The Effect of Different pH Conditions on Peptides' Separation from the Skipjack Dark Meat Hydrolysate Using Ceramic Ultrafiltration.

The conversion of Skipjack (Katsuwonus pelamis) dark meat into a hydrolysate via enzymatic hydrolysis is a promising approach to increase the value of tuna by-products as a source of bioactive peptides. Skipjack dark meat hydrolysate (SDMH) contains various sizes and sequences of peptides. To obtain and concentrate the targeted small peptides from SDMH, ultrafiltration, a key unit operation process, was employed to fractionate the protein hydrolysate due to its simplicity and productivity. The objective of this study was to investigate the effect of the feed pH on the membrane performance based on the permeate flux and the transmission of peptides. The fractionation of SDMH was performed using a ceramic membrane (molecular weight cut-off of 1 kDa) with three different pH values (5, 7, and 9) at various transmembrane pressures (TMP) (2.85, 3.85, and 4.85 bar). A high permeate flux and transmission were obtained at pH 9 due to the repulsive interactions between peptides and the membrane surface, leading to the reduction in concentration polarization that could promote high transmission. In addition, the combination of low TMP (2.85 bar) and pH 9 helped to even minimize the fouling formation tendency, providing the highest peptide transmission in this study. The fractionation process resulted in the enhancement of small peptides (MW < 0.3 kDa). The amino acid profiles were different at each pH, affirming the charge effect from the pH changes. In conclusion, the performance of the membrane was affected by the pH of the hydrolysate. Additionally, the ultrafiltration method served as an alternate method of peptide separation on a commercial scale.

A dual near-infrared and dielectric spectroscopies strategy to monitor populations of Chinese hamster ovary cells in bioreactor.

OBJECTIVE: to develop a new strategy combining near-infrared (NIR) and dielectric spectroscopies for real-time monitoring and in-depth characterizing populations of Chinese hamster ovary cells throughout cultures performed in bioreactors. RESULTS: Spectral data processing was based on off-line analyses of the cells, including trypan blue exclusion method, and lactate dehydrogenase activity (LDH). Viable cell density showed a linear correlation with permittivity up to 6 × 10(6) cells ml(-1), while a logarithmic correlation was found between non-lysed dead cell density and conductivity up to 10(7) cells ml(-1). Additionally, partial least square technique was used to develop a calibration model of the supernatant LDH activity based on online NIR spectra with a RMSEC of 55 U l(-1). Considering the LDH content of viable cells measured to be 110 U per 10(9) cells, the lysed dead cell density could be then estimated. These calibration models provided real-time prediction accuracy (R(2) ≥ 0.95) for the three types of cell populations. CONCLUSION: The high potential of a dual spectroscopy strategy to enhance the online bioprocesses characterization is demonstrated since it allows the simultaneous determination of viable, dead and lysed cell populations in real time.

28S rRNA is inducibly pseudouridylated by the mTOR pathway translational control in CHO cell cultures.

The mTOR pathway is a conserved master regulator of translational activity that influences the fate of industrially relevant CHO cell cultures, yet its molecular mechanisms remain unclear. Interestingly, rapamycin specific inhibition of the mTOR pathway in CHO cells was found to down-regulate the small nucleolar RNA U19 (snoRNA U19) by 2-fold via translatome profiling. snoRNA U19 guides the two most conserved pseudouridylation modifications on 28S ribosomal RNA (rRNA) that are important for the biogenesis and proper function of ribosomes. In order to further understand the role of snoRNA U19 as a potential player in the mTOR pathway, we measured 28S rRNA pseudouridylation upon rapamycin treatments and/or snoRNA U19 overexpression conditions, thereby characterizing the subsequent effects on ribosome efficiency and global translation by polysome profiling. We showed that 28S rRNA pseudouridylation was increased by rapamycin treatment and/or overexpression of snoRNA U19, but only the latter condition improved ribosome efficiency toward higher global translation, thus implying that the mTOR pathway induces pseudouridylation at different sites along the 28S rRNA possibly with either positive or negative effects on the cellular phenotype. This discovery of snoRNA U19 as a new downstream effector of the mTOR pathway suggests that cell engineering of snoRNAs can be used to regulate translation and improve cellular growth in CHO cell cultures in the future.

Understanding translational control mechanisms of the mTOR pathway in CHO cells by polysome profiling.

The mammalian target of rapamycin (mTOR) pathway plays essential roles in the regulation of translational activity in many eukaryotes. Thus, from a bioprocessing point of view, understanding its molecular mechanisms may provide potential avenues for improving cell culture performance. Toward this end, the mTOR pathway of CHO cells in batch cultures was subjected to rapamycin treatment (inhibition) or nutrient supplementation (induction) and translational activities of CHO cells producing a monoclonal antibody (mAb) were evaluated with polysome profiling technology. Expectedly, rapamycin induced a shift of mRNAs from polysomes towards monosomes, thus reducing maximum cellular growth rate by 30%, while feeding additional nutrients extended mTOR pathway activity during the stationary growth phase in control batch culture, thereby contributing to an increase in global translation activity by up to 2-fold, and up to 5-fold higher specific translation of the heavy and light chains of the recombinant mAb. These increases in translation activity correlated with a 5-day extension in cellular growth and a 4-fold higher final product titer observed upon nutrient feeding. This first study of the relationship between the mTOR pathway and translational activity in CHO cultures provides key insights into the role of translational control in supporting greater productivity, which will lead to further enhancement of CHO cultures.

Translatome analysis of CHO cells to identify key growth genes.

We report the first investigation of translational efficiency on a global scale, also known as translatome, of a Chinese hamster ovary (CHO) DG44 cell line producing monoclonal antibodies (mAb). The translatome data was generated via combined use of high resolution and streamlined polysome profiling technology and proprietary Nimblegen microarrays probing for more than 13K annotated CHO-specific genes. The distribution of ribosome loading during the exponential growth phase revealed the translational activity corresponding to the maximal growth rate, thus allowing us to identify stably and highly translated genes encoding heterogeneous nuclear ribonucleoproteins (Hnrnpc and Hnrnpa2b1), protein regulator of cytokinesis 1 (Prc1), glucose-6-phosphate dehydrogenase (G6pdh), UTP6 small subunit processome (Utp6) and RuvB-like protein 1 (Ruvbl1) as potential key players for cellular growth. Moreover, correlation analysis between transcriptome and translatome data sets showed that transcript level and translation efficiency were uncoupled for 95% of investigated genes, suggesting the implication of translational control mechanisms such as the mTOR pathway. Thus, the current translatome analysis platform offers new insights into gene expression in CHO cell cultures by bridging the gap between transcriptome and proteome data, which will enable researchers of the bioprocessing field to prioritize in high-potential candidate genes and to devise optimal strategies for cell engineering toward improving culture performance.