L4-7628 Continuous chromatographic isolation isoforms of monoclonal antibodies

Development of a continuous system for isolation of different mAbs isoforms.

Monoclonal antibodies (mAbs) represent today most important biological drugs, enabling treatment of several serious diseases such as cancer etc. mAbs are proteins of around 150 kDa with biologic function highly dependent on posttranslational modifications. Because of that, the same antibody can be present in over 1000 different isoforms and only single isoform with target biologic function is to be isolated, increasing substantially complexity of isolation process. Purification can be achieved using combination of different chromatographic techniques using shallow gradients for elution, increasing isolation costs on preparative level.

One possible way to decrease isolation costs is to switch from batch to continuous operation that results in decrease of chromatographic column volume but further increases system complexity. The most frequently used mode of continuous chromatographic operation is simulated moving bed (SMB). By switching valves between chromatographic columns, movement of stationary phase is simulated. Such system is frequently used for separation of chiral compounds in isocratic mode. Only very recently gradient continuous chromatographic systems were introduced (MCSGP) but they are predominantly still in development phase.

Recent studies of ion-exchange (IEX) chromatographic columns revealed, that shallow reproducible pH gradient can be generated as a consequence of IEX group titration by a stepwise shift of the mobile phase ionic strength. This phenomenon opens possibility to construct rather simple continuous system for isolation of different mAbs isoforms.

Project is divided in four segments. First segment is development of continuous chromatographic system, similar to MCSGP, but much simpler due to absence of gradient pumps. It will consist of two columns, on one column sample is to be loaded while on the other there is elution of mAbs charged variants via induced pH gradient and recycling of fractions containing product and impurities. Main goal will be to provide robust and reproducible operation.

Second segment is development of chromatographic column bearing weak and strong IEX groups. For efficient utilization of induced pH gradient on preparative level, we need strong IEX groups for binding of mAbs and weak IEX groups, inaccessible for mAbs, for generation of robust induced pH gradient. Therefore, we will test different ratios of resins bearing weak and strong IEX groups to find optimal conditions for a particular mAbs system.

Third segment is development of grafted monolithic support bearing weak and strong IEX groups. Advantage of monoliths is in flow independent properties enabling operation at high linear velocity and performing high number of cycles per time unit. Grafted monolith will be prepared by introduction of weak IEX groups on a monolith skeleton and strong IEX groups by grafting of polymer chains. If density of polymer chains is high enough they do spontaneously form so called brush regime, meaning that all chains extend perpendicular from pore surface toward the pore center. As mAbs are present originally in mobile phase they are adsorbed on strong IEX before they can reach weak IEX groups and have therefore no impact on induced pH gradient. Properties of such support can be varied by rate of weak IEX group conversion into polymer chains and polymer chain length.

Fourth segment is development of continuous chromatographic process protocols for real mAbs samples. Both types of chromatographic supports will be used. Proper analytics to determine quality and purity of product will be implemented, optimal chromatographic columns and protocols will be derived.

The project is co-funded by the Slovenian Research Agency ARRS and Lek Pharmaceutical Company d.d.