Pub Date : 2025-02-18DOI: 10.1016/j.chroma.2025.465789
Haiyue Zuo , Dandan Zhou , Chunli Gao , Yang Yang , Lin Cao , Sha Liao , Junjie Ou , Yangyang Bian
This study aimed to evaluate the separation efficiency and loading capacity of four commercially available micro-flow liquid chromatography (micro-flow LC) columns with 1.0 mm i.d. (PepMap™ C18, HALO® ES-C18, YMC-Triart™ C18 and Acquity UPLC Peptide BEH C18, abbreviated as PepMap, HALO, YMC and BEH) for mass spectrometry-based proteomics analysis. Samples including cytochrome c (cyt-c), human plasma, and HeLa protein digest were used for the tests. The YMC showed much wider peak widths than the other columns, exhibited relatively poor identification results. However, the other three columns showed similar identification performance. Among them, the PepMap was the optimal choice for plasma proteomics as it had a high loading capacity and exhibited the most symmetrical peak shape with a symmetry factor closest to 1.0. In general, our results provided valuable and solid support for the choice of a chromatographic column, which could potentially contributes to the wider application of micro-flow LC-MS/MS in proteomics research.
{"title":"Evaluation of commercial micro-flow liquid chromatography columns for mass spectrometry-based proteomics","authors":"Haiyue Zuo , Dandan Zhou , Chunli Gao , Yang Yang , Lin Cao , Sha Liao , Junjie Ou , Yangyang Bian","doi":"10.1016/j.chroma.2025.465789","DOIUrl":"10.1016/j.chroma.2025.465789","url":null,"abstract":"<div><div>This study aimed to evaluate the separation efficiency and loading capacity of four commercially available micro-flow liquid chromatography (micro-flow LC) columns with 1.0 mm i.d. (PepMap™ C18, HALO® ES-C18, YMC-Triart™ C18 and Acquity UPLC Peptide BEH C18, abbreviated as PepMap, HALO, YMC and BEH) for mass spectrometry-based proteomics analysis. Samples including cytochrome c (cyt-c), human plasma, and HeLa protein digest were used for the tests. The YMC showed much wider peak widths than the other columns, exhibited relatively poor identification results. However, the other three columns showed similar identification performance. Among them, the PepMap was the optimal choice for plasma proteomics as it had a high loading capacity and exhibited the most symmetrical peak shape with a symmetry factor closest to 1.0. In general, our results provided valuable and solid support for the choice of a chromatographic column, which could potentially contributes to the wider application of micro-flow LC-MS/MS in proteomics research.</div></div>","PeriodicalId":347,"journal":{"name":"Journal of Chromatography A","volume":"1746 ","pages":"Article 465789"},"PeriodicalIF":3.8,"publicationDate":"2025-02-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143445222","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-02-18DOI: 10.1016/j.chroma.2025.465792
Yufeng Shen, Xiaomei Zhu, Feifang Zhang, Bingcheng Yang
A surface-carboxylated polymer substrate-based hyperbranched anion exchanger has been described for ion chromatography (IC). It is prepared by grafting maleic anhydride onto porous polystyrene-divinylbenzene (PS-DVB) microspheres, then hydrolyzed to generate carboxylate groups being anchor points for electrostatic attachment of positively hyperbranched polymer obtained by cyclic reaction of diepoxides and amines. The obtained anion exchanger shows high hydrophilicity and good chromatographic performance, as demonstrated by the separation of common inorganic anions, including polarizable anions, disinfection byproducts and organic acids. High efficiency and good peak shape were achieved, as indicated by the theoretical plate counts of 45,000/m, 51,000/m and asymmetry factors of 1.12, 1.08 for nitrite and bromide, respectively. A desirable feature of such carboxylated groups-based anion exchanger is elimination of possible bleed of sulfate encountered in common sulfonated-based ones, avoiding sulfate blank in the case of the analysis of trace anions in pure water.
{"title":"A hyperbranched anion exchanger obtained by carboxylate surface modified polymer substrate for ion chromatography","authors":"Yufeng Shen, Xiaomei Zhu, Feifang Zhang, Bingcheng Yang","doi":"10.1016/j.chroma.2025.465792","DOIUrl":"10.1016/j.chroma.2025.465792","url":null,"abstract":"<div><div>A surface-carboxylated polymer substrate-based hyperbranched anion exchanger has been described for ion chromatography (IC). It is prepared by grafting maleic anhydride onto porous polystyrene-divinylbenzene (PS-DVB) microspheres, then hydrolyzed to generate carboxylate groups being anchor points for electrostatic attachment of positively hyperbranched polymer obtained by cyclic reaction of diepoxides and amines. The obtained anion exchanger shows high hydrophilicity and good chromatographic performance, as demonstrated by the separation of common inorganic anions, including polarizable anions, disinfection byproducts and organic acids. High efficiency and good peak shape were achieved, as indicated by the theoretical plate counts of 45,000/m, 51,000/m and asymmetry factors of 1.12, 1.08 for nitrite and bromide, respectively. A desirable feature of such carboxylated groups-based anion exchanger is elimination of possible bleed of sulfate encountered in common sulfonated-based ones, avoiding sulfate blank in the case of the analysis of trace anions in pure water.</div></div>","PeriodicalId":347,"journal":{"name":"Journal of Chromatography A","volume":"1746 ","pages":"Article 465792"},"PeriodicalIF":3.8,"publicationDate":"2025-02-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143463901","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-02-17DOI: 10.1016/j.chroma.2025.465788
Wan-Chih Su, Raymond Lieu, Yige Fu, Trevor Kempen, Zhixin Yu, Kelly Zhang, Tao Chen, Yuchen Fan
Nucleic acid-based medicines have achieved significant advancements in recent years, with lipid nanoparticles (LNPs) being a pivotal platform for their delivery. However, the complexity of LNP presents significant challenges, requiring analytical methods to identify and quantify individual components to guide formulation development and ensure quality and safety. Current approaches often perform nucleic acid and lipid analysis separately and focus on a single type of formulation, highlighting the need for a simple platform method that can be applied to diverse formulations. We present a platform ion-pair reversed-phase HPLC method with UV and charged aerosol detection (CAD) to simultaneously separate and quantify lipid and nucleic acid components in LNPs. The method separated and quantified 12 lipid species and three types of nucleic acids (antisense oligonucleotide, single-guide RNA, and mRNA), covering a broad range of therapeutic cargoes. Notably, this can be achieved for the first time by one HPLC run with one-step facile sample preparation. Specifically, we used a simple buffer containing Triton and heparin to enable the single-step, simultaneous extraction of both nucleic acid and lipid components from LNPs, achieving quantification recoveries of 90–110 %. We further applied this method and addressed process and quality control challenges of LNPs, including the recovery rate of individual LNP components after purification and simultaneous quantification of co-loaded, different nucleic acid species for potential gene editing applications. This new platform method offers a robust and widely applicable tool to assess the quality of lipid-based nucleic acid therapies.
{"title":"A platform method for simultaneous quantification of lipid and nucleic acid components in lipid nanoparticles","authors":"Wan-Chih Su, Raymond Lieu, Yige Fu, Trevor Kempen, Zhixin Yu, Kelly Zhang, Tao Chen, Yuchen Fan","doi":"10.1016/j.chroma.2025.465788","DOIUrl":"10.1016/j.chroma.2025.465788","url":null,"abstract":"<div><div>Nucleic acid-based medicines have achieved significant advancements in recent years, with lipid nanoparticles (LNPs) being a pivotal platform for their delivery. However, the complexity of LNP presents significant challenges, requiring analytical methods to identify and quantify individual components to guide formulation development and ensure quality and safety. Current approaches often perform nucleic acid and lipid analysis separately and focus on a single type of formulation, highlighting the need for a simple platform method that can be applied to diverse formulations. We present a platform ion-pair reversed-phase HPLC method with UV and charged aerosol detection (CAD) to simultaneously separate and quantify lipid and nucleic acid components in LNPs. The method separated and quantified 12 lipid species and three types of nucleic acids (antisense oligonucleotide, single-guide RNA, and mRNA), covering a broad range of therapeutic cargoes. Notably, this can be achieved for the first time by one HPLC run with one-step facile sample preparation. Specifically, we used a simple buffer containing Triton and heparin to enable the single-step, simultaneous extraction of both nucleic acid and lipid components from LNPs, achieving quantification recoveries of 90–110 %. We further applied this method and addressed process and quality control challenges of LNPs, including the recovery rate of individual LNP components after purification and simultaneous quantification of co-loaded, different nucleic acid species for potential gene editing applications. This new platform method offers a robust and widely applicable tool to assess the quality of lipid-based nucleic acid therapies.</div></div>","PeriodicalId":347,"journal":{"name":"Journal of Chromatography A","volume":"1746 ","pages":"Article 465788"},"PeriodicalIF":3.8,"publicationDate":"2025-02-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143463899","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-02-17DOI: 10.1016/j.chroma.2025.465784
Julie Robinson , Mayank Vats , Michael Hartmann
Flow through anion exchange chromatography (AEX) has provided reliable process-and product-related impurities removal as well as viral clearance for monoclonal antibodies (mAbs). The application of AEX to molecules with more complex impurity profiles or non-platform characteristics such as a low pI can become challenging because viral clearance considerations often constrain the AEX step design space. Multimodal anion exchange chromatography (MMAEX) can address the limitations of the platform AEX step while still allowing a “platform-like” manufacturing process. This work presents a case study on polishing step development for a Fc-fusion protein with pI < 6.5, high surface hydrophobicity, and aggregate content of up to 35% in the harvested cell culture fluid. An integrated computational and experimental high throughput screening (HTS) workflow was implemented to rapidly identify the MMAEX resin Capto Adhere at pH 5.5 as a viable alternative to the conventional AEX flow through polishing step. This work presents the impact of pH, conductivity, load, and load impurity level/composition on MMAEX step performance. Step performance was measured based on yield, HMW clearance, residual HCP and DNA clearance, as well as viral clearance. HMW clearance varied based on the starting load HMW level (from 5 -35%) and the highest relative clearance was obtained at a moderate HMW load level. Residual HCP and DNA showed a strong dependance on load HMW level, highlighting the competitive adsorption behavior that can impact resin-protein interactions in complex mixtures. Both residual HCP and DNA were removed to below quantification at pH 5.5. Viral clearance of up to 4 logs of XMULV and MVM was demonstrated at pH 5.5 using process relevant feed streams. The workflow presented here demonstrates how the integration of in silico modeling and high throughput screening (HTS) can streamline process development and enable rapid polishing step optimization. These results also underscore the impact of feed impurity and impurity composition on MMAEX resin performance.
{"title":"Implementation of multimodal anion exchange chromatography to address product quality challenges and downstream platform limitations: A case study","authors":"Julie Robinson , Mayank Vats , Michael Hartmann","doi":"10.1016/j.chroma.2025.465784","DOIUrl":"10.1016/j.chroma.2025.465784","url":null,"abstract":"<div><div>Flow through anion exchange chromatography (AEX) has provided reliable process-and product-related impurities removal as well as viral clearance for monoclonal antibodies (mAbs). The application of AEX to molecules with more complex impurity profiles or non-platform characteristics such as a low pI can become challenging because viral clearance considerations often constrain the AEX step design space. Multimodal anion exchange chromatography (MMAEX) can address the limitations of the platform AEX step while still allowing a “platform-like” manufacturing process. This work presents a case study on polishing step development for a Fc-fusion protein with pI < 6.5, high surface hydrophobicity, and aggregate content of up to 35% in the harvested cell culture fluid. An integrated computational and experimental high throughput screening (HTS) workflow was implemented to rapidly identify the MMAEX resin Capto Adhere at pH 5.5 as a viable alternative to the conventional AEX flow through polishing step. This work presents the impact of pH, conductivity, load, and load impurity level/composition on MMAEX step performance. Step performance was measured based on yield, HMW clearance, residual HCP and DNA clearance, as well as viral clearance. HMW clearance varied based on the starting load HMW level (from 5 -35%) and the highest relative clearance was obtained at a moderate HMW load level. Residual HCP and DNA showed a strong dependance on load HMW level, highlighting the competitive adsorption behavior that can impact resin-protein interactions in complex mixtures. Both residual HCP and DNA were removed to below quantification at pH 5.5. Viral clearance of up to 4 logs of XMULV and MVM was demonstrated at pH 5.5 using process relevant feed streams. The workflow presented here demonstrates how the integration of <em>in silico</em> modeling and high throughput screening (HTS) can streamline process development and enable rapid polishing step optimization. These results also underscore the impact of feed impurity and impurity composition on MMAEX resin performance.</div></div>","PeriodicalId":347,"journal":{"name":"Journal of Chromatography A","volume":"1746 ","pages":"Article 465784"},"PeriodicalIF":3.8,"publicationDate":"2025-02-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143454775","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-02-16DOI: 10.1016/j.chroma.2025.465786
Ruixi Wang , Albert Kao , Lu Wang, Mi Jin
Affinity chromatography is a critical step in gene therapy for capturing adeno-associated virus (AAV) vectors. However, the high cost of affinity resin needs effective cleaning and sanitization strategies to enable multi-cycle usage. This study evaluated various combinations of cleaning reagents, including alcohol, low concentrations of sodium hydroxide (NaOH), and acids, against a broad range of microbial strains, particularly acid- and alkaline-resistant species, to identify enhanced sanitization protocols for both pre-use and post-use. A solution comprising 100 mM acetic acid with 2 % benzyl alcohol, alongside 10 mM NaOH with 2 % benzyl alcohol, was identified as effective. This new cleaning and sanitization strategy, incorporating both pre- and post-use cleaning, was successfully implemented, allowing for up to six column cycles without product carryover between cycles. Notably, this strategy does not compromise process yield or product quality. It provides effective microbial control during AAV purification using AVB Sepharose resin, while also preserving resin integrity, reducing the risk of microbial contamination and product carryover, lowering AAV manufacturing costs, and ultimately enhancing the quality and reliability of gene therapy product manufacturing.
{"title":"Enhancing sanitization for AVB Sepharose resin in AAV vector purification","authors":"Ruixi Wang , Albert Kao , Lu Wang, Mi Jin","doi":"10.1016/j.chroma.2025.465786","DOIUrl":"10.1016/j.chroma.2025.465786","url":null,"abstract":"<div><div>Affinity chromatography is a critical step in gene therapy for capturing adeno-associated virus (AAV) vectors. However, the high cost of affinity resin needs effective cleaning and sanitization strategies to enable multi-cycle usage. This study evaluated various combinations of cleaning reagents, including alcohol, low concentrations of sodium hydroxide (NaOH), and acids, against a broad range of microbial strains, particularly acid- and alkaline-resistant species, to identify enhanced sanitization protocols for both pre-use and post-use. A solution comprising 100 mM acetic acid with 2 % benzyl alcohol, alongside 10 mM NaOH with 2 % benzyl alcohol, was identified as effective. This new cleaning and sanitization strategy, incorporating both pre- and post-use cleaning, was successfully implemented, allowing for up to six column cycles without product carryover between cycles. Notably, this strategy does not compromise process yield or product quality. It provides effective microbial control during AAV purification using AVB Sepharose resin, while also preserving resin integrity, reducing the risk of microbial contamination and product carryover, lowering AAV manufacturing costs, and ultimately enhancing the quality and reliability of gene therapy product manufacturing.</div></div>","PeriodicalId":347,"journal":{"name":"Journal of Chromatography A","volume":"1746 ","pages":"Article 465786"},"PeriodicalIF":3.8,"publicationDate":"2025-02-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143454774","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-02-16DOI: 10.1016/j.chroma.2025.465787
Paweł Wiczling
Prior information about analyte retention is often implicitly incorporated into the method development workflow. This prior knowledge can stem from various sources, such as the analyte's structure, analyte's properties, existing literature, or the analyst's experience. Alternatively, prior information can be formally integrated into the method development workflow using Bayesian reasoning. In such cases, it can be represented through the model structure, covariate relationships (e.g. quantitative-structure retention relationships), and population-level parameters derived from multilevel models or other sources. Population-level parameters are the same for each analyte belonging to a certain set of analytes and as such help predict the individual-level (analyte-specific) parameters given any type of preliminary data. The use of prior information and multilevel modeling framework enables development of an experimental design that leads to the desired precision of chromatographic predictions across a wide range of conditions and for a diverse set of analytes. This approach offers greater accuracy compared to optimizing conditions for a single or typical analyte. In this study maximization of the Bayesian D-optimality criterion was employed to identify an optimal set of experiments for diverse set of analytes (acids, bases with a wide range of lipophilicity). The benefit of incorporating prior information was emphasized, and simulations based on a recently developed mechanistic model validated the benefits of combining optimal design theory, multilevel models, and prior information to obtain more efficient experimental designs in Reversed-Phase HPLC.
{"title":"Leveraging prior knowledge for improved retention prediction in reversed-phase HPLC","authors":"Paweł Wiczling","doi":"10.1016/j.chroma.2025.465787","DOIUrl":"10.1016/j.chroma.2025.465787","url":null,"abstract":"<div><div>Prior information about analyte retention is often implicitly incorporated into the method development workflow. This prior knowledge can stem from various sources, such as the analyte's structure, analyte's properties, existing literature, or the analyst's experience. Alternatively, prior information can be formally integrated into the method development workflow using Bayesian reasoning. In such cases, it can be represented through the model structure, covariate relationships (e.g. quantitative-structure retention relationships), and population-level parameters derived from multilevel models or other sources. Population-level parameters are the same for each analyte belonging to a certain set of analytes and as such help predict the individual-level (analyte-specific) parameters given any type of preliminary data. The use of prior information and multilevel modeling framework enables development of an experimental design that leads to the desired precision of chromatographic predictions across a wide range of conditions and for a diverse set of analytes. This approach offers greater accuracy compared to optimizing conditions for a single or typical analyte. In this study maximization of the Bayesian D-optimality criterion was employed to identify an optimal set of experiments for diverse set of analytes (acids, bases with a wide range of lipophilicity). The benefit of incorporating prior information was emphasized, and simulations based on a recently developed mechanistic model validated the benefits of combining optimal design theory, multilevel models, and prior information to obtain more efficient experimental designs in Reversed-Phase HPLC.</div></div>","PeriodicalId":347,"journal":{"name":"Journal of Chromatography A","volume":"1746 ","pages":"Article 465787"},"PeriodicalIF":3.8,"publicationDate":"2025-02-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143454792","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-02-16DOI: 10.1016/j.chroma.2025.465785
Xiao Han , Yuxiang Zhang , Yao Zhang , Zhaoyang Sun , Qiexin Chen , Luyao Zhang , Huan Meng , Rong Yu , Chun Zhang , Yongdong Liu
Immunoaffinity chromatography using traditional antibodies as ligands has been adopted as a key step for purification of recombinant hepatitis B surface antigen (rHBsAg) but with limitations of high cost, instability, and exclusivity. In this study, a nanobody against HBsAg (named NbHB1) was employed to develop an affinity resin for rHBsAg purification. NbHB1 was expressed in E. coli as inclusion bodies, which were efficiently refolded through a two-step strategy and then directly immobilized to a SpyCatcher-derived support via the SpyTag fused to its C-terminus, resulting in an affinity resin with a static binding capacity of 2.03 mg/mL and a dynamic binding capacity of 1.43 mg/mL for rHBsAg. Using this affinity chromatography as the initial purification step, rHBsAg was efficiently purified from the clarified CHO cell culture fluid, resulting in 81 % recovery and 96 % purity. Similar recoveries were observed when scaling up from a 5 mL to a 500 mL column. The chromatographic performance was consistent over 25 purification cycles with the ligand leakage below 1.7 ng/mL resin during the repeated usage. The stability of this affinity resin was validated by various strip reagents, including 0.1 M citric acid, 20 mM sodium hydroxide, and 2 M guanidine chloride. Overall, these results demonstrate that the NbHB1-liganded affinity chromatography is robust and highly feasible, highlighting its potential to enhance the downstream process in the industrial manufacturing of rHBsAg-VLP.
{"title":"Implementation of a nanobody-liganded affinity resin for improving the production of recombinant HBsAg-VLP in large scale","authors":"Xiao Han , Yuxiang Zhang , Yao Zhang , Zhaoyang Sun , Qiexin Chen , Luyao Zhang , Huan Meng , Rong Yu , Chun Zhang , Yongdong Liu","doi":"10.1016/j.chroma.2025.465785","DOIUrl":"10.1016/j.chroma.2025.465785","url":null,"abstract":"<div><div>Immunoaffinity chromatography using traditional antibodies as ligands has been adopted as a key step for purification of recombinant hepatitis B surface antigen (rHBsAg) but with limitations of high cost, instability, and exclusivity. In this study, a nanobody against HBsAg (named NbHB1) was employed to develop an affinity resin for rHBsAg purification. NbHB1 was expressed in <em>E. coli</em> as inclusion bodies, which were efficiently refolded through a two-step strategy and then directly immobilized to a SpyCatcher-derived support via the SpyTag fused to its C-terminus, resulting in an affinity resin with a static binding capacity of 2.03 mg/mL and a dynamic binding capacity of 1.43 mg/mL for rHBsAg. Using this affinity chromatography as the initial purification step, rHBsAg was efficiently purified from the clarified CHO cell culture fluid, resulting in 81 % recovery and 96 % purity. Similar recoveries were observed when scaling up from a 5 mL to a 500 mL column. The chromatographic performance was consistent over 25 purification cycles with the ligand leakage below 1.7 ng/mL resin during the repeated usage. The stability of this affinity resin was validated by various strip reagents, including 0.1 M citric acid, 20 mM sodium hydroxide, and 2 M guanidine chloride. Overall, these results demonstrate that the NbHB1-liganded affinity chromatography is robust and highly feasible, highlighting its potential to enhance the downstream process in the industrial manufacturing of rHBsAg-VLP.</div></div>","PeriodicalId":347,"journal":{"name":"Journal of Chromatography A","volume":"1746 ","pages":"Article 465785"},"PeriodicalIF":3.8,"publicationDate":"2025-02-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143454210","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
As a local pig breed, Mi pig (MP) is highly prized by Chinese consumers for its unique flavor and exceptional quality. This study aimed to investigate the differences in volatile profile between MP and commercial crossbred pigs (CP), as well as potential factors contributing to these variations. Results showed that MP had significantly higher intramuscular fat (IMF) content (7.75 %) and unsaturated fatty acid (UFA) content (56.44 %) compared to CP. Utilizing headspace solid-phase microextraction-gas chromatography tandem mass spectrometry (HS-SPME-GC–MS) and lipidomics, 14 volatile compounds and 310 lipids were identified as key factors influencing the flavor differences between the two breeds. Specifically, UFAs produced through glycerophospholipid and glycerolipid metabolic pathways play an essential role in developing the rich MP flavor. Our findings provide valuable insights into the underlying mechanisms responsible for the distinctive flavor of MP, highlighting the role of lipid metabolism in flavor formation.
{"title":"Analysis of volatile flavor and lipids in different breeds of pork using electronic noses, GC–MS and UPLC-MS/MS","authors":"Simin Xu , Wenxia Zheng , You Zeng , Xingguo Tian , Xiaoyan Xu","doi":"10.1016/j.chroma.2025.465783","DOIUrl":"10.1016/j.chroma.2025.465783","url":null,"abstract":"<div><div>As a local pig breed, Mi pig (MP) is highly prized by Chinese consumers for its unique flavor and exceptional quality. This study aimed to investigate the differences in volatile profile between MP and commercial crossbred pigs (CP), as well as potential factors contributing to these variations. Results showed that MP had significantly higher intramuscular fat (IMF) content (7.75 %) and unsaturated fatty acid (UFA) content (56.44 %) compared to CP. Utilizing headspace solid-phase microextraction-gas chromatography tandem mass spectrometry (HS-SPME-GC–MS) and lipidomics, 14 volatile compounds and 310 lipids were identified as key factors influencing the flavor differences between the two breeds. Specifically, UFAs produced through glycerophospholipid and glycerolipid metabolic pathways play an essential role in developing the rich MP flavor. Our findings provide valuable insights into the underlying mechanisms responsible for the distinctive flavor of MP, highlighting the role of lipid metabolism in flavor formation.</div></div>","PeriodicalId":347,"journal":{"name":"Journal of Chromatography A","volume":"1746 ","pages":"Article 465783"},"PeriodicalIF":3.8,"publicationDate":"2025-02-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143464030","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Micro-fraction bioactivity profiling and high speed countercurrent chromatography were performed for screening and isolation of components with α-glucosidase inhibitory activity from Eriobotrya japonica (Thunb.) leaves. Three sesquiterpene glycosides and a number of known pentacyclic triterpene acids, including a new compound, were successfully screened. Sesquiterpene glycosides were found to have α-glucosidase inhibitory activity for the first time. An efficient strategy for preparative separation of the three sesquiterpene glycosides from E. japonica leaves by column chromatography combined with two-step high speed countercurrent chromatographic separation was established. Two biphasic solvent systems, including n-hexane-ethyl acetate-methanol-water (5:95:5:95, v/v) and ethyl acetate-ethanol-water (100:20:80, v/v), were selected. In the first countercurrent chromatographic separation, 17.50 mg of sesquiterpene glycoside 1 with 92.9% purity and 15.11 mg of sesquiterpene glycoside 3 with 94.4% purity were isolated from 80.10 mg of partially purified fraction I, and in the second separation, 4.42 mg of new sesquiterpene glycoside 2 with 94.9% purity were isolated. Each screened compound was evaluated by α-glucosidase inhibition assay, and results showed that the new sesquiterpene glycoside 2 had high inhibitory activity with IC50 = 7.83±0.01 μM.
{"title":"Separation of sesquiterpene glycosides with α-glucosidase inhibitory activity from Eriobotrya japonica (Thunb.) leaves by high-speed countercurrent chromatography","authors":"Chenlei Ma , Honglei Bao , Beibei Zhu , Junchao Zhu , Songlin Chen , Huawei Lv , Chu Chu , Shengqiang Tong","doi":"10.1016/j.chroma.2025.465780","DOIUrl":"10.1016/j.chroma.2025.465780","url":null,"abstract":"<div><div>Micro-fraction bioactivity profiling and high speed countercurrent chromatography were performed for screening and isolation of components with <em>α</em>-glucosidase inhibitory activity from <em>Eriobotrya japonica</em> (Thunb.) leaves. Three sesquiterpene glycosides and a number of known pentacyclic triterpene acids, including a new compound, were successfully screened. Sesquiterpene glycosides were found to have <em>α</em>-glucosidase inhibitory activity for the first time. An efficient strategy for preparative separation of the three sesquiterpene glycosides from <em>E. japonica</em> leaves by column chromatography combined with two-step high speed countercurrent chromatographic separation was established. Two biphasic solvent systems, including n-hexane-ethyl acetate-methanol-water (5:95:5:95, v/v) and ethyl acetate-ethanol-water (100:20:80, v/v), were selected. In the first countercurrent chromatographic separation, 17.50 mg of sesquiterpene glycoside 1 with 92.9% purity and 15.11 mg of sesquiterpene glycoside 3 with 94.4% purity were isolated from 80.10 mg of partially purified fraction I, and in the second separation, 4.42 mg of new sesquiterpene glycoside 2 with 94.9% purity were isolated. Each screened compound was evaluated by <em>α</em>-glucosidase inhibition assay, and results showed that the new sesquiterpene glycoside 2 had high inhibitory activity with IC<sub>50</sub> = 7.83±0.01 <em>μ</em>M.</div></div>","PeriodicalId":347,"journal":{"name":"Journal of Chromatography A","volume":"1746 ","pages":"Article 465780"},"PeriodicalIF":3.8,"publicationDate":"2025-02-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143427811","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-02-15DOI: 10.1016/j.chroma.2025.465782
Wenbin Zhang , Yuzeng Li , Nurimangul Muntiza , Wenquan Ji , Zhen Fan , Qinran Li , Jin Zhao , Hongfeng Zhang , Qiliang Deng , Donglan Sun , Tianjun Liu
A dual-epitopes imprinted strategy for cytochrome c selective recognition assisted with γ-cyclodextrin host-guest interaction via N-terminal and C-terminal epitope's simultaneous imprinting and reversible addition-fragmentation chain transfer (RAFT) polymerization was developed. N-terminal and C-terminal nonapeptides of Cyt c (GI-9 and AE-9) were used simultaneously as the epitope to achieve collaborative recognition for cytochrome c. As a supramolecule, γ-cyclodextrin can encapsulate the aromatic functional groups of amino acid residues to capture the peptide and improve the corresponding spatial orientation for epitope or cytochrome c recognition by host-guest interaction. After the γ-cyclodextrin modification and dual-epitopes immobilization, the imprinted polymer was synthesized by RAFT polymerization with 4-cyano-4-(phenyl-carbonothioylthio) pentanoic acid as a chain transfer agent. After the template removal, the obtained dual-epitopes imprinted particles showed well binding ability to AE-9 (26.50 mg·g−1, IF= 4.13), GI-9 (7.36 mg·g−1, IF= 2.18) and cytochrome c (79.56 mg·g−1, IF= 3.27). With the successive addition of RAFT agent, the imprinting factor rising of epitope peptide and cytochrome c further illustrated the regulation of imprinted polymer chains. The imprinted particles had the advantage for cytochrome c recognition compared to other proteins and good reusability with 82.60 % repeated reproduction rate after six cycles of adsorption and desorption. Furthermore, the selective recognition for cytochrome c in bovine serum proved its potentiality to be applied in complex biological samples. It indicated that the combination of dual-templates epitope imprinting, γ-CD host-guest interaction and RAFT polymerization provided an efficient method for collaborative protein recognition with well selectivity, reusability and stability.
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