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From Left: Dr Yang Yuansheng, Dr Loh Han Ping, A/Prof Bi Xuezhi, Dr Zhang Wei, Nuruljannah Dzulkiflie, Wang Xinhui, Dr Nattha Ingavat, Dr Kok Yee Jiun

 

Science

Bispecific antibodies (bsAbs) represent the next generation of therapeutics, capable of targeting two antigens or epitopes simultaneously, making them promising candidates for treating various diseases, including cancer. To date, there are 12 FDA-approved bsAb drugs available on the market. Appended bsAbs are among the most popular formats in bsAb development. However, they are highly hydrophobic and flexible, with a strong tendency to aggregate. In this study, the team investigate how the hydrophobicity and structural flexibility of appended bsAbs affect their purification, along with the steps taken to address these challenges.

 

Societal Impact

Protein A chromatography is the most commonly used method for purifying antibodies in the biopharmaceutical industry; however, it is also the most expensive step in the entire purification process. In this study, the team elaborated on the unique challenges associated with purifying appended bsAbs in Protein A chromatography. Most importantly, they provide insights into resolving these challenges, thereby making the Protein A purification process more robust and productive. This, in turn, will help reduce the overall cost of manufacturing appended bsAb, a promising class of biotherapeutics.

 

Technical Summary

The study showed that, although the intrinsic hydrophobicity and flexibility of the appended bsAb led to increased aggregation during the Protein A chromatography run, the team were able to optimise the purification process, improving product purity from 29% to 93% in a single step. They also demonstrated that the structural flexibility of the single-chain variable fragment (scFv) linkers to the parental antibody resulted in different hydrodynamic sizes of the appended bsAb molecule, with its flexibility shown to be pH-dependent. These findings indicate how the complexity of these appended bsAbs affects their interactions with the column during purification, necessitating more extensive study during development.

Figure 1. Structure of an appended bispecific antibody. Two single-chain variable fragment with flexible linkers attached to an IgG. The appended bispecific antibody was purified in a single step to achieve high purity.

 

References

Wang X, Ingavat N, Liew JM, Dzulkiflie N, Loh HP, Kok YJ, Bi X, Yang Y, Zhang W. Effects of molecule hydrophobicity and structural flexibility of appended bispecific antibody on Protein A chromatography. J Chromatogr A. 2024 Aug 30;1731:465206. doi: 10.1016/j.chroma.2024.465206. Epub 2024 Jul 22. PMID: 39053253.