Identification of the modifying sites of mono-PEGylated salmon calcitonins by capillary electrophoresis and MALDI–TOF mass spectrometry

Na, Dong Hee; Park, Myung Ok; Choi, So Yoon; Kim, Yong Suk; Lee, Seung Seok; Yoo, Sun Dong; Lee, Hye Suk; Lee, Kang Choon

doi:10.1016/s0378-4347(00)00599-5

Cited by 39 publications

(34 citation statements)

References 17 publications

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“…The mono-PEG 2k -sCT used in this study was a mixture of the N-terminus-, lysine 11-, and lysine 18-modified sCT. 11,14) We previously reported a chemical modification of sCT by covalent linkage with relatively higher molecular size succinimidyl carbonate monomethoxy polyethylene glycol (SC-mPEG, MW 5000 and 12000), and further isolated positional isomers of PEGylated sCTs. [11][12][13][14] The systemic clearance was unaltered for mono-PEG5K-sCT but was reduced for di-PEG 5k -sCT over unmodified sCT in rats.…”

Section: Discussionmentioning

confidence: 99%

“…Unlike other previous studies [11][12][13][14] utilizing high molecular weight PEGs (MW 5000 and 12000) for chemical modification, this study used a lower molecular size succinimidylpropionated monomethoxy-propylene glycol (SP-mPEG, MW 2000). The nasal absorption of synthesized mono-PEG 2k -sCT was further examined in rats compared with that of unmodified sCT.…”

Section: )mentioning

confidence: 99%

“…Mono-and di-PEGylated sCTs exhibit substantially improved stability in rat liver and kidney homogenates over unmodified sCT 11) while retaining the biological activity similar to that of unmodified sCT as examined by the adenosine cyclic 3Ј,5Ј-phosphate (cAMP) assay. 12) Unlike other previous studies [11][12][13][14] utilizing high molecular weight PEGs (MW 5000 and 12000) for chemical modification, this study used a lower molecular size succinimidylpropionated monomethoxy-propylene glycol (SP-mPEG, MW 2000). The nasal absorption of synthesized mono-PEG 2k -sCT was further examined in rats compared with that of unmodified sCT.…”

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Nasal Absorption and Pharmacokinetic Disposition of Salmon Calcitonin Modified with Low Molecular Weight Polyethylene Glycol

Shin

Jung

Lee

et al. 2004

CHEMICAL & PHARMACEUTICAL BULLETIN

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Salmon calcitonin (sCT) is a therapeutic polypeptide hormone consisting of 32 amino acids (3432 daltons). It is currently marketed either as a solution for intramuscular or subcutaneous injection, or as a nasal spray in the treatment of postmenopausal osteoporosis, symptomatic Paget's disease of the bone, and hypercalcemia due to malignancy. Like other peptide therapeutics, sCT is easily degraded by proteolytic enzymes and exhibits a short elimination half-life and low bioavailability in humans. [1][2][3] The absolute bioavailability of sCT after subcutaneous injection has been reported as 11.2-23.1% in rats. 4,5) Administration of sCT via the nasal route results in a low bioavailability but is shown to be effective, decreasing the osteoclastic bone resorption in humans. [6][7][8] The presence of tryptic endopeptidase activities appears to be crucial for sCT cleavage in the nasal mucosa. 9,10) We previously reported a chemical modification of sCT by covalent linkage with polyethylene glycol (PEG).11-13) PEG may bind to sCT at lysine 11, lysine 18, and N-terminus (cysteine 1) positions, yielding mono-, di-, and tri-PEGylated sCTs depending on the number of attached PEG molecules per molecule of sCT. Therefore, PEG modification results in a heterogeneous mixture of mono-, di-, and tri-PEG-sCTs. Formation of mono-PEG-sCT appears to be favored over that of di-PEG-sCT, while the formation of tri-PEG-sCT is minimal. Mono-and di-PEGylated sCTs exhibit substantially improved stability in rat liver and kidney homogenates over unmodified sCT 11) while retaining the biological activity similar to that of unmodified sCT as examined by the adenosine cyclic 3Ј,5Ј-phosphate (cAMP) assay. 12)Unlike other previous studies [11][12][13][14] utilizing high molecular weight PEGs (MW 5000 and 12000) for chemical modification, this study used a lower molecular size succinimidylpropionated monomethoxy-propylene glycol (SP-mPEG, MW 2000). The nasal absorption of synthesized mono-PEG 2k -sCT was further examined in rats compared with that of unmodified sCT. The chemical modification of sCT with low MW PEG was anticipated to increase the nasal absorption due to improved metabolic stability in the nasal mucosa. Our findings showed that the nasal absorption kinetics of mono-PEG 2K -sCT was altered, exhibiting increased AUC, C max , t max , and t 1/2 as compared with those of unmodified sCT. ExperimentalChemicals Salmon calcitonin (synthetic cyclic sCT) and Na 125 I were purchased from BACHEM (Torrance, CA, U.S.A.) and NEN (Boston, MA, U.S.A.), respectively. Succinimidyl-propionated monomethoxy-poly(ethylene glycol) (SP-mPEG, MW 2000) was purchased from Shearwater Polymers (Huntsville, AL, U.S.A). IODO-GEN was purchased from PIERCE (Rockford, IL, U.S.A.), and ketamine and xylazine from Sigma (St. Louis, MO, U.S.A). Acetonitrile and trifluoroacetic acid (HPLC grades) were purchased from J.T. Baker (Phillipsberg, NJ, U.S.A.) and Acros Organics (Springfield, NJ, U.S.A.), respectively. Other chemicals used in the study were of analytical grade.P...

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Section: Discussionmentioning

confidence: 99%

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Nasal Absorption and Pharmacokinetic Disposition of Salmon Calcitonin Modified with Low Molecular Weight Polyethylene Glycol

Shin

Jung

Lee

et al. 2004

CHEMICAL & PHARMACEUTICAL BULLETIN

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“…[67][68][69][70][71][72][73][74][75][76][77][78][79][80][81][82][83] Only these two configurations are covered in this review, although, brief descriptions of the other three are given in the succeeding paragraphs.…”

Section: Ionization Methodsmentioning

confidence: 99%

Recent Developments in Capillary Electrophoresis–Mass Spectrometry of Proteins and Peptides

Monton

Terabe

2005

ANAL. SCI.

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Many researchers have invested considerable efforts toward improving capillary electrophoresis (CE)-mass spectrometry (MS) systems so they can be applied better to standard analyses. This review highlights the developments in CE-MS of proteins and peptides over the last five years. It includes the developments in interfaces, sample-enrichment techniques, microfabricated devices, and some applications, largely in capillary zone electrophoresis (CZE), capillary isoelectric focusing (CIEF) and capillary isotachophoresis formats.

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“…Previously, MALDI-TOF MS has been shown to be useful in resolving the polymeric distributions of various polymers and PEGylated molecules conjugated with relatively small size PEG [14,[42][43][44][45][46], where singly charged ions are formed and detector saturation is not significant [23]. However, it is important to be able to analyze and resolve PEGylated biomolecules conjugated with PEG of large size, i.e., 20 kDa or higher, which have proven to have significant therapeutic value.…”

Section: Esi-ms and Maldi-tof Ms Analysis Of Pegylated Peptidementioning

confidence: 99%

Toward top-down determination of PEGylation site using MALDI in-source decay MS analysis

Yoo

Suckau

Sauerland

et al. 2009

J. Am. Soc. Mass Spectrom.

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A novel matrix assisted laser desorption/ionization (MALDI)-based mass spectrometric approach has been evaluated to rapidly analyze a custom designed PEGylated peptide that is 31 residues long and conjugated with 20 kDa linear polyethylene glycol (PEG) at the side chain of Lys. MALDI-TOF MS provided sufficiently high resolution to allow observation of each of the oligomers of the heterogeneous PEGylated peptide (m/⌬m of ca. 500), while a typical ESI-MS spectrum of this molecule was extremely complex and unresolved. Reflector in-source decay (reISD) analysis using MALDI-TOF MS was attempted to identify the PEGylation site at intact molecular level without any sample treatment. An reISD spectrum of the free peptide was observed with abundant c-, y-, and [z ϩ 2]-fragment ion series, whereas, in the fragmented PEGylated peptide, the fragment ion series were truncated at the residue where PEG was attached. Therefore, a direct comparison of these top-down reISD spectra suggested the location of the PEGylation site. Results from this study demonstrate a clear analytical utility of the ISD technique to characterize structural aspects of heterogeneous biomolecules. (J Am Soc Mass Spectrom 2009, 20, 326 -333) © 2009 American Society for Mass Spectrometry A significant challenge is associated with development of biomolecules into effective therapeutic use due to their limited chemical and physical stability. In particular, short plasma half-life of biotherapeutics may lead to poor efficacy in humans, and, therefore, developing methods to achieve longer circulation times at low dosage volumes is required. To obtain such improved stability, biomolecules can be chemically modified. One of the most successful approaches currently employed is to covalently attach inert, nontoxic, and nonantigenic polymers, such as polyethylene glycol (PEG), to active molecules, a strategy termed PEGylation [1][2][3][4][5][6]. Due to its favorable properties, PEG is approved by FDA [5] for oral, injection, and dermal administration to humans. PEGylation technology has been successfully developed and applied to achieve prolonged in vivo circulation of biomolecules in the body for effective potency, as demonstrated by a series of marketed PEGylated therapeutics, including Neulasta®, or pegfilgrastim [5,7,8].Typically, the conjugation of PEG of ca. 20 kDa or larger to biotherapeutics has proven efficient by providing desired stability [4,9,10]. However, tremendous analytical challenges are associated with studying such heterogeneous molecules [11], where confident determination of the PEGylation site is one of the important requirements to fully characterize and understand PEGylated molecules. Previously, several groups have reported on the identification of the site at which PEG was conjugated [12], where PEG of relatively small size were commonly used [13][14][15]. Due to a lower degree of polymerization associated with smaller PEG, an analysis was readily performed in these cases. Also, these studies mostly involved enzymatic digestions to sel...

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Identification of the modifying sites of mono-PEGylated salmon calcitonins by capillary electrophoresis and MALDI–TOF mass spectrometry

Cited by 39 publications

References 17 publications

Nasal Absorption and Pharmacokinetic Disposition of Salmon Calcitonin Modified with Low Molecular Weight Polyethylene Glycol

Nasal Absorption and Pharmacokinetic Disposition of Salmon Calcitonin Modified with Low Molecular Weight Polyethylene Glycol

Recent Developments in Capillary Electrophoresis–Mass Spectrometry of Proteins and Peptides

Toward top-down determination of PEGylation site using MALDI in-source decay MS analysis

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