Oxidation of recombinant methionyl human granulocyte colony stimulating factor

Reubsaet, Léon; Beijnen, Jos H.; Bult, A.; Hop, E.; Scholten, Serge D.; Teeuwsen, J.; Underberg, W.J.M.

doi:10.1016/s0731-7085(97)00199-4

Cited by 21 publications

(12 citation statements)

References 21 publications

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“…Isolated Met 122 has been reported by Reubsaet et al [12] in H 2 O 2 -stressed samples. However, data on G-CSF oxidation behavior published more recently as well as our data strongly suggest that the oxidized variant denoted as oxidized Met 122 by Reubsaet and colleagues is actually oxidized Met 1 .…”

Section: Resultsmentioning

confidence: 81%

Top-down MS for rapid methionine oxidation site assignment in filgrastim

Holzmann¹,

Hausberger²,

Rupprechter³

et al. 2013

Anal Bioanal Chem

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Protein therapeutics have emerged as a major new class of pharmaceuticals. One important shelf-life-limiting factor of biopharmaceuticals is methionine oxidation, and therefore, it is important that analytical methods are able to thoroughly characterize all possible oxidized variants. Here, we present a fast and sensitive method to perform online methionine oxidation site assignment using granulocyte colony-stimulating factor (filgrastim) as a model. The method is based on top-down MS using the all-ion fragmentation mode of the Exactive benchtop mass spectrometer. Conditions that provide information on the intact mass of the protein as well as on fragment ions that allow unambiguous site assignment of methionine oxidation in filgrastim variants as low as 0.12 % of total peak area in a chromatographic time scale were identified. Using this method, we performed methionine oxidation site assignment in H2O2-stressed filgrastim and in filgrastim which was stored at intended conditions, respectively. We show that the relative abundance of oxidation species observed in filgrastim stored under intended conditions differs strikingly from the oxidized species observed after H2O2 stress. Additionally, we report an oxidized filgrastim variant that has not been previously described in the literature.FigureA top-down approach on an Exactive benchtop mass spectrometer in all-ion fragmentation mode is a highly attractive alternative to the traditional approach of isolation/bottom-up analysis for methionine oxidation site assignement in biopharmaceuticals. With a sensitivity as low as 0.12 % of total peak area and a throughput of about one sample per hour, the method is highly suitable for a thorough characterization of oxidized methionine residues

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Section: Resultsmentioning

confidence: 81%

Top-down MS for rapid methionine oxidation site assignment in filgrastim

Holzmann¹,

Hausberger²,

Rupprechter³

et al. 2013

Anal Bioanal Chem

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“…This implication is supported by early papers in the literature that demonstrated that trace catalytic metals up to 20 M are ubiquitous in buffer solutions (23,24). G-CSF has an isoelectric point (pI) of 6.2 (25), and its residues that may be charged include four Asp (pKa ‫ס‬ 3.9), nine Glu (pKa ‫ס‬ 4.07), five His (pKa ‫ס‬ 6.04), four Lys (pKa ‫ס‬ 10.79), and five Arg (pKa ‫ס‬ 12.48) (26). EDTA has four carboxylic acid groups (pKa's ‫ס‬ 0.032, 1.0, 2.0, 2.66) and two amine groups (pKa's ‫ס‬ 6.16, 10.26), while citric acid has three acidic sites (pKa's ‫ס‬ 3.13, 4.77, 6.40) (27).…”

Section: Effects Of Edta and Citratementioning

confidence: 83%

Effects of Excipients on the Hydrogen Peroxide-Induced Oxidation of Methionine Residues in Granulocyte Colony-Stimulating Factor

et al. 2005

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“…Susceptibility to oxidation has been linked with the methionine residues in rHuG-CSF and conversion of its sulfur-containing residues into sulfoxide resulted in loss of biological activity [16,29,30]. It should be noted that oxidized forms are not only possible degradation products but also product-related impurities that should be tested for in the final preparation [11,26].…”

Section: Rp-hplcmentioning

confidence: 99%