Characterization of IgG1 Fc Deamidation at Asparagine 325 and Its Impact on Antibody-dependent Cell-mediated Cytotoxicity and FcγRIIIa Binding.

Antibody-dependent cell-mediated cytotoxicity (ADCC) is an important mechanism of action for many therapeutic antibodies. A therapeutic immunoglobulin (Ig) G ₁ monoclonal antibody lost more than half of its ADCC activity after heat stress at 40 °C for 4 months. Size-exclusion and ion-exchange chromatography were used to fractionate various size and charge variants from the stressed IgG ₁ . Physicochemical characterization of these fractions revealed that a rarely seen crystallizable fragment (Fc) modification, N325 deamidation, exhibited a positive correlation with the loss of ADCC activity. A further surface plasmon resonance study showed that this modification disrupted the binding between the IgG ₁ Fc and Fcγ receptor IIIa, resulting in decreased ADCC activity of the IgG ₁ antibody. Mutants of N325/D and N325/Q were made to confirm the effect of N325 deamidation on ADCC. We hypothesize that N325 deamidation altered the local three-dimensional structure, which might interfere with the binding and interaction with the effector cell. Because of its impact on biological activity, N325 deamidation is a critical quality attribute for products whose mechanism of action includes ADCC. A thorough understanding of the criticality of N325 deamidation and appropriate monitoring can help ensure the safety and efficacy of IgG ₁ or Fc-fusion products.