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Feature Articles : May 15, 2012 ( )
Simplifying Complex Biological Matrices
Protein therapeutics is a fast-growing segment within the global pharmaceutical industry. Six thousand biologic drugs were in clinical trials in 2009 compared to 1,200 in 2005. By 2014, six of the top 10 drugs on the market will be biologics, according to a 2011 Thompson Reuters–Newport study of U.S. drug trends.
For more than a decade, LC-MS has been used for small molecule absolute quantitation. As the role of protein-based biologics grows, this technology is becoming attractive for absolute quantitation of proteins. The challenge is the selective isolation of the protein of interest from the large amount of irrelevant protein species naturally present in biological matrices. This issue was one of the main topics discussed at the recent “Pittcon” conference in Orlando.
“Complicated samples contain a lot of proteins, especially high-abundance proteins. The mixtures are so complex that you need to clean them up before you use the LC-MS,” explains Richard King, Ph.D., laboratory director, PharmaCadence Analytical Services.
Absolute quantitation of proteins is difficult, and even more so, because there are no real standards as with small molecules. The assumption is every molecule of protein generates one molecule of peptide but, in reality, the peptides can come from inactive versions of the protein, fragments, precursors, or degradants. PharmaCadence Analytical Services uses electrophoresis as a protein sample-preparation technique to distinguish between those species.
The advantage of electrophoresis is that almost all proteins will run through a gel with consistent, high recovery rates. In addition, phospholipids, a main cause of matrix effects in the MS, run right through the gel. The limitation is loading capacity so electrophoresis might not be the preferred technique for a low-abundance protein in plasma.
“Our goal is separation at the whole protein level. The Expedeon/Protein Discovery 8100 Fractionation Gelfree® System (gel-eluted liquid fraction entrapment electrophoresis system from Protein Discovery), eight-channel, parallel-tube SDS-PAGE allows us to track our protein of interest with molecular markers, elute our fractions of interest off the gel, keep them in solution, then do the digestion. As long as we catch the protein we are interested in, we can live with impurities at that point; we will select against those with the LC-MS,” continues Dr. King.
The Gelfree system is an antibody-free separation technique and can fill in when there are no antibodies or antibodies are under development. Antibody development can be a daunting and time-consuming task, taking from two months to two years.
PharmaCadence Analytical Services combines electrophoresis with reverse-phase LC and MS. Each of the techniques has a different separation mechanism and, when combined, produces a very powerful system when working with complex mixtures.
More than One Approach
“Sample preparation is challenging because you are looking for a specific protein in a global protein soup. There is more than one approach. It depends on the variables, such as the concentration level, the location, solubility, and the tools available in the laboratory,” discusses Nalini Sadagopan, Ph.D., south-west regional LC-MS product specialist, Agilent Technologies.
“Many protein sample-preparation techniques have been around for a while. Manufacturers continually evaluate the market needs and develop new formats, such as 96-well formats for high-throughput screening and parallel processing. Plus there have been considerable improvements in abundant protein removal protocols and immunoaffinity purification in the last several years.”
“The next hurdle is to marry the sample-preparation techniques to the LC-MS workflow because of the desire to quantitate very low-level proteins, unknown proteins, or proteins for which antibodies do not exist.”
The two broad approaches to protein sample preparation are to remove the extraneous high-abundance proteins and to let the protein of interest pass through or to enrich the protein subgroups by capturing, then eluting, the protein of interest.
For human serum, specific removal of six high-abundance proteins depletes approximately 85–90% of the total protein mass. The Multiple Affinity Removal System, spin cartridges, and LC columns can be used to remove the top 7 or 14 high-abundance proteins from a sample.
The AssayMAP® platform for high-throughput micro-chromatography allows integrated protein sample-preparation workflows from crude samples and incorporates purification, digestion, and peptide clean-up using automated liquid-handling systems. The disposable cartridges contain a 5 μL bed, which can be packed with 15–100 μm particle-size chromatography resins or immobilized enzymes, such as protein A, streptavidin or trypsin, for use in subgroup enrichment.
Advances in MS technology have also simplified protein sample preparation. The high sensitivity of the Agilent 6490 triple-quadrupole LC-MS with iFunnel technology provides attogram (10–18) limits of detection and zeptomole (10–21) sensitivity with six logs of linearity. This allows, in some circumstances, the detection of proteins at low levels without extensive sample preparation by just diluting the sample.
Emerging Trend for LC-SPE Sorbents
“We are starting to see the use of LC-SPE (solid-phase extraction) sorbents to perform separations in the protein and proteomics areas. It is an emerging trend,” comments Rob Freeman, business manager, chromatography, SGE Analytical Science.
Conventional SPE started with disks and tubes for sample volumes from 50 mL to 1 L, using either gravity or a vacuum to move the solvent through the large sorbent bed. SGE Analytical Science has combined the advantages of SPE with automation by developing a digital syringe, eVol®, with an embedded miniaturized SPE cartridge.
MEPS™, micro-extraction by packed sorbent, incorporates a cartridge, which holds a few mg of sorbent, into a removable needle syringe. This solid-phase-extraction technique can be used for protein sample preparation manually, just like a standard syringe, or interfaced with gas or liquid chromatography systems through an autosampler. In addition, recently, the combination of eVol and MEPS was used to infuse extracted analytes directly into the MS-ESI (electrospray ionization) source, without any additional modifications.
The specific sorbents and particle pore sizes (pore size corresponds to surface area), impact the sizes of proteins and peptides that are retained. The typical MEPS pore size is 120 angstroms, which is larger than the conventional SPE pore sizes of 60–80 angstroms.
The miniaturized format works with small-volume biological samples, 10–1,000 µL, improves efficiencies, and virtually eliminates solvent use and waste.
The sorbent, a spherical silica particle with an attached polymer such as C4, C8, or C18, separates and extracts through hydrophobicity and retentive interaction. SGE Analytical Science is working on extending MEPS capabilities for protein sample preparation using polymer monoliths in collaboration with Emily Hilder, Ph.D., at the Australian Centre for Research on Separation Science.
Still a Long Way to Go
There is still significant work to be done in the area of protein sample preparation and absolute protein quantification.
According to Dr. King, “We have to understand what we are really measuring and what we are talking about. Results from a ligand-binding assay are not going to be the same as from a MS with a surrogate peptide workflow, which is not going to give you the same answer as a top-down measurement, looking at the whole intact protein in a MS.”
Small Molecules vs. Proteins
If the focus is on small molecules and not protein-based biologics, the HybridSPE®-Phospholipid can be used to clean up both proteins and phospholipids in just one step.
“The conventional procedures for small molecule sample preparation from biological samples include protein precipitation, liquid-liquid extraction, and solid-phase extraction. None of these techniques specifically target the removal of phospholipids, which are a well-known cause of matrix effects in LC-MS analysis of biological samples,” explains Dr. Xiaoning Lu, Ph.D., senior scientist, Supelco division of Sigma-Aldrich.
In conventional SPE the compound of interest is absorbed onto the sorbent and then eluted off. In HybridSPE-Phospholipid the compounds of interest go through the phase.
In a typical procedure, users load samples on the HybridSPE-Phospholipid device, add organic solvents and mix it with the samples, then pull the sample through the device by vacuum or positive displacement pressure. The precipitated proteins are filtered by a 5 µm PTFE frit and a 0.2 µm filter membrane. Phospholipids attach to the sorbent, a zirconia-coated silica bed. The protein- and phospholipid-free samples are then ready for injection and analysis in the LC-MS.
HybridSPE-Phospholipid is available in a variety of formats, 96-well plates, cartridges, and microliter pipette tips, for implementation into automated processes. Although unique in phospholipid removal, the HybridSPE-Phospholipid technique is very similar to the standard protein precipitation method and easily incorporated into the laboratory workflow.
Sample-Prep Changes for Production
USP Method 233, Elemental Impurities Procedures, is the proposed laboratory procedure to replace the antiquated USP Method 231, Heavy Metals, for sample preparation and analysis of raw materials and finished products for pharmaceutical drug products, including those from natural sources and rDNA.
USP Method 233 uses current analytical instrumentation, ICP-OES (inductively coupled plasma—optical emission spectroscopy), ICP-MS and microwave, instead of hot plates, muffle furnaces, and the technician’s eyes for visual determinations. Advances in ICP require cleaner sample to achieve lower detection limits.
The cost to the pharmaceutical industry for the method changeover is significant: instrument purchases, microwave digestion and ICP method development, determination of which analytes to analyze, training and documentation.
“Some samples are very simple to digest; others, like raw materials containing biological molecules, can be more challenging. CEM’s goal is to make it as easy as possible for a technician to walk up to the microwave, safely put their vessels together, and add the right amount of sample and acid. Our extensive built-in training aids and pre-programmed methods help achieve this,” explains Jason Keith, product manager, CEM.
The Discover SP-D digests one sample at a time and can handle, in an automated process, varying samples where different acids and digestions temperatures are needed.
The MARS 6 with One Touch™ Technology batch processes similar samples and has nearly 80 preprogrammed methods. Sensors count the number and type of vessels to determine the amount of microwave power needed to reach the digestion temperature for the selected method.
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