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Degradation pathways for recombinant human macrophage colony-stimulating factor in aqueous solution
J A Schrier1, R A Kenley, R Williams
1Genetics Institute, Andover, Massachusetts 01810.
Pharmaceutical Research
|July 1, 1993
Summary
Recombinant human macrophage colony-stimulating factor (rhM-CSF) is most stable at pH 7-8. Acidic conditions cause peptide cleavage, while alkaline conditions lead to cross-linking, but N-terminal fragments retain activity.
Area of Science:
- Biochemistry
- Protein Chemistry
- Pharmaceutical Science
Background:
- Recombinant human macrophage colony-stimulating factor (rhM-CSF) is crucial for macrophage proliferation and activity.
- Current clinical trials necessitate a stable pharmaceutical formulation of rhM-CSF.
- Understanding rhM-CSF degradation is vital for developing effective drug products.
Purpose of the Study:
- To investigate the impact of pH on rhM-CSF degradation.
- To identify and characterize the degradation products of rhM-CSF.
- To determine optimal pH conditions for rhM-CSF stability.
Main Methods:
- Incubation of rhM-CSF at varying temperatures (30-50°C) and pH levels (2-10).
- Analysis using SDS-PAGE, reverse-phase HPLC, size exclusion HPLC, scanning microcalorimetry, and murine bone marrow assays.
- Characterization of degradation products via N-terminal sequencing, mass spectrometry, and peptide mapping.
Main Results:
- Maximal rhM-CSF stability observed between pH 7 and 8.
- Acidic pH (2-6) induced peptide cleavage at Asp169-Pro170 and Asp213-Pro214.
- Alkaline pH (>6) resulted in parallel cleavage and beta-elimination-mediated cross-linking.
Conclusions:
- The optimal pH range for rhM-CSF stability is 7-8.
- Distinct degradation pathways occur in acidic and alkaline environments.
- N-terminal degradation fragments of rhM-CSF retain biological activity, suggesting potential therapeutic utility.