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Calcium-dependent Affinity Domains for the Purification of Antibodies and Antibody Fragments

Time: Fri 2022-04-01 13.00

Location: Kollegiesalen, Brinellvägen 8, Stockholm

Language: English

Subject area: Biotechnology

Doctoral student: Julia Scheffel , Proteinteknologi, Hober lab

Opponent: Professor Alois Jungbauer, Universität für Bodenkultur Wien

Supervisor: Professor Sophia Hober, Centrum för Bioprocessteknik, CBioPT, Science for Life Laboratory, SciLifeLab, Albanova VinnExcellence Center for Protein Technology, ProNova, Proteinteknologi

QC 2022-03-08


Antibodies are essential proteins in both our bodies and biotechnological research, and hold outstanding therapeutic value. The market for antibody-based therapeutics has grown exponentially during the last decades, owing to several advantages over small molecule drugs, such as fewer undesirable side effects associated with a higher target specificity. To keep up with the increasing amounts of antibodies that are on demand, emphasis has been on the optimization of upstream processes for antibody production while the advances in downstream processing and the purification of antibodies have been limited. In the downstream process, the gold standard for the primary capture step is Protein A affinity chromatography. However, elution of the antibodies from the Protein A ligand is accomplished at a low pH, which can lead to antibody aggregation and impaired biological activity. The purification procedure therefore hinders the development of new antibodies that are acid-sensitive, despite promising therapeutic potential, and may pose a threat to the increasingly popular bispecific antibodies that tend to be more aggregation prone. Further, acidic elution conditions may be an even bigger concern in the purification of antibody fragments, which also represent promising therapeutic candidates, providing several advantages over full-length antibodies in certain applications.

The work in this thesis aimed to enable the purification of a more diverse group of antibodies and antibody fragments, regardless of their stability in a highly acidic environment. Efforts were also made to reduce the high antibody manufacturing costs to make these antibody therapeutics more easily accessible to patients. In order to elute the antibodies in the Protein A capture step under milder conditions, the protein ligand ZCa was developed. ZCa was isolated from a phage display library based on a Protein A domain with a grafted calcium-binding loop, and permits the calcium-dependent elution of antibodies at close to neutral pH by adding sodium chloride. The domain provides the foundation for this thesis, and was extensively optimized to achieve a high-capacity resin and an inexpensive purification strategy, yielding exceptional recoveries of pure antibody. Most importantly, ZCa was able to entirely prevent the formation of aggregates of an antibody through mild elution. Moreover, the optimized ZCa resin was applied in an integrated continuous biomanufacturing process, designed to entirely avoid the use of low pH. The implementation of the process at pilot scale for 17 days demonstrated the robustness of the novel resin along with many other promising process attributes. High productivity and yields were obtained in addition to negligible aggregate formation and low levels of residual DNA and host cell proteins, comparable to established processes.

Finally, this thesis presents a combinatorial library for calcium-regulated affinity constructed from ZCa, from which numerous binders with novel target specificities were isolated. The calcium-dependent binders to single chain variable fragments (scFvs) could be used to selectively capture and elute scFv at neutral pH. Binding analysis and optimization efforts indicated potential as a platform approach for the mild and efficient purification of different scFvs.

In conclusion, the purification strategies proposed in this thesis considerably improve the purification of antibodies and scFvs, and may encourage the future innovation of a wider range of antibody-based therapeutics. The continuous process supports the applicability of ZCa in a state-of-the-art commercial manufacturing process, and contributes to the more efficient manufacturing of antibodies, which can make them more affordable and accessible to the patients in need.