150

Chinese Hamster Ovary (CHO) cells are avaluable tool for producing recombinant proteinswith "native" mammalian glycosylation patterns.Following their introduction in the 1950s, CHOcell lines have become a workhorse formanufacturing recombinant proteins. Geneticengineers can modify CHO cells to produceproteins with "mammalian" post-translationalmodifications, and through gene amplificationcan selectively produce high levels ofrecombinant proteins.

Process development, using CHO cell lines,focuses on achieving the maximum amount ofactive product. Optimization of the amount ofactive product can be achieved in at least twobasic ways. The first way is to increase thespecific productivity (i.e., the product per cell)through cell line development. Cell linedevelopment may include both sub-cloning thecell line to select higher producing clones anduse of gene amplification. Drug resistance hasbeen the tool of choice in the biopharmaceuticalindustry to induce gene amplification.

By combining the gene of interest with aselectable gene, increased production levelscan be accomplished. Using productivity-per-cellapproaches, scientists have increasedexpression levels more than 1,000-fold(Schimke, 1982). Two gene amplificationsystems used in CHO cells are the dihydrofolatereductase (DHFR) system using methotrexate(MTX) resistance, and the glutamine synthetase(GS) system using methionine sulfoxamine(MSX) resistance. The DHFR enzyme catalyzesthe conversion of folate to tetrahydrofolate(Figure 1).

This precursor is necessary for the de novosynthesis of purines, pyrimidines, and glycine(Goeddel, 1990). Methotrexate is a drug which

is similar (i.e., an analog) to folate. MTX bindsto DHFR, thereby inhibiting the production oftetrahydrofolate. With insufficient levels ofDHFR, cells are deprived of nucleosideprecursors (hypoxanthine and thymidine) anddie. Gene amplification is a technique used bymolecular biologists. They transform cells withrecombinant DNA consisting of the gene ofinterest closely linked to the gene for DHFR.Then during gene amplification the cells arecultured in increasingly higher levels of MTX.Those CHO cells that have increased copies ofthe DHFR gene, and therefore higher levels ofthe enzyme, are selected.

The GS/MSX system is similar to theDHFR/MTX system. The GS enzyme catalyzesthe production of glutamine from glutamate andammonia (Figure 2). Methionine sulfoxaminebinds to the GS enzyme and prevents theproduction of glutamine. Gene amplificationoccurs when cells are subjected to increasingconcentrations of MSX.

A second way to achieve higher recombinantprotein yields is to increase the cell yield (i.e.,cells per volume) of the process. This may beaccomplished through process development(e.g., batch, fed-batch, perfusion, etc.) andmedium development. By increasing the cellsper volume per day, higher levels of productmay be produced.

To demonstrate the utility of this proteinproduction system, we used CHO cell line(ATCC® CRL-10154 CHO 5/9 M! 3-18), whichproduces human macrophage colony stimulatingfactor (hm-CSF). This cell line was producedusing a DHFR-CHO DG44 cell clone as theparental line. Gene amplification wasperformed using the DHFR/MTX system.The cell growth and protein production kineticsof this cell line were studied using a powdered

medium developed for CHO cells byThemo Scientific HyClone. Standardculturing procedures for CHO cellswere used for these evaluations(Table 1).

Initial evaluations comparedcommercially available serum-free

and protein-free media to Themo ScientificHyClone PF-CHOMPSq (SH30333) using theCHO 5/9 M! 3-18 cell line. Representativeresults obtained in these comparisons areshown in Table 2 and Figure 3.

Table 1: Evaluation Parameters foruse with Shaker and Spinner CulturesTemperature 37°CCO2 Atmosphere 5%Agitation Shaker 100–130 rpm

Spinner 60–80 rpmOxygen AtmosphericBuffering 2 g/L Sodium

Bicarbonate

Table 2: Comparison of SH30333 vsCompetitor MediumParameter SH30333Maximum CellDensity (Mean)

212.1% Higher

MaximumPopulation DoublingTime (Mean)

19.6% Higher

Maximum ProteinProduction (Mean)

74.6% Higher

Application-Specific Technical Information—Application Notes

Chinese Hamster Ovary Cells for the Productionof Recombinant GlycoproteinsCamire, Joseph. Art to Science, Vol. 19, No. 1; Logan, UT, 2000

Analysis of growth promotion and hm-CSFproductivity kinetics showed a maximumspecific productivity (Figure 4) of1.26 pg/cell/day. These data indicate thatincreased protein expression with this cellclone is cell-growth associated; that is, themaximum specific productivity is seen duringlog-phase growth. Additional studies analyzedextended cell performance (Figure 5).In the case of this clone, cells were culturedfor more than 70 days with approximately 68cumulative population doublings (CPD). TheCPD was linear over the course of the studyindicating good stability of the cell line in thismedium. Following these evaluations, cultureswere scaled up to increase the total proteinyield by increasing the cell population density.

PF-CHOMPSqmedium is a two-part powder,protein-free medium designed for suspensiongrowth of CHO cell lines. The medium wasdesigned to be "regulatory friendly" by limitinganimal-derived components to just two, i.e.,cod liver oil and cholesterol. This productcomes in a range of standard packagingconfigurations (Table 3).

HyClone_150:Layout 1 12/27/2007 3:16 PM Page 150

Some typical components thatmay be added at the time of liquidpreparation are: sodiumbicarbonate, L-glutamine (forcell clones which require thiscomponent for growth), andPluronic F68e. L-glutaminesupplementation is recommendedfor DHFR systems.

We anticipate that users will evaluate themedium prior to scale-up procedures. Werecommend using cells that have beenpreviously adapted to the medium to eliminatecarryover variables from prior sera or media.

Through customer collaborations, we havesuccessfully optimized PF-CHOMPS tospecific CHO clones. Contact your salesrepresentative to inquire about availabilityof these services.

We anticipate that users will evaluate themedium prior to scale-up procedures. Werecommend using cells that have beenpreviously adapted to the medium.

References:1. Schimke, R.T. (ed.). 1982. Gene AmplificationColdspring Harbor Laboratory, Cold SpringHarbor, N.Y.

2. Goeddel, D.V. (ed.). 1990. Methods inEnzymology, Vol. 185 pp. 543-551.

Pluronic, F68e is a registed trademark of BASF Corporation

The medium is designed to be used with awide range of CHO cell lines and productionprocesses, from straight batch to fed-batch tocontinuous perfusion cultures. The formulation,which is deficient in nucleic acids andprecursors (i.e., hypoxanthine and thymidine),is designed for use with the dihydrofolatereductase (DHFR) selection system andmethotrexate (MTX) induced gene amplification.The medium is also deficient in L-glutaminefor use with the glutamine synthetase (GS)selection system and the methioninesulfoxamine (MSX) gene amplification system.

PF-CHOMPS can be purchased in lot sizes aslarge as 400,000 L. This reduces QA/QC cost;by reducing the number of lots needing tests.

151

Tech

nic

alIn

form

atio

n

Table 3. Standard PackagingConfigurationCatalog Number Package SizeSH30333.01 5 LSH30333.02 10 LSH30333.03 50 LSH30333.04 100 LSH30333.05 1,000 L

Figure 3. Comparison of commercially available CHO medium andPF-CHO MPSq (SH30333), using CHO 5/9 Ma 3-18 cells.

Figure 4. Cell growth performance of CHO 5/9 Ma 3-18 cells usingPF-CHO MPSq. Specific productivity appears to be strongly growth-associated with this cell clone.

Figure 5. Adaptation of cell clones to a medium often results indecreased cell growth over multiple passages. The cell growthperformance of CHO 5/9 M! 3-18 was observed to be consistentover an extended period of greater than 70 days when usingPF-CHO MPSq.

HyClone_151-152:Layout 1 12/27/2007 3:17 PM Page 151