Flexible sensor patch for continuous carbon dioxide monitoring

Hetzler, Zach; Wang, Yan; Krafft, Danny; Jamalzadegan, Sina; Overton, Laurie K.; Kudenov, Michael W.; Ligler, Frances S.; Wei, Qingshan

doi:10.3389/fchem.2022.983523

“…Biomanufacturing process analytical technology (PAT) has seen an increased shift towards realtime monitoring since the FDA-encouraged increased adoption and focus on robust analytical technologies [17], [47]. AAV vector manufacturing has broadly not seen new analytical technologies co-develop with the emerging cell and gene therapy market.…”

Section: Discussionmentioning

confidence: 99%

Adeno-associated virus genome quantification with amplification-free CRISPR-Cas12a

Hetzler,

Marinakos,

Lott

et al. 2024

Gene Ther

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Efficient manufacturing of recombinant Adeno-Associated Viral (rAAV) vectors to meet rising clinical demand remains a major hurdle. One of the most significant challenges is the generation of large amounts of empty capsids without the therapeutic genome. There is no standardized analytical method to accurately quantify the viral genes, and subsequently the empty-to-full ratio, making the manufacturing challenges even more complex. We propose the use of CRISPR diagnostics (CRISPR-Dx) as a robust and rapid approach to determine AAV genome titers. We designed and developed the CRISPR-AAV Evaluation (CRAAVE) assay to maximize sensitivity, minimize time-to-result, and provide a potentially universal design for quantifying multiple transgene constructs encapsidated within different AAV serotypes. We also demonstrate an on-chip CRAAVE assay with lyophilized reagents to minimize end user assay input. The CRAAVE assay was able to detect AAV titers as low as 7e7 vg/mL with high precision (<3% error) in quantifying unknown AAV titers when compared with conventional quantitative PCR (qPCR) method. The assay only requires 30 minutes of assay time, shortening the analytical workflow drastically. Our results suggest CRISPR-Dx could be a promising tool for efficient rAAV genome titer quantification and has the potential to revolutionize biomanufacturing process analytical technology (PAT).

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“…Biomanufacturing process analytical technology (PAT) has seen an increased shift towards real-time monitoring since the FDA-encouraged increased adoption and focus on robust analytical technologies [17], [47]. AAV vector manufacturing has broadly not seen new analytical technologies co-develop with the emerging cell and gene therapy market.…”

Section: Discussionmentioning

confidence: 99%

Rapid Adeno-Associated Virus Genome Quantification with Amplification-Free CRISPR-Cas12a

Hetzler,

Marinakos,

Lott

et al. 2023

Preprint

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Efficient manufacturing of recombinant Adeno-Associated Viral (rAAV) vectors to meet rising clinical demand remains a major hurdle. One of the most significant challenges is the generation of large amounts of empty capsids without the therapeutic genome. There is no standardized analytical method to accurately quantify the viral genes, and subsequently the empty-to-full ratio, making the manufacturing challenges even more complex. We propose the use of CRISPR diagnostics (CRISPR-Dx) as a robust and rapid approach to determine AAV genome titers. We designed and developed the CRISPR-AAV Evaluation (CRAAVE) assay to maximize sensitivity, minimize time-to-result, and provide a potentially universal design for quantifying multiple transgene constructs encapsidated within different AAV serotypes. We also demonstrate an on-chip CRAAVE assay with lyophilized reagents to minimize end user assay input. The CRAAVE assay was able to detect AAV titers as low as 7e7 vg/mL with high precision (<3% error) in quantifying unknown AAV titers when compared with conventional quantitative PCR (qPCR) method. The assay only requires 30 minutes of assay time, shortening the analytical workflow drastically. Our results suggest CRISPR-Dx could be a promising tool for efficient rAAV genome titer quantification and has the potential to revolutionize biomanufacturing process analytical technology (PAT).Graphical Abstract

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“…As long ago as 1994, the Saylor group embedded a fiber optic tipped with bioluminescent bacteria in ground water to detect environmental pollutants [6]. In a similar configuration for a very different application, carbon dioxide-sensitive hydrogels located at the end of an optical fiber continuously monitor fermentation processes to optimize performance [25].…”

Section: Current Optical Biosensor Configurationsmentioning

confidence: 99%

Forty years of advances in optical biosensors—are “autonomous” biosensors in our future?

Ligler,

Ligler

2024

Anal Bioanal Chem

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Optical biosensors have employed at least three distinct system architectures over the last 40 years, moving from “sample in-answer out” systems to completely embedding the optical biosensor into the sample to embedding the recognition module in the sample and optically interrogating the recognition module from outside of the sample. This trends article provides an overview of the evolution of these three system architectures and discusses how each architecture has been applied to solve the measurement challenges of a wide variety of applications. A fourth biosensor system architecture, that of an “autonomous” biosensor which “takes the user out of the loop” while both detecting target analytes and responding to that measurement, is currently under development for applications initially including environmental cleanup and “smart therapeutics.” As is the case in many other areas of technology, it will be profoundly interesting to observe the further development and application of elegant, simpler (optical) biosensor systems to address tomorrow’s measurement needs. Graphical abstract

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Flexible sensor patch for continuous carbon dioxide monitoring

Cited by 9 publications

References 24 publications

Adeno-associated virus genome quantification with amplification-free CRISPR-Cas12a

Adeno-associated virus genome quantification with amplification-free CRISPR-Cas12a

Rapid Adeno-Associated Virus Genome Quantification with Amplification-Free CRISPR-Cas12a

Forty years of advances in optical biosensors—are “autonomous” biosensors in our future?

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