Hugo W.B., Russel A.D.(ed). Pharmaceutical Microbiology

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this immunity lasts for years but it may need to be reinforced by 'booster' doses of

vaccine given at relatively long intervals.

Immunosera and human immunoglobulins depend for their protective effects on

their content of antibodies derived, in the case of immunosera, from immunized

animals and, in the case of immunoglobulins, from humans who have been immunized

or who have high antibody titres consequent upon prior infection. This form of

immunity, known as passive immunity, is achieved immediately but is limited in its

duration to the time that protective levels of antibodies remain in the circulation: see

also Chapter 16.

A feature that is common to vaccines, immunosera and human immunoglobulins is

the marked specificity of their actions. Each provides immunity to only one infection.

This specificity has led to the development of vaccines and immunosera with several

different components such as are present in the widely used diphtheria/tetanus/pertussis

vaccines that are used to prevent the infectious diseases that commonly afflict infants

and young children.

In addition to the three types of immunological product that are generally available

there are two further types: synthetic peptide vaccines and monoclonal antibodies. Both

have been extensively investigated but neither has, as yet, a place in conventional

prophylaxis or therapeutics.

Principles of immunity were discussed in Chapter 14, whereas Chapter 16 describes

a vaccination and immunization programme.

2 Vaccines

The vaccines currently used for the prevention of the infectious diseases of humans all

originate, albeit in a variety of ways, from pathogenic microbes. The essence of vaccine

manufacture thus consists of procedures which produce from dangerous pathogens,

their components or their products, the immunogens that are devoid of pathogenic

properties but which, nonetheless, retain the property of inducing a protective response

in those to whom they are administered. The methods that are used by vaccine

manufacturers are constrained by costs, by problems of delivery to the vaccinee and,

most of all, by the biological properties of the pathogens from which vaccines are

derived. Even so, the vaccines used in conventional vaccination programmes today are

of only five readily recognizable types.

1 Live vaccines. Live vaccines are preparations of live bacteria or viruses which,

when administered in an appropriate way, cause symptomless or almost symptomless

infections. In the course of such an infection the constituents of the microbes in a

vaccine evoke an immune response which provides protection against a serious natural

disease. Live vaccines have a long history. The very first vaccine, smallpox vaccine,

was a live vaccine. It was introduced in 1796 by the Gloucestershire doctor, Edward

Jenner, who recognized that an attack of the mild condition known as cowpox protected

milkmaids from smallpox during epidemics of this dreaded disease. He therefore took

some fluid, the lymph, from a cowpox pustule on the hand of a milkmaid and used it to

inoculate a small boy. A little later he courageously inoculated the boy with lymph

from a case of smallpox—and nothing happened! The boy, protected as he was by the

cowpox infection, remained well. Jenner's use of the causative organism of one disease

Manufacture and quality control of immunological products 305

to provide protection against another is paralled by the use of the bacille Calmette-

Guerin (BCG) strain of bovine tubercle bacilli to protect against infections with the

human strain. However, in this case, there is the difference that the ability of the BCG

strain to cause disease, its pathogenicity or virulence, has been reduced by many

sequential subcultivations on laboratory media. Live vaccines such as smallpox and

BCG vaccines that rely on the phenomenon of 'cross-protection' are exceptions to the

generality that most vaccines are derived from the causative organisms of the diseases

against which each is intended to provide protection. Thus, a virulent typhoid bacillus

that was enzymically crippled by the action of nitrosoguanosine on its DNA gave rise

to the live typhoid vaccine Ty21a. Likewise polioviruses from human infections were

grown in the laboratory in such a way that it was possible to select infectious but

innocuous progeny viruses suitable for use in live (oral) polio vaccines. Comparable

procedures have been used to obtain the viruses that are currently used in live measles,

mumps, rubella and yellow fever vaccines. The microbes with the reduced ability to

cause disease that are used in live vaccines are said to have attenuated virulence and

are often referred to as attenuated or vaccine strains.

2 Killed vaccines. Killed vaccines are suspensions of bacteria or of viruses that have

been killed by heat or by disinfectants such as phenol or formaldehyde. Killed microbes

do not replicate and cause an infection and so it is necessary that, in each dose of a

killed vaccine, there are sufficient microbes to stimulate a vaccinee's immune system.

Killed vaccines have therefore to be relatively concentrated suspensions. Even so, such

preparations are rather poor antigens and, at the same time, tend to be somewhat toxic.

It is thus necessary to divide the total amount of vaccine that is needed to induce

protection into two or three doses that are given at intervals of a few days or weeks.

Such a course of vaccination takes advantage of the enhanced 'secondary' response

that occurs when a vaccine is administered to a person whose immune system has been

sensitized by a previous dose of the same vaccine. The best known killed vaccines are

whooping-cough (pertussis), typhoid, cholera, Salk type poliovaccine and rabies vaccine.

3 Toxoid vaccines. Toxoid vaccines are preparations derived from the toxins that are

secreted by certain species of bacteria. In the manufacture of such vaccines, the toxin

is separated from the bacteria and treated in a way that eliminates toxicity without

eliminating immunogenicity. Formalin (ca. 38% of formaldehyde gas in water) is used

for this purpose and consequently the treated toxins are often referred to as formol

toxoids. Toxoid vaccines are very effective in the prevention of those diseases such as

diphtheria and tetanus in which the harmful effects of the infecting bacteria are due to

the deleterious action of bacterial toxins on physiology and biochemistry.

4 Bacterial cell component vaccines. Several bacterial vaccines that consist, not of

whole bacterial cells as in conventional whooping-cough vaccine, but of components

of the bacterial cells, are now available. The potential advantage of such vaccines is

that they evoke an immune response only to the component, or components, in the

vaccine and thus induce a response that is more specific and effective. At the same time,

the amount of adventitous material in the vaccine is reduced and with it the likelihood

of adverse reactions. Among the vaccines prepared from cell components are various

acellular whooping-cough (pertussis) vaccines which have either a single component

or several bacterial components. Other vaccines based on bacterial components, in

each case on one or more capsular polysaccharides, are the Haemophilus influenzae

Type B vaccine, the Neisseria meningitidis Type A and C vaccine, the 23-valent

pneumococcal polysaccharide vaccine and an acellular typhoid vaccine.

5 Viral subunit vaccines. Three viral subunit vaccines are widely available, two

influenza vaccines and a hepatitis B vaccine. The influenza vaccines are prepared by

treating intact influenza virus particles from embryonated hens' eggs infected with

influenza virus with a surface acting agent. The virus particles are disrupted and release

the two virus subunits, haemagglutinin and neuraminidase, that are required in the

vaccine. The hepatitis B vaccine was, at one time, prepared from hepatitis B surface

antigen (HBsAg) obtained from the blood of the victims of hepatitis B. This very

constrained source of antigen has been replaced by yeast cells that have been genetically

engineered to express HBsAg during fermentation.

2.1 The seed lot system

The starting point for the production of all microbial vaccines is the isolation of the

appropriate microbe. Such isolates have been mostly derived from human infections

and in some cases have yielded strains suitable for vaccine production very readily; in

other cases a great deal of manipulation and selection in the laboratory have been

needed before a suitable strain has been obtained.

Once a suitable strain is available, the practice is to grow, often from a single

organism, a sizeable culture which is distributed in small amounts in a large number of

ampoules and then stored at ~70°C or freeze-dried. This is the seed lot. From this seed

lot, one or more ampoules are taken and used as the seed to originate a limited number

of batches of vaccine which are first examined exhaustively in the laboratory and then,

if found to be satisfactory, tested for safety and efficacy in clinical trials. Satisfactory

results in the clinical trials validate the seed lot as the seed from which batches of

vaccine for routine use can subsequently be produced.

2.2 Production of the bacteria and the bacterial components of bacterial vaccines

The bacteria and bacterial components needed for the manufacture of bacterial vaccines

are readily prepared in laboratory media by well-recognized fermentation methods.

The end-product of the fermentation, the harvest, is processed to provide a concentrated

and purified vaccine component that may be conveniently stored for long periods or

even traded as an article of commerce.

2.2.1 Fermentation

The production of a bacterial vaccine batch begins with the resuscitation of the bacterial

seed contained in an ampoule of the seed lot stored at -70°C or freeze dried. The

resuscitated bacteria are first cultivated through one or more passages in preproduction

media. Then, when the bacteria have multiplied sufficiently, they are used to inoculate

a batch of production medium. This is usually contained in a large fermenter, the contents

of which are continuously stirred. Usually the pH and the oxidation-reduction potential

of the medium are monitored and adjusted throughout the growth period in a manner

intended to obtain the greatest bacterial yield. In the case of rapidly growing bacteria

Manufacture and quality control of immunological products 307

the maximum yield is obtained after about a day but in the case of bacteria that grow

slowly the maximum yield may not be reached before 2 weeks. At the end of the growth

period the contents of the fermenter, which are known as the harvest, are ready for the

next stage in the production of the vaccine.

2.2.2 Processing of bacterial harvests

The harvest is a very complex mixture of bacterial cells, metabolic products and

exhausted medium. In the case of a live attenuated vaccine it is innocuous and all that

is necessary is for the bacteria to be separated and resuspended in an appropriate

menstruum, possibly for freeze-drying. In a vaccine made from a pathogen the harvest

may be intensely dangerous and great care is necessary in the following procedures.

1 Killing. The process by which the live bacteria in the culture are killed and thus

rendered harmless. Heat and disinfectants are employed. Heat and/or formalin are

required to kill the cells of Bordetella pertussis used to make whooping-cough vaccines,

and phenol is used to kill the Vibrio cholerae in cholera vaccine and the Salmonella

typhi in typhoid vaccine.

2 Separation. The process by which the bacterial cells are separated from the culture

fluid. Centrifugation using either a batch or continuous flow process is commonly

used, but precipitation of the cells by reducing the pH is an alternative. In the case of

vaccines prepared from cells, the fluid is discarded and the cells are resuspended in a

saline mixture; where vaccines are made from a constituent of the fluid, the cells are

discarded.

3 Fractionation. The process by which components are extracted from bacterial

cells or from the medium in which the bacteria are grown and obtained in a purified

form. The polysaccharide antigens of Neisseria meningitidis are separated from the

bacterial cells by treatment with hexadecyltrimethylammonium bromide and those of

Streptococcus pneumoniae with ethanol. The purity of an extracted material may be

improved by resolubilization in a suitable solvent and precipitation. After purification,

a component may be dried to a powder, stored indefinitely and, as required, incorporated

into a vaccine in precisely weighed amounts at the blending stage.

4 Detoxification. The process by which bacterial toxins are converted to harmless

toxoids. Formalin is used to detoxify the toxins of both Corynebacterium diphtheriae

and Clostridium tetani. The detoxification may be performed either on the whole culture

in the fermenter or on the purified toxin after fractionation.

5 Adsorption. The adsorption of the components of a vaccine on to a mineral adjuvant.

The mineral adjuvants, or carriers, most often used are aluminium hydroxide, aluminium

phosphate and calcium phosphate and their effect is to increase the immunogenicity

and decrease the toxicity, local and systemic, of a vaccine. Diphtheria vaccine, tetanus

vaccine, diphtheria/tetanus vaccine and diphtheria/tetanus/pertussis vaccine are

generally prepared as adsorbed vaccines.

6 Conjugation. The linking of a vaccine component that induces only a poor immune

response, with a vaccine component that induces a good immune response. The

immunogenicity for infants of the capsular polysaccharide of H. influenzae Type b is

greatly enhanced by the conjugation of the polysaccharide with diphtheria and tetanus

toxoids, and with the outer membrane protein of Neisseria meningitidis.

308 Chapter 15

2.3

Production of the viruses and the viral components of viral vaccines

Viruses replicate only in living cells so the first viral vaccines were necessarily made

in animals: smallpox vaccine in the dermis of calves and sheep; and rabies vaccines in

the spinal cords of rabbits and the brains of mice. Such methods are no longer used in

advanced vaccine production and the only intact animal hosts that are used are

embryonated hens' eggs. Almost all of the virus that is needed for viral vaccine

production is obtained from cell cultures infected with virus of the appropriate strain.

2.3.1 Growth of viruses

Embryonated hens' eggs are still the most convenient hosts for the growth of the viruses

that are needed for influenza and yellow fever vaccines. Influenza viruses accumulate

in high litre in the allantoic fluid of the eggs and yellow fever virus accumulates in the

nervous systems of the embryos.

2.3.2 Processing of viral harvests

The processing of the virus-containing material from infected embryonated eggs may

take one or other of several forms. In the case of influenza vaccines the allantoic fluid

is centrifuged to provide a concentrated and partially purified suspension of virus. This

concentrate is treated with ether or other disruptive agents to split the virus into its

components when split virion or surface antigen vaccines are prepared. The chick

embryos used in the production of yellow fever vaccine are homogenized in water to

provide a virus-containing puree. Centrifugation then precipitates most of the embryonic

debris and leaves much of the yellow fever virus in an aqueous suspension.

Cell cultures provide infected fluids that contain little debris and can generally be

satisfactorily clarified by filtration. Because most viral vaccines made from cell cultures

consist of live attenuated virus, there is no inactivation stage in their manufacture.

There are, however, two important exceptions: inactivated poliomyelitis virus vaccine

is inactivated with dilute formalin or /3-propiolactone and rabies vaccine is inactivated

with /3-propiolactone. The preparation of these inactivated vaccines also involves a

concentration stage, by adsorption and elution of the virus in the case of poliomyelitis

vaccine and by ultrafiltration in the case of rabies vaccine. When processing is complete

the bulk materials may be stored until needed for blending into final vaccine. Because

of the lability of many viruses, however, it is necessary to store most purified materials

at temperatures of-70°C.

2.4 Blending

Blending is the process in which the various components of a vaccine are mixed to

form a final bulk. It is undertaken in a large, closed vessel fitted with a stirrer and ports

for the addition of constituents and withdrawal of the final blend. When bacterial

vaccines are blended, the active constituents usually need to be greatly diluted and

the vessel is first charged with the diluent, usually containing a preservative such as

thiomersal. A single-component final bulk is then made by adding bacterial suspension,

Manufacture and quality control of immunological products 309

bacterial component or concentrated toxoid in such quantity that it is at the desired

concentration in the final product. A multiple-component final bulk of a combined

vaccine is made by adding each required component in sequence. When viral vaccines

are blended, the need to maintain adequate antigenicity or infectivity may preclude

dilution and tissue culture fluids or concentrates made from them are often used undiluted

or, in the case of multicomponent vaccines, merely diluted one with another. After

thorough mixing a final bulk may be broken down into a number of moderate sized

volumes to facilitate handling.

2.5 Filling and drying

As vaccine is required to meet orders, bulk vaccine is distributed into single dose

ampoules or into multidose vials as necessary. Vaccines that are filled as liquids are

sealed and capped in their containers, whereas vaccines that are provided as dried

preparations are freeze-dried before sealing.

The single-component bacterial vaccines are listed in Table 15.1. For each vaccine,

notes are provided of the basic material from which the vaccine is made, the salient

production processes and tests for potency and for safety. The multicomponent vaccines

that are made by blending together two or more of the single component vaccines are

required to meet the potency and safety requirements for each of the single components

that they contain. The best known of the combined bacterial vaccines is the adsorbed

diphtheria, tetanus and pertussis vaccine (DTPer/Vac/Ads) that is used to immunize

infants, and the adsorbed diphtheria and tetanus vaccine (DT/Vac/Ads) that is used to

reinforce the immunity of school entrants.

The single-component viral vaccines are listed in Table 15.2 with notes similar to

those provided with the bacterial vaccines. The only combined viral vaccine that is

widely used is the measles, mumps and rubella vaccine (MMR Vac). In a sense, however,

both the inactivated (Salk) poliovaccine (Pol/Vac (inactivated)) and the live (Sabin)

poliovaccine (Pol/Vac (oral)) are combined vaccines in that they are both mixtures of

virus of each of the three serotypes of poliovirus. Influenza vaccines, too, are combined

vaccines in that many contain components from as many as three virus strains, usually

from two strains of influenza A and one strain of influenza B.

2.6 Quality control

The quality control of vaccines is intended to provide assurances of both the efficacy

and the safety of every batch of every product. It is executed in three ways:

1 in-process control;

2 final-product control; and

3 a requirement that for each product the starting materials, intermediates, final product

and processing methods are consistent.

The results of all quality control tests are always recorded in detail as, in those

countries in which the manufacture of vaccines is regulated by law, they are part of the

evidence on which control authorities judge the suitability or otherwise of each batch

of each preparation.

310 Chapter 15

Manufacture and quality control of immunological products 311

Table 15.1 Bacterial

vaccines used for the prevention of infectious disease in humans. Vaccines marked * are those used in

conventional immunization schedules; those marked f are used to provide additional protection

indicate a need

Vaccine

Anthrax*

BCG*

Diphtheria

(adsorbed)*

Haemophilus

influenzae

type b*

Neisseria

meningitidis

Types A and Ct,

Pneumococcal

polysaccharidet

Tetanus

(adsorbed)*

Source material

Medium from

cultures of

B. anthracis

Cultures of live

BCG cells in

liquid or on

solid media

Cultures of

C. diphtheriae in

liquid medium

Cultures of

H. influenzae

type b

Cultures of

N. meningitidis

of serotypes

A and

Cultures of

23 serotypes of

Strep, pneumoniae

Cultures of

CI. tetani in

liquid medium

Processing

1 Separation of

protective

antigen from

medium

2 Adsorption

1 Bacteria

centrifuged

from medium

2 Resuspension

in stabilizer

3 Freeze-drying

1 Separation and

concentration of

toxin

2 Conversion of

toxin to toxoid

3 Adsorption of

toxoid to adjuvant

1 Separation of

capsular

polysaccharide

2 Conjugation

with a protein

1 Precipitation

with hexadecyl-

trimethyammonium

bromide

2 Solubilization

and purification

3 Blending

4 Freeze-drying

1 Precipitation

of polysaccharides

with ethanoi

2 Blending into

polyvalent

vaccine

1 Conversion of

toxin to toxoid

2 Separation and

purification of

toxoid

3 Adsorption to

adjuvant

Potency assay

3 + 3 quantal

assay in

guinea-pigs

using challenge

with B. anthracis

Viable count;

induction of

sensitivity to

tuberculin in

guinea-pigs

3 + 3 quantal

assay in

guinea-pigs

using intra-

dermal challenge

Estimation of

capsular poly-

saccharide

content

Estimation of

capsular poly-

saccharide

content

Physico-chemical

estimation of

polysaccharides

3 + 3 quantal

assay in mice

using subcutaneous

challenge

with tetanus

toxin

when circumstances

Safety tests

Exclusion of

live B. anthracis

and of

anthrax toxin

Exclusion of

virulent mycobacteria;

absence of

excessive dermal

reactivity

Inoculation of

guinea-pigs to

exclude residual

toxin

Inoculation of

guinea-pigs to

exclude presence

of untoxoided

toxin

continued on p. 312

Notes: Diphtheria and whooping cough vaccines are seldom used as single-component preparations but as components of

diphtheria/tetanus vaccines and diphtheria/tetanus/pertussis vaccines. A combined diphtheria/tetanus/pertussis/Hib vaccine

is available.

Bacterial vaccines less generally available than those listed in the table include botulism vaccine, necrotizing enteritis

(pigbel) vaccine, Pseudomonas aeruginosa vaccine and tularaemia vaccine.

2.6.1 In-process control

In-process quality control is the control exercised over starting materials and

intermediates. Its importance stems from the opportunities that it provides for the

examination of a product at the stages in its manufacture at which testing is most likely

to provide the most meaningful information. The WHO Requirements and national

authorities stipulate many in-process controls but manufacturers often perform tests in

excess of those stipulated, especially sterility tests (Chapter 23) as, by so doing, they

obtain assurance that production is proceeding normally and that the final product is

likely to be satisfactory. Examples of in-process control abound but three of different

types should suffice.

1 The quality control of both diphtheria and tetanus vaccines requires that the products

are tested for the presence of free toxin, that is for specific toxicity due to inadequate

detoxification with formalin, at the final-product stage. By this stage, however, the

toxoid concentrates used in the preparation of the vaccines have been much diluted

and, as the volume of vaccine that can be inoculated into the test animals (guinea-pigs)

312 Chapter 15

Table 15.1 Continued

Vaccine

Typhoidt

whole cell

Typhoid

Vi capsular

polysaccharide

antigent

Typhoid

live vaccinet

Whooping-cough

(Pertussis)

whole cell*

Whooping-cough

(Pertussis)

(acellular)t

Source material

Cultures of

Sal. typhi

grown in

liquid media

Cultures of

Sal. typhi

grown in

liquid medium

Cultures of

Sal. typhi strain

Ty21A

Cultures of

B. pertussis

grown in liquid

or on solid

media

Cultures of

Bord. pertussis

Processing

1 Killing with

heat or phenol

2 Separation and

resuspension of

bacteria in saline

Extraction of

capsular antigen

Encapsulation

1 Harvest

2 Killing with

formalin

3 Resuspension

1 Harvest

2 Extraction and

blending of cell

components

Potency assay

Induction of

antibodies in

rabbits

Estimation of

capsular

antigen

Estimation of

content of

live bacteria

3 + 3 quantal

assay in mice

using intra-

cerebral

challenge with live

Bord. pertussis

As for whole-cell

whooping-cough

vaccine

Safety tests

Exclusion of live

Sal. typhi

Estimation of

bacteria to limit

content to 20 x 10

per

human dose;

weight gain test in

mice to exclude

excess toxicity

Weight gain test in

mice to exclude

excess toxicity

Table 15.2 Viral vaccines used for the prevention of infectious disease in humans. The vaccines marked * are those used

in conventional immunization programmes, those marked t are used to provide additional protection when circumstances

indicate a need

continued on p. 314

Manufacture and quality control of immunological products 313

Vaccine

Hepatitis At

Hepatitis Bt

Influenza

(split virion)t

Influenza

(surface

antigen)t

Measles*

Mumps*

Poliomyelitis

(inactivated)t

(Salktype)

Source material

Human diploid

cells infected

with hepatitis

A virus

Yeast cells

genetically

modified to express

surface

antigen

Allantoic fluid

from embryonated

hens' eggs

infected with

influenza

viruses A and B

Allantoic fluid

from embryonated

hens' eggs

infected with

influenza

viruses A and B

Chick embryo

cell cultures

infected with

attenuated

measles virus

Chick embryo

cell cultures

infected with

attenuated

mumps virus

Human diploid

cell cultures

infected with

each of the

three serotypes

of poliovirus

Processing

1 Separation of

virus from

cells

2 Inactivation

with HCHO

3 Adsorption

toAI(OH)

gel

1 Separation of

HBsAg from

yeast cells

2 Adsorption to

AI(OH)

gel

1 Harvest of

viruses

2 Disruption

with surface

active agent

3 Blending of

components of

different serotypes

1 Inactivation

and disruption

2 Separation of

haemagglutinin

and neuraminidase

3 Blending of

haemagglutinins

and neuraminidase

of different

serotypes

1 Clarification

2 Freeze-drying

1 Clarification

2 Freeze-drying

1 Clarification

2 Inactivation

with formalin

3 Concentration

4 Blending of

virus of each

serotype

Potency assay

Assay of

antigen content

by ELISA

Immunogenicity

assay or HBsAg

assay by ELISA

Assay of haemagglutinin

content by

immunodiffusion

Assay of haemagglutinin

content by

immunodiffusion

Infectivity

titration in

cell cultures

Infectivity titration

in cell

cultures

Induction of

antibodies to

polioviruses

in chicks or

guinea-pigs

Safety tests

Inoculation of

cell cultures to

exclude presence

of live virus

Test for presence

of yeast DNA

Inoculation of

embryonated hens'

eggs to exclude

live virus

Inoculation of

embryonated hens'

eggs to exclude

live virus

Tests to exclude

presence of extraneous

viruses

Tests to exclude

presence of

extraneous

viruses

Inoculation of

cell cultures and

monkey spinal cords

to exclude live

virus

Table 15.2 Continued

Vaccine

Poliomyelitis

(live or oral)*

(Sabin type)

Rabiest

Rubella*

(German

measles)

Varicellat

Yellow fevert

Source material

Cell cultures

infected with

attenuated

poliovirus

of each of the

three serotypes

Human diploid

cell cultures

infected with

rabies virus

Human diploid

cell cultures

infected with

attenuated

rubella virus

Human diploid

cell cultures

infected with

attenuated

varicella virus

Aqueous homogenate

of chick embryos

infected with

attenuated yellow

fever virus 170

Processing

1 Clarification

2 Blending of

virus of three

serotypes in

stabilizing

medium

1 Clarification

2 Inactivation

with beta-

propiolactone

1 Clarification

2 Blending with

stabilizer

3 Freeze-drying

1 Clarification

2 Freeze-drying

1 Centrifugation

to remove cell

debris

2 Freeze drying

Potency assay

Infectivity titration

of each of three

virus serotypes

3 + 3 quantal

assay in mice

Infectivity titration

in cell cultures

Infectivity titration

in cell cultures

Infectivity-

titration in

cell cultures

by plaque

assay

Safety tests

Test for attenuation

by inoculation of

spinal cords of

monkeys and comparison

of lesions with

those produced by

a reference vaccine

Inoculation of

cell cultures to

exclude live virus

Tests to exclude

presence of

extraneous

viruses

Tests to exclude

presence of

extraneous

viruses

Tests to exclude

extraneous

viruses

Notes: Measles, mumps and rubella vaccines are generally administered in the form of a combined measles/mumps/rubella

vaccine (MMR vaccine).

Viral vaccines less generally available than those listed in the table include Congo Crimean haemorrhagic fever vaccine,

dengue fever vaccine, Japanese encephalitis B vaccine, smallpox vaccine, tick borne encephalitis vaccine, and Venezuelan

encephalitis vaccine.

ELISA, enzyme-linked immunosorbent assay.

is limited, the tests are relatively insensitive. In-process control, however, provides for

tests on the undiluted concentrates and thus increases the sensitivity of the method at

least 100-fold.

2 An example from virus vaccine manufacture is the titration, prior to inactivation,

of the infectivity of the pools of live poliovirus used to make inactivated poliomyelitis

vaccine. Adequate infectivity of the virus from the tissue cultures is an indicator of the

adequate virus content of the starting material and, since infectivity is destroyed in the

inactivation process, there is no possibility of performing such an estimation after

formolization.

3 A more general example from virus vaccine production is the rigorous examination

of tissue cultures to exclude contamination with infectious agents from the source animal

or, in the cases of human diploid cells or cells from continuous cell lines, to detect

314 Chapter 15