Browsing by Author "Willson, Richard C."

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    Fluorescence Correlation Spectroscopy Study of Protein Transport and Dynamic Interactions with Clustered-Charge Peptide Adsorbents
    (Wiley-Blackwell, 2012-08) Daniels, Charlisa R.; Kisley, Lydia; Kim, Hannah; Chen, Wen-Hsiang; Poongavanam, Mohan- Vivekanandan; Reznik, Carmen; Kourentzi, Katerina; Willson, Richard C.; Landes, Christy F.
    Ion-exchange chromatography (IEX) relies on electrostatic interactions between the adsorbent and the adsorbate, and is used extensively in protein purification. Conventional IEX utilizes ligands that are singly charged and randomly dispersed over the adsorbent, creating a heterogeneous distribution of potential adsorption sites. Clustered-charge ion exchangers exhibit higher affinity, capacity, and selectivity than their dispersed-charge counterparts of the same total charge density. In the present work, we monitored the transport behavior of an anionic protein near clusteredcharge adsorbent surfaces using Fluorescence Correlation Spectroscopy. We can resolve protein free diffusion, hindered diffusion and association with bare glass, agarose-coated, and agaroseclustered peptide surfaces, demonstrating that this method can be used to understand and ultimately optimize clustered charge adsorbent and other surface interactions at the molecular scale.
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    High ionic strength narrows the population of sites participating in protein ion-exchange adsorption: A single-molecule study
    (Elsevier, 2014) Kisley, Lydia; Chen, Jixin; Mansur, Andrea P.; Dominguez-Medina, Sergio; Kulla, Eliona; Kang, Marci; Shuang, Bo; Kourentzi, Katerina; Poongavanam, Mohan-Vivekanandan; Dhamane, Sagar; Willson, Richard C.; Landes, Christy F.
    The retention and elution of proteins in ion-exchange chromatography is routinely controlled by adjusting the mobile phase salt concentration. It has repeatedly been observed, as judged from adsorption isotherms, that the apparent heterogeneity of adsorption is lower at more-eluting, higher ionic strength. Here, we present an investigation into the mechanism of this phenomenon using a single-molecule, super-resolution imaging technique called motion-blur Points Accumulation for Imaging in Nanoscale Topography (mbPAINT). We observed that the number of functional adsorption sites was smaller at high ionic strength and that these sites had reduced desorption kinetic heterogeneity, and thus narrower predicted elution profiles, for the anion-exchange adsorption of ?-lactalbumin on an agarose-supported, clustered-charge ligand stationary phase. Explanations for the narrowing of the functional population such as inter-protein interactions and protein or support structural changes were investigated through kinetic analysis, circular dichroism spectroscopy, and microscopy of agarose microbeads, respectively. The results suggest the reduction of heterogeneity is due to both electrostatic screening between the protein and ligand and tuning the steric availability within the agarose support. Overall, we have shown that single molecule spectroscopy can aid in understanding the influence of ionic strength on the population of functional adsorbent sites participating in the ion-exchange chromatographic separation of proteins.
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    Navigating the challenges of initiating pediatric device trials – a case study
    (Cambridge University Press, 2024) Hunter, R. Brandon; Willson, Richard C.; Haridas, Balakrishna; Luk, Christine; Toman, Kara; Heffernan, Michael J.; Fischer, Gwenyth; Wettergreen, Matthew; Koh, Chester J.
    Introduction:Pediatric medical devices lag behind adult devices due to economic barriers, smaller patient populations, changing anatomy and physiology of patients, regulatory hurdles, and especially difficulties in executing clinical trials. We investigated the requirements, challenges, associated timeline, and costs of conducting a multi-site pivotal clinical trial for a Class II pediatric physiologic monitoring device.Methods:This case study focused on the negotiation of clinical trial agreements (CTAs), budgets, and Institutional Review Board (IRB) processing times for a pediatric device trial. We identified key factors contributing to delays in clinical trial execution and potential best practices to expedite the process while maintaining safety, ethics, and efficacy.Results:The total time from site contact to first patient enrollment averaged 14 months. CTA and budget negotiations were the most time-consuming processes, averaging nearly 10 and 9 months, respectively. Reliance and local IRB processing also contributed significantly to the timeline, overall adding an average of 6.5 months across institutions. Nearly half of all costs were devoted to regulatory oversight. The COVID-19 pandemic caused significant slowdowns and delays at multiple institutions during study enrollment. Despite these pandemic-induced delays, it is important to note that the issues and themes highlighted remain relevant and have post-pandemic applicability.Conclusions:Our case study results underscore the importance of establishing efficient and standardized processing of CTAs, budget negotiations, and use of reliance IRBs to expedite clinical trial execution for pediatric devices. The findings also highlight the need for a national clinical trials network to streamline the clinical trial process.