid false negative results, we confirmed the usefulness of the bovine viral diarrhoea virus (BVDV) for use as an internal control in the detection of hepatitis C virus (HCV) RNA in plasma-derived products. These products, which were spiked with BVDV, were extracted and then NAT was performed. Specificity and sensitivity were determined via the adjustment of primer concentrations and annealing temperatures. BVDV detection allows for validation in the extraction, reverse transcription, and amplification techniques used for HCV detection in plasma-derived products.

• Journal of the Korea Institute of Military Science and Technology
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• v.22 no.4
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• pp.575-580
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• 2019

In biological warfare, it is important to identify biological agents for proper treatment. We focused on developing a real-time RT-PCR kit that can detect multiple species of biological agents. AccuPower(R) Biothreat Real-Time RT-PCR Kit(v3.0) could detect Bacillus anthracis, Yersinia pestis, Vibrio cholerae, Francisella tularensis, Salmonella typhi, Rickettsia prowazekii, Variola virus, Hantaan virus, Yellow fever virus, Brucella spp., Shigella dysenteriae in a single reaction. The results showed that the kit was verified to be able to detect at least 0.005 ng of nucleotide and 10,000 CFU/ml of bacteria. Therefore, the kit is expected to be used as a rapid and sensitive detection kit for 11 species of biological agents within 2 hours.

• Microbiology and Biotechnology Letters
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• v.38 no.4
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• pp.420-427
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• 2010

A process for manufacturing virally-safe bovine amniotic membrane(BAM) has been developed for biological dressing. BAM was harvested from a healthy bovine placenta, and then the epithelium was removed. The remaining stromal layer was consecutively disinfected with 70% ethanol and 0.05% sodium hypochlorite. The stromal layer was incubated in a decellularization solution containing 0.25%(w/v) trypsin to remove the cellular components. The resulting acelluar BAM was lyophilized to preserve its biochemical and structural integrity. The BAM was packed and exposed to 25 kGy of gamma irradiation for sterilization purpose. Histological, electron microscopical, and biochemical observations showed that the acellualr BAM had intact structural integrity of three dimensional collagen fibers and contained several growth factors, accelerating wound healing, such as EGF (Epidermal growth factor), KGF (Keratinocyte growth factor), and FGF (Fibroblast growth factor). Bovine herpes virus (BHV), bovine viral diarrhoea virus (BVDV), bovine parainfluenza virus type 3 (BPIV-3), and bovine parvovirus (BPV) were chosen as the biological indicators for validation of viral safety of the acellular BAM. Samples from relevant stages of the production process were spiked with each virus and subjected to viral inactivation processes. Viruses were recovered from the samples and then titrated immediately. All the viruses tested were completely inactivated to undetectable levels within 1 h of 70% ethanol treatment. Enveloped viruses such as BHV, BVDV, and BPIV-3 were more effectively inactivated than BPV by 0.05% sodium hypochlorite treatment. BHV, BVDV, and BPIV-3 were completely inactivated to undetectable levels by 25 kGy of gamma irradiation. Also BPV was effectively inactivated by 25 kGy of gamma irradiation. The cumulative log reduction factors of BHV, BVDV, BPIV-3, and BPV were ${\geq}$13.30, ${\geq}$14.32, ${\geq}$15.22, and ${\geq}$7.57, respectively. These results indicate that the production process for acelluar BAM has a sufficient virus-reducing capacity to achieve a high margin of the virus safety.

• Membrane Journal
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• v.30 no.1
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• pp.9-20
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• 2020

Membranes are used in most processes of biopharmaceutical production. It is used for pretreatment of other processes, separation of impurities in the process, virus removal, control of products concentration and buffer solution exchange. Virus filters play an important role in ensuring product efficacy and stability because viral contamination of biopharmaceuticals for humans is a sensitive issue that is directly related to serious clinical outcomes. Virus filters typically have complex multilayer structures made of various polymers such as surface-modified PVDF, PES, CRC. Depending on the manufacturer, filters have different pore structures and shapes, such as symmetric or asymmetric, and is used in the form of pleated membrane, flat sheets or hollow fibers. Virus filters are exclusively supplied by few foreign companies such as Asahi Kasei, Millipore, Pall and Sartorius. Replacing virus filters can be time consuming and expensive, including approval from regulatory agencies through validation. As localization has become important due to Japan's recent export regulations, it is necessary to increase the degree of technical independence.

• Research in Plant Disease
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• v.26 no.3
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• pp.144-158
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• 2020

Quantitative reverse transcription PCR is used for gene expression analysis as the accurate and sensitive method. To analyze quantification of gene expression changes in apple plants, 10 housekeeping genes (ACT, CKL, EF-1α, GAPDH, MDH, PDI, THFs, UBC, UBC10, and WD40) were evaluated for their stability of expression during infection by Apple stem grooving virus (ASGV) or in cold-stress apple plant buds. Five reference-gene validation programs were used to establish the order of the most stable genes for ASGV as CKL>THFs>GAPDH>ACT, and the least stable genes WD40CKL>UBC10, and the least stable genes were ACT