INTRODUCTION
Automation gives researchers the ability to process and screen orders of magnitude higher numbers of samples than manual experimentation. Over several decades, microscale protein purification techniques have been sorted into five basic approaches: batch extraction, spin column, functionalized magnetic beads, and fixed-bed pipette extraction.
However, these technologies vary in their processing mechanics, necessary hardware, and investment of operator time and expertise. Automated high throughput (HTP) systems can reduce operator input or minimize centrifugation; however, to maintain consistent flow rates, backpressure must be kept low, and to achieve high purity, carryover between sample wells or wash steps must also be controlled. Combined, these limitations mean that none of the four purification methods listed above fully accommodates HTP automated affinity purification. Challenges with these four protein purification methodologies include limited throughput, sample carryover and automation incompatibilities.
There is a fifth approach to address these issues: dispersive solid-phase extraction (dSPE).
DSPE BACKGROUND
What is dSPE, and what are dSPE tips? dSPE stands for dispersive solid-phase extraction. From its name, the technique relies on the dispersion of a solid phase (i.e., functionalized chromatography resin) to facilitate batch extraction.
dSPE pipette tips such as IMCStips contain loosely packed resin stored between upper and lower porous filters. A blue ring, called a disperser, is placed within the disposable pipette tip to promote turbulent mixing. The resins within the pipette tip, each with their unique properties, influence the application’s scope and parameters.
PROBLEMS WITH OTHER TECHNOLOGIES
Batch Extraction
This method involves mixing loose resin with liquid sample in a single vessel, followed by sedimentation, washing and elution of captured protein. It has the lowest cost and can be performed at any scale and remains a widely used technique for processing large numbers of samples. However, it has limited throughput and the liquid cannot be removed completely because some of it is contained within the volume of porous bead pellet. Consequently, a portion of each fraction about equal to the volume of resin used is left behind in the pellet, making washes and elution somewhat inefficient.
IMCStips: As with batch chromatography, IMCStips suffer little or no backpressure. Its advantage over batch chromatography is that outside of the initial setup of the plate, IMCStips method requires few if any manual steps (spin down is eliminated), and blowout steps between wells minimize carryover.
Spin Column
Spin columns trap resin between filters in a spin column and pass liquid samples, washes, and eluents through the resin by centrifugation. It utilizes centrifuge or vacuum manifold (common equipment in laboratories) and reduces the carryover of resin into subsequent steps. However, there is limited throughput on centrifuge and poor control of flow rate. Additionally, vacuum manifolds may cause well-to-well contamination.
IMCStips: Unlike spin columns, IMCStips are high-throughput compatible and have customizable flow rates via automated liquid handlers. Individual tips and great control over sample pipetting eliminate carryover and contamination between wells as well.
Functionalized Magnetic Beads
This method employs functionalized magnetic beads to separate the bound proteins from solution. While it is compatible with high-throughput applications, it is slowed by backpressure/ It has a high cost, nonspecific adsorption, and lower capacities compared with standard agarose beads.
IMCStips: IMCStips are cheaper, reduce the backpressure due to loosened resin, and have a higher specific binding capacity.
Fixed-Bed Pipette Extraction
This extraction method uses resin packed in a pipette tip (compressed resin between two membranes). It is a miniaturized version of (bi)directional fast protein liquid chromatography. It is high-throughput compatible. However, upon the addition of a top filter to retain the resin, there is a noticeable increase in backpressure that necessitates lower aspiration and dispense speeds. It has channeling effects due to improper resin packing and air bubbles could be trapped in the resin bed.
IMCStips: The lower backpressure, ability to translate between automation methods, and high degree of reproducibility seen with IMCStips are advantageous for dispersive extraction. Additionally, methods typically run faster than fixed-bed pipette tip methods.
SOLUTION WITH IMCSTIPS
As the demand for HTP applications continues to rise, there is a great need for a micro-purification strategy that accommodates fully automated workflows. Such an approach requires compatibility with standard robotic liquid handlers, low sample carryover, reliability, high protein yields and less labor time - features fully met by IMCStips.
The following are the reasons why these dSPE tips are the future:
1: Loose Resin in dSPE Pipette Tips Facilitate Higher Analyte Recoveries: As mentioned earlier, dSPE tips contain loose resin. The repeated aspiration and dispense in sample wells enable turbulent mixing that is further enhanced by the disperser. Resin dispersion maximizes the surface area, making this technique more effective than traditional unilateral flow. Extractions with dSPE tips lead to higher analyte recoveries in less time than conventional methods such as spin column chromatography. The ability to control pipetting speed and to reset the resin bed by dispersion during pipetting steps negate any channel effects that may limit analyte interactions to the resin in traditional chromatography products.
The figure below shows results from an experiment that demonstrates increased protein recovery from IMCStips compared to the traditional spin column method.
Spin columns and IMCStips used in this experiment contained equal amounts of the same affinity resin.
The identical samples and sample volumes were processed three (3x) or five (5x) times to mimic the 3x or 5x aspiration and dispense (or binding cycles) of automation with a pipette tip.
2: Well-characterized Binding Kinetics Enable Tailored Workflows: Since dSPE tips are miniaturized chromatography columns, methods and models to characterize binding kinetics are within reach. Binding profiles for IMCStips are available, enabling users to select resin bed amounts and binding cycles based on available binding data. This automated approach cuts down the purification time of 96 samples to somewhere between 10 to 30 minutes.
The figure below shows that the binding rate of polyclonal human antibody (huIgG) to affinity IMCStips is dependent on protein concentration and ligand densities distributed throughout the porous resin. Based on the curve below, reducing the binding cycles for lower titers (< 0.5 mg/mL) with smaller resin beds will provide faster processing. Pushing for greater yields at higher titers (> 0.7 mg/mL) will require more binding cycles equivalent to longer residence times.
3: dSPE tips Demonstrate Consistency and Reproducibility when Paired with Automation: Ideal execution is leveraging the precise liquid aspiration and dispense speeds of automated pipetting systems, and the beauty of this technology lies in its compatibility with automation systems. Allowing the liquid handler to facilitate repeated pipetting throughout the purification workflow enables consistency and reproducibility.
An automated affinity purification workflow with IMCStips has been widely used by Kemp Proteins for their HTP micro-purification services. This workflow provides consistent results with high accuracy, precision, and repeatability for candidate protein screening. Other success stories include how Sanofi harmonized Protein A purifications across multiple sites and how SizeX IMCStips enabled Just-Evotec biologics to automate their multi-attribute method (MAM) sample preparation.
4: Tip-based Sample Preparation Has a Wide Variety of Applications: The applications with dSPE tips such as IMCStips are endless. Historically, dSPE tips have mostly been used for small molecule isolation and sample clean-up followed by analyte detection. Other examples include contaminant detection in pork and wine and catecholamine extraction in urine. With the foray of IMCStips in the biomacromolecule purification market, expanded applications include affinity purification, peptide desalting with reverse-phase resins, ion-exchange chromatography, phosphopeptide enrichment, and even automated multi-attribute method (MAM).
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