Pub Date : 2025-10-21DOI: 10.1016/j.pep.2025.106838
Dafeng Liu , Na Li
The pathogen Staphylococcus aureus threatens clinical and public health. Vancomycin, a former last-resort treatment, has waning efficacy against resistant strains (VISA) that alter their cell envelope using enzymes like VraA. However, the specific functional mechanism of VraA in VISA is still not understood. Here, we successfully obtained monomeric VraA and found that oleate is the optimal substrate for VraA. VraA was characterized as a peripheral membrane protein with a hydrodynamic radius of 5.7 ± 0.3 nm. Disruption of VraA significantly increased S. aureus susceptibility to vancomycin, as demonstrated by minimum inhibitory concentration (MIC) assays and RT-qPCR. The structural model of VraA predicted using AlphaFold2 was refined via ModRefiner, and active site residues were identified. Our findings underscore the pivotal role of VraA in mediating vancomycin resistance in S. aureus, providing valuable insights for developing targeted therapies against this pathogen.
{"title":"The long chain fatty acid-CoA ligase VraA plays a regulatory role in vancomycin resistance in Staphylococcus aureus","authors":"Dafeng Liu , Na Li","doi":"10.1016/j.pep.2025.106838","DOIUrl":"10.1016/j.pep.2025.106838","url":null,"abstract":"<div><div>The pathogen <em>Staphylococcus aureus</em> threatens clinical and public health. Vancomycin, a former last-resort treatment, has waning efficacy against resistant strains (VISA) that alter their cell envelope using enzymes like VraA. However, the specific functional mechanism of VraA in VISA is still not understood. Here, we successfully obtained monomeric VraA and found that oleate is the optimal substrate for VraA. VraA was characterized as a peripheral membrane protein with a hydrodynamic radius of 5.7 ± 0.3 nm. Disruption of <em>VraA</em> significantly increased <em>S. aureus</em> susceptibility to vancomycin, as demonstrated by minimum inhibitory concentration (MIC) assays and RT-qPCR. The structural model of VraA predicted using AlphaFold2 was refined via ModRefiner, and active site residues were identified. Our findings underscore the pivotal role of VraA in mediating vancomycin resistance in <em>S. aureus</em>, providing valuable insights for developing targeted therapies against this pathogen.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"238 ","pages":"Article 106838"},"PeriodicalIF":1.2,"publicationDate":"2025-10-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145355741","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Human granulocyte-colony stimulating factor (h-GCSF) is used to mitigate neutropenia caused by chemotherapy. E. coli is the most common host for h-GCSF production due to its high yield and versatile culture strategies. However, overproduction of h-GCSF in E. coli often results in the formation of inclusion bodies (IBs) in the cytoplasm. The efficiency of refolding and purifying recombinant h-GCSF (rh-GCSF) produced as IBs depends on the expression strategy, induction, and cell growth conditions. This study investigated how different culture media and fermentation processes affect the refolding of IBs and the purification of rh-GCSF. The purified samples were compared to Neupogen® and PDgrastim reference standards using Western blotting, size exclusion chromatography, and reverse-phase high-performance liquid chromatography. The analysis confirmed that all proteins were correctly refolded and exhibited a purity exceeding 90 % after purification. The highest protein recovery percentage and purity of rh-GCSF with the lowest endotoxin content was achieved using M9 media in fed-batch culture.
{"title":"Effect of culture media and fermentation process on the refolding and purification of rh-GCSF","authors":"Somayeh Abolghasemi , Fatemeh Poureini , Valiollah Babaeipour , Faezeh Farji , Mohammad Reza Mofid","doi":"10.1016/j.pep.2025.106837","DOIUrl":"10.1016/j.pep.2025.106837","url":null,"abstract":"<div><div>Human granulocyte-colony stimulating factor (h-GCSF) is used to mitigate neutropenia caused by chemotherapy. <em>E. coli</em> is the most common host for h-GCSF production due to its high yield and versatile culture strategies. However, overproduction of h-GCSF in <em>E. coli</em> often results in the formation of inclusion bodies (IBs) in the cytoplasm. The efficiency of refolding and purifying recombinant h-GCSF (rh-GCSF) produced as IBs depends on the expression strategy, induction, and cell growth conditions. This study investigated how different culture media and fermentation processes affect the refolding of IBs and the purification of rh-GCSF. The purified samples were compared to Neupogen® and PDgrastim reference standards using Western blotting, size exclusion chromatography, and reverse-phase high-performance liquid chromatography. The analysis confirmed that all proteins were correctly refolded and exhibited a purity exceeding 90 % after purification. The highest protein recovery percentage and purity of rh-GCSF with the lowest endotoxin content was achieved using M9 media in fed-batch culture.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"238 ","pages":"Article 106837"},"PeriodicalIF":1.2,"publicationDate":"2025-10-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145346688","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-10-20DOI: 10.1016/j.pep.2025.106828
Mengran Fang , Xinyue Chen , Yuan Liu, Hui Yuan
The inherent advantages of transgenic chickens, combined with the increasing demand for therapeutic proteins, are driving significant progress in transgenic chicken bioreactors. Primordial germ cells (PGCs) and embryonic stem cells (ESCs) are two pivotal carrier cells in the production of transgenic chicken bioreactors. Transgenic chickens have successfully produced therapeutic proteins using genetically modified and transferred PGCs. Meanwhile, considerable progress has been made in expressing therapeutic proteins using exogenous gene integration mediated by ESCs. Both carrier cells facilitate the efficient process of transgenic chickens. Here, we reviewed the characteristics, current applications, comparisons, and future directions of these two carrier cells, providing theoretical foundations and practical guidance for advancing development in the fields of chicken bioreactors.
{"title":"Protein production in transgenic chickens mediated through primordial germ cells and embryonic stem cells","authors":"Mengran Fang , Xinyue Chen , Yuan Liu, Hui Yuan","doi":"10.1016/j.pep.2025.106828","DOIUrl":"10.1016/j.pep.2025.106828","url":null,"abstract":"<div><div>The inherent advantages of transgenic chickens, combined with the increasing demand for therapeutic proteins, are driving significant progress in transgenic chicken bioreactors. Primordial germ cells (PGCs) and embryonic stem cells (ESCs) are two pivotal carrier cells in the production of transgenic chicken bioreactors. Transgenic chickens have successfully produced therapeutic proteins using genetically modified and transferred PGCs. Meanwhile, considerable progress has been made in expressing therapeutic proteins using exogenous gene integration mediated by ESCs. Both carrier cells facilitate the efficient process of transgenic chickens. Here, we reviewed the characteristics, current applications, comparisons, and future directions of these two carrier cells, providing theoretical foundations and practical guidance for advancing development in the fields of chicken bioreactors.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"238 ","pages":"Article 106828"},"PeriodicalIF":1.2,"publicationDate":"2025-10-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145337522","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-10-18DOI: 10.1016/j.pep.2025.106836
Huanlan Chen, Naiqi Ji, Maozhi Zhou, Yingyue Bu
The Knob-into-Hole (KiH) design is an effective strategy to promote the heterodimerization of heavy chains. In this research, through the integration of advanced analytical techniques, we made a critical finding regarding heterodimer formation. When mixing materials derived from different clones containing hole-hole homodimers and knob-knob homodimers, the resulting structures initially appear to form the desired knob-into-hole (KiH) configuration. However, detailed characterization by SDS-Caliper NR analysis revealed these were not genuine KiH heterodimers. While this phenomenon presents an optimal apparent outcome, it has significant impact on the optimization of process in heterodimer production. Additionally, a comprehensive assessment was carried out on the efficacy of cation exchange chromatography (CEX) in removing homodimers. This study presents a case for the optimization of the CEX process, utilizing bispecific - antibody materials sourced from different clonal bioreactors. By adjusting the operating pH of CEX, the knob-related impurities and hole-related impurities were reduced to below the detection limit, as shown by iCIEF analysis. Meanwhile, SEC-UPLC analysis indicated that purity increased to 99 %, and SDS-Caliper NR results showed that the percentage of functional KiH improved from 85 % in the input to 97 % after elution. These findings demonstrate that the CEX process contributes to an increase in the proportion of functional KiH.
{"title":"Study on the hole and knob related impurities in the purification of bispecific antibodies","authors":"Huanlan Chen, Naiqi Ji, Maozhi Zhou, Yingyue Bu","doi":"10.1016/j.pep.2025.106836","DOIUrl":"10.1016/j.pep.2025.106836","url":null,"abstract":"<div><div>The Knob-into-Hole (KiH) design is an effective strategy to promote the heterodimerization of heavy chains. In this research, through the integration of advanced analytical techniques, we made a critical finding regarding heterodimer formation. When mixing materials derived from different clones containing hole-hole homodimers and knob-knob homodimers, the resulting structures initially appear to form the desired knob-into-hole (KiH) configuration. However, detailed characterization by SDS-Caliper NR analysis revealed these were not genuine KiH heterodimers. While this phenomenon presents an optimal apparent outcome, it has significant impact on the optimization of process in heterodimer production. Additionally, a comprehensive assessment was carried out on the efficacy of cation exchange chromatography (CEX) in removing homodimers. This study presents a case for the optimization of the CEX process, utilizing bispecific - antibody materials sourced from different clonal bioreactors. By adjusting the operating pH of CEX, the knob-related impurities and hole-related impurities were reduced to below the detection limit, as shown by iCIEF analysis. Meanwhile, SEC-UPLC analysis indicated that purity increased to 99 %, and SDS-Caliper NR results showed that the percentage of functional KiH improved from 85 % in the input to 97 % after elution. These findings demonstrate that the CEX process contributes to an increase in the proportion of functional KiH.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"238 ","pages":"Article 106836"},"PeriodicalIF":1.2,"publicationDate":"2025-10-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145337454","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-10-14DOI: 10.1016/j.pep.2025.106829
Sina Tavakoli , Shohreh Zare Karizi , Jafar Amani
Emicizumab is a therapeutic antibody used in the management of Hemophilia A. It mimics the function of coagulation Factor VIII by bridging activated Factor IX (FXa) and Factor X (FX), thereby facilitating the activation of FX and promoting blood coagulation. Due to its prolonged half-life in patient plasma following administration, accurate detection of Emicizumab is clinically important. Enzyme-linked immunosorbent assay (ELISA) is a valuable method for monitoring Emicizumab levels, but it requires proteins that bind with high affinity to the antibody.
In this study, we engineered and expressed a recombinant fragment of Factor X containing the EGF-like 2 domain, designed to bind Emicizumab. A gene fragment encoding the EGF-like 2 domain of Factor X, previously identified as an Emicizumab-binding region, was codon-optimized and cloned into pUC57, then subcloned into the pET28a expression vector. Escherichia coli DH5α and E. coli Shuffle T7 (DE3) strains were used as cloning and expression hosts, respectively. The recombinant protein was purified and verified using 10 % SDS-PAGE, Bradford assay, and Western blot. Following expression, the recombinant protein was purified and quantified, with concentrations of 119 μg/mL for native and 81 μg/mL for denatured protein. These values correspond to total recoveries of 255 μg and 65 μg, respectively, from 50 mL cultures. These findings validate the use of this bacterially expressed FX-derived protein as a candidate component for incorporation into ELISA-based platforms aimed at Emicizumab detection.
{"title":"Development and expression of a factor X-derived recombinant protein for ELISA-based detection of Emicizumab","authors":"Sina Tavakoli , Shohreh Zare Karizi , Jafar Amani","doi":"10.1016/j.pep.2025.106829","DOIUrl":"10.1016/j.pep.2025.106829","url":null,"abstract":"<div><div>Emicizumab is a therapeutic antibody used in the management of Hemophilia A. It mimics the function of coagulation Factor VIII by bridging activated Factor IX (FXa) and Factor X (FX), thereby facilitating the activation of FX and promoting blood coagulation. Due to its prolonged half-life in patient plasma following administration, accurate detection of Emicizumab is clinically important. Enzyme-linked immunosorbent assay (ELISA) is a valuable method for monitoring Emicizumab levels, but it requires proteins that bind with high affinity to the antibody.</div><div>In this study, we engineered and expressed a recombinant fragment of Factor X containing the EGF-like 2 domain, designed to bind Emicizumab. A gene fragment encoding the EGF-like 2 domain of Factor X, previously identified as an Emicizumab-binding region, was codon-optimized and cloned into pUC57, then subcloned into the pET28a expression vector. <em>Escherichia coli</em> DH5α and <em>E. coli</em> Shuffle T7 (DE3) strains were used as cloning and expression hosts, respectively. The recombinant protein was purified and verified using 10 % SDS-PAGE, Bradford assay, and Western blot. Following expression, the recombinant protein was purified and quantified, with concentrations of 119 μg/mL for native and 81 μg/mL for denatured protein. These values correspond to total recoveries of 255 μg and 65 μg, respectively, from 50 mL cultures. These findings validate the use of this bacterially expressed FX-derived protein as a candidate component for incorporation into ELISA-based platforms aimed at Emicizumab detection.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"238 ","pages":"Article 106829"},"PeriodicalIF":1.2,"publicationDate":"2025-10-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145308822","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-10-08DOI: 10.1016/j.pep.2025.106827
Jie Liu , Wenjun Xu , Bingbing Xia , Yanfei He , Shiping Huang , Jun Zhao
Porcine epidermal growth factor (PoEGF) promotes intestinal epithelial development and enhances immune function in weaned piglets, offering potential as an alternative to in-feed antibiotics. However, large-scale application of recombinant PoEGF (rPoEGF) is hindered by high production costs and complex purification requirements. In this study, we constructed a recombinant Saccharomyces cerevisiae INVSc1 strain capable of expressing and secreting rPoEGF through the integration of an α-factor signal peptide-PoEGF-6 × His expression cassette. Expression was confirmed by SDS-PAGE and Western blot analysis, and bioactivity was validated using a BALB/c 3T3 fibroblast proliferation assay, yielding a maximum mitogenic activity of 10,963 U/mL. To improve production efficiency, a two-stage fermentation strategy was developed and optimized in a 4-L bioreactor. The process consisted of a glucose-based fed-batch growth phase followed by galactose-induced expression under optimized conditions (25 °C, pH 5.0, DO 30 %). This approach enabled high-cell-density cultivation (OD600 ≈ 71.5) and achieved a final secreted protein yield of 30.2 mg/L. Notably, the culture containing active rPoEGF could be directly applied as a functional feed additive without the need for downstream purification. These results provide a practical, scalable, and cost-effective approach for producing bioactive rPoEGF and support its future application in swine production as a safe, antibiotic-free growth promoter.
猪表皮生长因子（PoEGF）促进断奶仔猪肠上皮发育，增强免疫功能，有可能作为饲料中抗生素的替代品。然而，高生产成本和复杂的纯化要求阻碍了重组PoEGF （rPoEGF）的大规模应用。本研究通过整合α-因子信号peptide-PoEGF-6×His表达盒，构建了一株能够表达和分泌rPoEGF的重组酿酒酵母INVSc1菌株。通过SDS-PAGE和Western blot分析证实了表达，并通过BALB/c 3T3成纤维细胞增殖试验验证了生物活性，产生的最大有丝分裂活性为10,963 U/mL。为了提高生产效率，在4-L生物反应器中开发并优化了两阶段发酵策略。该过程包括葡萄糖为基础的分批补料生长阶段，然后在优化条件（25°C, pH 5.0, DO 30%）下进行半乳糖诱导表达。该方法实现了高密度培养（OD600≈71.5），最终分泌蛋白产量为30.2 mg/L。值得注意的是，含有活性rPoEGF的培养物可以直接用作功能性饲料添加剂，而无需进行下游纯化。这些结果为生产具有生物活性的rPoEGF提供了一种实用的、可扩展的、具有成本效益的方法，并支持其作为一种安全的、无抗生素的生长促进剂在猪生产中的应用。
{"title":"Secretory expression and fermentation optimization of recombinant porcine epidermal growth factor in Saccharomyces cerevisiae","authors":"Jie Liu , Wenjun Xu , Bingbing Xia , Yanfei He , Shiping Huang , Jun Zhao","doi":"10.1016/j.pep.2025.106827","DOIUrl":"10.1016/j.pep.2025.106827","url":null,"abstract":"<div><div>Porcine epidermal growth factor (PoEGF) promotes intestinal epithelial development and enhances immune function in weaned piglets, offering potential as an alternative to in-feed antibiotics. However, large-scale application of recombinant PoEGF (rPoEGF) is hindered by high production costs and complex purification requirements. In this study, we constructed a recombinant <em>Saccharomyces cerevisiae</em> INVSc1 strain capable of expressing and secreting rPoEGF through the integration of an α-factor signal peptide-PoEGF-6 × His expression cassette. Expression was confirmed by SDS-PAGE and Western blot analysis, and bioactivity was validated using a BALB/c 3T3 fibroblast proliferation assay, yielding a maximum mitogenic activity of 10,963 U/mL. To improve production efficiency, a two-stage fermentation strategy was developed and optimized in a 4-L bioreactor. The process consisted of a glucose-based fed-batch growth phase followed by galactose-induced expression under optimized conditions (25 °C, pH 5.0, DO 30 %). This approach enabled high-cell-density cultivation (OD<sub>600</sub> ≈ 71.5) and achieved a final secreted protein yield of 30.2 mg/L. Notably, the culture containing active rPoEGF could be directly applied as a functional feed additive without the need for downstream purification. These results provide a practical, scalable, and cost-effective approach for producing bioactive rPoEGF and support its future application in swine production as a safe, antibiotic-free growth promoter.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"237 ","pages":"Article 106827"},"PeriodicalIF":1.2,"publicationDate":"2025-10-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145259007","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-10-08DOI: 10.1016/j.pep.2025.106826
Taiji Oyama, Satoko Suzuki, Ken-ichi Akao
Circular dichroism (CD) spectroscopy is a rapid and versatile method for assessing the higher-order structures of proteins and peptides. Far-UV CD enables quantitative evaluation of secondary structures, while near-UV CD provides sensitive fingerprints of tertiary organization. With advances in recombinant protein expression, CD spectroscopy has become widely used for the characterization of novel generated proteins. This review focuses on the applications of CD spectroscopy in protein engineering and pharmaceutical sciences. Examples include strategies for reliable measurements with limited sample quantities and quality assessments such as lot-to-lot comparisons and biosimilar evaluations. CD spectroscopy also serves as a valuable tool for detecting conformational changes associated with protein–protein and protein–drug interactions, as well as for evaluating proteins and peptides in membrane-mimetic environments. Obtaining reliable CD spectra requires careful selection of buffers, water-miscible solvents, and excipients. Here, we summarize their properties and propose practical criteria for their selection to ensure high-quality CD measurements.
{"title":"Circular dichroism spectroscopy in protein engineering and pharmaceutical development: Applications in structural characterization and quality assessment","authors":"Taiji Oyama, Satoko Suzuki, Ken-ichi Akao","doi":"10.1016/j.pep.2025.106826","DOIUrl":"10.1016/j.pep.2025.106826","url":null,"abstract":"<div><div>Circular dichroism (CD) spectroscopy is a rapid and versatile method for assessing the higher-order structures of proteins and peptides. Far-UV CD enables quantitative evaluation of secondary structures, while near-UV CD provides sensitive fingerprints of tertiary organization. With advances in recombinant protein expression, CD spectroscopy has become widely used for the characterization of novel generated proteins. This review focuses on the applications of CD spectroscopy in protein engineering and pharmaceutical sciences. Examples include strategies for reliable measurements with limited sample quantities and quality assessments such as lot-to-lot comparisons and biosimilar evaluations. CD spectroscopy also serves as a valuable tool for detecting conformational changes associated with protein–protein and protein–drug interactions, as well as for evaluating proteins and peptides in membrane-mimetic environments. Obtaining reliable CD spectra requires careful selection of buffers, water-miscible solvents, and excipients. Here, we summarize their properties and propose practical criteria for their selection to ensure high-quality CD measurements.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"237 ","pages":"Article 106826"},"PeriodicalIF":1.2,"publicationDate":"2025-10-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145259018","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-09-26DOI: 10.1016/j.pep.2025.106825
Xu Shengnan , Wang Pan , Wang Xiaofei, Liu Xiaojing, Li Guozhu, Qin Guohong, Xu Dan
To ensure a high degree of similarity between biosimilars and reference drugs, it is crucial to perform comprehensive characterization and maintain rigorous control over glycosylation processes. Here,we aimed to optimize the glycosylation profile of a biosimilar of a bispecific antibody (BsAb) to closely resemble that of the reference drug through the synergistic use of glycosylation modulators. To identify the strongest modulators and appropriate concentration ranges, we first examined the effects of different concentrations of galactose (Gal) and manganese chloride (MnCl2) on the galactosylation rate in Shake Flask, as well as the influence of tris(hydroxymethyl)aminomethane (Tris) on the incorporation of mannose and fucose in 2 L Bioreactor. Importantly, the concurrent use of Tris and galactose did not result in any interaction effects on N-glycan modifications and had no detrimental impact on cell growth, metabolism, antibody charge variants or purity. In conclusion, The concurrent use of 0.75 mM Tris and 8 mM galactose yields a glycosylation profile of Bs-mAb1 that is highly comparable to that of the reference drug, thereby providing an effective strategy for optimizing glycosylation in biosimilars. These findings provide significant insights into the regulation of glycosylation in the production of therapeutic monoclonal antibodies and may contribute to enhancing the consistency and therapeutic performance of biosimilars.
为了确保生物类似药和参比药之间的高度相似性，进行全面的表征和严格控制糖基化过程至关重要。在这里，我们旨在优化双特异性抗体（BsAb）的生物类似物的糖基化谱，通过糖基化调节剂的协同作用，使其与参比药物非常相似。为了确定最强的调节剂和合适的浓度范围，我们首先研究了不同浓度的半乳糖（Gal）和氯化锰（MnCl2）对摇瓶中半乳糖基化速率的影响，以及三（羟甲基）氨基甲烷（tris）对甘露糖掺入和2 L生物反应器中的影响。重要的是，Tris和半乳糖同时使用不会对n -聚糖修饰产生任何相互作用，也不会对细胞生长、代谢、抗体电荷变异或纯度产生有害影响。综上所述，同时使用0.75 mM Tris和8 mM半乳糖可以产生Bs-mAb1的糖基化谱，这与参考药物的糖基化谱高度相似，从而为优化生物类似药的糖基化提供了有效的策略。这些发现为治疗性单克隆抗体生产中糖基化的调控提供了重要的见解，并可能有助于提高生物仿制药的一致性和治疗性能。
{"title":"Modulation of N-glycosylation in bispecific antibody biosimilars through combined modulators","authors":"Xu Shengnan , Wang Pan , Wang Xiaofei, Liu Xiaojing, Li Guozhu, Qin Guohong, Xu Dan","doi":"10.1016/j.pep.2025.106825","DOIUrl":"10.1016/j.pep.2025.106825","url":null,"abstract":"<div><div>To ensure a high degree of similarity between biosimilars and reference drugs, it is crucial to perform comprehensive characterization and maintain rigorous control over glycosylation processes. Here,we aimed to optimize the glycosylation profile of a biosimilar of a bispecific antibody (BsAb) to closely resemble that of the reference drug through the synergistic use of glycosylation modulators. To identify the strongest modulators and appropriate concentration ranges, we first examined the effects of different concentrations of galactose (Gal) and manganese chloride (MnCl<sub>2</sub>) on the galactosylation rate in Shake Flask, as well as the influence of tris(hydroxymethyl)aminomethane (Tris) on the incorporation of mannose and fucose in 2 L Bioreactor. Importantly, the concurrent use of Tris and galactose did not result in any interaction effects on N-glycan modifications and had no detrimental impact on cell growth, metabolism, antibody charge variants or purity. In conclusion, The concurrent use of 0.75 mM Tris and 8 mM galactose yields a glycosylation profile of Bs-mAb1 that is highly comparable to that of the reference drug, thereby providing an effective strategy for optimizing glycosylation in biosimilars. These findings provide significant insights into the regulation of glycosylation in the production of therapeutic monoclonal antibodies and may contribute to enhancing the consistency and therapeutic performance of biosimilars.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"237 ","pages":"Article 106825"},"PeriodicalIF":1.2,"publicationDate":"2025-09-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145186603","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Histidinol dehydrogenase (HisD, E.C. 1.1.1.23), encoded by the hisD gene and catalyzing the final two steps in the l-histidine biosynthesis, has emerged as a promising antibacterial target for several human pathogens, such as Brucella suis and Mycobacterium tuberculosis. Herein, biochemical properties on recombinant HisD from the human pathogen Neisseria gonorrhoeae (NgHisD) were characterized in detail. SDS-PAGE and size-exclusion chromatography revealed that recombinant NgHisD is a homodimer (∼105 kDa native size; ∼47 kDa subunit). Kinetic analysis identified this protein as a highly selective NAD+-dependent enzyme (specific activity: 17.3 ± 0.4 U mg−1), exhibiting minimal activity with NADP+. The KM values for NAD+ and l-histidinol were 0.9 ± 0.3 mM and 45 ± 6 μM, respectively. Maximal activity occurred at pH 9.5 and 45 °C in the presence of Mn2+. Heat-inactivation experiment showed that rapid inactivation began to occur following incubation at 45 °C for 30 min. The metal ions coordinated by the enzyme were not sequestered by 10 mM EDTA but were efficiently chelated by 1,10-phenanthroline. The recombinant NgHisD lost 85 % activity in the presence of 1 mM 1,10-phenantroline. Our study not only establishes a foundation for investigating the physiological function of NgHisD in vivo but also uncovers the absence of the canonical histidine biosynthesis pathway in Neisseria cinerea.
组氨酸脱氢酶（HisD， E.C. 1.1.1.23）由HisD基因编码，催化l-组氨酸生物合成的最后两步，已成为猪布鲁氏菌和结核分枝杆菌等几种人类病原体的有前途的抗菌靶点。本文对人淋病奈瑟菌（Neisseria gonorrhoeae, NgHisD）重组HisD的生化特性进行了详细的表征。SDS-PAGE和大小隔离层析显示重组NgHisD是一个同源二聚体（约105 kDa天然大小；约47 kDa亚基）。动力学分析表明，该蛋白是一种高度选择性的NAD+依赖酶（比活性：17.3±0.4 U mg-1），对NADP+的活性极小。NAD+和l-组氨酸二醇的KM值分别为0.9±0.3 mM和45±6 μM。在pH 9.5和45°C条件下，Mn2+的存在使活性达到最大。热失活实验表明，在45℃孵育30 min后，开始出现快速失活。该酶配位的金属离子不被10 mM EDTA隔离，而被1,10-菲罗啉有效地螯合。重组NgHisD在1mm - 1,10-phenantroline存在下失去85%的活性。我们的研究不仅为研究NgHisD在体内的生理功能奠定了基础，而且揭示了灰奈瑟菌中缺乏典型的组氨酸生物合成途径。
{"title":"Biochemical characterization of histidinol dehydrogenase from the human pathogen Neisseria gonorrhoeae","authors":"Shuping Fei , Jiatong Zhao , Wenwen Wu , Chaoqun Lian , Wanggang Tang","doi":"10.1016/j.pep.2025.106824","DOIUrl":"10.1016/j.pep.2025.106824","url":null,"abstract":"<div><div>Histidinol dehydrogenase (HisD, E.C. 1.1.1.23), encoded by the <em>hisD</em> gene and catalyzing the final two steps in the <span>l</span>-histidine biosynthesis, has emerged as a promising antibacterial target for several human pathogens, such as <em>Brucella suis</em> and <em>Mycobacterium tuberculosis</em>. Herein, biochemical properties on recombinant HisD from the human pathogen <em>Neisseria gonorrhoeae</em> (<em>Ng</em>HisD) were characterized in detail. SDS-PAGE and size-exclusion chromatography revealed that recombinant <em>Ng</em>HisD is a homodimer (∼105 kDa native size; ∼47 kDa subunit). Kinetic analysis identified this protein as a highly selective NAD<sup>+</sup>-dependent enzyme (specific activity: 17.3 ± 0.4 U mg<sup>−1</sup>), exhibiting minimal activity with NADP<sup>+</sup>. The <em>K</em><sub>M</sub> values for NAD<sup>+</sup> and <span>l</span>-histidinol were 0.9 ± 0.3 mM and 45 ± 6 μM, respectively. Maximal activity occurred at pH 9.5 and 45 °C in the presence of Mn<sup>2+</sup>. Heat-inactivation experiment showed that rapid inactivation began to occur following incubation at 45 °C for 30 min. The metal ions coordinated by the enzyme were not sequestered by 10 mM EDTA but were efficiently chelated by 1,10-phenanthroline. The recombinant <em>Ng</em>HisD lost 85 % activity in the presence of 1 mM 1,10-phenantroline. Our study not only establishes a foundation for investigating the physiological function of <em>Ng</em>HisD <em>in vivo</em> but also uncovers the absence of the canonical histidine biosynthesis pathway in <em>Neisseria cinerea</em>.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"237 ","pages":"Article 106824"},"PeriodicalIF":1.2,"publicationDate":"2025-09-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145186531","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-09-25DOI: 10.1016/j.pep.2025.106823
Gaoya Yuan, Meng Qu, Yifeng Li
Half-antibody is a common byproduct associated with the production of asymmetric bispecific antibody (bsAb). We previously demonstrated that Protein A column chromatography can effectively separate this byproduct from the bsAb product, as these two species exhibit different binding valencies (half-antibody and intact bsAb contain one and two Protein A binding sites, respectively). Specifically, half-antibody, which binds weaker, can be selectively removed by an appropriate wash prior to elution of the target bsAb. However, the performance of this wash step is sensitive to the loading density, making the process unrobust to variations in harvest titer. In the current study, we demonstrate that Sartobind Rapid A Protein A membrane effectively reduces half-antibody from approximately 16.5% (in the load) to 0.1% (in the eluate). In addition, using Protein A membrane also improves process robustness by avoiding the impact of loading density variation on performance. Thus, for bsAb purification where half-antibody removal is required, Protein A membrane is a better choice than resin-based Protein A column.
半抗体是与不对称双特异性抗体（bsAb）产生相关的常见副产物。我们之前证明了蛋白A柱层析可以有效地将这种副产物从bsAb产物中分离出来，因为这两个物种具有不同的结合价（半抗体和完整的bsAb分别含有一个和两个蛋白A结合位点）。具体来说，结合较弱的半抗体可以在洗脱目标bsAb之前通过适当的洗涤选择性地去除。然而，该洗涤步骤的性能对装载密度很敏感，使得该过程对收获滴度的变化不稳定。在目前的研究中，我们证明Sartobind Rapid A Protein A膜有效地将半抗体从大约16.5%（负载）降低到0.1%（洗脱液）。此外，使用蛋白A膜还可以避免负载密度变化对性能的影响，从而提高工艺稳健性。因此，对于需要去除半抗体的bsAb纯化，蛋白A膜是比基于树脂的蛋白A柱更好的选择。
{"title":"Effective and robust separation of half-antibody byproduct in bispecific antibody purification by Sartobind Rapid A Protein A membrane chromatography","authors":"Gaoya Yuan, Meng Qu, Yifeng Li","doi":"10.1016/j.pep.2025.106823","DOIUrl":"10.1016/j.pep.2025.106823","url":null,"abstract":"<div><div>Half-antibody is a common byproduct associated with the production of asymmetric bispecific antibody (bsAb). We previously demonstrated that Protein A column chromatography can effectively separate this byproduct from the bsAb product, as these two species exhibit different binding valencies (half-antibody and intact bsAb contain one and two Protein A binding sites, respectively). Specifically, half-antibody, which binds weaker, can be selectively removed by an appropriate wash prior to elution of the target bsAb. However, the performance of this wash step is sensitive to the loading density, making the process unrobust to variations in harvest titer. In the current study, we demonstrate that Sartobind Rapid A Protein A membrane effectively reduces half-antibody from approximately 16.5% (in the load) to 0.1% (in the eluate). In addition, using Protein A membrane also improves process robustness by avoiding the impact of loading density variation on performance. Thus, for bsAb purification where half-antibody removal is required, Protein A membrane is a better choice than resin-based Protein A column.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"237 ","pages":"Article 106823"},"PeriodicalIF":1.2,"publicationDate":"2025-09-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145182205","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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