PHARMACEUTICAL MICROBIOLOGY - V. A. Galynkin - 2015

PART III. MICROBIOLOGICAL ASPECTS OF PHARMACEUTICAL MANUFACTURING

CHAPTER 22. SOURCES AND ROUTES OF MICROBIAL CONTAMINATION IN PHARMACEUTICAL MANUFACTURING

22.1 General characteristics of Typical Sources of Microbial Contamination in Pharmaceutical Manufacturing

Major biopharmaceutical and chemical operations involve the manufacturing of active pharmaceutical ingredients (APIs), finished dosage products, and immunobiological agents for human and veterinary medicine, as well as various non-medicinal substances used across industries.

Biotechnological manufacturing is a complex, multi-stage process comprising The production of intermediates, API substances, and finished products.

An intermediate refers to partially processed raw Materials or medicinal substances that must undergo further manufacturing stages before becoming a final drug product.

A drug substance or API is a standardized chemical compound (substance) possessing therapeutic or prophylactic properties, approved for use, and intended for the formulation of medicinal products.

A finished drug product (FDP) — is a medicinal product intended for the end consumer in a convenient administration form (dosage form). In this final form, the active substance exerts its maximum therapeutic effect, exhibits minimal adverse reactions, and ensures ease of use and storage.

Active substances (APIs) are produced via chemical synthesis, extraction and chemical purification from plant materials, or through The Use of producer Cell lines (Bacteria, Fungi, plants, and animals) (Fig. 81). The specific Organization of these technological processes directly impacts the microbial contamination of the substance and, ultimately, the final product.

Class="center">Fig. 81. Main technological processes for manufacturing finished drug products (FDPs)

Despite the diverse range of Processing schemes used to obtain Water/28.html">Medicinal substances of various origins, the primary (typical) sources of microbial Introduction into biotechnological facilities include:

— processing equipment;

— raw materials and excipients at all stages of production, storage, and transportation;

— containers and packaging materials;

— water used in manufacturing;

— process and ventilation air;

— manufacturing personnel.

In processes involving producer cell lines, nutrient media and additives, antifoaming agents, and inoculum assume critical importance as additional sources of microbial contamination. Depending on The Nature of the Production Process and the specific manufacturing stage, the relative contribution of these factors to contamination can vary significantly.

22.2 Dependence of Microbial Contamination on the Proper Operation of Processing Equipment

The Role of equipment and piping systems in the potential contamination of Biosynthesis products is particularly crucial during the cultivation of producer Cells. This process must be carried out under aseptic conditions [42].

The Fermentation complex comprises the cultivation vessel (fermenter) along with its associated piping, Valves, instrumentation, and auxiliary systems. Delivery pipelines for material flows (sterile air, steam, nutrient media, and additives) span tens of kilometers. This entire system must guarantee the sterility of the fermenter's interior. Loss of containment within the fermentation complex during operation is a major cause of microbial contamination. This can occur if the technical standard of the equipment fails to meet effective sealing requirements, allowing ambient microorganisms to infiltrate the internal cavities of the apparatus and valves.

The Design Features of equipment and piping do not always ensure the complete sterilizability of all internal surface points. For instance, the bottom section of the fermenter, where condensate accumulates, as well as sampling ports and inoculation nozzles, requires special attention. During sterilization, temperatures in these zones may remain lower than throughout the bulk of the fermenter, potentially leading to non-sterility. Proper Selection of equipment materials is equally vital. It is essential to use materials whose internal surfaces are resistant to biodeterioration and biofouling. High-quality surface finishing (polishing) of internal equipment surfaces is a key prerequisite for preventing biofouling risks.

Replacing natural filter media with synthetic alternatives that are less favorable for microbial proliferation is highly advisable. For example, when using belting fabric for filtration, new samples contain fewer than 500 cells per cm², whereas samples laundered for reuse exhibit up to 10⁴ cells per cm² of surface area.

Other common causes of production contamination via processing equipment include inadequate equipment preparation (such as improper washing and disinfection, or ineffective sterilization) and failure to follow operational Procedures.

22.3 Dependence of Microbial Contamination on Raw Materials and Excipients

Raw materials used in drug manufacturing can be of mineral, plant, animal, or synthetic origin [43, 44]. Materials of animal and plant origin are typically the most heavily contaminated.

22.3.1 Microbiota of Animal-Derived Raw Materials

Animal-derived raw materials serve as primary sources for organotherapeutics. For instance, pancreatin is obtained from the Pancreas of pigs, thyreoididin from The Thyroid Gland of cattle, cytochrome c from Heart Muscle, and thymalin from the Thymus. Organs from slaughtered animals also provide the basis for numerous culture media. In viral vaccine production, cell cultures derived from the Kidneys of monkeys, guinea pigs, and dogs are utilized, along with embryonated eggs from chickens, ducks, and quails, among others.

Animal organs and Tissues can harbor high concentrations of microorganisms, including members of the animal's normal microbiota as well as pathogens that invaded specific organs hematogenously during the animal's life, often due to weakened Immunity caused by prolonged starvation, fatigue, or similar stressors. Microorganisms can also contaminate tissues post-mortem or during handling—via tools, hands, or workers' clothing—during initial carcass processing, storage, and transport.

In terms of qualitative composition, the microbiota of animal raw materials may comprise saprophytic, pathogenic, and opportunistic microorganisms, predominantly aerobic and facultatively anaerobic bacteria, coliforms (such as Proteus, Aeromonas, and Salmonella species, Clostridium perfringens, and Bacillus cereus). Their counts can reach 10–10⁵ cells per 1 cm³ of surface area. Many microorganisms remain viable in raw materials for extended periods during freezing; for example, salmonellae can survive in frozen meat for 13 months and in eggs for 12 months. Animal raw materials may also carry various Viruses. Notably, monkey cell cultures used to produce multiple Vaccines have been found to contain Adenoviruses, enteroviruses, and monkey Herpesviruses, all pathogenic to humans. Therefore, mandatory supplemental virological testing using specialized Methods is required.

22.3.2 Microbiota of Herbal Medicinal Raw Materials

Herbal medicinal raw materials (HMRM) refer to dried parts of medicinal plants that have not undergone chemical processing. These include roots, rhizomes, bark, flowers, leaves, fruits, and buds. Currently, approximately 200 plant species are utilized to derive medicinal products.

Like all plants, medicinal plants serve as a natural habitat for microorganisms (Chapter 3). Their microbiota is divided into epiphytic (from epi — upon, phyton — plant) and phytopathogenic (from pathos — disease, suffering). Epiphytes are microorganisms that normally develop on the plant surface without causing harm. They do not penetrate plant tissues, subsisting instead on normal plant exudates and organic surface contaminants. These microorganisms are resistant to phytoncides, desiccation, and ultraviolet radiation, and they help prevent phytopathogenic microorganisms from invading plant tissues. The predominant component of epiphytic microbiota is Erwinia herbicola, an antagonist of soft-rot vegetable pathogens. Normal flora also includes Pseudomonas fluorescens, Phytomonas spp., Chromobacterium spp., Bacillus mesentericus, and low levels of fungi.

Microbial composition depends on plant species, age, stem height, soil type, and growing conditions. Epiphytic populations increase with higher humidity and decrease under dry conditions. Phytopathogenic microorganisms include bacteria, fungi, and viruses. The most common bacterial plant diseases (bacterioses) and fungal diseases (mycoses) are listed in Table 46.

Table 46. Main pathogens causing plant bacterioses and mycoses

Plant viruses (viroses) cause mosaic symptoms (mottled coloration of leaves and fruits) and yellows, which manifest as plant stunting and The Development of abnormal lateral shoots and flowers.

Signs of microbial damage in HMRM include the following.

1. Dry and wet rot (softening and degradation of specific plant tissue areas) resulting from bacterial and fungal proliferation.

2. White coating (powdery mildew) on leaves and shoots caused by fungal multiplication.

3. Yellowing and spotting induced by fungi and bacteria.

4. Sooty mold, characterized by the appearance of a dark, easily removable film on leaves and shoots due to fungal growth.

5. Blight — blackening of shoots, leaves, fruits, and flowers driven by the proliferation of the bacterium Erwinia amylovora.

6. Deformation — alterations in plant organ Morphology (such as SHOOT curvature and leaf curl) resulting from fungal infection.

7. Tumors — localized enlargement of stems, branches, roots, and rhizomes caused by cell hyperplasia at sites damaged by bacteria, fungi, or mechanical injury.

8. Leaf mosaic — The Emergence of pale, angular spots alternating with normally colored areas, caused by viral infection.

Affected plants undergo alterations in Cellular Structures and tissue chemistry, accompanied by a decrease in biologically active compounds, rendering such raw materials unsuitable for use.

In addition to epiphytic and phytopathogenic microbiota, HMRM can become contaminated with extraneous microorganisms at any processing stage—namely, during harvesting, drying, grinding, granulation or briquetting, packaging, transportation, and storage.

During storage, high humidity (>60%) and elevated Temperature (>20°C) promote the active proliferation of microorganisms. This can lead to the deterioration of medicinal plant materials (MPM) due to the growth of fungi of the genus *Aspergillus*, *Penicillium*, and other widespread cosmopolitan microorganisms. Under such conditions, the content of active substances drops sharply. For example, digitalis leaves lose 50% of their potency due to microbiota action, while lily of the valley leaves lose 30%.

When contaminated MPM is used, pollutant microbes enter the substance and the finished dosage form. The use of such MPM is of particular concern when preparing aqueous extractions, such as infusions, decoctions, and other dosage forms at home.

Negative consequences of using contaminated MPM may include a reduction or complete loss of therapeutic value, allergic reactions, the onset of infectious diseases, and the introduction of toxic byproducts. Among the latter, fungal toxins (produced by *Fusarium*, *Penicillium*, *Aspergillus*, etc.) that cause mycotoxicoses are particularly dangerous.

To prevent the Adverse effects of microbial contaminants introduced via raw and auxiliary materials, microbiological purity requirements have been established for these pharmaceutical manufacturing components (Table 47).

22.4 Water as a Raw Material and Excipient

Water is used in drug manufacturing as both a primary and auxiliary material. It serves as a component of nutrient media and finished dosage forms. It is utilized in the technology of isolating and purifying BIOLOGICALLY ACTIVE SUBSTANCES (BAS), for the sanitary preparation of facilities and equipment, and for preparing disinfectant and antiseptic solutions. Technological processes employ drinking water from centralized municipal water supply systems, as well as purified water produced in manufacturing via distillation, Ion Exchange, reverse osmosis, and electrodialysis. The production of sterile medicinal products requires water for injection, which is derived from purified water.

The United States Pharmacopeia provides for the use of drinking water, purified water (for the production of non-sterile medicinal products), sterile purified water (for topical dosage forms); water for injection, sterile water for injection, sterile bacteriostatic water (with added biocides), and sterile water for inhalation products. This variety is due to the significant impact that water quality has on the quality of medicinal products.

Water quality, along with its preparation, distribution, and storage systems, constitutes a critical control point when evaluating factors that affect pharmaceutical safety.

Water quality is regulated by normative and technical documentation, including GOST standards, Sanitary Rules and Norms 2.1.4.1074-01, and pharmacopeial monographs for purified water (FS 42.2619.97) and water for injection (FS 42.2620.97). Purified water must not contain more than 100 microorganisms per 1 mL, excluding members of the *Enterobacteriaceae* family, *Staphylococcus aureus*, and *Pseudomonas aeruginosa*. Water for injection must be apyrogenic and contain no more than 10 CFU per 100 mL.

During storage in industrial reservoirs, the microbial count increases rapidly and can reach 10⁵–10⁶ bacterial cells per liter. *Legionella pneumophila* is capable of multiplying in water at 57⁰C. Fittings and equipment used for water storage and distribution (pumps, pipelines, valves, water meters, etc.) can be colonized by microorganisms that form biofilms on their inner surfaces; these biofilms are resistant to biocides and pose a threat as a source of microbial contamination. Therefore, special attention is paid to the design of the water Treatment system. Pipelines may be made of stainless steel, specially manufactured polymeric materials, or Glass. Their joints and layout must allow for sterilization by flushing with cleaning and sterilizing solutions at a velocity of at least 1.5 m/s in the system's largest-diameter pipes. Water must be stored at a temperature of no less than 80 °C and circulated through the distribution system at a velocity of 1–2 m/s to prevent biofilm formation. Recirculation regimes are employed to prevent biofouling on vessel walls.

Table 47. Microbiological purity of substances and excipients used in the manufacture of medicinal products [29].

Notes to Table 47:

1. Depending on the COMPOSITION OF THE medicinal product and the specifics of its manufacturing process, normative documents may exceptionally specify other limits.

2. If pathogenic bacteria other than those specified above are detected during testing, the medicinal products, substances, and excipients are considered to fail the requirements for "Microbiological Purity."

22.5 Dependence of Production Facility Microbial Contamination on Air

The air in production areas may be ambient air, entering without preliminary purification, and supply air, delivered via air-conditioning systems. Process air is used for aeration during the cultivation of producer cell lines, for transporting process liquids and bulk materials from one vessel to another, and for dry-heat sterilization of primary packaging materials (e.g., ampoules for injection solutions).

The causes of microorganism introduction into production objects via air include high initial contamination of ambient air—especially if it enters without preliminary purification—and inefficient operation of the air-conditioning system. Depending on the stringency of the air purity requirements, the required number of filtration stages is selected at the design stage.

22.6 Excipients in the Manufacture of Finished Dosage Forms (FDF)

Excipients are used in FDF manufacturing technology to ensure the maximum biological activity of the drug within The Human Body. Technologically, they serve as fillers to achieve the required tablet mass (glucose, lactose, sucrose, starch, sodium chloride, etc.); flavor enhancers (glucose, sucrose); sustained-release agents (certain oils, such as cotton-seed oil); and vehicles to impart the required dosage form (cocoa butter for suppositories or gelatin for capsules). Many of these can harbor significant numbers of microorganisms.

22.7 Packaging Materials and Their Role in FDF Contamination

A distinction is made between primary (or individual) packaging, which is in direct contact with the drug; secondary packaging, which groups a certain number of primary packages; and transport packaging, used to deliver products to storage or distribution sites.

Primary FDF packaging is a container or vessel that provides long-term Protection of the product from environmental factors, including exogenous contamination and moisture. Primary packaging materials include vials, glass ampoules, polyethylene dropper tubes, polyvinyl chloride blister packs, films, and aluminum foil. Bioburden levels depend on the Nature of the material, its microbial resistance, and its moisture content. The use of biodegradable-susceptible packaging materials (paper, cardboard, cork stoppers) is not recommended.

Packaging materials with smooth, impermeable surfaces typically exhibit low levels of microbial contamination, but under improper storage conditions, they can be actively colonized by microorganisms. For example, bacterial and fungal spores have been detected in glass containers stored in humid environments.

One of the primary reasons packaging materials can become a source of microbial contamination is the adaptive capacity of microorganisms to utilize them as substrates in metabolic processes.

22.8 Quarantine

To prevent the potential spread of microorganisms from a contaminated source, batches of raw materials, excipients, packaging and labeling materials, intermediates, and finished products are assigned a quarantine status. This entails storing them separately or using other methods that preclude their use or distribution until a formal release decision is made.

22.9 Dependence of Microbial Contamination on Personnel

Humans are a primary source of microbial and particulate contamination [40, 42]. The principal causes include:

1) the presence of diverse and abundant human body microbiota (resident and transient);

2) the execution of technological operations by individuals suffering from gastrointestinal, Skin, or respiratory disorders, those who are microbial carriers, or those with abnormally high perspiration rates or dry skin;

3) the absence or poor condition of cleanroom garments;

4) failure of personnel to comply with personal and production hygiene requirements, as well as non-observance of behavioral rules during the manufacturing process;

5) improper staff selection without regard for the nature of production and the individual CHARACTERISTICS OF THE worker.

Microorganisms can be introduced into the manufacturing environment from personnel via:

1) airborne droplet transmission through secretions from the Oral Cavity and Upper Respiratory Tract;

2) airborne dust and contact transmission from areas of skin not covered by clothing (face, neck, hands, Hair);

3) airborne dust and contact transmission from personal work garments.

A significant number of microorganisms are released from the human upper respiratory tract into the surrounding environment: for instance, a single sneeze disperses up to 100 × 10³ viable Bacterial cells and Viral Particles over a distance of 10 m or more; saliva contains up to 100 × 10⁶ cells/mL, and nasal secretions contain up to 10 × 10⁶ cells/mL.

The most heavily colonized skin areas are exposed regions: the hands, the skin beneath the fingernails, the face (around the nasal alae), and the neck. The quantity of mechanical and microbial particles depends on the nature of the movements performed during production. A motionless person sheds up to 10 × 10³ mechanical and microbial particles into the environment per minute; while sitting and making slight movements with the hands and HEAD, this figure rises to up to 500 × 10³, and during intense work, up to 10⁶.

The resident normal microbiota cannot be removed mechanically by washing. During work, it rapidly regenerates due to heavy sweating, which facilitates the emergence of microorganisms from the pores of the sebaceous and Sweat Glands. The hands are the most heavily contaminated areas, particularly the first Phalanges of the three working fingers, the palm depression, the skin near the wrist, and the interdigital spaces. Other skin areas vary in microbial load. The number of aerobic bacteria per 1 cm² of scalp is approximately 1.5 × 10⁶; in the axillary region, 2.4 × 10⁶; and on the forehead, 0.2 × 10⁶.

Conclusion

Raw materials of animal origin may be contaminated with saprophytic, pathogenic, and opportunistic microorganisms, such as coliforms, species of the genera Proteus, Bacillus, and Salmonella, among others, and may also harbor various viruses.

Medicinal plant materials may contain phytopathogenic bacteria (Pseudomonas, Xanthomonas, Erwinia, etc.), epiphytic bacteria, and Molds (Penicillium, Aspergillus, etc.).

To prevent Microbial spoilage of raw materials, storage conditions (temperature, humidity, etc.) must be strictly observed.

The microbiological purity of substances and excipients used in drug manufacturing is recommended by the Pharmacopoeia.

The quality of potable water is regulated by sanitary standards (SanPiN), while purified water and water for injection are regulated by Pharmacopoeial Monographs (FS).