Customize Solution for Refolding of Biotherapeutic Protein

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

Rashmi Sharma is a budding PhD scholar working in a prestigious institute Indian Institute of Technology,Delhi, under the supervision of the renowned Prof Anurag S. Rathore. She was born and brought up in the national capital, and acquired her scientific curiosity and endeavour through anime and cartoons. She has pursued bachelors in Biochemistry from Daulat Ram College, University of Delhi. She subsequently did her masters in Genetics from University of Delhi. Her interest in proteins led her to join the realm of biopharmaceuticals, which is her current field of study. Besides work she enjoys reading authors such as Murakami and taking coffee breaks.

Interview

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

Proteins are widely known for their functions in our body. These functions are generally the boon of their specific 3D structure in space (cells, solutions etc) called the ‘native structure’. Therefore, when cells make proteins, they are ‘folded’ to the correct form before being employed. Hence, the structure is extremely important and any change/s in it can render the protein useless or even toxic.

A variety of protein manufacturing processes have steps dedicated for the conversion of the proteins to their native, functional 3D states, before being processed further for consumption. This step is called the ‘Refolding step’, wherein a mis-folded protein is re-folded to correct shape. Specific chemicals and treatments are provided to the molecules to change their structures in the desired way. However, it is a very dynamic and sensitive reaction. Most of the protein is lost in misfolded states due to the complex folding pathway and incompetent chemical/physical environment. The refolding step is therefore dubbed as a bottleneck. The expensive chemicals and large amount of time add to the complexity.

Scanty information about the detailed folding steps in the reaction fails the researchers to produce appropriate refolding schemes.

The aim of our study was to analyse the details of the folding pathway of a biotherapeutic protein called ‘Ranibizumab’. It is used to treat age related macular degeneration (AMD) which leads to vision loss. We looked at the pathway using various sophisticated analytical tools which led us to decipher part of the process. We were able to dissect the process in two parts, where initial stages supported correct bond formation while the later stages corresponded to correct rearrangements of the bonds and shape.

This information was used to tailor suitable chemicals and precise physical (temperature, pH, time etc) set points for the different stages. We then compared this refolding scheme to a ‘conventional’ generic hit-and-trail approach and a significant increment (55%) was observed.

The usage of a multi-step segmented refolding approach led to the increase in the refolding yield for the Fab Ranibizumab. Segmented approach bisected the reaction after analysing the reaction using sophisticated tools such as RP-HPLC, zeta potential and intrinsic fluorescence analysis. The approach proved better than a conventional approach where the refolding was treated as a ‘black box’. The illustration describes the experimental set up that led to the formulation of a segmented refolding approach. The renaturation reaction of protein Ranibizumab was analysed using tools like RP-HPLC, intrinsic fluorescence and zeta potential change. This led to the observation that the refolding realm exhibited the presence of native structured species almost halfway through the reaction, which later became the point of division. The reaction was then treated at different conditions in both the segments which increased the yield by 55%. It was hypothesized that the different conditions were optimised according to the reaction phases and specific chemical microenvironment was provided to aid the refolding of unstable structures.

How do these findings contribute to your research area?

These findings provide insights about the refolding pathway for Ranibizumab, which was previously considered a ‘black-box’.

The unconventional technique of segmenting the refolding pathway, using various analytical tools, can be applied to various other molecules and replicated for efficiency.

It also highlighted the need for intensive research of the refolding pathway, which can navigate the manufacturing process from the traditional hit and trial approach to a tailored new generation methodology.

“These findings provide insights about the refolding pathway for Ranibizumab, which was previously considered a ‘black-box’.”

What was the exciting moment during your research?

The exciting moment during my research was, performing the pH switches which would visually change the turbidity of the solution and setting up the finalised ‘unconventional’ refolding scheme with the team.

What do you hope to do next?

I hope to deduce the pathway further by seeking other analytical tools and construct an even fine-tuned refolding approach. I also plan to deconstruct the various intermediate protein folding stages through which the molecular structure goes in order to reach certain targeted conformations.

Where do you seek scientific inspiration from?

The story of Gregor Mendel found me highly motivated. His keen observations to things as simple as different coloured flowers and peas as what I’ve tried to imbibe in day to day life.

I’ve sought great inspiration from my supervisor for his zeal and scientific rigour. Also, Dr. Nikhil Kateja is my current inspiration. His innovative and smart approach to tricky problems set a great example. However, his perspective on life and how to cope with the stress that comes with research is what a young researcher needs.

How do you intend to help Indian science improve?

By contributing as much as I can and highlighting the negative results as well.

I believe adequate counselling should be necessary for all the young research aspirants before they start their scientific journey.

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.

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