Nano applications, mass spectrometry-based tissue imaging, and top-down proteomics are playing a role in the progress of this tool
Genentech Inc. was founded in 1976 following the development of the then-new scientific field of recombinant DNA technology. Over the years, the company has introduced a number of protein-based products and now has more than 30 in development for cancer, immunological diseases, and vascular medicine. In March, Genentech’s stock price soared after it announced preliminary results of a phase III clinical trial for its oncology drug Avastin (bevacizumab). Researchers concluded that patients treated with Avastin and chemotherapy for non-small lung cell cancer survived longer than did patients treated with chemotherapy alone. Avastin was approved last February by the US Food and Drug Administration (FDA) for the treatment of colorectal cancer.
Its track record of producing cutting-edge drugs makes Genentech an important biotechnology company, but the drugs are discovered and developed using many technologies that have been in use for decades, albeit ones that have been updated in various ways. One of these is mass spectrometry (MS), which continues to evolve since the introduction of the first commercial gas chromatograph/mass spectrometer (GC/MS) system in 1966. Over the last decade, MS has become even more sophisticated with the introduction of tools such as high-resolution mass spectrometers, electrospray ionization techniques, and hybrid instruments. Researchers in MS labs across Genentech’s 100-acre campus in South San Francisco continually strive to integrate these tools into their work while also developing new tools.
|Rethinking Mass Spec from the Top Down
Before they start an analysis using mass spectrometry (MS), researchers typically digest proteins with enzymes. While this bottom-up approach is widely used, information is lost when the protein sequence is broken down. That is a shortcoming that a newer approach called top-down proteomics seeks to address.
“We’re intrigued by the possibilities of top-down proteomics, working with large peptides or intact proteins where formerly we would have to digest the protein into small pieces before we could do MS,” says David Arnott, PhD, a senior scientist in the protein chemistry group at Genentech. “This is an area that is becoming increasingly important, not just to us but in modern MS . . . We’re just getting into it.”
Miryam Kadkhodayan, PhD, a scientist in Genentech’s bioanalytical research and development department, says interest in the technology has reached such a level that there were whole sessions dedicated to it at meetings of the American Society for Mass Spectrometry. “Before, nobody could even imagine doing that, partially because the technology wasn’t there. But the MS companies have come up with the technology that makes it available for people.”
One of the places where the top-down approach was pioneered was at Cornell University by Fred McClafferty, PhD, and Neil Kelleher, PhD, who is now an assistant professor of chemistry at the University of Illinois, Urbana-Champaign. Researchers began to recognize that a top-down approach could make the entire sequence available for examination, making it easier to completely characterize the protein and any associated posttranslational modifications.
One system that can be used to perform the top-down approach is the Finnigan LTQ FT mass spectrometer from Thermo Electron Corp., Waltham, Mass. Last year, Bruker Daltonics Inc., Billerica, Mass., introduced TopPro capability on its Apex-Qe Q-q-FTMS system which allows for identification, primary structure determination, and localization of posttranslation modifications directly at the protein level. Kadkhodayan says having such tools available will have a great influence on the field. “Because of the capability of being able to do top-down proteomics, I think we will get a lot better coverage of sequencing in the future, and we will probably get much more sophisticated libraries of proteins.”
“We are always trying to get the most information out of the least amount of sample, so we’re always pushing sensitivity, we’re always pushing throughput, and we’re always pushing the comprehensiveness of our analyses. So we are always trying to do a better job at identifying posttranslational modifications of proteins from limited samples,” says David Arnott, PhD, senior scientist, protein chemistry, in Genentech’s basic research department. Company researchers are also interested in integrating newer technologies such as top-down proteomics and MS-based tissue imaging, or tools based on nanotechnology.
Miryam Kadkhodayan, PhD, a Genentech scientist in bioanalytical research and development, says that when people were doing nano-liquid chromatography in the technology’s early days, the equipment had shortcomings that would result in flow problems and column carryover, problems that have largely been solved over the last decade. In particular, she points to a commercially available nanospray interface that uses a chip for automated nanospray infusion. The NanoMate 100 and ESI Chip System from Advion BioSciences Inc., Ithaca, N.Y., combine automated sample-handling robotics and an array of 100 microfabricated electrospray nozzles that individually ionize a liquid sample before introducing it into a mass spectrometer.
From art to science
“Nanotechnology until recently was an art, because people would have to pull their own nanotips,” says Kadkhodayan, who heads a small group that performs mass spectrometry. “When you pull these glass tips, you can’t be exactly reproducible, so the person doing that had to be very careful and use due diligence as to how they were doing it to generate reproducibility.” With this new system, there is no need to pull tips. The chip was designed using the same technology that Silicon Valley uses to edge silicon chips, so all the nozzles have the exact same dimension as the nozzle next to them. “It makes the work very reproducible, and it’s a robotic system, which makes life easy,” she says.
The scale of the system means researchers can set it to rates of 100 nL/min, thereby making sparing use of samples. Kadkhodayan says 5 µL will last several hours, allowing scientists to perform many different types of experiments as the system is collecting data. She adds that one of its biggest applications is in metabolism and the identification of metabolites. For example, collecting radiolabel fractions requires a few hundred microliters, which with traditional techniques means one or two injections. “Good luck if you make a mistake the first time,” Kadkhodayan says, laughing. Using the nanosystem, however, means that 100 µL would last a very long time.
In a poster presented at last year’s meeting of the American Society for Mass Spectrometry (ASMS), Kadkhodayan described use of the NanoMate 100 for infusion into a mass spectrometer in order to characterize and quantitate intact and reduced recombinant monoclonal antibodies. She concluded that the NanoMate provided the sensitivity and accuracy needed for characterization, and chromatic separation was not required because researchers were able to selectively observe the light or heavy chain by modifying the solvent system. The NanoMate also allowed them to miniaturize sample volume, thus greatly reducing consumption of reagents and antibody samples. In addition, analysis times were reduced to one minute per sample, down from seven to 30 minutes in the past, and no carryover was observed. In 2003, Advion and Thermo Electron Corp., Waltham, Mass., announced the launch of what they said was the world’s first fully automated chip-based nanoelectrospray MS system, the NanoMate-LCQ Deca XP Plus. The system combines the NanoMate 100 and the ESI Chip with Thermo’s Finnigan LCQ Deca XP Plus Ion Trap mass spectrometer.
Adopting tools that automate processes related to MS has increased the efficiency of Kadkhodayan’s lab, which comprises her and two research assistants. “Another piece of equipment we use for plasma samples is an online extractor. When we have small molecules that we need to characterize, instead of doing a traditional [precipitation], we just dilute the plasma sample and put it in the autosampler and press a button. The system is programmed to take a sample, extract it on a solid-phase extractor, and divert it to the LC/MS.” These tools allow her small team to handle the workload of five people. “When people find out how many people report to me and how many projects we’re on, they just shake their heads.”
For mass analysis tools Kadkhodayan’s lab uses triple quad MS and hybrid quadrupole time-of-flight (TOF) MS. When it comes to clean up and/or chromatography technologies, they use, among other things, online and off-line plasma cleanup techniques using HPLC, such as online solid-phase clean up, online affinity chromatography, and reverse-phase chromatography. For sample preparation, they use various enzymatic reactions, solvent exchange columns, and various trap columns.
Another part of the MS equation at Genentech are groups like protein chemistry, which is part of the company’s basic research department. Arnott and Jennie Lill, PhD, are both members of the group, which provides molecular weight verification of intact proteins, peptide mapping, tandem MS protein verification, and amino acid analysis. “Identifying protein-protein interactions is a large part of what we do, meaning amino precipitation of a big protein and identification of the other proteins that bind to it. We also do posttranslational modification, identification, and mapping,” says Arnott.
|Tissue Imaging Determines Drug Metabolism, Distribution
Tools for spectrometry are in a constant state of evolution, and many in the field believe that MS-based tissue imaging is one of the more interesting and promising technologies now emerging. This tissue-imaging approach, which was developed by Richard Caprioli, PhD, director of the Mass Spectrometry and Proteomics Research Center at the Vanderbilt University School of Medicine, Nashville, Tenn., can be used to examine drug metabolism and drug distribution in tissue to determine if a drug is effectively reaching its target.
Caprioli teamed with Applied Biosystems, Foster City, Calif., and last year the company released new MALDI imaging software and an extremely fast high-repetition laser for use with its Qstar XL Hybrid LC/MS/MS System. The new tools generate 2D and 3D images of low-molecular-weight drug compounds in tissue samples and display the spatial distribution of drugs, thus helping determine if the drug is reaching its intended target or simply accumulating in tissue. Its laser fires at speeds up to 30 times faster than existing MALDI QTOF-based lasers, so it accelerates the data collection and interpretation process.
“I think the reason that this work is so significant is that now, if you’re looking for small molecules in tissue, you can just directly look at them rather than having to homogenize the tissue and look at it,” says Miryam Kadkhodayan, PhD, a scientist in Genentech’s bioanalytical research and development department. “I think it’s going to become routine.” At the time of the system’s release, Caprioli said in a statement that it had a number of potential applications in drug discovery and development: “This research method could eventually lead to the recommendation of more appropriately prescribed treatment regimens, the ability to determine a drug’s efficacy, as well as lead to the avoidance of unnecessary side effects from overly aggressive treatment regimens.”
Lill says the lab’s workhorses are two ion trap mass spectrometers and a linear ion trap, which are used primarily for identifying unknown proteins, confirming sequences of known proteins, and looking at posttranslational modifications. They also have a quadrupole TOF instrument to measure the molecular weight of intact proteins, another quadrupole TOF with a matrix-assisted laser desorption/ionization (MALDI-TOF) source in use most of the time, as well as a regular MALDI-TOF instrument. The lab also has an open-source mass spectrometer to train researchers to perform full MS and protein sequence database-based analyses. In the coming months, they will receive a triple-quad mass spectrometer, to be used for performing multiple reaction monitoring for quantitation of small biomolecules, and a hybridized Fourier transform ion cyclotron resonance linear ion-trap instrument. They believe this last tool will be capable of extremely good mass accuracy, sensitivity and resolution, and will also become a high-throughput instrument.
Lill says the department is unique because it is continuously being assigned new projects for which they may not already have methods in place. “We have to do method development to accommodate each individual project, so we don’t work on one specific antibody or one specific protein,” she says. Arnott finds the variety enjoyable. “There’s always something new. It also means that we have to have people in the lab with a whole variety of backgrounds and skills. We have people who are experts in chromatography or peptide separations, we have somebody in the lab who does tissue cultures and can work with cells. That’s important, because sample preparation is really the key to the success of a lot of experiments. MS is only the end of the line for the technology. There are a lot of other techniques and technologies that we buy.”
Arnott says that one technology which has become increasingly important since first emerging five or six years ago is quantitative MS. “The first attempts to use this were for quantitating large molecules like proteins and peptides. There are also the ICAT (Isotope-Coded Affinity Tags) reagents and other isotopic labeling strategies. Basically, there’s just an increasing interest from the time these techniques first emerged to the present.” Lill says they have been evaluating their amino acid analysis procedures in order to make them more high throughput. “I’ve been doing different vendor evaluations, that kind of thing. Our throughput needs to increase in most of the areas that we work on, so that’s always a challenge. It’s been a big project in the lab recently.” The researchers are also investigating the use of high-mass accuracy instruments so they can attain more accurate protein and peptide molecular weights more quickly.
Viswanatham Katta, PhD, is a senior scientist in early-stage analytical development at Genentech. His group uses various MS tools to help identify and define molecules. “We come at it from a slightly different angle because early on, materials are available in very small amounts, so we have to use analytical techniques to define things.” His group provides information on molecule and cell conditions that allow other groups to develop formulation and purification processes. Like Kadkhodayan, Katta has been using the NanoMate 100 because it provides rapid characterization that enables the analysis of a number of samples very quickly.
Katta’s lab uses a triple quadrupole mass spectrometer equipped with turbo ion spray for measuring intact molecular masses of recombinant antibodies. For peptide mapping, one of the tools that they use extensively is a linear ion-trap mass spectrometer equipped with an electrospray ion source. They also use linear MALDI-TOF MS systems for glycan profiling and intact protein analysis, and reflector MALDI-TOF systems for proteolytic digest. His lab is also investigating the use of MALDI-TOF in proteomic applications to identify product impurities, as well as a one-step process to map proteins.
The next link in the chain is Victor Ling, PhD, a scientist in late-stage early development. He manages one of his group’s MS labs, and they provide analytical data to a variety of areas, including post-development, purification processes, formulation development, and quality control. “We define the structure and the physicochemical characteristics of materials made by our process. We look at the comparability of materials resulting from process changes and different types of manufacture.”
Ling’s group also supports efforts to identify what factors affect critical structural characteristics of the product. This allows other groups to work on things like purification and fermentation with a better understanding of what critical parameters to manipulate. “We investigate discrepancies and oddities, which is for the most part possibly our most interesting work . . . It takes some creative thinking and digging. It is detective work, so it’s fun.” Ling says the most important thing that his section does is deliver the characterization section for the drug license application.
Investigating new techniques
In addition to using available technologies and tools, many of the MS groups at Genentech are investigating new techniques. “Part of our mission is technology development, finding new applications for MS or sequencing that can be applied to important biological questions here in research,” says Arnott.
Ling is working on applying methods from MS and proteomics to create a bioassay that can act as a substitute for more typical bioassays such as enzyme-linked immunosorbent assay (ELISA). “For instance, we can use this to measure the level of antibody drugs in serum, for pharmacokinetic studies, for situations where the ELISAs are not giving true results,” he says. They are investigating orthogonal methods using MS to substitute for other assays and are also developing software that will automatically and objectively compare peptide mass.
Kadkhodayan says one of the advantages of being a scientist at Genentech is the company’s collaborative environment, something which has allowed her to evolve scientifically and develop new tools. When she first came to Genentech, she was part of a group that was supposed to develop liquid chromatography/MS assays for small molecules. That evolved into developing assays to validate in a good laboratory practices (GLP) setting and transferring it to a contract research organization, so she had to learn GLP. Because of her varied experience, Kadkhodayan was assigned to a project with people in research developing a method for examining and quantitating intact antibodies or proteins in plasma using MS. That work, which she says she cannot discuss in more detail, is the subject of two patent applications.
Kadkhodayan believes the technology covered by the patent applications will allow researchers to use MS for many tasks now done with ELISA. “What makes it interesting is that a lot of people doing quantitation of proteins with MS are either digesting the antibody or protein and looking at a specific peptide, or they are labeling it with ICAT technology. I’m not using any of those. I’m just looking at a bare-bones protein. What that allows you to do is see some of the changes that could happen to the intact protein a lot easier, like posttranslations or modifications and things like that.”
As for the future, she believes nano-technology tools will improve as competition drives their development. “There are so many people who are working on it and so much
good work that I’ve seen at ASMS meetings, that I think the technology is going to become very routine, which is interesting, because the idea of lab on a chip was something that was imagined years ago and now it’s becoming somewhat of a reality.” Kadkhodayan and others at Genentech note there are two other emerging trends that will play vital roles in the future of MS: top-down proteomics and MS-based tissue imaging (see related stories on pages 29 and 31).
Lill says her group is investigating the use of high-mass accuracy mass spectrometers, which allow researchers to attain more accurate protein and peptide molecular weights more quickly. Ling believes what is needed are more robust, simpler mass spectrometers that can be put online to monitor production processes on an automatic basis, and also software to objectively evaluate data and provide a picture of product quality at every step of the process. “Hopefully, we can get this information in time for us to make corrections so that we don’t end up looking at the final product and having to throw out a whole 12,000-liter run.”
Katta is looking forward to developments in informatics in the form of data-analysis tools and presentation. While these tools have improved over the last few years, they require more advanced software. Ling agrees and says his group is working on software that will automatically generate data, make comparisons, and evaluate the LC/MS type of peptide mass. “We’ve spent an awful lot of time going through data files, raw data, manually. Aside from the time, it also tends to be subjective, so it would really be good to have quick and objective evaluations, particularly for the online, real-time type of applications.”
Filed Under: Drug Discovery