PhyTip Columns and Automation

What are PhyTip columns?

PhyTip columns are part of a unique protein purification and enrichment system offered by PhyNexus. The PhyTip tips are novel column protein capture devices employing various affinity resins maintained at the base outlet of a pipette tip. PhyTip columns capture the protein of interest by bi-directional flow of sample volume through the column bed. By using back and forth flow, the capture interaction of the protein with the column is driven to completion: capture is complete.

PhyNexus provides the power of chromatography in a pipette tip column in automated liquid handlers for protein and plasmid purification. We provide life science researchers the solution to high through put sample processing in drug discovery, drug screening, assays, and structure work – any process where very high concentrations of very pure product are desired and where it is important that the biological molecule remains undamaged and active. For proteins, PhyTip columns can be used for purification from culture and modifying reactions, expression analysis, purification and immunogenicity screening, protein to protein measurements, CoIP, IP, ChIP, target immobilization, buffer exchange, glycosylation analysis, process development, and biofunctional assays. Through our flexible, custom manufacturing process, PhyNexus can pack most customer resin and chemistries. For plasmids, PhyNexus has developed a proprietary silica resin to purify transient transfection quality plasmids.

Our PhyTip chromatography pipette tip platform enables chromatography for up to 12 samples in parallel and for up to 96 samples in parallel. A wide range of sample sizes can be processed from a broad selection of column bed sizes and chemistries, including affinity, ion exchange, gel filtration, normal phase, and reverse phase.

Can PhyTip columns be used on full automation robotics?

PhyTip pipette columns are compatible with all major robotic systems including Tecan, Agilent, Beckman, Perkin Elmer/Caliper, Dynamic Devices and Hamilton robotic systems. As we understand the goal and the sample provided for the process, we provide the total solution including scripts, robotic set up, installation, etc.

For customers with limited budgets and limited bench space desiring automation, bench top MEA and MMG systems are available. The PhyNexus AutoPlasmid MMG benchtop system is available to produce automated large scale transient transfection quality plasmid.

How are PhyTip columns used?

Virtually any liquid handling robot can use PhyTip columns. The multi-channel head functions simply as a pump. PhyTip columns process is equilibration, sample load, a series of washes and recovery of the sample in final elution step. In addition to fully automated walk-away advantages, the samples can be tracked by analysis of the flow through solutions and washes. The small elution buffer volume maintains the target protein at high concentrations, making the columns especially useful for downstream assays requiring high concentrations.

PhyTip columns are used for leads screening, purification prior to protein analytics, to study protein-protein interactions, and to screen expression and purification conditions.

What are the current competitive technologies used for affinity purification?

Current affinity purification tools include spin columns, gravity columns, magnetic beads, and the FPLC chromatography platform. For small samples, PhyTip tips outperform all of these technologies with higher protein concentrations, higher purity, and higher protein activity. By incorporating Dual Flow Chromatography, the PhyTip sample purification preparation system provides a level of performance unmatched by standard techniques. Purification conditions developed for PhyTip columns are scaleable to preparative and large scale columns. Further information throughout this question and answer section.

How does PhyTip technology compare to FPLC chromatography?

The FPLC chromatography platform performs well and is sometimes considered the “gold standard for protein purification. However, the throughput is limited (samples are run one at- a-time). FPLC set-up and clean-up is time consuming and elution volumes can be large, diluting the final product.

Why would I use PhyTip columns?

PhyTip columns have been optimized to produce the high yield, purity, and activity of sample proteins. They have also been optimized to be able to perform these functions with low volume samples, thus saving valuable resources. Sample work flow is seamlessly integrated with protein express and assays with complete sample and solution tracking. The columns can operate rapidly in parallel operation to perform purification in minimal time, frequently in as little as 15 minutes. The columns are easy to set up and are disposable after use making buffer preparation and cleanup very easy.

PhyTip Column Hardware

What materials are used in the PhyTip column body and frit?

All PhyTip column bodies are manufactured from polypropylene. The column frits are mesh or screen retainers composed of a hydrophilic, bio compatible polymer. The column hardware is designed to have minimal contact with the sample protein to reduce protein denaturation. This is accomplished by having protein compatible surfaces and by reducing the overall exposed surface area of the column. The frits have extremely low exposed surface area.

What are the resins that are available for PhyTip columns?

PhyNexus has a wide variety of resins for affinity chromatography, plasmid purification, ion
exchange gel filtration, reverse phase, normal phase and other types of separations. For protein purification, available resins include: ProA, ProG, ProPlus, ProPlus LX, BAC Capture Select, IMAC, Streptavidin, GST, all configurations of ion exchange, gel filtration, etc. In addition, PhyNexus provides a custom packing service whereby virtually any customer-sourced commercial or inhouse resin may be sent to PhyNexus to be packed into the PhyTip columns.

Are PhyTip columns sterile?

No, but as they are supplied, they are resistant to bacteria growth. PhyTip columns are shipped in glycerol which is a bactericide. The columns may be stored at room temperature prior to processing for several hours with no degradation to the column. Depending on the resin, the columns may be treated or conditioned with ethanol/water if desired.

How are PhyTip columns QC tested?

PhyTip columns are tested at multiple steps in manufacturing for, structure, dimensions, backpressure and flow. Each column is tested. The resins in the column have been tested for capacity.

Can I reuse my PhyTip columns?

PhyTip tip columns are priced to be disposable, and we do not recommend the tips be reused.
We cannot guarantee that contaminating proteins will not be eluted into the new experiment.
Nor can the capture efficiency or the recovery of enriched protein from a used column be guaranteed. After use, columns will dry out harming column capacity and subsequent column flow. The resin may become contaminated with bacteria.

What are the resin bed volumes available for the PhyTip column?

PhyNexus Bench benchtop robotic columns have bed volumes of 5, 10, 20, 40, 80, 160, or 320 μL. Bed volumes for the various other robotics can be found in the product section.

Dual Flow Chromatography

What is Dual Flow Chromatography?

Dual Flow Chromatography (DFC) separations are performed with back and forth flow of the fluids through the column. This is different and distinct conventional unidirectional flow through chromatography. However, chromatographic principles still control the separations. Physical and mechanical properties of the pipette tip column control the amount and rate of fluid flow through the column. Selectivity coefficients and Langmuir adsorption isotherms control the separation chemistry properties of the column and dictate the mobile phase conditions needed to achieve separation.

The pipette tip columns using DFC have a real advantage over conventional columns because the kinetic rates of diffusion and interaction of mobile phase molecules with the stationary phase, column channeling and, other column properties are not applicable. Unlike convention flow through columns such as spin columns and plates, complete interaction of the buffers with the column is achieved through back and forth flow. The result is the possible use of very small columns that can be operated in parallel to perform rapid method development, design of experiments (DOE), and quality by design (QbD) or high throughput chromatographic purification. Chromatographic conditions developed with DFC can be scaled to any size including lab and industrial preparative columns. One practical use of DFC is in the pharmaceutical industry where multi-dimensional method development and high through put parallel processing has been used to improve R&D productivity and decrease the time to manufacturing of bio drugs.

What affects the rate binding or capture of the sample to the column?

In general, biomolecules adsorb or are taken up slowly by affinity columns. The kinetics of diffusion of molecules to and into the stationary phase bead and to functional groups is slow. The kinetics of the actual binding interactions is slow. The problem is exasperated with small columns, especially spin columns and plates where flow through is extremely rapid. The time of interaction of the sample with the column is difficult to control and may be insufficient. Linear velocity of mobile phases traveling through the column increase as the column diameter decreases. Small columns are used in 96 well format. This means the columns must have 9 mm center-to-center spacing and the column diameters have to be well under 9 mm, most often well below 9 mm.

Because of this, the ability to achieve complete isotherm equilibrium of the sample with the stationary phase and mobile phase interaction can be difficult to achieve. But high performance parallel processing requires a convenient and practical solution for achieving this.

Increasing the time of interaction will compensate for slow reaction kinetics. The time of column interaction is easily controlled with DFC simply by controlling the number of back and forth flow cycles until equilibrium interaction of sample and column are achieved. The smallest column having the fastest linear velocity of fluid flow takes the longest time to achieve complete interaction. In any case, within 3 minutes of back and forth flow interaction, the capture is complete regardless of the bed size.

What is the Tip Concentrating Effect?

The Tip Concentrating Effect is a process that allows the recovery of high concentrations of proteins in a single purification procedure compared to competitive techniques such as spin columns, gravity columns, magnetic beads and FPLC.

The Tip Concentrating Effect relies on the ability of PhyTip columns to capture protein on very small column bed volumes. Proteins are captured to completion in an equilibrium process. The column is completely loaded. Then proteins are recovered in a highly efficient and effective process. With all conditions being equal, with any two different resin bed sizes of PhyTip columns, the smaller resin bed size will produce the highest concentration of recovered protein

How does the Tip Concentrating Effect affect recovered protein concentration?

It seems counter intuitive to increase the concentration of a protein by using a smaller column.

How is this possible?

With other technologies, the recovered protein becomes more diluted as the resin bed size is decreased. PhyTip columns are unique. The columns are designed to use the same elution volume to bed ratio as the bed volume is decreased, usually a 3 x ratio. Even with resin bed sizes as low as 5 μL (a 100 fold decrease over a “normal” 0.5 mL resin bed) a 3 x (or even a 2 x) elution volume can be used. Thus an 80 μL bed uses a 240 μL elution volume; a 20 μL bed uses a 60 μL elution volume; a 5 μL bed uses a 15 μL elution volume and so on. In one example, to increase the concentration of a recovered sample, all capture and wash conditions are kept the same but the resin bed volume of the column is decreased from 80 μL to 20 μL. Essentially, the same protein is captured with 20 μL bed vs. the original 80 μL bed, but now is eluted with 60 μL of buffer rather than the original 240 μL. Thus, the concentration of the recovered protein can be increased up to 4 fold over the original larger bed size purification process.

What does “optimizing and enhancing the capture step and elution step” mean? Is this part of the Tip Concentrating Effect process?

PhyTip pipette tip columns are unique because the resins always capture the maximum possible amount of protein that is dictated by the selectivity of the resin and concentration of the protein and volume of the sample. First, PhyTip columns operate under a back and forth flow process, continuously flowing the sample through the resin bed. Target proteins that are present in the sample are transported actively to the affinity resin bed with sufficient cycles to increase contact time to fully capture the protein. This active transport process increases the capture kinetics (over diffusion processes) and also pushes capture equilibrium reaction to completion.

By optimizing and enhancing the capture process, even small resin beds can be used to capture the protein contained in the sample. Resin beds are loaded to a very high percentage of available capture sites and substantial amounts of protein can be captured with small bed volumes. Finally, after washing to remove contaminants from the resin bed, a very efficient, small volume elution process is used to enrich the sample. Since only very small elution volumes are needed to elute the proteins from the resin bed, the recovered protein concentration is very high. As the elution volume is decreased, the concentration of protein is increased. PhyTip columns can be eluted with extremely small volumes of buffer. The concentrations of recovered proteins are 5-20 times higher than what can be produced from competitive technologies.

If a smaller bed volume PhyTip tip gives higher concentration, why not always use the smallest bed column available?

Because many times, the total mass of recovered protein is important too. In samples where
the initial protein concentrations are high, higher bed volumes should be used to prevent overloading of the column. The back and forth loading method allows the resin to be loaded with protein to very high levels. However if too much protein is present, some protein can be lost in the column flow through. Overloading can be monitored by performing an analysis of the capture flow-through before and after capture.

Why do spin columns, gravity columns and FPLC use large bed volumes?

Proteins need sufficient time to interact with the resin to be captured. Increasing the bed size of a spin column, gravity column or FPLC column bed increases the opportunity for capture due to increased time of interaction as the protein flows through the larger bed. In fact, these competitive techniques have less efficient capture process (than dual flow chromatography), and it is difficult or even impossible to use very small capture columns.

Larger buffer elution volumes are needed for these larger bed volumes. These large buffer volumes dilute the recovered protein.

PhyTip tips are able to use very small elution volumes recovering the protein at higher concentrations. Incidentally is why the elution process for PhyTip tips is sometimes called the enrichment process.

Why do PhyTip columns outperform magnetic beads?

For the same amount of magnetic bead resin, smaller elution volumes can be used with PhyTip columns making the recovered protein at a much higher concentration. But there are other reasons
PhyTip columns outperform magnetic beads. Magnetic beads are low capacity – just the surface is derivatized so capacity is lower. Magnetic beads cannot use back and forth flow, the active transport mechanism of PhyTip tips so the capture process is slower. The washing/purification process for PhyTip columns is more effective; therefore, the recovered protein is purer.

Could you summarize the Tip Concentrating Effect phenomena?

As the bed size of the column is decreased the concentration of the eluted protein is increased provided:

a) the same elution volume to column bed ratio is maintained for the smaller column
b) the affinity resin capacity is sufficient so that the loading of protein is increased with the smaller column bed volume. Affinity resins used in PhyTip columns with back and forth flow have extremely high loading capacity.