(M1430-09-58) Improvements to PS80 Detection in Drug Product Formulations: A Mixed Mode High-Performance Liquid Chromatography Method for the Quantification and Improvement in the Detection of Polysorbates

Purpose: Analysis of Excipients in drug products is necessary for safe release of effective biotherapeutics. Some of the most common excipients found in biotherapeutics are non-ionic surfactants. These small molecules are used to stabilize proteins against aggregation and prevent surface absorption. Polysorbates, specifically PS80 and PS20 (Tween 80 and 20), are included in a growing number of drug formulations. For quality control and overall public health and safety, it is important to have chemical assays that can accurately measure these excipients. Unfortunately, the analysis of these small molecules is conventionally difficult. Polysorbates are a complex mixture of different isomeric structures and adducts. Additionally, these molecules lack a chromophore making UV detection impractical. The current validated reverse phase HPLC (RP-HPLC) method is suitable for the quantification of PS80 as an excipient in a recombinant protein therapeutic but suffers from interference of the protein, multiple PS80 peaks contributing notable difficulties when integrating and quantitating, and carryover detectable in the baseline from samples in early versus late runs. To address the resolution and interference issues, solid phase extraction is employed but adds time and variability to the method. The purpose of this study is to develop a more robust method that can be executed in less time with similar or better precision and accuracy.

Methods: The drug product is reconstituted and prepared at a minimum required dilution into an acidic buffer to ensure compatibility with the mixed mode (MM) column. The samples are injected followed by a step gradient to remove all matrix components including the protein therapeutic from the polysorbate. The polysorbate elutes as a single peak with baseline resolution. A charged aerosol detector (CAD) produces the signal that allows for peak determination and quantitation. The reverse phase method is similar; however, matrix and protein therapeutic components are removed through a separate SPE process. The elution is then done through a gradual gradient and quantified using signal from CAD.

Results: The two methods have similar precision (%RSD ≤ 10%), accuracy (80 – 120% recovery), and linearity (R² ≥ 0.98). However, the mixed mode method decreases run time from 12.5 hours to 6.5 hours by eliminating the SPE step. Baseline resolution of PS80 as a single peak with no carryover streamlines the integration process and makes the method more robust.

Conclusion: The MM method exhibits valuable improvements to a traditional reverse phase HPLC (High Performance Liquid Chromatography) method used to measure polysorbates, specifically PS80 (Polysorbate 80). Mixed mode HPLC has a shorter run time, better signal resolution, little to no sample preparation and fewer consumables needed for assay completion. These improvements not only improve data analysis, but also reduce costs versus the reverse phase method, and alleviate potential errors brought on by extensive sample handling and preparation. The enhancements demonstrated from the mixed mode method make the analysis of polysorbates, specifically PS80, more practical, efficient, and robust by removing external sources of variability.