Enhancing Recombinant Monoclonal Antibody Production in CHO Cell Cultures: Leveraging the Power of Multivariate Analysis and Chemical Additives in Upstream Bioprocessing

Biopharmaceutical manufacturing is a complex process requiring in-process quality control to ensure product yield and quality. Monitoring key parameters, such as viable cell density (VCD), metabolite levels, and antibody titer, is critical for addressing irregularities and optimizing production outcomes. With the increasing demand for monoclonal antibodies (mAbs), more research has focused on improving upstream bioprocessing. This thesis focuses on two critical aspects of mAb production: (1) employing multivariate data analysis (MVDA) with spectroscopic data to determine key parameters during mAb production in CHO cells, and (2) using chemical additives to enhance mAb production through cell proliferation regulation. In the first aspect, Chapter 2 demonstrates the use of MVDA with UV-Vis spectroscopy for monitoring critical parameters such as viable cell density (VCD), antibody titer, and cell viability during mAb production. Partial least squares (PLS) models were developed to predict these parameters accurately. The study showed the feasibility of using minimal volumes (as low as 2 µL) for rapid evaluation of small-scale cultures. Compared to advanced instruments such as Fourier-transform infrared (FTIR) and near-infrared spectroscopy (NIR), UV-Vis spectroscopy offers a cost-effective alternative with broad applicability, particularly in resource-limited settings. Moreover, Chapter 3 expanded on this approach by utilizing microvolume UV-Vis spectroscopy for fast screening of high-producing clones. This method eliminated the reliance on traditional assays such as trypan blue staining and ELISA, thereby reducing time and complexity, particularly advantageous for laboratory-scale operations in low-to-middle-income countries. The second aspect focused on the regulation of cell proliferation using chemical additives, emphasizing cell cycle arrest mechanisms. Chapter 4 investigated the effects of adenosine and cordycepin (adenosine derivative) on mAb production in two CHO cell lines, CHO-DHFR– and GS-KO CHO cells. Adenosine significantly increased antibody titer in GS-KO CHO cells under fed-batch conditions with glucose feeding. Conversely, cordycepin enhanced antibody titer only in CHO-DHFR– cells in batch cultures without glucose feeding. These findings highlighted the metabolic diversity among CHO cell lines, emphasizing the need for cell line-specific supplementation strategies and a deeper understanding of additive mechanisms. Chapter 5 explored ectoine as a novel additive for enhancing mAb production in fed-batch cultures with glucose feeding. Ectoine improved cell viability and prolonged culture duration through cell cycle arrest mechanisms, as confirmed by RNA sequencing. This compound demonstrated its potential to increase antibody titers, particularly in CHO-DHFR– cells, by modulating gene expression related to cell proliferation and homeostasis. Besides these two critical aspects, an additional study in Appendix A examined thermal stability differences between erythropoietin (EPO) formulations with different additives. Hemax®, containing human serum albumin (HSA), exhibited greater thermal stability compared to the polysorbate-80-based Eprex®. These findings highlighted the importance of additive composition in ensuring the safety and efficacy of biopharmaceutical products. In conclusion, this study highlights innovative approaches for enhancing mAb production, emphasizing the utility of microvolume UV-Vis spectroscopy assisted with MVDA and the potential of novel chemical additives in mAb production.