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

Abstract

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.

Quantitation of Trastuzumab and an Antibody to SARS-CoV-2 in Minutes Using Affinity Membranes in 96-Well Plates

“… 53 − 55 In prior work, we immobilized 25 ± 4 mg of the mimotope Tra19 per mL of a spongy nylon membrane. 42 The immobilization method includes adsorption of a PAA-containing film, activation of the −COOH groups of PAA, and reaction of primary amines on the mimotope, Tra19, with active esters. Continuous recirculation of solutions through 96-well plates is not feasible, so we add solutions to the wells and allow them to incubate for 5–10 min prior to vacuum filtration.…”

“…Our group developed nylon membranes modified with peptide mimotopes for capture and analysis of mAbs using spin columns. 42 , 43 In general, membrane adsorbers are attractive for rapid capture of various molecules due to convection and short radial diffusion distances in micron-sized pores. 43 − 47 Adsorption of a poly(acrylic acid) (PAA)-containing film in membrane pores followed by activation of the film enables immobilization of affinity groups at high densities.…”

“… 43 − 47 Adsorption of a poly(acrylic acid) (PAA)-containing film in membrane pores followed by activation of the film enables immobilization of affinity groups at high densities. 42 , 43 , 48 − 50 Modification of pores with either polymer brushes 45 − 47 or layer-by-layer films 42 , 43 , 51 can create affinity membranes with high binding capacities, and membrane modification using layer-by-layer deposition of a few polyelectrolyte layers is very convenient.…”

Iminodiacetic Acid (IDA) Cation-Exchange Nonwoven Membranes for Efficient Capture of Antibodies and Antibody Fragments

“…As a result of these factors, several alternative, and potentially more efficient separation approaches for mAb purification are being considered [ 9 , 10 , 11 , 12 , 13 , 14 ]. Membrane chromatography is widely regarded as a promising alternative to resin chromatography [ 15 , 16 ].…”

Bio-inspired incorporation of phenylalanine enhances ionic selectivity in layer-by-layer deposited polyelectrolyte films

“…towards a specific application where crosslinking can make a film more ion selective 32,35 but can also be controlled to make a film more impermeable [41][42][43] if desired for a given application. Surface functionalization can make a membrane capture selective proteins 44 or anitbodies 45 for isolation and purification. Besides post-coating crosslinking, the films' properties can also be tailored by incorporation of different constituents.…”