Centrifugation
With the advent of low-shear centrifuges, the use of continuous disk-stack centrifugation coupled with depth filtration became the industry-preferred method for harvest recovery.
Scale up of centrifugation has historically been performed using the Sigma concept. Maintaining the ratio of the flow rate (Q) to the equivalent clarification or settling area (∑) constant enables scaling up the process. However, there is a limitation to this scale up approach, often requiring a reduction in Q/∑ to maintain the clarification efficiency. This limitation is attributed to the significant difference in the equipment design at different scales, such as the lack of a partial discharge functionality for the lab scale centrifuge.
Various centrifuge operating parameters (g-force, residence time, and discharge frequency) and cell culture feedstock quality (e.g., cell density, viability at harvest) have an impact on the clarification efficiency of the centrifuge. Some of the process impurities, including the cell culture media components and host cell impurities, are negatively charged at harvest pH values and can form colloids during the cell culture and harvest processes.
Shear forces within a centrifuge could also generate additional particles. The wide size distribution of these particles may result in insufficient clarification. This decrease in clarification efficiency can have a negative impact on the performance of the subsequent chromatographic capture step, as well as the sterile filterability of process intermediates.
kSep Systems’ patented kSep technology provides an opportunity to consider the use of single-use centrifugation systems to either harvest cells as product or discard cells as by-product like in the case of mAb manufacturing. Through the balance of centrifugal and fluid flow forces, the kSep retains particles, such as cells or microcarriers, as a concentrated fluidized bed under a continuous flow of media or buffer. kSep Systems are fully automated, easily scalable, and have reduced processing steps and time. kSep Systems’ low-shear process ensures reduced downstream contamination and high product quality, while achieving robust cell clarification efficiencies for high cell density cultures (10–50×106cells/mL). Higher recoveries, while achievable are usually associated with increased pool volumes. Many biopharma companies have evaluated kSep Systems for various different applications, but to our knowledge it has not yet replaced currently used harvest methods in a commercial mAb manufacturing process.
Depth Filtration
There is a limit to the particle density that can be removed by a disk-stack centrifuge depending on the cell culture properties, centrifuge feed rate, bowl geometry, and rotational speed. Because of this size limitation, further clarification by depth filtration is typically used to remove smaller solid particulates that still remain in the centrifuge product. Commonly used depth filters consist of a thick, porous matrix of cellulose fibers with inorganic filter aids bound to them by a positively charged resin. The thick matrix provides a tortuous path to retain a range of particle sizes, and the positive charge imparts adsorptive properties to the filter. The minimum particle size that can be effectively removed solely by the sieving mechanism of a depth filter is about 0.1μm. The adsorptive mechanism, however, can remove much smaller, soluble and negatively charged impurities such as DNA and host cell proteins to further improve product quality. These cellulosic depth filters release relatively high levels of water-soluble contaminants into the system. These high levels of organic and inorganic contaminants are reduced to acceptable levels through extensive preflushing prior to use. In subsequently available commercial depth filters, cellulosic fibers were eliminated and water-soluble thermoset resin binders were substituted with water-soluble binders to reduce the amount of extractables, thereby lowering the risk of product contamination.
The use of depth filtration is not limited to the secondary harvest step; it could potentially replace disk-stack centrifugation for processes based entirely on disposables. Depth filters have been effective in the removal of impurities when positioned at various stages of bioprocessing. In addition to improving the product quality, depth filters aid in protecting and prolonging the life of sterilizing filters and chromatography resins. The use of depth filters with relatively large pore sizes, primarily targeting retention of cells and cell debris, seems feasible for high viability and low cell density cell cultures. High cell density cultures with variable cell viability have increased the risk of filter fouling, thereby making the implementation of depth filters challenging. Using depth filters for harvesting high cell density cultures may require significant filter areas and a large footprint. Since depth filtration is a single-use technology, the undesired side effect of simplifying the process by replacing the centrifuge with this approach could lead to increased manufacturing costs.
