This chapter deals with protein aggregation, which is a key issue in biopharmaceutical processes. Several experimental techniques to characterize the aggregate size and content are presented and fundamentals on the kinetic modelling of aggregation mechanisms are provided. The impact of operating conditions on the aggregation rate is reviewed and the steps critical for aggregate formation in biopharmaceutical processes are identified. Finally, methods aiming at reducing the aggregate content are proposed. These methods focus either on improving protein stability or on removing the formed aggregates. The former can be achieved by synthesizing aggregation-resistant proteins, tuning operating conditions, or designing processes with a shorter residence time (e.g. perfusion bioreactors or counter-current chromatography). The latter method is mainly achieved by filtration and chromatography. In particular, the simulated moving bed process is shown to be very advantageous for aggregate removal with size exclusion chromatography: it allows improving productivity, decreasing eluent consumption and increasing the outlet protein concentration as compared to single-column processes.

This chapter deals with protein bioconjugation, which has emerged as an attractive technique to enhance therapeutic properties of protein-based drugs (e.g. in-vivo circulation, potency). The first part of this chapter aims at answering questions regarding the coupling chemistry and the modelling of the kinetics of bioconjugation. Then, the purification of conjugated proteins is addressed. In particular, chromatography is presented as a technique to purify conjugated proteins according to their extent of conjugation on the one hand and to the position of the conjugated groups on the other hand. Moreover, continuous technologies for both the conjugation reaction and the purification by chromatography are discussed. Finally, the potential benefits from the integration between the reaction and the purification steps in bioconjugation processes are examined. This chapter combines theoretical aspects, model simulations and experimental results from practical examples, for instance dealing with PEGylated proteins and antibody-drug conjugates.