(T1430-09-60) 2D-LC as A Novel Analytical Platform for the Characterization of Complex Drug Substances: A Case Study with Glatiramer Acetate

Purpose: The objective of this work was to investigate two-dimensional liquid chromatography (2D-LC) for its characterization capabilities of complex drug substances by using glatiramer acetate (GA) as a model sample.

Methods: 2D-LC methods were developed using an Agilent 1290 Infinity II 2D-LC system operated in high resolution mode. Chromatographic separations based on molecular weight were performed in the first dimension using a Tosoh biosciences TSKgel G3000 SWXL column (7.8 mm x 30 cm, 5 μm) in sequence with a TSKgel Guard SWXL analytical column (6.0 mm x 4.0 cm, 7 μm). The method utilized isocratic elution of mobile phase A (water containing 0.1% formic acid) and mobile phase B (acetonitrile containing 0.1% formic acid) held at a ratio of 90% A: 10% B. The flow rate was set to 0.3 mL/min, column temperature at 30°C and an injection volume of 10 μL. Second dimensional chromatographic separations based on hydrophobicity was achieved using an Agilent SB-C18 column (2.1 mm x 100 mm, 1.8 μm). Gradient elution (0.00 min at 5% B to 1.40 min at 20% B) of the same mobile phases used in the first dimension was applied. The flow rate was set to 0.6 mL/min, with a transfer volume from first dimension to second dimension of 14 μL.

Results: Complex polymeric drug substances, such as glatiramer acetate (GA), represent some of the most challenging drug substances to characterize due to their inherent molecular and compositional complexity. The increased separation power of multi-dimensional analytical techniques is critical for the characterization of compositional profiles of complex drug substances. 2D-LC was utilized for advanced characterization of a complex drug, and its application was demonstrated through GA characterization. A more comprehensive characterization of GA will benefit the development of generic counterparts, improve quality control approaches, and promote the identification of critical quality attributes (CQA) which could affect clinical performance. Broader application and continued exploration of this technique may lead to more in-depth characterization of complex drugs to support the assessment and guidance development of complex generic drugs. The combination of size exclusion chromatography (SEC) on the first dimension (1D) and reverse phase chromatography (RP) on the second dimension (2D) were evaluated for the separation and characterization of glatiramer acetate by 2D-LC. A total of 50 1D fractions, or “cuts” were transferred with a 4 second delay for 2D analysis, which ran simultaneously with the ongoing 1D analysis. Regardless of the location of the cut sampled from the 1D, only one peak was observed in the 2D, and its retention time remained consistent (% RSD < 0.0046) although the peak area varied. The 2D chromatographic peak area for each cut was plotted against the order of the cuts for one lot each of two GA drug products: Product A and Product B. The peak areas for the first ten cuts display a large amount of variation (% RSD < 3.21), which decreases greatly for the remaining cuts (% RSD < 0.92). The difference in variation between the cuts could be due to size differences of the eluted polypeptides. The polypeptides that eluted earlier by SEC were larger than the ones that eluted later and had a tendency to elute as broad peaks by RP in the second dimension; broad peaks are often challenging to integrate consistently. Additionally, the elution of the 1D Product B peak occurs slightly earlier (0.052 min) than the Product A peak in SEC, leading to a lower UV absorbance signal for cuts sampled at the beginning of the 1D peak for the same time points (Product A = 92 maU, Product B = 86 maU), producing 2D peaks with larger peak areas for Product A in both dimensions. These data demonstrate the difficulty in distinguishing two GA drug products based on peak area of the larger polypeptides. Once the region containing broad peaks due to larger polypeptides is excluded, it becomes evident that the peak areas of all the remaining cuts from Product B are smaller than their corresponding Product A cuts (Figure 1). In addition, the peak areas change with the cut number in a manner consistent with the UV absorbance change of the SEC peak in the 1D.

Conclusion: Two-dimensional liquid chromatography (2D-LC) was investigated as a tool for the characterization of glatiramer acetate (GA) drug products from two different sources. A 2D-LC method was developed by separating GA according to the molecular weights of its components in the first dimension by size exclusion chromatography (SEC) and their hydrophobic nature in the second dimension by reverse-phased chromatography. Further optimization and development work is needed to fully explore the application and potential of 2D-LC in GA and complex drug product characterization.

Figure 1. 2D RP peak areas for Product A (blue) and Product B (orange).