How to pack Cytiva™ Protein Select™ resin using verified packing methods
Cytiva™ Protein Select™ resin is an affinity chromatography resin for purification of recombinant proteins using the Cytiva™ Protein Select™ tag. During an affinity step performed with the resin, the protein self-cleaves from the tag and elutes with no residual tag amino acids. This technology simplifies tagged protein purification when used in research. It standardizes purification during process development and manufacturing of any protein that does not have an affinity binding partner. The resin is based on high-flow agarose and offers excellent resolution in bioprocessing.
You can pack Cytiva™ Protein Select™ resin in AxiChrom™ columns to allow high flow velocities and increase the productivity and flexibility in your process design. To achieve effective purification, you need to be sure that the resin is efficiently packed. Poorly packed columns can lead to costly disruptions and loss of your valuable product. Robust and verified packing and testing methods can eliminate such concerns and risks.
This article explains best practices to help you efficiently pack Cytiva™ Protein Select™ resin in AxiChrom™ chromatography columns.
Example results are shown in the accordions at the bottom of this page.
Cytiva™ Protein Select™ chromatography resin characteristics
Cytiva™ Protein Select™ resin is based on the established high-flow agarose matrix, which gives excellent pressure/flow properties. The rigid matrix allows high-flow velocities in recombinant protein purification processes. High physical and chemical stability is essential for increasing productivity in large-scale operations. The ligand is expressed in Escherichia coli (E. coli). Fermentation and subsequent purification are performed without using animal-derived products.
Cytiva™ Protein Select™ resin has an average bead size of 60 μm. A fast sample load and wash (i.e, high flow rate) is preferred to minimize the loss of cleaved protein during sample load and wash. However, for slow-cleaving protein the flow rate can be decreased during those steps. The flow performance in AxiChrom™ columns is 520 cm/h at less than 0.3 MPa (3 bar, 43.5 psi) at a 10 cm bed height (20% ethanol at 20°C). The viscosity for 20% ethanol is in the same region as the viscosity for E. coli lysate and can simulate a sample, having a similar pressure flow profile. When scaling up the process, ensure the chosen bed height and velocity do not conflict with the pressure/flow limitations for the large-scale columns.
Properties of Cytiva™ Protein Select™ resin in various packing solutions
Cytiva™ Protein Select™ resin settles quickly in water, in 20% ethanol, and in salt solutions for packing. When you use these solutions, remember to rinse tubing and nozzles directly after packing to prevent clogging of the flow path.
Adding salt to packing solutions slows the settling of the resin beads and allows them to settle less tightly. Therefore, it is very difficult to measure slurry concentration by gravity with salt-based solutions. However, the slurry concentration method described below allows for quick and accurate determination of slurry concentration.
For example, when Cytiva™ Protein Select™ resin is settled at 60 cm/h, the consolidated bed height will be up to 4% higher in salt solution and up to 0.5% higher in 20% ethanol when compared to settling in water. The effect can be compensated for by using different packing factor (PF). However, for optimal performance of the packed bed, we recommend 0.15 M NaCl as a packing solution, which is further described in this article.
General packing considerations
Definitions
The height of a gravity-settled bed differs from the height of a bed settled under low flow (consolidated) due to the properties and the behavior of the resin under different conditions. Therefore, you should separate the compression factor (CF) from the PF (Equations 1 and 2). For example, if you are packing AxiChrom™ 50 to 200 mm columns with the resin using 0.15 M NaCl, the PF is 1.28 and the CF 1.32 when consolidated at 60 cm/h.
Equations to calculate CF, PF, and column volume (CV) are shown below:
Eq 1:
Compression factor, CF = Lsettled / Lpacked
Eq 2:
Packing factor, PF = Lcons / Lpacked
where
Lsettled = bed height measured after settling by gravity
Lcons = consolidated bed height measured after settling the resin at a given flow velocity
Lpacked = packed bed height
CV = Lpacked × AC
where
AC = cross-sectional area of the column
When packing AxiChrom™ columns, PF is used in the packing procedure to calculate the final packed bed height after the consolidation step. CF is used in the chromatography resin preparation step to calculate the resin volume needed to pack a desired bed height.
Slurry preparation
Cytiva™ Protein Select™ resin is supplied in 20% ethanol. Before packing, transfer the resin to the packing solution as described in the instructions for the column that you intend to pack.
Preparing the resin to form a slurry can be performed manually by shaking/stirring or using a mixer such as the Media Wand™ slurry mixing and transfer tool. Shaking gives good results but is often not practical for larger volumes. When stirring, it is preferable to use soft stirrers without sharp edges. Media Wand™ suspends the resin directly in the container and transfers the slurry to the BioProcess™ resin mixer in one operation, which makes it suitable for large-scale packing.
To achieve the correct amount of chromatography resin for packing to target bed height or compression, it is important that you measure slurry concentration correctly. When you prepare the slurry, start by calculating the chromatography resin volume (V) needed to pack the desired bed height. You can determine the slurry concentration in several ways, but to get an accurate slurry concentration of Cytiva™ Protein Select™ resin, we recommend the Slurry Concentration Kit, which contains all the materials required for determination of slurry concentration.
PACKING CYTIVA™ PROTEIN SELECT™ RESIN IN AXICHROM™ COLUMNS
AxiChrom™ columns are low-pressure, axial mechanical compression chromatography columns designed for process development and biopharmaceutical manufacturing. Mechanical axial compression enables accurate and reproducible control of the packing, even with large diameter columns.
The Intelligent Packing function makes column operation straightforward and gives you very high packing success rates. The packing methods we describe here apply to bed height 10 cm.
When packing AxiChrom™ 50 to 200 mm columns for use with ÄKTA™ chromatography systems, Intelligent Packing control is a feature managed by UNICORN™ control software. For AxiChrom™ 300 to 1600 mm columns, Intelligent Packing is performed by the AxiChrom™ column controller, a separate unit that comprises of a touchscreen operated user interface, or from UNICORN™ control software on the ÄKTA process™ chromatography system.
Intelligent Packing in AxiChrom™ columns – general considerations
Packing methods are created by entering values for the packing variables in the Intelligent Packing wizard, for example, resin, slurry concentration, and target bed height.
Contact your Cytiva representative for more information about automated AxiChrom™ functionality.
Specific considerations for packing Cytiva™ Protein Select™ resin in AxiChrom™ columns
The general bed height for Cytiva™ Protein Select™ resin in AxiChrom™ columns is 10 cm. Packing variables and recommended packing buffers are shown in Table 1. For packing in diameters above 300 mm, contact your Cytiva representative.
Table 1. Packing variables for packing Cytiva™ Protein Select™ resin in AxiChrom™ 100 and 300 columns
| Packing variable | AxiChrom™ 100 and AxiChrom™ 300 columns |
| Packing solution | 0.15 M NaCl |
| Packing flow velocity | 60 cm/h |
| Packing factor | 1.28 |
Using ÄKTA™ process™ chromatography system for packing and unpacking Cytiva™ Protein Select™ resin
When you pack Cytiva™ Protein Select™ resin in AxiChrom™ 300 column via the ÄKTA process™ chromatography system, the UNICORN™ Intelligent Packing wizard is used. When you apply this wizard, the correct packing variables such as PF and adapter velocity are applied.
TESTING THE PERFORMANCE OF THE PACKED COLUMN
Process-scale packed columns must perform with a high degree of efficiency over many processing cycles (that is, display very high stability). The efficiency of a packed column can be expressed in terms of height equivalent to a theoretical plate (HETP) and asymmetry factor (AS). Efficiency testing should be repeated regularly to monitor the state of the packed bed throughout the working life of the column. To ensure comparable test results over time, conditions such as flow velocity (cm/h), liquid pathway, sample composition, and elution buffer should be kept constant.
Our article on column efficiency testing provides details on setting test parameters.
To compare the performance of columns packed with chromatography resins of different particle diameters, the reduced plate height (h = HETP/average bead diameter [dp]) is typically used. As a guideline, a value of h < 3 is achieved when testing a well-packed bed with an optimized set-up of column and system under optimal test conditions.
Test conditions used in this study
Sample: 2% v/v acetone in water
Sample volume: 1% of the column volume (CV)
Flow velocity: 30 cm/h
Eluent: Water
RESULTS IN AXICHROM™ COLUMNS
Packed with the methods outlined above, the columns were tested for plate number, asymmetry, pressure-flow velocity, and stability.
Table 2 highlights examples of efficiency range and stability results for Cytiva™ Protein Select™ resin packed in AxiChrom™ 100 column and AxiChrom™ 300 column. Pressure flow curves for AxiChrom™ 100 and 300 columns are shown in Figure 1.
Figure 2 highlights pressure flow curves for HiScale™ columns for scale-down studies. When scaling up to AxiChrom™ from smaller columns formats, ensure the chosen bed height and velocity do not conflict with the largescale pressure/flow limitations in AxiChrom™ columns. For small-scale column pressure/flow curves, see instruction-for-use 29711002.
Table 2. Results for Cytiva™ Protein Select™ resin packed in AxiChrom™ 100 and AxiChrom™ 300 columns
| Column | Bed height (cm) | Average N/m* | Reduced plate height (h) range* | Asymmetry factor (As) range* |
Flow velocity for stability test (cm/h)† |
Change after stability test (%)† | |
| h | As | ||||||
| AxiChrom™ 100 | 10 | 9800 | 1.6 to 1.8 | 1.0 to 1.1 |
660 | 7 | 6 |
| AxiChrom™ 300 | 10 | 9500 | 1.6 to 1.7 |
1.2 to 1.3 |
640 | -3 | 7 |
† Stability tests were run in 20% ethanol for 16 h at given velocity.
* Performed at optimal test conditions. Average and ranges of upflow and downflow tests for at least three packs.
Fig 1. Pressure flow curves with 20% ethanol for Cytiva™ Protein Select™ resin in AxiChrom™ 100 and 300 columns, normalized to 10 cm bed height and 20°C. 1 bar = 0.1 MPa = 14.5 psi (20% ethanol was used to simulate a sample i.e, viscosity similar to an E. coli lysate)
Fig 2. Pressure flow curves with 20% ethanol for Cytiva™ Protein Select™ resin in HiScale™ columns at 10 cm bed height. 1 bar = 0.1 MPa = 14.5 psi (20% ethanol was used to simulate a sample i.e., viscosity similar to an E. coli lysate)
Conclusions
To utilize the full flow potential of the Cytiva™ Protein Select™ resin, we recommend using AxiChrom™ columns in your process. We have described packing Cytiva™ Protein Select™ resin in AxiChrom™ columns using the custom mode feature of Intelligent Packing. We have also described each packing method in relation to a specific packing solution. Deviation from use of the packing solutions described may have a significant impact on the PF and subsequently on the packing result. The packing result and bed performance are well within normal acceptance criteria for a well-packed bed and ensure robust and stable process performance.
Related resources
- Product page for AxiChrom™ 300 to 1600 mm columns
- Product page for AxiChrom™ 50 to 200 mm columns
- Process scale column packing resources
- FAQ for Cytiva™ Protein Select™ technology
- Data file for Cytiva™ Protein Select™ resin
- Tag sequence for Cytiva™ Protein Select™ tag
- Product page for Cytiva™ Protein Select™ resin and HiTrap™ Protein Select™ column
- Instructions for use for Cytiva™ Protein Select™ resin
- Instructions for use for HiTrap™ Protein Select™ columns
TR31134267, TR31140640 and TR31172805
CY48610