Field flow fractionation for assessing neonatal Fc receptor and Fcγ receptor binding to monoclonal antibodies in solution

Pollastrini, Joey; Dillon, Thomas M.; Bondarenko, Pavel V.; Chou, Robert Y.-T.

doi:10.1016/j.ab.2011.03.001

Cited by 30 publications

(24 citation statements)

References 48 publications

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“…Recently, AF4 has been suggested as a powerful method for investigating weak protein-protein interactions (K d 4mM) in solution 43 . In the AF4 elution profiles of a PS solution (Fig.…”

Section: Resultsmentioning

confidence: 99%

Hyperthin nanochains composed of self-polymerizing protein shackles

et al. 2013

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Protein fibrils are expected to have applications as functional nanomaterials because of their sophisticated structures; however, nanoscale ordering of the functional units of protein fibrils remains challenging. Here we design a series of self-polymerizing protein monomers, referred to as protein shackles, derived from modified recombinant subunits of pili from Streptococcus pyogenes. The monomers polymerize into nanochains through spontaneous irreversible covalent bond formation. We design the protein shackles so that their reactions can be controlled by altering redox conditions, which affect disulphide bond formation between engineered cysteine residues. The interaction between the monomers improves their polymerization reactivity and determines morphologies of the polymers. In addition, green fluorescent protein-tagged protein shackles can polymerize, indicating proteins can be stably attached to the nanochains with its functionality preserved. Furthermore we demonstrate that a molecular-recognizable nanochain binds to its partner with an enhanced binding ability in solution. These characteristics are expected to be applied for novel protein nanomaterials.

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“…Recently, AF4 has been suggested as a powerful method for investigating weak protein-protein interactions (K d 4mM) in solution 43 . In the AF4 elution profiles of a PS solution (Fig.…”

Section: Resultsmentioning

confidence: 99%

Hyperthin nanochains composed of self-polymerizing protein shackles

et al. 2013

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“…17,18 Using asymmetric flow field flow fraction and liquid chromatography-mass spectrometry, IgG:FcRn:albumin binding was determined to be at a 1:2:1 molar ratio. 19 Data regarding whether FcRn can mediate albumin transcytosis or ©2 0 1 1 L a n d e s B i o s c i e n c e .…”

Section: Fundamental Biology Of Fcrnmentioning

confidence: 99%

Neonatal Fc receptor and IgG-based therapeutics

Kuo

Aveson²

2011

mAbs

206

199

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“…Some of the methods used to assay FcRn-rhumAb interactions include surface plasmon resonance (SPR) (Biacore, ProteOn), 13,17,21,22,23,24,25,26 biolayer interferometry (Octet), 26 isothermal titration calorimetry, 27 enzyme-linked immunosorbent assays (ELISA), 28,29 cellbased assays, 30,31 AlphaScreen, 32 affinity chromatography, 33 and asymmetrical flow field flow fractionation. 34 Among these technologies, SPR-based biosensor assays are the most commonly used, likely due to the fact that they can provide real-time quality data on binding specificity and kinetics over a wide range of binding affinities.…”

Section: Introductionmentioning

confidence: 99%

Impact of SPR biosensor assay configuration on antibody: Neonatal Fc receptor binding data

Wang¹,

McKay²,

Yee³

et al. 2016

mAbs

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Binding interactions with the neonatal Fc receptor (FcRn) are one determinant of pharmacokinetic properties of recombinant human monoclonal antibody (rhumAb) therapeutics, and a conserved binding motif in the crystallizable fragment (Fc) region of IgG molecules interacts with FcRn. Surface plasmon resonance (SPR) biosensor assays are often used to characterize interactions between FcRn and rhumAb therapeutics. In such assays, generally either the rhumAb (format 1) or the FcRn protein (format 2) is immobilized on a biosensor chip. However, because evidence suggests that, in some cases, the variable domains of a rhumAb may also affect FcRn binding, we evaluated the effect of SPR assay configuration on binding data. We sought to assess FcRn binding properties of 2 rhumAbs (rhumAb1 and rhumAb2) to FcRn proteins using these 2 biosensor assay formats. The two rhumAbs have greater than 99% sequence identity in the Fc domain but differ in their Fab regions. rhumAb2 contains a positively charged patch in the variable domain that is absent in rhumAb1. Our results showed that binding of rhumAb1 to FcRn was independent of biosensor assay configuration, while binding of rhumAb2 to FcRn was highly SPR assay configuration dependent. Further investigations revealed that the format dependency of rhumAb2-FcRn binding is linked to the basic residues that form a positively charged patch in the variable domain of rhumAb2. Our work highlights the importance of analyzing rhumAb-FcRn binding interactions using 2 alternate SPR biosensor assay configurations. This approach may also provide a simple way to identify the potential for non-Fc-driven FcRn binding interactions in otherwise typical IgGs.

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Field flow fractionation for assessing neonatal Fc receptor and Fcγ receptor binding to monoclonal antibodies in solution

Cited by 30 publications

References 48 publications

Hyperthin nanochains composed of self-polymerizing protein shackles

Hyperthin nanochains composed of self-polymerizing protein shackles

Neonatal Fc receptor and IgG-based therapeutics

Impact of SPR biosensor assay configuration on antibody: Neonatal Fc receptor binding data

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