Initial Evaluation for Clarification
Successful clarification of mammalian cell culture fluid is the first step in preparation for downstream purification. When you are evaluating the optimal clarification technology, consider not only the performance of the primary and secondary clarification steps but also the performance of the sterile filter immediate downstream of the depth filtration.
Since sterile filtration is an important part of the clarification train, the optimization and selection of a sterile filter are also critical .
Regardless of the technologies you evaluate, when conducting small-scale experiments for your initial evaluation, it is preferable to mimic your large-scale operation as much as possible. Doing everything possible to emulate your future large-scale operation will help ensure your success in achieving your goals and objectives. Tip: To learn more about primary and secondary clarification, visit the Clarification Primer section.
The choice of the depth filter grade is typically guided by the properties of the starting material and the required filtrate quality. A cell culture harvest featuring a high particle load, low viability, and high cell density typically represents a more challenging situation for a normal flow filter. In this instance, it may be necessary to use a centrifuge for primary clarification followed by a one- or two-step depth filtration clarification scheme. However, a high viability cell culture (>80%) with a relatively low cell density (< 5x10^6 cells/ml) may be easily processed using only a single stage depth filtration process
Depth Filter Capacity
The capacity of a depth filter is defined as the volume of filtrate that can be processed on the surface of a given pre-filter until one of the following occurs:
- A maximal working pressure is achieved (typically 20-30 psi or 1.5 bars at small and pilot scale)
- The instantaneous turbidity of the filtrate reaches approximately 20% of the initial turbidity
- TFF modules with an open or suspended screen channel design are best suited for large bioreactors (>1,000 L) or higher batch frequency (>10 batches/year) and situations involving high cell densities.
- TFF separates particles by size exclusion. Membrane pore sizes can range from 0.1 to 0.65 microns; a tighter membrane (0.1 to 0.2 microns) can facilitate the removal of lipids and other colloids. Very open UF membranes (1000 kD) can be used as well.
- Permeate flux control or low TMP (Trans Membrane Pressure) control allows volume reduction as fluid becomes concentrated. If cost is a concern at the clarification step, remember that the robust construction of TFF modules allows for multiple uses.
The outcome at this stage of the process will be the initial selection of depth filtration media grade(s) and/or TFF membrane that best meets the objectives established above. However, confirmation of filter selection and sizing at the pilot scale are required once the cell culture fluid has been optimized.
Once you’ve selected your depth filtration media grades and/or TFF membranes, the next steps are process optimization and optimizing your cell culture fluid. When you have completed cell culture fluid optimization, begin confirmatory sizing trials (also known as pilot testing). Regardless of the technology option or options selected, a yield assay is typically performed on the preferred clarification option to verify yield targets are met.
Merck offers a full line of clarification products and services to help speed your MAb and plasma-derived products to market faster and more cost effectively.