Development of an automated method for monoclonal antibodies purification and analysis

Two major problems in biotherapeutics are aggregation of and the presence of variants of active pharmaceutical ingredients. These product-related substances can have different efficacy 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.

This article discusses the development of an automated solution for purification and separation (by ion exchange or sizeexclusion chromatography) of antibodies in a single method. In this process the autosampler of the HPLC was configured to performs the injection, high volume fraction collection, and reinjection.

During various stages in the development of biopharmaceuticals, purification, and analytical characterisation of the product are required. In the early phase of process optimisation of recombinant antibodies, a large number of samples must be screened for titer, aggregation, and protein variants.

These protein aggregates and variants of the active pharmaceutical ingredient can have different efficacies than the main product and may cause serious side effects, for example, antidrug- antibody formation. Protein aggregates are mostly the consequence of suboptimal production, purification, or handling conditions (temperature or 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. While the purification on affinity columns yields information on the titer of the product, it is unselective with respect to related substances.To verify the sample purity or antibody quality, techniques, such as ion exchange chromatography (IEX) or size-exclusion chromatography (SEC) are needed. SEC 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 article discusses an automated solution for purification followed by a separation (IEX or SEC) of antibodies using the UltiMate® 3000 Titanium HPLC system (Figure.1). In this process, multiple protein separation steps are performed automatically.

The autosampler performs the injection, high-volume fraction collection, and reinjection of the collected fractions for analysis.