IMAC Resin Selection
Overcome Decision Fatigue: Embrace Goal-Driven IMAC Resin Selection
- Fig. 1a: Dynamic binding capacity vs. flow velocity. Comparison of DBCs of five commercially available IMAC Resins. The resins were packed into 1 mL columns (0.5 x 5 cm). Histidine-tagged green fluorescent protein (GFP) (1.2 mg/mL) in 50 mM sodium phosphate, 5 mM imidazole and 300 mM NaCl, pH 7.5, was loaded onto each column until 10% breakthrough (BT) was observed.
- Fig. 1b: Pressure/flow performance of Nuvia IMAC Resin. Uncharged Resin slurry prepared in water was packed into a 20 x 20 cm column by axial compression with a compression factor of 1.2.
- Fig. 1c: Reusability of Nuvia IMAC Resin. Histidine-tagged GFP was loaded onto a 0.5 x 5.1 cm column packed with a compression factor of 1.25. The column was run at 300 cm/hr. The binding buffer was 50 mM sodium phosphate, 5 mM imidazole, and 300 mM NaCl, pH 7.5, and the elution buffer contained 250 mm imidazole. The column was stripped with 50 mM EDTA, pH 8.0, cleaned with 1 N NaOH, and recharged with 0.1 M Ni2SO4.
- Tab. 1.: Packed column performance. Nuvia IMAC Resin was packed in three different sized columns and the performance of the columns was tested by performing Asymmetry (As) and reduced HETP analysis (rHETP) analysis. a: flow pack, b: axial compression
Immobilized metal affinity chromatography (IMAC) is one of the most widely used affinity chromatography techniques for protein purification. It leverages the affinity of transitional metal ions such as Ni2+, Cu2+, Zn2+ and Co2+, for the amino acids histidine (His) and cysteine (Cys). To create an IMAC resin, transitional metal ions are immobilized using chelating ligands on solid supports or resin backbones. When protein samples are run on IMAC resins, His/Cys-rich proteins preferentially bind to the resin and are purified. To exploit IMAC’s potential, the past few decades have seen the emergence of genetically engineered proteins with histidine tags attached at their N- or C-terminus for easier purification on IMAC resins. Furthermore, the technique has been expanded to purify not only histidine-rich native proteins and histidine-tagged recombinant proteins, but also numerous antibodies, phosphoproteins, metalloproteins, membrane proteins, and zinc finger motif containing proteins.
The success of IMAC resins is due to its ability to provide a single-step purification platform that yields relatively pure/homogeneous target protein. It is a robust and simple to use technique that allows numerous downstream applications. However, the use of IMAC resins does pose some challenges. The charged metal ions on the resin increase the potential of contamination by co-purifying non-target proteins. The presence of the immobilized metal ions also limits the compatibility of IMAC resins with commonly used chelating agents. Such drawbacks were overcome in the past by carrying out a battery of optimization runs before the target purification.
Current vendors have taken these drawbacks into account to design superior resins that provide far more specificity and broader chelating agent compatibility than the previous versions. Over the years, several modifications have been made to IMAC resins and modern IMAC resins allow fast, specific and highly productive protein purifications without lengthy optimization. With multiple IMAC resins on the market, the process of selecting the right IMAC resin for a specific purification project is now the challenge. We describe a goal-driven approach to selecting IMAC resins that takes into consideration the scale of purification and the downstream application for which the protein is being purified.
Small-Scale IMAC Protein Purification for Qualitative Analysis
Small-scale protein purification is often required for downstream applications such as the study of protein-protein interactions, mass spectrometry analysis and other downstream protein characterization assays.
The primary criterion to be considered for such purifications is the final purity level that can be achieved with a given IMAC resin. Most of the available commercial IMAC resins can deliver the purity required at small scale.
For downstream applications that require a relatively concentrated sample, a resin with a good resolution capacity, that can produce a sharp elution peak for the target protein, should be chosen. Resins with a relatively small particle size of ~50 µm are the ideal choice in this scenario. Unlike large-scale purifications, small-scale purifications are not necessarily limited by factors such as pressure spikes during the run, flow rate compatibility, scalability and reusability of the resin.
Process-Scale IMAC Purification for Protein Therapeutics and Enzyme Manufacture
Large-scale purifications have to be performed to manufacture protein-based drugs, therapeutics and vaccines. This purification scale demands highly optimized and efficient process design to yield a protein of highest purity, at minimal cost and time input, in the minimal number of steps possible. For such processes, a workflow is usually designed in which the IMAC is often followed by an ion exchange or mixed-mode chromatography step. The selection of an IMAC resin for process-scale purifications thus has to take into account multiple features and capabilities that do not need to be considered for small-scale protein purification, including:
Flow rate: Regardless of whether an IMAC resin is used as part of a single step purification platform or as the first step of a multi-step purification process, it is loaded with large quantities of feed stock/cell lysates. An IMAC resin that allows fast flow rates ensures that purification of large volumes can be performed within reasonable times. This helps maintain process time efficiency.
Dynamic binding capacity (DBC): DBC is the target binding capacity of a resin at a given flow rate before a given percentage of breakthrough of the unbound protein occurs. When the DBC of a chosen resin is low at required flow rates, yields of the purified protein can be low, reducing cost effectiveness of the purification process.
Resin particle size and backpressure: Bead size has to be considered carefully before selecting a resin for process-scale purifications, as it impacts both the DBC and the column backpressure generated during the purification run. A resin with a large bead size (>90 µm) may not generate high backpressure at the required flow rates, but runs the risk of having a low DBC that is not ideal for process purification. A resin with a small bead size (<35 µm), on the other hand, may have a relatively high DBC but runs the risk of generating high backpressures at the flow rates required for process purifications. This can be detrimental, as high backpressures can compromise the integrity of the resin bed and lead to column collapse. Process-scale purifications thus require a resin with an optimized pore size that retains a high DBC while allowing high flow rates without generating excessive backpressure. An example of such a resin is Nuvia IMAC; it shows high DBC (>40 mg/mL) (Figure 1A) and a column pressure below 2 bar (Figure 1B) at a linear flow rate of 300 cm/hr.
Reusability: The greater the number of times a particular resin can be washed, regenerated and used without a significant loss in its performance parameters, the more cost efficient a process can be. Therefore a resin which shows no significant loss in its DBC, even after more than 100 cycles of repeated use (Figure 1C), can prove to be a better choice than a resin which costs less initially, but cannot survive repeated washes and regenerations.
Chemical compatibility and stability: The selected resin should demonstrate compatibility with and stability in the presence of a broad range of reducing agents, denaturing agents, additives, buffer substances and chemicals, especially the most commonly used reagents for protein expression and purification. One of the concerns with IMAC is metal leaching in the presence of chelating agents such as EDTA. Therefore, care should be taken to select a resin that is compatible with the levels of chelating agents required for a given process purification.
Packing reproducibility: Typically, depending on the column size, column performance can vary, despite similar packing. This warrants an upfront investment of cost and time to optimize process-scale column packing for the most efficient column performance. Resins show reproducibility in packing efficiency at small and large scale as measured by Asymmetry (As) and reduced height equivalent of theoretical plate (rHETP) (Table 1). This enhances the efficiency of the purification.
These and other features, such as the resin backbone, resolution capacity, scalability and recovery, have tremendous effect on large-scale protein purification process efficiency.
IMAC resins offer numerous advantages including low costs relative to other affinity chromatography techniques, simplicity of use and robust purification efficiencies for various proteins, in particular histidine-tagged proteins. Due to significant innovations and improvements in the building blocks of the IMAC resins – the resin backbones and the ligand structures – numerous resins are now commercially available. This paradox of choice can be overcome by using goal-based IMAC resin selection based on the scale and the downstream requirements for the purified proteins.
We are thankful to our colleagues [Dr. Xuemei He and her team] who provided the data included in this article.
Author: Payal Khandelwal
1Bio-Rad Laboratories, Hercules, CA, USA
Dr. Payal Khandelwal
Hercules, CA, USA
Optimized Resin for High Productivity in Downstream Purification Processes (Bulletin # 6859). Bio-Rad Laboratories.
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