Plant-based systems (whole plants and plant cell suspension cultures) have been investigated as alternative for the production of recombinant proteins, especially for human therapeutics (biopharmaceuticals) due to their intrinsic product safety, cost-effective bioprocessing, and the capacity for proper protein post-translational modification. Unlike whole plant systems, plant cell cultures can be grown under controlled conditions in bioreactors to reproduce product yield and quality, particularly if the products can be targeted to and purified from the cell culture broth, making it as an attractive platform for sustained biopharmaceutical production. In this study, different aspects related to the expression of recombinant human alpha-1-antitrypsin (rAAT) glycoprotein in transgenic tobacco (Nicotiana benthamiana) cell cultures were investigated. Three alternative gene expression systems were developed for expressing rAAT, including a Cauliflower mosaic virus (CaMV) 35S constitutive promoter expression system (35S), a chemically inducible, estrogen receptor-based promoter expression system (XVE), and a novel Cucumber mosaic virus inducible viral amplicon (CMViva) expression system. The results demonstrated the CMViva system gives a higher titer of extracellular functional rAAT (27 μg/L) and a higher rAAT specific activity (ratio of functional to total rAAT) (22%) than that in XVE or 35S system in a batch bioreactor operation. A biphasic bioreactor strategy involving pH adjustment was proposed for significantly improving the production of functional rAAT by minimizing proteolytic degradation of and maintaining inherent stability of rAAT protein, resulting in a 4-folds increase in functional rAAT yield (100 μg/L) and a 2-folds increase in rAAT specific activity (48.6%) in transgenic CMViva cell culture bioreactor. For the optimization of semicontinuous culture bioreactor, it demonstrated that the transgenic CMViva cells exhibit higher oxygen uptake rate, lower phenolics content and lower protease activity, leading to a 22-folds increase in functional rAAT yield (603 μg/L) and a higher rAAT specific activity (85.7%). The impact of post-transcriptional gene silencing (PTGS) on transgene expression was further investigated by evaluating the effectiveness of viral gene silencing suppressors (VGSS) for improving the rAAT protein production in transgenic plant cell cultures. In transgenic CMViva cell cultures, the functional rAAT as a percentage of total soluble protein was increased 5.7 folds with the expression of P19 and 17.2 folds with the co-expression of CP, P19 and P24, suggesting that PTGS triggered in the transgenic CMViva cell cultures can be highly suppressed by the co-expression of VGSS of different modes of action and further highlighted the importance of VGSS on enhancing the recombinant protein production. Finally, the optimization of cell culture medium using statistical design of experiment demonstrated that the supplementation of critical amino acids (cysteine and isoleucine) was beneficial for improving the functional rAAT production, leading to a 9-folds increase in functional rAAT yield, implying that cysteine and isoleucine can possibly prevent AAT protein from proteolytic degradation and/or stabilize the inherent stability of AAT protein. These results lay for a foundation for developing plant cell cultures as a platform of biopharmaceuticals production in industry.