Blog References: What does the FDA's new biosimilars guidance actually require?

Requirement	Typical Analytical methods	Role of BLI
Primary Sequence and post-translational modifications	MS	–
Higher-order structure	CD, HDX-MS, FTIR	–
Size/charge variants	SEC, AUC, cIEF	–
*Target antigen binding: Affinity (K_D) and kinetics (k_off* and k_on)**	BLI, SPR	Primary method
Full Fc receptor panel (FcRn, FcγRI/IIa/IIb/IIIa/IIIb)	BLI	Primary method
Cell-based functional assays (ADCC, ADCP, CDC)	Varies	Supports via receptor binding data
Glycan Profiling linked to Fc receptor results	LC-MS	Orthogonal validation

ASSAY	CAPTURE AND LIGAND	LIGAND	ANALYTE	KEY PARAMETER REPORTED	REFERENCES
Antibody-antigen kinetics	Human Fc Gen II	Biosimilar, Reference	Antigen	Affinity (K_D), kinetics (k_off, k_on)
FcRn pH 6.0 binding	SA	Biotinylated FcRn	Biosimilar, Reference	k_on, response at end of association	[2] [3] [4]
FcRn pH 7.2 Release	SA (same tip and assay as above)	Biotinylated FcRn	Biosimilar, Reference	k_off	[2] [3] [4]
FcγRIIIa (CD16a) affinity	HIS XT	FcγRIIIa-His	Biosimilar, Reference	Relative binding response; K_D	[4]
FcγRIIa (CD32a) affinity	HIS XT	FcγRIIa-His	Biosimilar, Reference	Relative binding response; K_D	[4] [5]
FcγRI (CD64) affinity	HIS XT	FcγRI-His	Biosimilar, Reference	Relative binding response; K_D	[4]
C1q	ProL (direct capture), SA (antigen capture then Ab)	Biosimilar, Reference	C1q	Response	[6, 7, 8]
Epitope Binning	ProA or HFC Gen II (Sandwich); HIS XT (Tandem)	Sandwich: Ab -> Antigen -> Ab Tandem: Antigen -> Ab -> Ab		Blockage %	[Epitope Binning Guidebook]

References

[1]	U. D. o. H. a. H. Services, “new and Revised Draft Q&As on Biosimilar Development and the BPCI Act (Revision 4) Guidance for Industry,” 9 March 2026. [Online]. Available: https://www.fda.gov/media/119278/download.
[2]	S. Ko, M. Jo, M. Kyung, W. Lee, W. H. Ko, J.-H. Na, Y. S. Chun, B. J. Ko and S. t. Jung, “Engineering FcRn binding kinetics dramatically extends antibody serum half-life and enhances therapeutic potential,” Journal of Biological Engineering, vol. 19, 2025.
[3]	A. Bajardi-Taccioli, A. Blum, C. Xu, Z. Sosic, S. Bergelson and M. Feschenko, “Effect of protein aggregates on characterization of FcRn binding of Fc-fusion therapeutics,” Molecular Immunology, vol. 67, no. 2, pp. 616-624, 2015.
[4]	K. P. Geuijen, C. Oppers-Tiemissen, D. F. Egging, P. J. Simons, L. Boon, R. B. Shasfoort and M. H. Eppink, “Rapid screening of IgG quality attributes – effects on Fc receptor binding,” FEBS Open Bio, vol. 7, no. 10, pp. 1557-1574, 2017.
[5]	A. Bajardi-Tacciolo, C. Co, C. Bond, A. Masci, T. Brantley, C. Zu, S. Bergelson and M. Feschenko, “Biolayer Interferometry-based FcγRIIa binding assay for a therapeutic antibody with strong effector function,” Analytical Biochemistry, vol. 611, 2020.
[6]	W. Shou, S. Lin, R. Chen, J. Liu and Y. Li, “haracterization of antibody-C1q interactions by Biolayer Interferometry,” Anal Biochem., vol. 549, pp. 143-148, 2018.
[7]	A. Chouquet, A. J. Pinto, J. Hennicke, w. L. Ling, I. Bally, L. T. N. Schwagerlehner, R. Kunert and J.-B. Reiser, “Biophysical Characterization of the Oligomeric States of Recombinant Immunoglobulins Type-M and Their C1q-Binding Kinetics by Biolayer Interferometry,” Front Bioeng Biotechnol., vol. 10, 2022.
[8]	D. Jiao, Y. Hu, L. Liao, H. Zhang, L. Wang, H. Xiao and J. Tian, “Qualification and robustness study of a bio-layer interferometry-based C1q binding assay for therapeutic antibodies,” Journal of Pharmaceutical and Biomedical Analysis, vol. 268, 2026.
[9]	T. Neuber, K. Frese, J. Haehrling, S. Jager, D. F. K. Daubert, M. Linnemann, A. Hohne, S. Kaden, J. Kolin, T. Tiller, B. Brocks, R. Ostendorp and S. Pabst, “Characterization and screening of IgG binding to the neonatal Fc receptor,” mAbs, vol. 6, no. 4, pp. 928-942, 2014.
[10]	J. A. A. E. Golay and I. Cattaneo, “Role of the Fc Core Fucosylation in the Effector Function of IgG1 Antibodies,” Front. Immunol., vol. 13, 2022.
[11]	N. Pereira, K. F. Chan, P. C. Lin and S. Song, “The “less-is-more” in therapeutic antibodies: Afucosylated anti-cancer antibodies with enhanced antibody-dependent cellular cytotoxicity,” MAbs, vol. 10, no. 5, pp. 693-711, 2018.
[12]	M. B. J. Meudt, E. Machal, M. Knape, B. Miziakoff, S. Ebert, F. Rosenau, M. Blech and F. Higel, “Spotlight on Glycan Pairing: The Generation and Impact of Monoclonal Antibody Asymmetrial Fc N-glycan Pairs on Fc Receptor Interaction,” ACS Pharmacology & Translational Science, vol. 8, pp. 1756-1767, 2025.
[13]	S. Kohns, J. Shu, C. Xiang, R. De Guzman, Q. Zhang, W. Bretzlaf, N. Miscalichi, K. Kalenian and M. Joubert, “Differential influence on antibody dependent cellular phagocytosis by different glycoforms on therapeutic Monoclonal antibodies,” J Biotechnol., vol. 20, no. 317, pp. 5-15, 2020.
[14]	e. a. Rathore, “Analytical Similarity Assesment of Biosimilars: Global Regulatory Landscape, Recent studies and Major Advancements in Orthogonal Platforms,” Front. Bioeng. Biotechnol., vol. 10, 2022.