Purification of novel antibody formats: A toolbox approach Antibody-based therapies offer targeted treatment options for a range of diseases, from cancer to autoimmune disorders. As the field continues to evolve, antibody formats such as bispecific and fragment antibodies have emerged as important new modalities, enabling novel mechanisms of action—while also introducing new challenges in purification.
Antibody fragments, such as Fab, scFv, and individual light chain (VL) or heavy chain (VH) variable domain fragments, are an attractive therapeutic modality due to their smaller size, ease of production, potential for better tissue penetration and reduced immunogenicity compared to full-size mAbs. Bispecific antibodies (BsAbs) represent another breakthrough in therapeutic design, with their ability to simultaneously engage different antigens. By simultaneously binding to a cancer cell and a T cell, bispecific antibodies draw immune cells closer to tumors, amplifying the body’s immune response beyond the capabilities of traditional mAbs. Other forms of bispecifics engage targets to block aberrant signaling pathways that drive a range of pathological conditions. According to the United States Food and Drug Administration (FDA), more than 100 BsAbs are in clinical development. Since 2014, the FDA has approved nine BsAb marketing applications to treat cancer, as well as hematologic and ocular diseases.i
Novel modalities present new purification challenges The unique properties of antibody fragments and BsAbs not only expand the potential of biologic therapies, but also pose different challenges in purification and manufacturing that must be addressed for successful clinical and commercial development.
the Fc region, making Protein A ineffective. While Protein L is useful for capturing antibody fragments lacking an Fc region, it has several limitations. It selectively binds kappa light chains, making it ineffective for lambda or light chain-free fragments. The lower binding capacity and pH sensitivity can affect efficiency and purity, while its high cost and reduced stability limit reusability. Additionally, Protein L may exhibit non-specific binding, necessitating further polishing steps. For bispecific antibodies, purification is complicated by the potential for mispaired chains, unwanted byproducts, and the formation of aggregates. Mispairing of antibody chains occurs during production when the two heavy and two light chains fail to pair correctly, leading to product-related impurities such as homodimers, half-antibodies, and mixed light chain antibodies. Mispaired chains can lead to misfolding, exposing hydrophobic regions that promote protein aggregation. The complexity of purification workflows for BsAbs and fragments makes process development a critical aspect of manufacturing, requiring customized approaches tailored to the specific structure and properties of each therapeutic candidate.
Standard mAb purification workflows typically rely on Protein A affinity chromatography, which efficiently binds to the Fc region of antibodies, enabling a straightforward and high-yield purification process. However, many antibody fragments lack
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