The enhancement of recombinant protein expression of a transfected cell line is essential for the development of an efficient large-scale bioprocess. The effect of various media additives and temperature conditions were studied in an attempt to optimize protein production, stability, and protein glycosylation from a Chinese hamster ovary (CHO) cell line producing human β-interferon (Hu-β-IFN). We observed a decrease in the ELISA response of the glycoprotein in the later stages of batch cultures, which was attributed to molecular aggregation. Cells were subjected to various concentrations of glycerol, dimethyl sulfoxide (DMSO), and sodium butyrate (NaBu) in a variety of culture systems and conditions. The addition of both NaBu and DMSO resulted in higher specific productivities but reduced growth rates that resulted in a net reduction of interferon produced. Glycerol appeared to stabilize the secreted β-IFN, resulting in reduced aggregation, despite a decrease in cell growth rate. Glycosylation analysis of isolated β-IFN showed a time-dependent decrease in sialylation in batch culture that was ameliorated by the presence of glycerol. Low-temperature conditions (30 °C) had the greatest effect on productivity with a significant increase in β-IFN titer as well as a reduction in the degree of molecular aggregation.