Cleland J L, Mac A, Boyd B, Yang J, Duenas E T, Yeung D, Brooks D, Hsu C, Chu H, Mukku V, Jones A J
Department of Pharmaceutical Research & Development, Genentech, Inc., South San Francisco, California, USA.
Pharm Res. 1997 Apr;14(4):420-5. doi: 10.1023/a:1012031012367.
The development of a sustained release formulation for recombinant human growth hormone (rhGH) as well as other proteins requires that the protein be stable at physiological conditions during its in vivo lifetime. Poly(lactic-co-glycolic acid) (PLGA) microspheres may provide an excellent sustained release formulation for proteins, if protein stability can be maintained.
rhGH was encapsulated in PLGA microspheres using a double emulsion process. Protein released from the microspheres was assessed by several chromatrographic assays, circular dichroism, and a cell-based bioassay. The rates of aggregation, oxidation, diketopiperazine formation, and deamidation were then determined for rhGH released from PLGA microspheres and rhGH in solution (control) during incubation in isotonic buffer, pH 7.4 and 37 degrees C.
rhGH PLGA formulations were produced with a low initial burst (< 20%) and a continuous release of rhGH for 30 days. rhGH was released initially from PLGA microspheres in its native form as measured by several assays. In isotonic buffer, pH 7.4 and 37 degrees C, the rates of rhGH oxidation, diketopiperazine formation, and deamidation in the PLGA microspheres were equivalent to the rhGH in solution, but aggregation (dimer formation) occurred at a slightly faster rate for protein released from the PLGA microspheres. This difference in aggregation rate was likely due to the high protein concentration used in the encapsulation process. The rhGH released was biologically active throughout the incubation at these conditions which are equivalent to physiological ionic strength and pH.
rhGH was successfully encapsulated and released in its fully bioactive form from PLGA microspheres over 30 days. The chemical degradation rates of rhGH were not affected by the PLGA microspheres, indicating that the internal environment of the microspheres was similar to the bulk solution. After administration, the microspheres should become fully hydrated in the subcutaneous space and should experience similar isotonic conditions and pH. Therefore, if a protein formulation provides stability in isotonic buffer, pH 7.4 and 37 degrees C, it should allow for a safe and efficacious sustained release dosage form in PLGA microspheres.
开发重组人生长激素（rhGH）以及其他蛋白质的缓释制剂，要求蛋白质在其体内生存期内于生理条件下保持稳定。如果能维持蛋白质稳定性，聚乳酸-乙醇酸共聚物（PLGA）微球可为蛋白质提供优良的缓释制剂。
采用复乳法将rhGH包封于PLGA微球中。通过多种色谱分析、圆二色性分析和基于细胞的生物分析来评估从微球中释放的蛋白质。然后测定在pH 7.4、37℃的等渗缓冲液中孵育期间，从PLGA微球释放的rhGH和溶液中的rhGH（对照）的聚集、氧化、二酮哌嗪形成及脱酰胺化速率。
制备的rhGH PLGA制剂初始突释率低（<20%），rhGH持续释放30天。通过多种分析测定，rhGH最初以天然形式从PLGA微球中释放。在pH 7.4、37℃的等渗缓冲液中，PLGA微球中rhGH的氧化、二酮哌嗪形成及脱酰胺化速率与溶液中的rhGH相当，但从PLGA微球释放的蛋白质聚集（二聚体形成）速率略快。这种聚集速率的差异可能归因于包封过程中使用的高蛋白浓度。在这些等同于生理离子强度和pH的条件下孵育期间，释放的rhGH始终具有生物活性。
rhGH成功地被包封于PLGA微球中，并在30天内以完全生物活性形式释放。rhGH的化学降解速率不受PLGA微球影响，表明微球的内部环境与本体溶液相似。给药后，微球应在皮下空间完全水化，并应经历相似的等渗条件和pH。因此，如果一种蛋白质制剂在pH 7.4、37℃的等渗缓冲液中具有稳定性，那么它应能制成安全有效的PLGA微球缓释剂型。
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