Impact of mAb Aggregation on its Biological Activity

Dr. Rozaleen Dash’s interview with Bio Patrika hosting “Vigyan Patrika”, a series of author interviews. Dr. Dash completed her PhD in Biochemistry from All India Institute of Medical Sciences, New Delhi then, she joined as Scientist- B at Maulana Azad Medical College, New Delhi. Currently, she is doing Post-Doc as a ICMR-RA fellow in the Professor Anurag S Rathore Lab, Department of Chemical Engineering, Indian Institute of Technology Delhi, India. She has expertise in Functional Characterization of biotherapeutics, Comparability of Biosimilars and Drug immunogenicity. Here, she talks about her co-first author research article titled “Impact of mAb Aggregation on Its Biological Activity: Rituximab as a Case Study” published in the Journal of Pharmaceutical Sciences (2020).

How would you explain your papers’ key results to the non-scientific community?

Monoclonal antibody products (mAbs) are prone to molecular instabilities like aggregation, fragmentation, oxidation, and reduction. Aggregation phenomena is a common issue for technological advancement in mAbs production which can induce adverse immune responses in patients that may affect safety and efficacy. The factors that influence the mAbs aggregation, including physical, mechanical, and chemical stresses. These stresses may form during manufacturing, storage, filling, formulation development, and shipping. Growing data suggested that mAbs aggregation can enhance immunogenicity, yet underlying mechanisms are not fully understood. Further understanding mAbs aggregation phenomena helps develop robust analytical techniques for monitoring the aggregational level and precise nature of mAbs aggregation. Our recent paper includes the physical characterization of protein aggregates generated by different stress treatments by a host of analytical techniques like DLS (Dynamic light scattering), SE-HPLC (Size-exclusion chromatography), TEM (Transmission electron microscopy), and Fluorescence spectroscopy. Apart from physicochemical characterization, these aggregates were also characterized to evaluate their impact on the biological activity of mAb molecule by a wide variety of tools such as SPR (Surface Plasmon Resonance), FACS (Fluorescence-activated cell sorting), ADCC (Antibody-dependent cell cytotoxicity) and CDC (Complement-dependent cytotoxicity). We demonstrate the extent of aggregation, the nature of aggregates, and the impact of aggregates on biological activity all depend on the source of aggregate formation. This study illustrates the ability of different analytical tools in characterizing aggregate species as well as in determining their impact on biological activity. The results affirm that understanding the mechanism of aggregation is critical for achieving consistent product quality.

Figure 1: The workflow of mAb aggregates with biological potency resulting in immunogenicity. Our studies show extreme pH (pH 3.5 and pH 11.0), stirring (1 d and 3 d stir) and oxidation via CuSO4 have the most impact on aggregate formation and the biological potency of the therapeutics.

“This study illustrates the ability of different analytical tools in characterizing aggregate species as well as in determining their impact on biological activity.”

What are the possible consequences of these findings for your research area?

There is a possibility that the impact of aggregates on the bioassays could be analytical artifacts, suggesting hyperactivity. Without a doubt, further studies are required to acquire a deeper and more accurate understanding of aggregation phenomena. These could include in vivo testing of the immunogenic potential of these mAb aggregates formed under the extreme pH, stirring, and oxidizing stress conditions. However, we hope that the results presented in this study would be of significance to those in academia and industry working on protein formulation and stability.

What was the exciting moment (eureka moment) during your research?

Our results related to physicochemical and biological characterization tools for mAb aggregates were highly rewarding, and the SPR study was relatively more challenging. We found no or minimal binding affinity with the receptor in SPR studies. The KD value increased with percentage aggregates and was very clear from the SPR sensorgrams. This moment proved to be a “Eureka” moment as executing SPR studies successfully for challenging mAb aggregates.

What do you hope to do next?

Multi-domain monoclonal antibodies are far more complex than small molecule drugs with various intrinsic heterogeneities that are typically well characterized for identity and bioactivity. The critical quality attributes (CQAs) of a mAbs, include product-related variants i.e., aggregation, charge variant and glycosylation. Keeping this in view, my next proposed work intended to understand the precise molecular mechanism underlying the structure-function relationship during the aggregation process.

Where do you seek scientific inspiration?

Science is a way of life, a zestful drive that allows researchers to fuel their dreams into reality. Since childhood, I have been motivated by a fascination for science and a burning desire for enquiry. I gained my scientific inspiration from my mentors Prof. Nibhriti Das (PhD guide) and Prof. Anurag S Rathore (Postdoc guide). They are amazing persons I ever met, and they are my continuous inspirational source in my research career.

How do you intend to help Indian science improve?

I believe my passion, expertise, knowledge, and skills will help develop and further advance the Indian scientific vision in the domains of affordable biotherapeutics and drug discovery. I hope that my research reaches out to a broader audience and is accessible to all.

Reference

Bansal R#, Dash R#, and Rathore AS. Impact of mAb Aggregation on its Biological Activity: Rituximab as a Case Study”. J Pharm Sci. 2020; 109(9): 2684- 2698. doi.org/10.1016/j.xphs.2020.05.015. #equal contribution.

Prof. Anurag S Rathore’s lab webpage: http://biotechcmz.com and https://cbt.iitd.ac.in

Edited by: Govinda Raju Yedida (Volunteer, Bio Patrika)

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