Tools and Techniques for mAb Structure, Function, and Purity Characterization

There are over 200 approved antibody drugs to date and an increasing number of monoclonal antibodies (mAbs) are approved each year. But before antibodies are approved, they must go through a rigorous testing, clinical trial, and regulatory approval process. One such assessment is antibody characterization, which assesses the structure, function, and purity of the antibody.

“mAb characterization occurs throughout the development lifecycle starting in early-stage R&D and continuing through production QC,” says Kyle Luttgeharm, Senior Product Manager (ProteoAnalyzer system) at Agilent Technologies. Early on in the process, mAb characterization focuses on structure and function. “Biochemical characterizations, such as binding and affinity assessments, happen first to triage down the number of mAbs that are then screened for functional characterizations,” adds Kalyani Mondal, Associate Director (Biosensors) at Charles River Laboratories. Later on, for example during commercialization, mAb characterization focuses on ensuring product quality and consistency.

“Achieving consistent and reliable analytical methods for mAb characterization across different stages of development and production presents a challenge due to the complexity and sensitivity of these molecules,” explains Sayuri Otaki, Global Product Manager and Segment Lead (Protein Separations) at MilliporeSigma (the life science business of Merck KGaA, Darmstadt, Germany in the U.S. and Canada).

What characteristics does mAb characterization assess?

Characterizing a mAb includes inquiring about a wide range of properties and behavior of the antibody. For example:

• Do antibodies have the expected primary, secondary, and tertiary structure?
• What post-translational modifications exist?
• What is the binding between the antibody and the antigen like?
• What is the mechanism of function of the mAb?
• Do aggregates form during manufacturing or storage?
• Are there impurities like variants and host cell protein contaminating the mAb?

To answer these questions and more, antibody characterization labs turn to a variety of techniques to assess antibodies.

Techniques for characterizing monoclonal antibodies

With so many attributes to characterize, using methods that evaluate multiple attributes at once can save time and resources, says Otaki. “For example, some advanced chromatographic methods can separate mAb variants and impurities while also providing information on glycosylation patterns.”

Chromatography-based methods can be used to identify post-translational modifications based on the separation of molecules that have different characteristics. For example, chromatography can be used to identify contaminants that are a different size than the mAb. “For mAb purity assessments, accurate quantification of both low molecular weight and high molecular weight impurities are needed,” notes Luttgeharm. To accomplish this, size exclusion chromatography, which accurately quantifies high molecular weight impurities, can complement capillary electrophoresis SDS (CE-SDS), which better quantifies low molecular weight contaminants.

Mass spectrometry, a popular method for characterizing mAbs, is used to measure the molecular weight of the antibody and to assess its primary structure, post-translational modification, and glycosylation. mAbs can be analyzed as intact antibodies or as peptide fragments in a method called peptide mapping. This technique separates peptides by HPLC and then detects fragments using mass spectrometry.

Other spectroscopy-based techniques, such as circular dichroism, NMR, and Raman and Fourier transform infrared spectroscopy, can be used to assess higher order structure of mAbs.

Capillary electrophoresis techniques are fast, high resolution, and require minimal sample input. These techniques, which include CE-SDS and capillary zone electrophoresis (CZE), can be used to understand the heterogeneity in the mAb’s size, glycosylation, and charge.

Ligand binding assays measure the binding kinetics between antibody and antigen and include tools such as fluorescence-based ELISAs, bio-layer interferometry, and SPR-based methods. ELISAs, and LC-MS can also be used to detect contaminating host cell proteins. “The advantage of biosensor-based assays is that these are semi-automated and require minimal human intervention,” says Mondal.

To characterize mAb function, cell-based assays, such as antibody-dependent cellular cytotoxicity assays and complement-dependent cytotoxicity (CDC) assays, help determine if the mAb induces immune effector cells to kill target cells coated with mAbs or if the mAb induces complement-mediated cell killing, respectively.

Emerging technologies for mAb characterization

With so many properties to assess, many in the field have pointed toward increasing throughput and automation for mAb characterization. “In the last decade high-throughput biosensors have made a significant impact on screening capabilities,” Mondal points out.

“Two-dimensional electrophoresis is not generally an emerging technology, but it is regarded as an emerging technology for mAb characterization,” says Otaki. Two-dimensional electrophoresis (2DE) has high separation capabilities, so it’s useful in the early stage of mAb development when samples are variable and complex, Otaki adds. MilliporeSigma has recently developed an automatable 2DE system to facilitate reproducible and rapid mAb characterization.

To increase throughput of mAb analysis, Agilent has developed a parallel CE-SDS system called the ProteoAnalyzer. “The major advantages of this type of system are the efficiency of running multiple samples simultaneously as well as the reliability gained through rejuvenation of the capillaries between samples,” explains Luttgeharm.

“Several new startups are taking novel approaches for function first characterizations of challenging cell-surface targets,” adds Mondal.

Download the Monoclonal Antibody Characterization Methods infographic  to learn more