Bioconjugate Chem. 1995, 6, 62-69
62
A Branched Monomethoxypoly(ethy1ene glycol) for Protein Modification Cristina Monfardini, Oddone Schiavon, Paolo Caliceti, Margherita Morpurgo, J. Milton Harris,+a n d Francesco M. Veronese" Department of Pharmaceutical Sciences, Centro di Studio di Chimica del Farmaco e dei Prodotti Biologicamente, Attivi del CNR, University of Padua, Via F. Marzolo 5, 35131 Padua, Italy, and Chemistry Department, University of Alabama in Huntsville, Huntsville, Alabama 35899. Received July 27, 1994@
Procedures are described for linking monomethoxypoly(ethy1ene glycol) (mPEG) to both E and a amino groups of lysine. The lysine carboxyl group can then be activated as a succinimidyl ester to obtain a new mPEG derivative (mPEG2-COOSu) with improved properties for biotechnical applications. This branched reagent showed in some cases a lower reactivity toward protein amino groups than the linear mPEG from which it was derived. A comparison of mPEG- and mPEG2-modified enzymes (ribonuclease, catalase, asparaginase, trypsin) was carried out for activity, pH and temperature stability, K, and Kcatvalues, and protection to proteolytic digestion. Most of the adducts from mPEG and mPEG2 modification presented similar activity and stability toward temperature change and pH change, although in a few cases mPEG2 modification was found to increase temperature stability and to widen the range of pH stability of the adducts. On the other hand, all of the enzymes modified with the branched polymer presented greater stability to proteolytic digestion relative to those modified with the linear mPEG. A further advantage of this branched mPEG lies in the possibility of a precise evaluation of the number of polymer molecules bound to the proteins; upon acid hydrolysis, each molecule of mPEG2 releases a molecule of lysine which can be detected by amino acid analysis. Finally, dimerization of mPEG by coupling to lysine provides a needed route to monofunctional PEGs of high molecular weight.
INTRODUCTION
With improved chemical and genetic methods many new peptides and proteins are now available for potential application as new drugs or specific biocatalysts. Limitations, however, still exist to more extensive use (1-6). As therapeutic agents, peptides and proteins are often rapidly cleared from circulation or give rise to immunological problems, sometimes when they apparently have the same structure as the homologous natural product. Also, application in biocatalysis is mostly restricted to hydrophilic substrates because of the low stability and solubility of enzymes in organic solvents. Linking suitable hydrophilic or amphiphilic polymers to peptides and proteins presents the potential of overcoming these problems since the polymer cloud surrounding the protein increases stability toward proteolysis and reduces renal excretion and immunological complications (1-4). Moreover, if the polymer is soluble in organic solvents, the enzyme conjugates may acquire this same solubility, and biocatalysis may be extended to organic media (5, 6). Monomethoxypoly(ethy1ene glycol) (mPEGY has been the polymer most used for these applications thus far, with linear polymers of molecular weights (MWs) in the range of 2000-5000 being preferred, although branched
* Author to whom correspondence
should be addressed. University of Alabama in Huntsville. Abstract published in Advance ACS Abstracts, December 1, 1994. Abbreviations: MW, molecular weight; mPEG, monomethoxypoly(ethy1ene glycol); mPEG-Nle-OSu, mPEG carrying a norleucine spacer, activated a s succinimidyl ester; mPEG2COOH, mPEG-lysine dimer; mPEG2-COOSu, mPEG-lysine dimer activated as succinimidyl ester; TEA, triethylamine; PITC, phenyl isothiocyanate; and TAME, Nu-p-tosylarginine methyl ester. +
@
PEGs and high MW mPEGs have been used. Recent work by Somak et al. has shown the utility of high MW mPEGs for protein modification (7). A branched form of mPEG has been prepared by substitution of two chloride atoms of trichloro-s-triazine with mPEG, while using the third chloride to bind to protein; this procedure, however, presents severe limitations because of lack of specificity in protein modification, inactivation of many enzymes, and toxicity of the intermediates (8). In a related preparation of a branched mPEG, Yamsuki and coworkers coupled mPEG succinimidyl succinate to norleucine, activated the resulting acid, and coupled two molecules of the activated mPEG to lysine (9). The resulting branched acid can be activated and coupled to amines. This approach offers the advantage of providing percent modification from amino acid analysis, but disadvantages are hydrolytic instability of the succinate ester linkage, overall complexity of the synthetic procedure, and the large linker region beween mPEG and conjugated protein that may provide an antigenic site. In view of the great utility of large or branched monofunctional PEGs for increasing the polymer cloud volume surrounding a protein while maintaining the same number of binding sites, we have prepared a new branched mPEG derivative devoid of the above disadvantages. This derivative also presents the important advantage of easy analytical characterization of the adduct. This new polymer preparation is based on direct linkage of mPEGs to the a and E amino groups of lysine (to give mPEG2-COOH), followed by activation of the carboxyl group as the succinimidyl ester (to give mPEG2COOSu), Figure 1. This paper reports also the use of this new derivative in modifying model proteins, and it reports comparison of the properties of proteins modified with linear and branched mPEGs. Furthermore, results are given on evaluation of degree of enzyme modification
1043-1802/95/2906-0062$09.00/00 1995 American Chemical Society
Bioconjugafe Chem., Vol. 6,No. 1, 1995 63
Proteins Modified by Branched Polymer
0
It % mPEG-0-C-NH pH=8
11 + NH2-(CH2),-CH-COOH I NH2
’
111 c m P E G - 0 - C - O
I11
I (CHI), I CH
N& ‘COOH 0
mPEG-0-C-NH II
*NO=2‘
I (CHI),
0 II mPEG-0-C-NH
IV I
0
IV
Dcc
V
0
0
It mPEG-0-C-NH
V
NH2-Protein
0 II mPEG-0-C-Nd
I (YHd4 CH
VI
‘C-NH-Protein It 0
Figure 1. Scheme of synthesis of activated mPEG2-COOSu (V) through a “two-step” procedure and reaction of V with protein amino groups.
by both mPEG and the new dimer, based on amino acid analysis after acid hydrolysis. Two procedures for preparation of mPEG2-COOH are described. The first procedure uses mPEG activated as thep-nitrophenyl carbonate and takes place in two steps (the first in water, the second in methylene chloride), Figure 1. The same polymer may be more easily prepared by a “single-step’’ procedure using more active mPEG succinimidyl carbonate (10). The single step procedure was used to prepare a dimer of MW 40 000. The methodology reported here for preparing this branched mPEG dimer, carrying a single point of attachment, may be considered useful not only for PEGenzyme chemistry but also for PEG chemistry in general since it allows doubling the amount of PEG that may be linked to the same site of drugs to obtain polymeric prodrugs (111, surfaces to increase their blood compatibility (12) and to avoid protein adsorption (12),bioactive components for which the two phase partitioning may be useful (14-16), and liposomes to obtain long-lasting preparations (17,18). Also, this chemistry provides ready access to monofunctional PEGS of high molecular weight by dimerization of low-diol mPEG (note that the usual polymerization of ethylene oxide to yield mPEGs is limited to about 20 000 g/mol) (19). EXPERIMENTAL SECTION
Materials. mPEG, M, 5000,4-nitrophenyl chloroformate, N,N-dicyclohexylcarbodiimide,N-hydroxysuccinimide, norleucine, lysine, and phosgene solution were purchased from Fluka (Buchs, Switzerland). mPEG of M, 20 000, mPEG succinimidyl carbonate, and mPEG 5000 p-nitrophenyl carbonate were obtained from Shearwater Polymers (Huntsville, AL). Salts and solvents were purchased from Carlo Erba (Milan, Italy). Dimethyl sulfoxide-da was from Janssen (Geel, Belgium). Ribonuclease, catalase, trypsin, pronase, elastase, subtilisin, cytidine 2‘:3‘ cyclic monophosphate, N-p-tosylarginine methyl ester, casein, and asparagine were from Sigma
Chemical Co. (St. Louis, MO). Asparaginase from Erwinia Caratiuora was from PHLS, Porton Down (Salisbury, England). a-Ketoglutaric acid, glutamic-oxalacetic transaminase, and malate dehydrogenase were from Calbiochem (San Diego, CAI. For pH titration, a Radiometer autoburette ABU 80 (Copenaghen, Denmark) with titrator TTT 80 and titrigraph REA 160 was used. UV-vis analysis was performed on a Perkin-Elmer Lambda 2 spectrophotometer. lH-NMR spectra were performed on a 200 MHz Bruker spectrometer. Analytical gel filtration chromatography was performed with a Waters Ultrahydrogel column. Activation of mPEG (mPEG-Me-OSu). Linear mPEG 5000, with norleucine as a n amino acid spacer arm and activated as the succinimidyl ester, was synthesized according t o the method previously reported from our laboratory (20). Alternatively, the compound was purchased from Shearwater Polymers. Two-step Procedure for mPEG2-COOH. (a) Preparation of mPEG p-Nitrophenyl Carbonate (II). Five g (1mmol) of mPEG-OH, M, 5000, was dissolved in 120 mL of toluene and dried by removal of the water-toluene azeotrope. The solution was cooled to room temperature and concentrated, and 20 mL of anhydrous methylene chloride, 0.28 mL (2 mmoles) of triethylamine (TEA), and 0.4 g (2 mmoles) of p-nitrophenyl chloroformate were added under stirring a t 0 “C; the pH was maintained a t 8 with TEA (this pH measurement was made by placing a drop of solution on a piece of wet pH paper). After being allowed to stand overnight a t room temperature, the reaction mixture was concentrated under vacuum to about 10 mL, filtered, and added drop by drop into 100 mL of stirred diethyl ether. The resulting precipitate was collected by filtration and crystallized twice from ethyl acetate. mPEG activation, calculated spectrophotometrically on the basis of the released 4-nitrophenol absorption a t 400 nm in alkaline media, after 15 min, was 98% (6 of p-nitrophenol a t 400 nm = 17 000 M-l cm-’1. Alternatively, this material was purchased from Shearwater Polymers. (b) Preparation of mPEG-Monosubstituted Lysine (III). Lysine (353 mg, 2.5 mmol) was dissolved in 20 mL of water at pH 8.0-8.3, and then 5 g of mPEGp-nitrophenyl carbonate (1mmol) was added in portions for 3 h, while the pH was maintained at 8.3 with 0.2 N NaOH. After being stirred overnight at room temperature, the solution was cooled to 0 “C and brought to pH 3 with 2 N HC1. Impurities were extracted with diethyl ether, the mPEGLys substituted a t the 6 amino group was extracted three times with chloroform, and the solution dried. After concentration, the solution was added drop by drop to diethyl ether. The precipitate was collected and then crystallized from absolute ethanol. The percentage of modified amino groups, calculated by colorimetric analysis (211, was 53%. (c) Preparation of mPEG-DisubstitutedLysine (mPEG2COOH) (N).TEA was added to 4.5 g (0.86 mmol) of the above product, dissolved in 10 mL of anhydrous methylene chloride, to reach pH 8.0. mPEG p-nitrophenyl carbonate (4.9 g,1.056 mmol) was added over 3 h, while the pH was maintained a t 8.0 with TEA. The reaction mixture was refluxed for 72 h, brought to room temperature, concentrated, filtered, precipitated with diethyl ether, and finally crystallized in a minimum amount of hot ethanol. The excess of activated mPEG was hydrolyzed in pH 9-10 buffer by stirring overnight a t room temperature. The solution was then cooled to 0 “C and brought to pH 3 with 2 N HC1. This solution was extracted with diethyl ether to remove p-nitrophenol. mPEG2-COOH and the remaining traces of mPEG were
64 Bioconjugate Chem., Vol. 6,No. 1, 1995
extracted from the mixture three times with chloroform, dried, concentrated, precipitated with diethyl ether, and crystallized from ethanol. No unreacted lysine amino groups remained in the polymer mixture as assessed by colorimetric analysis (21). mPEG2-COOH was purified from mPEG by gel filtration chromatography using a Bio Gel PlOO (Bio-Rad) column (5 x 50 cm), 100-200 mesh, and water as eluent; 10 mL fractions were collected. (According to these conditions no more than 200 mg of material could be purified for each run). The fractions corresponding to mPEG2-COOH, revealed by iodine reaction (22), were pooled, concentrated, dissolved in ethanol, and concentrated. The product was dissolved in methylene chloride, precipitated with diethyl ether, and crystallized from ethanol. Alternatively, the purification of the desired product from unmodified mPEG was performed by ion exchange chromatography on a QAE Sephadex A50 column (5 x 80 cm) (Pharmacia) using 8.3 mM borate buffer pH 8.9 (23). This procedure permitted fractionation of a greater larger of material for each run (4 g). In both cases, purified mPEG2-COOH, titrated with NaOH, gave 100% of free carboxyl group, considering a polymer of MW 10 000. The product was also characterized by lH-NMR on a 200 MHz Bruker instrument in dimethyl sulfoxide-ds, at a 5% W N concentration. 'HNMR data confirmed the expected M , of 10 000. Note that we now believe our original NMR method can give reasonable M , values up to 20 000 g/mol (24). The chemical shifts and assignments of the protons in mPEG2-COOH are as follows: 1.2-1.4 ppm (multiplet, 6H, methylenes 3,4,5 of lysine); 1.6 ppm (multiplet, 2H, methylene 6 of lysine); 3.14 ppm (s, 3H, terminal mPEG methoxy); 3.49 ppm (s, mPEG backbone methylene); 4.05 ppm (t, 2H, -CHZOCO-) 7.18 ppm (t,lH, -NH- lysine); 7.49 ppm (d, l H , -NH-lysine). (d)Activation of mPEG2-COOH by N-Hydroxysuccinimide (v). mPEG2-COOH (6.2 g, 0.6 mmol) was dissolved in 10 mL of anhydrous methylene chloride, cooled to 0 "C, and 0.138 g (1.2 mmol) of N-hydroxysuccinimmide and 0.48 g (1.2 mmoles) of N,N-dicyclohexylcarbodiimide were added under stirring. After the mixture was stirred overnight a t room temperature, precipitated dicyclohexylurea was removed by filtration and the solution was concentrated and precipitated with diethyl ether. The final product was crystallized from ethyl acetate. The yield of esterification, calculated on the basis of N hydroxysuccinimide absorption at 260 nm (produced by hydrolysis), was over 97% (6 of hydroxysuccinimide a t 260 nm = 8000 M-l cm-'). The NMR spectrum was identical to that of the preceding acid except for the new succinimide singlet a t 2.80 ppm (2H). One-StepProcedure for mPEG2-COOHPreparation. a. Preparation of mPEG-Disubstituted Lysine (mPEG2-COOH) . A 10.8 g portion of mPEG succinimmol) (Shearwater idyl carbonate 20 000 (VIII)(5.4 x Polymers, Huntsville, AL) was added to 40 mL of lysine HC1 solution in borate buffer, pH 8.0, a t a concentration of 0.826 mg/ml (1.76 x lo-* mol). After addition of 20 mL of the same buffer, solution pH was maintained a t 8.0 with aqueous NaOH solution for the following 8 h. The reaction mixture was allowed to stir a t room temperature for 24 h. For dimerization of mPEG 5000 a molar excess of carbonate of only 10% is required. After dilution with 300 mL of deionized water, the solution was adjusted to pH 3.0 by addition of oxalic acid and extracted three times with dichloromethane. The combined dichloromethane extracts were dried with anhydrous sodium sulfate. The filtrate was concentrated
Monfardini et al.
to about 30 mL and the product precipitated with about 200 mL of cold ethyl ether. The yield was 90%. Nine g of the above reaction product was dissolved in 4 L of distilled water and loaded onto DEAE Sepharose FF (500 mL of gel equilibrated with 1500 mL boric acid, sodium hydroxide buffer, 0.5%, pH 7.0 and then washed with water). Impurities of mPEG-lysine and mPEG were washed off the column with water, whereas the desired mPEG2-COOH was eluted with 10 mM NaC1. The pH of the eluate was adjusted to 3.0 with oxalic acid and the product extracted with dichloromethane, dried with sodium sulfate, concentrated, and precipitated with ethyl ether. A total of 5.1 g of the desired product was obtained. M,, was determined to be 38000 by gel filtration chromatography and 36 700 by potentiometric titration (as described above). (b) Preparation of mPEG2-COOSu. The procedure previously described for preparing the branched 5000 x 2 polymer was followed for activating this high-MW, branched (20 000 x 2) polymer. Yield was over 95%. Enzyme Modification. To obtain a similar extent of amino group derivatization for each enzyme, different procedures were used for enzyme modification depending upon the type of enzyme and the polymer used (linear mPEG, activated as mPEG-Nle-OSu, or branched mPEG2-COOSu). Larger molar ratios of mPEG2COOSu were generally required. Common conditions were 0.2 M, pH 8.5 borate buffer to dissolve proteins, and addition of polymers in small portions to facilitate dissolution (approximately 10 min required), followed by stirring for 1 h. The amount of polymer used for modification was calculated on the basis of available amino groups in the enzyme. a. Ribonuclease (1.5 mg/ml) was modified a t room temperature, and mPEG-Nle-OSu or mPEG2-COOSu was added a t a molar ratio of polymer to protein amino groups of 2.5/1 and 511, respectively. Ribonuclease has a molecular weight of 13 700 D and 11 available amino groups. b. Catalase (2.5 mg/ml) was modified a t room temperature using mPEG/protein-NHz and mPEG21proteinNHZ molar ratios of 5/1 and lO/l, respectively. Catalase has a molecular weight of 250 000 D with 112 available amino groups. c. Trypsin (4 mg/mL) modification was performed a t 0 "C using a mPEG/protein-NHz or mPEG2/protein-NHz molar ratio of 2.5/1. Trypsin has a molecular weight of 23 000 D and 16 available amino groups. d. Asparaginase (6 mg/ml) was modified with a mPEG/ protein-NHz molar ratio of 3/1 a t room temperature, while derivatization with mPEG2-COOSu (mPEG2/ protein-NHz molar ratio of 3.3/1) was carried out a t 37 "C. Erwinia Caratiuora asparaginase has a molecular weight of 141 000 D and 92 free amino groups. Polymer-enzyme conjugates were purified by ultrafiltration and concentrated in an Amicon system with a PM 10 membrane (cutoff 10000) to eliminate N hydroxysuccinimide. The conjugates were further purified from unreacted polymer by gel filtration chromatography on a Pharmacia Superose 12 column (on an FPLC) using 10 mM phosphate buffer, pH 7.2, 0.15 M in NaCl, as eluent. Protein concentration of the native forms of ribonuclease, catalase, and trypsin was evaluated spectrophotometrically using molar extinction coefficients of 9.45 x lo3, 1.67 x lo5, and 3.7 x lo4 M-l cm-l a t 280 nm, respectively. The concentration of asparaginase was evaluated by biuret assay. Biuret assay was also used to evaluate concentrations of modified proteins.
Proteins Modified by Branched Polymer
The extent of protein modification was evaluated colorimetrically by the method of Habeeb (25)or by amino acid analysis after acid hydrolysis, following the postcolumn procedure of Benson et al. (26) or precolumn derivatization by phenyl isothiocyanate (PITC) according to Bidlingmeyer et al. (27). For amino acid analysis, the amount of bound mPEG was evaluated from norleucine content with respect to other protein amino acids, and the amount of mPEG2 was determined from the increase in lysine content, since for each bound polymer one additional lysine is present in the hydrolysate (28). Enzymatic activity of native and modified enzyme was evaluated as follows: For ribonuclease, the method of Crook et al. (29) was used. Catalase activity was determined by the method of Beers and Sizer (30). The esterolytic activity of trypsin and its derivatives was determined by the method of Laskowsky (31),while the proteolytic activities of the conjugates were assayed according to the method of Zwilling and Neurath (32). Native and modified asparaginase were assayed according to a method reported by Cooney et al. (33): namely, 1.1mL containing 120 pg of a-ketoglutaric acid, 20 UI of glutamic-oxalacetic transaminase, 30 UI of malate dehydrogenase, 100 pg of NADH, 0.5 pg of asparaginase, and 10 pmol of asparagine were incubated in 0.122 M Tris buffer, pH 8.35, while the NADH absorbance decrease a t 340 nm was followed. Proteolytic Digestion of Free Enzyme and mPEGor mPEG2-Enzyme Conjugates. Proteolytic digestion was performed in 0.05 M phosphate buffer, pH 7.0, as follows: a. For ribonuclease and its adducts, 0.57 mg of protein was digested a t room temperature with 2.85 mg of pronase or 5.7 mg of elastase or with 0.57 mg of subtilisin in a total volume of 1 mL. b. Native and modified catalase, 0.58 mg of protein, were digested at room temperature with 0.58 mg of trypsin or 3.48 mg of pronase in a total volume of 1mL. c . Autolysis of trypsin and its derivatives a t 37 “C was evaluated by esterolytic activity of protein solutions a t 25 mg/mL of TAME. d. For native and modified asparaginase, 2.5 pg was digested a t 37 “C with 0.75 mg of trypsin in a total volume of 1mL. From each enzyme solution, aliquots were taken at various time intervals and enzyme activity was assayed spectrophotometrically. Thermal Stability of Free and Conjugated Enzymes. Thermal stability of native and mPEG- or mPEG2-modified ribonuclease, catalase, and asparaginase was evaluated in 0.5 M phosphate buffer, pH 7.0, a t 1, 9, and 0.2 mg/mL, respectively. The samples were incubated a t the specified temperatures for 15, 10, and 15 min, respectively, cooled to room temperature and assayed spectrophotometrically for activity. pH Stability of Free and Conjugated Enzymes. Unmodified or polymer-modified enzymes were incubated for 20 h in the following buffers: sodium acetate, 0.05 M, pH 4.0-6.0, sodium phosphate, 0.05 M, pH 7.0, and sodium borate, 0.05 M, pH 8.0-11. Enzyme concentrations were 1 mg/mL, 9 pg/mL, and 5 ,ug/mL for ribonuclease, catalase, and asparaginase, respectively. Stability to incubation a t various pHs was evaluated on the basis of enzyme activity. RESULTS AND DISCUSSION
Preparation of mPEG2-COOH by a Two-Step Procedure. The structure of the branched mPEG obtained by coupling two polymer chains to lysine amino groups is shown in Figure 1. This polymer was first
Bioconjugate Chem., Vol. 6, No. 1, 1995 65
prepared by a “two-step” procedure. In the first step of this method a single lysine amino group is modified by reaction with p-nitrophenyl carbonate-mPEG in aqueous buffer. Both lysine and the mPEG are soluble in aqueous medium, but the mPEG derivative undergoes hydrolysis; lysine is not soluble in organic solvents in which the activated PEG is stable. The product is readily extracted into chloroform. Modification of the second lysine amino group is achieved by reaction in dry methylene chloride, where mPEG-substituted lysine is soluble and the activated mPEG is soluble and stable. NMR analysis shows that the first mPEG chain is bound to the €-amino group. Although either gel filtration or ion exchange chromatography give high degrees of purification from unreacted mPEG, ion exchange is preferred because large amounts of material can be applied to the column. The mPEGZ-COOH was characterized by -COOH titration and ‘H-NMR analysis. The NMR spectrum is consistent with the assigned structure. For example, two different carbamate NH signals are observed, with the first (at 7.18 ppm) showing a triplet for coupling with the adjacent methylene group, and the second (at 7.49 ppm) showing a doublet from coupling with the a-CH of lysine. The intensity of these signals relative to the mPEG methylene peak is consistent with the 1:l ratio between the two carbamate groups and the expected M , of 10,000 of the branched polymer. For protein modification, mPEG2-COOH was activated as the succinymidyl ester according to known methods (34). Preparation of mPEG2-COOH by a “One-Step” Procedure. The two-step procedure is somewhat time consuming. A more straightforward one-step method may be employed in which lysine is reacted with 2 mol of highly active mPEG succinimidyl carbonate to produce acid IV directly, Figure 1(IO). One particular advantage of mPEG dimerization with lysine is that monofunctional PEGs of high MW can be obtained. To demonstrate this application we applied the one-step procedure to dimerize mPEG 20 000, thus preparing a clean, monofunctional mPEG2-COOH of MW 40 000. To appreciate the importance of this application, one need only recall the great difficulty in obtaining low-MW monofunctional mPEGs by direct ethylene oxide polymerization (19, 24). One disadvantage of the one-step method, relative to the twostep method, is that the latter procedure allows the attachment of PEGs of two different molecular weights. Comparison of Properties of Enzymes Modified by Linear and Branched Polymer. To evaluate the influence of linear mPEG and branched mPEG2 attachment on enzyme properties, four different model enzymes, ribonuclease, catalase, asparaginase, and trypsin, were modified with mPEG-Nle-OSu 5000 or mPEG2COOSu (2 x 50001, and the catalytic properties of the enzymes were determined. To facilitate comparison, each enzyme was modified with the two polymers to a similar extent by a careful choice of polymer to enzyme ratios and reaction temperature (Table 1). The purpose of this comparison is to see if the larger mPEG2 will confer improved conjugate properties without increasing the number of attachment sites, as would be necessary with the smaller, linear mPEG. Recall that high MW linear mPEGs are difficult to obtain. Future studies will present more extensive comparison of various MWs of linear and branced mPEGs. Ribonuclease. Ribonuclease with 50% and 55% of the amino groups modified with mPEG and mPEG2, respectively, presented 86% and 94% residual activity with respect to the native enzyme. In Figure 2 the stability to proteolytic digestion by pronase (a), elastase (b), and
Monfardini et al.
66 Bioconjugate Chem., Vol. 6,No. 1, 1995
Table 1. Properties of Enzymes Modified by mPEG and mPEG2 and Molar Ratios Used in Enzyme Modification NH2:
enzymea ribonuclease RN RP1 RP2 catalase CN CP1 CP2 trypsinb TN TP1 TP2 asparaginase AN AP1
AP2c
polymer molar % modifi- % actiratio cation vitv
l:o 1:2.5 1:5 l:o
1:5 1:lO l:o
1:2.5 1:2.5
l:o 1:3 1:3.3
K, (M)
Kcat
(min-')
0 50 55
100 86
0 43 38
100 100 90
0 50 57
100 120 125
8.2 x 7.6 x 8.0 x
830 1790 2310
0 53 40
100 110 133
3.31 x 3.33 x 3.30 x
523 710 780
94
a N = native enzyme, P1= enzyme modified with mPEG, P2 = enzyme modified with mPEG2. Reaction temperatures in text. For trypsin, only the esterolytic activity is reported.
subtilisin ( c ) is reported for native and the two modified enzymes. Polymer modification greatly increases the stability to digestion by all three proteolytic enzymes, but the protection offered by branched mPEG2 is more effective than linear mPEG. Increased thermal stability was found for the modified forms relative to the unmodified enzyme (pH 7.0,15 min incubation), but no significant difference between mPEG and mPEG2 modification was observed (Figure 3a). Decreased stability for the branched mPEG2 derivative was found after incubation a t acid and alkaline pH values (Figure 3b). Catalase. Catalase was modified with mPEG and mPEG2 to obtain 43% and 38% modification of protein amino groups, respectively. Enzyme activity was not significantly changed after modification. However, proteolytic stability was much greater for the mPEG2 derivative than for the mPEG derivative, particularly toward pronase and trypsin where no digestion took place, Figure 4. Catalase thermal stability was not influenced by polymer modification, but the stability toward low-pH incubation of the modified forms was superior as compared to the native enzyme, Figure 5. Asparaginase. Asparaginase with 53% and 40% modified protein amino groups was obtained by coupling with mPEG and mPEG2, respectively. Enzymatic activity was increased, relative to the free enzyme, to 110% for the mPEG conjugate and to 133%for the mPEG2 conjugate. An increase in enzyme activity following polymer derivatization has been observed for other enzymes and is also seen below for trypsin modification (IO, 35, 36). A possibility is that polymer coupling results in conformational modification which gives either a more active form or increased affinity for substrate. In the present case it appears that a more active form is produced because the K, values of the modified forms were not changed upon modification: 3.31 x 3.33 x lo+, and 3.3 x M for the free, mPEG-, and mPEG2-asparaginase, respectively. The Kcatvalues for these enzyme forms are 523, 710, and 780 min-l, respectively. Modification with mPEG2 had an impressive influence on stability toward proteolytic enzymes. Increased protection was achieved a t a lower extent of modification with respect to the derivative obtained with the linear polymer (Figure 6). A limited increase in thermal
I
0
15
-
50
-
25 25
-
1
1
I
I
50 I b
------tO 100
1
Figure 2. Time course of native ribonuclease, RN (01,mPEG ribonuclease, RP1 (01, and mPEG2 ribonuclease, RP2 (W) digestion as assessed by enzyme activity upon incubation with pronase (a), elastase (b), and subtilisin (c).
stability and stability to acid and alkaline pH values was observed for both adducts (data not shown). Trypsin. Trypsin was modified a t the level of 50%and 57% of amino groups with mPEG and mPEG2, respectively. Esterolytic activity for mPEG- and mPEG2modified enzyme, assayed on the small substrate TAME, was 120% and 125%)respectively. Modification did not change affinity for the substrate; native and modified forms gave K, values of 8.2 x 7.6 x and 8.0 x M. Catalytic activity, Kcat,increased from 830 min-l for native trypsin to 1790 min-l for mPEG-trypsin and 2310 min-' for mPEG2-trypsin. Presumably a more active conformation is produced by polymer modification. Modification with mPEG and mPEG2 reduced proteolytic activity of trypsin toward casein, a high molecular weight substrate: activity relative to the native enzyme was found, after 20 min incubation, to be 64% for the mPEG conjugate and only 35% for the mPEG2 adduct. In agreement with these results, the trypsin autolysis rate, evaluated by enzyme esterolytic activity, was totally prevented in mPEG2-trypsin but only reduced in mPEGtrypsin, Figure 7. To prevent autolysis with mPEG,
Bimonjugate Chem., Vol. 6,No. 1, 1995 67
Proteins Modified by Branched Polymer
c
100 41
b
g
75-
a2
50-
-a 4
Be 25
b
a 0
o
I
w
1
s
I
%
I
a
T
g
Temperature (“C)
PH
Figure 3. Heat (a) and pH (b) stability of native ribonuclease,RN (01,mPEG ribonuclease,RPl(O), and mPEG2 ribonuclease,RP2 (0) following 15 min incubation at the indicated temperatures or 20 h at different pH values.
0 ^
Time (min)
=
1
^
z
I
^
i
I
R
T
g
Time (min)
Figure 4. Time course of native catalase, CN (O), mPEG catalase, CP1 (O), and mPEG2 catalase, CP2 (W), digestion as assessed by enzyme activity upon incubation with pronase (a) and trypsin (b).
modification of 78%of the available protein amino groups was required. These data are all consistent with a n increased hindrance to access to the active site by the polymer, as discussed in our previous work (36). CONCLUSIONS The results reported in this paper demonstrate that new, branched mPEG dimers may be prepared by a “twostep” procedure, using mPEG p-nitrophenyl carbonate, or by a “single-step” procedure, using more reactive mPEG succinimidyl carbonate. The branched polymer, activated as the succinimidyl ester (mPEG2-COOSu)) reacts under mild aqueous conditions, compatible with the stability of most enzymes, to give a stable amide linkage with protein amino groups. Here the branched mPEG2 was studied for its utility in enzyme modification, but it has a general applicability in several areas of PEG chemistry. Single-point attachment is always maintained. Modification of four model enzymes with mPEG2 was accompanied by no appreciable loss of activity. Asparaginase, an enzyme of significant therapeutic interest, was found to undergo major loss of activity when modified
with branched chlorotriazine mPEG (81, but exhibited increased activity using this new mPEG2-COOSu. Protein adduds obtained by modification with mPEG2COOSu, having two polymer chains bound at the same reactive amino group, present increased hindrance to approaching macromolecules in comparison to the smaller, linear mPEG derivative. This is shown by the larger increase in protection to proteolysis of three proteins modified by mPEG2-COOSu compared to modification with linear mPEG-Nle-OSu. Also, the reduced hydrolytic activity of modified trypsin towards casein, and its slower autolysis rate, provides additional support for this conclusion. Enzyme structural stability toward denaturating agents such as temperature or pH, or the substrate binding properties of the bound polymer-enzyme conjugates, do not differ significantly between mPEG- and mPEG2enzyme adducts. A further advantage in the use of mPEG2 in protein modification is the possibility of easy and direct evaluation of the number of bound polymer chains by amino acid analysis after acid hydrolysis. Unlike analytical methods that monitor the percentage of modified lysines,
Monfardini et al.
68 Bioconjugate Chem., Vol. 6, No. 1, 1995
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Figure 7. Time course of native trypsin, TN (01, mPEG !
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PH Figure 5. Native catalase, CN (O), mPEG catalase, CP1 (m), and mPEG2 catalase, CP2 (01, stability toward 20 h incubation at the indicated pH values.
trypsin, TP1 (m), and mPEG2 trypsin, TP2 (A), autolysis evaluated as residual activity towards TAME.
by the Italian Minister0 della Pubblica Istruzione and the U.S.National Science Foundation, the Army Research Office, and the National Institutes of Health. LITERATURE CITED
(1) Nucci, M. L., Shorr, R., and Abuchoswki, A. (1991) The
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Figure 6. Time course of native asparaginase, AN (O), mPEG asparaginase, Ap1 (01, and mPEG2 asparaginase, Ap2 (m), digestion as assessed by enzyme activity assay upon trypsin incubation.
mPEG2 modification will release lysine when the polymer is bound to the a-amino group of a terminal amino acid or to an amino acid other than lysine (e.g., histidine or tyrosine) (37). Preliminary data obtained in our laboratory suggest improved immunological properties as well as increased body residence time of mPEG2 conjugates relative to mPEG conjugates. The pharmacokinetics and immunological behavior of enzymes of potential therapeutic interest, as well as the effect on solubility and activity in organic solvents, are under active investigation and will be reported soon. ACKNOWLEDGMENT
The authors wish to thank Prof. T. Atkinson from PHLS Centre for Applied Microbiology and Research (Porton Down, Salisbury, England) for the kind supply of asparaginase. This research was partially supported
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