Increased t-PA Yields Using Ultrafiltration of an Inhibitory Product from CHO Fed-Batch Culture

Abstract

Fed-batch operation for the production of t-PA using Chinese Hamster Ovary (CHO) cells was optimized using serial and parallel experimentation. The feed, an isotonic concentrate, was improved to obtain 2- to 2.5-fold increases in integrated viable cell days versus batch. With a low glucose inoculum train, the viability index was further increased up to 4.5-fold. Hydrolysates were substituted for the amino acid portion of the concentrate with no significant change in fed-batch results. The concentrate addition rate was based on a constant 4 pmol/cell.day glucose uptake rate that maintained a relatively constant glucose concentration (approximately 3 mM). Increased viable cell indices did not lead to concomitant increases in t-PA concentrations compared to batch. The fed-batch concentrate and feeding strategy were shown to be effective in hybridoma culture, where a 4-fold increase in viable cell index yielded a 4-fold increase in antibody concentration. The half-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 extracellular t-PA concentrations upon exogenous additions. Instead, intracellular t-PA levels were increased, revealing a possible secretory pathway limitation. A new reactor configuration was developed using an acoustic filter to retain 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 increased over 2-fold, up to a yield of 80 mg/L.

Host–Guest Chemistry of Aromatic‐Amide‐Linked Bis‐ and Tris‐Calix[4]pyrroles with Bis‐Carboxylates and Citrate Anion

“…Since the feeding was begun only after cell densities had reached ∼1 × 10 6 cells/mL, the feeding was initiated at different times for different supplements. Finally, glucose (Sigma) was added to the fed‐batch cultures to maintain ≥3 g/L44, 46, 50 on an as needed basis.…”

“…The t‐PA titer was determined using a colorimetric enzymatic activity assay as described previously 44, 50. A conversion factor of 630,000 U/mg converted the activity units to concentrations 44. The overall cell specific productivity ( q p ) was determined from the slope of plots of integrated viable cell density versus t‐PA concentration.…”

“…A Multiparameter Bioanalytical System YSI 7100 (YSI Life sciences, Yellow Springs, OH) was used to measure glucose, lactate, glutamine, and glutamate concentration in the culture supernatant. Amino acid and ammonia concentrations were determined by the AccQ‐tag method followed by separation on a Waters (Milford, MA) C‐18 reverse‐phase column using a Waters HPLC system (600S controller and Millenium) according to Dowd et al 2000 44…”

“…In this study, a model CHO cell line expressing t‐PA was used to investigate glutamine replacement strategies to reduce ammonia production and to improve recombinant protein productivity in fed‐batch culture. t‐PA levels in fed‐batch cultures have been reported up to 45 mg/L using serum‐free medium40, 44 and up to 250 mg/L of t‐PA using serum containing medium 45. Recently, we have reported over 500 mg/L fed‐batch production using inhibition of autophagy by 3‐methyl adenine 46.…”

Colorimetric pH measurement of animal cell culture media

“…CHO cells are widely used to produce valuable therapeutic proteins (Trill et al 1995;Dowd et al 2000;Kaszubska et al 2000). In this study, suspension CHO cells expressing recombinant human EPO (R-CHO-S) and its exclusive culture medium, CHO-S-SFM II, were used.…”