WO1989011899A1

WO1989011899A1 - Tandem hollow fiber cell culture product harvest system

Info

Publication number: WO1989011899A1
Application number: PCT/US1989/002490
Authority: WO
Inventors: William R. Tolbert; Chester S. Ho; Mark Baumgartener
Original assignee: Invitron Corporation
Priority date: 1988-06-08
Filing date: 1989-06-07
Publication date: 1989-12-14
Also published as: AU3868889A

Abstract

A method for continuous removal of product from cell culture or continuous performance of other cross-flow separations, while maintaining the clearance of the separation barrier is described. Tandem cross-flow separators (A, B), to restore the barrier, are employed in cyclic processes to retain continuous effectiveness in separation.

Description

TANDEM HOLLOW FIBER CELL CULTURE PRODUCT HARVEST SYSTEM

Technical Field

The invention relates to large scale fermenta¬ tion and cell culture methods. In particular, it relates to methods of continuous cell culture and product harvest using a hollow fiber harvest system.

Background Art

The production of substances by individual cells in culture is nearly as old as civilization. The industrialization of such production is relatively more recent, and has become fairly suddenly more complex with the advent of hybridoma and recombinant technology. These technologies have required the extension of cell culture techniques beyond microorganisms to mammalian cell cul¬ ture, which is inherently more sensitive to culture condi- tions. Because mammalian cells are also more demanding nutritionally, and hence more expensive, procedures for efficient production' of target substances are even more significant than was the case with bacterial cultures. It has been clear for some time that the productivity of mammalian cell cultures is markedly increased if the cells are maintained and the product harvested in a continuous rather than batch culture ap¬ proach. A number of studies have shown that productivity is greatly enhanced in perfusior as opposed to batch culture. See, for example, Martin, N. et al,

Biotechnology (1987) 5_:838-840. This is particularly true of cells that are sensitive to shear, which sensitivity may preclude the use of standard techniques such as spin¬ ner or shake flasks (Brennan, A.J. et al, Biotechnology Techniques (1987) l.:169-174). This has led to the development of a number of approaches directed to continuous replacement of medium and product harvest.

Brown, D.E. et al, Process Biochemistry (August 1987) pp. 96-101 review the state of the art with regard to cross-flow filtration to remove product. As described in this publication, rather than replacing medium and harvesting product by allowing the cells to settle, either by simple discontinuance of agitation or aided by centrifugation, and rather than single step filtration whereby the cells are retained on a filter and the medium removed, the cell culture suspension is passed through a continuous filtration system. The sedimentation and simple filtration processes are inherently batch methods and do not permit the continuous maintenance of the cell suspension in contact with nutrients and surrounding medium. However, by use of cross-flow separation techniques, a dilute suspension of cells can be concentrated and the medium continuously replenished while product is harvested, both without interruption in the cells' life and growth cycle. The Brown article, which is incorporated herein by reference, lists a substantial number of manufacturers of filtering materials of use in cross-flow separation techniques.

In principle, cross-flow separations involve passiner the dilute cell culture past a membrane (which may include—a planar, tubular or fiber membrane) wherein the product of the cell culture is able to transit the membrane but the particulates remain confined on one side of the membrane barrier. The "filtrate" which is capable of transiting the barrier is recovered and the cell suspension, which is confined on the side of the membrane, where it has been introduced, is rediluted with medium and returned to the culture vessel. While this continuous approach greatly enhances the productivity of the cell culture, it is not without its own problems. The process can be continued only for a fairly brief period of time before the membrane becomes clogged because of intermedi¬ ate size particles present in the suspension. In order to continue the process, the membrane must either be replaced (which is expensive) or clarified by backflushing with the filtrate. Either approach introduces an inherent dis- continuity in the culture system.

A review of one such general approach as applied to hollow fiber cross-flow separations was given by Breslau, B.R. et al at the 79th National Meeting of the American Institute of Chemical Engineers, Houston, Texas (1975). This general process is illustrated in Figure 1. As shown in the figure, in the normal process mode, the suspension enters a cartridge containing hollow fibers so that the suspension passes through the lumen of the individual fibers. The fibers are capable of passaging through their walls only the supernatant and not the cells. The permeate which has passed through to the outside of the fibers is harvested as shown, while the contents of the lumen are returned to the culture vessel from the top of the cartridge. Two approaches to clearing the fiber membranes are shown. In the first, shown as part (b) of Figure 1, the cartridge is disconnected from the culture vessel and the permeate is used to flow in the opposite direction from the outside of the membrane into the liwe and the backflushed material is discarded. As pointed-out in the paper, the backflushing medium has to be of "permeate quality", and backflushing of a portion of the permeate is suggested. It is clear that this approach creates a discontinuity as the entire cartridge is dis¬ connected from the culture vessel while cleaning takes place. This particular problem is avoided by the clean¬ ing method shown in part (c) of Figure 1 which entails simply staunching the flow of permeate, thus creating back pressure so that at least those particles which have adhered to the interior walls of the fibers are carried through the cartridge and back to the culture vessel. This is due simply to the back pressure exerted when no flow through the membrane is permitted. This approach is effective only to the extent that the fiber walls constitute the primary barrier to passage of particles and to the extent that the particles are not embedded more deeply into the fiber materials. As is made clear in the cited report, this requirement makes necessary the design of fibers which have large pores through the thickness of the wall with the interior wall being relatively imperme¬ able. Thus, the solution of Breslau et al to the dis¬ continuity problem is of relatively limited application.

The invention disclosed herein, on the other hand, is universally applicable to systems of cross-flow separations and effectively assures a continuous stream of product without waste of permeate for cleansing purposes.

Disclosure of the Invention

The invention provides a tandem system whereby cell cultures can be maintained under conditions which provide for their healthy maintenance and product- producing capacity and which permit the continuous harvest of product. The invention method accomplishes this by provia±ng^*- at least two cross-flow filtration systems in tandeπr,-thus permitting the maintenance in a functional condition of at least one cross filtration system continuously. The use of the invention method prevents a disconnect of product harvest, and thus provides a constant product harvest which not only maintains cellular vigor, but also prevents decomposition of the product. In one aspect, the invention is directed to a method to provide continuous cross-flow separation from an untreated dispersion of components of differing sizes, such as a cell culture suspension, which method comprises passing the untreated dispersion alternately through one of at least a first and second cross-flow separation means, and regenerating the other of said first or second means by reverse flow of a barrier-clearing fluid. In the conduct of the method, the dispersion to be treated is passed through the retentate side of the first cross-flow separation means to obtain a permeate and a retentate. The permeate is withdrawn from the permeate side of the barrier, and the retentate returned to the source of the untreated dispersion. Simultaneously, the barrier-clear- ing fluid is passed from the permeate side of the barrier in the second cross-flow separation means, to be recovered from the retentate side. This completes one phase of the cycle, and is followed by activating a switching means to introduce the untreated dispersion to the retentate side of the barrier of the second cross-flow separation means and effecting the flow of clearing fluid in reverse direc¬ tion through the first separation means. The cycle is then repeated by activating the switching protocol so that the first cross-flow separation means is employed in the recovery of retentate and harvest of product, while the second separation means is subjected to backflow with bar¬ rier clearing fluid. The cycles are repeated for an arbitrary number of times, thus maintaining a continuous procesTaring of the dispersion. In another aspect, the invention is directed to apparatus suitable for conducting the method of the inven¬ tion. Brief Description of the Drawings

Figure 1 shows a system for cross-flow filtra¬ tion and for its maintenance by discontinuous cleaning processes. Figure 2 shows a diagram of a typical apparatus useful in the method of the invention.

Modes of Carrying Out the Invention

A. Definitions

As used herein, "cross-flow separation means" refers to an apparatus or material into which is introduced a nonhomogeneous fluid mixture containing at least one component which is capable of transiting a bar- rier past which it is flowing and at least one other component which is retained on the same side of the bar¬ rier as that of its passage. This initial mixture containing these inhomogeneous components will be referred to herein as the "untreated dispersion" . The side of the barrier past which the untreated dispersion flows is designated the "retentate side of the barrier"; the other side is the "permeate side of the barrier".

While most of the "untreated dispersions" discussed herein are in fact suspensions — i.e., the components incapable of transiting the barrier are of such dimensions as to prevent true solution in the fluid (notably cells in suspension in culture), other smaller components which nevertheless are incapable of passaging the ba_Eri-er in question are also included. In particular, high moie^'cular weight particles such as proteins which are capable of segregation by molecular sieving can form the component incapable of transiting the membrane. In the case of such high molecular weight materials, a true solu¬ tion or colloid may in fact be formed — hence the more generic term "dispersion". The cross-flow separation means comprises at least a barrier which effects the separation of the untreated dispersion into a "permeate" which has transited the barrier and a "retentate" which has not. Of course, substantially all of the component incapable of transiting the barrier will be included in the retentate; depending on the efficiency of the separation, some of the components, including the fluid itself, which are capable of transiting the barrier may in fact be included in the retentate. The permeate, however, contains substantially only the components capable of barrier transition as well as the supporting fluid.

The barrier may vary both in composition and in physical form. Exemplified and preferred herein is a series of hollow fibers wherein the walls of the fibers constitute the barrier. It is preferred that the untreated dispersion be introduced into the lumen of the fibers so that the retentate remains in the interior of the fibers while the permeate passes through the fiber walls to the exterior. It is not impossible, although relatively impractical, to introduce the untreated disper¬ sion to the exterior of fibers contained in a cartridge and to harvest the permeate from their interior; the practicality of this approach increases as the diameter of the fiber approaches that of a tubule. Other physical configurations include a series of flat panels wherein the interstitial spaces between the panels are not in physical communication. Other means for providing volumes separated by these semipermeable membrane barriers can also be—envisioned.

The material of the barrier must be such that it is, in regard to the components of the untreated disper¬ sion, a semipermeable membrane -- i.e., the pore size or other means of transit must be such that some components are capable of passing through the membrane while others are not. The compositional nature of the barrier will therefore vary with the nature of the untreated disper¬ sion. For use in treating cell culture suspensions to harvest molecular products, the variety of materials disclosed by the Brown et al reference cited above may be suggested. These materials include acrylic polymers, borosilicate glass, cellulose esters, polysulfones, sintered metals , polypropylenes, porcelains, cellulose acetates and nitrates. Depending on the manner of manufacture, the discrimination size can be varied. For use in segregating individual molecules according to size, materials generally employed for dialysis separation are useful. These include cellophane, polyvinylidene difluoride, microporous Teflon, polysulfones, cellulose derivatives and polyether sulfone. The method and apparatus of the invention are applicable to a wide variety of untreated dispersions/barrier combinations.

Suitable untreated dispersions include not only cells suspended in media, but also a variety of dispersions encountered in various contexts such as sewage treatment, food processing, pharmaceutical and chemical manufacturing, isolation and purification of natural products, paper manufacture, recycling operations, and many others. The method and apparatus of the invention are applicable to any process or protocol which involves, or advantageously could involve, cross-flow separation of the components in a liquid mixture based on size.

As used herein, "barrier-clearing fluid" refers to a fluid which contains only components which are capable c-f readily transiting the barrier. If the untreated^" dispersion is a cell culture suspension, a suit¬ able barrier-clearing fluid is fresh medium.

As is well understood, while the barriers useful in the invention have theoretical cutoff sizes for their ability to discriminate among the components of the untreated dispersion, these values are never precise.

Most untreated dispersions will contain components which are quite clearly too large to transit the barrier, components which freely transit the barrier, and "inter¬ mediate" components which have dimensions comparable in size to the dimensions of the passageways through the bar- rier. Some of these intermediate particles will in fact pass through the barrier into the permeate; others will be retained. However, many will be trapped in the passage¬ ways of the barrier and it is these particles which, in fact, constitute the problem the invention is designed to solve. One of the advantages of the invention is the use of a barrier-clearing fluid which is free of intermediate components, which, of course, the permeate is not.

The apparatus of the invention comprises at least two interconnected compartments capable of effecting cross-flow separation of the components of the untreated dispersion. In the method, the untreated dispersion is passed alternately through one or more of these compartments in a manner so as to effect cross-flow separation so that the larger components are retained on the retentate side of a barrier and the smaller ones pass through to the permeate side. Simultaneously, in the alternate compartment(s) the barrier clearing fluid is passed from the permeate side of the barrier to be recovered from the retentate side. If the barrier- clearing fluid is heated prior to use, it may be advantageous to include a gas bleed connection on the permeate side of the barrier to prevent gas build up due to change in gas solubility of the fluid. The roles of the various compartments are then repeatedly reversed. The reversal cycles can be set up to be timed on an arbitrary schedule, which is preferably adjusted to effect the reversal as clogging begins to occur in the "separation" compartments, or can be triggered by the onset of the clogging. Both types of timing can be automated, the first simply using a timer; the second a sensor and response means. At each reversal, the "separation" compartment(s) become "barrier clearing" compartments, and vice versa.

B. Detailed Description of a Preferred Embodiment The operation of the method of the invention and a preferred embodiment of an apparatus employs hollow fiber cross-flow separation units as illustrated in Figure 2.

The system illustrated in Figure 2 is designed for application of the invention process to a cell suspen¬ sion from a reactor. The nature of the reactor is not critical to the method of the invention, and can be a simple shaker, stirred reactor, or static maintenance re¬ actor as described in U.S. patent 4,537,860 or may itself be a hollow fiber reactor as described in U.S. 4,201,845. In the case of the hollow fiber reactor, of course, the barrier composition will differ between that in the product harvest apparatus shown in the figure and that in the reactor itself. The configuration in Figure 2 is, of course, applicable to other untreated dispersions such as high MW protein solutions, particle size separations and the like, as set forth above.

Referring to Figure 2, for example, as it applies to treatment of cell suspensions, the tandem separation units A and B are cartridges containing packages of hollow fibers whose walls are of appropriate pore size to permit the passage of product, but not of cells. in each cartridge, the inlet ports 1A and IB and outlet^"ports 2A and 2B are in communication with the lumen of each of the packaged fibers. The inlet ports 3A and 3B and the outlet ports 4A and 4B ere in communication with the surroundings and exterior surfaces of the fibers. The system is controlled by a series of on/off valves labeled C1-C6. The tandem separations and cleaning operations employ fluids from two sources: the untreated dispersion is pumped from the reactor by the pump PI as shown; the barrier-clearing fluid, which in this embodiment is identical with replacement medium is supplied by a second pump P2 as shown. A third pump, P3, is employed to remove the permeate from the appropriate hollow fiber cartridge. The series of valves C1-C6 provide a switching means to permit either A or B in one phase of a cycle to be provided with the untreated dispersion and the other to be in the barrier clearing phase. An additional valve, C7, is common to both phases of each cycle and remains closed through normal operations. It permits the supply of additional medium to the reactor. In the first of a typical cycle, wherein cartridge A will be employed for product removal by cross-flow separation and cartridge B will be subjected to barrier clearance, the switching means is configured as shown for condition 1 in the figure. Valves Cl, C4 and C5 will be open; valves C2, C3, C6 and C7 will be closed. Under this condition, the cell suspension will be pumped by PI through valve Cl into the inlet 1A for cartridge A and the retentate will continue through the lumen of the fibers through the outlet means 2A and through the attached conduit IT and then into the common conduit 3T for return to the reactor. Backflow through the medium-bearing conduit 4T is prevented because valve C7 is closed. The product which exits cartridge A throuqh the outlet 4A is permitted to pass by open valve C5 anri- is^* pumped out of the system by the pump P3. Closed valve C2- ^"prevents flow through cartridge B.

During the phase of the cycle in which cartridge A is employed for product harvest, cartridge B is sub¬ jected to barrier clearance using fresh medium using the medium pump P2. Open valve C4 permits flow into cartridge B at inlet 3B which is in communication with the exterior of the fibers, closed valve C3 prevents flow into cartridge A. As valve C6 is closed, the medium is forced into the lumen of the fibers, carrying with it any "inter¬ mediate" size components which have lodged in the pores and the fluid exits through the outlet in communication with the fiber lumen, 2B, through the conduit 2T to merge with the retentate from cartridge A in conduit 3T for return to the reactor. Thus, the incoming medium serves not only to clear the carrier in cartridge B, but also to replenish medium lost in the cross-flow separation in cartridge A. The valve C7 can be opened if needed to provide additional replacement medium.

At such time as it is considered that the pres¬ sure caused by clogging of the fiber pores in A needs remediation, the apparatus is converted to condition 2 wherein the fibers of cartridge A will be cleared and those of cartridge B employed for product harvest. Under this condition, valves Cl, C4, C5 and C7 are closed, but valves C2, C3 and C6 are open. The functions of cartridges A and B are thereby reversed, and the second phase of the cycle is entered.

The cycle can, of course, be effected at arbitrary intervals and can be preset and automated. Suitable intervals for the duration of each phase of the cycle will vary with the untreated dispersion, the nature of the barrier, and the physical parameters of the apparatus. Suitable intervals for harvest of product from cell culture will vary from a few (e.g., 1-2) minutes to a few (e.g., 1-8) hours, to a few (e.g., 1-4) days for each phase.. These intervals can, of course, be set using a timer. —Intervals determined by condition of the barrier can also be automated by employing sensors and response means thereto.

Under condition 2 the untreated suspension flows through C2 into inlet IB where the cells are retained within the fibers, but the product appears in the perme¬ ate. The permeate containing product exits at 4B through valve C6 and is pumped away by pump P3. The retentate then exits at 2B through conduit 2T for return to the re¬ actor. Closed valve Cl prevents flow of cell suspension to cartridge A. Fresh medium is pumped by pump P2 past valve C3 into inlet 3A so that fresh medium now is provided to the outside of the hollow fibers in cartridge A. It is devi¬ ated from cartridge B by closed valve C4, and is prevented from exiting from the permeate side of the barrier in A by the closed valve at C5. The medium must therefore enter the lumen of the fibers carrying along with it the inter¬ mediate components embedded in the barrier, these particles are carried along with the clearing fluid through outlet 2A into conduit IT. The fluid containing both intermediate particles and replacement medium then is combined with the effluent from the lumen of cartridge B in conduit 3T for return to the reactor.

The cycle can then be repeated an indeterminate number of times alternating cartridges A and B from separation phase to barrier clearing phase. At all times, the untreated cell suspension is subjected to uniform conditions of product harvest and media replacement.

The illustration in Figure 2 shows only one unit or cartridge for separation and one for barrier clearance, but, of course, cartridges A and B can be replaced by a multiplicity of units — two, three, several or dozens if needed. While it is preferred that the number of "A" units equal the number of "B" units, this is not a neces¬ sary csndition for practice of the invention. While the invention has been illustrated to show a preferred embodiment and to explain the manner of its operation, the invention is not limited thereby, but rather is defined by the following claims.

Claims

1. An apparatus for continuous cross-flow separation from an untreated dispersion of at least one barrier-transiting component in a permeate from at least one barrier-retained component in a retentate which comprises: a first cross-flow separation means containing a first barrier and a second cross-flow separation means containing a second barrier; means for providing the untreated dispersion to the retentate side of the barrier, and for collecting the permeate from the permeate side of the barrier and the retentate from the retentate side of the barrier, in the first cross-flow separation means; means for simultaneously providing barrier- clearing fluid to the permeate side of the barrier and for collecting said barrier clearing fluid from the retentate side of the barrier in said second cross-flow separation means; and switching means whereby the untreated dispersion is diverted to said second cross-flow separation means in the manner previously provided to the first, and whereby said barrier clearing fluid is supplied said first separa¬ tion means as in the manner previously supplied to the second.

- 2. The apparatus of claim 1 wherein the first and second separation means are cartridges containing hol¬ low fibers, and wherein the switching means is a multiplicity of valves.

3. The apparatus of claim 1 which includes a multiplicity of said first and second separation means.

4. A method for continuously separating at least one barrier-retained component from at least one barrier-transiting component in an untreated dispersion which method comprises a cycle consisting essentially of: in the first phase of the cycle, passing the untreated dispersion through the retentate side of a barrier in a first cross-flow separation means to provide a retentate and a permeate; removing the permeate from the permeate side of the barrier and retentate from the retentate side of the barrier in said first separation means; simultaneously providing barrier-clearing fluid to the permeate side of the barrier of a second cross-flow separation means and removing said barrier-clearing fluid from the retentate side of said second cross-flow separa¬ tion means; followed by in the second phase of the cycle, altering the flow of the untreated dispersion and the barrier-clearing fluid so as to provide the untreated dispersion to the retentate side of the barrier of said second cross-flow separation means to obtain a permeate and a retentate, and removing the retentate from the retentate side of the barrier and the permeate from the permeate side of the barrier of said second separation means and simultaneously providing barrier-clearing fluid to the permeate side of the barrier in the first separa¬ tion means and removing said barrier-clearing fluid from the retentate side of said first separation means.

^"

5. The method of claim 4 wherein the untreated dispersion is a cell suspension and the barrier-clearing fluid is fresh medium.

6. The method of claim 4 wherein the retentate from the untreated dispersion and the barrier-clearing fluid removed from the retentate side of the barrier are recombined and returned to a common vessel.

7. The method of claim 4 wherein the first and second separation means are provided as multiplicities of units.

8. The method of claim 4 wherein said units are hollow-fiber cartridges.

9. The method of claim 4 wherein the cycle is conducted a multiplicity of times.