A genetically engineered Dihydrofolate Reductase (DHFR)-/ - DG44 Chinese Hamster Ovary (CHO) cell line with Methotrexate (MTX) as a selective agent, expressing a recombinant monoclonal antibody was used. Cells were cultivated for 14 days in a fed-batch mode in a chemically defined medium and fed according to process description.
Culture systems: Different bioreactor scales were used in this study : ambr™48 (TAP -Sartorius Biosystems), an automated system with 48 disposable microbioreactor vessels, 2L stirred tank glasses bioreactors with Biostat B-DCUII control systems (Sartorius Stedim), 80L and 400L stainless-steel bioreactors (Zeta).
Data was analysed using JMP statistical (SAS) program.
All the experiments were conducted using the same cell bank at the same cell age at bioreactor inoculation.
pH (7.0 +/- 0.2) was controlled using CO2 and base addition.
The scale independent factors (pH, DO set point, seeding density, temperature, culture duration, media and feed composition), were the same for all the scales.
The scale dependent factors (culture start volume, feed volumes) were linearly adapted. Agitation speed and aeration that were determined theoretically or though experiment.
Sampling plans and sample volumes were especially adapted to the ambr™ system to take into account the low bioreactor volume.
A miniaturised metabolite assay was developed to allow daily measurements glucose, ammonium, lactate and osmolality using low analyte volumes.
Viable Cell Density (VCD) and cell viability were measured using a ViCell® XR cell counter (Beckman Coulter) directly connected to the ambr™ system. Metabolites (glucose, lactate, ammonium) concentrations were measured by an enzymatic assay using a UV-method (R-Biopharm) for the ambr™ vessels and by a BioProfile Analyser 400 (Nova Biomedical) for larger scale bioreactors. For all the systems, pH measurement was obtained with a BioProfile pHOx pH/Gas Analyser (Nova Biomedical). And osmolality was obtained using a Osmometer (Advanced Instruments). Production titers were measured throughout the culture using an Octet QK (ForteBio) and after 14 days with protein A HPLC (Waters).
Product quality attributes (PQA) of the produced monoclonal antibodies were analysed as follows: Cell culture fluid samples were centrifuged and filtered to remove cell debris. The monoclonal antibodies were purified by ÄKTA-express (GE Healthcare) Protein-A purification. The neutralized eluate was used for product quality analysis. Charge variant analysis determined the relative percentage of acidic, basic and main isoform species of the mAb by means of an imaged capillary isoelectric focusing system (Protein Simple, iCE3) after a Carboxypeptidase B digestion. Size exclusion analysis method determined the percentage of aggregates, monomers and fragment levels by size exclusion chromatography (SE-UPLC).