The ph-dependent formation of PEGylated bovine lactoferrin by. branched polyethylene glycol (PEG)-N-hydroxysuccinimide (NHS)

Transcription

1 Biol. Pharm. Bull. Notes Miscellaneous The ph-dependent formation of PEGylated bovine lactoferrin by branched polyethylene glycol (PEG)-N-hydroxysuccinimide (NHS) active esters Yasuhiro NOJIMA, 1) Kazuma IGUCHI, Yosuke SUZUKI, Atsushi SATO* School of Bioscience and Biotechnology, Tokyo University of Technology, Katakura, Hachioji, Tokyo , Japan *To whom correspondence should be addressed.

2 Summary PEGylation is an established method for improving the pharmacokinetic properties and pharmacodynamic effects of therapeutic proteins. Amino conjugation by N-hydroxysuccinimide activated polyethylene glycol (PEG-NHS) represents the most common modification of the target proteins. In this study, we compared the reaction velocities of ph-dependent preparations of bovine lactoferrin modified with branched PEG-NHS conducted at ph 7.4 to ph 9.0. PEGylation reaction rates were dependent on ph value, but not on PEG molecular mass. At ph 7.4, reactions advanced gradually and reached steady state by 2 h, whereas at ph 9.0, reactions advanced very quickly and reached steady state within 10 min. Hydrolysis half-life of PEG-NHS exceeded 120 min at ph 7.4, but was below 9 min at ph 9.0. Thus, the ph value is one of the most important factors to obtain the optimal condition for PEGylation by NHS esters. Keywords: N-hydroxysuccinimide (NHS); PEGylation ; ph; Kinetics; Lactoferrin

3 Introduction Lactoferrin (LF) is an 80-kDa member of the transferrin family of iron-binding glycoproteins and is found in various biological fluids, especially milk. 2) The multifunctional LF protein exhibits a wide variety of activities including immunomodulation, 3) iron binding, 4) anti-microbial, 5) anti-inflammatory, 6,7) cell proliferation 8) and anti-oxidation. 9) This multitude of biological activities has created great interest in the use of LF in drug discovery. 10) Recently, we have developed PEGylated LF with high biological activities and enhanced pharmacokinetics properties. 11) PEGylation refers to the modification of biological molecules by the linking of one or more polyethylene glycol (PEG) groups. This approach is utilized in many pharmaceutical and biotechnical applications. 12,13) N-hydroxysuccinimide (NHS) active esters of PEG (PEG-NHS) are frequently used for the amino group modification of target proteins. NHS activated esters produce stable amide linkages between PEG and primary amines such as N-terminal α-amine and lysine ε-amine residues. PEG-NHS typically couples to the free amino group of the targeted protein at physiological ph, ranging from ph 7 to 9. NHS active esters are known to possess highly hydrolytic activities under basic conditions.

4 Although bioconjugation by NHS active esters is generally considered to be the mechanism of PEGylation, detailed reports of actual PEGylated reactions by NHS active esters are rare. Thus, the aim of this study was to investigate the kinetics of the conjugation reaction between branched PEG-NHS and bovine lactoferrin (blf), focusing on ph dependency. The present investigation demonstrates the importance of the ph values in the PEGylated reactions.

5 Materials and methods ph-dependent PEGylation of bovine lactoferrin (blf) PEGylated reaction mixtures contained blf (MG nutritionals, Melbourne, Australia) and branched 40-kDa PEG-NHS reagent (SUNBRIGHT GL2-400GS2, NOF Corporation, Tokyo, Japan) at a 1:10 molar ratio. Reaction mixtures were dissolved in the following buffers at the indicated ph: 50 mm acetate buffer (ph 4.0 and 5.0); 50 mm phosphate buffer (ph 6.0, 7.0 and 8.0); or 50 mm borate buffer (ph 9.0). The final blf concentration was 0.5 mg/ml. Reactions were carried out for 1 h at 25 C and stopped by the addition of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer. The reaction mixtures were then subjected to 7.5% SDS-PAGE under non-reducing conditions and visualized by Coomassie Brilliant Blue (CBB) staining. Conjugation reaction kinetics in buffers of different ph values Two kinds of branched PEG-NHS with average molecular weights of 20 kda (SUNBRIGHT GL2-200GS2, NOF Corporation, Tokyo, Japan) or 40 kda (see above) were

6 used. Conjugation reactions were carried out as described above except that the incubation time was 24 h, and the buffers used were phosphate-buffered saline (PBS, ph 7.4), 50 mm borate (ph 9.0) and Good s buffers (BICINE, CHES and TAPS, ph 9.0). Reaction samples (corresponding to 2 µg blf) were taken at indicated times and subjected to 7.5% SDS-PAGE. After CBB staining, intensities of CBB-stained bands were analyzed by Image J software and represented as graphs. Hydrolysis half-life of PEG-NHS at ph 7.4 and 9.0 Conjugation reactions were carried out as described above except that each PEG-NHS was pre-incubated at ph 7.4 (PBS) or 9.0 (50 mm borate buffer) for the indicated times, and then mixed with blf (final concentration 0.5 mg/ml) to start the coupling reactions. PEGylation was carried out for 2 h at ph 7.4 (PBS) and for 1 h at ph 9.0 (50 mm borate buffer). Reactions were stopped by the addition of SDS-PAGE loading buffer. Reactants (corresponding to 2 µg blf) were analyzed by 7.5% SDS-PAGE and visualized by CBB

7 staining. The intensities of CBB-stained bands were analyzed by Image J software and represented as graphs

8 Results and discussion The ph-dependent conjugation of blf with branched PEG-NHS We used two kinds of branched PEG-NHS with average molecular weights of 20 kda and 40 kda. The reaction formula for the PEGylation of blf is illustrated in Fig. 1. First, conjugation of 40-kDa PEG-NHS to blf at various ph values was investigated. Fig. 2 shows the SDS-PAGE profile of PEGylation reaction mixtures in various buffers at different ph values. Although PEGylated blf was scarcely formed under acidic conditions (ph 4.0, 5.0 and 6.0), it readily formed at ph 7.0 and above (as indicated by black arrows). The mono-pegylated blf migrated on SDS-PAGE with a higher apparent molecular mass (approximately 200 kda) than the calculated one (40 kda for the branched PEG and 80 kda for blf). This increase in apparent molecular mass is due to the contribution of PEG s large hydrodynamic volume. 14) The conjugation yield of PEGylated blf increased with increasing ph. Furthermore, oligo-pegylated blf by-product (as indicated by gray arrows) was observed at ph 8.0 and above, which suggested that the activation of NHS active esters occurred in a ph-dependent manner. Similar results were obtained using the 20-kDa

9 PEG-NHS derivative (data not shown). Thus, PEG-bLF formation using PEG-NHS derivatives occurred in a ph dependent manner. Kinetic analysis of PEGylated blf with branched PEG-NHS at ph 7.4 and 9.0 Next, we compared the kinetics of conjugation reactions performed in a buffer at neutral ph (PBS at ph 7.4) to reactions at a higher ph (50 mm borate buffer at ph 9.0). Reaction products were analyzed by SDS-PAGE, as shown in Fig. 3A-D. Intensities of the CBB-stained bands were analyzed by Image J software (Fig. 3E and F). At ph 7.4, the reactions involving 20-kDa and 40-kDa PEG-NHS proceeded smoothly up to 2 h, with reaction curves reaching a plateau thereafter (Fig. 3E). In contrast, at ph 9.0, the 20-kDa and 40-kDa PEG-NHS reactions were so fast that steady states were reached within 10 min (Fig. 3F). Regardless of the molecular mass of the PEG, very similar conjugation kinetics were observed (Fig 3E and F). To determine whether the rapid reaction rate at ph 9.0 depended on ph value or on a component of the borate buffer, we carried out conjugation reactions of 20-kDa and 40-kDa PEG-NHS to blf in three different Good s buffers (BICINE, CHES and

10 TAPS, all at ph 9.0) under the same experimental conditions as used for the borate buffer (ph 9.0). For both 20-kDa and 40-kDa PEG-NHS, reaction rates of PEG-NHS with blf in BICINE, CHES and TAPS buffers (ph 9.0) were very similar to those observed in borate buffer (ph 9.0) (data not shown), which suggested that rapid PEGylated reactions at ph 9.0 are simply dependent on ph value. Hydrolysis half lives of branched PEG-NHS at ph 7.4 and 9.0 The PEG-NHS reaction rate is the result of the balance between the gain in reaction velocity and the loss of activated NHS esters by hydrolysis. Therefore, we determined the hydrolysis half-lives of branched 20-kDa and 40-kDa PEG-NHS at ph 7.4 and 9.0 (Fig. 4A-C). The hydrolysis half-lives of 20-kDa and 40-kDa PEG-NHS at ph 7.4 (PBS) were estimated to be approximately 128 min and 166 min, respectively. However, at ph 9.0 (borate buffer), the hydrolysis half-lives of 20-kDa and 40-kDa PEG-NHS were approximately 9.0 min and 5.0 min, respectively. Therefore, hydrolysis rates of 20-kDa and 40-kDa PEG-NHS at ph 9.0 were and 33.2-fold higher, respectively, than those at ph

11 7.4. In summary, reaction and hydrolysis rates of PEG-NHS are relatively slow at near neutral ph (ph 7.4), but were much faster under alkaline conditions (ph 9.0). The reaction and hydrolysis rates of PEG-NHS were ph-dependent, but not dependent on the molecular mass of the PEG. As conjugation reaction rates were relatively slow at a near neutral ph, PEGylation could be easily regulated. However, the reaction and hydrolysis rates of PEG-NHS were fast under alkaline conditions, resulting in completion of the conjugation reaction in a relatively short time. Understanding of the ph-dependent properties of PEG-NHS is important to determine the optimal conditions for PEGylation. It is worthwhile to note that other parameters, such as temperatures, molecular ratios of PEG-NHS to the target protein and protein concentrations in the reaction mixture, are also essential for obtaining the optimal conditions for PEGylation (data not shown).

12 Acknowledgement We thank Mr. Hirohiko Shimizu (NRL Pharma, Inc., Kanagawa, Japan) for helpful suggestions. This work was supported in part by grants from New Energy and Industrial Technology Development Organization (NEDO).

13 References 1) Present address: Department of Virology, Kitasato Research Center of Environmental Sciences, Kitazato, Sagamihara, Kanagawa , Japan 2) Levay P. F., Viljoen M., Haematologica, 80, (1995). 3) Yamauchi K., Wakabayashi H., Hashimoto S., Teraguchi S., Hayasawa H., Tomita M., Adv. Exp. Med. Biol., 443, (1998). 4) Chierici R., Sawatzki G., Tamisari L., Volpato S., Vigi V., Acta Paediatr., 81, (1992). 5) Arnold R. R., Brewe M., Gauthier J. J., Infect. Immun., 28, (1980). 6) Mattsby-Baltzer I., Roseanu A., Motas C., Elverfors J., Engberg I., Hanson L. A., Pediatr.

14 Res., 40, (1996). 7) Håversen L., Ohlsson B. G., Hahn-Zoric M., Hanson L. A., Mattsby-Baltzer I., Cell Immunol., 220, (2002). 8) Nichols B. L., McKee K. S., Henry J. F., Putman M., Pediatr. Res., 21, (1987). 9) Ambruso D. R., Johnston R. B. Jr., Clin. Invest., 67, (1981). 10) Jonasch E., Stadler W. M., Bukowski R. M., Hayes T. G., Varadhachary A., Malik R., Figlin R. A., Srinivas S., Cancer, 113, (2008). 11) Nojima Y., Suzuki Y., Iguchi K., Shiga T., Iwata A., Fujimoto T., Yoshida K., Shimizu H., Takeuchi T., Sato, A., Bioconjugate Chem., 19, (2008). 12) Roberts M. J., Bentley M. D., Harris J. M.,, Adv. Drug Deliv. Rev., 54, (2002).

15 13) Veronese F. M., Pasut G., Drug Discov. Today, 10, (2005). 14) Bailon P., Palleroni A., Schaffer C.A., Spence C.L., Fung W.J., Porter J.E., Ehrlich G.K., Pan W., Xu Z.X., Modi M.W., Farid A., Berthold W., Graves M., Bioconjugate Chem., 12, (2001).

16 Figure Legends Fig. 1. Reaction mechanism of the synthesis of PEGylated bovine lactoferrin (blf). The α- and ε-amino groups of blf react with the N-hydroxysuccinimide (NHS) ester derivatives of branched polyethylene glycol (20 kda and 40 kda) forming an amide bond. Fig. 2. SDS-PAGE profiles of PEGylated bovine lactoferrin (blf) modified with 40-kDa PEG-NHS at various ph conditions. Proteins (2 µg) were electrophoresed on a non-reducing 7.5% gel and stained with Coomassie Brilliant Blue (CBB). Lanes: M, molecular weight markers; blf, unmodified bovine lactoferrin (2 µg); 4-9, reaction ph. Gray and black arrows indicate PEG-modified blf. White arrow indicates unmodified blf. Fig. 3. Time-dependent changes in PEG modification reactions at 25 ºC for 24 h. Proteins (2 µg) were electrophoresed on a non-reducing 7.5% gel and stained with Coomassie Brilliant Blue (CBB). (A, C) Reactions carried out at ph 7.4 (PBS). (B, D) Reactions carried out at ph

17 9.0 (50 mm borate buffer). Lanes: M, molecular weight markers; blf, unmodified bovine lactoferrin (2 µg); numerals, incubation time. Oligo-PEGylated blfs are marked by asterisks. (E, F) Graphs exhibit the kinetics of PEGylation. Proteins bands (see A-D) were quantified using Image J software. Open and closed circles indicate 20-kDa and 40-kDa PEG-NHS, respectively. Concentration of PEG-bLfs at 6 h was arbitrarily set at 100%. Inset in F shows the time-dependent formation of PEG-bLF at ph 9.0 in the first hour. Fig. 4. Hydrolysis rates of the PEG-NHS derivatives. Each PEG-NHS was pre-incubated in PBS (ph 7.4) or 50 mm borate buffer (ph 9.0) at 25 ºC for the indicated times and then reacted with blf for PEGylation. Reactants were analyzed by 7.5% SDS-PAGE and visualized by Coomassie Brilliant Blue (CBB). The intensities of CBB-stained bands were analyzed by Image J software and represented as graphs.

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