Establishing a Microbial Control Strategy for Active Pharmaceutical Ingredients

In the pharmaceutical industry, defining the control strategy for a new active pharmaceutical ingredient (API) is a main goal during development of the process for its commercial manufacture. This task includes defining measures to limit microbial content and specifying microbial quality attributes as needed to ensure patient safety. International Conference on Harmonization (ICH) Guideline Q6A1 provides a framework for determining on a case-by-case basis when microbial limits should be established. The guidance document identifies information to be assembled during development, and provides a decision tree for determining the need for microbial testing of the drug substance. This paper illustrates how such data may be collected and evaluated to ensure a robust microbial control strategy that is consistent with ICH Guideline Q6A.

Microorganisms may be introduced into APIs via the materials used in their manufacture and through various environmental sources during processing. Once microbes are present, proliferation may occur when conditions are favorable. Likewise, there are key control points that can effectively limit bioburden (the number of microorganisms present in a material) and control microbial growth. A comprehensive microbial control strategy involves limiting the potential for contamination during API manufacture, relying on the bactericidal conditions of the chemical synthesis to limit viability of microorganisms, and appropriately controlling water in the API that is available to sustain microbial growth during storage. These opportunities for control are discussed in the context of establishing a microbial control strategy for nonsterile APIs produced through chemical synthesis.

Engineering controls provide the means to minimize the potential for microbial contamination during processing. In general, materials are manufactured in a high containment facility. The room air is filtered (bag filter with a high-efficiency particulate air [HEPA] prefilter), heated as required, and distributed throughout the building. Personnel wear protective equipment that protects the API from human-sourced microbial contamination. Personnel gowning areas and equipment air locks are part of the facility design to control the flow of airborne particles. Equipment is designed to minimize dead legs and is sloped to allow drainage to reduce opportunities for water to be trapped in the equipment, which could permit microbial growth. Manufacturing equipment is typically cleaned using antimicrobial agents and dried by purging with nitrogen.

The manufacture of an API via chemical synthesis involves a sequence of operations that transform starting material(s) into the desired drug substance. Raw materials including organic solvents may be utilized at various points in the chemistry. Processing conditions such as temperature or pH are often manipulated to control reactions and isolate products. Steps in the synthesis are typically performed in an oxygen-free environment to ensure safety and minimize oxidative by-products. While essential for control of the chemical synthesis, each of these variables also offers the potential to affect bioburden of the API.

Table 1 - Solvent–microbial viability relationship*

Microorganisms cannot thrive in nonaqueous environments. In fact, many of the organic solvents commonly used in the manufacture of nonsterile APIs are bactericidal. The solvents listed in Table 1 were evaluated for their antimicrobial properties. The microorganisms tested represent various classes of organisms commonly found as contaminants in the manufacturing environment. In general, the solvents inoculated with Gram-positive bacterial endospores did not show a significant antimicrobial effect toward these microorganisms. Bacterial endospores contain dipicolinic acid in the spore coat, which enables the spores to resist some heating, chemicals, and desiccation effects when other microorganisms are killed by these conditions. Vegetative bacteria and the fungal spores experienced nearly instantaneous lethality due to the organic solvents’ antimicrobial activity.

Processing conditions such as temperature and pH can have a significant effect on microbial viability.2–4 In general, microorganisms commonly found in the API pharmaceutical manufacturing environment thrive at pH 6–8. Conditions outside this pH range are usually deleterious to organisms. While the temperature required to destroy a microorganism depends upon the individual organism and the other environmental conditions, temperatures above 60 °C are effective in killing most vegetative bacterial cells, and temperatures above 100 °C kill most microorganisms. In 100% aqueous media, microorganisms grow very little or not at all at temperatures of 0–5 °C.

Many of the common microorganisms require oxygen for growth. Executing the chemical process in an oxygen-free environment obtained by purging with nitrogen and processing under a nitrogen headspace effectively reduces oxygen content and limits microbial viability.

Packaging of the active pharmaceutical ingredient is a seemingly simple but important parameter to consider in protecting the material from microbial proliferation during storage. All microorganisms require water to grow and survive.4–6 Water is the one key ingredient needed for metabolic and nutritive purposes and to maintain the structural integrity of the cell wall. Packaging the drug substance with a foil-based outer liner provides an effective moisture barrier.

Given the means to limit bioburden and control microbial growth afforded through engineering controls, synthetic process conditions, and packaging components, there remains potential for microbial proliferation in the API during storage when water is present. Water may be part of the crystalline lattice (“bound” water) or adsorbed to the surface (“free” water). Of the two types of water described, it is the free water, not the bound water, that is required for microbial growth. Since total water in a compound is not necessarily directly related to the potential for microbial proliferation, a technique to measure the free water is needed to assess the quality of material.

Water activity describes the amount of free water in a material that is available for microbial or chemical reactions. It is defined as the ratio of partial pressure of water above the material (p) to the partial pressure of pure water (pw) at the same temperature.4,5,7

aw = p/pw

Water activity instruments measure the equilibrium relative humidity of a material in a sealed or enclosed container (as compared to hygrometers, which measure relative humidity under dynamic conditions). The test material is placed in a closed cell fitted with humidity and temperature probes. Following an equilibration period, the instrument measures the temperature and vapor pressure generated by moisture present in the test material to determine the water activity.

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