Investigation of the interactions of critical scale-up parameters (pH, pO2 and pCO2) on CHO batch performance and critical quality attributes

Abstract

Understanding process parameter interactions and their effects on mammalian cell cultivations is an essential requirement for robust process scale-up. Furthermore, knowledge of the relationship between the process parameters and the product critical quality attributes (CQAs) is necessary to satisfy quality by design guidelines. So far, mainly the effect of single parameters on CQAs was investigated. Here, we present a comprehensive study to investigate the interactions of scale-up relevant parameters as pH, pO₂ and pCO₂ on CHO cell physiology, process performance and CQAs, which was based on design of experiments and extended product quality analytics. The study used a novel control strategy in which process parameters were decoupled from each other, and thus allowed their individual control at defined set points. Besides having identified the impact of single parameters on process performance and product quality, further significant interaction effects of process parameters on specific cell growth, specific productivity and amino acid metabolism could be derived using this method. Concerning single parameter effects, several monoclonal antibody (mAb) charge variants were affected by process pCO₂ and pH. N-glycosylation analysis showed positive correlations between mAb sialylation and high pH values as well as a relationship between high mannose variants and process pH. This study additionally revealed several interaction effects as process pH and pCO₂ interactions on mAb charge variants and N-glycosylation pattern. Hence, through our process control strategy and multivariate investigation, novel significant process parameter interactions and single effects were identified which have to be taken into account especially for process scale-up.

Keywords: CHO cell culture; Design of experiments; Monoclonal antibody CQA; Process parameter; Scale-up.

Fig. 1

Viable cell density (a) and cell viability (b) over process time for all batch fermentations. (Black symbols represent processes at pH 7.0, blue symbols at pH 6.8, red symbols at pH 7.2; closed symbols represent processes at pCO₂ 5 %, half-closed at 12.5 % and open symbols at 20 %; triangles represent processes at pO₂ 25 %, squares at 10 %, circles at 40 %). High pH values led to high viable cell densities but concurrently to shorter process time due to faster depletion of the main c-source. Cell viabilities stayed at high values as long as glutamine was available

Fig. 2

Metabolite profiles for all conducted batch processes. (Black symbols represent processes at pH 7.0, blue symbols at pH 6.8, red symbols at pH 7.2; closed symbols represent processes at pCO₂ 5 %, half-closed at 12.5 % and open symbols at 20 %; triangles represent processes at pO₂ 25 %, squares at 10 %, circles at 40 %). a Glucose became limiting in almost all fermentations before reaching the harvest criteria of 75 % viability. b Glutamine profiles showed glutamine limitations after 100–150 h of process time for all batches. c Ammonia concentrations showed process phases of production and consumption for almost all runs at pH values of 6.8 and 7.0, whereas only production and steady-state values were derived from fermentation runs at pH 6.8. d Lactate was produced and consumed during all processes

Fig. 3

IgG concentration over process time (a) and integral viable cell density (b) for all batch fermentations. (Black symbols represent processes at pH 7.0, blue symbols at pH 6.8, red symbols at pH 7.2; closed symbols represent processes at pCO₂ 5 %, half-closed at 12.5 % and open symbols at 20 %; triangles represent processes at pO₂ 25 %, squares at 10 %, circles at 40 %). Highest process titers were obtained for fermentation runs conducted at pH 7.0 and 6.8, mainly due to the highest IVCD values at these process conditions

Fig. 4

Relative area out of cation exchange chromatography (CEX) for various charge variants and one sum parameter at different pH set points for pCO₂ 12.5 % and pO₂ 25 %

Fig. 5

Correlations and trends of glycosylation variants. (Black symbols represent processes at pH 7.0, blue symbols at pH 6.8, red symbols at pH 7.2; closed symbols represent processes at pCO₂ 5 %, half-closed at 12.5 % and open symbols at 20 %; triangles represent processes at pO₂ 25 %, squares at 10 %, circles at 40 %). Antibody galactosylation level (GI) over a sialylation (SI) and b afucosylation (aFI). Antibody galactosylation seemed to correlate positively with afucosylation and sialylation. A strong correlation between sialylation and mannosylation 8 were derived from C. In D, mannose 8 levels are plotted over mannose 6, it can be derived that highest mannosylation levels only occurred for processes at pH 6.8 and 7.2