June 1, 2012 (Vol. 32, No. 11)
Fabien Rousset Novasep
Product Designed for Improved Performance and Cost Effectiveness
With ever increasing cell culture titers, the most common challenge for biopharmaceutical manufacturers is to continually find new debottlenecking tools to speed up the downstream process. Moreover, growing pressure by health authorities and the emergence of biosimilars have driven the biopharmaceutical industry towards lower cost of production, which can be achieved though greater productivity and/or increased yields.
The capture step in the downstream processing of antibodies is often the bottleneck and the most expensive operation due to the use of protein A media.
In recent years, several new generation protein A media have been launched on the market claiming improved mAb capture. Such improvements address dynamic binding capacity (DBC), cleaning, and sanitization resistance.
Novasep recently commercialized a novel protein A media under the brand name AbSolute® High Cap. The product, which features both uniform particle size and homogeneous pore size distribution, maintains DBCs at low and high velocities, thus exhibiting excellent performance. As a result, productivity is improved and operating costs are reduced.
As an example, DBCs measured at 10% breakthrough are equal to 65, 55, 35, and 30 mg/mL at 250, 500, 1,000 and 1,500 cm/hr, respectively (Figure 1). Columns of 5 mm I.D. and 100 mm bed height were packed with AbSolute High Cap.
DBCs at 10% breakthrough also were determined for AbSolute High Cap and several industry standards with human IgG in PBS at 0.5 mg/mL. All these media were packed in columns of 5 mm I.D. and 50 mm bed height (bed volume of 0.98 mL).
AbSolute High Cap illustrated a higher DBC at 10% breakthrough than many market standards at all linear velocities (Figure 2). As an example, using AbSolute High Cap compared to the third-generation agarose-based media at 90, 150, and 375 cm/hr, the overall performance is improved by 100%, 125%, and 230% respectively.
Excellent performance was observed for a number of biomolecules, showing consistency among several human IgGs; between IgG and mAb 1; and for one IgG at different concentrations. Figure 3 shows the wide applicability of AbSolute High Cap.
As for selectivity, studies with AbSolute High Cap provide clear evidence that its selectivity is identical to the industry standard. Indeed, IgG purified by AbSolute High Cap has similar purity and impurity profiles compared to the same IgG purified by standard media.
AbSolute High Cap is rigid and incompressible, leading to a linear relationship between back pressure drop and velocity. The combination of rigid structure and uniform particle size allows operation at low and high velocities with a bed length higher than 20 cm.
Moreover, AbSolute High Cap can be used with most chromatography equipment (low, medium, and high pressure) from lab to production scale.
Cleaning and Sanitization
AbSolute High Cap is based on specially modified silica and has multiple-point protein A attachment to the matrix. Its resistance to high pH levels makes it stable through alkaline cleaning and sanitization. Indeed, AbSolute High Cap remains stable after 400 cycles of use with alkali washing using 100 mM NaOH + 0.5 M NaCl (10 min contact time) every 10 cycles.
The evolution of DBC was evaluated on a column of 5 mm I.D. and 50 mm bed height with a loading of 115 mL of a serum human IgG solution at 0.5 mg/mL. The elution step was performed with 0.1 M Glycine- HCl, pH=3.0. The alkali CIP was then applied (100 mM NaOH + 0.5 M NaCl, 2 column volumes at 0.2 mL/min).
After 400 cycles with repeated alkali washing every 10 cycles, the DBC remains at 88% of initial DBC (Figure 4).
Based on experimental data obtained at lab scale, an affinity chromatographic step was designed to process 10,000 L of feed at 1.4 g/L of mAb in 16 hours. AbSolute High Cap is incompressible (meaning that a 1 L column is packed with 1 L of media). This feature represents an economical benefit in comparison with soft gels such as agarose-based media, which have compression factors of 1.25 and thus require a higher quantity of media to pack a 1 L column.
The geometry of all columns has been optimized, thus optimizing the volume of protein A required to perform the step, in order to maximize the productivity. In these conditions, performing the step at industrial scale using AbSolute High Cap allows a volume reduction in the media by up to 2.5-fold and an increase in productivity by 3.6-fold compared to other industry standards (Table).
This comparative study illustrates the real potential of AbSolute High Cap for large-scale processes and various biomolecule separations (mAbs and IgGs). It allows manufacturers to accelerate their antibody capture processes and makes them more cost-effective.