Strategies for enhancing mature human $TGF-\beta 1$ production in recombinant CHO cells

Abstract

Transforming growth factor $(TGF)-\beta 1$, belonging to the $TGF-\beta$ superfamily, is a multifunctional cytokine involved in the regulation of cell proliferation, differentiation, apoptosis, and extracellular matrix (ECM) regeneration. The clinical potential of $TGF-\beta 1$ in wound healing, bone and cartilage formation, and the treatment of autoimmune diseases has been recognized in numerous studies. Functionally active ligand of recombinant human $TGF-\beta$ superfamily, produced primarily in Chinese hamster ovary (CHO) cells for clinical applications, are considered to be difficult-to express proteins because they undergo maturation process, signaling pathway, or endocytosis. The $TGF-\beta$ superfamily of cytokines consists of proteins with highly conserved mature domains but low prodomain sequence similarity. Extensive efforts have been made to enhance the production of the $TGF-\beta$ superfamily cytokines in CHO cells, but the properties of the $TGF-\beta$ superfamily of cytokines are highly variable, and any general strategy to enhance their production in CHO cells remains unavailable. For most $TGF-\beta$ superfamily members, the precursor passes through the proprotein convertases (PCs) located trans-Golgi network (TGN), the functionally active dimeric mature domain is separated from the prodomain. However, for $TGF-\beta 1$, the prodomain still binds non-covalently to the mature domain after proteolytic cleavage by PCs and secreted as inactive latent complex. Therefore, understanding the properties of recombinant human $TGF-\beta 1$ ($rhTGF-\beta 1$) is essential for improving $rhTGF-\beta 1$ production in CHO cells. However, the properties of $rhTGF-\beta 1$ in CHO cells have not yet been well characterized. To enhance the production of $rhTGF-\beta 1$ in CHO cells, $rhTGF-\beta 1$ was first characterized for endocytosis, signaling pathway, and overall maturation process. The mature $rhTGF-\beta 1$ used for clinical application was internalized into CHO cells and inhibited the growth of CHO cells in a dose-dependent manner. However, mature $rhTGF-\beta 1$ was mostly produced in the form of latent $rhTGF-\beta 1$ in cultures of recombinant CHO (rCHO) cells producing $rhTGF-\beta 1$ ($CHO-rhTGF-\beta 1$). The concentration of active mature $rhTGF-\beta 1$ in the culture supernatant of $CHO-rhTGF-\beta 1$ cells was not high enough to compromise yield. In addition, a significant amount of unprocessed precursors was produced by $CHO-rhTGF-\beta 1$ cells. Overexpression of PACEsol, a soluble form of furin, in $CHO-rhTGF-\beta 1$ cells was effective for the proteolytic cleavage of unprocessed precursors. The highest mature $rhTGF-\beta 1$ concentration was obtained with the PACEsol-expressing clone, which was approximately $45%$ higher than that of the parental clone ($P < 0.01$). Thus, a comprehensive understanding of the intrinsic properties of $rhTGF-\beta 1$ with respect to the overall maturation process, signaling pathway, and endocytosis is essential for effectively enhancing the production of mature $rhTGF-\beta 1$ in CHO cells. Additionally, we explored chemical additives to increase specific productivity (qp) of mature $rhTGF-\beta 1$ in CHO cells. However, these chemical additives significantly inhibited cell growth, thereby preventing an increase in the yield of mature $rhTGF-\beta 1$ in $CHO-rhTGF-\beta 1$ cells. By effectively suppressing chemical-induced apoptosis through Bax and Bak double knockout engineering in $CHO-rhTGF-\beta 1$ cell line, the addition of the HDAC inhibitor (iHDAC) $MS-275$ increased the yield of mature $rhTGF-\beta 1$. Unlike $CHO-rhTGF-\beta 1$, the Bax/Bak-knockout clone consistently exhibited increased production of mature $rhTGF-\beta 1$ at various concentrations of $MS-275$. Bax/Bak-knockout clones showed a minimum $38%$ increase in mature $rhTGF-\beta 1$ yield induced by $MS-275$ ($P < 0.01$). Therefore, the combined use of chemical additives and Bax/Bak knockout engineering represents a synergistic strategy suitable for enhancing the production of challenging proteins like $rhTGF-\beta 1$. In summary, understanding the intrinsic properties of $rhTGF-\beta 1$ and utilizing a combination of approaches, including anti-apoptotic engineering and chemical additives, can significantly enhance $rhTGF-\beta 1$ production in CHO cells, paving the way for therapeutic protein producing of $rhTGF-\beta 1$.