NEWSLETTER
ISSUE
Sep to Dec, 2017 Volume 4
BRAIN WAVE
SkanfogĀ® STI - Monitoring Program
Dr. Hans-Juergen Baessler
SKAN AG – 2017
INTRODUCTION
In aseptic production, the masterplan provides insightful insights into the quality of the direct environment of aseptic production. Over a longer period of time (trending), consistent monitoring makes it possible to detect changes in the “germ-free” status of the isolator. The monitoring of the isolator should help to find possible contamination routes quickly, in order to initiate appropriate corrective measures.
āWhere aseptic operations are performed monitoring should be frequent using methods such as settle plates, volumetric air and surface sampling (e.g. swabs and contact plates). Sampling methods used in operation should not interfere with zone protection. Results from monitoring should be considered when reviewing batch documentation for finished product release.ā [1]
Appendix 1 of the FDA aseptic processing guide explicitly states āan appropriate environmental monitoring program should be established that routinely ensures acceptable microbiological quality of air, surfaces and gloves (half-suits) as well as particle level within the isolator.ā [2] A clearly defined, written program with scientifically based methods is the basis for every monitoring of the quality of air and surfaces in the isolator. The monitoring program includes all persons involved in the manufacturing process.
It includes air, walls, ceilings, floor of the isolator, all surfaces of built-in systems, including the product-contacting critical surfaces. The monitoring program contains a list of all sampling points. The frequency, time and location of the sampling are carefully selected and exactly described, taking into account the production or testing process. An in-depth statement of reasons is provided for the election. The samples cover the entire area of the isolator. The size of the specimens and the frequency of the removal are sufficient to provide an optimum detection of impurities as expected for the clean room class in the isolator. The focus of the program is the positions in the isolator, which have the highest particulate and microbiological risk.
In order to verify the status “germ free” there are two systems in the isolator, the physical and the microbiological monitoring. The physical monitoring parameters include:.
1. The differential pressure to the environment
2. The number of air changes
3. The air velocity
4. The particle concentration
5. The H2O2Ā concentration during and after decontamination
In isolators where products are processed with solvent, the following parameters may additionally be used:
1. The oxygen concentration
2. The solvent concentration
A microbiological monitoring is carried out in aseptically operated isolators, the objective of which is to control the germ-freeness in the isolator. Frequent sampling of samples is necessary, the frequency and locations of which are determined on the basis of a risk analysis. [3] In this risk analysis the weak points of the isolator technology, the material transfers as well as the glove impacts are to be evaluated. For all measures one has to take into account that not all contaminations can be detected due to temporal and spatial limitations as well as due to the recovery rate of the methods. The results of the microbiological monitoring over time are the basis for the assessment of the microbiological situation in the isolator. Another important prerequisite for a qualified, microbiological monitoring system is the adequate training of the sample takers.
The area in an isolator in the aseptic mode which is used for the sample materials must be accessible to the operator (Fig. 7.147). In this case, the material should be stored in a gas-tight packaging in such a way that the surfaces of the packaging do not touch each other and the chamber wall and thus their adequate decontamination is ensured.
Where microbiological samples should be taken? Basically in places where there is an increased risk of contamination:
1. On transfer systems (RTP, pass box)
2. Close to places where the product is processed openly
3. Surfaces with high D values [4]
4. In the event of an accident (gloves, sleeves, etc.)
A written justification should be drawn up for each sampled area. The points of sampling should be checked at regular intervals:
1.Changes in the production process
2.On new insights about the isolator
3. With regard to the meaningfulness of the data collected so far
Particle Count
Particle counting in isolators for the aseptic production in the pharmaceutical industry is mandatory according to ISO 14644 guideline.[5] For counting particles in the Skanfog STI an isokinetic probe is installed on the left side wall or the isolator floor behind the Steritest pump (Fig. 1 (1)). After each session the isokinetic probe is dismantled and taken to an autoclave in order to be sterilized in an adequate sterilization pouch. Loading the isolator with the new test samples, the isokinetic probe should also be loaded in the pouch. After decontamination of the loaded chamber with H2O2, the isokinetic probe is unpacked and installed on the Triclamp (1). The cover of the Triclamp on the outside of the chamber is removed and the tube to the particle counter is installed. Then the cover on the isokinetic probe (2) can be removed.
After aeration is completed, the particles in the isolator should be counted with an adequate device for 10 minutes before the testing session starts. The testing session can begin if the particle count is within the limits. At the end of the session, before opening the chamber a second particle count can be performed. The isolator is running in test mode for 10 minutes without glove movement followed by a 5 minute particle count. If the particle count is within the limits, the counter is switched off and the cover for the probe is put on. Now the door can be opened and the samples as well as the garbage can be removed.
Total particulate counts using electronic devices are likely to give more direct and more immediate indications of changes in isolator performance status than microbiological sampling.[2]
Airborne viable sampling
Settle Plates
Airborne viable sampling is performed either with settle plates and/or with active viable sampler. The settle plates are placed near by the Steritest pump left and right on the lower shelf and/or on the holder near the side walls (Fig. 1. (3)) and (Fig. 3). It is good practise to use Agar plates with double layer if they are exposed longer than 2 hours. If single layer plates are used it is advisable to validate the exposition time with the vegetative germs described in the Pharmacopeia.
Ā
Active Air Sampler
The SAS Super 90 system corresponds in principle to a single-stage Andersen collector. The device consists of a perforated plate through which the air is sucked. Underneath the perforated plate, the culture medium is placed in a 90 mm plastic dish and underneath this dish an air fan with a defined volume of 180 l or alternatively 100 l/min sucks up. The perforated plate can optionally be provided with 219 or 487 round nozzles. The nozzle diameter is about 2Ī¼m. Before the sample taking, the perforated plate is removed and a 90 mm agar plate is inserted into a holder. The device is then closed again with the perforated plate and the air quantity or time for sampling is entered. The collector is not suitable for high germ concentrations because of the collecting principle, which is not a disadvantage for use in the isolator since only a very small germ load is expected.
It is advisable to use active air samplers which can be easily decontaminated with H2O2Ā inside the isolator chamber (Fig. 1. (2)) and (Fig. 4). The SAS Super Isolator should run during the decontamination cycle without Agar plate and sampler head in order to ensure the proper decontamination of the surfaces inside. Like the SAS Super Isolator the collector should be furnished with spacers, so there is a small distance to the isolator floor, which allows H2O2Ā to circulate between isolator floor and sampler. The head of the sampler is removed and sterilized in an autoclave in an adequate pouch. After the aeration is completed, the Agar plate is set up and the head is closed with a bayonet or screw coupling. No more than two air samples 1m3 each need to be taken in a reasonably sized testing isolator over a four-hour period. The first sample should be taken immediately after aeration and the second at the end of the session before opening the isolator. Indications are that active air sampling is more sensitive than settle plates so there is no good reason to do settle plates at all. [3]
Continuous Sampler (LASER based)
The control of microorganisms in the pharmaceutical manufacturing processes and environments is one of the most important tasks in ensuring products are safe and effective. This is true for conventional pharmaceuticals, biotech products, compounding pharmacy drugs, gene and cell therapy products and radiopharmaceuticals. However, conventional microbiology methods are antiquated and do not support the need for faster, accurate and reproducible results. Therefore, the modern laboratory should develop innovative approaches for the detection, quantification and identification of microorganisms using alternative and rapid microbiological methods (RMM).[8]
The development of instruments that are capable of performing microbiological monitoring continuously and in real time has been made possible by the application of laser-induced fluorescence (LIF). LIF instruments utilize a high intensity light source (e.g., 405 nm LASER) to measure scattered light on particles and additionally to measure the fluorescence of particles containing potentially viable cells. The real-time detection is successful for bacteria, yeast and mold fungi.
The sample is passed through the optic chamber and illuminated by a 405 nm laser source. Light scattering occurs when any particle crosses the laser beam inside the measuring cell. Only particles with fluorescent molecules, however, also give a signal at the second detector (photomuliplier). Interference filters ensure that only the fluorescence of the relevant wavelength is detected at the second detector and that the purely physical particles and their scattered light do not disturb the signal.
The fluorescence is originated from molecules such as NADH and riboflavin, which are found in all cells. Dipicolic acid (DPA), a molecule contained in spores, is also recognized by this technology. As a rule, particles in the range from 0.5 Ī¼m to 5.0 Ī¼m are detected. The Results are counted as bio fluorescence counts. [12]
Surface sampling
SSurface samples from plants and facilities are important components of the microbiological monitoring program in critical production areas. Both contact plates and wipers (SWABS) are often used for sampling [6].
Contact plates methods are based on the direct contact of a solid culture medium with the surface to be tested and subsequent incubation. A part of the microorganisms present on the test surface adheres to the culture medium. The results are obtained, after a sufficiently long incubation period, by visual detection and counting of the colonies grown on the medium. These data are then documented as Colony Forming Units (CFU) per volume or mass.
The RODAC plate consists of a transparent nutrient carrier made of plastic. The culture medium is then applied with a convex curvature. It can be transported and stored with a lid. The microbiological monitoring for the determination of the microbiological purity of the surfaces in an isolator is performed on different surfaces. These surfaces such as, aluminum, plastic, glass or stainless steel can behave very differently to the decontaminating agent like H2O2Ā due to their surface structure and porosity. [4]
Contact plates
In isolators with H2O2Ā decontamination, the RODAC (Replicate Organism Detection and Counting) plates (Fig. 5) are entrained in gas-tight three-layer packaging, sterilized by gamma-irradiation, containing an inactivation agent for H2O2. The media types which should be used are TSA ā Soybean-casein Digest Agar for bacteria and yeast and SDA ā Sabouraud Dextrose Agar which favors the growth of fungi. Before loading the package in the isolator the outer layer is removed. The package is than exposed to the decontamination agent. After aeration the second and third shells are removed, the plates are labeled and sampling can begin.
For sampling the lid is removed and the agar is pressed on the surface for 10 seconds. Afterwards the plate is covered by the lid and is transferred to the incubator. Incubate TSA at 30 ā 35Ā°C and SDA at 20 ā 25Ā°C for 7 days. The media should be evaluated for the suitability of the intended use. All materials present in the isolator should be sampled in at least in one place. Also important is the sampling of the gloves on the finger tips, which were currently in use (Fig. 1 (4/5). In addition, preferably areas of frequent interventions and areas around the open product are sampled. The risk analysis for the aseptic process provides justifications for the definition of the surfaces to be patterned. After sampling, it is important not to forget to clean the site with 70% isopropanol (sterile filtered).
In addition to the samples of surface monitoring which are aerobically incubated, samples should also be anaerobically incubated at regular intervals. Microbiological sampling of surfaces that have been decontaminated with H2O2Ā it is unlikely to yield anything, so the number should be minimized. [3]
SWAB Test
Many suppliers of microbiological test systems also provide ready to use SWAB test sets in their portfolio. Surfaces which cannot be sampled with plates for spatial reasons are taken as SWAB samples for example in drains (Fig. 1 (6). A SWAB is then used to wipe an area of approximately 100 cm2 and transfer the stick into a small tube with Letheen broth. [6] The medium contains beef extract and peptone to provide a nutrient rich medium supporting the growth of a variety of microorganisms. Quaternary ammonia compounds are neutralized by lecithin while phenolic disinfectants and hexachlorophene are neutralized by Tween Ā® 80. Together, lecithin and Tween Ā® 80 neutralize ethanol. [5] From this device the liquid is transferred in the microbiology lab on an RODAC plate. The incubation and evaluation is carried out as with the RODAC plates.
The frequency of sampling is again determined by the risk analysis for the aseptic process and by the long-term results of the measurement to identify a trend. While samples are frequently taken at the beginning of the introduction of a container, the sampling intervals can be selected longer with increasing experience. “Frequent” means, for example, that at the end of each test session or production, surface samples are taken at the designated locations. If results outside the specification (OOS) are observed (1 germ = alarm limit) the reaction must follow immediately.
IThe use of SWABS has several advantages over the use of contact plates, especially in an ISO-5 / Class A range. Since the contact plates leave agar residues on the tested surfaces and have to be cleaned afterwards, a cleaning validation is required, which is usually not required for SWABS during sampling. In addition, wipers are particularly useful for sampling on often irregular surfaces in Class A areas (e.g., in filling machines etc.). The advantages of using a wipe-based method against the use of contact plates become apparent in carrying out a risk analysis of the critical point for the manufacturing process in an isolator. Interestingly, a rapid microbiological method (RMM), which has combined the use of SWABS combined with the detection of a microbial contamination by oxygen consumption, has recently been released onto the market.
SurCaptā¢
SurCaptā¢ is a microbiological rapid test for surfaces in clean rooms and aseptic isolators, based on oxygen-induced phosphorescence suppression. [12] Further possible applications of this technology include tests of microbial contamination (bioburden) in raw materials, excipients, pharmaceuticals, pharmaceutical water and environmental monitoring in the pharmaceutical industry. The system detects microbiological impurities based on measurements of oxygen consumption caused by microbiological growth during the incubation period in liquid nutrient media like TSB. The technology works as follows:
1. Bacteria in a SurCaptā¢ tube incubated, consume oxygen dissolved in the nutrient medium.
2. A polymer sensor on the inside of the sample bottle responds to oxygen consumption.
3. The amount of bacterial O2 consumption corresponds to the microbiological load of the sample. The larger the initial quantity, the faster the result. An increase in the O2 concentration causes a reversible decrease in the phosphorescence signal. Conversely, when oxygen is consumed by the microbiological load, the sensor generates a stronger phosphorescence signal.
The readerās internal LED sends green light to the polymer sensor located in the kit vial. The graphical plot of the phosphorescence signal versus time represents a typical microbial growth curve.
Validation studies show that this rapid technology is reliable and sensitive to the detection of microbiological contamination, as well as to the standard tests.
LITERATURE
[1] EU-GMP Guideline Annex 1, (Manufacturing of steril medicinal products)
[2] Food and Drug Administration (FDA) (2004) āGuidance for Industry – Sterile Drug Products Produced by Aseptic Processing – Current Good Manufacturing Practiceā, Maryland (MD 20993), USA.
[3] Risk and Scientific Considerations in the Environmental Monitoring of Isolators in Aseptic Processing James Akers, American Pharmaceutical Review, Posted: January 1, (2010)
[4] Sigwarth, V. and Staerk, A. Effect of Carrier Materials on the resistance of Spores of Bacillus stearothermophilus to Gaseous Hydrogen Peroxide; PDA Journal of Pharmaceutical Science and Technology, Vol. 57, No. 1 (2003)
[5] ISO 14644
[6] Hardy Diagnostics, Instructions for use of Letheen Media (2017)
[7] United States Pharmacopoeia (USP) (2013) āMicrobiological Control and Monitoring of Aseptic Processing Environmentsā In: USP Vol 36, Chapter 1116, Rockville, Maryland, USA Rockville, MD 20852-1790, USA
[8] Markarian, J. (2013) āEmerging Sterilization and Disinfection Technologies Offer Alternative Solutionsā, PharmTech.com, 16/Oct/2013 issue
[9] FDA (2015) Draft Guidance, āAdvancement of Emerging Technology Applications to Modernize the Pharmaceutical Manufacturing Baseā Silver Spring, Maryland, USA
[10] PDA. (2013). āEvaluation, Validation and Implementation of Alternative and Rapid Microbiological Methodsā, Technical Report No. 33 (Revised 2013), Parenteral Drug Association, Bethesda, MD, USA
[10] Ph. Eur. (2006). Chapter 5.1.6 āAlternative Methods for Control of Microbiological Quality, European Pharmacopeia, European Directorate for the Quality of Medicines & HealthCareā. 5.5:4131. EDQM – Council of Europe,, Strasbourg, France
[12] Denoya,C and Jansen,R. Schnelle mikrobiologische Testmethoden, Wiley, ChemManager; 20.01.2017
[13] SurCaptā¢ is a Trade Mark of Particle Measuring Systems Inc. (2016)
