High cell density perfusion process for high yield of influenza A virus production using MDCK suspension cells

Abstract

Similar to the recent COVID-19 pandemic, influenza A virus poses a constant threat to the global community. For the treatment of flu disease, both antivirals and vaccines are available with vaccines the most effective and safest approach. In order to overcome limitations in egg-based vaccine manufacturing, cell culture-based processes have been established. While this production method avoids egg-associated risks in face of pandemics, process intensification using animal suspension cells in high cell density perfusion cultures should allow to further increase manufacturing capacities worldwide. In this work, we demonstrate the development of a perfusion process using Madin-Darby canine kidney (MDCK) suspension cells for influenza A (H1N1) virus production from scale-down shake flask cultivations to laboratory scale stirred tank bioreactors. Shake flask cultivations using semi-perfusion mode enabled high-yield virus harvests (4.25 log 10 (HAU/100 μL)) from MDCK cells grown up to 41 × 10 6 cells/mL. Scale-up to bioreactors with an alternating tangential flow (ATF) perfusion system required optimization of pH control and implementation of a temperature shift during the infection phase. Use of a capacitance probe for on-line perfusion control allowed to minimize medium consumption. This contributed to a better process control and a more economical performance while maintaining a maximum virus titer of 4.37 log 10 (HAU/100 μL) and an infectious virus titer of 1.83 × 10 10 virions/mL. Overall, this study clearly demonstrates recent advances in cell culture-based perfusion processes for nextgeneration high-yield influenza vaccine manufacturing for pandemic preparedness. Key points• First MDCK suspension cell-based perfusion process for IAV produciton was established.• "Cell density effect" was overcome and process was intensified by reduction of medium use and automated process control.• The process achieved cell density over 40 × 10 6 cells/mL and virus yield over 4.37 log 10 (HAU/100 μL).

Cell culture-based production of defective interfering influenza A virus particles in perfusion mode using an alternating tangential flow filtration system

“…We previously reported the batch production of pure DI244 particles in shake flasks for the use as an antiviral (Hein et al 2021a). To evaluate if process intensification strategies that are already used for non-modified MDCK suspension cells and IAV STV production (Wu et al 2021) would be applicable, a manually adjusted perfusion process in a STR was implemented. For this cultivation, the perfusion rate was adjusted every 12 h, according to a pre-calculated profile.…”

“…One manually adjusted and three automated perfusion cultivations were performed in the STR. The manually adjusted and one automated perfusion cultivation were conducted with a commonly used HFM to compare against results obtained with non-modified MDCK suspension cells and IAV STR production (Wu et al 2021). For the following two automated perfusion cultivations, the VHU was evaluated as a new cell retention membrane to test options for direct harvesting of virus particles through the membrane.…”

“…For higher perfusion rates, damage of the VHU was observed in previous perfusion cultivations (data not shown). After the medium exchange, the cultivation temperature was set to 32°C, as this was shown to have a positive impact on virus replication (Wu et al 2021). For infection, a pure DI244 seed virus at MODIP 1E-3 and 20 U/mL trypsin (5000 U/mL in PBS; 27250018, Gibco) were added.…”

“…However, uptake rates are relatively high and consequentially a high CSPR of 200 pL/cell/day was needed for perfusion cultivations. For comparison, when a perfusion process with the same medium but a different MDCK cell line (descendent from a MDCK cell line originally obtained from ATCC) was established, a lower CSPR from 60 pL/cell/day was sufficient (Wu et al 2021). Furthermore, no oxygen limitations were observed in cultivations with the latter and the optimal pH value for cell growth was much lower (pH 7.5 for MDCK-PB2(sus) and its parental cell line (ECACC); pH 7.0 for MDCK cells obtained from ATCC).…”

Production of a Foot-and-Mouth Disease Vaccine Antigen Using Suspension-Adapted BHK-21 Cells in a Bioreactor

“…This phenomenon is thought to be caused by a limitation of key nutrients, by-product inhibition, or cell-cycle arrest at high cell densities, although the exact mechanism(s) involved remain unknown [ 51 , 52 ]. Some strategies have been developed to overcome the cell-density effect, such as perfusion culture and medium replacement before virus infection [ 53 , 54 , 55 , 56 ]; thus, we performed batch mode cultivation with fresh medium replacement with the aim of developing a simple process. We tested 16 prototype media (eight CDMs and eight PFMs) and screened CDM-2, which showed the highest virus titer even at a low cell density ( Figure 4 ).…”