Companies are addressing requests for higher throughput with higher resolution in chromatography by developing more sophisticated platforms. In addition, many are introducing nanoscale instruments for protein separation and addressing niche markets like Mab separation, while others are developing turn-key solutions with advanced software capabilities.
GE Healthcare (www.gehealthcare.com) recently introduced several new chromatography products. DeCyder MS software compares chromatogram and mass-spectra data by plotting it as a 2-D or 3-D map. It makes it easier to look at trends, because the software makes the patterns more recognizable by the human eye, explains Jasmine Gray, Ph.D., director of marketing for the Americas, protein science business.
The DeCyder automatically compares data sets and answers differential analysis questions to determine relative quantification of proteins, important for biomarker discovery research, according to Dr. Gray. She also notes that data from any chromatography and mass spectrometry system can be imported into the software.
The Ettan nanoLC is a nanoscale-dedicated 1-D LC peptide separation chromatography system for early biomarker discovery, used upstream from electrospray ionization MS. It enables elution at flow rates as low as 200 nL/min and accommodates a range of sample amounts. Integrated, automated sample handling allows injection of as little as 100 nL from vials or microtiter plates. Reversed-phase separation can be done directly into MS. Unicorn software controls and evaluates the chromatography, or the system can be controlled by a virtual interface with the mass-spectrometer software.
The Ettan MDLC (multidimensional liquid chromatography) provides several chromatography separation methods and is designed for full flexibility peptide nanoseparation, prior to protein identification by MS/MS. Three selectable preprogrammed column configurations allow a high-throughput approach. The Ettan MDLC can perform 1-D separation (reversed-phase) or 2-D LC online or offline ion exchange first and then reversed-phase, providing more flexibility depending on the samples.
The main bottleneck in chromatography is dealing with complex samples like plasma. The low-abundance proteins are the most relevant to biomarker discovery, and the multidimensional system provides a way to focus on these proteins, explains Dr. Gray.
High-Throughput Sample Prep
Millipore’s (www.millipore.com) Multi-SPE 96-well extraction plates provide enhanced sample preparation prior to liquid chromatography. Solid-phase extraction is a preferred method for isolating and concentrating analytes at low levels because it eliminates interferences that contribute to signal suppression. The key advantage is that it features 3M’s Empore membrane technology, which includes integrated prefilters that are ideal for samples of biological fluids, explains Jeff Busnach, bioscience product manager.
Each plate well contains an Empore membrane, providing clean, concentrated samples for enhanced LC-MS, the company notes. There are five, solid-phase extraction sorbents for various applications. You can cover a broad range of molecules with the different sorbents, biomarkers, small peptides, small chemical compounds, and their metabolites, states Busnach. Dense particle packing within the membrane allows for less sorbent mass and lower elution volumes.
A line of small volume chromatography products for HPLC called Vantage L Chromatography Columns is for concentrations in the picomole or nanomole range. This is integrated in the HPLC system as part of the online preparation and stays in the system over the course of many samples.
Microparallel Liquid Chromatography
Nanostream (www.nanostream.com) has developed a high-throughput, parallel-format, microfluidic liquid chromatography system. The key to this technology is the Brio cartridge, consisting of 24 columns packed with stationary-phase material for reversed-phase separations. Three different cartridges are available with more in development.
The company offers three productsthe original CL system for high-throughput chemical analysis, the LD system for assay detection, and the CX system for chromatographic sample preparation. One benefit of our systems is that we are using a flow rate of 240 microliters/minute and dividing that among 24 different separations, explains Surekha Vajjhala, vp, marketing. Sample sizes are usually 0.5 microliters up to 5 microliters.
The LD system obtains quantitative information about hits while offering additional information on compound purity and solubility. Historically, HPLC has been used to develop biochemical assays. With the emphasis on higher throughput and larger compound libraries, researchers moved away from HPLC. Now they are turning back to separation-based assays because they want a generic screening platform that can be used for assays that are otherwise difficult to do, states Vajjhala.
The CX system is for chromatographic sample preparation for drug metabolism and pharmacokinetic profiling. It also has UV absorbance detection and a time-triggered parallel fraction collector.
Analysis software for all three systems provides real-time visualization of chromatograms from sets of 24 samples as they are separated.
Eksigent Technologies’(www.eksigent.com) product line for drug discovery includes the Express LC-100 and the Express LC-800. Both use special small-scale columns, called ChromXP, and reportedly provide high-resolution and high-speed separations that can’t be achieved with conventional HPLC. The Express LC-100 is a single-channel HPLC that runs assays five-times faster, says the company, and is optimized for 300-µm internal diameter columns and flow rates of 0.20 to 30 microliters/minute. Inline flow meters actively measure actual flow in each mobile phase and provide adjustments to deliver accurate and consistent gradient.
The Express LC-800 is a parallel HPLC with eight channels that the company says runs ten-times faster than conventional HPLC. It allows the user to try eight different separation methods simultaneously. High-throughput applications include AMDETox, analytical method development, and peptide separations.
In addition, the company is developing a system in partnership with two pharmaceutical companies for chiral analysis, says Phillip DeLand, senior product marketing manager. This uses molecule confirmation to differentiate two compounds.
You often find an ingredient with two structuresone is biologically active and the other is inert, or worse-case scenario, toxic, DeLand explains. The company anticipates a launch date for the chiral system in the third quarter of 2006.
Enhancing Protein Analysis
Waters (www.waters.com) has a new Ultra Performance Liquid Chromatography (UPLC) system with components for high resolution and fast separations. The Acquity SQD is a single quadrapole mass spectrometer that allows scanning at 10,000 amu/second. It features IntelliStart diagnostic software to tune and calibrate the mass detector and conduct system-performance checks. Applications include peak purity/impurity profiling, synthesis confirmation, and enhanced methods development through the use of molecular weight-based peak tracking.
Additional detectors for the Acquity UPLC include a photodiode array detector, an evaporative light scattering detector, and an enhanced tunable detector. Getting it to go fast is good but it is not enoughall these detectors are designed to minimize band broadening in the detector, states Tom Wheat, Ph.D., principal scientist and manager of the life sciences application lab. Excess volume causes the bands to broaden, requiring the flow rate to be slowed down, which hampers high-throughput capability.
The company also recently introduced two new turn-key solutions for the Acquity UPLC Systemthe UPLC amino acid analysis application solution and the UPLC peptide mapping application solution. This is a different approach. We’re using components we’ve had but are putting them together for application solutions, explains Dr. Wheat.
The amino acid analysis solution contains a quality-controlled AAA column, eluents, derivatization reagents and standards, and software templates. The peptide-mapping solution contains a UV-based Acquity UPLC System, peptide-mapping columns, and a system PQ kit with a column and two peptide standards.
Another company focusing on proteins, Pall (www.pall.com), recently introduced two protein purification kitsthe Enchant life science kit for IgG purification and the Enchant life sciences kit for albumin depletion. The IgG kit is focused on purification of antibodies from small quantities of serum. The idea behind the kit is to give the end user all the tools they need to go from the beginning of the purification process for an antibody to the end, states John Jenco, Ph.D., senior staff scientist and chromatography applications manager.
The kit includes protein A or protein G columns for selective binding and a small desalting column. The albumin-depletion kit eliminates this abundant protein from serum samples to enable detection of smaller quantity proteins. So you can find the needle in the haystack by eliminating the haystack, says Dr. Jenco.
The company says it will soon introduce its physiological hydrophobic interaction sorbents. Traditional hydrophobic interaction chromatography works by requiring high salt to bind proteins but this also causes proteins to precipitate out. Based on its HyperCel chromatography bead, the company has designed two ligands that allow hyperphobic interaction without high saltthe PPA-HyperCel (aromatic ligand) and the HEA-Hypercel (alophatic ligand).
Proteins can bind to these sorbents under physiological conditions (pH 7.4) using only 150-millimolar sodium chloride. These will help to identify a protein target that may be interacting with a drug and to purify sufficient amounts of that protein for mass spectroscopy.
Membranes Add Flexibility
The Sartobind Membrane developed by Sartorius (www.sartorius.com) is a cellulose-based membrane that is pH stable. Ligands are attached directly to it and can be cationic or anionic, strong or weak. One of the advantages of a cellulose-based membrane is that it is very low protein binding, so when you want to bind a protein and present conditions for elution, the protein will elute nicely, says Jeff Mora, product manager, membrane chromatography.
There is a 96-well plate format for drug discovery on a small scale. The membrane is located in each well of the plate, and the bottom is open to apply a vacuum to pull material through it. Other formats include small centrifuge devices, a syringe filter device, and a capsule design for removal of protein and virus contaminants during drug production.
A new purification tool for gene therapy, the Adeno-Pack, uses the Sartobind membrane in a syringe-disc format. Adeno-Pack allows users to quickly screen different sera types of viral vectors or different expressed proteins in the late discovery phase. It would take several hours for one column prep versus several minutes with Adeno-Pack, adds Mora.
Addressing customer demand for more sophisticated and turn-key solutions for complicated methods, such as 2-D LC or parallel LC, Dionex (www.dionex.com) has continued its UltiMate 3000 Systems with additional modules.
Demands are more focused on finding an analyte in complex matrices. You want to have a sophisticated LC system for that, meaning automation of sample prep along with separation, detection, and quantification. Based on the UltiMate 3000, we provide these turn-key solutions for tandem operations; it is one system with a dual concept that switches from one run to the other, explains Dietrich Hauffe, vp, corporate marketing and business development.
The LC series has seven standard systems, five dual-gradient systems, and several new modules. The advantage of the X2 Dual LC System, Hauffe notes, is that it has multiple flow paths that can be used independently of one another and allows column switching for parallel or tandem LC. It can perform 2-D or multidimensional LC, as well as online SPE-LC. The wide range of modules cover various applications from nanoflow LC/MS to automative semipreparative purification.
Integrated Chromeleon software offers enhanced features like the Virtual Column Separation Simulator that optimizes gradient IC and isocratic separations. Waveforms can be switched during analysis to optimize detection for different analytes. An integrated database locates samples matching any combination of sequence, sample, or result criteria, and its Excel-compatible spreadsheet provides flexibility in reporting results.
For chromatography applications in drug discovery, Beckman Coulter (www.beckmancoulter.com) offers its System Gold HPLC platform and Agilent Technologies (www.agilent.com) markets the 1200 Series LC systems.