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| 2. |
- Jönsson, Malin, et al.
(författare)
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CaRA – A multi-purpose phage display library for selection of calcium-regulated affinity proteins
- 2022
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Ingår i: New Biotechnology. - : Elsevier B.V.. - 1871-6784 .- 1876-4347. ; 72, s. 159-167
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Tidskriftsartikel (refereegranskat)abstract
- Protein activity regulated by interactions with metal ions can be utilized for many different purposes, including biological therapies and bioprocessing, among others. Calcium ions are known to interact with the frequently occurring EF-hand motif, which can alter protein activity upon binding through an induced conformational change. The calcium-binding loop of the EF-hand motif has previously been introduced into a small protein domain derived from staphylococcal Protein A in a successful effort to render antibody binding dependent on calcium. Presented here, is a combinatorial library for calcium-regulated affinity, CaRA, based on this domain. CaRA is the first alternative scaffold library designed to achieve novel target specificities with metal-dependent binding. From this library, several calcium-dependent binders could be isolated through phage display campaigns towards a set of unrelated target proteins (IgE Cε3-Cε4, TNFα, IL23, scFv, tPA, PCSK9 and HER3) useful for distinct applications. Overall, these monomeric CaRA variants showed high stability and target affinities within the nanomolar range. They displayed considerably higher melting temperatures in the presence of 1 mM calcium compared to without calcium. Further, all discovered binders proved to be calcium-dependent, with the great majority showing complete lack of target binding in the absence of calcium. As demonstrated, the CaRA library is highly capable of providing protein-binding domains with calcium-dependent behavior, independent of the type of target protein. These binding domains could subsequently be of great use in gentle protein purification or as novel therapeutic modalities.
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| 3. |
- Kanje, Sara, 1986-, et al.
(författare)
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Engineering of Protein A for improved purification of antibodies and Fc-fused proteins
- 2020
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Ingår i: Approaches to the Purification, Analysis and Characterization of Antibody-Based Therapeutics. - : Elsevier. ; , s. 35-54
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Bokkapitel (övrigt vetenskapligt/konstnärligt)abstract
- In industrial scale downstream processing of antibodies and Fc-fusion proteins, Protein A chromatography is the most commonly used purification method. As the treatments for many severe diseases shift toward biological drugs, in particular antibodies, the need for robust purification methods has increased. Since the 1970s, when Protein A debuted as an affinity ligand, the full protein and its domains have been extensively studied. Even if matrices based on unmodified Protein A historically met the process demands of industrial antibody manufacturing, there is room for improvement. The cleaning in place (CIP) methods required for safe reuse of the matrix in therapeutic protein purification pose a problem since the harsh conditions required can be harmful to the proteinaceous purification ligand. Further, the low pH used for elution can sometimes be detrimental to the target protein. Here, efforts to improve these features as well as capacity improvements for Protein A resins will be discussed.
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| 4. |
- Kanje, Sara, 1986-, et al.
(författare)
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Protein engineering allows for mild affinity-based elution of therapeutic antibodies
- 2018
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Ingår i: Journal of Molecular Biology. - : Elsevier. - 0022-2836 .- 1089-8638. ; 430:18, s. 3427-3438
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Tidskriftsartikel (refereegranskat)abstract
- Presented here is an engineered protein domain, based on Protein A, that displays a calcium-dependent binding to antibodies. This protein, ZCa, is shown to efficiently function as an affinity ligand for mild purification of antibodies through elution with ethylenediaminetetraacetic acid. Antibodies are commonly used tools in the area of biological sciences and as therapeutics, and the most commonly used approach for antibody purification is based on Protein A using acidic elution. Although this affinity-based method is robust and efficient, the requirement for low pH elution can be detrimental to the protein being purified. By introducing a calcium-binding loop in the Protein A-derived Z domain, it has been re-engineered to provide efficient antibody purification under mild conditions. Through comprehensive analyses of the domain as well as the ZCa–Fc complex, the features of this domain are well understood. This novel protein domain provides a very valuable tool for effective and gentle antibody and Fc-fusion protein purification
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| 5. |
- Scheffel, Julia
(författare)
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Calcium-dependent Affinity Domains for the Purification of Antibodies and Antibody Fragments
- 2022
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- 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.
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| 6. |
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| 7. |
- Scheffel, Julia, et al.
(författare)
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Calcium-dependent affinity ligands for the purification of antibody fragments at neutral pH
- 2023
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Ingår i: Journal of Chromatography A. - : Elsevier BV. - 0021-9673 .- 1873-3778. ; 1694, s. 463902-
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Tidskriftsartikel (refereegranskat)abstract
- The emerging formats of antibody fragments for biotherapeutics suffer from inadequate purification methods, delaying the advances of innovative therapies. One of the top therapeutic candidates, the single -chain variable fragment (scFv), requires the development of individual purification protocols dependent on the type of scFv. The available approaches that are based on selective affinity chromatography but do not involve the use of a purification tag, such as Protein L and Protein A chromatography, require acidic elution buffers. These elution conditions can cause the formation of aggregates and thereby greatly com-promise the yield, which can be a major problem for scFvs that are generally unstable molecules. Due to the costly and time-consuming production of biological drugs, like antibody fragments, we have en-gineered novel purification ligands that elute the scFvs in a calcium-dependent manner. The developed ligands are equipped with new, selective binding surfaces and were shown to efficiently elute all cap-tured scFv at neutral pH with the use of a calcium chelator. Further, two of three ligands were proven not to bind to the CDRs of the scFv, indicating potential for use as generic affinity ligands to a range of different scFvs. Multimerization and optimization of the most promising ligand led to a 3-fold increase in binding capacity for the hexamer compared to the monomer, in addition to highly selective and efficient purification of a scFv with > 95% purity in a single purification step. This calcium-dependent ligand could revolutionize the scFv industry, greatly facilitating the purification procedure and improving the quality of the final product.
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| 8. |
- Scheffel, Julia, et al.
(författare)
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Design of an integrated continuous downstream process for acid-sensitive monoclonal antibodies based on a calcium-dependent Protein A ligand
- 2022
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Ingår i: Journal of Chromatography A. - : Elsevier BV. - 0021-9673 .- 1873-3778. ; 1664, s. 462806-462806
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Tidskriftsartikel (refereegranskat)abstract
- Monoclonal antibodies (mAb) are used as therapeutics and for diagnostics of a variety of diseases, and novel antibodies are continuously being developed to find treatments for new diseases. Therefore, the manufacturing process must accommodate a range of mAb characteristics. Acid-sensitive mAbs can severely compromise product purity and yield in the purification process due to the potential formation of aggregates. To address this problem, we have developed an integrated downstream process for the purification of pH-sensitive mAbs at mild conditions. A calcium-dependent Protein A-based ligand, called ZCa, was used in the capture step in a 3-column periodic counter-current chromatography operation. The binding of ZCa to antibodies is regulated by calcium, meaning that acidic conditions are not needed to break the interaction and elute the antibodies. Further, the virus inactivation was achieved by a solvent/detergent method, where the pH could remain unchanged. The polishing steps included a cation and an anion exchange chromatography step, and screening of the capture and polishing steps was performed to allow for a seamless integration of the process steps. The process was implemented at laboratory scale for 9 days obtaining a high yield, and a consistently pure drug substance, including high reduction values of the host cell protein and DNA concentrations, as well as aggregate levels below the detection limit, which is attributed to the mild conditions used in the process.
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| 9. |
- Scheffel, Julia, et al.
(författare)
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Highly selective Protein A resin allows for mild sodium chloride-mediated elution of antibodies
- 2021
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Ingår i: Journal of Chromatography A. - : Elsevier BV. - 0021-9673 .- 1873-3778. ; 1637
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Tidskriftsartikel (refereegranskat)abstract
- The manufacturability of therapeutic monoclonal antibodies is limited by the harsh conditions that antibodies are subjected to during the purification procedure, which in turn restricts the development of novel acid-sensitive antibodies. The gold standard for antibody purification, Protein A affinity chromatography, offers the selective capture of antibodies with great yields, but also poses a threat to the quality of the antibodies. Antibodies and Fc-fusion proteins risk forming aggregates as a consequence of the acidic elution from the Protein A ligands, compromising the potency and safety of the drug. Here, we present a novel, mild purification strategy based on a calcium-dependent ligand derived from Protein A, called Z(c)(a). Antibodies captured on a high-capacity tetrameric Z(ca) resin in the presence of calcium can be eluted by removing the calcium ions through the addition of a chelator, and we describe the strive to find a sustainable alternative to the previously applied chelator EDTA. The naturally occurring chelator citrate is shown to seamlessly replace EDTA. Further buffer optimization reveals that the elution can be considerably improved by increasing the conductivity through the addition of 300 mM sodium chloride, leading to a very concentrated eluate. Remarkably, merely sodium chloride at a concentration of 50 mM is proven to be sufficient for calcium-dependent antibody release in a cost-efficient manner. Antibodies of subclasses IgG2 and IgG4 are eluted with sodium chloride at neutral pH and IgG1 at pH 6, due to varying affinities for the tetrameric Z(Ca), ranging between 90-780 nM. The mild elution of an IgG4 antibody eliminated the formation of aggregates, which constituted as much as 34% of all eluted antibody from MabSelect SuRe at pH 3. This novel purification strategy thus combines the valuable qualities of a Protein A resin, by providing high selectivity and a recovery of 88-99%, with an exceptionally mild elution step similar to ion-exchange chromatography, rendering considerably more functional antibody.
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| 10. |
- Scheffel, Julia, et al.
(författare)
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Optimization of a calcium-dependent Protein A-derived domain for mild antibody purification
- 2019
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Ingår i: mAbs. - : TAYLOR & FRANCIS INC. - 1942-0862 .- 1942-0870.
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Tidskriftsartikel (refereegranskat)abstract
- As reported here, we developed and optimized a purification matrix based on a Protein A-derived domain, Z(Ca), displaying calcium-dependent antibody binding. It provides an alternative to the acidic elution conditions of conventional Protein A affinity chromatography for purification of sensitive antibodies and other Fc-based molecules. We describe the multimerization of Z(Ca) to generate a chromatography resin with higher binding capacity. The highest order multimeric variant, Z(Ca)TetraCys, demonstrated a considerably high dynamic binding capacity (35 mg IgG/ml resin) while preserving the specificity for IgG. High recovery was obtained and host cell protein and DNA content in purified fractions proved to be comparable to commercial MabSelect SuRe and MabSelect PrismA. Various elution conditions for use of this domain in antibody purification were investigated. The purification data presented here revealed variations in the interaction of different subclasses of human IgG with Z(Ca)TetraCys. This resulted in diverse elution properties for the different IgGs, where complete elution of all captured antibody for IgG2 and IgG4 was possible at neutral pH. This optimized protein ligand and the proposed purification method offer a unique strategy for effective and mild purification of antibodies and Fc-fusion proteins that cannot be purified under conventional acidic elution conditions due to aggregation formation or loss of function.
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