SEARCH

SEARCH BY CITATION

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References and Notes

Large-scale transient gene expression (TGE) in mammalian cells is a rapid method to generate recombinant proteins, but the volumetric productivity for secreted proteins is still more than an order of magnitude lower than the yields typically achieved with recombinant cell lines. Here transient recombinant protein production in Chinese hamster ovary cells transfected with linear 25 kDa polyethylenimine was significantly enhanced by incubation of the cells at temperatures ranging from 29 to 33 °C after DNA delivery. With this approach, transient recombinant antibody yields of 60–80 mg/L were achieved within 6 days of transfection. The increase in TGE correlated with the accumulation of cells in the G1 phase of the cell cycle, increased cell size, higher cell viability, higher steady-state levels of transgene mRNA, reduced consumption of nutrients, and decreased accumulation of waste products. The enhancement of TGE was not vector-dependent, but the presence of the woodchuck hepatitis virus post-transcriptional regulatory element in the 3′ untranslated region of the transgene mRNA increased transient recombinant antibody expression more than 3-fold at 31 °C as compared to expression at 37 °C. The yields achieved by the low-temperature enhancement of TGE in CHO cells makes this technology feasible for the rapid production of gram amounts of secreted recombinant proteins at large scale (up to 100 L).


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References and Notes

Large-scale transient gene expression (TGE) is currently being developed for the rapid production of milligram to gram quantities of recombinant proteins for biochemical and preclinical studies (13). Chinese hamster ovary (CHO) and human embryo kidney (HEK293) cells are the major hosts for large-scale TGE as they are readily transfected with low-cost delivery agents such as calcium phosphate (CaPi) and polyethylenimine (PEI) (49). Reported volumetric yields for TGE, however, have only been in the range of 1−25 mg/L for batch cultures at volumes up to about 100 L (2, 3). In contrast, volumetric productivities ranging from 0.5 to 5 g/L have been observed in commercial bioprocesses for the production of therapeutic proteins from recombinant cell lines (10). Considerable improvements in TGE are therefore necessary to match the yields from stable cell lines.

In standard bioprocesses, mammalian cells are cultivated at 37 °C, but it is well-known that mild hypothermic conditions (27−34 °C) can induce an increase in protein production in some recombinant cell lines (1116). More recently, hypothermic conditions have been shown to enhance TGE in CHO cells (17, 18). The reason(s) for the low-temperature enhancement of protein production from mammalian cells are not yet fully understood. Mild hypothermia causes an accumulation of cells in the G1 phase of the cell cycle and an increase in mRNA stability (19, 20). Changes in cell metabolism and gene expression also occur at low temperatures (14, 21, 22). For example, the consumption of glucose and glutamine is reduced, leading to a lower production of cytotoxic metabolites such as lactate and ammonium; proteins able to stabilize mRNA such as cold-induced RNA binding protein (Cirp) and Rbm3 are expressed; and the overall activity of the transcriptional-translational machinery is reduced (16, 23).

By performing TGE under mild hypothermic conditions, we have achieved a substantial increase in volumetric productivity of a recombinant antibody in CHO cells. The increase in production correlated with accumulation of the transfected cells in G1, reduced cellular metabolism, increased steady-state levels of transgene mRNAs, increased cell size, and increased cell viability. The effect of hypothermia on TGE was time-dependent, with the highest yields obtained when cells were transferred to low temperature at 4 h after DNA addition. A large part of the enhancement in antibody production resulted from the inclusion of the woodchuck hepatitis virus post-transcriptional element (WPRE) in the 3' untranslated region of the transgene mRNA. The results contribute to the understanding of low-temperature enhancement of recombinant gene expression and demonstrate the feasibility of producing gram quantities of secreted recombinant proteins in CHO cells by TGE.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References and Notes

Cell Culture. Suspension-adapted CHO DG44 cells (dhfr-/-) were routinely cultivated in ProCHO5 CDM medium (Lonza, Verviers, Belgium) supplemented with 0.68 mg/L hypoxanthine, 0.194 mg/L thymidine, and 4 mM glutamine (SAFC Biosciences, St. Louis, MO) in orbitally shaken 250-mL square-shaped bottles as previously described (24). Biomass was determined by the packed cell volume (PCV) method using ValuPac tubes (Sartorius AG, Göttingen, Germany) as previously described (25). Under standard cultivation conditions at 37 °C, a cell density of 1 × 106 cells/mL was equivalent to a PCV of 0.35% for CHO DG44 cells (25).

Transfection. One day prior to transfection, cells were seeded at 1 × 106 cells/mL in the appropriate volume of fresh ProCHO5 CDM in a square-shaped glass bottle. On the day of transfection, the cells were centrifuged at 800 rpm for 5 min and resuspended in fresh, supplemented ProCHO5 CDM at a cell density of 2 × 106 cells/mL. Transfections were performed in CultiFlask 50 tubes (Sartorius AG) (26). Each transfection was performed in 2.5 mL of culture using 6.25 μg of DNA and 25 μg of linear 25 kDa PEI (27). The DNA and PEI were each diluted in 125 μL of 150 mM NaCl, mixed, and then allowed to stand for 10 min at room temperature prior to addition to the cells. The transfected cultures were incubated at 37 °C in 5% CO2 and 85% humidity with agitation at 180 rpm (27). After 4 h, the cultures were diluted with 2.5 mL of ProCHO5 CDM with supplements. For temperatures other than 37 °C, the cultures were maintained in an ISF-4-W incubator-shaker (Adolf Kühner AG, Birsfelden, Switzerland) in the presence of 5% CO2 and 85% humidity.

Nutrient and Metabolite Quantification. After transfection, 500-μL aliquots of culture were centrifuged, and the supernatant was used to quantify the levels of glucose, glutamine, ammonium, and lactate using the RX Daytona chemistry analyzer (Randox Laboratories Ltd., Crumlin, U.K.).

Cell Size Analysis. After transfection, 20-μL aliquots of cells were diluted with 10 mL of CasyTon solution (Innovatis AG, Durmersheim, Switzerland), and the mean cell volume and cell diameter were quantified with a Casy Counter (Innovatis AG).

Plasmids. pEGFP-N1 expressing the enhanced green fluorescent protein (GFP) gene was purchased from ClonTech (Palo Alto, CA). The expression vectors for the human IgG light (pEAK8-LH39 and pKML) and heavy chain (pEAK8-LH41 and pKMH) genes have been described previously (6, 28). The IgG light and heavy chain cDNAs from pKML and pKMH and the GFP gene from pEGFP-N1 were individually subcloned into pXLGHEK to produce pXLGHEK-RhLC, pXLGHEK-RhHC, and pXLGHEK-EGFP, respectively, as described (29). pXLGHEK-ΔWPRE-RhLC and pXLGHEK-ΔWPRE-RhHC were generated through removal of the WPRE by restriction digestion of pXLGHEK-RhLC and pXLGHEK-RhHC, respectively, with BamHI and HindIII followed by religation. pMYKEF1-puro and pMYKEF1-EGFP-puro have been described previously (30). For the pXLGHEK vectors and pEGFP-N1, the recombinant genes were transcribed from the human cytomegalovirus (hCMV) immediate early promoter; for the pEAK8 vectors, the transgene was transcribed from the human elongation factor-1 alpha (EF-1α) promoter; and for pKML, pKMH, and pMYKEF1-EGFP-puro, the transgene was transcribed from a hybrid promoter composed of the mouse cytomegalovirus (mCMV) immediate early promoter and the EF-1α first intron. Plasmid DNA for transfection was purified using a NucleobondAX anion exchange column (Macherey-Nagel, Düren, Germany) according to the manufacturer's protocol and stored in TE buffer (50 mM Tris-HCl, 5 mM EDTA, pH 7.4).

Cell Cycle Analysis. Cells were transfected with herring sperm DNA (Invitrogen, Basel, Switzerland) as described above. At the times indicated, 1 × 106 cells were mixed with 1 mL of DNACon3 (ConsulAR, Villeneuve, Switzerland) and analyzed with a CyAn ADP High-Performance Research flow cytometer (Dako, Glostrup, Denmark).

mRNA Extraction and Quantification. At day 6 post-transfection, 5 × 106 cells were collected by centrifugation and washed twice with sterile PBS. Total RNA was extracted using a NucleoSpin RNA II kit (Macherey-Nagel, Düren, Germany) according to the manufacturer's protocol. First-strand cDNA synthesis was performed with the M-MLV reverse transcriptase (Invitrogen AG, Basel, Switzerland) using oligo dT (New England Biolabs, Ipswich, MA) as the primer. Each reaction was diluted 10-fold in RNAse-free water for quantitative PCR. The oligonucleotide primers for the amplification of the β-actin (foward 5'-GCTCTTTTCCAGCCTTCCTT-3'; reverse 5'-GAGCCAGAGCAGTGATCTCC-3'), IgG heavy chain (foward 5'-AAGGCTTCTATCCCAGCGACA-3'; reverse 5'-GCATCACGGAGCATGAGAAG-3'), and IgG light chain (foward 5'-TGTCTTCATCTTCCCGCCA-3'; reverse 5'-GCGTTATCCACCTTCCACTGT-3') cDNAs were purchased from SAFC Biosciences (St. Louis, MO). The samples from the first-strand cDNA synthesis were mixed with the appropriate oligonucleotide primer pair and the reaction mix from the Quanti Tect SYBR Green Kit (Qiagen AG, Basel, Switzerland) and amplified according to the manufacturer's protocol. All experiments were performed on an ABI 7700 Real Time PCR system (Applied Biosystem, Foster City, CA). All samples were analyzed in triplicate. Collected data were processed using SDS software (Applied Biosystem, Foster City, CA) to obtain Ct values. The comparative Ct-values method was used to calculate the relative quantity of IgG light and heavy chain mRNAs (31). The quantity of β-actin mRNA was used as an internal control for normalization.

Protein Quantification. For GFP analysis, 100 μL of culture was lysed by addition of 1 vol of 1% Triton X-100 in PBS. After incubation at 37 °C for 1 h with agitation, the fluorescence was measured using a TECAN Saphire II plate-reading fluorometer (TECAN, Männedorf, Switzerland). GFP was excited at 485 nm with a bandwidth of 10 nm, and the emission fluorescence was measured at 515 nm with a bandwidth of 10 nm. IgG concentration was determined by sandwich ELISA using a goat anti-human kappa light chain antibody for capture and alkaline phosphatase-conjugated goat anti-human IgG (BioSource, Lucerne, Switzerland) for detection (32).

Partial Purification and Characterization of Recombinant Antibody. Recombinant antibody was partially purified by affinity chromatography using Streamline Protein A (GE Healthcare, Uppsala, Sweden). The PolyPrep column (BioRad, Hercules, CA) was equilibrated with 5 column volumes (CVs) of 50 mM sodium phosphate (pH 7.0), and then 10 mL of the clarified culture medium was loaded. The column was washed with 10 CVs of 50 mM sodium phosphate (pH 7.0), and IgG was eluted with 2 CVs of 0.1 M sodium citrate (pH 3.0) and immediately buffered with 10% (v/v) 1 M Tris-HCl (pH 8.0). The neutralized eluate was desalted, and the buffer was replaced with deionized water using a Microcon Y-10 centrifuge filter (Millipore, Bedford, MA). The antibody was treated with PNGase F (QAbio, Palm Desert, CA) according to the manufacturer's protocol and analyzed on a 4−12% polyacrylamide gradient gel (Invitrogen) in reducing conditions. Proteins were visualized by Coomassie Blue staining.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References and Notes

Transient Gene Expression under Mild Hypothermic Conditions. To determine if low-temperature exposure enhanced TGE, CHO cells were transfected in agitated 50-mL CultiFlask tubes with a mixture of pXLGHEK-RhLC, pXLGHEK-RhHC, pXLGHEK-EGFP at a mass ratio of 49:49:2 for the expression of a recombinant antibody and GFP. At different times after DNA addition, the cultures were transferred to 31 °C or maintained at 37 °C as a control. The effect of the temperature shift on recombinant protein yield was most pronounced when the cells were transferred to 31 °C 4 h after DNA addition (Figure 1). Transfer of the cultures to 31 °C after being maintained at 37 °C for more than 2 days had little effect on transient recombinant protein yield (Figure 1). For the culture at 31 °C, the antibody yield was about 80 mg/L by 6 days post-transfection. Similar results were obtained for transfections with the same plasmids in 3-L stirred tank bioreactors at culture volumes of 1.5 L with a temperature shift to 31 °C at 4 h post-transfection (data now shown). In addition 31 °C hypothermia resulted in 5- to 10-fold yield improvement with a chimaeric Fc-receptor fusion protein expressed by TGE in round-shaped 10-L bottles with a working volume of 3.5 L (data not shown). The same three expression vectors were then used to determine the effect of temperatures other than 31 °C on TGE. Cells were transfected as described above and shifted to 29, 31, or 33 °C at 4 h post-transfection. At all three temperatures tested, there was enhancement of antibody and GFP production relative to the control culture at 37 °C, with the highest yields of GFP (Figure 2A) and antibody (Figure 2B) at 31 and 33 °C, respectively. Overall, recombinant antibody yield was enhanced 12- to 18-fold and GFP yield was enhanced 2- to 4-fold compared to levels at 37 °C by exposure of cells to mild hypothermia after transfection (Figure 2).

thumbnail image

Figure Figure 1.. Effect of mild hypothermia on transient gene expression. Cells were transfected with pXLGHEK-RhLC, pXLGHEK-RhHC, and pXLGHEK-EGFP at a 49:49:2 ratio (w/w/w). The transfected cells were shifted to 31 °C at the times indicated. The control transfection (C) was maintained at 37 °C. GFP-specific fluorescence (A) and antibody concentration in the medium (B) were measured at 6 days post-transfection. The error bars represent the standard deviation from three independent experiments.

Download figure to PowerPoint

thumbnail image

Figure Figure 2.. Enhancement of transient gene expression at different temperatures. Cells were transfected with pXLGHEK-RhLC, pXLGHEK-RhHC, and pXLGHEK-EGFP as in Figure 1 and either shifted to low temperature (29, 31, or 33 °C) at 4 h post-transfection or maintained at 37 °C (control) as indicated. GFP-specific fluorescence (A) and antibody concentration in the medium (B) were measured at 6 days post-transfection. The fold increase over the control is indicated above the bar representing the reporter protein yield at low temperature. The error bars represent the standard deviation from three independent experiments.

Download figure to PowerPoint

Vector Influence on the Low-Temperature Enhancement of TGE. To determine if the low-temperature enhancement of TGE was dependent on the expression vector, the GFP and IgG cDNAs were subcloned into three different plasmids (see Materials and Methods) and cells were transfected with three different combinations of plasmids: (1) pXLGHEK-RhLC, pXLGHEK-RhHC, and pXLGHEK-EGFP at a ratio of 49:49:2 (w/w/w); (2) pEAK8-LH39, pEAK8-LH41, and pEGFP-N1 at a ratio of 29:69:2 (w/w/w); and pKML, pKMH, and pMYKEF1-EGFP-puro at a ratio of 29:69:2 (w/w/w). The ratios for the IgG light and heavy chain plasmids for the different vector sets were individually optimized previously for transient expression at 37 °C (data not shown). The cells were shifted to 31 °C at 4 h post-transfection while the control transfections were maintained at 37 °C. For all three sets of vectors, enhanced antibody and GFP production at 31 °C relative to that of the control transfection was observed (Figure 3). Among the three GFP vectors, GFP yield was clearly highest for pEGFP-N1 (Figure 3A), while the highest antibody yield was seen with pXLGHEK-RhLC and pXLGHEK-RhHC (Figure 3B).

thumbnail image

Figure Figure 3.. Vector influence on low-temperature enhancement of transient gene expression. Cells were transfected with (1) pEAK8-LH39, pEAK8-LH41, and pEGFP-N1 at a ratio of 29:69:2 (w/w/w); (2) pKML, pKMH, and pMYKEF1-EGFP-puro at a ratio of 29:69:2 (w/w/w); and (3) pXLGHEK-RhLC, pXLGHEK-RhHC, and pXLGHEK-EGFP at a ratio of 49:49:2 (w/w/w). The cells were either shifted to 31 °C at 4 h post-transfection or maintained at 37 °C. GFP-specific fluorescence (A) and antibody concentration in the medium (B) were measured at 6 days post-transfection. The fold increase over the control is indicated above the bar representing the reporter protein yield at low temperature. The error bars represent the standard deviation from three independent experiments.

Download figure to PowerPoint

To further investigate the low-temperature enhancement of antibody production from cells transfected with pXLGHEK-RhHC and pXLGHEK-RhLC, the two vectors were modified to eliminate the post-transcriptional regulatory element WPRE to create pXLGHEK-ΔWPRE-RhLC and pXLGHEK-ΔWPRE-RhHC. Cells were transfected with the two sets of light and heavy chain vectors at a 1:1 (w/w) ratio. At 4 h post-transfection the cultures were shifted to 31 °C, while the control transfections were maintained at 37 °C. The low-temperature enhancement of antibody yield was about 18-fold in the presence of the WPRE but only about 6-fold in its absence (Figure 4A). For each transfection, the steady-state levels of the IgG light and heavy chain mRNAs were determined by quantitative RT-PCR. Mild hypothermic conditions enhanced the levels of both the heavy (Figure 4B) and light chain (Figure 4C) mRNAs at 6 days post-transfection regardless of the presence or absence of the WPRE. However, the light to heavy chain mRNA ratio at both 31 and 37 °C was higher in the presence of the WPRE than in its absence (Figure 4D).

thumbnail image

Figure Figure 4.. Effect of the WPRE on low-temperature enhancement of transient gene expression. Cells were transfected with a 1:1 ratio (w/w) of pXLGHEK-RhLC and pXLGHEK-RhHC (designated WPRE) or pXLGHEK-ΔWPRE-RhLC and pXLGHEK-ΔWPRE-RhHC (designated ΔWPRE). (A) Antibody concentration in the medium was measured by ELISA at 6 days post-transfection. Antibody levels from temperature-shifted cultures were normalized to those of the control cultures at 37 °C. Quantitative RT-PCR was used to determine the steady-state levels of (B) the IgG heavy chain (HC) mRNA and (C) the IgG light chain (LC) mRNA at 6 days post-transfection. The levels of these two mRNAs were normalized to that of β-actin (BA) mRNA. (D) The ratios of the relative levels of LC and HC mRNA for each transfection were determined. The error bars represent the standard deviation from three independent experiments.

Download figure to PowerPoint

Characterization of Low-Temperature Effects on Transfected Cells. To better understand the mechanism of low-temperature enhancement of TGE, CHO cells transfected with pXLGHEK-RhLC, pXLGHEK-RhHC, and pXLGHEK-EGFP were partially characterized by periodically analyzing biomass, cell viability, glucose and glutamine consumption, lactate and ammonium production, and cell size. The cells transferred to 31 °C at 4 h post-transfection were compared to those maintained at 37 °C. Surprisingly, the biomass accumulation for the two cultures was similar at times up to 5 days post-transfection (Figure 5A). This could be explained in part by lower cell viability for the cultures at 37 °C (Figure 5B) and an increase in the average cell size at 31 °C as compared to cells at 37 °C. For the cells at 31 °C, both the cell diameter (Figure 5C) and the cell volume (Figure 5D) were increased as compared to cells at 37 °C. At 31 °C, cell viability was >95% throughout the cultivation period, while cell viability at 37 °C decreased to 40% by 6 days post-transfection (Figure 5B). In addition, the levels of glucose and glutamine consumption were lower at 31 °C than at 37 °C (Figure 5E, F), and the level of ammonium production was less at 31 °C than at 37 °C (Figure 5G). Lactate production was also lower at 31 °C than at 37 °C, but only up to 3 days post-transfection (Figure 5H).

thumbnail image

Figure Figure 5.. Effects of mild hypothermia on cell growth and metabolism. Cells were transfected with pXLGHEK-RhLC, pXLGHEK-RhHC, and pXLGHEK-EGFP at a ratio of 49:49:2 (w/w/w) and shifted to 31 °C at 4 h post-transfection (○) or maintained at 37 °C (·). At the times indicated, biomass (A), cell viability (B), cell diameter (C), cell volume (D) glucose (E), glutamine (F), ammonium (G), and lactate (H) were measured as described in Materials and Methods. Each point represents the average of three independent experiments.

Download figure to PowerPoint

To compare the cell cycle phase distribution of cells maintained at 31 °C and 37 °C, CHO cells were transfected with herring sperm DNA and either shifted to 31 °C at 4 h post-transfection or maintained at 37 °C. At different times the cells were stained with DNACon3 and analyzed by flow cytometry. By 3 days post-transfection, the cell cycle distribution at 31 °C was distinctly different from that at 37 °C, with a higher percentage of cells in the G1 phase for the 31 °C culture (Figure 6). By 6 days post-transfection, almost all of the cells at 31 °C were in G1, but this was not the case for the culture maintained at 37 °C (Figure 6).

thumbnail image

Figure Figure 6.. Effects of mild hypothermia on cell cycle phase distribution. Cells were transfected with herring sperm DNA and either shifted to 31 °C at 4 h post-transfection (left panels) or maintained at 37 °C (right panels). Flow cytometry analysis was performed at the times indicated. The positions of cells in G1, S, and G2 plus M (M) are indicated in the upper left-hand panel.

Download figure to PowerPoint

Effect of Low Temperature on Recombinant Antibody Glycosylation. Recombinant antibody transiently produced at 31 or 37 °C was partially purified by affinity chromatography. As a control, the same antibody produced from a recombinant CHO cell line (CHO AMW) (Maria De Jesus, unpublished data) at 37 °C was purified in the same way. Samples were treated with PNGase F, and then analyzed by SDS-PAGE to estimate the extent of heavy chain glycosylation. A polyclonal human IgG was included in the analysis as a control. The heavy chain of each sample had about 3 kDa of glycosylation judging from the band shift after PNGase F treatment (Figure 7). Thus, the exposure of transfected cells to 31 °C did not affect the level of heavy chain glycosylation.

thumbnail image

Figure Figure 7.. SDS-PAGE analysis of antibody glycosylation. Affinity purified antibody produced from CHO AMW cells (lanes 1 and 2) and from CHO cells transiently transfected with pXLGHEK-RhLC, pXLGHEK-RhHC, and pXLGHEK-EGFP at 37 °C (lanes 5 and 6) or 31 °C (lanes 7 and 8) along with a commercial IgG antibody (lanes 3 and 4) were deglycosylated with PNGase F. Protein samples before and after treatment with PNGase F were electrophoresed on a 4−12% polyacrylamide gradient gel. A molecular weight marker is shown (M). The positions of the glycosylated and unglycosylated heavy chain [HC(+) and HC(−), respectively] and the light chain (LC) are indicated.

Download figure to PowerPoint

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References and Notes

This study demonstrated that a shift in cultivation temperature from 37 to 31 °C at 4 h after transfection of CHO cells with PEI increased transient recombinant antibody yield up to 18-fold to 60−80 mg/L by 6 days post-transfection. This represents the highest level of transient antibody production in mammalian cells reported to date for a batch culture (2). These results have been extended in that low-temperature cultivation of transfected CHO cells could yield 50−60 mg/L within 10 days in orbitally shaken bottles of 5−20 L (33). The enhancement of TGE at 31 °C correlated with an accumulation of cells in the G1 phase of the cell cycle, reduced cellular metabolism, greater cell viability, increased steady-state levels of transgene mRNAs, and increased cell size as compared to cells kept at 37 °C. The extent of the increase in protein yield was partly dependent on the expression vector used for transfection. More specifically, WPRE, which has a post-transcriptional effect on heterologous gene expression, was particularly effective in increasing transient recombinant protein yield at low temperatures.

The enhancement of TGE by mild hypothermia was found to be time-dependent, with the greatest effect observed when cells were shifted to 31 °C 4 h after transfection. In contrast, little effect on protein yield was observed when the cells were shifted at 3 or more days after transfection. In a separate study on PEI-mediated transfection kinetics, it was shown for CHO cells in suspension that most of the DNA uptake occurs within 60 min of DNA-PEI addition (Bertschinger and Wurm, unpublished data). Thus, exposure of cells to low-temperature probably did not have a significant effect on DNA uptake since the protein yields following temperature shifts at the time of DNA addition or 4 h later were about the same. Instead, the temperature shift may have affected one or more subsequent events including DNA uptake into the nucleus, disassembly of PEI-DNA complexes, plasmid DNA stability in the nucleus, transcription, mRNA processing and transport, mRNA stability, translation, and protein modification and transport. Further experiments are still necessary to elucidate the mechanism(s) involved.

Experiments addressing the importance of the WPRE indicated that post-transcriptional events play a significant role in the low-temperature enhancement of TGE in CHO cells. This RNA element is known to have effects on polyadenylation, mRNA transport, and translation (34). Here we showed that the ratio of the IgG light to heavy chain mRNA was at least 2-fold higher in the presence of the WPRE than in its absence at both 31 and 37 °C. In the absence of the WPRE, the light to heavy chain mRNA ratio was nearly 1:1 at 31 °C, while this ratio in the presence of the WPRE was about 5:1. It has previously been shown that the level of the light chain polypeptide is limiting for antibody assembly (35, 36). Thus, the effect of the WPRE may be to increase the steady-state level of the light chain mRNA, resulting in higher light chain protein levels and higher antibody secretion. Interestingly, the overexpression in CHO cells of two cold-shock proteins (cold-induced RNA binding protein and Rbm3) that are expected to stabilize mRNA under hypothermic conditions (23) failed to enhance transient recombinant protein production (Wulhfard, unpublished data), even though other studies showed an increase in recombinant protein yield when either one of these proteins was stably expressed in a recombinant CHO-derived cell line (37, 38).

We also observed that mild hypothermia has an impact on the viability, size, and metabolism of cells and on the cell cycle. The accumulation of cells in the G1 phase of the cell cycle as the result of mild hypothermia has previously been described for recombinant CHO cell lines (13, 21, 39). Even though most of the cells shifted to 31 °C were no longer dividing by 3 days post-transfection, the average cell volume increased about 2-fold relative to cells at 37 °C. Thus, the biomass levels of the transfected cultures at 31 and 37 °C were approximately the same up to 5 days post-transfection. After this time point, cell viability at 37 °C decreased sharply. A similar increase in cell size and recombinant protein production was reported for transfected CHO cells blocked in the G2/M phase of the cell cycle following treatment with nocodazole (9). The difference in viability described here between the cultures at 31 and 37 °C was already evident by 3 days post-transfection, and this difference was increased by 6 days post-transfection. Cells at 31 °C maintained a high viability (>90%) for a longer time than at 37 °C without feeding. This may have been due to the reduced consumption of glucose and glutamine and the consequent reduction of lactate and ammonium production. A similar reduction in cellular metabolism has been observed for recombinant CHO cells lines maintained under mild hypothermic conditions (21, 22). Subsequent transfections of CHO at scales up to 50 L demonstrated that the viability of CHO cells can remain above 90% for up to 11 days post-transfection at 31 °C (33). The changes in cell growth, division, and physiology due to mild hypothermia are likely to be important for enhancing transient recombinant protein yield, but additional studies are necessary to determine the precise pathways involved.

Although the different mammalian expression vectors described in this study responded differently to low-temperature conditions, in all cases recombinant protein production was increased under mild hypothermia. Only the total protein yields and the extent of the enhancement of recombinant protein production varied. pEGFPN1 and pXLGHEK-RhLC/pXLGHEK-RhHC provided the highest absolute levels of GFP and antibody, respectively. The level of enhancement over the control transfection was 10-fold for pEGFP-N1 and 18-fold for pXLGHEK-RhLC/pXLGHEK-RhHC. Regardless of the fact that for all three of these plasmids the recombinant gene was under the control of the hCMV immediate early promoter, we cannot conclude that the hCMV promoter is superior to the other promoters used in the study since the various plasmids included other elements which may have affected transgene expression at low temperatures. For example, the pXLGHEK-based plasmids included the adeno-associated virus inverted terminal repeats which have intrinsic promoter activity and the wild-type WPRE which encodes a truncated form of the woodchuck hepatitis virus X protein (40-42). Furthermore, the two reporter proteins also responded differently to hypothermic conditions which may signify differential low-temperature effects on secreted and intracellular proteins.

Hypothermic cultivation of mammalian cells is widely used in the biotechnology industry (1115). However, to date no substantial difference was detected in the glycosylation of recombinant proteins expressed under conditions of mild hypothermia from recombinant cell lines or transiently transfected cells as compared to production of the same antibody at 37 °C (12, 18, 43). In the present work we analyzed only the extent of glycosylation of the anti-Rhesus D antibody transiently produced at 31 and 37 °C, and we compared it to the glycosylation of the same antibody recovered from a recombinant CHO cell line grown at 37 °C. The same levels of heavy chain glycosylation were observed for all the samples, but additional experiments will be necessary to chemically define the glycans for antibodies produced transiently at 31 and 37 °C.

The recombinant antibody levels observed here were the highest ever reported for transiently transfected CHO cells, and the present studies have been extended by performing transfections at scales up to 50 L in disposable containers agitated by orbital shaking (33). Thus, low-temperature TGE is a very promising technology for the rapid production of gram quantities of secreted recombinant proteins from CHO cells. Further improvements in volumetric productivity are anticipated for cultures that are maintained longer than the 6-day limit used in the experiments reported here.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References and Notes

We thank Dr. Markus Hildinger for pXLGHEK-RhHC, pXLGHEK-RhLC, and pXLGHEK-EGFP; Fanny Delegrange and Dr. Myriam Adam for reagents and suggestions for quantitative RT-PCR; and Dr. Lucia Baldi for critically reading the manuscript.

References and Notes

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References and Notes
  • 1
    Wurm, F. M.; Bernard, A. Large-scale transient expression in mammalian cells for recombinant protein production. Curr. Opin. Biotechnol. 1999, 10, 156159.
  • 2
    Baldi, L.; Hacker, D. L.; Adam, M.; Wurm, F. M. Recombinant protein production by large-scale transient gene expression in mammalian cells: state of the art and future perspectives. Biotechnol. Lett. 2007, 29, 677684.
  • 3
    Pham, P. L.; Kamen, A.; Durocher, Y. Large-scale transfection of mammalian cells for the fast production of recombinant protein. Mol. Biotechnol. 2006, 34, 225237.
  • 4
    Jordan, M.; Schallhorn, A.; Wurm, F. M. Transfecting mammalian cells: optimization of critical parameters affecting calcium-phosphate precipitate formation. Nucleic Acids Res. 1996, 24, 596601.
  • 5
    Schlaeger, E. J.; Christensen, K. Transient gene expression in mammalian cells grown in serum-free suspension culture. Cytotechnology 1999, 30, 7183.
  • 6
    Durocher, Y.; Perret, S.; Kamen, A. High-level and high-throughput recombinant protein production by transient transfection of suspension-growing human 293-EBNA1 cells. Nucleic Acids Res. 2002, 30, E9.
  • 7
    Girard, P.; Jordan, M.; Tsao, M.; Wurm, F. M. Small-scale bioreactor system for process development and optimization. Biochem. Eng. J. 2001, 7, 117119.
  • 8
    Derouazi, M.; Girard, P.; Van Tilborgh, F.; Iglesias, K.; Muller, N.; Bertschinger, M.; Wurm, F. M. Serum-free large-scale transient transfection of CHO cells. Biotechnol. Bioeng. 2004, 87 (4), 537545.
  • 9
    Tait, A. S.; Brown, C. J.; Galbraith, D. J.; Hines, M. J.; Hoare, M.: Birch, J. R.; James, D. C. Transient production of recombinant proteins by Chinese hamster ovary cells using polyethyleneimine/DNA complexes in combination with microtubule disrupting anti-mitotic agents. Biotechnol. Bioeng. 2004, 88, 707721.
  • 10
    Wurm F. M. Productionof recombinant protein therapeutics in cultivated mammalian cells. Nat. Biotechnol. 2004, 22, 13931398.
  • 11
    Yoon, S. K.; Choi, S. L.; Song, J. Y.; Lee, G. M. Effect of culture pH on erythropoietin production by Chinese hamster ovary cells grown in suspension at 32.5 and 37.0 °C. Biotechnol. Bioeng. 2005, 89, 345356.
  • 12
    Yoon, S. K.; Choi, S. L.; Song, J. Y.; Lee, G. M. Effect of low culture temperature on specific productivity, transcription level, and heterogeneity of erythropoietin in Chinese Hamster Ovary cells. Biotechnol. Bioeng. 2003, 82, 289298.
  • 13
    Kaufmann, H.; Mazur, X.; Fussenegger, M.; Bailey, J. E. Influence of low temperature on productivity, proteome and protein phosphorylation of CHO cells. Biotechnol. Bioeng. 1999, 63, 573582.
  • 14
    Baik, J. Y.; Lee, M. S.; An, S. R.; Yoon, S. K.; Joo, E. J.; Kim, Y. H.; Park, H. W.; Lee, G. M. Initial transcriptome and proteome analyses of low culture temperature-induced expression in CHO cells producing erythropoietin. Biotechnol. Bioeng. 2006, 93, 361371.
  • 15
    Fox, S. R.; Patel, U. A.; Yap, M. G. S.; Wang, D. I. Maximizing interferon-γ production by Chinese Hamster Ovary cells through temperature shift optimization: experimental and modelling, Biotechnol. Bioeng. 2004, 85, 177184.
  • 16
    Al-Fageeh, M.; Smales, C. M. Control and regulation of the cellular responses to cold shock: the responses in yeast and mammalian systems. Biochem. J. 2006, 397, 247259.
  • 17
    Underhill, M. F.; Marchant, R. J.; Carden, M. J.; James, D. C.; Smales, C. M. On the effect of transient expression of mutated eIF2alpha and eIF4E eukaryotic translation initiation factors on reporter gene expression in mammalian cells upon cold-shock. Mol. Biotechnol. 2006, 34, 141149.
  • 18
    Galbraith, D. J.; Tait, A. S.; Racher, A. J.; Birch, J. R.; James, D. C. Control of culture environment for improved polyethylenimine-mediated transient production of recombinant monoclonal antibodies by CHO cells. Biotechnol. Prog. 2006, 22, 753762.
  • 19
    Fussenegger, M.; Bailey J. E. Control of mammalian cell proliferation as an important strategy in cell culture technology, cancer therapy and tissue engineering. In Cell Engineering I; Al-Rubeai, M., Ed.; Kluwer Academic Publishers: Dordrecht, 1999; pp 186219.
  • 20
    Fox, S. R.; Yap, M. X.; Yap, M. G.; Wang, D. I. Active hypothermic growth: a novel means for increasing total interferon-gamma production by Chinese hamster ovary cells. Biotechnol. Appl. Biochem. 2005, 41, 265272.
  • 21
    Moore, A.; Mercer, J.; Dutina, G.; Donahue, C. J.; Bauer, K. D.; Mather, J. P.; Etcheverry, T.; Ryll, T. Effects of temperature shift on cell cycle, apoptosis and nucleotide pools in CHO cell batch cultures. Cytotechnology 1997, 23, 4754.
  • 22
    Yoon, S. K.; Hwang, S. O.; Lee, G. M. Enhancing effect of low culture temperature on specific antibody productivity of recombinant Chinese hamster ovary cells: clonal variation. Biotechnol. Prog. 2004, 20, 16831688.
  • 23
    Sonna, L. A.; Fujita, J.; Gaffin, S. L.; Lilly, C. M. Effect of heat and cold stress on mammalian gene expression, J. Appl. Physiol. 2002, 92, 17251742.
  • 24
    Muller, N.; Girard, P.; Hacker, D. L.; Jordan, M.; Wurm, F. M. Orbital shaker technology for the cultivation of mammalian cells in suspension, Biotechnol. Bioeng. 2005, 89, 400406.
  • 25
    Stettler, M.; Jaccard, N.; Hacker, D. L.; De Jesus, M.; Wurm, F. M.; Jordan, M. New disposable tubes for rapid and precise biomass assessment for suspension cultures of mammalian cells. Biotechnol. Bioeng. 2006, 95, 12281233.
  • 26
    De Jesus, M.; Girard, P.; Bourgeois, M.; Baumgartner, G.; Jacko, B.; Amstutz, H.; Wurm, F. M. TubeSpin satellites: a fast track approach for process development with animal cells using shaking technology. Biochem. Eng. J. 2004, 17, 217223.
  • 27
    Muller, N.; Derouazi, M.; Van Tilborgh, F.; Wulhfard, S.; Hacker, D. L.; Jordan, M.; Wurm, F. M. Scalable transient gene expression in Chinese hamster ovary cells in instrumented and non-instrumented cultivation systems. Biotechnol. Lett. 2007, 29, 703711.
  • 28
    Pick, H. M.; Meissner, P.; Preuss, A. K.; Tromba, P.; Vogel, H.; Wurm, F. M. Balancing GFP reporter plasmid quantity in large-scale transient transfection for recombinant anti-human Rhesus-D IgG1 synthesis. Biotechnol. Bioeng. 2002, 79, 595601.
  • 29
    Backliwal, G.; Hildinger, M.; Wulhfard, S.; De Jesus, M.; Wurm F. M. Rational vector design and multi-pathway modulation of HEK 293 cells yield recombinant antibody titers exceeding 1 gram per liter by transient transfection under serum-free conditions. Submitted for publication.
  • 30
    Derouazi, M.; Flaction, R.; Girard, P.; De Jesus, M.; Jordan, M.; Wurm, F. M. Generation of recombinant Chinese hamster ovary cell lines by microinjection. Biotechnol. Lett. 2006, 28, 373382.
  • 31
    Livak, K.; Schmittgen, T. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 2001, 25 (4), 402408.
  • 32
    Meissner, P.; Pick, H.; Kulangara, A.; Chatellard, P.; Friedrich, K.; Wurm, F. M. Transient gene expression: recombinant protein production with suspension-adapted HEK293-EBNA cells. Biotechnol. Bioeng. 2001, 75, 197203.
  • 33
    Stettler, M.; Zhang, X.; Hacker, D. L.; De Jesus, M.; Wurm, F. M. Novel orbital shake bioreactors for transient production of CHO derived IgGs. Biotechnol. Prog. 2007, 23, 13401346.
  • 34
    Donello, J. E.; Loeb, J. E.; Hope, T. J. Woodchuck hepatitis virus contains a tripartite posttranscriptional regulatory element. J. Virol. 1998, 72, 50855092.
  • 35
    Schlatter, S.; Stansfield, S. H.; Dinnis, D. M.; Racher, A. J.; Birch, J. R.; James, D. C. On the optimal ration of heavy to light chain genes for efficient recombinant antibody production by CHO cells. Biotechnol. Prog. 2005, 21, 122133.
  • 36
    Leitzgen, K.; Knittler, M. R.; Haas, I. G. Assembly of immonuglobulin light chains as a prerequisite for secretion. A model for oligomerization-dependent subunit folding. J. Biol. Chem. 1997, 272, 31173123.
  • 37
    Dresios, J.; Aschrafi, A.; Owens, G. C.; Vanderklish, P. W.; Edelman, G. M.; Mauro, V. P. Cold stress-induced protein Rbm3 binds 60S ribosomal subunits, alters microRNA levels, and enhances global protein synthesis. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 18651870.
  • 38
    Tan, H. K.; Lee, M. M.; Yap, M. G.; Wang, D. I. Over-expression of cold-inducible RNA-binding protein (Cirp) increases interferon-gamma production in CHO cells. Biotechnol. Appl. Biochem. 2007, Epub ahead of print; doi 10.1042/BA20070032.
  • 39
    Hendrick, V.; Winnepenninckx, P.; Abdelkafi, C.; Vandeputte, O.; Cherlet, M.; Marique, T.; Renemann, G.; Loa, A.; Kretzmer, G.; Werenne, J. Increased productivity of recombinant tissue plasminogen activator (t-PA) by butyrate and shift of temperature: a cell cycle phase analysis. Cytotechnology 2001, 36, 7183.
  • 40
    Flotte, T. R.; Afione, S. A.; Solow, R.; Drumm, M. L.; Markakis, D.; Guggino, W. B.; Zeitlin, P. L.; Carter, B. J. Expression of the cystic fibrosis transmembrane conductance regulator from a novel adeno-associated virus promoter. J. Biol. Chem. 1993, 268, 37813790.
  • 41
    Chen, H.; McCarty, D. M.; Bruce, A. T.; Suzuki, K.; Suzuki, K. Gene transfer and expression in oligodendrocytes under the control of myelin basic protein transcriptional control region mediated by adeno-associated virus. Gene Ther. 1998, 5, 5058.
  • 42
    Kingsman, S. M.; Mitrophanous, K.; Olsen, J. C. Potential oncogenic activity of the woodchuck hepatitis post-transcriptional regulatory element (WPRE). Gene Ther. 2005, 12, 34.
  • 43
    Bollati-Fogolín, M.; Forno, G.; Nimtz, M.; Conradt, H. S.; Etcheverrigaray, M.; Kratje, R. Temperature reduction in cultures of hGM-CSF-expressing CHO cells: effect on productivity and product quality. Biotechnol. Prog. 2005, 21, 1721.