Evaluation of strategies to control Fab light chain dimer during mammalian expression and purification: A universal one‐step process for purification of correctly assembled Fab

Spooner, Jennifer; Keen, Jenny; Nayyar, Kalpana; Birkett, Neil R.; Bond, Nicholas J.; Bannister, David; Tigue, Natalie J.; Higazi, Daniel R.; Kemp, Benjamin; Vaughan, Tristan J.; Kippen, Alistair D.; Buchanan, Andrew

doi:10.1002/bit.25550

Cited by 12 publications

(14 citation statements)

References 20 publications

(18 reference statements)

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“…29 The formation of LC dimer-related contaminants would also potentially increase in cases where the LC-Fc plasmid is more efficiently transfected or expressed than the HC plasmid. 30 Conversely, cell secretion of HC dimers has been observed for some engineered mAbs in the absence of paired LC and could also contribute to the 100-kDa fraction. 31 However, the strong affinity of the native HC-LC interaction would presumably favor the formation of heterodimers rather than homodimers.…”

Section: Discussionmentioning

confidence: 99%

Design and characterization of novel dual Fc antibody with enhanced avidity for Fc receptors

et al. 2019

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Monoclonal antibodies (mAbs) have become an important class of therapeutics, particularly in the realm of anticancer immunotherapy. While the two antigen‐binding fragments (Fabs) of an mAb allow for high‐avidity binding to molecular targets, the crystallizable fragment (Fc) engages immune effector elements. mAbs of the IgG class are used for the treatment of autoimmune diseases and can elicit antitumor immune functions not only by several mechanisms including direct antigen engagement via their Fab arms but also by Fab binding to tumors combined with Fc engagement of complement component C1q and Fcγ receptors. Additionally, IgG binding to the neonatal Fc receptor (FcRn) allows for endosomal recycling and prolonged serum half‐life. To augment the effector functions or half‐life of an IgG1 mAb, we constructed a novel “2Fc” mAb containing two Fc domains in addition to the normal two Fab domains. Structural and functional characterization of this 2Fc mAb demonstrated that it exists in a tetrahedral‐like geometry and retains binding capacity via the Fab domains. Furthermore, duplication of the Fc region significantly enhanced avidity for Fc receptors FcγRI, FcγRIIIa, and FcRn, which manifested as a decrease in complex dissociation rate that was more pronounced at higher densities of receptor. At intermediate receptor density, the dissociation rate for Fc receptors was decreased 6‐ to 130‐fold, resulting in apparent affinity increases of 7‐ to 42‐fold. Stoichiometric analysis confirmed that each 2Fc mAb may simultaneously bind two molecules of FcγRI or four molecules of FcRn, which is double the stoichiometry of a wild‐type mAb. In summary, duplication of the IgG Fc region allows for increased avidity to Fc receptors that could translate into clinically relevant enhancement of effector functions or pharmacokinetics.

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Section: Discussionmentioning

confidence: 99%

Design and characterization of novel dual Fc antibody with enhanced avidity for Fc receptors

et al. 2019

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“…While increasing HC to LC expression ratio significantly improved Fab1 titer, the relationship between gene copy number and productivity does not seem to be linear and position of the transgene(s) within the integrated site also plays an important role in gene expression 24 . Increasing HC gene copy number ratios in a transient expression system has also been shown to reduce the levels of LC dimers, 26 likely in a similar fashion observed in this study, where excess HC molecules can complex with the LC molecules, reducing the levels of unpaired LC and improving effective titers.…”

Section: Discussionmentioning

confidence: 99%

Expressing antigen binding fragments with high titers in a targeted integration CHO host by optimizing expression vector gene copy numbers and position: A case study

Tang¹,

Gunson²,

Tran³

et al. 2022

Biotechnology Progress

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Antigen binding fragments (Fab) are a promising class of therapeutics as they maintain high potency while having significantly smaller size relative to full-length antibodies. Because Fab molecules are aglycosylated, many expression platforms, including prokaryotic, yeast, and mammalian cells, have been developed for their expression, with Escherichia coli being the most commonly used Fab expression system. In this study, we have examined production of a difficult to express Fab molecule in a targeted integration (TI) Chinese Hamster Ovary (CHO) host. Without a need for extensive host or process optimization, as is usually required for E. coli, by

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“…These fragments can be expressed and purified to homogeneity, and used in a complex with the protein of interest to promote crystallization. Importantly, Fab fragments produced as described in Section 10 can have a tendency to form non-functional LC dimers86. These dimers are contaminants and should be removed during purification.…”

Section: Discussionmentioning

confidence: 99%

Characterization of Glycoproteins with the Immunoglobulin Fold by X-Ray Crystallography and Biophysical Techniques

Ereño‐Orbea

Sicard

Cui

et al. 2018

JoVE

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Glycoproteins on the surface of cells play critical roles in cellular function, including signalling, adhesion and transport. On leukocytes, several of these glycoproteins possess immunoglobulin (Ig) folds and are central to immune recognition and regulation. Here, we present a platform for the design, expression and biophysical characterization of the extracellular domain of human B cell receptor CD22. We propose that these approaches are broadly applicable to the characterization of mammalian glycoprotein ectodomains containing Ig domains. Two suspension human embryonic kidney (HEK) cell lines, HEK293F and HEK293S, are used to express glycoproteins harbouring complex and high-mannose glycans, respectively. These recombinant glycoproteins with different glycoforms allow investigating the effect of glycan size and composition on ligand binding. We discuss protocols for studying the kinetics and thermodynamics of glycoprotein binding to biologically relevant ligands and therapeutic antibody candidates. Recombinant glycoproteins produced in HEK293S cells are amenable to crystallization due to glycan homogeneity, reduced flexibility and susceptibility to endoglycosidase H treatment. We present methods for soaking glycoprotein crystals with heavy atoms and small molecules for phase determination and analysis of ligand binding, respectively. The experimental protocols discussed here hold promise for the characterization of mammalian glycoproteins to give insight into their function and investigate the mechanism of action of therapeutics.

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Evaluation of strategies to control Fab light chain dimer during mammalian expression and purification: A universal one‐step process for purification of correctly assembled Fab

Cited by 12 publications

References 20 publications

Design and characterization of novel dual Fc antibody with enhanced avidity for Fc receptors

Design and characterization of novel dual Fc antibody with enhanced avidity for Fc receptors

Expressing antigen binding fragments with high titers in a targeted integration CHO host by optimizing expression vector gene copy numbers and position: A case study

Characterization of Glycoproteins with the Immunoglobulin Fold by X-Ray Crystallography and Biophysical Techniques

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