Author:

BO person Lin, PhD, SHA dish NI MRI?Sean Arabian?kin that day Watson, PhD i 1open TR ONS lab works, Inc., bio him LLC.

AbstractThis application was developed for the automation of dual-flow chromatography using BiotagePhyTip° columns on the Opentrons OT-2 automated pipetting workstation. Two types of PhyTip columns were tested: Ni-IMAC columns for His-tagged protein extraction and Protein A series columns for human IgG purification, including Protein A, ProPlus, and ProPlus LX. The results show that the combination of OT-2 and PhyTip columns using 96-well plates for small-scale protein sample processing can achieve excellent sample yield and concentration.

Key Findings When purified using a PhyTip Ni-IMAC column, OT-2 was able to purify His-tagged GAPDH protein while maintaining activity. OT-2 also demonstrated excellent purification capabilities for human IgG using Protein A, ProPlus, and ProPlus LX PhyTip columns. Multi-step purifications, such as immunoprecipitation, can be run fully automatically on the OT-2 using ProPlus, ProPlus and ProPlus LX PhyTip columns. ● Protein analysis verified that the extracted protein was the target protein and the finished product was in line with expectations. ● In an experimental setting, this application protocol can handle up to 96 samples with minimal hands-on time.

Introduction In recent years, due to the rapid development of protein characterization analysis and functional determination, the industry's demand for protein analysis is increasing day by day. As a result, many techniques have emerged that can be used to expand the throughput of protein separations through affinity chromatography, which has become an important tool in early drug discovery. However, there are few devices on the market suitable for small-scale protein purification. And the lack of programming and automation expertise among researchers may lead to a lot of unnecessary overhead when establishing these processes, which will bring additional cost burdens when optimizing workflows.

PhyTip columns are chromatographic columns that work based on pipette tips and separate target biomolecules from non-target molecules through the bidirectional flow of a mobile phase (such as a sample solution) on a stationary phase (i.e., a resin filled in the tip). This process is called dual-flow chromatography. Purified samples can be eluted in small volumes, resulting in highly concentrated samples. Two-flow chromatography is a gentle purification process that produces proteins with high biological activity. These PhyTip columns utilize protein A for antibody purification or enrichment. Protein A is a 49 kDa protein with high affinity for the Fc region of most IgGs.

ProPlus (MabSelectTM SuReTm, Cytiva) and ProPlus LX (MabSelect SuRe, Cytiva) use modified protein A tips. Protein A has a stronger ability to bind target IgG and is often used for antibody purification and enrichment.

Metal affinity chromatography (IMAC) is another common method used to purify or enrich target proteins from crude samples. This method uses metal ions to extract recombinant proteins with genetically engineered tags, which are usually peptide sequences capable of chelating metal ions. PhyTip columns are combined with nickel ion (Ni)-IMAC affinity resin to efficiently purify polyhistidine (His)-tagged proteins through the strong affinity between Ni2+ and histidine bases.

OT-2 is a biologist-friendly automated platform. PhyTip columns use tips that are compatible with the OT-2's single-channel and 8-channel pipettes, allowing fully automated protein purification to be performed on the OT-2. This study evaluates the performance of PhyTip columns on OT-2 and demonstrates the platform's ability to adapt to a variety of applications. By combining PhyTip columns with the OT-2 platform, researchers can automate protein purification runs, improving operational convenience and consistency. This provides a more suitable solution for a variety of different applications and supports high-throughput purification and enrichment.

Materials and Methods Overview of the workflow for protein purification using PhyTip columns on the Opentrons OT-2 platform The first part of the process is "Reagent Plate Preparation", which prepares the PhyTip column rack and reagent plate . These include an equilibration buffer plate, two plates for different wash buffers, and an elution buffer plate. The second part of the process is "protein purification", which captures and enriches the protein of interest by performing dual-flow chromatography using PhyTip columns. This is achieved by passing the sample or buffer through the resin inside the column (i.e. pipetting up and down). Setup includes cycle number, flow rate, and buffer volume, which are adjusted for the specific application (Table 1).

Table 1: PhyTip Column tested on Opentrons OT-2 Setup and runtime

Schematic layout of the OT-2 platform for reagent plate preparation and protein purification protocols (Figure 1)PhyTip column models tested in this study include:● Pro A 20μL PTX-93-20-01● ProPlus 20 μL PTX-93-20-07● ProPlus LX 20 μL PTX-93-20-26● Ni-IMAC 20 μL PTX-93-20-03

Protein purification process1. Take out the PhyTip column 2. Equilibrate 3. Capture the target protein 4. Wash (first time) 5. Wash (second time) 6. Elution

Experimental Results Isolation of His-tagged GAPDH while retaining activity Through SDS-PAGE and protein quantitative analysis, we concluded that the His-tagged GAPDH sample extracted in PBS has ideal yield and consistency. Conclusion (average yield 79%, coefficient of variation 3%) (upper part of Figure 2).

A common method for isolating His-tagged GAPDH from bacterial lysates to produce recombinant proteins is to transform E. coli OT-2 with plasmids. The use of a PhyTip column on the platform did not destroy the enzyme activity, which is the result of the GAPDH activity assay (middle of Figure 2) . In addition, the results also show that His-tagged GAPDH can be successfully separated from BSA of non-target proteins in samples of two different proteins (Figure 2, lower part).

Original address:Automated protein purification using Biotage dual-flow chromatography PhyTip columns on OT-2