Screening for protein–protein interactions with asymmetrical flow field-flow fractionation

Published:February 24, 2021DOI:


      We describe a new method for screening protein-protein interaction of biopharmaceutical molecules at dilute concentrations to predict development issues at high concentration. The method is based on Asymmetrical Flow Field-Flow Fractionation (AF4) measurements using well known effects of protein-protein attraction on the fractionation profile due to elevated protein concentrations occurring close to the membrane. We explore the effect for 4 different monoclonal antibodies and show that the profiles obtained are quite different. Interestingly, we find that the recovery in AF4 correlates with the diffusion interaction parameter, which is a standard method for the analysis of protein-protein attraction. The results are insensitive to the protein concentration and buffer composition of the sample solution and only depend on the absolute amount of protein loaded and on the running buffer. This makes the method highly suitable for developability assessment in a compound discovery workflow.
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        • Jarasch A
        • Koll H
        • Regula J T
        • Bader M
        • Papadimitriou A
        • Kettenberger H.
        Developability assessment during the selection of novel therapeutic antibodies.
        J Pharm Sci. 2015; 104: 1885-1898
        • Jain T
        • Sun T
        • Durand S
        • Hall A
        • Houston N R
        • Nett J H
        • Sharkey B
        • Bobrowicz B
        • Caffry I
        • Yu Y
        • Cao Y
        • Lynaugh H
        • Brown M
        • Baruah H
        • Gray L T
        • Krauland E M
        • Xu Y
        • Vásquez M
        • Wittrup KD
        Biophysical properties of the clinical-stage antibody landscape.
        PNAS. 2017; 114: 944-949
        • Wolf Pérez A-M
        • Sormanni P
        • Andersen J S
        • Sakhinini L I
        • Rodriguez-Leon I
        • Bjelke J R
        • Gajhede A J
        • De Maria L
        • Otzen D E
        • Vendruscolo M
        • Lorenzen N
        In vitro and in silico assessment of the developability of a designed monoclonal antibody library.
        mAbs. 2019; 11: 388-400
        • Connolly B D
        • Petry C
        • Yadav S
        • Demeule B
        • Ciaccio N
        • Moore J M R
        • Shire S J
        • Gokarn YR.
        Weak interactions govern the viscosity of concentrated antibody solutions: high-throughput analysis using the diffusion interaction parameter.
        Biophys J. 2012; 103: 69-78
        • Starr C G
        • Tessier P M
        Selecting and engineering monoclonal antibodies with drug-like specificity.
        Curr Opin Biotechnol. 2019; 60: 119-127
        • Kingsbury J S
        • Saini A
        • Auclair S M
        • Fu L
        • Lantz M M
        • Halloran K T
        • Calero-Rubio C
        • Schwenger W
        • Airiau C Y
        • Zhang J
        • Gokarn YR.
        A single molecular descriptor to predict solution behavior of therapeutic antibodies.
        Sci Adv. 2020; 6: eabb0372
        • Yadav S
        • Shire S J
        • Kalonia D S
        Viscosity behavior of high-concentration monoclonal antibody solutions: correlation with interaction parameter and electroviscous effects.
        J Pharm Sci. 2012; 101: 998-1011
        • Wu J
        • Schultz J S
        • Weldon C L
        • Sule S V
        • Chai Q
        • Geng S B
        • Dickinson C D
        • Tessier P M
        Discovery of highly soluble antibodies prior to purification using affinity-capture self-interaction nanoparticle spectroscopy.
        Protein Eng Des Sel. 2015; 28: 403-414
        • Liu Y
        • Caffry I
        • Wu J
        • Geng S B
        • Jain T
        • Sun T
        • Reid F
        • Cao Y
        • Estep P
        • Yu Y
        • Vásquez M
        • Tessier P M
        • Xu Y.
        High-throughput screening for developability during early-stage antibody discovery using self-interaction nanoparticle spectroscopy.
        MAbs. 2014; 6: 483-492
        • Wahlund K-G
        • Giddings J C
        Properties of an asymmetrical flow field-flow fractionation channel having one permeable wall.
        Anal Chem. 1987; 59: 1332-1339
        • Wahlund K-G.
        Chapter 18 Asymmetrical flow field-flow fractionation in.
        in: Schimpf M E Caldwell K Giddings J C. Field-Flow Fractionation Handbook. Wiley-Interscience, New York2000: 279-294
        • Wahlund K-G.
        Flow field-flow fractionation: critical overview.
        J Chromatogr A. 2013; 1287: 97-112
        • Yohannes G
        • Jussila M
        • Hartonen K
        • Riekkola ML
        Asymmetrical flow field-flow fractionation technique for separation and characterization of biopolymers and bioparticles.
        J Chromatogr A. 2011; 1218: 4104-4116
        • Fraunhofer W
        • Winter G.
        The use of asymmetrical flow field-flow fractionation in pharmaceutics and biopharmaceutics.
        Eur J Pharm Biopharm. 2004; 58: 369-383
        • Leeman M
        • Storm M U
        • Nilsson L.
        Practical applications of asymmetrical flow field-flow fractionation (AF4): a review.
        LCGC. 2015; (December): 642-651
        • Gigault J
        • Pettibone J M
        • Schmitt C
        • Hackley VA
        Rational strategy for characterization of nanoscale particles by asymmetric-flow field flow fractionation: a tutorial.
        Anal Chim Acta. 2014; 80
        • Cao S
        • Pollastrini J
        • Jiang Y.
        Separation and characterization of protein aggregates and particles by field flow fractionation.
        Curr Pharm Biotechnol. 2009; 10: 382-390
        • Caldwell KD
        • Brimhall S L
        • Gao Y
        • Giddings JC
        Sample overloading effects in polymer characterization by field-flow fractionation.
        J Appl Polym Sci. 1988; 36: 703-719
        • Arfvidsson C
        • Wahlund K-G.
        Mass overloading in the flow field-flow fractionation channel studied by the behaviour of the ultra-large wheat protein glutenin.
        J Chromatogr A. 2003; 1011: 99-109
        • Litzén A
        • Wahlund K-G.
        Effects of temperature, carrier composition and sample load in asymmetrical flow field-flow fractionation.
        J Chromatogr. 1991; 548: 393-406
        • Benincasa MA
        • Giddings JC
        Separation and molecular weight distribution of anionic and cationic water-soluble polymers by flow field-flow fractionation.
        Anal Chem. 1992; 64: 790-798
        • Benincasa MA
        Chapter 27 Synthetic polymer-water soluble in.
        in: Schimpf ME Caldwell K Giddings JC Field-Flow Fractionation Handbook. Wiley-Interscience, New York2000: 407-432
        • Moon MH
        • Park I
        • Kin Y.
        Size characterization of liposomes by flow-field flow fractionation and photon correlation spectroscopy, effect of ionic strength and pH of carrier solution.
        J Chromatogr A. 1998; 813: 91-100
        • Marioli M
        • Kok W T
        Recovery, overloading, and protein interactions in asymmetrical flow field-flow fractionation.
        Anal Bioanal Chem. 2019; 411: 2327
        • Wolf Pérez A M
        • Sormanni P
        • Andersen J S
        • Sakhnini L I
        • Rodriguez-Leon I
        • Bjelke J R
        • Gajhede A J
        • De Maria L
        • Otzen D E
        • Vendruscolo M
        • Lorenzen N
        In vitro and in silico assessment of the developability of a designed monoclonal antibody library.
        mAbs. 2019; 11: 388-400
        • Kopp M R G
        • Wolf Pérez A M
        • Zucca M V
        • Palmiero U C
        • Friedrichsen B
        • Lorenzen N
        • Arosio P
        An accelerated surface-mediated stress assay of antibody instability for developability studies.
        mAbs. 2020; 12e815995
        • Neergaard M S
        • Kalonia D S
        • Parshad H
        • Nielsen A D
        • Møller E H
        • van de Weert M.
        Viscosity of high concentration protein formulations of monoclonal antibodies of the IgG1 and IgG4 subclass - prediction of viscosity through protein-protein interaction measurements.
        Eur J Pharm Sci. 2013; 49: 400-410
        • Ma D
        • Martin N
        • Tribet C
        • Winnik F M
        Quantitative characterization by asymmetrical flow field flow fractionation of IgG thermal aggregation with and without polymer protective agents.
        Anal Bioanal Chem. 2014; 406: 7539-7547