Projects / Programmes
Continuous chromatographic isolation izoforms of monoclonal antibodies
| Code |
Science |
Field |
Subfield |
| 4.06.06 |
Biotechnical sciences |
Biotechnology |
Finishing processes in biotechnology |
| Code |
Science |
Field |
| T490 |
Technological sciences |
Biotechnology |
| Code |
Science |
Field |
| 2.09 |
Engineering and Technology |
Industrial biotechnology |
continuous chromatography, separation of MAbs charge variants, induced pH gradient, grafted monoliths with hierarchically organized functional groups
Researchers (16)
Organisations (3)
Abstract
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.
Significance for science
During the project we expect to provide important contributions on different scientific fields. Development of continuous chromatographic system for separation of biologic macromolecules without implementation of gradient pumps is completely novel approach that can substantially contribute to further development of downstream processing. Application of induced pH gradient is also an inventive approach and one can anticipate that once successfully applied for isolation of mAbs charged variants, its implementation will be extended to other complex systems like e.g. PEGylated proteins, enzyme isoforms and others. More efficient purification of described molecules can facilitate their application and characterization and therefore accelerate knowledge of their properties.
Second important segment is preparation of chromatographic supports with hierarchically organized functional groups. This is an entirely new field that can lead to novel separation mechanisms and selectivity during separation processes. There will be contribution in synthesis procedures to obtain hierarchically organized functional groups that can be further transferred to particulate supports but especially important can be elucidation of adsorption mechanisms on such supports. There is almost no information available about the effect of organization of functional groups on adsorption properties and progress in this field can lead to novel chromatographic stationary phases with superior chromatographic properties.
Significance for the country
Biopharmaceuticals, where the largest group is represented by monoclonal antibodies (mAbs), present today a large part of the pharmaceutical market. Due to prolonged life expectancy and aging populations in the developed countries, the incidence of the autoimmune diseases and cancer steadily increases. High doses and long-term therapies make the treatments very expensive and even the richest countries of the world are facing the problems of very expensive or too expensive health care and services. Our approaches could lead to cost effective therapeutics, broadly available to the whole population and contribute to the improvement of health and quality of life.
In short this should eventually lead to:
- Broader patients’ accessibility to highly efficient biopharmaceuticals;
- Smaller Health Care System burden;
- Highly efficient as well as more sustainable production processes;
A
iming to create new and breakthrough technologies, the action will also boost competitiveness and growth of the companies involved and creation of jobs with high added value.
Most important scientific results
Interim report,
final report
Most important socioeconomically and culturally relevant results
Interim report,
final report
