NMR spectroscopic evaluation of the internal environment of PLGA microspheres.

The internal environment of poly(lactide-co-glycolide) (PLGA) microspheres was characterized using 31P and 13C solid-state and solution NMR spectroscopy. Physical and chemical states of encapsulated phosphate- and histidine-containing porogen excipients were evaluated using polymers with blocked (i.e., esterified) or unblocked (free acid) end groups. Spectroscopic and gravimetric results demonstrated that the encapsulated porogen deliquesced upon hydration at 84% relative humidity to form a solution environment inside the microspheres. Dibasic phosphate porogen encapsulated in unblocked PLGA was partially titrated to the monobasic form, while in the same formulation 13C NMR showed partial protonation of the histidine imidazole. Similarly, encapsulated monobasic phosphate was partially converted to phosphoric acid. Coencapsulation of monobasic and dibasic phosphate porogens resulted in a single peak on hydration, indicating chemical exchange between discrete excipient microphases. Exogenous buffer addition differentiated external from internal, nontitratable, excipient populations. Microspheres containing dibasic phosphate porogen were hydrated with fetal calf serum, incubated at 37 degrees C, and characterized by 31P NMR through the polymer erosion phase. Within 48 h the 31P chemical shift moved over 2 ppm upfield and the line width narrowed to <60 Hz; there was little additional change through day 14. This indicated complete conversion to the monobasic phosphate form throughout the polydisperse sample and that pH remained below 4 but above the phosphoric acid p K a during matrix erosion.

Poly(lactide-co-glycolide) microsphere formulations of darbepoetin alfa: spray drying is an alternative to encapsulation by spray-freeze drying.

PURPOSE: The purpose of this work was to evaluate spray-freeze drying and spray drying processes for encapsulation of darbepoetin alfa (NESP, Aranesp). METHODS: Darbepoetin alfa was encapsulated in poly(lactide-co-glycolide) by spray-freeze drying and by spray drying. Integrity was evaluated by size-exclusion chromatography and Western blot. Physical properties and in vitro release kinetics were characterized. Pharmacokinetics and pharmacodynamics were evaluated in nude rats. RESULTS: Microspheres produced by spray drying were larger than those produced by spray-freeze drying (69 microm vs. 29 microm). Postencapsulation integrity was excellent for both processes, with < 2% dimer by size-exclusion chromatography. In vitro release profiles were similar, with low burst (< 25%) and low cumulative protein recovery at 4 weeks (< or = 30%), after which time covalent dimer (< or = 6.5%) and high molecular weight aggregates (< or = 2.3%) were recovered by denaturing extraction. After a single injection, darbepoetin alfa was detected in serum through 4 weeks for all microsphere formulations tested in vivo, although relative bioavailability was higher for spray-freeze drying (28%) compared with spray drying (21%; p = 0.08) as were yields (73-82% vs. 34-57%, respectively). For both processes hemoglobin was elevated for 7 weeks, over twice as long as unencapsulated drug. CONCLUSIONS: Spray drying, conducted at pilot scale with commercial equipment, is comparable to spray-freeze drying for encapsulation of darbepoetin alfa.

Protein powders for encapsulation: a comparison of spray-freeze drying and spray drying of darbepoetin alfa.

PURPOSE: To evaluate spray-freeze drying and spray drying processes for fabricating micron-sized particles of darbepoetin alfa (NESP, Aranesp) with uniform size distribution and retention of protein integrity, requirements for encapsulation. METHODS: Darbepoetin alfa was spray-freeze dried using ultrasonic atomization at 120 kHz and 25 kHz and spray dried at bench-top and pilot scales. Reconstituted powders were evaluated by size exclusion chromatography and UV/VIS spectroscopy. Powder physical properties were also characterized. RESULTS: Spray-freeze drying resulted in aggregation of darbepoetin alfa. Aggregates (primarily insoluble) formed on drying and/or reconstitution. Particle size distributions were broad (span > or = 3.6) at both nozzle frequencies. Annealing before drying reduced aggregate levels slightly but increased particle size over 5-fold. Spray drying at inlet temperatures up to 135 degrees C (and outlet temperatures up to 95 degrees C) showed little impact on integrity. Integrity at bench-top and pilot scales was identical, with 0.2% dimer and no high molecular weight or insoluble aggregates detected. Particle size was small (< or = 2.3 microm) with uniform distribution (span < or = 1.4) at both process scales. CONCLUSIONS: Under the conditions tested spray drying, conducted at bench-top and pilot scales with commercially available equipment, was superior to spray-freeze drying for the fabrication of darbepoetin alfa particles for encapsulation.