Development and partial validation of a bioanalytical assay for the detection of a novel bone-targeting parathyroid hormone conjugate using metal-free LC-MS/MS

• Author / Creator

  Wajda, Benjamin

• In an effort to address shortcomings with current drug treatments for osteoporosis (OP) and improve the efficacy of human parathyroid hormone (hPTH(1-34)), we have employed a bisphosphonate-conjugation strategy by tethering hPTH(1-34) to a non-nitrogenated bisphosphonate (BP) moiety (BP-PTH) in order to enhance targeted drug delivery to bone. Conjugation of hPTH (1-34) to a BP imparts mineral affinity to the peptide hormone, allowing it to localize to positively-charged calcified tissues in bone. Improved drug targeting will lead to administration of lower therapeutic doses, resulting in low systemic exposure and lower concentrations being observed in plasma. Therefore, it is essential to develop a sensitive bioanalytical assay for future pharmacokinetic studies of this conjugate.
  This thesis describes the development and partial validation of an LC-MS/MS assay for the sensitive measurement of BP-PTH in rat plasma. A combination of protein precipitation and solid phase extraction was used for sample preparation, allowing for concentrated samples without the need of evaporation. Chromatographic separation was performed on a reverse-phase Phenomenex bioZen PS-C18 column with a total run time of 10.50 min. The calibration curve was linear (r2=0.996) in the range of 5 ng/mL to 25 ng/mL, the lower limit of quantification was 2.5 ng/mL. The intra- and inter-assay precision over two days (percent coefficient of variation) calculated from quality control samples was less than 10%, and the mean accuracy (percent deviation from nominal) for all samples was between 102.22%. Despite achieving satisfactory validation parameters, the chosen analytical column proved to have a short column lifetime before becoming irreversibly damaged. The method will be re-optimized with a more appropriate analytical column in the future. To our knowledge, this is the first reported method for the detection and quantification of BP-conjugated peptides in a biological matrix.
  In an effort to achieve lower limits of detection of BP-PTH, TMSD derivatization was used to methylate the phosphonic acid groups present in the BP moiety of BP-PTH. Lack of specificity with the derivatization agent resulted in the creation of numerous BP-PTH derivatives, with the most abundant corresponding to derivatives containing 11-15 methylations. The generation of numerous derivatives caused by the various number of possible methylation sites on the conjugate resulted in poorer sensitivity in comparison to non-derivatized BP-PTH.
  The use of a highly specific commercial hPTH (1-34) ELISA was explored as an alternative to LC-MS/MS for the detection of BP-PTH. The ELISA was unable to establish a concentration-response relationship for unmodified BP-PTH. Modifications to BP-PTH were undertaken with the aim to generate variants that were more chemically and structurally similar to hPTH (1-34) to allow for ELISA detection. Neutralization of the phosphate groups present in the BP portion of the conjugate were unsuccessful and suitable ELISA detection was not achieved.

• Subjects / Keywords

  • LC-MS/MS
  • Osteoporosis
  • PTH
  • Bioanalysis

• Graduation date

  Fall 2020

• Type of Item

  Thesis

• Degree

  Master of Science

• DOI

  https://doi.org/10.7939/r3-fp1k-zj43

• License

  Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. Where the thesis is converted to, or otherwise made available in digital form, the University of Alberta will advise potential users of the thesis of these terms. The author reserves all other publication and other rights in association with the copyright in the thesis and, except as herein before provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission.