UNIT OPERATION / PRODUCTION PROCESS


Introduction

• Standards of Sterility
  • While there is general agreement that sterilization of the final filled container as a dosage form or final packaged device is the preferred process for assuring the minimal risk of microbial contamination in a lot, there is a substantial class of products that are not terminally sterilized but are preferred process for assuring the minimal risk of microbial contamination in a lot, there is a substantial class of products that are not terminally sterilized but are prepared by a series of aseptic steps.
  • These pharmacopeial procedures are not by themselves designated to ensure that a batch of product is sterile or has been sterilized. This is accomplished primarily by method suitability of the sterilization process or of the aseptic processing procedure.
• Why Perform Sterility Tests?
  • For each batch of drug product purporting be sterile and/or pyrogen-free, there shall be appropriate laboratory testing to determine conformance to such requirements.
• Points to Consider for Sterility Testing
  • The test may be carried out using the technique of Membrane Filtration or by Direct Inoculation of the Culture Medium with the product to be examined. Appropriate negative controls are included. The technique of membrane filtration is used whenever the nature of the product permits; that is for filterable aqueous preparations, for alcoholic or oily preparations, and for preparations miscible  with, or soluble in, aqueous or oily solvents, provided these solvents do not have an antimicrobial effect in the conditions of the test.
  • Use membrane filters having a nominal pore size not greater than 0.45 micrometer, in which the effectiveness to retain microorganisms has been established.

Regulatory Requirements for the Materials Used

  • Soybean Casein Digest Broth (SCDB) and TSB (Tryptic Soy Broth, Trypticase Soy Broth, Tryptone Soy Broth)
    • pH of 7.3 +/- 0.2 after sterilization
    • Store at 2°-25°C in a sterile well-closed container, unless it is intended for immediate use.
    • Do not use the medium for a longer storage period than has been validated.

  • Fluid Thioglycollate Medium (FTM)
    • pH of 7.1 +/- 0.2 after sterilization
    • Store at 2°-25°C in a sterile well-closed container, unless it is intended for immediate use.
    • If more than the upper 1/3 of the container is pink, it can be restored once by heating the containers in a water-bath or in free-flowing steam until the pink color disappears and by cooling it quickly.
    • Do not use the medium for a longer storage period than has been validated.
  • Alternative Fluid Thioglycollate (CTM)
    • pH of 7.1 +/- 0.2 after sterilization
    • Heat in water bath prior to use and incubate at 30-35°C under anaerobic conditions.
  • Other Media
    • Equivalent commercial media may be used provided that they comply with the growth promotion test.
    • Products containing mercurial preservative that cannot be tested by membrane filtration method: FTM at 20-25°C may be used instead of TSB.
    • Where prescribed or justified and authorized alternative FTM may be used (without agar and resazurin sodium solution).
    • For Direct Innoculation: Media for Penicillins or Cephalosporins
    Culture Media Growth: Regulatory Requirements USP
  • Sterility: No growth after 14 days
  • Growth Promotion Test of aerobes, anaerobes and fungi
    • SCDB/TSB: Aspergillus brasilliensis, Bacillus subtilis, Candida albicans
    • FTM: Clostridium sporogenes or Bacteroides vulgaris, Pseudomonas aeruginosa or Micrococcus luteus (Kocuria rhizophila), Staphylococcus aureus.
    • Alternative FTM: Clostridium sporogenes
    • Not more than 100 CFU in separate portion of medium.
    • Strains used for inoculation are not more than 5 passages removed from the original master seed-lot.
    • Not more than 3 days in case of bacteria.
    • Not more than 5 days in case of fungi.

    • Suitability Test

    • The suitability test have to be carried out before, or in parallel, with the test on the product to be examined.
    • Pharmacopoeias require that sterility testing should be performed to confirm the sterility of the microbiological medium.

    • Growth promotion Test

    • To confirm the ability of the test medium to support the growth and reproduction of selected microorganisms.
    • Test each lot of readily prepared medium and each batch of medium prepared either from dehydrated medim or from ingredients.

    • Sterile Buffers

    • Purpose:
      • Assure the sterility of rinsing fluid.
      • Prevent the occurrence of false positive results during the sterility test of the test article.
    • The test method used: membrane filtration
    • The rinse buffer is considered sterile if after filtration there is no microbial growth observed within 14 days.

    • Method Suitability Test

    • Certain products may contain bacteriostatic or fungistatic agents which if not neutralized, will inhibit the growth of viable microorganisms present in the product, producing false negative results.
    • Neutralization of these products may be achieved via:
      • Dilution
      • Chemical neutralization
      • Filtration and Rinsing
      • Enzyme activity
      • Or the combination of the above four methods.

    Test for Sterility

    • Membrane Filtration Method

    • Precautionary Measures: The tests for sterility must always be carried out under highly specific experimental parameters so as to avoid any least possible accidental contamination of the product being examined, such as:
      – a sophisticated laminar sterile airflow cabinet (provided with effective hepa-filters);
      – necessary precautionary measures taken to be such so as to avoid contamination that they do not affect any microbes which must be revealed duly in the test;
      – ensuing environment (i.e., working conditions) of the laboratory where the ‘tests for sterility’ is performed must always be monitored at a definite periodical interval by:
      • sampling the air of the working area,
      • sampling the surface of the working area, and
      • perforing the stipulated control tests.
    • Methodology : In usual practice, it is absolutely urgent and necessary to first clean meticulously the exterior surface of ampoules, and closures of vials and bottles with an appropriate antimicrobial agent ; and thereafter, the actual access to the contents should be gained carefully in a perfect aseptic manner. However, in a situation where the contents are duly packed in a particular container under vacuum, introduction of ‘sterile air’ must be done by the help of a suitable sterile device, for instance: a needle duly attached to a syringe barrel with a non-absorbent cotton.

    • Direct Inoculation Method

    • Nutrient Broth
      — Importantly, it is exclusively suitable for the ‘aerobic microorganisms’.
      • Oxidation-reduction potential (Eh) value of this medium happens to be quite high to enable the growth of the anaerobes specifically.
      • Importantly, such culture media that particularly allow the growth of fastidious microorganisms, such as: soy bean casein digest broth, Hartley’s digest broth.
    • Cooked Meat Medium and Thioglycollate Medium
      — These two different types of media are discussed briefly as under :

      (a) Cooked Meat Medium: It is specifically suited for the cultivation (growth) of clostridia.

      (b) Thioglycollate Medium: It is particularly suited for the growth of anaerobic microbes. It essentially comprises of the following ingredients, namely :

      • Glucose and Sodium thioglycollate
      • that invariably serve as an inactivator of mercury compounds,
      • to augment and promote reducing parameters,
      • an oxidation-reduction indicator.
      • Agar
    • Sabouraud Medium
      — It is a medium specifically meant for fungal species. It essentially bears two vital and important characteristic features, such as :
      • an acidic medium, and
      • contains a rapidly fermentable carbohydrate e.g., glucose or maltose

    Sterility Test Isolators / Sterility Testing Isolators

    INTRODUCTION

    The sterility test is an important end-product test for medicinal products that are required to be sterile (including those that are aseptically filled and many that are terminally sterilized, unless regulatory approval has been granted for parametric release). The sterility test, as a culture based method, is described in the harmonized pharmacopoeias: United States Pharmacopeia (USP) (<71>), European Pharmacopeia (EP) (2.6.1), and Japanese Pharmacopeia (4.06). The US Food and Drug Administration, through the Code of Federal Regulations, allows for alternative rapid methods to be used as a replacement to the culture method. The focus of this paper is the culture-based method.

    One of the concerns with the sterility test, especially when high-value products are handled, is the risk of false positives that can lead to expensive and lengthy failure investigations (and where proving a false positive is extremely difficult, therefore, the outcome can often be batch rejection). A false positive can be described as a contaminating microorganism that has been transferred into the test media through cross-contamination by personnel or from the test environment; in contrast, to microbial contamination being present in the product under test. 

    Where the sterility test is conducted within a conventional cleanroom, the risk of cross-contamination is arguably higher than if the test is conducted within an isolator. The advent of isolation technology for sterility testing, since the 1990s, has, in theory, lowered the incidence of false positives.

    An isolator is an arrangement of physical barriers that are integrated so that the workspace (an enclosed environment) within the isolator is sealed from the outside environment. The barrier to the outside is measured in terms of a routine leak test and the maintenance of pressure differential, both of which are assessed within specified limits. The isolator allows manipulations to be performed within the workspace in a way that does not compromise the integrity of the isolator (usually through glove ports).

    To maintain the barrier with the external environment, isolators consist of either a flexible film for the outer wall or have a solid wall envelope. With the exception of handling certain medicinal products, such as cytotoxic drugs, isolators used for the sterility test, when operative, are at a positive pressure relative to the room and have a high efficiency particulate air (HEPA) filtered airflow. The pressure level of the isolators is normally monitored by pressure alarms and readings.

    As a general concept, the aims of a sterility test isolator or sterility testing isolator are to:

    • Provide a testing environment free from contamination, through routine sanitization using a validated cycle and confirmed by environmental monitoring.
    • Enable the isolation between the operator and the process.
    • Provide a non-corrosive, drainable, and easily cleaned enclosure.
    • Have doors to provide access to the equipment inside the isolator.
    • Have glove ports situated to allow users access to machinery and product during testing.
    • Have a mechanism by which materials can be sanitized and then be securely placed within the isolator. This can be through the use of transfer isolators or through the use of a rapid gassing port. Some users elect to load all materials into the isolator each time and run a full sanitization cycle.
    • Provide an in-feed opening to introduce testing materials and product to the system.
    • Be equipped with an exit opening to allow finished product and test materials to exit the system.

     

    MAINTAINING THE STERILITY TEST ISOLATOR OR STERILITY TESTING ISOLATOR

    Parameter

    Limit/Requirement

    Justification

    Frequency of Testing

    Pressure

    Positive Pressure (20-250 Pascals (Pa))

    Positive pressure relative to the isolator room is required to prevent the ingress of contamination from the outside environment into the enclosed clean zone.

    The minimum specification of +20 Pa is the lowest setting to prevent this occurrence while establishing a good margin of safety.

    The maximum specification of 250 Pa relates to the integrity of the isolator chamber.

    Monitored each working week prior to the isolator system being sanitized.

    In addition, the pressure is checked prior to each test. Pressure gauges are also calibrated biannually.

    Leak Rate Test

    From a starting pressure of 150 Pa, a time sequence of monitoring leakage over a 90 second period is undertaken. By the end of the 90 seconds, the pressure must not fall by more than 25 Pa.

    Recommended by ISO 14644 Part 7.

    Monitored each working week prior to the isolator system being sanitized.

    Airflow

    Airflow pattern

    Although laminarity is not required for a sterility test isolator or sterility testing isolator, there is a theoretical argument that the airflow should demonstrably remove contamination, at faster rate, away from the critical zone.

    This was assessed as a one-off airflow visualization (smoke) study.

    Air speed

    0.45 meters per second (ms-1) at the filter face (±20%)

    Required by ISO 14644 Part 7 and EU GMP for the effective removal of any contamination,

    Assessed biannually.

    Air supply

    HEPA filtered with >99.995% retention total.

    Required by ISO 14644 Part 7.

    HEPA filter based on European norm: EN 1822:2009

    HEPA filter will theoretically filter all but 0.01% of 0.3 mm particle size (defined as the most penetrating particle size).

     

    Air changes

    >20 air-changes per hour

    A set number of air changes above 20 is necessary for the dilution and removal of any contaminants from the isolator environment.

    Recommended by Midcalf et al, 2004:27.

    In practice, the isolator achieved considerably more than 20 air changes per hour.

    Particle counts

    0.55 mm = 3, 520 counts per cubic meter

     

    5.0 mm = 20 counts per cubic meter.

    EU GMP limit. This is tighter than the ISO 14644 Part 1 specification.

    Assessed biannually as per ISO 14644.

    Static monitoring is performed weekly during normal operation as part of an on-going check.

    Sanitization

    Validated decontamination cycle.

    Validated method for a minimum six log reduction of Geobacillus stearothermophilus spores using hydrogen peroxide vapor.

    Run weekly for an isolator. This interval is considered to be suitable to prevent bioburden build up.

    Sanitizing Agent

    Hydrogen peroxide liquid (30% w/w)

    -

    -

    Gloves: Leakage

    Low level of leakage

    Recommended by USP <1208>

    Tested as part of the leak test of the isolator system (each working week).

    Gloves are change each week. Following a change of gloves, a water test is conducted to assess leaks and replacement gloves are re-tested as part of the isolator leakage test.

    Gloves are additionally visually inspected prior to each sterility test being performed. At the end of the sterility test, finger plated are taken to assess microbial growth.

    In the event of gloves requiring changing, the isolator will be sanitized as a precautionary measure.

     

    SANITIZATION CYCLE: THE KEY STEPS

    For the operation of an isolator for sterility testing a, number of steps must be followed. It is important to present these steps prior to discussing the validation and operational requirements of the isolator.

    Pre-cycle Disinfection

    Prior to final product items (and other material that is not sterilized and contained within double wrapping) being loaded into the isolator or gassing port, some users elect to wipe items with 70% isopropyl alcohol (IPA) using a low particulate wipe (observing the required contact time for the alcohol). Surface gaseous disinfection requires a starting condition wherein a surface is visibly clean of soiling. Visible contamination or soiling may occlude microorganisms from the disinfection process and if uncovered in-process may present a bio-contamination risk. Any surface contamination is removed through the 70% IPA wiping step.

    Gassing Port and Disinfection Cycles

    The gassing port is a chamber connected to the isolators via interlocking doors; it can be opened to the room environment for loading without compromising the integrity of the isolators. The gassing port is designed to sanitize the different sterility test loads in a faster time than would be possible if loads were placed inside an isolator for sanitization. The gassing port is connected to a gas generator. Because hydrogen peroxide in the passive state is very poor at diffusion, the function of the gas generator is to heat the hydrogen peroxide to produce a vapor and to provide the generated vapor to the port. The function of the port is to actively distribute the vapor via a distribution nozzle. The function of the rotating nozzle is to prevent hot spots arising from an uneven distribution of the gas. The dosage of the hydrogen peroxide is controlled by the user prior to placing the chemical into the gas generator. This will provide a controlled volume of the chemical, provided the correct cycle is selected by the user.

    Sanitization Cycle

    The sanitization process occurs by an aqueous solution of hydrogen peroxide being evaporated in such a way as to produce the same weight ratio in the vapor phase as in the source liquid (starting with 30% wt/wt of hydrogen peroxide, which is evaporated into the heated carrier gas stream to produce hydrogen peroxide concentrations. This vapor is transported to the chamber to be bio-decontaminated in a heated carrier gas (initially sterile compressed air). Vapors are circulated around the chamber, then through a catalysing filer at the end of sanitization to break down the hydrogen peroxide.

    There are four key steps to ensure an optimal hydrogen peroxide vapor disinfection process:

    1. Vaporization of liquid to small molecules–gas phase delivery to target volume.
    2. Development of the gas concentration in the target environment to saturated vapor conditions, past dew point, and transition into liquid phase.
    3. At saturated vapor conditions, the gas concentration can hold no more molecules, thus, the process of condensation formation and disinfection agent surface deposition starts.
      Micro-condensation forms on surfaces by merging molecules. Initially, nuclei form on any surface contaminants, before full condensation occurs over the entire available surface, eventually forming a disinfectant monolayer (from gas to liquid phase). 
    4. Re-evaporation of the surface condensate and removal of residual gas to target endpoint concentration.

    The sanitization cycle operates through the following steps:

    1. The generator initially dehumidifies the ambient air (conditioning). Here, initial temperature and relative humidity is optimized before vapor injection. Hot dry air is exchanged with the target environment to achieve required starting humidity conditions.
    2. The generator then produces hydrogen peroxide vapor by passing aqueous hydrogen peroxide over a vaporizer. The gas distribution is an active process controlled through a nozzle for a pre-determined volume of hydrogen peroxide. The gassing phase is validated to deliver a disinfection agent dose volume that will reach required micro-condensation conditions in the target area and on target surfaces. During validation, an expected time range for the gassing or dosage phase is determined (based on gassing pump speed); cycle times for routine operations must fall within this range.
    3. The vapor is then circulated at a programmed concentration in the air and held for a set period of time (dwell or hold phase). The dwell time is optimized to maintain micro-condensation conditions throughout the complete dwell phase for assured disinfectant contact time.
    4. After the hydrogen peroxide vapor has circulated in the enclosed space for a pre-defined period of time, it is circulated and broken down into water and oxygen by a catalytic converter until concentrations of hydrogen peroxide vapor fall to safe levels, at 1 parts per million (ppm) (aeration phase) (less than 1 ppm is below the occupational exposure level and deemed safe for personnel). Gas residual removal is typically achieved with integral or supporting HEPA-catalysts and/or supporting dilution via barrier air being vented to the environment.

    Primary process variables for hydrogen peroxide vapor disinfection include:

    • Starting relative humidity (RH)
    • Surface and environmental temperatures
    • Gas distribution for a homogeneous deposition of vapor and resulting surface condensate
    • The amount of evaporated hydrogen peroxide solution dosed into the target volume (g/min) to reach saturated vapor conditions and provide a sufficient monolayer of disinfectant
    • Surface area for condensate distribution together with amount of surface absorbency (e.g., packaging).

    OTHER OPERATIONAL DECISIONS

    Further decisions, based on the validation outcomes and the process cycles selected, are required for the daily operation of an isolator system. These include setting the frequency of sanitizing the entire system and the policy for glove changes. These aspects are discussed below.

    Frequency of Sanitization of the Isolators

    The frequency of sanitizing the entire isolator system needs to be established. This was a separate activity to the gassing of each load required for the sterility test in the gassing port. In part, this decision was based on environmental monitoring data.

    Routine Cleaning and Disinfection Agents

    The cleaning and disinfection agents should be carefully selected. For cleaning, the detergent selected should be a sterile neutral detergent because any residues are less likely to react with the disinfectant. The disinfectant of choice, given that most isolators are constructed from stainless steel, is 70% IPA. This is because other disinfectants could damage the stainless steel inner chamber of the isolator (by causing scratching, rouging, and etc.). Seventy percent IPA may not be appropriate for cleaning perspex visors; sterile filtered water can then be used to remove any debris. Where a more efficacious disinfectant is needed, such as a sporicide, then due to many sporicides being aggressive, 70% IPA should be used to remove any residues of the sporicide post-application. 

    Frequency of Glove Changes 

    Isolator gloves are prone to leakage, and this represents a weak point with any isolator system. Some users test all gloves for leakage before and after each test. Additional supporting data for a glove change policy can be provided from post-sterility test and post-sanitization microbial finger plate monitoring. The use of a water intrusion test for gloves that have been changed can provide further data as to the frequency that leaks occur. 

    Requirements for Environmental Monitoring

    Environmental monitoring should be conducted for each sterility test. The testing isolator environment should be monitored every time a sterility test is performed. The typical environmental samples taken are: air-samples (using a volumetric sampler) and settle plates (during the test); and finger plates, swabs, or contact plates (immediately post-test).

    Isolator Housing Room

    Many isolators are held within a cleanroom or a controlled environment. The isolator described in this study protocol was held within an EU GMP Grade D/ISO 14644 class 9 (‘in operation’) cleanroom.

    Where a cleanroom is used, the cleanroom will have to meet various operational parameters itself. These parameters will include: 

    • Positive pressure differential to the outside corridor
    • Controlled temperature, given that this can affect isolator gassing parameters
    • High efficiency particulate air (HEPA) (with filters of 99.99% efficiency)
    • Controlled air-changes
    • Particles monitored in the dynamic state
    • Room subject to viable environmental monitoring
    • Room falls under a routine maintenance plan including classification.