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Two major problems in monoclonal antibody biopharmaceuticals are aggregation of and the presence of variants of active pharmaceutical ingredients. These product-related substances can have different efficacies than the main product and may cause serious side effects, for example, anti-drug-antibody formation. Protein aggregates are mostly the consequence of suboptimal production, purification, or handling conditions (e.g., temperature, pH). In the purification of antibodies, a protein-affinity separation is generally the first step. Affinity chromatography on protein A or G columns typically yields a purity of more than 95% in a single step. To verify the purification efficiency (or sample purity or antibody quality), a technique such as ion-exchange or size-exclusion chromatography (SEC) is needed. The SEC technique provides the necessary selectivity to identify agglomerates and size-based variations of the main component. Ion-exchange stationary phases provide good selectivity for separation of charge variants of the protein biopharmaceutical. The variations may be very subtle or small, and finding the optimal chromatographic conditions requires optimization. This work discusses the development of an automated solution for purification and separation (by ion-exchange or size-exclusion chromatography) of monoclonal antibodies in a single method. In this process, the autosampler of the HPLC was configured to perform the injection, high-volume fraction collection, and reinjection of the collected fractions.