(M0930-01-05) Modernization of Compendial Size-Exclusion Chromatography Aggregation Analysis Methods in Compliance with Updates to USP General Chapter < 129 >

Purpose: Aggregation in monoclonal antibody (mAb) drugs is considered a critical quality attribute that must be monitored throughout the drug product lifecycle. Aggregate formation can occur during the fermentation, purification, and storage steps of the manufacturing process and may lead to immunogenic reactions or loss of drug efficacy. Size-Exclusion Chromatography (SEC) is the gold standard for monitoring mAb aggregation. The United States Pharmacopeia (USP) provides instruction on the size-exclusion analysis of aggregates in mAbs through USP General Chapter < 129 >. Traditionally this chapter has only been provided for High-Performance Liquid Chromatography (HPLC) separation methods using 7.8 mm Internal Diameter (ID) columns. On May 1^st, 2025 the USP is introducing updates to General Chapter < 129 > detailing the use of Ultra-High Performance Liquid Chromatography (UHPLC) systems and 4.6 mm ID columns for mAb aggregate analysis. In this work a HPLC SEC method is modernized to UHPLC following the requirements in USP General Chapter < 129 >. System dispersion of each system is determined and a 6-protein standard is used to baseline system performance in each case. A mAb drug is then analyzed for aggregation amount on each instrument and instrument characteristics are analyzed for impacts on separation quality.

Methods: The originator system used was a bioinert HPLC system. Receiver systems included the same bioinert HPLC and a bioinert binary UHPLC system. Each system used a buffered mobile phase of 60.3 mM dibasic potassium phosphate, 140.3 mM monobasic potassium phosphate, and 249.5 mM potassium chloride at pH 6.2. HPLC analysis used a 7.8 mm X 300 mm, 250Å, 2.5 µm column with a 0.5 mL/min flow rate. UHPLC analyses used a 4.6 mm X 300 mm, 250Å, 2.5 µm column with a 0.3 mL/min flow rate. Columns were kept at room temperature during analysis. A 6-protein standard mix was reconstituted to 500 µL in mobile phase and injected at 10 µL and 5 µL for HPLC and UHPLC respectively. Infliximab was diluted to 10 mg/mL with water, injected at 20 µL and 10 µL for HPLC and UHPLC respectively, and analyzed past expiry. Samples were kept at 6°C in an autosampler. UV detection was performed at 10 Hz at 280 nm.

Results: 5σ system dispersion was measured as 21 µL for the HPLC system and 25 µL for the UHPLC system. Chromatographic comparisons of the 6-protein mix are presented in Figure 1 and Infliximab in Figure 2. Looking at the standard, chromatographic resolution declines when moving to the 4.6 mm ID column however retention time repeatability and peak tailing remain comparable. In terms of aggregate determination of the mAb, modernization of the method to the 4.6 mm ID column resulted in equivalent results to that achieved with the 7.8 mm ID column on both systems. (Table I)

Conclusion: A successful modernization of a SEC mAb aggregate analysis method from a 7.8 mm ID HPLC column to a 4.6 mm ID UHPLC column was performed following USP General Chapter < 129 >. A low dispersion bioinert HPLC system was used as an originator system and as a receiver system, while a bioinert UHPLC system was used as an additional receiver system. A protein standard mix demonstrated lower resolution on the 4.6 mm ID column on both receiver systems, while maintaining peak shape (USP tailing) and retention time repeatability. Both receiver systems demonstrated comparable results to the originator system for mAb aggregate determination. Modernization of compendial methods to smaller ID columns results in lower sample consumption, lower analysis time, and reduction in waste production. Being able to maintain chromatographic quality while achieving these benefits is important towards moving the industry toward a more sustainable and efficient future.


Figure 1: Overlayed duplicate injections of a 6-protein mixture. Peaks are labeled in the left chromatogram. The y-axis on the left applies to all chromatograms.


Figure 2: Single Infliximab injections. Peaks are labeled in the left chromatogram.


Table I: Relative area comparisons of Infliximab. HMWS = high molecular weight species. LMWS = low molecular weight species.