Optimization strategies for high performance purification and analysis of recombinant proteins

Introduction

In the post genomic era of drug discovery, the need to prepare and analyze large numbers of recombinant proteins and antibodies has increased significantly, which in turn is driving the process of efficient expression and purification to higher throughput and lower volume technologies. PhyTip® columns are unique separation columns designed to facilitate the purification and evaluation of recombinant proteins and antibodies from relatively low volumes of starting material in a high throughput format. The ability to pack any separation resin such as ion exchange or affinity resins in the PhyTip column and process from any sample type with automation such as the liquid handling systems provided by Caliper Life Sciences allows for efficient purification and screening of resins, early stage constructs, and purification conditions in a completely automated process. Subsequent data analysis using Caliper Life Sciences LabChip GXII eliminates time consuming assays and gels and provides a total solution for bioprocess development applications and expedites the drug discovery process. Purification with PhyTip columns is a simple process that includes capturing the protein of interest on the resin, purifying the protein by washing away the non specific binding products and finally eluting with a small volume of enrichment buffer as low as 10 μL. The flexibility of using various resins, buffer and elution conditions on liquid handling systems with the Labchip GXII system in a single experiment allows for unprecedented amounts of scalable information for complex data generation and analysis never before achievable with a single automated high throughput method.

Studies show that every recombinant protein or antibody has an affinity for each of the specific affinity resins. The ability of each affinity resin to capture and subsequently elute the protein of interest is dependent on a number of factors which when using good laboratory practices should be optimized. As an example, his-tagged Ubiquitin is easily captured by a Ni-NTA resin and does not require as many cycles to load onto a Ni-NTA column, compared to a less tightly held protein, and because it is tightly held by the resin it can be washed extensively at high imidazole concentrations without fear of losing the Ubiquitin. However since the his-tagged Ubiquitin binds tightly, the elution process may require stronger elution conditions. Other proteins in the lysate containing Histidine residues may also bind to the Ni-NTA column though not as tightly and so may be washed of by imidazole in the washes. Protein that is lost in this step will result in better percent recovery in the elution step of the Ubiquitin yielding a product that is more pure for analysis. As more proteins are expressed and optimal purification conditions need to be quickly and efficiently obtained, the combined flexibility of the PhyTip columns and automation provided by Caliper Life Sciences allows for exploration of various binding, washing and elution conditions to both provide optimal expression selection and scalable purification conditions for improved proteins for analytical studies.

In order to facilitate this optimization of the capture, wash and elute process, automation such as that provided by the Caliper Life Sciences Sciclone and Zephyr Liquid Handling Workstaton with the ability to process 96 columns simultaneously allows for easy manipulation of buffers and plates in an easily programmed, cost effective, compact and flexible deck. The LabChip GX family of instruments is the most advanced nucleic acid and protein separations system available today. Like its predecessor the LabChip90, the GX utilizes Caliper’s innova- tive microfluidics technology to perform reproducible, high- resolution, eletrophoretic separations. For assessing protein quality and quantitating proteins, the LabChip GX instrument accelerates research and helps generate more meaningful data, faster. This combination of PhyNexus and Caliper Life Sciences platforms is ideally suited to rapidly and automatically perform the necessary steps involved in optimizing the protocols for resins and proteins of interest.

Optimization of capture and purification of a recombinant protein using PhyTip® columns with Ni-NTA affinity resin and eluting with buffer containing imidazole

This experiment demonstrates how replicates of capture conditions, wash conditions and elution conditions within a single experiment can be processed and analyzed in less than 2 hours. Here, optimum conditions were studied for the purification of his-tagged ubiquitin (10 kDa) from an E. coli lysate. A pure standard of Ubiquitin (10 µg) was spiked into 200 µL of an E. coli lysate, with the pH adjusted to 7.4 by the addition of a volume of 5x capture buffer (25 mM imidazole, 50 mM NaH2PO4, 1.5 M NaCl, pH 7.4) equal to ¼ the total volume of ubiquitin-spiked lysate. The purification method was optimized with regard to three independent variable factors:

• The number of capture cycles,
• The concentration of imidazole in the wash, and
• The concentration of imidazole in the elution buffer.

The entire purification process was performed using the Caliper Life Sciences Zephyr Liquid Handling Workstation with 200+ PhyTip® columns containing 5 μL of Ni-NTA affinity resin and subsequently analyzed with the LabChip GXII.

Equilibration

PhyTip columns are shipped ready to use and stabilized with a low vapor pressure, water-miscible liquid. Although not necessary, to remove it the columns were rinsed in a reservoir filled with 1X Capture Buffer (5 mM imidazole, 10 mM NaH2PO4, 300 mM NaCl, pH 7.4) for two cycles at a flow rate of 4.15 μL/sec. A single cycle involves passing liquid from the sample container, over the resin bed and into the column chamber, pausing for 20 seconds, followed by expelling the same volume of liquid back into the original sample container and pausing for 20 seconds. The Zephyr was programmed to process 190 μL for each cycle.

Capture

A standard 96-well microplate was arrayed with 48 aliquots of pH-adjusted, ubiquitin-spiked E. coli lysate as described. The 96 well plate is depicted schematically below, with row and column identifiers to define the location of each of the 96 wells (Matrix 1). A box of 48 PhyTip columns containing 5 μL Ni-NTA affinity resin was placed on the Zephyr deck. For the Capture step, the instrument was programmed to perform either 2 or 4 capture cycles with 190 μL, all at a flow rate of 4.15 μL/sec. Since cycles need independent programming the capture, wash and elution experiment was done with 2 capture cycles first and repeated with 4 capture cycles.

Matrix 1. Capture step.

Purification step 1

The wash process for purification was optimized by varying the concentration of imidazole in the wash buffer. A 96 well microplate was placed on the instrument deck and arrayed with 200 µL aliquots of wash buffers in each well. The concentration of imidazole in the wash buffer for row A was 0mM imidazole (10 mM NaH2PO4, 140 mM NaCl, pH 7.4), for row B it was 5 mM (5 mM imidazole, 2.5 mM NaH2PO4, 7.5 mM NaCl, pH 7.4), for row C it was 10 mM (10 mM imidazole, 5 mM NaH2PO4, 15 mM NaCl, pH 7.4), and for row D it was 20 mM (20 mM imidazole, 10 mM NaH2PO4, 30 mM NaCl, pH 7.4), as depicted below (Matrix 2). The Zephyr was programmed to run 2 cycles of 180 μL wash buffer through each tip column, at flow rates of 4.15 μL/sec.

Purification step 2

A second microplate arrayed with an identical set of wash buffers as in purification step 1 was placed on the instrument deck, and the MEA instrument was programmed to repeat the same protocol as used in purification step 1.

Matrix 2. Purification step 1 and 2.

Elution

The final elution step was optimized by varying the concentration of imidazole in the elution buffer. A 96 well microplate was placed on the instrument deck and arrayed with 40 µL aliquots of PBS elution buffers varying in the concentration of imidazole. The wells in rows A–H columns 1–3 were prepared with 150 mM imidazole/PBS buffer (6 mM NaH2PO4, 84 mM NaCl, pH 7.4) and rows A–H columns 4–6 were prepared with a 250 mM imidazole/ PBS buffer (10 mM NaH2PO4, 140 mM NaCl pH 7.4). The Zephyr was programmed to run 4 cycles of elution buffer through each tip column at flow rates of 8.3 µl/sec (Matrix 3).

Matrix 3. Elution step.

Assay

Following the automated purification protocol for the 96 samples, the amount of total purified Ubiquitin from each extraction was run and quantified by Caliper Life Sciences LabChip GXII.

Results

In total, 16 distinct conditions were tested. This is an example of a factorial design experiment. There are 2 capture cycle condi- tions (2 or 4 capture cycles), 4 wash buffer conditions (1, 5, 10 or 20 mM imidazole), and 2 elution buffer conditions (2x4x2=16). Three replicates of each condition were run. The results are shown in Tables 1–4.

Each table represents a specific set of elution and capture conditions (e.g., Table 1 reports protein recovery for 2 capture cycles and 150 mM imidazole in the elution buffer), and each column in the table represents the 3 replicates at each wash buffer imidazole concentration (0, 5, 10 or 20 mM). Recovery is reported in terms of μg of ubiquitin, as determined by LabChip GXII analysis using titer of his-ubiquitin standard. For example, recovery for 2 capture cycles and 150 mM imidazole elution buffer, with no imidazole in wash, was 3.54, 3.48, and 3.19 μg over 3 replicates, for an average recovery of 3.40 μg and a standard deviation of 0.19 μg.

Table 1. 2 cycle capture, 150 mM imidazole.

Table 2. 2 cycle capture, 250 mM imidazole.

Table 3. 4 cycle capture, 150 mM imidazole.

Table 4. 4 cycle capture, 150 mM imidazole.

Figure 1. Optimization of purification conditions for his-tagged ubiquitin. Comparison of number of capture cycles and elution buffer.

Figure 2. To evaluate the protein profile, 7 µL of Caliper Protein Express Sample Buffer was added to 5 µL of the protein eluate and heated at 100 °C for 5 minutes. 32 µL of water was added and the samples were run with the LabChipGX Protein Express 200 Assay. The red arrow is the his-ubiquitin expected. The data below demonstrates that the 4 cycle binding is preferable to the 2 cycle for all proteins and that increasing amounts of imidazole in the wash buffer removes undesirable proteins and does not deplete the his-ubiquitin improving purity.

Figure 3. Multiple overlay electropherogram generated by the LabChip GXII demonstrating enrichment of 10kDa his-Ubiquitin from original spiked lysate and with 0 mM and 20mM imidazole wash.

Figure 4. Comparison of the effect of imidazole in the wash buffers on the purity of his-ubiquitin, for protein purified with 2 cycles.

Conclusion

This application note demonstrates the ability to generate quanti- ties of data in a single experiment to optimize protein quickly, easily, and with high reproducibility with the PhyTip® columns and Caliper instrumentation. Some proteins like his-tagged ubiquitin are easily captured and bind tightly to the affinity resin, they can be washed extensively with higher concentrations of imidazole without protein loss, but are harder to recover at the elution step as they require higher concentrations of imidazole for elution.

For other proteins like the endogenous lysate proteins that also have histidine residues and bind to the Ni-IMAC, they can be washed away with varying amounts of imidazole to yield a higher purity protein of interest at elution. The flexibility of the PhyTip columns allows for combinations of resins, protein, and buffers to be simultaneously explored to generate optimal conditions for larger scale purification. The ability to use the columns on an automated liquid handling system and to analyze the data by an automated method such as those provided by Caliper Life Sciences greatly increases throughput and expedites the protein discovery process.

Literature Number: AN142