INSTRUCTIONS FOR THE DESIGN, EQUIPPING AND · PDF fileINSTRUCTIONS FOR THE DESIGN, EQUIPPING...

WORLD HEALTH ORGANIZATION

ORGANISATION MONDIALE DE LA SANTE

DISTR. : LIMITED DISTR. : LIMITEE

WHO/Rabies/90.201 Rev.1

ORIGINAL: FRENCH

INSTRUCTIONS FOR THE DESIGN, EQUIPPING AND STAFFING OF A LABORATORY FOR THE PRODUCTION OF ANIMAL RABIES VACCINE

PREPARED ON CELL-CULTURE

by Dr P. Reculard, Short term consultant to Veterinary Public Health Unit, WHO

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WHO/Rabies/90.201 Rev.1

This document describes the requirements for establishing a unit capable of an annual production of about half a million doses of cell-cultured rabies vaccine for veterinary use. It is intended as a guide for decision makers wishing to study the feasibility of such a project, and at the same time provides instructions for laboratory personnel planning to improve or establish such a unit.

The use of roller bottles for antigen production is described. However, replacement by classical fermentor technology is recommended if a higher annual output is required. This techonology, however, calls for a high level of investment.

New technologies are currently being tested and may become available in the not too distant future. These technologies may be less demanding in terms of space and laboratory environment control. Nevertheless, the cycles of manufacture and control remain more or less the same, whichever procedure is used., Therefore, conversion should be possible without any major modification to either the design or the specifications of the laboratories described in this document.

This document was prepared under the direction of Dr F.-X. Meslin, Veterinary Public Health,

Division of Communicable Diseases, WHO, Geneva, under the auspices of the

Mediterranean Zoonoses Control Programme

WHO/Rabies/90.201 Rev.1 page 1

INSTRUCTIONS FOR THE DESIGN, EQUIPPING AND STAFFING OF FACILITIES FOR PRODUCTION OF RABIES VACCINE FOR VETERINARY USE FROM CELL CULTURES

LIST OF CONTENTS Page

FOREWORD ................................... . .................................. 2

OBJECTIVES .................................................................... 3

Chapter 1: Production of rabies vaccine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

1.1 Introduction ........................................................ 4 1.2 Production site ..................................................... 4 1.3 Buildings ........................................................... 5 1.4 Equipment ........................................................... 9 1.5 Staff ............................................................... 10

Chapter 2: Control of rabies vaccine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

2.1 Introduction ........................................................ 13 2.2 Buildings ........................................................... 13 2.3 Equipment ........................................................... 14 2.4 Staff ............................................................... 15

Chapter 3: Central Services . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

3.1 Introduction ........................................................ 16 3.2 Central Washing Plant ............................................... 16 3.3 Media Preparation Unit .............................................. 17 3.4 Filling and Packaging Unit .......................................... 19 3.5 Other General Services .............................................. 21

Chapter 4: Animal houses ..................................................... 24

4.1 Introduction ........................................................ 24 4.2 Central animal houses for small laboratory animals .................. 24 4.3 Animal houses for testing of rabies vaccine for veterinary use ...... 24

Annex 1 List of equipment and materials used . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

Annex 2 Plans and figures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

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FOREWORD

The development of cell culture techniques and progress made in virology over the last few decades have led to the development of new viral vaccines that use antigens (complete virions or subunits) harvested in culture after the infection of cells with the chosen virus.

Human and animal rabies vaccines are among those that have benefited from this technological progress.

However, in spite of such progress, most of the rabies vaccines used throughout the world are still prepared from nerve tissue taken from experimentally infected animals. They consist of a suspension of cerebral matter obtained from homogenized brains of sheep, lambs and newborn mice. Large quantities of vaccine can be produced in a conventional laboratory with simple, rudimentary of equipment, which means they are produced cheaply.

On the other hand, and whatever the scale of production, manufacture of rabies vaccine from cell cultures requires purpose-built, protected premises for each of a number of different functions. It calls for special equipment and infrastructure, considerable resources, and staff qualified and experienced in cell and virus culture techniques.

Nevertheless, the World Health Organization (WHO) recommends that inactivated rabies vaccines prepared from cell cultures be used wherever possible, because of the advantages they offer. These are:

1. Safety and innocuitv

The absence of nerve tissue considerably reduces the risks of allergic reactions and serious neurological damage, which is most often caused by myelin. Furthermore, it is easy to check the complete inactivation of virus from cell cultures, to ensure freedom from residual pathogens.

2. Potencv

Successive improvements in manufacturing procedures now yield inactivated, stabilized and adjuvanted rabies vaccines from cell cultures with potency higher than that of brain tissue vaccines. Animals can be immunized for at least two years with a single dose of these new vaccines.

Special attention is therefore given in these Instructions to the resources required for the production of rabies vaccines from animal cells cultivated and infected in vitro.

Furthermore, the Instructions concern only the requisite conditions for preparation of liquid, adjuvanted rabies vaccine for the protection of animals. Tl=Y therefore do not deal with the installations and equipment used for purifying or freeze-drying the virus, features which are indispensable to the production of rabies vaccine for human use.


OBJECTIVES

These Instructions aim to help Member States to:

(i) decide what size of unit they need for production of rabies vaccine for veterinary use;

(ii) devise the production unit as part of a larger facility that should have sufficient infrastructure and resources to guarantee the quality of the work done there;

(iii) choose the form and layout of buildings, and also the equipment, materials and staff that provide the best in terms of safety and effectiveness.

Repeated reference is made in these Instructions to the technical recommendations that have appeared in WHO reports, especially the following:

WHO Technical Report Series, No. 709, 1983 (Seventh report of the WI-IO Expert Committee on Rabies);

WHO Technical Report Series, No. 658, 1981 (Thirty-first report of the WHO Expert Committee on Biological Standardization), Annex 3 - Requirements for rabies vaccine for veterinary use.

Those reports should be consulted in conjunction with these instructions.

Finally, the mention of manufacturers or suppliers in these Instructions (see list of equipment and main supplies, pages 28-38) should not be taken as an expression of preference by WHO: they are cited only by way of example.

These Instructions comprise four parts:

Chapter 1: Production of rabies vaccine

Chapter 2: Control of rabies vaccine

Chapter 3: Central services (in common with other production units):

central washing plant media preparation unit bottling and packaging unit central warehouse administration essential/common services

Chapter 4: Animal houses

central animal houses animal houses for testing of rabies vaccine for safety and efficacy.

WHO/Rabies/90.201 Rev.1 -page 4

CHAPTER 1. PRODUCTION OF RABIES VACCINE

1.1 INTRODUCTION

1.1.1 The general provisions set out in the report of the WHO Expert Committee on Biological Standardization (WHO Technical Report Series, No. 760, 1987, page 171, paragraph 2) for manufacturing establishments and control laboratories, apply also to laboratories for the production of rabies vaccines for veterinary use.

1.1.2 The size of the premises, the quantity of equipment and the principles of operation are determined by the manufacturing capacity selected, which in our example is between 400 000 and 500 000 ready-to-use doses of vaccine per annum.

This objective can be achieved at. the "semi-industrial" level, using cell and virus culture technology in roller bottles, within a compact space, with no need for a mechanized production line.

1.1.3 The production of viral vaccines from cell cultures is based on the seed lots systems (see WHO Technical Report Series, No. 709, 1983, paragraph 4.3), namely seed viruses from a rabies virus strain and, when cell lines are used, cell seeds.

The quality and quantity of rabies virus produced in culture can vary greatly depending on the cell and virus strain pair used.

It is therefore important to note that in these Instructions the data are based on average yields obtained using one of, the best known systems for production of rabies vaccine for veterinary use: BHK (baby hamster kidney) cell lines infected with the Pasteur-PV strain of rabies virus adapted to those cells.

In that system, 3 to 5 x lo6 infected cells provide in 1 ml of harvest enough viral suspension to make a dose of finished vaccine that conforms to WHO recommendations (potency >l.O IV) (see WHO Technical Report Series, No. 709, 1983, paragraph 5.4).

The premises are therefore designed and equipped for the annual production of 500 litres of cell and virus culture in roller bottles, divided into 20 batches of 25 litres, to be produced 10 months in the year.

NOTE. -* For production on an industrial scale of 1 million doses per annum or more, the procedure of culture in fermentors is best. Such technology involves very heavy investments. It also calls for maintenance engineers, and scientists and technicians who are highly qualified and experienced in this field. Yet, in this case, the structures are the same and the cycle of vaccine production is identical so that if a simplified method of producing cultures in fermenting vats were devised in the future, the production premises described in these Instructions could still be used, without significant alterations.

1.1.4 In these Instructions, the facility for production of rabies vaccine for veterinary use is envisaged as part of a larger set-up in which other viral and bacterial vaccines are produced. This is because the infrastructure and general facilities required are such that their cost could not be reasonably borne by production of rabies vaccine alone.

1.2. THE PRODUCTION SITE

It should of course be borne in mind that before any construction work begins, the site must be properly surveyed, even for a simple extension to existing buildings. The survey of a site for biological productions must examine the following:

1 .

1

topography and planned or existing activities in the neighbourhood;

access roads;

the nature of the soil and substrate;

the risk of flooding and other climatic hazards;

water supply - quality and quantity;

electricity supply - voltage, available power, risks of power cuts, etc.;

access to supplies of liquid nitrogen, dry ice, gas, oxygen, carbon dioxide, pure nitrogen, etc.;

fire prevention facilities;

telephone, telex, etc.;

facilities for decontamination and evacuation of liquid and gaseous effluents, and solid waste.

3 BUILDINGS

,.3.1 Inclusion in an overall nlan


1.3.la In view of the risks involved in handling rabies virus, the premises used for manufacture of the vaccine must be independent from the other buildings and reserved exclusively for the use of the rabies unit for as long as production continues. Between two runs of production, however, if the premises are free, they may be used (subject to total disinfection) for preparation of another inactivated viral vaccine. The premises should then be duly disinfected before rabies vaccine production is resumed.

1.3.lb As stated in paragraph 1.1.4, the building should be part of a facility where several vaccines or other biological products are manufactured. In this way it shares certain services with other units: central laundry, building management, distribution, packaging, storage of finished products, general services, and control laboratories.

A practical way of building such a complex would be to put all the production and control buildings inside a hangar, which would isolate them from the outside (see plan for biological production unit, plan no. 1, page 39). Such a system facilitates links between the units and allows the installations to be maintained without entry into the work areas. The buildings must have air conditioning. This system is particularly recommended in countries where temperatures can be very high. The hangar creates a buffer area between the buildings and the outside environment.

1.3.2 Choice of tvne.of construction

1.3.2a The buildings on the site can be:

purpose-built with permanent materials;

built from prefabricated panels assembled and perfectly sealed on a concrete platform;

or built of assembled or transportable modules (see figure 1, page 40), equipped either as laboratories or as animal houses.

WI-lO/Rabies/90.201 Rev.1 page 6

1.3.2b Traditional buildings allow free choice of shape, size and configuration in the interests of rational connection of one area to another. This type of construction is most suited to the building that houses the essential/common services, in view of safety regulations and the number of different machines.

For all the other buLldings, in each country concerned, the cost of traditional construction must be weighed against that of prefabricated buildings. It must always be ensured that the firms contacted have the experience and the resources to provide the quality and standard of work required by building specifications.

1.3.2~ Buildings made from prefabricated panels are easily extended and modified. Building is quick. Figures 2 and 3 on page 41 show a laboratory built of panels 1.2 m wide and 3.1 m high. Such panels can be blank, or fitted with panes of glass, windows or doors. Interior partitions also are made with panels which are joined with brackets and fixtures In such a way as to seal each room. The flexibility of the system allows for different sizes of rooms, configurations of corridors and airlocks.

The examples annexed to these Instructions show several buildings, including one for the production of rabies vaccine, which measures approximately 30 x 13 m. In 1.2 m panels, this amounts to 2 x 25 panels long and 2 x 11 panels wide.

1.3.2d Another system for prefabricated construction is based on assembly and juxtaposition of modules that come in a variety of sizes and can be combined to suit requirements. Large modules can be partitioned as described above.

Modules can also be purchased fully equipped but even several modules together, will not be big enough for the production of a rabies vaccine. The modules marketed by the IFFA-CREDO Company, for example, are 13.5 m long and 3 m wide, which allows only for a row of two or three communicating rooms, with no corridor (see figures 4 and 5, and specifications, on pages 42 and 43). Such modules can nevertheless be used in production centres, for special functions such as small, independent control units or separate, isolated animal houses.

1.3.3 Main specifications of the buildine: for nroduction of veterinarv rabies vaccine

1.3.3a The size and layout are determined by the objective of approximately 500 000 annual doses of vaccine produced from BHR cell line cultures infected by an adapted Pasteur strain. The plan presented in this document (see plan of unit for production of rabies vaccine for veterinary use, plan no. 2, page 45) shows a rectangular building 30 m by 13m.

1.3.3b The building and interior installations must provide safe operating conditions, preventing the escape of rabies virus and external contamination.

1.3.3~ Whatever type of structure is chosen, the height of the ceiling should be at least 3 m to allow bulky equipment to be installed and shifted. Floor coverings should be non-slip, smooth and continuous, not giving rise to dust. Walls and ceilings should be painted or plastered with a smooth, waterproof finish that is easy to clean and free of cracks.

Worktops (mobile tables and laboratory benches) should be made of shock absorbing material, to reduce the risk of breaking glass. They should be easily disinfected and resistant to bleach and alcohol.

Periodic disinfection of the premises must be possible.

WHO/Rabies/go.201 Rev.1 page 7

1 3 3d The "sterile" . . areas must be sealed and independent units equipped with their own airlock entrances. They are ventilated with air that is sterilized by passage through absolute filters, and introduced through indeoendent and easily accessible ventilation shafts.

Only pure, sterile water should be supplied to these sterile areas, which must contain neither sinks nor drains nor gully traps: spilled liquid must be sponged and emptied into autoclavable containers.

Lighting should be either electric or natural, through perfectly airtight, sealed panes of glass.

Finally, it is recommended that care and maintenance work on the plant within these areas be reduced to a minimum by having electrical junction boxes, stopcocks for fluids and gases, temperature controls etc. housed in service areas accessible from outside.

1.3.4 Descrintion of nremises and annexes

1.3.4a The building houses three separate areas. Plan no. 2 on page 45 shows the layout of rooms in each area and how staff should move from one area to another:

- Area 1 is a clean area, ventilated with dust-free air, in slight Pl pressure;

- Area 2, accessible via airlocks Sl and S2, is a m-virulent sterile area, with air-conditioning, air filtered through absolute filters (HEPA quality) and kept under positive pressure P2 > Pl;

- Area 3 is a sterile area where the rabies virus is handled before inactivation; it is therefore a virulent area, accessible by airlocks S3 and s4. It, too, has air-conditioning that circulates sterile air. The best way to prevent the virus escaping this area is by using neeative pressure. Alternatively, since such a system is very expensive, the sterile air can also be propelled by using superpressure P3 less than superpressure P2, as long as the ventilation shafts for each area are independent, and if the air extracted from the virulent area is decontaminated (e.g. by passage over elements heating it to 180°C).

1.3.4b Area 1

This comprises an entrance hall, changing rooms, two toilets, an office, a store for small equipment and supplies in use, and two corridors leading to the airlocks where the staff enter: Sl and S2 to the non-virulent sterile area, S3 and S4 to the virulent area. There are therefore two airlock doors for each section, one for men and the other for women. Within these airtight entrances the staff undress, shower and dress in the protective clothing prepared for them in sterile packaging (overalls, boots, bonnets, protective glasses, disposable masks and gloves) (see figures 6 and 7, page 44).

Area 1 also contains an assembly and sterilization room, since although there is a central washing plant for that purpose, it is verv stronnlv recommended that equipment be sterilized in this area with a double-entry autoclave and oven opening directly on the protected, sterile areas where supplies can be stored. Experience has shown that this prevents many kinds of "subtle" contamination that tend to plague laboratories which prepare cell cultures in large quantities. The central washing plant washes and rinses equipment, and the final cycle of preparation, assembly and sterilization takes place in Area 1 of the "rabies" building.


To avoid contamination the media should also be filtered and put in small containers. The central media preparation service weighs and mixes ingredients and prepares solutions, then sends them, unsterile, to Area 1 of the rabies building to be placed in containers in equipment airlock EAl. A tube connects this area to sterile Area 2, where the media are sterilized, filtered and put in small receptacles, under a laminar airflow extractor fan.

1.3.4~ Area 2

This comprises:

- A room for storage of sterile material coming out of the autoclave (Al) and the oven (F);

- A room for filtration and distribution of media coming through supply airlock Ml . This is equipped with a "Biohazard" 2-position extractor fan with vectical laminar airflow cabinet for two workers;

- A cold +8"C);

- A room

room for the storage of filtered media (temperature between +2" and

for incubation of the media before use (temperature 37" + 0.5"C);

- A room for cell cultures, equipped with Biohazard laminar airflow cabinets for four people (2.50 m), communicating with an incubation room at 37" (+ 0.5"C) containing the mobile "Roller" frames for roller bottles (see figure 8, page 46);

- A room where the virus is transferred after inactivation, through a supply airlock SM3, which is equipped with a decontamination vat. This room has a laminar airflow cabinet for one person;

- A room for preparation of the finished vaccine (addition of stabilizer and adjuvant). This room has a 2-man vertical airflow cabinet and communicates with a cold room for bulk storage of the finished product.

1.3.4d Area 3

In this plan the virulent area is quite separate from the others, being linked to them by a special corridor. This corridor leads to staff airlocks S3 and S4 and double entry autoclave A2, through which all virulent material must pass for decontamination before it leaves the area. Sterile equipment enters a 3 through supply airlock SM2, from area 2.

In area 3 there is a room for cell and virus cultures that communicates with an incubation chamber whose temperature can be set between 30°C and 38°C with an accuracy of + 0.5"C. This chamber contains the mobile roller frames that hold the culture bottles.

The culture room communicates also with a cold room (temperature between +2' and + 8'C) for the viral harvest which is in the process of becoming inactive. The bottles of inactivated virus are passed through airlock EA3 to the transfer room (end of inactivation) in non-virulent sterile area 2.

NOTE. The incubation rooms and cold rooms of the three areas can be either built-infrom the start or installed by manufacturers of collapsible and convertible chambers. The latter option is more flexible, making it easier to switch to the production of culture with fermenters, or to use.the building for a different purpose altogether.


1.4. EQUIPMENT

The equipment required for production of inactivated, absorbed rabies vaccine is listed below for each area.

1.4.1 Area 1

1.4.la Room for assembly and sterilization:

1 double-entry autoclave with its own steam supply, a vacuum pump, a purger, and a display showing the cycles. Accessory equipment: 2 trolleys and 2 baskets;

1 oven for sterilization at 180°C. Minimum dimensions: 100 x 60 x 120 cm, with fan extraction of hot air, Temperature programmable between 50" and 250°C with recording of heating cycles;

2 trolleys: 120 x 80 cm, and 80 cm high;

1 mobile bench for assembly, 200 x 80 cm, 90 cm high;

1 peristaltic pump;

1 machine for sealing plastic packaging.

1.4.lb Room for liquid nitrogen storage:

4 wide aperture, 40-litre containers equipped with canisters (4 x 6) and racks for tubes or ampules of 2 and 5 ml (for cell seeds);

1 tank of liquid nitrogen of 200 litres capacity, situated outside the room and connected to it with protected piping;

1.4.1~ Room for frozen stocks:

2 freezers at -85°C (for viral seeds) of approximately 350 litres capacity, equipped with a liquid nitrogen or liquid carbon dioxide safety device;

1 freezer at -20°C (+ 5°C) of approximately 300 litres capacity for storage of sera, antibiotics, beta-propiolactone etc.

1.4.ld Room for small equipment:

1 machine for crushed ice;

1 dry-ice chest (1 m3).

1.4.2 Area 2

1.4.2a Storage room for sterile supplies:

1 "Milli Q" or equivalent system for production of pure water;

2 trolleys (120 x 80 cm, 80 cm high).

1.4.2b Room for filtration and distribution of media:

1 Biohazard vertical laminar airflow cabinet for two people, with at least 75 cm clearance over worktop;


2 stainless steel, pressurized filter canisters, 20 litres each;

1 pressurized filter canister (5 litres), also stainless steel (pressurized by filtered compressed air or, better, pure anhydrous nitrogen);

2 stainless steel, flat disc filters (293 mm diameter), with joints and connectors;

2 stainless steel, flat disc filters (142 mm diameter), with joints and connectors;

4 magnetic stirrers (2 large and 2 small);

1 pH meter;

1 water bath (20 to 6OyC), with thermostat, 30 litres capacity;

1 refrigerator (+2" to +8"C), of 100 litres;

1 metering pump to fill containers (adjustable between 10 ml and 1 litre).

1.4.2~ Cell culture room:

1 Biohazard vertical laminar airflow cabinet for four workers, with at least 75 cm clearance above the worktop. Alternatively, a laminar ceiling could be used in the centre of the room (working surface of 200 x 150 cm);

1 roller system: see figure 8, page 46, showing a set of rollers on a Bellco unit (e.g. Bellco rollers, 2 units of 9 modules each, for 180 bottles of 2 litres each - 76 x 62 cm, 2.5 m high; or 3 units of 6 modules each, 1.75 m high);

1 standard microscope;

1 inverted microscope;

1 pH metre;

1 shaker for homogenization of cell suspensions;

1 water bath with thermostat, 30 litres;

2 magnetic shakers;

1 table top, refrigerated centrifuge, 6000 rpm, capacity 4 x 300 ml;

1 Dyana electronic balance, capacity 200 kg;

1 refrigerator (+2 to +8"C), 100 litres.

1.4.2d Room to which virus is transferred after inactivation:

1 vertical airflow cabinet for one.worker;

1 temperature exchanger, circulation principle, with thermostat;

1 pH meter;

1 magnetic stirrer.


1.4.2e Room for final preparation of vaccine:

1 vertical airflow cabinet for two workers;

1 magnetic stirrer.

1.4.3 Area 3

Room for cultures and harvest of virus:

1 Biohazard vertical laminar airflow cabinet for four people. It could be replaced with a central laminar ceiling;

1 peristaltic pump;

1 metering pump, as described in 1.4.2b;

1 standard microscope equipped for fluorescence;

1 inverted microscope;

1 pH meter;

1 water bath with thermostat;

4 magnetic stirrers (2 small and 2 large);

1 Dyana electronic balance, 200 kg capacity;

1 refrigerated centrifuge, 4000 rpm (6 x 1 litre capacity);

2 stainless steel pressurized filtration canisters, capacity 20 litres;

4 filter cases for filter cartridges of 0.753 m, with joints and connectors;

1 stainless steel flat disc filter, 293 mm diameter, with joints and connectors;

roller units as described in 1.4.2~ (3 units of 9 modules or 5 units of 6 modules) for 300 two-litre bottles;

1 ultrafiltration device equipped with cassettes (cut-off point at 100 000) with connections and variable speed volumetric pump;

1 freezer, -15" to -2O"C, 100 litres capacity;

1 double entry autoclave, as in 1.4.la.

1.4.4 Various containers for all areas

1.4.4a 12 stainless steel drums, 30 litres capacity, with wide necks and stainless steel screw tops and 3 pipes, with inlet at the top, outlet at the bottom, and air supply.

1.4.4b Borosilicate Pyrex glassware between 5 ml and 15 litres capacity.

1.4.4~ Polypropylene or teflon flasks with airtight lids for storage of viral seeds.

-


1.4.4d Airtight cryotubes of 2 and 5 ml for storage of cell seed in liquid nitrogen, or sealable phials of glass that can withstand temperatures down to -180°C.

1.4.5 Miscellaneous

Glassware and other small pieces of equipment used in virology

1.5. STAFF (Number and qualifications)

1.5.1 In accordance with the general manufacturing requirements (WHO Technical Report Series, No. 658, 1981, page 103, paragraph 2), rabies vaccine shall be produced by staff who do not have to handle any other infectious agents during the production. The health of the staff should be verified and certified. All those working in the sectors of vaccine production and control must be immunized and have an antibody titre of at least 0.5 IU per ml of serum. Those who are not involved in production or control shall not have access to the sterile working areas.

1.5.2 The qualifications of those responsible for production and control, and thus for the signing of protocols, should be forwarded to the national authority for approval.

1.5.3 The production staff will comprise at least:

1 senior scientist (the unit chief) with a postgraduate diploma in biology followed by specialist research and practical experience in microbiology and virology. He will also have trained in a rabies laboratory (preparation of virus and cell cultures, analysis and control);

1 highly qualified technician with a degree in biology and considerable experience in cell cultures, virology and microbiology;

4 technicians specially trained in cell culture techniques, working mainly in the sterile areas. It is preferable to have two men and two women, since a number of delicate operations on cell cultures are often better done by women, whereas other more strenuous tasks are more suitable for men;

2 laboratory staff for the non-sterile area (Area 1).


CHAPTER 2. CONTROLS

2.1 INTRODUCTION

2.1.1 A number of the vaccine controls of cell cultures must be performed in the production unit: naked eye and microscope examination of cell cultures before and after infection with the virus, and of "control" cells. Sterility control by incubation of the filtered media before their use is also performed in situ.

2.1.2 With those exceptions, all controls of vaccine under preparation and of the finished product must be made in a unit which is separate from the production unit.

NOTE: The control unit must also be able to control the raw materials.

2.1.3 The laboratory plans, the qualifications of the staff, and the control protocols, must receive prior approval from the national control authority.

2.1.4 When several vaccines are manufactured on the same site, as described in Chapter 1 (1.1.4), the laboratory controls of all the vaccines can take place in one and the same building.

2.1.5 The animal controls must be conducted in separate installations. These are described in Chapter 4, animal houses.

2.1.6 All the controls (in vitro tests, animal testing and controls on the production site) come under the authority of the scientific officer responsible to the relevant national authority.

2.2 BUILDINGS

2.2.1 Control building

2.2.la As in the case of the production buildings, there is a choice to be made between a purpose-built construction and one made of prefabricated elements or modules set together.

2.2.lb There is no need for sterile areas. The unit is designed as an air-conditioned building, of the same materials as the production buildings, with equally smooth and easily disinfected floors, walls and ceilings.

2.2.1~ In the sterile operations room, either a Bunsen burner or a laminar airflow cabinet can be used for protection.

2.2.ld The building is not necessarily supplied with steam, nor does it need a washing plant or sterilization equipment, since the small volumes that need to be decontaminated can be treated with antiseptics. After use, the recipients and other pieces of equipment are placed in air-tight containers and sent to the central washing plant. The washing plant also provides the control laboratories with sterile equipment. Ready-to-use media can be supplied by the central unit for media preparation.

2.2.2 Layout of the control laboratorv

2.2.2a The plan of the control unit, plan no. 3 on page 47, shows one possible layout for the laboratory.


2.2.2b In this example, all rooms open on a central corridor. There are several sectors:

1 sterility control laboratory (testing for bacteria, fungi and mycoplasmas);

1 bacterial control laboratory (2 rooms);

1 viral vaccine control laboratory (3 rooms);

1 laboratory for physical, chemical and'biochemical analyses.

The corridor also provides access to an incubation chamber with two compartments (the temperatures of which can be regulated separately) a cold room, a room for decontamination and for storage of air-tight containers holding equipment to be put through the autoclave, and a room for storage of samples.

On one side of the entrance to the building are the offices and on the other is a double air lock in which the staff change their clothes before going into the work area.

2.2.2~ In each section the size of the working surfaces are calculated from the volume of control activities to be undertaken, which in its turn depends on the range of vaccines to be controlled and the number of lots of each vaccine received.

2.2.2d As regards viral vaccines, irrespective of the volumes concerned, it is better to have at least three non-communicating control rooms:

- one room for cell cultures into which no viral strain is brought;

- two rooms for tests involving virulent material.

Such a division makes for flexible working arrangements and safe control, avoiding the situation where several strains of different viruses are handled in the same room in the same day.

2.3 EQUIPMENT

2.3.1 Each section is provided with the basic equipment needed for the discipline concerned.

2.3.2 Each room has work benches supplied with gas, compressed air, electricity, a sink, hot and cold water.

2.3.3 The building has a demineralized water supply as well as a "Milli-Qn or equivalent machine to produce pure, sterile water.

2.3.4 Each room used for cell culture and virology has a Biohazard vertical laminar airflow cabinet with space for two workers.

2.3.5 The control laboratory has the equipment necessary for control of rabies vaccine for veterinary use.

Controls require, in addition to the usual equipment for virology (see list of equipment and raw materials, pages 28-38):

1 immunofluorence microscope;

2 adjustable incubation compartments - one at 37"C, one at 33°C (+ 0.5");


in the chemistry and biochemistry section: equipment for immunoenzyme tests with a multiscan photometer to measure viral nucleocapside and glycoprotein, as well as a spectrophotometer that can register ultraviolet light.

2.3.6 It is worth acquiring a CO2 incubator and an ultracentrifuge for sedimentation of rabies virus and for saccharose gradient separation in the interests of qualitative analysis of the viral antigen obtained.

2.3.7 Finally, sufficient space must be reserved in the sample room for samples taken at different stages of production. Vertical cabinets with drawers are the best for this purpose. The sample room should contain at least three cabinets: 1 at +4"C, 1 at -30°C and 1 at -80°C.

Samples of rabies virus before inactivation are preserved at -80°C; the inactivated virus and the final adjuvanted vaccine are kept at +4"C.

2.4. STAFF

2.4.1 Staffing levels are determined by the total volume of controls to be performed.

2.4.2 The person responsible for these controls should be a scientist with a degree and considerable experience in quality control of biological products.

2.4.3 This person should have two assistants, also scientists with experience in the usual control techniques employed in bacteriology, virology, chemistry and biochemistry. At least one of them should have done some work in a rabies vaccine control laboratory.

2.4.4 The specific controls of rabies vaccine can be conducted by two part-time technicians. One must have training in analysis techniques (both chemical and biochemical), the other in cell and virus cultures.

2.4.5 These staff members should be vaccinated against rabies and serologically tested at regular intervals (antibody titre of at least 0.5 IU per ml of serum).


CHAPTER 3. CENTRAL SERVICES

3.1 INTRODUCTION

These services offer facilities for every unit in the production centre. They are therefore of great importance. Their design, installation and equipment must be carefully studied and selected, since faults and shortcominge jeopardize the objectives. These services are:

a central washing plant;

a media preparation service;

a distribution and packaging service that stores and distributes the vaccines;

a central store;

the general administration;

the essential/common services. This is the heart of the unit, which sees to the running not only of the production and control units but also of the rest of the central sesvices.

3.2 CENTRAL WASHING PLANT

3.2.1 Specifications

3.2.la It is important to plan for space that is big enough for the washing and sterilization of equipment from the production and control units. This area should include space for equipment waiting to be washed, and space for storage after sterilization. A rule of thumb is that the total area of a washing plant should be approximately a fifth of that of the laboratories served. Thus on the production site illus

5 rated on plan no. 1, page 39, the planned surface area of the washing plant is

390 m , one fifth of the surface of the buildings located under the central hangar.

3.2.lb This approximation will have to be amended in accordance with the volume of equipment passing through the washing plant.

3.2.1~ The washing plant (plan no. 4, page 48) consists of four main parts:

a room for reception of soiled equipment;

a soaking and washing room;

an assembly and sterilization room;

a room for storage of clean equipment.

Each of these rooms must be big enough to allow the trolleys and vehicles carrying equipment to circulate freely.

3.2.2 Rauinment

3.2.2a The washing plant is supplied with electricity, steam, deionized water, and if necessary gas, by the general services. It also needs distilled water. It is therefore often worth equipping it with single or double distillation apparatus, so that water can be used directly from the machines or stored in stainless steel containers at a temperature of 80°C.


3.2.2b The washing room is equipped with tubs either made of stainless steel or lined with inert, heat-resistant plastic. Some are used for soaking the equipment in detergent, others for scrubbing and rinsing with demineralized water and distilled water. The room also has automatic pipette washers.

An automatic washing machine with several programmes might be considered, when there is a large quantity of equipment including bulky objects such as large glass bottles and stainless steel drums, especially in countries where labour costs are high.

3.2.2~ The sterilization room contains a large rack for preparation of the washed equipment before sterilization, which involves several operations: putting the pipettes in cylinders, putting stoppers in boxes, wrapping bottle seals in aluminium foil, assembling of tubing and connecting systems, assembling of filters etc.

Sterilization is then done either by autoclave (drums, flexible tubes, stoppers, filters etc.) or by dry heat in an electric oven (glassware). Two autoclaves of different sizes offer greater flexibility. The autoclaves and the oven have the same specifications as those in the building for production of rabies vaccine (paragraph 1.4.1.a).

3.2.2d Special notes on washing of equipment from rabies vaccine unit.

The equipment that has been used for cell and virus cultures is decontaminated in an autoclave before being sent to the washing plant. Proteins and cell debris coaguate on the walls of vessels which have been autoclaved. Therefore, they must be soaked in tubs containing an appropriate detergent solution (such as 7X) or be treated with trypsine solution before they can be properly washed. Disposable plastic bottles for cultures (see list of equipment and raw materials pages 38-48), offer the best material for cells to anchor on and considerably reduce the quantity of equipment passing through the washing plant in the process of vaccine production.

For cells to grow and multiply in vitro and survive in good physiological condition, it is essential that the equipment used is perfectly clean. After washing, the glassware should therefore be rinsed once with an acid solution, several times with abundant, deionized water, and finally with distilled or double distilled water.

Excellentresults are produced by machines that are purpose built for washing and rinsing; these range in size from laboratory to industrial models.

3.2.3 Staff

The team leader organizes and plans the work in the light of requests from other units, and supervises operations;

The size of the team working under the leader is dictated by the workload, which divides into several occupations: reception of equipment, soaking and washing, sterilization, assembly and packaging.

It is always good to have staff capable of doing more than one job, and it is especially useful to have at least two people who are well-versed and practised in use of the machines (washing machines, autoclaves, ovens, etc.).

3.3. MEDIA PREPARATION UNIT

3.3.1 Snecifications

3.3.la The media preparation unit is considerably smallyer than the washing plant,

WHQ/Rabies/90.201 Rev.1 page 18

and can be housed in one of-the "standard" buildings of the-production and control centre. In the arrangement shown in plan no. 1 on page 39, it is part of the extension of the viral vaccine unit,' near the central washing plant.

3.3.lb The premises for preparation of cultures consist of several rooms (see plan no. 5, Media preparation unit, page 49):

an office:

a store room for raw materials;

a cold room;

an incubation room (temperature 37°C + 0.5");

a room for weighing, mixing and preparation of solutions;

a room for filtration and filling.

The filtration room is a protected area (shaded in plan no. 5, page 49). It has a supply of filtered air (through absolute filters) and is kept at higher than atmospheric pressure. Staff members gain access through an airlock, where they don protective clothing.

The store room contains all raw materials except those that must be kept at a' temperature between +2" and +8"C. The raw materials that can be checked must be given a clearly visible label to show that they have passed inspection.

The cold room is for the storage both of heat sensitive raw materials and media ready for use.

The incubation room is for incubation of filtered media before samples are taken for inspection.

3.3.1~ Media that are to be heat-sterilized are autoclaved at the central washing plant.

3.3.ld Culture media to be used in preparation of rabies vaccine on cell cultures should if possible be sent unfiltered to the production building, where the sterilization is carried out.

3.3.2 Rauioment

The main items of equipment are:

1 refrigerator, temperature +2' to +8'C, capacity 200 litres;

1 freezer, temperature -15" to -2O"C, capacity 200 litres;

several filtration units, with supports, for stainless steel, flat disc filters 293 mm and 142 mm in diameter, with joints and connectors;

several stainless steel, pressurized filtration canisters, capacity 2 litres and 20 litres;

magnetic stirrers;

1 water bath for heating to between 20' and 6O'C, thermostated, capacity 30 litres;

,-


I-.

heating vats, capacity 40 litres and 120 litres;

2 pH metres (accuracy of + 0.05 pH);

1 stainless steel mixing vat (capacity 150 litres) with turbine;

balances of various capacities (10 kg, 200 g and one precision balance (accuracy of + l/10 mg);

1 peristaltic pump;

2 trolleys.

In the sterile area where filtration is conducted:

a horizontal laminar airflow cabinet for 2 workers;

apparatus with a metered pump for distribution of filtered media.

3.3.3 Staff

3.3.3a The number of people preparing media can be halved if commercially available powdered media are used; these contain most of the relevant ingredients and have the advantage of constant quality.

3.3.3b This unit must be run by a scientist who is experienced in large-scale preparation of media.

3.3.3~ The subordinate technicians must be specially trained, in situ if possible,

A rule of thumb for calculating the number of interchangeable staff required for the various posts is one person for every 2000 litres of media and reagents to be produced each year.

3.4 FILLING AND PACKAGING UNIT

3.4.1 Introduction

This unit fills all the inactivated vaccines produced on the site in containers for distribution. Live vaccines should be packed at the end of the production line or in a separate room within the unit.

The filling unit must ensure that the products are free of contamination from within or without; the environment must therefore be sterile and protected.

3.4.2 Soecifications of the buildinz

3.4.2a The general specifications of this building are the same as for building lE, for production of rabies vaccine (see Chapter 1, paragraph 1.3.2).

3.4.2b The sterile area is supplied with filtered and conditioned air. It must be airtight and kept at higher than atmospheric pressure.

As in the rabies building, the area is entered through double airlocks (for men an women) where the staff dons protective clothing (ready for them in sterile wrapping).

All surfaces must be smooth, dust free and easily disinfected.


Apparatus for disinfection with formalin vapour must be available for separate treatment of each room.

The rooms are supplied with electricity, gas, oxygen and compressed air.

3.4.2~ The building has neither washing plant nor sterilization equipment, since it is near the central washing plant, which provides sterile material in air-tight containers

3.4.3 Rooms and Annexes

3.4.3a The plan of the filling and packaging unit (plan no. 6, page 50) shows the lay-out of the filling and packaging rooms within the sterile area, where access to the rooms is from a side corridor.

3.4.38 In the same building, on either side of the sterile area, are:

At the entrance, an office and a cold room for products in bulk awaiting packaging, and access to the airlock into the sterile area;

At the air-locked exit from the sterile area, a room where the final products are packed for delivery.

3.4.3~ On one side of that room is a cold store room, and on the other is a loading bay for the finished products.

3.4.3d The size of the premises should be determined by the total volume of products to be packaged. In dealing with rabies vaccine for veterinary use, 500 000 doses per annum are made from a series of 20 batches of 25 litres each and presented in the form of individual, 1 ml vials, or multidose bottles (the latter should be used & in mass vaccination campaigns). At times 2 ml doses are preferred in order to minimize loss of vaccine in use, and in such cases the quantities of vaccine to be packaged are doubled.

3.4.3e Rooms in the sterile area

If there is a central packaging unit, at least two rooms should be provided so that different vaccines can be bottled at the same time. For safety's sake, the entrance to these rooms should be a double-doored airlock.

Beyond the bottling rooms is an inspection room. Vials and bottles containing cloudy suspensions in liquid such as rabies vaccine are inspected visually. Only products in transparent solution should be inspected by sorting machines, which are expensive.

The labelling and packaging room is the last room in the sterile area. The work can be done either by hand or by simple or sophisticated machinery. The number of operations to be conducted in this room can be reduced if the filling rooms have machinery that can also label the recipients.

3.4.3f There must be space in the packaging room for the storage of boxes, cartons or crates in which the vaccine is delivered.

3.4.4 gauinment

3.4.4a Bottling rooms

These rooms contain machinery adapted to the type of containers to be filled, the type of sealing to be used, the unit volumes to be prepared as well as to the working conditions.


The supply of sterile air to those rooms should enable vaccines to be bottled in complete asepsis. Sterile air, once it has passed through absolute filters, can therefore be circulated in the rooms through conventional vents, although a vertical laminar air "tent" above and around the bottling machine is a more effective method. Another less complicated method is that of placing the machines under Biohazard airflow cabinets placed on rollers and equipped with removable lateral flaps.

The bottling room should also contain small pieces of equipment, eg. magnetic shakers, double-skinned containers for the refrigeration of heat sensitive vaccines, et

3.4.4b Optical inspection room:

This contains a magnifying viewer and an automatic sorting machine if necessary.

3.4.4~ Packaging room:

This contains equipment for hand-labelling, and perhaps for automatic labelling also.

-. 3.4.4d Packing room:

If the packing is done by hand, a large table is sufficient. The cold room next door should have separate sets of shelves, coded for easy identification, so that stocks of different products are not mixed up.

3.4.4e Trolleys must be available: at least one at the entrance, two in the sterile area and one in the packaging and despatch area.

3.4.5 Staff

The number of staff employed depends on the annual volume of work. In a small team, employees should preferably be able to do more than one of the jobs involved.

A team leader supervises all activities and monitors stocks of vaccine. It is good if the team leader or another employee of the unit is able to make routine adjustments to the machinery.

3.5 OTHER GENERAL SERVICES

3.5.1 Uenot

Premises that are separate (as in plan no. 1 page 39) or incorporated in one of the buildings must be set aside for reception and storage of raw materials, small equipment, and the main spare parts needed for repair and maintenance of heavy plant.

Arrangements must also be made for keeping some products at low temperature.

Un-inspected goods must be kept separate from goods passed by the inspection unit of the facility and labelled with inspection codes and numbers.

A facility producing several types of vaccine should have at least two people to manage the depot.

3.5.2 Administration

The administration building should be near the site entrance if possible, away from unpleasant smells or noises.


It should have enough offices for the management, the secretariat, the accounts and archives departments. The archives should include all records of the main data and results in accordance with production and inspection protocols. A record should be kept for each batch of every vaccine manufactured. Standard records for rabies vaccine for veterinary use are presented in WHO Technical Report Series, No. 658, 1981, Annex 3.

The administration facilities should also include a meeting room that could double as library, and a medical service containing an infirmary equipped for emergencies and a surgery for physicians' visits.

Finally, if there are no external food services, a canteen should be provided, since the basic rules of hygiene and safety forbid food in the laboratories and annexes.

3.5.3 Essential services

3.5.3a This is the heart of the establishment, providing the crucial supplies of energy, water, ventilated air, heat, cold, etc., as well as equipment maintenance and emergency repairs.

3.5.3b A detailed study of the requirements of the essential services must, be made as soon as the production targets are known. It is recommended that capacity be increased immediately if increased production or the creation of new units is planned in the short- or medium-term.

In view of the special skills involved,, these needs should be estimated, the machinery and equipment chosen and the plans for the centre and distribution networks drawn up in close collaboration with specialist engineers for each of those areas.

3.5.3~ The main activities of this department fall under the following headings:

Electricitv surely. This usually comes from an external, high-tension power line; there is a transformer at the entrance to the site. Power is transmitted to the other buildings from the central control unit of the central building of essential services. In each building power lines for all rooms emanate from protected cupboards with triple-phased current. Appropriate safety devices must be installed in each one.

The service must be prepared to cope with any power failure. The essential services building should contain several generators with enough power to supply the incubation rooms, cold rooms, freezers, culture apparatus and animal houses. The back-up generators require a nearby oil.supply.

Steam SUDD~Y. A group of boilers in the building provides the necessary steam especially for sterilization of equipment (autoclaves, or in situ fermenters).

Water SUDD~V. Ordinary and deionized water, the latter produced through ion-exchangers, should also be supplied by the'main building of the essential services.

Ventilation. Because of the complexity of the ventilation equipment, only specialist technicians and engineers are able to recommend the most appropriate systems according to climatic conditions, volume of the premises and special features of the various laboratories, including the sterile areas that must be supplied with air filtered through independent circuits.

Waste disposal. Waste water drains to a sewage, and solid wastes are incinerated.


3.5.3d The staff of the essential services unit carries out the above tasks and maintains the plant. The technicians are also responsible for emergency repairs and for manufacturing certain light apparatus and various tools. The unit therefore needs workshops (number 10 on the plan for a biologicals production unit - plan no. 1, page 39).

The head of unit must be a highly qualified and experienced engineer, assisted by one or more maintenance engineers.

Essential services also need a team consisting of one or more electricians, electro-technicians, mechanics, heating engineers, refrigeration engineers, plumbers and carpenters.

They should be trained in fire-fighting techniques, at the work place if possible.

.-


CHAPTER 4: ANIMAL HOUSES

4.1 INTRODUCTION

Control tests performed on animals come under the central control unit (see 2.1.6).

However, these tests are conducted in animal houses that must be at a physical distance from the laboratories, in a separate building if possible, away from the production and control centre, (blocks 14a and 14b in the overall plan - plan no. 1 - on page 39).

The animal handlers should not enter the laboratories.

4.2 CENTRAL ANIMAL HOUSES FOR SMALL LABORATORY ANIMALS (see eg, plan no. 7, page 51)

4.2.1 The control tests call for the use of mice, guinea pigs, rabbits and other species which are either bought as needs arise or bred at the centre; numbers and species are determined by the vaccines produced. The choice is governed by local resources and by cost.

4.2.2 For each species of animal, separate rooms should be devoted to (a) breeding (if necessary), (b) storage of animals prior to use and (c) observation of animals under inspection.

4.2.3 The animal houses should be ventilated so as to renew the air 10 to 15 times per hour, keeping relative humidity between 40% and 65% and atmospheric temperature between 18" and 24°C.

4.2.4 There should also be an annex large enough for washing and sterilization of soiled equipment, and for storage of clean equipment, feed and bedding.

4.2.5 Guidelines on design, equipping and running of such anima'l ho'uses are to be found in WHO Manual BLG/UNDP/78.1, on production and control of bacterial vaccines.

4.3 ANIMAL HOUSES FOR TESTING OF RABIES VACCINE FOR VETERINARY USE

4.3.1 Tests on animals

4.3.la The rabies vaccine tests are described in WHO Technical Report Series, No. 658, 1981; they include:

a test for the absence of abnormal toxicity, on mice and guinea pigs;

tests for safety (inactivation and innocuity) on mice;

a potency test on mice using the NIH test, by vaccination followed by challenge with rabies virus.

4.3.lb The same technique will be used for stability tests (WHO Technical Report Series, No. 790, 1983, paragraph 5.4).

4.3.1~ It is also necessary to evaluate the immunogenicity of the vaccine on the target species - the dog - by vaccination followed by blood testing to ascertain titres of specific antibodies. It is also important to estimate the duration of protection conferred by a newly manufactured vaccine on the first batches produced. For this purpose, vaccinated dogs and control animals must be kept for one, two and even three years before they are challenged with a potent wild virus strain.


4.3.2. Specifications of the animal houses used for testing of rabies vaccine

4.3.2a In view of the risk of contamination with rabies vaccine, the only test that can be conducted in the central animal house is the test for abnormal toxicity of the vaccine produced.

4.3.2b All tests involving inoculation of the virus-vaccine before inactivation, and/or challenge strains must be performed in separate parts of the central animal house used for that purpose alone (see plan no. 1 of a centre for biologicals production, page 39).

4.3.2~ Two types of animal house are needed for testing of rabies vaccine for veterinary use: housing for mice and kennels.

4.3.2d The building and running of kennels equipped for performing challenges with a wild strain of rabies vaccine puts a heavy burden on the production process. Tests on dogs in general are only conducted with a few batches of vaccine, at the start of production; such tests show what protection is given to the species concerned by the vaccine, which is produced in accordance with a finalized manufacturing protocol. Each country should therefore explore the possibility of conducting such tests in a national or foreign establishment equipped with an isolation animal house that has the proper safeguards and accepts the responsibility (e.g. a regional centre for rabies studies).

4.3.2e If testing on dogs is restricted to vaccination and testing of blood samples for titres of antibodies, a conventional kennel for 15 to 20 dogs is sufficient. The animals can be placed in cages in a closed room or in niches opening on to a cemented area surrounded by wire mesh. Adjacent o the kennels are a store room for equipment and food and a room of approximately 8 m 5 with a table for restraining animals while blood samples are taken.

4.3.2f If, on the other hand, a kennel for performing challenges on dogs is needed, it must be installed in an enclosed building, as an isolated area in airtight conditions similar to those in the building where rabies vaccine is produced from cell cultures. For practical reasons it is worth installing the animal house for tests on mice in the same building, in a second area separate from the kennels, without the same degree of protection (unless a wild strain of virus is being used). Plan no. 8 on page 52 shows one arrangement for such facilities in an animal house for the testing of rabies vaccine on mice and dogs.

To increase security, the building itself is surrounded by a wall some 4 m in height, with two sliding doors for an entrance. It must be designed to prevent "wild" animals (especially small rodents) from going in.

4.3.3 Housine for mice

4.3.3a This comprises:

an entrance corridor with airlock where the staff put on working clothes and shoes. New mice, equipment and feed are brought in through the same airlock;

a storeroom for feed and bedding;

two rooms where mice are kept on shelves in boxes of 5 and 10, both with access to an inoculation box. The first room is for vaccinations, the second for challenges. There is no place to keep mice in readiness: they must be brought in just before they are used;


at the exit an airlock for decontamination, using formalin or another active process, where discarded bedding, waste or dead animals are kept in bags before incineration. Equipment is immersed in an antiseptic solution of formalin or bleach before being sent to the washing plant.

4.3.3b To test the annual production of the 20 batches of vaccine assumed in these Instructions (and irrespective of the size of the batches), the animal house should be able to accommodate 300 mice in 60 cages at any one time. Cages consisting of a plastic container with a stainless steel mesh lid, feed tray and drinking water, that can be sterilized in an autoclave (see figure 9, page 53) are easiest to use.

4.3.3~ The animal house should be ventilated by maintaining higher than atmospheric air pressure, the outgoing air being decontaminated by passage through a conduit where the temperature is raised to 180°C (or any other active decontamination process.

4.3.4 Isolation kennel for vaccination followed by challenge

4.3.4a The kennels and annexes must be strictly isolated. It takes the form of a protected animal house in plan no. 8, page 52. It is airtight, and ventilation is adjusted to produce negative pressure.

Extracted air must be sterilized by burning in a conduit at 180°C.

Used water (especially water used for washing the kennel) must pass through a decontamination vat before expulsion.

These safety measures are justified by the high virulence of challenge strains of the virus used for challenge.

4.3.4b The kennels comprise:

an airlock entrance for staff with shower and room for dressing in full protective clothing;

an airlock entrance for clean equipment, food, and experimental animals;

the animal house, with a central corridor and floors that slope to drain off the water used for cleaning, and several pressurized water taps. The dogs are kept on either side of the corridor in individual kennels of metal (see figures 10 and 11, page 54);

the central passageway with openings: one laboratory equipped with a biohazard vertical airflow cabinet for one person, who prepares the challenge specimens of rabies virus; one room with a table on which the dogs are restrained during inoculation and taking of blood samples; a spare room for equipment and food;

- a room at the exit with a double-entry autoclave (capacity 1 to 2 m3) for decontamination of equipment and clothing which is also used for the sterilization of solid wastes and dead animals which arrive in plastic bin bags.

4.3.4~ Outside the kennels, at the outer door of the autoclave, sterilized products are taken through a special airlock where the outside of the wrappings are decontaminated; this airlock is also used for the mouse housing. Once they have been taken outside the surrounding wall, the disposal bags and the dead animals are incinerated, and the equipment is sent to the washing plant.

WRO/Rabies/90.201 Rev.1 page 27

4.3.5 Staff

4.3.5a Staff must be vaccinated against rabies and their levels of specific neutralizing antibodies should be checked every 8 to 12 months.

4.3.5b If the mice and dogs are kept in the same area, the unit can be run by two people.

4.3.5~ At least one of the handlers should have completed a training session in an animal testing centre, preferably in a unit that tests for or studies canine rabies.

,-.

WRO/Rabies/90.201 Rev.1 page 28

ANNEX1

List of eauinment and materials used in Droduction and control of rabies vaccine for veterinarv use

This list features only the main plant and equipment, and the most commonly used supplies.

Some names of manufacturers or suppliers are provided. There are, of course, other suppliers and alternative products. The listing does not necessarily imply special recommendation by WHO.

Prices, where available, are quoted in French Francs

. ../...


A: EOUIPMENT

ITEM SUPPLIER APPROXIMATE UNIT PRICE

(1986 - less tax) in French francs

-\

1. Sterilization

- Autoclaves (l-2 m3) with automated programmes

AMSCO, USA' OLSA, Italy LEQUEUX, France

- Pasteur ovens LEQUEUX, France HERAEUS GmbH, Germany

2. Washing

- Laboratory washing machines

HAM0 by Flobio France - distributors GALIAY, Switzerland

Industrial washing machines BETTER BUILT Machinery Corp., USA

- Machines for washing virus ampoules or bottles

ARENCO GmbH, Germany STRUNCK & Co., Germany STRUERS, Denmark

- Machines for washing BETTER BUILT Machinery animal cages Corp., USA

3. Water Droduction

- Separate bed deionizers (100 1. per hour)

- Distillation apparatus

- "Milli Q" Pure water generators (100 1. per hour)

WATCO, France

OLSA, Italy

MILLIPORE CORPORATION, USA

4. Filling

- Filling machines ARENCO GmbH, Germany STRUNCK & Co., Germany STRUERS, Denmark

- Printing machines HAPA A.G.

- Perifill sequential metering pumps

BIOBLOCK Scientific, France 18 000

300 000

100 000

25 000

100 000

50 000

-


A: EOUIPMENT (cont.)



5. Refrigeration

- Liquid nitrogen containers for storing seeds, 40 1. capacity (for 180 vials of 2 ml)

- Freezers -30°C (300 1.)

- Freezers -85°C (380 1.)

- Crushed ice machines INCO-ZIEGRA, Germany

- Refrigerators +2 - +8"C (200 1.)

FRIGIDAIRE KELVINATOR (Flobio France - distributors)

6. Heating

- Laboratory incubators (110 1.)

- CO2 incubators

- Thermostatic baths (20 1.) and thermostat

- Double-hulled stainless steel tanks with heat exchangers

7. Laminar airflow cabinets

BIOHAZARD (0.3~) with HEPA absolute filters (UV, recycled air and pull-down shutter)

Societe AIR LIQUIDE, France BIOBU>CK Scientific, France

6 000

FRIGIDAIRE KELVINATOR (Flobio France - distributors)

4 000

FROID-LAB0 - REVCO (Flobio France - distributors)

50 000

15 000

4 000

ASSAB (Flobio France - distributors)

6 000

38 000

BIOBLOCK Scientific, France ROUCAIRE, France

5 000

ROUCAIRE, France 25 000 to 35 000

FLOW G., Luc. Company, USA GEIMAN Instrument, Italy E.S.I., France A.D.S., France N.E.U., France

30 000 (1 place) 40 000 (2 places)

100 000 (3 places)

I -.

,T--

WHO/Rabies/90.201 Rev;1 page 31

ITEM


APPROXIMATE UNIT PRICE SUPPLIER (1986 - less tax)

in French francs

8. Filtration

- Flat filters with membranes SARTORIUS, France and accessories 142 mm diameter and 293 mm diameter

SARTORIUS:WERKE, Germany MILLIPORE, France

- Filter holders with accessori s

5 for cartridges

of 0.75 m

PALL, France

- Device for checking SARTORIUS, France membranes SARTORIUS-WERKE, Germany

- Pressurized filtration canisters

20 1. 5 1.

SARTORIUS, France SARTORIUS-WERKE, Germany

9. Concentration

Ultrafiltration device with packs of membranes: cut-off point - 100 000

Variable speed metering PumP

MILLIPORE, France 80 000

BIOBLOCK Scientific, France

10. Mixing

Stainless steel mixing vat (200 1.) + turbine

LABO. MODERNE, France

11. Balances

- Universal balances, METTLER, Switzerland (3-4 kg, accuracy + 0.1 g) SARTORIUS, France

- Analytical balances SARTORIUS-WERKE, Germany (200 g, accuracy & 0.1 mg)

- DYANA industrial balances (150 kg, accuracy of + 1 g)

15 000 (142 mm) 20 000 (293 mm)

30 000

15 000

10 000 8 000

5 000

12 000

7 000

18 000

7 000





12. Centrifuges

- Refrigerated centrifuges SIGMA, 6000 4x300 rpm, ml 35 000 6000 6 x 1 rpm, 1. 70 000

- Ultracentrifuge BECKMAN Instrument Inc., USA 130 000

13. Cell and virus cultures

- Basic roller system BELLCO Equipment System, USA 25 000 (10 x 2 litre bottles)

- Extra stages BELLCO Equipment System, USA

- Glass bottles BELLCO Equipment System, USA

14. MicroscoDes

- Binoculars with 3 lenses

- Normal or inverted stand

- Equipment for fluorescence

OLYMPUS, France

ZEISS, Germany

ZEISS, Germany

8 000 to 12 000

20 000

15. pH metres

- Traditional digital pH metre

BECKMAN Instrument Corp., USA 3 500

- pocket pH meter BIOBLOCK Scientific, France 1 300

16. Immunoenzvme tests

- Titertek equipment with FLOW Laboratory, USA multiscan photometer and printer

50 000





17. Homoeenizers

- Airtight vibration system CHENAP A.d., Switzerland 10 1. capacity

18. Animal caees

- Mice

- Dogs IFFA-CREDO, France


19. Miscellaneous Light eauioment

Such equipment is made by many manufacturers and can be obtained from the main suppliers of laboratory equipment.

- Magnetic stirrers

- Vortex stirrers

- Microplate stirrers - Masterflex transfer peristaltic pumps

- 30-litre stainless steel drums (beer barrel type)

- Meker burners

- Hemostatic clamps

- Screw-on clamps

- Countering cells

- Electrically operated pipettes

- Eppendorf micropipettes

20. Glassware

Main Suppliers: CORNING GLASS WORKS, USA JENAIR GLASSWERK SCHOTT U.G. FRG

Pyrex quality borosilicate glass

- Traditional cylindrical bottles, (capacity from 2 ml to 500 ml)

- Graduated cylindrical bottles of (capacity of 1, 2, 5, 10 and 15-litres)

- Various test tubes, pipettes etc.

21. Autoclavable elastics

- Cylindrical bottles, test tubes, funnels etc.

- Polycarbonate and polypropylene bottles with airtight screw-on lids for storage at -85°C

- Airtight screw top tubes for samples (5, 10, 20 ml)

- Rhodorsil silicon stoppers of various sizes (24 to 61 mm diameter)

- Grooved polypropylene tubing, I, T and Y shaped

- Flexible silicon tubing in several diameters

22. Disoosable nlastics

- Special airtight tubes for storing the seed immersed in liquid nitrogen

- Pipettes in sterile wrapping (l-10'ml)

- Sterile wrapped cones for Eppendorf pipettes

- 96-hole microplates with lid (sterile) - Disposable syringes (sterile, with needles of 1, 2 and 5 ml)

WHOjRabies/90.201 Rev.1 page 35

22. Disnosable nlastics (cont.)

- Vacutainers for blood samples

- Rhodonsil flexible pipe (8 x 12 and 8 x 14 diameter)

- Sterile latex surgical gloves

- "Pneumofilter" disposable air filters with grooved

- Autoclavable bin bags

- Bottles for cell cultures ("TC" specially treated)

Main suppliers:

FALCON CORNING GREINER

nozzles, sterile

- Flat bottles of 25, 75 and 150 cm2 surface area

- Bottles for rollers, 850 cm2 surface area (bulk price between 30 and 40 French francs)

23. Protective clothing

Societe ADS, France

- Overalls (plastified, non-flammable)

- Shoes and boots

- Hoods, masks, caps

- Goggles

(Cost approximately 2000 French francs for each suit)


B: MEDIA AND MAIN RAW MATERIALS

1. Virus and cell culture media

Manufacturer: FLOW or GIBCO

Ouantitv ner batch Ouantitv in 20 Annroximate total nrice in French Francs

(1986)

MEM medium powder to make 50 litres 100.0 1. 2 000 1. 24 000

Calf foetus serum 3.5 1. 70 1. 56 000

Newborn calf serum 3.5 1. 70 1. 14 000

FV bovine albumin serum (powder) 75.0 g 1 500 g 15 000

Tryptose Ph. broth 5.0 1. 100 1. 5 000

2. Trvosin

l/250

Sunnliers: FLOW-DIPCO-GIBCO, etc.

Soluble in PBS without Ca-Mg 15 g. 300 g. 6 000

WHO/Eabies/90.201 Rev.1 page 37

3. Additives: amino acids. sugars. mineral salts etc.

Manufacturer: MERE

Annual reauirements

Cuantitv oer batch Quantitv in 20

Tricine 2 kg 40 kg

L-glutamine 500 g 10 kg

L-histidine 50 g 1 kg L-alanine 25 g 500 g

Glycine 100 g 2 kg Saccharose 150 kg

Glucose 2 kg Sorbitol 4 kg Tween 80 1 1.

Tween 20 1 1.

Sodium bicarbonate 5 kg Calcium chloride 1 kg Magnesium sulfate 500 g

Magnesium chloride 500 g

Monosodium phosphate 1 kg Disodium phosphate 500 g

Sodium chloride 20 kg

Trisodium citrate 2 kg

300 ml

4.,Virus inactivation aeent

Manufacturer: FLUKA

- Betapropiolactone 65 ml (to be kept at -20°C)

5. Adluvant

Manufacturer: SUPFXFOS Speciality Chemicals, Denmark

- Alhydrogel 3.5 1. 70 1.

ADDrOXhEite

total nrice in French Franca

(1986)

approx.

16 000

13 000

3 000


6. Miscellaneous

- 7 x detergent for soaking containers (FLOW)

- 90" alcohol

- Caustic soda

- Hydrochloric acid

- 40% formalin

- Ammonia etc.

7. Reagents for immunoenzvme and immunofluorescence tests

- Oxygenated water

- 0-phenylendiamine

- Antibodies conjugated with peroxydase

7

Manufacturer: - Reference antigens -

Institut Pasteur, Paris, France - Fluorescent antinucleocapside antibodies

Approximate cost of all media and reagents used in a year is 170 000 French francs for 500 000 doses, or 0.32 French francs per dose (1986).

c: BUILDINGS

1. Makers and installers of nrefabricated buildines

- St& TRANSLCXO, France

- Ste IFFA, France

2. Builders and installers of ventilation eauinment

- Ste ESI, France

*********

WJJO/Rabies/90.201 Rev.1 page 39

Plan No. 1 PLAN FOR A BIOLOGICAL PRODUCTIONS FACILITY

(Housed in a hangar)

,

1 = Control laboratory 2 and 3 = Bacterial vaccines 4 = Central washing plant 5 = Media preparation 6 = Viral vaccines (except

rabies) 7 = Rabies vaccine for

veterinary use 8 = Bottling and packaging 9 = Storage of finished

products and dispatch IO = Workshops 11 = Essential common

services 12 = General store 13 = Incinerator 14 = Rabies animal compounds

(a) mice (b) kennels

15 = Washing plant and supplies for animal house

16 = Animal house (mice: rabbits, guinea pigs)

17 = Administrative building 18 and 19 = Watchmen’s iodgings 20 = High-tension line and

transformer

11

r-----d I /!a / !i-- r: 1 I

Exit Entrance

Shaded areas = buildings under hangar l Dimensions are given in metres WHO 91246


FIGURE 1

Placement of a modular laboratory


FIGURES 2 & 3

Example of structure assembled using prefabricated panels


FIGURES 4 & 5

Examples of modular laboratories


-.

SPECIFICATIONS OF A MODULE (to house any type of animal house equipment)

Technical snecifications

External dimensions (in metres):

length: 13.5 breadth: 3 height: 2.885

Internal dimensions (in metres):

length: 12.410 breadth: 2.910 height: 2.5 (at lowest)

Panels 45 mm thick in polyester/glass fibre/polyurethane foam Fittings for the installation of equipment Self-supporting floor 2 m airlock entrance with benches, wash basin, water heater, serving:

a) one room for standard A module b) two rooms for standard B module

Day/night lighting controlled by timer

Air conditioning:

5 micron filtration Superpressure 40% new air (standard A) 100% new air (standard B)

Maintenance of 22"C, 50% relative humidity (RR) by:

a) 10°C and 80% RH in winter b) 32°C and 40% RH in summer c) With possible animal input of 1200 Kcal.

Safeguarded ventilation, high temperature, low temperature, refrigeration circuit, electrical heating elements.

Very rapid installation.

* Example from IFFA-CREDO, France


FIGURES 6 & 7

Protection of Personnel in sterile areas eg, during distribution of media into bottles


Plan No. 2

PLAN FOR A FACILITY FOR THE PRODUCTION OF RABIES VACCINE FOR VETERINARY USE l

Entrance

Production procedure: cultures in roller tubes (system: BHK cells infected with PASTEUR PV/BHK rabies virus)

Annual production capacity: 400 000 to 600 000 doses.

Area 1

1 = Entrance hall v = Dressing rooms 2 = Room for storage in liquid nitrogen 3 = Room for storage in freezers (- 80°C) 4 = Office 5 = Toilets 6 = Store 6.1 = Dry ice cabinets 6.2 = Crushed ice cabinets 7 = Assembly room and sterilizers Al = sterilization autoclave F = sterilization oven

Area 2

SI, S2 = Staff entrance airlock SMl = Airlock for media containers 8 = Room for storage of sterile equipment 9 = Media filtration room CEl = Media incubation room CFI = Media cold room 10 = Cell culture room CE2 = Cell culture incubator r = Sliding doors 11 = Room for transfer of inactivated

virus vaccine SM3 = Transfer airlock 12 = Room for addition of adjuvant CF2 = Cold room for bulk vaccine Hl, H2, H3, H4 = Laminar airflow cabinets EP = Pure water production

Zone 3

S3 - S4 = Staff entry airlock SM2 = Equipment entry airlock A2 = Autoclave for decontamination of

outgoing equipment 13 = Viral culture room CE3 = Incubator for viral culture CF3 = Cold room for storage of

virulent harvests r = Roller doors H5 = Laminar airflow cabinet

Outside the building:

LTI and LT2 = Maintenance centre and controls

l Dimensions given in metres. WHO 91247


FIGURE 8

Set of rollers on a Bellco unit


Plan No. 3

INSPECTION UNIT l

* Dimensions given in metres

Entrance

1 = Office 2 = Cloakrooms and toilets 3 = Decontamination room 4 = Cell culture room 5 and 6 = Viral vaccine control rooms 7 = Cold room 8 = Biochemistry cold room 9 = Room for physical, chemical and

biochemical analyses

IO = Room for physical, chemical and biochemical analyses

11 = Incubation room (37”)C 12 = Incubation room (30” to 35°C) 13 and 14 = Bacterial vaccine control rooms SI, s2 = Airlock with staff changing room E = Sample library H = Laminar airflow cabinets

WHO 91248


1 = Office 2 = Products store room 3 = Toilets 4 = Changing rooms

Plan No. 4

CENTRAL WASHING PLANT l

13.00 1 Exit

A

.

Entrance

* Dimensions given in metres

A = Distillation equipment B = Soaking and washing vats C = Washing machines D = Worktop for preparation, packaging and assembly

5 = Room for deposit of dirty equipment 6 = Washing room 7 = Room for preparation, assembly and

sterilization of clean equipment 8 = Store room for sterile equipment

E = Autoclaves and oven

WHO 91249


Plan No. 5

MEDIA PREPARATION UNIT l

ti

. -1

l Dimensions given in metres

1 = Office 6 = Rooms for weighing, mixing etc. 2 = Raw material store 7 = Filtration rooms 3 = Changing rooms and toilets Si = Staff entry airlock 4 = Cold room S2 = Airlock for equipment and media to be filtered 5 = Incubation room (37°C) H = 3-p!ace laminar airflow cabinet

WHO 91250


Plan No. 6

FILLING AND PACKAGING UNIT*

1 = Office

Entrance

8 = Packing room 2 = Cold room for bulk products 3 and 4 -= Filling rooms 5 = Visual inspection room 6 = Labelling and packaging room 7 = Cold room for storage before dispatch

a i-l

7

Shaded area is protected zone


9 = Loading bay (outside) Sl , S2 = Airlocks with staff changing space V = Changing rooms T = Toilets

WHO 91251


Plan No. 7

CENTRAL ANIMAL HOUSE *


I3 = Office Entrance

VT = Changing rooms and toilets ;;, L2 = Rabbits (breeding)

= Rabbits (keeping) SAS 1 and SAS 2 = for men and women in the clean area L4 = Inspection of rabbits IAV = Washing plant Cl, C2 = Guinea pigs (breeding) ML = Area for washing of cages c3 = Guinea pigs (keeping) A = Double-entry autoclave c4 = Inspection of guinea pigs Rl and R2 = Storage rooms for sterile equipment, Sl, S2, S3 = Mice (breeding)

feed and bedding s4 = Mice (keeping) Tl = Clean corridor S5, S6 = Inspection of mice T2 = Dirty corridor WHO 9 1252


Plan No. 8

PLAN OF PROTECTED ANIMAL HOUSE FOR CONTROL.OF RABIES VACCINE FOR VETERINARY USE ON MICE AND DOGS, INCLUDING

CHALLENGE WITH VIRULENT STRAINS OF RABIES *

1 I ! I

‘, I - 1 I E

I

1

!

1

I -

I

I

Area outlined in red = protected kennel, ventilated with negative air pressure

t

3 3 3

E = Wall surrounding the building 1 = Corridor leading to kennels Sl = Airlock, entrance for animals or equipment SP = Airlock, entrance for staff

(with showers and protective clothing) 1 to 20 = Individual kennels Rl = Store room for equipment and dog food LA = Laboratory for preparation of rabies inoculums

(wild strains) for challenges H = BIOHAZARD vertical laminar airflow cabinet


I

11 = Room for inoculation of dogs A = Decontamination autoclave 2 = Corridor leading to housing for mice Cl and C2 = Housing for mice 12 and 13 = Cubicles for inoculation of mice R2 = Spare cages, bedding and feed for mice 52 = Decontamination airlock/exit

(for both kennels and mouse housing) 3 = Sliding doors BU = Office WHO 91253


FIGURE 9

Example of cage for S-10 mice (autoclavable stainless steel mesh lid, feed tray and drinking bottle)


FIGURES 10 & 11

Examples of individual dog cages

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