Characterization Of The Final Conjugate Materials

Published Date: 02 Nov 2017

Many reagents are used to derivatize and conjugate the vaccines. Unlike proteins, the preparation of saccharide components, adding, and then removing reagents require complex procedure. Purified bulk saccharides are trimmed and sorted into size classes to get the proper specifications of the product. This process is done by size exclusion chromatography. But it may lead to bigger volumes of dilute material that has to be concentrated by means of ultrafiltration.

Unwanted reagents are also removed from the protein by a similar process. The unreacted conjugation chemicals are to be removed after the completion of conjugation by passing the conjugate over a small porosity ultrafiltration membrane. This also helps the conjugate to concentrate or diafilter once again to reach the final formulation strength and carrier fluid prior to further formulations which may be required with any adjuvants and/or preservatives.

In case of multivalent product that is, it is comprised of multiple saccharide serotypes. In such cases, each serotype can be prepared and conjugated separately, and then in the final formulation the serotypes are mixed together. An example of such a vaccine is Prevnar (Wyeth). It is with seven different polysaccharides – serotypes 4, 6B, 9V, 14, 18C, 19F and 23F, representing the most frequently encountered. The vaccines which are in developmental stages addresses even more serotypes. Thus it further complicates manufacturing process, as each type must be developed and controlled separately. This complexity is one constrain to transfer the technology of this type of vaccine in a cost effective way to the developing areas of the world where they could use such vaccines.

CHARACTERIZATION OF THE FINAL CONJUGATE MATERIALS:

The final purified conjugates are characterized for their molecular properties. The test include structural analysis such as

Estimation of molecular weight of Carrier Protein

Molecular size distribution

Total protein content in the final purified conjugate sample

Total Polysaccharide content and free content in the final product.

Endotoxin

The purity of each conjugate lot is assessed through the Analytical testing program and thepurification process was also shown to efficiently and consistently eliminate the conjugation reagents. These specification are required for the releasing of the conjugate material

Production and Control Materials/Intermediates for Pneumococcal Conjugate Vaccines

S.PneumoniaPolysachharide

Seed Lot System:

The bacteria used for the preparation of the polysaccharides are strains of S. pneumoniae isolated from patients with otitis media. Master and working seeds from these strainswere derived and have been grown and stored in the absence of any material of animal origin.

Fermentation and Purification:

The production of the polysaccharides of the different serotypes is very similar and differs just in a few aspects of the purification process, depending on the nature of the PS:

acid or neutral PS. The content of a working seed vial is used to inoculate the fermentor; the fermentation occurs undercontrolled temperature, air-flow rate conditions and stirring, and it is stopped according to opticaldensity (OD) measurement.

At the end of the fermentation, inactivation of bacteria is achieved using phenol added to the fermentation broth.

Characterization:

Characterization analysis of polysaccharide bulks documenting identity, structure,

size, integrity and content of functional groupswere performed.

Specification:

The release of the polysaccharides are based upon the water content, alcohol content, molecular size distribution, residual protein, nucleic acid and endotoxin content.The specifications applied to most of the tests are those recommended by the applicable guidelines (WHO guideline ‘Recommendations for production and control of S. pneumoniae conjugate vaccines)

Stability:

The stability of purified S. pneumoniae polysaccharides (PS) is assessed by longterm,

real-time stability studies (up to 84 months) on three batches per polysaccharide

serotype. Batches are stored at -20°C or -70°C depending on the serotype.

Purified Diptheria Toxoid

Seed Lot System:

The strain of Corynebacteriumdiphtheriaethat is used to manufacture purified diphtheria toxoid.The seed lot system was established using meat-free culture media.

Fermentation and Purification:

The manufacturing process of the purified diphtheria toxoid consists of 3 main stages: fermentation, detoxification and purification.The production of purified diphtheria toxoid is performed following the WHO and general GMP requirements. Purified diphtheria toxoid, complies with WHO (TRS 800), (Ph. Eur) requirements.

Characterization:

Characterisation was conducted on the purified DT bulks including SDS gel electrophoresis and other physicochemical tests were performed.

Specification:

The tests and specifications for release of the DT bulks should be complying with WHO (TRS 800) and Ph. Eur. Requirements.

REVIEW OF LITERATURE

1.1 Vaccines

The term "vaccine" was coined by Louis Pasteur to commemorate first successful immunization against small poxby Edward Jenner. The term vaccine was derived from "vacca", meaning cow, since Edward Jenner used cowpoxvirus (Vaccinia) to prevent smallpox infection(Rao 2006).T-cell memory is very important for long-lasting immunity, because T-cells control both humoral and cell mediated immunity. When the immune system recognizes a foreign antigen for the first time, an immune response is produced. When T cells are involved, immunological T-cell memory is produced. When the body encounters same antigen subsequently, a stronger immune response is produced. This is because of existing immunological memory against that antigen. Further antigenic stimulus increases the immune response. First antigenic stimulus is "priming" whereas subsequent stimuli are "booster". This is the principle of active immunization(Rao 2006).Vaccination is aimed at inducing active immunity in an individual, so that subsequent contact with the microorganism following natural infection induces strong protective immune response. The protective immunity may involve secretion of neutralizing antibodies or production of memory CTL or Th1 cells. The use of vaccines is now being extended to immunize against tumors or to block fertilization (contraceptive vaccines). Today the term ‘vaccine’ applies to all biological preparations, produced from living organisms, that enhance immunity against disease and either prevent (prophylactic vaccines) or, in some cases, treat disease (therapeutic vaccines), Vaccines are administered in liquid form, either by injection, by oral, or by intranasal routes(WHO 2012).Vaccines are composed of either the entire disease-causing microorganism or some of its components. They may be constructed in several ways.

From living organisms that have been weakened, usually from cultivation under sub-optimal conditions (also called attenuation), or from genetic modification, which has the effect of reducing their ability to cause disease(WHO 2012).

From whole organisms that have been inactivated by chemical, thermal or other means also known as Killed Vaccines.

From components of the disease-causing organism, such as specific proteins and polysaccharides, or nucleic acids(WHO 2012).

From inactivated toxins of toxin-producing bacteria.

From the linkage (conjugation) of polysaccharidesto proteins (this increases the effectiveness of polysaccharide vaccines in young children.

1.2 Properties of an Ideal Vaccine:

Provide long lasting immunity.

Should induce both humoral and cellular immunity.

Should not induce autoimmunity or hypersensitivity.

Should be inexpensive to produce, easy to store and administer.

Vaccines must also be perceived to be safe.

The vaccine vial may contain relevant antigen, adjuvant (usually alum), preservatives and/or traces of protein derived from the cells in which the vaccine agent was cultured e.g. egg protein(Rao 2006).

Types Of Vaccines

Vaccines Containing Live, Attenuated Micro-organisms -These vaccines are composed of live, attenuated microorganisms that cause a limited infection in their hosts sufficient to induce an immune response, but insufficient to cause disease. To make an attenuated vaccine, the pathogen is grown in foreign host such as animals, embryonated eggs or tissue culture, under conditions that make it less virulent. The strains are altered to make it non-pathogenic, for example, by changing its tropism the pathogen can no longer grow at a site where it can cause disease. Some mutants will be selected that are capable of growing in a foreign host. For the original host this will be non pathogenic in nature. These vaccines may be given by injection or by the oral route. A major advantage of live virus vaccines is that because they cause infection, the vaccine very closely reproduces the natural stimulus to the immune system. Examples include yellow fever, measles, rubella, and mumps. A related strain called BCG [Bacillus Calmette-Guérin] is a weakened version of the live tuberculosis vaccine which can cause tuberculosis in cows but it is not dangerous like the contagious live tuberculosis strain for example measles, mumps, and rubella ("German measles") vaccines. The Sabin oral polio vaccine (OPV) is another example.

Vaccines containing KILLED microorganisms - These pathogenic microorganisms are chemically killed or killed by heat to make it harmless. These are preparations of the normal (wild type) infectious, pathogenic microorganisms that have been rendered non-pathogenic.Examples are vaccines against flu, cholera, bubonic plague, and hepatitis A, typhoid etc. The viral vaccines contain whole virus particles which are inactivated (usually with formaldehyde) so that they cannot replicate in the host cells. They still keep some unchanged immunogenic properties which can induce an immune response for example polio vaccine IPV (Salk vaccine).

SUBUNIT - Subunit vaccines contain purified antigens instead of whole organisms. Such a preparation consists of only those antigens that elicit protective immunity. Subunit vaccines are composed of toxoids, subcellular fragments, or surface antigens. Administration of whole organism, as in case of pertussis was found unfavorable immune reactions resulting in severe side effects. The effectiveness of subunit vaccines is increased by giving them an adjuvant. Adjuvants help to slow down release of antigen thereby giving more prolonged immune stimulation. An adjuvant can increase the effectiveness of subunit vaccine. Examples are the subunit vaccine against HBV and the virus like particle vaccine against HPV. Vaccine against HBV is composed of surface proteins of virus(produced in yeast) and the virus like particle(VLP) vaccine against human papillomavirus (HPV) is composed of the viral major capsid protein.

Advantages:

They can safely be given to immunosuppressed people

They are less likely to induce side effects.

Dis-Advantages:

Sometimes antigens may change their native conformation, and then antibodies produced against the subunit may not be able to recognize the same protein on the pathogen surface.

Isolated protein does not stimulate the immune system as well as a whole organism vaccine.

TOXOIDS - These are inactivated bacterial toxins which are no longer toxic but can induce a strong immunogenic response and thereby providing immunity towards a specific disease. Diseases like diphtheria and tetanus are caused not by the growth of the bacterium itself but due to the toxic proteins liberated by the causative organisms. These toxins can be denatured with certain chemicals for example, formaldehyde. These denatured toxic proteins are no longer dangerous, but continue to keep some epitopes on the molecules sothat they can induce immunogenic response. Examples of toxoid-based vaccines include tetanus and diphtheria.

RECOMBINANT VACCINES:

Genetic engineering is also used to produce vaccines. In this desired antigen producing gene of a microbe is isolated and inserted in to a suitable cloning vector. Different strategies are used to produce recombinant vaccine.

Genetically modified vector (e.g., Vaccinia virus) that is expressing desired antigen is used to create a vaccine. The modified vector (e.g., yeast) is grown in a suitable medium to express desired antigen. This antigen is purified and used as a subunit vaccine. Other expression vectors include the bacteria Escherichia coli, mutant Salmonella spp. and BCG.

Introduction of a mutation by deleting a portion of DNA such that they are unlikely to revert can create an attenuated live vaccine. Attenuated live vaccines can be produced by mutation. In order to use as an attenuated vaccine, a part of DNA is deleted. This prevents its reversion to its original state.

Genomes of virulent and avirulent strains are reasserted to produce live attenuated vaccine.

Fruits bearing foreign antigens may be produced by introducing antigens coding genes in to respective plants. Experiments on such edible vaccines are still going on.

Examples:

Hepatitis B Virus (HBV) vaccine is a recombinant subunit vaccine. Hepatitis B surface antigen is produced from a gene transfected into yeast (Saccharomyces cerevisiae) cells and purified for injection.

Vaccinia virus may be engineered to express protein antigens of HIV, rabies etc. Foreign genes cloned into the viral genome are expressed on the surface of infected cells in association with class I MHC molecules. The antigen-MHC complex induces a Tc cell response.

B subunit of cholera toxin, the B subunit of heat-labile E. coli enterotoxin (LT), and one of the glycoprotein membrane antigens of the malarial parasite are being developed using this technique.

Salmonella typhimurium engineered to express antigens of Vibrio cholerae.

BacilleCalmette-Guérin vaccine strain engineered to express genes of HIV-1.

Reassortment of genomes between human and avian strains to create Influenza vaccine. Human and swine strains to create Rotavirus vaccine.

Advantages:

Vectors which are safe as well as easy to grow and store can be chosen.

Antigens which do not induce protective immunogenic response or which cause damaging responses are eliminated from the vaccine. Example Cholera toxin A can be safely removed from cholera toxin.

Disadvantages:

Locating and cloning of the gene for desired antigen and its efficient expression in the new vector increases the cost of production.

When engineered vaccinia virus is used to vaccinate, care must be taken to spare immunodeficient individuals.

DNA VACCINES - These vaccines are still in experimental stage. Like recombinant vaccines, genes for the desired antigens are located and clonedThe "gene gun" with compressed gas blows the DNA in to the muscle cells of the animals to be vaccinated. DNA-coated gold particles are used in bombarding the skin and thereby introducing DNA in to the tissues. DNA can also be introduced in to nasal tissue using nasal drops. Some muscle cells express the pathogen DNA to stimulate the immune system. DNA vaccines have induced both humoral and cellular immunity.

Advantages:

DNA is very stable, it resists extreme temperature and hence storage and transport are easy.

A DNA sequence can be changed easily in the laboratory.

The introduced DNA only encodes the proteins of interest but does not replicate.

Since protein components are absent there is no immune response against the vector itself.

Because of the way the antigen is presented, there is a cell-mediated response that may be directed against any antigen in the pathogen.

Disadvantages:

Insertional mutagenesis is caused due to potential integration of DNA into host genome.

Induction of autoimmune responses: Against introduced DNA, anti-DNA antibodies may be produced.

Induction of immunologic tolerance: The expression of the antigen in the host may lead to specific non-responsiveness to that antigen.

ANTI-IDIOTYPIC VACCINE – In an antibody the antigen binding site (paratope) is a reflection of the three-dimensional structure of part of the antigen (epitope). This unique structure in the antibody is known as the idiotype., it is considered as a mirror of the epitope in the antigen. By introducing the antibody into another animal, antibodies can be produced against the idiotype. This anti-idiotype antibody mimics part of the three dimensional structure of the antigen. This can be used as a vaccine. When the anti-idiotype antibody is introduced into a vaccinee, antibodies (antianti - idiotypeantiobodies) are formed that identify a structure similar to part of the virus and may help to neutralize the virus.

Advantage:

Antibodies against potentially significant antigen can be produced.

Disadvantage:

Only humoral immunity is produced. There is no cellular immunity and poor memory. Identification and preparation of idiotypes is labor intensive and difficult

WORKING PRINCIPLE OF VACCINES:

Disease causing organisms have at least two clear effects on the body:

The first effect exhibiting symptoms such as fever, vomiting, nausea, rash, diarrhea, and many others, and the second effect is the effect that generally leads to final recovery from the infection: the disease causing organism elicit an immune response in the infected host. As the response increases in strength over time, the number of infectious agents are slowly reduced till symptoms disappear and recovery is complete.

The immune response is stimulated by an ‘antigen’ which is a protein present on disease causing orgamism. The resulting multi-fold immune response includes the synthesis of proteins called "antibodies." These antibodies bind to the antigens present on the disease causing organisms and lead to their final destruction. In addition, "memory cells" are also produced as an immune response. Memory cells remain in the blood stream, sometimes for the entire life span of the host, produce a quick protective immune response against subsequent attack by same disease causing agents. If there is a repeated infection, the memory cells would help to induce a quick immune response to inactivate the disease causing agents and to prevent appearance of symptoms.