(M0930-08-48) Comparative Evaluation of Automated Protein Purification Technologies

Purpose: Developing effective protocols for sample preparation, extraction, and purification requires careful consideration of multiple factors. Optimized protocols should be reproducible, scalable, flexible, gentle on the sample, and efficient. In addition, protocols need to be a high-throughput and easily automatable to enable efficient processing of multiple samples and different workflows. To achieve this, several platform options are available, including magnetic beads, filter plates, spin columns, and tip-based technology. This poster evaluates these different platforms for their suitability in high-throughput and automated sample prep, extraction, and purification workflows. We compared performance data across platforms based on yield, binding capacity, reproducibility, and scalability

Methods: This comparative study evaluated tip-based technology (Biotage® PhyTip® columns), filter plates (Cytiva), spin columns (ThermoFisher Science Piece™), and magnetic beads (ThermoFisher Scientific Pierce™) for protein purification. All platforms were investigated using Biotage IMAC and ProPlus buffer kits, for their respective experiments. Yield consistency was assessed by purifying 300µg of His-eGFP on 20 µL Ni-IMAC resin (25 µL for filter plates) across 48 samples per platform, comparing percentage recovery and CV values. Binding capacity was determined by purifying His-eGFP expressed in BL21 cells at concentrations ranging from 0.3-1.0mg/mL with 40 µL Ni-IMAC resin, as well as human IgG from human serum spiked in CHO media at concentrations ranging from 1.5-4.0mg/mL with 20 µL ProPlus resin. Both were purified in triplicate and eluted with a total volume of 600 µL. Samples were analyzed by fluorescence spectroscopy (NanoDrop™ at 490nm) and SDS-PAGE gel for qualitative assessment.

Results: PhyTip® columns consistently yielded significantly higher human IgG and His-eGFP protein yields when compared to filter plates, spin columns, or magnetic beads across various concentrations (Figure 1, Left). The gentle aspiration and dispense of sample onto the PhyTip® column over multiple cycles maximized the binding capacity of the resin. PhyTip® columns achieved the highest average yield (216.6 µg, 72.2% recovery) with the lowest CV (7.8%) (Figure 2). Unlike filter plates, spin columns, and magnetic beads, PhyTip® columns exhibit higher reproducibility because they are independent of external modules such as magnetic strength, vacuum, or centrifugation. SDS-PAGE gel analysis shows higher intensity band for samples eluted on PhyTip® columns (Figure 3, lane 2) demonstrating how they efficiently eluted high-concentrated samples in a very small volume. Purified samples were efficiently eluted on PhyTip® columns in as low as 120 μL elution buffer, whereas other platforms typically required 200 μL of elution buffer.

Conclusion: Four different platforms for high-throughput, automated sample preparation and purification technologies were evaluated: magnetic beads, filter plates, spin columns, and tip-based technology. Each technology presents distinct advantages and limitations that should be carefully considered when developing or optimizing protocols/methods. Among these, tip-based purification demonstrated superior performance in terms of recovery, reproducibility, scalability, and overall consistency across samples, making it a strong candidate for streamlined, automated workflows.

Acknowledgements: The authors declare no competing financial interest. However, salaries of all authors are paid by Biotage.

Figure 1. (Left) Human IgG spiked with CHO media for capacity testing on 20 µL of ProPlus (MabSelect Sure™) resin. Each sample was performed in triplicate for each dilution of 4 different starting sample concentrations. (Right) The eGFP capacity testing on 40 µL of IMAC resin represents the average of triplicates for each sample shown above. The dilutions were prepared with 1x PBS for 4 different starting sample conc.

Figure 2. Consistency yields for Ni-IMAC resin with 40uL volume, tested on 300mg of His-eGFP protein for each respective protein purification platform at standard conditions.

Figure 3. SDS-PAGE gel image highlights final concentration of eluted sample for each respective purification platform. Tip concentrating effect of PhyTip® Columns allows for efficient elution compared to other platforms.