CA1134296A - Use of surface of motionless mixer as cell culture propagator - Google Patents

Abstract

ABSTRACT OF THE INVENTION:
Use of a static mixing system device, con-sisting of a shaped assembly of paralles corrugated metal or plastic sheets, as a tissue culture propa-gator. The configuration of the device provides uniform flow across all surfaces for precise control of growth conditions and maximum cell growth.

Description

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~SE OF SURF~CE OF MOTIONLESS MIXER AS CELL CULTURE
P~OPAGAT~R
BACKGRO~WD ~r 5~E lN~ENlroN
The invention relates to a process for producing cells and vaccines.
~ re particularly, this invention relates to a process for producing cells and vaccines which utilizes a motionless mixer as a tissue culkure propagator. The motionless mixer is patented, U.S.
Pa~nt 3,785,620, U.S. Patent 3,918,688 and U.S.
3,871j624. I~ i~ commercially available as the "Koch Static Mixing Sy~tem", Koch Engineering Co., Inc., New York, a licensee of Sulzer Brothers~ Ltd., Winterthur, Switzerland. Briefly, the motionless mixer is a shaped assembly containing a large number of parallel corrugated metal or plastic ~heets~ The corrugation angle of adjacent layers in the element is reversed wi~h respect to the mixer axis~ ~nclined corrugations o~ adjacent sheets thus intersect and form a multitude of mixing cells. Str~ams entering a mixing cell are rearranged due to extensional and shearing forces and ~ubse~uently divided into two new streams, each of which l~a~es the mixing ~ a different direction~ This rearrangement of streams con-, ` ;

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-2- 16~36 tinues through ~he apparatus. The static mixing system is used for blending, mixing and dispersing.
In addition, the Bulletin KSM-2 discusses the use of the Koch Static Mixing System as a catalyst support, commenting that the geometrical arrangement of the system "is ideally suited to serve as a catalyst support for any kind of catalytic reaction requiring a narrow residence time distribution and minimized lateral temperature and concentration gradients".
Human and animal vaccines have been com-mercially produced by growing the desired virus in primary cells which must be grown on surfaces.
Commercial processes were initially developed in Brockway bottles and, as production techniques evolved, the Brockway bottles were replaced by roller bottles.
More recently, mass culture systems have been de-veloped, including those which utilize a series of concentric rings or tanks having a plurality of stacked plates. The most recent mass culture system which has been developed is the multiplate machine produced by Biotec A. B. of Sweden which contains a series of titanium discs or plates which axe mounted on a rotatable shaft in a cylindrical glas~ vessel.
The advantage of the Koch Static Mixer System in the production of live cell~ for vaccine use is that the unique mixer element design acts as a simple stationery baffle that utilizes the energy of the flowing fluids to produce mixing resulting in con-sisten~ performance regardless of flow rate and equipment dLmensions. ~hese well-defi~ed flow characte characte ristics and compactness permit operation in circulating modes with wide range of flow rates and excellent control of growth conditions. These characteristics are also beneficial when washing cellq of undesirable materials.

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~3- 16036 The procexs of the present invention may be used to produce viral ~accines such as mumps, measles, rubella, M~rek's disease, Herpes I and II, influenza, parainfluenza, varicella, cytomegalo and respiratory syncitia and cells such as chick embryo and duck embryo cells and cell lines such as WI-38, VIRO, Hela, Standard cells, sera, and media may be used to charge the propagator. For example, primary cells such as chick embryo fibroblasts, green monkey kidney, bovine kidney, dog kidney cells or diploid cells such as WI-38 may be utilized as may standard sera such as fetal calf, calf, bovine, G-G-free new born calf, a-gamma calf or a-gamma bovine and standard media such as Eagles Basel Medium, Medium l99, Medium EBME and Eagle~s Miminum Essential Medium (EMEM).
The process of the pxesent invention will be better understood by an examination of the accompanying drawing in which.
FIG. l is a schematic view of the cell pro-pagation system using the Koch Static Mixing System.
The growth medium reservoir 10 i8 fitted with a medium conduit ll, inlet 27, return l9, and vent 28. The inlet 27 provides an air inlet 23 a~d an air/CO2 inlet 25, connected through two flow meters 24 and 25 respect-ively, so that gas intake into the medium reservoir is controllable. The medium conduit 11 can be divided into a multiple of feed streams. Two are illustrated, going through pumps 12 and 13 to two Koch Static Mixer System ~pparatuses 14 and 15. A ~ampling outlet is provided at 16. Following residence in the Koch apparatus, and return l9 which is fitted with a sampling point 20, dissolved oxygen monitor 21, and inlet 22 (for fresh media or viruses) the medium is r~turned to the medium reservoir 10 via return l9, providing the recyclable closed system.

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The operation o the system involves pumping a previously prepared cell suspe~sion into the Koch apparatus, then pausing to let the cells settle on the urface. Following a residence time of 5 - 30 hours, 5 during which the units are shifted and rotated occasionally, cell growth is initiated. The end of the attachment is determined by monitcring cell counts of the fluid recovery sample outlet 16. Cell growth is initated and supported by circulating the desired medium 10 through the system. M~nitoring control of the growth phase is achieved by controlling of dissolved oxygen levels at l to 10 ppm and pH a~ 5 to 8 throughout. The end point is reached by monitoring the glucose concen~
tration, which drops from l to 0.01 mg/ml. The cells 15 are then stripped from the Roch appsratus by charging with trypsin and recovering following usual procedures.
This invention also relates to production of vaccines by growing the cells in the static mixer, then seeding with the desired virus, propagating and 20 harvesting.
m is invention is illustrated further by _;~e following examples.

Eleven day chicken ~mbryos are aseptically re-25 moved, decapitated, wa~hed, minced and then trypsinized,~or 2 hours at 37C. The ~lurry is strained to remove debris after which the slurry is centrifuged to collect the cells. The supernate is decanted and the cells (pellet) resu~pended in Medium "O" wîth 10% fetal calf 30 serum. A total of 12 embryos are processed yielding 1200 x 106 cells. These are further diluted with Medium "O" containing 2% fetal calf serum for a final concen-~ration of 1.8 x 106 cells/ml ready fox addition to the static mixing units.

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-5~ 16036 The static mixing unitC are a~sembled by inserting 5 and 6 2inch titanium elemenSs (Type CY = 900 cm2/ element) in each of two 12"
sections of 2 n glass pipe with Pnd plates. These units have been cleaned with acid and base and ~lushed with copious quantities of distilled water.
Tubing is attached to the end o the fetal calf serum and incubated at 37C for 3 days. Medium is drained from units and retained ~s sterility test.
Cell suspension i~ now introduced into the mixing units with 275 ml and 300 ml charged to units con~aining 5 and 6 elements respectively. The partially filled units contain ca. 200 ml. of air. Uhiform cell attachment is carried out by shifting unit~ to hori-zontal position and rotating at 6 minutes perrevolution for 6 and 17 hours for 6 and 5 element units respectively. Subse~uently, arP turned to vertical position and medium with unattached cells drained. Cell coun~s of this fluid recover 15%
and 33% of input cells from 5 and 6 element units respectively. Cell growth is now initiated by con-necting units to a medium reservoir containing 2.61 of Medium "O" with 2% fetal calf serum and medium is circulated ~hrough units as initial r~te of 40 ml/min. Medium reservoir i9 aerated with air/CO~
mixture to maintain pH and dissolved oxygen. Growth phase is continued for 72 hours during which time medium circulation is gradually increased to 70 ml/min for a nominal residence time of 7 minutes. Excellent control is attained as dissolved oxygen remains at 5 to 6 ppm and pH is ~ontrollable at pH 7.0 to 7.3 by varying aeration rate Glucose conc ntration in medium drops ~rom 0.9 to 0.4 mg/ml during grow~h phase. Cells are ~tripped from surface by charging :. .. .. ..

300 ml. of ~rypsin at 37C ~o units and rotating at l/Ç RPM for 15 minutes. 'rrypsin was n~utralized with fetal calf serum and detachment completed by rotating units end over end ca~ 6 times ~or additional mixing. Cell count or this solution shows recovery of 1.1 x 10 cells and cell densities of 200,000 cells/cm2 .

A cell suspension is prepared by the process of Example 1. A total of 30 embryos are processed yielding 3.45 x 109 cells which are diluted to a final concentration of 4.8 x 10~ cells/ml for planting.
Static mixing units are again assembled with 6 2-inch Type CY t~anium~e~ements in two sections of pipe, one glass a~d one stainless steel. Tubing is attached to the clean units. After autoclaving, units are preincubated at 37C for 3 days. Uhits are each charged with 300 ml. of cell suspension (equiv-alent to 13 embryo) and rotated at 1/6 RPM for 7 hours at 37C. to complete attachmenk. Uhits are sub-sequently drained to remove unattached cells (ca.
16% of input). Growth phase is then continued by fixing units in vertical position and con~ecting to medium reservoir containing 3.0 1 of Medium "O" with 2% fetal calf serum. Medium is circulated through units in parallel at initial rate of 50 ml/min which is gradually increased to 80 ml/min. Medium reservoir is aerated with air/CO2 mixture to control - pH 7.0 to 7.4 and dissolved 2 at ca. 5 ppm during growth cycles. Rapid consumption o~ glucose at these high loadings necessitates complete replacement of medi~m after 50 hours and 68 hours of growth.
Uhits are harvested with trypsin a~ter 89 hours.
Cell yields are 2.3 and 2.5 x 109 cells for cell de~sities of 450,000 cells/cm2.

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~7- 16036 A cell suspension is prepared from 11 day chicken embryos by the procedure of Example 1.
A total of 30 embryos axe processed yielding 3.0 x 10 cells which are diluted to a final concen-tration of 4.3 x 106 cells/ml for planting.
Static mixing units are assembled by in-serting 6 2-dn~h Type CY titanium elements in each of two 12.5" sections of 316 stainless steel pipe.
As usual, alternate elements are rotated 90 to achieve optimal mixing. Units are cleaned in place, autoclaved and preincubated at 37C. Units are charged with 300 ml of cell suspension (equivalent to 13 embryos) and rotated 1.6 RPM for 7 hours at 37C
in a horizontal position. Units are then returned to a vertical position, drained and connected to medium reservoix containing 3.0 1 of Medium "O" with ~% fetal calf serum. Medium i~ circulated through each unit at rate of 30 ml/min which is gradually increased to 80 ml/min corresponding to 15 minutes and 6 minutes nominal residence time respectively.
Medium reservoir is aerated with air/Co2 to control pH in range of S.9 to 7.5 during growth phase that is continued ~or 96 hours (4 days). Units are drained and fresh medium added after 53 hours and 78 hours to maintain adequate nutrient supply for cells. After 91 hours, one unit is ~arvested with trypsin yielding 1.8 x 109 cells for cell density of 350,000 cells/cm3.
~ After 96 hours the ~econd unit i8 drained and seeded with Herpes Simplex Type I virus. Seed i9 prepared by disrupting a sample of infected cells by sonication and diluting in Medium Q so that 1.5 x 108 PFU of virus are available in~500 ml of medium. Mixing units are charged in vertical position and lncubated at 37C for one hour for virus attachment to cell layer. M~dium is then diluted to 1,000 ml in medium r~servoir and .

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Inected cells are harvest~d at this time by draining one-half of medium from unit and then turning unit end over so that the two phase solution (medium/air) rapidly rushes through elements. Medium is now drained for assay. Solution contains 2 x 10" PFU
for a substantial virus production.

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Claims (2)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A process for propagating cells comprising attaching cells to the surfaces of a motionless mixer element disposed within a housing, the motionless mixer element comprising an assembly of parallel sheets shaped to provide a plurality of channels which converge and diverge to form a plurality of mixing cells, each cell being formed and bounded by the juncture of two inlet channels which converge toward each other at about right angles in one plane, and two outlet channels which diverge from each other at about right angles in another plane, the planes being rotated about 90° with respect to each other, supplying nutrients into the mixer and maintaining the cells under conditions which permit cell growth and division, and harvesting the cells.

2. A process for replicating a virus com-prising attaching virus-infected cells to the surfaces of a motionless mixer element disposed within a housing, the motionless mixer element comprising an assembly of parallel sheets shaped to provide a plurality of channels which converge and diverge to form a plurality of mixing cells, each cell being formed and bounded by the juncture of two inlet channels which converge toward each other at about right angles in one plane, and two outlet channels which diverge from each other at about right angles in another plane, the planes being rotated about 90° with respect to each other, supplying nutrients into the mixer and maintaining the cells under conditions which permit viral replication, and harvesting the virus.