Process for the manufacture of an adsorbent filter media devoid of cellulose for the removal of biological contaminants in process liquids

This application claims the priority of US Patent Provisional Application No. 60 / 774,773 filed on February 17, 2006, the disclosure of which is referenced herein.

The present invention relates to an adsorptive filter material for the removal of biological contaminants in process liquids.

Cellulose depth filters, such as filters commercially available from Millistak® + Millipore, are commonly used in the production of biopharmacy obtained from mammalian cell cultures to purify various crude product fluids. .. These composite filters include a tightly constructed layer of cellulose depth material and can be optimized for specific applications such as retention of colloidal particles and necrotic tissue fragments, or retention of whole cells and larger necrotic tissue fragments. They combine consecutive grades of material in a single filter cartridge. These filters are typically used in polishing or secondary purification steps to remove small amounts of suspended solids from the water-soluble product (protein) stream. The first function of these filters is to preserve or extend the useful life of more expensive downstream separation steps such as sterilization and affinity chromatography. That is, a common use for these filters is a "pre-filter" that protects downstream equipment and filter materials from colloidal contaminants and other necrotic tissue fragments. In addition, such depth filters are also used to protect virus removal filters by removing trace amounts of aggregated proteins.

Composite depth filters are also known to retain to varying degrees some of the soluble contaminants commonly found in mammalian cell culture, such as nucleic acids, host cell proteins, lipids, detergents, and the like. This retention function for soluble contaminants is based on the adsorption properties of the depth filter material.

Filter materials commonly used in these depth filters include purified cellulose fibers (wood pulp and / or derived cotton), diatomaceous earth, and water-soluble thermosetting resin binders. The diatomaceous soil (the natural form of silica containing trace amounts of various silicates) in these composites is generally 40-60% by weight and is a fundamental component: cell fragments, organelles, and aggregates. It appears to be absorbable, colloid-sized biological materials such as proteins, as well as various soluble biochemical materials such as proteins, lipids, and nucleic acids.

However, one of the main drawbacks of using these cellulose depth filters for the production of parenteral drugs and other medicines is the relatively high level of water solubility that the cellulose depth filters release into the system. It is a pollutant. In practice, extensive pre-cleaning is required to reduce the levels of these organic and inorganic contaminants to acceptable levels prior to use. In addition, the maximum loading of the diatomaceous earth adsorbent inside the depth filter material is limited to about 60% by weight, and the minimum particle size for the adsorbent retained in the fiber matrix is about 10 micrometers.

<p> Therefore, an object of the present invention is to reduce or eliminate the release of contaminants from the adsorptive filter.</p><p> Another object of the present invention is to increase the content or loading of the adsorbent in the filter material.</p><p> Yet another object of the present invention is to provide a filter with smaller adsorbent particles in order to maximize the surface area available for adsorption.</p><p> Other objects and advantages of the present invention will become apparent from the following detailed description and accompanying drawings.</p>

<p> The problems of the prior art are overcome by the present invention, which provides an adsorptive filter material particularly suitable for the removal of biological contaminants in process liquids. A porous fixed layer of adsorbent material is formed using only granular adsorbents and water-insoluble thermoplastic binders. The resulting composite filter allows for larger amounts of adsorbent with smaller adsorbent particles than conventional depth filters. Removal of cellulose fibers and removal of water-soluble thermosetting binders results in a reduction of contaminants in the process liquid. As a result, extensive pre-cleaning to reduce exogenous contaminants is no longer necessary. Improved material performance is obtained by increasing the content of the adsorbent material and / or using a smaller adsorbent material to maximize the surface area available for adsorption.</p>

<p> The resulting composite filter performance is substantially or completely devoid of cellulose and thermosetting binders and can be placed in purification systems downstream of the bioreactor and upstream of the sterile filter. Other uses include pretreatment of cell culture streams prior to virus removal filtration and chromatographic separation.</p>

The filter material of the present invention includes an adsorbent material and a water-insoluble thermoplastic binder. This combination can be used to form a fixed layer of porous adsorbent material with mechanical properties suitable for this application, including permeability, tensile strength, and flexural strength. The filter material is particularly useful as a depth filter.

Suitable adsorbent materials include diatomaceous earth, silica, porous glass, zeolites, and activated carbon. Diatomaceous earth is particularly preferred. In addition, any form of chromatographic material (beads, fine powders, etc.) and interfacial chemistry (ion exchange, hydrophobicity, etc.) can be used as an adsorbent in such porous materials. Suitable binders include thermoplastic binders such as polypropylene, preferably polypropylene, or mixtures thereof, preferably polyolefins, polyethylene. The binder is preferably used in the form of beads, powders, or fibers. By properly selecting the binder (in terms of a sufficiently high melting point or softening point), the material is sterilized under pressure to aid in the reduction or removal of any biological contaminants therein. , Steam sterilized, or gamma-ray emitted.

The material manufacturing process depends on the form in which the binder is used. The material can be prepared by mixing the binder with the adsorbent material and then fusing the adsorbent particles by partially melting or softening the binder. For example, polyethylene powder can be dried (by shaking / tumbling for only a few minutes) with adsorbent particles such as diatomaceous earth or silica beads and a binder: adsorbent by weight at a ratio of about 1: 1 to about 1: 3. And mixed. The resulting mixed material is placed in a mold and at a temperature suitable for fusing the adsorbent particles (eg, in a heated hydraulic press), eg 130 ° C to 160 ° C, Is heated up to. Diatomaceous earth, which is hardened to a thickness of 2-4 mm using ultra-high molecular weight polyethylene powder (registered trademark Mipelon) available from Mitsui Chemicals, and silica beads have an average bead diameter of 20-30 micrometers, this method. Has been formed by. As the material heats and softens in the press, the compressive force should be adjusted periodically to maintain a constant force during the heating cycle (about 5-10 minutes).

Alternatively, a wet process can be used to form the material, in particular the binder is in the form of fibers. For example, fine polyethylene fibers (registered trademark Fybrel, Mitsui Kagaku) are dispersed in isopropanol and water and fused into a slurry, and the ratio of diatomaceous earth powder and binder: adsorbent is about 1: 1 to about 1: 3 by weight. It is mixed with. The slurry is transferred to a Büchner funnel that holds a nominal 1 micrometer non-woven support material as a base so that the fibers and adsorbent do not pass through the perforations of the funnel. Most of the liquid then flows out through the vacuum flask. The formed discs are transferred to an oven for thermal bonding by drying the adsorbent particles and partially melting or softening the polyethylene fibers.

The composite material constructed as described above exhibits extremely high transmittance (high porosity) and low particle retention properties as compared with conventionally available cellulose depth filter materials. Gravity sedimentation of polyethylene fibers, or diatomaceous earth particles with powder, results in a low density composite structure with relatively large gaps.

In order to improve the separation properties of the diatomaceous earth composites of the present invention, the material should be compacted or compressed before and / or during heating. For example, using an air press in the range of 689475.7 to 2240796.025 Pascal (100 to 325 lbs / inch), it proved effective to compress the filter material sample for about 30 seconds. The material sample does not substantially relax after compression. The material maintains a compressed thickness in order to melt the structure into a monolith after heating. Depending on the composition and compressive force, the composite can be compressed to about 50-70% of its original volume.

The application of mechanical compression to the composite can also be used to adjust the water permeability, which can be close to that of a standard cellulose depth filter pad. Figure 1 shows the water permeability measurements (flow rate relative to pressure difference) of a composite material made from Celpure (registered trademark, Advanced Minerals) diatomaceous earth and Fybrel (registered trademark, Mitsui Chemicals) polyethylene ultrafine fibers. (Registered Trademark) + A1HC Depth Filter Shown in comparison with the sample. FIG. 1 shows that the water permeability of the composite material of the present invention is in a range consistent with that of a commercially available cellulose depth filter material.

The third granular or fibrous component can be added to the adsorbent / binder mixture as a means of manipulating the permeability of the product to allow the adsorbent contained to be used more effectively. The additive may be a functional adsorbent or an inert material, but must be of a size and amount that has a measurable effect on the permeability of the material. Test results show that affecting material permeability in this way does not substantially compromise (decrease) the adsorbent capacity of the material. Selective creation of larger channels within the material allows deeper / wider permeation of the process fluid inside the porous matrix that can efficiently compensate for the overall low adsorbent content of the material. To do.

In addition to particle capture, the adsorbent porous monoliths of the present invention have significant advantages over conventional cellulose adsorbent depth filters, especially with respect to water extraction. Any extractable material is of great concern in the manufacture of parenteral medicine. Conventional cellulose depth materials are known to have a relatively high extractable loading that requires extensive cleaning prior to use. The inventor of the present application has demonstrated that the extract that contributes to conductivity (inorganic) is not derived alone or primarily from diatomaceous earth. In practice, the composites of the invention lacking cellulose and thermosetting binders result in a 75-90% reduction in effluent conductivity compared to conventional cellulose materials. Removing cellulose and replacing thermosetting water-soluble resin binders with water-insoluble binders such as polyethylene results in DE-based materials with dramatically lower amounts of inorganic extracts. For users of such materials, there are considerable advantages of reducing cleaning requirements with a low risk of product contaminants.

In general terms, one preferred method for preparing preferred filter composites of the present invention is as follows. Ultra-high molecular weight polyethylene powder with an average particle size of 25 micrometers and natural diatomaceous earth with a particle size in the range of 0.2 to 25 micrometers are batch united in a rotary V blender containing a high speed internal stirrer. Dry and mix in. The mixture is transferred from the blender to a powder dispenser (or applicator). The applicator supplies the mixed powder to a moving web of porous non-woven polyester material at room temperature with a controlled thickness of up to 1.27 cm (0.5 inch). Multiple different powder mixtures can be applied in a similar manner to produce a gradient composition adsorbent material.

The loose mixed powder layer is then lightly compacted and flattened by contact with an overhead roller before being heated from below by an electrically heated plate and from above by an IR lamp that simultaneously softens the polyethylene powder. To. The temperature of the heated plate rises along the manufacturing path to a final temperature of approximately 340 ° F. The temperature of the powder mixture is maintained at up to about 340 ° F for a few minutes before applying the additional non-woven polyether web to the surface.

The composite is then passed through two heated calendar rollers set to a temperature of approximately 340 ° F to 0.254 to 0.508 centimeters (0.10 to 0.20) at approximately 689475.7 pascals (100 lbs / inch). It is continuously compressed to a thickness of inches). The finished material can then be cooled on a metal plate exposed to the air.

Samples of various mixtures of diatomaceous earth and polyethylene microfibers melted inside a pad approximately 2-4 mm thick were tested in standard purification steps related to protein product recovery from mammalian cell cultures. .. The test validated the adsorbent material with a suspension of E. coli lysate (in buffer) under constant flow conditions and was associated with increased pressure on the material sample (rate of clogging) and volume of treated fluid. The quality of the effluent was monitored. The quality of the effluent, or filtrate, was measured by directly filtering the fluid through a 0.2 micrometer sterilization grade thin film filter, in this case Durapore® GV. These experimental composite samples were recompared with Millistak® + Cellulose Depth Filter Material.

2 and 3 show pressure properties for adsorbent material samples and Millistak® + pads, as well as related sterile filter properties. Throughput is reported as the volume of processed fluid relative to the volume (total volume) of depth filter material utilized.

As shown in the figure, DE / PE fiber composites can match the tightest or most holding grade Millistak® + DE material (grade 75) for both throughput and retention. done. The various composite samples have a pressure rise rate equal to or less than the 75DE Millistak® + material pressure rise rate. In addition, the rate of pressure rise, or pressure rise, in the downstream sterile filter is almost equivalent to the composites of the invention compared to the 75DE Millistak® + material, which indicates a comparative level of particle retention. ..

A sample of diatomaceous earth melted inside a fixed layer pad using polyethylene powder (Mipelon registered trademark, Mitsui Chemicals) was tested for its ability to protect the virus-carrying thin film, NFP Viresolve 180. In this test, DE / PE composites (thickness, approximately 3 mm) were validated at a concentration of 0.5 gm / L using a solution of polyclonal human IgG protein. In general, Viresolve® thin films are approximately 150 L / m for this feed material.²Brings the ability of. Viresolve thin film capacity is 750-1500 L / m using Millistak® + A1HC depth filter pretreating this feed material for protein aggregation removal²Can be improved to the range of. Two samples of diatomaceous earth (Celpure 25 and Celpure 300 (Advanced Minerals)) are mixed with Mipelon PE powder and formed into 2-3 mm pads after heating (without compression). The IgG feed material was then pretreated using the DE / PE composite sample and the filtrate was treated again through Viresolve 180 to determine its effect on thin film capacity.

Celpure 25 (fine grade DE) is 440L / m²Produces a Viresolve of the ability, Celpure 300 (larger coarse particle DE) is 1000 L / m²Produced a greater ability of Viresolve. These tests show that diatomaceous earth monoliths formed with PE powder binders are applied to virus-carrying thin films such as those currently provided by cellulose depth filters such as Millistak® + Depth Filter (removal of protein aggregation). Indicates that the same level of protection can be provided.

To measure the cleanliness of the composites of the invention, the material samples were washed with clean deionized water and the conductivity of the effluent was measured after the washing. Conductivity values are taken to represent the level of soluble metal present in the filter material. FIG. 4 shows the conductivity values obtained for various DE / PE composite samples associated with a commercially available Millistak® + depth filter sample.

Millistak® + DE material is a composite material of cellulose and diatomaceous earth + a water-soluble thermosetting resin binder. The CE material contains only cellulose fibers and binders. It is clear from these measurements that those extracts that contribute to conductivity (inorganic) are not primarily derived from diatomaceous earth. Comparing these values to the tested DE / PE composites, there is a 75-90% reduction in effluent conductivity.

When added to a 2: 1 mixture of powdered polyethylene (20-30 micrometers) and diatomaceous soil (0.5-10 micrometers), 10-20% of the 75-100 micrometer porous glass beads are subject to particle capture and processing. The hydraulic permeability of the finished material can be reduced by 10-30% without measurable loss in adsorbent capacity when measured by volume.

<figref num="1">It is a graph which compares the water permeability of the composite material of this invention with the conventional composite material.</figref><figref num="2">Prefilter pressure difference-throughput graph.</figref><figref num="3">Aseptic filter pressure difference-throughput graph.</figref><figref num="4">It is a graph of the conductivity value of the effluent passing through various materials.</figref>