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First of all, agreement needs to be reached onthe definition of a “biocatalytic process”. Inprinciple, it covers a wide range of variants

(de novo fermentations, precursor fermentations,bioconversions, enzymatic reactions and so on). Inthis article, the focus will be on biotransformations -independent of the type of biocatalyst employed; ingeneral terms, it will deal with the “bio-conversion”of an educt A (a non-renewable carbon source) intoa product B. The second issue to reach a consensuson is which types of processes are going to beconsidered; they can either be aimed at already-existing, well-established products (for example,6-APA or D-pHPG) or at new chemical entities(NCEs) and the intermediates for their synthesis.Emphasis will be given to the second kind ofprocess; meanwhile, however, the stage will be setby saying a few words on the first category.

Biocatalytical processes forexisting products

Many of the products covered here have - at sometime in the past - been manufactured by means ofa synthetic chemical process. Most of them arebroadly established products with large marketsand are likely to remain available for some decadesto come (for example, some vitamins, amino acids,building blocks for antibiotics, food/feedadditives). With the advances in biological/biochemical knowledge, it has become possible toenvisage and establish new biotechnologicalprocesses with improved economics on the one

hand, and improved environmental acceptabilityon the other - or both at the same time. The largesales and correspondingly high profits generated bythese products has meant that time- and money-consuming R&D programmes could be undertaken,enabling production processes to be implementedon an industrial scale. These programmes includedbasic and applied research, and new engineeringconcepts, and frequently spanned time-frames of10-20 years. Quite often, they involved some kind oftechnological “quantum leap” coupled with numerousincremental improvements - leading finally to thehighly competitive processes in use today.

A typical example is the biocatalytic processdeveloped by Lonza for the production ofL-carnitine; this involved several “processgenerations” before becoming mature and highlycompetitive, requiring nearly ten years of sustainedwork at various different levels. The media usedwere optimised, downstream processing (DSP) wasimproved and productivity was increased by morethan 100% - leading to better product quality anddrastically lower manufacturing costs. But, as inother similar cases, the upfront investmentrequired was enormous.

Biocatalytical processes fordevelopmental products

The situation is dramatically different whenconsidering the optimisation of biocatalyticalprocesses for developmental products, as this tendsto be performed by custom manufacturers, such as

Strategies for theoptimisation ofbiocatalytical processesThe optimisation of biocatalytical processes is a long and complex task,requiring many decisions to be made along the way - from R&D to industrial-scale production.

Dr Claude Chassin, Lonza Ltd

The situationis dramaticallydifferent when

considering theoptimisation of

biocatalyticalprocesses for

developmentalproducts …

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ApplyBiocatalysis?

AvailabilityBiocatalyt?

Microbiologicalscreening?

Optimisationrequired?

Strainimprovement?

Convenientstrain(s)?

Convenientstrain(s)?

No

No

No

Yes

Yes

No Yes

YesNo

YesNo

Yes No

Yes

C.S.

H.T.S.

I.S.

H.D.S.

ProcessDesign

Cloning

Evolution

Mutagenesis

Figure 1. Decision tree for the development of a biocatalytic strain.

The process design exercise includes decisions about use of the strain as such,or development of an enzymatic process, as well as connected issues such asthe type of bioreactor to be used.

Lonza Ltd and, in particular, Lonza Biotechnology.Here, in a standard situation, a client wishes tooutsource the manufacturing of a new activepharmaceutical ingredient (API) or a penultimatekey intermediate; usually, a route selection hasalready been performed which either includes abiocatalytic step or has the potential to utilise one.

As a general rule of thumb, at that stage, themolecule is undergoing early clinical evaluationand the customer has already set up a well-definedschedule for filing an NDA with the regulatoryauthorities. Assuming that the required product ispart of the corresponding DMF filing, then there isusually about 18 months to two years’ time available tohave a process up and running towards validation.

Once faced with this challenge, the necessarysteps to be taken can be described as a decision tree(Figure 1):

• Check the opportunities available to applya biocatalytic reaction, in case it has not yetbeen included as an option,

• Check the approximate value-added of such a step,

• Establish what type of biocatalytic activityis required,

• Check the commercial availability, priceand quality of the biocatalytic activities needed,

• Assess the technical feasibility of thereaction to be performed (for example,co-factor requirements, physico-chemicalproperties of the educt/product and anypotential inhibitory actionon the biocatalyst to be used), and

• Apply personal judgment - based onexperience - to assess whether the proposedbiocatalytic pathway has a fair chanceof being economically feasible (“theguesstimate act”).

Assuming this assessment of the general frame-work opens up a reasonable possibility to develop acompetitive process, the next step is to review theintellectual property estate in the field. It is acritical pre-requirement since, quite often, prior artcan influence the choice of route selection.

If - as is often the case - an off-the-shelfenzymatic activity is either not available or notefficient enough, then the next step is to perform amicrobiological screening. Once a sensitive assayhas been developed, then there are several routeswhich can be followed:

• Use of existing banks of micro-organisms(conventional screening, CS),

• Blind screening (here, techniques like highthroughput screening, HTS, might be useful),

• “Japanese screening” - this corresponds to atargeted and highly skilled and intelligentconventional screening (IS), and

• High diversity screening (HDS), forexample, extremophiles.

The next step is to select for the mostappropriate candidate(s). Depending on the levelof biocatalytic activity shown by the native strain,there is a wide range of further actions to be under-taken. In the case of a high efficiency candidate,these include:

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• Identification of the best possible inductionsystems (when required),

• Optimisation of culture conditions, and• Optimisation of the growth medium.In the event that natural expression levels of the

desired activity are too low, then there are severaloptions to be considered, for example: cloning andover-expressing the gene(s) coding for the biocatalyticactivity in a homologous or heterologousrecombination; or applying evolutionary techniqueslike random mutagenesis, site-directed mutagenesisand gene shuffling to increase the original activity.

Once the “ideal biocatalyst” has been identifiedand developed, there is still a further decision to bemade, namely the form in which the biocatalyst isto be used. There are several possibilities:

• Whole biomass (growing or resting),• Immobilised whole cells,• Crude/purified soluble enzyme, and• Immobilised enzyme.Only when all these different issues have been

answered satisfactorily has the time come for theindustrial microbiologists and process engineers totackle issues such as piloting, process design andscale-up. Here, too, there is a wide range ofoptions to be chosen from, including:

• A purely aqueous versus a multi-phase process,• A continuous process with biocatalyst

recycling, or• A column design (where there are no

drastic pH shifts within the reaction).While these activities are taking place, the target

product is moving through the developmentpipeline. Two extreme scenarios can be envisagedwith regard to the product’s progress: first, theproduct does not fulfil expectations and suffers a”sudden death”, and second, the product does betterthan expected and is given “fast track” status, which

further increases the pressure on timeschedules. During this limited “time window”,piloting must be concluded, know-how transfercompleted, regulatory actions taken and the supplychain secured.

The manufacturer:client relationship

When one looks at the complex chain of eventsrequired on the way to developing a biocatalyticprocess, then it becomes obvious that therelationship between the custom manufacturer andthe client needs to be very close and based on trust- coupled with an interactive cross-fertilisation ofideas, and an intensive and unbiased communicationat all levels. The client must be included in allelements of the decision process, as the choice offinal parameters can have a strong influence on thequality and economics of the product to be made.

Such a relationship enables a number ofalternative strategies to be envisaged, allowingbetter compliance with time constraints. One ofthe possibilities given here - and one which is oftenjudged attractive by the client base - is as follows.As a first step, a “quick fix” solution is adopted;this allows the client to file for an NDA as soon aspossible, while using a less than fully optimisedprocess. (This is usually called a first generationprocess.) In parallel, work is started on allconceivable improvements, yielding a trulyoptimised system. While development of a“second generation” process will most probablynecessitate a repeat filing of some regulatory data,as well as an amended/new DMF, the economicadvantages can often offset the additional costsinvolved, if the target molecule makes it worth-while. Within such a procedure, one has to takecare that issues relating to IPR (intellectualproperty rights) are dealt with fairly and on acommonly agreed basis, and also within acollaborative effort aimed at development of such a“second generation” process.

Up to now, considerations have been limited toa single biocatalytic reaction. Most of the time,such a reaction forms part of a multi-step processinvolving synthetic organic chemistry as well. Thisbrings an additional dimension of complexity:

• The need for different parts of the processdevelopment (chemical, biological) to cometogether at the same time,

• Quality aspects of the educt to be used forthe biocatalytic step, as well as of theproduct utilised in the following processsequence, and

• The economics of the substrate to be used -as well as the costs of the biocatalytic step -integrated within the whole value chainleading to the desired product.

Figure 2. Pilot facilities at Visp, Switzerland.

Once the “idealbiocatalyst”

has beenidentified and

developed,there is still a

further decisionto be

made - namelythe form inwhich it isto be used

… the choiceof final

parameters canhave a strong

influence onthe quality and

economics ofthe product to

be made

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Conclusion

As one can see, the development and optimisationof a biocatalytic process is a complex undertaking,requiring a highly versatile, experienced andunbiased group of multi-disciplinary specialistsworking in unison. This might explain why thereare still only relatively few established biocatalyticprocesses within the life science industry.Nevertheless, the trend is growing and it can besafely predicted that the new generations ofscientists - having a knowledge and understandingof both the chemical and biological worlds - will bein a better position to make a contribution to theproblem-solving process. Furthermore, thedifferent technologies in use will become moremature, predictable and scaleable, resulting inmore efficient process selection and development.The development of biocatalytic processes willremain a complex exercise, requiring perseveranceand a lot of decision-making along the way - fromresearch and development down to industrial-scaleoperations. In addition, numerous interfaces willcontinue to be of vital necessity, requiring

experienced, well-trained specialists with amulti-disciplinary background.

Dr Claude Chassin received aPhD in organic chemistry fromTübingen University (Germany),and then spent three years inpost-doctoral fellowships at theUniversity of Wisconsin and theMassachusetts Institute ofTechnology (US), and UniversityCollege, London (UK). He

started his professional career with BoehringerMannheim (now Roche Diagnostics) in Germany,and then moved on to Transgène (France) beforejoining Lonza (Switzerland), where he contributedto the start-up and growth of their biotechnologicalactivities. Lonza Biotechnology is a custommanufacturing organisation to the life scienceindustry, working in the field of the developmentand production of products obtained bybiotechnological methods.

Figure 3. 5 x 15m3 fermenters at Kourim.

… the newgenerationsof scientists …will be in abetter positionto make acontributionto theproblem-solvingprocess