Recombinant Methionyl Human Growth Hormone Research Articles

Selective removal of N-terminal methionine from recombinant human growth hormone by an aminopeptidase isolated from Aspergillus flavus

An aminopeptidase was isolated from a soil fungus, which specifically cleaves the unnatural N-terminal methionine in recombinant human growth hormone. Reaction mixtures with different ratios of aminopeptidase to recombinant methionyl human growth hormone showed that the removal of N-terminal methionine was complete at 1:200 (w/w), and more than 90% complete at ratios up to 1:2000 (w/w) when incubated for 24 h at 37°C. The data indicate that the aminopeptidase we have purified can be used for the efficient conversion of unnatural recombinant proteins to their natural form.

Feasibility study on spray-drying protein pharmaceuticals: recombinant human growth hormone and tissue-type plasminogen activator.

The feasibility of spray-drying solutions of recombinant methionyl human growth hormone (hGH) and tissue-type plasminogen activator (t-PA) was investigated. hGH was formulated in a mannitol phosphate buffer and t-PA was used in an arginine phosphate formulation containing 0.004% (w/v) polysorbate 80. Using filtered air (90-150 degrees C) as the drying medium, hGH could be dried to a residual moisture content of < or = 4%. However, approximately 25% of the protein was degraded during the processing. Results of atomization studies suggest that surface denaturation at the air-liquid interface of the droplets in the spray plays a major role in the degradation of the protein. The addition of 0.1% (w/v) polysorbate 20 into the hGH formulation reduced the formation of soluble and insoluble aggregates by approximately 90% during atomization. During spray-drying the addition of 0.1% (w/v) polysorbate 20 reduced the formation of soluble and insoluble aggregates by approximately 70 and 85%, respectively. In contrast, t-PA remained intact upon atomization. Depending on the spray-drying conditions, product powders with a residual moisture content between 5 and 8% were obtained. No oxidation, aggregation, or denaturation occurred in the protein under several operation conditions. Overall, this study demonstrates that it is feasible to spray-dry t-PA in the current marketed formulation.

Long-term treatment with growth hormone of children with short stature and normal growth hormone secretion

Children with short stature but normal growth rate and/or normal growth hormone response to sleep and secretagogues were treated with recombinant methionyl human growth hormone, 0.3 mg/kg per week. In each year of treatment, about 80% of the subjects maintained an increase in growth rate greater than the defined limit (greater than 1 cm/yr above pretreatment growth rate) for continuation of human growth hormone treatment. Comparison of the group that continued to respond to human growth hormone with the group that did not maintain an accelerated growth rate did not reveal differences in bone age delay, sleep or secretagogue-stimulated human growth hormone secretion, degree of short stature either absolute or relative to target height, and somatomedin C concentration before or after initiation of therapy. The group that failed to respond to the human growth hormone treatment in the first year of treatment was younger and had a higher pretreatment growth rate. Review of the longitudinal growth curves revealed five patterns of response to human growth hormone treatment: (1) failure to increase growth rate in two subjects with height SD scores within 1 SD of target height, (2) failure to increase growth rate in five subjects with height SD scores greater than 1 SD less than the target height, (3) acceleration in growth rate in three subjects that was not maintained until achievement of a height within 1 SD of the target height, (4) acceleration of growth rate in five subjects that was maintained until achievement of a height within 1 SD of the target height, and (5) acceleration in growth rate that was maintained during the 3 years of treatment in 15 subjects who had not attained a height within 1 SD of the target height. We conclude that human growth hormone treatment of some but not all short children with "normal" growth hormone secretion will result in sustained acceleration of growth rate and attainment of prepubertal heights that are closer to but do not exceed their genetic height potential. A clinical trial of human growth hormone may be necessary to determine which subjects will benefit from the treatment.

Isolation and characterization of a succinimide variant of methionyl human growth hormone

Deamidation of asparagine and glutamine residues, isomerization of aspartic acid side chains, and racemization of the L- to the D-form of the amino acids are common spontaneous chemical reactions known to occur in proteins. Previous studies have implicated succinimides as intermediates in these reactions; however, the evidence has been indirect. Our results demonstrate, for the first time, the presence of a succinimide intermediate in an intact protein. The succinimide (cyclic imide) variant was isolated from thermally stressed recombinant methionyl human growth hormone (hGH) by high performance anion-exchange chromatography, further purified by reversed-phase high performance liquid chromatography, and analyzed by tryptic mapping. A later eluting tryptic peptide, compared with the native T12 peptide (residues 128-134, Leu-Glu-Asp-Gly-Ser-Pro-Arg), was analyzed by mass spectrometry (MS). This variant had a protonated molecular mass of 755.3 atomic mass units (u), as compared with 773.3 u for the native T12 peptide. A difference of 18 u, a loss of water, is consistent with the formation of a succinimide intermediate at Asp-130 of methionyl hGH. MS/MS analysis of the cyclic imide-containing peptide verified that the modification occurred at Asp-130. A difference of 18 u was also observed for the intact cyclic imide methionyl hGH variant (22,238 u), as measured by electrospray mass spectrometry, compared with native methionyl hGH (22,256 u).

Absorption of recombinant methionyl-human growth hormone (Met-hGH) from rat nasal mucosa

The absorption of recombinant methionyl human growth hormone (Met-hGH) from the nasal mucosa into the systemic circulation was studied in anesthetized rats. Met-hGH was administered intranasally (i.n.), intramuscularly (i.m.) and intravenously (i.v.) to determine the relative and absolute bioavailability of an intranasal Met-hGH formulation. In the absence of detergent enhancers, the absolute bioavailability of meth hGH was <1%. Hypotonicity enhanced absorption slightly. The absolute bioavailability of Met-hGH increased markedly in the presence of the non-ionic surfactant, polyoxyethylene 9-lauryl ether (laureth-9). The histological changes in the nasal mucosa of rats treated with 1% laureth-9 were severe, however, and included complete removal of the nasal epithelium in places. The bile salt sodium glycocholate was also evaluated for its permeation enhancing abilities. At 0.5% glycocholate there were few noticeable histological changes relative to controls and the absolute biovailability of Met-hGH was approximately 7–8%. While absorption of Met-hGH from the nasal epithelium is demonstrated, the effects of permeation enhancing detergents on the delicate nasal mucosa must be better understood before the intranasal route of administration may be considered suitable for delivering Met-hGH to the systemic circulation.

Absorption enhancement of growth hormone from the gastrointestinal tract of rats

An increased interest in non-injectable dosage forms for polypeptides has led to identification of some compounds which can increase absorption of these drugs. Sodium salicylate administered in rectal suppositories with recombinant methionyl human growth hormone (met-hGH) lead to measurable serum concentrations and demonstratable biological activity of the polypeptide. Sodium salicylate and mineral oil were tested in the ligated stomach, duodenum, ileum and colon of rats for their ability to enhance absorption of met-hGH. Rats were injected in one of the segments of the gastrointestinal tract with 3 mg kg of met-hGH with or without sodium salicylate in aqueous buffer or mineral oil vehicle. Blood samples were collected at various time points and assayed for hGH. Absolute bioavailabilities calculated for each formulation in each intestinal segment indicated that the only treatment with greater than 3% bioavailability was injection of sodium salicylate in mineral oil into the ileum (7%) and colon (9.5%). Statistical analysis proved the surprising interaction of salicylate and mineral oil to be synergistic in absorption enhancement. This is a promising finding but several issues must be addressed and pharmaceutical formulations must be optimized before it can be developed into an oral dosage form for polypeptide drugs.