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Apr 1, 2006 (Vol. 26, No. 7)

Protein Characterization Applications Grow

Emphasis on Biologics Leads to a Wider Adoption of the Technology

  • As the demands for more detailed protein characterization increase, many companies are working to meet those demands through new tools, technologies, and assays.

    Alistair Kippen, Ph.D., head of protein analysis at Covance (www.covance.com), says that the current focus on biologics is changing regulatory testing requirements for such products.

    “Recent focus includes immunogenicity testing at early-stage product development, looking at antibody detection and neutralization effects from preclinical studies and onward through clinical trials,“ he explains. In addition, more detailed protein drug characterization is occurring earlier and incorporating powerful techniques such as peptide mapping and mass spectrometry.

    “You now include extensive protein characterization studies for product comparability, release, and stability assessment in support of preclinical studies.“ Methods should be fully evaluated prior to regulatory testing for product purity, protein structure, concentration, and potency; in addition to the requirement for manufacturing GMP-grade material entering clinical trails that can add up to a year additional development time.

    Another area where regulatory requirements are tightening is in detection and characterization of impurities. “If you are producing a biologic from a cell line, you have to determine levels of any residual host cell protein, residual DNA, protein aggregates, protein breakdown, or container leachables in the product,“ adds Dr. Kippen.

    Biological potency assessment is also a key assay. “If you have a product going into clinical trials you need to have developed an effective biopotency assay, and this often requires cell-based assays to look at functional biological activity. Such assays can determine whether the native structure of the molecule has changed, which can have direct affect on biological activity and potency,“ continues Dr. Kippen.

    Often potency is a key measurement of the final active structure, which can’t always be measured by other physio-chemical characterization methods. Biological assays should be set up through initial discovery and early preclinical/toxicology testing of a protein. “The earlier the biological assay is set up, the more efficient it will be to have it validated and ready for clinical trials,“ he emphasizes.

  • New Assays

    A growing trend that Beckman Coulter’s (www.beckman.com) proteomics business group recently recognized is the adoption of protein-characterization methods, including Beckman’s, into the quality control field.

    “For example, biopharma is taking our capillary electrophoresis technology as an additional assay to determine product quality. The ultimate goal is to determine if the product being developed is exactly what it should be—without impurities or degradation,“ explains Mark Lies, strategic marketing manager.

    The core technology is the ProteomeLab PF 2D, which enables protein fractionation in 2-D. A complex protein sample is broken down into several hundred fractions and then run through reversed-phase liquid chromatography.

    “The liquid fractions are significant because you can go to a downstream process, like mass spectroscopy, and you can recover exactly which fraction you want to look at,“ says Lies. This allows for differential display analyses where it’s possible to take two proteomes (one control sample and one drug-treatment sample) and differentiate between the two—look for proteins that are up- or down-regulated, proteins that are missing, or discover biomarkers.

    The company recently released a new product for sample prep prior to analysis on the ProteomeLab PF-2D platform. This proteome partitioning system, is based on IGY affinity chromatography, which uses chicken-derived antibodies. The IGY-12 column consists of a mixture of antibodies against the 12 most abundant proteins in serum plasma, removing these to allow discovery of low-abundant proteins.

    Beckman’s main system for protein characterization is the ProteomeLab PA 800. It determines protein purity and critical biochemical characteristics. It also provides information relating to molecular weight, relative charge, post-translational modifications, and heterogeneity.

    “Customers are doing molecular weight analysis to replace gels, and many are using this system for monoclonal antibody analysis,“ points out Lies, adding that the main advantage of this system is that it’s automated and quantitative, thus allowing for many different assays.

    “You can switch out the modular detectors and perform UV detection, diode array detection, or fluorescent-based detection. You can also couple directly to mass spectroscopy and use that as your detector,“ he says.

  • Novel Applications

    Charles River Laboratories (www.crl.com) has a contract testing facility based in Edinburgh, U.K. where the main focus is release testing for marketed products in Europe. Niall Dinwoodie, head of product characterization, bioservices group, says their testing is more GMP-oriented for biopharmaceutical products than clinical-stage testing.

    “These are more robust assays to characterize the final product and not quite as in-depth as you would use for earlier stages of drug development. It tends to involve HPLC, electrophoretic techniques, and biological assays for potency. Within those, we’re beginning to see an increase in the amount of biological-based assays for determining product potency,“ he explains. This is accomplished by a response within a cell line.

    Another shift that Dinwoodie’s group is seeing is that traditional gel methods (i.e., SDS-Page and IEF gels) are being converted from slab gels to capillary electrophoresis. This has advantages because it can be automated and is more readily quantitative.

    In addition, data analysis and statistical interpretation are becoming more restricted. “Any data manipulation that is more complex than standard linear regression will be challenged more by the regulatory agencies and they will be looking for explanations why a particular statistical program is used,“ maintains Dinwoodie.

    Since the Clinical Trial Directive changed regulations in Europe last year, Dinwoodie notes that methods must now be validated earlier in the development process.

    “That does put a lot of strain on getting a method developed and validated early on [Phase I trials instead of Phase III] when you’re still trying to understand the product. It can take 12 to 18 months to design and validate a biological assay,“ says Dinwoodie.

  • Protein Complexes and Interactions

    Many researchers are interested in analyzing protein complexes and identifying the interaction between proteins, says Charles Piazza, vp, protein analysis, at Invitrogen (www.invitrogen.com).

    “People have been interested in looking at complexes because they represent some of the more interesting phenomenon within cells, either cell receptors or ion channels. Cell signaling is usually mediated by protein complexes on the surface of cells and communicated within the cell also by these complexes,“ according to Piazza.

    Invitrogen offers the NativePAGE Novex gels for high-resolution analysis of membrane protein complexes and large proteins that can’t be easily analyzed using other methods, he explains. The newest gel of this line, the NativePAGE Novex Bis-Tris Gel system, enables analysis of membrane-protein complexes in their native conformations and provides better resolution than with traditional Tris-glycine native electrophoresis, he claims, adding that its neutral pH provides maximum stability of the proteins and the gel matrix.

    There are two additional products the company recently released for detecting protein-protein interactions. These are the ProQuest Two-Hybrid System with Gateway Technology and the Pro-Quest Reverse Two-Hybrid System. The first is a yeast-based system for detecting protein-protein interactions by using three reporter genes and low-copy number of vectors. This helps to minimize false positives.

    The second is designed to eliminate background noise by using the company’s SureFrameAllele Library Construction Kit to facilitate in vitro allele library production and select for full-length proteins in E.coli prior to analysis in yeast.

    A new approach to analyze protein-protein interactions is being addressed by Stratagene (www.strategene.com). Although traditionally analyzed by genetic methods, such as two-hybrid screening, this approach is limited to two individual interacting proteins and cannot analyze protein complexes. There are two kits available: the Mammalian InterPlay TAP System and the newer InterPlay Adenoviral TAP System.

    The first kit reportedly can be used in any cell line difficult to transfect with traditional methods. There are two purification steps, and each uses a different affinity purification tag fused to at least one known component of a protein complex combined with two purification resins specific for each tag. The initial tag is streptavidin-binding peptide and the second is calmodulin-binding peptide.

    “By doing two rounds, you get very pure protein at the end. That’s why the kit is called Tandem Affinity Purification or TAP,“ says Melissa Stolow, Ph.D., director of product marketing, functional biology.

    The second part of the process involves taking the protein and performing mass spectroscopy or Western blot or 2-D gels to identify the proteins interacting with the protein of interest.

    The Adenoviral TAP System contains the two purification tags, but also includes the AdEasy XL Adenoviral Vector System, a viral-based gene delivery system. This has the ability to transfect additional cell types, such as primary cells, which are known to be difficult to transfect.

    “Now customers can get the DNA into cells at a high efficiency rate and into more cell types. It provides more options in terms of looking for interacting proteins in different cell types,“ explains Dr. Stolow. In addition, recombinant adenoviruses can infect both dividing and nondividing cells and can insert up to 6.6 kb of foreign DNA.

  • Protein Modifications

    According to Tina Settineri, Ph.D., product line director, Applied Biosystems (www.appliedbiosystems.com), there is a trend to move back to more careful protein analysis.

    “Proteomics overshadowed protein research for a while by trying to identify every protein in a sample. But the real science is done more with protein characterization than with just protein identification,“ she maintains..

    Applied Biosystems’ new software, ProteinPilot, can automatically search for over 150 peptide modifications and substitutions (biological, chemical, or amino acid substitutes) simultaneously from LC/MS/MS or LC/MALDI experiments. It can also identify protein isoforms, which have slight differences in amino acid sequences, often causing different biological activities.

    There is also increased interest in post-translational modifications (PTMs). The MIDAS Workflow software on the 4000 Q TRAP mass spectrometer system performs selective detection and sequencing of PTMs in a single LC/MS/MS experiment, according to the company.

    “We use something called a MRM-scan (Multiple Reaction Monitoring), that provides quantitative information between samples. This is combined with MS/MS data to specifically target certain phosphorylation sites on a protein,“ explains Dr. Settineri.

    This scan method has low noise because the mass spectrometer only transmits the parent ion of interest for fragmentation and then selects only a single daughter ion. This allows peptides to be detected at lower concentrations. The software can also specifically target low-level phosphorylation sites of proteins to identify or confirm modification locations and quantify proteins/peptides with accompanying MS/MS for identity confirmation.

    The QSTAR Elite Hybrid LC/MS/MS system addresses demands for analyzing large protein complexes, according to Dr. Settineri. The product performs noncovalent protein analysis (where the proteins are not chemically bound but are associated with each other).

    “We can analyze these complexes because of a feature called the noncovalent sleeve, that improves sensitivity for analyzing these proteins, which can be bigger than a million Daltons,“ adds Dr. Settineri. The technology helps researchers understand how proteins act biologically in the body and assists pharmaceutical companies in determining if a drug is binding to a protein.



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