Gel Filtration PhyTip® columns packed with 5k size exclusion resin: automated high-throughput desalting and buffer exchange of functional proteins

High performance:

• Remove >95% of salts with >80% yield of protein
• Retain protein functionality
• <10% CV in volume and concentration
• Ready for downstream assay

Automated desalting/buffer exchange of functional proteins:

• Fully automated process with MEA Personal Purification System
• 12 samples desalted/buffer exchanged in ~30 minutes.

High throughput format:

• Available in 96 sample format for high throughput processing

Introduction

For biopharmaceutical companies whose focus is on antibody and recombinant protein molecules, there is an increasing desire to perform more screens of potential leads where the data generated are more physiologically relevant to the issues of potency, toxicity, and other factors. These assay formats all require that the antibodies and recombinant proteins are well purified, enriched and functional. By utilizing high performance micro scale functional protein separations, it is now possible to obtain more physiologically relevant data at the high content screening step and thus make the decision making power available at the earliest stages in the discovery process. Biotage's PhyTip® column technology has been developed for high throughput preparation of antibodies and recombinant proteins to facilitate the process of preparing large numbers of potential leads that are ready for functional assays without the need for scale-up.

In many cases differing elution conditions are required for various downstream functional assays. Furthermore, the final purification product may require an additional preparation step to either reduce toxic buffer salts or to completely exchange buffer conditions. This latter step can now be achieved through fully automated desalting and buffer exchange with Gel Filtration Phytip® columns used in conjunction with the MEA 2 System. These unique columns allow for high recovery of the functional antibody of interest while removing greater than 95% of salts.

The design of the Gel Filtration Phytip® columns (Figure. 1) is based upon a unique manufacturing process where pipette tips are filled with gel filtration resin. Thin screens are placed above and below the resin bed that retain the gel filtration media within the column structure, while allowing the passage of liquids. The top frit is a larger liquid interface barrier that helps facilitate the transfer of liquid to the top of the column. Gel Filtration Phytip columns retain small molecules while molecules over 5 KDa will pass through the gel filtration media.Figure 1.

Desalting or buffer exchange process

Using the MEA 2 System, a row of either 200 μL or 600 μL resin bed Gel Filtration Phytip columns are conditioned in 1 mL of buffer. After the columns are conditioned, samples are added and processed followed by differing volumes of chaser buffer addition and processing. The samples can be collected in fractions to determine the optimal sample recovery and salt removal conditions. Using the MEA 2 System, each row of
12 samples requires 30 minutes to process while the entire plate of 96 samples can be processed in 2.5 hours.

Results

The process of desalting or buffer exchange using the Gel Filtration Phytip columns and the MEA 2 System have been carefully optimized to ensure both maximum recovery of the protein of interest and removal of salt.

Consistent volume recovery

Most pharmaceutical companies streamline the processes of antibody/protein production, purification, and desalting/ buffer exchange for cell-based assays. In order to efficiently process the samples from production to cell-based assays, high throughput solutions are desirable. However, in practice, achieving a true high throughput solution requires a level of precision not easily achieved. High throughput sample preparation is not advantageous if subsequent quantification is required prior to the next assay. The solution requires consistent mass and concentration recovery.

To determine the consistency of volume recovery from 200 μL resin bed Gel Filtration PhyTip columns, 80 μL or 130 μL of buffer was loaded on top of 11 or 12 conditioned columns. Flow through was collected and the volume was measured. In three replicate experiments, recovered volumes varied by less than 10% CV (Figure. 2A and 2B).

Figure 2A.
Figure 2B.

Separation by Gel Filtration PhyTip® Column

Sample (far left microfuge tube) containing brown myoglobin protein (16.7kDa) and yellow DNPglutamate salt (313Da) was loaded onto a 600 µL Gel Filtration Phytip® column. The same PhyTip column at different steps of desalting is pictured along with the collected flow through.

Figure 3.

From Left:

1) Column conditioned by PBS buffer prior to sample loading
2) Column after 200 μL sample has entered the resin bed
3–6) Column after 100 μL PBS buffer is applied
7) Column after a final chase of 400 µL PBS buffer

This example illustrates a typical separation allowing the user to discard sample flow through (2) to maintain concentrated purifications. Additionally, salt is retained until fraction (6), while most of the protein is released by fraction (4).

HPLC Chromatogram (Figure 4) of His-Tagged ubiquitin at 0.05mg/mL concentration spiked with 250mM imidazole. 300 μL samples (red) were applied to 600 μL columns. The resulting desalted sample was collected and adjusted to 300 μL with water (blue) to compare with the starting sample. Water was injected as a blank (green). The Post-desalting sample shows both recovery of protein and complete removal of imidazole.

Figure 4.

Myoglobin recovery and salt removal

After columns are conditioned, samples are first added and processed followed by different volumes of chaser buffer addition and processing. The samples can be collected in fractions to determine the maximum sample recovery. In this experiment, we used myoglobin and DNP-glutamate (20 μL sample volume) and separated myoglobin from DNP-glutamate using 80 μL chaser volume (Table 1) and 200 μL resin bed gel filtration columns. As shown in the table below, we were able to recover ~80% of myoglobin and remove ~99.8% of DNP-glutamate. Using the MEA 2 System each row of 12 samples can be separated in about 30 minutes. A full plate of 96 samples takes about 2.5 hours to process.

Table 1.

IgG separation and recovery

IgG was separated from free dye using a 200 μL resin bed gel filtration column. In this experiment, Gel Filtration PhyTip® columns were first conditioned with 800 μL of PBS. After conditioning, 40 μL of [2 mg/mL] sample was added and processed followed by 130 μL of chaser (PBS) buffer to the Gel Filtration PhyTip column to collect and separate IgG from unlabeled dyes.

Figure 5 and Table 2 demonstrate that we were able to recover an average of 76% of IgG (Table 2, Figure 5 red) with an average of 0.5mg/mL concentration (Table 2, Figure 5 blue) in an average final volume of 121 μL (Table 2, Fig 5 black). As shown in Table 2, the percent IgG recovery, concentration, and final recovered volume had very small CV values. These SD and CV values show the reliability and consistency of the Gel Filtration PhyTip columns.

Figure 5.
Table 2.

Conclusion

Gel Filtration PhyTip columns and the MEA 2 System represent a high throughput solution for rapid desalting and buffer exchange. Gel Filtration PhyTip columns can process 12 samples in 30 minutes compared to the laborious and time consuming dialysis procedure. Using Gel Filtration PhyTip columns, on the MEA 2 system, and 96 samples in 30 minutes on a 96-channel system equipped with a gripper arm, researchers can save hours while obtaining a high level of performance. Unlike spin columns, PhyTip columns simulate traditional chromatography and can perform fractionation of protein samples. This allows researchers to optimize maximum protein recovery and salt removal in an automated, high throughput manner.
 
Gel Filtration PhyTip columns can be used in combination with other types of PhyTip purification columns on the MEA 2 System. This allows researchers to combine purification with desalting and buffer exchange. For example, 6-His-tagged proteins can be purified by PhyNexus IMAC PhyTip columns and eluted using high concentrations of imidazole, which can be removed by Gel Filtration PhyTip columns. These processes allow researchers to perform multiple sample processing steps in a high throughput, walk-away scenario.

Literature Number: AN136