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  • EDITORIAL

    UCL Biochemical Engineering

    When Jack Drummond, the earliest UCL Professor of Bio-chemistry, isolated Vitamin A in the 1930s, he needed toprocess large quantities of fish liver oils and later, wheatgerm. Drummond, whose chair was funded by the Rocke-feller Foundation, was helped by Maxwell Donald, a younglecturer in Chemical Engineering. So began the linkage ofdepartments that created UCL Biochemical Engineering.Following war service, Donald, now Head of Department,worked with Ernest Baldwin, Head of Biochemistry, to es-tablish a joint Diploma, later a Masters course in Biochemi-cal Engineering and put forward a visionary biochemist,Eric Crook, as first Professor of Biochemical Engineering.The less visionary University of London, which then regu-lated these matters, blocked the appointment. Not to be putoff, Donald and Crook set about establishing a journal. Theydiscovered that Elmer Gaden was pursuing a similar goalagainst a background of rapid recognition in the USA of thesignificance of the field. The Journal of Biochemical andMicrobiological Technology and Engineering, now Bio-technology and Bioengineering, was formed under theirjoint editorship. Meanwhile, a young biochemist, MalcolmLilly, had finished a PhD in microbiology with Pat Clarke in1962 in the UCL Biochemistry Department and was expect-ing to work under Eric Crook. When Crook was obliged totake a Biochemistry Chair elsewhere in London, the futurehung in the balance. However, a joint grant from the White-hall Foundation of New York to Eric Crook and MalcolmLilly, now in the Chemical Engineering Department, savedthe day. It was for a study of enzymes on solid supports.Malcolm Lilly created a broad program of biochemical en-gineering research while an industrial biochemist, FifeWebb, built the Diploma course with Pat Clarkes biologi-cal input and wrote one of the fields first monographs(Webb, 1964). Within a short space of time, Malcolm Lillyhad initiated research on separation of nucleotides (Lilly,1965), single-cell protein production from hydrocarbons(Ertola et al., 1965), biological fuel cells (Gray Young et al.,1966), continuous culture (Baidya et al., 1967), and mam-malian cell culture (Self et al., 1968).

    For reasons of space as well as the context of MalcolmLillys death, this article focuses just on the subsequentdevelopments at UCL. Therefore, it is important to note atthe outset that these developments were part of the birth ofa new subject, initially in just a few departments in theworldsubsequently in many. The exciting field today isthe summation of contributions from a host of laboratories,and its central foundations have been built by a group ofdistinguished pioneers of whom Malcolm Lilly was one.

    ENZYME TECHNOLOGY

    There had been a few early demonstrations that enzymescould be usefully immobilized as catalysts. The Tate andLyle sugar company had even operated a 6 meter-deep bonechar immobilized invertase column in the 1940s. However,there was no rigorous kinetic or engineering foundation forthe field, and immobilization was often erratic. In the 1960s,Ephraim Katchalski in Israel began to address the biophys-ics, George Mannecke in Germany the immobilizationchemistry, and Malcolm Lilly and his students the kinetics(Hornby et al., 1966) and the engineering (Sharp and Lilly,1968). The frustration that Malcolm Lilly suffered in beingobliged to study the few enzymes available from catalogswas also shared by Peter Dunnill, who had been workingunder Sir Lawrence Bragg on the first protein and enzymestructures by x-ray diffraction. They had begun to examinehow to isolate large quantities of commercially unavailableenzymes at UCL. He decided to combine their interests andcreate methods of large-scale isolation of intracellular en-zymes for subsequent immobilization and study. One earlyoutcome was research on penicillin amidase (acylase) forthe conversion of benzyl penicillin to 6-amino penicillanicacid (Self et al., 1969) to produce semi-synthetic penicillins.The work was quickly taken up by the company now knownas SmithKline Beecham and today is the procedure mostoften used worldwide in the production of semi-syntheticpenicillins. This success encouraged the development ofimmobilization research on soluble supports to tackle largesubstrates (Wykes et al., 1971b), cofactors on soluble sup-ports (Wykes et al., 1972) to allow interaction with enzymesimmobilized on particles, and magnetic supports to permitaction in fouling environments (Robinson et al., 1973).

    The Escherichia coli source of penicillin acylase was nothard to disrupt but many microorganisms were, and sostarted a series of studies of cell rupture that still continues(Keshavarz et al., 1990). A young Australian, Peter Heth-erington, did painstaking research on the rupture of bakersyeast in milk homogenizers to the point where a precisemathematical expression for rupture was defined (Hether-ington et al., 1971). This persuaded the UK engineeringcommunity that biological material processing was suscep-tible to rigorous analysis after all. Malcolm Lilly and PeterDunnill were helped in this by a new Head of the ChemicalEngineering Department, Peter Rowe, who not only took ascientific interest (ONeill et al. 1971), but provided spaceand encouragement. Elmer Gaden, who was Biotechnologyand Bioengineering in these years, was also a huge sourceof encouragement with his handwritten editors comments

    Biotechnology and Bioengineering, Vol. 60, Pp. 527533 (1998) 1998 John Wiley & Sons, Inc.

    CCC 0006-3592/98/050527-07

  • of firm but warm guidance. Together with the engineeringinputs, the early enzyme technology phase initiated thestrong interaction with the UCL biomedical community.Isolation of enzymes from human blood (Fisher et al., 1968)and from cattle brains (Street et al., 1973) would raise safetyissues today that could make them hard to address. How-ever, with the work on production of L-DOPA using immo-bilized tyrosinase (Wykes et al., 1971a), links were estab-lished that are now crucial. A third external influence wasArthur Humphrey who, by providing a sabbatical period forMalcolm Lilly with the challenge of giving a course onenzyme technology, helped the crystallization of ideas andgave faith to the endeavor.

    The defining moment of early worldwide enzyme tech-nology studies was the first Enzyme Engineering meeting in1971 in New Hampshire, sponsored by the EngineeringFoundation. With attendees such as Jerker Porath, EphraimKatchalski, Arthur Humphrey, Elmer Gaden, Arnold De-main, and Daniel Wang, it was a truly seminal meeting andcreated an international community. Subsequent participa-tion by Malcolm Lilly and Peter Dunnill in the MIT summercourses on fermentation and enzyme technology led to anAnglo-American co-authored book on fermentation and en-zyme technology (Wang et al., 1979).

    DOWNSTREAM PROCESSINGSuccess in defining cell rupture kinetics encouraged similarapproaches to other downstream operations such as precipi-tation (Foster et al., 1976) and affinity chromatography(Robinson et al., 1974). With this, an area of research wasset in motion that has since become a particular UCL inter-est. The studies of precipitation embraced a range of agents,and the work [which was reviewed in Bell et al. (1983)] hasinevitably become increasingly concerned with precise de-scriptions of the engineering mechanisms (Ayazi Shamlouet al., 1994a). It also led to studies directed at more selectiveflocculation (Milburn et al., 1990) and to crystallization asa useful large-scale operation for proteins (Jacobsen et al.,1998). The natural corollary of this research has been along-term concern with particleliquid separation. This hasembraced filtration (Gray et al., 1973), centrifugation (e.g.,Rumpus and Hoare, 1992), and membrane-based methods(e.g., Taylor et al., 1993). As the recombinant era has de-veloped, the focus has increasingly switched to the study ofmaterials such as inclusion bodies (Taylor et al., 1986) andvirus-like particles (Milburn et al., 1994). High-resolutionseparation has taken two paths. In parallel with seeking todefine conventional large-scale chromatography in processterms (Aguilera Soriano et al., 1996), new approaches suchas raised beds (De Luca et al., 1994) and sub-micron mag-netic affinity materials have been explored (OBrien et al.,1996) to allow selective separation to be brought forward inthe separation sequence.

    BIOTRANSFORMATIONIt was clear even in the 1960s that to be fully effective,biocatalytic technology had to link up to chemical catalysis.

    One major barrier was that many key chemical reactantswere poorly soluble in water. The isolation of cholesteroloxidase from a Nocardia species by Barry Buckland for hisdoctoral project had shown the organism to be extraordi-narily resistant to mechanical rupture. Fortunately, the en-zyme was in the outer cell envelope and was releasable bydetergent. The project at that point of the doctorate wasalready successful, providing a route (Buckland et al., 1974)to a diagnostic test for cholesterol in blood serum. It becameone of the largest income earners for the UK governmentagency that then controlled university intellectual property.In the time remaining for thesis research, it was interestingto know whether the robust Nocardia could be used forbiotransformation of cholesterol at higher concentrations.The sterol was hardly soluble and so, fearing the worst, thecells were plunged into solvents such as carbon tetrachlo-ride and toluene mixed with water. Catalysis proceeded forup to 10 useful cycles, drawing cholesterol at 150 times theaqueous level from the organic solvent reservoir into theaqueous phase (Buckland et al., 1975). So began a newcycle of studies. Other researchers were coming to a parallelrealization, and many have now contributed. However, Mal-colm Lilly with his students, and especially John Woodley,have had a particularly active role in placing this option inthe broader context of bioreactor selection and operation. Arecent review (Woodley and Lilly, 1992) introduced theidea of using experimental tools to define reaction proper-ties that relate directly to process design and scale-up. Theindustrial interesterification of fats and oils is one practicaloutcome of this work (Ison et al., 1988).

    The field that Malcolm Lilly particularly shaped and gaveimportant structure to was that of biochemical reactor de-sign and operation. This began with a classification of therelatively limited classes of biocatalyst, immobilizationmethod, and reactor types in the early 1970s (Lilly andDunnill, 1971, 1972). It progressed through a series of moresophisticated concepts to embrace two liquid-phase systems(Woodley and Lilly, 1992) and the strategies for dealingwith less favorable reactions exhibiting reactant or productinhibition/toxicity as well as unfavorable thermodynamics(Woodley and Lilly, 1994; Lilly and Woodley, 1996). Muchof the philosophy of this approach was surveyed in a Danck-werts Lecture of the UK Institution of Chemical Engineers(Lilly, 1994). The biotransformation research has empha-sized, as in fermentation and downstream processing, thevital need to take account of potential damage by the engi-neering environment. In particular, for two-phase systems,the combined impact of solvents and interfaces can be verysignificant (Woodley et al., 1991). It has also emphasizedthe need to study the reaction in the context of productrecovery, and from this a new phase of work on in-situproduct removal has developed (Freeman et al., 1993).

    FERMENTATION

    An early drive to make enzymes and to examine them aspotential industrial catalysts was always in danger of reduc-

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  • ing fermentation to a servant status. However, it becameclear very quickly that robust, well-understood, fermenta-tion was an imperative, and that the interaction with down-stream processing was critical. In this respect Malcolm Lil-lys early breadth of work was a great help, and articles suchas that on control of dissolved oxygen tension in the 1960s(Flynn and Lilly, 1967) set the stage for a detailed concernwith key parameters. The continuing influence of PatClarkes insight into microbial genetics was also critical inincorporating this element into fermentation thinking(Clarke et al., 1968). The fermentation aspects were put tothe test when at the end of the 1960s a new continuous1000-L vessel was used to feed a continuous downstreamprocess for research on the production of enzymes (Gray etal., 1972). The fermentation had to be brought speedily frombatch to continuous mode and held stable. The continuousprocessing research at that time was ahead of both adequatemonitoring and control and of modern recombinant genet-ics, but it was a useful forcing ground for new approaches.Malcolm Lilly returned at intervals to deepen the fermen-tation research (Suphantharika et al., 1994). Work on theeffect of cycling dissolved oxygen tension on product for-mation and on scaling issues as examined in airlift systems(Vardar and Lilly, 1982; Pollard et al., 1998) are good ex-amples of studies that are of broad importance today. Thefermentation studies have, like those of downstream pro-cessing, been concerned with the impact of the engineeringenvironment on delicate biological materials (Lilly et al.,1992) and the way in which consequent morphologicalchanges then affect nutrient transfer (Warren et al., 1995;Ayazi Shamlou et al., 1994b). Much of the accumulatedfermentation thinking was assembled recently in an articleby Barry Buckland and Malcolm Lilly (1993).

    WHOLE BIOPROCESS RESEARCH

    The 1990s saw UCL become a national center with a con-sequent need to create a research program that was distinc-tive and yet complemented that of other groups. Manyteams examine the individual operations and the history ofUCL biochemical engineering suggested that the centercould make a particular contribution to the understanding ofwhole bioprocesses for small and macromolecular ma-terials. The theme was consistent with the construction of anew facility that provides for the linked study of fermenta-tion, downstream processing, and biotransformation at up tofull pilot scale for those natural and recombinant materials,which can be examined safely at up to B3 containmentlevel. This together with a flameproof quality environmentin one suite meant that Malcolm Lilly was able to see in anew phase of research to verify at scale, the process frame-work he had originated for two liquid-phase biocatalysis.

    Whole bioprocess research has had two goals. Thefirst has been to accelerate the selection of the best processand its design. The second has been to speed the acquisitionof information about the product and its key contaminants.The examination of a first whole process demanded test

    targets for which UCL already had a significant amount ofprocess information. The intracellular enzyme alcohol de-hydrogenase, in fact, a family of delicate tetrameric pro-teins, from Saccharomyces cerevisiae was chosen as thefirst macromolecular target. In a series of parallel pilot-scalestudies the influence of fermentation on cell disruption (Sid-diqi et al., 1996), of disruption on centrifugation (Clarksonet al., 1993), and of centrifugation on precipitate recovery(Clarkson et al., 1996) were examined, and allowed lessconventional methods such as raised-bed absorption to beevaluated in a process context (Smith et al., 1996). With thisframework, it was then possible to examine how to acquirethe same information at a much faster rate by ultra scale-down beyond the smallest industrial homogenizers (Siddiqiet al., 1997) and centrifuges (Maybury et al., 1998). Bylinking the ultra scale-down to a set of experimentally vali-dated process models, it was subsequently possible to pre-dict the performance of a process for a recombinant yeastwith a protein-engineered alcohol dehydrogenase by acquir-ing a minimum of data on the new system (Varga et al.,1998). The alcohol dehydrogenase target provided the basisfor whole bioprocess studies of plasmid genes (Ciccolini etal., 1998) and their complexes with delivery and cell tar-geting molecules such as liposomes and antibody frag-ments.

    The first small-molecule test system chosen for the wholebioprocess approach was carboncarbon bond formation us-ing a transketolase enzyme. The study which Malcolm Lillyled in a collaboration with the Universities of Exeter andEdinburgh assembled genetics, crystallography, chemistry,and biochemical engineering methods to examine catalystformation and enhancement (Lilly et al., 1996), togetherwith optimal use (Mitra et al., 1998) and product recovery(Chauhan et al., 1996). The second group of small moleculetargets for whole bioprocess studies encompassed the re-combinant polyketides emerging from combinatorial biol-ogy. Here, erythromycin was a useful base case (Lye andStuckey, 1994; Pollard et al., 1998). For process models tobe used, it is important for the choices they suggest to beeasily understoodfor both small and macromolecular ma-terials the use of a windows of operation approach hasbeen developed (Woodley and Titchener-Hooker, 1996).

    One of the severe constraints in creating new biopro-cesses was the time taken for analysis of the target materialsand key contaminants. It meant that each cycle of processexperiments essentially proceeds blindly. The problem wascritical for early continuous process studies, and rapid moni-toring approaches were begun (Dunnill and Lilly, 1972).Subsequently, both the fermentation stages (Royce andThornhill, 1992) and the downstream steps such as precipi-tation (Holwill et al., 1997), flocculation (Habib et al.,1997), and chromatography (Bracewell et al., 1998) werethe subjects of study. In addition to analysis of the productand contaminants, there was a major concern with measur-ing cell morphology (Packer et al., 1992) and with productauthenticity (James et al., 1994).

    AYAZI SHAMLOU ET AL.: EDITORIAL 529

  • A linking theme that grew to connect all of the studies ofbiological materials at UCL was the impact of the engineer-ing environment on delicate large entities. The earliest workshowed that globular proteins are surprisingly resistant toshear, but very susceptible to shear-induced interfacial ef-fects (Thomas and Dunnill, 1979). On the other hand, largermembrane-associated enzymes are acutely sensitive to shearitself (Talboys and Dunnill, 1985). Later, shear effects cameto dominate the work on protein precipitates and their re-covery (Hoare et al., 1982). A second theme that developedwas product design for processability. The advances inrecombinant science meant that it was increasingly possibleto change the properties of biomolecules to enhance theirprocessing, for example, by pre-derivatizing them to easeseparation (Sloane et al., 1996). Finally, bioprocess safetyhas been a long-term concern (Dunnill, 1982), and one in-creasingly capable of precise study (Ferris et al., 1995) tobest address public concerns about recombinant products.

    BIOCHEMICAL ENGINEERING EDUCATIONAND LEADERSHIP

    From the beginning, the UCL ethos was that research andteaching are inseparable. There was also a recognition thatto attract the best young people into bioprocess engineering,

    communicating the importance of the activity to a broaderaudience is required. From popular articles in the 1950s(Webb, 1959) to one of Malcolm Lillys last articles, (Lilly,1997), educational issues have been a constant theme, andall staff have shared in the evangelizing role (Harrison,1998; Turner, 1995). In 1973 UCL began to teach a fullundergraduate degree and in 1988, became the first UKDepartment of Biochemical Engineering.

    THE FUTURE

    The challenge and excitement of making available at afford-able prices the materials being defined in the explosion ofbiological discoveries are immense. The sensitivities to theprocessing environment, which were first observed withproteins, are severe for much larger entities such as plasmidgenes for therapy (Levy et al., 1998), nematodes for bio-pesticides (Young et al., 1998), and fibronectin fibers fortissue repair (Underwood et al., 1998). Some of the keyfuture medicines using genes, cells, and replacement tissueare likely to be targeted at much smaller subgroups in thepopulation and achieving their production at realistic costswill demand approaches to mass customization as radi-cal as any in the automotive industry. For example, one

    Figure 1. In the early 1960s, UCL Biochemical Engineering was just a few tanks and rigs in a part of the main pilot plant.

    530 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 60, NO. 5, DECEMBER 5, 1998

  • UCL project is examining the biochemical and other engi-neering fundamentals of disposable whole bioprocesses tominimize stainless steel equipment, steam, and revalidationof process surfaces. Computer-based decisional tools arebeing developed that can link whole bioprocess models tobusiness process models so that it is possible to place pro-cessing in the context of clinical trials and to provide link-age to regulatory and financial decisions. At UCL, meta-bolic modeling is seen as a crucial adjunct to process plan-ning where, though experiments are still vital, time can besaved and experiments focused (Sheridan et al., 1998).

    The increase in complexity that marks developments withmacromolecular and tissue targets is also a feature ofsmaller chemical entities. The challenge of achieving selec-tive action, whether with drugs or pesticides is calling forcomplex and often multi-chiral centered molecules. Againstthis background, the potential of biocatalysis, which Mal-colm Lilly had such a major part in laying biochemicalengineering foundations for, is growing strongly.

    The UCL team is grateful to the editors of Biotechnologyand Bioengineering for giving it a place to describe its workover the years, and for allowing us to pay tribute to our

    distinguished colleague. We look forward to an exciting21st century with the Journal.

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    AYAZI SHAMLOU ET AL.: EDITORIAL 531

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    Parviz Ayazi Shamlou Gary J. LyePeter Dunnill Nigel J. Titchener-HookerMichael Hoare Michael K. TurnerAndrew P. Ison John M. WoodleyEli Keshavarz-Moore Barry C. Buckland (Fellow

    and Visiting Professor)

    AYAZI SHAMLOU ET AL.: EDITORIAL 533

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