Gander, A; (2012) Development of a Quality System to Control DNA, Endotoxin and Particulates as part of an Extracorporeal Bioartificial Liver Medical Device. Doctoral thesis , UCL (University College London).

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Abstract

The Bioartificial Liver Devices (BAL) could provide treatment for acute liver failure by supporting patients awaiting transplantation or aid the process of liver regeneration. For use within a clinical setting, a number of regulatory criteria must be met, including controlling DNA, endotoxins and particulates. The aim of this thesis was to begin the development of a system to control plasma quality returning to the patient. Methods for DNA, endotoxin and particulates detection were established with human plasma to measure sensitivity for use with the BAL system. DNA detection by QPCR using Alu repeats were validated for use as an analytical method to demonstration DNA removal, achieving a Limit of Quantification (LoQ) of 0.1ng/ml DNA. Endotoxin analysis utilised a fluorescent derivative of the widely used LAL assay to increase sensitivity, enabling 2EU/ml to be detectable. Particulates down to 1μm were measured using laser light obscuration. Initially the removal of particulates from alginate as a starting material (alginate prior to encapsulation) was shown, using filtration by depth charge filter, sand bed filtration and gas solid cyclonic filtration. Encapsulated bead integrity, cell function and growth were compromised with all techniques of filtering alginate in solution, including depth charged and sand bed filtration. Conversely, gas solid cyclonic filtration maintained bead integrity, cell growth and function. Testing potential DNA levels in the large scale BAL system required the development of a scaled down model of the BAL treatment phase, replicating the large scale BAL system with cell number to plasma volume ratio. This provided an indication of the DNA challenge a removal system at a large scale would need to contend with, predicted to be 68ng/ml for a full scale BAL. A scaled down filtration model was then established to model the DNA removal capability of different 3M® Cuno® DNA depth charged filters. This established a requirement for a predicted surface area of 1300cm2 to achieve complete DNA removal. The volumetric capacity of the filters were calculated using established filter blockage models, in order to scale the capacity to the full BAL system size. Finally, the chosen depth charge filter was tested at a large scale with the extracorporeal BAL system, spiking human plasma with DNA and endotoxin, whilst measuring endotoxin and DNA removal over 8 hours of treatment. Page

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