Identification of a Single Tryptophan Residue as Critical for Binding

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Anal. Chem. 2007, 79, 2797-2805

Identification of a Single Tryptophan Residue as Critical for Binding Activity in a Humanized Monoclonal Antibody against Respiratory Syncytial Virus Ziping Wei, Jinhua Feng, Hung-Yu Lin, Sombabu Mullapudi, Eric Bishop, Guillermo I. Tous,‡ Jose Casas-Finet, Fadi Hakki, Robert Strouse, and Mark A. Schenerman*

Department of Analytical Biochemistry, MedImmune, One MedImmune Way, Gaithersburg, Maryland 20878

We have identified a single tryptophan (Trp) residue responsible for loss of binding and biological activity upon ultraviolet (UV) light irradiation in MEDI-493, a humanized monoclonal antibody (MAb) against respiratory syncytial virus (RSV). This finding provides a better understanding of structure-function relationship in a 150-kDa protein. Irradiation of MEDI-493 with UV light resulted in spectral changes typical of Trp photoproducts and in a progressive loss of MEDI-493 binding and biological activity as measured by ELISA, Biacore, and cell-based assays. Mass spectrometric characterization of the proteolytic peptides generated from the UV irradiated MEDI493 confirmed that most methionine (Met) and a few Trp residues were oxidized to various extents upon exposure to UV light. Among Trp residues, only Trp-105, containing the most solvent-exposed indole moiety in MEDI-493 and residing in a complementary-determining region (CDR) of the heavy chain, was significantly oxidized. When bound to a synthetic antigenic peptide, MEDI-493 showed significant resistance toward binding activity loss during UV irradiation. A second MAb (MEDI-524) with Trp-105 replaced by phenylalanine (Phe) showed a similar pattern of Met oxidation, but no loss of binding and biological activity following irradiation. Treatment of both MAbs with Met- and Trp-specific oxidizing reagents showed that oxidation of Trp-105 correlated with the activity loss, whereas Met oxidation did not affect the activity. These results demonstrate that Trp-105 in MEDI-493 is responsible for the UV light-induced effects. Single amino acid changes in proteins have been studied through chemical modification or amino acid substitution to identify the residues responsible for binding activity and structure changes of proteins.1-7 It has been shown that modification of a * To whom correspondence should be addressed. Tel: (301) 398 4288. Fax: (301) 398 9288. E-mail: [email protected]. ‡ Current address: Phyton Biotech Inc., 279 Princeton-Highstown Rd., East Windsor, NJ 08520. (1) Russell, J.; Katzhendler, J.; Kowalski, K.; Schneider, A. B.; Sherwood, L. M. J. Biol. Chem. 1981, 256, 304-307. (2) Martinez del Pozo, A.; Merola, M.; Ueno, H.; Manning, J. M.; Tanizawa, K.; Nishimura, K.; Asano, S.; Tanaka, H.; Soda, K.; Ringe, D.; Petsko, G. A. Biochemistry 1989, 28, 510-516. 10.1021/ac062311j CCC: $37.00 Published on Web 02/24/2007

© 2007 American Chemical Society

single amino acid residue can affect the stability, structure, and activity of proteins. For instance, chemical oxidation of a single Trp in human placental lactogen resulted in conformational changes and reduced lactogenic activity.1 A single Trp-139 in the thermostable D-amino acid transaminase was critical for its activity, as proved by site-directed mutagenesis.2 Modifications of a single Trp-187 residue of human recombinant annexin V exhibited significant changes in protein stability, folding cooperativity, biological activity, and fluorescence properties compared to the wild-type protein.3 Similarly, for large proteins such as MAbs, a single amino acid change can significantly affect binding specificity. One amino acid change in the binding pocket of the anti-integrin MAb AP7.4 altered its specificity, as revealed by the crystal structure of its Fab.4 A single amino acid replacement at position 35 in the heavy chain of a MAb with specificity for p-azophenylarsonate conferred specificity for the structurally related hapten p-azophenylsulfonate, while abolishing specificity for p-azophenylarsonate.5 A single amino acid substitution from Leu-235 to Glu-235 in the Fc segment of humanized OKT3 MAb generated a 100-fold decrease in the affinity of the MAb for the Fc receptor on U937 cells, without affecting antigen binding.6 A single amino acid substitution at position 103 of the heavy chain of a MAb B1-8. δ1 drastically altered idiotypic, but not antigen-binding specificity, of the molecule.7 Besides non-photochemical modification and amino acid substitution, one of the commonly studied protein modifications is photooxidation through UV irradiation. Oxidation reactions may take place through different mechanisms of action and can be quite complex. Trp in proteins merits special attention because, as a principal chromophore of proteins in the near UV, it is thought (3) Minks, C.; Huber, R; Moroder, L.; Budisa, N. Biochemistry 1999, 38, 1064910659. (4) Vasudevan, S.; Celikel, R.; Ruggeri, Z. M.; Varughese, K. I.; Kunicki, T. J. Blood Cells Mol. Dis. 2004, 32, 176-181. (5) Kussie, P. H.; Parhami-Seren, B.; Wysocki, L. J.; Margolies, M. N. J. Immunol. 1994, 152, 146-152. (6) Alegre, M. L.; Collins, A. M.; Pulito, V. L.; Brosius, R. A.; Olson, W. C.; Zivin, R. A.; Knowles, R.; Thistlethwaite, J. R.; Jolliffe, L. K.; Bluestone, J. A. J. Immunol. 1992, 148, 3461-3468. (7) Radbruch, A.; Zaiss, S.; Kappen, C.; Bru ¨ ggemann, M.; Beyreuther, K.; Rajewsky, K. Nature 1985, 315, 506-508.

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to contribute to photosensitivity.8 Photolytic oxidation occurs when Trp absorbs energy from an irradiation source and produces an excited intermediate, leading to photoionization, essentially dissipating the free radical through transfer to dissolved oxygen.9 One of the important products formed from UV light absorption by Trp is N-formylkynurenine (NFK), which can act as a photosensitizer in proteins by producing reactive oxygen species, including singlet oxygen, superoxide, and hydroxyl radicals.10,11 Trp photosensitivity has been reported to be dependent on the amino acids surrounding the Trp residues,12 and the location of these residues (exposed or buried) within the three-dimensional structure of a protein.13,14 Trp oxidation resulting from UV light irradiation affects the structure and folding of proteins, and may also influence the stability and bioactivity of proteins. For instance, the photooxidation of Trp causes significant cross-linking and conformational changes in lens crystallins.15 UV irradiation of lac repressor leads to a photooxidation of Trp, a decrease in the fluorescence of the protein, and a loss of inducer activity.16 It has also been reported that certain MAbs display significantly decreased fluorescence emission intensity after UV light exposure as a result of photolysis of single Trp residues in the variable regions.17 In order to have a better understanding of structure-function relationship, we have studied a humanized MAb (MEDI-493) against RSV F protein for its photooxidation sensitivity and the effect on binding activity after UV light exposure. Our study identifies Trp-105 in MEDI-493 as the amino acid responsible for the loss of antigen binding and biological activity. Inspection of the X-ray crystal structure of MEDI-493 Fab shows that Trp-105, located in the heavy chain CDR3 region, has the most solventexposed indole side chain of all Trp amino acids in the antibody.18 Additionally, with the exception of two Trp residues (Trp-54 and Trp-55) in the heavy chain CDR2 region, which adopt a stacked ring conformation, all other Trp residues are shielded from solvent. For comparison, we also tested another MAb (MEDI524) with less than a fifteen amino acid difference from MEDI493 in the variable region; Trp-105 in MEDI-493 was replaced by a Phe in MEDI-524. None of the other seven Trp residues present in MEDI-493 were subjected to mutational substitution. Given the increased probability of photoionization by a solvent-exposed Trp as a relaxation from the excited-state following UV irradiation, we investigated whether there were measurable differences between the rates of UV-induced oxidation of the two MAbs. Because modification of a subset of Trp residues in the CDRs by UV light may affect MAb function, potential differences in binding activity were also assessed following UV irradiation. The modifica(8) Creed, D. Photochem. Photobiol. 1984, 39, 537-562. (9) Griffiths, H. R. Free Rad. Res. 2000, 33, 47-58. (10) Walrant. P.; Santus, R. Photochem. Photobiol. 1974, 19, 411-417. (11) Pileni, M. P.; Santus, R.; Land, E. J. Photochem. Photobiol. 1978, 28, 525529. (12) Tassin, J. D.; Borkman, R. F. Photochem. Photobiol. 1980, 32, 577-585. (13) Pigault, C.; Gerard, D. Photochem. Photobiol. 1984, 40, 291-296. (14) Grossweiner, L. I.; Kaluskar, A. G.; Baugher, J. F. Int. J. Radiat. Biol. 1976, 29, 1-16. (15) Abdley, U. P.; Clark, B. A. Curr. Eye Res. 1988, 7, 571-579. (16) Spodheim-Maurizot, M.; Charlier, M.; Helene, C. Photochem. Photobiol. 1985, 42, 353-359. (17) Volkin, D. B.; Mach, H.; Middaugh, C. R. In Protein Stability and Folding; Shirley, B. A., Ed.; Humana Press: Totowa, NJ, 1995; pp 35-36. (18) Johnson, L. S.; Braden, B. Crystals and structures of Synagis Fab. US Patent 6,955,717, 2005.

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tions induced by chemical reagents specific to Met and Trp residues were studied to confirm the effect observed in the UV irradiation study. Our results support that oxidation of Trp-105 in the CDR region is a rate-limiting step in determining the photostability of MEDI-493. The mutant of MEDI-493 with a Trp105 to Phe-105 substitution is considerably more resistant to UV light oxidation. EXPERIMENTAL SECTION Materials. MEDI-493 and MEDI-524 were humanized MAbs (IgG1) against RSV F protein produced using hybridoma technology. They consist of the CDR regions specific for the targeted antigen and the constant regions of a human γ1 heavy chain and κ light chain.19 Preparation of Light-Irradiated MAb. MEDI-493 in 10 mM His and 1.6 mM Gly buffer, pH 6.0, and MEDI-524 in 10 mM His buffer, pH 6.0, at high concentrations (100 mg/mL) were exposed to a 300-700 nm light source with UV light intensity of 6.6 W/m2 and cool white light intensity of 9.3 Klux, in a photostability chamber (Model PST54SD, Powers Scientific, Pipersville, PA) for up to 7 days. The MAb control samples were wrapped in aluminum foil and marked as control. The controls and samples were placed at the marked positions, where the light intensity was measured. An aliquot (100 µL) from each time point (initial, 1-day, 2-day, 3-day, and 7-day) was taken for testing. The peptide protection experiment was executed as follows: MEDI-493 at 0.15 mg/mL in the presence and absence of a 24mer F peptide at 1 or 5 mg/mL was exposed to UV light. The MAb control samples in the presence and absence of the 24-mer peptide were wrapped in aluminum foil. An aliquot (100 µL) from each time point (initial, 4, 8, 12, 24, 36, 48, and 60 h) was taken for ELISA testing. Preparation of Oxidized MAb Using tert-Butyl Hydroperoxide (t-BHP). To generate oxidized antibody, MAb (10 mg/ mL) was first exchanged into 50 mM sodium acetate, 150 mM sodium chloride, 0.01% Tween 80, at pH 5.0 by Centricon ultrafiltration. t-BHP was added to the solution to a final concentration of 1% and incubated in the dark at room temperature.20,21 MAb aliquots (200 µL) were taken at 1, 3, and 24-h time points and immediately dialyzed against 1 L of 100 mM tris(hydroxymethyl)aminomethane (Tris) buffer, pH 7.5, for 2 h. The oxidized antibody was dialyzed for an additional 2 h in 1 L of fresh 100 mM Tris buffer, pH 7.5. Preparation of Oxidized MAb Using Ozone. To generate ozone-oxidized antibody, MAb (10 mg/mL) was first exchanged into 50 mM sodium phosphate, pH 6.0, by dialysis for 2 h and equilibrated to 0 °C. Another 50 mM sodium phosphate buffer was ozonized for 4 h at 0 °C by an ozone generator (Water Purifier XT301, AirZone Inc., Suffolk, VA). Then, the dialyzed monoclonal antibody (100 µL) was mixed with the ozonized sodium phosphate buffer at the ratios of 1/0.5, 1/1, 1/2, 1/4, 1/7, and 1/11 (v/v) and incubated at 0 °C for 14 h. Circular Dichroism. MAb circular dichroism spectra were collected with a Jasco 810 spectropolarimeter (Jasco, Inc., Easton, (19) Wu, H.; Pfarr, D. S.; Tang, Y.; An, L. L.; Patel, N. K.; Watkins, J. D.; Huse, W. D.; Kiener, P. A.; Young, J. F. J. Mol. Biol. 2005, 350, 126-144. (20) Shen, F. W.; Kwong, M. Y.; Keck R. G.; Harris, R. J. In Techniques in Protein Chemistry VII; Marshak, D. R., Ed.; Academic Press: San Diego, CA, 1996; pp 275-284. (21) Keck, R. G. Anal. Biochem. 1996, 236, 56-62.

MD) from 260 to 190 nm with a 1 nm bandwidth, a data density of 10 points/nm, a scan rate of 10 nm/min, and a time constant of 16 s. MAbs in 10 mM sodium phosphate, pH 7.0, were placed in quartz Suprasil cuvettes (Uvonic Instruments, Plainview, NY) with a path length of 0.5 mm and measured. Peptide Mapping with On-Line Tandem Mass Spectrometry (MS). Two different enzymatic digestion methods were used for peptide mapping: trypsin and trypsin/Glu-C digestions. The tryptic digestion was performed by mixing MAbs (500 µg in 100 µL) with 200 µL of 8 M guanidine-HCl, 130 mM Tris, and 1 mM EDTA, pH 7.6, and 10 µL of 500 mM DTT and incubating at 37 °C for 1 h. Then, 25 µL of 500 mM iodoacetamide was added and the mixture incubated at ambient temperature for 1 h in the dark. The reduced and alkylated protein was dialyzed into 6 M urea and 100 mM Tris (pH 8.0) in 10,000 MWCO dialysis cassettes at 4 °C for 2 h. The dialyzed protein (200 µL) was mixed with 400 µL of 50 mM Tris buffer (pH 8.0) and 12 µL of 1 mg/mL trypsin in 1 mM HCl and incubated at 37 °C for 2 h. A second aliquot of trypsin (12 µL) was then added to the mixture, and the incubation was continued at 37 °C for an additional 2 h. The digestion was quenched by the addition of 12 µL of trifluoroacetic acid (TFA). The trypsin/Glu-C digestion was performed as described above, with the following modifications. The reduction step prior to trypsin digestion was carried out in 8 M guanidine-HCl, 100 mM sodium phosphate, 1 mM EDTA, pH 7.6, and the reduced and alkylated MAb was dialyzed into 6 M urea containing 100 mM sodium phosphate buffer, pH 8.0. Then, the dialyzed samples were diluted with 25 mM sodium phosphate buffer, pH 7.8. No TFA was added to the tryptic digests. After storage at 4 °C overnight, the trypsin digest was further diluted with 25 mM phosphate buffer (pH 7.8) at a sample to buffer ratio of 1:0.85. Glu-C was immediately added at a substrate to enzyme ratio of 10:1. The digestion was allowed to proceed for 3 h at 30 °C, and then a second aliquot of Glu-C was added to the enzymatic reaction. After 3 h at 37 °C, the Trypsin/Glu-C digest was stored at -20 °C until analysis. The on-line LC-MS/MS analysis of the digested peptides was performed using an Agilent 1100 HPLC system (Agilent Technologies, Palo Alto, CA) and a ThermoElectron LTQ ion-trap mass spectrometer (Thermo Electron, San Jose, CA). The HPLC system was equipped with a Varian Polaris C18-A reversed-phase column (5 µm, 250 × 4.6 mm) connected to the UV detector, followed by mass spectrometry. Mobile phase A was 0.02% TFA in HPLC water and mobile phase B was 0.02% TFA in acetonitrile. The peptides were eluted using a gradient of 0-20% mobile phase B over 70 min and 20-36% over 90 min at a flow rate of 0.7 mL/min. The eluted peptides were monitored by UV detection at 220 nm and using a LTQ ion-trap mass spectrometer in positive ion mode. For the analysis of tryptic peptides, the mass spectrometer was operated in data-dependent “triple play” mode with dynamic exclusion enabled. In this mode, the instrument continuously acquired full scan mass spectra (m/z 300-2000). When the signal exceeded a predefined threshold, a high resolution “zoom scan” and an MS/MS scan were acquired. The MS data were searched against the known protein sequences for confirmation and for posttranslational modifications using the ThermoElectron Bioworks software. The relative percent of the unmodified peptide was determined by the peak area of the unmodified peptide versus

the total peak area of the modified and unmodified species for each peptide. For a tryptic peptide map, peak areas were calculated from extracted ion chromatograms of full scan MS data. For the tryptic/Glu-C peptides, the mass spectrometer was operated in MS and MS/MS scan mode of selected parent ions from the peptides of interest. The peptide peak areas were calculated from the extracted ion chromatograms of full scan MS data. F Protein Binding ELISA. Each well of the assay plate was coated with 50 µL/well of a 1 µg/mL soluble F protein antigen overnight at 2-8 °C. After the plate was aspirated and washed with phosphate-buffered saline with 0.05% Tween-20 buffer, it was blocked by incubating with phosphate-buffered saline with 0.05% Tween-20 and 0.5% bovine serum albumin buffer for 1 h at ambient temperature. The plate was washed and protein standard curve samples, test samples, protein standard, and negative control were added to the washed plate. Following a 1-h incubation at ambient temperature, the plate was washed, and 50 µL per well of a goat anti-human IgG-horseradish peroxidase at 1:16 000 dilution was added to the plate. After washing, 100 µL/well of 3,3′,5,5′tetramethylbenzidine (TMB) substrate was added to the plate and incubated at ambient temperature protected from light for 10 min. The enzymatic reaction was stopped by adding 50 µL/well of 2 N sulfuric acid, and the absorbance at 450 nm was measured using a microplate reader. The slope of the log-log transformation of the protein standard curve was determined, and parallelism (90% confidence limit) of the test sample curve to the standard curve was tested. After meeting the criteria of the parallelism test, the ED50 ratio of the test sample to the reference standard was calculated, and the results were expressed as a percentage of reference standard binding. Microneutralization Assay. The microneutralization assay measures the ability of the antibody to prevent RSV virus from infecting a susceptible target cell line, Hep-2.19 To determine whether a MAb had virus neutralizing activity, a fixed amount of virus was added to varying dilutions of the MAb. After mixing the antibody and virus, and dispensing the mixtures into 96-well plates, the plates were incubated for 2 h at 37 °C to allow the antibody time to bind to the epitopes present on the virion. Then, 2.5 × 104 Hep-2 cells were added to each assay well, and the plates were incubated for five additional days at 37 °C. During this time, the virus infected and replicated within the cells in wells containing no neutralizing antibody, and infectivity was reduced in a manner proportional to neutralizing antibody titer in wells containing neutralizing antibodies. The cells were fixed to the plate after several wash steps, and the virus was detected with a mouse MAb specific for the F protein, followed by a horseradish peroxidaselabeled anti-mouse antibody and TMB substrate. The absorbance was then measured spectrophotometrically, and the ED50 of the test sample, as well as the ratio of the ED50 values of the test sample compared to the reference standard was calculated. Biacore. This study was performed using a CM5 sensor chip (Biacore AB, Uppsala, Sweden) which contains a carboxymethyl (CM) dextran matrix and a Biacore 3000 surface plasmon resonance biosensor (Biacore AB, Uppsala, Sweden). RSV F protein (420 pg) was covalently coupled to an N-hydroxysuccinimide-N-ethyl-N′-(3-diethylaminopropyl)carbodiimide-activated CM5 sensor chip at a low protein density, according to the manufacturer’s protocol. Unreacted active ester groups were blocked with Analytical Chemistry, Vol. 79, No. 7, April 1, 2007

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1 M ethanolamine. For reference purposes, a blank surface containing no antigen was prepared under identical immobilization conditions. For kinetic measurements, MAbs were diluted to 100 nM with HBS-EP buffer (10 mM HEPES, pH 7.4, 0.15 M sodium chloride, 3 mM EDTA, 0.005% Surfactant P20). MAbs were injected for 4 min in a serial-flow fashion over the reference and F protein antigen surfaces at a flow rate of 10 µL/min. Dissociation of bound MEDI-493 was monitored for 240 s at a flow rate of 10 µL/min in the presence of HBS-EP buffer. Due to the high affinity of MEDI-524, the dissociation of bound MEDI-524 was monitored for 5400 s at a flow rate of 10 µL/min in the presence of HBS-EP buffer. After the dissociation period, the remaining bound sample was removed with one (for MEDI-493) or two (MEDI-524) 4-min injections of 100 mM HCl at a flow rate of 10 µL/min across both reference (blank) and antigen protein surfaces. The dissociation data was fit to a 1:1 Langmuir dissociation model using BIAevaluation software (Biacore, Inc., Piscataway, NJ) to calculate a dissociation rate constant (kd). The association data was fit to a 1:1 Langmuir association model using BIAevaluation software to calculate an association rate constant (ka). The calculated kd was used in the fitting to determine ka. The affinity constant (KD) was derived as a ratio of kd/ka. RESULTS AND DISCUSSION Photochemical Modification of MAb. Irradiation of MEDI493 and MEDI-524 with UV light caused changes in their UVvisible spectra, characterized by a decrease of the maximal absorbance near 280 nm (ascribed to Trp residues) and the appearance of a new and weakly absorbing band in the near-UV, centered near 340 nm. Excitation of UV irradiated MAb at this wavelength in a spectrofluorometer cell resulted in the emission of visible light, with maximal emission around 430 nm. These features have previously been observed as the result of the photochemical oxidation event for indole and the production of Trp photoproducts.16 A dose-response analysis of the evolution of intrinsic Trp fluorescence at 340 nm showed that it decreased exponentially with increasing time of UV exposure. Similar studies using only the visible light source in the irradiation chamber did not induce any spectral or functional alteration of MEDI-493 (data not shown). The fluorescence intensity of the band emitting at 430 nm showed a more complex temporal evolution, a red-shift of the wavelength of maximal emission with irradiation time, and a lack of isoemissive point with the intrinsic fluorescence band under conditions where both intact and photoreacted Trps can be excited simultaneously (data not shown). MEDI-493 showed consistently stronger fluorescence intensity at 430 nm compared to MEDI524 at all irradiation time points. These results strongly suggest a multiplicity of Trp photoproducts, some of which are likely to be themselves subjected to further photochemical reactions. There was no significant change in secondary structure of the antibody following irradiation, as assessed by circular dichroism spectroscopy (data not shown). Primary Structure Characterization of UV-Irradiated MAb. It has been reported that Trp and His typically photooxidize faster than other amino acids at neutral pH, while at lower pH, Trp and Met are the predominant photoreactive amino acids.22 Two MAbs were reduced, alkylated, and digested by trypsin followed by peptide mapping with on-line ESI-MS detection to identify the 2800

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modification sites during UV irradiation. The tryptic peptide maps of MAbs photoirradiated for 1, 2, 3, and 7 days were compared to that of the unexposed control MAb. Figure 1 compares the peptide maps of MEDI-493 before (panel A) and after UV irradiation for 7 days (panel B). The oxidized peptides normally elute several minutes prior to the parent peptides, as the resulting products are more hydrophilic. The on-line tandem mass spectrometry was capable of detecting and identifying all modification sites based on the fragment ions in the MS/MS spectra.23 We found that most of the Met residues were oxidized, albeit to different extents, with increasing irradiation time. The oxidation products mostly contained Met sulfoxide, with a small amount of Met sulfone. These Met residues are as follows: Met-34, Met-101, Met-255, Met-361, and Met-431 on the heavy chain, as well as Met-4 on the light chain. The only detectable Trp oxidation came from two CDR regions of the heavy chain: Trp-105 in the CDR3, and Trp-54 or Trp-55 in the CDR2. MEDI-524 has the same oxidation sites as MEDI-493, except for Trp-105, because Trp-105 is mutated to a Phe in MEDI-524. The extent of oxidation at each active site was determined by selected ion trace analysis of the oxidized product and its parent peptide. The extent of MEDI-493 and MEDI-524 oxidation at each site was calculated based on the respective tryptic peptide, except for Met-101 and Trp-105 of MEDI-493. Due to the large size of the peptide containing these two amino acids (25-mer) and the closeness of Met-101 to the N-terminal end of the peptide, a Glu-C digestion step was added after trypsin digestion to obtain a smaller peptide and accurately distinguish the oxidation of Met-101 and/ or Trp-105 in multiple photodegradation products with a 4, 20, 16, 32, or 48 Da increase. In the trypsin/Glu-C digest, the unmodified peptide (SMITNWYFD) with m/z 1176.7 was first identified and was confirmed by the fragment ions in the MS/ MS mass spectrum (Figure 2A). The numbering system used has been described by Roepstorff and Fohlman.24 Figure 2 also shows the fragmentation ions for two oxidized peptides with a 16 and 32 Da MW increase, due to the presence of oxidized Trp-105. The oxidized peptide with a 16 Da MW increase at Trp-105 was identified as either 5-OH-Trp or oxindolylalanine (Oia), and that with a 32 Da increase was either NFK or dioxindolylalanine (DiOia).25,26 The masses of the N-terminal fragment b series ions, b6 to b8, and the C-terminal fragment y series ions, y4 to y9, of the oxidized peptides are 16 and 32 Da higher than that of the parent peptide, respectively. The characteristic fragment ions of WY (b5/ b7), NWY (b4/b7), and TNWY (b3/b7) of H12′ shifted 16 and 32 Da higher in the oxidized peptides, respectively. Similarly, a total of eight oxidized peptides with the combination of Met-101 and Trp-105 oxidation were identified by the MS/MS data. The multiple degradation products were a combination of Met sulfoxide and Met sulfone (1%) at Met-101, with Oia, 5-OH-Trp, NFK, DiOia, and kynurenine (Kyn, 4 Da MW increase, 1%) at Trp-105. UV exposure for 7 days resulted in 71% oxidation of Met-101 and 61% of Trp-105 in MEDI-493. (22) Cleland, J. L.; Powell, M. F.; Shire, S. J. Crit. Rev. Ther. Drug Carrier Syst. 1993, 10, 307-377. (23) Finley, E.; Busman, M.; Dillon, J.; Crouch, R. K.; Schey, K. L. Photochem. Photobiol. 1997, 66, 635-641. (24) Roepstorff, P.; Fohlman, J. Biomed. Mass Spectrom. 1984, 11, 601. (25) Simat, T.; Meyer, K.; Steinhart, H. J. Chromatogr. A 1994, 661, 93-99. (26) Reubsaet, J. L. E.; Beijnen, J. H.; Bult, A.; van Maanen, R. J.; Marchal, J. A. D.; Underberg, W. J. M. J. Pharm. Biomed. Anal. 1998, 17, 955-978.

Figure 1. Tryptic peptide map profiles of MEDI-493 before (A) and after (B) 7-day photoirradiation. The susceptible residues are labeled. * indicates that the residue is oxidized. Met-431 appears on two different peptides due to nonspecific cleavage. The inserts are expanded views of the chromatograms. Met/Trp and Met*/Trp* represent nonoxidized and oxidized Met-101/Trp-105.

Figure 3 (A, B) shows the plot of the log fractions of intact residue for the susceptible peptides versus the irradiation time of MEDI-493 and MEDI-524 during a 7-day study. The slopes, corresponding to the oxidation rates, indicate that MEDI-493 and MEDI-524 showed comparable levels of Met oxidation; the various sites ranked in the following order: Met-255 > Met-101, Met-431 > Met-361. Met-34 and Met-4 showed lower levels of oxidation (data not shown). Our results indicate that Met-255, Met-101, and Met-431 are the major oxidation sites; it is known that they are more exposed in the three-dimensional MAb structure18 and, thus, more susceptible to oxidation. Met oxidation was also reported as the major degradation pathway following UV exposure for recombinant humanized MAb HER2, with Met-255 in the heavy chain in the Fc region as the primary site of oxidation and Met431 as another major oxidation site.27 Besides Met photodegradation, a major Trp oxidation site was also found at Trp-105 for MEDI-493, while Trp-54 and Trp-55 showed only a low level of oxidation for both MAbs. This is because Trp-105 is fully exposed and susceptible to photooxidation, while Trp-45 and Trp-55 are only partially exposed to the solvent environment.18 Functional Characterization of UV-Irradiated MAb. The binding activity of MEDI-493 and MEDI-524 was assessed using a binding ELISA assay to evaluate the effect of oxidation. The ELISA results showed that the binding activity of MEDI-493 (27) Lam, X. M.; Yang, J. Y.; Cleland, J. L. J. Pharm. Sci. 1997, 86, 1250-1255.

decreased with the photoirradiation time and became 28% of the initial value upon UV irradiation for 7 days, while MEDI-524 did not show any significant activity loss in the same UV irradiation study (Figure 3 A, B). There are several oxidized amino acids located in the CDR regions: Met-34 in the heavy chain CDR1 region; Trp-54 or Trp-55 in the CDR2; Met-101 and Trp-105 in the CDR3. MEDI-493 and MEDI-524 have identical sequences and similar extents of oxidation for Met-34, Met-101, and Trp-54 or Trp-55 in the CDR regions, and for the other oxidized residues (Met-255, Met-431, Met-361, Met-4) that are not in the CDR region (Figure 3 A, B). The only amino acid vulnerable to oxidation events that was present in MEDI-493 and not in MEDI-524, and could be linked to the loss in binding activity in MEDI-493, was Trp-105. As shown in Figure 3 (A, B), Trp-105 oxidation in MEDI493 correlated well with the decreased binding activity observed during UV irradiation, while Met residues showed the same rate of oxidation for MEDI-493 and MEDI-524. This finding suggests that the oxidation of Trp-105 directly affects the binding activity of the MEDI-493 molecule. To further support that the loss of binding activity was due to a photochemical event in the CDR region, MEDI-493 was photooxidized in the presence of a 24-mer peptide mimic of the RSV F protein antigen. Because the peptide exhibited about 1000-fold less binding affinity than the F protein antigen,28 it was added in large excess to MEDI-493. Figure 4 shows the rate of photochemiAnalytical Chemistry, Vol. 79, No. 7, April 1, 2007

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Figure 2. MS/MS spectra and assignment of fragment ions of the trypsin/Glu-C peptide (SMITNWYFD): (A) unmodified, (B) oxidized containing Oia or 5-OH-Trp, and (C) oxidized containing NFK or DioOia. 2802

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Figure 3. Plot of log fraction of intact susceptible residues versus photoirradiation time, t-BHP oxidation time, and ozonized buffer ratio for MEDI-493 (A, C, and E) and MEDI-524 (B, D, and F). Intact susceptible residues are labeled as 9 Met-101, ( Trp-105, 2 Met-255, ∆ Met-361, and O Met-431. The log fraction of ELISA activities is shown in vertical bar format for the initial and last samples.

cal inactivation of MEDI-493 and MEDI-493 in the presence of an excess of a synthetic peptide carrying a specific antigen. The experiment revealed that addition of the 24-mer F-peptide resulted in a significant protection of MEDI-493 from UV photooxidation, in a dose-response manner. Protection of the CDR region, including Trp-105, preserves the binding activity during UV irradiation and further demonstrates the importance of Trp-105 in MEDI-493 biological function. The UV-irradiated MEDI-493 and MEDI-524 were also evaluated for their ability to prevent RSV from infecting a susceptible (28) Johnson, S.; Oliver, C.; Prince, G. A.; Hemming, V. G.; Pfarr, D. S.; Wang, S. C.; Dormitzer, M.; O’Grady, J.; Koenig, S.; Tamura, J. K.; Woods, R.; Bansal, G.; Couchenour, D.; Tsao, E.; Hall, W. C.; Young, J. F. J. Infect. Dis. 1997, 176, 1215-1224.

target cell line (Hep-2), using a cell-based microneutralization assay.19 MEDI-493 decreased to 77% potency after being photoirradiated for 1 day and to 26% activity after 7 days, while MEDI524 did not show any significant activity loss throughout the photostability study. This result further confirmed the loss of binding activity observed during the ELISA study. The effect of light exposure on the binding affinity to the target protein and binding kinetics of MEDI-493 and MEDI-524 was studied by surface plasmon resonance analysis (Biacore). By day 7 of the photostability study, the binding affinity of MEDI-524 decreased 2-fold while that of MEDI-493 showed a 7-fold drop in binding. The decrease in affinity for MEDI-524 is mainly accounted for by a 2-fold increase in the dissociation rate constant. The Analytical Chemistry, Vol. 79, No. 7, April 1, 2007

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Figure 4. Rate of photochemical inactivation of MEDI-493 and MEDI-493 in the presence of an excess of a synthetic peptide carrying a specific antigen.

decrease in affinity for MEDI-493 is not only due to an increase in the dissociation rate constant, but also to a decrease in the association rate constant. The Biacore results support the conclusions from the ELISA and microneutralization assays that MEDI493 has subsequent bioactivity and binding affinity loss upon UV irradiation and is more susceptible to UV irradiation than MEDI524. Characterization of Chemically Oxidized MAbs. In order to unequivocally demonstrate that the oxidation event of Trp-105 modification results in loss of bioactivity, the contribution to binding activity from Met (if any) needed to be ascertained. We reasoned that, if a Met-specific reagent was used for oxidation and no activity loss was seen, the Trp-105 oxidation event must be the critical factor. Thus, MEDI-493 and MEDI-524 were oxidized by a Met-specific oxidant, t-BHP,20,21 which is capable of selectively oxidizing the exposed Met residues to the sulfoxide form. The oxidized MAbs were analyzed after 1, 3, and 24 h by tryptic peptide mapping using on-line tandem mass spectrometry. As expected, only Met residues were oxidized and the oxidation products were Met sulfoxide, as confirmed by MS/MS analysis. Oxidation was not detected for Trp-105 in MEDI-493. Figure 3 (C, D) shows the plot of the log fraction of intact susceptible residues versus reaction time. The facility of t-BHP oxidation was governed by the solvent accessibility of the Met residues, while the extent of photooxidation was dependent on the amount of light energy imparted to the susceptible residues.29 Both oxidation methods (UV and chemical) are dependent on the exposure level of the oxidized residues. Similarly to the UV irradiation study, our results indicate that Met-101, Met-255, and Met-431 are more susceptible to oxidation, whereas Met-361 and Met-4 showed only low levels of oxidation. The order of oxidation rates at the various sites were similar to that observed in the light exposure study. ELISA results were also obtained and showed that MEDI-493 did not lose binding activity, as observed in the UV study, although the extent of Met oxidation at each site reached similar levels and yielded the same degradation product (Figure 3 C, D). MEDI524 showed similar levels of Met oxidation and no loss of activity during this study. These results indicate that the biological activity loss of MEDI-493 observed during the UV irradiation study was not caused by Met oxidation. (29) Duenas, E. T.; Keck, R.; DeVos, A.; Jones, A. J. S.; Cleland, J. L. Pharm. Res. 2001, 18, 1455-1460.

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To further verify that the bioactivity loss was solely caused by Trp-105 oxidation, a selective reagent for Trp oxidation (ozone) was chosen. Ozone can oxidize Trp to NFK or Kyn in proteins.30 Modification of a single Trp residue by ozone in an immunoglobulin light chain, ribonuclease T1, or hen egg white lysozyme produced a large decrease in the stability of these proteins to guanidine chloride and heat.30 It was reported that ozonization of one out of six Trp residues in lysozyme resulted in concomitant loss of the enzymatic activity.31 To study the effect of Trp modification, MEDI-493 was treated with different amounts of the ozonized solution at 0 °C and was analyzed by peptide mapping with on-line mass spectrometry. The oxidation events were monitored using LC-MS from tryptic peptides, except for Met-101 and Trp-105, which were monitored using tryptic/Glu-C digestion, as previously discussed. Trp-105 was the only oxidized Trp residue (Figure 3 E, F) with NFK as the major degradation product and Kyn, 5-OH-Trp, and Oia as minor products (1-2%). Several exposed Met residues, such as Met-101, Met-255, Met-431, and Met-361, were also significantly oxidized to Met sulfoxide; while Met-34 and Met-4 were oxidized to a lesser extent. Because we have already demonstrated that Met oxidation alone did not significantly affect the binding activity, the detected bioactivity loss was ascribed to Trp-105 oxidation. Similar to the observation in the photostability study, Trp-105 oxidation in MEDI-493 correlated well with the significantly decreased binding activity following ozone treatment. The chemical modification by ozone further supports the conclusion from the photostability study that a single amino acid (Trp-105) is responsible for MEDI-493 bioactivity loss following photooxidation. CONCLUSIONS An oxidation event in MEDI-493 involving Trp-105 in the CDR3 region of the heavy chain results in a reduction in binding affinity and biological activity of MEDI-493 to its specific antigen (RSV F protein). The binding activity of MEDI-493 was diminished to 28% of the nonirradiated antibody as measured by the ELISA assay, its KD decreased 7-fold as measured by Biacore, and its potency was decreased to 26% in a microneutralization assay after exposure to UV light for one week. The trypsin/Glu-C digestion and mass spectrometric analysis correlated the photooxidation of Trp-105 to the loss of binding activity of MEDI-493 in the ELISA, microneutralization, and Biacore assays. A closely related antibody (MEDI-524), containing a Phe residue substituted for Trp-105 in the CDR3, was considerably more resistant to UV light irradiation as measured by ELISA, microneutralization, and Biacore assays. Furthermore, when UV degradation studies were performed in the presence of a 24-mer synthetic F protein mimetic that bound to the CDR regions of the MEDI-493, the antibody was protected from activity loss; the peptide shielded the antibody from the Trp105 oxidation event when it bound to MEDI-493 CDR region. The analysis of the antibodies following oxidation by t-BHP, a Met-specific reagent, further proves that the loss of binding and biological activity of MEDI-493 is due to Trp oxidation and not to Met oxidation, because MEDI-493 was not affected by the treatment with t-BHP. In contrast, the ozone-treated MEDI-493, producing both Trp-105 and Met oxidation products, showed a (30) Okajima, T.; Kawata, Y.; Hamaguchi, K. Biochemistry 1990, 29, 9168-9175. (31) Kuroda, M.; Sakiyama, F.; Narita, K. J. Biochem. 1975, 78, 641-651.

significant loss in binding and biological activity. The results of the chemical treatment confirmed the critical role that Trp-105 plays in MEDI-493 biological activity. Given that the UV-exposed MEDI-493 produces multiple Trp105-derived degradants, it is difficult to assign the loss of activity to any particular Trp degradation species (Oia, 5-OH-Trp, NFK, or DioOia). However, our data support the hypothesis that Trp105 in the CDR3 region of MEDI-493 heavy chain is responsible for the initial binding of the RSV F protein in the CDR3 region of the MAb, and that its oxidation critically impairs the function of the MAb. We have demonstrated that a single Trp in the CDR of MEDI493 is responsible for the MAb susceptibility to UV light and is critical for biological activity (RSV neutralization). There are several approaches to protecting proteins from oxidation, including inspecting the amino acid sequence for photosensitive residues

in the CDR region and using antioxidants in pharmaceutical products.27,32 Further studies of the structure-function relationship may lead to MAb molecules that are more resistant to UV light and, hence, more stable biopharmaceutical products.

(32) Akers, M. J.; Parenter, J. J. Parenter. Sci. Technol. 1982, 36, 222-228.

AC062311J

ACKNOWLEDGMENT The authors would like to thank Kenneth Miller for Biacore analysis, and Nicholas Knoepfle and Elisa Mitchell for microneutralization analysis. We would also like to thank Gail Wasserman and James Young for their critical review of this manuscript. NOTE ADDED AFTER ASAP PUBLICATION This article was released ASAP on February 24, 2007 with a minor error in the caption for Figure 3. The correct version was posted on February 28, 2007. Received for review December 5, 2006. Accepted January 18, 2007.

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