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Quantitatively investigating monomethoxypolyethylene glycol modification of protein by capillary electrophoresis
J. Biochem. Biophys. Methods 59 (2004) 65 – 74 www.elsevier.com/locate/jbbm
Quantitatively investigating monomethoxypolyethylene glycol modification of protein by capillary electrophoresis Weijun Li 1, Zhiguo Su * State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, P.O. Box 353, Beijing 100080, PR China Received 22 June 2003; received in revised form 10 November 2003; accepted 17 November 2003
Abstract Capillary electrophoresis (CE) was applied to study quantitatively protein modification with succinimidyl succinate-activated monomethoxypolyethylene glycol (MPEG-SS). The heterogeneous distribution of modified proteins and the average modification degree were determined by CE, and the latter met with the results from 2,4,6-trinitrobenzenesulfonic acid (TNBS) spectrometric assay. It was found that the optimal buffer pH for the modification was between pH 7.4 and 8.4, and the modification degree decreased when the modified sample was preserved in high pH solutions. The protein fractions attached with different number of polyethylene glycols (PEGs) were monitored along the process of protein modification. CE was proved to be efficient to evaluate quantitatively several factors of the protein modification, including the modifier/protein molar ratio, the stability of conjugates in different pH environments, and the time course of modification process. D 2003 Published by Elsevier B.V. Keywords: Capillary electrophoresis; Chemical modification; Polyethylene glycol; Protein
* Corresponding author. Tel.: +86-10-6256-1817; fax: +86-10-6256-1813. E-mail addresses: [email protected] (W. Li), [email protected] (Z. Su). 1 Present address: Department of Biopharmaceutical Sciences, School of Pharmacy, University of California, Room HSE-1145, 513 Parnassus Avenue, San Francisco, CA 94143-0446, USA. 0165-022X/$ - see front matter D 2003 Published by Elsevier B.V. doi:10.1016/j.jbbm.2003.11.004
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1. Introduction As a selection of site-directed chemical modification, polyethylene glycol (PEG) has been widely used to modify pharmaceutical proteins to lower their immunogenicity and/or prolong their blood retention time for therapeutic use [1]. Protein PEGylation generates a heterogeneous mixture of proteins with different number of PEG chains attached. The linear PEG polymers are usually a mixture within certain ranges of molecular weight distribution. Their hydrodynamic radius is equal to those of the globular proteins having molecular weights four to five times of PEG [2]. These two properties of PEG complicate the analytical methodology of PEGylated proteins. Methods for the analysis of PEGylated proteins, such as 2,4,6-trinitrobenzenesulfonic acid (TNBS) spectrometry [3], SDS-PAGE [4], HPLC [5], MALDI-MS [6] and NMR [7], have been explored with varying success, but none was accurate and convenient enough for general acceptance [8,9]. For examples, HPLC is routinely used to separate the PEGylated proteins from the native proteins and free PEG, but its resolution is generally not satisfactory to resolve the heterogeneous fractions with different PEG chains attached [5,10]. MALDI-MS can determine the accurate molecular weights of the PEGylated proteins or their trypsin-digested fragments to investigate the exact sites of the PEGylation on the proteins [10]; however, its ability for quantitative analysis is not fully identified since the protein fractions with different PEG chains attached might have different intensities to be ionized from the matrix [11] and its signal/noise ratio needs to be improved further [6,12]. A relatively new choice is using capillary electrophoresis (CE) for the purpose [9,13]. Compared to other methods, there are several advantages to use CE to analyze the PEGylated proteins, including its high resolution, short operation time, and tiny sample requirement (picomole level of sample required every injection). More recently, a spectrophotometric analysis for determination of the average number of PEG chains attached onto proteins were developed by utilizing pronase to digest the PEGylated proteins and release the attached PEG chains before the biphasic assay of the PEG amount [14]. Although the analysis gave similar accuracy to the CE method on the analysis of the average number of PEG chains attached to proteins [14], the pronase digestion step complicates the analysis, and the spectrophotometric method cannot give information on the heterogeneous distributions of the PEGylated fractions compared to the CE method [9], which is important for the quality control of protein PEGylation. Nevertheless, quantitative studies on the reaction characteristics of protein PEGylation by CE are far from accomplishment to date. In this study, we used CE to quantitatively characterize ribonuclease A (RNase) modified with succinimidyl succinate-activated monomethoxypolyethylene glycol (MPEG-SS), which is one of the most widely accepted modifier for its fast reaction rate and low toxicity [15]. RNase is not only a well-characterized model for protein chemistry research, but also developed as a potential antivirus reagent after PEGylation [16,17]. The average modification degree of the PEGylated mixture was calculated from CE analysis, which met with the result given by TNBS assay. The influence of the reaction molar ratio of activated MPEG to protein on the PEGylation degree was investigated quantitatively as well as the influence of pH value of the reaction buffer. The time course of protein PEGylation was resolved by CE.
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2. Materials and methods 2.1. Materials Bovine pancreatic ribonuclease A (RNase) with Mw 13,700 Da, N-hydroxysuccinimide (NHS) and 2,4,6-trinitrobenzenesulfonic acid (TNBS) were purchased from Sigma (St. Louis, MO, USA). Methoxypolyethylene glycol (MPEG) (Mw 5000 F 250 Da) was obtained from Union Carbide (Danbury, CT, USA). Polyethylene oxide (PEO) of 8000 kDa was from Aldrich (Milwaukee, WI, USA). Acetonitrile of HPLC grade was from Fisher (Fair Lawn, NJ, USA). 2.2. PEGylation of proteins The MPEG with one hydroxyl group should be activated to methoxypolyethylene glycolyl N-succinimidyl succinate (MPEG-SS) before reaction with proteins (Fig. 1). MPEG-SS was prepared according to the previous procedure [9]. For protein modification, special amount of MPEG-SS was added to 2 mg/ml of RNase dissolved in 0.05 M borate buffer at particular pH. The mixture was agitated rapidly under room temperature for half an hour. Various PEGylation degrees were achieved by adding MPEG-SS to RNase at different molar ratio. The PEGylation process could be terminated by using a 1:1 dilution of the reaction mixture with 0.05 M sodium phosphate buffer, pH 3.0.
Fig. 1. Mechanism of protein PEGylation (a), protein de-PEGylation (b) and MPEG-SS hydrolysis (c). The ester linkage between MPEG and protein was circled by a broken line.
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2.3. CE operation CE was performed on a P/ACEk 5000 capillary electrophoresis system (Beckman, Fullerton, CA, USA) equipped with a fused silica capillary (inner diameter 50 Am; 37 cm total length; 30 cm effective length from the sampling end to the detector, Yongnian Optical Fiber Factory, Hebei, China). UV detection was set at 200 nm and separation voltage at + 30 kV. The capillary temperature was maintained at 25 F 0.1 jC by the thermostated coolant from Beckman. Between every run, the capillary was thoroughly rinsed by 0.1 M NaOH for 4 min and water for 4 min. Then, 2 mg/ml of PEO 8000 kDa in 0.1 M HCl was used as a dynamic coating solution to rinse the capillary for 4 min. The sodium phosphate buffer (0.025 M, pH 2.5) prepared with the binary solvent of acetonitrile – water (1:1 v/v) was applied as the separation buffer after being used to rinse the capillary for 3 min. The rinse pressure was 20 psi (1 psi = 6894.74 Pa). Sample solutions were injected hydrodynamically at 0.5 psi pressure for 8 s (injection volume 13 nl, calculated with Beckman CE Expert 1.0 software). More details on the CE operation, the accuracy and reproducibility were described elsewhere [9]. 2.4. TNBS assay RNase has 10 q-amino groups on the lysine residues and one a-amino group on the Nterminal which are available for the PEGylation. The average number of MPEG chains attached per protein was calculated by multiplying the total number of reactive amino groups on a protein molecule (11 for RNase) with the fraction of amines consumed by the PEGylation, which was determined by TNBS assay [3].
3. Results and discussion 3.1. CE analysis of PEGylated proteins We used capillary zone electrophoresis (CZE) mode to characterize the mixture of PEGylated RNase. Typical electropherograms of the mixture sampled by different PEGylation time are shown in Fig. 2. In CZE mode, the PEGylated proteins move towards the cathode in the following order: the native protein, protein with one MPEG attached, protein with two MPEG attached, and so on [9,13,18]. Along with the increase of PEGylation degree, the resolution of PEGylated proteins decreased (Fig. 2). However, the peak area could be calculated according to the peak Fig. 2. Capillary electropherogram of PEGylated RNase sampled at different reaction times. The reaction time is 0.5 min (a), 7 min (b), 15 min (c) and 35 min (d), respectively. Protein PEGylation was performed with 2 mg/ml of RNase in 0.05 M borate buffer at pH 8.4. The MPEG-SS/RNase molar ratio for the reaction was 5:1. Samples were prepared by using an equivolume dilution of the PEGylation mixture with 0.05 M sodium phosphate buffer (pH 3.0) at particular time intervals. Numbers (i) over peaks correspond to the number of MPEG chains per protein molecule. The peaks are divided from the peak shoulders by the perpendicular broken lines (d) to illustrate the quantitative calculation on the peak area.
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shoulder integration method by dropping perpendiculars from peak shoulders to baseline [13]. The corrected peak area, calculated by dividing the raw peak area by the peak migration time, was used to quantitatively evaluate the protein fractions in the PEGylated mixture. The average degree of protein PEGylation determined by CE was calculated from the following equation: X a¼ i ðAi %Þ ð1Þ where a is the average number of MPEG chains attached to a protein molecule; Ai is the percent of corrected peak area of peak i, corresponding to the protein with i MPEG chains attached (Fig. 2). 3.2. Influence of reaction molar ratio on protein PEGylation degree In Fig. 3, we compared the PEGylation degree (a) given by CE analysis (Eq. (1)) and the corresponding result from TNBS assay. The protein PEGylation degree increased along with the ratio of MPEG-SS/RNase. On the contrast, the enzymatic activity of PEGylated RNase decreased along with the increase of the PEGylation degree (data not shown). When the ratio of MPEG-SS/RNase exceeded 5:1, almost all the native RNase was modified, which could be detected by CE. Overall, both these two methods gave similar results on the average PEGylation degree. However, CE could further give the distribution of protein fractions attached by different number of MPEG chains (Fig. 2), which might be more useful in drug quality control of PEGylated proteins
Fig. 3. Influence of MPEG-SS/RNase molar ratio on PEGylation degree (a). The PEGylation degree was determined by CE (5) and TNBS assay (n), respectively. Protein PEGylation was performed with 2 mg/ml of RNase at different MPEG-SS/RNase molar ratio in 0.05 M borate buffer, pH 8.4 for 0.5 h. The molar ratio of MPEG-SS/RNase in the initial mixture for PEGylation was correlated to the protein PEGylation degree (a).
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compared to the spectrometric methods, including TNBS assay [3] and fluorescamine assay [19]. 3.3. Influence of pH on PEGylation degree of proteins and its stability We investigated the PEGylation degree of proteins modified in the pH range from 5.4 to 10.4 and the stability of corresponding samples after incubation for 24 h at defined pH environments (Fig. 4). When MPEG-SS/RNase molar ratio was 5:1, the PEGylation degree of samples before incubation increased from pH 5.4 to 9.4 and decreased from pH 9.4 to 10.4. All the PEGylation degree decreased after 24 h incubation of the samples in pH range 5.4 – 10.4, but slightly in pH 5.4 –8.4 and distinctly in pH 9.4 –10.4. The pH range 7.4 –8.4 kept highest PEGylation degree than others after 24 h incubation. When the MPEG-SS/RNase molar ratio was lowered to 3:1, the PEGylation degree was found lower than that of the molar ratio 5:1. The influence of pH on PEGylation degree could be explained by the reaction mechanism shown in Fig. 1. The PEGylation of proteins with MPEG-SS is based on nucleophilic substitution. Only the deprotonated amino groups (mainly consisted of qamino groups of lysine residues) on proteins could react with MPEG-SS [20]. Theoretically, the PEGylation degree would increase along with the buffer pH because the deprotonated amino groups on the protein would increase at high pH (Fig. 1). However, too high a pH would hydrolyze the succinimidyl ester bond of MPEG-SS before it reacted with proteins (see pathway c in Fig. 1), which limited the effective concentration of MPEG-SS and led to a low PEGylation degree at pH f 10.4. It is necessary to consider the stability of PEGylated proteins in different pH for pharmaceutical applications. We found that the PEGylation degree decreased after 24
Fig. 4. Influence of buffer pH on PEGylation degree (a). Protein PEGylation was performed with 2 mg/ml of RNase in 0.05 M borate buffer in pH 5.4 – 10.4. The molar ratio of MPEG-SS/RNase in the initial mixture for PEGylation was 5:1 (n, .) or 3:1 (E). The PEGylation degree was determined by CE, 0.5 h (n) or 24.5 h (., E) after PEGylation.
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h incubation (Fig. 4). This could be explained by the slow hydrolysis of the ester linkage between MPEG and the amino groups of the protein, which was named as ‘‘dePEGylation’’ process (see pathway b in Fig. 1). According to general theories, the ester linkage hydrolyzes more rapidly in high pH solution than in low pH solution after 24 h incubation (Fig. 4). It seems that the PEGylated protein is more stable in acidic or neutral solution than in alkaline solution. 3.4. Time course of protein PEGylation The kinetic time course of RNase PEGylation was monitored by CE (Fig. 5). It was found that the PEGylation process finished in f 30 min and the native protein decreased rapidly in the initial stage (0 –10 min). The production rates of the five PEGylated components decreased one by one in the initial stage, as MPEG1-RNase>MPEG2-RNase>MPEG3-RNase>MPEG4-RNase>MPEG5-RNase. The protein fraction with one MPEG chain (MPEG1 – RNase) had a maximum percent at f 5 min. The fraction with two MPEG chains (MPEG2 – RNase) had a maximum percent at f 10 min. The proportion of the PEGylated components kept steadily after PEGylation for about 30 min. For pharmaceutical proteins, when the PEGylation time course of each protein fraction is obtained, further optimization on the reaction could be done to let the desirable fractions achieve their maximum percent in the whole PEGylated mixture by evaluating the modifier/protein molar ratio or terminating the PEGylation process in specific time point when necessary. These optimizations would be beneficial to increase the yield and the recovery ratio of the specific PEGylated fractions in the later isolation steps.
Fig. 5. Time course of RNase PEGylation with MPEG-SS. The PEGylation process was monitored by CE with the same conditions as presented in Fig. 2. Protein fractions are expressed as their molar percentage (%) in the whole protein mixture including both the modified and unmodified proteins. The protein fractions with different number (0, 1, 2, 3, 4 and 5) of PEG chains attached per protein molecule are named as Native RNase (n), MPEG1-RNase (.), MPEG2-RNase (5), MPEG3-RNase (z), MPEG4-RNase (o), and MPEG5-RNase (E).
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We believe that the CE method could not only be considered as a quantitative tool to characterize the protein modification with PEG, but also with other types of chemical modifiers. Acknowledgements Special acknowledgement is given to Mrs. Yi Zhong (Science Press, Beijing) for her technical support. References [1] Lundblad RL, Bradshaw RA. Applications of site-specific chemical modification in the manufacture of biopharmaceuticals: I. An overview. Biotechnol Appl Biochem 1997;26:143 – 51. [2] Jackson CJ, Charlton JL, Kuzminski K, Lang GM, Sehon AH. Synthesis, isolation, and characterization of conjugates of ovalbumin with monomethoxypolyethylene glycol using cyanuric chloride as the coupling agent. Anal Biochem 1987;165:114 – 27. [3] Snyder SL, Sobocinski PZ. An improved 2,4,6-trinitrobenzenesulfonic acid method for the determination of amines. Anal Biochem 1975;64:284 – 8. [4] Kurfurst MM. Detection and molecular weight determination of polyethylene glycol-modified hirudin by staining after sodium dodecyl sulfate – polyacrylamide gel electrophoresis. Anal Biochem 1992;200:244 – 8. [5] McGoff P, Baziotis AC, Maskiewicz R. Analysis of polyethylene glycol modified superoxide dismutase by chromatographic, electrophoretic, light scattering, chemical and enzymatic methods. Chem Pharm Bull (Tokyo) 1988;36:3079 – 91. [6] Watson E, Shah B, DePrince R, Hendren RW, Nelson R. Matrix-assisted laser desorption mass spectrometric analysis of a pegylated recombinant protein. Biotechniques 1994;16:278 – 81. [7] Banci L, Bertini I, Caliceti P, Monsu Scolaro L, Schiavon O, Veronese FM. Spectroscopic characterization of polyethyleneglycol modified superoxide dismutase: 1H NMR studies on its Cu2Co2 derivative. J Inorg Biochem 1990;39:149 – 59. [8] Roberts MJ, Harris JM. Attachment of degradable poly(ethylene glycol) to proteins has the potential to increase therapeutic efficacy. J Pharm Sci 1998;87:1440 – 5. [9] Li W, Zhong Y, Lin B, Su Z. Characterization of polyethylene glycol-modified proteins by semi-aqueous capillary electrophoresis. J Chromatogr, A 2001;905:299 – 307. [10] Lee H, Park TG. A novel method for identifying PEGylation sites of protein using biotinylated PEG derivatives. J Pharm Sci 2003;92:97 – 103. [11] Mann M, Hendrickson RC, Pandey A. Analysis of proteins and proteomes by mass spectrometry. Annu Rev Biochem 2001;70:437 – 73. [12] Pettit DK, Bonnert TP, Eisenman J, Srinivasan S, Paxton R, Beers C, et al. Structure-function studies of interleukin 15 using site-specific mutagenesis, polyethylene glycol conjugation, and homology modeling. J Biol Chem 1997;272:2312 – 8. [13] Bullock J, Chowdhury S, Severdia A, Sweeney J, Johnston D, Pachla L. Comparison of results of various methods used to determine the extent of modification of methoxy polyethylene glycol 5000-modified bovine cupri-zinc superoxide dismutase. Anal Biochem 1997;254:254 – 62. [14] Barker TH, Klinger MM, Feldman DS, Fuller GM, Hagood JS. Spectrophotometric analysis for determining the average number of poly(ethylene) glycol molecules on PEGylated proteins utilizing a protein digestion step. Anal Biochem 2001;290:382 – 5. [15] Zalipsky S, Lee C. Use of functionalized poly(ethylene glycol)s for modification of polypeptides. In: Harris JM, editor. Poly(ethylene glycol) chemistry: biotechnical and biomedical applications. New York: Plenum; 1992. p. 347 – 70. [16] Laznicek M, Schiavon O, Caliceti P, Veronese FM. Pharmacokinetics and distribution of ribonuclease and its monomethoxypoly(ethylene glycol) derivatives in rats. Pharmacol Res 1993;28:153 – 61.
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[17] Schiavon O, Caliceti P, Sartore L, Veronese FM. Surface modification of enzymes for therapeutic use: monomethoxypoly (ethylene glycol) derivatization of ribonuclease. Farmaco 1991;46:967 – 78. [18] Cunico RL, Gruhn V, Kresin L, Nitecki DE, Wiktorowicz JE. Characterization of polyethylene glycol modified proteins using charge-reversed capillary electrophoresis. J Chromatogr, A 1991;559:467 – 77. [19] Stocks SJ, Jones AJ, Ramey CW, Brooks DE. A fluorometric assay of the degree of modification of protein primary amines with polyethylene glycol. Anal Biochem 1986;154:232 – 4. [20] Lundblad RL, Noyes CM. Chemical reagents for protein modification. Boca Raton: CRC Press; 1984. p. 127.
Quantitatively investigating monomethoxypolyethylene glycol modification of protein by capillary electrophoresis Weijun Li 1, Zhiguo Su * State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, P.O. Box 353, Beijing 100080, PR China Received 22 June 2003; received in revised form 10 November 2003; accepted 17 November 2003
Abstract Capillary electrophoresis (CE) was applied to study quantitatively protein modification with succinimidyl succinate-activated monomethoxypolyethylene glycol (MPEG-SS). The heterogeneous distribution of modified proteins and the average modification degree were determined by CE, and the latter met with the results from 2,4,6-trinitrobenzenesulfonic acid (TNBS) spectrometric assay. It was found that the optimal buffer pH for the modification was between pH 7.4 and 8.4, and the modification degree decreased when the modified sample was preserved in high pH solutions. The protein fractions attached with different number of polyethylene glycols (PEGs) were monitored along the process of protein modification. CE was proved to be efficient to evaluate quantitatively several factors of the protein modification, including the modifier/protein molar ratio, the stability of conjugates in different pH environments, and the time course of modification process. D 2003 Published by Elsevier B.V. Keywords: Capillary electrophoresis; Chemical modification; Polyethylene glycol; Protein
* Corresponding author. Tel.: +86-10-6256-1817; fax: +86-10-6256-1813. E-mail addresses: [email protected] (W. Li), [email protected] (Z. Su). 1 Present address: Department of Biopharmaceutical Sciences, School of Pharmacy, University of California, Room HSE-1145, 513 Parnassus Avenue, San Francisco, CA 94143-0446, USA. 0165-022X/$ - see front matter D 2003 Published by Elsevier B.V. doi:10.1016/j.jbbm.2003.11.004
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1. Introduction As a selection of site-directed chemical modification, polyethylene glycol (PEG) has been widely used to modify pharmaceutical proteins to lower their immunogenicity and/or prolong their blood retention time for therapeutic use [1]. Protein PEGylation generates a heterogeneous mixture of proteins with different number of PEG chains attached. The linear PEG polymers are usually a mixture within certain ranges of molecular weight distribution. Their hydrodynamic radius is equal to those of the globular proteins having molecular weights four to five times of PEG [2]. These two properties of PEG complicate the analytical methodology of PEGylated proteins. Methods for the analysis of PEGylated proteins, such as 2,4,6-trinitrobenzenesulfonic acid (TNBS) spectrometry [3], SDS-PAGE [4], HPLC [5], MALDI-MS [6] and NMR [7], have been explored with varying success, but none was accurate and convenient enough for general acceptance [8,9]. For examples, HPLC is routinely used to separate the PEGylated proteins from the native proteins and free PEG, but its resolution is generally not satisfactory to resolve the heterogeneous fractions with different PEG chains attached [5,10]. MALDI-MS can determine the accurate molecular weights of the PEGylated proteins or their trypsin-digested fragments to investigate the exact sites of the PEGylation on the proteins [10]; however, its ability for quantitative analysis is not fully identified since the protein fractions with different PEG chains attached might have different intensities to be ionized from the matrix [11] and its signal/noise ratio needs to be improved further [6,12]. A relatively new choice is using capillary electrophoresis (CE) for the purpose [9,13]. Compared to other methods, there are several advantages to use CE to analyze the PEGylated proteins, including its high resolution, short operation time, and tiny sample requirement (picomole level of sample required every injection). More recently, a spectrophotometric analysis for determination of the average number of PEG chains attached onto proteins were developed by utilizing pronase to digest the PEGylated proteins and release the attached PEG chains before the biphasic assay of the PEG amount [14]. Although the analysis gave similar accuracy to the CE method on the analysis of the average number of PEG chains attached to proteins [14], the pronase digestion step complicates the analysis, and the spectrophotometric method cannot give information on the heterogeneous distributions of the PEGylated fractions compared to the CE method [9], which is important for the quality control of protein PEGylation. Nevertheless, quantitative studies on the reaction characteristics of protein PEGylation by CE are far from accomplishment to date. In this study, we used CE to quantitatively characterize ribonuclease A (RNase) modified with succinimidyl succinate-activated monomethoxypolyethylene glycol (MPEG-SS), which is one of the most widely accepted modifier for its fast reaction rate and low toxicity [15]. RNase is not only a well-characterized model for protein chemistry research, but also developed as a potential antivirus reagent after PEGylation [16,17]. The average modification degree of the PEGylated mixture was calculated from CE analysis, which met with the result given by TNBS assay. The influence of the reaction molar ratio of activated MPEG to protein on the PEGylation degree was investigated quantitatively as well as the influence of pH value of the reaction buffer. The time course of protein PEGylation was resolved by CE.
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2. Materials and methods 2.1. Materials Bovine pancreatic ribonuclease A (RNase) with Mw 13,700 Da, N-hydroxysuccinimide (NHS) and 2,4,6-trinitrobenzenesulfonic acid (TNBS) were purchased from Sigma (St. Louis, MO, USA). Methoxypolyethylene glycol (MPEG) (Mw 5000 F 250 Da) was obtained from Union Carbide (Danbury, CT, USA). Polyethylene oxide (PEO) of 8000 kDa was from Aldrich (Milwaukee, WI, USA). Acetonitrile of HPLC grade was from Fisher (Fair Lawn, NJ, USA). 2.2. PEGylation of proteins The MPEG with one hydroxyl group should be activated to methoxypolyethylene glycolyl N-succinimidyl succinate (MPEG-SS) before reaction with proteins (Fig. 1). MPEG-SS was prepared according to the previous procedure [9]. For protein modification, special amount of MPEG-SS was added to 2 mg/ml of RNase dissolved in 0.05 M borate buffer at particular pH. The mixture was agitated rapidly under room temperature for half an hour. Various PEGylation degrees were achieved by adding MPEG-SS to RNase at different molar ratio. The PEGylation process could be terminated by using a 1:1 dilution of the reaction mixture with 0.05 M sodium phosphate buffer, pH 3.0.
Fig. 1. Mechanism of protein PEGylation (a), protein de-PEGylation (b) and MPEG-SS hydrolysis (c). The ester linkage between MPEG and protein was circled by a broken line.
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2.3. CE operation CE was performed on a P/ACEk 5000 capillary electrophoresis system (Beckman, Fullerton, CA, USA) equipped with a fused silica capillary (inner diameter 50 Am; 37 cm total length; 30 cm effective length from the sampling end to the detector, Yongnian Optical Fiber Factory, Hebei, China). UV detection was set at 200 nm and separation voltage at + 30 kV. The capillary temperature was maintained at 25 F 0.1 jC by the thermostated coolant from Beckman. Between every run, the capillary was thoroughly rinsed by 0.1 M NaOH for 4 min and water for 4 min. Then, 2 mg/ml of PEO 8000 kDa in 0.1 M HCl was used as a dynamic coating solution to rinse the capillary for 4 min. The sodium phosphate buffer (0.025 M, pH 2.5) prepared with the binary solvent of acetonitrile – water (1:1 v/v) was applied as the separation buffer after being used to rinse the capillary for 3 min. The rinse pressure was 20 psi (1 psi = 6894.74 Pa). Sample solutions were injected hydrodynamically at 0.5 psi pressure for 8 s (injection volume 13 nl, calculated with Beckman CE Expert 1.0 software). More details on the CE operation, the accuracy and reproducibility were described elsewhere [9]. 2.4. TNBS assay RNase has 10 q-amino groups on the lysine residues and one a-amino group on the Nterminal which are available for the PEGylation. The average number of MPEG chains attached per protein was calculated by multiplying the total number of reactive amino groups on a protein molecule (11 for RNase) with the fraction of amines consumed by the PEGylation, which was determined by TNBS assay [3].
3. Results and discussion 3.1. CE analysis of PEGylated proteins We used capillary zone electrophoresis (CZE) mode to characterize the mixture of PEGylated RNase. Typical electropherograms of the mixture sampled by different PEGylation time are shown in Fig. 2. In CZE mode, the PEGylated proteins move towards the cathode in the following order: the native protein, protein with one MPEG attached, protein with two MPEG attached, and so on [9,13,18]. Along with the increase of PEGylation degree, the resolution of PEGylated proteins decreased (Fig. 2). However, the peak area could be calculated according to the peak Fig. 2. Capillary electropherogram of PEGylated RNase sampled at different reaction times. The reaction time is 0.5 min (a), 7 min (b), 15 min (c) and 35 min (d), respectively. Protein PEGylation was performed with 2 mg/ml of RNase in 0.05 M borate buffer at pH 8.4. The MPEG-SS/RNase molar ratio for the reaction was 5:1. Samples were prepared by using an equivolume dilution of the PEGylation mixture with 0.05 M sodium phosphate buffer (pH 3.0) at particular time intervals. Numbers (i) over peaks correspond to the number of MPEG chains per protein molecule. The peaks are divided from the peak shoulders by the perpendicular broken lines (d) to illustrate the quantitative calculation on the peak area.
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shoulder integration method by dropping perpendiculars from peak shoulders to baseline [13]. The corrected peak area, calculated by dividing the raw peak area by the peak migration time, was used to quantitatively evaluate the protein fractions in the PEGylated mixture. The average degree of protein PEGylation determined by CE was calculated from the following equation: X a¼ i ðAi %Þ ð1Þ where a is the average number of MPEG chains attached to a protein molecule; Ai is the percent of corrected peak area of peak i, corresponding to the protein with i MPEG chains attached (Fig. 2). 3.2. Influence of reaction molar ratio on protein PEGylation degree In Fig. 3, we compared the PEGylation degree (a) given by CE analysis (Eq. (1)) and the corresponding result from TNBS assay. The protein PEGylation degree increased along with the ratio of MPEG-SS/RNase. On the contrast, the enzymatic activity of PEGylated RNase decreased along with the increase of the PEGylation degree (data not shown). When the ratio of MPEG-SS/RNase exceeded 5:1, almost all the native RNase was modified, which could be detected by CE. Overall, both these two methods gave similar results on the average PEGylation degree. However, CE could further give the distribution of protein fractions attached by different number of MPEG chains (Fig. 2), which might be more useful in drug quality control of PEGylated proteins
Fig. 3. Influence of MPEG-SS/RNase molar ratio on PEGylation degree (a). The PEGylation degree was determined by CE (5) and TNBS assay (n), respectively. Protein PEGylation was performed with 2 mg/ml of RNase at different MPEG-SS/RNase molar ratio in 0.05 M borate buffer, pH 8.4 for 0.5 h. The molar ratio of MPEG-SS/RNase in the initial mixture for PEGylation was correlated to the protein PEGylation degree (a).
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compared to the spectrometric methods, including TNBS assay [3] and fluorescamine assay [19]. 3.3. Influence of pH on PEGylation degree of proteins and its stability We investigated the PEGylation degree of proteins modified in the pH range from 5.4 to 10.4 and the stability of corresponding samples after incubation for 24 h at defined pH environments (Fig. 4). When MPEG-SS/RNase molar ratio was 5:1, the PEGylation degree of samples before incubation increased from pH 5.4 to 9.4 and decreased from pH 9.4 to 10.4. All the PEGylation degree decreased after 24 h incubation of the samples in pH range 5.4 – 10.4, but slightly in pH 5.4 –8.4 and distinctly in pH 9.4 –10.4. The pH range 7.4 –8.4 kept highest PEGylation degree than others after 24 h incubation. When the MPEG-SS/RNase molar ratio was lowered to 3:1, the PEGylation degree was found lower than that of the molar ratio 5:1. The influence of pH on PEGylation degree could be explained by the reaction mechanism shown in Fig. 1. The PEGylation of proteins with MPEG-SS is based on nucleophilic substitution. Only the deprotonated amino groups (mainly consisted of qamino groups of lysine residues) on proteins could react with MPEG-SS [20]. Theoretically, the PEGylation degree would increase along with the buffer pH because the deprotonated amino groups on the protein would increase at high pH (Fig. 1). However, too high a pH would hydrolyze the succinimidyl ester bond of MPEG-SS before it reacted with proteins (see pathway c in Fig. 1), which limited the effective concentration of MPEG-SS and led to a low PEGylation degree at pH f 10.4. It is necessary to consider the stability of PEGylated proteins in different pH for pharmaceutical applications. We found that the PEGylation degree decreased after 24
Fig. 4. Influence of buffer pH on PEGylation degree (a). Protein PEGylation was performed with 2 mg/ml of RNase in 0.05 M borate buffer in pH 5.4 – 10.4. The molar ratio of MPEG-SS/RNase in the initial mixture for PEGylation was 5:1 (n, .) or 3:1 (E). The PEGylation degree was determined by CE, 0.5 h (n) or 24.5 h (., E) after PEGylation.
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h incubation (Fig. 4). This could be explained by the slow hydrolysis of the ester linkage between MPEG and the amino groups of the protein, which was named as ‘‘dePEGylation’’ process (see pathway b in Fig. 1). According to general theories, the ester linkage hydrolyzes more rapidly in high pH solution than in low pH solution after 24 h incubation (Fig. 4). It seems that the PEGylated protein is more stable in acidic or neutral solution than in alkaline solution. 3.4. Time course of protein PEGylation The kinetic time course of RNase PEGylation was monitored by CE (Fig. 5). It was found that the PEGylation process finished in f 30 min and the native protein decreased rapidly in the initial stage (0 –10 min). The production rates of the five PEGylated components decreased one by one in the initial stage, as MPEG1-RNase>MPEG2-RNase>MPEG3-RNase>MPEG4-RNase>MPEG5-RNase. The protein fraction with one MPEG chain (MPEG1 – RNase) had a maximum percent at f 5 min. The fraction with two MPEG chains (MPEG2 – RNase) had a maximum percent at f 10 min. The proportion of the PEGylated components kept steadily after PEGylation for about 30 min. For pharmaceutical proteins, when the PEGylation time course of each protein fraction is obtained, further optimization on the reaction could be done to let the desirable fractions achieve their maximum percent in the whole PEGylated mixture by evaluating the modifier/protein molar ratio or terminating the PEGylation process in specific time point when necessary. These optimizations would be beneficial to increase the yield and the recovery ratio of the specific PEGylated fractions in the later isolation steps.
Fig. 5. Time course of RNase PEGylation with MPEG-SS. The PEGylation process was monitored by CE with the same conditions as presented in Fig. 2. Protein fractions are expressed as their molar percentage (%) in the whole protein mixture including both the modified and unmodified proteins. The protein fractions with different number (0, 1, 2, 3, 4 and 5) of PEG chains attached per protein molecule are named as Native RNase (n), MPEG1-RNase (.), MPEG2-RNase (5), MPEG3-RNase (z), MPEG4-RNase (o), and MPEG5-RNase (E).
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