3.4: Sterile Compounding & Pharmaceutical Microbiology

3.4: Sterile Compounding & Pharmaceutical Microbiology

Cleanroom Classifications and Airflow Systems

Sterile compounding involves the preparation of medications that must be completely free of viable microorganisms and particulate matter. In Australia, sterile compounding in hospital and manufacturing pharmacies must adhere to the PIC/S Guide to Good Practices for Preparation of Medicinal Products in Healthcare Establishments (PE 010) and the Pharmacy Board of Australia guidelines.

Cleanroom Grading (PIC/S vs. ISO)

The compounding environment is controlled using particulate-filtered air and classified into different grades depending on the level of cleanliness required.

Laminar Airflow Hoods (LAFH)

Laminar airflow hoods provide a Grade A working environment by passing air through a High-Efficiency Particulate Air (HEPA) filter, which removes 99.97% of particles $\ge 0.3\ \mu\text{m}$ in size. Air flows in parallel lines at a constant velocity (~0.45 m/s).

• Horizontal LAFH: Air is drawn through a pre-filter, passes through the HEPA filter at the back of the hood, and blows horizontally forward across the work surface toward the operator.
  • Use: Preparation of non-hazardous sterile products (e.g., intravenous fluids, total parenteral nutrition [TPN]).
  • Precaution: Never use for hazardous drugs (cytotoxics, hormones, radiopharmaceuticals), as air is blown directly at the operator.
• Vertical LAFH (Cytotoxic Cabinets / Biosafety Cabinets): Air passes through a HEPA filter at the top of the hood and blows downward onto the work surface, where it is drawn into grilles at the front and back.
  • Use: Compounding hazardous substances (e.g., chemotherapy agents like methotrexate). This setup protects the product from contamination and the operator from inhaling aerosolised drug particles.

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Aseptic Technique and the First Air Principle

Aseptic technique refers to the methods used to maintain the sterility of components and pathways during compounding.

The First Air Principle

"First air" is the clean, undisturbed air exiting the HEPA filter. It is free of particulate matter and microorganisms.

• Critical Sites: The parts of sterile components that must not be touched or obstructed (e.g., the needle shaft, needle hub, syringe plunger, ampoule neck, and vial rubber septum).
• Obstruction and Turbulence: Placing hands, vials, or waste paper between the HEPA filter and a critical site blocks the first air. This causes turbulence and creates a low-pressure wake that pulls contaminated room air onto the critical site, compromising sterility.
  • In a horizontal hood: Never place hands or objects behind a critical site.
  • In a vertical hood: Never place hands or objects directly above a critical site.

Gowning and Cleaning Protocols

1. Gowning Sequence (Anteroom Grade D to Buffer Room Grade B): Remove jewelry, perform hand hygiene (using chlorhexidine or isopropyl alcohol-based scrubs), don shoe covers, hairnet, mask, gown, and finally, sterile gloves. Sanitise gloves frequently with sterile 70% Isopropyl Alcohol (IPA) during compounding.
2. Hood Sanitation: Clean the LAFH at the start of each shift, before each batch, and after any spill. Wipe with sterile water to remove residues, followed by sterile 70% IPA. Wipe surfaces from top to bottom, and from back (nearest the HEPA filter) to front. Avoid spraying the HEPA filter directly, as this can damage the paper membrane.

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Methods of Sterilisation

Sterilisation is the complete destruction or removal of all viable microorganisms, including bacterial spores. The choice of method depends on the chemical and physical characteristics of the formulation.

1. Moist Heat Sterilisation (Autoclaving)

• Mechanism: Coagulates and denatures microbial proteins.
• Standard Cycle: 121°C at 15 psi pressure for a minimum of 15 minutes.
• Applications: Terminally sterilised aqueous solutions, glassware, surgical instruments.
• Limitations: Cannot be used for oils, dry powders (moisture cannot penetrate), or thermolabile drugs.

2. Dry Heat Sterilisation

• Mechanism: Dehydrates and oxidises microbial cell components.
• Standard Cycle: 160°C for 2 hours or 180°C for 30 minutes.
• Applications: Non-aqueous materials (oils, petrolatum, waxes, silicone, dry powders) and glassware.
• Depyrogenation: Glassware requires $250^\circ\text{C}$ for at least 30 minutes to destroy pyrogens.

3. Sterilisation by Filtration

• Mechanism: Physical removal of microorganisms by passing the solution through a sterile membrane filter with a nominal pore size of 0.22 µm.
• Applications: Thermolabile solutions (e.g., protein formulations, certain eye drops, vitamins).
• Limitations: Does not remove viruses, mycoplasmas, or pyrogens.
• Quality Control: A bubble point test must be performed on the filter post-filtration. This test measures the pressure required to force air bubbles through a water-saturated membrane. If the bubble point pressure is lower than the manufacturer's specification, the filter is ruptured, and the batch is not sterile.

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Pyrogens and Pyrogen Testing

Nature of Pyrogens

Pyrogens are fever-producing substances. The most common pyrogens are endotoxins, which are lipopolysaccharides (LPS) derived from the outer cell wall of Gram-negative bacteria (e.g., Pseudomonas aeruginosa, Escherichia coli).

• Stability: Endotoxins are highly water-soluble, heat-stable, and can survive standard autoclaving or 0.22 µm filtration.
• Clinical Impact: Injection of pyrogens into the bloodstream induces cytokine release (IL-1, TNF-alpha), leading to fever, chills, hypotension, and potentially fatal septic shock.

Pyrogen Testing Methods

1. Limulus Amebocyte Lysate (LAL) Test:
   • Principle: An in vitro test utilizing the blood (amebocytes) of the horseshoe crab (Limulus polyphemus). Endotoxins cause a gel clot or chromogenic change in the lysate due to an enzymatic cascade.
   • Pros: Highly sensitive, rapid, quantitative, and specific for Gram-negative endotoxins.
   • Cons: Does not detect non-endotoxin pyrogens (e.g., chemical pyrogens, viral/fungal pyrogens).
2. Rabbit Pyrogen Test:
   • Principle: An in vivo test where the sample is injected intravenously into three rabbits, and their rectal temperatures are monitored. A specified temperature rise indicates the presence of pyrogens.
   • Pros: Detects all pyrogens (both endotoxin and non-endotoxin).
   • Cons: Expensive, slow, qualitative, and involves animal testing.