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September 15, 2010 (Vol. 30, No. 16)

High-Velocity Affinity Chromatography

Silica-Based Protein A Media Designed to Reduce Bottlenecks in Antibody-Capture Process

  • Better Productivity

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    Productivity results for industrial applications

    The optimized particle size and uniform pore size distribution enable rapid mass transfer, resulting in higher dynamic capacities over a wide range of velocities. Combined with a low pressure drop and the rigid nature of the homogenous pore base matrix, this delivers high productivity.

    Based on experimental data obtained at lab scale, an affinity chromatographic step was designed to process 14,000 L of feed at 1 g/L of mAb in 16 hours. In performing this step, the use of AbSolute at industrial scale allows a volume reduction in the media by 1.7 fold and an increase in productivity by twofold compared to agarose-based protein A media (Table).

  • Cleaning and Sanitization

    Silica-based media are perceived as having limited resistance to alkaline conditions and, therefore, to typical sanitization procedures used in biomanufacturing processes.

    In the case of protein A media, the alkaline resistance is limited by the intrinsic fragility of the protein A itself and by the nature of the coupling chemistry, rather than by the solid support itself (except for glass bead-based protein A media, for which the use of NaOH is prohibited by the manufacturer).

    A new agarose-based protein A media was introduced to address this issue, but its improved chemical stability is correlated with lower DBC, and it obviously still suffers from the intrinsic mechanical limitations of agarose.

    AbSolute is based on specially modified silica and features multiple-point epoxy-type couplings of protein A to the matrix. It is stable through alkaline cleaning and sanitization. Indeed, the modified silica-based protein A media remains stable after 150 cycles of repeated alkali washing using 50 mM NaOH with a residence time of 20 minutes.

    A test was performed on a 4.6x10 mm column with a flow velocity of 300 cm/hr. A solution of 0.2 mg/mL polyclonal human IgG in PBS (pH=7.4) was loaded for 20 minutes. The elution step was performed with 0.15 M citric acid containing 0.15 M NaCl (pH=2.2).

    The alkali-CIP was then applied (50 mM NaOH, 20 minutes). The overlay of chromatograms obtained during 150 cycles (not shown) illustrates the reproducibility of the tested method. After 150 cycles with repeated alkali washing, a decrease of only 13% of the DBC was measured.

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