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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 antibod, Denna avhandling grundar sig i proteiner, vilka är nödvändiga för vår överlevnad och alla former av liv. De är involverade i nästintill alla processer i kroppen och gör allt från att transportera syre till att bryta ner maten vi äter. Ett av de mest välkända proteinerna är antikroppen, som bär på den tunga uppgiften att skydda oss från smittämnen såsom bakterier och virus, som en del av vårt immunförsvar. Vi kan bilda antikroppar mot i stort sett vilka inkräktare som helst, vilket leder till att andra delar av immunförsvaret i sin tur kan förstöra smittämnet. Förmågan att skapa antikroppar som binder starkt till många olika molekyler på dessa smittämnen har utnyttjats inom bland annat medicinsk forskning. Många av de så kallade biologiska läkemedlen utgörs av antikroppar eller fragment av antikroppar, vilka erbjuder många fördelar gentemot traditionella syntetiska läkemedel. Ett exempel är färre oönskade biverkningar på grund av mer målinriktade specifika interaktioner. Marknaden för dessa biologiska läkemedel är ständigt växande, men är emellertid begränsad av betydligt mer kostsamma produktionsmetoder och högre priser jämfört med andra typer av läkemedel. Antikroppar är dock bara en grupp av alla proteiner som kan binda till någon typ av molekyl. Intressant nog finns det, på ytan av flera typer av bakterier, proteiner som har utvecklats till att binda specifika delar av antikroppar. På så sätt kan bakterierna gömma sig från immunförsvaret och förhindra antikropparna från att tillkalla hjälp, och de klarar sig därmed längre i våra kroppar. Ett av dessa bakteriella ytproteiner kallas för Protein A, och detta protein spelar en central roll i denna avhandling. Det används vanligtvis i tillverkningsprocessen för antikroppar, i steget där antikropparna ska renas, på grund av förmågan att binda till dessa. Rening av proteiner är nödvändigt då även andra proteiner bildas under tillverkningen. Proteiner kan tillverkas med hjälp av särskilda celler som har modifierats geno, QC 2022-03-08

In this study, we demonstrated the first, to our knowledge, integrated continuous bioprocess (ICB) designed for the production of acid-sensitive monoclonal antibodies, prone to aggregate at low pH, on pilot scale. A high cell density perfusion culture, stably maintained at 100 × 106 cells/ml, was integrated with the downstream process, consisting of a capture step with the recently developed Protein A ligand, ZCa; a solvent/detergent-based virus inactivation; and two ion-exchange chromatography steps. The use of a mild pH in the downstream process makes this ICB suitable for the purification of acid-sensitive monoclonal antibodies. Integration and automation of the downstream process were achieved using the Orbit software, and the same equipment and control system were used in initial small-scale trials and the pilot-scale downstream process. High recovery yields of around 90% and a productivity close to 1 g purified antibody/L/day were achieved, with a stable glycosylation pattern and efficient removal of impurities, such as host cell proteins and DNA. Finally, negligible levels of antibody aggregates were detected owing to the mild conditions used throughout the process. The present work paves the way for future industrial-scale integrated continuous biomanufacturing of all types of antibodies, regardless of acid stability., QC 20220523

Therapeutic antibodies are at the forefront of modern medicine where high purity, which is typically obtained by Protein A-based affinity purification, is of utmost importance. In this chapter, we present a method for neutral and selective purification of antibodies by utilizing an engineered affinity ligand, ZCa, derived from Protein A. This domain displays a calcium-dependent binding of antibodies and has been multimerized and immobilized to a chromatography resin to achieve an affinity matrix with high binding capacity. IgG antibodies can be eluted from the tetrameric ZCa ligand at pH 7 with the addition of EDTA, or at pH 5.5 with EDTA for purification of monoclonal IgG1, which is significantly milder than the low pH (3–4) required in conventional Protein A affinity chromatography. Here, a protocol for selective capture of IgG with elution at neutral pH from a ZCa tetramer ligand immobilized on a chromatography resin is described., QC 20210217

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., QC 20210609

The proteins secreted by human cells (collectively referred to as the secretome) are important not only for the basic understanding of human biology but also for the identification of potential targets for future diagnostics and therapies. Here, we present a comprehensive analysis of proteins predicted to be secreted in human cells, which provides information about their final localization in the human body, including the proteins actively secreted to peripheral blood. The analysis suggests that a large number of the proteins of the secretome are not secreted out of the cell, but instead are retained intracellularly, whereas another large group of proteins were identified that are predicted to be retained locally at the tissue of expression and not secreted into the blood. Proteins detected in the human blood by mass spectrometry-based proteomics and antibody-based immuno-assays are also presented with estimates of their concentrations in the blood. The results are presented in an updated version 19 of the Human Protein Atlas in which each gene encoding a secretome protein is annotated to provide an open-access knowledge resource of the human secretome, including body-wide expression data, spatial localization data down to the single-cell and subcellular levels, and data about the presence of proteins that are detectable in the blood.

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., QC 20191008

In dieser Arbeit wurde der bakterieninduzierte Zelltod in Phagozyten und dessen Auswirkung auf eine anti-bakterielle Immunantwort untersucht. Der erste Teil der Arbeit widmete sich dem Zelltod in Makrophagen nach der Aufnahme von E.coli Bakterien. Hierbei zeigte sich, dass nach einem effektiven Verdau der Bakterien - bereits nach 4-6 Stunden - ein Teil der Phagozyten ihre Plasmamembran-Integrität verliert, ohne Hinweise auf apoptotischen Zelltod zu zeigen: Die Zellen sterben durch Nekrose. Erst nach 16-20 Stunden treten zusätzlich auch apoptotische Zellen auf. Nekrose tritt dabei unabhängig von Apoptose auf: Bei Verwendung hitzeinaktivierter Bakterien (die nicht mehr phagozytiert werden), Blockierung von Caspasen durch z-VAD.fmk oder Überexpression von Bcl-2 konnte zwar Apoptose blockiert werden, die Zellen starben aber dennoch durch Nekrose. Diese Form des Zelltods, die sonst oft nur unter extremen in vitro Bedingungen, bei schwerer Zell-Traumatisierung oder Blockierung von Apoptose auftritt, scheint somit ein relevanter Teil der zellulären Antwort von Phagozyten auf den Kontakt zu Bakterien oder bakteriellen Bestandteilen zu sein. Im zweiten Teil der Arbeit wurde die Bedeutung des nekrotischen Zelltods für eine Aufnahme der toten Zellen durch dendritische Zellen evaluiert. Hierbei konnten wir feststellen, dass der Zelltod durch Nekrose ausreichend ist für eine schnelle und effektive Aufnahme durch phagozytierende Zellen: Es konnten keine großen Diskrepanzen in der Effektivität der Phagozytose apoptotischer und nekrotischer Zellen festgestellt werden. Wie bereits für apoptotische Zellen bekannt, könnte auch bei der Aufnahme nekrotischer Zellen eine – wenn auch vielleicht nur passive – Exposition von Phosphatidylserin eine Rolle spielen. Der früh auftretende nekrotische Zelltod führt somit zu einer schnellen Weitergabe von bakteriellen Antigenen an dendritische Zellen. Diese nehmen eine Schlüsselstellung im Immunsystem ein, da sie sowohl Toleranz als auch die Entwic, In this work we analysed the role of bacteria-induced cell death in phagoycytes for the development of an anti-bacterial immune response. In the first part of this thesis we investigated the cell death in macrophages after ingestion of E.coli bacteria. We could demonstrate that, having succesfully digested the bacteria after 4-6 hours, some of the phagocytes loose plasma membrane integritiy without showing morphological hallmarks of apoptotic cells: These cells die by necrosis. After 16-20 hours, we additionally found apoptotic cells. Necrosis appeared to be a process separate from apoptosis: Using heat-inactivated bacteria (which are poorly taken up my macrophages), inhibiting caspases by z-VAD.fmk or overexpressing bcl-2, we were able to block apoptosis – regardlessly the cells were dying by necrosis. This form of cell death, otherwise seen only under extreme conditions in vitro (e.g. physical injury), seems to be a relevant part of the cellular response of phagoytes after contact to bacteria. In the second part of the thesis we evaluated the impact of the necrotic cell death for the uptake of dead cells by dendritic cells. We could demonstrate that cell death by necrosis is sufficient for a quick and efficient clearance by phagocytes: We did not find any difference in the efficiency of phagocytosis of apoptotic or necrotic cells. As already known for the uptake of apoptotic cells, an exposure of phosphatidylserine could also be the trigger for phagocytosis of necrotic cells. Cell death by necrosis is occuring early after ingestion of bacteria. As the dead cells are efficiently taken up by dendritic cells, bacterial antigens will end up in a potent antigen-presenting cell, and this process may thus impact on the generation of an adaptive immune response. These dendritic cells have a key position for the development of an immune response: They have the capability to induce either tolerance or a specific immune response. Whether tolerance or an immune response is tr

In dieser Arbeit wurde der bakterieninduzierte Zelltod in Phagozyten und dessen Auswirkung auf eine anti-bakterielle Immunantwort untersucht. Der erste Teil der Arbeit widmete sich dem Zelltod in Makrophagen nach der Aufnahme von E.coli Bakterien. Hierbei zeigte sich, dass nach einem effektiven Verdau der Bakterien - bereits nach 4-6 Stunden - ein Teil der Phagozyten ihre Plasmamembran-Integrität verliert, ohne Hinweise auf apoptotischen Zelltod zu zeigen: Die Zellen sterben durch Nekrose. Erst nach 16-20 Stunden treten zusätzlich auch apoptotische Zellen auf. Nekrose tritt dabei unabhängig von Apoptose auf: Bei Verwendung hitzeinaktivierter Bakterien (die nicht mehr phagozytiert werden), Blockierung von Caspasen durch z-VAD.fmk oder Überexpression von Bcl-2 konnte zwar Apoptose blockiert werden, die Zellen starben aber dennoch durch Nekrose. Diese Form des Zelltods, die sonst oft nur unter extremen in vitro Bedingungen, bei schwerer Zell-Traumatisierung oder Blockierung von Apoptose auftritt, scheint somit ein relevanter Teil der zellulären Antwort von Phagozyten auf den Kontakt zu Bakterien oder bakteriellen Bestandteilen zu sein. Im zweiten Teil der Arbeit wurde die Bedeutung des nekrotischen Zelltods für eine Aufnahme der toten Zellen durch dendritische Zellen evaluiert. Hierbei konnten wir feststellen, dass der Zelltod durch Nekrose ausreichend ist für eine schnelle und effektive Aufnahme durch phagozytierende Zellen: Es konnten keine großen Diskrepanzen in der Effektivität der Phagozytose apoptotischer und nekrotischer Zellen festgestellt werden. Wie bereits für apoptotische Zellen bekannt, könnte auch bei der Aufnahme nekrotischer Zellen eine – wenn auch vielleicht nur passive – Exposition von Phosphatidylserin eine Rolle spielen. Der früh auftretende nekrotische Zelltod führt somit zu einer schnellen Weitergabe von bakteriellen Antigenen an dendritische Zellen. Diese nehmen eine Schlüsselstellung im Immunsystem ein, da sie sowohl Toleranz als auch die Entwic, In this work we analysed the role of bacteria-induced cell death in phagoycytes for the development of an anti-bacterial immune response. In the first part of this thesis we investigated the cell death in macrophages after ingestion of E.coli bacteria. We could demonstrate that, having succesfully digested the bacteria after 4-6 hours, some of the phagocytes loose plasma membrane integritiy without showing morphological hallmarks of apoptotic cells: These cells die by necrosis. After 16-20 hours, we additionally found apoptotic cells. Necrosis appeared to be a process separate from apoptosis: Using heat-inactivated bacteria (which are poorly taken up my macrophages), inhibiting caspases by z-VAD.fmk or overexpressing bcl-2, we were able to block apoptosis – regardlessly the cells were dying by necrosis. This form of cell death, otherwise seen only under extreme conditions in vitro (e.g. physical injury), seems to be a relevant part of the cellular response of phagoytes after contact to bacteria. In the second part of the thesis we evaluated the impact of the necrotic cell death for the uptake of dead cells by dendritic cells. We could demonstrate that cell death by necrosis is sufficient for a quick and efficient clearance by phagocytes: We did not find any difference in the efficiency of phagocytosis of apoptotic or necrotic cells. As already known for the uptake of apoptotic cells, an exposure of phosphatidylserine could also be the trigger for phagocytosis of necrotic cells. Cell death by necrosis is occuring early after ingestion of bacteria. As the dead cells are efficiently taken up by dendritic cells, bacterial antigens will end up in a potent antigen-presenting cell, and this process may thus impact on the generation of an adaptive immune response. These dendritic cells have a key position for the development of an immune response: They have the capability to induce either tolerance or a specific immune response. Whether tolerance or an immune response is tr

QC 20220316

QC 20220317