41
Significant fractionation of the remaining compounds can be observed, highlighting the need for the polishing step; fractions 7-13 were analysed for bivalirudin and impurity content (Table 4).
The results show a marked increase in the purity of bivalirudin from the IEX-purified sample, with purity > 99 % and recovery > 95%. A combined IEX-RP process can therefore be exploited for significant gains in the efficiency and cost of a peptide purification process.
To demonstrate that the purification could not be achieved without the initial ion exchange step, a comparison reverse phase-only purification of the crude bivalirudin sample was carried out using the Chromalite®
15AD2 column, Figure 5 shows the chromatogram.
In this purification, we can see that some separation is occurring, however there appear to be somewhat more species present in the solution than was observed in Figure 4. The fractions 8-15 were analysed by HPLC, shown in Table 5.
In this purification, only one fraction was purified to a greater standard than the loaded crude sample, and this represented <30 % of the recovered bivalirudin, indicating that the initial IEX step significantly contributes to the final purity of the bivalirudin.
Conclusions
A novel combination of a low pressure IEX step followed by standard reverse phase polishing step in peptide purification was demonstrated using Chromalite® PCG1200FS and 15AD2. This simplified approach is both cost-effective and highly efficient when compared with a traditional RP-RP purification process, as the larger particle size of the IEX resin facilitates a higher flow rate and lower backpressure, which reduces both the cost of the resin and of the process equipment.
The flexibility of the Chromalite® range
was demonstrated by the purification of bivalirudin from a semi-crude feed by a sulfonic acid-functionalised resin, with purity enhanced from ca. 80% to >90% with removal of organic scavengers and other aqueous impurities and complete recovery of the peptide loaded, followed by a reverse phase polishing step that increased the purity from >90% to >99%. A single reverse phase step following the same procedure as the polishing step could only improve the purity from 86% to 92% with a recovery of <30%.
Fraction Load RP 7 RP 8 RP 9
RP 10 RP 11 RP 12 RP 13 Total
Bivalirudin PA Impurity PA Purity Bivalirudin (mg) Recovery 1705140 0 0 0 0
106913 5757 6045 5961 4577
1725051 207443 31799
12107 43663 6148
94.1% 0% 0% 0% 0%
99.3% 82.6% 83.8%
0.677 0 0 0 0
0.685 0.078 0.008 0.771
Table 5: HPLC Data for fractions of crude bivalirudin purification by reverse phase Fraction
Load RP 8 RP 9
RP 10 RP 11 RP 12 RP 13 RP 14 RP 15 Total
Bivalirudin PA Impurity PA Purity 5817532 0 0 0
946965 4694
11973 61698
2717931 8565461 524012 56043 1423
223177 1439039 449183 11342 6707
86% 0% 0% 0%
92% 86% 54% 83% 18%
0% 0% 0% 0%
101% 12% 1%
114%
Figure 4: Chromatogram of the purification of IEX-purified bivalirudin by Chromalite® 15AD2. 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 4: HPLC data for fractions of IEX-purified bivalirudin purification by reverse phase using Chromalite® 15AD2.
Bivalirudin (mg) Recovery 4.064 0 0 0
0% 0% 0%
1.082 3.421 0.205 0.018 0
4.726
27% 84% 5%
<1% 0%
116%
Figure 5: Chromatogram of the purification of semi-crude bivalirudin by Chromalite®
15AD2. 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.
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