Aggregation of recombinant bovine granulocyte colony stimulating factor in solution.
Aggregation of recombinant bovine granulocyte colony-stimulating factor (rbG-CSF) was examined by the techniques of size exclusion chromatography (SEC), multiangle laser light scattering (MALS), and SDS-PAGE. Solutions of rbG-CSF in different buffers and pH were exposed to an elevated temperature of 50 degrees C to induce aggregation. The formation of noncovalent soluble aggregates with molecular weight in the millions of Daltons was observed when a solution of rbG-CSF at pH 2.9 was exposed to 50 degrees C. Precipitated protein was the main product of rbG-CSF aggregation in citrate and phosphate buffers at a pH greater than 4. It was demonstrated that precipitant was a mixture of covalent and noncovalent aggregates. The ratio of covalent to noncovalent binding increased with increase in pH of the protein solution. The covalent binding that occurred was primarily due to disulfide linkages via intermolecular disulfide scrambling as demonstrated by SDS-PAGE.
Bartkowski
Richard
R
Pfizer Global Manufacturing, Quality Operations, Groton, Connecticut 06340, USA. richard.e.bartkowski@pfizer.com
Kitchel
Ryan
R
Peckham
Niomi
N
Margulis
Leonid
L
D000818:Animals; D002021:Buffers; D002417:Cattle; D011232:Chemical Precipitation; D002850:Chromatography, Gel; D003545:Cysteine / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000737:chemistry / Q000378:metabolism; D016179:Granulocyte Colony-Stimulating Factor / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D011994:Recombinant Proteins; D012542:Scattering, Radiation; D013056:Spectrophotometry, Ultraviolet; D013696:Temperature
D002021:Buffers; D011994:Recombinant Proteins; D016179:Granulocyte Colony-Stimulating Factor; D003545:Cysteine