Dissertation/Thesis Abstract

Structural and Functional Characterization into the Impact of Non-enzymatic Protein Glycation on a Therapeutic Monoclonal Antibody
by Geist, Brian J., Ph.D., University of the Sciences in Philadelphia, 2013, 174; 3586350
Abstract (Summary)

Various posttranslational modifications (PTMs) occurring systemically or during manufacture/production/formulation can impact the structure and function of therapeutic monoclonal antibodies (mAbs). Non-enzymatic protein glycation is highly associated with hyperglycemia and elevated glycation levels of long-lived proteins, such as hemoglobin, albumin, immunoglobulin G (IgG), have been reported in diabetic populations. Although data has demonstrated that glycation can alter the structure and function of proteins, little is known regarding the association between in vivo protein glycation and the potential impact on therapeutic mAb interaction with endogenous targets as well as the result on the pharmacokinetic (PK) profile and biological activity. The following dissertation summarizes research aimed at characterizing the impact of non-enzymatic protein glycation on the structure and function of a therapeutic mAb, ustekinumab (CNTO 1275).

The current research investigated the potential effect that protein glycation could have on the interaction between a therapeutic mAb and the anticipated biological targets. Of particular interest with regard to PK was the functionality of the mAb within the FcRn receptor salvage pathway. To replicate glycation under the in vivo setting, an in vitro reaction model was conducted in buffer and serum under appropriate physiological conditions. Positive incorporation of glycation adducts (i.e. addition of +162 daltons (Da)) on the therapeutic mAb structure were identified separately for both light and heavy chains as well as for the distinct Fab and Fc domains. The in vitro buffer glycation model demonstrated the modification to be both concentration and time-dependent, but differed in profile from the serum glycation model. The extent of glycated IgG incorporation was also shown to be dependent on the degree of solvent-exposed residues (Fc domain ≥ light chain > Fab heavy chain). No significant biophysical alteration to structural stability or increased aggregation was noted between the highly glycated and non-modified mAb. Investigation into the FcRn:mAb complex through a comprehensive analytical approach did reveal a significant difference toward decreased FcRn interaction dependent on the extent of glycation on the mAb. However, these results were only observed through the competitive binding analysis. Furthermore, incorporating oxidized residues in the Fc domain negatively impacted receptor affinity, stability of the complex, and overall binding to FcRn when compared to the highly glycated variants or non-modified controls. Evaluation of target (interleukin-12/23 p40) and Fc gamma receptor (FcγRI and FcγRIIIa) binding displayed minor differences between the highly glycated and non-glycated samples. In summary, this research suggests that non-enzymatic glycation shows a limited capacity to influence the structural stability of a therapeutic mAb even at very high incorporation levels. In addition, in vitro molecular interaction between IL-12, Fcγ receptors, and FcRn with highly glycated mAb remained relatively unaffected. Nevertheless, under conditions resembling the in vivo environment, protein glycation and the subsequent affect on mAb function appears to exhibit distinct behavior when compared to that observed during in vitro analysis.

Indexing (document details)
Advisor: Li, Zhiyu
Commitee: D'Mello, Anil P., Davis, Darryl, Harrison, Peter J., Pendley, Charles
School: University of the Sciences in Philadelphia
Department: Pharmacology and Toxicology
School Location: United States -- Pennsylvania
Source: DAI-B 75/07(E), Dissertation Abstracts International
Subjects: Pharmacology
Keywords: Fc gamma receptor, Monoclonal antibody, Non-enzymatic glycation, Pharmacokinetics, Posttranslational modifications, Ustekinumab
Publication Number: 3586350
ISBN: 978-1-303-86727-9
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