As the field of analytics continues to evolve, methods guided by regulatory general chapters are increasingly seen as outdated due to their limited resolving power and speed. These compendial methods were originally written when larger particle sizes and systems with larger system dispersion were the norm. In response, regulatory agencies have lifted restrictions on method parameters such as flow rate, column dimensions, and particle size. This allows scientists using compendial methods to leverage modern instruments and particle sizes for a significant enhancement in performance. In this study, a HPLC system and several columns were constructed with bio-inert materials for modernizing biotherapeutic compendial methods. To assess the benefits of this next-generation HPLC instrument platform, the modernized methods were compared to the regulatory general chapter method analysed on a legacy HPLC system, evaluating them based on speed and recovery.

The compendial methods evaluated on both systems were USP General Chapters <129> and <121.1>. For USP <129>, size exclusion chromatography was deployed for size variant analysis of a mAb standard. The general chapters requirements were adhered to on a legacy HPLC system before modernizing it using a method scaling calculator for the newly designed bio-inert HPLC system. By utilizing the next-generation HPLC in conjunction with a hydroxy terminated polyethylene oxide bonded particle, the 30-minute USP method was scaled down to a 7.5-minute method which provided a 75% reduction in runtime and 87.5% reduction in sample and mobile phase consumption. Due to lower system dispersion, the next-generation HPLC system also reduced monomer tailing by 10% which translated to an approximate 20% increase in resolution. For USP <121.1>, the same systems were utilized and evaluated on peptide mapping for insulin using reversed phase chromatography. The next-generation HPLC leverages a new column with a smaller particle size and bio-inert column hardware to reduce the analysis time by 75% and sample and solvent consumption by 50%. Sample vials with a bio-inert surface further reduces enzyme or protein/peptide adherence to the vial and ensures enzyme digestion efficiency and eliminate the complexity from intact protein/peptide in the sample.

The next-generation HPLC instrument platform has been developed with a host of new software features, making the system easier to operate and requiring less maintenance. A touchscreen on the front panel provides dynamic guidance on errors and visible alerts to scientists using the instrument. Several pre-run checks can also be enabled to have the system verify the correct column, vial placements, and mobile phase expiration to alert users of errors before acquisition starts. Overall, the next-generation HPLC platform empowers scientists to keep pace with the advancements made in biotherapeutics to yield more accurate results.