Reversed-phase purification

The productivity of a typical reversed phase purification of a peptide is summarized in Table 4.3. For this particular separation the column loading is relatively low and a large number of separations are carried out in order to deliver the annual production target. On the face of it, the productivity of this separation is poor in practice the column size was chosen to balance the output with the rate of synthesis of crude peptide. The actual combined costs of synthesis and purification for this particular peptide was only a tiny fraction of the value of the formulated drug. On occasions it may be better to purify a valuable product in smaller portions rather than risk a proportionally high loss from failed separation. 

PEG MAL-GV-N reverse-phase purification 1414 PEG-staphylokinase, preformulation 1409 Polyethyleneimine 1316... 

Finally, the transcripts were completely digested by RNase Ti at 37°C for 10 min with MALDI matrix (3-HPA) added as a denaturant. Briefly, 5 pL of RNA transcript (up to 20 pg) is added to 4 pL 3-HPA in 50% ACN/water and 1 pL of RNase Tj (1000 units) and reacted for 10 min and then placed on ice for MALDI preparation. For highest quality spectra, samples are desalted by reverse-phase purification in ZipTips according to manufacturer s directions for nucleic acid purification [96]. The final step in this process is elution of purified RNA oligonucleotide fragments onto the MALDI target using 2 pL of the MALDI matrix itself. Samples (MALDI spots ) are allowed to air dry or may be rapidly dried under vacuum. 

Reversed-phase chromatography is widely used as an analytical tool for protein chromatography, but it is not as commonly found on a process scale for protein purification because the solvents which make up the mobile phase, ie, acetonitrile, isopropanol, methanol, and ethanol, reversibly or irreversibly denature proteins. Hydrophobic interaction chromatography appears to be the least common process chromatography tool, possibly owing to the relatively high costs of the salts used to make up the mobile phases. 

Another example is the purification of a P-lactam antibiotic, where process-scale reversed-phase separations began to be used around 1983 when suitable, high pressure process-scale equipment became available. A reversed-phase microparticulate (55—105 p.m particle size) C g siUca column, with a mobile phase of aqueous methanol having 0.1 Af ammonium phosphate at pH 5.3, was able to fractionate out impurities not readily removed by hquid—hquid extraction (37). Optimization of the separation resulted in recovery of product at 93% purity and 95% yield. This type of separation differs markedly from protein purification in feed concentration ( i 50 200 g/L for cefonicid vs 1 to 10 g/L for protein), molecular weight of impurities (<5000 compared to 10,000—100,000 for proteins), and throughputs ( i l-2 mg/(g stationary phasemin) compared to 0.01—0.1 mg/(gmin) for proteins). 

If the hGH is exported to the culture medium the product can easily be collected by removal of the cells from the culture medium by centrifiigation. Purification of hGH from the culture medium is faciUtated by low amounts of contaminating proteins present. In fact, it has been shown that hGH can be purified on a laboratory scale by a single purification step on a reversed-phase hplc column (43). Mammalian cells growing in tissue culture have also been used as hosts to produce hGH, which is exported into the culture media (44). 

Recovery and Purification. The dalbaheptides are present in both the fermentation broth and the mycelial mass, from which they can be extracted with acetone or methanol, or by raising the pH of the harvested material, eg, to a pH of 10.5—11 for A47934 (16) (44) and A41030 (41) and actaplanin (Table 2) (28). A detailed review on the isolation of dalbaheptides has been written (14). Recovery from aqueous solution is made by ion pair (avoparcin) or butanol (teicoplanin) extraction. The described isolation schemes use ion-exchange matrices such as Dowex and Amberlite IR, acidic alumina, cross-linked polymeric adsorbents such as Diaion HP and Amberlite XAD, cation-exchange dextran gel (Sephadex), and polyamides in various sequences. Reverse-phase hplc, ion-exchange, or affinity resins may be used for further purification (14,89). 

This group, used for 5 -phosphate protection, has hydrophobicity similar to that of the dimethoxytrityl group and thus was expected to assist in reverse-phase HPLC purification of product from failure sequences in oligonucleotide synthesis. The group is cleaved with Bu4N F in DMSO at 70°. ... 

This highly lipophilic group is cleaved with isoamyl nitrite in Pyr/AcOH. The use of a lipophilic 5 -phosphate protective group aids in reverse-phase HPLC purification of oligonucleotides. 

Purification of the activation products (PMs). The methylamine activation product dissolved in methanol is purified by chromatography, first on a column of silica gel using a mixed solvent of chloroform/ethanol, followed by reversed-phase HPLC on a column of divinylbenzene resin (such as Jordi Reversed-Phase and Hamilton PRP-1) using various solvent systems suitable for the target substance (for example, acetonitrile/water containing 0.15% acetic acid). 

Chromatography. A number of HPLC and TLC methods have been developed for separation and isolation of the brevetoxins. HPLC methods use both C18 reversed-phase and normal-phase silica gel columns (8, 14, 15). Gradient or iso-cratic elutions are employed and detection usually relies upon ultraviolet (UV) absorption in the 208-215-nm range. Both brevetoxin backbone structures possess a UV absorption maximum at 208 nm, corresponding to the enal moeity (16,17). In addition, the PbTx-1 backbone has an absorption shoulder at 215 nm corresponding to the 7-lactone structure. While UV detection is generally sufficient for isolation and purification, it is not sensitive (>1 ppm) enough to detect trace levels of toxins or metabolites. Excellent separations are achieved by silica gel TLC (14, 15, 18-20). Sensitivity (>1 ppm) remains a problem, but flexibility and ease of use continue to make TLC a popular technique. 

Sixteen solid-phase materials were tested on a laboratory scale and the antho-cyanin and sugar content of collected fractions were determined. Among these, reverse-phase silica gels and macroreticular non-ionic acrylic polymer adsorbents such as Serdolit PAD IV or Amberlite XAD-7 turned out to be most suitable. SPE was used to investigate these materials on an enlarged scale, improving elution gradient and column purification. Amberlite XAD-7 was successfully applied in a middle-scale separation. ... 

In the current era many medicinal chemists are unaware of the very important role of compound soUd state properties on aqueous solubility and therefore to oral absorption. In many organizations compound purification by crystallization has disappeared being replaced by automated reverse-phase HPLC purification. If medicinal chemists isolate a compound as a white powder from evaporation of... 

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