Background Cryopreserved conduit valved homografts （CVH） have been widely used in surgical treatment of cardiac disease. This study aimed to determine the extent of host cell ingrowth and the durability and immunoge...Background Cryopreserved conduit valved homografts （CVH） have been widely used in surgical treatment of cardiac disease. This study aimed to determine the extent of host cell ingrowth and the durability and immunogenicity of CVH,and to compare the performance of CVH stored at 4℃ and CVH cryopreserved in liquid nitrogen at -196℃.Methods Heterotopic transplants of canine CVH stored at 4℃ （n=14） and cryopreserved in liquid nitrogen （n=14） were made onto the abdominal aorta of recipient dogs. Animals were sacrificed at 7 and 15 days and at 1, 3, 6, 9, and 12months after transplantation to excise the implanted CVHs. Tissue DNA extraction and quantitative polymerase chain reaction （PCR） were performed to calculate the ratio of donor cells and host cells in the CVH. The tissue viability of CVH after implantation was analyzed by detecting alkaline fibroblast growth factor 2 （FGF-2） using immunohistochemical staining and by observation under transmission electron microscope and scanning electron microscope.Results All the animals survived and recovered well. There were few repopulating host cells （0.04-0.83%） in the implanted CVH at 7 or 15 days. The ratio of ingrowing host cells into the CVH continued rising after implantation and reached 40%-47% in the 12th month postoperation. Histology, transmission electron microscopy and FGF-2immunohistochemical staining indicated that fibroblasts and the host＇s endothelial cells were the main cellular elements invading the CVH. There were no significant differences in results between CVH stored at 4℃ and CVH cryopreserved in liquid nitrogen.Conclusions Host cells growing into CVH are very important for maintaining the long-term structure and function of the implanted CVH. There is no significant difference between CVH storing at 4℃ or in liquid nitrogen in regard to the ingrowth of host cells or of morphologic features after CVH allografting.展开更多
Process analytical technology(PAT) is gaining more interest in the biomanufacturing industry because of its potential to improve operational control and compliance through real-time quality assurance.Currently, biopha...Process analytical technology(PAT) is gaining more interest in the biomanufacturing industry because of its potential to improve operational control and compliance through real-time quality assurance.Currently, biopharmaceutical producers mainly monitor chromatographic processes with ultraviolet/visible(UV/Vis) absorbance. However, this measurement has a very limited correlation with purity and quantity. The current study aims to determine the concentration of monoclonal antibody(mAb) and host cell proteins(HCPs) using a build-in UV/Vis monitoring during Protein A affinity chromatography and to optimize the separation conditions for high purity of mAb and minimizing the HCPs content. The eluate was analyzed through in-line UV/Vis at 280 and 410 nm, representing mAb and HCPs concentration,respectively. Each 0.1 column volume(CV) fraction of UV/Vis chromatogram peak area were calculated,and different separation conditions were then compared. The optimum conditions of mAb separation were found as 12 CV loading, elution at pH 3.5, and starting the collection at 0.5 CV point, resulting in high m Ab recovery of 95.92% and additional removal of 49.98% of HCP comparing with whole elution pool. This study concluded that UV/Vis-based in-line monitoring at 280 and 410 nm showed a high potential to optimize and real-time control Protein A affinity chromatography for mAb purification from HCPs.展开更多
Monitoring of host cell proteins(HCPs)during the manufacturing of monoclonal antibodies(mAb)has become a critical requirement to provide effective and safe drug products.Enzyme-linked immunosorbent assays are still th...Monitoring of host cell proteins(HCPs)during the manufacturing of monoclonal antibodies(mAb)has become a critical requirement to provide effective and safe drug products.Enzyme-linked immunosorbent assays are still the gold standard methods for the quantification of protein impurities.However,this technique has several limitations and does,among others,not enable the precise identification of proteins.In this context,mass spectrometry(MS)became an alternative and orthogonal method that delivers qualitative and quantitative information on all identified HCPs.However,in order to be routinely implemented in biopharmaceutical companies,liquid chromatography-MS based methods still need to be standardized to provide highest sensitivity and robust and accurate quantification.Here,we present a promising MS-based analytical workflow coupling the use of an innovative quantification standard,the HCP Profiler solution,with a spectral library-based data-independent acquisition(DIA)method and strict data validation criteria.The performances of the HCP Profiler solution were compared to more conventional standard protein spikes and the DIA approach was benchmarked against a classical datadependent acquisition on a series of samples produced at various stages of the manufacturing process.While we also explored spectral library-free DIA interpretation,the spectral library-based approach still showed highest accuracy and reproducibility(coefficients of variation<10%)with a sensitivity down to the sub-ng/mg mAb level.Thus,this workflow is today mature to be used as a robust and straightforward method to support mAb manufacturing process developments and drug products quality control.展开更多
Ensuring the removal of host cell proteins(HCPs) during downstream processing of recombinant proteins such as monoclonal antibodies(m Abs) remains a challenge.Since residual HCPs might affect product stability or safe...Ensuring the removal of host cell proteins(HCPs) during downstream processing of recombinant proteins such as monoclonal antibodies(m Abs) remains a challenge.Since residual HCPs might affect product stability or safety,constant monitoring is required to demonstrate their removal to be below the regulatory accepted level of 100 ng/mg.The current standard analytical approach for this procedure is based on ELISA;however,this approach only measures the overall HCP content.Therefore,the use of orthogonal methods,such as liquid chromatography-mass spectrometry(LC-MS),has been established,as it facilitates the quantitation of total HCPs as well as the identification and quantitation of the individual HCPs present.In the present study,a workflow for HCP detection and quantitation using an automated magnetic bead-based sample preparation,in combination with a data-independent acquisition(DIA) LC-MS analysis,was established.Employing the same instrumental setup commonly used for peptide mapping analysis of m Abs allows for its quick and easy implementation into pre-existing workflows,avoiding the need for dedicated instrumentation or personnel.Thereby,quantitation of HCPs over a broad dynamic range was enabled to allow monitoring of problematic HCPs or to track changes upon altered bioprocessing conditions.展开更多
The restricted neurogenesis limits the brain ability to overcome neuronal cell death following ischemic lesion：Failure of the damaged brain to regenerate following cerebral ischemia results in functional deficits tho...The restricted neurogenesis limits the brain ability to overcome neuronal cell death following ischemic lesion：Failure of the damaged brain to regenerate following cerebral ischemia results in functional deficits those are most often irreversible and can further deteriorate,causing mortality and severe disability,progressive memory loss and cognitive impairments,known as dementia.展开更多
Bacteriophages are viruses that infect bacteria and can choose any one of the two alternative pathways for infection,i.e.,lysis or lysogeny.Phage lysis is one of the conventional biological processes required to sprea...Bacteriophages are viruses that infect bacteria and can choose any one of the two alternative pathways for infection,i.e.,lysis or lysogeny.Phage lysis is one of the conventional biological processes required to spread infection from one bacterium to another.Our analysis suggests that in the paradigm bacteriophage Mu,six proteins might be involved in host cell lysis.Mu has a broad host range,and Mu-like phages were found in both Gram-negative and Gram-positive bacteria.An analysis of the genomes of Mu and Mu-like phages could be useful in elucidating the lysis mechanism in this group of phages.A detailed review of the various mechanisms of phage lysis and different proteins associated with the process will help researchers understand the phage biology and their life cycle in different bacteria.The recent increase in the number of multidrug-resistant(MDR)strains of bacteria and the usual long-term nature of new drug development has encouraged scientists to look for alternative strategies like phage therapy and the discovery of new lysis mechanisms.Understanding the lysis mechanism in the Mu-like phages could be exploited to develop alternative therapeutics to kill drug-resistant pathogenic bacteria.In this review article,we have analyzed the phage Mu-mediated host lysis system,which is unknown till now,and our analysis indicates a possibility of the existence of a new lysis mechanism operating in Mu.展开更多
Objective To investigate the distribution and clonality of the T-cell receptor (TCR) Vβ repertoire in chronic graft versus host disease (cGVHD).Methods The complementarity determining region 3 (CDR3) of the TCRβ gen...Objective To investigate the distribution and clonality of the T-cell receptor (TCR) Vβ repertoire in chronic graft versus host disease (cGVHD).Methods The complementarity determining region 3 (CDR3) of the TCRβ gene with 24 variable regions was amplified in peripheral blood mononuclear cells drawn from one cGVHD patient after allogenic bone marrow transplantation (allo-BMT) 35, 39, 43 or 45 months respectively, using RT-PCR, to observe the expression of TCR Vβ repertoire T cells. The PCR products were further analyzed by genescan to evaluate clonality of T cells. Ressults Fourteen or 16 TCR Vβ subfamily T ceils were detected in each sample of cGVHD case. Oligoclonal T cells were identified in TCR Vβ 6, 16, 17, 19 and 21 subfamilies. The stable clonal T cells in all samples were identified in Vβ6, Vβ17 and Vβ21 subfamilies.Conclusion Skewing distribution and stable clonal expansion of T cells can be found in cGVHD cases and it may be related to the initiation of cGVHD.展开更多
The first event in viral infection is the attachment of a virus to specific receptors on the host cell surface. This will trigger conformational changes of the viral surface protein. For
The recent outbreak of the human Zaire ebolavirus(EBOV)epidemic is spiraling out of control in West Africa.Human EBOV hemorrhagic fever has a case fatality rate of up to 90%.The EBOV is classified as a biosafety level...The recent outbreak of the human Zaire ebolavirus(EBOV)epidemic is spiraling out of control in West Africa.Human EBOV hemorrhagic fever has a case fatality rate of up to 90%.The EBOV is classified as a biosafety level 4 pathogen and is considered a category A agent of bioterrorism by Centers for Disease Control and Prevention,with no approved therapies and vaccines available for its treatment apart from supportive care.Although several promising therapeutic agents and vaccines against EBOV are undergoing the Phase I human trial,the current epidemic might be outpacing the speed at which drugs and vaccines can be produced.Like all viruses,the EBOV largely relies on host cell factors and physiological processes for its entry,replication,and egress.We have reviewed currently available therapeutic agents that have been shown to be effective in suppressing the proliferation of the EBOV in cell cultures or animal studies.Most of the therapeutic agents in this review are directed against non-mutable targets of the host,which is independent of viral mutation.These medications are approved by the Food and Drug Administration(FDA)for the treatment of other diseases.They are available and stockpileable for immediate use.They may also have a complementary role to those therapeutic agents under development that are directed against the mutable targets of the EBOV.展开更多
The rabies virus is a neurotropic virus that causes fatal diseases in humans and animals.Although studying the interactions between a single rabies virus and the cell membrane is necessary for understanding the pathog...The rabies virus is a neurotropic virus that causes fatal diseases in humans and animals.Although studying the interactions between a single rabies virus and the cell membrane is necessary for understanding the pathogenesis,the internalization dynamic mechanism of single rabies virus in living cells remains largely elusive.Here,we utilized a novel force tracing technique based on atomic force microscopy(AFM)to record the process of single viral entry into host cell.We revealed that the force of the rabies virus internalization distributed at(65±25)pN,and the time was identified by two peaks with spacings of(237.2±59.1)and(790.3±134.4)ms with the corresponding speed of 0.12 and 0.04µm/s,respectively.Our results provide insight into the effects of viral shape during the endocytosis process.This report will be meaningful for understanding the dynamic mechanism of rabies virus early infection.展开更多
Since the beginning of the COVID-19 pandemic,multiple SARS-CoV-2 variants have emerged.While some variants spread only locally,others,referred to as variants of concern,disseminated globally and became drivers of the ...Since the beginning of the COVID-19 pandemic,multiple SARS-CoV-2 variants have emerged.While some variants spread only locally,others,referred to as variants of concern,disseminated globally and became drivers of the pandemic.All SARS-CoV-2 variants harbor mutations relative to the virus circulating early in the pandemic,and mutations in the viral spike(S)protein are considered of particular relevance since the S protein mediates host cell entry and constitutes the key target of the neutralizing antibody response.As a consequence,mutations in the S protein may increase SARS-CoV-2 infectivity and enable its evasion of neutralizing antibodies.Furthermore,mutations in the S protein can modulate viral transmissibility and pathogenicity.展开更多
文摘Background Cryopreserved conduit valved homografts （CVH） have been widely used in surgical treatment of cardiac disease. This study aimed to determine the extent of host cell ingrowth and the durability and immunogenicity of CVH,and to compare the performance of CVH stored at 4℃ and CVH cryopreserved in liquid nitrogen at -196℃.Methods Heterotopic transplants of canine CVH stored at 4℃ （n=14） and cryopreserved in liquid nitrogen （n=14） were made onto the abdominal aorta of recipient dogs. Animals were sacrificed at 7 and 15 days and at 1, 3, 6, 9, and 12months after transplantation to excise the implanted CVHs. Tissue DNA extraction and quantitative polymerase chain reaction （PCR） were performed to calculate the ratio of donor cells and host cells in the CVH. The tissue viability of CVH after implantation was analyzed by detecting alkaline fibroblast growth factor 2 （FGF-2） using immunohistochemical staining and by observation under transmission electron microscope and scanning electron microscope.Results All the animals survived and recovered well. There were few repopulating host cells （0.04-0.83%） in the implanted CVH at 7 or 15 days. The ratio of ingrowing host cells into the CVH continued rising after implantation and reached 40%-47% in the 12th month postoperation. Histology, transmission electron microscopy and FGF-2immunohistochemical staining indicated that fibroblasts and the host＇s endothelial cells were the main cellular elements invading the CVH. There were no significant differences in results between CVH stored at 4℃ and CVH cryopreserved in liquid nitrogen.Conclusions Host cells growing into CVH are very important for maintaining the long-term structure and function of the implanted CVH. There is no significant difference between CVH storing at 4℃ or in liquid nitrogen in regard to the ingrowth of host cells or of morphologic features after CVH allografting.
基金supported by the National Key Research & Development Program of China (2021YFE0113300)the National Natural Science Foundation of China (22078286 and 21878263)+1 种基金Zhejiang Universitythe Talent-Introduction Program of China for the Postdoctoral Researcher for the financial support。
文摘Process analytical technology(PAT) is gaining more interest in the biomanufacturing industry because of its potential to improve operational control and compliance through real-time quality assurance.Currently, biopharmaceutical producers mainly monitor chromatographic processes with ultraviolet/visible(UV/Vis) absorbance. However, this measurement has a very limited correlation with purity and quantity. The current study aims to determine the concentration of monoclonal antibody(mAb) and host cell proteins(HCPs) using a build-in UV/Vis monitoring during Protein A affinity chromatography and to optimize the separation conditions for high purity of mAb and minimizing the HCPs content. The eluate was analyzed through in-line UV/Vis at 280 and 410 nm, representing mAb and HCPs concentration,respectively. Each 0.1 column volume(CV) fraction of UV/Vis chromatogram peak area were calculated,and different separation conditions were then compared. The optimum conditions of mAb separation were found as 12 CV loading, elution at pH 3.5, and starting the collection at 0.5 CV point, resulting in high m Ab recovery of 95.92% and additional removal of 49.98% of HCP comparing with whole elution pool. This study concluded that UV/Vis-based in-line monitoring at 280 and 410 nm showed a high potential to optimize and real-time control Protein A affinity chromatography for mAb purification from HCPs.
基金supported by the“Association Nationale de la Recherche et de la Technologie”and UCB Pharma S.A.(Belgium and France)via the CIFRE fellowship of Steve Hessmannsupported by the“Agence Nationale de la Recherche”via the French Proteomic Infrastructure ProFI FR2048(ANR-10-INBS-08-03).
文摘Monitoring of host cell proteins(HCPs)during the manufacturing of monoclonal antibodies(mAb)has become a critical requirement to provide effective and safe drug products.Enzyme-linked immunosorbent assays are still the gold standard methods for the quantification of protein impurities.However,this technique has several limitations and does,among others,not enable the precise identification of proteins.In this context,mass spectrometry(MS)became an alternative and orthogonal method that delivers qualitative and quantitative information on all identified HCPs.However,in order to be routinely implemented in biopharmaceutical companies,liquid chromatography-MS based methods still need to be standardized to provide highest sensitivity and robust and accurate quantification.Here,we present a promising MS-based analytical workflow coupling the use of an innovative quantification standard,the HCP Profiler solution,with a spectral library-based data-independent acquisition(DIA)method and strict data validation criteria.The performances of the HCP Profiler solution were compared to more conventional standard protein spikes and the DIA approach was benchmarked against a classical datadependent acquisition on a series of samples produced at various stages of the manufacturing process.While we also explored spectral library-free DIA interpretation,the spectral library-based approach still showed highest accuracy and reproducibility(coefficients of variation<10%)with a sensitivity down to the sub-ng/mg mAb level.Thus,this workflow is today mature to be used as a robust and straightforward method to support mAb manufacturing process developments and drug products quality control.
基金funding from Thermo Fisher Scientific as part of a funded collaborative agreement with NIBR。
文摘Ensuring the removal of host cell proteins(HCPs) during downstream processing of recombinant proteins such as monoclonal antibodies(m Abs) remains a challenge.Since residual HCPs might affect product stability or safety,constant monitoring is required to demonstrate their removal to be below the regulatory accepted level of 100 ng/mg.The current standard analytical approach for this procedure is based on ELISA;however,this approach only measures the overall HCP content.Therefore,the use of orthogonal methods,such as liquid chromatography-mass spectrometry(LC-MS),has been established,as it facilitates the quantitation of total HCPs as well as the identification and quantitation of the individual HCPs present.In the present study,a workflow for HCP detection and quantitation using an automated magnetic bead-based sample preparation,in combination with a data-independent acquisition(DIA) LC-MS analysis,was established.Employing the same instrumental setup commonly used for peptide mapping analysis of m Abs allows for its quick and easy implementation into pre-existing workflows,avoiding the need for dedicated instrumentation or personnel.Thereby,quantitation of HCPs over a broad dynamic range was enabled to allow monitoring of problematic HCPs or to track changes upon altered bioprocessing conditions.
文摘The restricted neurogenesis limits the brain ability to overcome neuronal cell death following ischemic lesion：Failure of the damaged brain to regenerate following cerebral ischemia results in functional deficits those are most often irreversible and can further deteriorate,causing mortality and severe disability,progressive memory loss and cognitive impairments,known as dementia.
基金Hallym University Research Fund and by Basic Science Research Program through the National Research Foundation of Korea(NRF)Funded by the Ministry of Education(NRF-2020R1C1C1008694&NRF-2020R1I1A3074575).
文摘Bacteriophages are viruses that infect bacteria and can choose any one of the two alternative pathways for infection,i.e.,lysis or lysogeny.Phage lysis is one of the conventional biological processes required to spread infection from one bacterium to another.Our analysis suggests that in the paradigm bacteriophage Mu,six proteins might be involved in host cell lysis.Mu has a broad host range,and Mu-like phages were found in both Gram-negative and Gram-positive bacteria.An analysis of the genomes of Mu and Mu-like phages could be useful in elucidating the lysis mechanism in this group of phages.A detailed review of the various mechanisms of phage lysis and different proteins associated with the process will help researchers understand the phage biology and their life cycle in different bacteria.The recent increase in the number of multidrug-resistant(MDR)strains of bacteria and the usual long-term nature of new drug development has encouraged scientists to look for alternative strategies like phage therapy and the discovery of new lysis mechanisms.Understanding the lysis mechanism in the Mu-like phages could be exploited to develop alternative therapeutics to kill drug-resistant pathogenic bacteria.In this review article,we have analyzed the phage Mu-mediated host lysis system,which is unknown till now,and our analysis indicates a possibility of the existence of a new lysis mechanism operating in Mu.
文摘Objective To investigate the distribution and clonality of the T-cell receptor (TCR) Vβ repertoire in chronic graft versus host disease (cGVHD).Methods The complementarity determining region 3 (CDR3) of the TCRβ gene with 24 variable regions was amplified in peripheral blood mononuclear cells drawn from one cGVHD patient after allogenic bone marrow transplantation (allo-BMT) 35, 39, 43 or 45 months respectively, using RT-PCR, to observe the expression of TCR Vβ repertoire T cells. The PCR products were further analyzed by genescan to evaluate clonality of T cells. Ressults Fourteen or 16 TCR Vβ subfamily T ceils were detected in each sample of cGVHD case. Oligoclonal T cells were identified in TCR Vβ 6, 16, 17, 19 and 21 subfamilies. The stable clonal T cells in all samples were identified in Vβ6, Vβ17 and Vβ21 subfamilies.Conclusion Skewing distribution and stable clonal expansion of T cells can be found in cGVHD cases and it may be related to the initiation of cGVHD.
文摘The first event in viral infection is the attachment of a virus to specific receptors on the host cell surface. This will trigger conformational changes of the viral surface protein. For
文摘The recent outbreak of the human Zaire ebolavirus(EBOV)epidemic is spiraling out of control in West Africa.Human EBOV hemorrhagic fever has a case fatality rate of up to 90%.The EBOV is classified as a biosafety level 4 pathogen and is considered a category A agent of bioterrorism by Centers for Disease Control and Prevention,with no approved therapies and vaccines available for its treatment apart from supportive care.Although several promising therapeutic agents and vaccines against EBOV are undergoing the Phase I human trial,the current epidemic might be outpacing the speed at which drugs and vaccines can be produced.Like all viruses,the EBOV largely relies on host cell factors and physiological processes for its entry,replication,and egress.We have reviewed currently available therapeutic agents that have been shown to be effective in suppressing the proliferation of the EBOV in cell cultures or animal studies.Most of the therapeutic agents in this review are directed against non-mutable targets of the host,which is independent of viral mutation.These medications are approved by the Food and Drug Administration(FDA)for the treatment of other diseases.They are available and stockpileable for immediate use.They may also have a complementary role to those therapeutic agents under development that are directed against the mutable targets of the EBOV.
基金This work was supported by the Project of the Education Department of Jilin Province,China(No.JJKH20220666KJ)the Talent of Jilin Province Development Fund Project,China(No.2021Y004)National Natural Science Foundation of China(No.21773017).
文摘The rabies virus is a neurotropic virus that causes fatal diseases in humans and animals.Although studying the interactions between a single rabies virus and the cell membrane is necessary for understanding the pathogenesis,the internalization dynamic mechanism of single rabies virus in living cells remains largely elusive.Here,we utilized a novel force tracing technique based on atomic force microscopy(AFM)to record the process of single viral entry into host cell.We revealed that the force of the rabies virus internalization distributed at(65±25)pN,and the time was identified by two peaks with spacings of(237.2±59.1)and(790.3±134.4)ms with the corresponding speed of 0.12 and 0.04µm/s,respectively.Our results provide insight into the effects of viral shape during the endocytosis process.This report will be meaningful for understanding the dynamic mechanism of rabies virus early infection.
基金SP acknowledges funding by BMBF(01KI2006D,01KI20328A,01KI20396,01KX2021)the Ministry for Science and Culture of Lower Saxony(14-76103-184,MWK HZI COVID-19)+3 种基金the German Research Foundation(DFGPO 716/11-1,PO 716/14-1)MSW received unrestricted funding from Sartorius AG,Lung Research.HMJ received funding from BMBF(01KI2043,NaFoUniMedCovid19-COVIM:01KX2021)Bavarian State Ministry for Science and the Arts and Deutsche Forschungsgemeinschaft(DFG)through the research training groups RTG1660 and TRR130.
文摘Since the beginning of the COVID-19 pandemic,multiple SARS-CoV-2 variants have emerged.While some variants spread only locally,others,referred to as variants of concern,disseminated globally and became drivers of the pandemic.All SARS-CoV-2 variants harbor mutations relative to the virus circulating early in the pandemic,and mutations in the viral spike(S)protein are considered of particular relevance since the S protein mediates host cell entry and constitutes the key target of the neutralizing antibody response.As a consequence,mutations in the S protein may increase SARS-CoV-2 infectivity and enable its evasion of neutralizing antibodies.Furthermore,mutations in the S protein can modulate viral transmissibility and pathogenicity.
