464 S.K. Yoon et al. / Journal of Biotechnology 122 (2006) 463–4721. Introduction may be able to alleviate growth suppression of rCHO cells at low culture temperature through adaptation, Chinese hamster ovary (CHO) cell culture is becom- and thereby, further increase the yield as well as theing increasingly important for the production of human volumetric productivity of recombinant protein at lowrecombinant proteins especially in the pharmaceuti- culture temperature.cal ﬁeld. Increasing recombinant protein yields, and Previously, we investigated the effect of culturethereby, reducing production costs is a major biotech- temperature on rCHO cell growth and erythropoietinnological target. Culture temperature is one of the most (EPO) production and found the beneﬁcial effect ofimportant parameters to be optimized for increasing lowering culture temperature on EPO production (Yoonrecombinant protein yields. CHO cells, like most mam- et al., 2003). Here, we adapted rCHO cells producingmalian cells, are cultivated at 37 ◦ C to simulate the body EPO to low culture temperature to determine whetherenvironment. the beneﬁcial effect of low culture temperature on EPO Lowering culture temperature below 37 ◦ C production can be enhanced through adaptation. Wedecreases speciﬁc growth rate (µ). It has been also monitored the CIRP content of the cells duringsuggested that the induction of cold-inducible RNA- adaptation to low culture temperature. Furthermore, tobinding protein (CIRP) is responsible in part for establish generality of the effect of adaptation to lowthe growth suppression at low culture temperature culture temperature on CHO cell growth and recombi-(Nishiyama et al., 1997). Despite growth suppression, nant protein production, rCHO cells producing follicle-a number of studies demonstrate the beneﬁcial effects stimulating hormone (FSH) were also adapted to lowof low culture temperature. Recombinant CHO culture temperature.(rCHO) cell culture at low temperature improves cellviability and reduces contamination by endogenousCHO cell proteins for a longer period (Furukawa 2. Materials and methodsand Ohsuye, 1998; Yoon et al., 2003). Moreover,lowering the culture temperature often increases 2.1. Cell lines and culture mediaspeciﬁc productivity (q), though its effect on q isvariable among rCHO cell lines (Yoon et al., 2004). As The rCHO cells producing human EPO (CHO-a result, recombinant protein yields can be increased EPO, LGE10-9-27) were used in this study (Yoonsigniﬁcantly by lowering the culture temperature et al., 2003). They were established by transfection(Bollati-Fogolin et al., 2005; Fox et al., 2004; Yoon of a vector containing the dihydrofolate reductaseet al., 2003). However, due to the decreased µ, no (dhfr) and human EPO gene into dhfr-deﬁcient CHOsubstantial increase in a volumetric productivity, cells (DUKX-B11, ATCC CRL-9096) and subsequentwhich is also an important factor considered in process DHFR/methotrexate (MTX)-mediated gene ampliﬁca-optimization, was obtained by lowering culture tion. The stable rCHO cell line producing EPO wastemperature. Accordingly, to maximize the beneﬁcial selected at 5 M MTX. The rCHO cells producing FSHeffect of low culture temperature, growth suppression (CHO-FSH) were established in a similar manner, butat low culture temperature needs to be alleviated. selected at 0.32 M MTX. Adaptation of cells to low culture temperature is The proprietary protein-free media, LG-PF5 con-probably the simplest approach to alleviate the growth taining 5 M MTX and LG-D/F4 containing 0.32 Msuppression to some extent. When subjected to envi- MTX (LG Life Science, Daejon, Korea), were usedronmental changes, mammalian cells improve their for suspension cultures of CHO-EPO and CHO-cell growth through adaptation. A successful case FSH cells, respectively. Prior to the suspension cul-of adaptation was achieved in hyperosmotic culture tures, rCHO cells were maintained in 125 ml spin-of hybridoma cells for antibody production. Through ner ﬂasks (Bellco Glass, Vineland, NJ) containingadaptation to hyperosmolar medium, hybridoma cell the corresponding protein-free medium for severalgrowth was improved at elevated osmolality, resulting weeks on a magnetic stirrer plate (Bellco Glass) atin the increased ﬁnal antibody concentration in hyper- 60 rpm, placed in a 5% CO2 incubator, humidiﬁedosmotic batch culture (Chua et al., 1994). Likewise, we at 37.0 ◦ C.
S.K. Yoon et al. / Journal of Biotechnology 122 (2006) 463–472 4652.2. Cell culture ity (qEPO ) and speciﬁc FSH productivity (qFSH ) were based on the data collected during the growth phase and For batch cultures, exponentially growing cells at were evaluated from a plot of EPO and FSH concen-37.0 ◦ C were inoculated at 2 × 105 cells/ml into spinner trations against the time integral values of the growthﬂasks with a working volume of 80 ml. The spinner curve, respectively (Renard et al., 1988). The volumet-ﬂasks were agitated at 60 rpm on a magnetic stirrer ric productivity was calculated by dividing the productplate (Bellco Glass) placed in 5% CO2 incubators, and concentration by the culture time.humidiﬁed at either 32 ◦ C or 37 ◦ C. For adaptation experiments, cells were cultivated 2.5. Southern blot hybridizationin a repeated batch mode. When viable cell con-centration reached (1.5–2.0) × 106 cells/ml, cells were For Southern blot analysis, chromosomal DNA washarvested by centrifugation and were re-inoculated extracted from rCHO cells using genomic DNA puriﬁ-at 2 × 105 cells/ml into the spinner ﬂasks containing cation kit (Promega, Madison, WI). After 2 g of chro-fresh media. Approximately 0.5 ml of culture sample mosomal DNA was treated with EcoRI at 37 ◦ C forwere taken daily from the spinner ﬂasks. Cell con- 18 h, it was loaded on 1% (w/v) agarose gel for elec-centration was estimated using a hemacytometer and trophoresis. The separated DNA was characterized byviable cells were distinguished from dead cells using Southern blot hybridization with the correspondingthe trypan blue dye exclusion method. Culture super- probe. For preparation of the EPO probe, human EPOnatants, after centrifugation, were aliquoted and kept cDNA (579 base pairs) was used as a template. For thefrozen at −70 ◦ C until needed for recombinant protein preparation of FSH probes, human FSH cDNAs (351assays. base pairs for ␣-unit and 390 base pairs for ␤-unit of FSH) were used as templates. The probes were then2.3. EPO and FSH assays radioactively labeled by random primed incorporation of [␣-32 P]dCTP (Amersham, Amersham, UK). Mem- The secreted EPO concentration in the medium was brane transfer, prehybridization and hybridization werequantiﬁed by enzyme-linked immunosorbent assay performed using the protocol described (Sambrook and(ELISA), as described previously (Yoon et al., 2003). Russell, 2001). After hybridization and washing, theIn brief, 96-well plates (Nunc) were coated with mon- blots were exposed to a phosphor screen (Molecularoclonal mouse anti-human EPO antibody (R&D Sys- Dynamics, Sunnyvale, CA) for 3 h and the band inten-tems, Minneapolis, MN) and blocked with bovine sity was quantitated using PhosphorImager (Molecularserum albumin (BSA) and Tween 20. The internal EPO Dynamics).standard and culture supernatants diluted with blockingbuffer were loaded on wells and treated with poly- 2.6. Reverse transcriptase-polymerase chainclonal rabbit anti-human EPO antibody (R&D Sys- reaction (RT-PCR) analysistems) in diluent solution. The horseradish peroxidase(HRP) conjugated goat anti-rabbit IgG (ICN, Costa The mRNA levels of CIRP and ␤-actin in the cellsMesa, CA) was used as an enzyme–antibody conju- were quantiﬁed by RT-PCR analysis. For RT-PCR, totalgate. The secreted FSH concentration in the medium RNA from cells was extracted with TRI reagentTMwas quantiﬁed using an ELISA kit (IBL, Hamburg, (Sigma) according to the protocol provided by theGermany), according to the protocol provided by the manufacturer. First-strand cDNA was synthesized frommanufacturer. 5 g of total RNA using the SuperScriptTM synthe- sis system (Invitrogen, Carlsbad, CA). CIRP mRNA2.4. Calculation of µ, q and volumetric was ampliﬁed using the primer pair 5 -CCATGG-productivity CATCAGATGAAGGC-3 (NCBI gene no. AY359860, 30–50) and 5 -ATGGAAGGACGATCTGGACG-3 The µ was calculated by plotting the logarithm of (NCBI gene no. AY359860, 561–580) for 26 cyclesviable cell concentration versus culture time during the (94 ◦ C for 30 s, 54 ◦ C for 30 s, 72 ◦ C for 60 s). ␤-exponential growth phase. The speciﬁc EPO productiv- actin mRNA was ampliﬁed using the primer pair 5 -
466 S.K. Yoon et al. / Journal of Biotechnology 122 (2006) 463–472CCAGTTCGCCATGGATGACG-3 (NCBI gene no.NM007393, 71–90) and 5 -GCAGCTCAGTAACAG-TCCGC-3 (NCBI gene no. NM007393, 1209–1228)for 26 cycles (94 ◦ C for 30 s, 56 ◦ C for 30 s, 72 ◦ C for90 s). The reaction products were then resolved on a1% agarose gel. DNA bands were visualized using anultraviolet light source after staining with 1 g/ml ofethidium bromide.3. Results3.1. Growth and EPO production of CHO-EPOcells unadapted to low culture temperature To characterize cell growth and EPO production atlow culture temperature, CHO-EPO cells, which hadbeen growing at 37 ◦ C, were cultivated at two differenttemperatures, 32 ◦ C and 37 ◦ C. Fig. 1 shows cell growth and EPO production pro-ﬁles during batch cultures. When subjected to low cul-ture temperature, cell growth was suppressed (Fig. 1A).At 32 ◦ C, cells started to grow after a long lag period(∼ h) and the maximum viable cell concentration =80obtained was only 1.9 × 106 cells/ml. On the otherhand, cells at 37.0 ◦ C started to grow exponentiallywithout a lag period and reached a maximum viable cellconcentration of 4.2 × 106 cells/ml. Despite depressed Fig. 1. Effect of culture temperature on cell growth and EPO pro- duction of CHO-EPO cells ( : 32 ◦ C, ᭹: 37 ◦ C). (A) Viable cellcell growth, the maximum EPO concentration at 32 ◦ C concentration, (B) EPO concentration and (C) volumetric EPO pro-was much higher than that at 37 ◦ C (Fig. 1B). The max- ductivity.imum EPO concentration at 32 ◦ C was 106.2 g/ml,which was 1.7-fold higher than that at 37 ◦ C. Extended 3.2. Sequential adaptation of CHO-EPO cells toculture longevity as well as enhanced qEPO at 32 ◦ C was low culture temperatureresponsible for increased maximum EPO concentrationat 32 ◦ C. However, the volumetric EPO productivity To determine whether adaptation of cells to low cul-was rather decreased at 32 ◦ C because of slowed growth ture temperature can alleviate growth suppression, cellsrate (Fig. 2B). The µ, qEPO and maximum volumetric were cultivated at 32 ◦ C in a repeated batch mode usingEPO productivity at 32 ◦ C and 37 ◦ C were summarized spinner ﬂasks. Cells were also cultivated at 37 ◦ C as ain Table 1. control.Table 1Effect of adaptation to low culture temperature on µ, qEPO and maximum volumetric productivity of CHO-EPO cellsCulture Temperature (◦ C) Speciﬁc growth Speciﬁc EPO productivity, Maximum volumetric rate, µ (h−1 ) qEPO (g/(106 cells h)) productivity (g/(ml h))Unadapted cells 37 0.027 0.26 0.39 32 0.011 0.39 0.28Adapted cells 32 0.019 0.20 0.18
S.K. Yoon et al. / Journal of Biotechnology 122 (2006) 463–472 467Fig. 2. Changes of growth of CHO-EPO cells during adaptation at Fig. 3. Changes of EPO production of CHO-EPO cells during adap-32 ◦ C ( ) and 37 ◦ C (᭹). (A) Viable cell concentration and (B) µ. tation at 32 ◦ C ( ) and 37 ◦ C (᭹). (A) EPO concentration and (B) qEPO . Fig. 2 shows adaptive growth responses of cells tolow culture temperature. When cells were cultivated at32 ◦ C, cell growth rate appeared to increase during ear- (mean ± standard deviation, n = 10) up to 35 genera-lier culture passages. On the other hand, cell growth rate tions.at 37 ◦ C appeared to be constant throughout repeated Fig. 3 shows changes of EPO production proﬁlesbatch cultures (Fig. 2A). To quantitate changes in cell during adaptation to low culture temperature. Duringgrowth rate, µ at each batch culture was calculated and earlier culture passages at 32 ◦ C, decrease in EPO pro-plotted as a function of generations (Fig. 2B). At 32 ◦ C, duction was apparent despite improved cell growthµ gradually increased from 0.011 h−1 to 0.019 h−1 for rate (Fig. 3A). At 32 ◦ C, qEPO gradually decreasedthe ﬁrst 15 generations, and thereafter, did not increase from 0.39 g/(106 cells h) to 0.20 g/(106 cells h) forfurther. These results imply that growth suppression the ﬁrst 15 generations, which was even lower thanat low culture temperature can be alleviated by an qEPO at 37 ◦ C. Thereafter, qEPO at 32 ◦ C remainedadaptation. However, the µ at 32 ◦ C was still lower almost constant (Fig. 3B). In contrast, qEPO at 37 ◦ Cthan that at 37 ◦ C, even after cells were fully adapted. was almost constant at 0.26 ± 0.02 g/(106 cells h) upAt 37 ◦ C, µ was almost constant at 0.027 ± 0.001 h−1 to 35 generations.
468 S.K. Yoon et al. / Journal of Biotechnology 122 (2006) 463–472 growth rate achieved by adaptation did not increase EPO production (Fig. 4B and C). In fact, the adaptation decreased the maximum EPO concentration and the maximum volumetric productivity by 59% and 35%, respectively. The µ, qEPO and maximum volumetric EPO productivity of adapted cells at 32 ◦ C and 37 ◦ C were also summarized in Table 1. 3.3. Growth and FSH production of CHO-FSH cells unadapted to low culture temperature To accept generality of the effect of adaptation to low culture temperature on growth and recombinant protein production of CHO cells, CHO-FSH cells were also adapted to low culture temperature. First, CHO- FSH cells, which had been growing at 37 ◦ C, were cul- tivated at two different temperatures, 32 ◦ C and 37 ◦ C to characterize cell growth and FSH production at low culture temperature. Like CHO-EPO cells, growth of CHO-FSH cells was also suppressed at 32 ◦ C, but to a lesser extent. No signiﬁcant lag period was observed and the max- imum viable cell concentration achieved at 32 ◦ C (4.7 × 106 cells/ml) was comparable to that at 37 ◦ C (4.9 × 106 cells/ml). Despite depressed cell growth, the maximum FSH concentration at 32 ◦ C (7.7 g/ml) wasFig. 4. Effect of adaptation of cell growth and EPO production of 4.2-fold higher than that at 37 ◦ C (1.8 g/ml) becauseCHO-EPO cells at 32 ◦ C (adapted cells: , unadapted cells: ). (A) of both extended culture longevity and enhanced qFSHViable cell concentration, (B) EPO concentration and (C) volumetric at 32 ◦ C. Moreover, unlike CHO-EPO cells, the vol-EPO productivity. umetric FSH productivity was also increased signiﬁ- cantly at 32 ◦ C, but not as much as the maximum FSH To evaluate the effect of adaptation to low culture concentration. The µ, qFSH and maximum volumetrictemperature on cell growth and EPO production, a FSH productivity at 32 ◦ C and 37 ◦ C were summarizedbatch culture of adapted cells (11 culture passages at in Table 2.32 ◦ C) was carried out at 32 ◦ C. Fig. 4 shows the pro-ﬁles of cell growth and EPO production of adapted cells 3.4. Sequential adaptation of CHO-FSH cells toas well as unadapted cells used as a reference (Fig. 1). low culture temperatureAdaptation to low culture temperature signiﬁcantlyreduced a lag period (Fig. 4A). The µ also increased Like CHO-EPO cells, CHO-FSH cells were culti-from 0.011 h−1 to 0.019 h−1 . However, improved cell vated at 32 ◦ C and 37 ◦ C in a repeated batch mode.Table 2Effect of adaptation to low culture temperature on µ, qFSH and maximum volumetric productivity of CHO-FSH cellsCulture Temperature (◦ C) Speciﬁc growth Speciﬁc FSH productivity, Maximum volumetric rate, µ (h−1 ) qFSH (ng/(106 cells h)) productivity (ng/(ml h))Unadapted cells 37 0.026 8.2 10.3 32 0.015 21.0 27.7Adapted cells 32 0.018 16.3 17.9
S.K. Yoon et al. / Journal of Biotechnology 122 (2006) 463–472 469 At 37 ◦ C, cell growth rate appeared to be constant perature did not increase FSH production. In fact, thethroughout repeated batch cultures. On the other hand, adaptation decreased the maximum FSH concentrationcell growth rate at 32 ◦ C appeared to slightly increase and the maximum volumetric productivity by 49% andduring subsequent culture passages. Average µ of the 35%, respectively. The µ, qFSH and maximum volu-ﬁrst three batches was 0.145 ± 0.001 h−1 , while that of metric FSH productivity of adapted cells at 32 ◦ C andthe last three batches was 0.177 ± 0.001 h−1 . Unlike 37 ◦ C were also summarized in Table 2.EPO production, a signiﬁcant decrease in FSH produc-tion was not observed during repeated batch cultures 3.5. Analysis of CIRP mRNA by RT-PCRat 32 ◦ C because qFSH was not decreased as muchas qEPO . At 32 ◦ C, qFSH gradually decreased from To investigate whether the alleviation of growth sup-21.0 ng/(106 cells h) to 16.1 ng/(106 cells h) for the ﬁrst pression at low culture temperature by adaptation was15 generations, which was still signiﬁcantly higher related to down-regulation of CIRP, mRNA contents ofthan qFSH at 37 ◦ C. Thereafter, qFSH at 32 ◦ C remained CIPR at different generations of CHO-EPO and CHO-almost constant. At 37 ◦ C, qFSH was slightly decreased FSH cells were quantiﬁed by RT-PCR. The mRNAfrom 8.2 ng/(106 cells h) to 6.0 ng/(106 cells h) during content of ␤-actin was used as an internal control forrepeated batch cultures. normalizing mRNA level of CIRP. As observed in CHO-EPO cells, improved cell Fig. 5 shows the mRNA content of CIRP at differ-growth rate achieved by adaptation to low culture tem- ent generations. Regardless of cell lines used, the levelFig. 5. RT-PCR analysis of CIRP in CHO-EPO cells (A and B) and CHO-FSH cells (C and D) at different generations. The RT-PCR of ␤-actinmRNA was conducted for the normalization of CIRP mRNA level. The relative CIRP mRNA content was estimated as a ratio of the CIRPmRNA content to ␤-actin mRNA content for each cell sample.
470 S.K. Yoon et al. / Journal of Biotechnology 122 (2006) 463–472Fig. 6. Southern blot analysis of EPO genes (A and B) and FSH genes (C and D) at different generations. Arrows indicate the positive controlprepared by digesting the plasmids containing EPO or FSH gene with EcoRI. The relative gene copy numbers were estimated as a ratio of theDNA content at different generations to that at earliest generations (three generations for EPO and nine generations for FSH).of CIRP mRNA signiﬁcantly increased at 32 ◦ C. Its extracted from the cells at different generations oflevel was over 4-fold higher than that at 37 ◦ C. Since it CHO-EPO and CHO-FSH cells were characterized byhas been suggested that the induction of CIRP is partly Southern blot hybridization. The band intensities ofresponsible for growth suppression at low culture tem- samples prepared at different generations were normal-perature (Nishiyama et al., 1997), it is expected that the ized by that at the earliest generation.mRNA level of CIRP decreases during the adaptation As shown in Fig. 6, both EPO and FSH gene con-to low culture temperature. However, it was observed tents were not changed signiﬁcantly with increasingthat the CIRP mRNA level of both cell lines, despite generations, suggesting that the decreased qEPO duringimproved cell growth, did not decrease during adapta- the adaptation was not due to the loss of foreign genetion at 32 ◦ C. Thus, improved cell growth at low culture copies.temperature by adaptation did not appear to be relatedto changes in CIRP transcription level. 4. Discussion3.6. Analysis of foreign gene copies by Southernblot hybridization Lowering culture temperature, which is easily exe- cuted in cell culture processes, has become a popular To investigate whether decreased q during adapta- method for increasing the product titer of rCHO cells intion to low temperature was related to changes in for- commercial processes. However, the low culture tem-eign gene content in CHO cells, chromosomal DNAs perature suppresses cell growth, and therefore it does
S.K. Yoon et al. / Journal of Biotechnology 122 (2006) 463–472 471not increase volumetric productivity substantially in during adaptation to low culture temperature, suggest-batch cultures of rCHO cells. Accordingly, improve- ing that CIRP expression may not be the only cause forment of hypothermic cell growth rate may further growth suppression at low culture temperature.extend the beneﬁcial effect of low culture temperature Unfortunately, the recombinant protein productionto include enhanced volumetric productivity. of both cell lines was not increased during adapta- At the low culture temperature (32 ◦ C), growth of tion because of decreased qs. The qEPO and qFSH wereboth CHO-EPO and CHO-FSH cells used in this study decreased by 49% and 22%, respectively, by adapta-was signiﬁcantly depressed, but to a different degree. tion. The q in CHO-FSH cells is more than 10-foldDespite depressed cell growth, the maximum EPO and lower than in CHO-EPO cells, which may accountFSH concentrations obtained at 32 ◦ C were 1.7-fold for the higher relative productivities in adapted cellsand 4.2-fold higher than those at 37 ◦ C, respectively. producing FSH when compared to those producingHowever, because of slowed growth rates, the volu- EPO. Thus, adaptation to the low culture tempera-metric productivities were not increased as much as the ture increased neither the volumetric productivitiesmaximum product concentrations. In fact, the volumet- nor the maximum product concentrations of both cellric EPO productivity was rather decreased, compared lines because the detrimental effect of decreased q out-with that at 37 ◦ C. weighed the beneﬁcial effect of increased µ. When cells are continuously exposed to unfavor- For rCHO cells producing a chimeric antibody,able growth conditions, they often change the levels of decrease in speciﬁc antibody productivity (qAb ) wasmany enzymes and accumulation of metabolites to sur- observed in the presence of MTX with increasing gen-vive, and therefore, improve their growth (Christie and erations and was found to be related to the loss ofButler, 1999; Chua et al., 1994). Adaptation of BHK-21 antibody gene copies (Kim et al., 1998). To determinecells to glutamate-based medium was associated with whether the decrease in q during adaptation was relatedchanges in the activities of enzymes involved in glu- to changes in foreign gene copies, Southern blot analy-tamate or glutamine metabolism (Christie and Butler, ses of CHO-EPO and CHO-FSH cells were carried out1999). Accordingly, the adaptation of rCHO cells to and showed that foreign gene copies of both cell linesthe low culture temperature is likely to improve their did not decrease during adaptation. Thus, the decreasehypothermic cell growth rates. In this study, we investi- in q of CHO-EPO and CHO-FSH cells during adapta-gated whether an adaptation of rCHO cells (CHO-EPO tion was not due to the loss of foreign gene copies.and CHO-FSH) to the low temperature can alleviate The decrease in q of CHO-EPO and CHO-FSHgrowth suppression at low culture temperature while cells during adaptation may be related to changes inmaintaining enhanced q. µ. When µ and q values collected during adaptation As expected, hypothermic cell growth of both CHO- were plotted against each other, µ was inversely pro-EPO and CHO-FSH cells gradually improved during portional to q (Fig. 7). However, in order to determineadaptation to the low culture temperature. The µs ofCHO-EPO and CHO-FSH cells at 32 ◦ C were increasedby 73% and 20%, respectively, by adaptation. Although the molecular mechanisms of slow growthrate associated with lower culture temperature have notbeen completely understood (Glofcheski et al., 1993;Matz et al., 1996), a discovery of the cold-inducibleRNA-binding protein (CIRP) in mouse ﬁbroblasts byNishiyama et al. (1997) suggests that growth suppres-sion at hypothermic conditions is due to an activeresponse by the cell rather than to passive thermaleffects. Thus, we inferred that the improved hypother-mic growth by adaptation might be related to changesin CIRP expression. However, the CIRP mRNA levels Fig. 7. Relationship between µ and q (CHO-EPO cells: , CHO-of both rCHO cell lines did not change signiﬁcantly FSH cells: ). The values were collected during adaptation.
472 S.K. Yoon et al. / Journal of Biotechnology 122 (2006) 463–472whether this inverse relationship between µ and q Chua, F.K., Yap, M.G., Oh, S.K., 1994. Hyper-stimulation of mon-during adaptation to the low culture temperature is a oclonal antibody production by high osmolarity stress in eRDFcommon phenomenon, more rCHO cell lines need to medium. J. Biotechnol. 37, 265–275. Christie, A., Butler, M., 1999. The adaptation of BHK cells to abe tested. Recently, the relationships between µ and non-ammoniagenic glutamate-based culture medium. Biotech-q were reviewed and discussed in depth (Dinnis and nol. Bioeng. 64, 298–309.James, 2005). Dinnis, D.M., James, D.C., 2005. Engineering mammalian cell facto- Improvement of hypothermic cell growth by adapta- ries for improved recombinant monoclonal antibody production:tion does not appear to be a good strategy for increasing lessons from nature? Biotechnol. Bioeng. 91, 180–189. Fox, S.R., Patel, U.A., Yap, M.G., Wang, D.I., 2004. Maximizingvolumetric productivity because of decreased q. Alter- interferon-gamma␥ production by Chinese hamster ovary cellsnatively, a biphasic process, wherein cells are ﬁrst cul- through temperature shift optimization: experimental and mod-tivated at 37 ◦ C in the growth phase for high µ and then eling. Biotechnol. Bioeng. 85, 177–184.temperature is shifted to low culture temperature in the Furukawa, K., Ohsuye, K., 1998. Effect of culture temperature on a recombinant CHO cell line producing a C-terminal ␣-amidatingproduction phase for high q, may be used in order to enzyme. Cytotechnology 26, 153–164.increase the volumetric productivity (Bollati-Fogolin Glofcheski, D.J., Borrelli, M.J., Stafford, M., Kruuv, J., 1993. Induc-et al., 2005; Fox et al., 2004; Rodriguez et al., 2005). tion of tolerance to hypothermia and hyperthermia by a common In conclusion, an adaptation of CHO-EPO and mechanism in mammalian cells. J. Cell. Physiol. 156, 104–111.CHO-FSH cells to low culture temperature can allevi- Kim, S.J., Kim, N.S., Ryu, C.J., Hong, H.J., Lee, G.M., 1998. Char- acterization of chimeric antibody producing CHO cells in theate hypothermic growth suppression. This alleviation course of dihydrofolate reductase-mediated gene ampliﬁcationof growth suppression for adapted cells was not related and their stability in the absence of selective pressure. Biotech-to the down-regulation of CIRP expression. However, nol. Bioeng. 58, 73–84.since the q of both rCHO cell lines decreased during Matz, J.M., LaVoi, K.P., Moen, R.J., Blake, M.J., 1996. Cold-inducedadaptation to the low culture temperature, improvement heat shock protein expression in rat aorta and brown adiposeof hypothermic cell growth by adaptation was not appli- tissue. Physiol. Behav. 60, 1369–1374. Nishiyama, H., Itoh, K., Kaneko, Y., Kishishita, M., Yoshida, O.,cable for enhanced recombinant protein production. Fujita, J., 1997. A glycine-rich RNA-binding protein mediating cold-inducible suppression of mammalian cell growth. J. Cell Biol. 137, 899–908.Acknowledgments Renard, J.M., Spagnoli, R., Mazier, C., Salles, M.F., Mandin, E., 1988. Evidence that monoclonal antibody production kinetics is related to the integral of viable cells in batch systems. Biotechnol. This work was supported in part by grants from Lett. 10, 91–96.the Ministry of Commerce, Industry and Energy Rodriguez, J., Spearman, M., Huzel, N., Butler, M., 2005. Enhanced(10006913), the National Research Laboratory Pro- production of monomeric interferon-␤ by CHO cells through thegram (2000-N-NL-01-C-228) and the Brain Korea 21 control of culture conditions. Biotechnol. Prog. 21, 22–30.Program. Sambrook, J., Russell, D.W., 2001. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, New York. Yoon, S.K., Song, J.Y., Lee, G.M., 2003. Effect of low culture temper- ature on speciﬁc productivity, transcription level, and heterogene-References ity of erythropoietin in Chinese hamster ovary cells. Biotechnol. Bioeng. 82, 289–298.Bollati-Fogolin, M., Forno, G., Nimtz, M., Conradt, H.S., Etchever- Yoon, S.K., Hwang, S.O., Lee, G.M., 2004. Enhancing effect of low rigaray, M., Kratje, R., 2005. Temperature reduction in cultures culture temperature on speciﬁc productivity of recombinant Chi- of hGM-CSF-expressing CHO cells: effect on productivity and nese hamster ovary cells: clonal variation. Biotechnol. Prog. 20, product quality. Biotechnol. Prog. 21, 17–21. 1683–1688.