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Preparation and Quality Control of Immunological Products

M.Sc. Biotechnology Part-IIMumbai UniversityPaper III - Unit IV

By: Mayur D. Chauhan

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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.

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Types of Immunological Products

Vaccines

In-vivo Diagnostics

Immune seraImmmunoglobulins

MAbs

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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.

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History of VaccinesEdward 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

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Types of Vaccines

Live

Killed

ToxoidConjugate

Subunit

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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)

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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.

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• 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.

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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

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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.

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

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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.

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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

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Preparation of Vaccines

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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.

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• 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.

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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.

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• 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.

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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.

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• 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.

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• 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

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• 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.

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

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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

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Disadvantage

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

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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)

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Growth of Viruses

• Influenza Virus accumulates in high titre in the allantoic fluid.

• Yellow fever virus accumulates in the nervous system of the embryos.

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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.

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• 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.

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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

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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.

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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

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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.

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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.

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3 + 3 quantal dose assay

Test

Std

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Test

Std

16

Test

Std

16

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• 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

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• 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.

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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.

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• 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.

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• 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.

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3. Tests of general Applications

Sterility

Toxicity

Pyrogenicity

Free formaldehyde

(<0.02%)

Phenol Concentration(0.25% w/v)

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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.

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Name of the Protein

Source Disease detected Causative Agent

TUBERCULIN(Protein)

M. tbM. bovisM. avium

Tuberculosis M. tbM. bovisM. 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

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Preparation of TuberculinsGrow Mycobacterium tuberculosis strain in a protein-free medium for several weeks.

The culture supernatant is recovered by centrifugation and further concentrated by evaporation and sterile filtered to form OT

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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.

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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.

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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.

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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.

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PreparationThe 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.

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Two or more monovalent immune sera may be blended together to provide a multivalent immune serum.

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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

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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.

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• 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.

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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.

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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.

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MABs

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Quality control and quality assurance of sterile products

Bioburden

Test for Sterility

Parametric release

Pyrogens

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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.

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• 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.

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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.

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• 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.

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• 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.

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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.

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• 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

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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.

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

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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.

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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.

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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.

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2 parts of pyrogenic LPS

Hydrophilic polysacchari

de chain•Has Antigenic regions

Hydrophobic Lipid group

•Termed as Lipid A which has many biological activities.

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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.

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Measurement of Pyrogens

2 methods

LAL testPyrogen Test

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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.

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• 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.

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Criteria for Pass or Fail

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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.

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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.

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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.

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•Limulus polyphemusL

•AmoebocytesA

•LysateL

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ProtocolProduct samples are incubated with LAL reagent at 37o

C

If endotoxins are present, a solid gel forms which stays intact

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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.

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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.

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• 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.

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Reference Books

Hugo and Russell’s Pharmaceutical Biotechnology

Immunology by Kuby

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