Abstract
Purpose. To separate and characterize the different positional isomers of mono-PEGylated salmon calcitonins (mono-PEG-sCTs) and to evaluate the effects of the PEGylation site on the stability of different mono-PEG-sCTs in rat kidney homogenate.
Methods. Mono-PEG-sCTs were prepared using succinimidyl carbonate monomethoxy polyethylene glycol (5,000 Da) and separated by gel-filtration HPLC followed by reversed-phase HPLC. To characterize PEGylated sCTs, matrix-assisted laser desorption ionization time of flight mass spectrometry (M ALDI-TOF MS) and reversed-phase HPLC of the trypsin digested samples were performed. Mono-PEG-sCTs and sCT in rat kidney homogenates were measured by column-switching reversed-phase HPLC with on-line detection of the radioiodinated samples using a flow-through radioisotope detector.
Results. Three different mono-PEGylated sCTs were separated by reversed-phase gradient HPLC. From the MALDI-TOF MS analysis, the average molecular weight of mono-PEG-sCTs was confirmed as around 8650 Da. The presence of PEG moiety in the mono-PEG-sCTs was also manifested by the fact that the distance between two adjacent mass spectum lines was 44 Da which corresponds to PEG monomer unit. Tryptic digestion analysis demonstrated that these mono-PEG-sCTs are 3 positional isomers of N-terminus, Lys18- and Lys11-residue modified mono-PEGylated sCTs. The degradation half-life of these 3 positional isomers in rat kidney homogenates significantly increased in order of the N-terminus (125.5 min), Lys11- (157.3 min), and Lysl8-residue modified mono-PEGylated sCT (281.5 min) over the native sCT (4.8 min).
Conclusions. Three positional isomers of mono-PEGylated sCTs were purified and characterized. Of these, the resistance to proteolytic degradation was highest for the Lysl8-residue modified mono-PEG-sCT. These studies demonstrate that the in vivo stability of PEGylated sCTs is highly dependent on the site of PEG molecule attachment.
Similar content being viewed by others
REFERENCES
M. Azria. The Calcitonins. Physiology and Pharmacology, Karger, Basel, 1989.
K. C. Lee, E. E. Soltis, P. S. Newman, K. W. Burton, R. C. Mehta, and P. P. DeLuca. In vivo assessment of salmon calcitonin sustained release from biodegradable micropsheres. J. Contr. Rel. 17:199–206 (1991).
E. Compston. Prevention and management of osteoporosis. Current trends and future prospects. Drugs 53:727–35 (1997).
G. Segra and P. Dal Pra. Calcitonin pharmacokinetics. In A. Pecile (ed.), Calcitonin, Excerpta Medica, Amsterdam, 1985, pp. 99–107.
K. C. Lee, Y. J. Lee, H. M. Song, C. J. Chun, and P. P. DeLuca. Degradation of synthetic salmon calcitonin in aqueous solution. Pharm. Res 9:1521–1523 (1992).
H. S. Lee, J. S. Lee, H. Lee, Y. S. Jung, P. P. DeLuca, and K. C. Lee. Reversed-phase high-performance liquid chromatography of salmon calcitonin and its degradation products in biological sample using column switching and flow-through radioisotope detection. J. Chromatogr. B. 673:136–141 (1995).
J. Hysing, J. O. Gordeladze, G. Christensen, and H. Tolleshaug. Renal uptake and degradation of trapped-label calcitonin. Biochem. Pharmacol. 41:1119–1126 (1991).
R. E. Simmons, J. T. Hjelle, C. A. Mahoney, L. J. Deftos, W. Lisker, P. Kato, and R. Rabkin. Renal metabolism of calcitonin. Am. J. Physiol. 254:F593–F600 (1988).
S. R. Lang, W. Staudenmann, P. James, H. Manz, R. Kessler, B. Galli, H. Moser, A. Rummelt, and H. P. Merkle. Proteolysis of human calcitonin in excised bovine nasal mucosa: Elucidation of the metabolic pathway by liquid secondary ionization mass spectrometry (LSIMS) and matrix assisted laser desorption ionization mass spectrometry (MALDI). Pharm. Res. 13:1679–1685 (1996).
V. Windisch, F. DeLuccia, L. Duhau, F. Herman, J. J. Mencel, S. Tang, and M. Vuilhorgne. Degradation pathways of salmon calcitonin in aqueous solution. J. Pharm. Sci. 86:359–364 (1997).
G. E. Francis, C. Delgado, and D. Fisher. PEG-modified proteins. In T. J. Ahern and M. C. Manning (eds.), Stability of Protein Pharmaceuticals, Part B: In vivo pathways of degradation andstrategies for protein stabilization. Plenum Press, New York, 1992, pp. 235–263.
N. V. Katre. The conjugation of proteins with polyethylene glycol and other polymers (Altering properties of proteins to enhance their therapeutic potential). Adv. Drug Del. Rev. 10:91–114 (1993).
Y. Inada, A. Matsushima, M. Hiroto, H. Nishimura, and Y. Kodera. Modification of proteins with polyethylene glycol derivatives. Methods Enzymol. 242:65–90 (1994).
S. Zalipsky. Chemistry of polyethylene glycol conjugates with biologically active molecules. Adv. Drug Del. Rev. 16:157–182 (1995).
F. M. Veronese, C. Monfardini, P. Caliceti, O. Schiavon, M. D. Scrawen, and D. Beer. Improvement of pharmacokinetic, immunological and stability properties of asparaginase by conjugation to linear and branched monomethoxy poly(ethylene glycol). J. Contr. Rel. 40:199–209 (1996).
Y. Tsutsumi, S. Tsunoda, H. Kamada, T. Kihira, S. Nakagawa, Y. Kaneda, T. Kanamori, and T. Mayumi. Molecular design of hybrid tumor necrosis factor-α II: the molecular size of polyethylene glycol-modified tumor necrosis factor-α affects its antitumor potency. Brt. J. Cancer 74:1090–1095 (1996).
P. McGoff, A. C. Baziotis, and R. Maskiewicz. Analysis of polyethylene glycol modified superoxide dismutase by chromatographic, electrophoretic, light scattering, chemical and enzymatic methods. Chem. Pharm. Bull. 36:3079–3091 (1988).
M. Kunitani, G. Dollinger, D. Johnson, and L. Kresin. On-line characterization of polyethylene glycol-modified proteins. J. Chromatogr. 588:125–137 (1991).
J. Snider, C. Neville, L. Yuan, and J. Bullock. Characterization of the heterogenicity of polyethylene glycol-modified superoxide dismutase by chromatographic and electrophoretic techniques. J. Chromatogr. 599:141–155 (1992).
D. H. Na, B. H. Woo, and K. C. Lee. Quantitative analysis of derivatized proteins prepared with pyridyl disulfide-containing cross-linkers by HPLC. Bioconj. Chem. 10: in press
R. J. Goodson and N. V. Katre. Site-directed PEGylation of recombinant interleukin-2 at its glycosylation site. Biotechnology 8:343–346 (1990).
H. F. Gaertner and R. E. Offord. Site-specific attachment of functionalized poly(ethylene glycol) to the amino terminus of proteins. Bioconj. Chem. 7:38–44 (1996).
O. B. Kinstler, D. N. Brems, S. L. Lauren, A. G. Paige, J. B. Hamburger, and M. J. Treuheit. Characterization and stability of N-terminally PEGylated rhG-CSF. Pharm. Res. 13:996–1002 (1995).
A. M. Felix, Y. Lu, and R. M. Campbell. Pegylated peptides IV. Enhanced biological activity of site-directed pegylated GRF analogs. Int. J. Peptide Protein Res. 46:253–264 (1995).
S. P. Monkarch, Y. Ma, A. Aglione, P. Bailon, D. Ciolek, B. DeBarbieri, M. C. Graves, K. Hollfelder, H. Michel, A. Palleroni, J. E. Porter, E. Russonman, S. Roy, and Y. E. Pan. Positional isomers of monopegylated interferon α-2a: Isolation, characterization, and biological activity. Anal. Biochem. 247:434–440 (1997).
K. C. Lee, K. K. Tak, M. O. Park, J. T. Lee, B. H. Woo, S. D. Yoo, H. S. Lee, and P. P. DeLuca. Preparation and characterization of polyethylene glycol-modified salmon calcitonins. Pharm. Dev. Tech. 4:269–275(1999).
K. C. Lee, T. S. Kang, B. H. Woo, J. T. Lee, H. S. Lee, and P. P. DeLuca. Reversed-phase high-performance liquid chromatography of radioiodinated salmon calcitonins. J. Chromatogr. B. 694:31–37 (1997).
J. Kajihara, K. Shibata, Y. Nakano, S. Nishimuro, and K. Kato. Physicochemical characterization of PEG-PPG conjugated human urokinase. Biochim. Biophys. Acta 1199:202–208 (1994).
R. Clark, K. Olson, G. Fuh, M. Marian, D. Mortensen, G. Teshima, S. Chang, H. Chu, V. Mukku, E. Canova-Davis, T. Somers, M. Cronin, M. Winkler, and J. A. Wells. Long-acting growth hormones produced by conjugation with polyethylene glycol. J. Biol. Chem. 271:21969–21977 (1996).
K. C. Lee, J. T. Lee, B. H. Woo, S. C. Moon, and P. P. DeLuca. Analysis of PEGylated salmon calcitonins by radioimmunoassay and HPLC with flow through radioisotope detection. Pharm. Res. 14:S350–351 (1997).
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Lee, K.C., Moon, S.C., Park, M.O. et al. Isolation, Characterization, and Stability of Positional Isomers of Mono-PEGylated Salmon Calcitonins. Pharm Res 16, 813–818 (1999). https://doi.org/10.1023/A:1018861616465
Issue Date:
DOI: https://doi.org/10.1023/A:1018861616465