• KSBB Journal
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• v.23 no.4
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• pp.303-310
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• 2008

Bovine blood, cell, tissue, and organ are used as raw materials for manufacturing biologics such as biopharmaceuticals, tissue-engineered products, and cell therapy. Manufacturing processes for the biologics have the risk of viral contamination. Therefore viral validation is essential in ensuring the safety of the products. Bovine parainfluenza virus type 3 (BPIV3) is one of the common bovine pathogens and has widely been known as a contaminant of biologics. In order to establish the validation system for the BPIV3 safety of biologics, a real-time RT-PCR method was developed for quantitative detection of BPIV3 contamination in raw materials, manufacturing processes, and final products. Specific primers for amplification of BPIV3 RNA was selected, and BPIV3 RNA was quantified by use of SYBR Green I. The sensitivity of the assay was calculated to be 2.8 $TCID_{50}/mL$. The real-time RT-PCR method was validated to be reproducible and very specific to BPIV3. The established real-time RT-PCR assay was successfully applied to the validation of Chinese hamster ovary (CHO) cell artificially infected with BPIV3. BPIV3 RNA could be quantified in CHO cell as well as culture supernatant. Also the real-time RT-PCR assay could detect 7.8 $TCID_{50}/mL$ of BPIV3 artificially contaminated in bovine collagen. The overall results indicated that this rapid, specific, sensitive, and robust assay can be reliably used for quantitative detection of BPIV3 contamination during the manufacture of biologics.

• 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.

• Clinical and Experimental Reproductive Medicine
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• v.34 no.3
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• pp.179-188
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• 2007

Objectives: Many neurological diseases are known to be caused by expansion of trinucleotide repeats (TNRs). It is hard to diagnose the alteration of TNRs with single cell level for preimplantation genetic diagnosis (PGD). In this study, we describe methods optimized for PGD of TNRs related diseases such as Huntington's disease (HD), spinocerebellar ataxia 3 (SCA3) and fragile X syndrome (FXS). Methods: We performed the preclinical assays with heterozygous patient's lymphocytes by single cell PCR strategy. Fluorescent semi-nested PCR and fragment analysis using automatic genetic analyzer were applied for HD and SCA 3. Whole genome amplification with multiple displacement amplification (MDA) method and fluorescent PCR were carried out for FXS. Amplification and allele drop-out (ADO) rate were evaluated in each case. Results: The fluorescent semi-nested PCR of single lymphocyte showed 100.0% of amplification and 14.0% of ADO rate in HD, and 94.7% of amplification and 5.6% of ADO rate in SCA3, respectively. We could not detect the PCR product of CGG repeats in FXS using the fluorescent semi-nested PCR alone. After applying the MDA method in FXS, 84.2% of amplification and 31.3% of ADO rate were achieved. Conclusions: Fluorescent semi-nested PCR is a reliable method for PGD of HD and SCA3. The advanced MDA method overcomes the problem of amplification failure in CGG repeats of FXS case. Optimization of methods for single cell analysis could improve the sensitivity and reliability of PGD for complicated single gene disorders of TNRs.

• Korean Journal of Microbiology
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• v.44 no.1
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• pp.14-21
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• 2008

Bovine blood, cell, tissue, and organ are used as raw materials for manufacturing biopharmaceuticals, tissue engineered products, and cell therapy. Manufacturing processes for the biologicals using bovine materials have the risk of viral contamination. Therefore viral validation is, essential in ensuring the safety of the products. Bovine herpesvirus type 1 (BHV-1) is the most common bovine pathogen found in bovine blood, cell, tissue, and organ. In order to establish the validation system for the BHV-1 safety of the products, a real-time PCR method was developed for quantitative detection of BHV-1 in raw materials, manufacturing processes, and final products as well as BHV-1 clearance validation. Specific primers for amplification of BHV-1 DNA was selected, and BHV-1 DNA was quantified by use of SYBR Green I. The sensitivity of the assay was calculated to be $2\;TCID_{50}/ml$. The real-time PCR method was validated to be reproducible and very specific to BHV-1. The established real-time PCR assay was successfully applied to the validation of Chinese hamster ovary (CHO) cell artificially infected with BHV-1. BHV-1 DNA could be quantified in CHO cell as well as culture supernatant. Also the real-time PCR assay could detect $10\;TCID_{50}/ml$ of BHV-1 artificially contaminated in bovine collagen. The overall results indicated that this rapid, specific, sensitive, and robust assay can be reliably used for quantitative detection of BHV-1 contamination during the manufacture of biologics.

• 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 Animal Science and Technology
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• v.50 no.4
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• pp.561-572
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• 2008

Responses of real-time control parameters, such as ORP, DO and pH, to the conditions of biological animal wastewater treatment process were examined to evaluate the stability of real-time control using each parameter. Also an optimum index for supplemental carbon source addition based on NOx-N level was determined under a consideration of denitrification rate by endogenous respiration of microorganism and residual organic matter in liquor. Experiment was performed with lab-scale sequencing batch reactor(SBR) and working volume of the process was 45L. The distinctive nitrogen break point(NBP) on ORP-and DO-time profiles, which mean the termination of nitrification, started disappearing with the maintenance of low NH4-N loading rate. Also the NBP on ORP-and DO-time profiles was no longer observed when high NOx-N was loaded into the reactor, and the sensitivity of ORP became dull with the increase of NOx-N level. However, the distinctive NBP was constantly occurred on pH(mV)-time profile, maintaining unique profile patterns. This stable occurrence of NBP on pH(mV)-time profile was lasted even at very high NOx-N:NH4-N ratio(over 80:1) in reactor, and the specific point could be easily detected by tracking moving slope change(MSC) of the curve. Revelation of NBP on pH(mV)-time profile and recognition of the realtime control point using MSC were stable at a condition of over 300mg/L NOx-N level in reactor. The occurrence of distinctive NBP was persistent on pH(mV)-time profile even at a level of 10,000mg/L STOC(soluble total organic carbon) and the recognition of NBP was feasible by tracing MSC, but that point on ORP and DO-time profiles began to disappear with the increase of STOC level in reactor. The denitrfication rate by endogenous respiration and residual organic matter was about 0.4mg/L.hr., and it was found that 0.83 would be accepted as an index for supplemental carbon source addition when 0.1 of safety factor was applied.

• Ecology and Resilient Infrastructure
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• v.8 no.1
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• pp.32-43
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• 2021

Multichannel automated chamber systems (MCACs) were developed for the continuous monitoring of soil CO2 efflux in forest ecosystems. The MCACs mainly consisted of four modules: eight soil chambers with lids that automatically open and close, an infrared CO2 analyzer equipped with eight multichannel gas samplers, an electronic controller with time-relay circuits, and a programmable logic datalogger. To examine the stability and reliability of the developed MCACs in the field during all seasons with a high temporal resolution, as well as the effects of temperature and soil water content on soil CO2 efflux rates, we continuously measured the soil CO2 efflux rates and micrometeorological factors at the Nam-san experimental site in a Quercus mongolica forest floor using the MCACs from January to December 2010. The diurnal and seasonal variations in soil CO2 efflux rates markedly followed the patterns of changes in temperature factors. During the entire experimental period, the soil CO2 efflux rates were strongly correlated with the temperature at a soil depth of 5 cm (r2 = 0.92) but were weakly correlated with the soil water content (r2 = 0.27). The annual sensitivity of soil CO2 efflux to temperature (Q10) in this forest ranged from 2.23 to 3.0, which was in agreement with other studies on temperate deciduous forests. The annual mean soil CO2 efflux measured by the MCACs was approximately 11.1 g CO2 m-2 day-1. These results indicate that the MCACs can be used for the continuous long-term measurements of soil CO2 efflux in the field and for simultaneously determining the impacts of micrometeorological factors.