Very high density of Chinese hamster ovary cells in perfusion by alternating tangential flow or tangential flow filtration in WAVE bioreactor™—part II: Applications for antibody production and cryopreservation

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

Perfusion Processes

“…XD Process Technology at DSM, achieving very high cell densities (Zijlstra et al 2008), however experimental details have not been disclosed. In a wave bioreactor study, Clincke et al observed a similar cell growth until 10 8 cells/ mL density using microfiltration and using 50 kDa UF membrane connected as TFF or as ATF (Clincke et al 2013a). In this UF process, the permeate was pumped at a much lower negative pressure due to the smaller fiber pore size, eventually causing a complete fiber fouling around 10 8 cells/mL density.…”

“…Furthermore the alternating flow creates a back flush in the filter membrane, which is favourable to reduce the filter fouling. We compared the ATF and TFF performances and showed that the POI retention by the membrane was systematically less severe using the ATF (Clincke et al 2013a). The product retention was however not totally absent.…”

“…The spin-filter consists of a cylindrical screen, which is continuously rotating. The cell-free supernatant enters in the spin-filter from where it is pumped into the harvest line using TFF or ATF using polysulfone HF (Clincke et al 2011(Clincke et al , 2013a. They established steady state cultures at % 25 Â 10 6 cells/mL or 1-1.2 Â 10 8 cells/mL during more than 2 weeks and reached a maximal density of 2.14 Â 10 8 cells/mL.…”

“…We performed a series of experiments in a wave bioreactor connected to an ATF2 using a MAb producing CHO-K1 DHFR À cell line (Clincke et al 2011(Clincke et al , 2013aChotteau et al 2014a). We developed and studied this system in several runs maintained at 20 Â 10 6 cells/mL by cell bleeding during up to 2 weeks.…”

“…Applications of perfusion mode other than for the manufacturing of biopharmaceuticals have recently been reported and are increasingly used in the industry, such as cell bank manufacturing (Tao et al 2011;Seth et al 2013;Clincke et al 2013a) or high cell density seeding of production bioreactors, i.e. 'N-1 step', (Pohlscheidt et al 2013;Padawer et al 2013;Yang et al 2014;Hecht et al 2014).…”