Adsorptive Properties of Depth Filters
The high surface area and the possibility of charged interactions imply that depth filters can possess adsorptive properties
in addition to their ability to trap larger particles. Positively charged depth filters have been used for a variety of applications,
including the removal of endotoxins from water; the removal of virus particles that were smaller than the effective pore size
of the filter; removing DNA from a buffer solution; and removing retrovirus and parvovirus during viral spiking studies into
a solution of pure protein.20–23 More recently, depth filters were shown to reduce host cell protein impurities that would otherwise precipitate during subsequent
Protein A column elution.24 Thus, in addition to clarifying the mammalian broth, depth filters also can adsorb some otherwise soluble impurities from
the feed streams.
Placement of Depth Filters in a Harvest and Clarification Scheme
In a harvest and clarification scheme for mammalian cell culture broths, depth filters typically are placed after a centrifugation
step.25 Because centrifugation (unlike MF) cannot efficiently remove all particulates from the broth, a secondary clarification step
is needed. This niche is nicely filled by depth filtration, because the use of dead-end microfilters is often costly and prone
to failure in the event of an excursion in particle counts from the centrifugation operation. Depth filters can be used as
the sole harvest and clarification step, but this is not usually the most robust or cost-effective solution for large-scale
operations. Because most depth filters do not come with an absolute pore size cutoff rating, a dead-ended microfilter is used
in-line after the depth filter to effectively remove any residual particulates that might clog subsequent chromatographic
Flocculants have been used for many years to improve the filtration of fermentation broths.2 These agents, ranging from simple electrolytes to synthetic polyelectrolytes, can act by several means to cause clumping
of smaller particulates to form larger solids that can be filtered more effectively.
Filter aids for mammalian cell culture harvest are most often diatomaceous earths or perlites. More recently, chitosan, a
nontoxic food-grade material, has been used as a flocculant for mammalian cell culture clarification.26 The use of chitosan was reported to increase depth filter volumetric throughput by six to seven fold.
The use of a calcium chloride and potassium phosphate combination as a flocculant also has been reported for MAb harvest.27 These two compounds, when combined, produce calcium phosphate, which is insoluble and can interact with proteins through
ionic and metal-chelate interactions. This was applied for removing impurities from the cell culture broth.
Flocculation for MAb cell culture harvest and clarification has not been applied very widely and should draw further interest
in the coming years given that it can not only be an aid to cell removal but also reduce impurity levels, thus reducing the
burden on the downstream chromatographic steps.
Absolute microfilters operated in the dead-end mode with pore sizes ranging from 0.2–1.0 μm can be applied for the removal
of cells and cell debris from mammalian cell culture fluids. In practice, it is rare to see such filters used as the sole
harvest technique beyond the laboratory scale because the surface area needed for filtration can be prohibitive. For large-scale
operations, these filters are commonly used as a terminal polishing step during clarification to ensure the absence of particulates
in the load material for the capture chromatographic step.
Expanded-bed adsorption chromatography (EBA) has drawn significant interest over the years because of its potential to eliminate
the need for separate harvest and clarification steps.28 –30 In this technique, the cell broth containing cells and cell debris is introduced into a column packed with the EBA resin
in the upward flow direction. The flow itself fluidizes the resin beads, causing them to float, thus allowing the cells and
cell debris to pass through and exit through the top adaptor. The product adsorbs onto the resin. Following completion of
loading, the column is washed and then allowed to settle. Product elution can take place in the downward flow direction.
Some of the important considerations during the development of EBA operations and some of the technique's limitations have
been reviewed recently.31 Although this technique has elicited significant interest, practical issues with uniform flow distribution from the bottom
of the column as column diameters increase and issues with frit and resin fouling have kept this from being adapted for commercial-scale
operations. If these engineering issues are addressed in the future, EBA could perhaps re-emerge as a technique of choice
for large-scale mammalian cell culture harvest.