Research On The Stability Of Recombinant Human Growth Hormone Of Aqueous Formulation

Recently,the application of recombinant DNA techniques forclone and express the gene of rhGH has made it possible to obtainmore purified and an unlimited amount of the hormone ,so as to makeits applications such as growth stimulation and Turner's syndromeand chronic renal disease,etc. wider and wider.However, one of themost challeng tasks is how to solve the problems of its physicaland chemical instability of protein pharmaceuticals.Proteininstability is one of the major reasons for protein pharmaceuticalsadministered traditionally through injection rather than takenorally like most small chemical drugs.Protein pharmaceuticalsusually have to be stored under cold conditions or freeze-dried toachieve an acceptable shelf life .RhGH consists of a single polypeptide chain with 191 amino acidresidues and two disulfide bridges . the molecular weight is22kDa.The formulation of rhGH undergoes several degradationpathways especially in aqueous solutions. Two derivatives of humangrowth hormone, a sulfoxide and a mixture of two monodesamidoisomers, have been isolated and characterized. The sulfoxidederivative arises from an oxidation of Met-14. The major site ofdeamidation is at Asn-149 with a minor site at Asn-152.Human growth hormone formulations known are all lyophilizedforms.The procedure of lyophilization usually leads to denature ofsome protein pharmaceuticals, Also it needs long time to prepareit which would limted the productivity.The lyophilizedformulations require reconstitution before adminstration,someindirect manipulation method,for example,shaking and shearing canmake proteins denaturalized.It is necessary to develope an aqueousformulation for rhGH in order to eliminate reconstitutionerrors,and increase dosing accuracy as well as simplify the use ofthe product clinically.To optimize the aqueous formulation,some research works haveto be done,including:At first,we confirmed the peak of rhGH-related substances(e.g.sulfoxide and desamido derivatives) in RP-HPLC through isolationand characterization of the accelerated oxidization anddeamidation products by SDS-PAGE and IEF.The RP-HPLC chart showsthat the elution sequence: sulfoxide derivative is first,then thedeamido derivative,and the main peak is at last.SEondly,we discussed the constitutions of weak band by SDS-PAGEin rhGH.The weak band appears the front of main zone bynon-reduction electrophoresis,which would be degraded two zonesunder the main band by reduction electrophoresis and we can obtainits'Mr sequential is 14000 and 8000 through calculating Rfvalue.The weak band would be the hydralization of rhGH,but thedisulfide bridges is not damaged.To characterize it needs more and...