Monoclonal Antibody Aggregation Associated with Free Radical Induced Oxidation.

Oxidation is an important degradation pathway of protein drugs. The susceptibility to oxidation is a common concern for therapeutic proteins as it may impact product efficacy and patient safety. In this work, we used 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH) as an oxidative stress reagent to evaluate the oxidation of therapeutic antibodies. In addition to the oxidation of methionine (Met) and tryptophan (Trp) residues, we also observed an increase of protein aggregation. Size-exclusion chromatography and multi-angle light scattering showed that the soluble aggregates induced by AAPH consist of dimer, tetramer, and higher-order aggregate species. Sodium dodecyl sulfate polyacrylamide gel electrophoresis indicated that inter-molecular disulfide bonds contributed to the protein aggregation. Furthermore, intrinsic fluorescence spectra suggested that dimerization of tyrosine (Tyr) residues could account for the non-reducible cross-links. An excipient screening study demonstrated that Trp, pyridoxine, or Tyr could effectively reduce protein aggregation due to oxidative stress. This work provides valuable insight into the mechanisms of oxidative-stress induced protein aggregation, as well as strategies to minimize such aggregate formation during the development and storage of therapeutic proteins.

Characterization and stability study of polysorbate 20 in therapeutic monoclonal antibody formulation by multidimensional ultrahigh-performance liquid chromatography-charged aerosol detection-mass spectrometry.

Polysorbate 20 is a nonionic surfactant commonly used in the formulation of therapeutic monoclonal antibodies (mAb) to prevent protein denaturation and aggregation. It is critical to understand the molecular heterogeneity and stability of polysorbate 20 in mAb formulations as polysorbate can gradually degrade in aqueous solution over time by multiple pathways losing surfactant functions and leading to protein aggregation. The molecular heterogeneity of polysorbate and the interference from proteins and the excipient in the formulation matrix make it a challenge to study polysorbate in protein formulations. In this work, the characterization and stability study of polysorbate 20 in the presence of mAb formulation sample matrix is first reported using two-dimensional liquid chromatography (2DLC) coupled with charged aerosol detection (CAD) and mass spectrometry (MS) detection. A mixed-mode column that has both anion-exchange and reversed-phase properties was used in the first dimension to separate protein and polysorbate in the formulation sample, while polysorbate 20 esters were trapped online and then analyzed using an reversed-phase ultrahigh-performance liquid chromatography (RP-UHPLC) column in the second dimension to further separate the ester species. The MS served as the third dimension to further resolve as well as to identify the polysorbate ester subspecies. Another 2DLC method using a cation-exchange column in the first dimension and the same RP-UHPLC method in the second dimension was developed to analyze the degradation products of polysorbate 20. Stability samples of a protein drug product were studied using these two 2DLC-CAD-MS methods to separate, identify, and quantify the multiple ester species in polysorbate 20 and also to monitor the change of their corresponding degradants. We found different polysorbate esters degrade at different rates, and importantly, the degradation rates for some esters are different in the protein formulation compared to a placebo that has no protein. The multidimensional UHPLC-CAD-MS approach provides insights into the heterogeneous stability behaviors of polysorbate 20 subspecies in real-time stability samples of a mAb formulation.

Metal-catalyzed oxidation of protein methionine residues in human parathyroid hormone (1-34): formation of homocysteine and a novel methionine-dependent hydrolysis reaction.

The oxidation of PTH(1-34) catalyzed by ferrous ethylenediaminetetraacetic acid (EDTA) is site-specific. The oxidation of PTH(1-34) is localized primarily to the residues Met[8] and His[9]. Beyond the transformation of Met[8] and His[9] into methionine sulfoxide and 2-oxo-histidine, respectively, we observed a hydrolytic cleavage between Met[8] and His[9]. This hydrolysis requires the presence of Fe(II) and oxygen and can be prevented by diethylenetriaminepentaacetic acid (DTPA) and phosphate buffer. Conditions leading to this site-specific hydrolysis also promote the transformation of Met[8] into homocysteine, indicating that the hydrolysis and transformation of homocysteine may proceed through a common intermediate.

Toward understanding molecular heterogeneity of polysorbates by application of liquid chromatography-mass spectrometry with computer-aided data analysis.

Polysorbates (PS) are widely used as oil-in-water emulsifiers, stabilizers, wetting agents, solubilizers, and dispersants in the agricultural, food, personal care, and pharmaceutical industries due to their cost effectiveness, biocompatibility, formulation flexibility, low toxicity, and good stabilizing and protecting properties. The polysorbates are often pictured as polyoxyethylated sorbitan monoesters of saturated and/or unsaturated fatty acids. In reality, polysorbates are complex mixtures of multiple components, as follows from the reactions involved in their production. In this work, we report a novel application of liquid chromatography-mass spectrometry (LC-MS) for the characterization of polysorbates. This method takes advantage of accurate mass measurements and information on the identity of a fatty acid from "in-source" generated characteristic dioxolanylium ions. The method allowed us to perform quick profiling of fatty acids in PS 20 and 80 which, combined with a computer-aided peak assignment algorithm, facilitated detailed characterization of their constituents. As a major finding, we determined that different samples of PS 20 varied from 0% to 15% in relative amounts of unsaturated oleic acid. Although the consequences of this difference were not fully evaluated in this work, one might expect that PS 20 with larger amounts of oleic acid will be more prone to autoxidation, thus potentially having greater impact on the oxidative degradation of the biotherapeutics it formulates.

Characteristics of rhVEGF release from topical hydrogel formulations.

PURPOSE: To study recombinant human vascular endothelial growth factor (rhVEGF), the release characteristics from topical gel formulations, and its interaction with the gelling agents. METHODS: The release kinetics were followed by quantifying rhVEGF that diffused into the receptor chamber of Franz cells. Analytical ultracentrifuge (AUC) was used to characterize the sedimentation velocity of rhVEGF experienced in the gel. The interactions were characterized by isothermal calorimetry (ITC), and rhVEGF conformation was assessed by circular dichroism (CD). RESULTS: The fraction of protein released was linear with the square root of time. The release rate constants did not show significant change within a wide range of bulk viscosities created by different concentrations of hydroxypropyl methylcellulose (HPMC) or MC gels. Sedimentation velocity determined by AUC generated comparable sedimentation coefficients of protein in these gels. AUC and ITC revealed no significant interaction between rhVEGF and HPMC and some change on secondary structure of the protein by Far UV CD, which was not the case with carboxymethyl cellulose (CMC). CONCLUSIONS: Microviscosity, not bulk viscosity, was the key factor for the release of rhVEGF from cellulosic gels such as HPMC. Interaction between rhVEGF and CMC resulted in slower, and reduced amount of, release from the gel.

Effect of EDTA and methionine on preventing loss of viscosity of cellulose-based topical gel.

Methylcellulose and hydroxypropylmethylcellulose (hypromellose) are used in topical formulations of a protein to form a viscous hydrogel. Five lots of hypromellose raw material were made into 3% gel; all showed viscosity loss after sterilization by autoclave. EDTA (edetate disodium) minimized the viscosity loss caused by autoclaving in the presence of up to 100 ppm H(2)O(2). These results suggest that EDTA may prevent loss of viscosity of the hydrogel when peroxide is present. H(2)O(2) at low levels (2-50 ppm) caused significant viscosity loss over time at either 40 degrees C or 5 degrees C in 3% methylcellulose or hypromellose gel. EDTA slowed the rate of viscosity loss during storage under stress by H(2)O(2) but did not completely prevent the loss. Methionine was effective in completely preventing gel-viscosity loss during storage in the presence of up to 50 ppm H(2)O(2). On the basis of these results, it is recommended that methionine be added to the protein topical formulation as a stabilizer against viscosity loss.

Hydrolysis of Polysorbate 20 and 80 by a Range of Carboxylester Hydrolases.

Degradation of the surfactant polysorbate (PS) by enzyme impurities has been previously suggested as a mechanism for the formation of visible and subvisible particles that affect product quality. Although chemical degradation pathways of PS, such as oxidation and acid/base hydrolysis, have been previously characterized, enzymatic degradation of PS remains poorly understood. In this report, enzyme-mediated hydrolysis of the major components of PS was monitored using an evaporative light scattering detection-high-performance liquid chromatography method. PS20 and PS80 tested contained 99% of laurate and 98% oleate esters, respectively, were heterogeneous with respect to head group, and contained a distribution of ester types. Carboxylester hydrolases tested included those from Pseudomonas cepacia, Thermomyces lanuginosus, Candida antarctica, rabbit liver, and pig pancreas. PS hydrolysis was monitored by observing the change in the peak area of major PS components over time and quantified using a parameter called t50, which was defined as the time required for each peak to reach 50% of its initial value. Time course experiments suggested that PS hydrolysis was dependent on the order of esters (mono-, di-, or triester), the identity of the hydrophilic head group (sorbitan or isosorbide), and the identity of the fatty acid ester tail (C12 vs C18:1). In addition, the pattern of PS hydrolysis was unique to the type of enzyme used. Importantly, we observed that no PS component was completely resistant to the carboxylester hydrolases tested here. Our results illustrate a potential fingerprint approach that could be useful in verifying enzyme-mediated PS degradation in drug substance and provide an improved understanding of the complexity of PS degradation in the presence of enzymes. LAY ABSTRACT: Degradation of the non-ionic surfactant polysorbate (PS) has been reported to lead to the formation of visible and subvisible particles that affect product quality. Chemical degradation pathways of PS, such as oxidation and acid/base hydrolysis, have been previously studied, but enzymatic degradation of PS remains poorly understood. In this study, enzyme-mediated hydrolysis of the major components in a heterogeneous mixture of PS20 or PS80 was monitored using an evaporative light scattering detection-high-performance liquid chromatography method. Carboxylester hydrolases from a broad range of organisms were tested, including enzymes from Pseudomonas cepacia, Thermomyces lanuginosus, Candida antarctica, rabbit liver, and pig pancreas. Time course experiments suggested that PS hydrolysis was dependent on the order of esters (mono-, di-, or triester), the identity of the hydrophilic head group (sorbitan or isosorbide), the identity of the fatty acid ester tail (C12 vs C18:1), and the identity of the enzyme. Importantly, no PS component was completely resistant to all the carboxylester hydrolases tested here. Our results illustrate a potential fingerprint approach that could be useful in verifying or identifying enzyme-mediated PS degradation in drug substance and provide an improved understanding of the complexity of PS degradation in the presence of enzymes.

Oxidative Degradation of Polysorbate Surfactants Studied by Liquid Chromatography-Mass Spectrometry.

Polysorbates (PSs), as acquired from manufacturing processes and chemical nature of fatty acids (FAs) used in production of biotherapeutic formulations, are heterogeneous mixtures of structurally related compounds, covering a wide range of physicochemical properties. Such complexity presents a certain challenge for analysis of these important surfactants and demands the use of methods offering sufficient resolution to monitor individual classes of species and detect changes upon stress. A liquid chromatography mass spectrometry method, benefiting from the use of low m/z marker ions, simplifies profiling of PSs by providing detailed information on FA composition even of chromatographically overlapping peaks. The ability of the method to monitor individual components and follow their changes because of oxidative stress was explored. A water-soluble azo compound was used as a model oxidizer. Major degradation products of PS 80, because of reactions involving double bond, were identified as oxo-C9:0, keto-C18:1, hydroxyl-C18:1, epoxy-C18:0, and hydroperoxy-C18:1. Stability of PS 20 components was found to depend on the carbon number of polyethoxylated (POE) sorbitan FA ester and its order. Rates of oxidative degradation increased with the length of the FA ester and, moreover, POE sorbitan diesters degraded significantly faster in comparison to the corresponding monoesters upon the oxidative stress. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association.

Oxidative degradation of polysorbate surfactants studied by liquid chromatography-mass spectrometry.

Polysorbates (PSs), as acquired from manufacturing processes and chemical nature of fatty acids (FAs) used in production of biotherapeutic formulations, are heterogeneous mixtures of structurally related compounds, covering a wide range of physicochemical properties. Such complexity presents a certain challenge for analysis of these important surfactants and demands the use of methods offering sufficient resolution to monitor individual classes of species and detect changes upon stress. A liquid chromatography mass spectrometry method, benefiting from the use of low m/z marker ions, simplifies profiling of PSs by providing detailed information on FA composition even of chromatographically overlapping peaks. The ability of the method to monitor individual components and follow their changes because of oxidative stress was explored. A water-soluble azo compound was used as a model oxidizer. Major degradation products of PS 80, because of reactions involving double bond, were identified as oxo-C9:0, keto-C18:1, hydroxyl-C18:1, epoxy-C18:0, and hydroperoxy-C18:1. Stability of PS 20 components was found to depend on the carbon number of polyethoxylated (POE) sorbitan FA ester and its order. Rates of oxidative degradation increased with the length of the FA ester and, moreover, POE sorbitan diesters degraded significantly faster in comparison to the corresponding monoesters upon the oxidative stress.

Effect of individual Fc methionine oxidation on FcRn binding: Met252 oxidation impairs FcRn binding more profoundly than Met428 oxidation.

The long serum half-lives of mAbs are conferred by pH-dependent binding of IgG-Fc to the neonatal Fc receptor (FcRn). The Fc region of human IgG1 has three conserved methionine residues, Met252, Met358, and Met428. Recent studies showed oxidation of these Met residues impairs FcRn binding and consequently affects pharmacokinetics of therapeutic antibodies. However, the quantitative effect of individual Met oxidation on Fc-FcRn binding has not been addressed. This information is valuable for defining critical quality attributes. In the present study, two sets of homodimeric site-directed IgG1 mutations were generated to understand how individual Fc Met oxidation affects FcRn binding. The first approach used Met to Leu mutants to block site-specific Met oxidation. In the other approach, Met to Gln mutants were designed to mimic site-specific Met oxidation. Both mutagenesis approaches show that either Met252 or Met428 oxidation alone significantly impairs Fc-FcRn binding. Met252 oxidation has a more deleterious effect on FcRn binding than M428 oxidation, whereas Met428 oxidation has a bigger destabilization effect on the thermal stability. Our results also show that Met358 oxidation does not affect FcRn binding. In addition, our study suggests that Met to Gln mutation may serve as an important tool to understand Met oxidation.

Compatibility, physical stability, and characterization of an IgG4 monoclonal antibody after dilution into different intravenous administration bags.

The physical stability of an immunoglobulin G4 monoclonal antibody (mAb) upon dilution into intravenous (i.v.) bags containing 0.9% saline was examined. Soluble aggregates and subvisible particles were observed by size-exclusion high-performance liquid chromatography (SE-HPLC) and light obscuration when formulated with suboptimal levels of polysorbate 20. The formation of soluble aggregates and particulates was further characterized by a combination of SE-HPLC, nanoparticle tracking analysis (NTA), microflow-digital imaging (MFI), and turbidity measurements. With sufficient PS20 levels, particle formation was minimized, although quantification of submicron sized particles by NTA was not possible because of the interference from PS20. Intravenous bags composed of polyvinyl chloride caused more protein particle formation than polyolefin bags. Differences between bag types were affected by removing headspace and by transferring the saline solution into glass vials. Characterization studies with Fourier transform infrared microscopy and extrinsic fluorescence spectroscopy demonstrated that isolated particles contained native-like secondary structure with partially altered tertiary structure, compared with heat-denatured and nonstressed controls. Transmission electron microscopy and MFI analysis showed particles had an amorphous morphology of varying sizes. Particles contained some non-native disulfide bond crosslinks, potentially initiated by low levels of free thiol in the native mAb. The critical role of proper formulation design to stabilize proteins against physical instability during i.v. administration is discussed.

Analysis of 2,2'-azobis (2-amidinopropane) dihydrochloride degradation and hydrolysis in aqueous solutions.

2,2'-Azobis(2-amidinopropane) dihydrochloride (AAPH), a free radical-generating azo compound, is gaining prominence as a model oxidant in small molecule and protein therapeutics, namely for its ability to initiate oxidation reactions via both nucleophilic and free radical mechanisms. To better understand its degradation pathways, AAPH was degraded at 40°C in aqueous solutions over a wide pH range. Samples were analyzed via liquid chromatography-ultraviolet spectroscopy and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The thermal decomposition rate of AAPH to form radical species averaged 2.1 × 10(-6) s(-1) and did not vary significantly with pH. The hydrolysis rate increased exponentially with pH, showing hydroxide ion dependence. A mechanism for AAPH hydrolysis is proposed. The LC-MS/MS results provided evidence that the alkoxyl radical is a major radical species in solution. The LC-MS/MS results also showed a radical disproportionation reaction and enabled the generation of an overall reaction scheme showing the various side and termination products of AAPH degradation.

The relative rate of immunoglobulin gamma 1 fragmentation.

The physicochemical stability of protein therapeutics is of significant pharmaceutical interest. Immunoglobulin gamma (IgG) hinge region fragmentation has recently garnered attention as an important degradation route of therapeutic monoclonal antibodies. In this work, the rates and relative amount of fragment species are compared for five different IgGs (IgG1-5) with widely varying solution properties. Native size-exclusion chromatography (SEC), sodium dodecyl sulfate (SDS)-based SEC, and capillary electrophoresis-SDS were used to characterize IgG1 fragmentation after storage at 30°C, 40°C, and 50°C. Two-dimensional correlation analysis of the chromatograms as a function of time was used to illustrate the relative rates of cleavage. Interestingly, the relative rate of Fab cleavage was greater than that of other species. An average apparent energy of activation for IgG1 fragmentation was also measured for all five molecules. This work suggests that IgG1 fragmentation is primarily hinge sequence dependent and other IgG1 molecules should behave similarly within the limits of the solution conditions used.

Compatibility of a protein topical gel with wound dressings.

The compatibility between several dressing materials and a recombinant human vascular endothelial growth factor (rhVEGF) topical methylcellulose gel formulation was investigated. The dressings being studied were Adaptic, Non-stick Dressing, Conformant 2, Opsite and Tegapore. The criteria to select a compatible dressing include protein stability, absence of leachables from the dressing, and ability to retain gel on wound. An LC-MS method with sample treatment using cellulase was developed to determine protein oxidation in gel formulations. Results showed that rhVEGF was significantly oxidized by Adaptic dressing in 24 h. Protein oxidation was likely due to the peroxides, as determined by FOX assay, released into the protein solution from the dressing. Furthermore, Adaptic dressing caused protein adsorption loss, formation of high MW protein adducts, and released leachables as determined by RP-HPLC, LC-MS, and SEC. No protein oxidation or loss was observed after exposure to the other four alternative dressings. However, unknown leachables were detected in the presence of Opsite and Non-stick Dressing. The pore sizes of the Conformant 2 and Non-stick dressings were too large to hold the topical gel within the wound area, making them unsuitable for patient use. No rhVEGF bioactivity loss was observed in the presence of Tegapore. In conclusion, Tegapore was considered suitable for the rhVEGF topical gel.

Methionine, tryptophan, and histidine oxidation in a model protein, PTH: mechanisms and stabilization.

Recent oxidation events on monoclonal antibody candidates prompted us to investigate the mechanism of oxidation of Met, Trp, and His residues and to search for suitable stabilizers. By using parathyroid hormone (1-34), PTH, as a model protein and various oxidants, aided by liquid chromatography, peptide mapping, and mass spectrometry, we identified and quantified the oxidation of these vulnerable residues. Whereas H(2)O(2) and t-butyl hydroperoxide (t-BHP) primarily oxidized the two Met residues, 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH), and H(2)O(2) + Fe(II) oxidized Met and Trp residues, with AAPH more capable of generating oxidized Trp species than the latter. H(2)O(2) + Fe(III) generated results comparable to those with H(2)O(2) + Fe(II), except that there was a lesser amount of hydroxylated Phe. Oxidation of the His residue in PTH occurred when copper was used instead of iron. AAPH, a free-radical generator, produced alkylperoxides, which simulated the oxidizing species from degraded polysorbate, commonly found in protein formulations. It is prudent to screen stabilizers by using H(2)O(2), H(2)O(2) + Fe(II), and AAPH because these agents represent potential assaults from the H(2)O(2) commonly present in degraded polysorbate, the residue of aseptic agents and the metal from stainless steel surfaces, and alkylperoxides from degraded polysorbate, respectively. Free Met protected the Met residues in PTH from oxidation by H(2)O(2) and H(2)O(2) + Fe(II). Mannitol and EDTA were effective against H(2)O(2) + Fe(II). Free Trp protected only the Trp residue in PTH from oxidation by AAPH, the combination of Trp and Met was effective against all three oxidant conditions. By using AAPH to generate oxidant, Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) and pyridoxine were also found to exhibit good free-radical scavenging activity and thus protected Trp in PTH against oxidation.