Antibody-drug conjugates (ADCs) are promising biopharmaceuticals. They combine the high selectivity and affinity to cancer cells with the toxicity of chemotherapeutics in one molecule.
ADCs consist of a monoclonal antibody, covalently bound via a linker to a highly potent cytotoxic drug. ADC-mimics contain a non-toxic payload with similar structure and physiochemical properties as the toxic payload of an ADC. Therefore, they can be used as a model to develop a suitable purification process or analytical method. The ADC-mimic in this work consists of Adalimumab bound to Fluorescein 5-isocyanate (FITC).
The purification process of ADCs is complex due to the heterogeneity of the conjugates. One of the main challenges is the separation in different drug-antibody-ratio (DAR), which correlates with the potency of the ADC. High DARs are associated with high cytotoxic levels. On the other hand, low DARs affect the efficacy of the therapeutics.
Due to the very hydrophobic payload, an increasing DAR results in an increase in the hydrophobicity of the ADC.
Using the relatively hydrophilic HIC resin, Toyopearl PPG-600M, results in high recovery together with high binding capacity, and wide working pH range.
The ADC-Mimic was loaded to a Toyopearl PPG-600M column (6.6mm ID x 10cm) to separate the ADC-Mimic in fractions of low, medium and high DAR. The concentration of low salt buffer in each step can be adjusted to modify the separation. Detailed information about the buffer used in this study can be found elsewhere.
The eluate for each elution step was fractionated and analysed on TSKgel Butyl-NPR. Due to the different absorption maxima between FITC (495nm) and antibody (280nm), it is possible to calculate an UV-estimated DAR according to the equation shown in the image above.
As displayed in the graph above, each bar represents the absorbance of the ADC-Mimics in blue for the antibody and red for FITC. The analysis was performed on TSKgel Butyl NPR.
Toyopearl PPG-600M resin offers sufficient selectivity in step gradient to separate the ADCs according to their DARs. The low DAR fraction has an average DAR of 1, the medium DAR fraction of 3 and the high DAR fraction a medium DAR of 5. All approved ADCs exhibit DARs between 2 and 4. By slight adjustments of the concentration during the step gradient, ADCs can be isolated within target DAR range.