Glycation of monoclonal antibodies impairs their ability to bind antigen

Abstract

SUMMARYAs elevated levels of glycated IgG have been detected in the plasma of patients with diabetes mellitus, a disease associated with increased susceptibility to infection, we have investigated whether glycation of MoAbs affects the kinetics and/or affinity of antigen binding. Three mouse MoAbs were incubated with 0-5 M glucose at pH 7-4 for 14-21 days at 37°C. Control MoAbs were incubated using identical conditions but with no added glucose. Using a surface plasmon resonance technique we found that glycation significantly increased the rate of dissociation (kdiss) of the antigen-antibody complex for all three MoAbs (P < 0*05, n = 4), but had no significant effect on the rate of association (kass). For one of the MoAbs, against human IgG (Fab), we also measured kdiS. by an alternative method utilizing radiolabelled antigen, which confirmed that glycation of the antibody significantly increases kdiss (P < 0001, n = 8). We also found using an ELISA-based method that glycation of the same MoAb significantly increased the equilibrium dissociation constant (Kd) (P < 0 05, n = 6). A significant increase in kd was observed after glycation using glucose concentrations consistent with those found in poorly controlled diabetics (P < 0 02, n = 5). We conclude that in vitro glycation can significantly lower the affinity of an antibody for its antigen, and significantly increases the rate of dissociation of the antigenantibody complex.

Advanced Glycation End Products (AGEs) Damaged IgG, a Target for Circulating Autoantibodies in Patients with Type 1 Diabetes Mellitus

“…Glycation of IgG is of special interest due to its influence on the functionality of immunoglobulin's and overall immunocompetence. Several studies showed the extent of glycation of IgG and its influence on the biological functionality of IgG [5][6][7][8][9][10][11][12][13], but the immunological role of AGEs damage IgG (AGE-IgG) in patients with type 1 DM is still not fully understood. Proper extensive clinical investigation of this aspect with a high level of confidence are needed.To meet this need, we hypothesized that AGE-IgG helps to initiate autoimmunity in type 1 diabetes mellitus patients.…”

Non enzymatic glycosylation of IgG and their urinary excretion in patients with diabetic nephropathy

“…The levels of nonenzymatically glycosylated total plasma proteins, albumin and IgG have been measured in the diabetics and non-diabetics patients in past (26)(27)(28)(29) and a significant increase in both glycosylated albumin and IgG, in diabetics were reported. These evidences also support our present findings.…”

Glycation of antibodies: Modification, methods and potential effects on biological functions

“…Glycation occurs at a number of sites and can be produced by forced stress conditions (95.4% overall glycation), and physiological cell culture antibody manufacturing conditions (10-40% glycation). 5,8,37,64,65 It is evident that glycation levels increase in stress exposure conditions with increased sugar concentration in the formulation, higher storage temperature, and time in storage. The glycation sites in mAbs manufactured under normal cell culture conditions with standard physiological parameters, such as temperature and pH, with elevated glucose levels, are similar to endogenous antibodies.…”

“…The glycation sites in mAbs manufactured under normal cell culture conditions with standard physiological parameters, such as temperature and pH, with elevated glucose levels, are similar to endogenous antibodies. 8,37 The effect of glycation on antibody activities ranged from no effect 5,8,37,65 to loss of activity, 64,66 although typically mAb glycation does not appear to substantially affect antibody binding activities. The effects of AGEs on antibodies have not been reported; however, it was reported that AGEs could potentially reduce the activity of the therapeutic protein interferon alfacon-1.…”

“…67 Glycation did not affect potency. Lysine mutation to arginine had no effect on potency Kennedy, et al 64 In vitro glycation can significantly lower the affinity of an antibody for its antigen, and significantly increases the rate of dissociation of the antigen-antibody complex Whole mouse IgG glycated for 21 days at 37 C using glucose concentrations of 0-, 15-, 50-, and 500-mM…”