Human IgG2 antibodies contain three classes of disulfide isoforms, based on the number of Fab arms having disulfide links to the heavy chain hinge region.  In the IgG2-B form, both Fab arms have interchain disulfide bonds to the hinge region, and in IgG2-A, neither Fab arm are disulfide linked to the hinge.  The IgG2-A/B is a hybrid between these two forms, with only one Fab arm disulfide linked to the hinge. Those isoforms can be separated and quantified by non-reducing RP-HPLC.  For both types of IgG2 molecules, IgG2? and IgG2?, four main peaks elute in the order of B, A/B and A1 and A2. Though the same elution order, IgG2? and IgG2? display different profiles in the intensities of those peaks.Studies of the IgG2 disulfide isofoms have revealed several interesting properties.  Changes in the relative levels of these forms over time were observed while IgG2 circulates in humans, suggesting IgG2- A1 -> IgG2-A/B -> IgG2-B conversion.  Further studies of conversion kinetics in vitro under physiological conditions have been completed using a flow-through dialysis system that mimics blood-like redox state. For two IgG2? antibodies, results in vitro closely matched the kinetics observed in vivo, indicating that the changes observed in vivo were solely conversions between isoforms, not differential clearance of specific forms.  Moreover, using material enriched in the IgG2-A1 forms, we were able to probe the isoform conversion in vivo and study the kinetics more extensively in vitro. The results unveiled that the IgG2-A1 -> IgG2-A/B rate was similar between IgG2? and IgG2? antibodies.  In IgG2? antibodies, conversion of IgG2-A/B -> IgG2-B was slower than the IgG2-A1-> IgG2-A/B reaction. On the other hand, in IgG2? antibodies, little IgG2-A/B ->IgG2-B conversion was detected under physiological conditions. Thus, small differences in the C-terminus of the light chain sequences affect the disulfide conversion kinetics and impact the IgG2 disulfide isoforms produced in vivo. In addition, the studies also uncovered that the IgG2-A2 did not appear to change over time in vivo or in vitro for both IgG2? and IgG2? antibodies.  A more in-depth study of A2 using enriched samples indicated that its hinge disulfides were protected from reduction and conversion.  The first two disulfides in the hinge are responsible for this protection.

In-depth study of human IgG2 disulfide isoform conversions

247th National Meeting (2014)

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