Separation of Monoclonal Antibodies by Analytical Size Exclusion Chromatography

Chakrabarti, Abhijit

doi:10.5772/intechopen.73321

Cited by 9 publications

(6 citation statements)

References 22 publications

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“…Detergent-solubilized membrane proteins elute differently than calibration proteins because their elution from SEC depends on the total size of the polypeptide-detergent-lipids complex rather than the oligomeric state and molar mass of the protein 25,26 . Column chemistry, pH and salt conditions all affect elution volumes of proteins with charged or hydrophobic surface residues 27,28 . SEC becomes much more versatile and reliable for MW determination when combined with multi-angle light scattering (MALS) and differential refractive index (dRI) detectors 3,4,11,29,30,31,32 .…”

Section: Introductionmentioning

confidence: 99%

Characterization of Proteins by Size-Exclusion Chromatography Coupled to Multi-Angle Light Scattering (SEC-MALS)

Some

Amartely

Tsadok³

et al. 2019

JoVE

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Analytical size-exclusion chromatography (SEC), commonly used for the determination of the molecular weight of proteins and protein-protein complexes in solution, is a relative technique that relies on the elution volume of the analyte to estimate molecular weight. When the protein is not globular or undergoes non-ideal column interactions, the calibration curve based on protein standards is invalid, and the molecular weight determined from elution volume is incorrect. Multi-angle light scattering (MALS) is an absolute technique that determines the molecular weight of an analyte in solution from basic physical equations. The combination of SEC for separation with MALS for analysis constitutes a versatile, reliable means for characterizing solutions of one or more protein species including monomers, native oligomers or aggregates, and heterocomplexes. Since the measurement is performed at each elution volume, SEC-MALS can determine if an eluting peak is homogeneous or heterogeneous and distinguish between a fixed molecular weight distribution versus dynamic equilibrium. Analysis of modified proteins such as glycoproteins or lipoproteins, or conjugates such as detergent-solubilized membrane proteins, is also possible. Hence, SEC-MALS is a critical tool for the protein chemist who must confirm the biophysical properties and solution behavior of molecules produced for biological or biotechnological research. This protocol for SEC-MALS analyzes the molecular weight and size of pure protein monomers and aggregates. The data acquired serve as a foundation for further SEC-MALS analyses including those of complexes, glycoproteins and surfactant-bound membrane proteins.

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Section: Introductionmentioning

confidence: 99%

Characterization of Proteins by Size-Exclusion Chromatography Coupled to Multi-Angle Light Scattering (SEC-MALS)

Some

Amartely

Tsadok³

et al. 2019

JoVE

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“…The rod-like structure of HA2* likely changes its retention time on the column compared to a globular protein with a similar MW, leading to an overestimate of its size from the calibration. 46 In summary, these results show that, while HA2* does form some degree of large aggregates at neutral pH, most is not aggregated, even without SRC. Catastrophic aggregation is observed only when HA2* is acidified, as discussed below.…”

Section: ■ Discussionmentioning

confidence: 99%

“…The second and more likely reason is that the shape of HA2 (and thus HA2*) is rod-like and very different from that of the globular size standards used in SEC calibration. The rod-like structure of HA2* likely changes its retention time on the column compared to a globular protein with a similar MW, leading to an overestimate of its size from the calibration . In summary, these results show that, while HA2* does form some degree of large aggregates at neutral pH, most is not aggregated, even without SRC.…”

Section: Discussionmentioning

confidence: 99%

Stability of HA2 Prefusion Structure and pH-Induced Conformational Changes in the HA2 Domain of H3N2 Hemagglutinin

2021

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Influenza hemagglutinin is the fusion protein that mediates fusion of the viral and host membranes through a large conformational change upon acidification in the developing endosome. The "spring-loaded" model has long been used to describe the mechanism of hemagglutinin and other type 1 viral glycoproteins. This model postulates a metastable conformation of the HA2 subunit, caged from adopting a lower-free energy conformation by the HA1 subunit. Here, using a combination of biochemical and spectroscopic methods, we study a truncated construct of HA2 (HA2*, lacking the transmembrane domain) recombinantly expressed in Escherichia coli as a model for HA2 without the influence of HA1. Our data show that HA2* folds into a conformation like that of HA2 in full length HA and forms trimers. Upon acidification, HA2* undergoes a conformational change that is consistent with the change from pre-to postfusion HA2 in HA. This conformational change is fast and occurs on a time scale that is not consistent with aggregation. These results suggest that the prefusion conformation of HA2 is stable and the change to the postfusion conformation is due to protonation of HA2 itself and not merely uncaging by HA1.

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“…Size exclusion chromatography (SEC) is another important method for the separation and purification of mAbs from the other heterogenic impurities which may cause immunogenic responses [ 108 ]. These impurities include components that are smaller and larger than the intact mAb; where smaller components are produced via enzymic or non-enzymic cleavage and the partial creation of uneven disulfide linkages.…”

Section: Analytical Techniques For Mabs Analysesmentioning

confidence: 99%

“…On the other hand, larger components result from molecular aggregation, association, or even through precipitation [ 109 ]. The biopharmaceutical industry uses SEC to monitor the purity level of mAbs for the quality control (QC) process, as more than 99% purity is required for their medicinal use [ 108 ]. The SEC used to separate the molecules depending on the molecule size leading to the elution of larger molecules first from the column.…”

Section: Analytical Techniques For Mabs Analysesmentioning

confidence: 99%

Analytical Techniques for the Characterization and Quantification of Monoclonal Antibodies

Alhazmi

Albratty

2023

Pharmaceuticals

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Monoclonal antibodies (mAbs) are a fast-growing class of biopharmaceuticals. They are widely used in the identification and detection of cell makers, serum analytes, and pathogenic agents, and are remarkably used for the cure of autoimmune diseases, infectious diseases, or malignancies. The successful application of therapeutic mAbs is based on their ability to precisely interact with their appropriate target sites. The precision of mAbs rely on the isolation techniques delivering pure, consistent, stable, and safe lots that can be used for analytical, diagnostic, or therapeutic applications. During the creation of a biologic, the key quality features of a particular mAb, such as structure, post-translational modifications, and activities at the biomolecular and cellular levels, must be characterized and profiled in great detail. This implies the requirement of powerful state of the art analytical techniques for quality control and characterization of mAbs. Until now, various analytical techniques have been developed to characterize and quantify the mAbs according to the regulatory guidelines. The present review summarizes the major techniques used for the analyses of mAbs which include chromatographic, electrophoretic, spectroscopic, and electrochemical methods in addition to the modifications in these methods for improving the quality of mAbs. This compilation of major analytical techniques will help students and researchers to have an overview of the methodologies employed by the biopharmaceutical industry for structural characterization of mAbs for eventual release of therapeutics in the drug market.

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Separation of Monoclonal Antibodies by Analytical Size Exclusion Chromatography

Cited by 9 publications

References 22 publications

Characterization of Proteins by Size-Exclusion Chromatography Coupled to Multi-Angle Light Scattering (SEC-MALS)

Characterization of Proteins by Size-Exclusion Chromatography Coupled to Multi-Angle Light Scattering (SEC-MALS)

Stability of HA2 Prefusion Structure and pH-Induced Conformational Changes in the HA2 Domain of H3N2 Hemagglutinin

Analytical Techniques for the Characterization and Quantification of Monoclonal Antibodies

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