O-Linked Glycosylation Leads to Decreased Thermal Stability of Interferon Alpha 2b as Measured by Two Orthogonal Techniques

Johnston, Michael; Frahm, Grant E.; Li, Xuguang; Durocher, Yves; Hefford, Mary Alice

doi:10.1007/s11095-011-0402-0

Cited by 14 publications

(8 citation statements)

References 29 publications

(27 reference statements)

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“…Thermal denaturation studies were carried out by monitoring CD (millidegrees) at 222 nm between 20°C and 90°C in increments of 2°C per minute. Due to the predominant alpha helical nature of HSA, monitoring the CD minimum at 222 nm - a characteristic of alpha helical structure - is ideal for assessing the thermal stability of albumin and other predominantly alpha helical proteins [3] , [19] , [23] . Samples were prepared in 1 mm quartz cuvettes.…”

Section: Methodsmentioning

confidence: 99%

Determination of Supplier-to-Supplier and Lot-to-Lot Variability in Glycation of Recombinant Human Serum Albumin Expressed in Oryza sativa

et al. 2014

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The use of different expression systems to produce the same recombinant human protein can result in expression-dependent chemical modifications (CMs) leading to variability of structure, stability and immunogenicity. Of particular interest are recombinant human proteins expressed in plant-based systems, which have shown particularly high CM variability. In studies presented here, recombinant human serum albumins (rHSA) produced in Oryza sativa (Asian rice) (OsrHSA) from a number of suppliers have been extensively characterized and compared to plasma-derived HSA (pHSA) and rHSA expressed in yeast (Pichia pastoris and Saccharomyces cerevisiae). The heterogeneity of each sample was evaluated using size exclusion chromatography (SEC), reversed-phase high-performance liquid chromatography (RP-HPLC) and capillary electrophoresis (CE). Modifications of the samples were identified by liquid chromatography-mass spectrometry (LC-MS). The secondary and tertiary structure of the albumin samples were assessed with far U/V circular dichroism spectropolarimetry (far U/V CD) and fluorescence spectroscopy, respectively. Far U/V CD and fluorescence analyses were also used to assess thermal stability and drug binding. High molecular weight aggregates in OsrHSA samples were detected with SEC and supplier-to-supplier variability and, more critically, lot-to-lot variability in one manufactures supplied products were identified. LC-MS analysis identified a greater number of hexose-glycated arginine and lysine residues on OsrHSA compared to pHSA or rHSA expressed in yeast. This analysis also showed supplier-to-supplier and lot-to-lot variability in the degree of glycation at specific lysine and arginine residues for OsrHSA. Both the number of glycated residues and the degree of glycation correlated positively with the quantity of non-monomeric species and the chromatographic profiles of the samples. Tertiary structural changes were observed for most OsrHSA samples which correlated well with the degree of arginine/lysine glycation. The extensive glycation of OsrHSA from multiple suppliers may have further implications for the use of OsrHSA as a therapeutic product.

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Section: Methodsmentioning

confidence: 99%

Determination of Supplier-to-Supplier and Lot-to-Lot Variability in Glycation of Recombinant Human Serum Albumin Expressed in Oryza sativa

et al. 2014

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“…IFN‐α‐2 contains one O‐glycosylation site at Thr106, and the effect of the O‐glycan on the thermal stability of IFN‐α‐2 is debated. Johnston et al . reported T m values of 65.7 and 63.8 °C for the non‐glycosylated and O‐glycosylated variants of IFN‐α‐2b, whereas Ceaglio et al .…”

Section: Modulation Of Stability Through Chemical Modificationmentioning

confidence: 99%

Effects of localized interactions and surface properties on stability of protein-based therapeutics

Mills

Chadwick

2016

Journal of Pharmacy and Pharmacology

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Physical and chemical stability of therapeutic proteins and antibody drug conjugates (ADCs) is of critical importance because insufficient stability prevents molecules from making it to market. Individual moieties on/near the surface of proteins have substantial influence on structure and stability. Seemingly small, superficial modification may have far-reaching consequences on structure, conformational dynamics, and solubility of the protein, and hence physical stability of the molecule. Chemical modifications, whether spontaneous (e.g. oxidation, deamidation) or intentional, as with ADCs, may adversely impact stability by disrupting local surface properties or higher order protein structure.

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“…Interestingly, and somewhat unexpectedly (compared with IFN-$), the glycosylation results in slightly decreased thermal stability of IFN-"-2b (melting temperature decreases from about 65 • C-66 • C to 64 • C). 45 Interferon-alpha is acid labile and undergoes structural changes and unfolding below pH 4. 46,47 IFN-"-2b is reported to have maximal conformational stability at pH 7, where it may be self-associated as dimers or even higher-order oligomers.…”

Section: Interferon Alphamentioning

confidence: 99%

“…The proteins are described water soluble in the manufacturers’ documentation. Interestingly, and somewhat unexpectedly (compared with IFN‐β), the glycosylation results in slightly decreased thermal stability of IFN‐α‐2b (melting temperature decreases from about 65°C–66°C to 64°C) …”

Section: Introductionmentioning

confidence: 97%

Formulation and Stability of Cytokine Therapeutics

Lipiäinen

Peltoniemi

Sarkhel

et al. 2015

Journal of Pharmaceutical Sciences

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O-Linked Glycosylation Leads to Decreased Thermal Stability of Interferon Alpha 2b as Measured by Two Orthogonal Techniques

Cited by 14 publications

References 29 publications

Determination of Supplier-to-Supplier and Lot-to-Lot Variability in Glycation of Recombinant Human Serum Albumin Expressed in Oryza sativa

Determination of Supplier-to-Supplier and Lot-to-Lot Variability in Glycation of Recombinant Human Serum Albumin Expressed in Oryza sativa

Effects of localized interactions and surface properties on stability of protein-based therapeutics

Formulation and Stability of Cytokine Therapeutics

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