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Abstract

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.

Paracellular permeation-enhancing effect of AT1002 C-terminal amidation in nasal delivery

“…The effects of dextrose on the stability of AT1002 terminally modified peptides are shown in Figure 3 . Dextrose is a stabilizing excipient of proteins, 19 which is used as an additive for nasal administration solutions during in vivo studies. 24 Our previous report indicated that dextrose has stabilizing effects on AT1002.…”

“…Chemical degradation and protein aggregation may be induced by a variety of physical factors such as temperature and ionic strength, or with time; 18 degradation products such as C-terminal fragments can change significantly with varying pH. 19 In addition, the stability of proteins can be modified by soluble and nontoxic excipients, such as dextrose, or by changing their structural characteristics towards those of thermophilic proteins by introducing negatively charged residues at the N-cap or positively charged residues at the C-cap of the helix. 18 The latter approach is effective because the helical structure of short peptides and proteins is stabilized by capping interactions between side chains and unfulfilled peptide groups at the N- and C-termini.…”

Minimization of Recombinant Human Flt3 Ligand Aggregation at the Tm Plateau: A Matter of Thermal Reversibility

“…Changes in the melting transition temperature ( T m ), or denaturation temperature of unfolding as it is sometimes called, can be a measure of a solution's influence on the stability of a protein ( − ). The T m is related to the change in free energy between the native and denatured states of a protein molecule (i.e., Gibbs−Helmholtz equation) and, therefore, is a thermodynamic parameter of conformational stability ( , ).…”

Stability of proteins in aqueous solution and solid state

“…Increase in soluble aggregates can result in change in immunogenicity of the therapeutic protein. Aggregates in certain cases can be visualized e.g., insulin and IL-1β 97,98 . Insoluble aggregates can be detected by FTIR, Raman, and electron spin resonance spectroscopy, or light scattering techniques (UV absorption).…”