Robotic Liquid Handling System Research Articles

A droplet robotic system enabled by electret-induced polarization on droplet

Robotics for scientific research are evolving from grasping macro-scale solid materials to directly actuating micro-scale liquid samples. However, current liquid actuation mechanisms often restrict operable liquid types or compromise the activity of biochemical samples by introducing interfering mediums. Here, we propose a robotic liquid handling system enabled by a novel droplet actuation mechanism, termed electret-induced polarization on droplet (EPD). EPD enables all-liquid actuation in principle and experimentally exhibits generality for actuating various inorganic/organic liquids with relative permittivity ranging from 2.25 to 84.2 and volume from 500 nL to 1 mL. Moreover, EPD is capable of actuating various biochemical samples without compromising their activities, including various body fluids, living cells, and proteins. A robotic system is also coupled with the EPD mechanism to enable full automation. EPD’s high adaptability with liquid types and biochemical samples thus promotes the automation of liquid-based scientific experiments across multiple disciplines.

Development and Validation of an Artificial Neural-Network-Based Optical Density Soft Sensor for a High-Throughput Fermentation System

Optical density (OD) is a critical process parameter during fermentation, this being directly related to cell density, which provides valuable information regarding the state of the process. However, to measure OD, sampling of the fermentation broth is required. This is particularly challenging for high-throughput-microbioreactor (HT-MBR) systems, which require robotic liquid-handling (LiHa) systems for process control tasks, such as pH regulation or carbon feed additions. Bioreactor volume is limited and automated at-line sampling occupies the resources of LiHa systems; this affects their ability to carry out the aforementioned pipetting operations. Minimizing the number of physical OD measurements is therefore of significant interest. However, fewer measurements also result in less process information. This resource conflict has previously represented a challenge. We present an artificial neural-network-based soft sensor developed for the real-time estimation of the OD in an MBR system. This sensor was able to estimate the OD to a high degree of accuracy (>95%), even without informative process variables stemming from, e.g., off-gas analysis only available at larger scales. Furthermore, we investigated and demonstrated scaling of the soft sensor’s generalization capabilities with the data from different antibody fragments expressing Escherichia coli strains. This study contributes to accelerated biopharmaceutical process development.

Automated liquid handling robot for rapid lateral flow assay development

The lateral flow assay (LFA) is one of the most popular technologies on the point-of-care diagnostics market due to its low cost and ease of use, with applications ranging from pregnancy to environmental toxins to infectious disease. While the use of these tests is relatively straightforward, significant development time and effort are required to create tests that are both sensitive and specific. Workflows to guide the LFA development process exist but moving from target selection to an LFA that is ready for field testing can be labor intensive, resource heavy, and time consuming. To reduce the cost and the duration of the LFA development process, we introduce a novel development platform centered on the flexibility, speed, and throughput of an automated robotic liquid handling system. The system comprises LFA-specific hardware and software that enable large optimization experiments with discrete and continuous variables such as antibody pair selection or reagent concentration. Initial validation of the platform was demonstrated during development of a malaria LFA but was readily expanded to encompass development of SARS-CoV-2 and Mycobacterium tuberculosis LFAs. The validity of the platform, where optimization experiments are run directly on LFAs rather than in solution, was based on a direct comparison between the robotic system and a more traditional ELISA-like method. By minimizing hands-on time, maximizing experiment size, and enabling improved reproducibility, the robotic system improved the quality and quantity of LFA assay development efforts.Graphical abstract

Fully automated peptide mapping multi-attribute method by liquid chromatography–mass spectrometry with robotic liquid handling system

The multi-attribute method (MAM) based on liquid chromatography (LC)–tandem mass spectrometry is emerging as a powerful tool to directly monitor multiple product quality attributes simultaneously. To better implement MAM, either for product characterization or for quality control (QC), there is a need for a robust, universal, and high-throughput workflow that can be broadly adopted in different laboratories with minimal barriers to implementation. Manual preparation of samples for MAM, however, is labor intensive and produces nontrivial variations across analysts and laboratories. We describe the development of a fully automated peptide mapping procedure with a high-throughput robotic liquid handling system to improve sample handling capacity and outcome reproducibility while saving analyst hands-on time. Our procedure features the automation of a “microdialysis” step, an efficient desalting approach prior to proteolytic digestion that optimizes digestion completeness and consistency each time. The workflow is completely hands-free and requires the analyst only to pre-normalize the sample concentrations and to load buffers and reagents at their designated positions on the robotic deck. The robotic liquid handler performs all the subsequent preparation steps and stores the digested samples on a chiller unit to await retrieval for further analysis. We also demonstrate that the manual and automated procedures are comparable with regard to protein sequence coverage, digestion completeness and consistency, and quantification of posttranslational modifications. Notably, in contrast to a previously reported automated sample preparation protocol that relied on customized accessories, all components in our automation procedure are commercial products that are readily available. In addition, we also present the high-throughput data analysis workflow by using Protein Metrics. The automation procedure can be applied cross-functionally in the biopharmaceutical industry and, given its practicality and reproducibility, can pave the way for MAM implementation in QC laboratories.

Multistep SlipChip for the Generation of Serial Dilution Nanoliter Arrays and Hepatitis B Viral Load Quantification by Digital Loop Mediated Isothermal Amplification.

Serial dilution is a commonly used technique that generates a low-concentration working sample from a high-concentration stock solution and is used to set up screening conditions over a large dynamic range for biological study, optimization of reaction conditions, drug screening, etc. Creating an array of serial dilutions usually requires cumbersome manual pipetting steps or a robotic liquid handling system. Moreover, it is very challenging to set up an array of serial dilutions in nanoliter volumes in miniaturized assays. Here, a multistep SlipChip microfluidic device is presented for generating serial dilution nanoliter arrays in high throughput with a series of simple sliding motions. The dilution ratio can be precisely predetermined by the volumes of mother microwells and daughter microwells, and this paper demonstrates devices designed to have dilution ratios of 1:1, 1:2, and 1:4. Furthermore, an eight-step serial dilution SlipChip with a dilution ratio of 1:4 is applied for digital loop-mediated isothermal amplification (LAMP) across a large dynamic range and tested for hepatitis B viral load quantification with clinical samples. With 64 wells of each dilution and fewer than 600 wells in total, the serial dilution SlipChip can achieve a theoretical quantification dynamic range of 7 orders of magnitude.

Chromatographic parameter determination for complex biological feedstocks.

The application of mechanistic models for chromatography requires accurate model parameters. Especially for complex feedstocks such as a clarified cell harvest, this can still be an obstacle limiting the use of mechanistic models. Another commonly encountered obstacle is a limited amount of sample material and time to determine all needed parameters. Therefore, this study aimed at implementing an approach on a robotic liquid handling system that starts directly with a complex feedstock containing a monoclonal antibody. The approach was tested by comparing independent experimental data sets with predictions generated by the mechanistic model using all parameters determined in this study. An excellent agreement between prediction and experimental data was found verifying the approach. Thus, it can be concluded that RoboColumns with a bed volume of 200 μL can well be used to determine isotherm parameters for predictions of larger scale columns. Overall, this approach offers a new way to determine crucial model input parameters for mechanistic modelling of chromatography for complex biological feedstocks. © 2018 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers Biotechnol. Prog., 34:1006–1018, 2018

Scope and limitations of carbohydrate hydrolysis for de novo glycan sequencing using a hydrogen peroxide/metallopeptide-based glycosidase mimetic

Acidic hydrolysis is commonly used as a first step to break down oligo- and polysaccharides into monosaccharide units for structural analysis. While easy to set up and amenable to mass spectrometry detection, acid hydrolysis is not without its drawbacks. For example, ring-destruction side reactions and degradation products, along with difficulties in optimizing conditions from analyte to analyte, greatly limits its broad utility. Herein we report studies on a hydrogen peroxide/CuGGH metallopeptide-based glycosidase mimetic design for a more efficient and controllable carbohydrate hydrolysis. A library of methyl glycosides consisting of ten common monosaccharide substrates, along with oligosaccharide substrates, was screened with the artificial glycosidase for hydrolytic activity in a high-throughput format with a robotic liquid handling system. The artificial glycosidase was found to be active towards most screened linkages, including alpha- and beta-anomers, thus serving as a potential alternative method for traditional acidic hydrolysis approaches of oligosaccharides.

A High-Throughput Mass-Spectrometry-Based Assay for Identifying the Biochemical Functions of Putative Glycosidases.

The most commonly employed glycosidase assays rely on bulky ultraviolet or fluorescent tags at the anomeric position in potential carbohydrate substrates, thereby limiting the utility of these assays for broad substrate characterization. Here we report a qualitative mass spectrometry-based glycosidase assay amenable to high-throughput screening for the identification of the biochemical functions of putative glycosidases. The assay utilizes a library of methyl glycosides and is demonstrated on a high-throughput robotic liquid handling system for enzyme substrate screening. Identification of glycosidase biochemical function is achieved through the observation of an appropriate decrease in mass between a potential sugar substrate and its corresponding product by electrospray ionization mass spectrometry (ESI-MS). In addition to screening known glycosidases, the assay was demonstrated to characterize the biochemical function and enzyme substrate competency of the recombinantly expressed product of a putative glycosidase gene from the thermophilic bacterium Thermus thermophilus.

High-throughput platform to investigate effects of dynamic presentation of mechanical and biochemical matrix cues on VIC phenotype

Event Abstract Back to Event High-throughput platform to investigate effects of dynamic presentation of mechanical and biochemical matrix cues on VIC phenotype Megan E. Schroeder1, Kelly M. Mabry2, Samuel Z. Payne2 and Kristi S. Anseth1, 2 1 University of Colorado Boulder, Materials Science and Engineering, United States 2 University of Colorado Boulder, Chemical and Biological Engineering, United States Introduction: Aortic stenosis occurs in heart valves when fibroblasts (valvular interstitial cells, VICs) transition to an activated myofibroblast state and engage in pathological matrix remodeling. Mechanotransduction is essential in maintaining valve homeostasis and contributes to the diseased state when misregulated[1]. To better understand how the extracellular matrix and biochemical cues influence VIC activation, we have implemented a high-throughput (HT) platform capable of producing and imaging cell-laden PEG hydrogels functionalized with adhesion peptides. The approach allows one to dynamically alter the culture environment (e.g., add cytokines, alter matrix adhesivity, stiffen the matrix) while simultaneously imaging cellular responses. This HT platform is used to investigate potential synergies between mechanical and biochemical cues on VIC phenotype. Materials and Methods: VICs were isolated from porcine hearts. Hydrogels were synthesized with a thiol-ene photo-click reaction of a norbornene end-functionalized PEG with a thiol containing MMP-degradable crosslinker (KCGPQG*IWGQCK). Concentrations of 0.75mM PEG and 1.875mM MMP led to pendant ene functionalities available for on-demand reactions with thiolated biomacromolecules. Dosing of gels with solutions containing CRGDS (1.25mM or 5mM) resulted in photoinitiated tethering of either 0.4mM or 1.5mM CRGD as determined by fluorescent labeling. A robotic liquid handling system (EpMotion M5073) dispensed 10μL gel droplets on 96-well plates that were then photopolymerized (2.5mW/cm2 @ 365 nm light). VIC morphology, proliferation and expression of α-smooth muscle actin (α-SMA), a marker of the myofibroblast phenotype, were observed with time. A high-content confocal imager (Operetta, Perkin Elmer) was used for all live cell and immunofluorescent imaging. Results and Discussion: VICs were encapsulated in gels of varying adhesivity (with 1mM or no CRGD (Day 0)). On Day 3, either 0.4mM or 1.5mM CRGD was tethered into a subset of the gels. Controls show VICs in gels without CRGD remained rounded, while those in gels with 1mM CRGD elongated up to Day 5. Upon dynamic addition of CRGD, VICs rapidly elongated in 24 hours, which may indicate local matrix degradation occurred prior to its addition. VIC activation to the myofibroblast required the presence of CRGD in the matrix to form contractile α-SMA stress fibers. Tethering CRGD at Day 3 resulted in slightly elevated levels of α-SMA compared to gels originally formed with 1mM CRGD, which relates to increases of fibronectin in diseased valves[2]. Deeper investigations to characterize the persistent vs. transient myofibroblast phenotype using cytokine cocktails and altering matrix modulus are in progress. We seek to understand factors that lead to an irreversible myofibroblast phenotype to identify potential pathways involved in aortic stenosis. Conclusion: This HT cell culture platform enables studies of the dynamic presentation of mechanical and biochemical matrix cues in directing VIC phenotype and assessing pathways involved in the myofibroblast transition. NSF Graduate Research Fellowship; Howard Hughes Medical Institute (HHMI)

Automating ChIP-seq experiments to generate epigenetic profiles on 10,000 HeLa cells.

Chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq) is a technique of choice for studying protein-DNA interactions. ChIP-seq has been used for mapping protein-DNA interactions and allocating histones modifications. The procedure is tedious and time consuming, and one of the major limitations is the requirement for high amounts of starting material, usually millions of cells. Automation of chromatin immunoprecipitation assays is possible when the procedure is based on the use of magnetic beads. Successful automated protocols of chromatin immunoprecipitation and library preparation have been specifically designed on a commercially available robotic liquid handling system dedicated mainly to automate epigenetic assays. First, validation of automated ChIP-seq assays using antibodies directed against various histone modifications was shown, followed by optimization of the automated protocols to perform chromatin immunoprecipitation and library preparation starting with low cell numbers. The goal of these experiments is to provide a valuable tool for future epigenetic analysis of specific cell types, sub-populations, and biopsy samples.

Automating ChIP-seq Experiments to Generate Epigenetic Profiles on 10,000 HeLa Cells

Chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq) is a technique of choice for studying protein-DNA interactions. ChIP-seq has been used for mapping protein-DNA interactions and allocating histones modifications. The procedure is tedious and time consuming, and one of the major limitations is the requirement for high amounts of starting material, usually millions of cells. Automation of chromatin immunoprecipitation assays is possible when the procedure is based on the use of magnetic beads. Successful automated protocols of chromatin immunoprecipitation and library preparation have been specifically designed on a commercially available robotic liquid handling system dedicated mainly to automate epigenetic assays. First, validation of automated ChIP-seq assays using antibodies directed against various histone modifications was shown, followed by optimization of the automated protocols to perform chromatin immunoprecipitation and library preparation starting with low cell numbers. The goal of these experiments is to provide a valuable tool for future epigenetic analysis of specific cell types, sub-populations, and biopsy samples.

Absorptive chemistry based extraction for LC-MS/MS analysis of small molecule analytes from biological fluids – an application for 25-hydroxyvitamin D

Already available sample preparation technologies for liquid chromatography-tandem mass spectrometry have substantial shortcomings with respect to automation. A novel approach is based on gel-like polymeric material with defined absorption chemistry, which is immobilized in micro-plate wells. It is referred to as Tecan Immobilized Coating Extraction™ (TICE™) technology and it enables easy automation on liquid handling systems. We aimed to study the performance of Tecan AC Extraction Plate™ based on this principle by addressing 25-hydroxyvitamin D (25OHD) as an exemplary analyte. A protocol for extraction of 25OHD from serum samples based on TICE™ technology was implemented on a robotic liquid handling system Freedom EVO® (Tecan). An isotope-dilution ultra-performance liquid chromatography-tandem mass spectrometry method was used for quantification. Performance was tested according to a comprehensive protocol. Linearity was found over a range from 4.3 to 65.8 ng/mL for 25OHD3. The coefficients of variation for the intra-day and inter-day precision were <6% and accuracy ranged between 96.9% and 99.8% for 25OHD3. Recovery was 84% and efficient control of matrix effects was verified. High sample throughput could be observed with 96 samples prepared in <60 min. Close agreement of results was found for clinical samples analyzed with a second tandem mass spectrometry method based on protein precipitation and two-dimensional ultra-performance liquid chromatography for sample preparation (r=0.988, n=73). The new TICE™ technology was found to be a useful process for sample preparation in clinical mass spectrometry. Full automation suited for routine analysis was achieved.

Multiplexed microfluidic viscometer for high-throughput complex fluid rheology

We report the first multiplexed microfluidic viscometer capable of measuring simultaneously the viscosity as a function of shear rate for multiple samples. The viscometer is based on a flow-comparator technique where the interface location between co-flowing streams of test and reference fluids is a sensitive function of the viscosity mismatch between the two fluids. We initially design a single microfluidic viscometer and study two different modes of comparator operation—the interface displacement (ID) mode and the interface compensation mode (IC). We find that both modes yield viscosity curves for Newtonian and polymeric fluids that are consistent with a conventional rheometer. Based on the results from the single microfluidic viscometer, we present an operating window that serves as a guide to assess accessible viscosities and shear rates. We then design a 4-plex and 8-plex viscometer based on the ID mode and show that it is capable of reliably measuring viscosity curves for Newtonian fluids, polymeric solutions and consumer products. Collectively, our results demonstrate that the multiplexed viscometer is capable of measuring in a parallel format, viscosities of fluids spanning nearly three orders of magnitude (≈10−3–1 Pa s) across a shear rate range of ≈1–1,000 s−1. We believe our multiplexed viscometer is a low cost and high-throughput alternative to conventional rheometers that analyze samples serially using expensive robotic liquid-handling systems. The multiplexed viscometer could be useful for rapidly analyzing a wide selection of complex fluids on-site during product formulation and quality control.

Automated Circular Dichroism Spectroscopy for Medium-Throughput Analysis of Protein Conformation

Circular dichroism (CD) spectroscopy is a powerful method for monitoring conformational changes of biomolecules. For peptides and proteins, it is highly sensitive to changes in secondary structure, which may be caused by alterations in amino acid composition or solution conditions (e.g., temperature, pH, salts, detergents, denaturants, and excipients), post-translational modifications, self-association, or ligand binding. The assets of CD spectroscopy are that the signal is directly linked to structure, the analyte is measured without labels and in solution, the technique requires low sample amounts, and data analysis is straightforward. However, CD spectroscopy has remained a low-throughput method because it imposes high requirements on the optical quality of sample cells and thus cannot be performed in microplate-reader format. Here, we introduce an automated CD spectrometer equipped with a low-birefringence flow-through cell that is coupled to a three-axis robotic liquid-handling system. This enables unattended CD measurements on up to 384 samples, including sample transfer from 96-well plates into the flow-through cell, data acquisition, and cell cleaning. We show that the accuracy, precision, and reproducibility afforded by the new instrument are excellent and exemplify how the advantages offered by automated CD spectroscopy can be exploited to quantify protein stability by titration with chemical denaturants.

Selective displacement chromatography in multimodal cation exchange systems

A library of displacer analogues with varying degrees of electrostatic, hydrophobic and hydrogen bonding moieties was evaluated for their ability to enhance the selectivity of multimodal (MM) chromatography under high loading conditions. The library was screened for displacement of model proteins using a robotic liquid handling system and selective batch separations were achieved for proteins that were inseparable with linear gradient chromatography. Trends in protein displacement were identified and displacers with higher hydrophobicity and net charge exhibited improved protein displacements. Proteins that interacted with the resins primarily via electrostatic interactions were more readily displaced than those that possessed a significant hydrophobic contribution to their binding. In addition, multimodal displacers were found to be more selective than single mode electrostatic displacers. Column chromatography studies were also carried out and baseline separations were achieved for model protein pairs using selective displacement. Finally, operation of these columns in the desorption mode resulted in baseline separation of model proteins which were not separable by selective displacement chromatography. This study indicates that the inherent selectivity of MM resins can be augmented by the selectivity of the displacer under non-linear competitive binding conditions, creating new opportunities for protein separations not possible using traditional gradient operations.

An Automation-Assisted Generic Approach for Biological Sample Preparation and LC–MS/MS Method Validation

Although it is well known that automation can provide significant improvement in the efficiency of biological sample preparation in quantitative LC-MS/MS analysis, it has not been widely implemented in bioanalytical laboratories throughout the industry. This can be attributed to the lack of a sound strategy and practical procedures in working with robotic liquid-handling systems. Several comprehensive automation assisted procedures for biological sample preparation and method validation were developed and qualified using two types of Hamilton Microlab liquid-handling robots. The procedures developed were generic, user-friendly and covered the majority of steps involved in routine sample preparation and method validation. Generic automation procedures were established as a practical approach to widely implement automation into the routine bioanalysis of samples in support of drug-development programs.

Purification of monomeric mAb from associated aggregates using selective desorption chromatography in hydroxyapatite systems

Selective desorption on ceramic hydroxyapatite (CHT) was implemented for the purification of monomeric monoclonal antibody (mAb) from associated aggregates and other post-protein A step impurities. A robotic liquid handling system was employed to carry out a parallel batch screen of selective desorbents on a post-protein A step mAb mixture. The effect of different phosphate concentrations was also investigated. Selective batch separations were achieved between monomeric mAb and associated aggregates/impurities. The batch screen results also established optimal mobile phase conditions for each selective desorbent. These initial batch results were then used to guide column separations, and baseline separation of monomeric mAb from associated aggregates and impurities was achieved, validating the screening results. Selective desorption also resulted in improved separations on CHT, with 100% yield of pure monomeric mAb as compared to 61% and 79%, respectively, for conventional linear and step gradient operations. This proof of concept study demonstrates selective desorption on CHT as an effective separation technique for the purification of monomeric mAb from associated aggregates and other post-protein A step impurities in a single process step.

Development of Standard Test Procedures for Quantifying Carry over from Fixed Pipetting Tips in Liquid-Handling Systems

Robotic liquid-handling systems using fixed reusable pipetting tips are used not only in fully integrated in vitro clinical diagnostic analyzers, but also in open platform robotic liquid-handling systems. These are not confined to particular diagnostic assays, as customers adapt a large variety of assays on these platforms. One major problem with the use of fixed, reusable tips is the carry over (CO) of analyte from sample to sample. Despite widespread use of fixed tips in open platform systems, systematic studies on procedures to quantify CO for analytes other than nucleic acids are missing. In a consortium with three liquid-handling system suppliers and one coating specialist, we developed test procedures for the quantification of CO. The procedures were standardized and tested with the analytes fluorescein, immunoglobulin G, and hepatitis B surface antigen as model substances for small organic molecules, antibodies, and complex biomolecules. The test procedures allow the reproducible quantification of the CO with intra- and interassay precisions of less than 6% coefficient of variation. They were used to investigate the effect of different tip coatings on the CO of the three analytes. Fluoropolymers, inorganic-organic nanocomposites, sodium-silicate glass, titanium dioxide, and silicone resins, which are used in special applications, showed only small differences in CO. The CO test procedures can be easily transferred to different liquid-handling systems and used with different analytes.

Reduction of Carry over in Liquid-Handling Systems with a Decontamination Step Integrated in the Washing Procedure

Robotic liquid-handling systems can be equipped with disposable pipetting tips or fixed reusable pipetting tips. The use of disposable tips is perceived as the best option to avoid carry over (CO) of analyte from sample to sample. We recently developed standardized CO test procedures that allow precise and reproducible quantification of CO for fixed reusable tips. We used these test procedures to reduce CO of the analytes fluorescein, IgG, and hepatitis B surface antigen (HBsAg) to minimal levels. Variations of washing intensity, using water as a washing solution, did not lower CO below acceptable target levels. These target levels would preclude a false-positive detection of IgG and HBsAg in human serum when a negative sample is measured subsequent to a sample with a high analyte concentration. We therefore integrated a decontamination step into the washing procedure. Screening of 12 decontamination solutions showed that sodium hypochlorite solution was very efficient in reducing CO. Optimization led to a final washing routine in which tips are exposed for 0.2 s to 0.17 M NaOCI and subsequently rinsed with 2 mL of water to remove any remaining decontaminant solution. The washing procedure only takes 15 s and is thus suitable for high-throughput applications. The procedure was able to lower CO of IgG and HBsAg in human sera below relevant levels. The decontamination step with hypochlorite can easily be integrated into different liquid-handling systems and is likely to be effective against CO of most proteins and peptides.

Unique selectivity windows using selective displacers/eluents and mobile phase modifiers on hydroxyapatite

A detailed study was carried out to combine the unique selectivity of ceramic hydroxyapatite (CHA) with the separation power of selective displacement chromatography. A robotic liquid handling system was employed to carry out a parallel batch screen on a displacer library made up of analogous compounds. By incorporating positively charged, metal chelating and/or hydrogen bonding groups into the design of the displacer, specific interaction sites on CHA were targeted, thus augmenting the selectivity of the separation. The effect of different mobile phase modifiers, such as phosphate, sulfate, lactate and borate, were also investigated. Important functional group moieties and trends for the design of CHA displacers were established. Selective batch separations were achieved between multiple protein pairs which were unable to be resolved using linear gradient techniques, demonstrating the applicability of this technique to multiple protein systems. The specific interaction moieties used on the selective displacer were found to dictate which protein was selectively displaced in the separation, a degree of control not possible using a mono-interaction type resin in displacement chromatography. Mobile phase modifiers were also shown to play a crucial role, augmenting the selectivity of a displacer in a synergistic fashion. Column separations were carried out using selective displacers and mobile phase modifiers identified in the batch experiments, and baseline separation of the previously unresolved protein pairs was achieved. Further, the elution order in these systems was able to be reversed while still maintaining baseline separations. This work establishes a new class of separations which combine the selectivities of multi-modal resins, displacers/eluents, and mobile phase modifiers to create unique selectivity windows unattainable using traditional modes of operation.