39 HPLC Quantification of Bivalirudin
HPLC was performed using an Ascentis RP- Amide 5 µm, 250 x 4.6 mm column attached to a Perkin-Elmer Flexar HPLC system, with detection by UV absorption at 214 nm. The mobile phases used were:
Mobile Phase A: 150 mM triethylamine set to pH 3.0 with phosphoric acid
Mobile Phase B: Acetonitrile
The mobile phase was run at 1 mL / min at 25°C following the below gradient:
25 minutes 80% A 1 minute 70% A 2 minutes 50% A 3 minutes 80% A
A calibration curve was generated from an external standard for bivalirudin to allow the quantification of the recovery levels:
Bivalirudin
Concentration (mg/mL) 1
0.75 0.5
0.25 0.1 0
Peak Area 8816916
6737482 4484665 2186559 905432 0
This gave the equation for determining bivalirudin concentration
Concentration (mg/mL) = (Peak Area * 0.0000001) - 0.0012
Recovery was defined as the proportion of bivalirudin present in the fractions as a percentage of the bivalirudin present in the load sample.
Regeneration Method for Chromalite®
PCG1200FS
The column was equilibrated with 5 CV of 1 M NaOH then continued until the UV absorbance at 280 nm was stable. The column was then washed with 5 CV of dH2O then continued until the UV absorbance at 280 nm was stable. The column was equilibrated with 5 CV of 1 M HCl then continued until the UV absorbance at 280 nm was stable. The column was then washed with 5 CV of dH2O then continued until the UV absorbance at 280 nm was stable.
Results and Discussions
Initial purification efforts were focused on the use of a cation exchange resin to conduct an initial clean-up of a crude preparation of bivalirudin, a 2.2 kDa
Figure 1: 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.
therapeutic peptide used as a thrombin inhibitor. These experiments were conducted using Chromalite®
PCG1200FS,
a styrene/DVB strong acid cation exchange resin of particle size 50 µm and a highly hydrophobic core that offers stabilisation of peptides after the initial ionic attraction.
A preliminary gradient elution was conducted at loading of 2.5 mg bivalirudin per mL of resin and the UV trace is shown in Figure 1.
Interestingly, most of the elution occurs during the water wash. This indicates that the mode of binding is not only cation exchange, but that there is also some hydrophobic character to the binding that is not disrupted until the salt concentration in the eluent decreases sufficiently for elution. This is a common observation for Chromalite®
Purity is determined by comparing the proportion of bivalirudin peak area with that of all non-bivalirudin peaks, which is suitable as a first approximation of purity when standards cannot be generated for specific impurities. For example, fraction 15 has a total peak area (not shown in the table) of 4661753 and the peak area of bivalirudin (4482532) divided by the total peak area gives 0.96 or 96 % pure. This first test demonstrates the increase of the bivalirudin purity from 86 % in the crude sample to 96% in fraction 15. This shows both that the desired purity, >95% in the first ‘capture’ step, can be achieved by this method of purification and confirms that the bivalirudin is not eluted until the salt concentration is reduced, as very little bivalirudin is present in fractions other than 14-16.
resins, as they are designed to
exploit the combination of ionic interactions for ion exchange purification, with a comparatively hydrophobic resin backbone to engage hydrophobic interactions simultaneously. The fractions 2, 6, 13, 14, 15, 16 and 17 were tested for bivalirudin content and purity by HPLC, giving the results reported in Table 1.
Based on the observed elution from this initial test, the method was adjusted to a more straightforward stepwise elution process. In this experiment, 5 mg bivalirudin was loaded per mL resin and the resulting chromatogram is shown in Figure 2.
Table 1: HPLC Data for fractions of crude bivalirudin purification by cation exchange.
Fraction Load 2 6
13 14 15 16 17
Bivalirudin Peak Area 1318733 37682 0
38378
708467 4482532 390895 33811
Total Impurity Peak Area 206085 88509
127158 99853
170344 179221 210561 263022
Purity 86% 30% 0%
28% 81% 96% 65% 11%
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