title: Effect of protein aggregates on characterization of FcRn binding of Fc-fusion therapeutics.
abstract: Recycling of antibodies and Fc containing therapeutic proteins by the neonatal Fc receptor (FcRn) is known to prolong their persistence in the bloodstream. Fusion of Fc fragment of IgG1 to other proteins is one of the strategies to improve their pharmacokinetic properties. Accurate measurement of Fc-FcRn binding provides information about the strength of this interaction, which in most cases correlates with serum half-life of the protein. It can also offer insight into functional integrity of Fc region. We investigated FcRn binding activity of a large set of Fc-fusion samples after thermal stress by the method based on AlphaScreen technology. An unexpected significant increase in FcR binding was found to correlate with formation of aggregates in these samples. Monomer purified from a thermally-stressed sample had normal FcRn binding, confirming that its Fc portion was intact. Experiments with aggregates spiked into a sample with low initial aggregation level, demonstrated strong correlation between the level of aggregates and FcRn binding. This correlation varied significantly in different methods. By introducing modifications to the assay format we were able to minimize the effects of aggregated species on FcRn binding, which should prevent masking functional changes of Fc-fusion protein. Biolayer interferometry (BLI) was used as an alternative method to measure FcRn binding. Both optimized AlphaScreen- and BLI-based assays were sensitive to structural changes in Fc portion of the molecule, such as oxidation of methionines 252 and 428, and therefore suitable for characterization of FcRn binding.
authors.affiliation: Department of Analytical Development, Biogen, Inc., 14 Cambridge Center, Cambridge, MA 02142, USA. Electronic address: adriana.bajardi@biogen.com.
authors.forename: Adriana
authors.initials: A
authors.lastname: Bajardi-Taccioli
authors.affiliation: Department of Analytical Development, Biogen, Inc., 14 Cambridge Center, Cambridge, MA 02142, USA. Electronic address: andy.blum@biogen.com.
authors.forename: Andrew
authors.initials: A
authors.lastname: Blum
authors.affiliation: Department of Analytical Development, Biogen, Inc., 14 Cambridge Center, Cambridge, MA 02142, USA. Electronic address: chongfeng.xu@biogen.com.
authors.forename: Chongfeng
authors.initials: C
authors.lastname: Xu
authors.affiliation: Department of Analytical Development, Biogen, Inc., 14 Cambridge Center, Cambridge, MA 02142, USA. Electronic address: zoran.sosic@biogen.com.
authors.forename: Zoran
authors.initials: Z
authors.lastname: Sosic
authors.affiliation: Department of Analytical Development, Biogen, Inc., 14 Cambridge Center, Cambridge, MA 02142, USA. Electronic address: Svetlana.Bergelson@biogen.com.
authors.forename: Svetlana
authors.initials: S
authors.lastname: Bergelson
authors.affiliation: Department of Analytical Development, Biogen, Inc., 14 Cambridge Center, Cambridge, MA 02142, USA. Electronic address: marina.feschenko@biogen.com.
authors.forename: Marina
authors.initials: M
authors.lastname: Feschenko
mesh_terms: D001681:Biological Assay; D002850:Chromatography, Gel; D015395:Histocompatibility Antigens Class I / Q000378:metabolism*; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000378:metabolism*; D007202:Indicators and Reagents; D007368:Interferometry; D008715:Methionine / Q000378:metabolism; D010084:Oxidation-Reduction; D066329:Protein Aggregates; D011485:Protein Binding; D011961:Receptors, Fc / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000627:therapeutic use*; D013312:Stress, Physiological; D013997:Time Factors; D017186:Titrimetry
chemical_list: D015395:Histocompatibility Antigens Class I; D007141:Immunoglobulin Fc Fragments; D007202:Indicators and Reagents; D066329:Protein Aggregates; D011961:Receptors, Fc; D011993:Recombinant Fusion Proteins; D008715:Methionine; C100688:Fc receptor, neonatal
keywords: Aggregates; AlphaScreen; Biolayer interferometry; Fc-fusion therapeutic; FcRn