Accumulation of toxic waste byproducts in hybridoma culture can limit cell growth and monoclonal antibody (MAb) productivity, and one of the major toxic metabolites is lactate produced via glycolytic pathway of glucose metabolism. The factors affecting the glucose to lactate conversion rate were investigated. The conditions of high initial glucose concentration and high growth rate stimulated glucose to lactate conversion rate. The glucose-controlled fed-batch culture was investigated, and 19% reduction in lactate formation and 41% enhancement of MAb titer could be achieved by fed-batch culture.