(T0930-09-57) In-Depth Characterization of PS-80 Degradation in Biologics Drug Products by Reversed Phase Liquid Chromatography Coupled with Charge-Reduction High Resolution Mass Spectrometry

Purpose: The complex composition of PS-80 poses a significant characterization challenge for its use in the pharmaceutical industry. PS-80 degradation could lead to particle formation in biologics drug product (DP), which directly impact the product quality and cause potential risk to patients. A Liquid chromatography–mass spectrometry (LC-MS) based method has been developed for characterization of the complex composition of PS-80. The method also offers a simple solution to identify the root causes of PS-80 degradation, which provides critical information to developPS-80 control strategy to ensure the shelf life of DP.

Methods: Experiments were performed on A Waters Acquity Premier UPLC coupled to a time‐of‐flight (TOF) mass spectrometer (Waters Xevo G2-XS) that was equipped with an ESI source. A Waters Symmetry 300 C4 column (2.1 x 150 mm, 300 Å, 3.5 mm) was used for reversed-phase chromatography. Initial conditions were set at 90% solvent A (0.1% formic acid in water) and 10% solvent B (0.1% formic acid in acetonitrile). Solvent B was then increased to 20% at 5.0 min, to 70% at 15.0 min, to 99% at 30.0 min and held for 10 min, followed by equilibration step of 10% B for 5 min. The flow rate was 0.3 mL/min and column temperature was set at 30°C. For the mass spectrometer, the following parameters were set for the ion‐source: capillary voltage 3.0 kV, sampling cone voltage 10 V, extraction cone voltage 3 V, source temperature 130°C, and desolvation temperature 450°C, and cone gas flow rate at 30 L/h, desolvation gas flow rate at 850 L/h. For PS-80 characterization, a fixed collision energy of 10 V was applied. For the fatty acid analysis, a collision energy ramping from 20 to 60 V was used for promoting in-source fragmentation to generate high ion counts of vinyl ester ions. These served as diagnostic and signature ions for PS-80 characterization and quantification according to the type of fatty acid esters. The full‐scan mass spectra were recorded over the m/z range of 120−6000 Da in positive mode. Charge reduction MS was achieved by post-column co-infusion of triethylamine, which focused the signal into mainly singly charged molecular ions and reduced the extent of in-source fragmentation. Waters MassLynx 4.2 software was used for MS data acquisition. Waters MassLynx 4.2 and Genedata Expressionist 16.0 were used for the analysis of PS-80 components and degradants. Typical sample load was five microliter of PS-80 (0.2 mg/mL) in buffers or in DP with protein concentration rang 60-150 mg/mL.

Results: An example of PS-80 component identification by charge-reduction LC-MS in neat solution is shown in Figure 1. This method offers advantages such as retaining intact polymeric structures and fast profiling of polymeric species. A data processing workflow using Genedata Expressionist has also been developed to automatically identify PS-80 component classes and reduce the burden of manually analyzing complex MS data. This novel approach offers a simple ion map for the comprehensive finger-print identification of PS-80 species.The two-dimensional graphical representation of the data helps visualize these features. This method has been successful applied to the studies of enzymatic degradation and metal induced oxidative degradation of PS-80 in DP. In case of this particular enzymatic degradation (Figure 2a), mono-esters were the primary targets. In case of metal induced degradation, PS-80 was randomly degraded to genernate a fuzzy elution pattern (Figure 2b). Together, these innovative methodologies enabled us to analyze components in PS-80 with unprecedented detail and have proven to be an extremely useful tool for formulation development and monitoring the stability of biologics DP .

Conclusion: With this approach, formulated drug substance or DP could be analyzed directly to keep the high integrity information of samples. Simplified workflow and computer-assisted data processing also helped to increase the throughput.

References: 1. R.-S. Yang, D. R. Bush, N. DeGraan-Weber, D. Barbacci, L.-K. Zhang, S. Letarte, D. Richardson. Advancing Structure Characterization of PS-80 by Charge-Reduced Mass Spectrometry and Software-Assisted Composition Analysis. J. Pharm. Sci. 111(2022) 314-322