Phytip® columns vs other technologies: Comparative analysis of protein purification efficiency and yield consistency

Abstract

PhyTip® columns were evaluated against other commonly used purification platforms for purifying recombinant proteins. The study assessed perfor- mance quality by examining yield consistency and protein purity using Ni-IMAC resin in PhyTip® columns, filter plates, magnetic beads and spin columns. PhyTip® columns outperformed filter plates, spin columns, and magnetic beads, providing higher yields with lower variability. The fully automated purification process ensured consistent recovery and batch-to-batch reproducibility. Additionally, the slow flow rates in PhyTip® columns prevented protein denaturation and aggregation, enabling efficient purification of small volume and low-titer samples.

Introduction

Automation plays a crucial role in small-scale purification during early-stage drug discovery and research. By efficiently processing a large number of protein samples in small volumes, researchers can gain valuable insights into protein expression, stability, and functionality.

Traditional protein purification platforms, including filter plates, spin columns, and magnetic beads encounter issues with protein aggregation and cross-contamination. These challenges often lead to inconsistent recovery and reproducibility, particu- larly when handling low-titer samples. PhyTip® columns, which operate at slow flow rates, prevent protein denaturation or aggregation, making them ideal for small volume and low-titer samples. The ability to fully automate the purification process with PhyTip® columns ensures consistent recovery and batch-to- batch reproducibility.

In high-throughput labs, maintaining consistent yield and purification efficiency is crucial for ensuring quality results. This study evaluates yield consistency by analyzing 48 samples processed under standard conditions. Additionally, purification efficiency is assessed by comparing eluted samples from the four different platforms using SDS-PAGE gel electrophoresis.

Materials used

The comparative study was performed using Biotage 40 µL Ni-IMAC resin bed, 1 mL Hamilton tip (P/N: PTH-91-40-03); Cytiva Hi MultiTrap FF Ni Sepharose 6 Fast Flow Filter plate (P/N: 28400990); ThermoFisher Scientific Pierce™ Spin Columns Snap cap (P/N: 69725); ThermoFisher Scientific Pierce™ High-Capacity Ni-IMAC MagBeads, EDTA compatible (P/N: A50591); All platforms used Biotage standard IMAC buffer kit (P/N: BUF-01-00-03) and Biotage standard ProPlus buffer kit (P/N: BUF-01-00-01) for respective experiments.

Methodology

Standard protocol conditions were followed for each platform. While optimization may yield different outcomes, in this study the performance of each platform was evaluated according to the respective manufacturer’s protocol. Four commonly used protein purification platforms were evaluated, each platform processed 300 µg of His-eGFP using 20µL of Ni-IMAC resin. For the filter plate 25 µL of Ni-IMAC resin was used as 20 µL resin bed filter plate was not commercially available.

For assessing yield consistency, the percentage recovery and coefficient of variation (CV) values were compared for 48 samples processed on each protein purification platform. Yield was measured using fluorescence spectroscopy of the expressed GFP on a ThermoFisher Scientific™ NanoDrop™ at 490nm wavelength.

For assessing purification efficiency, samples purified using PhyTip® columns were compared with those purified using filter plates, magnetic beads and spin columns via SDS-PAGE gel. Additionally, the imidazole wash efficiency was evaluated using a wash buffer gradient. Each platform was tested in duplicate for accuracy.

Results and discussion

Yield consistency and purification efficiency for His-eGFP samples were compared using different purification platforms: PhyTip® columns, filter plates, magnetic beads and spin columns containing Ni-IMAC resin. The goal of these experi- ments was to assess the performance of each protein purifica- tion platform in terms of protein yield, result reproducibility and purified protein quality.
Figure 1. 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

Table 1. Consistency experiment calculations.

Among the four platforms that were evaluated, PhyTip® columns provided the highest yield, averaging 216.60 µg with 72.2% recovery, with low sample-to-sample variation (Figure 1, Table 1). In comparison, filter plates providing the second- highest yields and recovery (189.21 µg; 60.45%), followed by magnetic beads (119 µg; 39.56%) and spin columns (103.86 µg; 34.53%). Notably, PhyTip® columns demonstrated the lowest sample-to-sample variability (CV<10%) and the highest consistency (Table 1) all while requiring less elution buffer than the competitive technologies resulting in higher protein concentrations.

The unique design of the PhyTip® columns incorporates a thin hydrophilic frit surrounding the packed resin bed, resulting in virtually no dead volume. A very small amount of liquid (sample or buffer) aspirated through the hydrophilic frit efficiently covers the entire resin bed. As a result, less elution buffer is required. For example, a 40 µL resin bed volume in a PhyTip® column can be effectively eluted with 120 µL elution buffer, whereas other platforms typically require 200 µL of elution buffer. This efficient elution process concentrates the final sample while minimizing contamination compared to other methods (see SDS-PAGE gel image in Figure 2). This phenomenon is known as the ‘tip concentrating effect’ of PhyTip® columns.

Figure 2. SDS-PAGE gel image of eluted samples from each platform. Lanes left to right: Ladder, PhyTip® column, spin column, magnetic beads, filter plate.

The improved performance of PhyTip® columns can be attributed to their Dual Flow Chromatography (DFC) mechanism. Unlike other protein purification platforms, the unique processing method in these columns maximizes interaction time with the resin. This enhances sample binding efficiency, ensures consistent yield, reduces sample-to-sample variation and enhances chromatographic selectivity, resulting in the desired separations¹.

Evaluating technology impact on consistency across four platforms

Liquid handling robots regulate sample flow through the PhyTip® column resin bed, minimizing sample-to-sample variation. Residence time for binding, washing, and elution of the target protein from the resin can be optimized by adjusting parameters like capture cycles and flow rates. Slow flow rates prevent protein denaturation and aggregation, making this technique ideal for small volume and low-titer samples. Additionally, slow flow rates preserve protein structure and functionality, resulting in highly functional proteins suitable for downstream applica- tions. SDS-PAGE gel analysis of imidazole wash buffer fractions at various concentrations demonstrates that PhyTip® columns effectively remove contaminants while maintaining the integrity of His-eGFP protein (Figure 3).
Figure 3. SDS-PAGE gel image of PhyTip column imidazole efficiency testing. Lanes left to right: Ladder, Fraction1, Fraction 2, Fraction 3, Fraction 4, Fraction 5, Ladder. Fractions are referring to concentrations of imidazole from table 1.

For Figures 3 and 4 seen here, Lanes 2-6 are separate fractions of sample eluted with various concentrations of imidazole. Fraction one is 10mM imidazole concentration, fraction two is 20mM imidazole concentration, fraction three is 60mM imidazole concentration, fraction 4 is 200mM imidazole concentration, and fraction five is 250mM concentration.
Figure 4. SDS-PAGE gel image of filter plate imidazole efficiency testing. Lanes left to right: Fraction1, Fraction 2, Fraction 3, Fraction 4, Fraction 5, Ladder. Fractions are referring to concentrations of imidazole from table 1.

Filter plates, on the other hand, rely on less precise external factors such as a vacuum pressure. Variations across different well positions significantly impact recovery and sample purity based on their location on the plate (Figure 3 and 4) this is demonstrated by the sharper band seen in lane 5 (Figure 3).

The performance magnetic beads relies heavily on their interac- tion with the magnetic module. This interaction can cause bead aggregation or sample/buffer carry-over, affecting purification consistency and yields. Demagnetized beads often become a source of impurity that is problematic in downstream analysis, particularly with mass spectrometry. These platform-specific considerations underscore the importance of choosing the right technique to achieve consistent and high-quality protein purification outcomes.

Conclusion

PhyTip® columns offer distinct advantages over other purifica- tion platforms, such as filter plates, spin columns, and magnetic beads. Having a 96-well format is ideal for parallel, multivariate method development. Users can fine-tune parameters affecting yield, purity, and protein activity/stability in a single run. The slow flow rates prevent protein denaturation and aggregation, making PhyTip® columns particularly suitable for low-titer and low volume sample screening. Additionally, the ability to fully automate the purification process using liquid handling robotics ensures consistent recovery and batch-to-batch reproducibility. Overall, PhyTip® columns outperformed all other technologies on the evaluation criteria of exceptional purity, highest yield and most consistent reproducibility. These columns are the optimal choice for biopharmaceutical industries seeking robust and efficient protein purification, especially when speed-to- outcome is crucial.

Reference

1. https://www.chromatographyonline.com/view/ dual-flow-chromatography

Supplementary material

Protein purification platform protocols

PhyTip® columns protocol: This protocol followed the pre-gener- ated automated script designed to run on liquid handlers. A cycle refers to one sequence of aspiration, pause, dispense and pause commands. Pre-generated script proceeds as follows: equilibration, sample capture, wash, elute.

Figure 5. Screenshot of the parameters set for PhyTip® columns for automated liquid-handling platform. The parameters can be altered to meet specific requirements of the experiment prior to each run.

Filter plate protocol: Followed manufacturer’s standard protocol. Protocol steps included: equilibration (x3), sample capture, incubation (30 mins), wash (x3), elution.

Spin columns protocol: Followed manufacturer’s standard protocol. The spin columns were filled with resin for respec- tive ProPlus and IMAC resin testing. Protocol steps included: equilibration, sample capture, wash, elution. The spin columns were centrifuged for 15 seconds at 10,000 x g for each step.

Magnetic beads protocol: Followed Opentrons pre-generated automation standard script. Protocol steps included: equilibra- tion, sample capture, incubation (30 minutes), wash (x2), elution.

Literature Number: PPS757