Switch to
| Predicate | Object |
|---|---|
| rdf:type | |
| lifeskim:mentions | |
| pubmed:issue |
7
|
| pubmed:dateCreated |
1993-10-21
|
| pubmed:abstractText |
Recombinant human macrophage colony-stimulating factor (rhM-CSF) promotes macrophage proliferation and activity. rhM-CSF clinical trials are currently in progress and require a stable, pharmaceutically acceptable dosage form. This report documents pH effects on rhM-CSF degradation profiles in aqueous solution, with an emphasis on identifying degradation products. Thus, highly purified rhM-CSF was maintained at 30 to 50 degrees C in solutions adjusted to pH 2 to 10. Stressed samples were analyzed by SDS-PAGE, reverse-phase HPLC, size exclusion HPLC, scanning microcalorimetry, and murine bone marrow activity. The results show maximal protein stability in the region pH 7 to 8. Degradation product chromatographic and electrophoretic analyses show distinctly different degradation product profiles in acidic versus alkaline solution. For samples stressed in acidic solution, degradation products were isolated chromatographically and electrophoretically. These degradation products were characterized by N-terminal amino acid sequencing, fast-atom bombardment mass spectrometry, and peptide mapping. The results show that the major degradation pathway in acidic solution involves peptide cleavage at two sites: aspartate169-proline170 and aspartate213-proline214. A third potential cleavage site (aspartate45-proline46) remains intact under conditions that cleave Asp169-Pro170 and Asp213-Pro214. In alkaline solution, degradation proceeds via parallel cleavage and intramolecular cross-linking reactions. A beta-elimination mechanism is proposed to account for the degradation in alkaline solution. Consistent with literature observations, the rhM-CSF N-terminal cleavage products retain biological activity.
|
| pubmed:language |
eng
|
| pubmed:journal | |
| pubmed:citationSubset |
IM
|
| pubmed:chemical | |
| pubmed:status |
MEDLINE
|
| pubmed:month |
Jul
|
| pubmed:issn |
0724-8741
|
| pubmed:author | |
| pubmed:issnType |
Print
|
| pubmed:volume |
10
|
| pubmed:owner |
NLM
|
| pubmed:authorsComplete |
Y
|
| pubmed:pagination |
933-44
|
| pubmed:dateRevised |
2009-11-19
|
| pubmed:meshHeading |
pubmed-meshheading:8378255-Alcaligenes,
pubmed-meshheading:8378255-Amino Acid Sequence,
pubmed-meshheading:8378255-Animals,
pubmed-meshheading:8378255-Bone Marrow,
pubmed-meshheading:8378255-Bone Marrow Cells,
pubmed-meshheading:8378255-CHO Cells,
pubmed-meshheading:8378255-Calorimetry, Differential Scanning,
pubmed-meshheading:8378255-Chromatography, Gel,
pubmed-meshheading:8378255-Chromatography, High Pressure Liquid,
pubmed-meshheading:8378255-Cricetinae,
pubmed-meshheading:8378255-Electrophoresis, Polyacrylamide Gel,
pubmed-meshheading:8378255-Humans,
pubmed-meshheading:8378255-Hydrogen-Ion Concentration,
pubmed-meshheading:8378255-Kinetics,
pubmed-meshheading:8378255-Macrophage Colony-Stimulating Factor,
pubmed-meshheading:8378255-Mice,
pubmed-meshheading:8378255-Molecular Sequence Data,
pubmed-meshheading:8378255-Peptide Mapping,
pubmed-meshheading:8378255-Protein Denaturation,
pubmed-meshheading:8378255-Receptor, Macrophage Colony-Stimulating Factor,
pubmed-meshheading:8378255-Recombinant Proteins,
pubmed-meshheading:8378255-Solutions,
pubmed-meshheading:8378255-Spectrometry, Mass, Fast Atom Bombardment
|
| pubmed:year |
1993
|
| pubmed:articleTitle |
Degradation pathways for recombinant human macrophage colony-stimulating factor in aqueous solution.
|
| pubmed:affiliation |
Genetics Institute, Andover, Massachusetts 01810.
|
| pubmed:publicationType |
Journal Article
|