Immobilized recombinant FcγRIIIa and FcRn receptors as a tool for characterization of human IgG antibodies.

Intravenous immunoglobulin (IVIg) therapeutics are complex biological mixtures of immunoglobulin G (IgG) requiring robust analytical methods to resolve their functional and structural heterogeneity. In this study, we evaluated the chromatographic performance of receptor affinity columns utilizing low-affinity immunoglobulin gamma Fc region receptor IIIa (FcγRIIIa) and neonatal fragment crystallizable receptor (FcRn) ligands to profile two commercial IVIg products. Using optimized pH-gradient elution on analytical nonporous resin (NPR) columns, we demonstrated that the two receptors display distinct separation selectivities toward specific IgG Fc-glycoforms. Retention of the IgG glycoforms on the FcγRIIIa column was consistent with sensitivity to steric constraints, where elution appeared to be modulated by the degree of galactosylation. Conversely, the FcRn column displayed a separation mechanism likely driven by charge heterogeneity. Despite the distal location of the glycosylation site relative to the binding interface with IgG, the pH-gradient elution resolved sialylated subpopulations based on pI-mediated retention shifts. Both receptor affinity separations yielded highly reproducible, product-specific chromatographic fingerprints that enabled the differentiation of the studied commercial preparations. The observation of distinct, product-specific fingerprints validates receptor affinity chromatography as a versatile, orthogonal tool for IVIg quality control, capable of assessing functional composition and potential batch consistency through chromatographic traces alone.