Large-scale transient gene expression in mammalian cells is being developed for the rapid production of recombinant proteins for biochemical and preclinical studies. Here, the scalability of transient production of a recombinant human antibody in Chinese hamster ovary (CHO) cells was demonstrated in orbitally shaken disposable bioreactors at scales from 50 mL to 50 L. First, a small-scale multiparameter approach was developed to optimize the poly(ethylenimine)-mediated transfection in 50 mL shake tubes. This study confirmed the benefit, both in terms of extended cell culture viability and increased product yield, of mild hypothermic cultivation conditions for transient gene expression in CHO cells. Second, the scalability of the process was demonstrated in disposable shake bioreactors having nominal volumes of 5, 20, and 50 L with final antibody yields between 30 and 60 mg L−1. Thus, the combination of transient gene expression with disposable shake bioreactors allows for rapid and cost-effective production of recombinant proteins in CHO cells.