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How do metal ions, hydrogen peroxide, and temperature increase monoclonal antibody fragmentation?

Work done in the lab of Prof. Anurag S Rathore at the Centre of Excellence, Biopharmaceutical Technology (DBT-COE-CBT), Indian Institute of Technology, Delhi.

About author

Surbhi Gupta

Surbhi Gupta is currently working on a doctoral thesis at the Department of Chemical Engineering, Indian Institute of Technology, Delhi, India, with the research group of Prof. Anurag S. Rathore. Her research areas of interest are understanding physical and chemical instabilities in biotherapeutics, improving the stability of biotherapeutics, and formulation studies. She had completed her master’s in Bioprocess Engineering from the Institute of Chemical Technology, Mumbai, India, and her undergraduate studies in Biotechnology from the University School of Biotechnology, GGSIPU, Delhi, India.

Interview

How would you explain your research outcomes to the non-scientific community?

Monoclonal antibodies (mAbs) are protein-based medicines used to treat various life-threatening diseases, including cancers. However, the proteinaceous nature of these medicines makes them susceptible to degradation through multiple pathways, including aggregation, fragmentation, oxidation, isomerization, deamidation, and so on. These degradation products reduce the medicine’s effectiveness and induce side effects in the patients. Fragmentation is one of the degradation pathways and is defined as the disruption of chemical bonds, including peptide bonds in a protein, via enzymatic or non-enzymatic reactions. Usually, peptide bonds of these protein-based medicines are extremely stable to non-enzymatic hydrolysis with a half-life of hundreds of years. However, various factors make them prone to hydrolysis, including pH, temperature, the flexibility of protein backbone, the side chain of amino acids, presence of metal ions and free radicals. The peptide bond of the hinge region of the mAbs is flexible and more susceptible to degradation. In this study, we have demonstrated how trace metal ion and hydrogen peroxide levels promote the degradation of the mAb hinge region and their synergistic effect on the degradation. This study shows that with an increase in the level of these impurities, the rate of fragmentation increases. Thus, it is important to avoid adding or reducing these impurities throughout the manufacturing of the product. This study also shows that lowering storage temperature reduces degradation.

Impact of metal ion and hydrogen peroxide on the fragmentation kinetics of the monoclonal antibody
Impact of metal ion and hydrogen peroxide on the fragmentation kinetics of the monoclonal antibody.

How do these findings contribute to your research area?

A trace level of metal ions and peroxides are introduced into the drug product either unintentionally during various steps of the manufacturing process, including excipients and buffer components, or added during production to increase the quantity of the product. This study suggests monitoring the level of these impurities throughout the manufacturing process and including spiking studies (i.e., adding these impurities on purpose) during formulation development to ensure the stability of the product. Further, this study recommends that buffer components of superfine grades should be used in drug manufacturing to control fragmentation.

“This study recommends that buffer components of superfine grades should be used in drug manufacturing to control fragmentation. “

What was the exciting moment during your research?

The exciting moment during the research was when we observed that the addition of both copper ions and hydrogen peroxide, increased the rate of fragmentation of mAb. Another exciting moment was fitting the linear regression model to the kinetics data. Kinetics data follow different orders depending on the reaction conditions.

What do you hope to do next?

We are currently working on understanding the impact of other chemical modifications, for example, deamidation, on fragmentation kinetics. In the future, we plan to develop a platform using spectroscopic tools to monitor these impurities throughout the manufacturing process as they significantly affect the stability of the drug product.

Where do you seek scientific inspiration from?

I have been lucky to be mentored by admirable mentors throughout my life. My master’s mentor, Prof. G. D. Yadav taught me that “Failure is just temporary whereas success is autocatalytic.” This quote of his inspired me during my failures and encouraged me to try again and again to succeed one day. My Ph.D. guide, Prof. Anurag S Rathore, inspired me on how to manage time and encouraged me to think of ideas independently, and supported me in pursuing those ideas. He has always motivated me to set new standards and excel those. Apart from them, my lab mates inspired and encouraged me to think scientifically through discussions during tea breaks.

How do you intend to help Indian science improve?

I want to contribute to Indian science by working in the field of biotherapeutics and making lifesaving medicines affordable. I also want to work towards making stable formulations that could especially benefit underdeveloped and developing countries where maintaining cold storage chains is not feasible.

Reference

S. Gupta, K. Upadhyay, C. Schöneich, A.S. Rathore, Impact of various factors on the kinetics of non-enzymatic fragmentation of a monoclonal antibody, European Journal of Pharmaceutics and Biopharmaceutics. 178 (2022) 131–139. https://doi.org/10.1016/j.ejpb.2022.08.002.

Copy Editor: Pragya Gupta

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