Designing monoclonal antibody fragment-based affinity resins with high binding capacity by thiol-directed immobilisation and optimisation of pore/ligand size ratio.

Monoclonal antibody (mAb) based affinity resins usually suffer from low binding capacity, most probably as a result of steric hindrance by the large 150kDa size of the mAb and a random immobilisation approach. The present work investigates the influence of a variety of factors on dynamic binding capacity (DBC) such as pore/ligand size ratio, accessibility of ligand and ligand density. The effect of pore/ligand size ratio was investigated using Fab and scFv fragments on various resins with different pore sizes. The accessibility of the ligand was investigated by a site-directed immobilisation approach, where three C-terminal tags, PPKPPK, FLAG™ and Cys, were introduced into the Fab fragments for immobilisation on resins via amino-, carboxyl- and thiol-groups, respectively. The scFv fragments were tagged at the C-terminal only with FLAG™ to enable a straight forward purification procedure, and were immobilised to resins via amino- and carboxyl-groups. The target protein had a molecular weight (MW) of 50kDa. A 3-fold higher dynamic binding capacity at 100% breakthrough (DBC100%) was observed for Fab wild-type (wt) on CNBr-activated Sepharose 4 FF relative to mAb on same resin at the same ligand density. However, no major difference in DBC100% was observed between Fab wt and scFv immobilised on CNBr-activated Sepharose 4 FF at the same ligand density. Thus, further increase of pore/ligand size ratio from Fab to scFv on a resin with average pore size of 300Å, did not seem to be beneficial. Among the tested tags, only the C-terminal Cys tag proved to site-direct the ligands during immobilisation as it allowed the DBC100% to increase 1.6-fold as compared to Fab wt immobilised via amino-groups on CNBr-activated Sepharose 4 FF and Actigel ALD Superflow at the same ligand density. The influence of ligand density was investigated by selecting immobilised Fab Cys on Sulfhydryl-reactive resin. Increasing ligand density from 0.103 to 0.202µmol/mL resulted in the same utilisation yield (82-85%), whereas a further increase in ligand density from 0.202 to 0.328µmol/mL resulted in a 20%-unit decrease in utilisation yield and less steep breakthrough curve, suggesting steric hindrance in the pores of the resin. In addition, site-directed affinity ligands resulted in a more pronounced, sigmoid-shaped breakthrough curve, leading to more efficient use of capacity. The highest DBC100% was obtained for Fab Cys on Sulfhydryl-reactive resin and scFv on Actigel ALD Superflow; 11mg/mL and 10mg/mL, respectively, as compared to the DBC100% of 0.8mg/mL for mAb on CNBr-activated Sepharose 4 FF. Pore/ligand size ratio of 3, which was achieved for Fab ligands on the studied resins, was shown to be feasible for capturing a protein in MW of 50kDa. Totally, a 13.8-fold improvement in DBC100% was achieved with the Fab-based affinity resin coupled via the C-terminal Cys as compared to the mAb-based affinity resin.

A thiol functionalized cryogel as a solid phase for selective reduction of a cysteine residue in a recombinant human growth hormone variant.

Site selective chemical modification is a preferred method, employed to prolong the circulation half-life of biopharmaceuticals. Cysteines have been used as attachment point for such modification, however, to be susceptible for chemical modification the involved thiol must be in its reduced form. Proteins often contain disulfides, which aid to maintain their tertiary structure and therefore must remain intact. Thus, methods for selectively reducing cysteine residues, introduced through site-directed mutagenesis, are of interest. In this study a macroporous, polymeric monolith was designed for selectively reducing a single cysteine residue inserted in recombinant human growth hormone (hGH). Advantages of such a material are the circumvention of the need to remove the reducing agent after reaction, as well as milder reduction conditions and a concomitant lower risk of reducing the native disulfides. The designed monolith showed very high capacity towards the selective reduction of an unpaired cysteine residue in a recombinant hGH variant. Factors influencing the selectivity and rate of reaction were investigated and it was found that monolith thiol loading, and buffer pH had an effect on the rate of reduction, whereas hGH variant concentration and buffer conductivity influenced both rate of reduction and selectivity. The developed system constitutes the basis for the development of a scalable platform for selective reduction of a capped cysteine residue in hGH.

Structure-based design of selective and potent inhibitors of protein-tyrosine phosphatase beta.

Protein-tyrosine phosphatases (PTPs) are considered important therapeutic targets because of their pivotal role as regulators of signal transduction and thus their implication in several human diseases such as diabetes, cancer, and autoimmunity. In particular, PTP1B has been the focus of many academic and industrial laboratories because it was found to be an important negative regulator of insulin and leptin signaling, and hence a potential therapeutic target in diabetes and obesity. As a result, significant progress has been achieved in the design of highly selective and potent PTP1B inhibitors. In contrast, little attention has been given to other potential drug targets within the PTP family. Guided by x-ray crystallography, molecular modeling, and enzyme kinetic analyses with wild type and mutant PTPs, we describe the development of a general, low molecular weight, non-peptide, non-phosphorus PTP inhibitor into an inhibitor that displays more than 100-fold selectivity for PTPbeta over PTP1B. Of note, our structure-based design principles, which are based on extensive bioinformatics analyses of the PTP family, are general in nature. Therefore, we anticipate that this strategy, here applied to PTPbeta, in principle can be used in the design and development of selective inhibitors of many, if not most PTPs.