Liquid purification or separation – With means to add treating material – Chromatography
Reexamination Certificate
1997-06-30
2001-02-27
Savage, Matthew O. (Department: 1723)
Liquid purification or separation
With means to add treating material
Chromatography
C210S321630, C210S321750, C210S413000, C210S314000, C210S317000
Reexamination Certificate
active
06193883
ABSTRACT:
The invention relates to an apparatus for the gentle continuous separation and purification of dissolved substances from suspensions, especially biologically active proteins from cell suspensions or from cultures, employing the principle of affinity filtration using surface-modified membranes.
Known processes for isolating and purifying proteins from cell suspensions frequently comprise a large number of steps which can be divided, in principle, into four sub-groups: 1: cell separation and concentration; 2: pre-enrichment; 3: fine purification, and 4: polishing. In particular, steps 1 and 2 are critical with respect to product yield and product stability. Typical processes in those steps are centrifugation, microfiltration, ultrafiltration, precipitation and extraction.
Gentle continuous cell separation and enrichment of the target products is required especially in the preparation of biologically active proteins using animal cell cultures. Whilst the conventional tangential flow processes frequently used for that purpose, such as, for example, microfiltration and ultrafiltration, yield a particle-free filtrate, their performance in terms of flow, service life and product yield is limited on account of the known problems of membrane fouling, and further product treatment is required for the purpose of enrichment, such as, for example, chromatography. Other processes for cell separation, such as centrifugation, do not usually yield a particle-free supernatant, which in turn limits the subsequent chromatography steps.
From the point of view of economic viability and technical procedure, it is desirable to reduce the number of work-up steps. One approach comprises processes that combine the steps of cell separation, pre-enrichment and concentration. Three variants have been described to date, which are: a) the use of membrane affinity filtration, b) the use of fluidized/expanded processes, and c) the use of “big beads” in conventional column chromatography processes. In principle, those processes are capable of processing solids-carrying or cell-containing media, but only the membrane filtration process yields a particle-free filtrate and it is the only process suitable for continuous cell feedback.
Membrane affinity filtration has hitherto been used primarily for the treatment of particle-free solutions, such as, for example, for enriching pharmaceutical proteins from culture solutions (Brandt, S., Goffe, R. A., Kessler , S. B., O'Connor, J. L., Zale, S. E.: “Membrane-Based Affinity Technology for Commercial Scale Purifications”, Bio/Technology 6, 779-782 (1988)). Typically, the membrane modules used are conventional tangential flow systems, such as, for example, hollow fibres, or filter stacks, which are used for direct filtration or membrane chromatography (Langlotz, P., Krause, S., Kroner, K. H.: “Affinitätsmembranen für die Bioproduktaufarbeitung”, F&S Filtrieren u. Separieren, 5 (2) 62-70 (1991) and Thömmes, J., Kula, M. R.: “Membrane Chromatography—An Integrated Concept in the Downstream Processing of Proteins”, Biotechnol. Progress, 11, 357-367 (1995)). The treatment of particle-containing suspensions has hitherto been described only in terms of an initial approach (Kroner , K. H.: “Cross-Flow Application of Affinity Membranes”, Membrane Processes in Separation and Purification, NATO ASI Series E, Applied Sciences, Vol. 272, Kluwer Academic Publishers, Dordrecht (1994)).
A particular problem of the membrane process is membrane fouling, that is to say blockage or formation of a deposit on the membrane, as a result of which the performance and especially also the separation-specific properties, such as the separation limit—and in the case of affinity membranes also the adsorption characteristics—are adversely affected (Langlotz, P., Krause, S., Kroner, K. H.: “Affinitätsmembranen für die Bioproduktaufarbeitung”, F&S Filtrieren u. Separieren, 5 (2) 62-70 (1991) and Kroner, K. H.: “Cross-Flow Application of Affinity Membranes”, Membrane Processes in Separation and Purification, NATO ASI Series E, Applied Sciences, Vol. 272, Kluwer Academic Publishers, Dordrecht (1994)). In order to reduce those problems, it is customary to operate the membrane modules at very high tangential cross-flow speeds, preferably in the turbulent flow region. However, that results in a high longitudinal pressure loss, which in turn results in a non-uniform distribution of the flow across the membrane. That leads to a reduction in the membrane capacity, also known by the term “premature breakthrough”, and in a broadening of the peak (Kroner, K. H.: “Cross-Flow Application of Affinity Membranes”, Membrane Processes in Separation and Purification, NATO ASI Series E, Applied Sciences, Vol. 272, Kluwer Academic Publishers, Dordrecht (1994)). The use of conventional tangential flow systems for membrane affinity filtration in chromatography-analogous operation is therefore possible only to a limited degree. A further problem of conventional tangential flow systems is the occurrence of high shear forces, which can result in cell destruction especially when such modules are used in the filtration of animal cell cultures. A consequence of this is the release of cell content substances and cell fragments, which limit the performance of the membrane filtration further and severely restrict its use in continuous cell feedback.
Also known are rotary modules, such as, for example, rotating disk filters, in which the shear at the membrane is produced mechanically. That is described, for example, in Murkes, J. et al.: “Crossflow Filtration”, Wiley, N.Y. (1988). Whilst those modules demonstrate higher filtration performances than tangential flow systems, the effective shear forces are, however, also higher and are distributed over the filter means non-uniformly as a function of the radius: their use with sensitive cells is therefore severely restricted.
The problem underlying the invention is accordingly to provide a filtration apparatus for the integrated separation and enrichment of dissolved substances from particle-containing suspensions, which apparatus achieves virtually optimum utilisation of the filter area combined with gentle treatment of the cell material and is capable of being scaled-up in simple manner.
The problem is solved by the features of claim
1
. Advantageous arrangements of the invention are the subject matter of the subsidiary claims.
The present invention relates to an apparatus for separating and/or enriching dissolved substances from a suspension, having at least one filter means through which flow takes place, there being arranged on the retentate side of the filter means a means for producing a shear field which produces a virtually homogeneous shear field across the entire inflow cross-section of the filter means.
Preferably, the means for producing a shear field is formed by a rotationally symmetrical rotor, the distance between the rotor and the filter means preferably increasing radially outwards in order to compensate for the increased circumferential speed, the homogeneous shear field thus being produced. A preferred construction of the rotationally symmetrical rotor is the simple conical rotor in which the distance (gap width) s is a linear function of the radius r. It is, of course, sufficient for the rotor to exhibit that linear dependency only in the region of the filter membrane, that is to say the cone may also be a truncated cone. The filter membrane is usually planar and the filter can be operated, depending on the use, as a cross-flow filter.
Preferably, the ratio of the gap width s between the filter membrane and the rotor to the rotor radius r is less than 0.2, that is to say s/r <0.2; preferably s/r <0.1 and >0.05. The cone angle &phgr; is in the region of <16°, preferably from 3 to 6°. Preferably, the cone angle &phgr; of the rotor cone is approximately 4°.
It is also possible for the retentate or concentrate to be discharged through an outlet opening arranged near the axle, or through the axle itself if the axle is in the form of a hollow shaft.
In orde
Kroner Karl-Heinz
Vogel Jens
Brooks & Kushman P.C.
Gesellscgaft fur Biotechnologische Forschung mbH (GRF)
Savage Matthew O.
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