Structural and Biophysical Characterization of the 40 kDa PEG

Bioconjugate Chem. 2005, 16, 504−517

504

ARTICLES Structural and Biophysical Characterization of the 40 kDa PEG-Interferon-r2a and Its Individual Positional Isomers Christophe Dhalluin, Alfred Ross, Luc-Alexis Leuthold, Stefan Foser, Bernard Gsell, Francis Mu¨ller, and Hans Senn* F. Hoffmann-La Roche AG Department of Pharma Research CH-4070 Basel, Switzerland. Received September 15, 2004; Revised Manuscript Received March 8, 2005

The human recombinant Interferon-R2a (IFNR2a) is a potent drug (Roferon-A) to treat various types of cancer and viral diseases including Hepatitis B/C infections. To improve the pharmacological properties of the drug, a new pegylated form of IFNR2a was developed (PEGASYS). This 40 kDa PEG-conjugated IFNR2a (40PEG-IFNR2a) is obtained by the covalent binding of one 40 kDa branched PEG-polymer to a lysine side chain of IFNR2a. 40PEG-IFNR2a is a mixture of mainly six monopegylated positional isomers modified at K31, K134, K131, K121, K164, and K70, respectively. Here we report the detailed structural and biophysical characterization of 40PEG-IFNR2a and its positional isomers, in comparison with IFNR2a, using NMR spectroscopy, analytical ultracentrifugation, circular dichroism, fluorescence spectroscopy, and differential scanning calorimetry. Our results show that the three-dimensional structure of IFNR2a is not modified by the presence of the polymer in all positional isomers constituting 40 PEG-IFNR2a. Regardless of where the PEG-polymer is attached, it adopts a very mobile and flexible random coil conformation, producing a shield for the protein without a permanent coverage of the protein surface. Hydrodynamic data indicate that the protein-attached PEG has a slightly more compact random-coil structure than the free PEG-polymer. Our results also provide evidence of significant structural and physicochemical advantages conferred by the pegylation: increase of the effective hydrodynamic volume and modification of the molecular shape, higher temperature stability, and reduced tendency for aggregation. These results are of tremendous pharmacological interest and benefit as was clinically shown with PEGASYS. This study constitutes a new standard for the characterization of pegylated proteins and enables an important step toward the understanding on a molecular level of the binding of 40PEG-IFNR2a and its positional isomers to its cellular receptors.

INTRODUCTION

In the 1970s, pegylation was developed to enhance the bioavailability of therapeutic proteins (1). In this process, an inert, nontoxic, and water-soluble poly(ethylene glycol) (PEG)-polymer,1 linear or branched, is attached to a therapeutic protein via a covalent linkage, mostly at lysine or histidine residues located on the surface of the native protein (2-4). This process has been shown to alter the biochemical and pharmacological properties of * To whom correspondence should be addressed. Tel: +41 (0)61 68 82028; fax: +41 (0)61 68 87408; e-mail: hans.senn@ roche.com. 1 Abbreviations: HCV, Hepatitis C Virus; IFNR, InterferonR; IFNR2a, Interferon-R2a (Roferon-A); 15N-IFNR2a, uniformly 15N-labeled Interferon-R2a; PEG, poly(ethylene glycol); 40PEG-IFNR , (PEGASYS) 40 kDa branched PEG-conjugated 2a Interferon-R2a, composed of monopegylated positional isomers; monopegylated positional isomer, one 40 kDa branched PEG molecule covalently attached at a lysine residue of InterferonR2a; 15N-isomer, uniformly15N-labeled isomer; pegylated lysine, lysine residue of Interferon-R2a linking the PEG-polymer to the protein; PEG-linker lysine, lysine molecule linking two PEG branches of 20 kDa each; NMR, nuclear magnetic resonance; AUC, analytical ultracentrifugation; CD, circular dichroism; DSC, differential scanning calorimetry; SAW, Self-AvoidingWalk; r.c., random-coil.

the resulting PEG-conjugated proteins (5, 6). It leads to a protein with improved solubility and temperature stability (7), enhanced stability against enzymatic degradation (8, 9), increased serum half-life (10), decreased renal clearance (11), and immunogenicity (12, 13) and thus enhanced biological effectiveness (14, 15) when compared with the unmodified protein. Today one of the major health concerns worldwide is the chronic Hepatitis C Virus (HCV) infection. It is estimated that 180 million people are infected with HCV (16). This infection is the leading cause of chronic liver disease and hepatocellular carcinoma, and the main indication for liver transplantation in the United States and Western Europe (17). Until recently the primary therapy for the treatment of chronic HCV infection remained subcutaneous injections of recombinant human Interferon-R (IFNR) in combination with the oral antiviral agent ribavirin (14, 18). It was realized that the pharmacokinetic properties of IFNR proteins are the major factors limiting the efficacy of the treatment: the protein is rapidly degraded, diffuses widely throughout the entire body and has a high rate of renal clearance (19, 20). As a result, after one subcutaneous injection the serum half-life of Interferon-R2a (IFNR2a) is short (ranging from 4 to 8 h) and the serum concentration decreases

10.1021/bc049781+ CCC: $30.25 © 2005 American Chemical Society Published on Web 05/03/2005

Characterization of PEG−Interferon-R2a

Figure 1. Schematic representation of a monopegylated positional isomer molecule of 40PEG-IFNR2a: one 40 kDa branched PEG-polymer is covalently attached to IFNR2a by an amide bond at the -amino group (in red) of a lysine residue of IFNR2a. The IFNR2a protein and the PEG-polymer are schematically separated by a dotted line. In the PEG-polymer, the two 20 kDa PEG branches (n ) 420-510) are linked by a lysine molecule (in blue), also called PEG-linker lysine, via urethane bonds, one at the R-amide group and the other at the -amide group of the PEG-linker lysine. IFNR2a contains 11 lysine residues (K23, K31, K49, K70, K83, K112, K121, K131, K133, K134, and K164). Potentially 11 monopegylated positional isomers plus one at the N-terminus may be formed. However, only six positional isomers constitute more than ∼98% of the mixture (28). In the 15N-labeled positional isomer samples, the nitrogen nuclei of the protein domain including the NH group of the pegylated lysine are 15N, whereas the one of the PEG-linker lysine are 14N.

below its detection limit after 24 h (21, 22). Thus, even though the current HCV treatment requires up to three subcutaneous injections weekly, the virus has extended periods when it is not exposed to a therapeutic concentration of the drug (14, 15). This results in a treatment regimen that places a significant burden on the patient, negatively impacting quality of life and often interfering with compliance. In the early 1990s, the pegylation of IFNR2a for the HCV treatment was first attempted with a 5 kDa linear PEG-polymer linked by a urea bond to lysine side chains of the protein.23 However, the pharmacological properties and efficacy of this first generation of pegylated IFNR2a were not significantly improved over that obtained with unmodified protein (24). Within the past several years, the influence of various PEG structures on the in vitro biological activity have been investigated (2, 25, 26). These results showed an important correlation between the PEG-polymer characteristics and the pharmacological properties. On the basis of this knowledge, second-generation pegylated IFNR drugs were developed by two companies. The biophysical charactersation for a 12 kDa PEG IFNR2b construct (Intron A) was reviewed by Wang et al. (27). Here we focus on the 40PEG-IFNR2a (PEGASYS) construct recently developed (24) in house. It is obtained by covalent binding of one 40 kDa branched PEG-polymer via an amide bond to a lysine side chain of the protein (Figure 1). 40PEG-IFNR2a is a mixture of mainly six monopegylated positional isomers (28). It exhibits a superior efficacy over IFNR2a with significantly increased serum half-life, and because of its increased size a reduced renal clearance, resulting in a strong antiviral response throughout a once weekly dosing schedule (29, 30). In patients with chronic HCV and liver cirrhosis, 40 PEG-IFNR2a monotherapy achieves a sustained virological response close to four times higher than conven-

Bioconjugate Chem., Vol. 16, No. 3, 2005 505

tional IFNR2a (14). In late 2002, PEGASYS was approved by the FDA for HCV treatment. On a structural level, the molecular bulk conferred by the PEG-polymer may effectively shield the protein surface (9). On one hand, these effects enhance serum half-life and reduce renal clearance, which in turn tend to potentiate the biological efficacy of the pegylated protein versus the unmodified form (10). On the other hand, the attached PEG may sterically and thermodynamically interfere with the ability of the protein to interact with its cellular receptors, which exert the biological effectiveness (9, 31-33). The overall biological response to the pegylated protein is consequently dependent upon a balance of these competing factors. Previous studies reporting biological activities showed an unavoidable reduction in specific activities upon pegylation (23, 32, 34). As far as 40PEG-IFNR2a is concerned, it exhibits an approximately 30-fold reduced in vitro biological activity in the antiviral assays relative to IFNR2a (24). However, substantial variations in the in vitro antiviral activity were observed when the individual positional isomers were isolated and compared (28). It is anticipated that these activity variations are due to differences in the kinetics and thermodynamics of the interaction between each positional isomer and Interferon receptors. These observations clearly highlight the need to investigate the structural, kinetic, and thermodynamic properties of 40PEG-IFNR2a and its positional isomers, that influence the interaction with its cellular receptors and therefore its biological activity (35). In view of the tremendous value of pegylated therapeutic proteins in medicine, it is quite surprising that few data are available on their structure, stability, and interaction with their receptors (23, 31-34). In this study we report the detailed structural and biophysical characterization of 40PEGIFNR2a and its positional isomers using NMR spectroscopy, analytical ultracentrifugation (AUC), circular dichroism (CD), fluorescence spectroscopy, and differential scanning calorimetry (DSC). The results are also compared with those obtained with unmodified IFNR2a. These data constitute the basis and form an essential collection of knowledge, on which the interaction of 40PEG-IFNR2a with its cellular receptors can be understood. The results of the study of the interaction between 40PEG-IFNR2a and its individual positional isomers with the extracellular domain of the receptor IFNAR2 are reported in the accompanying publication (36). Further improvements in the development of medically relevant therapeutic proteins critically depend on this type of knowledge and may lead to a third generation of structurally modified pegylated protein drugs. MATERIALS AND METHODS

Materials. The standard material of the recombinant human Interferon-R2a, Roferon-A (IFNR2a) (19.2 kDa, 165 amino acids) (37, 38) and the 40 kDa branched PEG-conjugated-IFNR2a, PEGASYS (40PEG-IFNR2a) (24), and the N-hydroxysuccinimide ester derivative of a 40 kDa branched PEG-polymer reagent (39) used for the pegylation reaction were obtained from the Biopharmaceuticals Production Department of Hoffmann-La Roche Inc.. For the purpose of the NMR analysis, uniformly 15 N-labeled IFNR2a (15N-IFNR2a) was produced in E. coli expression system and purified as previously reported (37, 40). The 15N-IFNR2a was subsequently pegylated using the standard pegylation procedure (24) that is only summarized here: 15N-IFNR2a is reacting with the 40 kDa branched PEG-polymer reagent. The PEG molecule

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is attached on surface exposed -amino group of lysine residues via an amide bond. The pegylation reaction product is subsequently purified to remove the excess of PEG-reagent, reaction byproducts, and pegylated 15 N-IFNR2a oligomers. This procedure results in a mixture of monopegylated 15N-IFNR2a conjugates (one IFNR2a molecule with one 40 kDa PEG molecule). As the PEG-reagent is reacting nonspecifically with any of the IFNR2a lysine side chains, the pegylation reaction results in potentially 11 positional isomers (plus the N-terminus) with different yield. Very recently, a combination of an analytical ion exchange separation procedure, peptide mapping and MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight) mass spectrometric analyses showed that 40PEG-IFNR2a is composed mainly of nine bioactive positional isomers of monopegylated IFNR2a: four major isomers modified at K31, K134, K131, K121; two minor isomers modified at K164 and K70; three very minor isomers modified at K83, K49, and K112 (28). The reaction product of monopegylated positional 15Nisomers, as well as the 40PEG-IFNR2a standard product, was subjected to preparative ion-exchange (IEX) separation methods that enabled the isolation of the four major and two minor unlabeled and 15N-labeled positional isomers. First, a cation exchange FPLC (fast-protein liquid chromatography) method (100 mg of material injected) was applied to separate pure positional isomers modified at K31, K134, and K70, respectively. With this method, an elution peak containing the positional isomers modified at K131, K121, and K164 was also collected. This elution peak was then further purified by a preparative (IEX) separation method (3 mg injected material per run). In final, the four major and two minor positional unlabeled and 15N-labeled isomers modified at K31, K134, K131, K121, K164, and K70, respectively, were separated in milligram amount (varying from 2 to 20 mg) with a purity over 97% as checked by analytical ionexchange HPLC. The molecular weight of IFNR2a protein, based on the amino acid content, is 19241.28 Da. IFNR2a and 40PEG-IFNR2a extinction coefficient is 1.00 mL‚mg-1‚cm-1 and 1.05 mL‚mg-1‚cm-1, respectively. Sample concentrations for 40PEG-IFNR2a are given in mass of protein per unit of volume. In the manuscript, IFNR2a and 40PEG-IFNR2a are referring to standard materials (see above) and the positional isomers to the four major and two minor monopegylated positional isomers modified at K31, K134, K131, K121, K164, and K70, respectively. Analytical Ultracentrifugation. Prior to the AUC measurement, IFNR2a and 40PEG-IFNR2a solutions were dialyzed in 10 mM potassium phosphate 0.02% NaN3 pH 3.7 and subjected to a preparative centrifugation (Beckman UZ Kontron apparatus, 2 h, 300 000 g, 6 °C) to ensure the removal of aggregates. The protein concentration was measured by UV absorbance (280 nm) as being 0.80 mg‚mL-1 and 1.60 mg‚mL-1 for IFNR2a and 40 PEG-IFNR2a, respectively. A solution of 40 kDa branched PEG-polymer 1.00 mg‚mL-1 was equally dialyzed against the buffer solution specified above. The knowledge of the partial specific volume vbar (mL‚gr-1) of IFNR2a and 40PEG-IFNR2a was required for the evaluation of the AUC data. In principle vbar for a protein can be calculated from its amino acid sequence (41), but for 40PEG-IFNR2a the presence of the PEGpolymer does not allow this calculation. vbar of IFNR2a, 40 PEG-IFNR2a, and the 40 kDa branched PEG-polymer was experimentally determined by measurement of the density of the solute and the buffer solution. Measure-

Dhalluin et al.

ments were performed using a five-digit precision densitymeter DMA-4500 Anton Paar at 20 °C ((0.01 °C). The eq 1 allowed the determination of vbar with c the total material concentration (g‚mL-1), F and F0 the density (g‚mL-1) of the solute and dialysis buffer solution, respectively.

vbar )

(

)

F - F0 1 1F0 c

(1)

Sedimentation equilibrium experiments were performed with a XL-A AUC apparatus (Beckman Instruments, Palo Alto, CA) at 20 °C equipped with UV absorption optics on IFNR2a and 40PEG-IFNR2a to determine the molecular weight MW and second virial coefficient B. The data were analyzed using the DISCREEQ (42) and Beckman software packages. The molecular weight of a protein can be determined in a sedimentation equilibrium experiment independently of the protein shape (43, 44). At the equilibrium between sedimentation and diffusion a radial gradient of concentration in the measurement cell is formed. By radial scanning (r ) 5.8-7.2 cm) of the sample UV absorbance at 280 nm, the molecular weight MW (Da) of the solute in an ideal and noninteracting system can be extracted from the eq 2,

MW )

∂(lnc) 2RT 2 (1 - F0vbar)ω ∂r2

(2)

with c the concentration, R (8.31 J‚K-1‚mol-1) the gas constant, T (K) the absolute temperature, vbar (mL‚g-1) the partial specific volume of the solute, F0 (g‚mL-1) the solvent density, and ω (rpm) the rotor angular velocity (22 000 rpm and 12 000 rpm for IFNR2a and 40 PEG-IFNR2a, respectively). The second virial coefficient B (L‚mol‚g-2) of IFNR2a and 40PEG-IFNR2a was evaluated as well. In solution the solute molecules can interact with one another (also called nonideality) (43, 44), because of the finite size and the charges they carry. With a real solution of a single solute, the measured molecular weight obtained from the slope of (ln c) versus r2 is an apparent molecular weight MWapp.. The dependency on the solute concentration is given in eq 3,

MWapp. )

MW ∂(ln γ) 1+c ∂c

(

)

(3)

with MW the true molecular weight and (ln γ) the logarithm of the activity expressed as polynomial of c with (ln γ ) B × MW × c + C × MW × c2 +...). For the low concentration range, higher orders can be neglected therefore the eq 3 becomes:

MWapp. ≈

MW 1 + B × MW × c

(4)

The apparent molecular weight MWapp. was measured at different solute concentration c (0.1, 0.2, 0.4, 1.0, 1.5, and 1.9 mg‚mL-1) of IFNR2a and 40PEG-IFNR2a. By the measurement of the second virial coefficient B of the solute, relevant information about the strength of intermolecular interaction is revealed. The sign of B discriminates repulsive from attractive interaction and describes the strength of the solute-solute interactions (45). For a solute with B positive, solute-solvent interactions are



preferred over solute-solute contacts. In contrast for a negative B the opposite holds true. The sedimentation coefficient S of IFNR2a and 40 PEG-IFNR2a was measured at 20 °C by AUC sedimentation velocity experiment using the apparatus as described above. S (in Svedberg unit [10-13 s]) represents the speed at which solute molecules are sedimenting to the bottom of the centrifuge cell under the influence of a strong force field during the experiment. The net sedimentation behavior of macromolecules in the centrifugal force is described by the Svedberg equation:

S)

( )

MW × (1 - F0vbar) ∂r 1 ) ) 2 ∂t rω Navf MW × DT × (1 - F0vbar) (5) RT

The rate of sedimentation dr/dt is dependent on the centrifugal force rω2. S is expressed with the partial specific volume of the molecule vbar, the molecular weight MW, the Avogadro number Nav, the shape of the macromolecule by its frictional coefficient f, the density of the buffer F0, the gas constant R, the absolute temperature T, and the translational diffusion coefficient DT. The Stokes-Einstein relationship (46) (eq 6) was used to derive the right-hand term in the Svedberg equation.

DT ) RT/Navf

(6)

Upon high angular velocity of the rotor (40 000 rpm), the sedimentation leads to formation of a solute concentration gradient in the form of boundaries recorded in UV absorbance at 280 nm by successive radial scanning in time (r ) 5.8-7.2 cm). For an ideal and noninteracting system, the sedimentation coefficient S can be extracted, using the second moment boundary spreading method with the Beckman software package, by the analysis of the rate of movement of the midpoint of the boundaries (43, 44). S of the 40 kDa branched PEG-polymer was measured at 20 °C by sedimentation velocity experiment using the apparatus as described above equipped with an interference optical device under an angular rotor velocity of 60 000 rpm. The sedimentation coefficient S provides a description of the hydrodynamic shape of the molecule by the frictional coefficient f. Using the Stokes-Einstein relationship (eq 6), the frictional coefficient for a sphere, f0, can be calculated with:

(

f0 ) 6πηR0 ) 6πη

)

3 × MW × vbar 4πNav

1/3

(7)

with η the viscosity of the solution and R0 the radius of the sphere defined with:

4π 3 MW × vbar R ) 3 0 Nav

(8)

Thus, the Svedberg relationship (eq 5) can be used to calculate the sedimentation coefficient of a sphere, S0, as function of MW, vbar, F0, and η to obtain:

S0 )

MW × (1 - F0vbar) 3 × MW × vbar 6πηNav 4πNav

(

)

1/3

(9)

Substituting the values of all the constants (η for water at 20 °C) using eq 9 yields the sedimentation value for a sphere in terms of MW, vbar, F0, and η.

S0 ) 0.012 × (1 - F0vbar) ×

( ) MW2 vbar

1/3

(10)

S0 is the maximum sedimentation coefficient that can be obtained for a molecule of a given mass, because a sphere has the minimum surface area in contact with solvent. Consequently a spherical molecule has a minimum frictional coefficient f0. The ratio of the sedimentation coefficient predicted for a sphere, S0, to the experimental S is f/f0 which can be interpreted for the molecule like its maximum shape asymmetry from a sphere. Differential Scanning Calorimetry. The DSC method was used to monitor the thermal unfolding transition of IFNR2a and 40PEG-IFNR2a. Prior to the measurement, the protein solutions were dialyzed in 20 mM sodium acetate/50 mM NaCl/0.02% NaN3, pH 5.0, and subjected to a preparative centrifugation (as described above) to ensure the removal of aggregates. The protein concentration was measured by UV absorbance (280 nm) as being 0.82 mg‚mL-1 and 0.60 mg‚mL-1 for IFNR2a and 40PEG-IFNR2a, respectively. DSC experiments were conducted with a MicroCal (Northampton, MA) microcalorimeter by measuring the difference of energy uptake between the sample cell and a reference cell that contains only dialysis buffer upon an increase of the temperature by 1 °C per minute from 20 °C to 85 °C. The denaturation melting temperature Tm was determined at the maximum of the heat capacity. Circular Dichroism. The CD measurements were performed for IFNR2a and 40PEG-IFNR2a using a JobinYvon CD-6 spectrometer equipped with a temperaturecontrolled water bath. Prior to the measurement, the protein solutions were dialyzed in 10 mM potassium phosphate/0.02% NaN3, pH 3.7, and subjected to a preparative centrifugation (as described above) to ensure the removal of aggregates. The protein concentration was measured by UV absorbance (280 nm) as being 0.80 mg‚mL-1 and 1.60 mg‚mL-1 for IFNR2a and 40PEGIFNR2a, respectively. The protein backbone absorption band (195-260 nm) and the protein aromatic absorption band (250-350 nm) were recorded at 20 °C in 0.05 and 1.00 cm path length cylindrical cell, respectively. The Cotton effect was plotted as ∆ that is related to the mean molecular ellipticity Θ [deg‚cm2‚dmol-1] by the relationship Θ ) 3298.8 × ∆. Buffer baseline recorded prior the CD measurement of the protein solution was subtracted from the protein data to minimize the effects of UV lamp drifts. The secondary structure content of the proteins was estimated from the absorption band (195-240 nm) using the Provencher method (47). It involves the simulation of the CD spectrum of the protein of interest as a linear combination of the CD spectra of 16 proteins whose secondary structures are known from X-ray crystallography. Fluorescence Spectroscopy. The fluorescence measurements were performed for IFNR2a and 40PEG-IFNR2a at room temperature with a SLM 8100 fluorometer. Prior to the measurement, the protein solutions were dialyzed in 10 mM potassium phosphate 0.02% NaN3 pH 3.7 and subjected to a preparative centrifugation (as described above) to ensure the removal of aggregates. The protein concentration was measured by UV absorbance (280 nm) as being 0.80 mg‚mL-1 and 1.60 mg‚mL-1 for IFNR2a and 40 PEG-IFNR2a, respectively. The fluorescence emission


spectra were recorded between 300 and 450 nm, with excitation at 283 nm. NMR Spectroscopy. NMR measurements were performed at 25 °C on a Bruker DRX-800 MHz spectrometer, equipped with xyz-axis pulse field gradient triple resonance probe-head, and on a DMX-600 MHz spectrometer, equipped with z-axis pulse field gradient triple resonance cryo-probe-head. Data were processed and analyzed with XWIN NMR 2.6 and NMRPipe (48). NMR samples of the following materials were prepared: 15N-labeled and unlabeled IFNR2a; 40PEG-IFNR2a; the 15N-labeled and unlabeled positional isomers modified at K31, K134, K131, K121, K164, and K70, respectively. The NMR samples were prepared using the following procedure: preparative centrifugation (as described above); dialysis in 50 mM acetic acid, pH 3.5, 0.02% NaN3, at 6 °C; concentration to 1 mL volume; dialysis in H2O, pH 3.5 (HCl), 0.02% NaN3, at 6 °C; dialysis in an aqueous solution containing protease inhibitors (H2O/D2O, 9/1) pH 3.5 (HCl), 0.02% NaN3, at 6 °C; preparative centrifugation for 1 h at 185 000 g at 6 °C; readjustment of the pH to 3.5 (HCl). The final concentration of the samples ranged from 0.35 mM to 0.60 mM in 5 mm NMR sample tubes. A NMR sample of the 40 kDa branched PEG-polymer was prepared. An amount of 8.5 mg of N-hydroxysuccinimide ester derivative of the 40 kDa branched PEG-polymer reagent used for the pegylation reaction was diluted in 550 µL of H2O, pH 12 (NaOH), 0.02% NaN3, and stirred overnight at 6 °C to hydrolyze the N-hydroxysuccinimide ester derivative. The solution was subsequently dialyzed in (H2O/D2O, 9/1) pH 3.5 (HCl), 0.02% NaN3, overnight at 6 °C. The resulting dialyzed solution of PEG-polymer free of N-hydroxysuccinimide derivative was then transferred with a concentration of ∼0.38 mM to a 5 mm NMR tubes. In all the NMR experiments the water signal suppression was performed using the WATERGATE scheme (49). For all the samples, a 1D 1H NMR spectrum was acquired at 600 MHz with 8990 Hz sweep-width (1024 scans) and 2048 acquisition points zero-filled to 4096 points and subjected to a squared sine-bell window function shifted by 60°. For the samples of PEG-polymer and the positional 15 N-isomers, 2D 1H-DQ and 2D 15N-filtered 1H-TOCSY were acquired at 600 MHz to derive the 1H assignment of the linker lysine in the PEG-polymer (Figure 1) (50, 51). The linker lysine remains unlabeled (14N) at the NR and N positions, as only IFNR2a was labeled with 15N. 2D 1H-DQ was recorded with 1536 t2 acquisition points (96 scans) and 512 t1 increments and spectral width of 14 and 24 ppm in F2 and F1 dimensions, respectively. 15 N-Decoupling was performed by a 180° pulse (carrier frequency at 118 ppm for 15N) and the GARP scheme (52) during t1 and t2 evolution period, respectively. 2D 15Nfiltered 1H-TOCSY with DIPSI-2 (53) for spin-lock (52 ms for mixing time) was recorded with 1536 t2 acquisition points (64 scans) and 512 t1 increments with a spectral width of 12 ppm in both dimensions. A postacquisition 15 N-filtration was performed by the implementation of a 15 N-filter during the experiment (carrier frequency at 118 ppm for 15N) combined with an interleaved acquisition method (54). Because of the 15N-filter, only 14N- and carbon-attached proton resonances are visible along the F2-dimension in the aromatic region of the spectrum. For the 2D 1H-DQ, 15N-filtered 1H-TOCSY and 2D [1H-15N]TROSY-HSQC (see below), the t2 and t1 time domains were zero-filled to the next power of two and subjected to a squared sine-bell window function shifted by 60°. For the samples of 15N-IFNR2a and the positional 15N-

Dhalluin et al.

isomers, 2D [1H-15N]-TROSY-HSQC was acquired at 800 MHz to derive the 1H and 15N chemical shift of the amides in the positional isomers (55). The experiments were recorded with 1536 t2 acquisition points (96 scans) and 512 t1 increments spectral width of 15 and 24 ppm in 1H and 15N (carrier frequency at 118 ppm), respectively. The 1 H and 15N chemical shift of the -amide group NH of the pegylated lysine in the protein domain of the positional isomers was derived from these experiments. The chemical shift perturbation ∆ppm of the backbone amides due to the PEG-polymer in each positional isomers was evaluated as follows (eq 11) where ∆(1H) and ∆(15N) are the backbone amide chemical shift differences in 1H and 15 N, respectively, between 15N-IFNR2a and the positional 15 N-isomers.

∆ppm ) 1/2[(∆(1H))2 + (∆(15N))2/25]1/2

(11)

To assess the shielding effect of the PEG-polymer on H/2H exchange of the protein, NMR saturation transfer experiments (56) were acquired by recording 2D [1H-15N]-TROSY-HSQC spectra with and without presaturation (50 Hz) of the water signal during 3 s. For 15N-IFNR2a and the positional 15N-isomers, 3D 15Nedited [1H-1H]-NOESY (57) was acquired to derive the NOE internuclear distance connectivities within the protein and between the former and the PEG-polymer. 3D 15N-edited [1H-1H]-NOESY were recorded at 800 MHz using 100 ms mixing time, 2048 acquisition points (8 scans), 90 increments for 1H and 40 increments for 15 N (carrier frequency at 118 ppm) with spectral widths of 15, 12, and 24 ppm, respectively. The indirect time dimension in 1H and 15N were doubled by linear prediction and zero-filled to the next power of two and subjected to a squared sine-bell window function shifted by 60° and 90°, respectively. For IFNR2a, 40PEG-IFNR2a and the 40 kDa branched PEG-polymer samples pulse field gradient-(longitudinal eddy current) PFG-(LED) (58) NMR experiment was recorded to derive the translational diffusion coefficient DT. Self-diffusion is defined as translational motion reflecting the random motions (Brownian movement) of a molecule in the absence of a concentration gradient. DT is indicative of hydrodynamic properties and molecular size. The PFG-(LED) NMR method is suitable for DT measurements of macromolecules because it is designed to minimize the T2 relaxation effects by storing the magnetization along the z-axis during the translational diffusion period, so that relaxation depends primarily on T1 which is much longer than T2 for macromolecules. The experiment was acquired with 1D 1Hspectra recorded at 600 MHz with 12000 Hz sweep-width (64 scans) and 32 k acquisition points zero-filled to 64 k and subjected to an exponential window function (linebroadening factor of 1 Hz) before FT. In the experiment the magnitude of z-gradient pulses is increased in successive 1D 1H-spectra where the signal intensity is attenuated due to the translational diffusion. The decrease in signal intensity I, normalized to the signal in the absence of gradient Io, is given by (59): 1

I ) exp(-γ2G2δ2DT(∆ - δ/3)) Io

(12)

with γ the 1H gyromagnetic ratio (2.68 104 Gauss-1‚s-1), ∆ the diffusion period (300 ms), δ the gradient duration (2 ms), and G the gradient magnitude varying from 0% to 100% (calibrated to 57.6 G‚cm-1). The determination of DT (cm2‚s-1) allows the calculation of a hydrodynamic



Figure 2. Aromatic (left) and aliphatic (right) regions of the 1D 1H NMR spectra recorded at 600 MHz for IFNR2a, 40PEGIFNR2a and the positional isomers modified at K31, K134, K131, K121, K164, and K70, respectively. In this representation, amplitudes in the aliphatic region are rescaled, with respect to the aromatic region, by a factor ∼4.

radius RH (nm) of a sphere with equivalent translational diffusion constant using the Stokes-Einstein equation:

RH )

kBT 6πηDT

(13)

with kB the Boltzmann constant, T (K) the absolute temperature, and η the viscosity taken to be that of water at 25 °C (46, 60). RESULTS

NMR Structural Analysis of the Protein Domain of Pegylated IFNR2as. The 1D 1H NMR spectra of IFNR2a and 40PEG-IFNR2a are extremely well superimposable in the resolved regions (Figure 2). The 1H linewidth for 40PEG-IFNR2a is broader (+5 Hz) as compared to IFNR2a, but much less broad than expected for a 60 kDa protein (61). The spectral identity in the 2D 1HNOESY spectra of the two samples is also remarkable. However, because 40PEG-IFNR2a is a mixture of positional isomers, NMR data do not resolve structural differences that may exist. Detailed NMR based structural comparison was achieved using NMR data of isolated positional isomers. The individual 1D 1H NMR spectra of the six isolated positional isomers of 40PEG-IFNR2a display a similar dispersion of signals in the aromatic and aliphatic regions with broader 1H-line-width (+5 Hz) as compared to IFNR2a (Figure 2). The perturbation of the PEG-polymer

on the IFNR2a protein structure (40) was evaluated by the mapping of the chemical shift perturbation ∆ppm (eq 11) of the backbone amide 1H- and 15N-resonances for each amino acid derived from 2D 15N-TROSY-HSQC spectra for the positional 15N-isomers as compared to 15NIFNR2a. This is illustrated in Figure 3 for the 15N-IsomerK31. The chemical shift perturbation plotted for all the amino acid sequence shows that the perturbation is small (maximum 0.12 ppm in the Isomer-K31) and localized to the attachment site of the PEG-polymer at K31 and surface regions close by in space (Figure 4). The same holds true for most of the positional isomers, as shown in Figure 4. Only the Isomer-K164 displays a chemical shift perturbation spread more widely over the protein surface. K164 is located at the C-terminal end of the polypeptide chain which is known to be highly flexible (40). For each positional 15N-isomer, a 3D 15N-edited [1H1 H]-NOESY spectrum was recorded. The analysis of the 3D spectra showed that all amino acid residues experiencing chemical shift perturbation display unaltered NOE internuclear 15NH-based distance connectivities when compared with IFNR2a. The same also holds true, as expected, for all the nonshifted 15NH resonances. This is illustrated in Figure 5 for the 15N-Isomer-K31 for which the residue H34 displays chemical shift perturbation because it is close to the pegylated lysine K31. The 1Hresonance of this amide displays the same NOE internuclear distance connectivities as in 15N-IFNR2a: the long-range NOE connectivity cross-peak between the resonance of the amide proton of H34 and the degenerated resonances of the two methyl groups of V142 is shown. For no positional isomer could a NOE internuclear distance connectivity be detected between the ethylene glycol units (OCH2CH2) or the two terminal groups (OCH3) of the PEG-polymer and the amides of the protein backbone. NMR Structural Analysis of the Pegylated Protein-lysine and the 40 kDa Branched PEG-polymer. For all the positional 15N-isomers, the 1H and 15N chemical shift of the -amide group of the pegylated lysine of the protein domain (Figure 1) was unambiguously assigned by the identification of additional cross-peaks (15N/15NH) in the 2D 15N-TROSY-HSQC spectra when compared to the IFNR2a reference spectrum (Figure 3 and

Figure 3. (A) Section of the superimposed 2D 15N-TROSY-HSQC spectra recorded at 800 MHz for 15N-IFNR2a (black) and the positional 15N-Isomer-K31 (red). As an example the amide of L30 close to the pegylated lysine K31 shows significant 1H and 15N chemical shift changes (black dotted arrow) whereas residues far away from the pegylated site do not display chemical shift perturbation. Two cross-peaks (15N/15NH) are observed for the -amide group of the pegylated lysine K31. Based on the relative intensity ratio (Table 1), these cross-peaks are assigned as the conformational State-A and State-B of the -amide group of the pegylated lysine. At the contour level of the spectrum for the positional 15N-Isomer-K31 the existing cross-peak for I53 is not visible. (B) Chemical shift perturbation ∆ppm (eq 11) of the backbone amides due to the PEG-polymer for the Isomer-K31 as a function of the amino acid sequence.


Dhalluin et al.

Figure 5. Region of a selected [1H-1H]-plane from the 3D 15N-edited [1H-1H]-NOESY spectra recorded at 800 MHz for 15N-Isomer-K31 (A), and 15N-IFNR (B). The planes correspond 2a to the 15N-shift (120.3 ppm) of the residue H34. This residue that is close to the pegylated lysine in the Isomer-K31 showed backbone amide chemical shift perturbation in 1H but no change in 15N. The 1H-resonance of the amide of H34 displays the same NOE internuclear distance connectivities in both molecules: a long-range NOE connectivity between the amide of H34 and the degenerated resonances of the two methyl groups of V142 is circled as an example.

Figure 4. Ribbon representation of IFNR2a structure (40) in the positional isomers modified at K31, K134, K131, K121, K164, K70, respectively. Amino acid residues that experience backbone amide chemical shift perturbation ∆ppm (eq 11) due to the PEG-polymer, are color-coded from dark blue for the most pronounced changes (0.12 ppm g ∆ppm g 0.08 ppm) gradually to light green for the small changes (0.04 ppm g ∆ppm g 0.01 ppm). The side chain of the pegylated lysine is colored in red. For each positional isomer, one possible r.c. conformation of the 40 kDa branched PEG-polymer (in yellow) was calculated but only partly represented for clarity. The polymer conformation was calculated as an on-lattice SAW (see also Figure 8).

Table 1). The 1H assignment to this position was further confirmed by the observation of direct and remote crosspeaks for 15NH in the 2D 1H-DQ spectra (50) (data not shown): these cross-peaks are again missing in the 2D 1H-DQ reference spectrum of 15N-IFNR2a. As shown in Figure 3, two (15N/15NH) cross-peaks are observed for the -amide group of the pegylated lysine K31 with a ∼6.5:3.5 relative intensity ratio. The same holds true for the 15N-Isomer-K134 and -K121 with a ∼5.5:4.5 relative intensity ratio in both isomers (Table 1). No correlation cross-peak is observed between the two 15NH signals in the 3D 15N-edited [1H-1H]-NOESY spectra. The structural origin of this peak duplication indicates a highly localized conformational heterogeneity of the pegylated lysine of the protein domain. For the 15N-Isomer-K131,

-K164, -K70 only one (15N/15NH) cross-peak is observed for the -amide group of the pegylated lysine (Table 1). The peak duplication is here either not resolved or not occurring (see Discussion). For all the positional 15N-isomers and the free PEGpolymer samples, the 1D 1H NMR spectra display for the two 20 kDa PEG branches of the polymer (Figure 1) a unique, very intense, and sharp resonance line (∼5 Hz) at 3.60 ppm for the ethylene-glycol units (OCH2CH2) and a sharp but much less intense resonance line at 3.27 ppm for the two terminal groups (OCH3). The 1H chemical shifts of the PEG-linker lysine for all the samples were obtained from analysis of the 2D 1H-DQ and 15N-filtered 1H-TOCSY spectra. In the aromatic region of the 15N-filtered 1H-TOCSY spectra along the F2-dimension, only the 14N-attached proton resonances, namely NRH and NH of the amide groups of the PEG-linker lysine, are seen. For all the samples, two 1H-resonances are observed for NRH and NH of the PEG-linker lysine (Table 1 and Figure 6). They derive from a cis/trans isomerization of the urethane bond (62-64) at the R- and -amide groups of the PEG-linker lysine (Figure 6). This isomerization was confirmed by the observation of conformational exchange cross-peaks between both conformations of the amide groups in 2D-1H-NOESY (65) and 2D-1H-ROESY (66) spectra (data not shown). The isomerization rate was estimated on the PEG-polymer sample from the signal separation (∼240 Hz) as lying in the millisecond time range (∼5 ms < 1/kex < ∼50 ms). The trans and cis conformations of each of the urethane bond at the R- and -positions were assigned to be populated in the ratio ∼9:1, based on the relative intensity ratio of (NRH/CRH) and (NH/CH2) cross-peaks, respectively (Table 1). This ratio is substantially different from the ratio derived from the localized conformational



Table 1. 1H Chemical Shift (ppm) of NrH, CrH, NEH, and CEH2 from the r- and E-Amide Groups of the Linker Lysine in the 40 kDa Branched PEG-polymer and the Positional Isomersa pegylated lysine in the protein domain

linker lysine in the PEG-polymer domain

15N /15N H

compound

State-A

40 kDa branched PEG polymer Isomer-K31

121.4/8.14

Isomer-K134

121.9/8.08 ∼55%b

Isomer-K131 Isomer-K121

121.1/8.07e 121.2/8.10 ∼55%c

Isomer-K164 Isomer-K70

120.8/8.05e 121.2/8.10e

∼65%b

N RH State-B

121.1/8.03 ∼35%b 121.1/8.08 ∼45%b 121.2/8.08 ∼45%c

C RH

N H

CH2

transd

cisd

transd

cisd

transd

cisd

transd

cisd

7.07

6.64

3.88

3.92

6.76

6.40

2.99,2.97

3.01,3.01

7.15 7.22 7.15 7.18 7.13e 7.13 7.16 7.14e 7.16e

6.77 6.77 6.76 6.76 6.74e 6.77 6.72 6.74e 6.75e

3.89 3.91 3.89 3.89 3.84e 3.89 3.89 3.87e 3.85e

3.91 3.91 3.89 3.89 3.87e 3.89 3.89 3.87e 3.87e

6.75 6.75 6.76 6.76 6.76 6.74 6.74 6.74 6.73

6.37 6.37 6.39 6.39 6.40 6.37 6.37 6.38 6.37

3.02,2.98 3.02,2.98 3.00,2.97 3.00,2.97 2.99,2.99 2.97,2.96 2.97,2.96 2.99,2.96 3.00,2.96

3.01,3.01 3.01,3.01 3.00,3.00 3.00,3.00 2.99,2.9 2.98,2.9 2.98,2.9 2.98,2.9 2.98,2.9

a Measured from 2D 15N-filtered 1H-TOCSY and 2D 1H-DQ spectra. 15N and 1H chemical shift (ppm) of 15N and 15N H, respectively, from the -amide group of the pegylated lysine in the protein domain of the positional isomers identified from 2D 15N-TROSY-HSQC and 1 b 15 15 15 2D H-DQ spectra. Populations based on the relative intensity ratio of the two cross-peaks for ( N/ NH) in the 2D N-TROSY-HSQC spectra: the cross-peaks with the highest and lowest intensity were assigned as deriving from the two conformational states called State-A and State-B, respectively, of the -amide group of the pegylated lysine. c Populations based on the relative intensity ratio of the two cross-peaks for (NRH/CRH)trans observed at the R-amide group of the PEG linker lysine in the 2D 15N-filtered 1H-TOCSY spectrum. d Trans and cis populations are in the ratio (∼9:1), respectively, according to the relative intensity ratio of the cross-peaks (NRH/CRH) and (NH/ CH2) of the PEG linker lysine observed in the 2D 15N-filtered 1H-TOCSY spectra. e Only one set of cross-peaks is observed. The signals are not split in the 2D 15N-TROSY-HSQC and 2D 15N-filtered 1H-TOCSY spectra.

heterogeneity of the pegylated lysine, where the conformational exchange is also slow. An additional duplication of 1H-resonances of the linker lysine of the polymer for NRH is also observed for the Isomer-K31, -K134, and -K121. This is illustrated in Figure 6 with the 15N-filtered 1H-TOCSY spectrum of 15 N-Isomer-K121: doubling of both NRHtrans and NRHcis is observed with a relative intensity ratio ∼5.5:4.5 in both cases. This ratio is identical to the one observed at the neighboring group 15NH of the pegylated K121 of the protein. For the Isomer-K31 and -K134 this doubling is observed only for the 1H-resonance of NRHtrans with a relative intensity ratio ∼6.5:3.5 and ∼5.5:4.5, respectively. For the Isomer-K131, -K164, and -K70, the 1Hresonance of NRHtrans and NRHcis, respectively does not show any doubling. Biophysical Characterization of IFNR2a, 40PEGIFNR2a and the 40 kDa Branched PEG-polymer. The data reflecting the biophysical characterization of IFNR2a, 40 PEG-IFNR2a and the free 40 kDa branched PEGpolymer are collected in Table 2. They are derived from the determination of the following parameters: the partial specific volume vbar by densitometry; the molecular weight MW, the second virial coefficient B, and the sedimentation coefficient S by AUC; the denaturation melting temperature Tm by DSC; the secondary structure content by CD; the translational diffusion coefficient DT by PFG-(LED) 1H NMR spectroscopy. The partial specific volume vbar of IFNR2a and the 40 kDa branched PEG-polymer (Table 2) are in good agreement with the value calculated from the amino acid content (0.739 mL‚gr-1) (41) and values reported for other PEG-polymers (67), respectively. 40PEG-IFNR2a presents a vbar that corresponds approximately to the mass weighted average of vbar of IFNR2a and the PEG-polymer. The knowledge of vbar for IFNR2a and 40PEG-IFNR2a allowed the determination of the molecular weights, by AUC to be 19.2 kDa and 58.9 kDa, respectively. The second virial coefficients B for IFNR2a and 40PEGIFNR2a are of opposite sign with a negative and positive value, respectively (Table 2). It is interesting to note that literature values of B for PEG-polymers are positive as well (68, 69), indicative that the B value of 40PEG-IFNR2a

is dominated by the properties of the PEG-polymer rather than IFNR2a. The sedimentation coefficient S of IFNR2a and the 40 kDa branched PEG-polymer are in the range of S values for globular proteins (43, 44) and other PEG-polymers (70, 71) of similar size. 40PEG-IFNR2a presents a S value between, but closer to, the one of the PEG-polymer than of IFNR2a (Table 2). For the positional isomers modified at K31, K134, K131, and K121, the sedimentation coefficient S was also measured and gave values comparable to 40PEG-IFNR2a (data not shown). The thermal unfolding transition from the native to the denaturated state monitored by DSC (Figure 7.A) is, for IFNR2a and 40PEG-IFNR2a, an irreversible process. For 40PEG-IFNR2a, the baseline of the DSC scan displays a normal profile. In contrast, the strongly dropping baseline observed for IFNR2a highlights an aggregation process that starts in the early phase of the unfolding transition and dominates beyond the denaturation melting temperature Tm. As compared to IFNR2a, 40PEGIFNR2a is more stable throughout the thermal denaturation with a slightly elevated Tm and a substantially reduced tendency for aggregation. This is indicated by the difference of the shape of the denaturation curve. The extraction of the enthalpy of unfolding is not possible due to the irreversibility of the denaturation process. The CD spectra for IFNR2a and 40PEG-IFNR2a recorded in the backbone and aromatic absorption region are identical over a broad range of pH values (pH 3.5 to 8). One example for sample conditions close to those of the NMR samples is shown in Figure 7, parts C and D, respectively. The same holds true for the fluorescence spectra (Figure 7B). Analysis of the CD spectra in the backbone absorption region shows that IFNR2a and 40 PEG-IFNR2a present ∼75% of R-helix secondary structure (40, 47). The unfolding and refolding process was monitored by the acquisition of CD spectra in the backbone absorption region during a thermal denaturation-renaturation cycle from 20 °C to 80 °C and back. These CD spectra showed ∼41% and ∼61% of R-helix secondary structure for IFNR2a and 40PEG-IFNR2a respectively, after completing the cycle. It must be noted


Dhalluin et al.

reveals important differences of hydrodynamic properties, i.e., molecular size and shape, between IFN-R2a, 40 PEG-IFNR2a and the 40 kDa branched PEG-polymer (Table 2). Surprisingly, RH of the 60 kDa pegylated protein is smaller than the one observed for the free 40 kDa PEG-polymer. DISCUSSION

Figure 6. Section of the 2D 15N-filtered 1H-TOCSY spectrum for the positional15N-Isomer-K121 recorded at 600 MHz. In this region of the spectrum along the F2-dimension only the 14Nattached NRH and NH proton resonances of the amide groups of the unlabeled PEG-linker lysine are seen. Because of the cis/trans isomerization of the urethane bond (in bold) at the R- and -amide groups of the PEG-linker lysine, two resonances for NRH and NH are observed: namely NRHtrans, NRHcis and NHtrans, NHcis, respectively. The cis/trans assignment is based on the relative intensity ratio of the cross-peaks (NRH/CRH) and (NH/CH2). Because of the concomitant existence of two conformational states for the neighboring -amide group of the pegylated lysine, an additional doubling is observed for NRHtrans resonance of the PEG-linker lysine as shown with the 1D trace at δ(CRH) ) 3.89 ppm: the two peaks have a relative intensity ratio ∼5.5:4.5. The same observation holds true for the resonance NRHcis. In contrast the 1D trace at δ(CH2) ) 2.96 ppm does not show any doubling for the resonance NHtrans and NHcis. This group is structurally more remote than the -amide group of the pegylated lysine. The chemical shift region for the 1H-resonances of C H , C H and C H is reported with a β 2 γ 2 δ 2 bracket. Below the spectrum the cis/trans isomerization of the urethane bond at the R- and -amide groups of the PEG-linker lysine is schematically shown.

however that R-helical recovery does not necessarily imply a return to correctly folded protein. The NMR-measured translational diffusion coefficients DT of IFNR2a and the 40 kDa branched PEG-polymer (Table 2) are in agreement with values reported for globular proteins and other PEG-polymers (72-74). In contrast, DT of 40PEG-IFNR2a corresponds, neither to a 60 kDa globular protein nor to a 60 kDa PEG-polymer. The DT of 40PEG-IFNR2a lies much closer to that of the 40 kDa branched PEG-polymer. The hydrodynamic parameters vbar, S and DT experimentally determined by independent methods for IFN-R2a, 40PEG-IFNR2a and the 40 kDa branched PEG-polymer (Table 2) are consistent with the corresponding molecular weight calculated using the Svedberg and Stokes-Einstein relationships (eqs 5 and 6 in Materials and Methods, respectively). The calculation of the hydrodynamic radius RH of the sphere with equivalent translational diffusion coefficient and the maximum shape asymmetry from a sphere f/f0

Overall Structure of the Protein Domain in Pegylated IFNR2a. The 1D 1H NMR spectra of the six isolated positional isomers and IFNR2a are fully superimposeable and almost identical in the resolved regions. The integrity of the IFNR2a fold in the positional isomers was also confirmed by fluorescence and CD spectra (data not shown). For each positional isomer, the chemical shift perturbation ∆ppm (eq 11) induced by the PEG-polymer on the IFNR2a protein structure is small and localized to the PEG-polymer attachment site and surface regions close by in space. Only the Isomer-K164 displays ∆ppm changes which are more spread over the protein surface (Figure 4). This may be caused by the higher flexibility of the PEG-attachment site at K164 that is located in the most mobile part of the protein at the C-terminus (40). The observed chemical shift perturbations on the protein structure probably do not reflect structural changes or differences to IFNR2a for any of the positional isomers. No structural differences in IFNR2a could be detected among the positional isomers, as the same NOE internuclear distance connectivities in the 3D 15N-edited [1H-1H]-NOESY spectra of the positional isomers are observed as in IFNR2a. This also holds true for the residues displaying PEG-polymer induced chemical shift perturbations. The NOE spectral analysis for all the positional isomers further showed that no NOEs could be identified between the PEG-polymer and the amides of the protein backbone, indicative that there is no stable contact between the protein surface and the two branches of the PEG-polymer. As no experimental result so far was found that indicates resolvable structural differences among and between the positional isomers and IFNR2a, the alteration of the protein structure by the attached 40 kDa branched PEG-polymer is very minor if at all. Nevertheless, the physical bulk of the PEG-polymer, although not interacting in a defined and stable manner, produces a shield around some areas of the protein merely by its exclusion volume. This shielding effect may interfere with the ability of the protein to bind to its cellular receptors (9, 31-33, 36). Indeed, the antiviral activity of the individual positional isomers of 40PEG-IFNR2a is reduced to various degrees depending on the PEG-attachment site (28). The acidic pH (3.5) of all the NMR samples causes a slow exchange rate, of the order of minutes, for the surface and solvent exposed amide protons of the IFNR2a protein domain with water (61). An acidic pH is needed to obtain resolved NMR spectra. These conditions do not allow the assessment of the differential shielding by the PEG-polymer of the protein surface by 1H/2H exchange NMR experiments (56). The moderately broader lines (+5 Hz) of the 1Hresonances for the positional isomers, when compared to IFNR2a, indicate that the molecular tumbling of the positional isomers does not at all correspond to that of a 60 kDa protein. With respect to 1H-dipolar relaxation, these pegylated IFNR2a isomers behave like a protein with ∼25 kDa apparent molecular weight. The modest line-broadening can be explained only by a highly independent motion of both the protein domain and the PEGpolymer (see below).


Characterization of PEG−Interferon-R2a Table 2. Hydrodynamic Properties and Thermal Stability of IFNr2a, PEG-polymer properties vbara

(ml‚g-1)

partial specific volume, molecular weight, MWb (kDa) second virial coefficient, Bb (10-6 L‚mol‚g-2) sedimentation coefficient, Sb (Sevdberg, 10-13s) maximum shape asymmetry from a sphere, f/f0c hydrodynamic radius, RHd (nm) translational diffusion coefficient, DTe (10-7 cm2‚s-1) secondary structure content (R-helix)f (%) R-helix content after thermal denaturationf (%) denaturation melting temperature, Tmg (°C)

40PEG-IFNr2a

40PEG-IFNR

IFNR2a 0.737 ( 0.002 19.2 ( 0.9 -1.49 ( 0.07 2.45 ( 0.10 1.02 2.73 ( 0.01 8.95 ( 0.03 75 ( 3 ∼41 66.1 ( 0.3

and the 40 kDa Branched

2a

0.806 ( 0.002 58.9 ( 2.9 2.71 ( 0.13 1.46 ( 0.06 2.59 9.46 ( 0.09 2.59 ( 0.02 74 ( 3 ∼61 67.4 ( 0.3

40 kDa branched PEG-polymer 0.824 ( 0.002 37.3-45.2h 2.21i 0.70 ( 0.03 3.76 10.65 ( 0.39 2.30 ( 0.08 -

a Determined by densitometry. v -1 (41). b Determined bar for IFNR2a was also calculated based on the amino acid sequence to 0.739 mL‚g by AUC. c Ratio of frictional coefficients f/f0 equal to the ratio of S0/S, with S0 the calculated value for a sphere with corresponding mass and S the experimental value (44). d Determined by PFG-(LED) 1H NMR spectroscopy (57). e Hydrodynamic radius of the sphere with equivalent translational diffusion coefficient DT calculated using the Stokes-Einstein equation. f Determined by analysis of the CD spectra at 20 °C (47). f Protein solutions experienced a thermal denaturation cycle from 20 °C to 80 °C and back to 20 °C monitored by CD. At this latter temperature the amount of R-helix content was evaluated (47). g Determined by DSC. h From ref 24. i From ref 68.

Figure 7. (A) DSC scans of the unfolding transition of IFNR2a (black) and 40PEG-IFNR2a (red). The denaturation melting temperature Tm is evaluated at the maximum of the heat capacity. (B) Fluorescence emission spectra recorded at room temperature after excitation at 283 nm of IFNR2a (black) and 40PEG-IFNR2a (red). (C) CD spectra recorded at 20 °C of IFNR2a (black) and 40PEG-IFNR2a (red) in the backbone absorption region, and (D) in the aromatic absorption region. Optical spectra as shown in B and C were also acquired for the individual positional isomers modified at K31, K134, K131, and K121, leading to identical results (data not shown).

Structure of the 40 kDa Branched PEG-polymer and the Pegylated Lysine. Weak coupling of motions of the protein domain and the PEG-polymer is further supported by the observation of an unique set of sharp resonance lines in the 1D 1H NMR spectra for the PEGunits and the two terminal groups (OCH3) with identical line-widths and chemical shifts in all the samples including free PEG-polymer. This shows that the polymer molecule adopts a very flexible and mobile random coil (r.c.) conformation, that seems neither to be influenced by IFNR2a nor depends on the pegylation site. Hydrodynamic data indicate, however, that the protein attachedPEG seems to be slightly more compact than the free PEG-polymer (see below). A snapshot of such a highly dynamic system is shown in Figure 8 based on the IFNR2a structure and Self-Avoiding-Walk (SAW) (75) model for the polymer conformation. Despite the very flexible structure of the PEG-polymer, local points of rigidity exist. In all the positional isomers and the free PEG-polymer a cis/trans isomerization was observed at the urethane bonds, one at the R- and the

other at the -position of the PEG-linker lysine (Figure 1). The same ratio ∼9:1 of trans and cis populations, respectively was observed in free PEG-polymer and for all the positional isomers independently of the pegylation site. Interestingly, in the Isomer-K121 an additional conformational heterogeneity was indicated by a doubling of the 1H-resonance for NRHtrans and NRHcis with relative intensity ratio of ∼5.5:4.5 reflected in both signals. For the Isomer-K31 and -K134, only the doubling of the 1Hresonance for NRHtrans was observed with the relative intensity ratio of ∼6.5:3.5 and ∼5.5:4.5, respectively. For the Isomer-K131, -K164, and -K70, the signal splitting is not resolved or not present. For all the three positional isomers which showed the additional splitting of the NRH linker signal, a corresponding doubling with identical intensity ratio was observed for the cross-peaks of the neighboring 15NH of the pegylated lysine (Figure 3). These cross-peaks could be assigned to two conformational states, arbitrarily called State-A and State-B, according to their higher and lower intensity, respectively. The observation of two (15N/15NH) cross-peaks for


Figure 8. Three-dimensional model of one positional isomer pegylated at K164 of 40PEG-IFNR2a. The model is based on the NMR solution structure of IFNR2a which is shown as a ribbon with R-helices in red and connecting loops and turns in blue (40). The r.c. conformation of the two 20 kDa branches of the PEG-polymer was calculated as an on-lattice SAW (75) using the molecular modeling package MOLOC (84, 85). It must be noted that the polymer molecule experiences a very high number of conformations, therefore this representation is only one snapshot of a single possible conformation. The polymer is reported as a solid Gaussian-Connolly molecular surface in white-grey computed with the MOE software (Chemical Computing Group Inc., Montreal, Canada). The impact of the timeaveraged structural envelope of the ensemble of r.c. conformations for the polymer is further evaluated in the accompanying publication with respect to the binding to the extracellular domain of the receptor

the -amide group of the pegylated lysine coincides with the duplication of the 1H-resonance for NRH of the PEGlinker lysine. The same relative intensity for the split lines of NRH of the PEG-linker lysine and of the neighboring 15NH of the pegylated lysine indicates that a common conformational process is responsible for both duplication of signals. As no conformational exchange was observed between State-A and -B on the time scale accessible by NMR, these two states are either in a very slow dynamic exchange or stable over time. This is in contrast with the cis/trans isomerization of the two neighboring urethane bonds, where for both R- and - positions, a conformational exchange takes place within the millisecond time range (∼5 ms < 1/kex < ∼50 ms). State-A and -B may be caused by a slow cis/trans isomerization of the peptide bond linking the IFNR2a protein to the polymer (Figure 1). Hydrodynamic Properties of 40PEG-IFNR2a As Compared To IFNR2a and the 40 kDa Branched PEG-polymer. The favorable pharmacological properties of 40PEG-IFNR2a are intimately related to the molecular structure and thermodynamic properties of the branched PEG, as discussed here and in the accompanying publication (36). The pegylation of the protein with the 40 kDa branched PEG-polymer alters the hydrodynamic properties of IFNR2a dramatically (Table 2): both sedimentation S and translational diffusion DT coefficients are substantially smaller in the modified protein. The hydrodynamic radius RH expands three to four times and the overall shape of the pegylated molecule becomes highly asymmetric (see f/f0 values). By the same token, the conformation of the protein domain remains unchanged, as was shown by detailed NMR structural analysis of the positional 15N-isomers as compared to IFNR2a.

Dhalluin et al.

The size difference of the pegylated protein when compared with the unmodified IFNR2a is clearly reflected in the change of DT. The observed decrease in the DT value, however, is much higher than expected from MW consideration alone. The 60 kDa pegylated IFNR2a shows diffusion properties very similar to globular proteins with MW’s of approximately 300 kDa (72, 73). If the effective hydrodynamic volumes are compared, the 40 kDa branched PEG-polymer is approximately 60 times larger (R3HPEG/R3HIFNR2a) and less densely packed (1/vbar) than IFNR2a. As the polymer has the unique property to ‘bind’ two to three water molecules per ethylene glycol unit (76), its r.c. structure is dynamically associated with a large cloud of water molecules. These hydrodynamic properties of the PEG-polymer are effectively transferred to IFNR2a upon pegylation, leading to a molecular particle with an effective hydrodynamic volume that has increased approximately 40 times. Unexpectedly, however, the 40 kDa PEG-polymer is self-diffusing ∼1.1 times slower than 40 PEG-IFNR2a that is 1.5 times higher in molecular weight. Attaching the polymer to the protein can be interpreted as leading to a reduction of the effective hydrodynamic volume of the PEG molecule by ∼30%. The protein attached-PEG, while keeping its very flexible and mobile r.c. conformation, seems to adopt a slightly more compact conformation than the free PEG-polymer in solution. Polymer branching can lead to compaction for entropic reasons (77, 78). In our case, attaching a protein to the two PEG-chains at its ‘branching’ site could be formally viewed as increasing the branching of the polymer. However, the compaction can also be explained enthalpically by assuming a small attractive interaction between the polymer and the protein. To decide between the two compaction mechanisms is not possible based on our current experimental evidence. The significant differences for the sedimentation coefficient S measured by AUC further show that pegylation changes the hydrodynamic shape of the pegylated protein. As expected, 40PEG-IFNR2a sediments faster than the 40 kDa PEG-molecule but, surprisingly, slower than IFNR2a, despite its bigger molecular weight. Therefore, the overall shapes, of IFNR2a and 40PEG-IFNR2a must be very different. In the sedimentation experiment with 40 PEG-IFNR2a, the PEG-moiety can be envisaged as a ‘parachute’ slowing down the sedimentation rate. The changes in hydrodynamic shape can be assessed from S by the ratio f/f0 that gives a measure of the maximum shape asymmetry from a sphere (44). The value f/f0 of 1.02 for IFNR2a indicates that the hydrodynamic shape of the protein is very close to a sphere. In contrast, the value f/f0 of 3.76 for the 40 kDa branched PEG-polymer shows that the hydrodynamic shape of the polymer alone is very asymmetric. This is in agreement with theoretical calculations of polymer structures by SAW showing highly asymmetric molecular shapes (79, 80). The attachment of the PEG to the protein creates a molecule with a hydrodynamic shape less asymmetric than the free PEG-polymer with a f/f0 value of 2.59. Further experimental evidence based on hydrodynamic modeling might allow a tentative approximation of the molecular shape of the 40 kDa branched PEG-polymer and 40PEG-IFNR2a to a hydrodynamic equivalent oblate or prolate ellipsoid of revolution. The uniquely tailored hydrodynamic properties and shape of 40PEG-IFNR2a are of extreme pharmacological interest and benefit as clinically shown with PEGASYS. The shielding of the protein by the polymer may effectively prevent, by steric hindrance, large molecules such as antibodies (9) or receptors (33, 36) from reaching


the protein surface. The recognition of pegylated proteins by the immune system is reduced, and in parallel also the immunogenicity of the protein drug (9, 81). Similarly, the increased resistance of pegylated proteins toward proteolytic degradation (9) is mediated by PEG through its large volume close to the protein surface which excludes other large molecules. Pegylation makes the molecular shape of a protein looks much larger than the actual MW of the pegylated protein might suggest. In this way, pharmacological properties related to size, e.g. the renal clearance of the drug, can critically be modulated and tuned. It is known that the size cutoff for a protein to be filtered out from the blood circulation by the kidney, is lower than 300 kDa (11, 82). The enhanced and prolonged bioavailability of the 40PEG-IFNR2a drug in the body (83) can thus be directly related to hydrodynamic properties of the pegylated protein. The second virial coefficient B is a description of the solute-solute interaction strength in solution. The opposite sign of B for IFNR2a and 40PEG-IFNR2a shows that the molecules have a completely different aggregation behavior (45). For IFNR2a, intermolecular solute-solute interactions are preferred, i.e., they are attractive, whereas for 40PEG-IFNR2a these interactions are repulsive, as solvent-solute interactions prevail. Again, these favorable properties are caused by the highly hydrated polymer and are effectively transferred to 40PEG-IFNR2a, creating a molecule with reduced tendency for aggregation. This becomes noticeable if the thermal unfolding of 40 PEG-IFNR2a is compared with IFNR2a. As shown by DSC and CD, 40PEG-IFNR2a has a significantly reduced tendency for heat induced irreversible aggregation, coupled with a slightly elevated denaturation melting temperature Tm (Table 2). At 20 °C, IFNR2a and 40PEG-IFNR2a present ∼75% of R-helix secondary structure. After a melting (80 °C) and cooling (20 °C) cycle, more R-helix secondary structure is recovered for 40PEG-IFNR2a (∼61% R-helix) than for IFNR2a (∼41% R-helix). The prevention of aggregation in protein drugs is crucial for the preservation of the native protein fold. The increased stability contributes to the favorable pharmacokinetic and immunological properties that are a consequence of the improved hydrodynamic properties of the pegylated protein. Summarizing the hydrodynamic properties (vbar, B, S, f/f0, DT, and RH), one can see that the covalent attachment of an unstructured, mobile, and highly flexible PEGpolymer molecule of appropriate size on a compact structured protein causes an efficient transfer of favorable hydrodynamic properties of the PEG-polymer to the pegylated protein. The modification of IFNR2a by the 40 kDa branched PEG-polymer provides a shield for the protein domain without changing the three-dimensional structure and conformation of the protein. The pegylation does alter the size and shape of the molecule, significantly increasing the effective hydrodynamic volume. It also improves the physicochemical properties of the protein, resulting in higher temperature stability and reduced tendency for aggregation. The small changes of the NMR 1 H-resonance line-width of IFNR2a and the unchanged line-width observed for PEG in 40PEG-IFNR2a show that the protein and PEG domains behave independently. CONCLUSION

Here, we reported the detailed structural and biophysical characterization by NMR spectroscopy, AUC, CD, fluorescence spectroscopy, and DSC of 40PEG-IFNR2a and its positional isomers modified at K31, K134, K131,


K121, K164, and K70, respectively, as compared to IFNR2a. Our results show that the three-dimensional structure of IFNR2a is not modified by the presence of the attached 40 kDa branched PEG-polymer in any positional isomer of 40PEG-IFNR2a. Regardless of where the PEG-polymer is attached, it adopts a very mobile and flexible r.c. conformation, producing a shield for the protein without a permanent coverage of the protein surface. This shield can be best envisaged by a large excluded volume close to the protein surface. This volume is visible to other large molecules, e.g. antibodies, proteases or receptors, approaching this part of space. Our results also provide evidence of significant structural and physicochemical advantages conferred by the pegylation to IFNR2a: alteration of the hydrodynamic properties by increase of the effective hydrodynamic volume and modification of the molecular shape, higher temperature stability, and reduced tendency for aggregation. These properties are of tremendous pharmacological interest and benefit as clinically shown with PEGASYS. Finally, our work represents a new standard for the characterization and quality-control of pegylated proteins, and it can be foreseen that this will have a significant impact in the pegylation area which plays an increasingly important role in new pharmaceutical applications. In the future, further investigations, particularly IFNR2a receptor binding studies, are clearly needed for a better understanding of the relationship between the structure and the biological function of 40PEGIFNR2a and its positional isomers. Ultimately, this knowledge might lead to selectively engineered positional pegylated IFNR2a with unique and optimized properties. ACKNOWLEDGMENT

We would like to thank D. Brugger, Dr. F. Danel, J. Kohler, E. Kusznir, and A. Schacher for their help in obtaining materials and for experimental work. We gratefully acknowledge Drs. T. Schreitmu¨ller, K. Weyer, and B. Wipf for constant support. For stimulating discussions and help many thanks to Drs. P. Gerber, S. E. Harding (Nothingham University), W. Huber, W. Klaus, and T. Schulz-Gasch. We thank Dr. D. Banner for a careful reading of the manuscript. LITERATURE CITED (1) Abuchowski, A., Van Es, T., Palczuk, N., and Davis, F. (1977) Alteration of immunological properties of bovine serum albumin by covalent attachment of poly-ethylene-glycol. J. Biol. Chem. 252, 3578-3581. (2) Bailon, P., and Berthold, W. (1998) Poly(ethylene glycol)conjugated pharmaceuticals proteins. Pharm. Sci. Technol. Today 1, 352-356. (3) Wang, Y. S., Youngster, S., Bausch, J., Zhang, R., McNemar, C., and Wyss, D. F. (2000) Identification of the major positional isomer of pegylated Interferon alpha-2b. Biochemistry 39, 10634-10640. (4) Harris, J. M., and Chess, R. B. (2003) Effect of pegylation on pharmaceuticals. Nat. Rev. Drug Discovery 2, 214-221. (5) Fuerteges, F., and Abuchowski, A. (1990) The clinical efficacy of poly-(ethylene-glycol)-modified proteins. J. Controlled Release 11, 139-148. (6) Inada, I., Matsushima, A., Kodera, Y., and Nishimura, H. (1990) Polyethylene-Glycol (PEG)-proteins conjugates: applications to biomedical and biotechnological processes. J. Bioact. Compat. Polym. 5, 343-364. (7) Katre, N. V., Knauf, M. J., and Laird, W. (1987) Chemical modification of Interleukin-2 by polyethylene glycol increases its potency in the murine Meth A Sarcoma model. Proc. Natl. Acad. Sci. U.S.A. 84, 1487-1491.

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Structural and Biophysical Characterization of the 40 kDa PEG

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