Microporous affinity membranes and their incorporation into microfluidic devices for monitoring of therapeutic antibodies.

Control of monoclonal antibody (mAb) concentrations in serum is important for maintaining the safety and efficacy of these lifesaving therapeutics. Point-of-care (POC) quantification of therapeutic mAbs could ensure that patients have effective mAb levels without compromising safety. This work uses mimotope-functionalized microporous alumina affinity membranes in vertical flow assays for detection and quantitation of therapeutic mAbs. Selective capture of bevacizumab from 1000:1 diluted serum or plasma and binding of a fluorescently labelled anti-human IgG secondary antibody enable fluorescence-based analysis of bevacizumab at its therapeutically relevant concentration range of ∼50-300 µg/mL. The assay results in a linear relationship between the fluorescence intensity of the antibody capture spot and the bevacizumab concentration. A simple prototype microfluidic device containing these membranes allows washing, reagent additions and visualization of signal within 15 min using a total of 5 mL of fluid. The prototype devices can monitor physiologically relevant bevacizumab levels in diluted serum, and future refinements might lead to a POC device for therapeutic drug monitoring.

Determination of the Serum Concentrations of the Monoclonal Antibodies Bevacizumab, Rituximab, and Panitumumab Using Porous Membranes Containing Immobilized Peptide Mimotopes.

Effective monoclonal antibody (mAb) therapies require a threshold mAb concentration in patient serum. Moreover, the serum concentration of the mAb Bevacizumab should reside in a specific range to avoid side effects. Methods for conveniently determining the levels of mAbs in patient sera could allow for personalized dosage schedules that lead to more successful treatments. This work utilizes microporous nylon membranes functionalized with antibody-binding peptides to capture Bevacizumab, Rituximab, or Panitumumab from diluted (25%) serum. Modification of the capture-peptide terminus is often crucial to creating the affinity necessary for effective binding. The high purity of eluted mAbs allows for their quantitation using native fluorescence, and membranes are effective in spin devices that can be used in any laboratory. The technique is effective over the therapeutic range of Bevacizumab concentrations. Future work aims at further modifications to develop rapid point-of-care devices and decrease detection limits.

Monoclonal Antibody Capture and Analysis Using Porous Membranes Containing Immobilized Peptide Mimotopes.

Rapid, convenient methods for monoclonal antibody (mAb) isolation are critical for determining the concentrations of therapeutic mAbs in human serum. This work uses porous nylon membranes modified with a HER2 peptide mimotope, KGSGSGSQLGPYELWELSH (KH19), for rapid affinity capture of Herceptin, a mAb used to treat breast cancer. Covalent linking of KH19 to poly(acrylic acid)-containing films in porous nylon leads to a Herceptin-binding capacity of 10 mg per mL of membrane and allows selective Herceptin capture from diluted (1:3) human serum in 5 min. Liquid chromatography-mass spectrometry demonstrates the high purity of eluted Herceptin. Moreover, the fluorescence intensity of the protein eluted from membranes increases linearly with the amount of Herceptin spiked in loading solutions containing diluted (1:3) human serum. These results demonstrate the promise of mimotope-modified membranes for Herceptin analysis that does not require secondary antibodies or derivatization with fluorescent labels. Thus, mimotope-containing membranes may form part of a simple benchtop analysis system for assessing the concentrations of therapeutic mAbs.