Impurity Evaluation of Heparin Sodium by Anion Exchange Chromatography

As a blood-thinning drug, heparin and its derivative, low-molecular-weight heparin (LMWH), have been widely used for decades during surgery and kidney dialysis. Heparin belongs to the group of linear polysaccharides called glycosaminoglycans (GAGs), and consists of alternating glucosamine and hexuronic acid residues. Heparin has tremendous heterogeneity, due to N-acetylation, and various sulfation patterns and chain lengths, making analytical characterizations extremely challenging.

Raw heparin material is extracted from mammalian tissues such as pig intestines. The heparin material must go through many treatment and purification steps before it can be used in the drug formula. Stringent quality control in the purification steps is important to ensure the quality of heparin as the final active pharmaceutical ingredient (API) of the drugs. Recent incidents, including severe allergic reactions and several deaths, have been attributed to adulterated heparin, resulting in a massive recall of heparin drugs by the manufacturer.1 Oversulfated chondroitin sulfate (OSCS) is a contaminant in heparin associated with the adverse clinical events.2 Because heparin is a drug commonly used in clinics, these adverse events have created a worldwide crisis and a call for an analytical method that can readily monitor the purity of heparin API before formulation of the drug.

A simple and easy high-performance liquid chromatography method has been developed (Waters Corp., Milford, MA) to separate and quantify OSCS in the presence of heparin. The method uses anion exchange chromatography to achieve complete resolution between heparin and OSCS, and UV absorption to quantify the concentrations of heparin and OSCS. The results demonstrate that the method not only generates reproducible, fast separations (10 min), but also detects OSCS at a concentration of less than 1% of overall content. The HPLC method addresses the limitations inherent with the capillary electrophoresis method for heparin analysis. The ability to quickly and unambiguously analyze the purity of heparin drugs can improve and accelerate the quality control of raw API materials in the pharmaceutical industry. The sensitive testing method can be used to screen for heparin quality and OSCS adulteration to protect patient health.

Experimental

Sample preparation

Heparin Sodium Identification RS (catalog #1304038) and Heparin Sodium System Suitability RS (catalog #1304049) were purchased from U.S. Pharmacopeia (USP, Rockville, MD). The Heparin Sodium System Suitability RS is a mixture that contains approximately 80% heparin and 20% OSCS.

Stock solutions (10 mg/mL) of heparin sodium standard or heparin sodium system suitability standard were prepared by reconstituting the samples in MilliQ® water (Millipore Corp., Billerica, MA). Samples with diluted concentrations were prepared by diluting the stock solutions to the desired concentration using Milli-Q water.

Anion exchange chromatography for heparin and OSCS

HPLC separations were performed on a Spherisorb® 5-µm strong anion exchange (SAX) column (4.6—250 mm) from Waters Corp. The column temperature was maintained at 40 °C during all experiments. Eluent A was 50 mM NaH2PO4, and eluent B was 2 M NaClO4 in 50 mM NaH2PO4. The mobile phase pH was adjusted to 2.5 with phosphoric acid. The sample (25 µL) was injected, and a linear gradient (from 10 to 90% eluent B in 10 min) at a flow rate of 0.5 mL/min was used for elution.

A Waters® Alliance® HPLC Bioseparations system equipped with a Waters 2998 photodiode array detector (Waters Corp.) was used in all LC-UV measurements. The elution profiles were monitored from 190 nm to 400 nm at a sampling rate of 2 pts per sec. Data acquisition and analysis was performed using MassLynx 4.1 (Waters Corp.).

Results

Figure 1 - Alliance HPLC Bioseparations system for ion
exchange chromatography.

Bioseparations via ion exchange chromatography typically involve the use of harsh salts and extreme pH conditions. To develop a robust and high-resolution anion exchange chromatography separation method for routine heparin analysis, an Alliance Bioseparations (AllianceBIO) system (Figure 1) featuring a titanium/ polymeric flow path was used to ensure the high-precision, reproducible delivery of mobile phases containing high amounts of salt.

Figure 2 shows an overlay elution profile for Heparin Sodium System Suitability RS and Heparin Sodium Identification RS from U.S. Pharmacopeia. The extracted chromatogram for the system suitability sample (at 202-nm wavelength) shows two distinct peaks with retention times of 8.25 and 11.43 min, whereas heparin sodium standard sample only gives one chromatographic peak at 8.25 min. Comparison between the two chromatographic traces indicates that heparin is eluted first (at 8.25 min) followed by OSCS at 11.43 min. This figure shows that SAX chromatography can be used to rapidly separate heparin from OSCS with a 10-min linear gradient.

Figure 2 - UV chromatograms (202 nm) of USP Heparin Standard RS (in purple) and USP Heparin System Suitability test sample RS (in red) using the Spherisorb 5-µm SAX 4.0 × 250 mm. In total, 25 µg of the materials was injected onto the column for each analysis.

Figure 3 - Overlay of UV chromatograms (202 nm) of four replicate injections of USP Heparin System Suitability test sample RS, showing the reproducibility of the separation by the Spherisorb 5 µm SAX 4.0 × 250 mm. In total, 25 µg of the materials was injected onto the column for each injection.

The chromatographic repeatability of the separation from run to run was investigated using a 1.0-mg/mL solution of Heparin Sodium System Suitability RS. To determine the reproducibility of the separation, the retention times at the peak top for corresponding heparin and OSCS peaks were collected for 10 consecutive injections, and the retention time variations were calculated. Figure 3 shows an example of the overlay of UV chromatograms obtained from four injections of the sample. The retention time RSD values for heparin and OSCS were found to be 0.08% and 0.05%, respectively.

Capillary electrophoresis (CE) was previously reported to separate heparin and OSCS. Only limited separation was achieved between heparin and OSCS in the initial CE experimental condition.3 Although improved separation was obtained using a different CE method, the separation required the use of high-molarity lithium phosphate buffer (600 mM) as the background electrolyte and a smaller i.d. (25 µm) capillary tubing to control the current, making implementation of the CE method more difficult. In comparison with Figure 2, the elution order of heparin and OSCS was also reversed in the electropherogram with OSCS migrating faster than heparin sodium in the CE analysis. The elution order difference of heparin and OSCS between CE and SAX confirms that SAX separation of heparin and OSCS is based on the negative charge density on the linear polysaccharide chain. OSCS typically has at least one extra sulfate group for every disaccharide repeat unit compared to heparin.

Figure - 4 Calibration curves of heparin and OSCS over the concentration range 0.1–10 mg/mL. The concentrations are given as the sum concentration of the two components.

One of the basic requirements to develop an analytical method for quality control purposes lies in quantitative impurity analysis. The method should entail simultaneous analysis of a large amount of parent compound and low-level impurity. On the basis of successful separation of heparin and OSCS by SAX, the linear dynamic range of the method was investigated. A stock solution of the system suitability sample was prepared (10 mg/ mL), and samples with a series of concentrations from 5.0 mg/mL to 0.1 mg/mL were prepared by sequential dilution of the stock solution. These solution standards were injected onto the SAX column in triplicate at an injection volume of 25 µL. Figure 4 shows the calibration curves generated from these injections. The calibration curves were generated by plotting the integrated respective peak areas of heparin and OSCS against the total concentrations of the two components. As shown in Figure 4, the calibration curves are linear over two orders of magnitude with R2 values in excess of 0.999.

Figure 5 - UV chromatograms showing the separation between heparin and OSCS when the concentration of OSCS is ~1% of heparin concentration. A UV trace of blank injection was also plotted in the graph to show the integrated peak areas of heparin and OSCS.

To test the applicability of the SAX method in impurity analysis, a heparin sample containing roughly 1% of OSCS was created by mixing the solution of heparin sodium standard (at 10 mg/mL) and the solution of heparin sodium system suitability standard (1.0 mg/mL) at a precalculated ratio. The calculation was based on the presumption that OSCS accounts for 20% of the total concentration in the solution of Heparin Sodium System Suitability RS. Figure 5 shows the chromatogram obtained from such a mixture. As can be seen, a small, well-defined chromatographic peak for OSCS is observed from the mixture. Integration of the chromatographic peaks for heparin and OSCS yields a peak area of 87,294 and 1889, respectively. Based on the calibration plot in Figure 4, the concentration of heparin and OSCS can be calculated as 4.61 mg/mL and 0.048 mg/mL. This indicates that heparin is 96.5-fold more concentrated than OSCS in the synthetic mixture, implying that the method can readily detect and quantify the concentration of OSCS with about 1% of heparin concentration.

Conclusion

The combination of the Alliance Bioseparations (AllianceBIO) system with the Spherisorb SAX column results in a powerful system for the separation and quantification of heparin and OSCS. It enables rapid, sensitive, and high-resolution separations, and generates data for the evaluation and determination of heparin purity. The wide linear dynamic range in conjunction with the effective separation of the system makes it well suited for quantitative impurity analysis. OSCS at 1% of heparin concentration is readily detected by the system. The result demonstrates that the system is well poised to detect the existence of OSCS as an adulterant to the heparin API.

References

  1. Kemsley, J. Chem. Eng. News 2008, 86, 46-7.
  2. Guerrini, M.; Beccati, D.; Shriver, Z.; Naggi, A.; Viswanathan, K.; Bisio, A.; Capila, I.; Lansing, J.C.; Guglieri, S.; Fraser, B.; Al- Hakim, A.; Gunay, N.S.; Zhang, Z.; Robinson, L.; Buhse, L.; Nasr, M.; Woodcock, J.; Langer, R.; Venkataraman, G.; Linhardt, R.J.; Casu, B.; Torri, G.; Sasisekharan, R. Nat. Biotechnol.2008, 26, 669-75.
  3. Wielgos, T.; Havel, K.; Ivanova, N.; Weinberger, R. J. Pharm. Biomed. Anal. 2009, 49, 319-26.

The authors are with the Biopharmaceutical Sciences Group, Waters Corp., 34 Maple St., Milford, MA 01757, U.S.A.; tel.: 508-482-2000; fax: 508- 482-3085; e-mail: [email protected].

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