2D NMR Analysis of the Effect of Asparagine Deamidation Versus Methionine Oxidation on the Structure, Stability, Aggregation, and Function of a Therapeutic Protein.

Two of the most common forms of chemical modifications that compromise the efficacy of therapeutic proteins are the deamidation of asparagine residues and oxidation of methionine residues. We probed how deamidation affects the structure, stability, aggregation, and function of interferon alpha-2a (IFNA2a), and compared with our earlier results on methionine oxidation. Upon deamidation, no significant changes were observed in the global secondary structure of IFNA2a with minor changes in its tertiary structure. However, deamidation destabilized the protein, and increased its propensity to aggregate under accelerated stress conditions. Cytopathic inhibition and antiproliferation assays showed drastic decrease in the functionality of deamidated IFNA2a compared to the wild-type. 2D NMR measurements showed structural changes in local protein regions, with no effect on the overall global structure of IFNA2a. These local protein regions corresponded well with the aggregation hot-spots predicted by computational programs, and the functional hot-spots identified by site-directed mutagenesis. When compared to the effects of methionine oxidation, deamidation caused lesser aggregation, because of lesser structural unfolding observed in aggregation hot-spots by 2D NMR. In comparison to oxidation, deamidation showed larger decrease in function, because deamidation affected key amino acid residues in functional hot-spots as observed by 2D NMR and structural modeling. Such quantitative comparison between the effects of deamidation and oxidation on a pharmaceutical protein has not been done before, and the high-resolution structural information on local protein regions obtained by 2D NMR provided a better insight compared to low-resolution methods that probe global protein structure.

Effect of Chemical Oxidation on the Higher Order Structure, Stability, Aggregation, and Biological Function of Interferon Alpha-2a: Role of Local Structural Changes Detected by 2D NMR.

PURPOSE: Oxidized interferons have been shown to aggregate and cause immunogenicity. In this study, the structural mechanisms underlying oxidation-induced interferon alpha-2a (IFNA2a) aggregation and loss of function were examined. METHODS: IFNA2a was oxidized using 0.037% vol/vol hydrogen peroxide. Oxidized protein was probed using biophysical methods that include denaturant melts, particle counting, proteolysis-coupled mass spectrometry, and 2D NMR. RESULTS: Oxidized IFNA2a did not show major changes in its secondary structure, but showed minor changes in tertiary structure when compared to the unoxidized protein. In addition, a significant loss of conformational stability was observed upon oxidation. Correspondingly, increased protein aggregation was observed resulting in the formation of sub-visible particles. Oxidized protein showed decreased biological function in terms of its anti-viral potency and cytopathic inhibition efficacy. Proteolysis-coupled mass spectrometry identified five methionine residues that were oxidized with no correlation between the extent of oxidation and their accessible surface area. 2D 15N-1H HSQC NMR identified residue-level local structural changes in the protein upon oxidation, which were not detectable by global probes such as far-UV circular dichroism and fluorescence. CONCLUSIONS: Increased protein aggregation and decreased function of IFNA2a upon oxidation correlated with the site of modification identified by proteolysis-coupled mass spectrometry and local structural changes in the protein detected by 2D NMR.

Effect of Peroxide- Versus Alkoxyl-Induced Chemical Oxidation on the Structure, Stability, Aggregation, and Function of a Therapeutic Monoclonal Antibody.

Current guidelines indicate that the effects of oxidation should be included as part of forced degradation studies on protein drugs. We probed the effect of 3 commonly used oxidants, hydrogen peroxide, tert-butyl hydroperoxide, and 2,2'-Azobis(2-amidinopropane) dihydrochloride (AAPH), on a therapeutic monoclonal IgG1 antibody (mAb8). Upon oxidation, mAb8 did not show noticeable changes in its secondary structure but showed minor changes in tertiary structure. Significant decrease in conformational stability was observed for all the 3 oxidized forms. Both hydrogen peroxide and tert-butyl hydroperoxide destabilized mainly the CH2 domain, whereas AAPH destabilized the variable domain in addition to CH2. Increased aggregation was found for AAPH-oxidized mAb8. In addition, a significant decrease in Fc receptor binding was observed for all 3 oxidized forms. Antibody dependent cell-mediated cytotoxicity, binding to target protein receptor, and cell proliferation activity were significantly reduced in the case of AAPH-oxidized mAb8. The presence of free methionine in the formulation buffer seems to alleviate the effect of oxidation. The results of this study show that the 3 oxidants differ in terms of their effects on the structure and function of mAb8 because of chemical modification of different sets of residues located in Fab versus Fc.

Effect of photo-degradation on the structure, stability, aggregation, and function of an IgG1 monoclonal antibody.

Photostability testing of therapeutic proteins is a critical requirement in the development of biologics. Upon exposure to light, pharmaceutical proteins may undergo a change in structure, stability, and functional properties that could have a potential impact on safety and efficacy. In this work, we studied how exposure to light, according to ICH guidelines, leads to photo-oxidation of a therapeutic IgG1 mAb. We also tested the ability of five different excipients to prevent such oxidation. In samples that were exposed to light, we found that the CH2 domain was considerably destabilized but there were no major changes in the overall structure of the protein. Aggregation of the protein was observed because of light exposure. Mass spectrometry identified that light exposure oxidizes two key methionine residues in the Fc region of the protein. In terms of function, a loss in binding to the neonatal Fc receptor, decreased antibody-dependent cell-mediated cytotoxicity and cell proliferation activities of the protein were seen. Combined analysis of the photo-oxidation effects on the structure, stability, aggregation, and function of the mAb has identified the underlying unifying mechanism. Among the sugars and amino acids tested, methionine was the most effective in protecting mAb against photo-oxidation.

Biophysical analysis of the effect of chemical modification by 4-oxononenal on the structure, stability, and function of binding immunoglobulin protein (BiP).

Binding immunoglobulin protein (BiP) is a molecular chaperone important for the folding of numerous proteins, which include millions of immunoglobulins in human body. It also plays a key role in the unfolded protein response (UPR) in the endoplasmic reticulum. Free radical generation is a common phenomenon that occurs in cells under healthy as well as under stress conditions such as ageing, inflammation, alcohol consumption, and smoking. These free radicals attack the cell membranes and generate highly reactive lipid peroxidation products such as 4-oxononenal (4-ONE). BiP is a key protein that is modified by 4-ONE. In this study, we probed how such chemical modification affects the biophysical properties of BiP. Upon modification, BiP shows significant tertiary structural changes with no changes in its secondary structure. The protein loses its thermodynamic stability, particularly, that of the nucleotide binding domain (NBD) where ATP binds. In terms of function, the modified BiP completely loses its ATPase activity with decreased ATP binding affinity. However, modified BiP retains its immunoglobulin binding function and its chaperone activity of suppressing non-specific protein aggregation. These results indicate that 4-ONE modification can significantly affect the structure-function of key proteins such as BiP involved in cellular pathways, and provide a molecular basis for how chemical modifications can result in the failure of quality control mechanisms inside the cell.

Missense mutation Lys18Asn in dystrophin that triggers X-linked dilated cardiomyopathy decreases protein stability, increases protein unfolding, and perturbs protein structure, but does not affect protein function.

Genetic mutations in a vital muscle protein dystrophin trigger X-linked dilated cardiomyopathy (XLDCM). However, disease mechanisms at the fundamental protein level are not understood. Such molecular knowledge is essential for developing therapies for XLDCM. Our main objective is to understand the effect of disease-causing mutations on the structure and function of dystrophin. This study is on a missense mutation K18N. The K18N mutation occurs in the N-terminal actin binding domain (N-ABD). We created and expressed the wild-type (WT) N-ABD and its K18N mutant, and purified to homogeneity. Reversible folding experiments demonstrated that both mutant and WT did not aggregate upon refolding. Mutation did not affect the protein's overall secondary structure, as indicated by no changes in circular dichroism of the protein. However, the mutant is thermodynamically less stable than the WT (denaturant melts), and unfolds faster than the WT (stopped-flow kinetics). Despite having global secondary structure similar to that of the WT, mutant showed significant local structural changes at many amino acids when compared with the WT (heteronuclear NMR experiments). These structural changes indicate that the effect of mutation is propagated over long distances in the protein structure. Contrary to these structural and stability changes, the mutant had no significant effect on the actin-binding function as evident from co-sedimentation and depolymerization assays. These results summarize that the K18N mutation decreases thermodynamic stability, accelerates unfolding, perturbs protein structure, but does not affect the function. Therefore, K18N is a stability defect rather than a functional defect. Decrease in stability and increase in unfolding decrease the net population of dystrophin molecules available for function, which might trigger XLDCM. Consistently, XLDCM patients have decreased levels of dystrophin in cardiac muscle.