Biotechnology

Cell Culture:
Is the complex process by which cells are grown under controlled conditions. In practice the term
"cell culture" has come to refer to the culturing of cells derived from multicellular eukaryotes, especially
animal cells. The historical development and methods of cell culture are closely interrelated to those of
tissue culture and organ culture.
Live attenuated vaccines
Live vaccines contain weakened forms of the organism that causes the disease. Such organisms are also
called attenuated. There are many examples of highly successful vaccines that have been developed
using a live attenuated organism or virus. The current vaccine for tuberculosis, BCG, contains an
attenuated form of a mycobacteria. Attenuated forms of many viruses have been prepared for use as
vaccines to control diseases such as polio, measles, and mumps.
Often, the attenuated form of the organism [or virus] is obtained by serial passage [or culture] of the active
organism in culture media or cells. In these cases, the molecular basis of attenuation is unknown. Today,
it is likely that regulatory agencies would require an understanding of the basis of attenuation. Therefore,
development of any new attenuated form of mycobacteria for use as a candidate vaccine is likely to
include the introduction of one or more specific mutations into the genome of the pathogen. Likely
candidates include mutations that interfere with synthesis of an amino acid or nucleic acid component
essential for the growth of the organism.
Some examples of such live vaccines have already been prepared and evaluated in preclinical trials. One
is a recombinant form of BGC called rBCG30. This strain overproduces and secretes a 30 kDa protein
from M. tuberculosis.
Inactivated viruses
Treated so that they are no longer able to produce evidence of growth or damaging effect on tissue.
Inactivation may be by physical means including heat, ultraviolet light, ultrasonic vibration, or by chemical
treatment. Used in inactivated viral vaccines.
Risk Assessment:
1. Risk group classification
2. Potential of Aerosol generation
3. Quantity
4. Concentration
5. Agent Stability in the environment (inherent biological decay rate)
6. Type of work proposed (e.g., In vitro, In vivo, aerosol challenge studies)
7. Use of recombinant organisms (e.g., gene coding for virulence factors or toxins, host
range alteration; oncogenicity, replication capacity, capabality, capability to revert to wild
type.

Vaccine
The development of vaccines to protect against viral disease is one of the hallmarks of modern medicine.
The first vaccine was produced by Edward Jenner in 1796 in an attempt to provide protection against
smallpox. Jenner noticed that milkmaids who had contracted cowpox, a relatively innocuous infection,
seemed to be resistant to smallpox, a disease of humans that regularly reached epidemic levels with
extremely high mortality rates.
Jenner theorized (correctly) that cowpox, a disease of animals, was similar to smallpox. He concluded
that the human reaction to an injection of cowpox virus would somehow teach the human body to respond
to both viruses, without causing major illness or death. Today, smallpox is totally eradicated. Only two
frozen samples of this virulent virus exist (one in the United States, the other in Russia), and as of mid1995 there is serious scientific debate about whether to destroy the samples, or keep them for further
laboratory study.
A virus is a small bit of RNA (Ribonucleic acid) and/or DNA (deoxyribonucleic acid), the material in all
living cells that instructs the cell how to grow and reproduce. Viruses cannot reproduce by themselves,
but only by taking over the nucleus of a host cell and instructing the cell to make additional viruses. When
a virus successfully invades an organism, it takes over the cell growth process in the host.
Under ordinary circumstances, the human body responds to viral invasion in several different ways.
Generalized immunity to a virus can be developed by the cells in the body that are targets of viral
invasion. In this situation, viruses are prevented from gaining access to host cells. A more common
protection is the body's ability to develop blood and lymph cells that destroy or limit the efficacy of the
invading virus. Often, an infected human body will "leam" how to respond to a specific virus in the future,
so that a single infection, especially from a relatively benign virus, usually teaches the body how to
respond to additional invasions from the same virus. The common cold, for example, is caused by one of
several hundred viruses. After recovering from a cold, most people are resistant to the particular virus that
caused the particular cold, although similar cold viruses will still cause similar or identical symptoms. For
some innocuous viruses, a person might even develop immunity without becoming visibly ill.

Virus Families
There are usually several variations or strains of any particular virus. Depending on the number of
varieties, a biologist might group viruses as types or strains. Vaccines frequently are made from more
than one group of related viruses; a preventive reaction to the multivalent vaccination will probably cause
immunity to almost all of the group's variants, or at least to those variants which a person is likely to
encounter. Choice of the specific members of the group to use in a vaccine are made with painstaking
care and deliberation.
The

Manufacturing
Process

Manufacturing an anti-virus vaccine today is a complicated process even after the arduous

task of creating a potential vaccine in the laboratory. The change from manufacturing a potential vaccine
in small quantities to manufacturing gallons of safe vaccine in a production situation is dramatic, and
simple laboratory procedure may not be amenable to a "scale up" situation.
The Seed

1 Manufacturing begins with small amounts of a specific virus (or seed). The virus must be free of
impurities, including other similar viruses and even variations of the same type of virus.
Additionally, the seed must be kept under "ideal" conditions, usually frozen, that prevent the virus

from becoming either stronger or weaker than desired. Stored in small glass or plastic containers,
amounts as small as only 5 or 10 cubic centimeters, but containing thousands if not millions of
viruses, will eventually lead to several hundred liters of vaccine. Freezers are maintained at
specified temperatures; charts and/or dials outside of the freezer keep a continuous record of the
temperature. Sensors will set off audible alarm signals and/or computer alarms if the freezer
temperature goes out of range.
Growing the virus

2 After defrosting and warming the seed virus under carefully specified conditions (i.e., at room
temperature or in a water bath), the small amount of virus cells is placed into a "cell factory," a
small machine that, with the addition of an appropriate medium, allows the virus cells to multiply.
Each type of virus grows best in a medium specific to it, established in pre-manufacturing
laboratory procedures, but all contain proteins from mammals in one form or another, such as
purified protein from cow blood. The medium also contains other proteins and organic
compounds that encourage the reproduction of the virus cells. As far as the virus is concerned,
the medium in a cell factory is a host for reproduction. Mixed with the appropriate medium, at
appropriate temperature, and with a predetermined amount of time, viruses will multiply.

3 In addition to temperature, other factors must be monitored, including the pH of the mixture. pH
is a measure of acidity or basicity, measured on a scale from 0 to 14, and the viruses must be
kept at a defined pH within the cell factory. Plain water, which is

neither acidic or basic (neutral) has a pH of 7. Although the container in which the cells are
growing is not very large (perhaps the size of a 4-8 quart pot), there are an impressive number of
valves, tubes, and sensors connected to it. Sensors monitor pH and temperature, and there are
various connections for adding media or chemicals such as oxygen to maintain the pH, places to
withdraw samples for microscopic analysis, and sterile arrangements for adding the components
of the cell factory and withdrawing the intermediate product when it is ready.

4 The virus from the cell factory is then separated from the medium, and placed in a second
medium for additional growth. Early methods of 40 or 50 years ago used a bottle to hold the
mixture, and the resulting growth was a single layer of viruses floating on the medium. It was
soon discovered that if the bottle was turned while the viruses were growing, even more virus
could be produced because a layer of virus grew on all inside surfaces of the bottle. An important
discovery in the 1940s was that cell growth is greatly stimulated by the addition of enzymes to a
medium, the most commonly used of which is trypsin. An enzyme is a protein that also functions
as a catalyst in the feeding and growth of cells.
In current practice, bottles are not used at all. The growing virus is kept in a container larger than
but similar to the cell factory, and mixed with "beads," near microscopic particles to which the
viruses can attach themselves. The use of the beads provides the virus with a much greater area
to attach themselves to, and consequently, a much greater growth of virus. As in the cell factory,
temperature and pH are strictly controlled. Time spent in growing virus varies according to the
type of virus being produced, and is, in each case, a closely guarded secret of the manufacturer.

Separation

5 When there is a sufficient number of viruses, they are separated from the beads in one or more
ways. The broth might be forced through a filter with openings large enough to allow the viruses
to pass through, but small enough to prevent the beads from passing. The mixture might be
centrifuged several times to separate the virus from the beads in a container from which they can
then be drawn off. Still another alternative might be to flood the bead mixture with another
medium which washes the virus off the beads.

Selecting the strain

The eventual vaccine will be either made of attenuated (weakened) virus, or a killed virus. The choice of
one or the other depends on a number of factors including the efficacy of the resulting vaccine, and its
secondary effects. Ru vaccine, which is developed almost every year in response to new variants of the
causative virus, is always an attenuated vaccine. The virulence of a virus can dictate the choice; rabies
vaccine, for example, is always a killed vaccine.

6 If the vaccine is attenuated, the virus is usually attenuated before it goes through the production
process. Carefully selected strains are cultured (grown) repeatedly in various media. There are
strains of viruses that actually become stronger as they grow. These strains are clearly unusable
for an attenuated vaccine. Other strains become too weak as they are cultured repeatedly, and
these too are unacceptable for vaccine use. Like the porridge, chair, and bed that Goldilocks
liked, only some viruses are "just right," reaching a level of attenuation that makes them
acceptable for vaccine use, and not changing in strength. Recent molecular technology has made
possible the attenuation of live virus by molecular manipulation, but this method is still rare.

7 The virus is then separated from the medium in which it has been grown. Vaccines which are of
several types (as most are) are combined before packaging. The actual amount of vaccine given
to a patient will be relatively small compared with the medium in which it is given. Decisions about
whether to use water, alcohol, or some other solution for an injectable vaccine, for example, are
made after repeated tests for safety, sterility, and stability.

Quality Control
To protect both the purity of the vaccine and the safety of the workers who make and package the
vaccine, conditions of laboratory cleanliness are observed throughout the procedure. All transfers of virus
and media are conducted under sterile conditions, and all instruments used are sterilized in an autoclave
(a machine that kills organisms by heat, and which may be as small as a jewel box or as large as an
elevator) before and after use. Workers performing the procedures wear protective clothing which
includes disposable tyvek gowns, gloves, booties, hair nets, and face masks. The manufacturing rooms
themselves are specially air conditioned so that there is a minimal number of particles in the air.

The Approval Process

In order for prescription drugs to be sold in the United States, a drug manufacturer must meet strict
licensing requirements established by law and enforced by the Food and Drug Administration (FDA).
All prescription drugs must undergo three phases of testing, although data from the second phase can
sometimes be used to meet third phase requirements. Phase 1 testing must prove that a medicine is safe,
or at least that no untoward or unexpected effects will occur from its administration. If a medicine passes
Phase I testing, it must next be tested for efficacyit must do what it is supposed to do; medicine cannot
be sold that is useless, or that makes claims for an effect that it does not have. Finally, Phase III testing is
designed to quantify the effectiveness of a medicine or drug. Although vaccines are expected to have
effectiveness close to 100%, certain medicines might well be acceptable even if they have limited
effectiveness, as long as the prescribing physician knows the odds.
The entire manufacturing process is reviewed carefully by the FDA which examines records of procedures
as well as visiting the manufacturing site itself. Each step in the manufacturing process must be
documented, and the manufacturer must demonstrate a "state of control" for the manufacturing process.
This means that scrupulous records must be kept for every step in the process, and there must be written
instructions for each step of the process. Except in cases of grievous error, the FDA does not determine if
each step in a process is correct, but only that it is safe and is documented sufficiently to be performed as
the manufacturer stipulates.
The Future
Producing a usable, safe antiviral vaccine involves a large number of steps which, unfortunately, cannot
always be done for each and every virus. There is still much to be done and learned. The new methods of
molecular manipulation have caused more than one scientist to believe that the vaccine technology is
only now entering a "golden age." Refinements of existing vaccines are possible in the future. Rabies
vaccine, for example, produces side effects which make the vaccine unsatisfactory for mass
immunization; in the United States, rabies vaccine is now used only on patients who have contracted the
virus from an infected animal and are likely, without immunization, to develop the fatal disease.
The HIV virus, which biologists believe causes AIDS, is not currently amenable to traditional vaccine
production methods. The AIDS virus rapidly mutates from one strain to another, and any given strain does

not seem to confer immunity against other strains. Additionally, a limited, immunizing effect of either
attenuated or killed virus cannot be demonstrated in either the laboratory or in test animals. No HIV
vaccine has yet been developed.

What is claimed is:

1. A process for the large-scale production of rabies vaccine comprising
(a) successively passing into biogenerators of increasing volume a cell stock comprising a VERO cell
strain and a liquid nutritive medium containing serum, said liquid nutritive medium having suspended
therein microcarriers present in an amount ranging from 1 to 10 grams per liter of said liquid nutritive
medium, each such passage being carried out with stirring at a rate not greater than 40 rpm and for a
period of time ranging from 5 to 8 days, the last of said passages being carried out in a biogenerator
having a volume of at least 150 liters,
(b) drawing off said liquid nutritive medium at the end of the final passage and replacing said liquid
nutritive medium with a serum-free liquid nutritive medium,
(c) inoculating said cell stock in the last passage biogenerator with virus and allowing the virus to develop
at a temperature between 35-37 C. at a pH of about 7.4 to 7.8 and at a partial oxygen pressure of about
10-50 percent while stirring at a rate not greater than 40 rpm,
(d) culturing the said virus for a period of at least 5 days,
(e) withdrawing the liquid suspension of cultured virus,
(f) filtering the withdrawn liquid suspension,
(g) ultrafiltering the filtered liquid suspension so as to concentrate the same at least 100 times,
(h) inactivating the concentrated suspension with beta-propiolactone and
(i) purifying the inactivated suspension by zonal centrifugation or chromatography.

2. The process of claim 1 wherein the liquid nutritive medium withdrawn in step (b) is contacted with a
dilute solution of purified protease so as to separate the cells from the microcarriers.
3. The process of claim 1 wherein said cell stock is inoculated in step (c) with a Pitman-Moore PM 1.5033 M rabies virus strain.
4. The process of claim 1 wherein the molecular separating power of the ultrafiltration in step (g) is at a
molecular point of 10,000 to one million.

Description:
The object of the present invention is to provide a process for the large-scale production of a rabies
vaccine which is economically satisfactory and easy to use.
Several types of vaccines have been produced since 1885, the date at which the first vaccine against
rabies developed by Pasteur was introduced.

There were first of all the vaccines produced on nerve tissue and then inactivated of the Fermi, Semple,
Hempt or Fuenzalida type. Then, in order to reduce the secondary effects, a great step was made with the
vaccine cultivated on the embryo of the duck (1955). This vaccine remained the reference vaccine for 25
years before the arrival of vaccines produced on tissue culture (1978).
The latter, which are still being used, made a distinct progress in the anti-rabies therapeutics by their high
antigenicity and their quasi-absence of secondary effects. The sole criticism which may be made of this
type of vaccine is their high cost and difficulty of producing them in a large quantity. All these criteria have
for result that the vaccines obtained on tissue cultures (mainly on human diploid cells) are reserved for
the curative treatment of rabies.
There is consequently a big place for a highly antigenic vaccine which is of a satisfactory price and can be
produced in large numbers.
Very considerable progress has been made in the field of the production of vaccines against viruses such
as poliomyelitis with, on one hand, the development of cell lines which are easily reproducible on a large
scale, and, on the other hand, microcarriers for cells which permit effecting cell cultures in suspension in
nutritive media at a large volume (see for example A. L. VAN WEZEL: New Trends of the Preparation of
Cell Substrates for the Production of Virus Vaccines. Progr. Immunobiol. Standard, Vol. 5, pp. 187-192
(Karger, Basel 1972) and Tissue Culture Technology in Virus Vaccine Production, Symposium on "Tissue
Culture Technology" at the 75th National Meeting of the American Institute of Chemical Engineers, Detroit,
Mich., U.S.A., June 3-6, 1973).
A few years ago, VAN WEZEL mentioned in a publication the use of a line issuing from cells of the kidney
of the African green monkey described by YASAMURA Y. in the Establishment of a Cell Strain Derived
from Monkey Kidney (C. Aethiops), 14th Meeting of the Jap. Tissue Culture Association October 1962 in
TOKYO and available at the American Type Culture Collection under number ATCC-CCL 81 (124th
passage). This line is known under the name VERO.
More recently, other processes for producing rabies vaccines have been published. This is the case of
VAN WEZEL, et al., "Vaccinations in the Developing Countries, La Guadeloupe 1978: New approach to
the production of concentrated and purified inactivated polio and rabies tissue culture vaccines" Develop.
Biol. Standard. Vol. 41, pp. 159-168 (S. Karger, Basel 1978). VAN WEZEL thus shows that it is possible to
prepare a vaccine against rabies from primary cells of the dog kidney trypsinized and cultivated on
microcarriers. However, the rate of harvest of the virus is not very high and the use of primary cells results
in high cost without allowing mass production. ATANASIU et al., 1981; Joint ESACT/LABS Meeting on the
use of Heteroploid and other cell substrates for the Production of Biologicals; Heidelberg, FR Germany,
1981 "Evaluation and comparative studies of inactivated rabies vaccine obtained in the heterologous
diploid and polyploid cells". Develop. Biol. Standard. 50, pp. 173-182 (S. Karger, Basel 1982), compare
the features of rabies antigens obtained by the culture in suspension on respectively HAK (Hamster adult
kidney), BHK (Baby hampster kidney) and VERO previously cultivated in monolayers. He concludes that,
while HAK and VERO form new candidates for the production of inactivated rabies vaccines, the VERO
line only provides a small production of virus if it is compared with the other cells.
Owing to these facts and probably to the difficulties of transposing knowledge acquired about one virus to
another virus of different type, the prior art has not suggested the mass production of rabies vaccine
which is purified and stable and has a high antigenic power from cell lines.
Among all these researches none has resulted up to the present time in a new vaccine which satisfies the
criteria mentioned in the introduction.
An object of the present invention is to provide an anti-rabies vaccine satisfying all these criteria.
Another object of the invention is to provide a process for providing a highly purified stable vaccine.

A further object of the invention is to provide such a process for producing a vaccine which has a high
antigenic value for a small volume.
The process according to the invention comprises the following steps: multiplying a cell line, preferably a
VERO line, from a working seed, by culture on microcarriers in suspension by successive passages in
biogenerators of increasing volume, the last passage being effected in a biogenerator whose vat is of at
least 150 liters, the ideal volume being 500 or 1000 liters, in a suitable nutritive medium, drawing off the
liquid medium at the end of the last passage and replacing it by a new liquid medium containing no
serum, inoculating the biogenerator of the last passage with virus, preferably maintaining the temperature
in the neighborhood of 35 C. to 37 C., the pH in the neighborhood of 7.4 to 7.8 and the partial pressure
of oxygen in the neighborhood of 10 to 50%, drawing off the liquid suspension after culture of the virus,
filtering the drawn-off suspension, concentrating at least 100 times by ultra-filtration the filtered
suspension, inactivating by the action of Beta-Propiolactone, purifying by zonal centrifugation or
chromatography in respect of each of the successive harvests.
The final stage of the process comprises mixing the purified harvests, effecting a dilution as a function of
the antigenic titration and adjusting the final formulation so as to prepare unit doses of anti-rabies vaccine.
Preferably, in starting with the VERO working seed, a passage is effected in a 1 liter biogenerator. The
cells are obtained by digestion with a very purified and diluted protease solution, then a passage is
effected in a 5 liter biogenerator; then a new passage is effected in a 25 liter biogenerator; then a new
passage is effected in a 150 liter biogenerator, and a last passage is effected with the use of a
biogenerator of very large volume (for example 1000 liters), or a plurality of biogenerators of smaller
volume (for example 150 liters), the inoculation by the virus being effected in this last passage.
A very important step from the point of view of production on an industrial scale resides in the passage
from one biogenerator to another and a very purified and diluted protease is used in situ in the vat itself,
from the 1 liter biogenerator up to that of 1000 liters. Preferably there is employed a very diluted
crystallized purified Trypsine solution at betwen 0.005% and 0.05%, and preferably 0.025%. For this
purpose, after having drawn off the culture medium, the culture is washed with a suitable buffer so as to
eliminate traces of serum coming from the culture medium, the purified and diluted Trypsine solution is
added and the product is allowed to incubate (for example for 10 to 15 minutes) while supervising the
detachment of the cells from the microcarriers and then the action of the enzyme is blocked by adding a
small quantity of serum concentrated medium.
The balls with the cells are recovered in a sterile receptacle with a stirring vibrator, the mixture being
passed through continuously with a moderate vibration so as to detach the cells without harming them.
The mixture is harvested with veal serum so as to inactivate the Trypsine, then inoculated in a suspension
of new balls in the following biogenerator either in a small volume, for example 1/10th of that of the
passage of culture proper, thereby facilitating a contact between the cells and the balls, namely in the
total volume of the new culture.
The microcarriers employed are preferably spherical balls having a mean diameter of about 50 to 300 m
in the dry state, having a density very slightly higher than 1, formed either by dextran polymers and
carrying on their surface grafted radicals of DEAE (diethyl-amino-ethyl), or by acrylic polymers carrying on
their surface peptidic groupings, or cross-linked insolubilized proteins. Preferably, the concentration of
microcarriers, expressed in weight, namely grams of microcarriers per liter of medium, is between 1 and
10 and preferably between 2 and 5 grams.
The culture temperature is kept at between 35 C. and 38 C. and may be lower so as to permit the
survival of the cells in a slowed-down growth phase (for example between 25 and 33 C.).
Preferably, each passage lasts 5 to 8 days, with a stirring, and there is usually observed at the end of the
culture a multiplication 8 to 30 times the cell growth.

The stirring is a critical point of the process in large-size biogenerators. Preferably, this stirring is carried
out at a rate of 10 to 40 rpm (for example with a stirrer rotating about a vertical axis and having triangular
blades one side of which is provided with a fin which makes a rather small angle with the plane of the fin),
such as that for example described in French patent No. 80 18608 filed on Aug. 27, 1980.
The culture medium for the passages of cells is preferably a usual medium, such as the Minimum
Essential Medium of Eagle enriched with Lactalbumine, Hydrolysate, Glucose, and Veal serum or the like.
or a Minimum Essential Medium of Eagle Dulbecco modified, Iscove modified, enriched with Hepes
(sulfonic ethane(2-[2-hydroxy-ethyl)-1-piperazinyl]-acid), Veal serum, Metal Salts.
For the viral infection, the culture medium employed both for effecting the cell growth and for the start of
the viral proliferation, is the Minimum Essential Medium of Eagle enriched with Glucose containing 1/5 of
Veal serum.
The viral inoculation is effected in the course of the last passage with the Pitman-Moore PM 1503-3 M
strain with a multiplicity of infection (MOI) between 1 and 0.001.
Two to three days after the infection, a rinsing is effected and the medium is changed with the viral culture
medium, Minimum Essential Medium enriched with human albumin serum at 0.05 to 0.3% and preferably
0.1%.
Five to seven days after the infection the viral harvest can be commenced. As the rabies virus is only
slighly lytic with respect to the VERO cell, multiple harvests are possible up to 10 but preferably 6.
Each of the harvests will be treated separately and in an identical way until purification.
The sequence of operations common to each harvest comprises a filtration of the raw harvest, a
concentration by ultrafiltration and then inactivation by Beta-Propiolactone and purification.
All of the checked, purified and inactivated harvests from the same batch will give a final bulk product.
The vaccine obtained according to the process described hereinbefore contains at least 2.5 International
Units per ml determined by the "NIH test" reference method.
By way of example, an individual dose at a volume of 0.5 ml after reconstitution contains, in addition to
the 2.5 UI of rabies antigen, 5% of human albumin serum, 5% of maltose, all of which is in the BME
medium of Eagle.
The antibiotics employed in the course of manufacture can no longer be measured in the finished product
owing to the purification effected.
The invention will now be described by way of a non-limiting example.
1. Cell Multiplication
The VERO line as distributed by the American Type Culture Collection under number ATCC-CCL 81 is
used. This cell is at its 124th passage. A cell working seed obtained during the 137th passage is prepared
in the conventional manner. The seed is divided up in phials and preserved in liquid nitrogen. Each phial
contains about 10010 6 cells.
Before preparing a working seed stock, a master seed stock is prepared. With the phial at the 124th
passage coming from the ATCC, subcultures are prepared by successive passages up to the 129th

passage. After trypsination of the cell strain the cell suspension is homogenized and then divided in phials
for storage in liquid nitrogen.
For preparing the working seed one starts with a phial of the the master seed at the 129th passage and
one effects a series of subcultures in Roux boxes up to and including the 135th passage by progressively
increasing the number of boxes. The 136th and 137th passages are effected in 20 liter and 150 liter
biogenerators. At this level the cell population, after trypsination and homogenization, is divided up in
phials in the proportion of 100 to 12010 6 cells per phial. Several hundreds of phials are sealed and deep
frozen in the liquid nitrogen at this stage.
The liquid medium used for the successive passages, including for the establishment of the cell stock, is
the Minimum Essential Medium of Eagle in a saline solution of Earle enriched with 0.2% of Lactalbumine
hydrolysate, 0.1% of Glucose or Fructose, 5% of Veal serum; it may also be in the Minimum Essential
Medium of Eagle in a saline solution of Earle according to the modifications of Dulbecco and Iscove,
enriched with 3% of Veal Serum, the osmolality is adjusted to a value between 300 and 350 mOsm/kg
and preferably 320 mOsm/kg per addition of sodium chloride and potassium chloride.
This medium may also be completed with metal ions, for example zinc, iron, nickel, copper, vanadium,
aluminum, manganese, cadmium, cobalt, chromium, molybdenum, rubidium, tin, titanium, zirconium.
Each milliliter of medium contains 75 units of Streptomycine, 14 units of Neomycine and 35 units of
Polymyxine B sulfate.
The microcarrier balls employed are DEAE Dextran balls sold under the trademark DYTODEX-1 by the
Swedish firm PHARMACIA. In the dry state, the balls have a mean diameter of about 67 micrometers,
with a density of 1.03. A gram of dry balls contains approximately 510 6 balls, which corresponds to a
total area per gram of about 0.6 m 2 . The concentration of balls per liter of medium is on the order of 1 to
5 g/l. These microcarrier balls may also be acrylic resin balls, covered with proteins rendered insoluble,
sold under the trademark MICARCEL by the French firm I.B.F. These balls have a mean diameter of 180
to 200 micrometers with a density of 1.03.
In order to prepare the balls, they are first of all allowed to inflate in a buffer solution, they are washed and
then sterilized, for example in a vat of at least 150 liters while stirring.
In order to effect the multiplication, the contents of a phial at the 137th passage is introduced into a
biogenerator containing a liter of medium with the balls according to the aforementioned density. The
culture is effected at a temperature of 37 C. for 6 to 7 days, while stirring at 20 revolutions per minute,
this stirring being gradually increased.
At the end of the growth, i.e. after the 6th or 7th day, the liquid medium is discharged and the cells are
retained, these cells being fixed on the balls. The cells are detached with an 0.025% solution of
crystallized Trypsine, buffered with 0.125M sodium citrate. The cells which have thus just undergone their
138th passage are then transferred into a 5 liter biogenerator where the 139th passage is effected in the
same way and under the same conditions. A 140th passage is then effected in a biogenerator containing
25 liters of medium and then the 141st passage is effected in a 150 liter biogenerator. The 142nd
passage is then effected in a 1000 liter (or 500 liter) vat containing 1000 liters (or 500 liters) of medium.
The indicated volumes are the useful volumes.
The stirring is carried out in the vats by means of a stirrer comprising a vertical rotary shaft plunging into
the liquid suspension and carrying adjacent to its lower end 2 or more blades each having the general
shape of a right-angled triangle, one of the sides of the right angle, preferably the larger, of which is
welded along a vertical generatrix of the shaft, the right angle being substantially at the lower end of the
shaft. The blades are consequently located in one or more radial vertical planes with the second side of
the right angle being substantially horizontal and extending radially at the lower end. Each second side is
extended downwardly by a rather short rectangular fin which makes an angle of 10 to 45 with the plane

of the blade, the fin having a rectangular shape, one of the larger sides of which conincides with said
second side of the right angle. The area of the fin is between 1/2 and 1/10 of the area of the triangular
blade.
The size of the stirrer of course increases with the volume of the vat. On the other hand, the speed of
rotation decreases each time one passes to a vat of larger volume, for example from 40 rpm for the vat of
1 liter to 7 rpm for the vat of 1000 liters.
2. Multiplication of the Virus
The viral strain is the same as that employed at the present time for the rabies vaccine produced on
human diploid cells, it concerns the strain PM-1503-3 M.
In order to produce the viral infection, the stirring is stopped, the balls covered with cells become
deposited at the bottom of the vat so that it is possible to draw off the culture medium. A sufficient quantity
of a maintenance medium, MEM, enriched with 1% of Veal serum, is introduced into the vat so as to
obtain a homogeneous stirring. At that moment, the viral seed can be introduced so as to start the contact
stage.
Two infection ways are possible:
(A) A direct infection of the biogenerator by the viral strain during the exponential stage of the growth of
the cells. By way of example, if a maximum cell concentration of 210 6 cells per ml is anticipated, the
infection will be effected when the cell concentration will be about 110 6 cells per ml. The quantity of viral
seed will be adjusted in such manner as to obtain a MOI of 1 to 0.001.
(B) An infection by cell mixing.
This mode of infection requires the starting up of a culture at the 142nd passage at the same time as the
last cell culture stage and with the same cells. This culture, to which is added a volume which is 1/30 th of
that of the principal culture, will be infected as soon as it is put into culture by the viral strain in the
proportion of a MOI of 0.001 to 0.1. The maintenance of the cell infection is effected by means of the
flourescent antibody method. When 100% of the cells are infected, the annex culture is used for infecting
the principal culture.
For an industrial production, the infection mode will preferably be that described at (A).
After the virus-healthy cells contact, the volume is completed to the total volume of the culture.
Two to three days after the infection, when the cell growth stage has finished, there are effected the
rinsings (3) of the balls plus the cells with the virus medium. The medium is replaced by the MEM medium
containing 0.1% of human albumin. The culture parameters are consequently adjusted: temperature 35
C., pH 7.50, PO 2 25%.
About 7 days after the day of infection, a first harvest, termed "R 1 ", is effected. The stirring of the
biogenerator is stopped, the microcarrier balls become deposited, the medium is drawn off by aspiration,
it is the harvest proper.
There is added the viral propagation, MEM with 0.1% of human albumin, the stirring is recommenced and
the product is held at 35 C. for 3 or 4 days until the following harvest, and so on. A succession of
harvests: R 1 -R 2 . . . R 6 . . . is consequently obtained.
3. Concentration-Inactivation

Each of the harvests is immediately filtered at 0.45 micron before the concentration step. The harvest is
subjected to an ultrafiltration on a Millipore membrane of polysulfone whose molecular separating power
is at a molecular weight of 10,000 or 100,000. It is also possible to contemplate the use of membranes
having a cutoff threshold in the neighborhood of one million. The filtering liquid, i.e. the ultrafiltrate, is
discarded and the retained part is kept. By this process, the volume of each harvest is thus reduced by a
factor 25. The inactivation is carried out on the concentrated viral product.
The inactivation step (less than 72 hours after the filtration) consists in putting the Beta-Propiolactone
(BPL) in contact with the viral suspension at a concentration of 1/4000, namely 0.025%.
By way of example, a schematic description of the method employed will now be given:
The BPL solution prepared extemporaneously on ice water is added to the viral suspension while
constantly stirring. Eight hours after the end of the addition of the BPL, a transfer is effected in a new
flask. After 234 hours at +4 C., the viral suspension is heated to 37 C. and maintained at this
temperature for 2 hours.
4. Purification
Each inactivated harvest is then concentrated a second time according to the procedure described in
paragraph 3 so as to reduce the volume to be purified by a factor 4. The concentration factor thus
obtained is 100 relative to the starting product.
At this stage, each of the harvests may be stored at the temperature of <+45 C. while awaiting
purification.
The purification is carried out by a zonal centrifugation in a sucrose density gradient. At the end of the
operation, the purified virus fractions are gathered and mixed: the volume thus obtained is diluted with a
Tris/EDTA/NaCl (TEN) buffer so as to obtain a concentration factor of 150 relative to the starting product.
The purification is carried out with a zonal rotor usually of the type B 15. The various sucrose solutions
are prepared with a TEN buffer, pH 7.8.
The centrifugation conditions are:
______________________________________
concentrated virus brought to 1200 ml 34% sucrose solution (P/P) 370 ml 60% sucrose solution (P/P) 100
ml centrifugation for 4 hours at 25,000 rpm at +4 C.
______________________________________
This purification method has a limiting factor, the number of cycles to be effected. This is why
chromatographic methods are preferred for mass production.
The chromatographic purification will associate a gelfiltration step with an ion exchange column.
The molecular screening column (gelfitration) containing an agarose gel, for example SEPHAROSE CL 4
B or TRISACRYL G 2000, or a modified Spherosil, permits the elimination of the proteins and other
impurities, the larger viral particles being excluded first of all.
In order to refine the purification, the addition of an ion exchange column DEAE-Dextran-Silice
(SPHERODEX) enables the remaining impurities to be fixed, the virus travelling freely through the
column.

5. Preparation of the Final Product

A plurality of satisfactory purified individual harvests are mixed. According to the antigenic titration of the
pool of the harvests, the dilution to be effected is determined. The final volume is adjusted in order to
have a concentration of maltose and human albumin of 5% in an Eagle BME medium at 1/2, in distilled
water and to have a dose at a volume of 0.5 ml.
The product is divided up and freeze dried in a sterile premises.
Each bottle of lyophilized vaccine is taken up with 0.5 ml of solvent (4 g/l NaC solution).
6. Checks
The vaccine was checked from the point of view of its antigenic activity, of the NIH potency at +4 C. and
37 C., its bacterial and fungus sterility and its toxicity.
Further, tumourigenicity of the VERO cells tests were carried out on newly-born rats. The tests were
negative.
The quantities of cell DNA detected after purification are extremely small.
7. Clinical Tests
Table I summarizes the side effects actively observed during the follow-up of 328 injections given in
France to 174 healthy adult volunteers. Local reactions observed included erythema at the injection site
(4%), and induration (3.3%), mainly seen during the two days following the injection. Slight local pain was
reported in 6.7% of cases, with a maximum effect 24 hours after the injection. General reactions observed
in the same subjects included:
fever of more than 38 C. in 0.9% of cases
slight asthenia for between 2 and 48 hours after one of the injections.
Table II shows the booster effect observed in 86 subjects previously immunized with traditional HDCS
vaccine, of whom approximately 84% presented residual antibodies at the time of injections with a single
0.5 ml dose of PVRV. A month after the booster injection, all subjects presented high antibody titres: the
range being from 1.6 to 189 with a geometric titre (GMT) of 18 IU per ml.
Table III summarizes one of the primo-immunization trials in 64 healthy volunteers who had never been
vaccinated against rabies. The pre-exposure vaccination schedule was that recommended by the
American authorities, and consisted of 3 subcutaneous injections of 0.5 ml each on days 0, 7 and 21. No
subject presented antibodies before the first injection.
TABLE I
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PVRV SAFETY - LOCAL AND GENERAL SIDE EFFECTS OBSERVED AFTER 328 INJECTIONS
NUMBER PERCENTAGE
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ERYTHEMA 13 4%

INDURATION 11 3.3%
SPONTANEOUS PAIN
22 6.7%
FEVER 3 0.9%
ASTHENIA 8 2.4%
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TABLE II
______________________________________
PVRV - BOOSTER EFFECT NUMBER OF GMT PERCENTAGE SUBJECTS IU/ML TITER > 0.5
______________________________________
BEFORE 86 2.15 83.7%
BOOSTER
1 MONTH AFTER
86 18.0 100%
BOOSTER
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TABLE III
______________________________________
PVRV RESULTS AFTER PRIMO-IMMUNIZATION USING 3 INJECTIONS (D 0. 7, 21) NUMBER OF GMT
PERCENTAGE SUBJECTS IU/ML TITER > 0.5
______________________________________
BEFORE 64 0 0%
IMMUNIZATION
1 WEEK 64 28.2 100%
AFTER 2
INJECTIONS
3 WEEKS 64 32.4 100%
AFTER 3
INJECTIONS