Moon Sue Lee

Proteome analysis of antibody-expressing CHO cells in response to hyperosmotic pressure.

To better understand intracellular responses to hyperosmotic pressure of recombinant Chinese hamster ovary (rCHO) cells expressing an antibody, we have taken a proteomics approach. Using two‐dimensional electrophoresis and mass spectrometry, a proteome profile of rCHO cells comprising 23 identified proteins was established. On the basis of this proteome profile, we found three proteins of which expression levels were significantly changed at 450 mOsm/kg. Compared to the results at 300 mOsm/kg, two glycolytic enzymes, glyceraldehyde‐3‐phosphate dehydrogenase and pyruvate kinase, were found to be up‐regulated, probably leading to an increased metabolic energy for antibody synthesis. The elevation of specific glucose consumption rate at 450 mOsm/kg agreed with the up‐regulation of these glycolytic enzymes. On the other hand, tubulin expression was down‐regulated, reflecting a depressed cell growth rate at 450 mOsm/kg. Taken together, this study shows the potential of the proteomics approach in understanding intracellular and physiological changes in cells and seeking a better insight into possible environmental or genetic manipulation approaches for increasing foreign protein production in rCHO cells.

Hyperosmotic pressure enhances immunoglobulin transcription rates and secretion rates of KR12H‐2 transfectoma

When subjected to hyperosmotic pressure resulting from NaCl addition, KR12H‐2 transfectoma, like most hybridomas, displayed a decrease in specific growth rate (μ) and an increase in specific antibody productivity (qAb). Elevation of medium osmolality from 285 to 425 mOsm/kg decreased μ by 20%, while it increased qAb by 376%. Although cell mass also increased at higher osmolality, it was not the main factor in increasing qAb. Hyperosmotic pressure was found to enhance transcription levels of immunoglobulin (Ig) mRNAs preferentially, compared with non‐IgG mRNA. The transcription levels of both heavy chain (HC) and light chain (LC) mRNAs were enhanced as much as qAb. This result suggests that enhanced qAb at higher osmolality was mainly due to enhanced transcription levels of Ig mRNA. However, these increased transcription levels of Ig mRNAs were not due to the enhanced stability of Ig mRNA. In fact, the stability of Ig mRNAs decreased at higher osmolality. Elevation of osmolality from 285 mOsm/kg to 425 mOsm/kg decreased the half‐lives of HC and LC mRNAs by 37% and 36%, respectively. A simple mathematical model revealed that transcription rates of Ig mRNAs increased by more than 476% at 425 mOsm/kg. These elevated transcription levels could, in turn, increase the translation rates of Ig polypeptides. However, the translation rates of Ig polypeptides were not enhanced as much as the transcription levels of Ig mRNAs and qAb. The elevation of osmolality from 285 mOsm/kg to 425 mOsm/kg increased HC and LC mRNA specific translation rates by 172% and 240%, respectively. Taken together, the data suggest that (1) enhanced qAb of KR12H‐2 transfectoma at higher osmolality is due to elevated transcription rates of Ig mRNAs and expedited post‐translational processing of Ig, and (2) antibody secretion by KR12H‐2 transfectoma is most likely controlled at the level of Ig translation, particularly HC translation.

Effects of Cloned Gene Dosage on the Response of Recombinant CHO Cells to Hyperosmotic Pressure in Regard to Cell Growth and Antibody Production

The effect of cloned gene dosage on growth and product formation under hyperosmotic conditions has been studied using recombinant Chinese hamster ovary (rCHO) cell lines producing chimeric antibody. Batch cultures of four rCHO cell lines carrying different numbers of antibody gene copies were carried out using the hyperosmolar medium. Depending on cloned gene dosage, hyperosmotic pressure decreased specific growth rate (μ) and increased specific antibody productivity (qAb) to a different degree. The cell line with lower cloned gene dosage displayed more significant enhancement in qAb and less reduction in μ at hyperosmolalities. However, the cell line with higher cloned gene dosage still yielded higher maximum antibody concentration at hyperosmolality up to 469 mOsm/kg. Northern blot analysis showed a positive relationship between immunoglobulin mRNA level per cell and qAb, indicating that transcriptional regulation was involved in the response of rCHO cells to hyperosmotic pressure. Cell cycle analysis showed that hyperosmotic pressure induced G1‐phase arrest, suggesting that the increase of cell population in G1‐phase may contribute in part to enhanced qAb at hyperosmolality. Taken together, although the cell line with lower cloned gene dosage displayed more significant enhancement in qAb at hyperosmolality, the factor that determined the maximum antibody concentration in hyperosmotic rCHO cell cultures was almost exclusively the gene dosage.

Effect of hypoosmotic pressure on cell growth and antibody production in recombinant Chinese hamster ovary cell culture

To determine the response of recombinant Chinese hamster ovary (rCHO) cells subjected to hypoosmotic pressure, rCHO cells (CS13*–1.0) producing a chimeric antibody were cultivated in the hypoosmolar medium resulting from NaCl subtraction. At hypoosmotic pressure, CS13*–1.0 cells displayed decreased specific growth rate (μ) and increased specific antibody productivity (qAb).When the medium osmolality was decreased from 300 mOsm kg-1(physiological osmolality) to 150 mOsm kg-1, μ was decreased by 68% and qAb was increased by 128%. To understand the mechanism of enhanced qAb resulting from hypoosmotic pressure, cellular responses of cells in the exponential phase of growth were observed at the transcription level. Total cytoplasmic RNA content per cell at 150 mOsm kg-1 was increased by 140%, compared with that at 300 mOsm kg-1. On a per μg RNA basis, immunoglobulin (Ig) mRNA levels at 150 mOsm kg-1 were comparable to those at 300 mOsm kg-1, indicating that hypoosmotic pressure did not lead to the preferential transcription of Ig mRNAs. Taken together, the data obtained here suggest that the increase in total RNA pool is primarily responsible for the enhanced qAb of CS13*–1.0 cells subjected to hypoosmotic pressure.

Targeting protein and peptide therapeutics to the heart via tannic acid modification

Systemic injection into blood vessels is the most common method of drug administration. However, targeting drugs to the heart is challenging, owing to its dynamic mechanical motions and large cardiac output. Here, we show that the modification of protein and peptide therapeutics with tannic acid—a flavonoid found in plants that adheres to extracellular matrices, elastins and collagens—improves their ability to specifically target heart tissue. Tannic-acid-modified (TANNylated) proteins do not adsorb on endothelial glycocalyx layers in blood vessels, yet they penetrate the endothelium to thermodynamically bind to myocardium extracellular matrix before being internalized by myoblasts. In a rat model of myocardial ischaemia-reperfusion injury, TANNylated basic fibroblast growth factor significantly reduced infarct size and increased cardiac function. TANNylation of systemically injected therapeutic proteins, peptides or viruses may enhance the treatment of heart diseases.The modification of protein and peptide therapeutics with tannic acid improves their ability to specifically target heart tissue, as shown with a rat model of myocardial ischaemia-reperfusion injury.

Inactivation efficiency of DNA and RNA viruses during chitin-to-chitosan conversion

Joseph P. Park, Mi-Young Koh, Pil Soo Sung, Keumyeon Kim, Min Sun Kim, Moon Sue Lee, Eui-Cheol Shin*, Ki Hong Kim*, and Haeshin Lee* 1Graduate School of Nanoscience and Technology, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 305-701, Korea 2Graduate School of Medical Science and Engineering, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 305-701, Korea 3R & D Center, InnoTherapy Inc., Daejeon 305-732, Korea 4Department of Aquatic Life Medicine, Pukyoug National University, Busan 608-737, Korea