Note: Descriptions are shown in the official language in which they were submitted.
~.3~
-1- 16036
~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-
, ` ;
,.
,.: .
~ ,.,
.,,
~3~
-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.
.
1,
~ ,
.. . . .
~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.
.
, " i ~ .
.~
. ... . .
; . . .
"
-4- 16036
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.
EXAMPLE 1
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.
.
.
'
. : .
. ~ ' :
-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
:. .. .. ..
-6- 16036
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 .
EXAMPLE 2
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.
.. .
;
~l3~
~7- 16036
EXAMPLE 3
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
.
: . . . . ~ , .
- . ' ', ': ' ' , ' , '
-8 16036
circulated through unit a~ 50 ml/min for another
hour. Medium volume is increased to 3,800 ml and
circulation main~ained at 60 ml/min for 48 hours.
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.
....
.
.
.. .; :, , . ~ :
