December 1, 2015 (Vol. 35, No. 21)

Hans J. Johansson Agarose Development Team Purolite
Hans Berg Agarose Development Team Purolite
Patrick Gilbert Agarose Development Team Purolite
Mark Hicks Agarose Development Team Purolite
Caroline Tinsley Agarose Development Team Purolite

Designing a Chromatography Platform with the Ability to Select an Optimal Particle Size

Designing a base matrix for large-scale purification of proteins is a delicate task. The ideal base matrix should have high dynamic binding capacity, low back pressure, and fast mass transfer during adsorption and desorption. In addition, the matrix should be hydrophilic, chemically and physically stable, but still be easily conjugated with relevant active groups. Several of the characteristics are contradictive, and the result will always be a compromise.

Agarose, a natural polymer derived from seaweed, is the most commonly used material for production of chromatography resins for preparative protein purification at any scale. This is due to the combination of extremely hydrophilic characteristics with excellent alkaline stability. The inherent softness of emulsified agarose beads can be greatly improved using sophisticated crosslinking chemistry.

The development of high-flow agarose allows the production of relatively small bead size resins that still suit the time constraints associated with manufacture of modern biologics.

A small bead will, however, always generate higher back pressure compared with a larger bead, irrespective of whether it is a very rigid silica bead or a compressible agarose bead. This could become an issue when scaling up to commercial scale, where availability and cost of equipment to work with small particle sizes could be prohibitive.


One could argue that all processes should be directly developed with a bead size that is fully scalable to commercial scale. However, it is a fact that only a fraction of molecules in preclinical evaluations make it all the way to commercial production, and the use of a small, high-resolution beads allows rapid process development and purification of clinical-grade proteins.

One way to overcome potential scalability issues is to create a chromatography platform with resins available in several particle sizes while maintaining porosity and ligand density. This will open up the possibility to select an optimal particle size for a given downstream process with respect to productivity, resolution, and pressure restraints.

Looking at a typical mAb downstream process in a production facility, stainless steel or disposable, pressure drop over large-scale columns is in most cases restricted by cost and available equipment to less than 3 bar. In practice, this sets a lower particle size limit to around 40 µm. At the other end of the range, particles larger than 100 µm will result in poor resolution due to mass transfer limitations.

With this in mind, Purolite has developed a high-flow agarose-based platform spanning from 45 to 90 µm, all with the same porosity (Figure 1). This approach allows a researcher to rapidly develop a process using the 45 µm beads, knowing that it is possible to transfer the process to a larger bead size if it is warranted by productivity and volume needs.


Figure 1. The selectivity (Kav) curves for plain agarose resin. The three different Praesto resins, 45, 65, and 90 µm, are compared with two established chromatography resins, Product X and Product Y. The curves show that the porosity for the Praesto Pure resins are similar for all three particle sizes and have a porosity between the two other resins. Experimental: Results were obtained by gel filtration analysis of each resin with different sized proteins. RNase, 13.7 kDa; Bovine serum albumin, 67 kDa; Ferritin, 440 kDa; Thyroglobulin, 670 kDa.

Optimal Particle Size

To illustrate the principle, a test was done using columns packed with 45 and 90 µm cation exchange resin from Purolite, and four agarose-based cation exchange resins from another vendor that differ both in particle size, porosity, ligand density, and immobilization strategy. A mixture of 25 mg polyclonal IgG and 5 mg lactoferrin was loaded to the resins and separated with a linear salt gradient over 10 column volumes.

The results (Figure 2) show that the Praesto™ cation exchangers with the same porosity and ligand density display a similar selectivity, irrespective of particle size. Resins with different ligand densities or using different grafting technologies vary significantly in selectivity. It is clear from the results that for this particular separation, dextran grafting has a detrimental effect on resolution (resin A). The polymer grafted resin (resin D) has a more defined structure and the impact is less pronounced but still noticeable under the conditions used when compared to the plain agarose resins.


Figure 2. A comparison of six different commercially available agarose-based strong cation exchange resins.

To further streamline process development and up-scaling, a current trend is to make resins available in pre-packed and pre-qualified formats (Figure 3). In particular, for early clinical manufacturing where a resin is rarely used for more than ten cycles, a pre-packed format makes sense. Savings come from avoiding time-consuming operations like packing, packing evaluation, and cleaning procedures.

In addition, investment costs for packing hardware as well as the risk associated with packing failures and microbial contamination are significantly reduced. To address this, Purolite has decided to make all process resins available in formats covering activities from process development to clinical manufacturing.

In summary, we believe this approach has the potential to facilitate rational process development and, in combination with the ability to supply the resins in a pre-packed format, further simplify and shorten the time and cost of producing material for clinical purposes as well as full-scale commercial manufacturing.


Figure 3. Praesto resins are available in bulk and in three prepacked formats. Prepacked columns are quality tested and documented.



























Hans J. Johansson (hans.johansson@purolite.com), Hans Berg, Patrick Gilbert, Mark Hicks, and Caroline Tinsley constitute the agarose development team at Purolite.

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