Investigation On The Deamidation And Disulfide-Bonds Of Pegylated Proteins

Pharmaceutical proteins have appealed wide attentions for their high bioactivity, specificity and explicit functions. However, the structural complex of proteins usually results in instability during transport and/or storage. The instability often causes protein degradation such as deamidation, dissociation of subunits and/or disulfide bond-mediated aggregation, which will decrease the pharmaceutical efficiency of the target proteins. In this work, deamidation and disulfide bond-mediated instability of proteins were investigated using high performance liquid chromatography mass spectrometry coupled with protein enzymatic hydrolysis. Two kinds of proteins, recombinant human endostatin (rhEndostatin) and recombinant human granulocyte colony stimulating factor (rhG-CSF), were used as model proteins. Then pegylate the two proteins and investigate the effect of PEGylation on the process of deamidation and disulfide-bond.The dissertation is divided into three parts to research the stability of pharmaceutical proteins. First, The deamidation process of Asn in recombinant human endostatin was studied under different storage conditions. It was found that Asn127 deamidated and Asp127 formed during storage, which is associated with Gly. The kinetic constants of deamidation reaction happened to Asn127 was determined under different conditions. The effect of pH and temperature of the protein solution on the kinetic constant of deamidation process was determined. The result showed that the reaction rate accelerated with the increasing pH and temperature.Then investigate the relation between stability and inter/intra-molecular disulfide bond of rhG-CSF. The inter-molecular disulfide bond, Cys18-Cys18, between rhG-CSF was found, and other disulfide bonds include Cys18-Cys37, Cys18-Cys65 or Cys37-Cys75. The concentration of protein increasing from 0.5mg/mL to 1.0mg/mL and 1.52mg/mL, the content of inter-molecular disulfide bond Cys18-Cys18 increased 15.25% and 105.08% basing on the concentration, which showed that the relative abundance of inter-molecular disulfide-bond was influenced by the concentration of the protein in a certain range. The increasing concentration promote the formation of disulfide bond. An inter-molecular disulfide bond containing peptide with molecular weight of 15710 Da was found in the digest mixture. The content of this peptide decreased with the concentration and precipitation appeared. It resulted that the disulfide bond was correlated with precipitation.The third part focused on the deamidation and disulfide bond of pegylated proteins. Use mPEG-ALD(20 kDa) to pegylate the protein and determine the linking site of PEG chain on protein surface. Then investigate the effect of PEGylation on the deamidation and disulfide bond. As to deamidation, the rate constant of deamidation showed that the effect of pH and temperature to the rate of deamidation decreased, but when the temperature reached to a certain conditions, the process of deamidation had been speeded up severely. As to the inter/intra-molecular disulfide bond, only the disulfide bond, Cys18-Cys18, was found in PEG-rhG-CSF solution and all other inter-molecular disulfide bonds were not found. The consent of Cys18-Cys18 is positive correlated with the concentration of PEG-rhG-CSF. Peptide Leu36-Arg147 was also found which was not found before pegylation and no precipitation was found after modification. Thus, it was concluded that the PEGylation will hamper the deamidation process and the formation of inter-molecular disulfide bond.