Bioanalysis of vinca-alkaloids and monoclonal antibodies : Challenges associated with the analysis of small and large molecules

Abstract

The research described in this thesis focused on two groups of anti-cancer agents. The group of ‘small’ molecules is represented by the vinca-alkaloids and the group of ‘large’ molecules is represented by the monoclonal antibody (mAb) trastuzumab. Both these groups require different analytical strategies. The bioanalysis of vinca-alkaloids In Chapter 2 different liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) assays were developed for vinca-alkaloids. In Chapter 2.2 and 2.3 assays were developed for vinorelbine and its metabolite 4-O-deacetylvinorelbine. Vincristine in combination with actinomycin-D is the preferred therapy to treat children with Wilms’ tumor. To support a pharmacokinetic study in Malawi we have developed a bioanalytical LC-MS/MS assay for the quantification of these drugs (Chapter 2.4). As vincristine experiences high plasma protein binding and protein levels are altered in malnourishment, the free drug concentration is also of interest and therefore an assay was developed as well. As frequent venipuncture in children is difficult we investigated in Chapter 2.5 whether dried blood spots can be used to study the pharmacokinetics of vincristine and actinomycin-D. The sampling procedure can be simplified as samples can be obtained by a simple finger prick. Structural characterization of monoclonal antibodies In Chapter 3.1 we have used a novel quadrupole ion-mobility time-of-flight mass spectrometer to study the N-glycosylation of trastuzumab. The new instrument offers a fast way for structural elucidation and batch-to-batch comparison. In the studied batches the same glycoforms were found, however their relative amount differed between batches. The quantification of monoclonal antibodies For the quantification of mAbs different approaches were investigated as described in Chapter 4. Chapter 4.1 gives a literature overview of the bioanalytical assays of the currently licensed therapeutic mAbs. In Chapter 4.2 the possibilities of using LC-MS for the quantification of trastuzumab were investigated. In Chapter 4.3 a bioanalytical method for the bioanalysis of trastuzumab in human serum is described. Different approaches were investigated to extract trastuzumab from human serum. The LLOQ of this assay is 5 g/mL in human serum, enough to support therapeutic drug monitoring. In Chapter 4.4 the development and validation of an enzyme-linked immunosorbent assay (ELISA) for trastuzumab quantification in human serum and plasma is described. The assay is very fast and specific with a validated LLOQ of 1.6 ng/mL. In Chapter 4.5 a novel approach of immunoassays is investigated; the precipitated enhanced immunoassay (PEIA). Conclusion For the bioanalysis of ‘small’ molecules LC-MS/MS is the analytical technique of first choice. We demonstrated that the dried blood spots strategy is very useful to execute pharmacokinetic studies with vincristine and actinomycin-D. It is reported that currently more than 100 therapeutic mAbs are in clinical development. Immunoassays can be expected to be the bioanalytical technique of first choice for this class of agents. However, a very specific catcher in the form of the antigen or anti-idiotype antibodies remains indispensable. Therefore, the search for alternatives will continue. The major challenge however is to obtain enough selectivity to find the ‘needle’ mAb in the ‘haystack’ of endogenous immunoglobulins.