Functionalized Microporous Membranes for Capture and Analysis of Therapeutic Monoclonal Antibodies

Efficient treatment with therapeutic monoclonal antibodies (mAbs) requires mAb concentrations above a lower limit in a patient’s serum. In some cases, such as Bevacizumab treatment, the concentration needs to be within a therapeutic window to ensure effective treatment without side effects. Rapid and simple methods to quantify mAbs in patients’ sera or plasma could enable personalized dosing regimens to increase the effectiveness of these treatments. The research described in this dissertation employed epitope-mimicking peptides (mimotopes) immobilized in porous nylon membranes to capture Bevacizumab, Rituximab, and Panitumumab. Terminal modifications and the positions of linkers on the mimotopes are important to ensure a high binding affinity. The target mAbs were captured from 25% serum and then eluted with high purity that allowed quantitation using native mAb fluorescence. This technique successfully quantified Bevacizumab across its therapeutic range.

Subsequent studies explored mimotope-modified alumina membranes and a fluorescent dye (Cy5)-labelled anti-human IgG secondary antibody to increase the sensitivity of mAb quantitation based on capture in a membrane. Lower detection limits are vital for analyses of most therapeutic mAbs. Selective capture of Bevacizumab followed by quantitation using the tagged secondary antibody allowed a >100-fold increase in sensitivity compared to analyses using native florescence of the mAb. This technique was effective in quantifying Bevacizumab between 0-400 ng/mL in buffer, as well as in 0.1% serum or plasma in under 30 minutes. Current work in collaboration with the Linnes lab at Purdue University focuses on incorporating these membranes into a microfluidics device. These devices will remove red blood cells and capture and detect Bevacizumab in less than 15 minutes. Such a prototype could prove valuable in developing a point-of-care device for therapeutic drug monitoring.