New core bead chromatography medium enables group separation and high productivity purification of large biomolecules

Transcription

1 New core bead chromatography medium enables group separation and high productivity purification of large biomolecules Patrik Adielsson, Anna Åkerblom, Fredrik Elwinger, Fredrik Larsson, Eric Routhier, John Schreffler, Tobias Söderman, Eric Wiltsie, Xun Zuo GE Healthcare Bio-Sciences AB, Uppsala, Sweden First presented at PREP 2012, the 25 th International Symposium July in Boston, MA

2 Abstract When large biomolecules such as IgM or influenza virus are purified, group separation with gel filtration is the most commonly used technique. Advantages of using gel filtration include the high purity and the high recoveries that can be achieved, while a drawback is the relatively low productivity. Capto Core 700 is a chromatography medium (resin) from GE Healthcare Life Sciences designed with core bead technology. The core bead technology enables double functionality for Capto Core size exclusion and binding separation in one bead. The benefits of this technology include high loading, high flow rates with short residence times, and robust performance in a wide window of operations, securing high productivity and straightforward process design. Here we show purification examples with Capto Core 700 on two examples of large biomolecules, IgM, and influenza virus. 2

3 Core beads Typically, chromatography beads have a homogenous design. In core beads the ligands are situated exclusively in the core (interior) and the outer layer is inactive (not functionalized with any ligands). A core bead design enables the bead to offer double functionality. Size exclusion (from the outer layer) and binding separation (through the ligands) in the core of the bead. The cutoff is defined by the pore size in the outer layer, while the binding separation is defined by the ligand used in the core of the bead. Depending on the size of the target a core bead could be designed and optimized for either bind-elute or flowthrough mode (Fig 1). 3

4 Core Beads 4

5 Capto Core 700 Capto Core 700 is a chromatography medium developed for purification of viruses and other large biomolecules. This medium is designed for group separation applications where the (large) targets go in the flowthrough while smaller contaminants bind in the core of the beads. Capto Core 700 has an inactive layer with a cutoff of Mr ~ The ligand in the core is octylamine, which allows for an efficient binding of a wide variety of impurities. This medium offers high loading, high flow rates (Fig 2), high yield and purity, leading to high productivity. Capto Core 700 has a wide window of operations allowing for straightforward process design. 5

6 Capto Core 700 6

7 Size exclusion of large targets The size exclusion performance and the cutoff of the Capto Core 700 beads is illustrated here, by measuring the dynamic binding capacity (DBC) on this medium for biomolecules with increasing molecular weight. Figure 3 shows that for biomolecules that are larger than the size cutoff for the beads, the DBC (at 3 min residence time) is zero (in Fig 3 illustrated by IgM). IgM (and any biomolecules larger than IgM) are hindered from entering through the pores in the outer layer and can therefore not bind to the ligands that are solely situated in the core of the beads. 7

8 Size exclusion of large targets 8

9 Robust binding separation Biomolecules that are smaller than the cutoff of the beads are able to enter the core and bind to the ligands in the core. The ligand in Capto Core 700 is octylamine, a multimodal ligand that shows both hydrophobic and anion exchange interactions. The multimodal characteristics of this ligand ensure a strong binding to various types of biomolecules. Using ovalbumin as a model protein (Fig 4), the ligand shows high static binding capacity over a range of ph and conductivity conditions. 9

10 Robust binding separation 10

11 IgM purification on Capto Core 700 Capto Core 700 was evaluated for IgM purification in collaborative studies with Morphotek Inc. Initial screening showed that residence time is an important parameter for recovery when working with target biomolecules close to the cutoff. As shown in Figure 5, loading is another important parameter to control in order to optimize on purity and recovery. HCP removal decreases with increased loading, while IgM recovery increases with the loading. These initial screening experiments show that Capto Core 700 can be a valuable tool in purification of IgM but that further optimization of loading and running conditions can be beneficial. 11

12 IgM purification on Capto Core

13 High productivity influenza virus purification on Capto Core 700 Figure 6 shows a comparison of purification of influenza virus on Capto Core 700 and on a typical gel filtration chromatography medium (Sepharose 4 Fast Flow) often used for this type of application. A one hundred times larger column load is applied on Capto Core 700, while still achieving similar HCP removal and influenza virus recovery as for Sepharose 4 Fast Flow (Table 1). Another benefit with Capto Core 700 is the possibility to avoid the twofold dilution that is typical with gel filtration. The higher loading, together with the possibilities to run short residence times (high flow rates, low bed heights), results in a significantly improved productivity for Capto Core 700 compared to gel filtration chromatography. 13

14 High productivity influenza virus purification on Capto Core

15 High productivity influenza virus purification on Capto Core

16 Conclusions Capto Core 700 is built with core-bead technology that allows double functionalitysize exclusion and binding separation in one bead. The size exclusion cutoff is in the range of Mr making Capto Core 700 an excellent tool for purification challenges for large biomolecules, such as IgM or influenza viruses. Capto Core 700 offers: High purity at high productivity with up to 100 times higher load than gel filtration Straightforward optimization thanks to flowthrough chromatography and robust performance in a large window of operations 16 productivity purification of large biomolecules

17 Acknowledgments GE, imagination at work, and GE monogram are trademarks of General Electric Company. Capto, and Sepharose are trademarks of GE Healthcare companies. All third party trademarks are property of their respective owners General Electric Company All rights reserved. First published All goods and services are sold subjects to the terms and conditions of sale of the company within GE Healthcare which supplies them. A copy of these terms and conditions is available on request. Contact your local GE Healthcare representative for the most current information. GE Healthcare Bio-Sciences AB, Björkgatan 30, Uppsala, Sweden. 17