Investigation of Pore Size Particle Size and Column Geometry Effects on SEC Analysis of Monoclonal Antibodies

Smaller particle size stationary phases have seen increased use in the modern analytical laboratory due to their ability to exhibit greater resolution while enabling the use of smaller column geometries. Shorter column lengths and narrower bores allow for faster run times and lower flow rates, decreasing laboratory costs in terms of solvent consumption and man-hours needed to run the analyses. This application note examines the effects of smaller particle sizes in various column geometries on size-exclusion chromatography (SEC) methods and the method variables affecting the characterization of antibody products.

This work focused on scaling-down a monoclonal antibody SEC method, starting with a typical analytical-size SEC column (YMC-Pack Diol 5 µm, 300 Å, 300 ??8.0 mm) and ending with a small particle, shorter length, narrower-bore column (YMC-pack Diol 2 µm, 300 Å, 150 ??4.6 mm). The purpose of this experiment was to detail the differing performance characteristics exhibited by each column as physical properties were altered, one variable at a time.

Operating Parameters

Sample: 1 mg/mL Avastin in PBS

Mobile Phase: 100 mM sodium phosphate with

200 mM sodium chloride at pH=7.0

Column Temp: 25 °C

Flowrate: 1.0 mL/min for 8.0 mm I.D columns

0.33 mL/min for 4.6 mm I.D. columns

Inj. Volume: 30 µL for 8.0 mm I.D. columns

10 µL for 4.6 mm I.D. columns

HPLC System: Agilent 1260

Changes in column geometry were addressed by scaling flow rate (1.0 mL/min to 0.33 mL/min) and injection volume (30 µL to 10 µL) accordingly as inner diameter was decreased from 8.0 mm to 4.6 mm. This allows for a method that is typically run at 1.0 mL/min for 15 min to be run at 0.33 mL/min for 10 min, cutting run time by 1/2 and solvent consumption by a factor of 6x. For resolution comparability see Table I.

As the results indicate, when applied to SEC analysis of monoclonal antibodies, smaller particle stationary phases in shorter columns can increase throughput, while still providing adequate resolution that rivals the standard 300 ? 8.0 mm, 5 µm columns.

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