pH dependent aggregation and conformation changes of rituximab using SAXS and its comparison with the standard regulatory approach of biophysical characterization

Narvekar, Aditya; Gawali, Santosh L.; Hassan, P. A.; Jain, Ratnesh; Dandekar, Prajakta

doi:10.1016/j.ijbiomac.2020.08.148

Cited by 12 publications

(6 citation statements)

References 28 publications

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“…The exact knowledge of these structural parameters is of paramount importance to simulate the crystallization behavior of the antibody in the framework of theoretical models for diffusion-limited nucleation of macromolecules in solution ( Lutsko, 2019 ). The R g value of rituximab at pH 6.5 is compatible with that recently reported by Narvekar et al (2020) .…”

Section: Discussionsupporting

confidence: 92%

Structural Characterization of the Full-Length Anti-CD20 Antibody Rituximab

Belviso

Mangiatordi

Alberga

et al. 2022

Front. Mol. Biosci.

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Rituximab, a murine–human chimera, is the first monoclonal antibody (mAb) developed as a therapeutic agent to target CD20 protein. Its Fab domain and its interaction with CD20 have been extensively studied and high-resolution atomic models obtained by X-ray diffraction or cryo-electron microscopy are available. However, the structure of the full-length antibody is still missing as the inherent protein flexibility hampers the formation of well-diffracting crystals and the reconstruction of 3D microscope images. The global structure of rituximab from its dilute solution is here elucidated by small-angle X-ray scattering (SAXS). The limited data resolution achievable by this technique has been compensated by intensive computational modelling that led to develop a new and effective procedure to characterize the average mAb conformation as well as that of the single domains. SAXS data indicated that rituximab adopts an asymmetric average conformation in solution, with a radius of gyration and a maximum linear dimension of 52 Å and 197 Å, respectively. The asymmetry is mainly due to an uneven arrangement of the two Fab units with respect to the central stem (the Fc domain) and reflects in a different conformation of the individual units. As a result, the Fab elbow angle, which is a crucial determinant for antigen recognition and binding, was found to be larger (169°) in the more distant Fab unit than that in the less distant one (143°). The whole flexibility of the antibody has been found to strongly depend on the relative inter-domain orientations, with one of the Fab arms playing a major role. The average structure and the amount of flexibility has been studied in the presence of different buffers and additives, and monitored at increasing temperature, up to the complete unfolding of the antibody. Overall, the structural characterization of rituximab can help in designing next-generation anti-CD20 antibodies and finding more efficient routes for rituximab production at industrial level.

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Section: Discussionsupporting

confidence: 92%

Structural Characterization of the Full-Length Anti-CD20 Antibody Rituximab

Belviso

Mangiatordi

Alberga

et al. 2022

Front. Mol. Biosci.

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“…However, there have been limited studies involving the use of X-ray crystallography (XRC), a traditional technique for protein structural studies at the near-atomic level, as it works well only with proteins that can be crystallized, it requires expensive instrumentation, trained operators, and extensive time for analysis ( Lerch et al, 2020 ). If we look at the evolution of HOS assessment in biosimilarity studies ( Figure 4 ), the timeline is peppered with multiple orthogonal techniques such as single dimension NMR ( Visser et al, 2013 ; Sörgel et al, 2015 ; Montacir et al, 2017 ), multi-dimension NMR ( Shekhawat et al, 2019 ; Bor Tekdemir et al, 2020 ; Kovács et al, 2020 ), HDX-MS ( Cho et al, 2016 ; Brokx et al, 2017 ; Brown et al, 2019 ), IM-MS ( Fang et al, 2016 ; Montacir et al, 2017 , 2018 ), antibody conformational array ( Jung et al, 2014 ; Hong et al, 2017 ), aptamer-based enzyme linked apta-sorbent assay ( Wildner et al, 2019 ), and small angle X-ray scattering (SAXS) ( Narvekar et al, 2020 ) ( Supplementary Table S4 ). Of these, NMR has emerged as the new gold standard for HOS assessment with more than 10 published biosimilarity studies published with either 1D, or 1D and 2D as a part of the analytical platform ( Supplementary Table S4 ).…”

Section: Global Landscape On Biosimilar Approvalsmentioning

confidence: 99%

Analytical Similarity Assessment of Biosimilars: Global Regulatory Landscape, Recent Studies and Major Advancements in Orthogonal Platforms

Nupur

Joshi

Gulliarme

et al. 2022

Front. Bioeng. Biotechnol.

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Biopharmaceuticals are one of the fastest-growing sectors in the biotechnology industry. Within the umbrella of biopharmaceuticals, the biosimilar segment is expanding with currently over 200 approved biosimilars, globally. The key step towards achieving a successful biosimilar approval is to establish analytical and clinical biosimilarity with the innovator. The objective of an analytical biosimilarity study is to demonstrate a highly similar profile with respect to variations in critical quality attributes (CQAs) of the biosimilar product, and these variations must lie within the range set by the innovator. This comprises a detailed comparative structural and functional characterization using appropriate, validated analytical methods to fingerprint the molecule and helps reduce the economic burden towards regulatory requirement of extensive preclinical/clinical similarity data, thus making biotechnological drugs more affordable. In the last decade, biosimilar manufacturing and associated regulations have become more established, leading to numerous approvals. Biosimilarity assessment exercises conducted towards approval are also published more frequently in the public domain. Consequently, some technical advancements in analytical sciences have also percolated to applications in analytical biosimilarity assessment. Keeping this in mind, this review aims at providing a holistic view of progresses in biosimilar analysis and approval. In this review, we have summarized the major developments in the global regulatory landscape with respect to biosimilar approvals and also catalogued biosimilarity assessment studies for recombinant DNA products available in the public domain. We have also covered recent advancements in analytical methods, orthogonal techniques, and platforms for biosimilar characterization, since 2015. The review specifically aims to serve as a comprehensive catalog for published biosimilarity assessment studies with details on analytical platform used and critical quality attributes (CQAs) covered for multiple biotherapeutic products. Through this compilation, the emergent evolution of techniques with respect to each CQA has also been charted and discussed. Lastly, the information resource of published biosimilarity assessment studies, created during literature search is anticipated to serve as a helpful reference for biopharmaceutical scientists and biosimilar developers.

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“…181 In the past years, numerous publications have used CD as an orthogonal method to study and characterize mAb aggregation. 88,97,120,183,184 Furthermore, Joshi et al employed this analytical method as a high-throughput screening tool for examining mAb stability in various buffers, proving that CD is a reliable method for rapid monitoring of mAb conformational changes.…”

Section: Circular Dichroism (Cd)mentioning

confidence: 99%

“…In the past years, numerous publications have used CD as an orthogonal method to study and characterize mAb aggregation 88,97,120,183,184 . Furthermore, Joshi et al .…”

Section: Miniaturizable Analytical Techniquesmentioning

confidence: 99%

Process analytical technique (PAT) miniaturization for monoclonal antibody aggregate detection in continuous downstream processing

Pedro

Klijn

Eppink

et al. 2021

J of Chemical Tech & Biotech

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The transition to continuous biomanufacturing is considered the next step to reduce costs and improve process robustness in the biopharmaceutical industry, while also improving productivity and product quality. The platform production process for monoclonal antibodies (mAbs) is eligible for continuous processing to lower manufacturing costs due to patent expiration and subsequent growing competition. One of the critical quality attributes of interest during mAb purification is aggregate formation, with several processing parameters and environmental factors known to influence antibody aggregation. Therefore, a real-time measurement to monitor aggregate formation is crucial to have immediate feedback and process control and to achieve a continuous downstream processing. Miniaturized biosensors as an in-line process analytical technology tool could play a pivotal role to facilitate the transition to continuous manufacturing. In this review, miniaturization of already wellestablished methods to detect protein aggregation, such as dynamic light scattering, Raman spectroscopy and circular dichroism, will be extensively evaluated for the possibility of providing a real-time measurement of mAb aggregation. The method evaluation presented in this review shows which limitations of each analytical method still need to be addressed and provides application examples of each technique for mAb aggregate characterization. Additionally, challenges related to miniaturization are also addressed, such as the design of the microfluidic chip and the microfabrication material. The evaluation provided in this review shows why the development of microfluidic biosensors is considered the key for real-time measurement of mAb aggregates and how it can contribute to the transition to a continuous processing.

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pH dependent aggregation and conformation changes of rituximab using SAXS and its comparison with the standard regulatory approach of biophysical characterization

Cited by 12 publications

References 28 publications

Structural Characterization of the Full-Length Anti-CD20 Antibody Rituximab

Structural Characterization of the Full-Length Anti-CD20 Antibody Rituximab

Analytical Similarity Assessment of Biosimilars: Global Regulatory Landscape, Recent Studies and Major Advancements in Orthogonal Platforms

Process analytical technique (PAT) miniaturization for monoclonal antibody aggregate detection in continuous downstream processing

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