Continuous Reaction and Separation of IgG

Abstract

Biopharmaceuticals are the fastest growing eld in the pharmaceutical sector. Among them, sca olds derived from the classical biopharmaceuticals, such as antibody drug conjugates, antibody fragments and PEGylated proteins, gain increasing attention. These are often derived from an already available product with the aim to give it new functionalities. This reaction is done in a step subsequent to the protein expression in cells. While continuous integrated biomanufacturing is an intensely discussed topic for the production of classical biopharmaceuticals, these alternative sca olds are left aside. Nevertheless, their production would bene t from the advantages of intensi ed processing, like robustness and improved economic performance. In this work we have a look into three di erent case studies dealing with the integration of the reaction and the subsequent separation. In the rst part, literature on the reaction of proteins with functionalized PEG is reviewed. PEGylation increases the blood circulation half-life time, which is an important factor for the drug administration frequency and ultimately for treatment convenience. The standard process consists of a batch reactor followed by a chromatographic puri cation. Innovative processes are introduced that have the aim to increase the selectivity of the reaction. This is relevant as the protein is a valuable reagent and a conversion to only the target product decreases the overall costs. Several process implementations with the reaction both in liquid phase and on a solid support are described and their assets and disadvantages are discussed. The second part is about the conjugation of an antibody with a small molecule. The idea is to have a model system for the synthesis of an antibody drug conjugate, which are potent drugs combining cytotoxic small molecules with the targeting abilities of antibodies. Here, a dye was used as conjugation partner due to safety and analytics. The dye has a reactive succinimidyl ester group, which conjugates primary amines on the surface of the antibody. The reaction was rst studied in both liquid and adsorbed state. With the results, a reactive multi-column countercurrent solvent gradient process was performed, in which the antibody reacts to the mono-conjugate, and simultaneously the product is separated from unconjugated protein. The process requires three columns. On one of these three, the reaction is carried out at all times. The process was run for 23 hours in total and 17 hours in a cyclic steady state. In the third part, a study on the digestion of an antibody into its Fab and Fc fragments is presented. Fab is a valuable fragment as it constitutes the part of the antibody with targeting capabilities. A tubular reactor containing papain bound on agarose was used for the fragmentation. The reaction conditions were studied at di erent temperatures and residence times. During the thesis, it was found that Fab is further digested, leading to smaller fragments. The reactor operation was optimized accordingly. Furthermore, the reaction bu er conditions lead to break down of the products. Hence, the reactor was integrated with the puri cation in order to immediately recover the product form these conditions. An adapted multi-column counter-current solvent gradient puri cation process was used for this purpose. It contains an additional column compared to the two column process in order to collect the reaction product continuously. A yield of 50 % Fab was achieved at a purity of 90 %. The use of multi-column processes to combine reaction and separation are a valuable addition to the repertoire of intensi ed processes, in particular by allowing higher safety and automation.