See also depth filtration for antibody purification

Companies:  3 M Purification

Depth filters refers to the use of a porous medium that is capable of retaining particulates throughout its matrix rather than just on its surface. These filters are frequently employed when the feed stream contains a higher content of particles. In such cases, depth filters can remove larger, insoluble contaminants prior to final filtraiton through a microfiltration membrane that would otherwise clog relatively quickly. Depth filters employed in bioprocessing are typically composed of a fibrous bed of cellulose or polypropylene fibers along with a filter aid (e.g., diatomaceous earth) and a binder that is used to create flat sheets of filter medium. (Yigzaw, Biotechnology progress, American Institute of Chemical Engineers, US, 22(1), 2006, 288-296).

Uses for Depth Filters

Clarification: Dept filters are traditionally used in the clarification of cell culture broths, to maintain capacity on membrane filters or to protect chromatography columns or virus filters. They are typically composed of cellulose, a porous filter aid such as diatomaceous earth and an ionic charged resin binder. For harvesting applications, dept filters can be applied directly with the whole cell broth where a filtration train containing three stages of filters is often employed. The primary stage uses a coarse or open depth filter with a pore size range of up to 10 um and removes whole cells and large particles. The secondary stage uses a tigther depth filter and clears colloidal and submicron particles. The last stage contains a membrane filter that is 0.2 um pore size in most cases. Debt filtration is frequently employed after the centrifugation process because there is a practical low limit to the particle size that can be removed by centrifugation (Liu, “Recovery and purification process development for monoclonal antibody production” mAbs,  2:5: 480-499 (2010).

Remvoal of endotoxins, viruses, DNA and HCP: Withe the addition of adsorptive surfaces, depth filters are now also being used for a variety of other applications involving removal of endotoxins, viruses, DNA nd HCP. (Rathore and Shirke, Preparative Biochemistry & Biotechnology 41:398-421, 2011). 

Due to the adsorptive mechanism of depth filters, they have been extensively used as a purification tool to remove a wide range of prcoess contaminants. Positively charged dept filters have been employed for the removal of E. coli derived and other dendogenous endotoxins and viruses many times smaller than the average pore size of the filter. They have also been used to remove prions form an immunoglobulin solution. (Liu, “Recovery and purification process development for monoclonal antibody production” mAbs,  2:5: 480-499 (2010).

Depth Filtration + Centriguation: Depth filters are traditionally used in the clarification of cell culture broths to maintain capacity on membrane filters or to protect chromatography columns or virus filters.  A depth filter is most frequetnly used after centrifugation because there is a practical lower limit to the particle size that can be removed by centrifugation. This type of depth filter has a pore size range of 0.1-4 um and is usually made of two distinct layers, with the upstream zone being a courser grade compared with the downstream. The larger particles are trapped in the coarse grade meida and smaller particles are trapped in the tighter media, reducing premature plugging and increasing filtration capacity.  (Liu, “Recovery and purificaiton process development for monoclonal antibody productn, mAbs 2:5, 480-499, 2010). 

Depth Filtration without Centrifugation: O’Connor (US13/880424) discloses purification of proteins such as antibodies using depth filters where the method does not include centrifuging. First, the mixture containing the protein is mixed with a solubilization buffer containing ethanolamine, arginine, EDTA, urea and DTE and then the protein is clarified with two or more depth filters. The method further includes refolding the protein with a refolding bufer having ethanolamine, EDTA and GSSG.

Composition

Cellulose + Diatomaceous earth: Depth filters are typically composed of cellulose, a porous filter aid such as diatomaceous earth and an ionic charge resin binder. A binding resin is often added to a small weight percent to covalently bind dissimilar construction materials together, giving the resultant media wet strenght and converring positive charge to the media surfaces. Because of this make up, depth filters rely on both size exclusion and adsorptive binding to effect separation. (Liu, “Recovery and purificaiton process development for monoclonal antibody production, mAbs 2:5, 480-499, 2010). 

Millipore Data Sheet, “Millistak+ Pod disposable depth filter system” 2009 discloses a depth filter system which is ideal for clarification of cell cultures which is composed of cellulose fiber and diatomaceous earth.

Posivitvely charged Depth Filters

Positively charged depth filters have been employed for the removal of endotoxins from water, virus particles that were smaller than the effective pore size of the filter, to remove DNA from a buffer solution and spiked prions from an immunoglobulin solution. Depth filters have also been sued to remove contaminant proteins in the cell culture harvest supernatant prior to Protein A chromatography for the purificaiton of antibodies. (Yigzaw, Biotechnology progress, American Institute of Chemical Engineers, US, 22(1), 2006, 288-296).

3 M purification, USA sells the Zetaplus encasulated filtraiton whystem which contains a hybrid depth filter media consisting of both size exclusion and anion exchange components. The primary application of this system is clarification of the fermnetation broth. However, published applicaitons have shown that the filter also provides capability to clear DNA and HCP from the protein A pool obtained during processing of CHO mAB product. (Preparative Biochemsitry & Biotechnology 41: 398-421, 2011).