48 BioProcess International APRIL 2004

LL orantis Ltd. (Cambridge,United Kingdom) is adiscovery companydeveloping products forantigen-specific

immunotherapy, ASPECT, based onthe Notch signaling pathway, whichhas been shown to play a key role inregulating immune responses. TheNotch receptor and its associatedligands represent a family of highlyconserved transmembrane proteinsexpressed during embryonicdevelopment in both vertebratesand invertebrates (1). Binding of aNotch ligand to a Notch receptoractivates signaling cascades that alterthe transcriptional program of thetarget cell, ultimately determiningits fate. Our ASPECT platformsexploit the company’s proprietaryposition covering the Notchsignaling pathway in immune cellsand have broad clinical applications.

The products in our currentpreclinical pipeline are derived fromthe Notch binding protein, Delta-1.To evaluate its biological efficacy,we chose to pursue severalapproaches. One was to produce acommercial cell line that secretes arecombinant protein derived fromthe extracellular domain of Delta-1fused to an antibody constantregion. This report summarizes thecell culture strategies we used tospeed up the development of asuitable cell line — and ultimatelythe time to clinic.

INITIAL CHOICES

Host Cells and Expression System:Production of a commercial cell lineinvolves several key decisions andactivities. First you must choose thehost cell type and expression system.We decided to use the glutamine

synthetase (GS) gene expressionsystem from Lonza Biologics(www.lonzabiologics.com) usingCHO-K1 cells. GS is the enzymeresponsible for biosynthesis ofglutamine using glutamate andammonium as substrates. When acell line expresses GS, it can survivein a glutamine-free medium. Theactivity of the GS gene can also beselectively inhibited by methioninesulphoxamide (MSX).

CHO-K1 cells produceendogenous GS. But they can beused to produce stable cell lines bytransfecting in a GS gene and usingglutamine-free medium plus MSX(at sufficient levels to inhibit theendogenous enzyme) to provideselection pressure. Use of amammalian host cell should giveour protein the appropriateposttranslational modifications.

PRODUCT: CELL-CULTURED PRODUCTS

PROCESS FOCUS: PRODUCTION,ANALYTICAL METHODS DEVELOPMENT

WHO SHOULD READ: PROCESS

ENGINEERS, MANUFACTURING,ANALYTICAL PERSONNEL

KEYWORDS: CELL CULTURE, SERUM-FREE MEDIA, ASSAYS, CHO CELL LINES

LEVEL: INTERMEDIATE

B I O P R O C E S S TECHNICAL

PHOTODISC (WWW.PHOTODISC.COM)

Strategies for ProducingCommercial Cell LinesOne Biotech Company’s Experience

Sue Clarke, Janette Dillon, Ann Smith, and Elizabeth Sotheran

The advantages of the GS geneexpression system are many. It isused throughout the industry andtherefore comes with a wealth ofaccumulated knowledge andavailable experience. A fullyqualified master cell bank isprovided, which is a critical startingmaterial for the whole process. GShas been used for licensed products,

and consequently, regulatoryauthorities are familiar with it.Although it carries an inherent cost,that is money well spent for a start-up company.

We continue to enhance thequality of our activities by sourcingraw materials, particularly those ofanimal origin, from reputablesuppliers that can provide necessarysupporting documentation. Themost critical, of course, is a BSE-free source of fetal bovine serum(FBS). Our batch was specificallychosen to create stable cell lines. Itis low in IgG, which aids thepurification process, it supportsclonal cell growth, and it is dialysedand confirmed to be glutamine free.

Tissue culture procedures for ourcell line are carried out in anisolated room under positive

pressure, maintaining segregationand thereby reducing the risk ofcross-contamination and theintroduction of adventitious agents.All materials are logged in by batchnumber, allowing full traceability.All such activities must be welldocumented to provide traceabilityand meet regulatory guidelines (2).A summary of data is not only

needed for clinicalsubmissions; it isalso extremelyuseful whentransferring the cellline to a contractmanufacturer.

Plasmid DNA:The second mostimportant startingmaterial is theplasmid DNA.Traditionally youwould want toconfirm that the

correct protein is secreted, usuallyby transient expression, beforeembarking on stable cell line work.Because we are confident in boththe vectors and our proteins, wenow set up a stable transfection anduse a bulk culture supernatant forthe initial analysis at the same timewe dispense a duplicate culture into96-well tissue culture plates (plateout the culture) at a variety of low-density concentrations and leave thecells to grow for at least threeweeks. While they grow, we fullysequence the plasmid and confirmthe quality and biological activity ofthe secreted protein. Running theseprocedures in parallel can thereforesave time, but it does carry a riskfactor. Several methods are availablefor the actual transfection, and wehave successfully used bothelectroporation and a lipid carriersuch as Lipofectamine from GibcoBRL (www.gibcoBRL.com).

ANALYTICAL METHODS

To be able to select the desiredclone in your cell line, you need theright analytical methods to measurethe quantity, quality, and biologicalactivity of the product. If you areworking with novel proteins, thatcan be challenging because standard

FFiigguurree 11:: Notch ligand signaling activty

TTaabbllee 11:: Plating efficiency in four 96-wellplates

Expected Frequency ofcells/well observed cells/well

0 1 >110 71 91 222

5 166 115 1031 332 42 10

Circle Reader Service No. 128

reagents may be unavailable.Reagent production must thereforebe considered in the overall projecttimeline.

Because our product is a fusionprotein, we have been able to usethe antibody constant region to aidus in both the quantitative analysisand subsequent purification. We usestandard Western blots as aqualitative assessment for Delta, andwe had to make our own polyclonalantibodies for that purpose.

The efficacy of the final moleculeis, of course, paramount. We made astable CHO reporter cell line to testfor that purpose. Cells areengineered to overexpress a Notch2membrane-bound protein and alsohave a core binding factor 1(CBF1)–driven luciferase constructfor easy monitoring of Notchsignaling (3). When the secretedrecombinant ligand or test productis mobilized to an assay plate, theDelta portion of this fusion proteinbinds the Notch presented by thereporter cells. That in turn allowscleavage and activation of theluciferase reporter gene (Figure 1).

Using the above assay, wedemonstrate good activation of thereporter cell, with culturesupernatants in the 10–30 µg/mLconcentration range. Furtherin vitro analysis of our later clonesdemonstrated that our fusionprotein delivers an active Notchsignal and modulates the cytokineprofiles of mouse CD4+ T cells (4).

As mentioned earlier, the plated-

out transfections are ready for assayafter several weeks of incubation.Ideally you would assay severalhundred clones and take areasonable number of them onthrough further selection andcloning steps. However, that processcan be extremely labor intensive. Amore realistic task (given the limitedresources of a small biotechcompany) would be to reducesignificantly the number of culturesassessed. The final endpoint doesnot change: You still need a high-producing, stable clone. Cloningand selection strategies thereforebecome vital.

CLONING AND SELECTION

Cloning: We tried two differentcloning and plating strategies toreach our endpoint. The firstapproach took about 12 monthsfrom gene to final stable cell line.We initially plated out the cells at anonclonal dilution and tested all thewells (12 � 96). We then expandeda selection of wells and performedfurther assays, while preparingcryopreserved stocks as back-up.The best clone was subjected to alimiting dilution cloning. The lasttwo steps were repeated, and four ofthe resulting clones were selected ascandidates to adapt to suspension.Each candidate can take up to twomonths to clone, select, and freeze.

The second method we usedaimed to achieve a confidence ofclonality at an earlier stage. Table 1shows the numbers of clones thatwere microscopically observed whenplating the cells three to five daysposttransfection. It can been seenfrom these results that the lowerdilutions — five and one expectedcells/well — gave us enough wellsthat appeared as a single colony. Theprobability that these single coloniescome from one cell is increased atthe lower dilutions. Transfectionefficiency also affects the actual

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FFiigguurree 22:: Typical growth profile

FFiigguurree 33:: Stability study

52 BioProcess International APRIL 2004

number of cells that survive in theselection medium at this initial phase.

We therefore took the highestproducing clones from the lowerdilution plates to increase the chanceof clonality. In this experiment, weperformed one additional round oflimiting dilution cloning to selectthe final clones. Note here that thebest producer in the static flask maynot be the best producer onceadapted to suspension, so a widerchoice is always the preference. Thissecond method reduced the timelineby six months and still yielded agood quality cell line.

Selection: Whenever possible,selection of the optimal cloneshould take into consideration theeventual production process. So thelast stage added to both of theabove methods is taking the finalclones and adapting them to bothsuspension and a protein-freemedium to make them more readilyscalable. Again, realistically only afew can probably be handled.

We adapted the clones byreducing the percentage of FBS inthe adherent culture to 2% and theninoculating a 500-mL spinner orshake flask with 4–5 � 105

cells/mL in protein-free Ex-Cell325 medium from JRH Biosciences(www.jrhbiosciences.com), giving afinal concentration of 1% FBS.Then, additions of 325 mediumalone were made, so the cells slowly(about three months) adapted tothe new culture system. Figure 2 is a

typical growth profile of an adaptedclone followed for 17 days.

Once adapted, the final clonesmust be tested for stability to ensurethat they are robust enough for amanufacturing campaign —normally at least 50–60 populationdoublings. A stock culture ismaintained and defined in terms ofpopulation doublings (5). At regularintervals (about 10 weeks apart), anormally prepared production vesselset up from the stock flask is used toassess productivity in relation to theoverall population generations.

Figure 3 shows that for a six-week study, two of the cloneslooked stable. However, when wecontinued the study for a furtherfew weeks, clone #LC06-0342 lostproductivity.

Analytical Cloning: A faster methodfor testing stability was to perform ananalytical cloning as an assessment ofpopulation drift. Ideally, 100% of agiven population produces the targetprotein. If a population hasnonproducers, they could outgrowthe producers, causing an instabilityin the culture over time. Ananalytical cloning therefore examinesthe distribution of cells at aconcentration that permits allapparently single-cell wells to betested for protein expression.

This method takes only threeweeks at most — two weeks for theclones to grow and a week foranalysis — instead of the ten weeksrequired by the method described

above. So we used analytical cloningfor both clones and found that#0304 was 96% positive, but #0342was only 56% positive for proteinproduction. Careful observation ofthe plates microscopically gave usmore information. Had we neededto, we could have subcultured theclones at a lower confluency rangebut higher productivity (Figure 4).

MAKE IT QUICK

In summary, building in quality fromthe start and having the appropriateassays available make it possible for asmall biotech company to produce asuitable cell line for a phase I study.Timelines can be significantlyreduced — even when resources arelimited — by the simple cloningstrategies described here.

REFERENCES1 McKenzie, GJ; et al. Notch Signaling

in the Regulation of Peripheral T-CellFunction. Seminars in Cell & DevelopmentalBiology 2003, 14: 127–134.

2 Center for Biologics Evaluation andResearch. Guidance for Industry: Q5DQuality of Biotechnological/Biological Products— Derivation and Characterisation of CellSubstrates Used for Production ofBiotechnological/Biological Products. US Foodand Drug Administration, 1998;www.fda.gov/cber/qdlns/qualbiot.txt.

3 Wong KK; et.al. Notch Ligation byDelta 1 Inhibits Peripheral ImmuneResponses to Transplantation Antigens By aCD8+ Cell Dependent Mechanism. J.Clinical Investigation, in press.

4 Young L; et al. The Notch LigandDelta 1 Selectively Enhances the Productionof IL-10 in Murine T Cells. Immunology2002, 107, Supplement 1: 1–7.

5 McAteer, JA; Davis JM. Basic CellCulture Techniques and the Maintenance ofCell Lines. In Basic Cell Culture, secondedition (University Press: Oxford, UK,2001), p. 166. ��

Corresponding author Sue Clarke ishead of manufacturing development;and Janette Dillon, Ann Smith, andElizabeth Sotheran are developmentscientists at Lorantis Ltd., 410Cambridge Science Park, CambridgeCB4 0PE, UK; 44-1223-702500, fax 44-1223-702599; sue.clarke@lorantis.com, www.lorantis.com.

FFiigguurree 44:: Analytical cloning