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.

Light-Induced Covalent Buffer Adducts to Histidine in a Model Protein.

PURPOSE: Light is known to induce histidine (His) oxidation and His-His crosslinking in proteins. The crosslinking is resulted from the nucleophilic attack of a His to a photooxidized His from another protein. The goal of this work is to understand if covalent buffer adducts on His residues can be generated by light through similar mechanisms in nucleophilic buffers such as Tris and His. METHODS: A model protein (DNase) was buffer exchanged into nucleophilic buffers before light exposure. Photogenerated products were characterized by tryptic peptide mapping with mass spectrometry (MS) analysis. Several buffer adductions on His residues were identified after light exposure. To understand the influencing factors of such reactions, the levels of adducts were measured for six nucleophilic buffers on all His residues in DNase. RESULTS: The levels of adducts were found to correlate with the solvent accessibility of the His residue. The levels of adducts also correlate with the structure of the nucleophile, especially the steric restrictions of the nucleophile. The levels of adducts can be higher than that of other His photoreaction products, including photooxidation and crosslinking. CONCLUSIONS: In nucleophilic buffers, light can induce covalently-linked adducts to His residues.

The Use of a 2,2'-Azobis (2-Amidinopropane) Dihydrochloride Stress Model as an Indicator of Oxidation Susceptibility for Monoclonal Antibodies.

Protein oxidation is a major pathway for degradation of biologic drug products. Past literature reports have suggested that 2,2-azobis (2-amidinopropane) dihydrochloride (AAPH), a free radical generator that produces alkoxyl and alkyl peroxyl radicals, is a useful model reagent stress for assessing the oxidative susceptibility of proteins. Here, we expand the applications of the AAPH model by pairing it with a rapid peptide map method to enable site-specific studies of oxidative susceptibility of monoclonal antibodies and their derivatives for comparison between formats, the evaluation of formulation components, and comparisons across the stress models. Comparing the free radical-induced oxidation model by AAPH with a light-induced oxidation model suggests that light-sensitive residues represent a subset of AAPH-sensitive residues and therefore AAPH can be used as a preliminary screen to highlight molecules that need further assessment by light models. In sum, these studies demonstrate that AAPH stress can be used in multiple ways to evaluate labile residues and oxidation sensitivity as it pertains to developability and manufacturability.

Photodegradation Pathways of Protein Disulfides: Human Growth Hormone.

PURPOSE: Comprehensive product characterization was performed for the photodegradation of protein disulfides, representatively of human growth hormone (somatotropin; hGH), in order to provide a product database, which will be useful for the general analysis of protein stability. METHODS: HGH was photo-irradiated at λ = 254 and λ > 295 nm and tryptic digests were analyzed by HPLC-MS to investigate light-induced disulfide degradation pathways. RESULTS: A total of 60 products were detected, and structures/tentative structures were assigned to the products by MS2 and MS3 analysis. The main products were reduced Cys residues, dithiohemiacetal, thioether and disulfide scrambling products. In addition, we detected Cys degradation products such as Cys thioaldehyde, dehydroalanine (Dha), Ala, Ser semialdehyde, Ser, S-sulfocysteine, and Gly. Frequently, the tryptic fragments contained more than one modification, i.e. a Cys degradation product in close proximity to a dehydrated amino acid. Several novel cross-links were detected between Cys and Tyr, Cys, Ser and Phe, Cys and Trp, and Trp and Tyr. Photo-induced protein fragmentation was detected specifically at or in close proximity to the disulfide bond between T6 and T16. An in-house packed 75 cm nano-column enabled us to resolve various isomers/diastereomers of the photo-degradation products. CONCLUSION: A comprehensive analysis of photodegradation products revealed a variety of novel photo-products, including cross-links, originating from disulfide degradation. The mechanisms of product formation are discussed.

Degradation Mechanisms of Polysorbate 20 Differentiated by 18O-labeling and Mass Spectrometry.

PURPOSE: To investigate the mechanisms of polysorbate (PS) degradation with the added objective of differentiating the hydrolysis and oxidation pathways. METHODS: Ultra-performance liquid chromatography mass spectrometry (UPLC-MS) was utilized to characterize all-laurate polysorbate 20 (PS20) and its degradants. 18O stable isotope labeling was implemented to produce 18O-labeled degradation products of all-laurate PS20 in H218O, with subsequent UPLC-MS analysis for location of the cleavage site on the fatty acid-containing side chain of PS20. RESULTS: The analysis reveals that hydrolysis of all-laurate PS20 leads to a breakdown of the ester linkage to liberate free lauric acid, showing a distinct dependence on pH. Using a hydrophilic free radical initiator, 2,2-azobis(2-amidinopropane) dihydrochloride (AAPH) to study the oxidative degradation of all-laurate PS20, we demonstrate that free lauric acid and polyoxyethylene (POE) laurate are two major decomposition products. Measurement of 18O incorporation into free lauric acid indicated that hydrolysis primarily led to 18O incorporation into free lauric acid via "acyl-cleavage" of the fatty acid ester bond. In contrast, AAPH-exposure of all-laurate PS20 produced free lauric acid without 18O-incorporation. CONCLUSIONS: The 18O-labeling technique and unique degradant patterns of all-laurate PS20 described here provide a direct approach to differentiate the types of PS degradation.

Photo-oxidation of IgG1 and Model Peptides: Detection and Analysis of Triply Oxidized His and Trp Side Chain Cleavage Products.

PURPOSE: Triply oxidized histidine in an IgG1 monoclonal antibody was noticed when exposed to ICH light conditions. In order to understand the role of light source, irradiation wavelengths and primary sequence, specifically those of a nearby tryptophan, we synthesized and exposed several peptides to ICH light conditions and analyzed the products using LC-MS analysis. METHODS: Protein and peptide samples were photo-irradiated under ICH conditions as well as with monochromatic light at λ = 254 nm and analyzed using either LTQ Orbitrap or a LTQ-FT ion cyclotron resonance mass spectrometer respectively. RESULTS: A triply oxidized His residue was detected along with a second doubly oxidized His residue in an IgG1. Both of these oxidized His residues are located near Trp residues. In order to investigate the role of Trp photosensitization in His oxidation we synthesized model peptides and Ala mutants. Peptides exposed to ICH light stress conditions revealed a small percent of triply oxidized His in the Trp-containing peptide sequences but not in their corresponding Ala mutants. CONCLUSIONS: The differences in product formation under different photo-irradiation conditions underline the importance of light source, irradiation wavelengths and primary sequence in the photosensitivity of proteins.

Evaluation of Heavy-Chain C-Terminal Deletion on Product Quality and Pharmacokinetics of Monoclonal Antibodies.

Due to their potential influence on stability, pharmacokinetics, and product consistency, antibody charge variants have attracted considerable attention in the biotechnology industry. Subtle to significant differences in the level of charge variants and new charge variants under various cell culture conditions are often observed during routine manufacturing or process changes and pose a challenge when demonstrating product comparability. To explore potential solutions to control charge heterogeneity, monoclonal antibodies (mAbs) with native, wild-type C-termini, and mutants with C-terminal deletions of either lysine or lysine and glycine were constructed, expressed, purified, and characterized in vitro and in vivo. Analytical and physiological characterization demonstrated that the mAb mutants had greatly reduced levels of basic variants without decreasing antibody biologic activity, structural stability, pharmacokinetics, or subcutaneous bioavailability in rats. This study provides a possible solution to mitigate mAb heterogeneity in C-terminal processing, improve batch-to-batch consistency, and facilitate the comparability study during process changes.

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.

Kinetic Modeling of Methionine Oxidation in Monoclonal Antibodies from Hydrogen Peroxide Spiking Studies.

UNLABELLED: When isolator technology is applied to biotechnology drug product fill-finish process, hydrogen peroxide (H2O2) spiking studies for the determination of the sensitivity of protein to residual peroxide in the isolator can be useful for assessing a maximum vapor phase hydrogen peroxide (VPHP) level. When monoclonal antibody (mAb) drug products were spiked with H2O2, an increase in methionine (Met 252 and Met 428) oxidation in the Fc region of the mAbs with a decrease in H2O2 concentration was observed for various levels of spiked-in peroxide. The reaction between Fc-Met and H2O2 was stoichiometric (i.e., 1:1 molar ratio), and the reaction rate was dependent on the concentrations of mAb and H2O2. The consumption of H2O2 by Fc-Met oxidation in the mAb followed pseudo first-order kinetics, and the rate was proportional to mAb concentration. The extent of Met 428 oxidation was half of that of Met 252, supporting that Met 252 is twice as reactive as Met 428. Similar results were observed for free L-methionine when spiked with H2O2. However, mAb formulation excipients may affect the rate of H2O2 consumption. mAb formulations containing trehalose or sucrose had faster H2O2 consumption rates than formulations without the sugars, which could be the result of impurities (e.g., metal ions) present in the excipients that may act as catalysts. Based on the H2O2 spiking study results, we can predict the amount Fc-Met oxidation for a given protein concentration and H2O2 level. Our kinetic modeling of the reaction between Fc-Met oxidation and H2O2 provides an outline to design a H2O2 spiking study to support the use of VPHP isolator for antibody drug product manufacture. LAY ABSTRACT: Isolator technology is increasing used in drug product manufacturing of biotherapeutics. In order to understand the impact of residual vapor phase hydrogen peroxide (VPHP) levels on protein product quality, hydrogen peroxide (H2O2) spiking studies may be performed to determine the sensitivity of monoclonal antibody (mAb) drug products to residual peroxide in the isolator. In this study, mAbs were spiked with H2O2; an increase in methionine (Met) oxidation of the mAbs with a decrease in H2O2 concentration was observed for various levels of spiked-in peroxide. The reaction between Met and H2O2 was 1:1, and its rate was dependent on mAb and H2O2 concentrations. Consumption of H2O2 by Met followed pseudo first-order kinetics; the rate was proportional to mAb concentration. Formulations containing trehalose or sucrose had faster consumption rates than formulations without the sugars, which could be due to excipient impurities. Based on H2O2 spiking study results, we can predict the amount of Met oxidation for a given mAb concentration and H2O2 level. Our modeling of the reaction between Fc-Met oxidation and H2O2 provides an outline to design a H2O2 spiking study that supports using VPHP isolators during manufacture of mAb products.

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.

Characterization of asparagine 330 deamidation in an Fc-fragment of IgG1 using cation exchange chromatography and peptide mapping.

Deamidation is one of the most common degradation pathways for proteins and frequently occurs at "hot spots" with Asn-Gly, Asn-Ser or Asn-Thr sequences. Occasionally, deamidation may occur at other motifs if the local protein structure can participate or assist in the formation of the succinimide intermediate. Here we report the use of a chymotryptic peptide mapping method to identify and characterize a deamidated form of an IgG1 which was observed as an acidic peak in the cation exchange chromatography (CEX). The antibody was formulated in sodium acetate buffer, pH 5.3 and this deamidated form was observed mainly under thermal stress conditions. It was found that the IgG1 molecule with deamidation in the Fc region at asparagine residue 330 (in a Val-Ser-Asn-Lys motif) is the predominant form in this CEX peak, and was missed by tryptic mapping because the peptides are hydrophilic and elute near the void volume. In addition, a domain-based CEX method using papain digestion was developed to monitor the Asn 330 deamidation. These methods revealed that the Fc deamidation occurs mainly at Asn 330 in the VSNK motif at pH 5.3, whereas at pH 7.5, deamidation occurs predominantly at Asn 389 and Asn 394 in the NGQPENNYK motif.

Effect of ionic strength and pH on the physical and chemical stability of a monoclonal antibody antigen-binding fragment.

Monoclonal antibody (mAb) fragments are emerging as promising alternatives to full-length mAbs as protein therapeutic candidates. Antigen-binding fragments (Fabs) are the most advanced with three Fab-based drug products currently approved. This work presents preformulation characterization data on the effect of pH, NaCl concentration, and various cationic excipients on the physical and chemical stability of a Fab molecule with multiple negatively charged Asp residues in the complementarity-determining region. Conformational stability was evaluated using an empirical phase diagram approach based on circular dichroism, intrinsic Trp and extrinsic 8-anilino-1-naphthalene sulfonate (ANS) fluorescence, and static light scattering measurements. The effect of NaCl concentration, various cationic excipients and pH on the Fab molecule's conformational stability, aggregation propensity, and chemical stability (Asp isomerization) was determined by differential scanning calorimetry, optical density measurements at 350 nm (OD350 ), and ion-exchange chromatography, respectively. Increasing NaCl concentration increased the overall conformational stability, decreased aggregation rates, and lowered the rates of Asp isomerization. No such trends were noted for pH or cationic excipients. The potential interrelationships between protein conformational and chemical stability are discussed in the context of designing stable protein formulations.

Photodegradation of human growth hormone: a novel backbone cleavage between Glu-88 and Pro-89.

The exposure of protein pharmaceuticals to light can cause loss of potency, oxidation, structural changes and aggregation. To elucidate the chemical pathways of photodegradation, we irradiated human growth hormone (hGH) at λ = 254 nm, λ ≈ 265-340 nm, and λ ≈ 295-340 nm (using the spectral cutoff of borosilicate glass) and analyzed the products by mass spectrometry. By means of LC-MS/MS analysis, we observed an unusual peptide backbone cleavage between Glu-88 and Pro-89. The crystal structure of hGH indicates that these residues are in proximity to Trp-86, which likely mediates this backbone cleavage. The two cleavage fragments observed by MS/MS analysis indicate the loss of CO from the amide bond and replacement of the Glu-C(═ O)Pro bond with a Glu-H bond, accompanied by double bond formation on proline. The reaction is oxygen-independent and likely involves hydrogen transfer to the Cα of Glu-88. To probe the influence of the protein fold, we irradiated hGH in its unfolded state, in 1:1 (v/v) acetonitrile/water, and also the isolated tryptic peptide Ile-78-Arg-90, which contains the Glu-88-Pro-89 sequence. In both cases, the cleavage between Glu-88 and Pro-89 was largely suppressed, while other cleavage pathways became dominant, notably between Gln-84 and Ser-85, as well as Ser-85 and Trp-86.

Asparagine deamidation dependence on buffer type, pH, and temperature.

The deamidation of asparagine into aspartate and isoaspartate moieties is a major pathway for the chemical degradation of monoclonal antibodies (mAbs). It can affect the shelf life of a therapeutic antibody that is not formulated or stored appropriately. A new approach to detect deamidation using ion exchange chromatography was developed that separates papain-digested mAbs into Fc and Fab fragments. From this, deamidation rates of each fragment can be calculated. To generate kinetic parameters useful in setting shelf life, buffers prepared at room temperature and then placed at the appropriate stability temperatures. Solution pH was not adjusted to the same at different temperatures. Deamidation rate at 40°C was faster in acidic buffers than in basic buffers. However, this trend is reversed at 5°C, attributed to the change in hydroxide ion concentration influenced by buffer and temperature. The apparent activation energy was higher for rates generated in an acidic buffer than in a basic buffer. The rate-pH profile for mAb1 can be deconvoluted to Fc and Fab. The Fc deamidation showed a V-shaped profile: deamidation of PENNY peptide is responsible for the rate at high-pH, whereas deamidation of a new site, Asn323, may be responsible for the rate at low-pH. The profile for Fab is a straight line without curvature.

Polar solvents decrease the viscosity of high concentration IgG1 solutions through hydrophobic solvation and interaction: formulation and biocompatibility considerations.

Low-volume protein dosage forms for subcutaneous injection pose unique challenges to the pharmaceutical scientist. Indeed, high protein concentrations are often required to achieve acceptable bioavailability and efficacy for many indications. Furthermore, high solution viscosities are often observed with formulations containing protein concentrations well above 150 mg/mL. In this work, we explored the use of polar solvents for reducing solution viscosity of high concentration protein formulations intended for subcutaneous injection. An immunoglobulin, IgG1, was used in this study. The thermodynamic preferential interaction parameter (Γ23 ) measured by differential scanning calorimetry, as well as Fourier transform infrared, Raman, and second-derivative UV spectroscopy, were used to characterize the effects of polar solvents on protein structure and to reveal important mechanistic insight regarding the nature of the protein-solvent interaction. Finally, the hemolytic potential and postdose toxicity in rats were determined to further investigate the feasibility of using these cosolvents for subcutaneous pharmaceutical formulations. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 102:1182-1193, 2013.

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.

Isomerization of Asp-Asp motif in model peptides and a monoclonal antibody Fab fragment.

Isomerization of aspartyl (Asp or D) residues is a critical degradation route to consider for stable monoclonal antibody formulations. Among the known hotspot sequences, the DD motif is relatively understudied. To gain mechanistic insights, we used model hexapeptides, YADXFK, YADDXK, and DIDDDM, as surrogates for the hotspots in a Fab protein (YADDFK and DIDDDM), to characterize the rate-pH profile of Asp isomerization. Compared with the YADGFK peptide, isomerization of D3 (the first D in the DD pair) in YADDFK was highly pH dependent. Comparison of rate-pH profiles of YADDFK, YADNFK, and YADHFK revealed a charge effect of the n + 1 residue-isomerization rate is accelerated by the positive side chain and reduced by negative side chain at n + 1 residue. Studies on YADDFK, YADDAK, and YADDGK indicated a mutual impact of D3 and D4 on their respective isomerization rates through charge effect. Comparison of rate-pH profile of DIDDDM sequence in peptide models with that in the complementary determining region of the Fab showed a faster rate in the Fab than in peptides, presumably because of contribution from structural factors in the former.

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.

Oxidation of human growth hormone by oxygen-centered radicals: formation of Leu-101 hydroperoxide and Tyr-103 oxidation products.

Human growth hormone (hGH) was exposed to oxygen-centered radicals generated through the thermolysis of AAPH in the presence of dioxygen. Such conditions mimic oxidative processes which protein pharmaceuticals can encounter during formulation in the presence of polysorbates. We detected the oxidation of Met to Met sulfoxide, the formation of protein carbonyls, the oxidation of Tyr to dityrosine and several additional Tyr oxidation products, the conformation-dependent oxidation of Trp, and the site-specific formation of protein hydroperoxides. The sensitivity of Met oxidation correlates with their solvent accessible surface, i.e. the yields of MetSO decreased in the order Met-14 > Met-125 > Met-170. Trp oxidation in native hGH was negligible, but was enhanced through denaturation. Dityrosine formed predominantly intramolecularly but did not contribute significantly to protein cross-linking. Hydroperoxides formed selectively on Leu-101 and were generated specifically by alkoxyl radicals, generated through the decomposition of peroxyl radicals. Tyr-103 was converted into a series of oxidation products characterized by mass shifts of Tyr + 14 Da and Tyr + 16 Da.