U.S. patent number 3,839,155 [Application Number 05/275,642] was granted by the patent office on 1974-10-01 for cell and vaccine production.
This patent grant is currently assigned to Merck & Co. Inc.. Invention is credited to William J. McAleer, Kenneth L. Posch, Raymond E. Spier.
United States Patent |
3,839,155 |
McAleer , et al. |
October 1, 1974 |
CELL AND VACCINE PRODUCTION
Abstract
Use of tumbling in a rotating disc propagator to evenly
distribute the cell slurry, obtain two side plating of cells,
increase the cell concentration and recover the cells from the
propagator in small volumes of medium, whereby the overall yield of
cells and vaccines is significantly increased and at substantially
reduced costs as compared to presently utilized procedures.
Inventors: |
McAleer; William J. (Ambler,
PA), Spier; Raymond E. (Lansdale, PA), Posch; Kenneth
L. (Lansdale, PA) |
Assignee: |
Merck & Co. Inc. (Rahway,
NJ)
|
Family
ID: |
23053246 |
Appl.
No.: |
05/275,642 |
Filed: |
July 27, 1972 |
Current U.S.
Class: |
435/298.2;
435/299.1 |
Current CPC
Class: |
C12M
27/10 (20130101); C12N 7/00 (20130101); C12M
25/06 (20130101); C12M 27/14 (20130101); C12N
3/00 (20130101); C12N 2760/18751 (20130101) |
Current International
Class: |
C12M
3/04 (20060101); C12N 3/00 (20060101); C12N
7/00 (20060101); C12b 001/00 () |
Field of
Search: |
;195/127,139,142,143 |
References Cited
[Referenced By]
U.S. Patent Documents
Primary Examiner: Tanenholtz; Alvin E.
Attorney, Agent or Firm: Deef; Francis H. Westlake, Jr.;
Harry E. Katz; Martin L.
Claims
1. A device for use in the production of at least one member
selected from the group consisting of cells and vaccines which
comprises;
a multiplate propagator comprising a substantially cylindrical
vessel having two end plates in which the desired cells and
vaccines are grown;
a plurality of spaced apart discs that are located within the said
propagator and are positioned in planes parallel to the end plates
of said propagator and are rotatively mounted on a shaft that is
positioned in the center of each of said end plates;
means for holding the propagator; and means for tumbling said
propagator
2. A device as in claim 1 which further comprises means to control
the
3. A device as in claim 1 wherein the tumbling means include a
chain driven sprocket wheel which is engaged by an arm on said
means for holding the propagator.
Description
This invention relates to the production of cells and vaccines.
More particularly, this invention relates to the use of tumbling in
rotating disc machines in order to produce cells and vaccines in
substantially increased yields, thereby greatly reducing production
costs.
Human and animal vaccines have been commercially produced by
growing the desired virus in primary cells which must be grown on
surfaces. Commerical processes were initially developed in Brockway
bottles. These processes required the use of thousands of
individual bottles to achieve the production of sufficient
quantities of vaccine. The use of such a large number of bottles or
production units is very time consuming and costly, and creates a
substantial risk of contamination. As production techniques
evolved, the original Brockway bottles were replaced by roller
bottles which only slightly reduced the number of bottles and the
handling problems associated therewith.
Some mass culture systems have been developed, such as the
multiplate unit disclosed in U.S. Pat. No. 3,407,120 and the Biotec
cylindrical rotating disc apparatus, but these units provide only
minimal advantages over the original individual bottle system. In
using a rotating disc apparatus, the greatest efficiency is
obtained by minimizing the space between the plates, and growing
cells on both sides of the plates. This however, creates a serious
problem in obtaining a uniform distribution of cells and/or virus
over both planar surfaces of the plates.
The present invention provides a method and device for overcoming
the aforementioned disadvantages of the prior art procedures in
which a cylindrical rotating disc apparatus is tumbled end over end
at controlled speeds during the cell and vaccine production cycles,
as for example during the cell plating, cell growth, cell washing,
virus seeding, virus replication, and harvesting cycles of
operation or during the addition of a chemical agent to the growth
unit. For example, during the cell plating cycle, the propagator
can be tumbled at a speed of from about 1 rev./5 min. to about 1
rev./15 min., preferably at a speed of about 1 rev./10 min., while
during the virus seeding cycle, the propagator can be tumbled at a
speed of from about 1 rev./3 min. to about 1 rev./8 min.,
preferably at a speed of 1 rev./5 min. and during the harvesting
cycle at a speed of from about 30 rev./min. to about 90 rev./min.,
preferably at a speed of about 60 rev./min.
An advantage of the present invention is the ability to
substantially increase the yield of cells and vaccines and thereby
reduce the production costs.
A further advantage of the present invention is the ability to
evenly distribute the cell slurry between the plates, thereby
obtaining greater uniformity in the cell distribution and a higher
yield of cells and vaccine.
A still further advantage of the present invention is the ability
to obtain plating of cells on both sides of the plates from a
single charge of cells without the necessity of a two-step
procedure which requires two separate charges of cells, thereby
increasing the yield of cells and reducing the risk of
contamination and the costs associated therewith.
Another advantage of the present invention is that extremely low
volumes of fluid can be used to bathe the cell sheet, and to
recover the cells from the tank, thereby increasing the yield of
cells or the titre/ml. of cell associated vaccine when compared to
prior art procedures.
These and other advantages of the present invention will be readily
understood by reference to the following detailed description when
considered in conjunction with the accompanying drawings in
which:
FIG. 1 is a perspective view of a typical multiplate propagator and
holding means for the propagator employed in the present
invention;
FIG. 2 is a cross sectional view of the propagator contained in the
holding means;
FIG. 3 is a sectional view taken along the line 3--3 of FIG. 2;
FIG. 4 is a front elevational view of the tumbling device of the
present invention;
FIG. 5 is a side elevational view of the tumbling device of the
present invention; and
FIG. 6 is a sectional view of another propagator which is exemplary
of those which can be utilized in accordance with the teachings of
this invention.
Referring to the drawings, FIG. 1 discloses the multiplate
propagator (1) and the holding means (2). The propagator (1)
includes a cylindrical stainless steel vessel (3) having top and
bottom flanges (4) and (5) and a top plate (6) and a bottom plate
(7). Clamps (8) are used to seal plates (6) and (7) to flanges (4)
and (5) respectively. Plates (6) and (7) also have fittings (9),
(10), (11), and (12) which connect to various lines when the
propagator is in use in order to cycle air and fluids through the
propagator. The holding means (2) also shown in FIG. 1 includes a
cage like structure having two semi-tubular portions (13) and (14)
which may be of an open or closed construction and are connected by
hinges (15). Portions (13) and (14) each have flanges (16) and (17)
which have holes (18) through which pins or clamps may be inserted
to close and latch the holding means (2) around the propagator (1).
The holding means (2) also has two cylindrical arms (19) and (20)
extending from opposite sides of the mid-point of the holding
means.
As illustrated in FIG. 2 the propagator (1) contains a series of
titanium discs (21) mounted on a bar (22), which supports the
plates (7) in a separated state due to the presence of cylindrical
spacers between each plate. One end of the bar is rotatably
supported by a bearing (23) which is mounted in a recess (24) in
the center of plate (7). The other end of the bar (22) is rotatably
supported by a bearing (25) which is mounted in a recess (26) in
the center of plate (6). A magnetic couple (25) which is fixedly
mounted on the bar (20) is engaged by magnetic drive means (not
shown) in order to rotate the plates. The holding means (2)
envelops the propagator and the edges of the semi-tubular members
(13) and (14) about the edges of flanges (4) and (5) in order to
hold the tank (1) in a fixed position.
FIG. 3 is a sectional view taken along line 3--3 of FIG. 2, and
shows the two semi-tubular portions (13) and (14) in the closed
position and a pin (28) inserted in the holes (18) in flanges (16)
and (17) to lock the holding means in the closed position around
the cylindrical portion (3) of the tank (1).
FIG. 4 is a front elevational view of the tumbling device of this
invention and shows the propagator (1) contained in the holding
means (2). Arm (19) is surrounded by a bearing (29) rotatably
mounted in hole (30) of supporting member (31) and arm (20) is
fixedly mounted in sprocket wheel (32) which in turn is rotatably
mounted on support member (33). Wheel (32) is connected to sprocket
wheel (34) of a motor (35) by a chain (36) in order to tumble the
propagator (1). The motor (35) has a variable speed transmission in
order to tumble the propagator at the desired speed. Support
members (31) and (33) are positioned on stand (37).
FIG. 5 is a side elevational view of the tumbling device of FIG. 4
and illustrates the manner in which the propagator is tumbled end
over end while positioned in the tumbling device.
Similarly in FIG. 6, there is disclosed another multiplate
propagator (51) which can be used in conjunction with the tumbling
device of this invention. This propagator (51) comprises a
cyclindrical stainless steel vessel (52) having a flange (53) at
one end thereof. Plate (54) is sealed to the flange (53) by clamps
(55). An air-carbon dioxide mixture can be pumped into the vessel
(52) from a reservoir (not shown) through a line (56) which extends
along the length of the wall of the vessel (52) to the back of the
vessel (52) where a portion of the line (56) extends along the back
of the vessel. This portion of the line (56) has openings (57)
which permit the egress of the air-carbon dioxide mixture. An
outlet line (58) is also used to keep the air pressure within the
vessel at a relatively constant level. Another line (59) may be
used to supply medium, serum and other nutrients and to withdraw
the expended medium and product. The plates (60) are mounted on a
bar (61), which supports the plates (54) in a separated state due
to the presence of cylindrical spacers between each plate. One end
of the bar (61) is rotatably supported by a bearing (62) which is
mounted in recess (63) in the bottom of the vessel (52). The other
end of the bar (61) is also rotatably supported by a bearing (64)
which is mounted in a recess (65) in plate (54). A magnetic couple
(66) which is fixedly mounted on bar (61) is engaged by a driven
magnet (67) to rotate the plates (60) through the medium (58)
during the cell growth and virus infection stages of the production
cycle.
The process and device of this invention may be used to produce
viral vaccines such as mumps, measles, rubella, parainfluenza,
Mareks and cells such as WI-38, chick embryo and duck embryo cells.
Standard cells, sera and media may be used to produce the
aforementioned vaccines. For example, primary cells such as chick
embryo fibroblasts, green monkey kidney, bovine kidney, dog kidney
or diploid cells such as WI-38 may be utilized as may standard sera
such as fetal calf, calf, bovine, G-G-free newborn calf,
.alpha.-gamma calf or .alpha.-gamma bovine and standard media such
as Eagles Basel Medium, Medium EBME, Medium 199, and Eagle's
Minimum Essential Medium.
The invention will be better understood by reference to the
following examples.
EXAMPLE 1
A rotating titanium disc propagator is charged with a mixture of 12
billion trypsinized chick embryo cells in Medium 199, 45 ml. 2.8%
NaHCO.sub.3 /L and 10% fetal calf serum. The charged propagator is
then locked into the tumbler and tumbled at 37.degree.C. end over
end at a speed of 1 rev./10 min. until two-side plating has
occurred at which time the propagator is removed from the tumbler.
The propagator is then positioned so that the plane of the discs is
in the vertical axis and a portion of the medium and serum is
discharged until the unit is about half full. The discs are then
rotated at a speed of 1 revolution/8 minutes and air or a mixture
of 95% air and 5% CO.sub.2 is passed through the unit at a rate of
100 cc/minute until the cell growth cycle has been completed at
which time the spent medium is discharged from the propagator, the
propagator is washed with Hank's solution and charged with fresh
Medium 199 containing 60 ml. 2.8% NaHCO.sub.3 /L 25% SPGA and 4
millilitres of a mumps virus suspension which has a -log.sub.10
TCID.sub.50 /0.1 ml. of 3.6. The discs in the unit are again
rotated at a speed of 1 revolution/8 minutes until there is no
further increase in the concentration of virus in the supernatant
fluids at which time the vaccine is harvested and frozen.
The yield of mumps vaccine when prepared by the above process is
substantially higher than the yield obtained by using conventional
procedures.
EXAMPLE 2
A rotating titanium disc propagator is charged with a mixture of 12
billion trypsinized duck embryo cells in Medium 199 containing 45
ml. NaHCO.sub.3 /L and 10% fetal calf serum. The charged propagator
is then locked into the tumbler and tumbled at 37.degree.C. end
over end at a speed of 1 revolution/10 minutes until two side
plating has occurred at which time the propagator is removed from
the tumbler. The propagator is then positioned so that the plane of
the discs is in the vertical axis and a portion of the medium and
serum is discharged until the unit is about half full. The discs
are then rotated at a speed of 1 revolution/8 minutes and air or a
mixture of 95% air and 5% CO.sub.2 is passed through the unit at a
rate of 100 cc/minute until the cell growth cycle has been
completed at which time the spent medium is discharged from the
propagator. The propagator is then recharged with 2 litres of
Medium 199 containing 60 ml. 2.8% NaHCO.sub. 3 /L, 2% .alpha.-gamma
calf serum and 170 ml. of a rubella virus suspension which has
-log.sub.10 IND.sub.50 /0.1 ml. of 3.5. The propagator and contents
are then locked into the tumbler and tumbled end over end at a
speed of 1 revolution/15 minutes for 2 hours after which the
propagator is removed from the tumbler and a further 6 litres of
Medium 199 containing 60 ml. 2.8% NaHCO.sub.3 /L and 2%
.alpha.-gamma calf serum is added to the propagator. The unit is
then set so that the discs rotate at one revolution in 8 minutes
with air or a mixture of air or 95% air and 5% CO.sub.2 passing
through it at 100 cc/minute at 37.degree.C. When the infection
process has been completed the spent medium is disharged and fresh
Medium 199 containing 60 ml. 2.8% NaHCO.sub.3 /L 10% SPGA is added
to the propagator. This medium in turn is discharged when the
concentration of rubella virus has reached a maximum
concentration.
This yield of rubella vaccine when prepared by the above process is
substantially higher than the yield obtained by using conventional
procedures.
EXAMPLE 3
A rotating titanium disc propagator is charged with a mixture of
Medium 199, F 10 and tryptose phosphate broth with 5% fetal calf
serum and 12 .times. 10.sup.9 cells from trypsinized 12 day duck
embryos and 14.4 .times. 10.sup.6 PFU Marek THV. The charged
propagator is then locked into the tumbler and tumbled at
37.degree.C. end over end at a speed of 1 revolution/ 20 minutes
until two side plating has occurred at which time the propagator is
removed from the tumbler. The propagator is then positioned so that
the plane of the discs is in the vertical axis and a portion of the
medium and serum is discharged until the unit is about half full.
The discs are then rotated at a speed of 1 revolution/8 minutes and
air or a mixure of 95% air and 5% CO.sub.2 is passed through the
propagator at a flow rate of 100 cc/minute. The pH is adjusted from
time to time with 7.5% NaHCO.sub.3 so that it remains within the
limits of pH 6.8-7.4. Also glucose is added to the system
periodically so that at no time should the glucose concentration to
outside the limits of 15-100 mg/100 ml. On the sixth day after
plating the spent medium is discharged and 6 litres of
KCl-citrate/Trypsin is transferred into the propagator. The discs
are rotated two complete revolutions and then the
KCl-citrate/Trypsin solution is voided. The propagator is then
locked into the tumbler and held with the plane of the discs in the
horizontal axis for 5 minutes after which it is rotated about its
long axis 90.degree. so that the plane of the discs is in the
vertical axis. While in this position 1.5 L of Eagle's Basel Medium
containing 15% fetal calf serum is pumped into the propagator. The
propagator and its contents are then rotated end over end at a
speed of 1 revolution/second for 10 minutes. Following this
tumbling the contents are discharged and assayed for Marek THV.
The yield of Marek's vaccine when prepared by the above process is
substantially higher than the yield obtained by using conventional
procedures.
EXAMPLE 4
A rotating titanium disc propagator is charged with a mixture of
300 .times. 10.sup.6 WI-38 cells in Medium EBME containing 10%
fetal calf serum and 10 ml. of glutamine/L. The propagator and its
contents are then held with the plane of the plates in the
horizontal axis at 37.degree.C until plating has been achieved. The
propagator is then positioned so that the plane of the discs is in
the vertical axis and a portion of the medium and serum is
discharged until the unit is about half full. The discs are then
rotated at a speed of 1 revolution/5 minutes and air or a mixture
of 5% CO.sub.2 and 95% air is passed through the unit at a rate of
100 cc/minute. Twenty-four hours later the medium is discharged
from the machine and the unit is refilled with an equal volume of
fresh Medium EBME containing 5% fetal calf serum and 10 ml.
glutamine/L. After a further 48 hours in the rotating and gassing
mode, the cell suspension is harvested from the unit. For this
operation the unit is voided of spent medium and is then half
filled with a solution containing trypsin. The plates are rotated
through the trypsin solution so that all parts of each plate
contact the solution. The trypsin is then voided. After waiting 5
minutes, 1 L of Medium EBME containing 15% fetal calf serum is
pumped into the propagator and the whole unit is then tumbled end
over end at a speed of 1 rev./sec. for 10 minutes, after which the
cell suspension is discharged and collected. By use of the above
procedure there is obtained an increase of cell yield of 300% over
the conventional procedure.
EXAMPLE 5
A rotating disc propagator is charged with a mixture of 3.0 billion
trypsinized chick embryo cells, Medium 199, 45 ml. 2.8% NaHCO.sub.3
/L and 5% fetal calf serum. The propagator is held in the vertical
position at a temperature of 37.degree.C and plating is effected.
After 3 hours the fluid in the propagator is discharged and a
further 3.0 billion trypsinized chick embryo cells are added to the
fluid. After mixing the fresh suspension is transferred back into
the propagator which is held in the opposite vertical position to
the first plating at a temperature of 37.degree.C in order to
effect plating on the second side of the discs. When this has been
accomplished, the propagator is positioned so that the plane of the
discs is in the vertical axis and a portion of the medium and serum
is discharged until the unit is about half full. The discs are then
rotated at a speed of 1 revolution in 5 minutes and air or a
mixture of 5% CO.sub.2 and 95% air is passed through the propagator
at a rate of 100 cc/min. When the cells have reached the confluent
state or the growth has ceased, the cells may be harvested.
The medium in the propagator is discharged and the propagator is
filled up to the halfway mark with a solution containing trypsin.
The discs are rotated twice so that all parts of the disc become
wetted with the trypsin.
The trypsin solution is then discharged and the unit is held with
the plane of the plates in the horizontal axis for 5 minutes. The
propagator is then charged with 1 L of fresh Medium 199 and 45 ml.
2.8% NaHCO.sub.3 /L and is tumbled end over end at a speed of 1
rev./min. for 10 minutes after which the cell suspension is
discharged.
In this way it was possible to prepare with great efficiency and
economy large quantities of chick embryo cells.
EXAMPLE 6
A rotating disc propagator is charged with a mixture of 3.0 billion
trypsinized duck embryo cells, Medium 199, F 10 5% fetal calf serum
and 30 ml. 2.8% NaHCO.sub.3 /L. The propagator is held in the
vertical position at a temperature of 37.degree.C and plating is
effected. After three hours the fluid in the propagator is
discharged and a further 3.0 billion trypsinized duck embryo cells
are added to the fluid. After mixing the fresh suspension is
transferred back into the propagator which is held in the opposite
vertical position to the first plating at a temperature of
37.degree.C in order to effect plating on the second side of the
discs. When this has been accomplished, the propagator is
positioned so that the plane of the discs is in the vertical axis
and a portion of the medium and serum is discharged until the unit
is about half full. The discs are then rotated at a speed of 1
revolution in 5 minutes and air or a mixture of 5% CO.sub.2 and 95%
air is passed through the propagator at a rate of 100 cc/min. When
the cells have reached the confluent state or the growth has
ceased, the cells may be harvested.
The medium in the propagator is discharged and the propagator is
filled up to the halfway mark with a solution containing trypsin.
The discs are rotated twice so that all parts of the disc become
wetted with the trypsin.
The trypsin solution is then discharged and the unit is held with
the plane of the plates in the horizontal axis for 5 minutes. The
propagator is then charged with 1 L of fresh Medium 199, F10 5%
fetal calf serum, 30 ml. 2.8% NaHCO.sub.3 /L and is tumbled end
over end at a speed of 1 rev./sec. for 10 minutes after which the
cell suspension is discharged.
In this way it was possible to prepare with great efficiency and
economy large quantities of duck embryo cells.
* * * * *