Qian-Hong Wan - Mixed-Mode Chromatography: Principles, Methods, and Applications

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Since the FDA approved the first monoclonal antibody drug in 1986, the therapeutic antibody drug market has experienced explosive growth. New antibody drugs have been approved to treat a variety of human diseases, including many cancers, autoimmune, metabolic, and infectious diseases. The manufacture of therapeutic antibody drugs usually involves a lengthy and expensive purification process based on protein A affinity chromatography. However, protein A chromatography has two main problems: ligands are easily cleaved by proteases present in cell lysates, and purified antibodies tend to aggregate under the low pH conditions required for elution. Therefore, considerable efforts have been made to mitigate these adverse effects.

Mixed-mode chromatography has emerged to simulate protein A chromatography for antibody purification. Synthetic ligands with multiple functional groups are used to replace natural biomolecules and bind to target molecules through a combination of hydrophobic, electrostatic, and hydrogen bonding interactions. Binding affinity and selectivity can be optimized by following the principles of molecular recognition, such as multivalency, complementarity, and geometric fitting. This book tends to introduce the principles, methods, and applications of mixed-mode chromatography. It starts from a historical perspective covering the development of chromatography in general and mixed-mode chromatography in particular. Then, theoretical considerations are provided, focusing on the mass transfer and band broadening in the packed bed, and the different separation modes and packing materials are described in detail. The following chapters deal with ligand design, retention mechanisms, stationary phases, and mobile phases in mixed-mode chromatography. A large number of examples are used to explain and illustrate the method development and application in biopharmaceutical manufacturing.

I would like to thank Tianjin University for its continuous strong support and the Springer Nature book project team, especially Lewis Liu and Kavitha Jayakumar, for their suggestions during the writing of this book.

Chromatography is a technique that uses the difference in the partition coefficient of a compound between the stationary phase and the mobile phase to separate the mixture. The so-called stationary phase refers to the solid-phase carrier that is immobile during the separation process and its ligands loaded on the surface of the carrier. The mobile phase refers to the fluid that moves relative to the stationary phase during the separation process, which can be liquid or gas. According to the state of the fluid used, chromatography can be divided into three important branches: liquid chromatography, gas chromatography, and supercritical fluid chromatography. According to the type of interaction involved in the separation process, chromatography can be divided into normal phase chromatography, reverse-phase chromatography, ion exchange chromatography, affinity chromatography, and so on. According to the way the separation process is realized, chromatography can be divided into packed-column chromatography, open-tubular column chromatography, electrochromatography, thin-layer chromatography, continuous chromatography, multidimensional chromatography, and so on. According to the scale of application, chromatography can be divided into two major branches: analytical chromatography and preparative chromatography. According to the application field, chromatography can be divided into ion chromatography, chiral chromatography, protein chromatography, nucleic acid chromatography, and so on. Chromatography can also be used in conjunction with other analytical techniques such as mass spectrometry to achieve component separation and structural characterization of analytical samples in one run. In short, the chromatographic technology has a wide variety, convenience, flexibility, and adaptability, making it unique among many separation technologies.