The Formulation Grope Of Recombinant Human Growth Hormone Liquid Formulation

More recently, with recombinant DNA technology development, the DNA encoding human growth hormone was cloned in bacterial plasmids, opening the way to the large-scale commercial production, as well as its ready acceptance into clinical usage, such as Children with idiopathic growth deficiency ,Turner's syndrome etc.The human growth hormone is a protein of 191 amino acids, synthesized and secreted from the anterior lope of the pituitary gland. The molecule is cross-linked by two disulphide bridges and the monomeric form has a molecular weight of 22kDa.Pharmaceutical formulations of growth hormone tend to be unstable . Degradation products such as deamidated or oxydated products and dimer or polymer forms are generated—especially in solution. Deamidation especially takes place at the Asn in position 149 and to a minor extent in position 152.rhGH is also rather easily oxidized in positions 14 and 125. In order to facilitate the handling to be performed by the patient and avoid the lyophilization step in the production of rhGH, it is essential to design a stable liquid formulation.First, we confirmed the chromatogram peak of oxide and deamidated products on a reversed-phase HPLC column by adding oxidant into rhGH aqueous solution and keeping rhGH solution at high temperature to accelerate degradation reaction. The liquid formulation of rhGH that we design includes buffer,non-ionic surfactant,preservative and additives.The preservative we select is phenol in a concentration of 2.5mg/ml.Buffer substance acts a key role on protein stability. Different buffers such as histidine, sodium citrate,and maleate is used in the formution that we design.We test the samples placed at 37℃through RP-HPLC and SEC-HPLC.The result shows that histidine in a concentration of 5mM can reduce the rate of degration processes of rhGH.Stability of rhGH formution is affected by pH.We compare with some samples in different pH and test them through RP-HPLC and SEC-HPLC.The result shows that pH5.8 adapt to the formuliton..Non-ionic surfactant is good for preventing the formation of dimer and polymer. poloxamer 188 is respective used in rhGH liquid formulation and in a concentration of 0.8mg/ml is preferred.And then some additives in rhGH formulation are compared with. We find that 1.5% glycerol is suitable for stabilizing rhGH in aqueous enviroment and other additives are not good enough or harmful to stabilization of rhGH.The final rhGH liquid formution includes 5mM histidine,0.8mg/ml poloxamer 188,2.5mg/ml phenol and 1.5% glycerol in pH5.8.The test result of RP-HPLC,SEC-HPLC and SDS-PAGE for the final formulion show that the liquid formution could be stable for more than one year at 4℃temperature.