【Case Analysis】Decoding the Stealth of Bispecific Antibody Fragment Impurities

Because bispecific antibodies (BsAbs) require the assembly of two different heavy chains (HC) and two different light chains (LC), factors during production (such as disulfide bond reduction and shear stress) can lead to mispairing or incomplete assembly, generating three typical classes of impurities:

Figure 1. Schematic of bsAb product-related impurities [1]

Purification Strategies for bsAb Fragment Impurities

For the major impurity types mentioned above, a multi-step chromatographic separation strategy based on differences in physicochemical properties is required:

Figure 2. Bispecific antibody purification strategy

Protein A affinity chromatography

As described in the previous article on aggregate removal, aggregates and fragments contain the Fc domain and can bind specifically to Protein A, with binding-elution characteristics similar to the target bispecific antibody. Given that different molecules exhibit varying binding affinities to Protein A, weakly binding fragments can be selectively removed by optimizing the elution pH conditions [2]. Using UniMab 50HC affinity chromatography to purify a bispecific antibody as an example (Figure 3): at pH 5.5, half-antibodies and fragments are eluted; at pH 5.0 and pH 4.5, the intact bispecific antibody and bispecific antibody aggregates are eluted, respectively.

Figure3. Purification of a bispecific antibody using UniMab 50HC affinity chromatography

Protein L affinity chromatography

Protein L affinity chromatography media can specifically bind to the light chain region of bsAbs. Notably, some bispecific antibody fragments exhibit significantly higher affinity for Protein L than the intact bsAb. By increasing the pH of the elution buffer, the elution ratio of low-molecular-weight impurities can be effectively reduced. Using NMab Prot L affinity chromatography media with stepwise pH elution enables efficient separation of the target bispecific antibody — the intact bsAb is selectively eluted under specific pH conditions, while low-molecular-weight bispecific antibody fragments remain bound to the media due to stronger binding. Finally, residual impurities are thoroughly removed through a cleaning-in-place (CIP) procedure, ensuring product purity with a yield of 80.3% (Figure 4).

Figure4. Removal of half-antibodies and other fragment impurities using NMab ProtL affinity chromatography

IEC

For the challenging removal of bispecific antibody fragments, UniGel-30CM weak cation exchange chromatography can be employed. By modulating pH and salt concentration gradients, fragments with weaker charges are eluted first. Adding a certain concentration of PEG or CaCl₂ can enhance resolution, achieving bispecific antibody monomer purity of 99% and yield above 80% (Figure 5).

Figure5. Purification of bispecific antibodies using UniGel-30CM cation exchange chromatography

HIC

Hydrophobic interaction chromatography is widely used for the removal of antibody fragments. Generally, compared to structurally intact, tightly folded BsAbs, fragment impurities lack complete pairing structures (e.g., missing a light or heavy chain), exposing hydrophobic regions that are normally buried within the molecule, resulting in stronger binding to the hydrophobic chromatography media. Using UniHR Phenyl-30L with stepwise salt elution, most fragment impurities can be effectively removed, increasing purity to 99.74% (Figure 6).

Figure6. Purification of a bispecific antibody using UniHR Phenyl-30L hydrophobic interaction chromatography

MULTI-MODE

In addition to traditional ion exchange and hydrophobic chromatography, multimodal chromatography can integrate multiple interaction forces for the synergistic separation of bispecific antibody fragments.

NW Rose Plus HCM HP is a multimodal media combining weak cation exchange and hydrophobic interactions. By optimizing the salt concentration gradient, this media enables high-resolution separation based on differences in hydrophobicity and charge between bispecific antibody fragments and the intact molecule, effectively removing target fragment impurities (Figure 7).

Figure7. Purification of a bispecific antibody using NW Rose Plus HCM HP multimodal chromatography

Summary

Efficient removal of bispecific antibody fragments is a critical challenge in ensuring drug quality. By deeply understanding the mechanisms of fragment impurity formation and targeting their specific characteristics — such as affinity differences, charge anomalies, and exposed hydrophobic regions — a multi-mechanism synergistic polishing strategy can be developed to effectively reduce bispecific antibody fragment levels. Below are our recommended resins for bispecific antibody fragment removal.

Recommended Resins

References :

[1] Q. Li, H. Zhao, X. Liang, Q. He, Z. Wang, G. Qin, G. Li, D. Xu, The downstream purification of bispecific antibodies, Analytical Biochemistry 696 (2025) 115692

[2] Y. Wen, Y. Ye, Z. Wang, J. Xu, Z. Meng, W. Luo, Y. Zhao, Y. Fang, Removal of antibody-related fragments during asymmetric bispecific antibody purification, Asian Journal of Complementary and Alternative Medicine 13 (2025) 1-15