Monoclonal antibodies and recombinant proteins have increased their importance and gained success as therapeutic agents in the treatment of various diseases.¹

Biomanufacturing a recombinant product by cell culture using animal cells follows a main route from a biology-driven upstream process to a downstream process strictly guided by technical and biochemical principles.¹

The biology-driven upstream process is the pacemaker and latest advances in cell culture revealed product titres of up to 5 mg/mL of an recombinant protein. Furthermore, both cell densities and cell viabilities have undergone significant changes, which have brought new challenges for the initial recovery process (IRP) of such a biopharmaceutical product.

Today's technologies in the IRP of the downstream process require the principal capability to handle cell densities of at least 10 mio cells/mL, product titres of up to 5 mg/mL and cell viabilities of <50%.²

Figure 1 Principle positioning of multilayer technologies.

Such recovery steps aim to remove cells and cell debris; however, this is now being challenged with cell culture processes increasingly featuring low cell viabilities. Instead, the focus has shifted to the recovery of the target protein out of cell debris such as feed stream, with a significantly increased amount of cell-derived contaminants, such as Chinese hamster ovary protein (CHOP), host cell DNA, lipoproteins and endogenous viruses. DNA levels in direct bioreactor offloads have increased up to 10⁶ppm and pose a major challenge to the entire chromatography strategy applied to a biopharmaceutical product.²

Cellulose-based filtration techniques have been used from early on in the IRP and are now advancing into the spotlight as the contaminant level needs to be tackled earlier to enable better intermediate purification and polishing of the target protein.

Figure 2 Filterability data for single layer and double layer depth filter technologies; post bioreactor offload application; 5 mio cells/mL and 75% viability; 11 μm/4 μm and 8 μm/1 mm filters are from Sartorius-Stedim Biotech.

Traditionally, one or more cellulose-based depth filter operations have been used in IRP.³Depth filter operations can feature either single layer depth filter media or a combination of various depth filter media and/or membranes into one process step.⁴Combining two or more depth filter media composites into one process element reduces the filtration steps from two to one. However, driven by the large amount of cells to be removed and cell debris-induced fouling mechanisms, multilayer technologies can act as single-stage operations that can be positioned after each other to tackle the overall challenge. In detail, bioreactor offloads with turbidities in excess of 1000 FNU might require a two-stage filtration each featuring multilayer depth filter configurations (Figure 1). Alternatively, a continuous centrifuge might be considered for the first removal step followed by a multilayer filtration step. The higher the cell density within a cell culture process the more likely it is a centrifuge will have to be positioned as the first recovery step taking into account that ultra large-scale volumes of >10000 L require handling by the IRP. Recent publications address this topic and discuss the use of two different multilayer depth filters in a post centrifuge application.⁵

The depth filtration operation in a post centrifuge application remains challenging as larger amounts of submicron particles are induced by either the cell culture process or the centrifuge process by shear forces or other mechanical stresses applied to the biological fluid.

Initial recovery is more than just applying filtration or centrifugation techniques to remove cells and cell debris. Bioburden control is another major task and thus 0.2 μm sterile grading filtration is integrated into the process chain, usually as a disposable technique.

The overall capacity of the sterile grading filtration operation is a direct function of the depth filter capability to reduce the bioreactor offload or post centrifuge turbidity.

The turbidity in both applications is a function of larger and submicron particles, which must be removed prior to sterile grade filtration.

Table 1 Comparison of filter effluent turbidity; post bioreactor offload application; 5 mio cells/mL, 75% viability, 1000 FNU filter inlet turbidity.

Multilayer depth filter technologies offer higher bed heights than single layer technologies of up to 11 mm and, therefore, significantly better control of breakthrough effects of bioreactor-derived contaminants. Thus, the protection of a subsequent 0.2 μm filter is improved and overall process costs are reduced.