(519d) Purification of Scfv Via Small Molecule Based Affinity Membrane Spin Column

Authors: 
Mejia, F., University of Notre Dame
Mustafaoglu, N., University of Notre Dame
Canonico, M., University of Notre Dame
Vrabel, M., University of Notre Dame
Bilgicer, B., University of Notre Dame
Recently, single-chain variable fragments (scFv) have emerged as an interesting alternative to antibodies for diagnostic and therapeutic purposes. They consist of variable regions of the heavy and light chains of antibodies joined by a flexible peptide linker. Owing to its significantly smaller size (~25 kDa vs ~150kDa for IgG antibodies) and lack of post-translational modifications they can be easily expressed in bacterial systems. Additionally, lack of an Fc region makes them less immunogenic while potentially having higher tissue penetration capacity than bigger antibody fragments and retaining antigen-binding capacity and high specificity.

However, several challenges in terms of conjugation and purification limit the potential of these molecules in academia and potential commercialization. Because they lack an Fc region, common purification methods such as Protein-A/G affinity-based chromatography are not useful. Instead, scFv are typically expressed as HIS-tagged proteins which can then purified through column chromatography with Ni+2-embedded resins. While this purification method yields relatively high purity products (typically >80%), it’s relatively expensive and requires hours of processing for purification. Presence of other histidine-containing proteins further affects the purity of these products. Furthermore, although regeneration and re-use is possible, it’s limited and requires extensive washing steps with a variety of solvents, often including highly acidic or basic solutions.

As an alternative, we’ve developed a novel affinity spin column capable of purifying scFv by targeting the Nucleotide Binding Site (NBS), initially identified in the variable domain of Fab and conserved in scFv. For this purpose, a small ring structured molecule with moderate affinity for the NBS was covalently conjugated to regenerated cellulose membranes, which allows scFv to be captured by the membranes. Our process allows sample purification in short times (<10 minutes) without the requirement of His-Tag or any post-purification processing while maintaining scFv activity. Multiple washing steps can be employed as required for further removal of contaminants without loss of sample and recovery can be achieved in a single elution step under mild conditions (0.5 M KCl in 20 mM TRIS pH 7.0). Here we present tests performed using several different scFv, consistently yielding >95% purity from pure or contaminated samples, including cell lysate. We study the effects of protein contaminants, amount of membranes in the column and column reusability as well as the binding activity of purified scFv. Importantly, our columns are reusable without the requirement of extensive washing or very high or low pH solutions. Additionally, compatibility of the system with resin-based separations and potential scale-up are evaluated. Overall, our system provides a simple, economic and fast tool for small-scale scFv purification without sacrificing purity or activity of the product.