40 August / September 2019 ADVERTORIAL


The elution is again observed in the water wash, in this case the increased load has resulted in a broadening of the elution peak as could be expected. The fractions 2, 7, 15, 16, 17 and 18 were tested for bivalirudin content and purity by HPLC, giving the results shown in Table 2 - a calibration was also carried out to quantify the amount of bivalirudin present and determine the % recovery of bivalirudin.


Figure 2: Chromatogram of the purification of crude bivalirudin by Chromalite® PCG1200FS. UV


absorbance at 280 nm shown in green, conductivity in red and % eluent in black. Fractions are identified by number at the top of the chromatogram.


Table 2: HPLC Data for fractions of crude bivalirudin purification by cation exchange Fraction


Load 2 7


15 16 17 18


Total


Bivalirudin Peak Area 58641336 5899 0


2312977 11002671 5077866 245512


Total Impurity Peak Area 11578990 168763 250355 72130


464518 272681 820174


Purity


84% 3% 0%


97% 96% 95% 23%


Bivalirudin (mg)


5.86 0.00 0.00 0.92 4.40 2.03 0.10 7.45


Recovery of Bivalirudin N/A 0% 0%


16% 75% 35% 2%


128%


The three fractions present in the large peak, 15; 16 and 17, show a very high level of purity for bivalirudin and also recover a significant majority of the initial bivalirudin mass. The recovery above 100% is likely due to insufficient dilution of the load and fraction 16, which resulted in values outside the calibration range, where more error is likely to result.


Pooling of the fractions 15, 16 and 17 results in a fraction with recovery >90% and purity of 96%, shows a very good result, allowing for the erroneously high recovery observed due to insufficient dilution.


Following this good result, the method was carried out at the target loading of 10 mg bivalirudin per mL of resin. The chromatogram is shown in Figure 3.


Table 3: HPLC Data for fractions of crude bivalirudin purification by cation exchange Fraction


Load 2 7


16 17


Total * Diluted 1/10 for accurate quantification.


Bivalirudin Peak Area 3198211* 220087 0


2852528* 2529208


Total Impurity Peak Area 445213* 87182


159244 217750* 1202963


Purity Bivalirudin (mg)


88% 72% 0%


93% 68%


12.75 0.15 0.00


11.36 1.01


12.52


Recovery of Bivalirudin N/A 1% 0%


89% 8%


98%


In this case the increased load has resulted in the bivalirudin elution significantly eclipsing other peaks present in the crude mixture. The fractions 2, 7, 16 and 17 were tested for bivalirudin content and purity by HPLC, giving the results shown in Table 3.


The results show good recovery of bivalirudin from the crude mixture at a high level of purity. To maximise recovery, pooling of fractions 16 and 17 could be carried out, which would give a total recovery of 97% but would reduce the purity to 90% - which remains an excellent level of purity.


Based on these results, Chromalite® PCG1200FS represents a potentially well- suited resin to the initial purification of bivalirudin when used in a cation exchange purification as described above. The elution in the water wash is particularly useful, as this facilitates the direct application of the eluted bivalirudin fraction to a reverse phase process, without the need for a desalting step to remove interfering salts.


Following this cation exchange first step, a reverse phase polishing step was carried out using Chromalite®


15AD2, in order to


Figure 3: Chromatogram of the purification of crude bivalirudin by Chromalite® PCG1200FS. UV absorbance at 280 nm shown in green, conductivity in red and % eluent in black. Fractions are identified by number at the top of the chromatogram.


gain further increases of purity with a target of >99% purity. A standard reverse phase gradient elution was carried out using 60% acetonitrile in 0.1% TFA in water and Figure 4 shows the chromatogram.


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