Comparison of Ensemble and Single Molecule Methods for Particle Characterization and Binding Analysis of a PEGylated Single-Domain Antibody

Lumelle A. Schneeweis, Linda Obenauer-Kutner, Parminder Kaur, Aaron P. Yamniuk, James Tamura, Neil Jaffe, Brian W. O'Mara, Stuart Lindsay, Michael Doyle, James Bryson

Research output: Contribution to journalArticle

Abstract

Domain antibodies (dAbs) are single immunoglobulin domains that form the smallest functional unit of an antibody. This study investigates the behavior of these small proteins when covalently attached to the polyethylene glycol (PEG) moiety that is necessary for extending the half-life of a dAb. The effect of the 40 kDa PEG on hydrodynamic properties, particle behavior, and receptor binding of the dAb has been compared by both ensemble solution and surface methods [light scattering, isothermal titration calorimetry (ITC), surface Plasmon resonance (SPR)] and single-molecule atomic force microscopy (AFM) methods (topography, recognition imaging, and force microscopy). The large PEG dominates the properties of the dAb-PEG conjugate such as a hydrodynamic radius that corresponds to a globular protein over four times its size and a much reduced association rate. We have used AFM single-molecule studies to determine the mechanism of PEG-dependent reductions in the effectiveness of the dAb observed by SPR kinetic studies. Recognition imaging showed that all of the PEGylated dAb molecules are active, suggesting that some may transiently become inactive if PEG sterically blocks binding. This helps explain the disconnect between the SPR, determined kinetically, and the force microscopy and ITC results that demonstrated that PEG does not change the binding energy.

Original languageEnglish (US)
Pages (from-to)4015-4024
Number of pages10
JournalJournal of Pharmaceutical Sciences
Volume104
Issue number12
DOIs
StatePublished - Dec 1 2015

Fingerprint

Single-Domain Antibodies
Atomic Force Microscopy
Surface Plasmon Resonance
Calorimetry
Hydrodynamics
Half-Life
Proteins
Light
Antibodies

Keywords

  • calorimetry (ITC)
  • light scattering (static)
  • microscopy
  • PEGylation
  • proteins

ASJC Scopus subject areas

  • Pharmaceutical Science

Cite this

Comparison of Ensemble and Single Molecule Methods for Particle Characterization and Binding Analysis of a PEGylated Single-Domain Antibody. / Schneeweis, Lumelle A.; Obenauer-Kutner, Linda; Kaur, Parminder; Yamniuk, Aaron P.; Tamura, James; Jaffe, Neil; O'Mara, Brian W.; Lindsay, Stuart; Doyle, Michael; Bryson, James.

In: Journal of Pharmaceutical Sciences, Vol. 104, No. 12, 01.12.2015, p. 4015-4024.

Research output: Contribution to journalArticle

Schneeweis, LA, Obenauer-Kutner, L, Kaur, P, Yamniuk, AP, Tamura, J, Jaffe, N, O'Mara, BW, Lindsay, S, Doyle, M & Bryson, J 2015, 'Comparison of Ensemble and Single Molecule Methods for Particle Characterization and Binding Analysis of a PEGylated Single-Domain Antibody', Journal of Pharmaceutical Sciences, vol. 104, no. 12, pp. 4015-4024. https://doi.org/10.1002/jps.24624
Schneeweis, Lumelle A. ; Obenauer-Kutner, Linda ; Kaur, Parminder ; Yamniuk, Aaron P. ; Tamura, James ; Jaffe, Neil ; O'Mara, Brian W. ; Lindsay, Stuart ; Doyle, Michael ; Bryson, James. / Comparison of Ensemble and Single Molecule Methods for Particle Characterization and Binding Analysis of a PEGylated Single-Domain Antibody. In: Journal of Pharmaceutical Sciences. 2015 ; Vol. 104, No. 12. pp. 4015-4024.
@article{3da5ddfa9add487ea418cced7514d540,
title = "Comparison of Ensemble and Single Molecule Methods for Particle Characterization and Binding Analysis of a PEGylated Single-Domain Antibody",
abstract = "Domain antibodies (dAbs) are single immunoglobulin domains that form the smallest functional unit of an antibody. This study investigates the behavior of these small proteins when covalently attached to the polyethylene glycol (PEG) moiety that is necessary for extending the half-life of a dAb. The effect of the 40 kDa PEG on hydrodynamic properties, particle behavior, and receptor binding of the dAb has been compared by both ensemble solution and surface methods [light scattering, isothermal titration calorimetry (ITC), surface Plasmon resonance (SPR)] and single-molecule atomic force microscopy (AFM) methods (topography, recognition imaging, and force microscopy). The large PEG dominates the properties of the dAb-PEG conjugate such as a hydrodynamic radius that corresponds to a globular protein over four times its size and a much reduced association rate. We have used AFM single-molecule studies to determine the mechanism of PEG-dependent reductions in the effectiveness of the dAb observed by SPR kinetic studies. Recognition imaging showed that all of the PEGylated dAb molecules are active, suggesting that some may transiently become inactive if PEG sterically blocks binding. This helps explain the disconnect between the SPR, determined kinetically, and the force microscopy and ITC results that demonstrated that PEG does not change the binding energy.",
keywords = "calorimetry (ITC), light scattering (static), microscopy, PEGylation, proteins",
author = "Schneeweis, {Lumelle A.} and Linda Obenauer-Kutner and Parminder Kaur and Yamniuk, {Aaron P.} and James Tamura and Neil Jaffe and O'Mara, {Brian W.} and Stuart Lindsay and Michael Doyle and James Bryson",
year = "2015",
month = "12",
day = "1",
doi = "10.1002/jps.24624",
language = "English (US)",
volume = "104",
pages = "4015--4024",
journal = "Journal of Pharmaceutical Sciences",
issn = "0022-3549",
publisher = "John Wiley and Sons Inc.",
number = "12",

}

TY - JOUR

T1 - Comparison of Ensemble and Single Molecule Methods for Particle Characterization and Binding Analysis of a PEGylated Single-Domain Antibody

AU - Schneeweis, Lumelle A.

AU - Obenauer-Kutner, Linda

AU - Kaur, Parminder

AU - Yamniuk, Aaron P.

AU - Tamura, James

AU - Jaffe, Neil

AU - O'Mara, Brian W.

AU - Lindsay, Stuart

AU - Doyle, Michael

AU - Bryson, James

PY - 2015/12/1

Y1 - 2015/12/1

N2 - Domain antibodies (dAbs) are single immunoglobulin domains that form the smallest functional unit of an antibody. This study investigates the behavior of these small proteins when covalently attached to the polyethylene glycol (PEG) moiety that is necessary for extending the half-life of a dAb. The effect of the 40 kDa PEG on hydrodynamic properties, particle behavior, and receptor binding of the dAb has been compared by both ensemble solution and surface methods [light scattering, isothermal titration calorimetry (ITC), surface Plasmon resonance (SPR)] and single-molecule atomic force microscopy (AFM) methods (topography, recognition imaging, and force microscopy). The large PEG dominates the properties of the dAb-PEG conjugate such as a hydrodynamic radius that corresponds to a globular protein over four times its size and a much reduced association rate. We have used AFM single-molecule studies to determine the mechanism of PEG-dependent reductions in the effectiveness of the dAb observed by SPR kinetic studies. Recognition imaging showed that all of the PEGylated dAb molecules are active, suggesting that some may transiently become inactive if PEG sterically blocks binding. This helps explain the disconnect between the SPR, determined kinetically, and the force microscopy and ITC results that demonstrated that PEG does not change the binding energy.

AB - Domain antibodies (dAbs) are single immunoglobulin domains that form the smallest functional unit of an antibody. This study investigates the behavior of these small proteins when covalently attached to the polyethylene glycol (PEG) moiety that is necessary for extending the half-life of a dAb. The effect of the 40 kDa PEG on hydrodynamic properties, particle behavior, and receptor binding of the dAb has been compared by both ensemble solution and surface methods [light scattering, isothermal titration calorimetry (ITC), surface Plasmon resonance (SPR)] and single-molecule atomic force microscopy (AFM) methods (topography, recognition imaging, and force microscopy). The large PEG dominates the properties of the dAb-PEG conjugate such as a hydrodynamic radius that corresponds to a globular protein over four times its size and a much reduced association rate. We have used AFM single-molecule studies to determine the mechanism of PEG-dependent reductions in the effectiveness of the dAb observed by SPR kinetic studies. Recognition imaging showed that all of the PEGylated dAb molecules are active, suggesting that some may transiently become inactive if PEG sterically blocks binding. This helps explain the disconnect between the SPR, determined kinetically, and the force microscopy and ITC results that demonstrated that PEG does not change the binding energy.

KW - calorimetry (ITC)

KW - light scattering (static)

KW - microscopy

KW - PEGylation

KW - proteins

UR - http://www.scopus.com/inward/record.url?scp=84954366727&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84954366727&partnerID=8YFLogxK

U2 - 10.1002/jps.24624

DO - 10.1002/jps.24624

M3 - Article

VL - 104

SP - 4015

EP - 4024

JO - Journal of Pharmaceutical Sciences

JF - Journal of Pharmaceutical Sciences

SN - 0022-3549

IS - 12

ER -