Robust Analytical Characterization:
Accurate, Reliable Quantitation for Biotherapeutics

Monoclonal antibodies have become one of the most important and fastest-growing classes of biopharmaceutical products in recent years. Since the approval of the first therapeutic antibodies in 1986, not only has genetic engineering enabled increased antibody specificity, but the manufacturing processes used for mAb production have evolved significantly. A pivotal improvement has been the ability to accurately measure antibody concentration (or titer). This has enabled researchers to select the most effective, high-yielding transfected cells, but also to monitor mAb concentration throughout the development process.

Affinity chromatography is ideally suited for mAb titer determination, using antibody-antigen interactions to separate mAbs from a complex mixture. The use of monolithic Protein A or Protein G affinity chromatography columns, such as the Agilent Bio-Monolith Protein A and Protein G LC columns, can provide the most effective separations. These columns provide analytical separation of all immunoglobulin (IgG) subclasses and the capture of mAb from complex matrices. These Agilent columns are compatible with HPLC and UHPLC systems.

Biomolecules such as mAbs are large, heterogenous macromolecules that can exist as multiple charged species, with different levels of amino acid PTMs, such as oxidation, deamidation, and glycosylation forming the net charge of a molecule. In conjunction, these attributes impact biomolecular function and ultimately efficacy and toxicity, and in the case of mAb-based biotherapeutics, affect antigen binding. These charge variants represent a core critical quality attribute and require strict monitoring during the biopharmaceutical manufacturing process.

Significant optimization is often required for each analyte of interest. Crucial to the process is the determination of the molecule isoelectric point (pI), and therefore the pH of mobile phase conditions, to aid in column retention. While strong cation exchange columns can provide improved ease of use, weaker cation exchange columns are often required for mAbs to enable improved peak shape and resolution through ion exchange chromatography.

During the biopharmaceutical manufacturing process, biomolecules are subject to multiple stress conditions, including changes in temperature, pH, concentration, and even mechanical shear stresses – all of which can make the molecule susceptible to aggregation. This can affect the quality and performance of the final product, making aggregate analysis a CQA for biopharmaceutical production and is typically monitored using size exclusion chromatography (SEC).

While SEC is an effective technique for the separation of protein monomers from their aggregates, in biopharmaceutical production, the process can often be made more complex due to the presence of hydrophobic cytotoxic drugs and secondary interactions. The use of bioinert, robust columns optimized for the accurate quantitation of aggregation and fragment analysis is therefore a key factor to consider.