The Experts below are selected from a list of 762 Experts worldwide ranked by ideXlab platform
Matiullah Khan - One of the best experts on this subject based on the ideXlab platform.
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Misfolded conformation dependent loss (MCDL) of N-CoR in AML-M5.
2013Co-Authors: Dawn Sijin Nin, Norio Asou, Chienshing Chen, Azhar Bin Ali, Koichi Okumura, Wee Joo Chng, Matiullah KhanAbstract:A, An aliquot of whole cell extract of various AML derived cells as mentioned on the top of each lane was resolved in SDS-PAGE and stained with N-CoR antibody. B, An aliquot of whole cell extract of AML-M5 patient samples was resolved in SDS-PAGE and stained with N-CoR antibody. N-CoR level in HL-60 was used as positive control. C, N-CoR is stabilized by AEBSF and genistein. Level of full length and cleaved N-CoR protein in THP-1 cells treated with AEBSF or genistein in a dose dependent manner was determined by western blotting assay using N-CoR antibody. D, Native N-CoR conformation is rescued by genistein but not by AEBSF. Relative solubility/insolubility of N-CoR protein in AEBSF or genistein treated THP-1 cells was determined by protein solubility assay. Soluble (S) and insoluble (I) fractions of AEBSF- or genistein-treated THP-1 cells were separated by high speed centrifugation and N-CoR level in each fraction was determined by western blotting assay using N-CoR antibody. A HMW (high molecular weight) variant of N-CoR protein, which was part of the insoluble fraction, was detected only in AEBSF treated cells. The relative solubility/insolubility of β-actin in each fraction was used as a control. The level of total of protein in each fraction was determined by coomassie blue staining. E, Subcellular distribution of N-CoR (red signal) in THP-1 cells treated with AEBSF (200 µM) or genistein (50 µM) was determined by confocal microscopy. DNA was stained with DAPI (blue signal). F, Level of serine/threonine phosphorylated N-CoR (upper panel) or total N-CoR (lower panel) in THP-1 cells treated with AEBSF or genistein was determined by staining the immunoprecipitated (IP) N-CoR with a generic phospho serine/threonine antibody or N-CoR antibody respectively. N-CoR protein level in each treated sample used in IP assay was determined by western blotting (right panel).
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AEBSF sensitize apl cells to upr induced apoptosis
Clinical Cancer Research, 2007Co-Authors: Matiullah KhanAbstract:B21 Acute promyelocytic leukemia (APL), which is caused by fusion protein PML-RARα, is characterized by accumulation of immature pro-myeloid cells in the blood and bone marrow. We have previously reported that PML-RARα-induced accumulation of mis-folded N-CoR in the endoplasmic reticulum (ER) triggers ER stress and unfolded protein response (UPR) in APL cells (1). Although intracellular accumulation of mis-folded protein and ER stress have been implicated in UPR-induced apoptosis of neuronal cells in various neurodegenerative diseases, APL cells escape the UPR-induced apoptosis due to proteolytic processing of mis-folded N-CoR protein by OSGEP, a glycoprotein endopeptidase selectively expressed in APL cells (2). This finding suggests that selective inhibition of OSGEP activity could restore the UPR-induced apoptosis pathway in APL cells. Based on this hypothesis, we tested several protease inhibitors on APL cells and identified AEBSF as a selective inhibitor of OSGEP activity and inducer of apoptosis of APL cells. Here, we demonstrate that AEBSF selectively inhibits the viability of APL cells and promotes UPR-induced apoptosis in a dose-dependent manner. AEBSF-induced apoptosis of APL cells is mediated by activation of Caspase 3 and 9, and cleavage of PARP. This is accompanied by a corresponding down-regulation of anti-apoptotic protein BCL2 and up-regulation of pro-apoptotic proteins BAX/ BAK, CHOP and GADD34. These results demonstrate that AEBSF could sensitize APL cells to UPR-induced apoptosis and highlights its therapeutic potential in APL. References: 1. Khan M.M., Nomura T., Chiba T., Tanaka K., Yoshida H., Mori K., and Ishii S. (2004) The Fusion Oncoprotein PML-RAR Induces Endoplasmic Reticulum (ER)-associated Degradation of N-CoR and ER Stress. The Journal of Biological Chemistry, 279 (12): 11814-24. 2. Ng A.P.P., Fong J.H., Nin D.S., Hirpara J.L., Asou N., Chen C.S., Pervaiz S. and Khan M. (2006) Cleavage of Misfolded Nuclear Receptor Co-repressor Confers Resistance to Unfolded Protein Response-Induced Apoptosis. Cancer Research, 60(22): 9903-12.
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cleavage of misfolded nuclear receptor corepressor confers resistance to unfolded protein response induced apoptosis
Cancer Research, 2006Co-Authors: Angela Ping Ping Ng, Jek Howe Fong, Jayshree L Hirpara, Norio Asou, Chienshing Chen, Shazib Pervaiz, Matiullah KhanAbstract:We have recently reported that accumulation of misfolded nuclear hormone receptor corepressor (N-CoR) as insoluble protein aggregates in acute promyelocytic leukemia (APL) cells induces endoplasmic reticulum (ER) stress and activates unfolded protein response (UPR). Although accumulation of misfolded proteins is known to trigger UPR-induced cytotoxic cell death in several neurodegenerative disorders, APL cells are notably resistant to UPR-induced apoptosis. The molecular basis for the paradoxical response of APL cells to UPR is not known. Here, we report that a glycoprotease, selectively expressed in APL cells, regulates the response of APL cells to UPR-induced apoptosis through processing of misfolded N-CoR protein. Results show that misfolded N-CoR is cleaved selectively in APL cells, and cellular extracts of APL cells and human primary APL cells contain activity that cleaves N-CoR protein. Purification and spectrometric analysis of N-CoR cleaving activity from an APL cell line reveals that it is a glycoprotein endopeptidase known as OSGEP. Furthermore, the cleavage of N-CoR in APL cells could be blocked by the broad-spectrum protease inhibitor AEBSF and by RNA interference–mediated down-regulation of OSGEP expression. AEBSF selectively inhibits growth and promotes apoptosis of APL cells possibly through a mechanism involving AEBSF-induced accumulation of insoluble N-CoR protein and by triggering ER stress. Taken together, these findings suggest that selective induction of protease activity in APL cells may represent a novel cytoprotective component of UPR, which could be exploited by tumor cells to survive the toxic insult of misfolded protein(s). (Cancer Res 2006; 66(20): 9903-12)
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cleavage of misfolded nuclear receptor corepressor confers resistance to unfolded protein response induced apoptosis
Cancer Research, 2006Co-Authors: Jek Howe Fong, Jayshree L Hirpara, Norio Asou, Chienshing Chen, Shazib Pervaiz, Dawn Sijin Nin, Matiullah KhanAbstract:We have recently reported that accumulation of misfolded nuclear hormone receptor corepressor (N-CoR) as insoluble protein aggregates in acute promyelocytic leukemia (APL) cells induces endoplasmic reticulum (ER) stress and activates unfolded protein response (UPR). Although accumulation of misfolded proteins is known to trigger UPR-induced cytotoxic cell death in several neurodegenerative disorders, APL cells are notably resistant to UPR-induced apoptosis. The molecular basis for the paradoxical response of APL cells to UPR is not known. Here, we report that a glycoprotease, selectively expressed in APL cells, regulates the response of APL cells to UPR-induced apoptosis through processing of misfolded N-CoR protein. Results show that misfolded N-CoR is cleaved selectively in APL cells, and cellular extracts of APL cells and human primary APL cells contain activity that cleaves N-CoR protein. Purification and spectrometric analysis of N-CoR cleaving activity from an APL cell line reveals that it is a glycoprotein endopeptidase known as OSGEP. Furthermore, the cleavage of N-CoR in APL cells could be blocked by the broad-spectrum protease inhibitor AEBSF and by RNA interference-mediated down-regulation of OSGEP expression. AEBSF selectively inhibits growth and promotes apoptosis of APL cells possibly through a mechanism involving AEBSF-induced accumulation of insoluble N-CoR protein and by triggering ER stress. Taken together, these findings suggest that selective induction of protease activity in APL cells may represent a novel cytoprotective component of UPR, which could be exploited by tumor cells to survive the toxic insult of misfolded protein(s).
Sandrine Silventepoirot - One of the best experts on this subject based on the ideXlab platform.
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identification and pharmacological characterization of cholesterol 5 6 epoxide hydrolase as a target for tamoxifen and aebs ligands
Proceedings of the National Academy of Sciences of the United States of America, 2010Co-Authors: Philippe De Medina, Marc Poirot, Michael Paillasse, Grégory Segala, Sandrine SilventepoirotAbstract:The microsomal antiestrogen binding site (AEBS) is a high-affinity target for the antitumor drug tamoxifen and its cognate ligands that mediate breast cancer cell differentiation and apoptosis. The AEBS, a hetero-oligomeric complex composed of 3β-hydroxysterol-Δ8-Δ7-isomerase (D8D7I) and 3β-hydroxysterol-Δ7-reductase (DHCR7), binds different structural classes of ligands, including ring B oxysterols. These oxysterols are inhibitors of cholesterol-5,6-epoxide hydrolase (ChEH), a microsomal epoxide hydrolase that has yet to be molecularly identified. We hypothesized that the AEBS and ChEH might be related entities. We show that the substrates of ChEH, cholestan-5α,6α-epoxy-3β-ol (α-CE) and cholestan-5β,6β-epoxy-3β-ol (β-CE), and its product, cholestane-3β,5α,6β-triol (CT), are competitive ligands of tamoxifen binding to the AEBS. Conversely, we show that each AEBS ligand is an inhibitor of ChEH activity, and that there is a positive correlation between these ligands’ affinity for the AEBS and their potency to inhibit ChEH (r2 = 0.95; n = 39; P < 0.0001). The single expression of D8D7I or DHCR7 in COS-7 cells slightly increased ChEH activity (1.8- and 2.6-fold), whereas their coexpression fully reconstituted ChEH, suggesting that the formation of a dimer is required for ChEH activity. Similarly, the single knockdown of D8D7I or DHCR7 using siRNA partially inhibited ChEH in MCF-7 cells, whereas the knockdown of both D8D7I and DHCR7 abolished ChEH activity by 92%. Taken together, our findings strongly suggest that the AEBS carries out ChEH activity and establish that ChEH is a new target for drugs of clinical interest, polyunsaturated fatty acids and ring B oxysterols.
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identification and pharmacological characterization of cholesterol 5 6 epoxide hydrolase as a target for tamoxifen and aebs ligands
Proceedings of the National Academy of Sciences of the United States of America, 2010Co-Authors: Philippe De Medina, Marc Poirot, Michael Paillasse, Grégory Segala, Sandrine SilventepoirotAbstract:The microsomal antiestrogen binding site (AEBS) is a high-affinity target for the antitumor drug tamoxifen and its cognate ligands that mediate breast cancer cell differentiation and apoptosis. The AEBS, a hetero-oligomeric complex composed of 3beta-hydroxysterol-Delta8-Delta7-isomerase (D8D7I) and 3beta-hydroxysterol-Delta7-reductase (DHCR7), binds different structural classes of ligands, including ring B oxysterols. These oxysterols are inhibitors of cholesterol-5,6-epoxide hydrolase (ChEH), a microsomal epoxide hydrolase that has yet to be molecularly identified. We hypothesized that the AEBS and ChEH might be related entities. We show that the substrates of ChEH, cholestan-5alpha,6alpha-epoxy-3beta-ol (alpha-CE) and cholestan-5beta,6beta-epoxy-3beta-ol (beta-CE), and its product, cholestane-3beta,5alpha,6beta-triol (CT), are competitive ligands of tamoxifen binding to the AEBS. Conversely, we show that each AEBS ligand is an inhibitor of ChEH activity, and that there is a positive correlation between these ligands' affinity for the AEBS and their potency to inhibit ChEH (r2=0.95; n=39; P<0.0001). The single expression of D8D7I or DHCR7 in COS-7 cells slightly increased ChEH activity (1.8- and 2.6-fold), whereas their coexpression fully reconstituted ChEH, suggesting that the formation of a dimer is required for ChEH activity. Similarly, the single knockdown of D8D7I or DHCR7 using siRNA partially inhibited ChEH in MCF-7 cells, whereas the knockdown of both D8D7I and DHCR7 abolished ChEH activity by 92%. Taken together, our findings strongly suggest that the AEBS carries out ChEH activity and establish that ChEH is a new target for drugs of clinical interest, polyunsaturated fatty acids and ring B oxysterols.
Cindy X Cai - One of the best experts on this subject based on the ideXlab platform.
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characterization of AEBSF antibody modifications for a protease inhibitor supplementation strategy
Analytical and Bioanalytical Chemistry, 2019Co-Authors: Cindy X Cai, Nicole A Schneck, Doug Harris, Daniel Blackstock, Vera B Ivleva, Weidong Zhao, Jiayan Cai, Deepika Gollapudi, Joe Horwitz, Frank J ArnoldAbstract:Application of a protease inhibitor, 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF), during the cell culture process was demonstrated to effectively reduce proteolytic activity at a specific amino acid site during the production of an HIV-1 broadly neutralizing antibody (bNAb). However, the addition of AEBSF could potentially introduce some modifications to the bNAb protein. Experimental design from sample preparation to LC-MS characterization was performed using middle-up and bottom-up approaches to identify AEBSF-modified species for the bNAb using an AEBSF supplementation in the cell culture media. Modified species along with the unmodified control sample were also subjected to binding activity assessment. The results showed that two amino acids (Tyr177 and Lys250) were susceptible to AEBSF modification in the bNAb test articles but at a negligible level and not in the CDR regions, which therefore did not reduce the in vitro binding activity of the bNAb.
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quantification of residual AEBSF related impurities by reversed phase liquid chromatography
Journal of Chromatography A, 2019Co-Authors: Cindy X Cai, Nicole A Schneck, Doug Harris, Daniel Blackstock, Vera B Ivleva, K C Cheng, Adam Charlton, Frank J Arnold, Jonathan W Cooper, Paula Q LeiAbstract:During research of a broadly neutralizing antibody (bNAb) for HIV-1 infection, site-specific clipping was observed during cell culture incubation. Protease inhibitor, 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF), was supplemented to the cell culture feeding to mitigate clipping as one of the control strategies. It led to the need and development of a new assay to monitor the free AEBSF-related impurities during the purification process. In this work, a reversed-phase liquid chromatography (RPLC-UV) method was developed to measure the total concentration of AEBSF and its major degradant product, 4-(aminoethyl) benzenesulfonic acid (AEBS-OH). This quantitative approach involved hydrolysis pre-treatment to drive all AEBSF to AEBS-OH, a filtration step to remove large molecules, followed by RPLC-UV analysis. The method was qualified and shown to be capable of measuring AEBS-OH down to 0.5 μM with good accuracy and precision, which was then applied for process clearance studies. The results demonstrated that a Protein A purification step in conjunction with a mock ultrafiltration/diafiltration (UF/DF) step could remove AEBSF-related impurities below the detection level. Overall, this study is the first to provide a unique approach for monitoring the clearance of free AEBSF and its related degradant, AEBS-OH, in support of the bNAb research.
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Hydrolysis-Kinetic Study of AEBSF, a Protease Inhibitor Used during Cell-Culture Processing of the HIV‑1 Broadly Neutralizing Antibody CAP256-VRC25.26
2018Co-Authors: Jesse L. Huang, Daniel Blackstock, Vera B Ivleva, Attila Nagy, Frank Arnold, Cindy X CaiAbstract:One approach to mitigate product clipping during HIV mAb CAP256-VRC26.25 cell-culture development is the addition of the protease inhibitor 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF) to the cell-culture media. AEBSF can undergo hydrolysis to form an inactive compound, 4-(2-aminoethyl) benzenesulfonic acid. Using mass-spectrometry detection, a kinetic profile of AEBSF hydrolysis was generated for conditions simulating those of cell culture at pH 7.0 and 37 °C. It was found that increasing the pH or the temperature could accelerate AEBSF hydrolysis. The kinetic-study results in this report provide an analytical characterization and guidance when optimizing an AEBSF-addition strategy for product-clipping control during cell-culture development and offer an alternative approach for AEBSF-related clearance studies post protein production
Daniel Blackstock - One of the best experts on this subject based on the ideXlab platform.
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characterization of AEBSF antibody modifications for a protease inhibitor supplementation strategy
Analytical and Bioanalytical Chemistry, 2019Co-Authors: Cindy X Cai, Nicole A Schneck, Doug Harris, Daniel Blackstock, Vera B Ivleva, Weidong Zhao, Jiayan Cai, Deepika Gollapudi, Joe Horwitz, Frank J ArnoldAbstract:Application of a protease inhibitor, 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF), during the cell culture process was demonstrated to effectively reduce proteolytic activity at a specific amino acid site during the production of an HIV-1 broadly neutralizing antibody (bNAb). However, the addition of AEBSF could potentially introduce some modifications to the bNAb protein. Experimental design from sample preparation to LC-MS characterization was performed using middle-up and bottom-up approaches to identify AEBSF-modified species for the bNAb using an AEBSF supplementation in the cell culture media. Modified species along with the unmodified control sample were also subjected to binding activity assessment. The results showed that two amino acids (Tyr177 and Lys250) were susceptible to AEBSF modification in the bNAb test articles but at a negligible level and not in the CDR regions, which therefore did not reduce the in vitro binding activity of the bNAb.
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quantification of residual AEBSF related impurities by reversed phase liquid chromatography
Journal of Chromatography A, 2019Co-Authors: Cindy X Cai, Nicole A Schneck, Doug Harris, Daniel Blackstock, Vera B Ivleva, K C Cheng, Adam Charlton, Frank J Arnold, Jonathan W Cooper, Paula Q LeiAbstract:During research of a broadly neutralizing antibody (bNAb) for HIV-1 infection, site-specific clipping was observed during cell culture incubation. Protease inhibitor, 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF), was supplemented to the cell culture feeding to mitigate clipping as one of the control strategies. It led to the need and development of a new assay to monitor the free AEBSF-related impurities during the purification process. In this work, a reversed-phase liquid chromatography (RPLC-UV) method was developed to measure the total concentration of AEBSF and its major degradant product, 4-(aminoethyl) benzenesulfonic acid (AEBS-OH). This quantitative approach involved hydrolysis pre-treatment to drive all AEBSF to AEBS-OH, a filtration step to remove large molecules, followed by RPLC-UV analysis. The method was qualified and shown to be capable of measuring AEBS-OH down to 0.5 μM with good accuracy and precision, which was then applied for process clearance studies. The results demonstrated that a Protein A purification step in conjunction with a mock ultrafiltration/diafiltration (UF/DF) step could remove AEBSF-related impurities below the detection level. Overall, this study is the first to provide a unique approach for monitoring the clearance of free AEBSF and its related degradant, AEBS-OH, in support of the bNAb research.
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hydrolysis kinetic study of AEBSF a protease inhibitor used during cell culture processing of the hiv 1 broadly neutralizing antibody cap256 vrc25 26
Analytical Chemistry, 2018Co-Authors: Jesse L Huang, Daniel Blackstock, Vera B Ivleva, Attila Nagy, Frank ArnoldAbstract:One approach to mitigate product clipping during HIV mAb CAP256-VRC26.25 cell-culture development is the addition of the protease inhibitor 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF) to the cell-culture media. AEBSF can undergo hydrolysis to form an inactive compound, 4-(2-aminoethyl) benzenesulfonic acid. Using mass-spectrometry detection, a kinetic profile of AEBSF hydrolysis was generated for conditions simulating those of cell culture at pH 7.0 and 37 °C. It was found that increasing the pH or the temperature could accelerate AEBSF hydrolysis. The kinetic-study results in this report provide an analytical characterization and guidance when optimizing an AEBSF-addition strategy for product-clipping control during cell-culture development and offer an alternative approach for AEBSF-related clearance studies post protein production.
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Hydrolysis-Kinetic Study of AEBSF, a Protease Inhibitor Used during Cell-Culture Processing of the HIV‑1 Broadly Neutralizing Antibody CAP256-VRC25.26
2018Co-Authors: Jesse L. Huang, Daniel Blackstock, Vera B Ivleva, Attila Nagy, Frank Arnold, Cindy X CaiAbstract:One approach to mitigate product clipping during HIV mAb CAP256-VRC26.25 cell-culture development is the addition of the protease inhibitor 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF) to the cell-culture media. AEBSF can undergo hydrolysis to form an inactive compound, 4-(2-aminoethyl) benzenesulfonic acid. Using mass-spectrometry detection, a kinetic profile of AEBSF hydrolysis was generated for conditions simulating those of cell culture at pH 7.0 and 37 °C. It was found that increasing the pH or the temperature could accelerate AEBSF hydrolysis. The kinetic-study results in this report provide an analytical characterization and guidance when optimizing an AEBSF-addition strategy for product-clipping control during cell-culture development and offer an alternative approach for AEBSF-related clearance studies post protein production
Vera B Ivleva - One of the best experts on this subject based on the ideXlab platform.
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characterization of AEBSF antibody modifications for a protease inhibitor supplementation strategy
Analytical and Bioanalytical Chemistry, 2019Co-Authors: Cindy X Cai, Nicole A Schneck, Doug Harris, Daniel Blackstock, Vera B Ivleva, Weidong Zhao, Jiayan Cai, Deepika Gollapudi, Joe Horwitz, Frank J ArnoldAbstract:Application of a protease inhibitor, 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF), during the cell culture process was demonstrated to effectively reduce proteolytic activity at a specific amino acid site during the production of an HIV-1 broadly neutralizing antibody (bNAb). However, the addition of AEBSF could potentially introduce some modifications to the bNAb protein. Experimental design from sample preparation to LC-MS characterization was performed using middle-up and bottom-up approaches to identify AEBSF-modified species for the bNAb using an AEBSF supplementation in the cell culture media. Modified species along with the unmodified control sample were also subjected to binding activity assessment. The results showed that two amino acids (Tyr177 and Lys250) were susceptible to AEBSF modification in the bNAb test articles but at a negligible level and not in the CDR regions, which therefore did not reduce the in vitro binding activity of the bNAb.
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quantification of residual AEBSF related impurities by reversed phase liquid chromatography
Journal of Chromatography A, 2019Co-Authors: Cindy X Cai, Nicole A Schneck, Doug Harris, Daniel Blackstock, Vera B Ivleva, K C Cheng, Adam Charlton, Frank J Arnold, Jonathan W Cooper, Paula Q LeiAbstract:During research of a broadly neutralizing antibody (bNAb) for HIV-1 infection, site-specific clipping was observed during cell culture incubation. Protease inhibitor, 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF), was supplemented to the cell culture feeding to mitigate clipping as one of the control strategies. It led to the need and development of a new assay to monitor the free AEBSF-related impurities during the purification process. In this work, a reversed-phase liquid chromatography (RPLC-UV) method was developed to measure the total concentration of AEBSF and its major degradant product, 4-(aminoethyl) benzenesulfonic acid (AEBS-OH). This quantitative approach involved hydrolysis pre-treatment to drive all AEBSF to AEBS-OH, a filtration step to remove large molecules, followed by RPLC-UV analysis. The method was qualified and shown to be capable of measuring AEBS-OH down to 0.5 μM with good accuracy and precision, which was then applied for process clearance studies. The results demonstrated that a Protein A purification step in conjunction with a mock ultrafiltration/diafiltration (UF/DF) step could remove AEBSF-related impurities below the detection level. Overall, this study is the first to provide a unique approach for monitoring the clearance of free AEBSF and its related degradant, AEBS-OH, in support of the bNAb research.
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hydrolysis kinetic study of AEBSF a protease inhibitor used during cell culture processing of the hiv 1 broadly neutralizing antibody cap256 vrc25 26
Analytical Chemistry, 2018Co-Authors: Jesse L Huang, Daniel Blackstock, Vera B Ivleva, Attila Nagy, Frank ArnoldAbstract:One approach to mitigate product clipping during HIV mAb CAP256-VRC26.25 cell-culture development is the addition of the protease inhibitor 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF) to the cell-culture media. AEBSF can undergo hydrolysis to form an inactive compound, 4-(2-aminoethyl) benzenesulfonic acid. Using mass-spectrometry detection, a kinetic profile of AEBSF hydrolysis was generated for conditions simulating those of cell culture at pH 7.0 and 37 °C. It was found that increasing the pH or the temperature could accelerate AEBSF hydrolysis. The kinetic-study results in this report provide an analytical characterization and guidance when optimizing an AEBSF-addition strategy for product-clipping control during cell-culture development and offer an alternative approach for AEBSF-related clearance studies post protein production.
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Hydrolysis-Kinetic Study of AEBSF, a Protease Inhibitor Used during Cell-Culture Processing of the HIV‑1 Broadly Neutralizing Antibody CAP256-VRC25.26
2018Co-Authors: Jesse L. Huang, Daniel Blackstock, Vera B Ivleva, Attila Nagy, Frank Arnold, Cindy X CaiAbstract:One approach to mitigate product clipping during HIV mAb CAP256-VRC26.25 cell-culture development is the addition of the protease inhibitor 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF) to the cell-culture media. AEBSF can undergo hydrolysis to form an inactive compound, 4-(2-aminoethyl) benzenesulfonic acid. Using mass-spectrometry detection, a kinetic profile of AEBSF hydrolysis was generated for conditions simulating those of cell culture at pH 7.0 and 37 °C. It was found that increasing the pH or the temperature could accelerate AEBSF hydrolysis. The kinetic-study results in this report provide an analytical characterization and guidance when optimizing an AEBSF-addition strategy for product-clipping control during cell-culture development and offer an alternative approach for AEBSF-related clearance studies post protein production