Anti-Human IgG3 monoclonal antibody, clone TJQD4496 [Biotin]

Immunoglobulin G (IgG) consists of four subclasses in humans: IgGl, IgG2, IgG3 and IgG4, which are highly conserved but have unique differences that result in subclass-specific effector functions. Though IgG1 is the most extensively studied IgG subclass, study of other subclasses is important to understand overall immune function and for development of new therapeutics. When compared to IgGl, IgG3 exhibits a similar binding profile to Fey receptors and stronger activation of complement. All IgG subclasses are glycosylated at N297, which is required for Fcy receptor and Clq complement binding as well as maintaining optimal Fc conformation. We have determined the crystal structure of homogenously glycosylated human IgG3 Fc with a GlcNAc2Man5 (Man5) high mannose glycoform at 1.8 A resolution and compared its structural features with published structures from the other IgG subclasses. Although the overall structure of IgG3 Fc is similar to that of other subclasses, some structural perturbations based on sequence differences were revealed. For instance, the presence of R435 in IgG3 (and H435 in the other IgG subclasses) has been implicated to result in IgG3-specific properties related to binding to protein A, protein G and the neonatal Fc receptor (FcRn). The IgG3 Fc structure helps to explain some of these differences. Additionally, protein-glycan contacts observed in the crystal structure appear to correlate with IgG3 affinity for Fcy receptors as shown by binding studies with IgG3 Fc glycoforms. Finally, this IgG3 Fc structure provides a template for further studies aimed at engineering the Fc for specific gain of function.

As therapeutic monoclonal antibodies (mAbs) become more humanized, traditional tryptic peptide approaches used to measure biologics in serum become more challenging since unique clonotypic peptides used for quantifying the mAb may also be found in the normal serum polyclonal background. An alternative approach is to monitor the unique molecular mass of the intact light chain portion of the mAbs using liquid chromatography-mass spectrometry (LC-MS). Distinguishing a therapeutic mAb from a patient's normal polyclonal immunoglobulin (Ig) repertoire is the primary limiting factor when determining the limit of quantitation (LOQ) in serum. The ability to selectively extract subclass specific Igs from serum reduces the polyclonal background in a sample. We present here the development of an LC-MS method to quantify eculizumab in serum. Eculizumab is a complement component 5 (C5) binding mAb that is fully humanized and contains portions of both IgG2 and IgG4 subclasses. Our group developed a method that uses Life Technologies CaptureSelect IgG4 (Hu) affinity matrix. We show here the ability to quantitate eculizumab with a LOQ of 5 mcg/mL by removing the higher abundance IgG1, IgG2, and IgG3 from the polyclonal background, making this approach a simple and efficient procedure.