IF 1.5 4区生物学

Biologicals Pub Date : 2025-05-01 DOI: 10.1016/j.biologicals.2025.101833

Negar Akhzari , Jafar Amani , Golnaz Tajadod , Shohreh Zare Karizi , Sedigheh Arbabian

{"title":"Construction and evaluation of a novel Stx2a-based immunotoxin against epidermal growth factor receptor (EGFR)","authors":"Negar Akhzari , Jafar Amani , Golnaz Tajadod , Shohreh Zare Karizi , Sedigheh Arbabian","doi":"10.1016/j.biologicals.2025.101833","DOIUrl":"10.1016/j.biologicals.2025.101833","url":null,"abstract":"<div><div>Immunotoxins (ITs) are chimeric proteins that combine the targeting specificity of a monoclonal antibody or antibody fragment with the cytotoxic properties of a toxin. They offer a promising strategy for cancer therapy by selectively delivering cytotoxic payloads to tumor cells. The epidermal growth factor receptor (EGFR) is frequently overexpressed in various cancers, making it an attractive target for IT-based therapies.</div><div>In this study, we designed and constructed a novel IT composed of a single-chain variable fragment (scFv) derived from Panitumumab, a humanized monoclonal antibody targeting EGFR, and the Shiga toxin A subunit 2 (Stx2a), a potent cytotoxic agent. The IT was designed using computational tools to optimize the linker region between the scFv and Stx2a domains. The recombinant IT was expressed in a prokaryotic host and purified to homogeneity.</div><div>The cytotoxic activity of the IT was evaluated in vitro against human colorectal carcinoma (HCT-116) and human embryonic kidney (HEK293) cells. The IT demonstrated significant cytotoxicity against HCT-116 cells, while exhibiting minimal toxicity to non-target cells. To enhance the delivery and efficacy of the IT, we encapsulated it in chitosan nanoparticles. The nanoparticle-based formulation showed improved cellular uptake and enhanced cytotoxicity compared to the free IT.</div><div>Our findings suggest that the designed IT has the potential to be a promising therapeutic agent against EGFR-expressing cancers. Further studies are warranted to evaluate its efficacy in vivo and to optimize its formulation for clinical applications.</div></div>","PeriodicalId":55369,"journal":{"name":"Biologicals","volume":"91 ","pages":"Article 101833"},"PeriodicalIF":1.5,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143891307","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}

引用次数: 0

Expression, purification, and immunogenicity study of human papillomavirus type 52 virus-like particles produced in Hansenula polymorpha 人乳头瘤病毒52型病毒样颗粒的表达、纯化及免疫原性研究

IF 1.5 4区生物学

Biologicals Pub Date : 2025-04-13 DOI: 10.1016/j.biologicals.2025.101831

Sheila Chairunnisa , Apon Zaenal Mustopa , Budiman Bela , Rosyida Khusniatul Arifah , Rifqiyah Nur Umami , Moh Egy Rahman Firdaus , Nurlaili Ekawati , Herman Irawan , Shasmita Irawan , Maritsa Nurfatwa , Ai Hertati , Sri Swasthikawati , Ela Novianti , Arizah Kusumawati , Huda Salahudin Darusman

{"title":"Expression, purification, and immunogenicity study of human papillomavirus type 52 virus-like particles produced in Hansenula polymorpha","authors":"Sheila Chairunnisa , Apon Zaenal Mustopa , Budiman Bela , Rosyida Khusniatul Arifah , Rifqiyah Nur Umami , Moh Egy Rahman Firdaus , Nurlaili Ekawati , Herman Irawan , Shasmita Irawan , Maritsa Nurfatwa , Ai Hertati , Sri Swasthikawati , Ela Novianti , Arizah Kusumawati , Huda Salahudin Darusman","doi":"10.1016/j.biologicals.2025.101831","DOIUrl":"10.1016/j.biologicals.2025.101831","url":null,"abstract":"<div><div>Human papillomavirus type 52 (HPV 52) infection is epidemiologically predominant in low-middle income countries in South-East Asia and remains a threat for global health. This study aims to assess the immunogenicity of prophylactic vaccine candidate formulated from HPV 52 L1 virus-like particles (VLPs). A codon-optimized and N-terminally truncated HPV 52 L1 gene was cloned using pHIPZ4 plasmid and expressed in <em>Hansenula polymorpha</em>. Protein purification was performed by one-step cation exchange chromatography and was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. The VLPs formation was confirmed by transmission electron microscopy (TEM) prior to formulation using AlPO<sub>4</sub> adjuvant and immunization of BALB/c mouse. The mouse sera were analyzed by enzyme-linked immunosorbent assay (ELISA), and green fluorescent protein (GFP) pseudovirion-based neutralization assay was performed for immunogenicity study. The purified HPV 52 L1 protein was successfully assembled into VLPs and showed a recovery yield of 51.76 %. The L1 antigen demonstrated a high antibody titer production, suggesting that the vaccine formulation induced humoral immune response in mouse model. Moreover, the antibody from mouse sera were able to neutralize HPV 52 pseudovirus infections in human embryonic kidney 293 (HEK293) cells. These findings suggest that the production of HPV 52 L1 VLPs using <em>H. polymorpha</em> expression system induces immune response, thus potentially can be developed as alternative HPV vaccine.</div></div>","PeriodicalId":55369,"journal":{"name":"Biologicals","volume":"90 ","pages":"Article 101831"},"PeriodicalIF":1.5,"publicationDate":"2025-04-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143824533","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}

引用次数: 0

Mass spectrometry analysis of bovine tuberculins - 3R potential in batch potency testing 牛结核菌素- 3R电位在批效检测中的质谱分析

IF 1.5 4区生物学

Biologicals Pub Date : 2025-04-05 DOI: 10.1016/j.biologicals.2025.101825

Elisabeth Balks , Jan-Hendrik Trösemeier , Michael D. Mühlebach , Andreas Reuter

{"title":"Mass spectrometry analysis of bovine tuberculins - 3R potential in batch potency testing","authors":"Elisabeth Balks , Jan-Hendrik Trösemeier , Michael D. Mühlebach , Andreas Reuter","doi":"10.1016/j.biologicals.2025.101825","DOIUrl":"10.1016/j.biologicals.2025.101825","url":null,"abstract":"<div><div>Tuberculin purified protein derivatives (tuberculin PPDs) are heat-treated, protein-enriched products of lysed mycobacteria. Tuberculin PPDs reveal a delayed hypersensitivity in individuals earlier sensitized to mycobacteria and are thereby used to detect tuberculosis. For batch potency testing of tuberculins, the European Pharmacopoeia advises intradermal injection of products into previously sensitized guinea pigs. Batch potency is reflected by the size of the resulting skin lesions as compared to a reference tuberculin. This procedure is quite compromising and results are highly variable, often requiring test repeats. In a proof of concept study, we evaluated the suitability of a combination of liquid chromatography and mass spectrometry (LC-MS<sup>E</sup>) to record qualitative protein profiles of bovine PPD tuberculin concentrates. Six batches of bovine PPD tuberculin concentrates from three manufacturers and the WHO International Standard for PPD of <em>Mycobacterium bovis</em> tuberculin were studied. In total, 35 proteins were identified by LC-MS<sup>E</sup> followed by MS database search. Eight proteins were consistently found in all batches of all bovine tuberculin PPD products. Our results suggest that LC-MS<sup>E</sup> can be used for the analysis of PPD tuberculin concentrates and that it can be further developed in the future into quantitative MS methods for batch analysis, i.e. consistency testing.</div></div>","PeriodicalId":55369,"journal":{"name":"Biologicals","volume":"90 ","pages":"Article 101825"},"PeriodicalIF":1.5,"publicationDate":"2025-04-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143783633","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

An alternative In vitro method for evaluation of inactivated infectious bronchitis (IB) vaccines 评估灭活传染性支气管炎（IB）疫苗的另一种体外方法

IF 1.5 4区生物学

Biologicals Pub Date : 2025-04-04 DOI: 10.1016/j.biologicals.2025.101829

Saleh E. Ali , Moustafa A. Zaghloul , Amina A. Radwan , Maha M. Sayed , Hala A. Said , Hanaa A. Moustafa , Osama Alaidi

{"title":"An alternative In vitro method for evaluation of inactivated infectious bronchitis (IB) vaccines","authors":"Saleh E. Ali , Moustafa A. Zaghloul , Amina A. Radwan , Maha M. Sayed , Hala A. Said , Hanaa A. Moustafa , Osama Alaidi","doi":"10.1016/j.biologicals.2025.101829","DOIUrl":"10.1016/j.biologicals.2025.101829","url":null,"abstract":"<div><div>We report a rapid <em>in vitro</em> method for the potency evaluation of oil-based inactivated Infectious Bronchitis virus (IBV) vaccines. The method is designed to be used by both, quality control laboratories during vaccine manufacturing and by authorizing national laboratories. The simple technique reduces the time and the number of live birds needed for vaccine potency evaluation, effectively promoting a clean environment. Further, the method is a convenient alternative to using the traditional vaccine potency test in which live animals are used. To illustrate a proof of concept, antigens from a total of ten commercial oil adjuvant infectious bronchitis vaccines from different manufacturers were chemically extracted using isopropyl myristate and an antigen capture ELISA test was used to quantify the antigen concentration in the aqueous extracts. The results from the conventional live birds’ tests, which determine the antibody titers after 3–4 weeks postvaccination, were compared to their corresponding antigen concentrations obtained by capture ELISA. The results indicate that, vaccines that contain a threshold amount of the specific IBV antigen (here determined to be > 1.26 pg/dose based on an antigen capture ELISA method), can be considered potent without the need to further test in live animals, provided that the concentration of the antigen can be reliably measured in its aqueous phase extract. Moreover, a linear relation between the antigen amount per dose and the antibody titer was found. Overall, the developed methods in this study are suited for high throughput vaccine potency evaluation.</div></div>","PeriodicalId":55369,"journal":{"name":"Biologicals","volume":"90 ","pages":"Article 101829"},"PeriodicalIF":1.5,"publicationDate":"2025-04-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143767755","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}

引用次数: 0

Regulatory changes during the Covid-19 pandemic: A comparative review of the European Union and Brazilian regulations for biological and biotechnological products 2019冠状病毒病大流行期间的监管变化：对欧盟和巴西生物和生物技术产品法规的比较审查

IF 1.5 4区生物学

Biologicals Pub Date : 2025-04-03 DOI: 10.1016/j.biologicals.2025.101827

Priscila Leone Nassur, Fernanda Borges de Almeida, Fernando Lucas Primo

{"title":"Regulatory changes during the Covid-19 pandemic: A comparative review of the European Union and Brazilian regulations for biological and biotechnological products","authors":"Priscila Leone Nassur, Fernanda Borges de Almeida, Fernando Lucas Primo","doi":"10.1016/j.biologicals.2025.101827","DOIUrl":"10.1016/j.biologicals.2025.101827","url":null,"abstract":"<div><div>The process for registration of biologic products is lengthy due to the high number of studies and data required by the health authorities. However, with the COVID-19 pandemic, and the urgent need for an alternative, the authorities reduce the time for registering new products with the challenge of maintaining all the necessary reliable standards. The objective of this paper is to compare the regulatory requirements for biologics in Brazil and the European Union in the context of the pandemic and evaluate the background differences in regulations before and after the emergency. The searches were conducted in the databases of EMA and Anvisa and the results were assessed for type of document/product, regulatory scope/process step, effectiveness, and year of publication/update. Both regulators foresaw the route of registration for emergency use and followed international standards, with strict requirements for quality, safety, and efficacy. After the end of the health emergency, while EMA gradually phased out the emergency regulations, Anvisa withdrew them. It was observed that the challenges faced by the Brazilian authority and industries were related to the lack of a centralized health monitoring system. The regulators were overall aligned in the approaches during the pandemic and both had a regulatory emphasis on vaccines but the measures taken after its end differed. The main difference observed was the slower phase-out and the adoption of lessons learned strategy in the EU, which should be learning points for Anvisa if targeting a continuous readiness strategy for health emergencies.</div></div>","PeriodicalId":55369,"journal":{"name":"Biologicals","volume":"90 ","pages":"Article 101827"},"PeriodicalIF":1.5,"publicationDate":"2025-04-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143761021","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}

引用次数: 0

Validation of a Next Generation Sequencing Method for adventitious agents detection in a live vaccine matrix 在活疫苗基质中检测不定因子的新一代测序方法的验证

IF 1.5 4区生物学

Biologicals Pub Date : 2025-04-02 DOI: 10.1016/j.biologicals.2025.101828

Alice Alston , Rebecca A. Bova , Bradley Hasson

{"title":"Validation of a Next Generation Sequencing Method for adventitious agents detection in a live vaccine matrix","authors":"Alice Alston , Rebecca A. Bova , Bradley Hasson","doi":"10.1016/j.biologicals.2025.101828","DOIUrl":"10.1016/j.biologicals.2025.101828","url":null,"abstract":"<div><div>Next Generation Sequencing (NGS) has proven itself as a suitable replacement technology for traditional viral safety assessment assays in the manufacturing of complex biologics. Most notably, its incorporation into ICH Q5A(R2) in November 2023 endorses the technology platform as a suitable alternative to traditional <em>in vivo</em>, <em>in vitro</em> and PCR-based testing for adventitious viruses based on the risk assessment of the product and its context for use. In addition to the finalization of ICH Q5A(R2), a separate European Pharmacopoeia chapter Ph. Eur. 2.6.41 (High Throughput Sequencing for the Detection of Viral Extraneous Agents) is under review to further outline and provide guidance for the application and validation of NGS-based methodologies. Within the context of avian-based quadrivalent influenza vaccine manufacturing, NGS provides an alternative viral safety assessment method to traditional <em>in vivo</em> based testing models which are requirements for every lot manufactured. However, prior to implementation of NGS alternative methodology for commercial product testing, suitability of the method must be demonstrated within the context of the product through appropriate assay validation.</div><div>In line with ICHQ5A (R2), 3.2.5.2 Next Generation Sequencing, non-targeted NGS can replace <em>In vivo</em> with broad virus detection for unknown or unexpected virus species without a head-to-head comparison. Therefore, complying with ICH Q5A (R2) and also AstraZeneca internal risk assessment for adventitious agents’ detection, a comparability study directly comparing <em>In vivo</em> to NGS was not completed.</div><div>This article summarizes the collaborative effort between AstraZeneca and MilliporeSigma to replace the <em>in vivo</em> adventitious virus test for Live Attenuated Influenza Vaccine (LAIV) with NGS for broad virus detection as part of a comprehensive virus testing strategy.</div></div>","PeriodicalId":55369,"journal":{"name":"Biologicals","volume":"90 ","pages":"Article 101828"},"PeriodicalIF":1.5,"publicationDate":"2025-04-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143746889","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Multiple factors shape technology transfer for the development and manufacture of vaccines in Latin America and the Caribbean 多种因素影响着拉丁美洲和加勒比疫苗开发和生产的技术转让

IF 1.5 4区生物学

Biologicals Pub Date : 2025-03-23 DOI: 10.1016/j.biologicals.2025.101826

Nelson Campos , María de los Ángeles Cortés , Tomás A. Pippo , Judit Rius , James Fitzgerald , Andrés Couve

{"title":"Multiple factors shape technology transfer for the development and manufacture of vaccines in Latin America and the Caribbean","authors":"Nelson Campos , María de los Ángeles Cortés , Tomás A. Pippo , Judit Rius , James Fitzgerald , Andrés Couve","doi":"10.1016/j.biologicals.2025.101826","DOIUrl":"10.1016/j.biologicals.2025.101826","url":null,"abstract":"<div><div>The COVID-19 pandemic highlighted significant inequalities in access to medicines and emergency supplies, including vaccines, that persist in Latin America and the Caribbean. From a regional perspective, it is necessary to improve the conditions to ensure more equitable and inclusive access to health technologies, both in normal scenarios and during future biological threats. Technology Transfer emerges as an effective tool to permanently avoid scarcity in global and regional vaccine supplies.</div><div>Here we describe the global and regional ecosystem of Technology Transfer, its actors, roles, interactions, and evolution through research of publicly available documents and interviews with experts from the region and international institutions. Additionally, we identify and analyze vaccine projects, characterize typologies of projects in the region, suggest an evolution of three temporal phases, reveal lessons from the COVID-19 pandemic and identify four drivers that expedite vaccine Technology Transfer in Latin America and the Caribbean. These drivers include (i) strengthening of regulatory capacities for vaccines; (ii) adoption of trade standards; (iii) increasing manufacture capacity, R&D, and human resources; and (iv) consideration of aggregated demand.</div><div>Finally, we present recommendations to maximize the potential of scientific-technological and vaccine production capacities in Latin American and the Caribbean. They relate to the four drivers, the promotion of complementary industries, data access and availability policies, inter-institutional dialogue and coordination, public health considerations, and future work in areas of information opacity.</div></div>","PeriodicalId":55369,"journal":{"name":"Biologicals","volume":"90 ","pages":"Article 101826"},"PeriodicalIF":1.5,"publicationDate":"2025-03-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143681696","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Global availability of critical reagents for biologicals testing - Current status, challenges and possible solutions 生物制品检测关键试剂的全球可用性——现状、挑战和可能的解决方案

IF 1.5 4区生物学

Biologicals Pub Date : 2025-03-22 DOI: 10.1016/j.biologicals.2025.101821

Laura Viviani , Joris Vandeputte , Dean Smith , Emmanuelle Coppens , Kutub Mahmood , Sunil Goel , Esther Wenzel , Le Sun , Catherine Milne , Quinton Meyer , Michelle Rubbrecht , Mic McGoldrick , Carmen Jungbaeck

{"title":"Global availability of critical reagents for biologicals testing - Current status, challenges and possible solutions","authors":"Laura Viviani , Joris Vandeputte , Dean Smith , Emmanuelle Coppens , Kutub Mahmood , Sunil Goel , Esther Wenzel , Le Sun , Catherine Milne , Quinton Meyer , Michelle Rubbrecht , Mic McGoldrick , Carmen Jungbaeck","doi":"10.1016/j.biologicals.2025.101821","DOIUrl":"10.1016/j.biologicals.2025.101821","url":null,"abstract":"<div><div>On July 2, 2024, the International Alliance for Biological Standardization (IABS) and Humane Society International (HSI) co-hosted a webinar on the global availability and affordability of critical reagents for vaccine and biologics production. Despite growing support for non-animal testing, significant barriers remain, especially in low-income countries facing financial and supply chain challenges.</div><div>This meeting showcased successful collaborations on reagent production and shared industry and regulatory perspectives. Key barriers included high reagent costs, import complexities, and the limited number of suppliers. Participants stressed the need for tailored risk-based testing, in-house assay validation, and stronger collaboration for standardised testing. The idea of regional hubs in Africa and Southeast Asia for reagent distribution was also discussed to address logistical challenges.</div><div>A central theme was advocating reliance strategies, which promote shared regulatory assessments and resource optimisation, as demonstrated by the EU/EEA OCABR Network activities and South African-European laboratory collaborations. Difficulties facing smaller national control laboratories in meeting international standards were highlighted, along with the need for further innovation in non-animal-derived reagents to address these challenges. Participants stressed the importance of continued global collaboration and adopting reliance practices to improve access to critical reagents and ensure sustainability in biologics testing.</div></div>","PeriodicalId":55369,"journal":{"name":"Biologicals","volume":"90 ","pages":"Article 101821"},"PeriodicalIF":1.5,"publicationDate":"2025-03-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143681701","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Development and optimization of multiplex PCR for rapid detection of type I-F1 and type I-F2 Cas cluster genes in Acinetobacter baumannii 快速检测鲍曼不动杆菌I-F1型和I-F2型Cas簇基因的多重PCR方法的建立与优化

IF 1.5 4区生物学

Biologicals Pub Date : 2025-03-13 DOI: 10.1016/j.biologicals.2025.101824

Gulshan Yadav , Amit Sharma , Umesh Prasad Sah Hathi , Rajni Gaind , Ruchi Singh

{"title":"Development and optimization of multiplex PCR for rapid detection of type I-F1 and type I-F2 Cas cluster genes in Acinetobacter baumannii","authors":"Gulshan Yadav , Amit Sharma , Umesh Prasad Sah Hathi , Rajni Gaind , Ruchi Singh","doi":"10.1016/j.biologicals.2025.101824","DOIUrl":"10.1016/j.biologicals.2025.101824","url":null,"abstract":"<div><div>Polymerase chain reaction (PCR), especially the multiplex PCR assay, enables simultaneous detection of multiple genes and is highly effective for diagnostic applications. The CRISPR-associated (Cas) system consists of several genes, and complete gene clusters are essential for its activity; multiplex PCR is an excellent method for detecting these multiple genes. This study focuses on the development and validation of a multiplex PCR protocol for the specific detection of CRISPR-Cas subtypes I-F1 and I-F2 found in <em>A. baumannii</em>, which is classified as a critical ESKAPE pathogen. The multiplex PCR method achieved a 100 % detection rate for isolates containing Cas subtypes I-F1 and I-F2 in clinical <em>A. baumannii</em> isolates. Testing across various genera and <em>Acinetobacter</em> species confirmed the high specificity of the assay, with no false positives, establishing it as a reliable tool for large-scale clinical applications. Of the 96 clinical <em>A. baumannii</em> isolates analysed, 29.167 % (n = 28) were multiplex PCR positive for a CRISPR-Cas system. Among these, 71.43 % (n = 20) had subtype I-F1, while 28.57 % (n = 8) had subtype I-F2. No clear association was found between Cas subtypes and resistance to the tested antibiotics or carbapenem genes. This study provides a valuable tool for monitoring CRISPR-Cas systems and can aid in various experimental and novel strategies to manage multidrug-resistant <em>A. baumannii</em>.</div></div>","PeriodicalId":55369,"journal":{"name":"Biologicals","volume":"90 ","pages":"Article 101824"},"PeriodicalIF":1.5,"publicationDate":"2025-03-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143619184","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}

引用次数: 0

Ultra-high performance liquid chromatography with tandem mass spectrometry (UPLC-MS/MS) for simultaneous estimation of residual glyphosate and its metabolite (amino methyl phosphonic acid - AMPA) in various vaccines 超高效液相色谱-串联质谱（UPLC-MS/MS）同时测定多种疫苗中残留草甘膦及其代谢物（氨基甲基膦酸- AMPA）

IF 1.5 4区生物学

Biologicals Pub Date : 2025-02-25 DOI: 10.1016/j.biologicals.2025.101822

Bharat Shinde , Dadasaheb Patil , Nitin Kadam , Manish Gautam , Kaushik Banerjee , Sunil Gairola , Pooja Doshi

{"title":"Ultra-high performance liquid chromatography with tandem mass spectrometry (UPLC-MS/MS) for simultaneous estimation of residual glyphosate and its metabolite (amino methyl phosphonic acid - AMPA) in various vaccines","authors":"Bharat Shinde , Dadasaheb Patil , Nitin Kadam , Manish Gautam , Kaushik Banerjee , Sunil Gairola , Pooja Doshi","doi":"10.1016/j.biologicals.2025.101822","DOIUrl":"10.1016/j.biologicals.2025.101822","url":null,"abstract":"<div><div>There is growing interest of monitoring of glyphosate (GLYP) and its active metabolite, aminomethylphosphonic acid (AMPA) in pharmaceuticals globally. Vaccines represents an important class of pharmaceuticals for human and veterinary use. In this work, a robust, sensitive and direct ultra-high-performance liquid chromatography coupled with mass spectrometry (UPLC-MS/MS) based method was developed and validated. This method enables simultaneous detection and quantification of GLYP and AMPA using a simple liquid-liquid extraction technique in complex vaccines. In absence of these residuals, the method was validated using spiked standards of GLYP and AMPA in the selected vaccines. The method demonstrated suitable linearity with r<sup>2</sup> > 0.997 over the wide concentration range of 2–50 ng/mL for GLYP and 2–100 ng/mL for AMPA respectively. LOD and LOQ of 0.5 ng/mL and 2 ng/mL for GLYP and AMPA was observed. The method showed precision (RSD of 14 %) and accuracy (83–108 %) in selected vaccines including diphtheria-tetanus-whole cell pertussis-hepatitis B and <em>haemophilus influenzae</em> type B conjugate, diphtheria-tetanus-whole cell pertussis-hepatitis B-<em>haemophilus influenzae</em> type B conjugate and inactivated polio virus, measles-mumps-rubella and pneumococcal polysaccharide conjugate vaccines. The study supports the suitability of the method for simultaneous monitoring of GLYP and AMPA in vaccine formulations.</div></div>","PeriodicalId":55369,"journal":{"name":"Biologicals","volume":"90 ","pages":"Article 101822"},"PeriodicalIF":1.5,"publicationDate":"2025-02-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143478700","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}

引用次数: 0

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