(458a) Transcriptome of CHO Cells Producers of Recombinant Tpa Cultured Under Moderate Hypothermia

The production of recombinant proteins carried out on Chinese hamster ovary (CHO) cells for pharmaceutical use, have great importance for the development of drugs with high added value. However, their manufacturing and processing are expensive, hence the need to improve their production. Thence, different methodologies have been implemented with the aim to increase specific productivity like the moderate hypothermia (Yoon et al., 2004). Therefore, to understand the effect of temperature decrease (24-34ºC) over the productivity, different molecular tools have been used. Here, a first transcriptome analysis by Massive sequencing is presented, to understand the effects of moderate hypothermia during biphasic cultures, diminishing the temperature from 37°C to 30°C after 48 h of culture. The kinetic parameters of the TF70R CHO cells, producing the recombinant tPA in biphasic and control conditions were evaluated. The transcriptome analysis was developed with mRNA samples collected at 48 h (5 minutes before the change from 37ºC to 30ºC), as well after 24 h and 48 h after temperature down shift (TDS). Samples were purified with Qiagen RNeasy kit, transformed to cDNA and sequenced with Illumina Genome Analyzer GAII. The samples obtained after 72 h were sequenced using single reads of 36 pb. While the samples obtained after 96 h were analyzed using paired ends lectures and reads of 72 pb. All experiments were performed using two biological replicas. Sequences were aligned with Cricetulus griseus and Mus musculus genome database. The differential expression was determined with the NOIseq nonparametric algorithms with reliability of 0.9. Data were ordered in gene ontology (GO) terms and classified considering the terms enrichment obtained by TopGo and Keeg database.

An initial grow rate (μ) of 0.023 h ^-1 was found in the first 48 h.  Reduction to 30ºC resulted in a decrease in μ (0.005h^-1). The cells at 37ºC reached a maximal viable cell density of 2.7±0.1x10⁶/mL at 72 h and cell viability declined over 50% at 240 h. While, under TDS the maximal viable cell density was obtained after 192 h and cell viability above 70% was maintained for 312 h representing around six days more than in 37ºC cultures. The glucose consumption and lactate production were lower in 11% and 19%, respectively; for the biphasic cultures compared with those at 37ºC. The consumption of glutamine was similar for both culture conditions. Under TDS, the heterologus tPA specific productivity increased by 1.6 folds, reaching the maximal recombinant protein accumulation (11 mg/L) after 10 days, that represents 2.2 folds more than in 37ºC cultures.

Cells respond to TDS, after 24 and 48 h of insult, overexpressing genes related with cold stress as cirp (3.3 and 7.7 folds, respectively) and rbm3 (5.8 and 13.55 folds, respectively). The expression level of those genes was confirmed by RT-PCR, presenting up-regulated similar values. Moreover, the gene coding for the recombinant t-PA did not presented changes by the effect of TDS. However, important changes at transcriptomic level were observed being affected more than 416 genes at 24 h after TDS and 3472 genes at 48 h after TDS.

In correlation with culture kinetics where cells under cold stress grew slowly compared with 37ºC cultures, transcripts related with the cell cycle arrest in G0/G1 and cyclin D were suppressed and some genes that are negative effectors such as p53 were overexpressed. Moreover, genes related with replication, DNA reparation and those chromosome-associated proteins, were down regulated after TDS. On the other hand, many transcripts (50) coding for proteins related with RNA processing and ribosome biogenesis were up-regulated. Metabolism also was affected at transcriptomic level by TDS. For example, around 4 and 8 genes associated with the ATP were down regulated at 24 and 48 h after TDS, respectively. While, 20 genes coding for proteins involved in central metabolism and lipid biosynthesis at 48 h after TDS, were down-regulated. However, 14 genes associated with carbohydrate metabolism were overexpressed, as lactate dehydrogenase.

Since cellular perturbations like changes in protein production can be detected by the endoplasmic reticulum (ER) (Rao et al., 2001), the expression changes on genes related with this compartment and with Golgi were analyzed in detail. For example, genes coding chaperones like Bip and calreticulin were overexpressed at 24 h after TDS, and the translocon Sec 61 also was up-regulated at 48 h after TDS. Furthermore, genes associated with the quality control in the (ER) like ERManI y EDEM were down-regulated at 48 h of culture.  Interestingly, 19 genes related to apoptosis were overexpressed, indicating that programed cell might be regulated at post-transcriptional or post-translational level, because, the culture cells survived for 7 days more. As well, genes coding effectors of apoptosis like Caspase 12 were repressed at 96 h.

In conclusion, several physiological changes were observed during cell cultivation under moderate hypothermia in biphasic cultures. The TDS prolonged culture cell survival more than 6 days, and total tPA production was increased 60%. The moderate hypothermia was detected by cells, which overexpressed genes coding for cold induced proteins like Cirp and Rbm3. Also genes related with cell cycle, replication and DNA reparation were down-regulated, indicating the slowly growth and replication. Nevertheless, genes coding for proteins that participates in transcription and translation were up-regulated, which suggest the biosynthesis of proteins necessary for survival and probably the increase of tPA production. As well, important changes in the pathway of synthesis, structuring and secretion of proteins were identified. Thus, this transcriptome approach that combine gene expression and physiological changes occurred under moderate hypothermia might provide a better understanding of responses that favor the increase in recombinant protein productivity accompanied by increase in the cell survival.

Acknowledgements. UNAM PAPPIT-IN-210013, IN-209113, CONACYT-INNOVAPYME 181895, CONACYT 178528, 104951-Z.

Rao RV, Hermel E, Castro-Obregon S, del Rio G, Ellerby LM, Ellerby HM, Bredesen DE. 2001. Coupling endoplasmic reticulum stress to the cell death program. J Biol Chem 276:33869-33847.

Yoon SK, Hwang SO, Lee GM. 2004. Enhancing effect of low culture temperature on specific antibody productivity of recombinant Chinese hamster ovary cells: clonal variation. Biotechnol Prog 20:1683-1688.