Capture of Green Fluorescent Protein on a High-Flow Agarose Anion Exchanger

Recent developments in upstream processing of biomolecules, driven by an increasing demand for biotherapeutics, have resulted in increased protein expression levels and larger feed volumes. As the total amount of protein increases, the capture and intermediate purification steps in the downstream process must be able to handle larger volumes with higher amounts of expressed protein in a fast and efficient way. This requires separation media with properties supporting both high flow velocity and high dynamic binding capacity.

The CaptoQ is a strong anion exchanger (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) based on a highly rigid agarose matrix. It combines high capacity with high flow rates at low backpressures.

This article describes the capture of green fluorescent protein (GFP) on Capto Q. Results from media screening, optimization, scaleup, as well as scale-up modeling and productivity calculations are presented. In addition, some general guidelines on optimizing a capture step when using the anion exchanger are given.

GFP is a 28-kDa protein with a pI of 6.2, in nature found in the pacific jellyfish AequoreaVictoria. In this study, recombinant GFP expressed in Escherichiacoli was purified. The goal of the study was to demonstrate that it is possible to capture 100 kg of product per 24 hr using Capto Q. As will be shown, the goal was reached.

Experimental and results

Method development and optimization work was performed using an ÄKTAexplorer system, while the scaleup was performed using an ÄKTApilot system (both from GE Healthcare Bio-Sciences AB). Since GFP absorbs light specifically at 490 nm, it was easy to follow the protein during process development and optimization.

Figure 1 - Screening of media selectivity. From top to bottom: Capto Q, Q Sepharose XL, Q Sepharose Fast Flow, DEAE Sepharose Fast Flow, and ANX Sepharose 4 Fast Flow (hs). Blue curve: A280, green curve: A490 (GFP). (Figures 1–4 © 2006 GE Healthcare Bio-Sciences AB. Reproduced with kind permission.)

An initial benchmarking of different anion exchange media (Capto Q, Q Sepharose Fast Flow, Q Sepharose XL, ANX Sepharose 4 Fast Flow [hs], and DEAE Sepharose Fast Flow [all from GE Healthcare Bio-Sciences AB]) was done at small scale using prepacked HiTrap columns (GE Healthcare Bio-Sciences AB). Figure 1 shows the result of the screening. It can be seen that Capto Q gave a selectivity profile that differed slightly from the other tested media and it was decided to use Capto Q for optimization of the capture step.

Optimization of the capture step was performed, determining both optimal pH and conductivity for binding and elution as well as residence time for optimal productivity. To save process time and minimize buffer consumption, the process  equilibration/reequilibration/wash/elution/cleaning in place (CIP) volumes were optimized without sacrificing purity and yield. It was found that the optimal loading buffer was 50 mM Tris/HCl, pH 8.2. Elution was accomplished by using a step gradient with an increasing amount of NaCl. Table 1 shows the optimized steps for the separation. One should be aware that for some proteins, the dynamic binding capacity on Capto Q does increase upon increased ionic strength up to a certain ionic strength, over which it again decreases. It is therefore recommended that both loading pH and loading conductivity, between 2 and 15 mS/cm, are optimized with respect to dynamic binding capacity as well as purity of the target protein.

Figure 2 - Capture of GFP on small-scale (2-mL) and pilot-scale (800-mL) columns. On the small scale, 30 mg, and on the pilot scale, 12 g of GFP were loaded per run. Note that in the pilot-scale chromatogram, the volume axis includes the equilibrium step; in the small-scale experiment, the start point is at the start of sample introduction.

The developed method was used for scaling up the separation to pilot scale. A 400-fold scaleup was performed from a Tricorn 5/100 column (GE Healthcare Bio-Sciences AB) (10-cm bed height, 2-mL volume) to a FineLINE 70 column (GE Healthcare Bio-Sciences AB) (20-cm bed height, 800-mL volume), keeping the residence time constant. Figure 2 shows a comparison of the results from the two scales. The purification factor was at both scales approx. 5, and yields in all runs were above 90%. Analysis by 2-D electrophoresis showed that the profile of the copurified contaminants was very similar at the two scales (data not shown). Scaleup of processes for biopharmaceuticals requires that the separation profile remain the same at the different scales. This includes parameters such as purification factor and yield as well as clearance of critical impurities like nucleic acids, host cell proteins, and viruses. Any of these factors may change when column bed height is altered, and should thus be validated using the final bed height. This is of great importance to remember, since in the early stages of a project there is often a shortage of material and work is carried out in columns with lower bed heights than will be used at the production scale.

Scale-up calculations

Figure 3 - Dynamic binding capacities for GFP on Capto Q, Q Sepharose XL, and Q Sepharose Fast Flow as a function of residence time.

Dynamic binding capacities (DBC) for GFP were determined at different residence times on Capto Q, Q Sepharose XL, and Q Sepharose Fast Flow (Figure 3). Based on these results and the chromatography cycle data of the three media (parameters used for Capto Q shown in Table 1), productivity calculations for each medium were performed.

Figure 4 - Calculated productivity with Capto Q compared to Q Sepharose XL and Q Sepharose Fast Flow. Note that Q Sepharose XL and Q Sepharose Fast Flow do not have the same rigidity as Capto Q; thus there are no values for these media at the shorter residence times attainable at the 20-cm bed height.

A theoretical scaleup was done to 20-cm bed height by keeping the respective residence times constant, i.e., operating Capto and Sepharose (Fast Flow and XL) resins at 600 cm/hr and 200 cm/hr, respectively. Exceptions were the loading step, run at different flow velocities, and the CIP, which was kept constant at 30 min. The cycle time when using Capto Q (1.5 hr) is considerably shorter compared to when using Q Sepharose XL or Q Sepharose Fast Flow (3.5 hr). The columns were loaded to 70% of DBC (QB 10%) and yield was measured to be 93.5%. The effect of varying the loading velocity on resin productivity is shown in Figure 4. Since Capto Q allows higher flow velocities to be used, the developed process can be run at shorter residence times compared to Q Sepharose XL or Q Sepharose Fast Flow. Thus, note that although the DBC is higher on Q Sepharose XL compared to Capto Q (Figure 3), the productivity is higher on Capto Q. The high productivity achieved on Capto Q stems from both a high dynamic binding capacity and the possibility of using high flow velocities, even at a large scale. As can be seen from Figure 4, the maximum productivity achieved on Capto Q is 10.4 kg/m3hr. This corresponds to the goal of 100 kg of product per 24 hr in a 400-L column (0.2 × 1.6 [i.d.] m).

Conclusion

A capture step was developed that gave a high yield of target protein as well as removal of bulk impurities in less than 2 hr. A 400-fold scaleup was demonstrated. Recoveries were above 90% at both scales. The purification factor, approx. 5, indicates that the majority of contaminants had been removed. Not only were the recoveries high, but the product eluted in a small volume (2.6 column volumes in the FineLINE 70 column), which is critical for a subsequent chromatography step. Finally, a short overall process time means improved product integrity by reducing exposure time to proteases and other contaminants detrimental to product quality and yield. Altogether, the results show that Capto Q is well suited for use in a capture step.

Ms. Åkerblom and Mr. Bryntesson are Research Engineers, Dr. Brekkan is a Scientist, and Dr. Eriksson is a Senior Scientist, GE Healthcare Bio-Sciences AB, Björkgatan 30, SE- 751 84 Uppsala, Sweden; tel.: +46 18 6120539; fax: +46 186121844; e-mail: [email protected]

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