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USP 797 Standards for Compounding Sterile Preparations | Aureobasidium

USP 797: A Brief Overview of Standards Issued by the United States Pharmacopoeia for Compounding Sterile Preparations

By Dr. Kamash Ramanathan, EMLab P&K West Regional Laboratory Director, and Dave Gallup, EMLab P&K Co-Founder

Sterile compounding procedures require clean facilities, specific training for operators, air quality evaluations, and a sound knowledge of sterilization and stability principles. The United States Pharmacopoeia (USP) General chapter <797>, titled "Pharmaceutical Compounding - Sterile Preparations," provides procedures and requirements for compounding sterile preparations. Originally issued several years ago, new standards were released on June 1st of this year.

USP <797> refers to the chapter 797 "Pharmaceutical Compounding - Sterile Preparations," the first set of enforceable sterile compounding standards issued by the United States Pharmacopeia (USP). USP Chapter 797 describes the procedures and requirements for compounding sterile preparations and sets the standards that apply to all settings in which sterile preparations are compounded. The standards in this chapter are intended to apply to all persons who prepare "compounding sterile preparations" (CSPs) and all places where CSPs are prepared (e.g., hospitals and other healthcare institutions, patient treatment clinics, pharmacies, physicians' practice facilities, and other locations and facilities in which CSPs are prepared, stored, and transported). USP<797> requirements affect all disciplines involved in sterile compounding, including the physicians, nurses, pharmacists, and pharmacy technicians.

USP <797> requires assigning manufactured products into one of three risk factors: low, medium, and high which then dictates what type of controls need to be demonstrably in operation. Products manufactured as an aseptic parenteral have the greatest risk, and therefore the greatest level of control over manufacturing must be proven. After determining the risk factor, a practice gap analysis of the compounding activities is performed followed by developing an action plan for compliance. The compliance action plan may include actions from incorporating simple procedures such as improved gowning, gloving, and hand washing procedures, to incorporating regular air quality monitoring of the compounding room for culturable and non-culturable particulates.

In critical areas, i.e. Class 100 or ISO 5 (the area in immediate proximity of exposed sterilized containers/closures and filling/closing operations), the particles per cubic meter must be no more than 3,520 particles/m3 in a size of 0.5 micrometers or larger when counted at representative locations normally not more than 1 foot away from the work site, within the airflow, and during filling/closing operations. Supporting areas, or clean room areas where the laminar flow stations are located, must meet at least Class 100,000 (ISO 8) air quality.

Clean Air Classification ISO Designation ≥ 0.5 µm particles/m3
100 5 3,520
1,000 6 35,200
10,000 7 352,000
100,000 8 3,520,000

The nature of the activities conducted in a supporting clean area determines its classification. The FDA recommends that the area immediately adjacent to the aseptic processing line meet, at a minimum, Class 10,000 (ISO 7) standards under dynamic conditions. Manufacturers can also classify this area as Class 1,000 (ISO 6) or maintain the entire aseptic filling room at Class 100 (ISO 5). An area classified as a Class 100,000 (ISO 8) air cleanliness level is appropriate for less critical activities (e.g., equipment cleaning).

Both air and surface samples may be taken based on the requirements of the individual situation. Impaction on a media plate is the preferred method for culturable air sampling. Settling plates, as previously suggested in earlier editions of USP<797>, is specifically discouraged in the June 1, 2008 guidelines, a change we support. Surface sampling is required in all ISO classified areas on a periodic basis. Surface sampling can be accomplished using contact plates and/or swabs. Also, sampling of operating personnel gear (clothing and gloves) must be performed at a regular frequency. In addition, "media-fill tests" must be conducted at least annually by each person authorized to make sterile compounds to help verify that they can do so aseptically.

Sampling for the airborne viable particles must be conducted based on a risk assessment of the compounding activities performed. Selected sampling sites must include multiple locations within each ISO Class 5 environment, ISO Class 7, ISO Class 8, and areas in the segregated compounding areas at greatest risk of contamination (e.g., work areas near the ISO Class 5 environment, counters near doors, pass-through boxes). For low, medium, and high-risk level compounding, perform air sampling at locations that are prone to contamination during compounding activities and during other activities such as staging, labeling, gowning, and cleaning. Locations must include zones of air backwash turbulence within laminar airflow workbenches (LAFW) and other areas where air backwash turbulence may enter the compounding area (doorways, in and around ISO Class 5 primary engineering control and environments). Consideration must be given to the overall effect the chosen sampling method may have on the unidirectional airflow within a compounding environment. For low-risk level CSPs with 12-hour or less beyond-use date prepared in a primary engineering control (laminar airflow workbench, biological safety cabinet, compounding aseptic isolator) that maintains an ISO Class 5, perform air sampling at locations inside the ISO Class 5 environment and other areas that are in close proximity to the ISO Class 5 environment. While sampling in addition to documenting sample location, volume of air collected, etc. also note the time of day as related to activity in the compounding area when the sample was collected.

For culturable sampling, a general microbiological medium such as Trypticase Soy Agar (TSA) or Soybean ­Casein Digest Medium should be used to support the growth of bacteria. For fungi, Malt Extract Agar (MEA) or some other media that supports the fungal growth must be used. Media used for surface sampling must be supplemented with additives to neutralize the effects of disinfecting agents (e.g., TSA with lecithin and polysorbate 80).

There are a variety of culturable sampling guidelines.
Air Sampling: Use an impaction sampler to collect as much air as possible without drying the media. The 2008 revision bulletin suggests a sampling volume of 400 to 1000 liters of air, subject to recommendations by the manufacturer of the sampling device. When sampling at these higher sampling volumes, we strongly suggest verifying that the manufacturer states that the device is capable of taking these volumes without drying out the media.

Surface Sampling: Surface sampling can be accomplished using contact plates and/or swabs. When swabbing is used in sampling, the area covered should be greater than or equal to 24cm2 but no larger than 30 cm2.

Gloved Finger Sampling: Contact agar plates must be used to sample gloved fingertips after compounding CSPs immediately after exiting the ISO Class 5 environment. Glove fingertip sampling must occur outside of the ISO Class 5 environment. Do not disinfect gloves with Isopropyl alcohol immediately prior to sampling. Personnel should "touch" the agar with the fingertips of both hands in a manner to create a slight impression in the agar.

USP<797> specifies culturable colony thresholds necessitating corrective actions. The table below lists the action levels adapted from the Microbiological Evaluation of Clean rooms and Other Controlled Environments , that when exceeded must initiate further investigation.

ISO Class Active Airborne (cfu*/m3) Glove Fingertip (cfu/contact plate) Inanimate Surfaces** (cfu/contact plate)
5 > 3 > 3 > 3
7 > 20 not required > 20
8 > 100 not required > 100

* cfu = colony forming units
** Contact plate areas vary from 24 to 30 cm2. When swabbing is used in sampling, the area covered should be at least 24 cm2 but no larger than 30 cm2.

Regardless of the cfu counts, corrective actions must be dictated by the identification of the microorganisms recovered. Highly pathogenic microorganisms regardless of cfu count must be immediately remedied.

Environmental monitoring data must be collected and trended as a means of evaluating the overall control of the compounding environment. Any colony forming unit (cfu) count that exceeds its respective action level should prompt a re-evaluation of the adequacy of personnel work practices, cleaning procedures, operational procedures, and air filtration efficiency within the aseptic compounding location. If highly pathogenic microorganisms are detected, irrespective of the cfu count, they must be immediately remedied with the assistance of a competent microbiologist, infection control professional, or industrial hygienist. An investigation into the source of the contamination must be conducted. Sources may include HVAC systems, damaged HEPA filters, and changes in personnel garbing or work practices. Once determined, the source of the problem must be eliminated, the affected area cleaned, and re-sampling performed.

For further information and guidance please refer to The United States Pharmacopoeia (USP) General chapter <797> "Pharmaceutical Compounding - Sterile Preparations," and Chapter "Microbiological Evaluation of Clean Rooms and Other Controlled Environments."

Microorganism of the Month: Aureobasidium

By Karen Santo-Pietro, EMLab P&K Analyst

Aureobasidium is a widely distributed fungal genus usually found in soil, fresh water, dead plant material, marine estuary sediments and wood. There are approximately 20 accepted species in this genus with the most commonly known being A. pullulans. This genus has also been observed to grow on textiles, foodstuffs, fruits and painted surfaces. In the indoor environment, Aureobasidium growth is commonly found in moist places such as bathrooms and kitchens, especially on shower curtains, tile grout and windowsills. The spores are usually disseminated by wind (when dry) and water.

Aureobasidium spores are difficult to identify on spore traps because of morphologic variation. Its most distinguishing feature is the production of primary blastospores (spores produced by a budding process) arising directly from pigmented, vegetative hyphae on short denticles (protuberances in the hyphae). The spores may be hyaline (colorless) or pigmented, variable in size, one-celled, ellipsoid or ovoid, and completely encased in a slimy coat. These primary spores can give rise to secondary or tertiary spores through yeast-like budding. The conidia (asexual spores) adhere together to form slimy heads. The brown hyphae can differentiate to form chlamydospores (resting spores) or arthroconidia (a spore type resulting from fragmentation of a hypha) at maturity. Generally, we report irregular clumps of dark brown hyphae, dividing in more than one plane to form chlamydospores, as A. pullulans. However, vegetative hyphae from other unrelated dematiaceous (pigmented) fungi, such as Cladosporium, may be indistinguishable from Aureobasidium when blastospores are absent. When chlamydospore-like structures are indistinguishable, we report them in the "other brown" category. Because this fungus is sticky and slimy, spores do not readily become airborne and are not commonly found on spore traps. In direct microscopic examination, it is recognizable if enough diagnostic structures have been preserved on tape lifts or swabs.

In culture, Aureobasidium species grow rapidly on Malt Extract Agar (MEA) and, at first, produce colonies that are yeast-like and cream or pink in color. As the colony ages, a slimy exudate appears and the coloration changes to dark brown or black on the surface. As seen from the reverse side of the agar plate the colony is a pale beige. The mycelium is characterized by irregular, dichotomous (two part) branching, with cells sometimes rounding off and separating, and is variable in thickness. Aureobasidium colonies exhibit distinct radial, "fan-shaped" growth that makes them recognizable among other colonies.

Aureobadisium pullulans has been used to produce pullulan, a biodegradable polysaccharide which, when processed, becomes a shiny and strong fiber used to package food and drugs. It has also been used industrially to remove unwanted components of raw textile materials. One of its negative economic impacts is that it has been associated with the deterioration of pears and oranges in storage or in transit.

Human pathogenicity is uncommon and Aureobasidum pullulans is generally considered a contaminant and not a primary human pathogen. Chronic human exposure via humidifiers/air conditioners can lead to hypersensitivity pneumonitis or "humidifier lung." It is also linked to a few other diseases such as keratomycosis, pulmonary mycosis with sepsis and other opportunistic infections such as cutaneous mycoses.


This article was originally published on September 2008.