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Wylie, David; Voloch, Marcio; Lee, Seoju; Liu, Yan‐Hui; Cannon-Carlson, Susan; Cutler, Collette; Pramanik, Birenda N.

doi:10.1023/a:1013006515587

Cited by 45 publications

(14 citation statements)

References 15 publications

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“…However, one exception is the inability to resolve by HPIEX the neighboring peaks of 5-kDa Lys 131 and 5-kDa Lys 164 , hence the lower purity estimation by analytical HPIEX relative to HPSEC. The HPIEX chromatography profiles and positional isomer elution order in this study matched those reported previously (12,15,19). In addition, the identity and purity of the purified positional isomers were confirmed by peptide mapping analysis.…”

Section: Confirmation Of Differential Activity Between 12-kda Monopegsupporting

confidence: 85%

“…This procedure, previously utilized to resolve positional isomers of 12-kDa PEG-IFN-␣2b (15), was optimized to improve the resolution of positional isomers with varying PEG molecular weights. The purified mono-PEG-IFN-␣2b size exclusion pools were dialyzed overnight at 5°C against HPIEX buffer A (1.8 mM citric acid monohydrate, 3.3 mM sodium phosphate dibasic heptahydrate, pH 5.3).…”

Section: Production and Isolation Of Pegylated Interferon Isomers Formentioning

confidence: 99%

“…Peptide Mapping-Characterization of purified positional isomer peaks was carried out as described previously (15). Peak fractions were concentrated to 0.5-1 mg/ml using a centrifugal spin filter (Ultrafree 15 ml, BioMaxx 50; Millipore Corp.).…”

Section: Production and Isolation Of Pegylated Interferon Isomers Formentioning

confidence: 99%

“…These monopegylated reaction products were then resolved into individual positional isomers by HPIEX (Fig. 1, A and B) to 98% purity by HPSEC and 91-99% purity by analytical HPIEX (15). Peptide mapping analysis can usually detect Ͼ5% impurities.…”

Section: Confirmation Of Differential Activity Between 12-kda Monopegmentioning

confidence: 99%

“…. His 34 and Cys 1 are most reactive at a neutral pH, whereas the ⑀-amine groups of lysine residues are most reactive at basic pH (15). Therefore, pegylation reactions utilizing SC-PEG 5 kDa, SC-PEG 12 kDa, SC-PEG 20 kDa, and SC-PEG 30 kDa were performed at room temperature at pH 6.5 to produce His 34 -and Cys 1 -modified positional isomers and pH 10 to produce the ⑀-amine-modified lysine positional isomers.…”

Section: Ifn-␣2b (Intron® A) and 12-kda Peg-ifn-␣2b (Peg-intron)mentioning

confidence: 99%

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Site of Pegylation and Polyethylene Glycol Molecule Size Attenuate Interferon-α Antiviral and Antiproliferative Activities through the JAK/STAT Signaling Pathway

Grace

Lee²,

Bradshaw

et al. 2005

Journal of Biological Chemistry

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Therapeutic pegylated interferon-␣s (IFN-␣) are mixtures of positional isomers that have been monopegylated at specific sites on the core IFN-␣ molecule. The pegylation results in lower in vitro specific activity associated with the core IFN-␣ molecule that is related to the site of pegylation and size of polyethylene glycol (PEG) attached. We prepared purified, homogeneous, positional pegylation isomers of IFN-␣2b that were monopegylated using 5-30-kDa linear PEG molecules attached at 7 primary reactive amino acid residues: Chronic hepatitis C is considered one of the major causes of chronic liver disease, cirrhosis, and hepatocellular carcinoma and is the most common reason for liver transplantation in the United States (1). It is estimated that there are 3 million chronically infected individuals in the United States and over 170 million worldwide (1). Treatment of hepatitis C has evolved from the use of interferon-␣ (IFN-␣), 1 either alone or in combination with ribavirin, to the newer pegylated interferons (PEGIFNs), which have provided a dramatic increase in virological response, especially in combination with ribavirin. Standard IFN-␣ therapy has a short (Ͻ12-h) half-life that requires subcutaneous injection three times weekly to maintain effective levels in the blood (2). The short half-life of IFN-␣ has led to the development of longer lasting preparations achieved by the attachment of a large polyethylene glycol (PEG) molecule directly to IFN-␣. Two different commercial preparations of PEG-IFN-␣ have been developed for clinical use, PEG-IFN-␣2b (PEG-INTRON®) and PEG-IFN-␣2a (Pegasys®); both have long half-lives (40 and 80 h, respectively) that permit once weekly administration (3). Both of these preparations have been demonstrated to be effective for the treatment of patients with hepatitis C (4), and clinical trial results have shown further that both of the pegylated molecules produce sustained viral response rates superior to those achieved with their respective standard IFN-␣s (5-7).Whereas pegylation has proven to be highly effective for slowing the clearance of biological molecules, including IFN-␣, and thus increasing serum half-life, it has been shown to also modify in vitro biological activity (8). For instance, we have reported that pegylation of IFN-␣2b with a 12-kDa linear PEG molecule results in a preparation that has a specific activity of 28% relative to IFN-␣2b; the loss in activity was not due to structural perturbation of the core IFN-␣2b core protein (9). Other groups have reported that pegylation of IFN-␣2a with a 40-kDa branched PEG molecule results in a preparation that contains from 1 to 7% relative specific activity compared with IFN-␣2a (10, 11). These two pegylated interferon-␣s (PEG-IFN␣s) differ substantially in their postpegylation constituent properties. PEG-IFN-␣2b has a 12-kDa linear PEG molecule attached using succinimidyl carbonate polyethylene glycol (SC-PEG) chemistry via a covalent urethane-like bond to the IFN␣2b protein (12). The pegylation linkage process results...

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Section: Confirmation Of Differential Activity Between 12-kda Monopegsupporting

confidence: 85%

Section: Production and Isolation Of Pegylated Interferon Isomers Formentioning

confidence: 99%

Section: Production and Isolation Of Pegylated Interferon Isomers Formentioning

confidence: 99%

Section: Confirmation Of Differential Activity Between 12-kda Monopegmentioning

confidence: 99%

Section: Ifn-␣2b (Intron® A) and 12-kda Peg-ifn-␣2b (Peg-intron)mentioning

confidence: 99%

See 3 more Smart Citations

Site of Pegylation and Polyethylene Glycol Molecule Size Attenuate Interferon-α Antiviral and Antiproliferative Activities through the JAK/STAT Signaling Pathway

Grace

Lee²,

Bradshaw

et al. 2005

Journal of Biological Chemistry

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PEGylation

Dhawan

Kapoor

Kapil

et al. 2011

Pharmaceutical Sciences Encyclopedia

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PEGylation involves the masking of the surface of proteins and peptides by covalent coupling with soluble PEG. It is a method for optimizing pharmacokinetic and pharmacodynamic properties of therapeutic small drug molecules such as peptides and proteins. Thus, this chapter will focus on PEGylation as the modification of the surface properties of proteins and peptide or nonpeptide molecules via covalent attachment using a chemical modifier, PEG, either alone or in combination with other moieties. PEGylation has been exploited to increase the half life ( t 1/2 ), solubility, stability and decrease of the immunogenicity, and toxicity of enzymes, proteins, peptides, and nonpeptide molecules.

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