The rapid growth of the biopharmaceutical industry has led to increasing demands on the protein production process. An important aspect is the yield of functional protein, which can be greatly affected by the choice of downstream purification. Purification based on acidic elution can be an issue for pH-sensitive proteins, since dramatic changes in pH can lead to protein aggregation and loss of function. The harsh, acidic elution conditions used in conventional purification of antibodies by Protein A affinity chromatography can thus be problematic. To address this, a calcium-dependent protein domain, called ZCa, has previously been developed for mild purification of antibodies, with elution close to physiological pH. Presented here are engineered variants of ZCa with novel affinity towards other biotherapeutics, which could also benefit from mild purification. Phage display selection, using a ZCa-based library, was applied to isolate promising ZCa-based binders against antigen-binding fragments, tissue plasminogen activator, and granulocyte colony stimulating factor, yet to be characterized. Additionally, three ZCa-based variants from a previous selection, with affinity for single-chain variable fragments (scFvs), have been identified and characterized. In a purification setup, they were shown to elute the scFv protein at neutral pH in a calcium-dependent manner. The reported results demonstrate that novel affinity can be introduced to the ZCa domain, while maintaining the calcium-dependent behavior that enables gentle purification. This offers a strategy for broadening the range of proteins that can be purified under mild conditions, with the benefit of reducing protein aggregation and thus increasing the yield of functional protein. / Den snabba tillväxten inom bioläkemedelsindustrin har lett till ökade krav på processen för proteinproduktion. En viktig aspekt är utbytet av funktionellt protein, där valet av reningsmetod kan ha stor påverkan. Proteinrening med syraeluering kan utgöra ett problem för pH-känsliga proteiner, då stora förändringar i pH kan leda till aggregering och försämrad funktionalitet. Det låga pH som används för eluering i den traditionella reningen av antikroppar med Protein A-baserad affinitetskromatografi kan därmed vara problematiskt. Som ett svar på detta har en kalciumberoende proteindomän, vid namn ZCa, tidigare utvecklats för mild rening av antikroppar med eluering nära fysiologiskt pH. I det här arbetet presenteras nya varianter av ZCa som modifierats för att binda till andra bioläkemedel, vilka också skulle kunna gynnas av mild proteinrening. Fagdisplay av ett ZCa-baserat bibliotek har applicerats för att isolera lovande ZCa-baserade bindare mot antikroppsfragment (Fab), vävnadsplasminogenaktivator och granulocytkolonistimulerande faktor, vilka ännu inte karaktäriserats. Utöver detta identifierades och karaktäriserades tre ZCa-baserade varianter från en tidigare selektion, med affinitet för enkelkedjiga antikroppsfragment (scFv). Då dessa varianter utvärderades för rening visade alla på kalciumberoende eluering av scFv vid neutralt pH. Det här demonstrerar att ny affinitet kan introduceras till ZCa-domänen, där det kalciumberoende beteende som möjliggör mild proteinrening bevaras. Detta erbjuder en strategi för att utöka antalet proteiner som kan renas under milda kalciumberoende förhållanden, vilket med fördel kan minska aggregering och därmed öka utbytet av funktionellt protein.

ZCa is an engineered three-helical affinity protein that through replacement of the loop between helix two and three with an EF-hand motif has a calcium-dependent binding to its target. By using the difference of calcium concentration in the plasma and the cell along with the favorable properties of ZCa, the interest in creating therapeutics toward different diseases based on the ZCa scaffold has come about. In this project novel variants from a phage display library based on the ZCa scaffold are characterized and their ability to bind to their intended target is evaluated. The targets used in this project are IgE for its involvement in allergic reactions and IL-23 for its ability to cause autoimmune diseases. Through several different analysis methods this project shows that novel variants towards different targets are capable of calcium-dependent target binding.

With an expanding life-science sector and growing production of recombinant proteins, the need for efficient downstream processing is increasing. Certain proteins are sensitive to the harsh conditions often used in protein purification, such as low pH, which can result in aggregation and denaturation. ZCa is a domain derived from Protein A that can be used for calcium-dependent purification of antibodies without the need for acidic pH. Based on this domain, the CaRA library has been constructed, which targets other therapeutic proteins than human antibodies. Four of the proteins isolated from the CaRA library, namely CaRA_scFv_1, CaRA_scFv_2, CaRA_G-CSF_1 and CaRA_G-CSF_3, are presented here for the purification of single chain variable fragment and granulate colony stimulating factor. The four proteins were produced as monomers, trimers and hexamers in an attempt to increase the binding capacity and attached to a matrix for purification using site-specific coupling. The successful binders CaRA_scFv_1 and CaRA_scFv_2 showed high affinity for their target protein scFv and were able to selectively capture an increased number of molecules through multimerization. Calcium-dependent binding was demonstrated by elution at neutral pH using the calcium chelator citrate, thus concluding that these multimerized CaRA variants can be used to considerably increase the efficiency in scFv purification while providing excellent purity and significantly reducing the risk of aggregation.