Gene therapies are part of a larger class of advanced therapies that aim to treat disease via delivery of recombinant genetic material. A gene therapy product has two components, the delivery system (viral vector or non-viral) and the transgene (DNA or RNA). These therapies act via replacement of a non-functional gene, silencing of a disease-causing gene, or introduction of a new or modified gene with the goal of generating a therapeutic response in patients. Gene therapy biodistribution and viral vector shedding must be evaluated during non-clinical testing. Polymerase chain reaction (PCR) has emerged as the technique of choice to quantify the gene therapy product and the transferred genetic material in study samples. With increasing numbers of gene therapies in pre-clinical development, there has been a concomitant increase in the use of PCR in bioanalytical laboratories. A major challenge in this space is the lack of formal guidance for the development, characterization, and validation of PCR assays. This article will focus on the opportunities and challenges in developing and characterizing non-GLP, digital PCR assays for AAV gene therapy products. AAV vectors are currently the most common viral delivery system, however many of the insights presented will be applicable to other delivery systems.
{"title":"Challenges in Development and Qualification of PCR/dPCR Assays for Gene Therapy Biodistribution and Viral Shedding Assessment","authors":"K. Pineault, B. Danis, Sabrina Lory, Daisy Yuill","doi":"10.17145/jab.23.003","DOIUrl":"https://doi.org/10.17145/jab.23.003","url":null,"abstract":"Gene therapies are part of a larger class of advanced therapies that aim to treat disease via delivery of recombinant genetic material. A gene therapy product has two components, the delivery system (viral vector or non-viral) and the transgene (DNA or RNA). These therapies act via replacement of a non-functional gene, silencing of a disease-causing gene, or introduction of a new or modified gene with the goal of generating a therapeutic response in patients. Gene therapy biodistribution and viral vector shedding must be evaluated during non-clinical testing. Polymerase chain reaction (PCR) has emerged as the technique of choice to quantify the gene therapy product and the transferred genetic material in study samples. With increasing numbers of gene therapies in pre-clinical development, there has been a concomitant increase in the use of PCR in bioanalytical laboratories. A major challenge in this space is the lack of formal guidance for the development, characterization, and validation of PCR assays. This article will focus on the opportunities and challenges in developing and characterizing non-GLP, digital PCR assays for AAV gene therapy products. AAV vectors are currently the most common viral delivery system, however many of the insights presented will be applicable to other delivery systems.","PeriodicalId":15014,"journal":{"name":"Journal of Applied Bioanalysis","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2023-08-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"79916607","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Current Challenges and Opportunities in using the LAL Assay for Endotoxin Testing","authors":"Timothy J. Francis","doi":"10.17145/jab.23.002","DOIUrl":"https://doi.org/10.17145/jab.23.002","url":null,"abstract":"","PeriodicalId":15014,"journal":{"name":"Journal of Applied Bioanalysis","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2023-04-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"86211560","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
M. Millan Fachi, Bruna Carolina Lui Dias, Jonata Augusto de Oliveria, Rosângela Gonçalves Peccinini, R. Pontarolo, M. D. de Campos
Objective: To achieve glycemic control, a combination of drugs is eventually necessary, especially the dual therapy of SGLT2 inhibitors with metformin. Despite the value of combination therapy, understanding the pharmacokinetic properties is critical. Therefore, this study aimed to conduct the combined and isolated administration of hypoglycemic drugs to understand their pharmacokinetic properties. Methodology: The study was performed by gavage in twenty-five rats that were divided into five groups: metformin alone (60 mg/kg), canagliflozin alone 20 mg/kg, canagliflozin and metformin (20 mg/kg and 60 mg/kg, respectively), dapagliflozin alone 2 mg/kg, and dapagliflozin and metformin (2 mg/kg and 60 mg/kg, respectively). Blood samples were collected between 0.25 and 36 hours postdose and quantified by an HPLC-MS/MS method. Results: The metformin pharmacokinetics showed values lower than those from literature, but the most relevant result was a significant change in Cmax (3400 ng/mL), AUC (872.4 ng.min/L) and CL/F (72 mL/min/kg) in the metformin with dapagliflozin group compared to metformin alone Cmax (523 ng/mL), AUC (106.8 ng.min/L) and CL/F (752 mL/min/kg). For canagliflozin, the Cmax of 6116.7 ng/mL observed in our study was similar to that observed in literature, while the clearance (5.1 mL/min/kg) was higher than that of literature, which was 3.5 mL/min/kg. Clearance of dapagliflozin CL/F was reported as 3.33 mL/min/kg, while our result was 4.6 mL/min/kg. The same study also published dapagliflozin half-life and MRT, which were slightly lower than our findings. In general, the parameters of canagliflozin and dapagliflozin were similar to the literature and did not change with simultaneous administration with metformin. Conclusion: Dapagliflozin significantly changed the pharmacokinetic disposition of metformin, while metformin coadministration had no influence on the pharmacokinetics of SGLT2 inhibitors
{"title":"Pharmacokinetic Profile of Metformin and SGLT2 Inhibitors alone and in Combination: a Pharmacokinetic Study in Wistar Rats","authors":"M. Millan Fachi, Bruna Carolina Lui Dias, Jonata Augusto de Oliveria, Rosângela Gonçalves Peccinini, R. Pontarolo, M. D. de Campos","doi":"10.17145/jab.23.001","DOIUrl":"https://doi.org/10.17145/jab.23.001","url":null,"abstract":"Objective: To achieve glycemic control, a combination of drugs is eventually necessary, especially the dual therapy of SGLT2 inhibitors with metformin. Despite the value of combination therapy, understanding the pharmacokinetic properties is critical. Therefore, this study aimed to conduct the combined and isolated administration of hypoglycemic drugs to understand their pharmacokinetic properties. Methodology: The study was performed by gavage in twenty-five rats that were divided into five groups: metformin alone (60 mg/kg), canagliflozin alone 20 mg/kg, canagliflozin and metformin (20 mg/kg and 60 mg/kg, respectively), dapagliflozin alone 2 mg/kg, and dapagliflozin and metformin (2 mg/kg and 60 mg/kg, respectively). Blood samples were collected between 0.25 and 36 hours postdose and quantified by an HPLC-MS/MS method. Results: The metformin pharmacokinetics showed values lower than those from literature, but the most relevant result was a significant change in Cmax (3400 ng/mL), AUC (872.4 ng.min/L) and CL/F (72 mL/min/kg) in the metformin with dapagliflozin group compared to metformin alone Cmax (523 ng/mL), AUC (106.8 ng.min/L) and CL/F (752 mL/min/kg). For canagliflozin, the Cmax of 6116.7 ng/mL observed in our study was similar to that observed in literature, while the clearance (5.1 mL/min/kg) was higher than that of literature, which was 3.5 mL/min/kg. Clearance of dapagliflozin CL/F was reported as 3.33 mL/min/kg, while our result was 4.6 mL/min/kg. The same study also published dapagliflozin half-life and MRT, which were slightly lower than our findings. In general, the parameters of canagliflozin and dapagliflozin were similar to the literature and did not change with simultaneous administration with metformin. Conclusion: Dapagliflozin significantly changed the pharmacokinetic disposition of metformin, while metformin coadministration had no influence on the pharmacokinetics of SGLT2 inhibitors","PeriodicalId":15014,"journal":{"name":"Journal of Applied Bioanalysis","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2023-02-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"90977458","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
The Journal of Applied Bioanalysis carried out an online survey on the following topic: “Microsampling: Opportunities and Challenges”. The survey was created to evaluate the attitudes of scientists from the bioanalysis community toward the application of microsampling technologies in the area of bioanalysis. Microsampling technologies have generated a huge amount of interest and technological developments resulting in the application of these technologies in drug development and (life-sciences) research in recent years. This online survey aimed to acquire a snapshot of attitudes and applications of microsampling technologies in the bioanalysis community and to learn about current opportunities and challenges of microsampling technologies
{"title":"Survey on Microsampling in Bioanalysis: Opportunities and Challenges-Results and Conclusions","authors":"R. Meesters","doi":"10.17145/jab.22.002","DOIUrl":"https://doi.org/10.17145/jab.22.002","url":null,"abstract":"The Journal of Applied Bioanalysis carried out an online survey on the following topic: “Microsampling: Opportunities and Challenges”. The survey was created to evaluate the attitudes of scientists from the bioanalysis community toward the application of microsampling technologies in the area of bioanalysis. Microsampling technologies have generated a huge amount of interest and technological developments resulting in the application of these technologies in drug development and (life-sciences) research in recent years. This online survey aimed to acquire a snapshot of attitudes and applications of microsampling technologies in the bioanalysis community and to learn about current opportunities and challenges of microsampling technologies","PeriodicalId":15014,"journal":{"name":"Journal of Applied Bioanalysis","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2022-12-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"80716890","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
OBJECTIVES: Validation and application of a fit-for-purpose PK assay used for quantitative analysis of acetaminophen in whole blood applying volumetric absorptive microsampling using MITRA® devices. METHODS: MITRA® devices were evaluated using whole blood samples prepared at different hematocrit values. PK assay parameters precision, accuracy, linearity, LOD, LLOQ, ULOQ, carry-over, and stability of analyte in dried whole blood on MITRA® device were validated following EMA guidelines. Acetaminophen was quantified by LC-HRMS. MITRA® devices were used on selected timepoints post-dose to collect capillary blood after finger prick. Acetaminophen was extracted using methanol containing acetaminophen-d4. RESULTS: Adsorbed whole blood volume ranged from 9.79 ± 0.18, 9.95 ± 0.34, and 10.10 ± 0.48 µL for 20, 45 and 65 % hematocrit. Highest intra-day precision and accuracy were 11.4 % CV and -19.7 %bias at 1 µg/ml. Highest inter-day precision and accuracy were 12.3% CV and -18.8 %bias at 1 µg/mL. Measuring range was 1-25 µg/mL and LOD of 0.30 µg/mL. ACP was stable on Mitra® device tip for up to 4 weeks. Obtained results from the pharmacokinetic study were not significantly different to parameters reported in the literature using plasma samples. CONCLUSIONS: MITRA® devices have a great potential use as an alternative whole blood collection device in comparison with other blood sampling techniques or devices.
{"title":"Fit-for-Purpose Validation of a PK Assay applying Blood Collection by Volumetric Absorptive Microsampling","authors":"J. R. Rincon Pabon, R. Meesters","doi":"10.17145/jab.22.001","DOIUrl":"https://doi.org/10.17145/jab.22.001","url":null,"abstract":"OBJECTIVES: Validation and application of a fit-for-purpose PK assay used for quantitative analysis of acetaminophen in whole blood applying volumetric absorptive microsampling using MITRA® devices. METHODS: MITRA® devices were evaluated using whole blood samples prepared at different hematocrit values. PK assay parameters precision, accuracy, linearity, LOD, LLOQ, ULOQ, carry-over, and stability of analyte in dried whole blood on MITRA® device were validated following EMA guidelines. Acetaminophen was quantified by LC-HRMS. MITRA® devices were used on selected timepoints post-dose to collect capillary blood after finger prick. Acetaminophen was extracted using methanol containing acetaminophen-d4. RESULTS: Adsorbed whole blood volume ranged from 9.79 ± 0.18, 9.95 ± 0.34, and 10.10 ± 0.48 µL for 20, 45 and 65 % hematocrit. Highest intra-day precision and accuracy were 11.4 % CV and -19.7 %bias at 1 µg/ml. Highest inter-day precision and accuracy were 12.3% CV and -18.8 %bias at 1 µg/mL. Measuring range was 1-25 µg/mL and LOD of 0.30 µg/mL. ACP was stable on Mitra® device tip for up to 4 weeks. Obtained results from the pharmacokinetic study were not significantly different to parameters reported in the literature using plasma samples. CONCLUSIONS: MITRA® devices have a great potential use as an alternative whole blood collection device in comparison with other blood sampling techniques or devices.","PeriodicalId":15014,"journal":{"name":"Journal of Applied Bioanalysis","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2022-11-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"86390410","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
OBJECTIVE: A high-performance liquid chromatography method for the estimation of Linezolid in human plasma was developed and validated. METHODS: Samples (100µµL) were deproteinized with acetonitrile and analyzed using LiChrospher 100, RP18e column with PDA detection at 254 nm. The flow rate of the isocratic mobile phase comprising of 0.1% formic acid in 1000 ml of water and acetonitrile in the ratio of 60:40 (v/v) was set at 1.0 ml/min. RESULTS: The calibration curve ranged from 0.50 to 20.0 µg/ml and was linear. The recovery ranged from 96% to 101%. The accuracy ranged from 98 to 101% and intra- and inter-day relative standard deviation was <4.58%. The method reliably eliminated interfering materials from plasma and R2 was 0.9973. The method described was applied to the determination of plasma LZD concentration in multi-drug-resistant tuberculosis patients who are treated with a dose of 600 mg LZD once daily. CONCLUSIONS: The developed method is suitable for determination of plasma LZD in routine care and considered feasible in less-resourced settings
{"title":"A simple HPLC-UV Method for Therapeutic Drug Monitoring of Linezolid in human Plasma in low-resourced settings","authors":"V. A, S. V, Alffenaar Jw, Jeyakumar Sm, H. Ak","doi":"10.17145/jab.21.008","DOIUrl":"https://doi.org/10.17145/jab.21.008","url":null,"abstract":"OBJECTIVE: A high-performance liquid chromatography method for the estimation of Linezolid in human plasma was developed and validated. METHODS: Samples (100µµL) were deproteinized with acetonitrile and analyzed using LiChrospher 100, RP18e column with PDA detection at 254 nm. The flow rate of the isocratic mobile phase comprising of 0.1% formic acid in 1000 ml of water and acetonitrile in the ratio of 60:40 (v/v) was set at 1.0 ml/min. RESULTS: The calibration curve ranged from 0.50 to 20.0 µg/ml and was linear. The recovery ranged from 96% to 101%. The accuracy ranged from 98 to 101% and intra- and inter-day relative standard deviation was <4.58%. The method reliably eliminated interfering materials from plasma and R2 was 0.9973. The method described was applied to the determination of plasma LZD concentration in multi-drug-resistant tuberculosis patients who are treated with a dose of 600 mg LZD once daily. CONCLUSIONS: The developed method is suitable for determination of plasma LZD in routine care and considered feasible in less-resourced settings","PeriodicalId":15014,"journal":{"name":"Journal of Applied Bioanalysis","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2021-12-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"82113624","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Yulia Benitex, Jonathan H. Davis, David L. Wensel, Tracy S. Mitchell, M. Krystal, D. Drexler
{"title":"Utility of LC-MS Surrogate Peptide Methodology in the Development of a Combinectin, a Unique Anti-HIV Biologic Drug","authors":"Yulia Benitex, Jonathan H. Davis, David L. Wensel, Tracy S. Mitchell, M. Krystal, D. Drexler","doi":"10.17145/JAB.21.007","DOIUrl":"https://doi.org/10.17145/JAB.21.007","url":null,"abstract":"","PeriodicalId":15014,"journal":{"name":"Journal of Applied Bioanalysis","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2021-07-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"84156181","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Perspectives on Business and Bioanalysis: An interview with Dr. Shane Needham","authors":"S. Needham","doi":"10.17145/JAB.21.006","DOIUrl":"https://doi.org/10.17145/JAB.21.006","url":null,"abstract":"","PeriodicalId":15014,"journal":{"name":"Journal of Applied Bioanalysis","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2021-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"88036683","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
crosampling techniques? Around 2009 I became involved with microsampling for bioanalysis research when working with clients in the field, helping to set up novel bioanalytical assays specifically around sample preparation. One area which was emerging at the time was the use of DBS for PK studies. The advantage of this technique was that remote samples could be taken without the need of a phlebotomist. Furthermore, once dried, the samples were often stable under ambient conditions so they could be posted in the regular mail without the need for cold-chain shipping. These were advantages for companies running clinical trial research projects, especially when access to phlebotomy services and refrigeration was limited. I spoke to Dr. Neil Spooner, who was pioneering quantitative DBS work for the DMPK department at GSK, and he told me of the drawbacks of DBS filter paper cards. One drawback was of sample quality and the second was of quantitation. He told me that efforts were being made to improve Neoteryx, Torrance, CA 90501, USA.
{"title":"Perspectives on Microsampling in Bioanalysis: Opportunities and Challenges in the era of the COVID-19 pandemic-an Interview with Dr. James Rudge","authors":"James Rudge","doi":"10.17145/JAB.21.004","DOIUrl":"https://doi.org/10.17145/JAB.21.004","url":null,"abstract":"crosampling techniques? Around 2009 I became involved with microsampling for bioanalysis research when working with clients in the field, helping to set up novel bioanalytical assays specifically around sample preparation. One area which was emerging at the time was the use of DBS for PK studies. The advantage of this technique was that remote samples could be taken without the need of a phlebotomist. Furthermore, once dried, the samples were often stable under ambient conditions so they could be posted in the regular mail without the need for cold-chain shipping. These were advantages for companies running clinical trial research projects, especially when access to phlebotomy services and refrigeration was limited. I spoke to Dr. Neil Spooner, who was pioneering quantitative DBS work for the DMPK department at GSK, and he told me of the drawbacks of DBS filter paper cards. One drawback was of sample quality and the second was of quantitation. He told me that efforts were being made to improve Neoteryx, Torrance, CA 90501, USA.","PeriodicalId":15014,"journal":{"name":"Journal of Applied Bioanalysis","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2021-03-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"88459125","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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