Depth Filters For Disposable Biotechnological Processes

Abstract

A process for the primary clarification of feeds, including chemically treated flocculated feeds, containing the target biomolecules of interest such as mAbs, mammalian cell cultures, or bacterial cell cultures, using a primary clarification depth filtration device without the use of a primary clarification centrifugation step or a primary clarification tangential flow microfiltration step. The primary clarification depth filtration device contains a porous depth filter having graded porous layers of varying pore ratings. The primary clarification depth filtration device filters fluid feeds, including chemically treated flocculated feeds containing flocculated cellular debris and colloidal particulates having a particle size distribution of approximately about 0.5 μm to 200 μm, at a flow rate of about 10 litres / m2 / hr to about 100 litres / m2 / hr. Kits and methods of using and making the same are also provided.

Description

CROSS-REFERENCED TO RELATED APPLICATIONS[0001]This application claims the benefit of U.S. Provisional Patent Application No. 61 / 571,994, filed on Jul. 8, 2011, the entire contents of which are incorporated by reference herein.DESCRIPTION OF THE INVENTION[0002]1. Field of the Invention[0003]In general, the present invention relates to the primary clarification of feeds. In certain specific embodiments, the invention provides a primary clarification depth filtration process of feeds, feedstreams, feedstocks, cell culture broths and the like, which utilizes a primary clarification depth filtration device without the use of a primary clarification centrifugation step or primary clarification tangential flow microfiltration step. In other embodiments, the invention provides primary clarification depth filtration process of chemically treated feeds in which the cell mass has been flocculated into larger aggregates.[0004]2. Background of the Invention[0005]Manufacturing pharmaceutical-grad...

AI Technical Summary

Benefits of technology

The patent text describes a process and device for using a depth filtration device to remove large particles and debris from mAbs, cell cultures, and other biomolecules without the need for a primary clarification centrifugation or tangential flow microfiltration step. The device includes a porous depth filter media with open graded layers that can filter out cellular debris and colloidal particles with particle sizes ranging from 0.5 μm to 200 μm, improving the throughput for unclarified feedstreams. The device has large pores for the large flocs of cellular debris and high depth to prevent internal floc bridging, which can cause breakdown of the floc. The invention is directed towards disposable primary clarification processes for efficient separation of biomolecules.

Problems solved by technology

As product molecule titers have increased, the higher cell mass and larger amounts of product create challenges for the downstream purification steps.

Higher cell densities result in difficulties during clarification and sterile filtration.

Higher product concentrations generally result in increased impurity load and the need for larger chromatography installations.

mAb manufacturers have invested a great deal of time and effort increasing the product titer of a feedstock.

However, while higher titers increase cell culture productivity, it also produces feedstocks with larger amounts of biomass and cell debris content.

Feeds containing such larger amounts of biomass and cell debris can produce high turbidity centrate after centrifugation.

High turbidity centrates often reduce the throughput of the secondary clarification depth filter and the subsequent sterile filter used downstream of the centrifuge.

The reduced throughput causes a range of problems from increased process cost to deviations in process procedures due to plugging of filters and long processing delays.

This is particularly problematic at pilot or clinical scale biotherapeutic production where it is desirable to process multiple products in a relatively short time.

The centrifuge cleaning procedures slow down the pilot plant's ability to change over to the production of a different biomolecule and greatly increase the risk of cross contamination between production runs.

In addition, centrifugation cannot efficiently remove all particulates and cellular debris from these feedstocks in the primary clarification step, hence the need for the secondary clarification step utilizing depth filtration after the centrifugation step, but prior to the subsequent chromatographic steps.

Alternatively, successive filtration runs have proven useful in removing different-sized cell and cellular debris from feedstocks, but typically the volumetric throughputs limit the application to smaller volumes (<1000 L) where the filter installation has a reasonable size.

Unfortunately, the low throughput requires a large number of filter units which can reduce filtration yields because each successive step results in the loss of a portion of the feed solution through hold-up volumes of the filter device and equipment.

However, flocculants have not been widely used in the clarification of mAbs, mammalian cells, and other bimolecular cellular materials of interest feedstocks.

While chemical flocculation is quite effective in agglomerating cellular debris and cellular contaminants (host cell proteins and DNA), the resulting flocculated suspension is generally not easily separable by ordinary filtration methods without the use of a centrifuge prior to filtration.

However, tangential flow microfiltration membranes used for cell culture harvests are often plagued with the problem of membrane fouling (i.e., irrecoverable declines in membrane flux) and typically require strict complex operating condition followed by a thorough cleaning regimen (as is also the case with a centrifuge) for the membranes after each use.

Therefore, flocculated cells and other biomolecules don't readily settle and often take a number of hours before settling occurs.

Another problem is the relatively low density of the flocculated cell mass which, typically form a fluffy mass that occupies significant part of the feed volume rather than forming a compacted cake.

Also, because of the biological origin of the particles, the flocs are fragile and tend to break down easily under pressure.

For this reason, most conventional solid-liquid separation methods while useful for solid particle systems, fail in flocculated cell masses such as mAb feedstocks.

However, depth filters are currently unable to handle the high solids feedstreams that are typical of high titer mAb processes, such that depth filters are therefore often used after centrifuging.

The high particulate load and high turbidity present in unclarified cell culture supernatant adds challenges to the primary clarification by depth filtration alone.

However, depth filters are currently unable to handle the primary clarification of high-solids feedstreams, and often must be used after centrifugation or tangential flow microfiltration.

The high particulate load and high turbidity present in unclarified cell culture supernatant adds challenges to primary clarification by depth filtration alone.

Currently, the limited throughput results in large installations of depth filters for primary clarification which results in yield losses due to the large hold up volume and scale-up issues as discussed above.

In addition, mAb feedstocks are challenging feed streams to clarify and filter because of the presence of a higher biomass content, and result in a high turbidity centrate after centrifugation.

Because of the need to remove large amounts of biomass, the high turbidity centrates shorten the life of the depth filter for clarification downstream.

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