BCHT Biotechnology Company

Changchun, China

BCHT Biotechnology Company

Changchun, China
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Xi H.,Jilin University | Yuan R.,BCHT Biotechnology Company | Chen X.,BCHT Biotechnology Company | Gu T.,Jilin University | And 5 more authors.
Protein Expression and Purification | Year: 2016

An anti-rabies virus single-chain antibody fragment of an anti-glycoprotein with the VL-linker-VH orientation, designated scFv57RN, was successfully and conveniently prepared in this study. The scFv57RN protein was mainly expressed in inclusion bodies in Escherichia coli. After washing and purification, the inclusion bodies were finally obtained with an on-column refolding procedure. Further purification by gel exclusion chromatography was performed to remove inactive multimers. About 360 mg of final product was recovered from 1 L of bacterial culture. The final product showed a high neutralizing titer of 950 IU/mg to the CVS-11 strain as measured using the rapid fluorescent focus inhibition test. Our study demonstrated a highly efficient method to mass produce scFV57RN with activity from inclusion bodies, which may be applied in the purification of other insoluble proteins. © 2016 Elsevier Inc. All rights reserved.


Sun B.,Jilin University | Zhao D.,Jilin University | Zhang X.,Jilin University | Gu T.,Jilin University | And 11 more authors.
Applied Microbiology and Biotechnology | Year: 2016

Here, we describe a process for expression, purification, and characterization of truncated human papillomavirus type-6 (HPV-6) L1 virus-like particles (VLPs). The scalable cultivation process in a WAVE Bioreactor at the 10-L scale was optimized to express HPV-6 L1 VLPs using the baculovirus insect expression system. A hollow fiber membrane system was used for the integrated operation, including concentration, diafiltration, extraction, and clarification. The HPV-6 L1 protein was further purified by anion-exchange chromatography and hydrophobic chromatography. The HPV-6 L1 protein could self-assemble into VLPs with a diameter of approximately 50–60 nm after removal of the reductant dithiothreitol (DTT). The final purified HPV-6 L1 VLPs product was characterized to estimate yield and purity, and exceeds the requirements for pharmaceutical-grade VLP vaccine. Immunization of mice demonstrated that the vaccine could elicit high titer neutralizing antibodies in vivo. This study confirms the feasibility of producing pharmaceutical-grade HPV type-6 L1 VLPs on an industrial scale for clinical trials. © 2015, Springer-Verlag Berlin Heidelberg.


Yuan R.,Jilin University | Chen X.,Jilin University | Chen Y.,Jilin University | Gu T.,Jilin University | And 9 more authors.
Applied Microbiology and Biotechnology | Year: 2014

Rabies virus (RABV) causes a fatal infectious disease, but effective protection may be achieved with the use of rabies immunoglobulin and a rabies vaccine. Virus-neutralizing antibodies (VNA), which play an important role in the prevention of rabies, are commonly evaluated by the RABV neutralizing test. For determining serum VNA levels or virus titers during the RABV vaccine manufacturing process, reliability of the assay method is highly important and mainly dependent on the diagnostic antibody. Most diagnostic antibodies are monoclonal antibodies (mAbs) made from hybridoma cell lines and are costly and time consuming to prepare. Thus, production of a cost-effective mAb for determining rabies VNA levels or RABV titers is needed. In this report, we describe the prokaryotic production of a RABV-specific single-chain variable fragment (scFv) protein with a His-tag (scFv98H) from a previously constructed plasmid in a bioreactor, including the purification and refolding process as well as the functional testing of the protein. The antigen-specific binding characteristics, affinity, and relative affinity of the purified protein were tested. The scFv98H antibody was compared with a commercial RABV nucleoprotein mAb for assaying the VNA level of anti-rabies serum samples from different sources or testing the growth kinetics of RABV strains for vaccine manufactured in China. The results indicated that scFv98H may be used as a novel diagnostic tool to assay VNA levels or virus titers and may be used as an alternative for the diagnostic antibody presently employed for these purposes. © 2013 Springer-Verlag Berlin Heidelberg.


Sun B.,Jilin University | Yu X.,Jilin University | Kong W.,Jilin University | Sun S.,Jilin University | And 8 more authors.
Applied Microbiology and Biotechnology | Year: 2013

A process for human influenza H1N1 virus vaccine production from Madin-Darby canine kidney (MDCK) cells using a novel packed-bed bioreactor is described in this report. The mini-bioreactor was used to study the relationship between cell density and glucose consumption rate and to optimize the infection parameters of the influenza H1N1 virus (A/New Caledonia/20/99). The MDCK cell culture and virus infection were then monitored in a disposable perfusion bioreactor (AmProtein Current Perfusion Bioreactor) with proportional-integral- derivative control of pH, dissolved O2 (DO), agitation, and temperature. During 6 days of culture, the total cell number increased from 2.0 × 109 to 3.2 × 1010 cells. The maximum virus titers of 768 hemagglutinin units/100 μL and 7.8 × 107 50 % tissue culture infectious doses/mL were obtained 3 days after infection. These results demonstrate that using a disposable perfusion bioreactor for large-scale cultivation of MDCK cells, which allows for the control of DO, pH, and other conditions, is a convenient and stable platform for industrial-scale production of influenza vaccines. © 2012 Springer-Verlag.


PubMed | Jilin University and BCHT Biotechnology Company
Type: Evaluation Studies | Journal: Applied microbiology and biotechnology | Year: 2016

Here, we describe a process for expression, purification, and characterization of truncated human papillomavirus type-6 (HPV-6) L1 virus-like particles (VLPs). The scalable cultivation process in a WAVE Bioreactor at the 10-L scale was optimized to express HPV-6 L1 VLPs using the baculovirus insect expression system. A hollow fiber membrane system was used for the integrated operation, including concentration, diafiltration, extraction, and clarification. The HPV-6 L1 protein was further purified by anion-exchange chromatography and hydrophobic chromatography. The HPV-6 L1 protein could self-assemble into VLPs with a diameter of approximately 50-60nm after removal of the reductant dithiothreitol (DTT). The final purified HPV-6 L1 VLPs product was characterized to estimate yield and purity, and exceeds the requirements for pharmaceutical-grade VLP vaccine. Immunization of mice demonstrated that the vaccine could elicit high titer neutralizing antibodies in vivo. This study confirms the feasibility of producing pharmaceutical-grade HPV type-6 L1 VLPs on an industrial scale for clinical trials.


PubMed | Jilin University and BCHT Biotechnology Company
Type: | Journal: Protein expression and purification | Year: 2016

An anti-rabies virus single-chain antibody fragment of an anti-glycoprotein with the VL-linker-VH orientation, designated scFv57RN, was successfully and conveniently prepared in this study. The scFv57RN protein was mainly expressed in inclusion bodies in Escherichia coli. After washing and purification, the inclusion bodies were finally obtained with an on-column refolding procedure. Further purification by gel exclusion chromatography was performed to remove inactive multimers. About 360mg of final product was recovered from 1L of bacterial culture. The final product showed a high neutralizing titer of 950IU/mg to the CVS-11 strain as measured using the rapid fluorescent focus inhibition test. Our study demonstrated a highly efficient method to mass produce scFV57RN with activity from inclusion bodies, which may be applied in the purification of other insoluble proteins.


Yang P.,BCHT Biotechnology Company | Sun Q.,BCHT Biotechnology Company | Sun S.-Y.,BCHT Biotechnology Company | Song Y.-S.,BCHT Biotechnology Company | And 2 more authors.
Chinese Journal of Biologicals | Year: 2016

Objective: To evaluate the cell-mediated immunity (CMI) of live attenuated varicella vaccine by interferon γ (IFNγ) release assay. Methods: The effect of various inactivated antigens (heat-inactivated, ultraviolet-inactivated and formaldehyde-inactivated), incubation time (24, 48 and 72 h) and individuals (12 volunteers before and after immunization with live attenuated varicella vaccine) on the release level of IFNγ were evaluated by quantitative ELISA kit for IKNγ. Results: The IFNγ release levels stimulated by ultraviolet-inactivated antigen and by fresh blood co-incubated with ultraviolet-inactivated antigen for 48 h were the highest. The GMTs of IFNγ in blood of 12 volunteers before and after immunization were 6. 13 and 234. 75 pg/ml respectively. However, the GMTs and their increasing folds showed significant difference in various individuals. Conclusion: IFNγ release assay was simple, rapid and quantitative, which was suitable for the evaluation of CMI of varicella vaccine.

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