Article pubs.acs.org/molecularpharmaceutics
Screening Methods to Identify Indole Derivatives That Protect against Reactive Oxygen Species Induced Tryptophan Oxidation in Proteins Parbir Grewal, Mary Mallaney, Kimberly Lau, and Alavattam Sreedhara* Late Stage Pharmaceutical Development, Genentech, Inc., 1 DNA Way, South San Francisco, California 94080, United States ABSTRACT: Oxidative damage to proteins is one of the most prominent chemical degradation pathways that are of concern for drug product development in the biotechnology industry. Especially susceptible to oxidation are the Met and Trp residues in proteins. While L-Met and L-Trp have been shown to act as antioxidants typically protecting proteins against Met and Trp oxidation, respectively, L-Trp has been shown to be particularly sensitive to light, thereby producing various reactive oxygen species (ROS), including H2O2. There is hence a need to identify nonphotosensitive molecules that can protect Trp oxidation in proteins so that they can be easily handled under drug product manufacturing conditions. A combination of screening methods, namely, cyclic voltammetry (CV) and hydrogen peroxide generation upon photoirradiation, was used to screen several molecules to identify compounds that can act as antioxidants. Specifically, indole and tryptophan with hydroxy groups on the six-membered aromatic ring were found to have lower oxidation potentials than the parent compounds and produced the least amount of H2O2 upon light exposure. These derivatives were also found to sufficiently protect tryptophan oxidation in mAb1 against a variety of reactive oxygen species such as alkyl peroxides, hydroxyl radicals, and singlet oxygen and may be useful as part of the formulation toolkit to protect against protein degradation via oxidation. KEYWORDS: tryptophan, antioxidants, 5-hydroxy tryptophan, 5-hydroxy indole, monoclonal antibodies, protein oxidation, reactive oxygen species
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INTRODUCTION Oxidative degradation of amino acid residues is a commonly observed phenomenon in protein pharmaceuticals. A number of amino acid residues are susceptible to oxidation, particularly methionine, cysteine, histidine, tryptophan, and tyrosine.1 Oxidation is typically observed when the protein is exposed to hydrogen peroxide, light, metal ions, or a combination of these during various processing steps.1 In particular, proteins exposed to light,2 azo-initiator 2,2'-azo-bis-(2-amidinopropane) dihydrochloride (AAPH), or Fenton reagents3 have shown increased levels of oxidation on tryptophan residues, whereas those exposed to hydrogen peroxide have typically shown only methionine oxidation.3 Light exposure can result in protein oxidation through the formation of reactive oxygen species (ROS) including singlet oxygen, hydrogen peroxide, and superoxide,1−4 whereas protein oxidation typically occurs via hydroxyl radicals in the Fenton mediated reaction5 and via alkyl peroxides in the AAPH mediated reaction.6 Oxidation of tryptophan leads to a myriad of oxidation products, including hydroxytryptophan, kynurenine, and N-formylkynurenine, and has the potential to impact safety and efficacy.1,3,4 Wei et al. reported that oxidation of a particular tryptophan residue in the heavy chain complementarity determining region (CDR) of a monoclonal antibody was correlated to loss of biological function.2 Trp oxidation mediated by a histidine coordinated metal ion has recently been reported for a Fab molecule.7 Autoxidation of polysorbate 20 in the Fab formulation, leading to the generation of various peroxides (probably alkyl and © 2014 American Chemical Society
hydrogen peroxide), has also been invoked in the same report. Autoxidation induced generation of these peroxides can also lead to methionine oxidation in the protein during long-term storage of the drug product since Met residues in proteins have been suggested to act as internal antioxidants8 and are easily oxidized by peroxides. Oxidation of amino acid residues has the potential to impact the biological activity of the protein. This may be especially true for monoclonal antibodies (mAbs). Methionine oxidation at Met254 and Met430 in an IgG1 mAb potentially impacts serum half-life in transgenic mice9 and also impacts the binding of human IgG1 to FcRn and Fc-gamma receptors.10 Protein formulation scientists need to evaluate the stability of proteins, especially in liquid state, during drug product manufacturing and storage. The development of pharmaceutical formulations sometimes includes the addition of antioxidants to prevent oxidation of the active ingredient. The addition of Lmethionine to formulations has resulted in the reduction of methionine residue oxidation seen during oxidation stress studies in proteins and peptides.3,11 Likewise, the addition of Ltryptophan has been shown to reduce the oxidation of tryptophan residues.3,7 L-Trp, however, possesses strong absorbance in the UV region (260−290 nm), making it a Received: Revised: Accepted: Published: 1259
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primary target during photo-oxidation.12 Trp has been hypothesized as an endogenous photosensitizer enhancing the oxygen dependent photo-oxidation of tyrosine13 and other amino acids.14 McCormick et al.15 and Wentworth et al.16 have shown that L-Trp can generate hydrogen peroxide when exposed to light. McCormick and Thomason have also shown that L-Trp under UV light produces hydrogen peroxide via the superoxide anion.17 Additionally, tryptophan is known to produce singlet oxygen upon exposure to light.18 Similar to the protein oxidation induced by autoxidation of polysorbate 20, it is possible that protein oxidation can occur upon ROS generation by other excipients in the protein formulation (e.g., L-Trp) under normal handling conditions. To understand the susceptibility of the protein to oxidation during formulation development, the formulation scientist relies on various stress models. Typical oxidation stress models used in peptide and protein formulation development include H2O2, tert-butyl hydroperoxide (tBHP), and the Fenton reactions.3 In the study by Ji et al., H2O2 and tBHP treatment led to Met oxidation in the absence of any metal ion. In the presence of metal ion, specifically Fe and Cu, various amino acids including Met, His, Tyr, and Trp were susceptible to oxidation, presumably by the ROS that are generated via the Fenton mechanism.3 Another useful oxidation stress model is the compound 2,2′azobis(2-amidinopropane) dihydrochloride, or AAPH, a hydrophilic free radical initiator which can be used as a tool for acceleration of free radical reactions that oxidize DNA.19 Decomposition of AAPH generates alkyl, alkoxyl, and alkyl peroxyl radicals6 that may mimic the products formed during the autoxidation of polysorbate. These degradation products have been shown to oxidize amino acid residues in proteins, including methionine, tyrosine, and tryptophan residues.3,20 Similarly, light exposure can be a valuable tool for the assessment of the oxidative sensitivity of proteins, especially when used under mild to moderate conditions. The potential for light induced damage to proteins is well reported in the literature and has been a topic of recent review.21 On the basis of their photosensitivity, it is recommended to study the effect of both visible and ultraviolet light on drug product quality during the formulation and clinical development of proteins.22 The ICH guideline recommends a total exposure of not less than 200 W h m−2 in the UV range (320−400 nm) and 1.2 million lx h in the visible range (400−800 nm) for confirmatory studies of photosensitivity. These conditions were developed to mimic sunlight and are thus extremely harsh on proteins; they may require revision for practical utility during formulation development. In addition to their use in the evaluation of the oxidative sensitivity of proteins, stress models can also be used as screening tools to identify potential antioxidants. Several screening methods have been elaborated in the literature.23 Of particular interest are methods that measure the scavenging activities of reactive oxygen species using fluorescent dyes and those that measure the redox potentials of new excipients using cyclic voltammetry. Cyclic voltammetry (CV) is an electrochemical technique that has been used extensively to understand the redox properties of various small molecules,24 pharmaceutical excipients,25 metalloproteins, and enzymes.26 In our application, the comparison of the oxidation potentials of several candidates can be used to guide antioxidant selection. In particular, it is desirable for the antioxidant to possess a lower
oxidation potential than the amino acid of interest so that the antioxidant is more readily oxidized.27 Ji et al. have demonstrated the antioxidant properties of LTrp using AAPH as the oxidation stress model and parathyroid hormone (PTH) as a model peptide.3 Additionally, they recommend the use of H2O2, H2O2 + Fe(II) or Fe(III), and AAPH as relevant stress models to screen for antioxidants. Since L-Trp is known to be photosensitive and produces H2O2 upon light exposure,15−17,28 it can potentially affect protein drug product stability via Met and Trp oxidation during routine manufacturing procedures where the drug product is exposed to ambient light. To find excipients that can be used as antioxidants without the detrimental effects of light induced peroxide generation, we screened a variety of compounds, including L-Trp derivatives and other known antioxidants, for their H2O2 producing ability during ambient and harsh light handling conditions. In addition to our preference for molecules which produced low amounts of H2O2 upon photoactivation, we hypothesized that molecules with lower oxidation potentials than L-Trp can potentially act as antioxidants by competing for ROS in solution. Compounds that, compared to L-Trp, produced less H2O2 upon light treatment as well as those with lower oxidation potentials than L-Trp were then evaluated for their possible antioxidant properties using an IgG1 mAb (“mAb1”) as a model protein and AAPH, light, and Fenton reaction as oxidative stress models. We have previously shown that mAb1 is susceptible to site-specific Trp oxidation at W53 in the heavy chain Fab and has been thoroughly characterized.29 Additionally, that study showed that H2O2 production acts as a good surrogate for 1O2 and other ROS formed during photoactivation studies. Our results clearly indicate that cyclic voltammetry can be used to screen for new compounds with lower oxidation potentials than L-Trp and that these compounds are potentially useful antioxidants that protect Trp oxidation in proteins. By using the cyclic voltammetry and light exposure screening approaches as complementary methods, we were able to identify promising antioxidants for use in protein formulation development.
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MATERIALS AND METHODS Materials. mAb1 (anti-DR5) is an IgG1 monoclonal antibody that was expressed in Chinese Hamster Ovary (CHO) cells and purified by a series of chromatography methods including affinity purification by protein A chromatography and ion-exchange chromatography. mAb1 was formulated at 20 mg/mL in 20 mM histidine acetate, 250 mM sugar, and 0.02% surfactant at pH 6.0 as a liquid formulation. L-Tryptophan, L-tryptophanamide, N-acetyl-L-tryptophan, Nacetyl-L-tryptophanamide, 5-fluoro-L-tryptophan, 5-methoxyDL-tryptophan, indole, 5-hydroxyindole, tryptamine hydrochloride, serotonin hydrochloride, melatonin, salicylic acid, 5hydroxysalicylic acid, anthranilic acid, 5-hydroxyanthranilic acid, and L-methionine were purchased from Sigma-Aldrich (St. Louis, MO). 5-Amino-DL-tryptophan, 5-nitro-DL-tryptophan, 4hydroxyindole, 5-hydroxyindole-3-acetic acid, 7-hydroxyindole, and 7-hydroxyindole-2-carboxylic acid were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). 5-Hydroxy-Ltryptophan (5-HT) and AAPH were purchased from EMD Biosciences (San Diego, CA). Indole-3-acetic acid and Trolox were purchased from Thermo Fisher Scientific (Waltham, MA). Pyridoxine HCl was purchased from Research Products 1260
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concentrations of 0.2 mM FeCl3 and 10 ppm H2O2 were added to each mAb1 solution and then incubated at 40 °C for 3 h. After incubation, each reaction was quenched by the addition of 100 mM L-Met (from a stock solution of 200 mM L-Met prepared in 20 mM histidine hydrochloride) and then prepared for analysis by reverse-phase HPLC. Sample Analysis by Reverse-Phase HPLC (RP-HPLC). mAb1 solution from the stress studies was prepared to 1.1 mg/ mL in 0.1 M Tris, 4.4 mM ethylenediaminetetraacetic acid (EDTA), and 1.1 mM cysteine. A sample of 150 μL of 0.1 mg/ mL papain was added to 1.35 mL of the mAb1 solution before incubation at 37 °C for 2 h. Following incubation, 900 μL of the solution was combined with 100 μL of 1 M dithiothreitol (DTT) and incubated for another 30 min at 37 °C. Samples were then run on an Agilent, Inc. 1100/1200 HPLC system (Santa Clara, CA) equipped with UV detection at 280 nm in conjunction with a Varian, Inc. Pursuit 3 μm, 2 mm ID × 250 mm diphenyl column (Palo Alto, CA). Mobile phase A was 0.1% TFA in water. Mobile phase B was 0.1% TFA in acetonitrile. The mobile phase gradient increased linearly from 34% B at 0 min to 43% B at 50.0 min, then to 95% B at 50.1 min. The gradient remained at 95% B until 60.1 min and then decreased linearly from 95% B to 34% B between 60.1 and 60.2 min. The gradient remained at 34% B until the end of the cycle at 80.2 min. The column temperature was 65 °C, the total flow rate was 0.2 mL/min, and the injection volume of each sample was 6 μL. Chromatograms were then integrated for the quantification of oxidation.
International (Mount Prospect, IL). All other reagents used were of analytical quality. Excipient Screening with Light Stress. Antioxidant candidates (1 mM) were prepared in 20 mM histidine acetate buffer at pH 5.5. These solutions were aliquoted into glass vials (2 mL/glass vial) and exposed to 4 h of light in an Atlas SunTest CPS+ Xenon Test Instrument (Chicago, IL). The total UV dose was 90 W h/m2, and the total visible dose was 0.22 million lx h over the 4 h period. Control vials were wrapped in aluminum foil and treated similarly. The amount of hydrogen peroxide generated after exposure to light was measured using the Amplex Ultra Red Assay (Invitrogen, Carlsbad, CA) following the manufacturer’s recommended procedure. On addition of horseradish peroxidase (HRP), the dye reacts 1:1 stoichiometrically with H2O2, resulting in the production of fluorescent oxidation product resorufin. In this study, fluorescence readings were obtained using a Spectra Max M2 Microplate Reader (Molecular Devices, Sunnyvale, CA) with excitation and emission set at 560 and 590 nm, respectively. Final H2O2 concentrations were determined using a standard curve ranging from 0 to 20 μM. Excipient Screening with Cyclic Voltammetry. Antioxidant candidates were dissolved in deionized water and then added to a 0.2 M lithium perchlorate electrolyte solution. Solutions were characterized with an EG&G Princeton Applied Research model 264A polarograph/voltammeter with a model 616 RDE glassy carbon electrode as working electrode. Solutions were scanned from −0.10 V to +1.50 V at a scan rate of either 100 or 500 mV/s. The analytical cell was purged for 4 min with nitrogen before the scanning of each antioxidant solution. mAb1 Light Stress Study. mAb1 was prepared to 5 mg/ mL in 20 mM histidine acetate, 250 mM sugar, and 0.02% surfactant at pH 6.0. L-Met was added to a final concentration of 1, 10, 25, 50, and 100 mM from a stock solution of 200 mM L-Met in the same buffer. All other antioxidant candidates were added to a final concentration of 1 mM from 10 mM stock solutions prepared in 20 mM histidine acetate. Glass vials containing these formulations were exposed to 250 W/m2 light in an Atlas SunTest CPS+ Xenon Test Instrument (Chicago, IL) at ambient temperature. Control vials were wrapped in aluminum foil and treated similarly. Afterward, light exposure solutions were prepared for analysis by reverse-phase highperformance liquid chromatography (HPLC). mAb1 AAPH Stress Study. mAb1 was prepared to 4 mg/ mL in a formulation of 20 mM histidine acetate, 250 mM sugar, and 0.02% surfactant at pH 6.0. Antioxidant candidates were added to a final concentration of 1 mM from 10 mM stock solutions prepared in 20 mM histidine acetate. A sample of 200 μL of 10 mM AAPH was added to 2 mL of each mAb1 solution and then incubated at 40 °C for 24 h. After incubation, each solution was buffer exchanged with formulation buffer (20 mM histidine acetate, pH 6.0) using a PD-10 column so that the final mAb1 concentration was 2.3 mg/mL. After buffer exchange, each solution was prepared for analysis by reversephase HPLC. mAb1 Fenton Stress Study. While typical Fenton reactions are carried out with Fe2+ and H2O2, we conducted our studies using a slightly modified method as reported earlier.3 mAb1 was prepared to 3 mg/mL in a formulation of 20 mM histidine hydrochloride at pH 6.0. Antioxidants were added to a final concentration of 1 mM from 10 mM stock solutions prepared in 20 mM histidine hydrochloride. Final
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RESULTS AND DISCUSSION Monoclonal antibodies have been shown to produce ROS through the antibody catalyzed water oxidation pathway (ACWOP) wherein antibodies potentially catalyze a reaction between water and singlet oxygen generating hydrogen peroxide.16,30 In the ACWOP, a variety of ROS, including superoxide anion, dihydrogen trioxide, ozone, and even hydrotrioxy radical are generated in the pathway toward the production of hydrogen peroxide.31 We have recently shown that surface exposed tryptophans in mAb1 act as substrate (1O2 and O2−) generators that facilitate ACWOP even under mild light conditions in a time- and concentration-dependent manner.29 As a result, controlled light exposure can be used as an accelerated stress model to study protein oxidation, specifically on proteins that have exposed tryptophans. That study has also shown that hydrogen peroxide can serve as a surrogate for a number of ROS, including superoxide and singlet oxygen. We utilized the same human monoclonal IgG1 antibody (mAb1) to screen and evaluate potential antioxidants. Our previous study29 demonstrated that mAb1 is particularly susceptible to oxidation during storage under pharmaceutically relevant conditions. Oxidation was shown to be site-specific and localized to Trp53 (W53) on the heavy chain CDR (Fab) as evaluated by tryptic peptide map. Additionally, a reverse-phase HPLC assay was used to measure the total oxidation in the HC Fab and Fc regions of mAb1. Peaks from RP-HPLC were identified using LC/MS and show a strong correlation with results of the tryptic peptide map, indicating that the RP method could be used as a surrogate for the detection of W53 (i.e., % Fab) oxidation.29 Ji et al. have demonstrated the effectiveness of free L-Trp against AAPH induced Trp oxidation and that of L-Trp plus LMet against peroxide, Fenton, and AAPH induced oxidation 1261
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Figure 1. Oxidation after eight hours of light exposure (250 W/m2) of (A) Fab and (B) Fc in mAb1 (5 mg/mL in 20 mM histidine acetate). All vials were placed in the lightbox except for the mAb1 ref Mat. Foil CTRL vials were covered in foil before placement in the lightbox. All samples are the average of three separate vials, except for 10 mM Met, 1 mM Trp (*), which was two vials, and mAb1 ref Mat, which was one vial with three independent injections on the HPLC. Error bars represent one standard deviation.
stress in PTH.3 Since L-Trp is known to be photosensitive28 and can produce H2O2 upon light exposure, we evaluated the sensitivity of mAb1 to L-Trp under light stress, with and without the addition of L-Met as a potential antioxidant. mAb1 at 5 mg/mL was incubated with 1 mM L-Trp and various concentrations of L-Met in a buffer of 20 mM histidine acetate and exposed to 8 h of light at 250 W/m2. We specifically chose L-His based buffers for our antioxidant screening studies because they are extensively used in protein formulations.32 Additionally, mAb1 was found to be stable in L-His based buffer at pH 6.0. The effects of L-Trp and L-Met on mAb1 Fab and mAb1 Fc oxidation are shown in Figure 1A and B, respectively. The mAb1 reference material (no light exposure) and the foil control show about 2% Fab oxidation. Since the foil control and the reference material show the same level of Fab oxidation, it is unlikely that heat alone is causing the oxidation of the Fab. When mAb1 is exposed to light (the “No Excipient” case), the Fab oxidation doubles to 4% (Figure 1A). With the addition of 1 mM L-Trp, the Fab oxidation increases to almost 9%, suggesting that free L-Trp is generating ROS under light exposure that may result in oxidation of W53 on the Fab. Further addition of 10, 25, 50, and 100 mM L-Met appears to reduce Fab oxidation slightly, but even 100 molar excess of L-
Met does not reduce Fab oxidation to the level of the foil control. Potentially L-His in the buffer can quench singlet oxygen with a quenching constant similar to that of L-Trp itself,33 but the reaction likely depends on proximity to the site of singlet oxygen generation. Oxidation in the Fc region of mAb1 is predominantly of Met residues Met 254 and Met 430.29 Figure 1B shows the effect of light exposure on Fc oxidation. mAb1 reference material and foil control show about 8% Fc oxidation even before exposure to light. Exposure to light results in only a minor increase in Fc oxidation (“No Excipient”) for mAb1 in formulation buffer. However, incubation with 1 mM L-Trp results in over 20% oxidation at these Met sites in the Fc region as seen by the RPHPLC assay. Addition of various concentrations of L-Met (10, 25, 50, and 100 mM) to formulations containing 1 mM L-Trp reduces the amount of Fc oxidation, although even 100 mM LMet does not reduce Fc oxidation to the level of the controls. Wentworth et al. reported that L-Trp produced H2O2 in a substoichiometric fashion, while antibodies under similar conditions were producing catalytic amounts.16 McCormick and Thomason showed that production of peroxide by L-Trp is mediated via superoxide ion.17 While these experiments were carried out predominantly using UV light, we wanted to test the 1262
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Figure 2. Dose-dependent H2O2 production by L-Trp. Blue diamonds, L-Trp alone; black triangles, L-Trp + SOD; black circles and blue squares, LTrp + NaN3 ± SOD.
susceptibility of free L-Trp under pharmaceutically relevant conditions, especially under both ICH and ambient light conditions. L-Trp (0.32−7.5 mM) was dissolved in sodium phosphate buffer at pH 7.1 and exposed for 3 h at 250 W/m2 UV light and about 150 000 lx visible light. Samples were taken and analyzed immediately via the Amplex assay to detect the amount of H2O2 generated under these conditions. A large quantity of H2O2 was generated by free L-Trp upon light exposure in a concentration-dependent manner (Figure 2). This H2O2 generation was reduced substantially in the presence of 50 mM sodium azide, a known quencher of singlet oxygen.34 When L-Trp was incubated with a combination of 50 mM NaN3 and 150 U superoxide dismutase (SOD) or SOD alone, significant amounts of H2O2 were still detected. This indicates that, in addition to singlet oxygen, superoxide ion was also generated upon photoirradiation that is converted to H2O2 by SOD. However, the H2O2 produced under our experimental (ICH light) conditions was substantially lower than previously reported in the presence of SOD.17 While confirming the photosensitivity of free L-Trp under ICH light conditions, we also studied the effect of ambient light that is typically seen in laboratories. Measurements using a DLM1 digital light meter in various laboratories indicated an average of 300 lx on a laboratory benchtop (with white fluorescent lighting), an average of 3000 lx in a laminar flow hood (with white fluorescent lighting), and about 10 000 lx for a windowsill exposed to southeast sunlight. Under these conditions, L-Trp in formulation buffers containing 50 mg/mL mAb1 produced hydrogen peroxide in the micromolar range as detected using the Amplex Ultra Red Assay (Figure 3A). Peroxide production increased with both luminosity (300, 3000, and 10000 lx) and time (1, 3, and 7 days). The protein samples were further analyzed using the mAb1 specific RP-HPLC assay and showed increased heavy chain Fab oxidation corresponding to oxidation in W53 (Figure 3B). At the same time, % Fc oxidation in mAb1 under these conditions increased from 5 to 40% between 300
Figure 3. (A) H2O2 production by 50 mg/mL mAb1 formulations containing 3.2 mM L-Trp when exposed to ambient light conditions in the laboratory for 1, 3, and 7 days; (B) % Fab oxidation in mAb1 formulations containing 3.2 mM L-Trp after 10 days of exposure to ambient light conditions in the laboratory.
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and 10000 lx, respectively (data not shown). These levels of light exposure and time are pharmaceutically relevant during drug substance handling under ambient light and temperature before fill/finish operations and potentially while inspecting drug product vials. These results support previous observations that L-Trp is photosensitive and that it produces several reactive oxygen species, including singlet oxygen, superoxide, and H2O216,17,28 that can be detrimental to mAb product quality and that care should be taken while handling and storing L-Trp containing buffers. Screening for Antioxidants. Tryptophan is an electron rich amino acid that undergoes oxidative and electrophilic addition reactions in the presence of ROS such as hydroxyl radicals and singlet oxygen. Any potential antioxidant to protect Trp oxidation in proteins should have similar if not superior reactivity toward these ROS. Trp residues in proteins have various functions including structural and functional roles, and oxidation at these sites could lead to detrimental effects.2 Given the importance of protecting Trp oxidation in proteins and peptides, we evaluated a series of compounds that are either based on the L-Trp structure or are known antioxidants. Compounds screened in this study included derivatives of tryptophan, indole, aromatic acids such as salicylic acid and anthranilic acid, and some vitamins. The chemical structures of the compounds tested are shown in Figure 4. While L-Trp may be an effective antioxidant under certain circumstances, its photosensitivity may limit its utility during normal processing without special precautions. Hence, we focused our attention on the photosensitivity of the above molecules and rated their H2O2 generation capability with respect to L-Trp. As a screening tool, antioxidant candidates were exposed to light (4 h), and the resulting H2O2 formation was quantified by the Amplex Ultra Red Assay (Figure 5). Hydrogen peroxide production ratios between the compounds tested and L-Trp are shown in Table 1. Figure 5A shows the hydrogen peroxide generation by tryptophan derivatives upon light exposure. Under similar conditions of light (corresponding to 0.22 million lx h visible light and 90 Wh/m2 UV dose over a 4 h period) and buffer (20 mM L-His-acetate, pH 5.5), 5-HT produced about one tenth of the H2O2, while kynurenine produced about one-fifth of the H2O2, when compared to L-Trp. Other tryptophan derivatives produced anywhere between 30% and 105% of the H2O2 produced by L-Trp. In comparison to L-Trp, Trolox (a watersoluble vitamin E derivative) produced 123 times more H2O2, and pyridoxine (vitamin B6) produced 5 times more H2O2 (Table 1). Various indole derivatives were also tested under similar light conditions and are shown in Figure 5B. It is interesting to note that indole, which has a basic structure like L-Trp, behaved similarly to L-Trp, but indole-3-acetic acid produced more than 2× as much H2O2 (Figure 5B). The hydroxy derivatives of indole behaved like 5-HT in that they produced negligible amounts of H2O2 upon light exposure. We also tested several biochemically relevant derivatives of L-Trp, namely, tryptamine, serotonin, and melatonin. Tryptamine produced about half as much H2O2 as L-Trp. Interestingly, serotonin (5-hydroxytryptamine) behaved much like the 5-OH derivatives of indole and tryptophan, producing very little H2O2 upon light exposure, while melatonin (N-acetyl-5-methoxytryptamine) produced less than a third of the H2O2 produced by L-Trp (Table 1). To understand the ROS formed during photoirradiation, we tested several of the Trp derivatives in the presence of NaN3, a known singlet oxygen quencher.34 All of
Figure 4. Structures of various compounds used in this study. (A) Tryptophan derivatives, (B) kynurenine, (C) indole derivatives, and (D) aromatic acid derivatives.
the compounds tested showed a substantial decrease in the amount of hydrogen peroxide generated under these conditions, indicating that singlet oxygen is a major ROS created upon photoirradiation of Trp and its derivatives (Figure 6). 1264
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Figure 5. Hydrogen peroxide generation under light stress (4 h at 250 W/m2). (A) Tryptophan derivatives and (B) indole derivatives.
We also investigated other aromatic compounds such as salicylic acid and derivatives based on their reported antioxidant properties.35 Ratios of hydrogen peroxide generated with respect to L-Trp are presented in Table 1. It is interesting to note that salicylic acid produced very little H2O2 upon light exposure, while its 5-OH derivative behaved like L-Trp. However, anthranilic acid produced twice as much H2O2 as L-Trp, but 5-OH-anthranilic acid produced half as much H2O2 compared to that of L-Trp. While we were unable to draw conclusions based on chemical structures alone, we became interested in redox properties because aromatic ring substitutions appeared to impact the amount of hydrogen peroxide generated. An understanding of oxidation/reduction characteristics can be useful in evaluating novel excipients; since the goal is preferential oxidation of the excipient rather than the protein drug, excipients that have low oxidation potentials may act as effective antioxidants.27 To protect Trp oxidation in proteins, we evaluated several compounds, including L-Trp and derivatives, using CV, and rank ordered them based on their oxidation potentials (Table 2). In this technique, the input is a linear scan of the potential of a working electrode, and the output is the measurement of the resulting current. As the potential is scanned (linearly increased or decreased), electrochemically active species in the CV cell undergo oxidation and reduction reactions at the surface of the working electrode that results in a current which is continuously measured. Redox
reactions are characterized by sharp increases or decreases in current (peaks). The potential at which an oxidation reaction occurs is known as the anodic peak potential (or oxidation potential), and the potential at which a reduction occurs is called a cathodic peak (or reduction) potential. The oxidation potentials of the excipients in this study ranged from 0.410 to 1.080 V vs Ag/AgCl as shown in Table 2. Under these conditions, L-Trp has an irreversible oxidation potential of 0.938 V vs Ag/AgCl. Nine compounds were found to have a lower oxidation potential than L-Trp, including all of the 5-OH compounds which had oxidation potentials between 0.535 and 0.600 V vs Ag/AgCl. Of all of the compounds tested, 5-aminoDL-tryptophan had the lowest oxidation potential at 0.410 V, while the N-acetyl compounds (0.730−0.880 V) and 5methoxy-DL-tryptophan (0.890 V) were also below L-Trp. Seven compounds had higher oxidation potential than L-Trp as shown in Table 2. These were indole-3-acetic acid, 5-fluoro-Ltryptophan, tryptamine, L-tryptophanamide, L-kynurenine, 5nitro-DL-tryptophan, and salicylic acid. Salicylic acid had the highest oxidation potential in this study (1.080 V vs Ag/AgCl). All of the tested compounds showed nonreversible CV, indicating that, once oxidized, the species do not tend to receive electrons and probably cannot be involved in further electrochemical reactions. A correlation was attempted between oxidation potential and light induced H2O2 generation for 16 compounds that had oxidation potentials above and below the oxidation potential of 1265
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Table 2. Oxidation Potentials of Excipientsa
Table 1. Hydrogen Peroxide Production Ratio between Tested Compounds and L-Trp compound L-Trp L-Trpamide N-acetyl-L-Trp N-acetyl-L-Trpamide 5-fluoro-L-Trp 5-hydroxy-L-Trp 5-methoxy-DL-Trp 5-amino-DL-Trp L-kynurenine trolox pyridoxine indole indole-3-acetic acid 4-hydroxyindole 5-hydroxyindole 5-hydroxyindole-3-acetic acid 7-hydroxyindole 7-hydroxyindole-2-carboxylic acid tryptamine serotonin (5-hydroxytryptamine) melatonin (N-acetyl-5-methoxytryptamine) salicylic acid 5-hydroxysalicylic acid anthranilic acid 5-hydroxyanthranilic acid
compound
(H2O2 produced by compound)/(H2O2 produced by L-Trp)
5-amino-DL-tryptophan 5-hydroxyindole-3-acetic acid 5-hydroxy-L-tryptophan 5-hydroxyindole serotonin HCl (5-hydroxytryptamine HCl) melatonin (N-acetyl-5-methoxytryptamine) N-acetyl-L-tryptophan N-acetyl-L-tryptophanamide 5-methoxy-DL-tryptophan L-tryptophan indole-3-acetic acid 5-fluoro-L-tryptophan tryptamine HCl L-tryptophanamide L-kynurenine 5-nitro-DL-tryptophan salicylic acid
1 0.43 0.31 0.34 0.71 0.09 1.05 0.29 0.20 122.75 5.16 0.95 2.40 0.00 −0.08 0.11 −0.03 0.15 0.53 0.03 0.28
oxidation potential (V vs Ag/AgCl) 0.410 0.535 0.565 0.580 0.600 0.730 0.875 0.880 0.890 0.938 0.948 0.965 1.010 1.015 1.040 1.055 1.080
a
Oxidation (anodic peak) potentials were measured using cyclic voltammetry with a glassy carbon working electrode in 0.2 M lithium perchlorate.
mechanism of photoactivation is not known, this data indicates that the C3 substitutions probably play a role in photoactivation and peroxide generation. The C3 substitutions did not affect the oxidation potentials of the molecules, whereas 5-substituted indole had significantly lower oxidation potential than L-Trp under our experimental conditions. While no clear conclusions could be drawn on oxidation potentials and H2O2 generation for C3 modified indole derivatives, substitutions at the C5 of the 6-membered aromatic ring behaved quite predictably. In general, compounds with electron-donating groups such as −NH2 and −OH had lower oxidation potentials than their parent compounds and also showed low levels of H2O2 production upon photoactivation (e.g., 5-amino-DL-tryptophan, 5-hydroxyindole-3-acetic acid, 5-HT, 5-hydroxyindole, and
0.03 0.84 2.50 0.44
L-Trp and H2O2 production levels above and below that of LTrp (Figure 7). Since indole and tryptophan behave similarly in H2O2 production under light exposure, we thought that substitutions on the C3 position of the five-membered ring probably do not affect this property. However, tryptamine with a −CH2CH2NH2 substitution and indole-3-acetic acid with a −CH2COOH substitution at the C3 position produced 2× less and 2× more H2O2, respectively, than L-Trp. While the
Figure 6. Effect of NaN3 on H2O2 production by various Trp derivatives upon light exposure. Data are shown as a ratio with respect to peroxide generated by L-Trp. 1266
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Figure 7. Correlation between oxidation potential and light induced peroxide formation. The quadrant shows molecules of interest to pursue for antioxidant work in this article.
serotonin). Similarly, compounds with high oxidation potential produced more H2O2 (5-methoxy-DL-tryptophan, L-Trp, indole3-acetic acid, and 5-fluoro-L-tryptophan) under these conditions. There were exceptions to this correlation; some compounds had high oxidation potential but did not produce much H2O2 (e.g., salicylic acid and L-kynurenine) indicating that there are potentially other mechanisms that play an important role for these six-membered aromatic compounds that may not be observed with compounds containing the indole backbone of L-Trp. Our area of interest was the quadrant (Figure 7) which contained compounds with lower oxidation potential and produced less H2O2 upon light exposure than LTrp. Compounds with these two qualities were considered interesting to pursue as potentially new antioxidants because they could (1) oxidize faster than Trp on the protein and (2) produce very little H2O2 during long-term storage and/or ambient processing during drug product production and therefore protect the protein from further oxidation under these conditions. Antioxidant Properties of Select Excipients. We used mAb1 to evaluate the effectiveness of select excipients to protect against Trp oxidation in the protein under the stress conditions shown in Table 3. Each stress model produces oxidation through a different mechanism, and therefore, each may be valuable in the assessment of biopharmaceutical stability. AAPH or 2,2′-azobis(2-amidinopropane) dihydrochloride is used as a stress model to mimic alkyl peroxides potentially generated from formulation excipients such as
degraded polysorbate. Decomposition of AAPH generates alkyl, alkoxyl, and alkyl peroxyl radicals that have been shown to oxidize amino acid residues in proteins, including methionine, tyrosine, and tryptophan residues.3,20 Similarly, controlled light can be used as a stress model to mimic ambient light exposure that drugs may experience during processing and storage. Light induced oxidation of biopharmaceuticals has been shown to proceed through a singlet oxygen (1O2) and/or superoxide anion (O2−) mechanism.29 The Fenton reaction is also commonly used as an oxidative stress model. This mixture of H2O2 and Fe ions generates oxidation through a metal catalyzed, hydroxyl radical mechanism5 and is used to model the metal residue from stainless steel surfaces used in drug manufacturing and storage. Tryptophan (W53) oxidation on mAb1 was thoroughly characterized previously using a RP-HPLC and a liquid chromatography/mass spectrometry (LC-MS) method.29 Oxidized versions of the HC Fab and HC Fc were found to elute earlier than their native counterparts. A comparison of the area integrated under the oxidized and native peaks was used to quantify HC Fab and Fc oxidation. In addition, LC-MS/MS peptide maps (by trypsin digestion and by Lys-C digestion) showed that oxidation of the HC Fab was primarily of a Trp residue, W53, while oxidation of the HC Fc was attributed predominantly to oxidation of two Met residues, M254 and M430. By using the papain digest RP-HPLC method in the present study, we were able to investigate Trp residue oxidation by quantifying HC Fab oxidation and Met residue oxidation by quantifying HC Fc oxidation. For simplicity, % Fab oxidation and % Fc oxidation were calculated as follows in the current article:
Table 3. Oxidation Stress Models Used in This Study stress model AAPH
mechanism
alkyl peroxides, alkyl radical catalyzed light singlet oxygen (1O2), superoxide anion (O2−), H2O2 Fenton hydroxyl radical, metal (H2O2 + Fe) catalyzed
purpose mimic alkyl peroxides from degraded polysorbate mimic ambient light exposure during processing and storage mimic metal residue from stainless steel surfaces
%Fab oxidation = 100 oxidized Fab peak area × Fab peak area + oxidized Fab peak area 1267
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Figure 8. Oxidation after AAPH incubation of (A) Fab and (B) Fc. All samples were incubated with AAPH except for mAb1 ref Mat and No AAPH. All samples were incubated at 40 °C except for mAb1 ref Mat. Data shown are the average of three samples ± 1 SD, except for mAb1 ref Mat, which is the average of six HPLC injections without error bars.
AAPH stress. None of the excipients appeared to impact the level of AAPH induced Fc oxidation (Figure 8B). The effect and mechanism of light induced mAb1 oxidation has been extensively studied,29 making mAb1 an excellent model for the investigation of novel antioxidants under photooxidative conditions. In that study, light exposure was found to induce site-specific oxidation of W53 that was analyzed by a variety of analytical techniques including H2O2 generation, LCMS, and RP-HPLC. Herein, we exposed mAb1 to 16 h of light at 250 W/m2 while testing the aforementioned seven excipients (Figure 9). Exposure of mAb1 to light (“No Excipient”) increased Fab oxidation 3.5 times over the control level (“mAb1 Ref Mat”, Figure 9A). Ji et al.3 showed that L-Trp can protect against Trp oxidation in the model protein parathyroid hormone (PTH). However, we found that addition of 1 mM LTrp to mAb1 increased the Fab oxidation over 11-fold, probably through the production of ROS such as singlet oxygen by light-exposed L-Trp (see Figure 2). Addition of the hydroxy compounds (5-HT, 5-hydroxyindole, 7-hydroxyindole, and serotonin) protected against light induced Fab oxidation, reducing Fab oxidation to near control levels (Figure 9A).
%Fc oxidation = 100 oxidized Fc peak area × Fc peak area + oxidized Fc peak area
Note that each antibody molecule has two Fabs; therefore, the % Fab oxidation obtained here does not reflect the % oxidized intact antibody containing Fab oxidation. Incubation of mAb1 with AAPH for 24 h at 40 °C resulted in 27% Fab (Trp residue) oxidation and 47% Fc (Met residue) oxidation (Figure 8). Seven excipients that had been previously screened using light stress and cyclic voltammetry were incubated with mAb1 under the AAPH conditions to evaluate antioxidant capabilities. Six of the seven compounds were found to significantly reduce AAPH induced Fab oxidation (Figure 8A). All six of these compounds contain the indole backbone. Moreover, all of the hydroxy derivatives tested (5-HT, 5hydroxyindole, 7-hydroxyindole, and serotonin) reduced Fab oxidation close to control levels (about 2%). Meanwhile, salicylic acid had almost no effect on Fab oxidation under 1268
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Figure 9. Oxidation after 16 h of light exposure (250 W/m2) of (A) Fab and (B) Fc. All vials were placed in the lightbox except for the mAb1 ref Mat. Foil CTRL vials were covered in foil before placement in the lightbox. All samples are the average of three separate vials, except for Ltryptophanamide (*), which was two vials, and mAb1 ref Mat, which was one vial with three independent injections on the HPLC. Error bars represent one standard deviation.
above. Data related to the antioxidant properties against the Fenton mediated reaction were analyzed using the RP-HPLC assay and are shown in Figure 10. Under the conditions tested in this study, the Fenton reaction produces about 4× the oxidation in the Fab region of mAb1 over the control. Most of the antioxidants tested, except salicylic acid, show similar hydroxyl radical quenching properties like L-Trp, which protected the Fab oxidation by about 25% with respect to the no excipient case (Figure 10A). In regards to protection against Fc oxidation, the tested excipients (other than salicylic acid) performed slightly better than L-Trp (Figure 10B). Role of Substitutions on Antioxidant Properties. Considerable interest in melatonin (N-acetyl-5-methoxytryptamine) and other Trp derivatives in quenching reactive oxygen species have been published since early 1990s. Melatonin has been reported to have an oxidation potential around 0.715 V and also to be a highly effective scavenger of hydroxyl radicals.36 Other indole derivatives have since been shown to interact with ROS and act as effective scavengers.37 Hardeland et al. have proposed electron donation from the indole ring to
However, salicylic acid performed similarly to L-tryptophanamide, increasing Fab oxidation 8-fold over the control level. Similar results were observed for Fc oxidation under light stress (Figure 9B). Light exposure of mAb1 resulted in a 40% increase in Fc oxidation over the control level, whereas addition of L-Trp increased Fc oxidation to 7 times the control level. Compared to the control (no excipient), L-tryptophanamide and salicylic acid also resulted in more Fc oxidation. The hydroxy compounds produced Fc oxidation similar to that of the no excipient control potentially because they produce much fewer ROS than L-Trp under light exposure. The light screening and NaN3 study results show a good correlation between the amount of H2O2 generated by an excipient and Fc Met oxidation of mAb1 as expected. The Fenton reaction, using a mixture of H2O2 and Fe ions, generates oxidation through a metal catalyzed, hydroxyl radical reaction.5 This reaction generated Fab, that is, tryptophan, oxidation in mAb1 as anticipated. The reaction was also carried out in the presence of select antioxidants that were useful against both AAPH and light induced oxidation as reported 1269
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Figure 10. Oxidation in 3 mg/mL mAb1 following the Fenton reaction using 10 ppm of H2O2 and 0.2 mM of Fe(III). The reaction was incubated at 40 °C for 3 h, quenched with 100 mM L-Met, and analyzed using RP-HPLC after papain digest. All samples are the average of three separate vials, and the mAb1 control (ref Mat) was one vial with five independent injections on the HPLC. Error bars represent one standard deviation. (A) Fab and (B) Fc.
demonstrated that compounds with lower oxidation potentials could be physically quenching 1O2.42,43 This supports the hypothesis and our observation that addition of electron donating groups such as hydroxyl groups on the aromatic ring produce lower amounts of H2O2 possibly by enhanced physical quenching of 1O2 as and when it is produced. Dad et al. have reported on the interesting photochemistry of 5-HT.44 While 5-HT is able to react with molecular oxygen to form 1O2 with a quantum yield of around 0.1, it was also demonstrated that 5-HT was an efficient quencher of 1O2 with a second-order rate constant of 1.3 × 108 dm3 mol−1 s−1. This quenching constant is about three times better than L-Trp (4.1 × 107 dm3 mol−1 s−1) and seems to correlate with the published one electron oxidation potential at pH 7 of the 5-indoxyl radical when compared to that of L-Trp.45 Electron donating substitutions on the aromatic ring may facilitate the formation
form the indolyl cation radical as the primary mechanism of scavenging hydroxyl radicals.38 The indolyl cation can further scavenge O2−. Besides OH* and O2−, melatonin has also been demonstrated to neutralize 1O2, NO*, and HOCl. Melatonin has been shown to protect Trp photooxidation in lysozyme, probably by scavenging the 1O2 formed during irradiation.39 Similarly, 5-HT has been shown to be better at scavenging hydroxyl radicals than vitamin C or melatonin.40 Additionally, 5-HT has been demonstrated to inhibit tert-butyl hydroperoxide induced oxidative damage, probably by scavenging various ROS.41 Igarashi et al. showed that light-exposed L-Trp generates significant amounts of 1O2 and O2− in a dosedependent manner.28 They also reported that irradiated L-Trp potentially quenches 1O2 through physical quenching as well as a chemical reaction, but these processes vary with the presence of substitutions on the indole ring of L-Trp. Abdel-Shafi et al. 1270
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activation of L-Trp leading to singlet oxygen and ultimately to H2O2 and the formation and quenching of singlet oxygen by 5HT is shown in Scheme 1.
of the indolyl radical cation and potentially faster reactivity with radicals. Hydroxyl groups attached to the aromatic rings are electron donors as the oxygen atom has a lone pair of electrons that can be involved in resonance structure leading to lower oxidation potentials and potentially more susceptibility to electrophilic attack. As seen in Table 2, the hydroxyl substitutions indeed lead to substantially lower oxidation potentials and could make these better antioxidants than LTrp and/or indole. Surprisingly, the hydroxyl substituted indole and Trp derivatives also produced the least amount of hydrogen peroxide upon light irradiation (Table 1). This may be due to the low quantum efficiency of these molecules in transferring light energy to molecular oxygen, coupled with their high quenching constants as demonstrated for 5-HT.44 As shown in Figures 8−10, the hydroxyl substituted indole and tryptophan derivatives provide considerable protection against AAPH, light, and Fenton induced oxidation to W53 in the Fab region of mAb1. However, none of these compounds provide substantial antioxidant protection to Met oxidation in the Fc region of mAb1 in the AAPH induced degradation. However, the indole and tryptophan derivatives behave as expected under light mediated oxidation. Molecules that produced higher amounts of peroxide upon photo activation (e.g., L-Trp and Trp-amide) also produced higher Met oxidation in the Fc region of mAb1, while the −OH derivatives produced lower H2O2, and also the lowest amount of Met oxidation in the Fc region under photo-oxidation conditions. Methionine is readily oxidized to methionine sulfoxide by H2O2 and alkyl peroxides through a nucleophilic substitution reaction.1 Photo-oxidation of methionine to methionine sulfoxide occurs via singlet oxygen, though this reaction occurs via a different intermediate.1 AAPH degrades under thermal stress to give both alkyl peroxides and alkoxyl radicals that have different reactivity toward Met and Trp, respectively.6 Ji et al. have shown previously that L-Trp was able to prevent Trp oxidation in PTH induced by AAPH and that L-Trp did not prevent Met oxidation in PTH under the same conditions.3 Similarly, L-Met was able to protect PTH against AAPH induced oxidation but did not protect Trp oxidation. These observations are in line with the reaction mechanisms wherein Met oxidation is predominantly via nucleophilic substitution reactions, whereas Trp oxidation is mainly via free radical mechanisms.
Scheme 1. Putative Mechanism of (A) L-Trp and (B) 5-HT Excitation and in Generating and Quenching 1O2a
a
1
k25C represents the second order rate constant for quenching of O2,44 while Eox is the oxidation potential of the molecule vs Ag/AgCl.
Finally, our results demonstrate that light exposure studies and CV are both valuable tools for the screening of potential antioxidants. The two approaches generate different but complementary pieces of information that can be combined to form a better understanding of antioxidant capability. For example, in the case of salicylic acid, we may have concluded that it would be an effective antioxidant based on its low production of H2O2 upon light exposure. Subsequent studies with mAb1, however, demonstrated that salicylic acid was not an effective antioxidant, which was predicted by its high oxidation potential in comparison to that of L-Trp. Conversely, Trolox has an oxidation potential of about 0.2 V,46 far lower than L-Trp, but produces more than 100-fold excess H2O2 in comparison to that of L-Trp upon photo-irradiation and probably is not ideal for protein formulations. We believe that indole and its derivatives, especially the hydroxyl substitutions, may provide significant antioxidant protection to proteins based on their electrochemical potentials and photoactivation studies reported herein.
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CONCLUSIONS Lam et al. have reported that peroxides are generated via the autocatalytic reaction of polysorbates in the protein formulation.7 They also report that the concentrations of these peroxides should be minimized to maintain protein stability over long-term storage in a liquid state. Our studies clearly indicate that peroxides can be generated via other formulation components, especially free L-Trp via a photoactivation pathway. The current studies hence reiterate the importance of considering mild light induced degradation during formulation development of proteins, especially proteins with oxidizable Trp residues, and of understanding the different degradation pathways not only in proteins but also in the excipients used in these formulations. To the best of our knowledge, this is the first report showing that indole derivatives, including hydroxyl substituted L-Trp derivatives, protect against various types of ROS (singlet oxygen, H2O2, hydroxyl radicals, and alkyl peroxides) induced oxidation of amino acids in proteins and could be an important tool in the protein formulation toolkit. A putative mechanism of photo-
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AUTHOR INFORMATION
Corresponding Author
*Phone: 650 4678488. E-mail:
[email protected]. Notes
All authors are employed by Genentech, Inc., a member of the Roche group. The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank Neelima Mantha for some experimental help. 1271
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