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Projects / Programmes source: ARIS

Continuous chromatographic isolation izoforms of monoclonal antibodies

Research activity

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 
Keywords
continuous chromatography, separation of MAbs charge variants, induced pH gradient, grafted monoliths with hierarchically organized functional groups
Evaluation (rules)
source: COBISS
Researchers (16)
no. Code Name and surname Research area Role Period No. of publicationsNo. of publications
1.  26055  PhD Gorazd Hribar  Biochemistry and molecular biology  Researcher  2016 - 2019  49 
2.  30700  PhD Rosana Hudej  Biotechnology  Researcher  2018 - 2019  26 
3.  33406  PhD Nikolaja Janež  Biochemistry and molecular biology  Researcher  2016 - 2018  86 
4.  34586  PhD Luka Jeromel  Physics  Researcher  2017 - 2019  48 
5.  16171  PhD Simona Jevševar  Biotechnology  Researcher  2016 - 2019  51 
6.  30674  PhD Menči Kunstelj  Pharmacy  Researcher  2016 - 2019  18 
7.  39257  Tanja Lukan  Chemistry  Technical associate  2017 - 2018  16 
8.  27882  PhD Miha Lukšič  Chemistry  Researcher  2016 - 2019  222 
9.  16327  PhD Matjaž Peterka  Biotechnology  Researcher  2016 - 2019  226 
10.  12728  PhD Aleš Podgornik  Chemical engineering  Head  2016 - 2019  713 
11.  20681  PhD Barbara Podobnik  Pharmacy  Researcher  2016 - 2019  62 
12.  35857  Edo Prašnikar  Chemical engineering  Researcher  2016 - 2017 
13.  30336  PhD Bojan Šarac  Chemistry  Researcher  2016 - 2019  105 
14.  39357  Helena Trnovec  Pharmacy  Researcher  2017 - 2019 
15.  37945  Jasmina Tušar  Biochemistry and molecular biology  Researcher  2016 - 2019  10 
16.  36120  Eva Zaletel  Biotechnology  Researcher  2017 - 2019  16 
Organisations (3)
no. Code Research organisation City Registration number No. of publicationsNo. of publications
1.  0103  University of Ljubljana, Faculty of Chemistry and Chemical Technology  Ljubljana  1626990  23,454 
2.  0258  Lek Pharmaceutical Company d.d.  Ljubljana  1732811  8,026 
3.  3030  Center of Excellence for Biosensors, Instrumentation and process Control  Ajdovščina  3660460  299 
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
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