Identification and refinement of a peptide affinity ligand with unique specificity for a monoclonal anti-tenascin-C antibody by screening of a phage display library

Journal of Chromatography A, 1107 (2006) 182–191

Identification and refinement of a peptide affinity ligand with unique specificity for a monoclonal anti-tenascin-C antibody by screening of a phage display library Piero Bellofiore a , Fiorella Petronzelli b , Tiziana De Martino a , Olga Minenkova c , Valentina Bombardi a , Anna Maria Anastasi b , Ragnar Lindstedt b , Franco Felici c,1 , Rita De Santis b , Antonio Verdoliva a,∗ a

Tecnogen S.C.p.A., Localit`a La Fagianeria, 81015 Piana di Monte Verna (CE), Italy b Sigma-Tau SpA R&D, via Pontina Km 30.400, 00040 Pomezia, Rome, Italy c Kenton Labs, c/o Sigma-Tau, Pomezia, Rome, Italy

Received 6 October 2005; received in revised form 15 December 2005; accepted 19 December 2005 Available online 18 January 2006

Abstract Using phage display technology, a 22-mer peptide was selected as a ligand with unique specificity for the murine monoclonal ST2146 antibody that recognizes the EGF repeats region of the human tumor-associated antigen tenascin-C. This peptide, synthesized in an 8-branched form to enhance its binding properties, is useful in replacing the native antigen in the affinity and immunoreactivity characterization of the ST2146 antibody and its biotinylated derivatives. Affinity resins, prepared by immobilizing the mimotope or its shorter 10-mer binding unit on a chromatographic support, were able to capture ST2146 directly from the hybridoma supernatant, with antibody recovery and host cell protein (HCP) reduction similar to or better than protein A sorbent, a purity degree exceeding 95%, and full recovery of antibody activity. The affinity constants of both peptides, as determined by frontal analysis of broad-zone elution affinity chromatography and BiaCore measurements, were very similar and included in a range suitable for affinity ligands. Column capacity, determined by applying a large excess of purified ST2146 to 1 mL of column bed volume, was close to 50 mg/mL for both resins. These matrices retain their ST2146 binding properties after various treatments, including sanitization, thus indicating very high stability in terms of ligand leakage and degradation. Moreover, the short form shows higher enzymatic stability, thus proving more suitable as ligand for ST2146 affinity purification. © 2006 Elsevier B.V. All rights reserved. Keywords: Affinity purification; Monoclonal antibody; Phage display technology; PAGRIT; Host cell proteins

1. Introduction Due to the exponential growth of regulatory restrictions, a biotechnological product takes at least 10 years to reach the market as an approved commercial biotherapeutic, with the final substance thoroughly characterized in terms of purity, efficacy, potency, stability, pharmacokinetics, pharmacodynamics, toxicity, immunogenicity and contaminants content. The need to reduce costs and fulfill the increasing quality assurance and regulatory compliance criteria has led to adoption of purifi∗

Corresponding author. Tel.: +39 0823 612 215; fax: +39 0823 612 230. E-mail address: [email protected] (A. Verdoliva). 1 Present address: Dept. Microbiology, Genetics, Molecular Biology, University of Messina, 98100 Messina, Italy. 0021-9673/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2005.12.064

cation strategies based on highly selective and sophisticated separation media, capable of improving yields and reducing processes through fewer chromatographic steps. At the moment, affinity chromatography represents the most effective fractionation technique for the purification of biotechnological products, including immunoglobulins, which are routinely used in biochemical and biological research as analytical reagents, and are gaining increasing interest as protein-based therapeutics. Both polyclonal and monoclonal antibodies, in fact, have become the basis for standard therapies in a number of malignancies [1–5], with large amounts of highly purified immunoglobulins currently produced in GMP facilities. Although antibodies of the G-class can be conveniently purified by affinity chromatography using immobilized protein A or G even on a large scale [6–9], synthetic affinity ligands, obtained by screening of synthetic or

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biological combinatorial libraries or by de novo design, represent a robust alternative to natural ligands [10–12]. Other than being less expensive, they pose fewer problems from both stability and regulatory points of view, in that they can be sanitized and regenerated under very stringent conditions. Moreover, these synthetic molecules help avoid the risk of contamination associated with natural ligands of human or animal origin [13]. To date, a number of synthetic derivatives have been proposed for immunoglobulin purification, such as peptides [14–19], amino acids [20], thiols [21], dyes [22], and triazine-based ligands [23], the majority of which is directed against shared epitopes, thus not able to discriminate between antibodies with different specificities. This represents a problem when purifying Fab fragments, scFv or full-size antibodies from cell culture supernatants containing serum antibodies, often present in cell culture media due to the fetal calf serum used as supplement. In this way, ligands with unique specificity for monoclonal antibodies, derived from known epitopes or selected from biological or synthetic libraries [24–29], allow to overcome these chromatographic limitations; in addition, they could replace the natural antigen in the antibody immunoreactivity and affinity characterization, since development of a monoclonal antibody as a biotherapeutic product relies on a number of tests where a pure, stable and low-cost specific antigen is recommended. Through the screening of a phage library displaying 12-mer random peptides with the ST2146 monoclonal antibody raised against the human tumor-associated antigen tenascin-C (Tn-C, MW ∼1800 kDa), we identified a peptide, initially synthesized in a 22-mer form comprising side pVIII sequences, with unique specificity for ST2146, as determined by ELISA and chromatographic experiments using several anti-Tn-C antibodies recognizing different protein epitopes, as well as Fab and Fc fragments of ST2146. To explore the possibility of using this peptide, called M[46-2], in the process development of the Tn-C-related pretargeted antibody-guided radioimmuno therapy (PAGRIT), the purification capability of the corresponding M[46-2] affinity resin was investigated and compared with the commercial protein A sorbent and the m[9-18] immobilized peptide; this latter is a 10-mer form deriving from the M[46-2] sequence, with binding properties to the ST2146 antibody similar to the starting peptide. Also, in order to evaluate the suitability of the peptide to replace the native antigen in the ST2146 immunoreactivity and affinity analysis, we investigated the ability of the mimotope, synthesized as the 8-branched M8 [46-2] multiple antigenic peptide (MAP) to enhance its binding properties, in recognition of the ST2146 antibody, at various biotinylation degrees, in comparison with tenascin-C, using ELISA and surface plasmon resonance (SPR) measurements.

The biotinylated ST2146 monoclonal antibody was used to select affinity binders from a phage-displayed random peptide library, according to the procedure described by Minenkova et al. [30]. The library is based on phagemid system pc89 [31] and composed of random 12-mer peptides, displayed on filamentous phage as fusion to the N-terminus of the major coat protein pVIII. Two micrograms of biotinylated ST2146 were incubated with an aliquot of the library containing 2 × 1010 TU (transducing units) in 0.2 mL of blocking buffer (PBS, 5% nonfat dry milk, 1% Triton X-100) for 1 h at 37 ◦ C under gentle agitation. Mixture of library and antibody was incubated with Magnetic Beads (100 ␮L), linked to streptavidin (Dynabeads M-280 Streptavidin, Dynal, Norway) for 10 min at room temperature (RT) under agitation. The beads were washed 10 times with 1 mL of washing solution (PBS, 1% Triton X-100). The bound phage was eluted with 100 ␮L of 0.1 M HCl, pH 2.2 adjusted by glycine, neutralized with 10 ␮L of 2 M Tris, pH 9.6 and amplified by infecting bacterial strain DH5␣F’ (supE44
lacU169 (φ80 lacZ
M15) hsdR17 recA1 endA1gyrA96 thi-1 relA1 F’ [traD36 proAB+ lacIq lacZ
M15]).

2. Experimental

2.4. ST2146 fragmentation

2.1. Chemicals and biologicals

The IgG2b ST2146 antibody was digested with papain to obtain Fab, Fc and a small amount of Fab/c fragments in absence of reducing agent. In this case, papain was preactivated with 10 mM Cysteine in 50 mM Na Phosphate, 2.0 mM EDTA, pH 7.0, for 30 min at 37 ◦ C, and the reducing agent was rapidly

Anti-mouse IgG (Fc-specific) alkaline phosphatase, antimouse IgG alkaline phosphatase, anti-mouse IgG horse radish peroxidase (HRP), ␣-chymotrypsin, trypsin, papain and

all chemicals were purchased from Sigma–Aldrich (Milan, Italy), unless otherwise stated. Prepacked recombinant protein A/Sepharose fast flow and Sepharose fast flow preactivated with N-hydroxysuccinimide (NHS) groups were purchased from Amersham Biosciences (Uppsala, Sweden). SP 2/0 host cell protein (HCP) ELISA kit was from Cygnus Technologies (Southport, NC, USA). BiaCore X instrument, CM5 dextran-coated and streptavidin precoated sensor chips, HBS buffer (10 mM Hepes, 0.15 M NaCl, 3.4 mM EDTA and 0.005% surfactant P20, pH 7.4), amino coupling kit (NHS, N-hydroxysuccinimide; EDC, N-ethylN dimethylaminopropylcarbodiimide and ethanolamine), were all obtained from Biosense (Milan, Italy). Tenascin and 7H3, ST1897, ST1485, ST1910 and ST2146 monoclonal antibodies were from Tecnogen (Piana di MonteVerna, CE, Italy). 2.2. ST2146 production Production of ST2146 was carried out through cultivation of cST2146 hybridoma cells in protein-free medium, in a 2 L perfusion MD2 bioreactor (Sartorius BBI Systems, Melsungen, Germany). The cellular supernatant was concentrated 10 times with the ProFlux M12 Tangential Filtration System (Millipore, Bedford, MA, USA) using a 50 kDa BioMax polyethersulfone membrane (Millipore), filtered through a 0.22 ␮m filter (Millipore) and stored at 4 ◦ C. 2.3. Affinity selection

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removed by a desalting step in the same buffer. The antibody (6 mg/mL) was then incubated in 50 mM Na-Phosphate, 2.0 mM EDTA, pH 7.0 at 37 ◦ C with an enzyme: IgG ratio of 1:200. The reactions, monitored by gel filtration chromatography (GF) on a Superdex HR200 10/30 column (Amersham Biosciences), were completed in 2–4 h and, to avoid further degradation, the enzyme was deactivated by addition of 10 mM iodoacetamide. 2.5. Peptide synthesis and stability All peptides were produced by solid-phase peptide synthesis following the Fmoc methodology using a fully automated 431A peptide synthesizer (Perkin-Elmer, Boston, MA, USA) as described previously [32]. After resin cleavage, peptides were purified by reverse phase chromatography (RP-HPLC) and their identity was confirmed by amino acid analysis and ESI mass spectrometry, which provided molecular weights identical to the expected values. The enzymatic stability of both M[46-2] and m[9-18] peptides was assessed by incubating 0.5 mL of a 1.0 mg/mL peptide solution with 5 ␮g of ␣-chymotrypsin, or trypsin in a ratio enzyme: peptide of 1:100 at room temperature. The extent of peptide degradation was monitored by RP-HPLC analysis, on a ␮RPC C2/C18 ST 4.6/100 column (Amersham Biosciences, Uppsala, Sweden), at different times, ranging from 30 min to 24 h. In addition, 50 ␮L of each peptide, dissolved in PBS solution at a concentration of 1.0 mg/mL, were added to 250 ␮L of ST2146 supernatant and incubated at 37 ◦ C. After 60, 120 and 180 min of incubation, 50 ␮L of the peptide-supernatant mixture were added to 450 ␮L of 0.1 M acetic acid, in order to detach the peptide from the supernatant, and stored at −80 ◦ C until analysis. The extent of peptide degradation was monitored by RP-HPLC analysis as described previously. 2.6. Immunoaffinity matrices Synthetic peptides were linked to the Sepharose fast flow (SFF) matrix, preactivated with NHS groups, employing a standard coupling ratio of 2 ␮mol peptide per mL of gel. As recommended by the manufacturer’s protocol, peptides were dissolved in 2 mL of 0.1 M NaHCO3 , pH 8.5, and mixed with 1 mL of preactivated matrix. The suspension was incubated for several hours at RT under gentle agitation, monitoring the extent of peptide incorporation by RP-HPLC analysis at different times. To deactivate residually active groups, the resin was incubated with 0.1 M Tris, pH 8.5 for 1 h at room temperature, and finally packed into a 6.6 mm I.D. glass column (Omnifit, Cambridge, United Kingdom). 2.7. Affinity purification The ST2146 cellular culture supernatant was loaded onto the M[46-2]/SFF and m[9-18]/SFF affinity columns equilibrated at a flow rate of 60 cm/h with the 50 mM Na Phosphate, pH 7.0 binding buffer. After elution of unbound material, the eluent was changed to 0.1 M Acetic Acid, pH 3.0, to elute bound material. Purifications on 1 mL protein A column were carried out as recommended by the manufacturer’s protocol. Sample was applied

to the affinity column equilibrated with 20 mM Na Phosphate, pH 7.0 as binding buffer, at a flow rate of 60 cm/h. After elution of unbound material, retained antibodies were eluted by lowering the pH with 0.1 M Gly, pH 3.0. All bound fractions were immediately neutralized with a few drops of 1 M Tris, pH 9.5, and characterized by UV (ε1% = 13.4 cm−1 ), GF and SDS-PAGE in order to determine IgG recovery and purity, and by RP-HPLC and ELISA to evaluate ligand leakage, immunoreactivity recovery and residual host cell proteins. 2.8. Chromatographic affinity constant In analytical affinity chromatography, frontal analysis of broad-zone elution provides a convenient and accurate way for the quantitative analysis of macromolecular interactions [33–35]. Experimentally, a solution containing a known concentration of solute is continuously applied to a column containing an immobilized ligand. As the solute binds to the ligand, the column becomes saturated and the amount of solute eluting from the column increases, forming a characteristic breakthrough curve. The result is essentially a titration of the number of active binding sites within the column. If fast association and dissociation kinetics are present, the mean position of the breakthrough curve can be related to the concentration of the applied solute, the amount of ligand in the column, and the dissociation equilibrium constant for solute-ligand, according to the equation: 1 KM/P [P0 ] = + V − V0 MT MT where V is the elution volume at which the affinity matrix is half-saturated; V0 , the void volume of the column; MT , the total amount of the immobilized M ligand; [P0 ], the initial concentration of the solute P and KM/P is the dissociation equilibrium constant of the complex MP. From a series of such elutions at varying [P0 ], the elution volume V can be plotted as 1/V − V0 versus [P0 ] to obtain a linear response with a slope equal to 1/MT and an intercept of KM/P /MT . V0 is an experimentally determined constant; V, the elution volume for half saturation, so KM/P (equilibrium dissociation constant) can be calculated by dividing the intercept by the slope. 2.9. SDS-PAGE analysis Characterization of the bound fractions from the affinity columns was performed by SDS-PAGE analysis under nonreducing conditions, on a 4–15% gradient gel (Bio-Rad, Milan, Italy) of acrylamide–bisacrylamide solution. About 7 ␮g of total proteins were analyzed by performing the electrophoretic runs on the Mini-PROTEAN II apparatus (Bio-Rad, Milan, Italy), following the manufacturer’s instructions. Detection of the protein bands was performed with the Coomassie Brilliant Blue R-250 (Merck, Darmstadt, Germany) staining method and purity degree was determined by electronic scanning of the gel on a HP scanjet 4400C, using the HP Precisionscan PRO 3.1 software (Hewlett-Packard Co.) and densitometric analysis with IMAGE PROPLUS software.

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2.10. Western-blotting analysis Bound materials (2 ␮g of total proteins) were run on a SDSPAGE gel, as previously described, and transferred to a nitrocellulose filter by the elettroblotting method. After protein transfer, the filter was incubated overnight at + 4 ◦ C in 0.1 M Tris, 0.15 M NaCl, 5% dried milk, as blocking buffer (B1). After five washing with B1, the membrane was incubated 1.5 h at RT with the goat anti-mouse IgG-HRP diluted 1:1000 with B1. The membrane was then left for 1 h at room temperature, washed three times with water and then soaked with chromogenic substrate solution consisting of 0.7 mg/mL 3,3 -diaminobenzidine, 0.17 Urea Hydrogen Peroxide in 60 mM Tris (Sigma–Aldrich, Milan, Italy). This substrate produces an intense brown–black precipitate at the site of the enzyme binding. 2.11. Gel filtration analysis Gel permeation analysis was performed using a Superdex HR 10/30 GF column (300 mm × 10 mm) equilibrated at a flow rate of 0.75 mL/min with PBS, 10 mM NaN3 , pH 6.8, with monitoring of the effluent at 280 nm. About 400 ␮g total protein deriving from crude and unbound material or 150 ␮g of affinity column purified immunoglobulins were 0.22 ␮m filtered and applied to the column. 2.12. ST2146 biotinylation In order to obtain an antibody:biotin molar ratio of 1:10, 1:15 and 1:20, respectively, 4, 6 and 8 mg of 2xAH-Biotin-NHS Ester (BIOSPA, Milan, Italy) were dissolved in 0.5 mL of DMSO, added, little by little on a stirrer, to 20 mL of 5 mg/mL antibody in 0.1 M NaHCO3, pH 8.5, and the reaction left at RT, under gentle agitation. Two hours later the free biotin was removed by tangential filtration with a LabScale TFF System (Millipore, Bedford, MA, USA), using a 50 kDa BioMax polyethersulfone membrane (Millipore, Bedford, MA, USA), and the biotinylated antibody concentrated to 5 mg/mL in 10 mM Na Phosphate, 150 mM NaCl, pH 7.0. 2.13. Enzyme-linked immunosorbent assay (ELISA) Polystyrene microtiter plates (Falcon, Becton Dickinson, NJ, USA) were incubated overnight at 4 ◦ C with 100 ␮L/well of a 0.5 ␮g/mL tenascin or 5 ␮g/mL M8 [46-2] solution in PBS, pH 7.2. After coating, the plates were washed with PBS, 0.1% Tween 20 (PBS-T, washing buffer) and blocked with 200 ␮L of PBS containing 3% BSA, for 2 h at 37 ◦ C. For immunoreactivity analysis the plates were filled with ST2146 anti-tenascin antibody at varying concentrations (0.1–10 ng/mL), previously diluted with PBS containing 0.5% BSA (PBS-B), and incubated at 37 ◦ C for 2 h. Each dilution was performed in duplicate. Plates were washed again with PBS-T and filled with 100 ␮L of an anti-mouse IgG (Fc-specific) alkaline phosphatase diluted 1:1000 with PBS-B. After 1.5 h of incubation at 37 ◦ C, 100 ␮L of chromogenic substrate solution, consisting of p-nitro-phenylphosphate (pNPP, Sigma–Aldrich, Milan, Italy), were added, the reaction stopped after 30 min at 37 ◦ C with 100 ␮L/well of 3 M

185

NaOH and the absorbance determined at 405 nm with a Model 3550 EIA Microplate Reader (Bio-Rad, Milan, Italy). For competitive ELISA, the monoclonal antibody ST2146 (5 ng/mL) was mixed with increasing concentrations of peptides as competitors (0.1–200 ␮g/mL), and 100 ␮L/well of this mixture was added to the tenascin-C-coated plates (0.5 ␮g/mL). Optimal concentrations of tenascin and ST2146 were determined in preliminary experiments. Incubation with secondary antibody and chromogenic development were performed as described above. 2.14. Phage-ELISA Multiwell plates were coated overnight at 4 ◦ C with 100 ␮L of the anti-pIII monoclonal antibody 57D1 [36] at a concentration of 1 ␮g/mL of antibody in 50 mM NaHCO3 , pH 9.6. A 1:1 mixture of phage supernatant and blocking buffer (PBS, 5% non-fat dry milk, 1% Triton X-100) was added to each well and allowed to bind for 1 h at 37 ◦ C. After washing with PBS, 1% Triton X-100 and 100 ␮L of blocking buffer containing the ST2146 antibody at a concentration of 1 ␮g/mL were added to wells. Plates were then washed and a 1:5000 dilution of antimouse IgG alkaline phosphatase-conjugated secondary antibody (Sigma–Aldrich, Milan, Italy) was used for developing ELISA as previously described. 2.15. Host cell protein-ELISA The immunoenzymetric assay for the measurement of SP 2/0 host cell proteins was performed according to the manufacturer’s protocol. Briefly, microtiter strips coated with an anti-SP 2/0 affinity-purified antibody were filled with 50 ␮L of SP 2/0 HCP standards at 0, 2, 8, 25, 75 and 200 ng/mL, and unknown samples. Simultaneously, 100 ␮L of a second horseradish peroxidase (HRP) enzyme-labelled anti-SP 2/0 antibody were added into each well, resulting in the formation of a sandwich complex of solid-phase antibody-HCP-enzyme-labelled antibody. The plate was covered and left at RT for 2 h under agitation at 180 rpm. After incubation, the strips were washed four times with 300 ␮L of washing buffer (Tris buffered saline with preservative) and filled with 100 ␮L of substrate solution, consisting of 3,3 ,5,5 tetramethyl benzidine (TMB). The reaction was stopped after 30 min by adding 100 ␮L of 0.5 N sulfuric acid into each well with the amount of hydrolyzed substrate determined on a microtiter plate reader at 450 nm. 2.16. BiaCore analysis Tenascin-C and the 8-branched M8 [46-2] peptide were immobilized on CM5 dextran-coated sensor chips by a standard amino coupling procedure, according to manufacturer’s instructions (Biosense, Milan, Italy). Briefly, the carboxymethylated dextran surface was activated by a 7-min injection of a solution containing 200 mM EDC and 50 mM NHS, followed by a manual injection of M8 [46-2] (100 ng/mL solution in 10 mM sodium acetate, pH 4.0—immobilization response 430 RU) or tenascin (82 ␮g/mL in 10 mM sodium acetate, pH 2.3—immobilization response 2800 RU). A continuous flow of HBS-EP at 5 ␮L/min

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was maintained, and capping of unreacted sites was achieved by injecting 1 M ethanolamine, pH 8.5, for 7 min. Blank surfaces were obtained in the same manner except that no protein was injected. M[46-2] and m[9-18] C-term biotinylated peptides were immobilized on streptavidin precoated sensor chips manually injecting a 50 ng/mL solution in a continuous flow of HBS-EP at 5 ␮L/min. Two chips were generated giving final immobilization responses of 55 and 60 resonance units (RU), respectively. Sensorgrams for kinetic measurements were generated by injection of ST2146 (at concentrations ranging from 3.9 to 500 nM) in HBS-EP at a flow rate of 30 ␮L/min. Kinetic data were collected in duplicate for each ST2146 concentration. The antibody was allowed to bind for 2 min and dissociate for another 2 min before final washing with HBS-EP. The chip was regenerated by injection of repeated pulses of 100 mM NaOH. Biosensor data were prepared, modelled and fitted by means of BIAevaluation 3.1 software using a bivalent-analyte model with simultaneous determination of association and dissociation constants. 3. Results 3.1. Ligand identification and characterization A phage library displaying 12-mer random peptides inserted at the amino terminus of 50-aa major coat pVIII protein of filamentous phage, was used to select specific anti-tenascin monoclonal ST2146 antibody binders. After two rounds of affinity selection, performed according to the protocol previously described, a pool of phages was amplified and single positive clones were identified by immunoscreening procedure [37]. Phage supernatants were prepared and tested for their capacity to recognize the ST2146 antibody in ELISA assays. As

shown in Fig. 1A, four positive clones, M[46-1], M[46-2], M[463], M[46-5], and one just above background, M[46-7], were selected and sequenced. Corresponding peptides were synthesized according to a general formula H2 N-AEGEF-[variable 12-mer epitope]-GDPAK-COOH, where the variable peptide is integrated into two 5-mer pVIII constant sequences (Fig. 2A), and tested for their ability to mimic the tenascin-C antigen in the interaction with the ST2146 antibody, by competitive ELISA assays. Results, shown in Fig. 1B, indicate that only the M[46-2] 22mer form can compete for ST2146 binding to tenascin, while there is no inhibition with all other peptides. To investigate ST2146/M[46-2] binding, preliminary experiments, including chromatographic and ELISA assays, were carried out using several monoclonal and polyclonal antibodies, including ST2146, ST1897, ST1910 and ST1485 noncrossreacting anti-Tn-C antibodies, the 7H3 anti-IL2 monoclonal, and mouse and human polyclonal immunoglobulins. Ten mg of each antibody were dialyzed against the binding buffer and loaded, at a concentration of 1 mg/mL, onto a 1 mL M[46-2]/SFF affinity resin, under the experimental conditions previously described. As expected, the ST2146 antibody is fully adsorbed and eluted from the resin, whereas there is no peak in the elution step with all other antibodies. These results were confirmed by competitive ELISA assays involving the anti-Tn-C antibodies recognizing different protein epitopes. The peptide can fully inhibit binding between the Tn-C antigen and the ST2146 antibody, whereas there is no reactivity with all other antibodies (Fig. 3A). Cumulatively, these findings underline the capacity of the M[46-2] form to recognize, with high specificity and selectivity, the ST2146 antibody and suggest that interaction occurs in a region overlapping its antigenic binding site. In strict agreement with this hypothesis, the affinity resin can only adsorb the ST2146 fragments containing at least

Fig. 1. (A) ELISA reactivity of single phage clones derived from second round of affinity selection with ST2146 antibody. Data are representative of two independent assays. (B) Inhibition of the binding between the ST2146 antibody and immobilized tenascin-C by M[46-1] (
), M[46-2] (), M[46-3] (♦), M[46-5] () and M[46-7] (䊉) soluble peptides.

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The performance of the mimotope, synthesized as the 8branched M8 [46-2] structure (Fig. 2B) to enhance its binding properties, and the natural antigen toward the ST2146 antibody were investigated by ELISA and SPR measurements. Table 1 shows kinetic values of ST2146 binding to M[46-2], M8 [462] and tenascin-C coated antigens. Despite similar association rates, the ST2146 binding to the mimotope exhibits a faster dissociation rate, more evident for the monomer, resulting in a 2 − 3 log KD reduction when compared to ST2146/tenascin affinity constant. With biotinylated ST2146 both MAP and TnC molecules show a reduction of immunoreactivity related to the increasing number of biotins per antibody. Nevertheless, while in ELISA experiments the trend of the residual immunoreactivity is very similar (Fig. 4A and B), in SPR the lower affinity towards the mimotope allows better discrimination of the binding characteristics at different antibody:biotin ratios. As shown in Fig. 5, the increasing number of biotins per antibody molecule gives rise to appreciable variations in both association and dissociation slopes of M8 [46-2]/ST2146 sensorgrams (5B), while it only marginally affects the binding to the native antigen (5A). 3.2. Affinity chromatography

Fig. 2. (A) Sequences of peptides deriving from positive clones identified by immunoscreening procedure. (B) Schematic representation of the 8-branched M[46-2] mimotope. (C) Sequences of 10-mer peptides deriving from the M[462] mimotope sequence.; italic letters are used for amino acids of the major coat pVIII protein of filamentous phage.

one Fab arm, while there is no binding with the corresponding Fc fragments (Fig. 3B). By sequence alignment of the selected peptide and the natural antigen, we find only three identities, thus suggesting that the 22-mer form is a mimotope of tenascin-C.

Starting from the N-terminus of the M[46-2] 22-mer, we selected 4 partially overlapping 10-mer peptides, called m[1-10], m[5-14], m[9-18] and m[13-22] (Fig. 2C), which were compared to the parent peptide for their ability to recognize the ST2146 antibody in competitive ELISA assays. Interestingly, only the m[9-18] form is able to fully inhibit the Tn-C/ST2146 binding in a dose- dependent manner similar to the 22-mer peptide, while there is no inhibition by all other peptides (data not shown), although the m[5-14] and the m[13-22] forms share, respectively, the first and the last six of the ten amino acids of the m[9-18] peptide.

Fig. 3. (A) Inhibition of the binding between immobilized tenascin-C and ST2146 (), ST1897 (
), ST1910 () and ST1485 (䊉) anti-tenascin antibodies by M[46-2] soluble peptide. (B) SDS-PAGE analysis (4–15% slab-gel under non-reducing conditions, Comassie staining) of bound (lane B) and unbound (lane U) materials deriving from mimotope sorbent for purification of ST2146 antibody (lane A) digested with papain (lane L).

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Table 1 Kinetic values of ST2146 binding to M[46-2], multimeric M8 [46-2] and tenascin-C-coated antigens Antigen

ka (M−1 s−1 ) [±SE]

M[46-2] M8 [46-2] Tenascin-C

1.24 × 105

[1.67 × 103 ]

1.6 × 105 [4.8 × 103 ] 3.15 × 105 [8.02 × 102 ]

Preliminary experiments comparing the M[46-2] and m[918] peptides, were performed to assess their stability to proteolytic enzymes. The simplified m[9-18] form is very stable to treatment with trypsin and ␣-chymotrypsin (there is no degradation after 24 h of incubation), while the 22-mer peptide is completely digested by ␣-chymotrypsin and trypsin after 2 and 22 h of incubation, respectively. We obtain similar results by incubating both peptides with the hybridoma culture supernatant, thus mimicking the purification environment. The amount of parent peptide in the mixture after 3 h of incubation is only 10% of the starting material, whereas the shorter form does not degrade after the same reaction time. To evaluate the chromatographic feasibility of M[46-2] and m[9-18] as affinity ligands, the binding of the ST2146 antibody to either peptide was characterized by frontal analysis of broad-zone elution affinity chromatography, which allows quantification of macromolecular interactions, expressing them as an equilibrium dissociation constant, independently from column-binding capacity [38]. Solutions at different concentrations of ST2146 immunoglobulins (ranging from 5 × 10−6 to 20 × 10−6 M) were passed through the affinity columns (0.5 ␮mol/mL ligand density), in each case until saturation was

Fig. 4. Immunoreactivity analysis of wild-type ST2146 antibody (
) and corresponding 1:10 (), 1:15 () and 1:20 (䊉) biotinylated forms to immobilized tenascin-C (panel A) and M8 [46-2] (panel B).

kd (s−1 ) [±SE] [2.28 × 10−3 ]

0.138 2.7 × 10−2 [1.3 × 10−3 ] 5.43 × 10−4 [6.44 × 10−5 ]

KD (M) 1.1 × 10−6 1.7 × 10−7 1.7 × 10−9

achieved and the mobile IgG had the same concentration in both eluate and applied solution. Fig. 6 shows the elution profiles for the M[46-2]/SFF resin and the corresponding straight line plot of 1/(V − V0 ) versus [P0 ], including the linear regression equation and the correlation coefficient shown in the inset. Results relating to the m[9-18] peptide are not shown but are similar, as demonstrated by the KM/T values determined by this procedure, which are 9.0 × 10−6 M for the M[46-2]/ST2146 complex and 8.5 × 10−6 M for the m[9-18]/ST2146 complex, both included in a range suitable for affinity ligands (10−4 –10−8 M). These results are confirmed by BiaCore analysis, immobilizing the M[46-2] and m[9-18] biotinylated peptides on streptavidincoated chip. The KD values of the ST2146 binding are 1.1 × 10−6 M and 1.47 × 10−6 M, respectively. The performance of both affinity matrices was then explored by assessing their capacity to adsorb the ST2146 monoclonal antibody under standard conditions of sample loading (90 cm/h), column size (1 mL of swollen gel in a 6.6 mm I.D. glass column),

Fig. 5. Ranking of BiaCore SPR sensorgrams for interaction of ST2146 at different antibody:biotin coupling ratios, with tenascin-C (panel A) and M8 [46-2] (panel B). Wild-type ST2146 (hatched lines) and corresponding 1:10 (– –), 1:15 (. . .) and 1:20 (- - -) biotinylated forms were injected at a concentration of 100 nM and a flow rate of 30 ␮L/min. A 1-min association and 2-min dissociation times were allowed.

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189

Fig. 6. (A) Frontal analysis experiments performed with ST2146 on the M[46-2] immobilized affinity resin. The concentrations of applied ST2146 were (a) 7.3 × 10−6 M; (b) 11.2 × 10−6 M; (c) 16.2 × 10−6 M and (d) 19.8 × 10−6 M. The flow rate was 0.5 mL/min and the column void time was 5 min. (B) Plot of 1/V − V0 vs. [P0 ], including the linear regression equation and the correlation coefficient shown in the inset.

ligand density (2 ␮mol per mL of gel) and elution (acetic acid 0.1 M, pH 3.0). Increasing amounts of starting material, ranging from 5 to 50 mL and containing from 3.0 to 30 mg of antibodies, were loaded onto 1 mL of each resin, including the rpA/SFF matrix, to investigate and compare their capacity to purify the immunoglobulins directly from the crude harvest. After washing of unbound material, retained antibodies were eluted by lowering the pH as previously described, and all bound fractions,

immediately neutralized with a few drops of 1 M Tris, pH 9.5, were characterized in terms of IgG content, immunoreactivity and purity. Data, collected in Table 2, confirm the similarity and the feasibility of both peptides as affinity ligands, with recovery properties better than recombinant-protein A. In working conditions, the M[46-2] and m[9-18] affinity matrices are, in fact, able to retain >90% of the immunoglobulins loaded onto the column at concentrations up to 25–30 mg of ST2146 per mL of resin,

Fig. 7. (A) SDS-PAGE comparison (4–15% slab-gel under non-reducing conditions, Comassie staining) of bound materials deriving from mimotope (lane M), simplified mimotope (lane m) and r-protein A (lane rpA) sorbents for ST2146 purification (30 mL loaded sample). About 7 ␮g of total proteins were loaded in each lane. (B) Gel filtration profiles of ST2146 crude harvest (400 ␮g of total proteins), unbound (400 ␮g of total proteins), and bound material (150 ␮g of total proteins) from the m[9-18]/SFF affinity matrix, on the Superdex HR 10/30 GF column (300 mm × 10 mm), equilibrated with phosphate buffer at a flow rate of 0.75 mL/min, monitoring the effluent at 280 nm.

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190

Table 2 Comparison of M[46-2]/SFF and m[9-18]/SFF resins with protein A sorbent for affinity purification of ST2146 Sample volume (mL)

5 10 15 20 25 30 40 50 a b c

IgG content in loaded sample (mg)

2.9 5.8 8.7 11.6 14.5 17.4 23.2 29.0

M[46-2]/SFF

m[9-18]/SFF

rpA/SFF

Recoverya

Purityb

HCPc

Recoverya

Purityb

HCPc

Recoverya

Purityb

HCPc

mg

%

%

ng/mg

mg

%

%

ng/mg

mg

%

%

ng/mg

2.9 5.7 8.6 11.2 13.6 16.1 20.4 24.9

100 98 99 96 94 92 88 86

>95 >95 >95 >95 >95 >95 >95 >95

ND 60 ND 53 ND 28 ND 34

2.8 5.7 8.5 11.3 13.7 16.0 20.7 25.2

97 98 98 97 94 92 89 87

>95 >95 >95 >95 >95 >95 >95 >95

ND 62 ND 75 ND 55 ND 38

2.8 5.6 8.1 10.4 12.8 15.0 19.2 23.5

97 96 93 90 88 86 83 81

>95 >95 >95 >95 >95 >95 >95 >95

ND 83 ND 50 ND 22 ND 48

IgG determination was done by ELISA. IgG purity was evaluated by SDS-PAGE. Values refer to intervals of 5%. Host cell proteins were determined by ELISA and expressed as ng HCPs/mg ST2146.

while the rpA/SFF affinity resin shows similar results in terms of recovery yield (90%) at lower concentrations (20 mg of loaded IgG per mL of matrix). In strict agreement with data relating to the IgG yield, the majority of immunoreactivity is recovered in the bound fractions, with only traces in the flow-through material, thus confirming the maintenance of antibody–antigen recognition after the affinity chromatography step. As shown in Table 2, significant Host Cell Protein removal is achieved with all affinity matrices. Starting from a concentration of 3.4 × 105 ng HCPs/mg antibody for the harvested cell culture supernatant, both mimotope affinity resins are able to reduce HCPs, in a single step, to levels comparable to the protein A sorbent (less than 100 ng/mg of IgG). Fig. 7A compares all retained materials deriving from the purification of 30 mL of crude harvest, by SDS-PAGE analysis, in non-reducing conditions. Column-bound fractions essentially display a protein band at the molecular mass of the IgG (≈150 kDa), and only faint bands corresponding to IgG fragments [17], as determined by western-blot analysis (data not shown). Data obtained from electronic scanning and densitometric analysis indicate that all affinity matrices are able to extract IgG from the supernatant concentrated 10 times at a comparable purity of over 95%. Gel filtration profile of fractions deriving from the m[9-18]/SFF resin (Fig. 7B) confirms the SDS-PAGE data. Bound fraction only shows the protein peak at the expected molecular mass corresponding to the IgG, while the flow-through material shows all contaminants and only traces of antibodies. The chromatographic stability of M[46-2] and m[9-18] affinity resins is evaluated by measuring matrix ligand leakage and IgG binding capacity after various treatments performed in column mode. One milliliter of resin was packed into a 6.6 mm I.D. glass column and treated with NaOH 0.5 M solution as sanitizing agent. After 30 min, the resin was washed with 10 volumes of PBS, equilibrated with running buffer and loaded with 30 mL of crude ST2146 harvest, containing 17.4 mg of IgG, according to the protocol previously described. After elution of flow-through material, the buffer was changed to 0.1 M acetic acid, pH 3.0 to elute the retained immunoglobulins. Data referring to ligand leakage and IgG binding capacity, determined by analyzing

acidic bonds and alkaline washing material for peptides and IgG content, underline the great stability of both resins in acetic acid and NaOH solutions. These treatments cause a significant peptidic leakage only during the first process (5–10% with NaOH and 0.1–0.2% with acetic acid), while we detect no material in the subsequent purification steps. However, this initial loss of peptide, presumably due to an inefficient immobilization onto the matrix rather than ligand instability, does not affect column capacity and stability, in that the amount of IgG recovered from both M[46-2] and m[9-18] affinity resins does not reduce after 10 purification steps, while purity is always greater then 95%. 4. Discussion BC2 and BC4 anti-tenascin antibodies described by Siri et al. [39] and Balza et al. [40], have been successfully used for clinical application, in both systemic and topical, pretargeted or direct therapeutic settings, in patients with brain tumors [41–44]. However, since both hybridoma clones were found unsuitable for process development because of the production of an additional, nonfunctional light chain, we generated a new cST2146 hybridoma clone producing the ST2146 monoclonal antibody directed against the EGF-like repeats of tenascin-C. This antibody, compared to other anti-tenascin antibodies, shows higher affinity and immunoreactivity, and similar selectivity by immunohistochemistry [5]. In addition, in biodistribution studies, biotinylated ST2146 showed the highest and most specific tumor localization in colon carcinoma [45]. The regulatory restrictions applied to the development and validation of biomolecules for clinical applications, in addition to cumbersome purification procedures and the high instability of the tenascin antigen [46–48], have led us to pursue a new affinity-based strategy for the purification and analytical control of the ST2146 antibody, in order to reduce time and costs. Using the phage display technology we identified the M[46-2] peptide as having unique specificity for ST2146. To enhance its binding properties, the mimotope was multimerized to produce the M8 [46-2] form, which showed characteristics similar to the natural antigen in ELISA, as demonstrated by ST2146’s compa-

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rable immunoreactivity. Despite the lower affinity for ST2146 with respect to Tn-C, determined by BiaCore measurements, the different kinetic properties of the mimotope allowed better detection of slight variations in antibody immunoreactivity due to a different biotinylation ratio, not easily detectable using the natural antigen. Through simple refinement of the mimotope we also obtained a 10-mer form, called m[9-18], which retained immunoaffinity and chromatographic properties of the starting 22-mer peptide. Corresponding affinity resins were used to purify ST2146 from the hybridoma culture supernatant in a single chromatographic step, with recovery yield and purity degree comparable or better than recombinant protein A, and with full recovery of antibody activity. Both matrices showed similar chromatographic affinity constants; binding capacities, determined by applying a large excess of purified ST2146 to 1 mL bed volume column, were close to 50 mg/mL. Both resins were very stable in all buffers normally used to sanitize, run and elute them, as determined by ligand leakage and IgG recovery, even after several purification cycles. Moreover, the simplified 10-mer peptide showed better properties in terms of enzymatic stability, thus suggesting its suitability as a ligand for ST2146 affinity purification. Cumulatively, these findings demonstrate the feasibility of using phage display technology to identify peptide mimics unique for monoclonal antibodies, and to modify these structures to make them suitable as immunoaffinity ligands in order to purify and control the corresponding antibody. The M[46-2] mimotope, in fact, represents, in both multimeric and simplified structures, a specific, stable and inexpensive surrogate antigen. References [1] R.J. Lories, J.A. Maertens, J.L. Ceuppens, W.E. Peetermans, Acta Clin. Belg. 55 (2000) 163. [2] A. Ohlsson, J.B. Lacy, Cochrane Database. Syst. Rev. 2 (2001) CD000361. [3] D.J. Slamon, B. Leyland-Jones, S. Shak, H. Fuchs, V. Paton, A. Bajamonde, T. Fleming, W. Eiermann, J. Wolter, M. Pegram, J. Baselga, L. Norton, N. Engl. J. Med. 344 (2001) 783. [4] M. Toi, H. Bando, S. Horiguchi, M. Takada, A. Kataoka, T. Ueno, S. Saji, M. Muta, N. Funata, S. Ohno, Br. J. Cancer 90 (2004) 10. [5] R. De Santis, A.M. Anastasi, V. D’Alessio, A. Pelliccia, C. Albertoni, A. Rosi, B. Leoni, R. Lindstedt, F. Petronzelli, M. Dani, A. Verdoliva, A. Ippolito, N. Campanile, V. Manfredi, A. Esposito, G. Cassani, M. Chinol, G. Paganelli, P. Carminati, Br. J. Cancer 88 (2003) 996. [6] M.A. Godfrey, P. Kwasowski, R. Clift, V. Marks, J. Immunol. Methods 160 (1993) 97. [7] J. Balthasar, H.L. Fung, J. Pharm. Sci. 84 (1995) 2. [8] S.F. Chou, C.Y. Chen, Hybridoma 20 (2001) 59. [9] Z. Yan, J. Huang, J. Chromatogr. B Biomed. Sci. Appl. 738 (2000) 149. [10] K. Huse, H.-J. B¨ohme, G.H. Scholz, J. Biochem. Biophys. Methods 51 (2002) 217. [11] C.R. Lowe, A.R. Lowe, G. Gupta, J. Biochem. Biophys. Methods 49 (2001) 561. [12] A.C.A. Roque, C.R. Lowe, M.A. Taipa, Biotechnol. Prog. 20 (2004) 639. [13] J.A. Bogdan, W. Yuan, K.O. Long-Rowe, J. Sarwar, E.A. Brucker, M.S. Blake, Appl. Environ. Microbiol. 69 (2003) 6272. [14] G. Palombo, A. Verdoliva, G. Fassina, J. Chromatogr. B Biomed. Sci. Appl. 715 (1998) 137. [15] A. Verdoliva, G. Basile, G. Fassina, J. Chromatogr. B 749 (2000) 233.

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