Katrin Köhler

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

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.

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%.

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 10

  ppm and pose a major challenge to the entire chromatography strategy applied to a biopharmaceutical product.

Figure 1 Principle positioning of multilayer technologies. 

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. 

Traditionally, one or more cellulose-based depth filter operations have been used in IRP.

  Depth 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.

  Combining 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.

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 Î¼m/4 Î¼m and 8 Î¼m/1 mm filters are from Sartorius-Stedim Biotech. 

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.  

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. 

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

The main goal of using cellulose-based depth filters in the post bioreactor application is to remove cells and cell debris, and allow for a subsequent bioburden reduction filtration. Figures 2–4 present data based on both low cell density (5 mio cells/mL) and high cell density (19 mio cells/mL) bioreactor offloads.  

In Figure 2, it is shown that a double layer depth filter used in a bioreactor offload application provides similar capacity to a single layer filter.  

All double layer depth filters tested provided significantly lower effluent turbidity and thus higher sterile grade filter capacity than any of the single layer depth filters tested.  

The lowest single layer effluent turbidity (8 μm filter) has been measured at 272 FNU, whereas the lowest effluent turbidity for the double layer filter is <5 FNU, which is summarized in Table 1. However, all filters tested showed a protein transmission of >95%.  

Figure 3 Breakthrough curves for single and double layer depth filter technologies; post bioreactor offload application; 5 mio cells/mL and 75% viability; 11 Î¼m/4 Î¼m filter is from Sartorius-Stedim Biotech. 

Figure 3 outlines breakthrough curves for single and double layer technologies used for the low cell density post bioreactor application described previously.  

For all single layer depth filter technologies tested the breakthrough curve starts earlier and ends in a higher final point and, thus, average effluent turbidity is greater than the values obtained for all double layer depth filters tested. 

Table 2 Comparison of depth filter effluent turbidities, taken from Figure 5. 

The data shown in Figure 4 outline decreased capacities for double layer technologies of <100 L/m

 . This is because of very high cell density (19 mio cells/mL) and ultra low cell viability (6%).  

For such applications featuring a cell density of >10 mio cells/mL and a cell viability of <15%, it is suggested to position a centrifuge as the first cell removal and clarification step.  

However, cell viabilities of <15% pose a major challenge to the subsequent purification regime as larger amounts of host cell-derived contaminants are loaded into the feed stream, which will have to be cleared by chromatography unit operations. 

Figure 4 Direct comparison of two double layer depth filter combinations used for the filtration of a high cell density (19 mio cells/mL) cell derived bioreactor offload fluid; 11 Î¼m/4 Î¼m filter is from Sartorius-Stedim Biotech. 

Within the post centrifuge application, the main goal of using cellulose-based depth filters is to remove residual cell debris and ideally other cell-derived contaminants.  

Figures 5–7 present filterability data based on both initial low cell density (3.8 mio cells/mL [Figure 5]) and high cell density (19.5 mio cells/mL [Figures 6 and 7]) bioreactor offloads subject to centrifugation and depth filtration at a tested capacity of 200 L/m

Within the post centrifuge application, the single layer depth filter does not match the capability of the double and multilayer filters with respect to filter effluent turbidity. Thus, protection of a subsequent sterile grading 0.2 μm membrane filter is improved using a double or multilayer depth filter. 

Figure 5 Comparison of filterability data of single layer, double layer and multilayer depth filter technologies in a post centrifuge low cell density (3.8 mio cells/mL) application; all filters are from Sartorius-Stedim Biotech. 

In this application, the post centrifuge inlet turbidity was measured at 128 FNU and the single layer 0.3 μm depth filter tested reduced this turbidity to an average of 59 FNU at a target capacity of 200 L/m

Changing from the single layer 0.3 μm depth filter to a double layer 0.3 μm/0.3 μm depth filter further decreased the turbidity to 9.3 FNU.  

Incorporating a 0.2 μm membrane into the double layer depth filter combination provides the lowest effluent turbidity of 4.5 FNU, which is outlined in Table 2. 

Results from another post centrifuge application with a filter inlet turbidity of 86 FNU (high cell density 19.5 mio cells/mL [Figure 6]) confirm the reduction of the depth filter effluent turbidity, significantly increasing the sterile grade membrane filter capacity by incorporating a membrane into the double layer depth filter unit operation.  

A combination of depth filters with a 0.1 μm membrane provides the lowest filter effluent turbidity value observed of 1.8 FNU. 

Figure 7 outlines breakthrough curves for double and multilayer technologies used for the high cell density post centrifuge application from Figure 6. 

Table 3 Comparison of depth filter effluent turbidities, taken from Figure 6. 

For the multilayer depth filter with a 0.1 μm membrane, the breakthrough curve ends in a lower final point and thus presents a lower average effluent turbidity than the multilayer depth filter with a 0.2 μm membrane and the double layer depth filter without a membrane.  

Figure 6 Direct comparison of double layer and multilayer depth filter combinations in a post centrifuge application (high cell density 19.5 mio cells/mL and 82% viability); all filters except product AXXX are from Sartorious-Stedim Biotech..

For post centrifuge applications, combining depth filter configurations with membranes significantly reduces the turbidity and effectively controls breakthrough effects.  

Determining the breakthrough point is essential as excessive breakthrough observed on any depth filter operation will lead to significant reduction in the subsequent membrane filter unit operation.  

Furthermore, excessive initial differential pressure or flow rates on the depth filter will significantly influence the overall depth filter capacity, as well as the depth filter effluent turbidity.

Therefore, it is important to carefully monitor the pressure differential and filter effluent turbidity during a process optimization run.  

With respect to the process design for the IRP, it is essential to consider all parameters that might significantly influence the process design.  

Cell density and cell viability are crucial and need to be considered as limiting parameters for the post bioreactor application.  

Katrin Köhler

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