Can you tell us why Glaucus failed?I think we were mostly a victim of last-minutebad luck in a deflated biotech market.Thingswere going exceptionally well, both scientificallyand with the financing. We had raisedUS$19.5m in a US$22m round of financing,and literally at closing, a German institutionalbanker pulled out, much to the surprise of ourfirst-round lead investor, Atlas Ventures (whichwas reinvesting) and also much to the surpriseof the new lead investors who had completeddue diligence and legals. I think maybe they hadseen Commerzbank and Deutschebank inSeptember [2002] cut back their investmentsector and lay off 20% of people, and thiscaught everyone unaware. Of course, due to alengthy closing of six months post-Term Sheet,we had been fighting to stay alive. Everyonewas still confident though, and the science hadbeen going brilliantly. I think one learnt a lot.Thats life though; if you live in a dynamic laneyou have to go with the flow of things.

Do you think it is more difficult to get venturecapitalists to invest in biotech right now?We have never had any trouble attracting theinterest of investors, and up until Glaucus failedI would have said there was no real problemwith biotech funding. However, there iscertainly not a dearth of money; I had seen a

group in California with a similar level offunding as Glaucus that had exactly the samedifficulties, namely a six-month Closing in thehome of fast capital.The money is not givenaway freely, and you work very hard for it, butit is still there to be had for good biotech.

So, what next for you?At the moment I am assessing my options, butthe Human Proteome Project has hardly gotoff the ground, if at all. Proteomics wont goaway for the next ten years, and I think its stilla very good sector to be in. So, hopefully onehas some expertise of relevance there!

How is the Human Proteome Organization(HUPO) progressing and what is your rolewithin the organization?Up until recently I was Vice President ofBusiness Development, but businessdevelopment seems to have been put on theback foot for the moment. So, I am currently acouncil member, and we have been focusing on

our five objective areas, and pushing forindustry involvement. Personally I think weshould be courting industry more actively, butthat is for the council as a collective to decide.I think generally HUPO has been progressingwell, and I am elated with the progress that hasbeen made. It was a lot of work getting HUPOinto existence I spent about six-months ofmy life trying to get a global consensus tostart! I thought that after almost a decade ofwilderness selling (no one listening to theprophets in the desert) and the human genomenearing completion, that everyone would beeager to start the Human Proteome Project.However, it was far from a unanimous decision.So one had to work to get people to give itthe go ahead. Now, I think things have matureda great deal and we have internationalinitiatives with clearly stated objectives, and sonow we must start to address those objectives.I think that is the challenge for HUPO in thenext 12 years: can we deliver someoutcomes, or even start to deliver? That iswhat people are criticizing us collectively for.So we have cleaned up our organization,having been in existence for over a year now,and we have groups keen to participate bothin industry and academia.

Do you think that biotech companies doingprotein arrays could suffer from a lack ofdifferentiation?I am a little surprised to see the proteinbiochip sector not taking off as quickly as manyhave predicted. It will be big one day, but, aspeople in the industry said last year, its notnecessarily going to be big in 2003. However,based on what weve learned from traditionalproteomics mass spectrometry (MS) andseparation-science based approaches you seeso little of the proteome and the technology isnot highly reproducible. So, there is only oneway forward and t hat is to use the lessonswe have learned from genomics. Parallelized,miniaturized, automated assays protein chips that is the route. So, are there enoughdifferentiators in the marketplace? Thatquestion is difficult to answer; at present noone has differentiated themselves by makingany significant sales. I think the only companiesthat do are Ciphergen (http://www.ciphergen.com) and Biacore (http://www.biacore.com), and in both cases they haventgotten to double-figures in terms of lookingat samples in parallel.

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UPDATE

INTERVIEW

Ian Humphery-Smith is Professor of Pharmaceutical Proteomics at Utrecht University,The Netherlands, and until recently was a Managing Director and Chief Scientific Officerof Glaucus Proteomics. After a PhD in Parasitology at the University of Queensland, hestudied virology and bacteriology in France as a post-doc, before returning to Australiaas Course-Coordinator in Medical Microbiology and Immunology at the University ofSydney. During this time, Humphery-Smith took up the posts of Executive Director ofAustralias second largest DNA sequencing facility and Director of the Center forProteomic Research and Gene-Product Mapping, which later became the worlds firstcenter to focus on studying the proteome. Humphery-Smith has devoted ten years ofresearch to analyzing proteins in health and disease, and it was his work that originallycoined the term proteomics. He was the first to publish the most complete analysis ofan entire proteome in 2000, that of the bacterium Mycoplasma genitalium. He currentlyserves as a council member of the Human Proteome Organization (HUPO) and hasbeen a prime mover in efforts to have the Human Proteome Project become a formally-ratified international initiative to follow-on from the Human Genome Project.

I am a little surprised to see theprotein biochip sector not taking offas quickly as many have predicted.

Ian Humphery-Smith oncurrent challenges inproteomics

Interview by Joanna Owens

Ian Humphery-Smith, Professor, PharmaceuticalProteomics, University of Utrecht

Up until Glaucus failed I wouldhave said there was no realproblem with biotech funding.

If you had your time at Glaucus again,would you do things differently?I should obviously be saying yes! I think wewere caught by the downturn in global biotechgenerally. When Atlas Ventures first invested inus, we were going to do the Human ProteomeProject. When there was a downturn [in theglobal biotech market], we refocused ourbusiness model to lead optimization addingvalue to the higher end of the value chain indrug discovery. We put that into practice withproteinprotein, antibodyprotein and small-moleculeprotein arrays, and it was all workingvery well. However, maybe at that time weshould have pulled back even more andfocused only on product. Unfortunately, thediscipline demands significant supportinginfrastructure. We had produced Beta chipsand had two successful assessment-of-productsby different biotech companies.Theseconsisted of production-ready biochipscontaining six replicates of 361 or 12 replicatesof 131 highly purified human recombinantproteins. But in the end, although we refocusedour business model, I think we should havebeen much more ruthless in the way we did it.

Can you give any advice to others abouthow to have a successful business model forgenomics and proteomics-based drugs?The sector we were in is highly CAPEX(Capital Expenditure) intensive, and you cannotgo and play in that sector with low-end capital.Genomics sciences are not a ten centendeavour! So even if you are well-funded as astart-up, you are still working on a tightbusiness model at every turn. If I were to do itagain, I would be wishing to do it withadequate pre-allocated funding.What one needs to ask next is whetherprojects that demand large amounts of capitalexpenditure will be done by the private orpublic sector, and I dont know the answer tothat. I think that HUPO has a goldenopportunity to drive the Human ProteomeProject forward, but as we saw at HUPOs firstWorld Congress, although the pharmaceuticalcompanies are happy to be involved somewhatphilanthropically, their business is to build newdrugs and have a commercial, patent-protectedposition. Public, philanthropic endeavours andbusiness endeavours are not highly compatible;substantial funding has to be backed up bypotential for substantial profits. But we spend alot of money on battleships and warplanes, ifwe had the money for one fighter jet then a lot

more would get done Im sure!I think we will see an interesting evolution overthe next 23 years. Six years ago, the leadinggenomics labs were academic, and thenindustry took over. In proteomics, there arestill no major academic groups leading theworld forward in the Human ProteomeProject. Instead, companies such as Geneprot(http://www.geneprot.com), Large Scale Biology(http://www.lsbc.com), MDS Proteomics(http://www.mdsp.com), and so on, are takingthe lead.

How do you think biotech companies thatidentify genes or proteins should go aboutprotecting their discoveries andcommercializing them?One cannot replace the patent system.No matter how much philanthropic good will there is, that system is still the status quo.I remember shortly after breast cancer genes were discovered, there was a lot of uproar about Myriad Genetics(http://www.myriad.com) owning BRCA1 andBRCA2. Eventually the same discoveries wouldhave been made elsewhere, but if Myriad hadntspent US$200m, the genes would not havebeen discovered as quickly and womens liveswould not have started to be saved. So there isgood and bad on both sides of the coin. Aspeople discussed at the recent HUPO WorldCongress, the pharma industry have made itclear that even if someone gave them a newdrug tomorrow, if it was generic, no one wouldwaste the US$500m to take it to market,because they cannot protect it. We cannot getaway from that dilemma unfortunately.

Will companies that are working onantibodies and proteins rather than genes find their discoveries easier tocommercialize?I would say yes. It is certainly the action endof the genome. What is missing at present,however, is a long list of recombinant proteins,and a long list of highly specific high-affinityantibodies. Some people have estimated that asmuch as 80% of off-catalogue antibodies arecross-reactive.There is a lot of work thatneeds to be done on content by that I meanboth recombinant proteins to generate theantibodies and the antibodies themselves. Onceyou have those antibodies or other affinityligands there are many, many applications in

diagnostics, therapeutics and the affinityenrichment of cellular soups to studymolecules of interest.

What made you decide to leave a career inAustralia and move to Holland?It was very clear-cut: first, we were too faraway in Australia to be genomically relevanton the globe.You had to be in the Northernhemisphere, and I had a lot of trouble going tothe USA and wanting to do proteomics whengenomics was very much the order of the day.So the question was, where in Europe? One ofthe major advantages of Holland was theaccess to very cost-effective supercomputinginfrastructure. We implemented a businessmodel based on accessing existinginfrastructure and thereby saved a fortune onnon-recoverable CAPEX. As a result, webecame part of Europes largest singlesupercomputing infrastructure. We knew thatwe had to be strong in informatics to succeedin genomics sciences. Holland was a good cost-effective solution for this.

How is the climate for genomics andproteomics research in Australia now,compared with when you left?It is improving all the time, but we still sufferfrom the tyranny of distance. It was the samewhen settlers went out there 200 years ago,and before I left I was spending one week outof six not on the ground! Its hard to sign adeal with someone without face-to-facecontact on 610 ocasions, and that lack ofclient interaction is still the problem forAustralians who are in this business; the clientsare all offshore, and we only have a smalldomestic market. Its a great place to live and agreat place to work, and if youre in mining orprimary industry its a different scenario.

How did you balance an academic careerat Utrecht University and a biotech careerat Glaucus?With difficulty, the two are not highlycompatible. I have a part-time association withthe university because biotech is an 8090 hourweek most weeks.There are always investorsneeding extra information or other urgentmatters and you are working flat-out all thetime. At Utrecht we learnt from Australia,

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Genomics sciences are not a tencent endeavour!

Public, philanthropic endeavoursand business endeavours are nothighly compatible.

I had a lot of trouble going to theUSA and wanting to do proteomicswhen genomics was very much theorder of the day.

and put in place a lengthy collaborativeagreement between the University of Utrechtand Glaucus on day one.The work in theacademic sector was subsidized by Glaucus,with much of the initial Glaucus work beingdone in the University. So we made Glaucus acost-effective business model by interactingwith academia. But unless those boundariesare set in legal contracts there is nothing butpotential problems.

What role do protein arrays have inexpression proteomics?You need highly reproducible assays. After twodecades of dealing with 2D gels, it would benice to go back to gel number two and find thesame spot in the same position. With arrayswe can put as many elements on a chip as wecan build or can have up to 12 replicates onthe same array, so with 360 different elementsyou can do a statistically valid experimentalmost on a single chip. Whereas if you useisotope-coded affinity tagging (ICAT)technologies or other mass adducts, it takesso much work to do one experiment that theyare not easily repeated. It would be nice to seepeople doing differential protein display ordifferential screening but on 2000 healthypeople and 1000 cancer patients, but that isbeyond the realm of current proteomicscapabilities. I think that proteomics throughreproducible protein arrays is the only waythat we will get good statistics to follow uppotentially useful leads.

What are the real limitations of proteinarrays at present?Content, content, content! No proteins andno antibodies. We have the building blocks interms of knowledge from the human genomeand we now have the mouse genome, so weare in a position where we know which genesto look for. By combining this genomicknowledge with what we have learnt fromtraditional proteomics over the past decade,we can add a lot of value. We can validatetargets from genomics, do pharmacogenomicsand patient cohorting all by differentialscreening using parallel analysis. With MS andother traditional proteomics approaches, thevariance between analyses can often be smallerthan the variance between two replicatesamples. So, the main advantage of arrays overMS is obviously their parallel nature.

How do you envisage the integration oftraditional proteomics technologies witharrays benefiting drug discovery?First, parallel array-based proteomics willprovide statistical confidence in which proteinsto follow-up by using more traditional

approaches. Collectively as a field over the pastdecade, we have learnt to process samplesmore rapidly in an MS environment. What weneed then is proteomic and protein coverage.The second is looking at the post-translationalmodifications on those proteins of interest, butin health and disease. We can already deliverthis, but the question is, where in the humanproteome do I start to look? There is low-hanging fruit and we will find it, hopefully fromboth academic and commercial ventures, butwe need to go further than this low-hangingfruit. In human cells approximately 80% ofproteins are low abundance, so we cannotdetect them using MS. However, if you addantibodies or other affinity ligands to enrichthe signal, you can then get strong signals fromyour MS experiment and get beautiful results.

How long do you think it will be beforeantibody arrays translate from being aresearch tool into a valuable diagnostic andscreening tool?The problem is the lack of infrastructure forusing antibody arrays, and the need to screenthem for cross-reactivity. Few people stress theimportance of a lack of cross-reactivityenough: if the antibodies are cross-reactivethey are useless. How long it will take dependson the resources given to the sector. I thinkinvestors still see the antibody array sector asattractive, but we dont know how muchmoney will go to public and private projects. Iam personally very keen to see the HumanProteome Project take on the challenge ofdeveloping affinity ligands for each and everygene in the human body, which will enable usto follow the output of that gene by a commondenominator strategy. For me, that project hasa clear-cut beginning and an end, in the sameway that the Human Genome Project did. Itcan also be attractive to both the public andprivate sectors depending upon resourcesavailable and associated business models.Once we have those affinity ligands, everythingis easier, from protein arrays to MS. Groupssuch as Somalogic (http://www.somalogic.com),more traditional antibody companies andphage display companies, are saying they areinterested in doing this.The biggest problem inproteomics is the lack of a PCR equivalent forproteins, and the closest thing we have isaffinity ligands to enrich for a protein.

What do you think are the likely lowerlimits of detection for array analysis of lowabundance proteins in tissue and body fluids?

Analysts often ask this question, andunfortunately there is not a simple answer.There are three things that make this complex:the concentration, the time, and the affinity ofthe ligand for its target. We do know thatsome housekeeping genes exist at very lowlevels. For example, the lac repressor inEscherichia coli works at four molecules per celland exists at about 1020 molecules per cell,so an array would have to be able to detectgenes at that concentration. I have discussed alot of these issues in an upcoming review [1].

Is the future bright for protein arrays asdiagnostic tools?Diagnostics are not always much-loved by theventure capital industry, because once youhave them in place then they are good moneyearners, but you have to register themindividually in each country. So there are hugecosts involved in having a new diagnosticapproved. If that can be reduced by doing 200or 300 tests at once, then it might be worthgoing ahead. But if an established company has300 diagnostic tests already that are workingwell, where is the economic incentive to makethem cheaper and in parallel? The registrationof diagnostics makes that sector difficult topenetrate for small biotechs with a newdiagnostic tool.

In diagnostics, if one protein is good andtwo proteins are better, how many proteinswill be best?Again, it comes back to p-values, if youcombine one protein that is highly specific withtwo or three proteins that are poorly specific,together they give a fantastic statisticalsignificance. Combinatorial sieving is whatthey call this in computer science. It worksbecause of the fantastic statistical confidenceyou can get by just combining really fuzzynumbers in the beginning. So, even if you havefour cross-reactive antibodies, if you bringthem together they can start to do good work.

What do you think of the approach ofanalyzing very small samples (e.g. 25l) andploughing through with expression analysis?Is the issue of going to lower and lowerdetection limits important?Here we have an antagonism. Nanotechnologyis where we would like to take arrays, but ifyou are looking for low abundance analytes,they do not exist in nanolitres. So, the dilemmathen is, how big a volume do I have to have tofind these low abundance analytes? The analytevolume needs to be quite large unless youenrich from a larger volume first using anaffinity ligand.

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INTERVIEW

I think investors still see theantibody array sector as attractive.

How do you think proteinproteinmicroarrays will fit into functional proteinanalysis, the limitation at the momentbeing that they only look at a singleprotein-protein interaction?That I would contest. What we learnt fromthe human genome is that there are very fewgenes, but they work in permutations andcombinations, and as soon as you usepermutations and combinations its known asa non-polynomially complete problem, whichmeans the interactions are exponentiallyexpanding. One protein is not doing one thingor interacting with one protein, it is interactingwith many proteins.

There is a tendency in biology to say, Whichare the partners for this protein? a yes/noanswer. No chemist would ever consider thatquestion it depends on the affinity of theinteraction. Biology does not do just one thingwith one gene, and within a cellular soup, eachbiomolecule interacts with every otherbiomolecule to a greater or lesser extent. Forexample, non-reversible signal transduction isvery dangerous in biological systems. In biology,you need to turn things on and off, so it isoften the subtle, lower-affinity interactions thatcan do vitally important work. If a protein needsto bind irreversibly, such as a ligand at a cellreceptor, then that might be high affinity, butlower down the signaling cascade proteins needto turn pathways on and off thousands of timesin a second.That cannot be done with highaffinity ligands, so this yes/no simplicity does notexist. I dont like that way of thinking in biology.

What do you see as an emerging techniqueabove and beyond MS and arrays thatmight revolutionize proteomics in ten years?I think that for the next ten years, the need forcontent will dominate. So, any technique thatenables us to rapidly and cost-effectively buildrecombinant proteins and pull pure proteinsout at the other end could revolutionize thefield.Genomics has taught us that any technologythat can miniaturize, parallelize, and automatewill be the future.Thus, provided theseattributes are combined, I remain open on theexact nature of the technological platform.

What do you think of antibody-like binders,such as those that Phylos and Pieris aredeveloping?Fantastic as long as they provide high affinityand lack of cross-reactivity.There is a list thatincludes polyclonals, monoclonals, phage-derived, aptamers, affibodies, antibody mimics,anticalins and any peptide or combinatorialcompound that will give highly specific targetrecognition. So they are all great, but thequestion is, can someone start to produce

them cost and time effectively on a genomicscale? That was what we were setting out todo at Glaucus. It is one of the five initiatives ofHUPO to look at building affinity ligands to allthe genes in the human genome, but it hasbeen the slowest HUPO initiative to get off theground, I think this is because of the interplaybetween the public and private sector. Large-scale public sector funding would make it moreattractive for everyone to participate, but thenit obviously kills the business model.

Who do you think is doing the mostinnovative work in proteomics at themoment?Right now, Richard Smith at Pacific NorthwestNational Laboratory (http://www.pnl.gov), whois developing Fourier transform ion cyclotronresonance MS and has just recently detailed asubstantial portion of the Deinococcusradiodurans proteome [2].The technology isnot easily accessible because it involves a lot ofcomputing, and it is neither parallel nor highlyreproducible as yet, but we are starting to see,at least in microbes, a relatively quick way toanalyze a whole proteome. I am veryimpressed with what Smith has been able toachieve with a whole proteome. Prior to this,the most complete proteome was our workpublished in 2000, but that was a very smallbacterium [3]. I think this is really great work.

What array technologies in developmentlook promising to you?I am very impressed with Molecular Staging(http://www.molecularstaging.com) at present.They are getting over this problem of cross-reactivity by separating cross-reactiveantibodies into different segments, and theyhave developed rolling circle amplificationtechnology (RCAT), which provides enoughsensitivity to detect low abundance analytes.However, for each analyte we still needantibodies. Molecular Stagings technology is

robust and scaleable, although as yet the affinityligands are not available for scaling-up thetechnology. However, on specific diseases ofinterest I think they will show the marketplacethat things can work well. I am very impressedwith their deliverables at this point in time.

In your view, how could the drug discoveryindustry best use proteomics to break thetarget validation bottleneck?Target validation is good, but for now I wouldbe concentrating on lead optimization. Wehave had a lot of potential targets fromgenomics. Proteomics as a whole, no matterwhat technology you use, has great potentialto validate those targets before you take themfurther down the drug discovery pathway. Buthow specific is the new drug? If you can laydown a substantial slab of the proteome onchips and expose them to your lead compoundseries, then during lead optimization you cantake the compound that is most specific foryour target. We have never had such tools,and so for a short-term revolution in new andbetter drugs from the Human GenomeProject, you need improvements in leadoptimization, in terms of target specificity andreduced toxicity prior to entering the clinic.This I believe is the new paradigm in drugdevelopment that registration authoritiesglobally are seeking. It is also the area mostlikely to be impacted by proteomics as a resultof a detailed knowledge of the human genome.

What do you think is the greatestachievement of your career, to date, andwhat is your biggest ambition?I have been happy to participate in the comingof age of proteomics. Now, I am still very keento have a role in improving healthcare andhealthcare delivery through proteomics.

References1 Humphery-Smith, I. Protein biochips and

array-based proteomics. In Protein Arrays,Biochips and Proteomics The Next Phase ofGenomic Discovery (Albala, J.S. andHumphery-Smith, I., eds) Marcel Dekker (in press)

2 Lipton, M.S. et al. (2002) Global analysis ofDeinococcus radiodurans proteome by usingaccurate mass tags. Proc. Natl. Acad. Sci. U. S. A. 99, 1104911054

3 Wasinger, V.C. et al. (2000) The proteome ofMycoplasma genitalium. Chaps-solublecomponent. Eur. J. Biochem. 267, 15711582

Ian Humphery-SmithPharmaceutical Proteomics

University of UtrechtSorbonnelaan16

UtrechtThe Netherlands

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UPDATE

INTERVIEW

There is a tendency in biology tosay,Which are the partners forthis protein? a yes/no answer.No chemist would ever considerthat question.

Genomics has taught us that anytechnology that can miniaturize,parallelize, and automate will bethe future.