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

Подождите немного. Документ загружается.

cells with abnormal characteristics. Monkey kidney cell cultures are tested for simian

herpes B virus, simian virus 40, mycoplasma and tubercle bacilli. Cultures of human

diploid cells and continuous line cells are subjected to detailed karyological examination

(examination of chromosomes by microscopy) to ensure that the cells have not

undergone any changes likely to impair the quality of a vaccine or lead to undesirable

side-effects.

2.6.2 Final-product control

Vaccines containing killed microbes or their products are generally tested for potency

in assays in which the amount of the vaccine that is required to protect animals from a

defined challenge dose of the appropriate pathogen, or its product, is compared with

the amount of a standard vaccine that is required to provide the same protection. The

usual format of the test is the 3 + 3 dose quantal assay that is used to estimate the

potency of whooping-cough vaccine {British Pharmacopoeia 1993). Three logarithmic

serial doses of the test vaccine and three logarithmic serial doses of the standard vaccine

are made and each is used to inoculate a group of 16 mice. In the case of both the

vaccine and the standard the middle dose is chosen, on the basis of experience, so that

it is sufficient to induce a protective response in about 50% of the animals to which it

is given. Each lower dose may then be expected to protect fewer than 50% of the mice

to which it is given and each higher dose to protect more than 50% of the animals to

which it is given. Fourteen days later all of the mice are infected ('challenged') with

Bordetella pertussis and, after a further 14 days, the number of mice surviving in each

of the six groups is counted. The number of survivors in each group is then used to

calculate the potency of the test vaccine relative to the potency of the standard vaccine

by the statistical method of probit analysis (Finney 1971). The potency of the test

vaccine may be expressed either as a percentage of the potency of the standard vaccine

but, as the standard vaccine will have an assigned potency in International Units (IU),

the potency of the test vaccine may be expressed in similar units. Tests similar to that

used to estimate the potency of whooping-cough vaccine are prescribed for the estimation

of the potencies of diphtheria vaccine and of tetanus vaccine. In the cases of these two

vaccines the bacterial toxins are used as the challenge material {British Pharmacopoeia

1993).

Vaccines containing live microorganisms are generally tested for potency by counts

of their viable particles. In the case of the only live bacterial vaccine in common use,

BCG vaccine, dilutions of vaccine are made and dropped in fixed volumes on to solid

media capable of supporting the microorganisms' growth. After a fortnight the colonies

generated by the drops are counted and the live count of the undiluted vaccine is

calculated. The potency of live viral vaccines is estimated in much the same way except

that a substrate of living cells is used. Dilutions of vaccine are inoculated on to tissue

culture monolayers in Petri dishes or in plastic trays, and the live count of the vaccine

is calculated from the infectivity of the dilutions and dilution factor involved.

Safety tests. Because many vaccines are derived from basic materials of intense

pathogenicity—the lethal dose of a tetanus toxin for a mouse is estimated to be 3 x

(ig—safety testing is of paramount importance. Effective testing provides a

Manufacture and quality control of immunological products 315

guarantee of the safety of each batch of every product and most vaccines in the final

container must pass one or more safety tests as prescribed in a pharmacopoeial

monograph. This generality does not absolve a manufacturer from the need to perform

'in-process' tests as required, but it is relaxed for those preparations which have a final

formulation that makes safety tests on the final product either impractical or meaningless.

Bacterial vaccines are regulated by relatively simple safety tests. Those vaccines

composed of killed bacteria or bacterial products must be shown to be completely free

from the living microbes used in the production process and inoculation of appropriate

bacteriological media with the final product provides an assurance that all organisms

have been killed. Those containing diphtheria and tetanus toxoids require in addition,

a test system capable of revealing inadequately detoxified toxins; inoculation of guinea-

pigs, which are exquisitely sensitive to both diphtheria and tetanus toxins, is always

used for this purpose. Inoculation of guinea-pigs is also used to exclude the presence of

abnormally virulent organisms in BCG vaccine.

Viral vaccines present problems of safety testing far more complex than those

experienced with bacterial vaccines. With killed viral vaccines the potential hazards

are those due to incomplete virus inactivation and the consequent presence of residual

live virus in the preparation. The tests used to detect such live virus consist of the

inoculation of susceptible tissue cultures and of susceptible animals. The cultures are

examined for cytopathic effects and the animals for symptoms of disease and histological

evidence of infection at autopsy. This test is of particular importance in inactivated

poliomyelitis vaccine, the vaccine being injected intraspinally into monkeys. At autopsy,

sections of brain and spinal cord are examined microscopically for the histological

lesions indicative of proliferating poliovirus.

With attenuated viral vaccines the potential hazards are those associated with

reversion of the virus during production to a degree of virulence capable of causing

disease in vaccinees. To a large extent this possibility is controlled by very careful

selection of a stable seed but, especially with live attenuated poliomyelitis vaccine, it is

usual to compare the neurovirulence of the vaccine with that of a vaccine known to be

safe in field use. The technique involves the intraspinal inoculation of monkeys with a

reference vaccine and with the test vaccine and a comparison of the neurological lesions

and symptoms, if any, that are caused. If the vaccine causes abnormalities in excess of

those caused by the reference it fails the test.

Tests of general application. In addition to the tests designed to estimate the potency

and to exclude the hazards peculiar to each vaccine there are a number of tests of more

general application. These relatively simple tests are as follows.

1 Sterility. In general, vaccines are required to be sterile. The exceptions to this

requirement are smallpox vaccine made from the dermis of animals and bacterial

vaccines such as BCG, Ty21A and tularaemia vaccine which consist of living but

attenuated microbes. WHO requirements and pharmacopoeial standards stipulate, for

vaccine batches of different size, the numbers of containers that must be tested and

found to be sterile. The preferred method of sterility testing is membrane filtration as

this technique permits the testing of large volumes without dilution of the test media.

The test system must be capable of detecting aerobic and anaerobic organisms and

fungi (see Chapter 23).

2 Freedom from abnormal toxicity. The purpose of this simple test is to exclude the

presence in a final container of a highly toxic contaminant. Five mice of 17-22g and

two guinea-pigs of 250-350 g are inoculated with one human dose or 1.0ml, whichever

is less, of the test preparation. All must survive for 7 days without signs of illness.

3 Presence of aluminium and calcium. The quantity of aluminium in vaccines

containing aluminium hydroxide or aluminium phosphate as an adjuvant is limited to

1.25 mg per dose and it is usually estimated compleximetrically. The quantity of calcium

is limited to 1.3 mg per dose and is usually estimated by flame photometry.

4 Free formalin. Inactivation of bacterial toxins with formalin may lead to the presence

of small amounts of free formalin in the final product. The concentration, as estimated

by colour development with acetylacetone, must not exceed 0.02%.

5 Phenol concentration. When phenol is used to preserve a vaccine its concentration

must not exceed 0.25% w/v or, in the case of some vaccines, 0.5% w/v. Phenol is

estimated by the colour reaction with amino-phenazone and hexacyanoferrate.

3 Immunosera

Immunosera are preparations derived from the blood of animals, usually from the blood

of horses. To prepare an immunoserum a horse is injected with a sequence of spaced

doses of an antigen until a trial blood sample shows that the injections have induced a

high titre of antibody to the injected antigen. A large volume of blood is then removed

by venepuncture and collected into a vessel containing sufficient citrate solution to

prevent clotting. The blood cells are allowed to settle and the supernatant plasma is

drawn off. The plasma is then fractionated by the addition of ammonium sulphate

and the globulin fraction is recovered and treated with pepsin to yield a refined

immunoserum, Harms (1948). This refined immunoserum contains no more than a

trace of the albumin that was present in the plasma. The refined immunoserum is

titrated for the potency of its antibody content, diluted to the required concentration

and transferred into ampoules. Two or more monovalent immunosera may be blended

together to provide a multivalent immunoserum.

The quality of immunosera is controlled by potency tests and by conventional tests

for safety and sterility. The potency tests have a common design in that, in the case of

all immunosera, the potency is estimated by comparing the amount of an immunoserum

that is required to neutralize an effect of an homologous antigen with the amount of a

standard preparation that is required to achieve the same effect. Serial dilutions of the

immunoserum and of a standard preparation are made and to each is added a constant

amount of the homologous antigen. Each mixture is then inoculated into a group of

animals, usually guinea-pigs or mice, and the dilutions of the immunoserum and of the

standard which neutralize the effects of the antigen are noted. As the potencies of the

standard preparations are expressed in IU the potencies of the immunosera are determined

in corresponding units per millilitre {British Pharmacopoeia 1993).

Table 15.3 lists the immunosera for which there is a need, or a potential need, today

and indicates the required potencies of these immunosera and the salient features of the

potency assay methods.

Manufacture and quality control of immunological products 317

In each of the assays of potency the amount of the immunoserum and the amount of a corresponding

standard antitoxin that are required to neutralize the effects of a defined dose of the corresponding

toxin are determined. The two determined amounts and the assigned unitage of the standard antitoxin

are then used to calculate the potency of the immunoserum in International Units (IU).

Human immunoglobulins

Human immunoglobulins are preparations of the immunoglobulins, principally

immunoglobulin G (IgG), that are present in human blood. They are derived from the

plasma of donated blood and from plasma obtained by plasmapheresis. Normal

immunoglobulin, that is immunoglobulin that has relatively low titres of antibodies, is

prepared from pools of plasma obtained from not fewer than a thousand individuals;

specific immunoglobulins, that is immunoglobulins with a high titre of a particular

antibody, are usually prepared from smaller pools of plasma obtained from individuals

who have suffered recent infections or who have undergone recent immunization and

who thus have a high titre of a particular antibody. Each contribution of plasma to a

pool is tested for the presence of hepatitis B surface antigen (HBsAg), for antibodies to

human immunodeficiency viruses I and II (HIV I and II) and for antibodies to hepatitis

C virus in order to identify, and to exclude from a pool, any plasmas capable of

transmitting infection from donor to recipient.

The immunoglobulins are obtained from the plasma pools by fractionation methods

that are based on ethanol precipitation in the cold with rigorous control of protein

concentration, pH and ionic strength (Cohn et al. 1946; Kistler & Nitschmann 1962).

Some of the fractionation steps may contribute to the safety of immunoglobulins by

inactivating or removing contaminating viruses that have not been recognized by testing

of the blood donations. The immunoglobulin may be presented either as a freeze-dried

or a liquid preparation at a concentration that is 10 to 20 times that in plasma. Glycine

may be added as a stabilizer and thiomersal as a preservative.

The quality control of immunoglobulins includes potency tests and conventional

tests of safety and sterility. The potency tests consist of neutralization tests that parallel

those used for the potency assay of immunosera, except that in the cases of some

immunoglobulins the assays are made in vitro. In addition to the safety and sterility

tests, total protein is determined by nitrogen estimations, the protein composition by

318 Chapter 15

Table 15.3 Immunosera used in the prevention of infections in

Immunoserum

Botulinum antitoxin

Diphtheria antitoxin

Tetanus antitoxin

Potency assay method

Neutralization of the lethal

effects of botulinum

toxins A, B and E in mice

Neutralization of the erythrogenic

effect of diphtheria toxin

in the skin of guinea-pigs

Neutralization of the

paralytic effect of tetanus

toxin in mice

humans

Potency requirement

500IUm|-

of type A

500IU mM of Type B

50IU ml-

of Type E

1000 IU ml"

if prepared

in horses

500IU mM if prepared in

other species

1000 IU ml"

for prophylaxis

3000 IU ml"

for treatment

In each of the assays of potency the amount of the immunoglobulin and the amount of a

corresponding standard preparation that are required to neutralize the infectivity or other biological

activity of a defined amount of virus or to neutralize a defined amount of a bacterial toxin are

determined. The two determined amounts and the assigned unitage of the standard preparation are

then used to calculate the potency of the immunoglobulin in International Units (IU). ELISA,

enzyme-linked immunosorbent assay.

cellulose acetate electrophoresis and molecular size by liquid chromatography. The

presence of immunoglobulins derived from species other than humans is excluded by

precipitin tests. Table 15.4 lists six human immunoglobulins and their requisite potencies

and indicates the methods in which the potencies are determined.

Tailpiece

Immunological products, notably vaccines, provide very secure protection from diseases

caused by small pathogenic entities such as bacterial toxins and many viruses. They

provide somewhat less secure protection from larger pathogens such as bacteria and

little protection, if any, from much larger pathogens such as malaria parasites. There

thus appears to be a rough inverse correlation between the efficacies of vaccines and

the sizes of the pathogens from which each vaccine is intended to provide protection.

This relationship may reflect the way in which vaccine-induced antibodies react with a

toxin or pathogen. Small homogeneous tetanus toxin molecules may be completely

invested by tetanus antitoxin molecules and thus wholly neutralized. In contradistinction

malarial parasites may be unaffected by antibodies that attach only to a cell component

that is not essential for the parasite's survival. It has recently been suggested (Beverly

1996) that in order to make effective vaccines against larger pathogens it may first be

necessary to identify those molecules in the pathogens that are essential for each

Manufacture and quality control of immunological products 319

Table 15.4 Immuno;

Immunoglobulin

Hepatitis B

Measles

Normal

Rabies

Tetanus

Varicella/zoster

globulins used in the prevention and treatment of infections in humans

Potency assay method

Radioimmunoassay or

enzymoimmunoassay

Neutralization of the

infectivity of measles virus

for cell cultures

Neutralization tests in cell

cultures or in animals

Neutralization of the

infectivity of rabies virus for mice

Neutralization of the

paralytic effect of tetanus toxin

in mice

ELISA in paralled with

a standard varicella-zoster

immunoglobulin

Potency requirement

Not less than lOOIUmh

Not less than 50IU ml"

Measurable amounts of one

bacterial antibody and of one

viral antibody for which there

are international standards

Not less than 150111ml-

Not less than SOIUml"

Not less than lOOIUmh

pathogen's survival. A vaccine containing such molecules might induce antibodies to a

pathogen's essential molecules and thus provide immunity against larger pathogens

comparable with that provided by the vaccines against toxins and small pathogens.

The cost of the vaccines used in the routine immunization of infants, children and

adolescents is roughly equivalent to the cost of 100 loaves of bread. In the industrialized

countries that is a small price to pay for what is virtually life-long protection from

diphtheria, tetanus, whooping-cough, H. Influenzae type B infection, poliomyelitis,

measles, mumps and rubella. In many developing countries it is a price far beyond the

reach of either individuals or health authorities but a price that is in large part borne by

the World Health Organization's Expanded Programme of Immunization.

Further reading

Vaccination and immunization 321

recognized that immunity developed towards one disease often brings with it cross-

immunity towards another related condition. Cowpox is a disease of cattle that can be

transmitted to man. Symptoms are similar but less severe than those of smallpox.

Material taken from active cowpox (Vaccinia) lesions was therefore substituted into

the variolation procedures. This conferred much of the protection against smallpox

that had become associated with variolation but without the associated risk. Edward

Jenner's discovery, made over two centuries ago, became known as vaccination

and heralded a new era in disease control. The term vaccination is now widely used

to describe prophylactic measures that use live microorganisms or their products

to induce immunity. The more general term immunization describes procedures that

induce immunity in the recipient but which do not necessarily involve the use of

microorganisms. Nowadays vaccination and immunization procedures are used not

only to protect the individual against infection but also to protect communities

against epidemic disease. Such public health measures have met with spectacular

success as illustrated in Fig. 16.1 for the incidence of paralytic poliomyelitis. In

instances, where there is no reservoir of the pathogen other than in infected

individuals, and survival outside the host is limited (i.e. smallpox, poliomyelitis and

measles) then such programmes, worldwide, have the potential to eradicate the disease

permanently.

Fig. 16.1 Reported incidence of paralytic poliomyelitis in England and Wales during the 1950s and

1960s. After introduction of vaccination programmes the incidence of disease dropped from an

endemic incidence of ca. 5000 cases per year to fewer than 10.

322 Chapter 16

Spread of infection

Infectious diseases may either be spread from a common reservoir of the infectious

agent that is distinct from diseased individuals (common source) or they might transfer

directly from a diseased individual to a healthy one (propagated source).

2.1 Common source infections

In common source infections, the reservoir of infection might be animate (i.e. insect

vectors of malaria and yellow fever) or they might be inanimate (infected drinking

water, cooling towers, contaminated food supply). In the simplest of cases the source

of infection is transient (i.e. food sourced to a single retail outlet or to an isolated event

such as a wedding reception). In such instances the onset of new cases is rapid,

phased over 1-1.5 incubation periods, and the decline in new cases closely follows the

elimination of the source (Fig. 16.2). This leads to an acute outbreak of infection limited

socially and geographically to those linked with the source. Such an incident was

epitomized by the outbreak of Escherichia coli 0157 infections, in Lanarkshire, in the

winter of 1996.

If the source of the infection persists, after onset, then the incidence of new cases is

maintained at a level which is commensurate with the infectivity of the pathogen and

the frequency of exposure of individuals. In this manner, if cases of the variant

Creutzfeldt-Jacob disease (vCJD), first recognized in the mid-1990s, relates to human

exposure to bovine spongiform encephalopathy-infected beef in the early 1980s, then

Incubation periods (time)

Fig. 16.2 Incidence pattern for common-source outbreaks of infection where the source persists (•)

and where it is short-lived (•).

Vaccination and immunization 323

2.2

the incidence of vCJD will increase over 1-1.5 incubation periods (i.e. 10-15 years)

and be sustained for many years before a decline. In such a scenario vCJD, related to a

single common-source outbreak, would persist well into the next millenium.

For those infectious diseases that are transmitted to humans via insect vectors the

onset and decline phases of epidemics are rarely observed other than as a reflections of

the seasonal variation in the prevalence of the insect. Rather, the disease is endemic

within the population group and has a steady incidence of new cases. Diseases such as

these are generally controlled by public health measures and environmental control of

the vector with vaccination and immunization being deployed to protect individuals

(e.g. yellow fever vaccination).

Propagated source infections

Propagated outbreaks of infection relate to the direct transmission of an infective

agent from a diseased individual to a healthy, susceptible one. Mechanisms of such

transmission were described in Chapter 4 and include inhalation of infective aerosols

(measles, mumps, diphtheria), direct physical contact (syphilis, herpes virus) and, where

sanitation standards are poor, through the introduction of infected faecal material into

drinking water (cholera, typhoid). The ease of transmission, and hence the rate of onset

of an epidemic (Fig. 16.3) relates not only to the susceptibility status, and general state

of health of the individuals but also to the virulence properties of the organism, the

route of transmission, the duration of the infective period associated with the disease,

Fig. 16.3 Propagated outbreaks of infection showing the incidence of new cases (•), diseased

individuals (•), and recovered immune (A). The dotted line indicates the incidence pattern for an

incompletely mixed population group.

324 Chapter 16