28 manufacturing chemist April 2018

Optimised biocatalysts for the pharmaceutical and food industries

Revolutionary advances in digital technology, genomics and sequencing have fuelledtremendous advances in protein engineering.Enhanced understanding of the complexrelationships between protein structure andfunction has unveiled virtually limitlessopportunities for engineering proteins that meetprecise performance specifications. Using highlyadvanced techniques that combine the disciplinesof biochemistry, chemistry, recombinant DNAtechnology, structural biology, biochemicalengineering and information technology, proteinengineering enables scientists to create enzymesthat are far superior to those available fromnature — and sometimes even absent in nature. A key result of the protein revolution is the

rapid development and production of high-performing enzymes that serve customapplications. Protein engineers are designing andproducing optimised proteins that dramaticallyreduce the cost and improve the quality ofpharmaceuticals and food ingredients, while alsocreating novel biotherapeutics and enabling thesequencing of minute quantities of DNA forin vitro diagnostics. As protein engineeringtechniques continue to evolve, suppliers of theseservices are increasingly able to deliver optimisedproteins that precisely match their clients’ desiredperformance specifications — and can do so fasterand less expensively than ever before.

The rationale for engineered enzymesScientists developing the manufacturing route toany chemical — be it an active pharmaceuticalingredient (API), a food ingredient, a fragrance ora flavour molecule, or even a commodity chemical— often need to assemble a series of chemicalreactions that convert an inexpensive rawmaterial to the desired commercial compound.These chemical reactions are catalysed bychemical or biological catalysts.A huge number of catalysts occur in nature and

have long been used in various ways to enable orspeed up reactions. Until recently, most catalystswere transition-metal based, with limited catalyticscope and selectivity. Moreover, metal-basedcatalysts are often toxic or rare and are usually notvery optimisable beyond their initial performance. Additionally, reaction conditions for

chemocatalysts are usually much harsher than forbiocatalysts, as the reactions must occur atelevated temperatures and at higher pressure.Chemocatalytic processes also generate morewaste and, thus, increase costs.By contrast, biological catalysts (enzymes) are

malleable and can be manipulated through the

Riding the wave of protein revolution

RESEARCH AND DEVELOPMENT

process of protein engineering. Engineeredenzymes have the significant advantages ofsimplicity, scalability and stereoselectivity,enabling the production of chirally purecompounds — an especially importantconsideration in API manufacture, as most drugsare chiral compounds. Additionally, biocatalyticprocesses use standard lab or plant equipment,take place within ambient temperature andnormal pressure ranges, and do not requirecomplex controls. These advantages provide amajor benefit for companies that outsourcemanufacturing to contract manufacturers. The most important benefit of biocatalysts may

lie in the fact that engineers can devise the mostcost-effective and sustainable route ofmanufacture, and then engineer the proteincatalyst to fit this “ideal” process; this feature isoften unavailable for chemocatalysts, for which asuboptimal process has to fit the catalyst.

Protein engineering technology CodeEvolver is a protein engineering technologyplatform developed by Codexis that enables themodification of proteins to enhance specificperformance characteristics. This technologyallows Codexis scientists to first identify the bestnatural enzyme that can perform the desiredchemical transformation, and then use theproprietary protein engineering platform toimprove the enzyme. Using bioinformatics tools, Codexis scientists

manipulate the sequence of the initial enzyme tocreate a library of variant enzymes that containmutations at specific positions. Screening thislibrary enables the identification of variantenzymes with improved performance for thedesired chemical reaction. Analysis of the libraryidentifies the specific advantageous mutations thatlead to these improved traits. With this information in hand, scientists can then combinethe beneficial mutations to produce the bestpossible variant enzyme.The CodeEvolver process consists of in silico

screening, the introduction of function-drivenmutations, high-throughput screening andsequencing, and machine learning to identify aproductive combination of mutations and, finally,the human expertise of the scientific team. Thisprocess is repeated iteratively for compoundedimprovements until all the desired traits areincorporated into the final enzyme. Thetechnology reflects an accelerated evolutionaryprocess — something that happens in nature butis harnessed in the lab for faster and specificimprovements at a molecular level.

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Another advantage of the CodeEvolver platformis the speed at which an optimised enzyme can bedelivered into a customer’s commercial process.Once an improved enzyme has been engineered,the technology enables rapid scale-up of theenzyme production, in some cases allowingcustomers to manufacture product at metric tonlevels using the biocatalytic route within a 3–6month period. Customers can also access Codexis’CodeEvolver technology by purchasing an off-the-shelf screening kit to assess the feasibility of abiocatalytic route in their own labs. One of the most powerful elements of the

CodeEvolver protein engineering technology isthat it is an additive construct, in that every cycleof variations and protein optimisation generatesmore data. This benefit allows Codexis toundertake each successive project with moreknowledge, facilitating the rapid identification ofcandidate proteins from nearly infinitepossibilities, as well as the engineering andoptimisation of those proteins to meet specificcustomer needs. Today, Codexis can accommodate15 unique protein engineering projects in parallel,with each project taking an average of 3–6 monthsto achieve its intended performance targets. Bycontrast, at the time Codexis was incorporated in2002, a single project might take 2 years or moreto reach fruition.

Evidence of customer benefitsEnzyme optimisation can yield benefits for avariety of companies in the life sciences, foodindustries and other applications. For example, abiocatalyst that had zero starting activity wastransformed into a commercially relevantbiocatalyst in less than 12 months, enabling amajor pharmaceutical company that faced capacityconstraints in its existing supply chain because ofincreasing demand for a blockbuster type 2diabetes medication to avoid building a new factory. In this case, Codexis developed a new, high-

performing enzyme catalyst to replace anexpensive, toxic chemocatalyst that was used toproduce the drug’s API. The biocatalytic solutioneliminated several steps from the manufacturingprocess, increased the measured productivity ofthe process by 53% and decreased energy usage by19%. Perhaps most importantly, use of the newbiocatalyst helped the pharmaceutical company toavoid the cost of building a second factory to meetthe rising demand for the drug, significantlyreducing capital expenditure requirements.Another notable example involves a leading food

industry supplier seeking a healthier ingredientthat would meet demanding caloric and sensorytargets. The new ingredient also needed to fulfilrequirements for speed of commercialisation,safety and lower production costs. UsingCodeEvolver, Codexis worked with the supplier toengineer a biocatalyst that would meet thecustomer’s goals and enable a simple, cost-efficient production process. The project resultedin commercial targets being reached in 7 months,and the engineered biocatalyst produced a 70-foldimprovement in catalyst stability underchallenging process conditions. The solution cut

Enzymeoptimisation canyield benefitsfor a variety ofcompanies inthe life sciences,food industriesand otherapplications

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costs by 90% and enabled commercial productionof the healthy ingredient less than 2 years afterthe first project discussion. In addition to engineering proteins as

biocatalysts, CodeEvolver is also being applied tocreate novel, targeted biotherapeutic solutions.Phenylketonuria (PKU) is a rare, inherited geneticdisorder that strikes approximately one in 15,000newborns in the United States. The disorder ischaracterised by a deficiency in the enzyme thatconverts the essential amino acid phenylalanineinto tyrosine, resulting in the accumulation ofhigh levels of phenylalanine in the brain, where itcauses serious neurological problems includingintellectual disability, seizures and cognitive andbehavioural problems. Whereas a screening testfor PKU has been available for more than 50years, treatment has essentially been limited todietary control. Codexis has engineered a biotherapeutic enzyme

candidate for the treatment of PKU thatcompensates for the absence of the patient’smissing natural enzyme that metabolisesphenylalanine in the body. The engineeredenzyme’s stability in the gastrointestinal tractenables convenient oral dosage, andpharmacokinetic analysis has revealed a greaterthan 50-fold improvement in stability in vitro(compared with the natural enzyme). Fourpreclinical models have demonstrated efficacy, andCodexis aims to initiate humantrials during 2018.Unsurprisingly, much of the

innovation in proteinengineering and enzymeoptimisation is driven bycustomers seeking a competitiveedge in a highly dynamiccommercial environment. In their quest to befirst-in-class or first-to-market, every customerthat approaches Codexis wants to increasemanufacturing efficiencies to drive costs down andimprove product quality, to create a bettertargeted biotherapeutic or to improve sensitivityand precision in their next-generationsequencing (NGS) workflows.Codexis’ success in the multiplemarket application of theirproprietary CodeEvolver technologyallows customers to seek a proteinengineering solution to deliver on thecustomer’s precise specifications. Moreover,greater customer awareness of the benefits ofadvanced protein engineering increases thedemand for engineered enzyme solutions,and the accumulated experience expandsCodexis’ knowledge base exponentially witheach project.

Future directionsAs the benefits of advanced proteinengineering and protein optimisationbecome more widely known, it isreasonable to expect continuedinnovation in this field. In additionto leveraging optimised enzymesolutions in the manufacture of APIs

and food and beverage ingredients, cost- andresource-conscious customers will also seek toapply protein engineering to renovate existing ordevelop new small-molecule drugs, conjugatedantibodies, animal feed enzymes, moleculardiagnostic enzymes and biotherapeutics.In particular, customers in the life sciences

fields will benefit from the maturation of methodsto produce more sensitive, fluid-based moleculardiagnostic tests from optimised NGS enzymes,which will enable the less invasive detection ofcancer and other diseases. Such developments maygive fresh momentum to the personalisedmedicine movement as greater numbers ofpatients and their physicians seek clearer, moreprecise diagnoses based on the molecularsignature of their diseases. Plus, proteinengineers’ initial forays into optimising proteinsas biotherapeutics, such as Codexis’ biologic leadcandidate for PKU, may yield advances in thetreatment of various diseases.Finally, advances in protein engineering and

evolving customer needs will launch exploration inapplications beyond biocatalysis, biotherapeuticsand medical diagnostics. As the protein engineeringtoolbox expands, so too will engineers’ ability toimprove the functionality and stability of proteinsof interest. Indeed, the range of possibilities is asvast as the protein landscape itself.

FOR MOREINFORMATIONDipnath BaidyaroyDirectorStrategic AlliancesBusiness Operations

Stefanie Ng MinorManagerProduct ManagementBusiness Operations

Codexis, Inc.www.codexis.com

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