Aseptic Techniques

Garbing

Sterile compounding requires that the compounder adhere to additional rules and requirements. Those regulations describe additional facilities and specialized equipment needed for this level of compounding. Special clothing is required. This garb can consist of aprons, sleeves, gloves, hoods, shoe covers, coveralls, head coverings, lab coats, smocks, shirts/pants, hats/caps, face masks, and beard covers. The entire process of garbing is designed to reduce the particle shedding count from personnel.

The garb worn during aseptic compounding also provides some level of microbial containment, but complete containment is not possible. Personnel cleansing and hygiene also play an important role in reducing the microbial burden. Hands must be cleansed carefully and personnel protective equipment worn correctly in order to minimize the microbial contamination of CSPs. Infected skin (e.g., rashes, cuts, sunburn, and weeping sores) or people with active respiratory infections shed particles at higher rates. When individuals wear cosmetics or makeup, more particles are shed.

There is an appropriate order of garbing that can be remembered as covering or clothing from the “dirtiest” to the “cleanest” areas of a person’s outside clothing. This order is given in the table below:

Hand Hygiene

Hand hygiene, which is the most effective method of preventing the transmission of pathogens that cause infection in the community and in health-care settings, is an essential component of good compounding practices. Waterless, alcohol-based hand sanitizers that exhibit persistent activity (that is, effectiveness for six hours) and comply with guidelines and/or criteria from the USP, the FDA, and the Centers for Disease Control and Prevention (CDC), are among the most effective products that ensure an appropriate level of hand hygiene during compounding. In studies of antimicrobial-resistant organisms, alcohol-based products reduce the number of multidrug-resistant pathogens recovered from the hands of health-care workers more effectively than hand washing with soap and water.

Most alcohol-based hand antiseptics contain isopropanol, ethanol, N-propanol, or combination of two of those agents. The antimicrobial activity of alcohols results from their ability to denature proteins and disrupt bacterial and fungal cell membranes and viral capsids. The resulting cell lysis causes intercellular material to enter the cell and cause cell death. Alcohols solutions that contain 60% to 95% alcohol are most effective; the higher concentrations are less potent because proteins need water for the denaturation process.

Alcohols demonstrate excellent in-vitro germicidal activity against gram-positive and gram-negative bacteria, including multidrug resistant pathogens such as methicillin-resistant Staphylococcus aureus, vancomycin-resistant enterococci, Mycobacterium tuberculosis, and various fungi. Some enveloped (lipophilic) viruses such as herpes simplex, human immunodeficiency, influenza, respiratory syncytial, and vaccinia have demonstrated susceptibility to alcohols when tested in-vitro. However alcohols exhibit very poor activity against protozoan oocysts, bacterial spores, and some non-enveloped (non-lipophilic) viruses. Alcohols are not effective against bacilli of the genus Clostridium, including those of Clostridium difficile.

A hand washing procedure is given in Appendix II of the USP-NF Chapter <797>. Below is a process validation check-off sheet that could be used alternatively.

Guidelines recommend that Low Risk Level compounding be done in a ISO Class 5 environment such as a LAWF. The High Risk categories require that a laminar flow hood be located in a Class 100,000 (Category 1) or 10,000 (Category 2) controlled area. In any of these cases, however, working in a laminar flow hood is not sufficient to ensure sterility. The hood does not provide sterility – just an ultraclean work area.

Products will be placed in the hood for assembly or compounding that are either sterilized before hand or will be sterilized by filtration while in the hood. Personnel carrying out these procedures must use techniques to minimize the potential contamination (microorganisms, particulate material, pyrogens) possible during these manipulations. Aseptic Techniques can be defined as the sum total of methods and manipulations required to minimize the contamination of sterile compounded formulations.

The following are considered minimum requirements for good aseptic technique:

• Conduct all manipulations inside a properly maintained and certified laminar flow hood. Allow the laminar flow hood to operate for at least 30 minutes before use in order to produce a particle free environment. Maintain a designated “clean” area around the hood.
• Remove all jewelry and scrub hands and arms to the elbows with a suitable antibacterial agent. Sterile gloves are worn in addition to scrubbing.
• Wear lint-free clothing or clothing covers, head and facial hair covers, and a mask.
• Clean all flat surfaces of the hood with 70% isopropyl alcohol, or other antibacterial scrub such as benzalkonium chloride solution, working from top to bottom, then from back to front.

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• Assemble all necessary supplies in the hood checking each for packaging damage, expiration dates, and particulate material. Use only pre-sterilized needles, syringes, and tubing for medication transfers.
• Remove the dust covering from supplies before placing them in the hood.
• Be sure there are no objects between the HEPA filter and the sterile surfaces, and that there is adequate space between objects. Place the smaller supplies closer to the HEPA filter and larger supplies farther away from the filter.
• Swab all surfaces that require entry (puncture) with 70% isopropyl alcohol or betadine solution. Avoid excess alcohol or lint that might be carried into the solution.
• Give close attention to hand position and the direction of air flow over injection ports or objects being manipulated. Minimize hand movements within the hood.
• To assemble needles and syringes, peel back the protective coverings and attach the needle and syringe, twisting to lock in place. When handling syringes and needles, be sure not to touch any surface that will come in contact with the sterile solution. Only the exterior of the syringe barrel, plunger tip and needle cap or sheath may be safely handled.
• Do all manipulations at least 6 inches inside the outer edge of the hood. Do not remove the hands from the hood until the compounding procedure is complete and the final inspection of the formulation has been made.
• Examine all formulations before removing them from the hood.
• Place all syringes and needles in puncture-proof containers and dispose of them according to institutional procedures.

Working in a specified laminar airflow workstation or clean room (collectively known as primary engineering controls, PECs) does not ensure sterility of a compounded preparation. These PECs provide an ultraclean work area but do not provide sterility. So procedures need to be followed that will minimize the microbial burden as much as possible. For example, products that are placed in these PECs for assembly or compounding are sterilized beforehand or the completed preparation will be sterilized in a final step. Aseptic techniques are used to minimize potential contamination by microorganisms, particulate material, endotoxins, and pyrogens during these manipulations. Many of the procedures that are required in compounding CSPs are mandated by USP–NF, but some of the procedures are best practice extensions of the requirements of USP–NF. Each compounding facility will consider what is required and what is “better” in their individual operation.

At a minimum, personnel should know how to transfer drugs in solution in vials or ampules into commonly utilized containers such as large volume parenterals (LVP) or small volume parenterals (SVP). Those techniques are highlighted in this section.

Discarded gloves, needles, syringes, ampules, vials, and prefilled syringes used in preparing sterile formulations pose a source of contamination and should be disposed of properly. Receptacles that are leak-proof, puncture proof, sealable, and easily identifiable should be used. Needles and syringes should be placed in “sharps” containers. They should not be clipped or recapped in order to prevent aerosolization or accidental needle sticks. Excess solutions should be returned to their original vial, an empty vial, or some other suitable closed container.

The compounded formulation should be inspected for:

• container integrity or leaks in flexible containers
• cracks or leaking stoppers in glass containers
• particulate material properties such as color, odor, fill volume, consistency, etc.
• unexpected precipitation, crystallization, or other physical property

Preparations in flexible containers should be squeezed to ensure the absence of unintended holes and slits. Glass bottles should be examined for cracks and leaking stoppers.

Visual inspection will show two of the six characteristics of parenteral solutions: (1) particulate material and (2) stability if such stability is physically characterized by precipitation or crystallization. The presence (or absence) of particulate material is best determined when the parenteral is held against an illuminated light/dark background.

Formulation properties should be known before hand and documented on the formulation record. The compounded product should be compared to these standards.

The pharmacist should also verify that the product was accurately prepared with respect to:

• ingredients (check used vials, ampules, diluents)
• quantities (check syringe volumes used)
• containers (check final solution container)
• instrumentation (check calibration and settings on repeat or automated dispensing devices)

During compounding, volumes of additives in syringes should be examined to confirm accurate measurements. The volumes of solutions remaining in vials and ampules should be determined to compare to the theoretical volumes required to make the formulation. A mass balance of materials should be evident. Also additive containers and syringes should be available (not discarded in the trash) until the product checks are completed. These inspections should be performed after each individual preparation is completed. Preparations that are not distributed promptly should be inspected again before leaving the pharmacy, the purpose of which is to check for defects such as precipitation, cloudiness, and leakage that may have developed during storage.

Formulations should be subjected to quality control tests as outlined in the formulation records and compounding records. These are generally the physical tests that can be conducted within the pharmacy and may involve weight variation, specific gravity, pH, filtration membrane integrity, etc.

A quality assessment review could involve the review of many items or steps, so it is important to focus on those items that may have significant impact on the entire compounding operation. Examples are:

1. Evaluate the compounding operation with the GMPs.
2. GMPs require information about “method suitability,” which assures that the test being conducted either in-process or at the completion of operations will provide a true outcome. For example, many compounded sterile preparations contain antibiotics and preservatives which could lead to false negative of a sterility test’s results.
3. Evaluate the effectiveness of the current microbiological applications in the compounding operation to a compliant microbiological program that is in compliance with USP-NF General Chapter <1116> Microbiological Control and Monitoring of Aseptic Processing Environments.
4. Preservatives such as antioxidants, antimicrobials, and antifungals often are added to the preparations to improve stability or to protect them from microbial contamination so evaluation of the effectiveness of the preservatives is important in those formulations.

Formulations that are not distributed promptly should be inspected again before leaving the pharmacy. The purpose of the pre-distribution inspection is to check for defects such as precipitation, cloudiness, and leakage which may have developed during storage.

In a general sense, validation is any mechanism that will establish a high degree of assurance that specific processes are achieving their objective. Its ultimate goal is to produce products that consistently meet predetermined specifications and quality attributes. Consistent quality (and improvement if possible) is a “must” for the health and well-being of the patient and should be an on-going process.

Several types of “quality control” can be developed for sterile compounding.

Media Fills

The Validation subsection of Section <1206> of the USP 24/NF19 describes an evaluation procedure commonly referred to as “media fills.” The evaluation involves an operator manipulating microbial growth media (usually soybean casein digest medium) according to a prescribed validation procedure. The procedure requires multiple aseptic transfers to multiple containers. It is recommended that the validation procedure by done at peak periods of fatigue, stress, and pacing demands (e.g., immediately after normal production activity).

The premise behind media fills is that the growth medium will support the growth of the contaminating microbe, and this growth can be detected. The other requirement of the validation is that the media must be manipulated using the same aseptic techniques actually being evaluated.

It is important to note that this validation is not intended to be a one time evaluation. The USP 24/NF19 recommends that competent operators be challenged quarterly. Other references suggest that 40 validation sample should be prepared for each 800 admixtures prepared, or that 10 validation samples be prepared each month. Regardless of the frequency, a competent sterile compounder will need to be evaluated on a regular and on-going basis.

Sterilization

There are several types of sterilization but the most appropriate method will depend on the physical and chemical properties of the preparation and its packaging. Typically, dry heat, filtration, and steam sterilization are used with compounded preparation.

Sterility Testing

Of the two general types of tests, one involves the use of culture medium and the other uses biological indicators. When using culture medium, the test sample can be introduced directly into the medium or the sample can be filtered and the filter transferred to the culture medium. There are several media that can be used in these tests, such as Soybean-Casein Digest Medium (SCDM), Fluid Thioglycollate medium (FTM), and dithionite-thioglycollate broth HS-T.

Biological indicator is usually the preferred method of verifying sterility. It is a preparation of a specific microorganism that is resistant to a particular sterilization process. The microorganisms are embedded either on paper or on plastic strips and are included with the materials being sterilized. USP-NF General Chapter <1035> Biological Indicators for Sterilization states that there are three types of biological indicators: 1) Type 1: a biological indicator that included spores that are added to a carrier and package to maintain the integrity and viability of the incubated carrier 2) Type 2: a spore suspension that is inoculated on or into representative units of the preparation to be sterilized. This represents an inoculated preparation; however, a simulated inoculated preparation may be used if it is not practical to inoculate the actual product 3) Type 3: a self-contained indicator. It is designed so that the primary package, intended for inoculation following sterilization processing, contains the growth medium for recovery of the process-exposed microorganism. This form of biological indicator, together with the self-contained growth media, can be considered a system.

Sterility Testing Failures

USP-NF Chapter <797> addresses the actions that should occur when a pharmacy encounters a sterility failure of a compounded sterile preparation. The chapter is not prescriptive on how to perform an investigation. However, there are certain basic points that should be covered and documented since the compounder may be asked to reproduce the information for future audits or inspections.

If the CSP has been dispensed before starting a failure investigation, a recall should be initiated by appropriate personnel, and patients and physicians should be immediately contacted. A recall should be followed and fully documented. If the CSP has not been dispensed, it should be quarantined until the investigation is complete.

The investigation should have a systemic procedure to review all of the items associated with the compounding of the CSP. Records of the compounding activity should be reviewed and other sterility failures or out of specification results examined for trends or consistent failure points. Further investigation may be needed and it has been suggested to conduct the investigation of the pharmacy operations using the acronym PEEMM, which represents people, environment, equipment, materials, and methods.

Depyrogenation and Endotoxin Testing

Pyrogens are organic compounds that are soluble in water and are not removed by filtration. They usually are endotoxins produced by gram-negative bacteria and are found in the cell wall. Endotoxins are at least 1000 times more potent than any other microbial agent and are stable over long periods of time. The endotoxins increase capillary permeability, and in small concentrations cause a variety of adverse effects such as fever, diarrhea, altered resistance to bacterial infections, complement activation, and leukopenia followed by leukocytosis. In greater concentrations, they cause septic shock that can lead to multiple organ failure and death.

The endotoxin test is the simplest test to perform in-house at the time of compounding. The test uses disposable Limulus Amebocyte Lysate (LAL) cartridges in a handheld spectrophotometer. During the testing procedure, kinetic chromogenic reagents and endotoxin controls are dried in the channels of the polystyrene cartridge. Diluted medication is pumped though the channels and mixed with reagents. The software monitors optical density changes in the solution, determined to reaction times, and interpolates the results against an archived endotoxin standard curve. The analysis is automatic, and the results can be read and interpreted in 15 minutes.

The types of preparations that should be screened for pyrogens include injectable preparations, intrathecal preparations, and all high-risk level sterile preparations made in batches of 25 or more. The decision to do endotoxin testing is often determined by the cost. When time is a factor and results are needed before a preparation is administered, an in-house endotoxin test kit is preferable to submitting a sample to an independent laboratory for testing. If contract laboratory testing is deemed preferable, then determining the reliability and accuracy of the endotoxin testing offered is essential.

Other Methods

• A process validation might involve sending formulations to contract analytical laboratories for testing. Analysis of drug content, sterility, and pyrogenicity can be routinely done using randomly selected samples.
• Process validation could be observing and testing formulation variables such as color, clarity, uniformity of dispersion, odor, consistency, pH, specific gravity, etc.
• The validation could also be documenting adherence to formulation records, policies and procedures, SOPs using compounding records, or techniques or procedures. Some example forms for Home Infusion Pharmacies have been published.

Routes of Administration Requiring Sterile Formulations

Some routes of administration demand that products do not bring microbial contamination with them into the body. This is required because some routes of administration by-pass the body’s natural defense mechanisms, or some tissues or organs are so sensitive and vital that such contamination could be serious.

All of these “sterility demanding” routes are parenteral routes.

NOTE: But not all parenteral routes are “sterility demanding” routes.

The term parenteral means next to or beside the enteral. Enteral refers to the alimentary tract, so parenteral means sites that are outside of or beside the alimentary tract. Since oral, sublingual, and rectal comprise the enteral routes of administration, any other route is considered a parenteral administration site. Topical administration is a parenteral route that does not require sterile formulations.

The parenteral routes of administration are used for various reasons.

• If a drug is poorly absorbed when orally administered or is degraded by stomach acid or the gastrointestinal enzymes, then a parenteral route would be indicated.
• The parenteral routes are also preferred when a rapid and predictable drug response is desired as in a emergency situation.
• Parenteral routes of administration are also useful when a patient is uncooperative, unconscious, or unable to take drug via an enteral route.
• Parenteral routes are used when localized drug therapy is desired.
• They provide a predictable and nearly complete bioavailability.

But there are major disadvantages.

• Most of these parenteral formulations are more expensive than enteral route formulations.
• Most these parenteral formulations must be sterile.
• Many formulations require that a skilled or trained person administer them.
• Once the drug is administered, it may be difficult to remove the dose is there if an adverse or toxic reaction.