Development of a novel sustained release formulation of recombinant human growth hormone using sodium hyaluronate microparticles.

A novel sustained release formulation of recombinant human growth hormone (SR-rhGH) was developed as a once-a-week injection formulation using sodium hyaluronate. SR-rhGH was produced in the form of solid microparticles by spray drying technology. A single administration of a prototype formulation of SR-rhGH with a ratio of hGH:HA=1:1 to cynomolgus monkeys through a fine 26-gauge needle induced continuous elevation of serum IGF-I level for 6 days demonstrating the bioactivity of hGH released from the prototype formulation. For expanded pre-clinical and clinical developments, a pilot-scale process under aseptic condition was established and used for the preparation of the optimized formulation of SR-rhGH with a ratio of hGH:HA=1:3. When the ratio of hGH to HA changed from 1:1 to 1:3, hGH released more slowly in vitro from SR-rhGH with almost complete release of hGH loaded. According to pharmacokinetic and pharmacodynamic studies in beagle dogs, sustained release of hGH from the optimized formulation of SR-rhGH continued for a more extended period longer than 72 h with a lower C(max) than those of prototype formulations. The single administration resulted in an elevation of serum insulin-like growth factor-I (IGF-I) level for 6 days with a maximum value higher than the baseline level by ca. 350 ng/mL, which supported the possibility of SR-rhGH as a once-a-week injection formulation of hGH. The bioavailability of both formulations was comparable to that of hGH daily injection formulation. Finally, toxicity studies revealed no evidence of adverse effect in both cynomolgus monkeys and beagle dogs.

Characterization of N alpha-acetyl methionyl human growth hormone formed during expression in Saccharomyces cerevisiae with liquid chromatography and mass spectrometry.

We found a new variant of human growth hormone (hGH) from the recombinant hGH expression process in Saccharomyces cerevisiae. The variant was identified as N(alpha)-acetyl methionyl hGH which may be formed by N(alpha)-acetylation of met-hGH during the intracellular expression of hGH in S. cerevisiae. The variant was isolated from manufacturing process of LG Life Sciences' hGH product. The variant was subjected to trypsin digestion and RP-HPLC analysis, resulting in a delayed retention time and an increased mass (173 Da) of T1 tryptic peptide. The amino acid composition and amino acid sequence of the peptide showed the same result with T1 peptide of met-hGH except the N-terminal modification on methionine in the variant peptide. With collision induced dissociation (CID) experiments of the variant T1 tryptic peptide, we found the sequence and the a(1) fragment of N-terminal residue matched with those of acetyl-methionyl hGH. Within our production process, we produce the methionyl hGH first and then use the aminopeptidase to cut the N-terminal methionine. So the acetylation may inhibit the aminopeptidase to remove methionine and produces N(alpha)-acetyl methionyl hGH. And the biological activity of the variant was comparable to one of the unmodified hGH when tested by rat weight gain bioassay.