Molecular profiling and weight determination of heparins and depolymerized heparins

Area of Science:

• Pharmaceutical Science
• Analytical Chemistry
• Biochemistry

Background:

• Accurate molecular weight (MW) determination is crucial for characterizing heparin and low molecular weight heparin (LMWH) therapeutics.
• High-performance size-exclusion chromatography (HPSEC) is a standard method for MW analysis of heparins.
• The suitability of calibrants directly impacts the accuracy of MW determination.

Purpose of the Study:

• To evaluate the performance of the calibrant LHN-1 (lot F537) for MW determination of heparins using HPSEC.
• To compare the accuracy of LHN-1 (F537) with a heparin mass calibrator (HMC) and a reference narrow-range-calibration method.
• To determine the optimal calibrant for accurate MW analysis across a range of heparin types.

Main Methods:

• High-performance size-exclusion chromatography (HPSEC) was employed for MW analysis.
• Molecular weight (MW), number-average MW, peak MW, and dispersity were calculated for various heparins.
• Calibrations were performed using LHN-1 (F537), heparin mass calibrator (HMC), and a reference narrow-range-calibration method.

Main Results:

• LHN-1 (F537) demonstrated a MW determination range too narrow for accurate analysis of low molecular weight heparins (LMWHs).
• Results obtained with HMC showed no significant difference from the reference method for substances up to 15.0 kDa.
• In contrast, LHN-1 (F537) yielded significantly different MW profiles compared to the reference method for samples above 7.5 kDa.

Conclusions:

• The calibrant LHN-1 (F537) is not suitable for accurate MW determination of all LMWHs due to its limited calibration range.
• Heparin mass calibrator (HMC) provides a broader and more accurate calibration range, suitable for both LMWHs and unfractionated heparins.
• HMC is a superior calibrant for HPSEC-based MW determination of heparins compared to LHN-1 (F537).