The present invention describes methods and processes for the production of proteins, particularly glycoproteins, by animal
cell or
mammalian cell culture, preferably, but not limited to, fed-batch
cell cultures. In one aspect, the methods comprise at least
two temperature shifts performed during the culturing period, in which the temperature is lower at the end of the culturing period than at the time of
initial cell culture. Throughout their duration, the culturing processes of the invention involving two or more downward shifts in temperature sustain a high viability of the cultured cells, and can yield an increased end
titer of
protein product, and a high quality of
protein product, as determined, e.g., by
sialic acid content of the produced
protein. In another aspect, the methods comprise the delayed addition of polyanionic compound during the culturing period. The delayed addition of polyanionic compound sustains a high viability of the cultured cells, and can extend the
growth phase,
delay the onset of the death phase, and arrest the death phase.