19-08-2014, 10:30 AM
For the purification of Leuprolide from synthetic mixture, screening of adsorbents was done on the basis of physicochemical properties of the adsorbents and target peptide. Of the several adsorbents tested, Diaion HP20ss showed maximum binding capacity. It was observed that separation is a monotonic function of differences in hydrophobicity of target peptides and related impurities. Results showed that use of polymeric material such as PS-DVB and polymethacrylate based reverse phase matrices gave good resolution and selectivity. The developed process not only could reduce the impurity level below the LOD limits but also gave purified product in recoveries of more than 95% and purity more than 98%.
Recent years have witnessed a revival in the field of peptides. The peptide therapeutics market is providing new commercial opportunities to biotechnology and pharmaceutical industries. Peptides play an important role in modulating many physiological processes in our body. Therapeutic peptides are now viable alternatives to other biopharmaceuticals, such as antibodies especially in the case of cancer treatment due to their ability to penetrate tumors. Today, more than 40 peptides are currently in the market, 200 are in clinical phases and more than 400 are in advanced preclinical trials. Despite successes in the synthesis, peptide drug discovery has been hampered by high manufacturing and purification costs. The main challenge in peptide production is to design purification strategies. Purification of peptides from natural sources as well as from synthetic reaction mixture is challenging because of the small differences in molecular properties between the target peptide, structural analogues and the failure sequences. Reverse Phase HPLC is the most popular method for peptide purification because of its high resolving power. Traditionally, most RP-HPLC peptide purifications are carried on octadecyl or octyl group grafted silica based sorbents. However, silica based adsorbents suffer from limitations such as low stability in alkaline conditions, high cost and fragility for application in large-scale systems.
There is thus need to develop purification methods that will be efficient, low cost and easily applicable to large scale systems. The present work is aimed at exploring the use of inexpensive and pH stable polymeric adsorbents media for chromatographic purification of peptides. Polymeric adsorbent based on polystyrene divinylbenzene have been used for purification of a synthetic peptide (Leuprolide), while ion exchange adsorbents have been used for purification of naturally derived bioactive peptide (Glycomacropeptide) at preparative scale. Leuprolide is a nona-peptide and a synthetic structural analogue of leutinizing hormone releasing hormone (LHRH) that regulates production of sexual hormones.
For the purification of Leuprolide from synthetic mixture, screening of adsorbents was done on the basis of physicochemical properties of the adsorbents and target peptide. Of the several adsorbents tested, Diaion HP20ss showed maximum binding capacity. It was observed that separation is a monotonic function of differences in hydrophobicity of target peptides and related impurities. Results showed that use of polymeric material such as PS-DVB and polymethacrylate based reverse phase matrices gave good resolution and selectivity. The developed process not only could reduce the impurity level below the LOD limits but also gave purified product in recoveries of more than 95% and purity more than 98%.
Glycomacropeptide (GMP) is a natural biologically active peptide present in milk. GMP is derived from -casein by the action of rennet and has received much attention due to its unique composition and health promoting characteristics. Work carried for purification of GMP demonstrated the feasibility of using two ion exchangers of opposite polarity in series to recover GMP and all other milk proteins from single stream of whey. The developed process gave purified GMP of purity more than 95%.