• Title, Summary, Keyword: virus validation

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Comparative Inactivation of Hepatitis A Virus and Murine Encephalomyocarditis Virus to Various Inactivation Processes (바이러스 불활화 공정에 대한 Hepatitis A Virus와 Murine Encephalomyocarditis Virus의 민감도 비교)

  • Kim, In-Seop
    • Korean Journal of Microbiology
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    • v.39 no.4
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    • pp.242-247
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    • 2003
  • Murine encephalomyocarditis virus (EMCV) has been used as a surrogate for hepatitis A virus (HAV) for the validation of virus removal and/or inactivation during the manufacturing process of biopharmaceuticals. Recently international regulation for the validation of HAV safety has been reinforced because of the reported cases of HAV transmission to hemophiliac patients who had received ntihemophilic factors prepared from human plasma. The purpose of the present study was to compare the resistance of HAV and EMCV to various viral inactivation processes and then to standardize the HAV validation method. HAV was more resistant than EMCV to pasteurization (60oC heat treatment for 10 hr), low pH incubation (pH 3.9 at 25oC for 14 days), 0.1 M NaOH treatment, and lyophilization. EMCV was completely inactivated to undetectable levels within 2 hr of pasteurization, however, HAV was completely inactivated to undetectable levels after 5 hr treatment. EMCV was completely inactivated to undetectable levels within 15 min of 0.1 M NaOH treatment, however, residual infectivity of HAV still remained even after 120 min of treatment. The log reduction factors achieved during low pH incubation were 1.63 for HAV and 3.84 for EMCV. Also the log reduction factors achieved during a lyophilization process of antihemophilic factor VIII were 1.21 for HAV and 4.57 for EMCV. These results indicate that HAV rather than EMCV should be used for the virus validation study and the validation results obtained using EMCV should be precisely reviewed.

Validation of a Real-Time RT-PCR Method to Quantify Newcastle Disease Virus (NDV) Titer and Comparison with Other Quantifiable Methods

  • Jang, Juno;Hong, Sung-Hwan;Kim, Ik-Hwan
    • Journal of Microbiology and Biotechnology
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    • v.21 no.1
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    • pp.100-108
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    • 2011
  • A method for the rapid detection and quantification of Newcastle disease virus (NDV) produced in an animal cell culture-based production system was developed to enhance the speed of the NDV vaccine manufacturing process. A SYBR Green I-based real-time RT-PCR was designed with a conventional, inexpensive RT-PCR kit targeting the F gene of the NDV LaSota strain. The method developed in this study was validated for specificity, accuracy, precision, linearity, limit of detection (LOD), limit of quantification (LOQ), and robustness. The validation results satisfied the predetermined acceptance criteria. The validated method was used to quantify virus samples produced in an animal cell culture-based production system. The method was able to quantify the NDV samples from mid- or late-production phases, but not effective on samples from the early-production phase. For comparison with other quantifiable methods, immunoblotting, plaque assay, and tissue culture infectious dose 50 ($TCID_{50}$) assay were also performed with the NDV samples. The results demonstrated that the real-time RT-PCR method is suitable for the rapid quantification of virus particles produced in an animal cell-culture-based production system irrespective of viral infectivity.

Optimization and Validation of a Virus Filtration Process for Efficient Removal of Viruses from Urokinase Solution Prepared from Human Urine

  • Kim, In-Seop;Choi, Yong-Woon;Lee, Sung-Rae
    • Journal of Microbiology and Biotechnology
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    • v.14 no.1
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    • pp.140-147
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    • 2004
  • Urokinase is an enzyme with fibrinolytic activity (plasminogen activator) isolated from fresh urine of healthy men. Viral safety is an important prerequisite for clinical preparation of the protein from urine. In order to increase the viral safety of a high purity urokinase in regard to non-enveloped viruses, a virus removal process using a novel polyvinylidene fluoride membrane filter (Viresolve NFP) has been optimized. Urokinase was able to pass through the filter with recoveries of 95% in the production scale process. No substantial changes were observed in physical and biochemical characteristics of the filtered urokinase in comparison with those of the enzyme before filtration. A 47-mm disk membrane filter was used to simulate the process performance of the production scale cartridges and tested if it could remove several experimental model viruses for human pathogenic viruses, including porcine parvovirus (PPV), human hepatitis A virus (HAV), murine encephalomyocarditis virus (EMCV), bovine viral diarrhoea virus (BVDV), and bovine herpes virus (BHV). Non-enveloped viruses (PPV, HAV, and EMCV) as well as enveloped viruses (BVDV and BHV) were completely removed during filtration. The log reduction factors achieved were $\geq$4.86 for PPV, $\geq$4.60 for HAV, $\geq$6.87 for EMCV, $\geq$4.60 for BVDV, and $\geq$5.44 for BHV. These results indicate that the virus filtration process successfully improved the viral safety of the final products.

Multiplex Reverse Transcription-PCR for Simultaneous Detection of Reovirus, Bovine Viral Diarrhea Virus, and Bovine Parainfluenza Virus during the Manufacture of Cell Culture-derived Biopharmaceuticals (세포배양 유래 생물의약품 제조공정에서 Reovirus, Bovine Viral Diarrhea Virus, Bovine Parainfluenza Virus 동시 검출을 위한 Multiplex Reverse Transcription-PCR)

  • Oh, Seon Hwan;Bae, Jung Eun;Kim, In Seop
    • Microbiology and Biotechnology Letters
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    • v.40 no.4
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    • pp.339-347
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    • 2012
  • Viral safety is an important prerequisite for clinical preparations of mammalian cell culture-derived biopharmaceuticals, because numerous adventitious viruses have been contaminated during the manufacturing process. In particular, Chinese hamster ovary (CHO) cells are highly susceptible to several RNA viruses including reovirus (Reo), bovine viral diarrhea virus (BVDV), and bovine parainfluenza virus (BPIV) and there have been reports of such viral contaminations. Therefore, viral detection during the CHO cell process is necessary to ensure the safety of biopharmaceuticals against viruses. In this study, a multiplex reverse transcription (RT)-PCR assay was developed and subsequently evaluated for its effectiveness as a means to simultaneously detect Reo, BVDV, and BPIV during the manufacture of cell culture-derived biopharmaceuticals. Specific primers for Reo, BVDV, and BPIV were selected, and a multiplex RT-PCR was optimized. The sensitivity of the assay for simultaneous amplification of all viral target RNAs was $7.76{\times}10^2\;TCID_{50}/ml$ for Reo, $7.44{\times}10^1\;TCID_{50}/ml$ for BVDV, and $6.75{\times}10^1\;TCID_{50}/ml$ for BPIV. The multiplex RT-PCR was proven to be very specific to Reo, BVDV, and BPIV and was subsequently applied to the validation of CHO cells artificially infected with each virus. It could detect each viral RNA from CHO cells as well as culture supernatants. Therefore, it was concluded that the multiplex RT-PCR assay can be applied to detection of the adventitious viruses during the manufacture of cell culture-derived biopharmaceuticals.

Evaluation of Viral Inactivation Efficacy of a Continuous Flow Ultraviolet-C Reactor (UVivatec) (연속 유동 Ultraviolet-C 반응기(UVivatec)의 바이러스 불활화 효과 평가)

  • Bae, Jung-Eun;Jeong, Eun-Kyo;Lee, Jae-Il;Lee, Jeong-Im;Kim, In-Seop;Kim, Jong-Su
    • Microbiology and Biotechnology Letters
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    • v.37 no.4
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    • pp.377-382
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    • 2009
  • Viral safety is an important prerequisite for clinical preparations of all biopharmaceuticals derived from plasma, cell lines, or tissues of human or animal origin. To ensure the safety, implementation of multiple viral clearance (inactivation and/or removal) steps has been highly recommended for manufacturing of biopharmaceuticals. Of the possible viral clearance strategies, Ultraviolet-C (UVC) irradiation has been known as an effective viral inactivating method. However it has been dismissed by biopharmaceutical industry as a result of the potential for protein damage and the difficulty in delivering uniform doses. Recently a continuous flow UVC reactor (UVivatec) was developed to provide highly efficient mixing and maximize virus exposure to the UV light. In order to investigate the effectiveness of UVivatec to inactivate viruses without causing significant protein damage, the feasibility of the UVC irradiation process was studied with a commercial therapeutic protein. Recovery yield in the optimized condition of $3,000\;J/m^2$ irradiation was more than 98%. The efficacy and robustness of the UVC reactor was evaluated with regard to the inactivation of human immunodeficiency virus (HIV), hepatitis A virus (HAV), bovine herpes virus (BHV), bovine viral diarrhea virus (BVDV), porcine parvovirus (PPV), bovine parvovirus (BPV), minute virus of mice (MVM), reovirus type 3 (REO), and bovine parainfluenza virus type 3 (BPIV). Non enveloped viruses (HAV, PPV, BPV, MVM, and REO) were completely inactivated to undetectable levels by $3,000\;J/m^2$ irradiation. Enveloped viruses such as HIV, BVDV, and BPIV were completely inactivated to undetectable levels. However BHV was incompletely inactivated with slight residual infectivity remaining even after $3,000\;J/m^2$ irradiation. The log reduction factors achieved by UVC irradiation were ${\geq}3.89$ for HIV, ${\geq}5.27$ for HAV, 5.29 for BHV, ${\geq}5.96$ for BVDV, ${\geq}4.37$ for PPV, ${\geq}3.55$ for BPV, ${\geq}3.51$ for MVM, ${\geq}4.20$ for REO, and ${\geq}4.15$ for BPIV. These results indicate that UVC irradiation using UVivatec was very effective and robust in inactivating all the viruses tested.

Real-Time PCR for Validation of Minute Virus of Mice Safety during the Manufacture of Mammalian Cell Culture-Derived Biopharmaceuticals (세포배양 유래 생물의약품 생산 공정에서 Minute Virus of Mice 안전성 검증을 위한 Real-Time PCR)

  • Lee, Dong-Hyuck;Cho, Hang-Mee;Kim, Hyun-Mi;Lee, Jung-Suk;Kim, In-Seop
    • Microbiology and Biotechnology Letters
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    • v.36 no.1
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    • pp.12-20
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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 minute virus of mice(MVM), and there are several reports of MVM contamination during the manufacture of biopharmaceuticals. In order to establish the validation system for the MVM safety, a real-time PCR method was developed for quantitative detection of MVM in cell lines, raw materials, manufacturing processes, and final products as well as MVM clearance validation. Specific primers for amplification of MVM DNA was selected, and MVM DNA was quantified by use of SYBR Green I. The sensitivity of the assay was calculated to be $6{\times}10^{-2}TCID_{50}/mL$. The real-time PCR method was proven to be reproducible and very specific to MVM. The established real-time PCR assay was successfully applied to the validation of Chinese hamster ovary (CHO) cell artificially infected with MVM. MVM DNA could be Quantified in CHO cell as well as culture supernatant. When the real-time 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 MVM.

Enhanced Virus Removal by Flocculation and Microfiltration

  • Han Binbing;Carlson Jonathan O.;Powers Scott M.;Wickramasinghe S. Ranil
    • Biotechnology and Bioprocess Engineering:BBE
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    • v.7 no.1
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    • pp.6-9
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    • 2002
  • In this work we have investigated the feasibility of virus clearance by flocculation and tangential flow microfiltration. Chinese hamster ovary cell feed streams were spiked with minute virus of mice and then flocculated using cationic polyelectrolytes prior to tangential flow microfiltration. Our results indicate that flocculation prior to microfiltration leads to more than 100 fold clearance of minute virus of mice particles in the permeate. Today, validation of virus clearance is a major concern in the manufacture of biopharmaceutical products. Frequently new unit operations are added simply to validate virus clearance thus increasing the manufacturing cost. The results obtained here suggest that virus clearance can be obtained during tangential flow microfiltration. Since tangential flow microfiltration is frequently used for bioreactor harvesting this could be a low cost method to validate virus clearance.

Real-Time RT-PCR for Quantitative Detection of Bovine Viral Diarrhoea Virus during Manufacture of Biologics (생물의약품 제조공정에서 Bovine Viral Diarrhoea Virus 정량 검출을 위한 Real-Time RT-PCR)

  • Cho, Hang-Mee;Lee, Dong-Hyuck;Kim, Hyun-Mi;Kim, In-Seop
    • Microbiology and Biotechnology Letters
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    • v.36 no.1
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    • pp.34-42
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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 using bovine materials have the risk of viral contamination. Therefore viral validation is essential in ensuring the safety of the products. Bovine viral diarrhoea virus (BVDV) is the most common bovine pathogen and has widely been known as a contaminant of biologics. In order to establish the validation system for the BVDV safety of biologics, a real-time RT-PCR method was developed for quantitative detection of BVDV contamination in raw materials, manufacturing processes, and final products. Specific primers for amplification of BVDV RNA was selected, and BVDV RNA was quantified by use of SYBR Green I. The sensitivity of the assay was calculated to be 1 $TCID_{50}/mL$. The rent-time RT-PCR method was validated to be reproducible and very specific to BVDV. The established real-time RT-PCR assay was successfully applied to the validation of Chinese hamster ovary (CHO) cell artificially infected with BVDV. BVDV RNA could be quantified in CHO cell as well as culture supernatant. Also the real-time RT-PCR assay could detect $10TCID_{50}/mL$ of BVDV artificially contaminated in bovine collagen.

Development of TaqMan Probe Real-Time RT-PCR for Quantitative Detection of Porcine Transmissible Gastroenteritis Virus During the Manufacture of Biopharmaceuticals (생물의약품 제조 공정에서 Porcine transmissible gastroenteritis virus 정량 검출을 위한 TaqMan Probe Real-Time RT-PCR 개발)

  • Lee, Jae Il;Han, Sang Eun;Kim, In Seop
    • Microbiology and Biotechnology Letters
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    • v.43 no.3
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    • pp.267-274
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    • 2015
  • Biopharmaceuticals and the cell substrates used for their manufacture are currently tested for porcine adventitious viruses due to the widespread use of porcine trypsin in cell culture. Porcine transmissible gastroenteritis virus (PTGV) is one of the major adventitious porcine viruses causing contaminated during the manufacture of biopharmaceuticals. Therefore, rapid and sensitive detection of PTGV is essential in ensuring the safety of biopharmaceuticals. A TaqMan probe real-time RT-PCR method was developed for the quantitative detection of PTGV contamination in cell substrates, raw materials, manufacturing processes, and final products, as well as PTGV clearance validation. Specific primers for the amplification of PTGV RNA were selected, and PTGV RNA was quantified by use of a specific TaqMan probe. Specificity, limit of detection (LOD), and robustness of the method was validated according to international guidelines on the validation of nucleic acid amplification tests. The sensitivity of the assay was calculated to be 1.10 × 100 TCID50/ml. The real-time RT-PCR method was validated to be reproducible, very specific to PTGV, and robust. The established real-time RT-PCR assay was successfully applied to the validation of Chinese Hamster Ovary (CHO)-K1 cells artificially infected with PTGV.