PHARMACEUTICAL MICROBIOLOGY - V. A. Galynkin - 2015

PART II. ANTIMICROBIAL AGENTS

CHAPTER 9. PRODUCTION OF CHEMOTHERAPEUTIC DRUGS

9.1 General Concepts of Industrial Drug Manufacturing

Industrial drug manufacturing involves the extensive use of machinery, apparatus, and mechanized or automated production lines. It provides for the mass, serial production of drugs according to standard formulations designed for the average consumer.

Large-scale pharmaceutical production consists of a complex of specialized workshops. A workshop is the primary production unit specialized in performing homogeneous processes (crushing, extraction, packaging, etc.) or manufacturing uniform types of products (tablets, ampoules, aerosols, etc.). Each workshop has several sections where similar operations constituting the technological process are carried out.

operations that make up the technological process. For example, the tablet workshop includes sections for ingredient mixing, granulation, granulate drying, compression, etc.

The operation of industrial enterprises is characterized by strict standardization and production planning. The manufacturing process is conducted under specific standard conditions stipulated by precise instructions compiled into a single consolidated document called the regulations (technological regulations). The regulations represent a set of rules that determine the operational Procedure of a pharmaceutical enterprise for the release of finished products. They provide characteristics of raw Materials, intermediates, and the finished product, indicate The sequence of technological process stages, the Processing mode for materials by stage, the apparatus diagram, Analytical Methods, safety rules, industrial hygiene, and other production conditions. The regulations are the law of production, and any deviation from them is unacceptable. Compliance with the regulations is monitored by the technical control department.

In pharmaceutical manufacturing, technological processes are subdivided into chemical processes, associated with the Chemical synthesis of medicinal substances, and physical processes. The latter include mechanical processes associated with the processing of solid materials (grinding, sifting, mixing, dispensing, compression), hydromechanical processes (liquid mixing, emulsification, filtration), thermal processes (evaporation, Condensation, melting), and mass transfer processes (dissolution, crystallization, drying, extraction, rectification). All these processes require appropriate equipment design, i.e., they are performed using special machines and apparatuses.

The main starting materials for the manufacture of medicinal products are active pharmaceutical ingredients, which can be obtained through chemical or biological synthesis, as well as through the processing of Medicinal plant raw materials or animal Tissues and Organs.

Medicinal products have a defined dosage form, i.e., a state convenient for application. There are solid (powders, tablets, granules), liquid (solutions, Suspensions, emulsions), and semi-solid (ointments, suppositories) dosage forms. The steps in the chemical synthesis of a specific medicinal substance may include mixing reaction ingredients, their thermal Treatment, extraction or chromatographic Separation of reaction products, evaporation, crystallization, drying, etc.

The MAIN STAGES OF the microbiological synthesis of Antibiotics, Enzymes, organic acids, etc. are shown in the diagram (Fig. 59).

Class="center">Fig. 59. Stages of obtaining products of microbiological synthesis.

Herbal remedies, powders, tinctures, extracts, as well as highly purified extraction preparations or individual substance preparations, are prepared from medicinal plant raw materials.

Hormones, enzymes, and non-specific action preparations are obtained from animal raw materials. They may represent dried, defatted, and ground tissues or extracts (highly purified or individual substance preparations).

Homeopathic pharmacy is based on THE PRINCIPLE OF potentiation (dynamization), a special technology for preparing homeopathic remedies. The Essence of this principle is that the process involves the stepwise reduction of the concentration of the starting homeopathic substance in a carrier (solution or powder) by 10 or 100 times at each step through intensive shaking, trituration, and mixing. As a result, preparations are obtained in which the content of the initial substance is reduced to negligible values.

The modern pharmaceutical industry is characterized by the continuous improvement and comprehensive application of new technological approaches based on an understanding of the MECHANISM OF ACTION and pharmacological effect of the medicinal substance, aimed at a common goal—the creation of more effective and safer medical drugs.

Modern pharmaceutical production requires personnel to understand the meaning and significance of each stage of the technological process and to strictly control compliance with regulatory requirements. In this regard, alongside chemistry and biotechnology, microbiology occupies a central place in the general direction of pharmaceutical technology development, and scientific research and progress in these areas determine the success of the entire industry.

A significant portion of the requirements for pharmaceutical product quality and production conditions is controlled by a microbiologist: sterility, microbial contamination of raw materials and non-sterile drugs, and compliance with the industrial hygiene rules stipulated by GMP. These requirements must be well known to all participants in the manufacturing process and strictly observed with an awareness of Structure/19.html">The Importance of carefully executing each of them.

With the advent of pharmaceutical preparations obtained using Introduction/32.html">Genetic Engineering METHODS, more than 80% of sterile dosage forms are prepared aseptically, since these substances are labile and cannot be sterilized in their finished form. Medicinal products prepared using aseptic technology surpass in quality the preparations produced previously.

Techniques for working under aseptic conditions must be mastered not only by microbiologists but also by chemists and all personnel upon whom the release of microbiologically safe products depends.

9.2 Antibiotic Production

The production of antibiotic preparations is a complex and multi-stage process. It consists of a set of sequential studies that can essentially be reduced to the following stages:

1) searching for antagonist microorganisms in nature and isolating them into pure culture;

2) studying the action spectrum and determining the antibiotic activity of the isolated antagonist cultures;

3) optimizing cultivation conditions for antibiotic producers;

4) preliminary identification of the antibiotic at the Cytology/cytology/16.html">Early stages of study;

5) isolation and chemical purification of the active principle from the culture liquid and Cells, as well as comparing the obtained antibiotic with already known preparations in terms of biological and chemical parameters to reveal novel Properties of the obtained substances;

6) studying The Mechanism of action and testing the toxicity and therapeutic properties of antibiotics in animals;

7) developing laboratory-scale production technology for the antibiotic and scaling it up to industrial manufacture;

8) generating new microbial genotypes with enhanced activity from parental strains via Mutations and recombinations using genetic and Cell engineering methods (Fig. 60).

To obtain new antibiotics, In addition to screening for new or genetically modified producers, the following methodological approaches are used:

1) deriving a preparation with novel properties from the starting antibiotic via chemical or biochemical modification of its molecule;

2) directed Biosynthesis through biochemical Modification of the structure obtained chemically;

3) chemical synthesis using natural structures as templates;

4) mutasinthesis. This method includes the following stages:

a) obtaining idiotrophic mutants that require a specific fragment of the molecule (a precursor) for antibiotic production;

b) synthesizing an analog of this precursor (a mutasinton) by chemical methods;

c) cultivating the idiotroph on a medium containing the mutasinton. In this process, the idiotroph incorporates the mutasinton into the molecule of the antibiotic it produces, resulting in novel (mutasynthetic) structures.

5) Obtaining hybrid antibiotics, i.e., substances produced by genetic hybrids; a hybrid antibiotic may contain structures of two different metabolites. They differ from antibiotics obtained by the methods listed above in that they are products of Gene combination.

The main stages of obtaining hybrid antibiotics include:

а) selecting a producer that synthesizes a known antibiotic;

б) searching for a new microorganism for Hybridization;

в) investigating the biochemical pathways of antibiotic synthesis, intermediates, and enzymes;

г) identifying the genes controlling The formation of biosynthetic enzymes and their regulators;

д) constructing recombinant DNA containing a gene combination favorable for the biosynthesis process;

е) cloning the new genetic structure into a recipient culture;

ж) chemical, microbiological, and pharmacological investigation of the new antibiotic.

Natural antibiotics are produced by cultivating a producer microorganism using biotechnological methods. In terms of production volume, the antibiotic industry represents the largest biotechnology-based manufacturing sector.

The goal of any biotechnology is to leverage the physiological and genetic properties of the producer strain

to achieve the maximum yield of the final product. The biotechnological Procedures required for this are carried out using dedicated equipment (Fig. 60).

Fig. 60. Process and instrumentation diagram of batch CULTIVATION OF MICROORGANISMS under sterile conditions: 1 — nutrient medium preparation Reactor; 2 — pump; 3 — medium heater / sterilization Column; 4 — holding section; 5 — medium cooler; 6 — individual air filter; 7 — seed fermenter; 8 — production fermenter; 9 — dosing tank. Water; .......steam; air

The metabolic processes of the producer can be controlled through the following approaches:

1) altering the COMPOSITION OF THE nutrient medium;

2) modifying environmental conditions (Temperature, pH, aeration);

3) designing the bioreactor (fermenter) configuration;

4) regulating the feeding schedule of additional substrates;

5) locking the physiological state of the culture using continuous cultivation methods;

6) employing genetically modified producer strains.

Implementing these methods requires specialized engineering and technological approaches to ensure the biochemical regulation of biosynthesis while preserving The properties of the producer population (avoiding cell damage, autolysis, infection, etc.).

Antibiotic Fermentation (Fig. 61) is generally an aerobic process that requires supplying air into the fermentation medium along with agitation.

Fig. 61. Fermenter. 1 — single-stage impeller; 2 — baffle; 3 — cooling jacket; 4 — agitator drive; 5 — top cover; 6 — air supply pipe (sparger); 7 — vessel body.

The antibiotic industry predominantly utilizes bioreactors with capacities ranging from 30 to 200 m³ equipped with mechanical agitators and automated process control and monitoring systems for fermentation. Fermentation temperature (typically 24-26°C) is maintained by a cooling system. Following fermentation, the biomass is separated, and the antibiotic is recovered from the filtrate (or from the producer cells for certain antibiotics) via extraction, Ion Exchange, ultrafiltration, precipitation, and crystallization. Inoculum preparation, fermentation, and many subsequent operations are performed under aseptic conditions.

Producer culture. The initial strain of the microorganism producing the antibiotic or other BIOLOGICALLY ACTIVE SUBSTANCES is isolated from natural sources (soil, plant substrates, etc.) using specialized screening methods. Because wild-type strains typically exhibit low activity, extensive genetic and Selection work is required—usually involving mutagens—to enhance their productivity. The resulting production strain is stored in an anabiotic state (e.g., lyophilized at low temperatures). Such cultures can be reactivated by inoculation onto a suitable nutrient medium and used to prepare the inoculum.

Inoculum preparation. The culture is transferred aseptically from a nutrient Agar slant into a flask containing the seed medium. When working with Fungi and actinomycetes, spore inoculum is used (500–5,000 spores per 1 L of medium). The flasks are incubated in a shaker incubator. The material from the flasks is then transferred to a 0.5–1 m³ inoculator (representing 0.1% of the medium volume) and grown for 1–4 days. Next, the inoculum is transferred aseptically into a 5–20 m³ seed fermenter (10–12% inoculum relative to the nutrient medium volume, with a cultivation time starting from 1 day). Samples are regularly collected for microbiological and biochemical analyses. The inoculum for the main fermentation is prepared in an amount equal to 5–10% of the nutrient medium volume. This stepped-up inoculum preparation allows for scaling up the volume required to ensure rapid and productive growth in the bioreactor while maintaining the culture in the logarithmic growth phase.

The nutrient medium is formulated to ensure rapid Microbial growth in the early stages and maximum product yield by the end of fermentation. The medium is sterilized with pressurized steam at 120–140°C, either directly within the fermenter or in a dedicated continuous sterilization unit.

Fermentation. The flowchart of a commercial batch process is illustrated in Fig. 61. Prior to filling with the medium, the fermenter and piping system are washed, leak-tested, and sterilized with live steam. To guarantee sterility, chemical disinfectants are often applied as a preliminary Treatment of the fermenter.

The volume of the sterile, cooled nutrient medium in the fermenter must not exceed 70% of its total capacity. The inoculum is introduced into the fermenter through the inoculation line using sterile air. Prior to inoculation, the temperature and pH of the nutrient medium must be adjusted to the optimal values for the specific culture.

Fermentation is carried out either with aeration (aerobic process) via sterile air sparging, or without aeration (anaerobic process), combined with agitation to facilitate oxygen dissolution in the liquid medium and ensure thorough contact between cells and nutrients. To prevent the ingress of unsterile ambient air into the apparatus, the headspace pressure above the liquid surface is maintained at 20–30 kPa (0.2–0.3 kgf/cm³). Chemical antifoaming agents are added when necessary.

During fermentation, the temperature and pH of the medium are automatically regulated, and additional nutrient medium components are fed according to a preset program. Control samples of the liquid are systematically withdrawn from the fermenter to determine required physicochemical parameters, activity levels, and the absence of contaminating microorganisms.

Fermentation is terminated once the maximum concentration of the target product has accumulated in the medium. Upon completion, the culture broth is cooled to 10–25°C and pumped into holding tanks for subsequent Downstream Processing.

Methods for antibiotic Isolation and Purification are specific to each compound and depend on its physicochemical characteristics. For example, penicillin is recovered from the culture broth by extraction (butyl acetate extraction), whereas streptomycin and tetracycline are isolated using Ion-exchange Chromatography.