Study On The Modification Site Of Non-specific Modified PEGylated Recombinant Human Growth Hormone

Recombinant human growth hormone has developed through five generations to form a preparation that we used.The primary sequence of the protein and the tertiary structure are completely consistent with the growth hormone.However,due to the short half-life of growth hormone,the patients need to be injected with drugs every day,which brings great pain and inconvenience to patients.Therefore,the development of long-acting recombinant human growth hormone has become a popular experimental subject.The use of modified methods to improve the half-life of the biological products research has a history of 40 years.PEGylation modification is also one of the most successful ways to extend the half-life of protein or peptide drugs.Polyethylene glycol has been affected by its immune inertness.FDA approved it as one of the few polymers that can be injected into the body,and its safety is basically confirmed.The first PEGrhGH injection was launched in China on 2014,which made more scientists come to the research of long-acting rhGH.In this study,we used the PEG-rhGH sample prepared by co-workers in our laboratory for the experiment.The sample is a non-specific modified protein prepared by covalently binding PEG with an average molecular weight of 30,000 Da and rhGH.First of all,the purity of the sample was confirmed by SDS-PAGE,and then we used MALDI-TOF to confirm the number of PEG that was connected to the protein.The MALDI-TOF results showed the molecular weight of the sample about 53000 Da,which is the same compared to the weight in theory,and no polymer and more PEG modified products were found.We confirmed the approximate isoelectric point of the sample by IEF,and we separated the protein with different position of polyethylene glycol by ion exchange chromatography.We used recombinant human growth hormone national reference substance to establish a linear regression model for quantitative experiments of the protein with different position of polyethylene glycol.Due to the limitation of the samples,only two compound P1 and P2 were enough to the next experiments.And then we used LC-MS to analyzed and characterized the main peptides of the rhGH using peptide map method,and we analyzed the P1 and P2 with the same method.The modification sites results showed that the position of the two positional isomers were located at the phenylalanine of position 1 and lysine of position 140,respectively.Afterwards,trypsin was used to cleave the PEG-rhGH samples.After then we used size exclusion chromatography to separate the pegylation peptides,and Edman degradation method to sequence the primary sequence of the pegylation peptides.The results showed P1 could not be sequenced due to nitrogen-terminal blocking,while the primary sequence of P2 pegylation peptides was the same comparing to the peptide mapping method,so we can sure that the modification sites of the P1 and P2 position isomers were the phenylalanine of position 1 and the lysine of position 140.Finally,the peptide mapping method is used to quantify the different position isomers.We used T12 peptide as standard to quantify the two positional isomers.The results show that the P1 positional isomer is about 54.1%,the P2 positional isomer is about 14.3%,and the others is about 31.6%.In summary,this paper established a method for modifying sites of PEG-rhGH using LC-MS,and performed relative quantification of two positional isomers.We hoped that it would be useful for the future research.