Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.677134
Title: Improving the sensitivity and specificity of LC/MS to enable bioanalysis of therapeutic and endogenous proteins and peptides
Author: Chambers, Erin
ISNI:       0000 0004 5368 3642
Awarding Body: King's College London
Current Institution: King's College London (University of London)
Date of Award: 2015
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Abstract:
This thesis establishes LC/MS as a viable, robust, and attractive alternative analytical platform to ligand binding assays for the quantification of therapeutic and endogenous peptides/proteins in biological fluids. A rigorous investigation of the parameters that affect assay sensitivity, specificity and robustness revealed that a careful combination of mixed-mode solid phase extraction, reversed-phase liquid chromatography utilizing sub 2μm charged surface solid core stationary phases, coupled to tandem quadrupole mass spectrometry can deliver a generic platform for assay development. By extending the innovations developed in the initial work to a micro-fluidic scale it was possible to both reduce sample consumption and increase assay sensitivity by up to 20-30 fold. The value of this approach was demonstrated with subsequent validation (to FDA guidelines) of 5 separate ultra-high sensitivity assays for large hydrophobic peptides using this approach. The initial studies focused on understanding the factors that influence LC/MS assay performance for the quantification of biotherapeutics and biomarkers in biofluids. This was divided into three discrete sections: sample preparation, liquid chromatography and mass spectrometry. This investigation produced a comprehensive set of rules and guidelines to be applied during LC/MS method development for biologics quantification. From an MS perspective, these included (but were not limited to) the recommendation to choose the highest precursor/product pairs possible, avoid immonium ions, monitor multiple MRM transitions during method development, and the importance of tuning at the chromatographic flow rate was also noted. The liquid chromatography studies revealed that sub-2 μm superficially porous particles with larger pore size, and/or positively charged surface stationary phases, produced the highest efficiency and most sensitive separations, at flow rates that yield throughputs compatible with routine bioanalytical work. In addition, it was discovered that decreasing the flow rate, lowering the gradient slope, and increasing temperature could all reduce carryover and increase sensitivity further for many peptid analyses. The rules derived from the basic research were then applied to the development of assays for teriparatide (an osteoporosis drug), amyloid beta peptides (putative Alzheimer’s disease biomarkers) and human insulin and five analogs. Implementing a protein precipitation plasma pre-treatment step to reduce endogenous background was combined with SPE and chromatography based on a charged surface column to yield a quantification limit of 15 pg/mL teriparatide from 200 μL human plasma. A key aspect of amyloid beta measurement in human cerebrospinal fluid included a guanidine HCl pre-treatment step which eliminated aggregation and protein binding, enabling accurate and precise quantification of total amyloid beta, with a quantification limit of < 40 pg/mL from 100 μL human cerebrospinal fluid. Protein precipitation and mixed-mode anion exchange SPE coupled to a multidimensional chromatographic system enabled differentiation of human insulin from Humalog even though they share many of the preferred product ions, have the same precursor masses, and co-elute. The achieved detection limit of 50 pg/mL (8.6 fmol/mL) enabled measurement of fasting insulin levels. The developed method was applied in the analysis of a cohort of Type I and Type II diabetic patients on a variety of combination therapies.
Supervisor: Smith, Norman William ; Legido Quigley, Cristina Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.677134  DOI: Not available
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