Method development for large molecules IEX separations

Liquid chromatography method development is defined by several key steps followed to end up with a suitable analytical process. Generally, one determines the goal of the procedure, sets out the approach, and after the practical development, moves on to prevalidation and validation of the experiments, while documenting all the actions. The case of ion-exchange chromatography (IEX) separations is no different; however, this chapter focuses on practical aspects of the method development specifically related to two class of large molecule biologics: proteins (i.e., antibodies and related products) and nucleic acids (i.e., mRNAs). Such a focus stems from the current momentum behind the development of these types of therapeutics in the biopharmaceutical industry and is aligned with our experience. Owing to their intrinsically charged nature, biopolymers are ideal substrates for IEX. Sequence and/or posttranslational modification caused differences in overall (effective or accessible) net charge and its distribution allows for an efficient fractionation of the biomolecules based on their electrostatic interactions with a charged stationary phase. Due to its usual mild and nondenaturing elution mode, it can routinely be used in many stages of the biomolecule lifecycle, starting from capture, intermediate purification to final polishing steps. Such at scale processes are discussed in detail in further chapters, while here we focus on elaborating general principles regarding the analytical scale method development.