Isolation, structural characterization, and antiviral activity of positional isomers of monopegylated interferon α-2a (PEGASYS)

Foser, Stefan; Schacher, Alfred; Weyer, Karl Aloys; Brugger, Doris; Dietel, Elke; Marti, Stefan; Schreitmüller, Thomas

doi:10.1016/s1046-5928(03)00055-x

Cited by 161 publications

(139 citation statements)

References 18 publications

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“…It is, therefore, quite likely that peak 1 and 2 in Fig. 1 are either Lys33 or Lys96 PEGylated lysozyme, although we have not confirmed this using the Lee-Park method [18] or the method by Nodake et al [11].…”

Section: Number Of Binding Sites (B)mentioning

confidence: 56%

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Interaction mechanism of mono‐PEGylated proteins in electrostatic interaction chromatography

Abe

Akbarzaderaleh

Hamachi

et al. 2010

Biotechnology Journal

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The retention and binding mechanisms in electrostatic interaction-based chromatography (ion-exchange chromatography) of PEGylated proteins (covalent attachment of polyethylene glycol chains to protein) were investigated using our previously developed model. Lysozyme and bovine serum albumin were chosen as model proteins. The retention volume of PEGylated proteins shifted to lower elution volumes with increasing PEG molecular weight compared with the non-modified (native) protein retention volume. However, PEGylation did not affect the number of binding sites appreciably. The enzyme activity of PEGylated lysozyme measured with a standard insoluble substrate in suspension decreased considerably, whereas the activity with a soluble small-molecule substrate did not drop significantly. These findings indicate that when a protein is mono-PEGylated, the binding site is not affected and the elution volume reduces due to the steric hindrance between PEGylated protein and ion-exchange ligand.

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Section: Number Of Binding Sites (B)mentioning

confidence: 56%

“…Major band of PEGylated reaction mixtures appeared around 30 kDa. The large hydrodynamic volume of PEG might contribute to the low mobility of PEGylated protein in SDS-PAGE [18]. Thus, the band showed a 1.5-fold higher molecular mass than the one calculated for mono-PEGylated lysozyme (19.5 kDa).…”

Section: Linear Gradient Elutionmentioning

confidence: 80%

Interaction mechanism of mono‐PEGylated proteins in electrostatic interaction chromatography

Abe

Akbarzaderaleh

Hamachi

et al. 2010

Biotechnology Journal

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“…Preliminary analysis of PE-GLA by MALDI-TOF mass spectrometry yielded a broad peak with an average molecular weight of Ϸ62,000 (data not shown), suggesting that LA was mono-PEGylated. It has been reported previously that, because of the large hydrodynamic volume of PEG, estimates of the molecular weight of PEGylated proteins by SDS/PAGE are significantly higher than those determined by mass spectrometry (23). PEGLA was capable of specifically inhibiting LIF-induced Ba/F3 cell proliferation but showed a 10-to 20-fold reduction in potency compared with the unmodified LA (Fig.…”

Section: Administration Of La Reduces Stat3 Phosphorylation In the Utmentioning

confidence: 86%

Blocking LIF action in the uterus by using a PEGylated antagonist prevents implantation: A nonhormonal contraceptive strategy

White¹,

Zhang

Salamonsen³

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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Blastocyst implantation is a critical stage in the establishment of pregnancy. Leukemia inhibitory factor (LIF) is essential for mouse blastocyst implantation and also plays a role in human pregnancy. We examined the effect of a potent LIF antagonist (LA) on mouse implantation. In mice, LIF expression peaks on day 3.5 of pregnancy (D3.5) (D0.5 ‫؍‬ day of mating plug detection) in the uterine glandular epithelium. LA (7 mg/kg per day) administered from D2.5 to D4.5 via four hourly i.p. injections plus continuous administration via miniosmotic pump resulted in complete implantation failure. To improve its pharmacokinetic properties, we conjugated LA to polyethylene glycol (PEG), achieving a significant increase in serum levels. PEGylated LA (PEGLA) (37.5 mg/kg per day) administered via three i.p. injections between D2.5 and D3.5 also resulted in complete implantation failure. PEGLA immunolocalized to the uterine luminal epithelium at the time of blastocyst implantation. Both LA and PEGLA reduced phosphorylation of the downstream signaling molecule STAT3 in luminal epithelial cells on D3.5. The effects of PEGLA were found to be endometrial, with no embryolethal effects observed. These data demonstrate that administration of a PEGylated LIF antagonist is an effective method of targeting LIF signaling in the endometrium and a promising novel approach in the development of nonhormonal contraceptives for women.embryo implantation ͉ leukemia inhibitory factor ͉ mouse

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“…Furthermore, each positional isomer is likely to have different efficacy, half-life, immunogenicity, and the like. Because chromatographic purification is not commercially viable (19), one is left with the challenge of optimizing the pharmacology of a heterogeneous mixture of drug substance (in contrast to the careful SAR studies historically used to optimize chemically defined small molecule drugs). An alternative is to exploit the low pKa of the amino terminus to direct conjugation, which is the basis of a mono-PEGylated GCSF drug (20).…”

Section: Discussionmentioning

confidence: 99%

Optimized clinical performance of growth hormone with an expanded genetic code

Cho

Daniel²,

Buechler

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

186

190

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The ribosomal incorporation of nonnative amino acids into polypeptides in living cells provides the opportunity to endow therapeutic proteins with unique pharmacological properties. We report here the first clinical study of a biosynthetic protein produced using an expanded genetic code. Incorporation of p-acetylphenylalanine (pAcF) at distinct locations in human growth hormone (hGH) allowed site-specific conjugation with polyethylene glycol (PEG) to produce homogeneous hGH variants. A mono-PEGylated mutant hGH modified at residue 35 demonstrated favorable pharmacodynamic properties in GH-deficient rats. Clinical studies in GH-deficient adults demonstrated efficacy and safety comparable to native human growth hormone therapy but with increased potency and reduced injection frequency. This example illustrates the utility of nonnative amino acids to optimize protein therapeutics in an analogous fashion to the use of medicinal chemistry to optimize conventional natural products, low molecular weight drugs, and peptides.protein engineering | endocrinology | bio-better

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Isolation, structural characterization, and antiviral activity of positional isomers of monopegylated interferon α-2a (PEGASYS)

Cited by 161 publications

References 18 publications

Interaction mechanism of mono‐PEGylated proteins in electrostatic interaction chromatography

Interaction mechanism of mono‐PEGylated proteins in electrostatic interaction chromatography

Blocking LIF action in the uterus by using a PEGylated antagonist prevents implantation: A nonhormonal contraceptive strategy

Optimized clinical performance of growth hormone with an expanded genetic code

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