PHARMACEUTICAL MICROBIOLOGY - V. A. Galynkin - 2015

PART II. ANTIMICROBIAL AGENTS

CHAPTER 20. IMMUNOBIOLOGICAL PREPARATIONS: PRODUCTION AND QUALITY CONTROL

The major achievements of immunology, stemming from the Structure/182.html">Practical Application of scientific findings, are aimed at the Prevention and Treatment of infectious and non-infectious diseases. In this regard, immunobiological preparations—particularly Vaccines—prove to be significantly more beneficial than any other pharmaceutical products.

Vaccination against smallpox, rabies, anthrax, diphtheria, poliomyelitis, pertussis, measles, tetanus, anaerobic infections, and other diseases has led to a dramatic decline in their incidence. Immunological Methods and The Use of immunobiological preparations are essential for addressing issues related to Blood transfusion, organ transplantation, hemolytic disease of the newborn, as well as the Diagnosis and therapy of numerous disorders.

20.1 Vaccines

Vaccines are preparations containing Antigens derived from one or more infectious agents, designed to induce artificial active Immunity for the prevention and treatment of corresponding diseases. The term was coined by Pasteur in honor of Jenner, who in 1796 demonstrated that inoculation with cowpox—vaccination (from *vaccina*, meaning cow-related)—effectively prevents smallpox. Jenner's vaccine was a brilliant empirical achievement; however, The Development of the immunology of infectious diseases as a science based on the understanding of their Etiology begins with Pasteur's discovery. Pasteur proved the feasibility of attenuating pathogen virulence while preserving their immunological properties, ultimately developing vaccines against rabies and anthrax. This breakthrough laid the foundation for the development of live vaccines containing microorganisms with attenuated virulence (attenuated strains). Today, A wide variety of vaccine preparations exist, which are discussed below.

Live vaccines are prepared from attenuated microbial strains, primarily obtained through the Selection of spontaneous mutants with reduced virulence. To achieve this, microorganisms are cultivated for extended periods under adverse conditions or passaged through susceptible animal hosts. For example, to produce the BCG (Bacillus Calmette-Guérin) vaccine strain, *Mycobacterium tuberculosis* was passaged for 13 years (230 subcultures) on a Bile-containing medium. The anti-rabies vaccine was developed by Pasteur through repeated (113 passages) serial passage of the infectious agent in rabbits until obtaining the so-called fixed virus—that is, a virus with a reliably defined virulent dose for rabbits yet safe for humans.

Vaccine strains generated via induced mutagenesis or genetic recombination are also utilized. Such strains require prolonged monitoring due to the potential risk of reversion to the original virulent phenotype.

Live vaccines are employed for the prophylaxis of bacterial infections (anthrax, tularemia, brucellosis, tuberculosis, plague, epidemic typhus, yellow fever) and viral infections (rabies, poliomyelitis, measles, smallpox, Influenza, mumps).

Killed (inactivated) vaccines are produced from Cells of highly immunogenic strains that have been inactivated via physical methods (heat, ultraviolet irradiation) or chemical agents (phenol, ethanol, acetone, formaldehyde). They are used to prevent bacterial infections (typhoid fever, pertussis, cholera, leptospirosis, *Pseudomonas aeruginosa* infection) and viral infections (tick-borne encephalitis, rabies, influenza). For the treatment of chronic conditions, vaccines prepared from killed Bacteria isolated directly from patients (such as staphylococci, gonococci, shigellae, and brucellae) are utilized.

Subunit (chemical) vaccines are formulated from antigenic fractions of microbial cells. Examples of bacterial chemical vaccines include the typhoid vaccine containing Cell wall Glycoconjugates of *Salmonella* spp., as well as capsular Polysaccharides from *Streptococcus pneumoniae*, *Haemophilus influenzae*, and *Neisseria meningitidis*. Viral chemical vaccines (targeting influenza, herpes, FOOT-and-Mouth disease, tick-borne encephalitis, rabies, etc.) contain surface Components of the viral capsid.

The advantage of chemical vaccines lies in their relatively low ballast content, high stability, low reactogenicity, and minimal risk of adverse side effects. The absence of Nucleic Acids eliminates the risk of reversion to virulence, a potential hazard associated with live vaccines. A key drawback of these vaccines is their lower immunogenicity compared to whole-cell (particulate) vaccines, owing to the rapid clearance of the antigen from the body. To prolong their duration of action, they are administered with adjuvants—substances that enhance the Immune Response (Table 42).

Class="center">Table 42. Adjuvants

Toxoids (anatoxins) are detoxified bacterial exotoxins (derived from agents of tetanus, diphtheria, anaerobic infections, botulism, cholera, Staphylococcal infections, etc.) that retain their immunogenic properties.

Ribosomal vaccines exhibit high protective efficacy coupled with comparatively low toxicity and broad cross-reactivity. This is of significant practical value as it allows for the development of broad-spectrum protective vaccines (effective against multiple serovars of a single species or even across several species within the same microbial genus). A notable practical application is the ribosomal polyvaccine used against Infections caused by *Klebsiella*, *Streptococcus*, *Staphylococcus*, *Proteus*, and *Haemophilus influenzae*.

Recombinant (Introduction/32.html">Genetic Engineering) vaccines are produced using recombinant host cells. A prime example is the hepatitis B vaccine containing the viral polypeptide HBsAg. The Gene encoding HBsAg is cloned into Yeast cells, which then synthesize the protein possessing protective activity. A major advantage of such vaccines is safety, as their manufacturing process requires no contact with live pathogens.

Synthetic vaccines are designed based on the structural mapping of pathogen antigenic determinants essential for immunity. Their primary advantages are chemical purity and safety. An illustrative example is a vaccine containing a synthetic analogue of a cell membrane protein from the malaria parasite (*Plasmodium*). This protein facilitates parasite-erythrocyte binding, and the resulting Antibodies effectively block the pathogen from invading the host's red Blood Cells.

DNA vaccines are engineered using Plasmids that incorporate segments of viral DNA. Inside the Cells of the immunized animal, these plasmids induce the expression of viral antigens, thereby triggering an immune response. These vaccines have not yet found widespread clinical application due to the theoretical risk of malignant cellular transformation mediated by the vaccine DNA.

Anti-idiotypic vaccines are developed based on anti-idiotypic antibodies (AIA). The idiotype of an immunoglobulin is defined by the unique structure of its antigen-binding site, which determines its antigenic Specificity. The hypervariable region of an immunoglobulin molecule structurally mirrors a specific antigenic determinant (e.g., a microbial antigen). Immunizing animals with specific IMMUNOGLOBULINS (idiotypes) yields AIAs that, reflecting the idiotype structure, act as a mirror image of the microbial antigen—essentially serving as an "internal image of the antigen." Thus, anti-idiotypic vaccines function as antigen-free vaccines. They are being developed against pathogens characterized by extensive cross-reactivity or those that are difficult to cultivate (such as hepatitis B virus, rabies virus, Fungi, etc.).

Combined (associated) vaccines contain antigens of diverse origins. For instance, the DTP (DTwP) vaccine comprises killed pertussis cells along with diphtheria and tetanus toxoids.

The stages of vaccine production include strain selection, establishment of storage and cultivation conditions, inoculum preparation, biomass accumulation in specialized bioreactors (fermenters), Separation of microbial cells from the culture medium, and subsequent Processing. Standardization and rigorous quality control represent essential final steps in vaccine manufacturing.

Bacteria are cultivated on nutrient media formulated to ensure the accumulation of sufficient biomass while preserving the strain's immunogenic activity and antigenic specificity.

Viral vaccine strains are propagated in embryonated eggs (chicken, duck, quail) and in cell cultures of human or animal origin. Cell cultures are derived from Tissues (such as human or monkey embryonic Kidneys) dispersed using Trypsin. The resulting cell suspension is transferred to a growth medium where the cells multiply to form a monolayer on the inner surface of the vessel (primary culture). Secondary cultures can be obtained by passaging primary cells into fresh nutrient medium; the viability of such cultures typically lasts 2–3 weeks. Continuous (immortalized) cell lines are capable of indefinite subculturing and generally consist of transformed or tumor-derived cell lines. Subunit (chemical) vaccines are produced by disrupting Bacterial cells and/or extracting antigenic components, followed by purification using appropriate Physicochemical methods. Toxoids are obtained from culture filtrates detoxified with formalin (0.4% formalin, 38°C, 21–25 days) and subsequently purified. Ribosomes are isolated from cells disrupted mechanically or via ultrasonication, using ultracentrifugation techniques. The final vaccine preparation typically contains ribosomes from several microbial species alongside *Klebsiella pneumoniae* peptidoglycan serving as an adjuvant.

Quality control of vaccine preparations is carried out at all stages of their manufacturing, including the control of the finished dosage form, in strict compliance with approved regulatory and technical documentation. During the manufacturing process, the absence of extraneous MICROORGANISMS AND THE preservation of properties (immunogenicity, toxigenicity) of the vaccine strain are monitored. When controlling finished preparations, the following parameters are evaluated: solubility and homogeneity (for dry vaccines upon The addition of a solvent); sterility (by inoculation onto nutrient media); safety (by animal testing); immunogenicity (sensitive animals are immunized with the vaccine and subsequently infected with a lethal dose of the pathogen of the given disease; the percentage of surviving animals indicates the degree of immunogenicity); the tolerability of each vaccine batch is tested on a group of 5 volunteers, assessing their general condition and local reaction; and the correctness of labeling and packaging.

The activity of an anatoxin is determined by its ability to react with a specific antitoxic serum in a flocculation reaction.

Viral vaccines are checked not only for the absence of bacteria and fungi, but also of extraneous Viruses, since production cultures may be contaminated with various microorganisms, including Oncogenic Viruses. Tissue cultures and nutrient media intended for the accumulation of viral material are monitored. The harvested virus is tested for identity, and its viral titer is determined.

20.2 Immunoglobulins

Immunoglobulins are used to induce artificial passive immunity for both the treatment and emergency prophylaxis of infectious diseases, especially in individuals with immunodeficiencies; furthermore, they are employed for diagnostic purposes to identify antigens of unknown origin.

Immunoglobulins are isolated from the serum or plasma of Donors or immunized animals. Plasma is the soluble fraction of blood that does not contain cells (erythrocytes, leukocytes); serum is the soluble fraction formed after Blood Coagulation.

Normal immunoglobulins (g-globulin, polyvalent immunoglobulins), containing antibodies of various specificities, are isolated from the plasma of non-immunized donors and placental blood serum. The main active component of normal immunoglobulins is IgG, with small amounts of IgM and IgA also present. Each batch of normal immunoglobulin preparation is manufactured from a plasma pool obtained from A large number of donors (at least 5,000 individuals), which neutralizes individual variations in antibody titers and ensures the standardization of immunological activity. The preparation contains a wide range of antibodies against the causative agents of bacterial and viral infections (hepatitis, measles, pertussis, poliomyelitis, influenza, etc.). Donor blood is collected into sterile polymeric containers containing an anticoagulant solution (5% sodium citrate solution) to prevent blood clotting. Blood cells are separated by centrifugation and returned to the donor, which significantly reduces the likelihood of adverse effects for them. Immunoglobulins are isolated from plasma by fractionated ethanol precipitation at temperatures below 0°C to prevent Protein Denaturation, lyophilized, diluted to a 10% concentration, sterilized by filtration, and ampouled. Due to the significantly increased requirements for intravenous immunoglobulin preparations, advanced technologies are used in their production: partial Cleavage by Proteolytic Enzymes, reduction, alkylation, additional chromatographic purification steps, etc.

All immunoglobulin preparations are tested for sterility, pyrogenicity, protein content, and safety.

Specific immunoglobulins are isolated from the Blood Plasma or serum of immunized volunteer donors or animals. For example, influenza immunoglobulin is derived from the blood of donors immunized with a live influenza vaccine; staphylococcal immunoglobulin is obtained from the blood of donors who have undergone a course of immunization with staphylococcal toxoid, or from the placental blood of women immunized during Pregnancy for prophylactic purposes.

To obtain certain antitoxic and antiviral immunoglobulins (against tetanus, botulism, diphtheria, anaerobic infection, rabies, measles, encephalitis, etc.), animals (horses, sheep, goats, etc.) are immunized repeatedly with escalating doses of the corresponding vaccine preparation. Once a sufficient antibody titer is reached, blood is drawn, and immunoglobulins are isolated from the serum According to the scheme described above.

Monoclonal Antibodies. Antibodies present in blood plasma are produced by multiple clones of antibody-forming cells and, being polyclonal, represent a heterogeneous group of immunoglobulins. The Diversity of polyclonal antibodies is irreproducible, which complicates the standardization of immunoglobulin preparations used for the diagnosis, prophylaxis, and treatment of corresponding diseases. Monoclonal antibodies possess the highest standardization and specificity; they are produced by a single cell clone and are therefore identical in antigenic specificity, as well as in the class and type of heavy and light chains within the molecule. Conventional antibody-forming cells are incapable of prolonged survival under in vitro conditions. Therefore, hybrid cells (hybridomas) are used as producers of monoclonal antibodies. These are obtained by fusing cells that produce antibodies against a specific antigen with myeloma cells capable of unlimited in vitro growth. Myeloma is a type of malignant tumor formed by the proliferation of plasma cells that synthesize strictly identical immunoglobulin molecules of unknown specificity. For Hybridization, mutagenized myeloma cells are used, which possess marker traits that allow hybrid cells to be separated from parental cells on specialized media. Antibody-synthesizing plasma cells are obtained from the spleens of immunized mice. Fusion is carried out in a medium containing polyethylene glycol, and hybridoma selection is performed on selective media; cells producing antibodies of the desired specificity are selected. They are cultured on specialized media that ensure cell growth and antibody production.

Monoclonal antibodies — Reagents unique in their specificity — are used in The Study of complex molecular structures, receptors, and cell surface markers, as well as in Affinity Chromatography for the Isolation and Purification of substances and cells. In addition, they can be used for the Radiological Diagnosis of tumor localization. To achieve this, antibodies against a tumor antigen are produced and conjugated with a radioactive isotope. Antitumor antibodies conjugated with a therapeutic agent can be useful in Cancer immunotherapy.

Immunoglobulins can be used not only for the therapy and prophylaxis of infectious diseases, but also as immunocorrective agents.

Antilymphocyte immunoglobulins block cytokine receptors On the surface of lymphocytes. They are used to reduce lymphocyte counts during the Immune Response to transplants. They are isolated from the blood serum of horses immunized with human lymphocytes.

Anti-Rh immunoglobulins (the IgG fraction of antibodies against human erythrocyte Rhesus (Rh) antigens) are used to prevent hemolytic disease of the newborn resulting from Rh incompatibility; they are obtained from the plasma or blood serum of Rh-negative female donors.

Monoclonal antibodies against lymphocyte receptors (CD3, CD4) and against IL-2 are designed to selectively suppress individual immune responses by blocking receptors or inhibiting cytokine Functions; they are obtained using hybridoma technology methods.

20.3 Biologically active Peptides

Transfer factor — a low-molecular-weight nucleopeptide that stimulates the cellular immune system and is used in DISEASES ASSOCIATED WITH immune suppression. It is obtained from the Blood Leukocytes of donors with a marked delayed-type hypersensitivity reaction to a specific antigen.

Myelopeptides are produced by Bone Marrow cells, stimulate hematopoiesis, T-lymphocyte activity, and antibody production. They are used in pathological conditions associated with immune disorders, such as Osteomyelitis.

20.4 Hormones

Thymic hormones — thymolin, tactividin, thymosin are isolated from the Thymus gland of cattle and used in cases of immune system weakening following irradiation, as well as in the treatment of ulcers, inflammations, and viral and bacterial infections. Preparations analogous to thymic peptides are obtained by chemical synthesis (thymulin, thymopentin, thymogen).

Pancreatic hormone — Insulin regulates Carbohydrate METABOLISM and stimulates the cellular immune system. It is isolated from the Pancreas of cattle or pigs or produced via Genetic engineering METHODS using recombinant strains of Saccharomyces spp. as producers.

Adrenal cortex hormone — glucocorticosteroids (cortisone and hydrocortisone) and their synthetic analogues (prednison and prednisolone) belong to immunosuppressive drugs; they are used to suppress the activity of lymphoid cells in inflammation, allergies, transplantation, and the treatment of autoimmune diseases.

20.5 Immunomodulators

Immunomodulators — are natural or synthetic preparations capable of exerting a regulatory effect on the functions of The Immune System; moreover, their influence extends to other body systems: vascular, nervous, endocrine, and hematopoietic.

Immunomodulators are synthesized by the body's cells (cytokines, hormones, biologically active peptides), and are also obtained via Biosynthesis or chemical synthesis, being used as therapeutic and preventive agents in various diseases and transplantation.

Interleukins are produced using normal lymphocyte or macrophage cultures, T-cell hybrid cultures, and recombinant microorganism cells. T-cell hybridomas are fusion products of T-lymphocytes producing a specific interleukin and tumor cells capable of unlimited growth. Recombinant producers include E. coli, Saccharomyces cerevisiae, and other microorganisms into whose genomes genes controlling interleukin synthesis (IL-1, IL-2) have been introduced using genetic engineering methods.

Among cytokines, interferon preparations were the first to enter medical practice, being used for viral (see Ch. 8) and certain malignant diseases.

Interferons (IFNs) are a group of proteins and Glycoproteins, each synthesized by specific body cells and performing specialized functions. About 20 natural IFNs are known, differing in structure and biological properties: α-IFN consists of 12 subspecies, β-IFN of 3-4 subspecies, and γ-IFN of 2-3 subspecies. Recombinant IFNs also have various subtypes.

The producer of α-IFN is human peripheral blood leukocytes, which are cultured in a special medium in the presence of a virus—the interferonogen. Native IFN is isolated from the culture fluid by precipitation and chromatography. This method has limited application due to the necessity of using large volumes of donor blood.

α-IFN is synthesized in a culture of fibroblasts (human Connective Tissue cells) in the presence of double-stranded RNA as an interferonogen. γ-IFN is produced in a culture of immune T- or B-lymphocytes; in both cases, the yield is low, making production using human cell cultures an expensive process. Obtaining IFN using recombinant cultures of microorganisms—such as Bacillus subtilis, Pseudomonas aeruginosa, and Saccharomyces cerevisiae—is economically justified. IFN purification is carried out by affinity chromatography using monoclonal antibodies.

Colony-stimulating factors (CSFs) are a type of cytokines with a predominant effect on hematopoiesis, serving as survival and growth factors for hematopoietic progenitors. Recombinant CSF preparations (leukomax, molgramostim, leucogen, lenograstim) are used to normalize suppressed hematopoiesis and activate the immune system, particularly against the Background of cytotoxic tumor therapy and induced immunosuppression during transplantation.

20.6 Immunomodulators of Microbial Origin

Prodigiosan, salmazan, pyrogenal are lipopolysaccharides from The Cell walls of Serratia marcescens and Salmonella spp. that stimulate phagocytosis, lymphocyte activity, antibody production, and interferon formation.

Muramyl dipeptide is a component of the Bacterial cell wall peptidoglycan and acts as a macrophage activator; its synthetic analogue is likopid (N-acetylmuramyl-alanyl-D-isoglutamine).

Fungal polysaccharides—β-glucans from Saccharomyces spp. (zymosan), Schizophyllum commune (schizophyllan), Lentinula edodes (lentinan), sulfated extracellular mannan from Rhodotorula rubra (ronasan), and extracellular glucan from Aureobasidium pullulans (aubasidan)—stimulate phagocytosis and activate immunocompetent cells, thereby increasing the body's Resistance to Infectious and non-infectious diseases.

Nucleic acid-based preparations are obtained from yeast and other microorganisms. Sodium nucleinate, a yeast RNA hydrolyzate, exerts a universal immunomodulatory effect and is effective in various diseases, with macrophages being its primary target.

Vaccine preparations can induce not only a specific immune response but also exert immunomodulatory and therapeutic effects. For example, the BCG vaccine is used not only for tuberculosis prevention but also as a nonspecific stimulator of cell-mediated immunity in tumor diseases.

Antibiotics such as actinomycin and cyclosporine possess immunosuppressive properties. The latter is used to suppress the immune response during transplantation.

20.7 Synthetic Immunomodulators

Immunomodulatory activity is exhibited by many nucleic acid analogues (synthetic polynucleotides), adaptogens, Surfactants (polysulfates, polycarbonates), pyran, imidazole, fluorene, and pyrimidine derivatives, among others. Out of a large number of immunomodulators, only a few find practical application, while the majority are not used due to high toxicity, cost, side effects, etc.

Immunostimulants such as levamisole, dibazole, chloridine, and methyluracil are used when immune system function is impaired, for example, As a result of radioactive irradiation, tumor Chemotherapy, or viral and other infections. Immunosuppressants such as cyclophosphamide, chlorambucil, and mercaptopurine are used to treat certain autoimmune diseases and in transplantology.

20.8 Diagnostic Preparations

Diagnostic methods for many diseases are based on serodiagnosis (serum)—determining the antibody titer in a patient's blood serum using a diagnostic antigen preparation, or detecting pathogen antigens or tumor cell antigens in body tissues using antibodies. Various serological reactions (agglutination, precipitation, Complement fixation), enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, and others are used for these purposes.

Antigen-based diagnostic preparations include vaccine strains of microorganisms, antigenic fractions isolated from pathogen cells, or those obtained through genetic engineering methods. Antibody-based diagnostic agents represent polyclonal or monoclonal immunoglobulins against the corresponding antigen. The sensitivity of Methods Based on monoclonal antibodies vastly exceeds that of reactions using polyclonal antibodies.