Abstract
A high cell density perfusion process of monoclonal antibody (MAb) producing Chinese hamster ovary (CHO) cells was developed in disposable WAVE Bioreactor™ using external hollow fiber (HF) filter as cell separation device. Tangential flow filtration (TFF) and alternating tangential flow (ATF) systems were compared and process applications of high cell density perfusion were studied here: MAb production and cryopreservation. Operations by perfusion using microfiltration (MF) or ultrafiltration (UF) with ATF or TFF and by fed-batch were compared. Cell densities higher than 108 cells/mL were obtained using UF TFF or UF ATF. The cells produced comparable amounts of MAb in perfusion by ATF or TFF, MF or UF. MAbs were partially retained by the MF using ATF or TFF but more severely using TFF. Consequently, MAbs were lost when cell broth was discarded from the bioreactor in the daily bleeds. The MAb cell-specific productivity was comparable at cell densities up to 1.3 × 108 cells/mL in perfusion and was comparable or lower in fed-batch. After 12 days, six times more MAbs were harvested using perfusion by ATF or TFF with MF or UF, compared to fed-batch and 28× more in a 1-month perfusion at 108 cells/mL density. Pumping at a recirculation rate up to 2.75 L/min did not damage the cells with the present TFF settings with HF short circuited. Cell cryopreservation at 0.5 × 108 and 108 cells/mL was performed using cells from a perfusion run at 108 cells/mL density. Cell resuscitation was very successful, showing that this system was a reliable process for cell bank manufacturing. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:768–777, 2013
Highlights
Fed-batch mode is widely used for the production of glycoprotein.[1]
Part II of this study focuses on the applications of perfusion by HF: the monoclonal antibody (MAb) production using MF alternating tangential flow (ATF) or MF Tangential flow filtration (TFF), the production obtained using the same perfusion settings with an external hollow fiber ultrafilter cartridge (UF HF), a comparison with fed-batch production, and cell bank manufacturing from high-cell-density bioreactor
Comparable amounts of MAb were produced by the cells in perfusion by ATF or TFF, MF or UF; the MAbs were partially retained by the MF
Summary
Fed-batch mode is widely used for the production of glycoprotein.[1]. This mode is typically based on the use of concentrated feeds and stoichiometric delivery of needed components in particular amino acids.[2,3] Often, a strategy of minimizing the by-product production is used.[4,5] Interestingly, simulation models have shown that the cost of goods per gram of produced antibody is the same for perfusion and fed-batch.[6,7] Lim et al.[6] pointed out that perfusion was more favorable economically owing to a lower initial investment but that looking at the failure and contamination risk fedbatch was preferred. They can be used for some benchmarking: 6-, 7-, and 1.4-fold higher production in perfusion than in fed-batch for HeLa, SF-9 insect, and Chinese hamster ovary (CHO) cells, respectively, has been reported.[8,9,10] Fed-batch process can result in higher level of protease, lower cell viability, and higher accumulation of lactate and ammonium, whereas perfusion provides a constant environment more favorable for the health of the cells and for the quality of the product of interest This is true when a cell-specific perfusion rate is applied.[11,12]
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