在合成 PEGylated Teriparatide方面，本研究藉由調控反應溶劑之pH值可得不同單一PEG5K接枝位置之Teriparatide位置異構物，經由Lysine C切割證明當反應溶劑在pH 6時，PEG5K大多接枝於Teriparatide 的N端；而反應溶劑在pH 8時，PEG5K多接於Teriparatide 的Lys13。此外也發現Teriparatide的結構螺旋性(Helicity)會因PEG5K的接枝而稍微降低，但此二位置異構物抵抗胰蛋白脢水解之能力也因PEG接枝而大幅提升。在逆相層析純化分離N端與Lys13接枝PEG5K的Teriparatide方面，結果發現隨著沖堤溶劑之pH 值提高，由於一級胺質子化程度之差異，導致於異構物間的極性不同，使得此二位置異構物有較佳之分離效果。進一步的以溶解度參數計算發現，可藉由調整溶劑之組成，使二位置異構物與溶劑間之極性項(delta P polarity term of solubility parameter)的差異增加，以利於基線分離，此結果與調整移動相溶劑pH值之結果相符。由本研究得知，溶劑組成與pH值之調整均可改變分子的極性差異，以利於N端與Lys13接枝PEG的胜?異構物達基線分離。 Teriparatide, a peptide drug for treating to osteoporosis by once-daily injection, is the 1-34 segment of recombinant human parathyroid hormone. However, Teriparatide is proteolytically instable in human serum resulting in short circulation half-life (less than 1 hour). Therefore, conjugation with polyethylene glycol (PEGylation) to Teriparatide may shield it from proteolysis to prolong the circulation half-life. For the PEGylated Teriparatide, the positional isomers are usually formed with random PEGylation. We obtained the Nter-PEGylated and K13-PEGylated Teriparatide while the synthesis conditions using pH 6.0 and pH8.0 phosphate buffer, respectively. In this investigation, we intended to directly separate the isomers by tuning mobile phase pH in reversed-phase chromatography (RPC) operation. The results showed that the baseline separation of two isomers can be achieved by tuning the pH value of mobile phase from 7.0 to 9.0, and Nter-PEGylated Teriparatide is much retained in RPC. Form the solubility parameter measurement, we examined that the key factor for the separation of these two isomers is the polarity difference rather than hydrogen bonding or dispersion force. From the circular dichroism measurement, the K13- PEGylated Teriparatide with higher helixity shows less retained in RPC. The results are coherent with our previously proposed structure-retention relationships for peptide isomer retention prediction in RPC.
