Insight into different environmental factors affecting Fab-scFv bispecific stability

Grp Photo_Bioresources and Bioprocessing_Nattha, Wei, Yuansheng, Say Kong

From Left: Dr Yang Yuansheng, Dr Loh Han Ping, Nuruljannah Dzulkiflie, Wang Xinhui, Dr Zhang Wei, Dr Nattha Ingavat, Dr Ng Say Kong, Dr Eunice Leong

Science

Bispecific antibodies (bsAbs) are a groundbreaking innovation in medicine. Unlike traditional antibodies, they can target diseases that were previously untreatable. Among hundreds of formats, asymmetric Fab-scFv stands out as exceptionally strong and stable. Excitingly, Ziihera, the first Fab-scFv bispecific antibody, was FDA-approved in November 2024! This research reveals how environmental factors, such as temperature, pH, salt, and protein concentration influence Fab-scFv stability and also shows its resilience at high temperatures. Using this knowledge, the manufacturing of bsAbs can be optimised, and this ensures safe and cost-effective treatment for the patients.

Societal Impact

The findings offer significant benefits to both the industry and the public by enhancing drug quality and safety. While bispecific antibodies are highly effective, drug aggregates can cause severe side effects, posing additional risks to patients already suffering from the disease. By understanding how environmental factors—such as temperature, pH, salt, and protein concentration—affect bispecific stability, we can optimise purification processes, formulations, and storage conditions to prevent aggregation and limit its buildup. This not only ensures safer, high-quality treatments for patients, but also reduces production costs by minimizing product loss from aggregation. Ultimately, our work helps deliver safer, more effective therapies to those in need, while improving efficiency within the pharmaceutical industry.

Technical Summary

The findings reveal that a representative bispecific antibody, engineered in an asymmetric Fab-scFv format, exhibits remarkable stability, even after one month of exposure to 40°C under mildly acidic and low ionic strength conditions. Notably, minimal aggregate formation was observed, with some reversible self-association aggregates showing a return to monomeric form over time.

However, extreme pH conditions trigger distinct aggregation mechanisms. At pH 3.5, structural fluctuations lead to protein unfolding, a well-known precursor to aggregation. Conversely, high pH near the isoelectric point (pI) promotes hydrophobic-driven aggregation, likely due to increased exposure of hydrophobic regions. These insights offer valuable implications for optimizing production processes and formulation strategies to ensure drug stability and safety for patients.

Fig 1_Bioresources and Bioprocessing_Nattha, Wei, Yuansheng, Say Kong

Figure 1. Graphical representation of the impact of environmental factors on the behaviours of the representative bispecific antibody in the asymmetric Fab-scFv format.

References

1. Ingavat, N., et al., Investigation on environmental factors contributing to bispecific antibody stability and the reversal of self-associated aggregates. Bioresour Bioprocess, 2024. 11(1): p. 82.
2. Pang, K.T., et al., Understanding and controlling the molecular mechanisms of protein aggregation in mAb therapeutics. Biotechnol Adv, 2023. 67: p. 108192.
3. Li, W., et al., Antibody Aggregation: Insights from Sequence and Structure. Antibodies, 2016. 5(3): p. 19.
4. Clarkson, B.R., A. Schön, and E. Freire, Conformational stability and self-association equilibrium in biologics. Drug Discovery Today, 2016. 21(2): p. 342-347.
5. https://www.accessdata.fda.gov/drugsatfda_docs/label/2024/761416s000lbl.pdf

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