Increased t-PA yields using ultrafiltration of an inhibitory product from CHO fed-batch culture

Fed-batch operation for the production of t-PA using Chinese Hamster Ovary (CHO) cells was optimized using serial and parallel experimentation. The fe ed, an isotonic concentrate, was improved to obtain 2- to 2.5-foId increase s in integrated viable cell days versus batch. With a low glucose inoculum train, the viability index was further increased up to 4.5-fold. Hydrolysat es were substituted for the amino acid portion of the concentrate with no s ignificant change in fed-batch results. The concentrate addition rate was b ased on a constant 4 pmol/cell(.)day glucose uptake rate that maintained a relatively constant glucose concentration (approximately 3 mM). Increased v iable cell indices did not lead to concomitant increases in t-PA concentrat ions compared to batch. The fed-batch concentrate and feeding strategy were shown to be effective in hybridoma culture, where a 4-fold increase in via ble cell index yielded a 4-fold increase in antibody concentration. The hal f-life of t-PA decreased from 43 to 15 days with decreasing cell viability (from 92% to 71%), but this was not sufficient to explain the apparent t-PA threshold. Instead, the CHO results were explained by a reduction in t-PA production at higher extracellular t-PA concentrations that limited the fed -batch maximum at 35 mg/L for the cell line investigated. Analysis of both the total and t-PA mRNA levels revealed no response to increasing extracell ular t-PA concentrations upon exogenous additions. Instead, intracellular t -PA levels were increased, revealing a possible secretory pathway limitatio n. A new reactor configuration was developed using an acoustic filter to re tain the cells in the reactor while an ultrafiltration module stripped t-PA from the clarified medium before the permeate was returned to the reactor. By adding this harvesting step, the t-PA fed-batch production was increase d over 2-fold, up to a yield of 80 mg/L.