NEW YORK, March 2 - With scores of businesses looking to develop protein chips, it would seem a company with years of experience mass-producing microarrays might have a leg up on the competition.
But Affymetrix, maker of the widely used GeneChip DNA microarrays, has no immediate plans to invest in protein chip research. "It's just too far in the future," a spokeswoman said.
Affymetrix's reluctance to enter the burgeoning market underscores the significant hurdles researchers face as they try to produce equivalent tools for studying proteins. Manufacturing a reproducible microarray of proteins will likely require advancements in surface chemistry, detection systems, and protein antibody synthesis, and, scientists say, knowing how to make a DNA array won't be much help.
However, with potential users hungering for an easy-to-use protein chip and market researchers such as Bioinsights predicting that the market for these tools will grow to an annual $490 million by 2006, there are plenty of players trying to overcome the obstacles. In fact, there are a number of early versions of a protein chip already on the market.
Ciphergen, of Fremont, Calif., sells an affinity chip with a surface that weakly binds proteins screened from a sample, and uses mass spectrometry to identify the protein profile. "Its main utility is discovery, especially if you're trying to find a biomarker and don't know the antigen or antibody," said Chris Pohl, vice president for research and development at Ciphergen.
Another type of chip-first introduced in 1990-comes from Sweden's Biacore. Instead of screening protein biomarkers, Biacore's chip uses a technique called surface plasmon resonance to investigate protein binding kinetics. The technique is good for measuring protein-protein interactions because no labeling of the proteins is required, a step that might influence how the proteins interact, said Ruedi Aebersold, a cellular biologist at the Institute for Systems Biology in Seattle.
Ultimately, however, scientists would like more than what is currently available. Ideally, said Aebersold, protein microarrays would be capable of identifying a large number proteins in a sample and quantifying how much of each protein is present. This type of high-throughput chip would allow researchers to determine which proteins a gene expresses in a sample, and give doctors a tool to gather large-scale protein profiles from patients.
How to Build a Protein Chip
In order for protein microarrays to fulfill their promise, however, researchers must overcome a number of hurdles, including a lack of information on what proteins are important to study.
Large Scale Proteomics, a subsidiary of Large Scale Biology, is one of the few players in the protein chip market with significant proteomics expertise-namely in the form of its index of protein expression levels from 157 tissues. In January, LSP of Germantown, Md., hired San Diego-based Biosite to manufacture between 2,000 and 5,000 antibodies for the proteins contained in LSP's index. Once LSP and Biosite have created their library of protein antibodies, the companies will either manufacture the chip themselves using Biosite's existing platform for making specialized diagnostic chips, or find a partner, said Ken Buechler, Biosite's vice president for research.
Other potential chipmakers are attempting to improve the techniques for attaching proteins to surfaces. In addition to an antibody-based approach developed by Biosite, many researchers are devising novel methods for capturing and binding proteins to a substrate.
Phylos, based in Lexington, Mass., is proposing a method for binding specific proteins that employs an artificial binder-a kind of pseudo-antibody-using the scaffold of a human protein called fibronectin. The artificial binders are about one quarter the size of regular antibodies, allowing more to be packed on a chip, and take only "days to a few weeks" to manufacture, said Phylos CEO Gustaf Christensen.
Meanwhile, scientists at SomaLogic of Boulder, Colo., have patented a method for using single strands of DNA to mimic antibodies. The technique takes advantage of oligonucleotides' ability to assume three dimensional shapes, said Todd Gander, senior director of marketing at SomaLogic, and relies on the SELEX process, a patented evolutionary approach to find strands of nucleic acid that, when folded, bind with a specific protein.
Somalogic's binding technique also involves photochemistry. When a protein initially binds with the oligonucleotide, only the protein's affinity for that shape keeps it there. But when the surface is exposed to UV light, active compounds placed in the oligonucleotide strand form cross-links with electron-rich areas of the protein, said Gander. This only happens, he said, if the protein is a perfect match. "The increased specificity is analogous to using two antibodies in a sandwich assay," he said.
Getting Proteins to Remain Intact
Finding a good binder for a protein, however, is complicated by the tendency of proteins to denature in less-than-optimal environments-such as near chip surfaces. Scientists at Zyomyx, in Hayward, Calif., are attempting to make proteins feel more at home when brought near to the surface of a chip, so that they will stay in their natural conformation and not denature.
To accomplish this, researchers at Zyomyx are applying surface chemistry derived from microscopy experiments carried out by chief technical officer Peter Wagner. The technique immobilizes proteins on the surface of the chip while surrounding them with a hydrophilic environment, said Zyomyx CEO Lawrence Cohen. "We provide the sticky points on a shag carpet," Cohen said. "We're interested in the binding sites and how the neutral sites contribute to the [protein's] environment."
Other chip technologies can get away with some surface inactivation because their arrays contain a relatively large number of antibodies, and some are bound to be in their natural conformation, Cohen added. "The problem comes with miniaturizing the devices," he said. "That's when you have to start thinking about surface chemistry."
While Zyomyx perfects its surface chemistry, others are focusing their attention on detection technologies. HTS Biosciences, an 80:20 joint venture between Applied Biosystems and Quantech, hopes to bring to market three technology platforms: a surface plasmon resonance system, a phase fluorescence platform, and an array-based chemiluminescence detection technology.
Researchers will want to use the SPR platform for drug target discovery, said HTS CEO Greg Freitag, while the phase fluorescence and chemiluminesence techniques will be good for more sensitive screening assays to identify where proteins have bound with the surface and how much of that protein is present.
"Most people have one technology that they have to develop into a system that can be all things for all people. Instead, we've been able to acquire a number of synergistic technologies and focus each detection system to its own use," said Freitag.
With many companies hoping to release beta versions of their systems by the end of this year, it may be only a matter of time before a shakeout proves which technologies will be successful.
"The proof is in the pudding," said Felicia Gentile, CEO of Bioinsights. "Can any of these guys make a product anyone will buy at a reasonable cost?"