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In previous chapters, the simple case of two-component systems, i.e., one single solute crystallizing in a solvent (or solvent mixture), was mainly considered. However, because crystallization is most often employed as a purification process, numerous impurities resulting from the upstream part of the process are necessarily present in solution, such as buffer components, residual reactants, intermediates, or by-products. These impurities may affect the crystallization process and the resulting crystal properties, even at low concentration. Besides, additives are sometimes placed intentionally in solution with a view to tuning certain crystal properties. The mechanisms by which impurities and additives dissolved in solution affect the crystallization process can be rationalized in a common framework, so they will both be placed under the umbrella of foreign species in this chapter. The species to be purified will instead be referred to as the host species.
This chapter provides a general introduction on biopharmaceutical processes. The first part presents a brief description of the single unit operations typically encountered during the manufacturing process, including cell culture, purification, viral inactivation, formulation. The second part addresses the potential benefits of continuous technologies in the biopharmaceutical industry both for the upstream and downstream parts of the process, focusing on perfusion bioreactors and continuous counter-current chromatographic processes, respectively. The chapter finishes with a discussion on process integration.
This chapter focusses on chromatographic processes for protein capture. The first part provides a detailed description of the mechanistic phenomena involved in protein capture, which can be regarded as a non-competitive binary system protein/impurity, where the protein of interest strongly interacts with the chromatographic medium. Several multi-column counter-current processes allowing the implementation of the sequence of operations necessary in protein capture (load, wash, recovery, regeneration) in a continuous manner are presented in the second part of the chapter. Two process design approaches for multi-column capture processes are provided: an empirical one, which allows to obtain a first guess of the operating conditions from breakthrough curves experiments, and a model-based one, which allows a more rigorous determination of the process variables. The model-based approach is used to compare the performance of multi-column and single-column processes.
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 provides fundamentals on protein chromatography. Different chromatographic media are described in terms of the solute-surface interactions that can be exploited to achieve the desired separation. Then, mechanistic models are presented to describe the three key physical phenomena involved in protein chromatography, namely the thermodynamics of fluid-solid equilibrium, the hydrodynamics and the kinetics of mass transfer. Simple methods to estimate model parameters are introduced as well as short-cut methods to design chromatographic processes. Although the main goal of this chapter is to bring theoretical basics about the modelling of protein chromatography, it is complemented by numerous experimental results for illustrative and pedagogical purposes.
This chapter provides basic concepts about multi-column counter-current chromatography. The benefit of counter-current contact in separation processes is demonstrated considering cascades of equilibrium stages. Based on this, the true moving bed (TMB) process and the simulated moving bed (SMB) process are introduced. Then, the design space of the TMB and SMB processes leading to a complete separation is identified, starting with simplistic systems and progressively introducing more complex effects, namely non-linear adsorption isotherms and mass transfer limitations. Finally, two process design approaches for multi-column chromatographic processes are presented: an empirical one, which allows to obtain a first guess of the operating conditions from a single-column experiment, and a model-based one, which allows a more rigorous determination of the process variables.
This chapter focusses on the polishing steps encountered during protein purification by chromatography. The first part provides a detailed description of the mechanistic phenomena at stake, whose complexity may greatly vary from one case to the other depending on the number and type of impurities, the selectivity for the target protein as well as non-linear and competitive effects. Several multi-column counter-current processes allowing the implementation of the polishing steps in a continuous manner are presented in the second part of the chapter. Modifications of the classical SMB process introduced to better cope with the specific needs of the biopharmaceutical industry are discussed. These modifications primarily aim at recovering more than two fractions and at implementing modifier concentration gradients. Two process design approaches for multi-column processes are presented: an empirical one, which provides a first guess of the operating conditions from single-column experiments, and a model-based one, which allows a more rigorous determination of the process variables. The model-based approach is used to compare the performance of multi-column and 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.
This innovative reference provides a coherent and critical view on the potential benefits of a transition from batch to continuous processes in the biopharmaceutical industry, with the main focus on chromatography. It also covers the key topics of protein stability and protein conjugation, addressing the chemical reaction and purification aspects together with their integration. This book offers a fine balance between theoretical modelling and illustrative case studies, between fundamental concepts and applied examples from the academic and industrial literature. Scientists interested in the design of biopharmaceutical processes will find useful practical methodologies, in particular for single-column and multi-column chromatographic processes.
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