The myriad of challenges of sample prep are being addressed in unique ways by many companies. These range from platform technologies, purification kits, automation, methods to stabilize samples, to validation approaches. The bottom line is that assay results are robust and reproducible.
At a Cambridge Healthtech Conference on proteomic sample prep held earlier this year, scientists from Amgen’s(www.amgen.com) medical sciences division talked about how the company is employing various biomarkers early in the drug development process. “There are few companies using biomarkers in Phase I studies,” said Scott Patterson, Ph.D., senior director, medical sciences. “Now that we have targeted therapeutics, we have the opportunity to determine whether or not the target is being modulated by the therapeutic when it’s first introduced into man.”
To focus on targeted therapeutics, stated Dr. Patterson, there must be appropriate samples and assays. It’s not always possible to look at different tissues; it depends on the disease state and what is clinically feasible. “We want to measure multiple analytes and therefore interrogate different pathways.” However, he added, current technologies do not always address the questions his group wants answered. Between these two issues exist preanalytical variables. This is what happens to the sample after it’s taken and before analysis. “That’s a big issue. There’s a lot of work being done around this,” Dr. Patterson pointed out.
Although the company has no proprietary technology to deal with such variables, his group performs rigorous qualification and validation of all assays. In addition, they are trying to address these challenges by bringing research-based technology into drug development. Although this poses its own problems, they work closely with vendors. “We want to apply the appropriate technology to the questions being asked—often with the technology not designed for a clinical setting. But, it doesn’t mean it can’t be done.”
However, he added what is important is results—how robust they are and how they are helping researchers make good decisions.
Proteomic Biomarker Discovery
Beckman Coulter (www.beckmancoulter.com) has developed sample prep methods for proteomic biomarker discovery. “The key problems when trying to find protein biomarker candidates in serum or plasma are the complexity and dynamic range of those proteins,” explained Jeffrey Feitelson, Ph.D., manager, strategic marketing, biomarker discovery.
In response, the company recommends partitioning and fractionation of the proteome. “We have introduced tools to remove the 12 most abundant proteins in serum or plasma in a targeted fashion using IgY-12 technology. We essentially shrink the haystack 20-fold without removing the biomarkers, substantially enriching the medium and low-abundant proteins,” he explained.
Up to 250 microliters of serum or plasma can be portioned in one run. “The rationale is that if you want to dig deep to find these rare protein biomarker candidates, you have to start with a lot of material,” added Dr. Feitelson.
The flow-through fraction from the IgY-12 column is then injected into the PF-2D (Proteome Fractionation in 2D), which generates hundreds of fractions with intact proteins for analysis. In addition, the posttranslational modifications can be analyzed, which are important in biomarker discovery. Another important feature of the PF-2D is its ability to quantitatively compare two or more proteome profiles, followed by analysis of differentially expressed or modified proteins by mass spectrometry, microarrays, or immunoassays. The PF-2D is flexible in that it works with cell and tissue extracts and soluble biofluids.
“We believe that the combined used of IgY and PF-2D technologies will result in a series of important biomarker discoveries in the near future,” summarized Dr. Feitelson.
Challenges in Proteomic Sample Prep
Some of the leading challenges in proteomic sample prep include biological diversity and the nature of proteins, which are subject to changes in the environment like stress and disease. “The set of criteria for managing samples in proteomic environment is very different from DNA, RNA, and classical cellular analysis,” noted Bruce Haywood, business development leader, clinical proteomics, Becton Dickinson (www.bd.com). He explained that the company’s approach is methodical to address the variability issues. “Our approach has been somewhat serial in learning, development, and interaction with customers to make products that improve work flows in proteomics.”
Part of the company’s approach is to develop tools that address broad issues first, Hayward said. Its first product, BD P100 blood collection kit, had a broad protease inhibitor. Offspring products, like the BD Vacutainer® P700 followed. This inhibits an enzyme that attacks GLP1 (glucagen-like peptide 1)—key to understanding the effects of certain drugs and titration.
Another area the company is focusing on is the future of clinical proteomics. “We’re looking at developing or acquiring technology that allows us to control pre-analytical variability.” The recently acquired Free-flow Electrophoresis product allows analysis of peptides, subpeptides, and whole cells. According to Haywood, two hot applications include organelle separations and membrane proteins.
He added that proteomics is difficult because there are so many different technologies to analyze proteins and new bioinformatic methods to relate proteins to RNA/DNA. In addition, the biology is dynamic, and it takes years to develop methods. “With proteomics, you need to be a bit out of the box,” he suggested.
Qiagen(www.qiagen.com) is focusing on enhancing globalization in the research environment. “There is more interaction between researchers and it’s important that processes become standardized,” said Wolfgang Theibinger, Ph.D., global business director, automated systems. “We also see emerging markets in molecular diagnostics, where standardization is key.”
Automated Nucleic Acid Purification
The company’s EZ1 technology was the first system on the market with a new approach to automating sample prep, said Dr. Theibinger. Although low-throughput (one to six samples), the various kits come with reagents in a prefilled cartridge. The customer selects an application (DNA or RNA purification) and the sample type (e.g., blood, tissue, cells). The system performs the purification process. “This is a paradigm shift in the way researchers and molecular diagnosticians were working with automation. The EZ1 brought the possibility of automated sample prep to the lab bench.”
The BioRobot Universal System provides automated high-throughput (96-well format) applications in systems biology. It also sets up accurate real-time PCR (RT-PCR) and PCR. Application packs (each containing software protocols and application-specific accessories) are available for gene expression and genotyping applications. New software, QIAsoft5, enables reproducible sample processing. A protein applications portfolio, including purification of recombinant proteins, will be launched early next year. “There are more players in this field trying to develop solutions to help customers standardize their processes,” noted Dr. Theibinger. The company is currently working with the Personalized Medicine Coalition and its Clinical Science Integration subcommittee to help drive standardization.
Measuring RNA Quality
Biorepositories contain frozen tissue samples for genomic and proteomic research. Since RNA is affected by sample collection and preparation, it’s important to know its quality. Researchers at Asterand (www.asterand.com) developed their own quality grading system and updated the results to the RNA Integrity Number (RIN) proposed by Agilent (www.agilent.com).
James Eliason, Ph.D., CSO, R&D, presented information on the results and several factors that affect RNA quality and yields. His group analyzed 35,000 samples from the company’s biorepository. “Agilent’s RIN is a mathematical way of looking at the whole RNA profile,” explained Dr. Eliason. “Our method was similar but semiquantitative, based on a five-point system.” The data is now being converted in their database so it will also supply the RIN.
Factors that affect RNA quality include time to freezing and tissue type. Almost 40% of all surgical samples and about 65% of post-mortem samples yield degraded RNA. Initially, researchers thought that only time to freezing affected the RNA. However, there are other unknown factors that occur even before the tissue sample is taken that causes degradation. “So it turns out the situation is more complex than we thought,” Dr. Eliason added.
The group is now trying to focus on which types of assays can be used for different quality RNA. “RT-PCR can use a lower quality RNA than Affymetrix chip assays, but you have to be careful in selecting your probe,” he explained. Research using fixed tissues with highly fragmented RNA in Illumina’s Dazzle assay is currently in review for publication.
Obtaining DNA from blood samples for various tests remains a challenge for the clinical lab. The safety issue of blood-borne pathogens represents one of the difficulties in handling laboratory specimens. Whatman (www.whatman.com) circumvents this issue through its FTA® Elute technology. This allows for sample collection, room temperature storage, and elution of DNA for various molecular-based amplification technologies, like PCR. It eliminates shipping of whole blood, freezer storage, use of organic reagents to isolate DNA, and nucleic acid purification kits.
“One of the major driving forces for FTA Elute,” informed Michael Harvey, Ph.D., director of development, microarrays and molecular biology, “was to provide a modified collection device that would make it simpler and safer to isolate DNA for amplification.”
The sample is applied to the FTA Elute Card and cell membranes and organelles are lysed. Released nucleic acids are trapped in the fibers of the card and remain immobilized and stabilized. Genomic DNA on the FTA Technology has been stored for up to 14 years and successfully amplified by PCR. Once the sample is dried on FTA Elute, a 3-mm punch is placed in a sterile microcentrifuge tube, water is added, and then heated for 30 minutes. The eluted DNA is measured by commercially available fluorescent kits.
Applications range from biobanking, pharmacogenomics, genotyping, rapid DNA isolation, molecular diagnostics, genetic identification, transgenics, and PCR. Dr. Harvey said the company has conducted studies demonstrating the FTA Elute’s use in genotyping for pharmacogenomics for personalized medicine. Another application Whatman is expanding upon is whole-genome amplification.