Depth Filtration

Despite low shear centrifuge designs, the centrate effluent may still contain appreciable amounts of smaller sized contaminants (e.g., aggregates and colloids). Depth filtration is most commonly used as a secondary clarification to remove the debris and to prevent plugging of downstream processes, such as chromatography. Charged depth filters are the preferred method for clarification and debris removal downstream of the primary harvest step, due to their ability to retain a large amount of contaminants using boht size exclusion and adsorption. (Low, J Chromatography B, 848 (2007) 48-63).

Pegal “Evaluating disposable depth filtraiton platforms for MAB harvest clarification” Bioprocess International, 2011, 52-55) showed that clarifying cell culture harvest with only single use depth filtraiton can be an attractive alternative to the traditional fed batch process that includes both centrifugation and filtration.

Types of Depth Filters

Charged Depth Filters:

Charged depth filters are available commercially from, for example, Cuno, Inc. (e.g., ZETA PLUS S series, ZETA PLUS SP series, ZETA PLUS LP series, ZETA PLUS CP series, ZETA PLUS LP BC series), EMD Millipore (e.g., DOHC, COHC, FOHC, A1HC, B1HC, XOHC) ), Sartorius AG, and Pall Corporation (e.g., SEITZ P series, SEITZ K series, SUPRADUR series, STAX series, SUPRACAP Series, SUPRAPAK series, SUPRADISC seires). (Hoang, US 15/751, 231, published as US 2018/02301800.

Depth Filtration in Combination with Other Types of Unit Operations

Rajendran (US 14/977,869, published as US2016/0176921) discloses a method of purifying a protein such as eculiumab by capturing the protein such as by protein A affinity chromatography, followed by one or more unit operations and then flowing the protien frough a depth filter to provide a filtrate.

Centrifugation + Depth Filtration:

The first step in the recovery of an antibody from mammalian cell culture is harvest or removal of cells and cell debris to yield a clarified, filtered fluid suitable for chromatography. This is generally accomplished through use of centrifugation, depth filtration and steril filtration. The depth filter is employed most frequently after the centrifugation process because there is a practical lower limit to the particle size that can be removed by centrifugation. The current preferred process for initial recovery is to use a disc-stack continuous centrifiguation coupled with depth filtration. Considerably thicker than a membrane filter, depth filters are about 2-4 mm in thickness. For harvesting applications, depth filters can be applied directly with whole cell broth or in conjunction with a primary separator such as TFF or centrifugation. The whole cell broth depth filter harvest is common for bench, pilot and smaller commercial scale applications. For this, a filtration tran containing three stages of filters is usually 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 tighter depth filter and clears colloidal and submicron particles. The alst stage contains a membrane filter that is 0.2 um pore size in most cases. (Liu, “Recovery and purification process development for monoclonal antibody production” mAbs, 2:5: 480-499 (2010).

Singh (WO/2013/009491) disclsoes a process for clarificaiton of feeds containing target iobmolecules such as mAbs using a pimary clarificaiton depth filtraiton device without the use of a primary clarificaiton centrifugation step or a primary clarificaiton tangential flow microfiltration step.

In combination with Protein A chromatography

–Protein A — Charged DF –Incubation:

Hoang (US 15/751231, published as US 2018/0230180) discloses a method that increases the re-oxidation of antibody molecules using a Millistak A1HC depth filtery at a throughput of 350 L/m2. There was more than two-fold decrease in % pre-peaks observed in the charged depth filtered filtrate compared to sterile filtered filtrate immediate following fitration. The DF was effective in enhacing re-oxidation of antibody molecules when performed prior to Protein A chromatography or after Protein A chormatography. Hoang disloses that incubating the fitlrate for at least four hours before being contacted with the chared dF or after DF. Hoang states that the structure and stability of antigen bining protein molecuels depends heavily on the disulfide bonds that link the two heavy chains and the heavy and light chains and that during production and purificaiton, one or more disulfide bonds can be reduced to free thiol groups. Thus reduction of the inter-chain dislfide bonds weakens the strucutral integrity of the antigen binding protein and can lead to antigen bidning protein fragments such as L and H chains as well as antigen-binding protein aggregates. Thus reducing the percentage of reduced antigen binding protein molecules with the DF step is important.

–Protein A – Unit operations (e.g., IEX, HIC, viral inactivation, viral filtraiton, adjustment of pH, etc) –DF

Sample -CEX – DF -AEX:

Kim (US 14/896,380, published as US 2016/0122384) discloses purifying an antibody with CEX followed by a multilayer filter (depth filtering) and then AEX

Conditions/Parameters

Isoelectric point:

Zou (WO 2008/036899) discloses a emthod for removing parvovirus or fragments thereof from a therpaeutic protein such as an antibody by passing the solution through a depth filter at a pH within 1 pH unit of the isolectric point (pi) of the virus such as a pH in the range of 4.0-6. In one embodiment the DF is an electropostively charged filter suchas a Millipore AIHC filter. The method can include the step of maintaining the solution at a pH for a lenght of tiem effective to inactivate virus in the solution.

Contacting solution with positive ion

Hoang (US 15/751231, published as US 2018/0230180) discloses aadding a positive ion to an aqueous solution with a charged depth filter. For example, copper can be added to the aquous solution or the solution can also be sparged with air or oxygen before, during and/or after contacting the solution with the cahrged dF.