Cross-interaction chromatography as a rapid screening technique to identify the stability of new antibody therapeutics.

Protein aggregation can be a major problem in the manufacturing of new biopharmaceuticals and there is a desirability for development of techniques that can predict the behaviour of new biopharmaceuticals early on in the development process. A technique that can be used to predict aggregation is self-interaction chromatography that is used to determine the second virial coefficient, B22, but one of the limitations includes the need to immobilise every protein of interest. In this study a related technique, cross interaction chromatography (CIC), is evaluated which overcomes this limitation. Three antibodies were studied across a range of NaCl concentrations with each antibody being studied as both a mobile phase and as the stationary phase - in total 6 different stationary-mobile phase combinations. The B22 values obtained for all three proteins correlated strongly with the B23 results obtained for the same protein in the mobile phase, and were significantly independent of the protein immobilised on the stationary phase. This observation allows the use of pre-prepared columns with known immobilised model proteins such as a polyclonal antibody or mAb, with other unknown monoclonal antibodies in the mobile phase. Preliminary experiments using a series of known immobilised mAbs columns with an unknown mAb in the mobile phase resulted in at least a 50 fold reduction in the amount of unknown protein needed and a rapid semi-quantitative assessment of aggregation propensity. CIC can speed up the screening process with minimum preparation time and therefore more rapidly be able to identify the aggregation stability of new antibody formulations.

Mapping the mAb Aggregation Propensity Using Self-Interaction Chromatography as a Screening Tool.

The osmotic second virial coefficient ( B2), which describes protein-protein molecular interactions in solution, was determined using self-interaction chromatography (SIC) for an IgG1-type mAb across a wide range of solution conditions. These data were compared with its time dependent aggregation behavior, as determined using size-exclusion chromatography (SEC), and its temperature dependent aggregation behavior using dynamic light scattering (DLS) over a four-week period (SEC) or overnight (DLS). DLS and SEC gave consistent data on aggregation behavior, which correlated well with experimental B2 trends across the wide pH (4-9) and NaCl concentration (0-1.0 M) ranges studied. The IgG aggregated at pH 4 for 0.5-1.0 M NaCl concentrations and for 0 M NaCl concentrations at pH 8. Best stability against aggregation was exhibited for the pH range from 5 to 8 at 0.8-1.0 M NaCl. SIC data were able to be classified within the one-day solution conditions for aggregation, which were not identified for 2-3 weeks in the accelerated SEC stability study. The ability of SIC to provide such data rapidly reflects the fundamentally thermodynamic nature of this parameter and of the aggregation process itself. Proteins with attractive protein-protein interactions and negative B2 coefficients in the range -3 to -6 clearly exhibit aggregation behavior, while B2 values in the range 0 to 2 showed good stability toward aggregation. SIC allows the rapid screening of solution conditions for which mAbs will exhibit stability to aggregation while requiring 90% less time and material compared with that required for a conventional SEC aggregation study.

Affinity ligands for immunoglobulins based on the multicomponent Ugi reaction.

This report describes a novel use of the four-component Ugi reaction to generate a solid-phase library suitable for the purification of immunoglobulins and their fragments by affinity chromatography. An aldehyde-functionalised Sepharose solid-support constituted one component in the four-component reaction, whereas the other three components (a carboxylic acid, a primary or secondary amine and an isonitrile) were varied in a combinatorial fashion to generate a tri-substituted peptoidal scaffold structure which provides a degree of rigidity and functionality suitable for rational investigation of immunoglobulin binding. The Ugi ligand library was initially screened chromatographically against whole human IgG and its fragments (Fc and Fab) to yield a Fab-specific lead ligand based on its ability to bind Fab differentially over Fc. Preparative chromatography of IgG from human serum showed 100% of IgG was adsorbed from the 20mg/ml crude stock and subsequently eluted with a purity of 81.0% as determined by SDS-PAGE analysis under non-optimised conditions. High purity Fab and IgG isolation was achieved from both yeast and E. coli host cell proteins according to silver-stained SDS-PAGE lane densitometry. The ligand density and spacer-arm chemistry of the immobilised ligand was optimised to define an affinity adsorbent which binds 73.06 mg IgG/ml moist gel (dynamic binding capacity at 10% breakthrough) and a static binding capacity of 16.1+/-0.25mg Fab/ml moist resin displaying an affinity constant K(d)=(2.6+/-0.3)x10(-6)M. The lead candidate was modelled in silico and docked into a human Fab fragment (PDB: 1AQK) to suggest a putative binding interface to the constant CH(1)-CL Fab terminal through six defined hydrogen bond interactions together with putative hydrophobic interactions.