pH dependent effect of glycosylation on protein stability.
The effect of glycosylation state on the thermal and storage stability of interleukin-2 mutein (IL-2 mutein) was investigated. The thermal stability of IL-2 mutein was studied by DSC and UV. An accelerated storage stability study was conducted at 40 degrees C in the dark and analyzed by UV, SDS-PAGE, and RP-HPLC. The unfolding temperatures (Tu) of both glycosylated and unglycosylated forms of IL-2 mutein are similar (within +/-1 degrees C) at pH 5.5 and 7.5. At pH 4.0, the Tu of glycosylated IL-2 mutein was 4 degrees C lower than that of the unglycosylated form. The precipitation temperature of glycosylated IL-2 mutein is similar to that of the unglycosylated form at pH 5.5 but 4 degrees C higher at pH 7.5. The precipitation temperature is not detectable for both forms at pH 4.0. During storage, both glycosylated and unglycosylated IL-2 mutein form aggregates (soluble and insoluble) and other degradation products. The aggregates are formed by both physical and chemical mechanisms. The major pathway of chemical aggregation appears to be disulfide bond formation/exchange. The glycosylated form is much less stable than the unglycosylated form at pH 4.0 and both forms are most stable at pH 5.5 in terms of thermal stability, precipitation rate and total degradation rate. This study clearly demonstrates that the effect of glycosylation on the stability of a protein is pH-dependent.
Wang
Wei
W
Pfizer Inc., Global Biologics, 700 Chesterfield Parkway West, Chesterfield, MO 63017, USA.
Antonsen
Kris
K
Wang
Y John
YJ
Wang
D Q
DQ
D019943:Amino Acid Substitution; D002152:Calorimetry, Differential Scanning; D011232:Chemical Precipitation; D003545:Cysteine / Q000737:chemistry / Q000235:genetics; D004356:Drug Storage; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007376:Interleukin-2 / Q000737:chemistry* / Q000235:genetics; D010084:Oxidation-Reduction; D011489:Protein Denaturation; D011506:Proteins / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D013056:Spectrophotometry, Ultraviolet; D013696:Temperature
D007376:Interleukin-2; D011506:Proteins; D011994:Recombinant Proteins; D003545:Cysteine