Untitled

Abstract

Formulation often has a dramatic effect on degradation of proteins during the freeze-drying process as well as impacting on the "shelf-life" stability of the freeze-dried product. This research presents the results of a formulation optimization study of the "in-process" and shelf-life stability of freeze-dried human growth hormone (hGH). Chemical decomposition via methionine oxidation and deamidation of asparagine residues as well as irreversible aggregation were characterized by HPLC assay methodology. In-process degradation and stability of low moisture freeze-dried solids were studied at 25 and 40 degrees C in a nominal nitrogen headspace (approximately 0.5% O2). Formulation variables included pH, level of salts, and the nature of the lyoprotectant. Studies of the effect of shear on aggregation in solutions indicated that shear comparable to that experienced during filtration does not induce aggregation. Irreversible changes in hGH during the freeze-drying process were minimal, but chemical decomposition via methionine oxidation and asparagine deamidation and aggregation did occur on storage of the freeze-dried solid. Decomposition via methionine oxidation was significant. A combination of mannitol and glycine, where the glycine remains amorphous, provided the greatest protection against decomposition and aggregation. It is postulated that an excipient system that remains at least partially amorphous is necessary for stabilization. However, the observation that dextran 40 formulations showed poor stability toward aggregation demonstrates that an amorphous excipient system is not a sufficient condition for stability. Stability of the solid was optimal when produced from solutions in the pH range, 7-7.5, with severe aggregation being observed at high pH. The level of sodium phosphate buffer affected stability of the solid, although this relationship was complex. Freeze-drying in the presence of NaCl produced severe aggregation and precipitation during the freeze-drying process as well as acceleration of oxidation and/or deamidation.

State diagrams for improving processing and storage of foods, biological materials, and pharmaceuticals (IUPAC Technical Report)

“…Chemical stability at extreme pH conditions may provide amorphous mannitol some advantages, among various other polyols and saccharides [159]. Amorphous mannitol is said to protect protein conformation during freeze-drying through hetero-solute molecular interaction with proteins (e.g., hydrogen bonding) [26,[160][161][162]. However, obtaining pure amorphous mannitol by rapid cooling of a hot solution or by freeze-drying of an aqueous mannitol solution, while possible, is not practical, because amorphous mannitol is physically unstable and will transform into a more stable crystalline form, even under ambient conditions [159,163].…”

“…Molecular-level mixing with other components may limit the spatial rearrangement of mannitol molecules required for crystallization. In fact, various solutes that can remain amorphous in frozen solutions and during freeze-drying (e.g., NaCl, K 2 HPO 4 , L-glycine 30 ) were reported to inhibit mannitol crystallization [23,[159][160][161][162][163][164][165][166][167][168]. Altering molecular mobility in an amorphous phase by complex formation and/or molecular interaction is another approach to prevent mannitol crystallization.…”

Analysis of recombinant human growth hormone by capillary electrophoresis with bilayer-coated capillaries using UV and MS detection

“…Several methods have been used for the analysis of content and purity of rhGH, e.g. reversedphase [11][12][13][14][15], ion exchange [14,16,17], and size-exclusion LC [12,18,19]. CE also shows great potential for the analysis of rhGH and its degradation products [20][21][22][23][24][25].…”

Molecular Mobility of Lyophilized Poly(vinylpyrrolidone) and Methylcellulose as Determined by the Laboratory and Rotating Frame Spin-Lattice Relaxation Times of 1H and 13C

“…Large-time-scale molecular motions have been determined by thermomechanical analysis and dynamic mechanical analysis at temperatures above glass transition temperature (T g ), 7) but such analyses are unsuitable for evaluation of motions at temperatures below T g . In contrast, NMR relaxation measurements, which have been used as a parameter of molecular mobility in food systems [8][9][10][11] and pharmaceutical formulations, 5,[12][13][14] can provide information regarding largetime-scale molecular motions that occur even in glassy polymer materials at temperatures below T g . Although NMR relaxation times reflect molecular motions on smaller time scales at temperatures substantially lower than T g , large-timescale molecular motions that begin to emerge with increasing temperature can be sensitively detected by NMR relaxation times.…”