Industrial therapeutic protein production has been greatly improved through fed-batch development. In this study, improvement to the productivity of a tissue-plasminogen activator (t-PA) expressing Chinese hamster ovary (CHO) cell line was investigated in shake flask culture through the optimization of the fed-batch feed and the reduction of ammonia generation by glutamine replacement. The t-PA titer was increased from 33 mg/L under batch conditions to 250 mg/L with daily feeding starting after three days of culture. A commercially available fed-batch feed was supplemented with cotton seed hydrolysate and the four depleted amino acids, aspartic acid, asparagine, cysteine, and tyrosine. The fed-batch operation increased the generation of by-products such as lactate and ammonia that can adversely affect the fed-batch performance. To reduce the ammonia production, a glutamine-containing dipeptide, pyruvate, glutamate, and wheat gluten hydrolysate, were investigated as glutamine substitutes. To minimize the lag phase as the cells adjusted to the new energy source, a feed glutamine replacement process was developed where the cells were initially cultured with a glutamine containing basal medium to establish cell growth followed by feeding with a feed containing the glutamine substitutes. This two-step feed glutamine replacement process not only reduced the ammonia levels by over 45% but, in the case of using wheat gluten hydrolysate, almost doubled the t-PA titer to over 420 mg/L without compromising the t-PA product quality or glycosylation pattern. The feed glutamine replacement process combined with optimizing other feed medium components provided a simple, practical, and effective fed-batch strategy that could be applied to the production of other recombinant therapeutic proteins. (c) 2012 American Institute of Chemical Engineers Biotechnol. Prog.29;165-175, 2013