The following presentation is only for quick reference. I would advise you to read the theoretical aspects of the respective topic and then use this presentation for your last minute revision. I hope it helps you..!! Mayur D. Chauhan

1. Preparation and Quality Control
of Immunological Products
M.Sc. Biotechnology Part-II
Mumbai University
Paper III - Unit IV
By: Mayur D. Chauhan
1

2. Immunological Products
Group of pharmaceutical preparations with
diverse origins but a common pharmacological
purpose: Modification of the Immune status of a
recipient, either to provide immunity to
infectious diseases or to aid in the detection of
such diseases.
2

3. Types of Immunological Products
Vaccines
In-vivo Diagnostics
Immune seraImmmunoglobulins
MAbs
3

4. Vaccines
• Vaccine is a biological preparation that
consists of either a whole organism or a part
of it against which immunization has to be
achieved.
• Vaccines provide active immunity as they
stimulate the immune system of the recipient
to produce T cells or antibodies that impede
the attachment of infectious agents and
promote their destruction.
4

5. History of Vaccines
Edward
Jenner (1798)
Dried crusts of pustules
inhaled or injected
Immunization against
Smallpox
Louis Pasteur
(1881)
Discovered the
bacterium causing fowl
cholera.
Confirmed by injecting
in Chickens
OC - Chickens became ill
& recovered
NC – Chickens were
protected
5

6. 6

7. Types of Vaccines
Live
Killed
ToxoidConjugate
Subunit
7

8. Live Vaccines
• These are preparations of live bacteria, viruses
or other agents which, when administered by
an appropriate route, cause subclinical or mild
infections. In the course of such an infection
the components of the microorganisms in the
vaccine evoke an immune response which
provides protection against the more serious
natural disease.
• Examples: Vaccinia (Smallpox), BCG (TB)
8

9. Killed Vaccines
• Killed vaccines are suspensions of bacteria,
viruses or other pathogenic agents, that have
been killed by heat or by disinfectants such as
phenol, ethanol or formaldehyde.
• Killed organisms cannot replicate and cause an
infection. Thus every dose of killed vaccine
must have an antigenic material to increase
the immunogenic response.
9

10. • Since all the components of the micro-
organism are present, it may be toxic to the
body. Thus it is recommended to divide the
vaccine into booster doses which may be
given at regular intervals of time.
• Examples: Polio, Typhoid, Pertussis, Cholera,
Plague, Rabies.
10

11. Adjuvants
• Heterogeneous collection of substances which
enhance the immune response.
• Examples: Aluminium hydroxide gel (hydrated
aluminium oxide) and aluminium phosphate are
the only ones in general use in human vaccines.
• A much wider range of substances including oily
emulsions, saponin, immune- stimulating
complexes (ISCOMS), monophosphoryl lipid A
and others are used in veterinary vaccines and
some are under investigation for use in human
vaccines
11

12. 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 and treated chemically
(Formaldehyde) to eliminate toxicity but not
immunogenicity.
• This process is called as toxoiding and the end
product is termed as Toxoid or Formol toxoids.
12

13. • Examples: Tetanus, Diphtheria, Botulism,
Clostridial infections of farm animals.
13

14. Cell components or Subunit Vaccines
• Instead of using whole cells which may consist of
undesirable reactogenic components, vaccines
are prepared from purified protective
components.
• 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.
• Examples: Hemophilus influenzae type b,
Neisseria meningitidis ACWY, Hepatitis b etc.
14

15. Conjugate Vaccines
• Some antigens which are used to prepare
vaccines are less immunogenic and do not
give appropriate responses.
• Such antigens are conjugated to certain
immunogenic carriers which improve the
immunogenic response.
• Example: Glyco- Conjugate Vaccine of
Neisseria meningitidis with carrier protein is
CRM197
15

16. Preparation of Vaccines
16

17. A] Seed Lot System
• The starting point for the production of all
microbial vaccines is the isolation of the
appropriate infectious agent.
• Bacterial strains may need to be selected for
high toxin yield or production of abundant
capsular polysaccharide; viral strains may
need to be selected for stable attenuation.
17

18. • Once a suitable strain is available, the practice
is to grow, often from a single viable unit, a
substantial culture which is distributed in
small amounts in a large number of ampoules
and then stored at -70°C or below, or freeze-
dried. This is the original seed lot.
• From this seed lot, one or more ampoules are
used to generate the working seed from which
a limited number of batches of vaccine are
generated.
• Full history of the seed lot should be known
including the media composition.
18

19. B] Production of Bacteria
• To obtain specific components from the bacteria,
generally fermentation methods are used.
• Production of bacterial vaccine begins with
resuscitation of bacterial strains from the seed
lot.
• Resuscitated bacteria are first cultivated through
one or more passages in pre-production media.
Then, when the bacteria have multiplied
sufficiently, they are used to inoculate a batch of
production medium.
19

20. • The medium in the fermenter should have an
optimum pH as well as it should be free of any
TSE agents. (Transmissible Spongiform
Encephalopathy)
• 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.
20

21. Processing of the Harvest
• Killing – Live bacteria are killed by either Heat
or by certain disinfectants like Formalin,
Thiomersal.
• Separation- Bacterial cells are separated from
the culture fluid and soluble products.
Centrifugation, Ultrafiltration and
Precipitation methods are commonly used.
21

22. • Fractionation – Components are extracted
from the bacterial cells or from the medium in
which they are grown in a purified from.
Example: The polysaccharide antigens of
Neisseria meningitidis are usually separated
from the bacterial cells by treatment with
hexadecyltrimethylammonium bromide
followed by extraction with calcium chloride
and selective precipitation with ethanol.
• The purity of an extracted material may be
improved by resolubilization in a suitable
solvent and re-precipitation.
22

23. • After purification, a component may be
freeze-dried, stored indefinitely at low
temperature and, as required, incorporated
into a vaccine in precisely weighed amount at
the blending stage
• Detoxification: Carried out by formaldehyde to
obtain toxoids. Detoxification may be
performed either on the whole culture in the
fermenter or on the purified toxin after
fractionation
23

24. • Further Processing – These may include many
physical and chemical treatments to modify the
product.
Example: Polysaccharides may be further
fractionated to produce material of a narrow
molecular size specification. They may then be
activated and conjugated to carrier proteins to
produce Glyco-conjugate vaccines
• Adsorption: It’s the process of adsorbing the
vaccine to the mineral adjuvant. It’s helps in
improving immunogenicity and decreasing
toxicity.
24

25. • Conjugation: The linking of a vaccine
component that induces an inadequate
immune response, with a vaccine component
that induces a good immune response.
25

26. Production of Viruses
• Viruses replicate only in living cells. Thus the
first viral vaccine was prepared in animals.
• Examples: Smallpox vaccine in the dermis of
calves and sheep; and rabies vaccines in the
spinal cords of rabbits and brains of mice.
• Such methods are no longer used in advanced
vaccine production.
• Embryonated Hen’s egg
26

27. 27

28. 28

29. 29

30. Disadvantage
• The site of inoculation varies with different
viruses. That is, each virus has different sites
for it’s growth and replication.
30

31. Cultivation of Viruses
• There are 3 main ways,
1. Inoculation of virus into animals.
2. Inoculation of virus into embryonated eggs
(Eggs should be from disease free flocks.)
3. Tissue culture (Media composition should
be known and it should be free of TSE agents)
31

32. Growth of Viruses
• Influenza Virus accumulates in high titre in the
allantoic fluid.
• Yellow fever virus accumulates in the nervous
system of the embryos.
32

33. Processing of Viruses
• 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 organic
solvent or detergent to split the virus into its
components when split virion or surface
antigen vaccines are prepared.
33

34. • The chick embryos used in the production of
yellow fever vaccine are homogenized in
sterile water to provide a virus-containing
pulp.
• Centrifugation then precipitates most of the
embryonic debris and leaves much of the
yellow fever virus in an aqueous suspension.
34

35. C] Blending
• Blending is the process in which the various
components of a vaccine are mixed to form a
final bulk.
• When bacterial vaccines are blended, the active
constituents usually need to be greatly diluted
and the vessel is first charged with the diluents,
usually containing a preservative.
• Thiomersal has been widely used in the past but
is now being phased out and replaced by
phenoxy ethanol or alternatives
35

36. D] Filling and Drying
• Bulk vaccine is distributed into single-dose
ampoules or into multi-dose 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.
36

37. Quality Control
• Mainly to provide assurances of both the
probable efficacy and safety of every batch of
every product.
• 3 main ways:
1. In-process control
2. Final product control
3. Consistency in every production step
37

38. 1. In-process Control
• In-process quality control is the control exercised
over starting materials and intermediates.
• 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) 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.
38

39. Final Product Control
1. Assays: Vaccines containing killed
microorganisms or their products are generally
tested for potency in assays in which the
amount of the vaccine that is require 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.
39

40. 3 + 3 quantal dose assay
Test
Std
16
Test
Std
16
Test
Std
16
40

41. • The number of survivors in each group is used
to calculate the potency of the test vaccine
relative to the potency of the standard vaccine
by the statistical method.
• The potency of the test vaccine may be
expressed as a percentage of the potency of
the standard vaccine
41

42. • Vaccines containing live microorganisms are
generally tested for potency by determining
their content of viable particles.
• Example: In the case of BCG vaccine, dilutions
of vaccine are prepared in a medium which
inhibits clumping of cells, and fixed volumes
are dropped on to solid media capable of
supporting mycobacterial growth. After a
fortnight the colonies generated by the drops
are counted and the live count of the
undiluted vaccine is calculated.
42

43. 2. Safety Tests
• 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 microorganisms used in the production
process.
43

44. • Those vaccines prepared from toxins, for
example, diphtheria and tetanus toxoids,
require in addition, a test system capable of
revealing inadequately detoxified toxins.
• This can be done by inoculation of guinea-
pigs, which are exquisitely sensitive to both
diphtheria and tetanus toxins.
• A test for sensitization of mice to the lethal
effects of histamine is used to detect active
pertussis toxin in pertussis vaccines.
44

45. • With killed vaccines the potential hazards are
those due to incomplete virus inactivation and
the consequent presence of residual live virus
in the preparation.
• 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
recipients.
45

46. 3. Tests of general Applications
Sterility
Toxicity
Pyrogenicity
Free
formaldehyde
(<0.02%)
Phenol
Concentration
(0.25% w/v)
46

47. In-vivo Diagnostics
• They are used to demonstrate an
Immunogenic response like previous exposure
to a pathogen.
• Helpful in the diagnosis of diseases.
• Examples: Tuberculin, Mallein, Histoplasmin,
Coccidiodin, Brucellin.
47

48. Name of the
Protein
Source Disease detected Causative Agent
TUBERCULIN
(Protein)
M. tb
M. bovis
M. avium
Tuberculosis M. tb
M. bovis
M. avium
MALLEIN
(Protein)
Burkholderia mallei Glanders in horses,
donkeys and mules
Burkholderia mallei
HISTOPLASMIN
(Protein)
Histoplasma
capsulatum
HIstoplasmosis Histoplasma
capsulatum
COCCIDIODIN
(Protein)
Coccidioides immitis Coccidiodomycosis
or Valley Fever
Coccidioides immitis
BRUCELLIN
(Protein)
Brucella species Brucellosis Brucella species
48

49. Preparation of Tuberculins
Grow Mycobacterium tuberculosis strain in a
protein-free medium for several weeks.
Culture is steamed for several hours to kill
the surviving bacteria and to facilitate
release of tubercular-proteins from the cells
The culture supernatant is recovered by
centrifugation and further concentrated by
evaporation and sterile filtered to form OT
49

50. Crude material is standardized against a
reference preparation by titration in the skin of
guinea-pigs sensitized to
M. tuberculosis.
In practice, further purification is usually
performed by precipitation with Trichloracetic
acid or other protein precipitant to produce
Purified Protein Derivative,which is standardized.
Concentrated preparations containing 100000 IU
per ml are used to formulate working strengths
such as 1000, 100 or 10 IU per ml. These have to
be diluted in a medium containing a Tween
surfactant to reduce adsorption to glass.
50

51. Quality Control
• Potency
• Material should be free from Mycobacteria
• The product is also checked for absence of
reactogenicity in unsensitized guinea-pigs and
if required by the regulatory authority, for
abnormal toxicity.
51

52. Major Disadvantage
• Analogous intradermal test reagents such as
mallein, histoplasmin and coccidioidin, are
produced by similar methods.
• Their use has declined however, as they, like
the tuberculin test, measure exposure and
sensitization to the antigens of the agent but
not necessarily active infection.
52

53. Immune Sera
• To prepare an immune serum, horses or other
animals are 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. An adjuvant may be used if required.
• The animals must be in good health, free of
infections and from sources free of TSEs, and
kept under veterinary supervision.
53

54. Preparation
Once sufficient antibodies are generated, A large volume of
blood is then removed by venipuncture 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 crude plasma can be sterilized by filtration and
dispensed for use, but it is preferable to fractionate it to
separate the immune globulin. This is done by fractional
precipitation of the plasma by the addition of ammonium
sulphate.
54

55. The globulin fraction is recovered and
treated with pepsin to yield a refined
immune product containing the Fab
fragment.
The antibody content of the refined
product is determined, the product is
diluted to the required concentration
and transferred into ampoules.
Two or more monovalent immune sera
may be blended together to provide a
multivalent immune serum.
55

56. Quality Control
• Potency
• Serial dilutions of the immune serum 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 immune serum and of the
standard, which neutralize the effects of toxin, are noted
• The quality of globulin fractions is usually monitored by gel
electrophoresis to detect contaminating proteins and
uncleaved immunoglobulin and by size exclusion high
performance liquid chromatography to detect aggregates
56

57. Human Immunoglobulins
• Source: Human immunoglobulins are preparations of
the immunoglobulins, principally (IgG) subclasses, that
are present in human blood. They are derived from the
plasma of donated blood and from plasma obtained by
plasmapheresis.
• 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.
57

58. • Each contribution of plasma to a pool is tested
for the presence of hepatitis B surface antigen
(HbsAg), for antibodies to human
immunodeficiency viruses 1 and 2 (HIV 1 and
2) and for antibodies to hepatitis C virus in
order to identify, and to exclude from a pool,
any plasma capable of transmitting infection
from donor to recipient.
58

59. Fractionation
• Ethanol precipitation in the cold with rigorous
control of protein concentration, pH and ionic
strength.
• The immunoglobulins may be presented
either as a freeze dried or a liquid preparation
at a concentration that is 10–20 times that in
plasma.
• Glycine may be added as a stabilizer and
thiomersal as a preservative.
59

60. Quality Control
• Potency - The potency tests consist of toxin or
virus neutralization tests.
• In addition to the safety and sterility tests,
total protein is determined by nitrogen
estimations, the protein composition by
sodium dodecyl sulphate-polyacrylamide gel
electrophoresis and molecular size by high
performance liquid chromatography.
60

61. MABs
61

62. Quality control and quality assurance
of sterile products
Bioburden
Test for Sterility
Parametric release
Pyrogens
62

63. Bioburden
• A successful sterilization process is dependent on
a product having a low pre-sterilization
bioburden. Sterilization should be considered as
the removal of the bioburden.
• This will also be true of the individual ingredients,
which must have low levels of microbial
contamination or else there is a danger that the
contaminants will find their way into the final
product or be a source of pyrogens.
63

64. • The bioburden is an estimate of the total
viable count of microorganisms present pre-
sterilization, and a knowledge of the
resistance characteristics of these organisms is
often an integral part of the sterility assurance
calculation.
• Sterilization process should be chosen in such
a way that all micro-organisms are highly
resistant.
64

65. Test for Sterility
• The broad basis of the test for sterility is that
it examines samples of the final product for
the presence of microorganisms. Theoretically,
the test for sterility should be applied to all
products that are designated as sterile.
• The test results can be valid only if all the
products of a batch are treated similarly.
65

66. • Clearly for products which are terminally
sterilized this might seem a reasonable
assumption but only if there is uniform heat
distribution in an autoclave or hot air oven or
uniform delivery of a radiation dose.
• A successful test only shows that no microbial
contamination was found.
• Extension of the result to a whole batch requires
the assurance that every unit in the batch was
manufactured in such a manner that it would also
have passed the test with a high degree of
probability.
66

67. • This highlights the weakness of the test for
sterility and why the controls of sterilization
processes are very important and probably of
greater assurance in confirming the sterility of
a batch.
67

68. Parametric Release
• As there are significant limitations with the
test for sterility, many authorities place
considerable reliance on the validation and
reliable performance of sterilizers and their
sterilization cycles.
• Parametric release takes this reliance a step
further by allowing batches of terminally
sterilized products to be released without
being subjected to the test for sterility.
68

69. • Validation studies would include heat distribution, heat
penetration, bioburden, container closure and cycle
lethality studies.
• For a product to be subject to parametric release, pre-
sterilization bioburden testing on each batch would be
completed, and the comparative resistance of isolated
spore-formers checked.
• In practice this requires confirmation that each part of
the manufacturing process has been satisfactorily
completed, the initial pre-sterilization bioburden is
within agreed limits, that the controls for the sterilizing
cycle were satisfactory and that the correct time cycles
were achieved.
• Clearly reproducibility, regular monitoring and
documentation are required
69

70. Pyrogens
• A pyrogen is any substance that, when injected
into a mammal, elicits a rise in body temperature,
and substances produced by some Gram-positive
bacteria, mycobacteria, fungi and also viruses
conform to this definition.
• Gram Negative bacteria produce pyrogens in the
form of endotoxins. They are termed as
Lipopolysaccharides (LPS) which are mainly found
in the cell wall.
70

71. • The presence of pyrogens in aqueous
solutions was first demonstrated by injection
into rabbits whose body temperature was
recorded.
71

72. Physiological effects of Pyrogens
• When injected into the body, there is elevated
temperature noted.
• Pyrogens elevate the circulating levels of
inflammatory cytokines, which may be
followed by fever, blood coagulation,
hypotension, lymphopenia, neutrophilia,
elevated levels of plasma cortisol and acute
phase proteins.
72

73. LOW DOSAGE
Induces asymptomatic
inflammatory reactions.
MODERATE DOSAGE
Induce fever and changes
in plasma composition
HIGH DOSAGE
Results in shock,
characterized by
cardiovascular dysfunction,
vasodilation,
vasoconstriction,
endothelium dysfunction
and multiple organ
dysfunction or failure and
death.
73

74. Characteristics of Bacterial endotoxins
• Release of LPS takes place after death and
Lysis.
• Many Gram-negative bacteria, e.g. Escherichia
coli and Proteus, Pseudomonas, Enterobacter
and Klebsiella species produce pyrogenic LPS.
74

75. 2 parts of pyrogenic LPS
•Has Antigenic
regions
Hydrophilic
polysaccharide
chain
•Termed as Lipid A
which has many
biological activities.
Hydrophobic
Lipid group
75

76. Sources of Pyrogens
• Water used at the end stages of the purification
and crystallization of the drug or excipients,
water used during processing; packaging
components; and the chemicals, raw materials or
equipment used in the preparation of the
product.
• If the drug is biologically produced, incomplete
removal of the microorganisms during
purification can result in high endotoxin levels.
76

77. Measurement of Pyrogens
2 methods
LAL testPyrogen Test
77

78. Pyrogen Test
• Samples of the product under test are injected
into the marginal ear vein at a dose no greater
than 10 ml/kg.
• The animals are monitored for the 3-hour
period immediately after injection, at 30-
minute intervals.
• The test assumes that the maximum rise in
temperature will be detected in this 3-hour
period immediately after injection.
78

79. 79
• Repeated use of animals leads to endotoxin
tolerance.
• Care must be taken in testing
radiopharmaceuticals, and certain drugs may,
themselves, elicit a rise in temperature on
administration. The test is therefore inadequate
radiopharmaceuticals, cancer chemotherapeutic
agents, hypnotics and narcotics, vitamins,
steroids and some antibiotics.
• There is low reactivity to the endotoxin produced
by certain species, e.g. Legionella.
• The rabbit test is insufficiently sensitive to detect
endotoxin in products where only low levels of
pyrogens are acceptable.

80. Criteria for Pass or Fail
80

81. LAL test
• More sensitive to Pyrogen test.
• Legionella endotoxin easily detected by LAL
test.
• It only detects endotoxins of Gram negative
bacteria and not all pyrogens.
81

82. Actual Test
• Test reagent comes from American horse-shoe
crab named Limulus polyphemus.
• Freshly obtained Blood from the Crab -
Amoebocytes are concentrated, washed and
lysed with endotoxin free water – LAL reagent
separated from the cellular debris and it’s
activity is optimized by metallic cations, pH
adjustment and then freeze dried.
82

83. Disadvantages of Inhibitors
• Chemical inhibitors may cause chelation of the
divalent cations necessary for the reaction,
protein denaturation or inappropriate pH
changes.
• Physical inhibition may result from adsorption
of endotoxin or be caused by viscosity of the
product.
83

84. •Limulus polyphemusL
•AmoebocytesA
•LysateL
84

85. Protocol
Product samples are
incubated with LAL reagent
at 37o C
If endotoxins are
present, a solid gel
forms which stays intact
85

86. Endotoxin Limit
• Endotoxin limit, EL, which represents the
maximum amount of endotoxin that is allowed in
a specific dose, is inversely related to the dose of
the drug; it may be assessed from the following
equation,
• EL = K/M,
where K is the threshold human pyrogenic dose
of endotoxin per kg body weight and M is the
maximum human dose of the product in kg body
weight that would be administered in a single 1-
hour period.
86

87. Depyrogenation
• Pyrogens and endotoxins are difficult to remove
from products once present and it is easier to
keep components relatively endotoxin-free rather
than to remove it from the final product.
• Rinsing or dilution is one way of eliminating
pyrogenic activity provided that the rinsing fluid
is apyrogenic.
• Pyrogens in vials or glass components may be
destroyed by dry heat sterilization at high
temperatures.
87

88. • A recommended condition for depyrogenation of
glassware and equipment is heating at 250°C for
45 minutes. Pyrogens are also destroyed at 650°C
in 1 minute or at 180°C in 4 hours.
• The removal of pyrogens from Water for
Injections may be effected by distillation or
reverse osmosis.
• Generally circulating hot water at temperatures
above 75°C provides an environment that is not
conducive to microbial growth and thus the
formation of endotoxin.
88

89. Reference Books
Hugo and Russell’s
Pharmaceutical Biotechnology
Immunology by Kuby
89

90. 90