Ligand-Bound Forced Degradation as a Strategy to Generate Functionally Relevant Analytical Challenge Materials for Assessment of CQAs

Abstract

Therapeutic monoclonal antibodies (mAbs) contain a variety of amino acids that are susceptible to enzymatic, chemical, and physical modifications. These modifications can happen throughout production, purification, formulation, and storage and many are known to affect the biological activity of a mAb. Methods that are able to characterize and evaluate these attributes are critical in order to understand how they might alter biological activity. Methods capable of site-specific monitoring of these critical quality attributes are extremely valuable to biopharmaceutical research but also require well-defined materials with site-specific attribute modifications. Here, we describe the development and application of a strategy to generate functionally relevant analytical challenge materials that have unique site-specific attributes. This method involves the use of a ligand that is bound to the mAb during oxidative stress resulting in unique oxidation patterns with some methionine residues protected while others are exposed to oxidation. These unique materials were used to develop a rapid surface plasmon resonance (SPR) assay that could detect methionine oxidation in both the Fab and Fc regions using specific molecular probes. The addition of uniquely oxidized materials to our data set enabled us to determine specific methionine residues vital to binding. Further analysis showed that antibody oxidation could also be rapidly detected in multiple domains from qualitative thermal melting using intrinsic tryptophan fluorescence. Methionine oxidation of an antibody was explored in this study, but we envision this method could be useful to explore structure function relationships of a variety of antibody modifications and modifications to other biologically relevant protein drugs.

Correlated analytical and functional evaluation of higher order structure perturbations from oxidation of NISTmAb

“…Met oxidation in mAbs can alter biological activity by diminishing ligand binding to both the Fc and Fab regions. 18 , 36 Here, to correlate the NMR and stability methods to a functional change, the binding capacity of oxidized NISTmAb was assessed by three binding partners that are known to have distinct binding sites on mAbs. Two of the partners are bacterial proteins, Protein A and Protein L, which bind to the Fc domain and to the variable region of the Fab light chain, respectively.…”

“…A peptide epitope of the NISTmAb, F peptide, with the sequence NSELLSLINDMPITNDQKKLMSNN and N-terminal acetylation, C-terminal amidation, and a C-terminal biotinylated lysine residue was previously synthesized by Genscipt. 18 …”

“…Binding measurements were performed following a previously developed method. 18 Briefly, biotinylated ligands (Protein A, Protein L, F peptide) were immobilized to a CAP sensor chip via biotin CAPture reagent consisting of streptavidin bound to an oligonucleotide that is complementary to the strand on the sensor chip. Protein A and Protein L ligands were diluted to 0.02 μg/mL with the running buffer, PBS pH 7.4, and supplemented with 0.1% BSA while F peptide was diluted to 0.05 μg/mL.…”

“… 16 , 17 Recently, a rapid SPR assay was developed to quickly detect Met oxidation in both the antigen-binding fragment (Fab) and Fc domains. 18 Using a model therapeutic IgG1κ mAb, NISTmAb, 19 , 20 specific molecular probes were used to assess the binding to one region in the Fc (Protein A) and two regions in the Fab (Protein L, F peptide). The chemical oxidation was performed freely in solution or while NISTmAb was bound to Protein A.…”

“…These analytical results were then correlated with binding data from a rapid SPR assay. 18 The observation of HOS variance that correlates with stability and functional changes underscore the utility of combining 2D 1 H- 13 C methyl NMR with other analytical tools to yield a more precise evaluation of mAb candidates during pharmaceutical development.…”