Related Experiment Video
Updated: Sep 17, 2025
Characterization of Glycoproteins with the Immunoglobulin Fold by X-Ray Crystallography and Biophysical Techniques
Published on: July 5, 2018
Characterization of free light chain impurity in a bispecific antibody
Monica Sadek1, Neelima Mantha2, Steffen Lippold1
1Analytical Development, Genentech Inc., South San Francisco, CA, USA.
Bispecific antibodies (bsAbs) target two distinct binding sites, which enable novel mechanisms of action for the treatment of disease. Due to this structural complexity, bsAbs exhibit greater heterogeneity as compared to monospecific antibodies and may require nonstandard purification strategies to remove undesired product-related impurities. However, the implementation of unique manufacturing processes may result in the observation of new impurities. When new impurities are identified, comprehensive characterization of these species is needed to understand the potential impact to the safety and efficacy of the product. In this study, the assessment of a unique purification mode, protein L affinity chromatography, for purification of a bsAb resulted in high levels of free light chain (LC) in the final product. It was discovered that the free LC formed oligomers of various sizes upon accelerated stress in a concentration-, time-, and temperature-dependent manner. This is the first time free LC oligomer has been reported as a product-related impurity. Characterization of the free LC and free LC oligomer species was performed using a variety of chromatographic, electrophoretic, and mass spectrometry tools to gain insight on the formation and behavior of these impurities in the product. These studies informed an impact assessment of the observed free LC and free LC oligomer species on safety, immunogenicity, pharmacokinetics, and bioactivity.
Bispecific antibodies (bsAbs) target two distinct binding sites, which enable novel mechanisms of action for the treatment of disease. Due to this structural complexity, bsAbs exhibit greater heterogeneity as compared to monospecific antibodies and may require nonstandard purification strategies to remove undesired product-related impurities. However, the implementation of unique manufacturing processes may result in the observation of new impurities. When new impurities are identified, comprehensive characterization of these species is needed to understand the potential impact to the safety and efficacy of the product. In this study, the assessment of a unique purification mode, protein L affinity chromatography, for purification of a bsAb resulted in high levels of free light chain (LC) in the final product. It was discovered that the free LC formed oligomers of various sizes upon accelerated stress in a concentration-, time-, and temperature-dependent manner. This is the first time free LC oligomer has been reported as a product-related impurity. Characterization of the free LC and free LC oligomer species was performed using a variety of chromatographic, electrophoretic, and mass spectrometry tools to gain insight on the formation and behavior of these impurities in the product. These studies informed an impact assessment of the observed free LC and free LC oligomer species on safety, immunogenicity, pharmacokinetics, and bioactivity.
More Related Videos
Related Concept Videos
01:10Antibody Structure
Antibodies, also known as immunoglobulins (Ig), are essential players of the adaptive immune system. These antigen-binding proteins are produced by B cells and make up 20 percent of the total blood plasma by weight. In mammals, antibodies fall into five different classes, which each elicits a different biological response upon antigen binding.
The Y-Shaped Structure of Antibodies Consists of Four Polypeptide Chains
Antibodies consist of four polypeptide chains: two identical heavy...
01:25Antibody Structure and Classes
The basic structure of an antibody consists of four protein chains: two identical heavy chains and two identical light chains. These chains are held together by disulfide bonds and other non-covalent interactions, forming a Y-shaped structure.
01:24B Cell Activation and Differentiation
When naive B cells encounter a specific antigen that can bind to the B cell receptor (BCR) on their surface, they undergo sensitization to respond to the antigen's presence. Sensitization begins with...