Mammalian cell culture technology has dramatically advanced over the past few decades because of the increasing demand for therapeutic proteins. A number of therapeutic proteins have been developed and produced in large‐scale manufacturing systems. To meet the increasing demand for high quality and quantity of therapeutic proteins, this technology requires optimization of a number of parameters. This chapter presents an overview of key aspects for processing optimization from upstream to downstream. We also provide recent advances in therapeutic protein production in mammalian cells, particularly Chinese hamster ovary (CHO) cells, and suggest new approaches for future developments.