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

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

With ever-increasing cell culture titers, the most common challenge for biopharmaceutical manufacturers is to continuously find new tools to debottleneck and, therefore, speed up the downstream process. Also, growing pressure from health authorities and the emergence of biosimilars is pushing the biopharmaceutical industry to lower the cost of production through greater productivity and/or increased yields.

The capture step in the downstream processing of antibodies is often the bottleneck and also the most expensive step due to the use of protein A media, an essential element in mAb purification.

Several generations of protein A media have been developed through the years in order to improve the mAb capture step. The improvements have typically addressed dynamic binding capacity (DBC), cleaning and sanitization resistance, and productivity with the same selectivity for the target molecule.

Agarose-based protein A media dominates the market. Although it has been improved over the years, agarose-based media continues to be limited in terms of operating at high velocities and pressures.

Novasep recently introduced AbSolute™ protein A media. AbSolute is a modified silica-based protein A media that maintains high DBC at higher velocities and provides improved productivity, thus reducing operating costs.

Dynamic Binding Capacity

AbSolute offers better DBC than third-generation agarose-based media. In fact, breakthrough with AbSolute occurs later than with agarose-based media. The breakthrough curve for AbSolute is steep—the result of uniform particles and homogenous pore size.

Breakthrough curves at 300 and 600 cm/h are shown in the Figure. The curves were generated with industrial mAb broth to determine DBC at 10% breakthrough.

Both types of media were packed in columns of 5 mm I.D. and 50 mm bed height; the bed volume was 0.98 mL. The concentration of IgG in industrial broth was 1.5 g/L. During the loading, fractions were collected and analyzed by HPLC to determine IgG content.

AbSolute provides much higher DBC at 10% breakthrough than the agarose-based protein A media. At 200, 300, and 600 cm/h, the DBC of AbSolute was, respectively, 38%, 76%, and 100% higher than the DBC of agarose-based protein A media. High dynamic capacity is reached at high linear velocities and low-residence time (Figure).

(A) Breakthrough curves, (B) DBC at 10% breakthrough of agarose-based protein A media, (C) evolution of DBC at 10% breakthrough f AbSolute and agarose-based protein A media with industrial mAb broth function of resident time. *Obtained with polyclonal iG.

Better Productivity

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).

Productivity results for industrial applications

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 4.6×10 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.


AbSolute silica-based media has the same selectivity as other types of media. For example, the purity of IgG eluted from AbSolute is similar to both the industrial reference and the IgG eluted from agarose-based protein A media.

Economics and Ease of Operation

Modified silica-based protein A media is incompressible, meaning that a 1 L column is packed with 1 L of media. This feature is an improvement over soft gels such as agarose-based media that have shrink factors of 0.75 and require a higher quantity of media to pack a 1 L column.

Moreover, silica-based media is easy to pack and can be used with most chromatography equipment (low, medium, and high pressure) from lab to production scale. For example, the packing of a 20 cm bed length in a 110 mm I.D. low pressure Prochrom® Bio column takes two minutes to obtain 5,900 plates at 160 cm/h.

This comparative study illustrates the benefits of AbSolute modified silica-based protein A media in large-scale mAb production. Its use allows biomanufacturers to speed up their antibody-capture processes, making them more cost-effective.

Jean Blehaut is marketing and communication director, Hélène Chochois ([email protected]) is biomolecules marketing manager, and Laurence Pegon is senior scientist biopharma at Novasep.

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