Like many large pharmaceutical and biotechnology companies, Genentech has made a point of delving into proteomics as a new approach to discovering potential drug targets and therapeutic proteins. The South San Francisco-based company has developed platforms for implementing differential proteomics experiments and mapping protein interactions, using primarily off-the-shelf multi-dimensional chromatography and mass spectrometry.
But classifying Genentechs strategy for applying proteomics as run-of-the-mill doesnt tell the whole story. In addition to its efforts in drug discovery, the 25-year old biopharmaceutical company has also taken steps to exploit proteomics techniques in later stages of the drug discovery pipeline, namely, the identification of potential impurities in proteins derived from cultured bacterial or mammalian cells.
Characterizing impurities is obviously important for a manufacturer of recombinant proteins. Kathleen Champion, an analytical chemist at Genentech, leads a group at the company that performs this service on therapeutic proteins in pre-clinical development using 2D gel electrophoresis and mass spectrometry.
Champion claims her group is the only one to apply standard proteomics technology to this downstream stage of the path of drug development, and has published a paper in the September issue of the journal Proteomics detailing her groups methods.
The 2D gel platform that Champions group uses in their analysis of recombinant proteins is not automated or particularly unique, she said, and was assembled from commercially available components. However, the significance is in its application.
As far as I know we are the only group doing this type of work, where we are looking at host-cell derived proteins and evaluating them, Champion said. We need to know what enters our purification processes and we need to monitor the removal of impurities.
Proteomics in Discovery
In addition to these more specialized applications of proteomics technology, another group of scientists at Genentech has developed proteomics technologies for more conventional drug discovery-related purposes. Led by John Stults, a senior scientist in protein chemistry, Genentech researchers have developed experimental protocols for verifying the results of DNA microarray experiments and identifying proteins involved in disease-related biological pathways.
But rather than use 2D gel electrophoresis, Stults group has focused on chromatography and advanced mass spectrometry techniques for its differential protein expression and protein-protein interaction studies. We did a lot of 2D gels a couple years ago, but our main mission is in target discovery, and we didnt find any results that found new targets, he said.
Instead, Stults group uses primarily 1D gels and online chromatography techniques for separating proteins, and a stable isotope tagging method coupled with mass spectrometry for measuring the relative expression levels of proteins derived from different samples.
The stable isotope tagging method developed at Genentech comes in handy for his groups work to verify that the relative levels of mRNA expression are accurately reflected in protein expression, Stults said. Typically, these kinds of validation experiments require researchers to generate antibodies to specific proteins, a process that can take a few weeks to several months. But using his stable isotope tagging method and mass spectrometry, Stults said the whole process can take three days.
Genentechs method for tagging and measuring the relative expression levels of proteins taken from separate samples is similar to the ICAT reagent technology, but uses a different molecule to tag the proteins. Instead of eight deuterium atoms, the tag developed by Stults team has three, and is attached to the protein differently. It makes the way we do the experiment just a little easier, Stults said.
To study protein-protein interactions, Stults group has developed a method for pulling out particular protein complexes using co-immunoprecipitation, a technique for obtaining proteins that bind to a tagged bait molecule that can be easily isolated from a sample. After separating the proteins in the complex using 1D gels or online chromatography, Genentech researchers then attempt to identify the components with a MALDI-TOF or tandem mass spectrometer, he said.
If a protein sample is relatively pure, Stults said his group often first attempts to identify the constituent peptides using its Applied Biosystems MALDI-TOF spectrometers. For more complex samples, his group uses tandem mass spectrometers such as Thermo Finnigan ion trap and Micromass QTOF instruments.
Moving Towards Chips
Despite the advances in mass spectrometry technology, the improvements represent only the initial steps in the development of techniques for analyzing the low-abundance proteins often associated with disease. We have a difficult time seeing the low-level proteins that are really at the control points that are good drug targets, Stults said. We can see the abundant proteins in cells, we can find lots of proteins that change, but few or none of them are proteins that are either the direct cause of the disease or at control points where a drug would be an effective interaction.
One way to tackle these issues is to reduce the complexity of the samples as they are prepared from tissue or cultured cells, Stults said. As his group begins to increasingly focus on clinical samples derived from actual patients, Stults is looking to develop his groups expertise in methods for isolating specific cell populations, such as laser capture microdissection. Other companies doing proteomics discovery, such as Celera Genomics, have made advances in similar techniques, he said, but Genentech hasnt decided whether to license these types of technology from other parties or develop them in-house.
At the same time, Genentech is also interested in other technologies, such as protein microarrays, that may offer other avenues for increasing the sensitivity and specificity of protein analysis techniques. Although the company is not devoting any resources to developing these techniques internally, Stults said that he has spoken with other companies that have made strides in this area, such as Large Scale Biology and Zyomyx.
Were looking a lot at protein arrays, he said. For specificity, ease of doing small amounts of lots of samples at high throughput, and especially for sensitivity, protein arrays have a lot potential.