Achieve fast, high throughput size exclusion chromatography of mAbs and ADCs using Agilent AdvanceBio SEC columns
M. Sundaram Palaniswamy, Agilent Application Scientist
Proteins frequently aggregate when exposed to stress conditions—such as changes in pH, temperature, or concentration—so aggregation can occur at many different stages of the production process: upstream, downstream, or simply during storage.
Size Exclusion Chromatography (SEC) is a method of choice for monitoring and characterization of aggregates of monoclonal antibodies and antibody drug conjugates (ADC). SEC separations, however, are usually carried out with large columns operated at comparatively slow flow rates, and therefore analysis times are often prolonged.
Another option, Ultra High Performance Liquid Chromatography (UHPLC) using a sub-2 µm column has been employed to overcome these challenges, demonstrating much shorter analysis time. However, when applying very fine particles and/or high flow rates, thermal as well as shearing forces might become critical for temperature or pressure sensitive proteins.
With the potential of immunogenicity, aggregate levels must be thoroughly controlled during production of recombinant proteins. This article examines the application of short and narrow Agilent AdvanceBio SEC columns for fast, high-throughput SEC analysis of mAbs and ADC.
Figure 1. SEC chromatographic profiles of native Rituximab innovator and biosimilar, Herceptin and ADC on an Agilent AdvanceBio SEC, 300Å, 7.8 x 150 mm, 2.7 µm column.
Figure 2. SEC chromatographic profiles of native Rituximab innovator and biosimilar, Herceptin and ADC on an Agilent AdvanceBio SEC, 300Å, 4.6 x 150 mm, 2.7 µm column.
Shorter column length and increased flow rate deliver faster SEC
In this study, the objective was to improve analysis throughput by reducing the run time. The run time in SEC for a given column dimension is governed by flow rate. At higher flow rates, however, resolution can be compromised. To achieve faster run times, the ratio of column void volume to the flow rate needs to be decreased.
Reducing the column length and increasing the flow rate are straightforward approaches for faster SEC.[2,3] Profiles of Rituximab biosimilar and innovator, Herceptin and ADC on an Agilent AdvanceBio SEC, 7.8 x 150 mm column demonstrates excellent elution of monomer in less than 4 minutes under chromatographic conditions as shown in Figure 1.
To achieve better sensitivity, the separation was carried out on an AdvanceBio SEC, 4.6 × 150 mm column, which also resulted in superior separation performance as shown in Figure 2. In both cases, absence of an early or late eluting peak suggests that the marketed mAb preparation is homogenous without any indication of aggregation or degradation.
Analysis of hydrophobic ADC with aqueous mobile phase using both of these columns resulted in a symmetrical peak indicating no secondary interactions of the hydrophobic payload with the stationary support.
AdvanceBio SEC provides accurate characterizations and superior peak shapes
SEC is widely used for highly accurate characterization of protein aggregates. Here we have shown the use of shorter and narrower Agilent AdvanceBioSEC columns packed with 2.7 µm particles for high throughput separation of monomer, aggregates, and fragments in less than 4 minutes. AdvanceBio SEC columns were able to provide superior peak shapes of hydrophobic ADC.
The precision of area and retention times was excellent and demonstrated the reliability of the method for routine use. Additionally, the Agilent AdvanceBio SEC shorter and narrower columns provided certainty of monitoring aggregates and fragments. This was achieved with forced stress studies for determining suitability of the column where high throughput and sensitivity are required.
Szabolcs Fekete, Alain Beck, Jean-Luc Veuthey and Davy Guillarme, “Theory and practice of size exclusion chromatography for the analysis of protein aggregates”, Journal of Pharmaceutical and Biomedical Analysis 101 (2014) 161–173
S. Park, H. Cho, Y. Kim, S. Ahn, T. Chang, “Fast size exclusion chromatography at high temperature”, J. Chromatogr. A 1157 (2007) 96–100.)
Andrew Coffey, “Fast, High-Resolution Size Exclusion Chromatography of Aggregates in Biotherapeutics”, Agilent Application note, 5991-6458EN, December 11, 2015.
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