Identifying Key Residues That Drive Strong Electrostatic Attractions between Therapeutic Antibodies.

Attractive electrostatic protein-protein interactions (PPI) necessarily involve identifying oppositely charged regions of the protein surface that interact favorably. This cannot be done reliably if one only considers a single protein in isolation unless there are obvious charge "patches" that result in extreme molecular dipoles. Prior work [ J. Pharm. Sci. 2019 , 108 , 120 - 132 ] identified three monoclonal antibodies (MAbs) that displayed experimental behavior ranging from net repulsive to strongly attractive electrostatic interactions. The present work provides a systematic computational approach for identifying the origin of diverse PPI, in terms of which sets of amino acids or individual amino acids are most influential, and determining if there are different patterns of pairwise amino acid interaction "maps" that result in different behaviors. The charge was eliminated computationally, one by one, for each charged residue in the wild-type sequences, which resulted in predicted changes in the second osmotic virial coefficient. The results highlight interaction "maps" that correspond to cases with qualitatively different net electrostatic PPI for the different MAbs and solution conditions, as well as key sets of residues that contribute to strongly attractive PPI. A more computationally efficient method is also proposed to identify key amino acids based on Mayer-weighted interaction energies.

Electrostatically Mediated Protein-Protein Interactions for Monoclonal Antibodies: A Combined Experimental and Coarse-Grained Molecular Modeling Approach.

Electrostatically mediated protein-protein interactions (PPI) can influence key product properties such as solubility, solution viscosity, and aggregation rates. Predictive models would allow for candidates/formulations to be screened with little or no protein material. Three monoclonal antibodies that display qualitatively different experimental PPI were evaluated at a range of pH and ionic strength conditions that are typical of product formulations. PPI parameters (kD, B22, and G22) were obtained from static and dynamic light scattering measurements and spanned from strongly repulsive to strongly attractive net interactions. Coarse-grained (CG) molecular simulations of PPI (specifically, B22) were compared against experimental PPI parameters across multiple pH and salt conditions, using a CG model that treats each amino acid explicitly. Predicted B22 values with default model parameters matched experimental B22 values semiquantitatively for some cases; others required parameter tuning to account for effects such as ion binding. Experimental PPI values were also analyzed for each monoclonal antibody within the context of single-protein properties such as net charge, and domain-based and global dipole moments. The results show that PPI predicted qualitatively and semiquantitatively by CG molecular modeling of B22 can be an effective computational tool for molecule and formulation assessment.

Biophysical characterization of influenza A virions.

Antigenic drift of the influenza A virus requires that vaccine production is targeted to the strains circulating each year. Live-attenuated influenza A vaccine manufacturing is used to produce intact virions with the surface antigens of the circulating strains. Influenza A typically contains a large percentage (>90%) of non-infective virions. The ribonucleoprotein (RNP) content, virion structure, and aggregation are factors that are thought to have an impact on infectivity. However, these factors are difficult to study because of the intrinsic variability in virion size, shape and overall structural integrity. Negative stain TEM for total particle counts and cryoTEM for detailed size/structural analysis are established benchmark techniques for virus characterization. Other methods may be valuable for certain sample types or circumstances. The aim of this work is to establish a benchmark comparison between orthogonal biophysical techniques for particle counts, population size distribution, structural integrity, and aggregate levels. NTA and FFF-MALS rapidly provided total counts, size distribution, and aggregate/elongated virion content. CryoTEM with size analysis and fraction counting yielded detailed information about the pleomorphism of the sample. The structural integrity of virions was inferred from multi-signal AUC-SV and CryoTEM. The current work provides a comparative assessment and a baseline for the selection of biophysical tools for the determination of particle counts, aggregation and pleomorphic characteristics of influenza A virus.

Feasibility of Freeze-Drying Oil-in-Water Emulsion Adjuvants and Subunit Proteins to Enable Single-Vial Vaccine Drug Products.

To generate potent vaccine responses, subunit protein antigens typically require coformulation with an adjuvant. Oil-in-water emulsions are among the most widely investigated adjuvants, based on their demonstrated ability to elicit robust antibody and cellular immune responses in the clinic. However, most emulsions cannot be readily frozen or lyophilized, on account of the risk of phase separation, and may have a deleterious effect on protein antigen stability when stored long term as a liquid coformulation. To circumvent this, current emulsion-formulated vaccines generally require a complex multivial presentation with obvious drawbacks, making a single-vial presentation for such products highly desirable. We describe the development of a stable, lyophilized squalene emulsion adjuvant through innovative formulation and process development approaches. On reconstitution, freeze-dried emulsion preparations were found to have a minimal increase in particle size of ∼20 nm and conferred immunogenicity in BALB/c mice similar in potency to freshly prepared emulsion coformulations in liquid form.

Impact of Mutations on the Higher Order Structure and Activity of a Recombinant Uricase.

This study explores the structural and functional changes associated with a low-temperature thermal transition of 2 engineered bacterial uricase mutants. Uricase has a noncovalent homotetrameric structure, with 4 active sites located at the interface of subunits. Using differential scanning calorimetry, a low-temperature transition was identified at 42°C for mutant A and at 33°C for mutant B. This transition was stabilized by the uricase inhibitor, oxonic acid, suggesting a strong structural relationship to the active site. For mutant B, there was a reversible loss of enzymatic activity above the low-temperature transition. Spectroscopic measurements demonstrated that there was also a reversible loss of secondary and tertiary structures and an increase in surface hydrophobicity. However, the hydrophobic core environment and the tetrameric structure were not altered over the low-temperature transition suggesting that the changes occurred primarily at the surface of the enzyme. The protein became aggregation-prone at temperatures approaching the cluster of higher-temperature melting transitions at 84°C, indicating these transitions represent a global unfolding of the protein. Our findings shed light on the structural changes that affect the uricase mechanism of action and provide new insights into how enzyme therapeutic development may be approached.

Analytical Characterization of an Oil-in-Water Adjuvant Emulsion.

Adjuvants are typically used in subunit vaccine formulations to enhance immune responses elicited by individual antigens. Physical chemical characterization of novel adjuvants is an important step in ensuring their effective use in vaccine formulations. This paper reports application of a panel of quantitative assays developed to analyze and characterize an oil-in-water adjuvant emulsion, which contains glucopyranosyl lipid A (GLA) and is a squalene-based emulsion. GLA is a fully synthetic analogue of monophosphoryl lipid A, which is a Toll-like receptor type 4 agonist and an FDA-approved adjuvant. The GLA-stable emulsion (GLA-SE) is currently being used for a respiratory syncytial virus vaccine in a phase 2 clinical trial. GLA was quantitated using reverse-phased high-performance liquid chromatography (RP-HPLC) coupled to a mass spectrometric detector, achieving higher assay sensitivity than the charged aerosol detection routinely used. Quantitation of the excipients of GLA-SE, including squalene, egg phosphatidyl choline, and Poloxamer 188, was achieved using a simple and rapid RP-HPLC method with evaporative light scattering detection, eliminating chemical derivatization typically required for these chromophore-lacking compounds. DL-α-tocopherol, the antioxidant of the GLA-SE, was quantitated using a RP-HPLC method with conventional UV detection. The experimental results compared well with values expected for these compounds based on targeted composition of the adjuvant. The assays were applied to identify degradation of individual components in a GLA-SE sample that degraded into distinct aqueous and oil phases. The methods developed and reported here are effective tools in monitoring physicochemical integrity of the adjuvant, as well as in formulation studies.

Identification of an IgG CDR sequence contributing to co-purification of the host cell protease cathepsin D.

Recombinant therapeutic monoclonal antibodies (mAbs) must be purified from host cell proteins (HCPs), DNA, and other impurities present in Chinese hamster ovary (CHO) cell culture media. HCPs can potentially result in adverse clinical responses in patients and, in specific cases, have caused degradation of the final mAb product. As reported previously, residual traces of cathepsin D caused particle formation in the final product of mAb-1. The current work was focused on identification of a primary sequence in mAb-1 responsible for the binding and consequent co-purification of trace levels of CHO cathepsin D. Surface plasmon resonance (SPR) was used to detect binding between immobilized CHO cathepsin D and a panel of mAbs. Out of 13 mAbs tested, only mAb-1 and mAb-6 bound to cathepsin D. An LYY motif in the HC CDR2 was common, yet unique, to only these two mAbs. Mutation of LYY to AAA eliminated binding of mAb-1 to cathepsin D providing confirmation that this sequence motif was involved in the binding to CHO cathepsin D. Interestingly, the binding between mAb-1 and cathepsin D was weaker than that of mAb-6, which may be related to the fact that two aspartic acid residues near the LYY motif in mAb-1 are replaced with neutral serine residues in mAb-6. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:140-145, 2017.

Technical decision making with higher order structure data: impact of a formulation change on the higher order structure and stability of a mAb.

Changes in formulation may be required during the development of protein therapeutics. Some of the changes may alter the protein higher order structure (HOS). In this note, we show how the change from a trehalose-based formulation to an arginine-based formulation concomitantly impacted the tertiary structure and the thermal stability of a mAb (mAb1). The secondary structure was not disrupted by the formulation change. The destabilization of the tertiary structure did not affect the long-term stability or the bioactivity of mAb1. This indicates that loss of conformational stability was likely compensated by improvements in the colloidal stability of mAb1 in the arginine-based formulation. The formulation-induced changes in HOS were reversible as proven by measurements after dilution in a common buffer (phosphate-buffered saline). For aggregation driven by assembly of aggregates (colloidally limited), small changes in conformational structure and stability as measured by HOS methods may not necessarily be predictive of long-term stability.

Live attenuated and inactivated viral vaccine formulation and nasal delivery: potential and challenges.

Vaccines are cost-effective for the prevention of infectious diseases and have significantly reduced mortality and morbidity. Novel approaches are needed to develop safe and effective vaccines against disease. Major challenges in vaccine development include stability in a suitable dosage form and effective modes of delivery. Many live attenuated vaccines are capable of eliciting both humoral and cell mediated immune responses if physicochemically stable in an appropriate delivery vehicle. Knowing primary stresses that impart instability provides a general rationale for formulation development and mode of delivery. Since most pathogens enter the body through the mucosal route, live-attenuated vaccines have the advantage of mimicking natural immunization via non-invasive delivery. This presentation will examine aspects of formulation design, types of robust dosage forms to consider, effective routes of delivery (invasive and noninvasive), and distinctions between live attenuated or inactivated vaccines.

Aggregation of anti-streptavidin immunoglobulin gamma-1 involves Fab unfolding and competing growth pathways mediated by pH and salt concentration.

Changes in non-native aggregation mechanisms of an anti-streptavidin (anti-SA) IgG1 antibody were determined over a wide range of pH and [NaCl] under accelerated (high temperature) conditions, using a combination of calorimetry, chromatography, static light scattering, dye binding, and spectroscopy (fluorescence, infra-red, and circular dichroism). Aggregation rates were strongly influenced by conformational stability of at least the Fab regions, but were only weakly affected by changes in electrostatic colloidal interactions. This was in contrast to the effects of electrostatic interactions on aggregate growth, as the dominant growth mechanism shifted dramatically with pH and [NaCl]. Pre-formed aggregates also displayed a reversible cloud-point boundary that quantitatively aligned with the overall pattern of aggregation mechanisms as a function of pH and [NaCl], suggesting an underlying thermodynamic transition may dictate whether molecular aggregates will coalesce into macroscopic particles. Structural changes upon unfolding and aggregation were also sensitive to pH and [NaCl]. Interestingly, Thioflavin T binding was essentially indistinguishable for aggregates formed in different pH and [NaCl] conditions, however, the other assays indicated notable differences across different solvent conditions. This suggests that the overall degree of conformational change during aggregation can be influenced by electrostatic interactions, but suggests caution in interpreting whether available techniques detect changes that are directly relevant to the mechanism(s) of aggregate formation and growth.

Perturbation of thermal unfolding and aggregation of human IgG1 Fc fragment by Hofmeister anions.

The thermal unfolding and subsequent aggregation of the unglycosylated Fc fragment of a human IgG1 antibody (Fc) were studied in the salt solutions of Na(2)SO(4), KF, KCl and KSCN at pH 4.8 and 7.2 below and at its pI of 7.2, respectively, using differential scanning calorimetry (DSC), far ultraviolet circular dichroism (far-UV CD), size exclusion chromatography (SE-HPLC) and light scattering. First, our experimental results demonstrated that the thermal unfolding of the C(H)2 domain of the Fc was sufficient to induce aggregation. Second, at both pH conditions, the anions (except F(-)) destabilized the C(H)2 domain where the effectiveness of SO(4)(2-) > SCN(-) > Cl(-) > F(-) was more apparent at pH 4.8. In addition, the thermal stability of the C(H)2 domain was less sensitive to the change in salt concentration at pH 7.2 than at pH 4.8. Third, at pH 4.8 when the Fc had a net positive charge, the anions accelerated the aggregation reaction with SO(4)(2-) > SCN(-) > Cl(-) > F(-) in effectiveness. But these anions slowed down the aggregation kinetics at pH 7.2 with similar effectiveness when the Fc was net charge neutral. We hypothesize that the effectiveness of the anion on destabilizing the C(H)2 domain could be attributed to its ability to perturb the free energy for both of the native and unfolded states. The effect of the anions on the kinetics of the aggregation reaction could be interpreted based on the modulation of the electrostatic protein-protein interactions by the anions.

Identification and quantification of Fc fusion peptibody degradations by limited proteolysis method.

An Fc fusion protein expressed in Escherichia coli contains Met1 and Asp2 residues at the N terminus and an active peptide attached to the C terminus of the Fc region. Due to the unique amino acid sequence of Fc, many commonly used proteolysis methods have severe drawbacks for characterizing degradations of Met1 and Asp2 residues. A novel method has been developed to effectively characterize the degradations by employing a limited endoproteinase Glu-C digestion. The limited digestion generates a dimeric peptide of (Met1-Glu14)2 due to specific cleavage at the residue Glu14 of the N terminus. This peptide together with its degraded products, including Met1 oxidation and Asp2 isomerization, can be identified and quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The optimization of digestion procedure and linearity of quantification are also described. This approach was successfully used in a photostability study to assess the product stability of an Fc fusion peptibody.

Stabilization by urea during thermal unfolding-mediated aggregation of recombinant human interleukin-1 receptor (type II): does solvation entropy play a role?

The protein denaturing properties of urea are well-known and still the subject of debate. It has been noted that in some cases where urea concentrations are relatively low stabilization is afforded against aggregation. An explanation for this unusual effect has seemingly remained elusive. Evidence is offered to propose urea stabilization is related to its influence on the solvation property of the protein molecules when in contact with an unfolded hydrophobic surface that tends to increase the entropy of the local aqueous solvent. This property of urea is expected to lower the entropic driving force of unfolded-mediated aggregation despite the increase in enthalpy. The data presented from toluene transfer experiments into 2 M urea + 0.1 M sodium phosphate solutions showed that the solvation free energy change was negative up to ∼75 °C. The associated ΔΔH was positive, leading to the conclusion that entropy drives the solvation process within the temperature domain from ∼20° to 75 °C. Using thermodynamic parameters from the toluene solvation experiments, it was possible to accurately determine the T(m) shift of recombinant human interleukin-1 receptor type II (rhuIL-1R(II)). Heating experiments above the apparent T(m) in the same urea/phosphate solution support the thesis that urea inhibits the entropy-driven aggregation process of rhuIL-1R(II), adding yet another molecule to the list of low urea concentration stabilized molecules.

Development of stable lyophilized protein drug products.

Freeze drying, or lyophilization is widely used for biopharmaceuticals to improve the long term storage stability of labile molecules. This review examines general theory and practice of rational lyophilization of biopharmaceuticals. Formulation development involving the selection of appropriate excipients, their associated physical properties, and mechanism of action in achieving a stable drug product are primary considerations for a successful lyophilization program. There are several parameters considered critical on the basis of their relationship to lyophilization cycle development and protein product stability. This along with the importance of analytical methods to provide insight toward understanding properties of drug product stability and cake structure are discussed. Also, aspects of instability found in lyophilized biopharmaceutical products, their degradation pathways and control are elucidated. Finally, container-closure requirements and drug product handling are described in context of the caveats to avoid compromising drug product quality.

Antibody solubility behavior in monovalent salt solutions reveals specific anion effects at low ionic strength.

Protein solubility was measured using the crystalline precipitate of a recombinant therapeutic antibody, in monovalent salt solutions containing KF, KCl, and KSCN (up to ∼ 0.7 M) at different pH conditions. For all three anions, the antibody solubility demonstrated complex behavior, both monotonic and nonmonotonic, with dependence on pH and salt concentration. At pH 7.1, close to the isoelectric point (pI) of 7.2, a typical salting-in behavior was observed with the salting-in constants of 12.7, 8.0, and 2.8 M for KSCN, KCl, and KF, respectively, suggesting that the anions follow the order of SCN(-) > Cl(-) > F(-) for increasing antibody solubility. Nonmonotonic behavior, as described by an initial solubility decrease followed by a solubility increase with ionic strength, was observed at pH 5.3, far below its pI. The effectiveness of the anion for reducing the solubility followed the order of SCN(-) > Cl(-) > F(-) . After the solubility reached the minimum, the anion's effectiveness for raising the antibody solubility was in agreement with that at pH 7.1. The mechanisms for the above phenomena are discussed based upon specific binding of the anions to the antibody surface. The mechanistic view of anion binding and its charge neutralization effect at pH 5.3 was supported by the results from the effective charge and zeta-potential measurements.

Opalescence of an IgG2 monoclonal antibody solution as it relates to liquid-liquid phase separation.

Opalescence for a monoclonal antibody solution was systematically studied with respect to temperature, protein concentration, ionic strength (using KCl), and pH conditions. Multiple techniques, including measurement of light scattering at 90° and transmission, Tyndall test, and microscopy, were deployed to examine the opalescence behavior. Near the vicinity of the critical point on the liquid-liquid coexistence curve in the temperature-protein concentration phase diagram, the enhanced concentration fluctuations significantly contributed to the critical opalescence evidently by formation of small liquid droplets. Furthermore, our data confirm that away from the critical point, the opalescence behavior is related to the antibody self-association (agglomeration) caused by the attractive antibody-antibody interactions. As expected, at a pH near the pI of the antibody, the solution became less opalescent as the ionic strength increased. However, at a pH below the pI, the opalescence of the solution became stronger, reached a maximum, and then began to drop as the ionic strength further increased. The change in the opalescence correlated well with the trends of protein-protein interactions revealed by the critical temperature from the liquid-liquid phase separation.

Liquid-liquid phase separation of a monoclonal antibody and nonmonotonic influence of Hofmeister anions.

Liquid-liquid phase separation was studied for a monoclonal antibody in the monovalent salt solutions of KF, KCl, and KSCN under different pH conditions. A modified Carnahan-Starling hard-sphere model was utilized to fit the experimental data, establish the liquid-liquid coexistence curve, and determine antibody-antibody interactions in the form of T(c) (critical temperature) under the different solution conditions. The liquid-liquid phase separation revealed the complex relationships between antibody-antibody interactions and different solution conditions, such as pH, ionic strength, and the type of anion. At pH 7.1, close to the pI of the antibody, a decrease of T(c) versus ionic strength was observed at low salt conditions, suggesting that the protein-protein interactions became less attractive. At a pH value below the pI of the antibody, a nonmonotonic relationship of T(c) versus ionic strength was apparent: initially as the ionic strength increased, protein-protein interactions became more attractive with the effectiveness of the anions following the inverse Hofmeister series; then the interactions became less attractive following the direct Hofmeister series. This nonmonotonic relationship may be explained by combining the charge neutralization by the anions, perhaps with the ion-correlation force for polarizable anions, and their preferential interactions with the antibody.

Ion-specific modulation of protein interactions: anion-induced, reversible oligomerization of a fusion protein.

Ions can significantly modulate the solution interactions of proteins. We aim to demonstrate that the salt-dependent reversible heptamerization of a fusion protein called peptibody A or PbA is governed by anion-specific interactions with key arginyl and lysyl residues on its peptide arms. Peptibody A, an E. coli expressed, basic (pI = 8.8), homodimer (65.2 kDa), consisted of an IgG1-Fc with two, C-terminal peptide arms linked via penta-glycine linkers. Each peptide arm was composed of two, tandem, active sequences (SEYQGLPPQGWK) separated by a spacer (GSGSATGGSGGGASSGSGSATG). PbA was monomeric in 10 mM acetate, pH 5.0 but exhibited reversible self-association upon salt addition. The sedimentation coefficient (s(w)) and hydrodynamic diameter (D(H)) versus PbA concentration isotherms in the presence of 140 mM NaCl (A5N) displayed sharp increases in s(w) and D(H), reaching plateau values of 9 s and 16 nm by 10 mg/mL PbA. The D(H) and sedimentation equilibrium data in the plateau region (>12 mg/mL) indicated the oligomeric ensemble to be monodisperse (PdI = 0.05) with a z-average molecular weight (M(z)) of 433 kDa (stoichiometry = 7). There was no evidence of reversible self-association for an IgG1-Fc molecule in A5N by itself or in a mixture containing fluorescently labeled IgG1-Fc and PbA, indicative of PbA self-assembly being mediated through its peptide arms. Self-association increased with pH, NaCl concentration, and anion size (I(-) > Br(-) > Cl(-) > F(-)) but could be inhibited using soluble Trp-, Phe-, and Leu-amide salts (Trp > Phe > Leu). We propose that in the presence of salt (i) anion binding renders PbA self-association competent by neutralizing the peptidyl arginyl and lysyl amines, (ii) self-association occurs via aromatic and hydrophobic interactions between the ..xxCTRWPWMC..xxxCTRWPWMCxx.. motifs, and (iii) at >10 mg/mL, PbA predominantly exists as heptameric clusters.

Anion binding mediated precipitation of a peptibody.

PURPOSE: Understand the underlying mechanism governing the salt-induced precipitation of a basic (pI = 8.8) protein, Peptibody A (PbA), in acidic solutions. METHODS: The rate, extent, and reversibility of PbA precipitation was monitored over 4-weeks as a function of pH (3.7-5.0), salt concentration (0-400 mM), and ion identity using a series of monovalent, Hofmeister anions (F(-), Cl(-), Br(-), I(-), ClO(4) (-), SCN(-)) and cations (Li+, Na+, K+, Rb+, Cs+). The effects of salt on conformational stability and reduced valence were determined using Fourier-transform infrared spectroscopy, circular dichroism, and capillary electrophoresis/analytical ultracentrifugation. RESULTS: PbA precipitation occurred upon salt addition and could be modulated with solution pH, salt identity & concentration. The precipitation was sensitive to anions, but not cations, and increased with anion size. A reverse Hofmeister effect (SCN(-) approximately ClO(4) (-)>I(-)>Cl(-)>Br(-)>F(-)) was observed with "salting-in" anions being the more effective precipitants. An increase in the precipitation rate below pH 4.3 indicated that protonation of aspartyl and glutamyl side-chains was also important for precipitation. The reversibility of precipitation was excellent (100%) at 4 degrees C but decreased upon storage at 25 degrees C and 37 degrees C; the loss in reversibility correlated with an increase in intermolecular beta-sheet content of the precipitate. CONCLUSION: Salts, employed as buffering, tonicifying, and viscosity modifying agents, may adversely affect the solubility of basic proteins formulated under acidic conditions.

Structure and stability changes of human IgG1 Fc as a consequence of methionine oxidation.

The Fc region has two highly conserved methionine residues, Met 33 (C(H)3 domain) and Met 209 (C(H)3 domain), which are important for the Fc's structure and biological function. To understand the effect of methionine oxidation on the structure and stability of the human IgG1 Fc expressed in Escherichia coli, we have characterized the fully oxidized Fc using biophysical (DSC, CD, and NMR) and bioanalytical (SEC and RP-HPLC-MS) methods. Methionine oxidation resulted in a detectable secondary and tertiary structural alteration measured by circular dichroism. This is further supported by the NMR data. The HSQC spectral changes indicate the structures of both C(H)2 and C(H)3 domains are affected by methionine oxidation. The melting temperature (Tm) of the C(H)2 domain of the human IgG1 Fc was significantly reduced upon methionine oxidation, while the melting temperature of the C(H)3 domain was only affected slightly. The change in the C(H)2 domain T m depended on the extent of oxidation of both Met 33 and Met 209. This was confirmed by DSC analysis of methionine-oxidized samples of two site specific methionine mutants. When incubated at 45 degrees C, the oxidized Fc exhibited an increased aggregation rate. In addition, the oxidized Fc displayed an increased deamidation (at pH 7.4) rate at the Asn 67 and Asn 96 sites, both located on the C(H)2 domain, while the deamidation rates of the other residues were not affected. The methionine oxidation resulted in changes in the structure and stability of the Fc, which are primarily localized to the C(H)2 domain. These changes can impact the Fc's physical and covalent stability and potentially its biological functions; therefore, it is critical to monitor and control methionine oxidation during manufacturing and storage of protein therapeutics.