An animal-component-free and chemically defined fed-batch process for GS-engineered cell lines producing recombinant antibodies has been developed. The fed-batch process relied on supplying sufficient nutrients to match their consumption, simultaneously minimizing the accumulation of by-products (lactate and osmolality). The proportionalities of nutritional consumption were determined by direct analysis. The robust, metabolically responsive feeding strategy was based on the offline measurement of glucose. The fed-batch process was shown to perform equivalently in GS-CHO and GS-NS0 cultures. Compared with batch cultures, the fed-batch technology generated the greater increase in cell yields (5-fold) and final antibody concentrations (4-8-fold). The majority of the increase in final antibody concentration was a function of the increased cell density and the prolonged culture time. This generic and high-yielding fed-batch process would shorten development time, and ensure process stability, thereby facilitating the manufacture of therapeutic antibodies by GS-engineered cell lines.