Cell Harvest Clarification Scaling Strategies
Goals of the Step
Extracellular expression of products, typical of mammalian cells and transgenic animals, requires primary clarification to remove cells, milk solids and colloids. Intracellular expression may require a cell concentration step, a cell lysis step to release product, and lysate clarification to remove cell debris. Insoluble products such as inclusion bodies require dissolution and refolding before clarification to remove aggregates.
Secondary clarification economically protects columns from plugging without losing product. Normal flow filters are preferred for small volumes and low particle loads; e.g. column or sterile filter protectors, haze removal, cell culture clarification. Tangential flow filters are used for large volumes with high particle loads. Filtration can also be used in tandem with centrifugation. Millistak+ Mini capsules are available in a broad range of secondary clarification media to facilitate the selection process.
Factors Influencing Filtration Process Design in the Clarification of Bioreactor Fluids provides some background and scope regarding the benefits of an efficient and robust process design for clarification of bioreactor fluids.
Methods and Protocols
- Filter Sizing Methods For normal flow filtration (NFF) applications -- AN1512EN00
- Using Millistak+™ HC Filters for Mammalian Cell Culture Clarification -- AN1100EN00
- Bioprocessing Tutorial: Selection & Sizing of Clarification Depth Filters -- RP1600EN00
- Strategies in Clarifying Mammalian Cultures -- RP1025EN00
- Vmax Filtration System Optimization -- VB010
Scale-Up Strategies
Scaling up secondary clarification assumes the capacity or volume of fluid that can be processed will be in proportion to the frontal area of the filter media. The challenge to scaling this process step is the variability in the consistency in the feed flow or in the filter medium. In most bioprocess applications where depth filters are employed, the flow rate per unit cross sectional area (the flux rate) will have a significant impact on the capacity of the filter. The higher the flux rate, the lower the capacity (L/m2). For this reason, constant pressure tests that allow variable flow rates over time will generally result in an underestimation of filter capacity. In such tests, initial surge in feed flow rate can create premature clogging. Further, the constant pressure test fails to reflect the fact that higher capacities could be achieved at lower flow rates. To get an accurate performance profile of a depth filter a constant flow rate test, Pmax, should be applied at selected flow rates including the lowest practical rate. (See GEN vol 22, no 9, May 1, 2002)Process compression possibilities should be evaluated when scaling up the clarification operation. Consolidation of clarification and prefiltration steps to fewer unit operations lowers operating costs through increased yield, reduced piping complexity and floor space, as well as reduced cleaning and flushing volumes. Millistak+ HC has been designed to achieve this goal with different product solutions to suit common process scenarios. For example, the following media grades have been optimized to follow these primary clarification steps:
| Grade | Post Primary Clarification |
| A1HC | TFF MF 0.65 micron |
| B1HC | Centrifuge |
| C0HC | Perfusion reactor spin filter |
Several factors not always apparent at small scale need to be considered when scaling-up. These include:
1. Consistency in the quality of the clarified intermediate product.
The process must be sufficiently robust to handle the natural batch-to-batch variation in the fermentation broth. During scale-up, the primary approach is to ensure that there is enough filter surface area based on lab scale testing, with an appropriate safety factor applied. Increased filter area increases equipment size and piping complexity which can impact the hydraulic flow through the filter. Any degradation of broth quality may increase suspended solids, DNA, and polysaccharides which can degrade downstream process steps.2. Process flow conditions affect filter performance
The flow through large systems is typically implemented with a complex piping network between the filter housings. As the size and number of filter housings is increased, flow through the filter elements can change due to hydraulic head and changing pressure profiles as the filtration continues. Since cleaning and preparation can require a significant amount of time, the clarification step can become a bottleneck in harvesting a fermenter. By selecting optimized clarification/prefiltration filters, system complexity is reduced and yield is improved. Millistak+ HC will achieve this goal through process compression.
3. Safety concerns
Large scale clarification operations require room for handling new disposable filter elements and used cartridges. Significant crowding of equipment may occur which would impeded operations and compromise operational safety. Consideration should be given to proper design and fabrication of equipment for steaming to the physical aspects of removing spent filter elements that can weigh up to 20 kg.
