D-optimal mixture design of experiments to optimize a monoclonal antibody production process

VALENTINA WANDEL-PETERSEN; RICARDO KRATJE; DR. MARCOS OGGERO EBERHARDT; MARÍA DE LOS MILAGROS BÜRGI

Lisboa

Congreso; 27th ESACT Meeting; 2022

European Society for Animal Cell Technology (ESACT)

Background and novelty: The purification and characterization processes of newly prepared erythropoietin-based neurotherapeutic candidates require large amounts of a specific monoclonal antibody (mAb), which heretofore, was obtained in vivo. Due to ethical, scientific and safety concerns inherent in laboratory work with animals, the producer hybridoma was adapted to a serum-free media (SFM) to enable the in vitro large-scale production of mAb avoiding the drawbacks of using fetal bovine serum (FBS). Aiming to optimize the mAb production process increasing its efficiency, product quality and minimizing production costs, a design of experiments (DoE) approach was carried out.Experimental approach Hybridomas underwent a step-wise adaptation involving a progressive decrease of FBS concentration in DMEM/Ham’sF12 culture media until 1.25% FBS was reached. In a further step, cells were adapted to grow in a SFM composed by DMEM/Ham’sF12 and EXCELL-620 media mixture supplemented with cholesterol, via direct and sequential adaptation protocols.SFM formulation was optimized using a D-optimal mixture DoE towards minimizing production costs and improving yield. We evaluated the following mixtures of DMEM/Ham´sF12:EXCELL-620: 50:50, 56:44, 63:37, 75:25, 81:19, 87:13, 94:6.Cell cultures were compared by evaluating specific parameters: growth rate, death rate, productivity and mAb half maximal effective concentration (EC50) as estimator of the mAb binding properties.Results and discussion Sequential adaptation decreased hybridoma productivity by 96%. However, the direct approach maintained a growth rate and productivity comparable to the initial condition. This clone yielded 200 mg of antibodies (average concentration 120 μg/ml) which preserved the initial EC50, in a single batch culture. DoE allowed the elimination of FBS and cholesterol as nutritional supplements. The study revealed two different optimal SFM formulations: the 67:33 and 94:6 mixtures of DMEM/Ham’sF12:EXCELL-620, that reduced SFM cost by 6 and 22 times, respectively, in relation to the initial culture medium. Both conditions maintained the growth rate of the initial condition (mean value of 0.04 h-1) and increased clone productivity 1.7-fold and 1.5-fold. Also, they allowed conserving the EC50 mAb and increased its purity by 1.3 times compared with its in vivo production. The optimized process significantly outperformed the in vivo production method, allowing us to replace it by an economical, reproducible and scalable process, free from animals and animal derivatives. Future work will focus in scaling up the mAb production, utilizing a biphasic process in bioreactor, in which the 67:33 mixture will be used as growth medium whereas the 94:6 mixture will be used as perfusion one.