Development and evaluation of analytical techniques for antibodies and antibody-drug conjugates : From verification of conjugation to stability testing

Abstract: Antibody-drug conjugates (ADCs) are becoming increasingly important in oncology. ADCs are inherently heterogeneous analytes. For a successful drug candidate to go through to the clinic, extensive characterization and evaluation of the constructs are necessary. The drug-to-antibody ratio (DAR) directly determines the toxicity and efficacy of the product and has to be closely monitored during the entire process.In this thesis, several analytical techniques suitable for DAR determination in early-stage development have been evaluated against two more readily available options. The main focus in all studies has been on cysteine-linked ADCs, due to their prevalence in successful designs and their higher demand on the analytical set-up.The comparisons showed that mass spectrometry (MS) derived DAR values did conform well with hydrophobic interaction chromatography derived values, irrespective of which MS instrument was used. For MS instruments, overall the desolvation was found to have a bigger impact on the apparent DAR values than the resolution of the instrument and low-resolution instruments, such as triple quadrupoles, can be viable options for DAR determination in early-stage development. Furthermore, it also could be concluded that MS-derived DAR values are susceptible to alterations in the sample preparation workflow.Stability testing is vital to ensure a safe product. In this thesis, different compositions of mobile phases in size exclusion chromatographic (SEC) were tested for their ability to elute ADC aggregates. It was concluded that sufficient ionic strength to elute ADC aggregates from a SEC column purely by adding ammonium acetate cannot be achieved without exceeding salt concentrations compatlible with MS sources.Finally, two new analytical workflows have been designed by applying a proteomic desalting protocol and supercharging reagents to mAb and ADC samples. Both techniques showed promise; the magnetic beads as a more flexible desalting alternative for mAbs and ADCs and the addition of selective supercharging reagents for improved sensitivity and peak shapes in MS spectra (without significant alterations of the derived DAR values).Taken together, this thesis provides guidance on many aspects of ADC analysis from DAR determination to aggregate detection. The gathered knowledge can help setting up faster or more reliable quality checks for new candidates.