Scholarly article on topic 'Protein folding and glycosylation process are influenced by mild hypothermia in batch culture and by specific growth rate in continuous cultures of CHO cells producing rht-PA'

Protein folding and glycosylation process are influenced by mild hypothermia in batch culture and by specific growth rate in continuous cultures of CHO cells producing rht-PA Academic research paper on "Biological sciences"

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Academic research paper on topic "Protein folding and glycosylation process are influenced by mild hypothermia in batch culture and by specific growth rate in continuous cultures of CHO cells producing rht-PA"

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POSTER PRESENTATION Open Access

Protein folding and glycosylation process are influenced by mild hypothermia in batch culture and by specific growth rate in continuous cultures of CHO cells producing rht-PA

Mauricio Vergara , Silvana Becerra , Julio Berrios , Juan Reyes , Cristian Acevedo , Ramon Gonzalez , Nelson Osses , Claudia Altamirano1,2

From 23rd European Society for Animal Cell Technology (ESACT) Meeting: Better Cells for Better Health Lille, France. 23-26 June 2013

Background

CHO cells are the primary host for the production of different biopharmaceuticals, including recombinant proteins, monoclonal antibodies, vaccines, etc. Primarily due to their ability to perform properly folding and gly-cosylation processes required for these proteins acquire adequate biological functionality.

However, culturing of these cells in the bioreactor still presents a number of disadvantages, among which can be mention: nutrient depletion, toxic byproducts accumulation, limited oxygen transfer, etc. These issues limit the cell growth and early onset of programmed cell death, which restricts the longevity of cultures and jointly specific productivity of recombinant protein.

To overcome these limitations, different approaches have been made to maximize the productivity of these cultures. One of these approaches, that has gained importance during the last 20 years is the use of mild hypothermic temperatures, within a range of 33°C to 30° C. This strategy has been demonstrated to reduce the rate of growth and metabolism of cells but in turn increases the longevity of cultures and increase in specific productivity of a wide range of recombinant proteins in batch cultures [1,2].

One possible cause involved in the increase of specific productivity of recombinant proteins, is the increase in folding capacity and expression of chaperones from endoplasmic reticulum [3,4]. However, the intracellular

* Correspondence: Claudia.altamirano@ucv.cl

1Escuela Ingeniería Bioquímica, Pontificia Universidad Católica de Valparaíso,

Valparaíso, 2362806, Chile

Fulllist of author information is available at the end of the article

mechanisms underlying the effect of temperature on the stages of post-translational protein synthesis are still poorly understood.

In this regard, the study of endoplasmic reticulum processes (folding, assembly and glycosylation of proteins, and degradation of misfolded proteins through ERAD pathway) has reached a high interest in recent years [4,5]. Reports show that the expression of several

Table 1 Intracellular rht-PA content (% of control) on CHO cells by inhibition of translation and glycosylation prosesses and ERAD I and II pathways at 37°C and 33°C.

Dilution rate (h-1) 0.014 0.012

Temperature Temperature Temperature

Batch Cultures 37°C 33°C Continuous Cultures 37°C 33°C 37°C 33°C

CC 1001 1002 SS 1003 1004 1005 1006

TM* 107 140 CHX/ERAD 1** 120 117 185 139

TM/ERAD 87 201 CHX/ERAD 107 115 242 150

TM/ERAD 79 176

CC: Control Culture; TM: Culture inhibited glycosylation; TM/ERAD I or II:

Culture inhibited glycosylation and ERAD I or II; SS: Steady State; CHX/ERAD I

or II: Culture inhibited translation and ERAD I or II; *Values at 24 hours after

perturbation with inhibitors respect to CC value at 0 h. **At 48 hours after

perturbation with inhibitors respect to value at SS.

'Concentration (8,8 ng/106 cells).

Concentration (8,6 ng/106 cells).

Concentration (7,9 ng/106 cells).

Concentration (6,5 ng/106 cells).

Concentration (4,2 ng/106 cells).

Concentration (6,1 ng/106 cells).

Central

© 2013 Vergara et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2-0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

proteins associated with the various processes that take place in the ER, are affected under conditions of mild hypothermia. However, this phenomenon has not been analyzed from a process perspective.

Thus, this study investigated the effect of mild hypothermic temperatures (33°C) on the process of protein folding of rht-PA expressed in CHO cells. For this, inhibitors of protein translation, glycosylation and endo-plasmic reticulum associated degradation pathways (ERAD I: via the ubiquitin/proteasome and ERAD II:

autophagosome/Lysosome) were used. Two experimental approaches were evaluated: batch culture and continuous culture.

Materials and methods

Batch Culture

CHO cells were cultured in HyClone SFM4CHO medium with out glucose, supplemented with 20 mM glucose, at 95% relative humidity in an atmosphere of 5% CO2, at temperatures of 37°C or 33°C. The inhibitors

Figure 1 First principal plane and Loads of first and second principal component of Batch and Continuous cultures. A: First principal plane of Batch culture B: First principal plane of continuous culture; C: Loads of first and second principalcomponent of Batch cultures. D: Loads of first and second principalcomponent of Continuous cultures.

used to block processes in the endoplasmic reticulum were: cycloheximide (Sigma, C4859)-protein translation; tunicamycin (Sigma, T7765)-N-glycosylation of proteins, MG132 (Merck, 474790)-ERAD I pathway; Pepstatin A (Merck, 516485), Leupeptin (Merck, 108976) and E64d (Sigma, E8640)-ERAD II pathway.

Continuous culture

The bioreactor was inoculated and operated in batch-mode during 48 h and it was then supplied with sterile feed throughout the period of operation. A series of four experiments was performed, in duplicate, at 37°C or 33°C, keeping D, at 0.014 and 0.012 h-1. Cultures were considered to reach steady-state (SS) when, after at least four residence times, both, the number of viable cells and lactate concentration, were constant in two consecutive samples.

Cell growth was measured by counting cells by trypan blue method; consumption and production of metabolites were measured by biochemical analyzer (YSI 2700); protein rht-PA was measured by ELISA (Trinilize tPA antigen) and enzymatic activity of the protein was measured by amidolytic assay (S-2288 peptide, Chromogenix Italy). The results were analyzed by the mathematical technique of PCA (Principal Component Analysis).

Results

The results of the batch cultures may indicate that the process of protein folding is sensitive to mild hypothermia. Inhibition of glycosylation process and ERAD pathways (ERAD I or II), under conditions of low temperature, promotes the accumulation of intracellular deglycosylated rht-PA as shown in Table 1. This response may indicate that the protein folding process is attenuated under conditions of mild hypothermia, promoting unfolded protein degradation by both ERAD pathways in CHO cells.

Recent reports [6,7] show that the effect of mild hypothermia condition in batch culture is associated predominant with a decrease on specific cell growth rate rather a decrease on culture temperature. To evaluate this fact, we carried out continuous cultures at different dilution rates.

These results show that the degradation of the protein would be more related to the decrease in specific growth rate than the temperature decrease. Also show that the temperature decrease would promote an increase in protein folding capacity of the endoplasmic reticulum. This fact is clearly observed at low specific growth rate (Table 1).

The cell behavior was evaluated using the technique of principal component analysis (PCA) in both, batch and continuous culture Figure 1.

The first principal plane (PC1 axis and PC2 axis) of batch cultures (Figure 1A) shows that there are only two values whose behavior is significantly away from the origin (P < 0,05). These correspond to the behavior of the tested batch cultures at 24 h at 37°C and 33°C, respectively. This indicates the great influence of culture temperature on cell behavior. The first principal plane of continuous culture (Figure 1B) shows the behavior of cells organized into two major groups, which are correlated with both dilution rates tested.

PC1 loads of batch cultures (Figure 1C) suggest that low temperature reduces the ability of the protein folding; this would explain the accumulation of intracellular deglycosylated rht-PA. However, loads of PC1 from continuous cultures (Figure 1D) shows that increasing of intracellular rht-PA content is associated with the reduction in the rate of dilution and is not associated with a lower temperature.

Conclusions

Experimental approach of continuous culture revealed that reduction on specific growth rate is associated to an increase ERAD activity on rht-PA while the temperature reduction may have a positive effect on protein folding. Moreover, PCA analysis indicated that specific growth rate is also responsible for general behavior exposed by CHO cells.

Authors' details

1Escuela Ingeniería Bioquímica, Pontificia Universidad Católica de Valparaíso, Valparaíso, 2362806, Chile. 2Centro Reg ional En Alimentos Saludables (CREAS), Valparaíso, 2340025, Chile. 3Instituto Química, Pontificia Universidad Católica de Valparaíso, Valparaíso, 2340025, Chile. 4Centro de Biotecnología, Universidad Técnica Federico Santa María, Valparaíso, 2390123, Chile. 5Department of Chemicaland Biomolecular engineering, RICE University, Houston, 77055, USA.

Published: 4 December 2013 References

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doi:10.1186/1753-6561-7-S6-P108

Cite this article as: Vergara et al.: Protein folding and glycosylation process are influenced by mild hypothermia in batch culture and by specific growth rate in continuous cultures of CHO cells producing rht-

PA. BMC Proceedings 2013 7(Suppl 6):P108.

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