Presentation Information
[1ACCE-08]Bioengineered Heparin Production Using CHO Cells
○Masamichi Kamihira1, Yoshinori Kawabe1, Razia Sultana1 (1. Kyushu Univ. (Japan))
Keywords:
CHO cells,glycoengineering,heparin-like glycans,bioprocessing,biopharmaceutical production
[Purpose]
Heparin is widely used as an anticoagulant and for coating medical devices, but it is primarily obtained from porcine intestinal tissues. This raises concerns regarding variability, impurities, limited supply, and inadequate quality control. Therefore, alternative production methods are needed. Cell culture offers a more stable and controllable approach. In this study, we aimed to develop a method for large-scale production of heparin-like glycans using genetically engineered Chinese hamster ovary (CHO) cells, which are widely used in biopharmaceutical manufacturing.
[Methods]
We introduced two sulfotransferase genes, Hs3st1 and NDST2, into CHO cells to enhance glycan sulfation. To enable secretion of the glycans into the culture medium, cells were engineered to express the extracellular domain of syndecan (SDC), a core protein bearing glycosaminoglycans (GAGs). We further introduced Hs6st3 to enhance specific activity, generating CHO/3F_S cells. In addition, knockout of CS GalNAcT2, an enzyme involved in chondroitin sulfate biosynthesis, resulted in CHO/3F_S-CKO cells with more than a twofold increase in anti-FXa specific activity.
[Results]
The engineered CHO/3F_S-CKO cells produced heparin-like glycans with enhanced anti-FXa and anti-FIIa activities. Although the degree of sulfation was lower than that of commercial heparin, the apparent specific activity reached approximately 90% of that of commercial heparin, and the ratio of anti-FXa to anti-FIIa activity was comparable. Culture conditions also affected production. Lowering the temperature to 30°C significantly increased activity, with anti-FXa and anti-FIIa specific activities increasing by up to 5-fold and 18-fold, respectively. Using semi-continuous culture at a high cell density (2 × 107 cells/mL), stable and continuous production was achieved over a 30-day culture period.
[Discussion]
CHO cells naturally produce heparan sulfate, which shares a similar structure with heparin but has a lower degree of sulfation. By introducing sulfotransferase genes, we enhanced sulfation and enabled the production of heparin-like glycans with anticoagulant activity. These glycans are typically produced on the cell surface; however, expression of the extracellular domain of SDC1 enabled their secretion into the culture medium. The glycans appeared to be released from the carrier protein, although the underlying mechanism remains unclear. Low-temperature culture markedly improved production efficiency and enabled stable long-term production.
[Conclusion]
We developed a method for producing heparin-like glycans with structures and activities comparable to commercial heparin using genetically engineered CHO cells. Through genetic modification and optimization of culture conditions, high activity and stable production were achieved, demonstrating the potential of a cell-based platform as a reliable alternative to animal-derived heparin.
Heparin is widely used as an anticoagulant and for coating medical devices, but it is primarily obtained from porcine intestinal tissues. This raises concerns regarding variability, impurities, limited supply, and inadequate quality control. Therefore, alternative production methods are needed. Cell culture offers a more stable and controllable approach. In this study, we aimed to develop a method for large-scale production of heparin-like glycans using genetically engineered Chinese hamster ovary (CHO) cells, which are widely used in biopharmaceutical manufacturing.
[Methods]
We introduced two sulfotransferase genes, Hs3st1 and NDST2, into CHO cells to enhance glycan sulfation. To enable secretion of the glycans into the culture medium, cells were engineered to express the extracellular domain of syndecan (SDC), a core protein bearing glycosaminoglycans (GAGs). We further introduced Hs6st3 to enhance specific activity, generating CHO/3F_S cells. In addition, knockout of CS GalNAcT2, an enzyme involved in chondroitin sulfate biosynthesis, resulted in CHO/3F_S-CKO cells with more than a twofold increase in anti-FXa specific activity.
[Results]
The engineered CHO/3F_S-CKO cells produced heparin-like glycans with enhanced anti-FXa and anti-FIIa activities. Although the degree of sulfation was lower than that of commercial heparin, the apparent specific activity reached approximately 90% of that of commercial heparin, and the ratio of anti-FXa to anti-FIIa activity was comparable. Culture conditions also affected production. Lowering the temperature to 30°C significantly increased activity, with anti-FXa and anti-FIIa specific activities increasing by up to 5-fold and 18-fold, respectively. Using semi-continuous culture at a high cell density (2 × 107 cells/mL), stable and continuous production was achieved over a 30-day culture period.
[Discussion]
CHO cells naturally produce heparan sulfate, which shares a similar structure with heparin but has a lower degree of sulfation. By introducing sulfotransferase genes, we enhanced sulfation and enabled the production of heparin-like glycans with anticoagulant activity. These glycans are typically produced on the cell surface; however, expression of the extracellular domain of SDC1 enabled their secretion into the culture medium. The glycans appeared to be released from the carrier protein, although the underlying mechanism remains unclear. Low-temperature culture markedly improved production efficiency and enabled stable long-term production.
[Conclusion]
We developed a method for producing heparin-like glycans with structures and activities comparable to commercial heparin using genetically engineered CHO cells. Through genetic modification and optimization of culture conditions, high activity and stable production were achieved, demonstrating the potential of a cell-based platform as a reliable alternative to animal-derived heparin.
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