Mesenchymal stem cells (MSCs) are one of the main stem cells that have been used for advanced therapies and regenerative medicine. To carry out the translational clinical application of MSCs, their manufacturing and administration in human must be controlled; therefore they should be considered as medicine: stem cell-based medicinal products (SCMPs). The development of MSCs as SCMPs represents complicated therapeutics due to their extreme complex nature and rigorous regulatory oversights. The manufacturing process of MSCs needs to be addressed in clean environments in compliance with requirements of Good Manufacturing Practice (GMP). Facilities should maintain these GMP conditions according to international and national medicinal regulatory frameworks that introduce a number of specifications in order to produce MSCs as safe SCMPs. One of these important and complex requirements is the environmental monitoring. Although a number of environmental requirements are clearly defined, some others are provided as recommendations. In this review we aim to outline the current issues with regard to international guidelines which impact environmental monitoring in cleanrooms and clean areas for the manufacturing of MSCs.
Mesenchymal stem cells (MSCs) hold considerable promise as a source of cells for novel therapies treating many serious diseases and injuries, including metabolic, degenerative, and inflammatory diseases, repair and regeneration of damaged tissues, and cancer. MSCs can be isolated from different tissues of the human body, expanded and/or differentiated
The manufacturing of MSCs for translational clinical research should be performed with appropriate controls that ensure their safety and quality. In this context, new regulatory regimes for advanced and complex treatments such as cell therapies, tissue engineering, and gene therapies have grown substantially in importance in developing countries because they offer ground-breaking new opportunities for the treatment of disease and injury [
This review provides all the necessary requirements to manufacture MSCs as medicine in order to present themselves as a new therapeutic alternative.
The current state of legislation and methodology for the environmental control monitorization are described.
The processing of MSCs for use in cell therapy protocols requires a specific environment in which air quality is controlled, in order to minimize the risk of contamination of cells. To control air quality monitorization of viable and nonviable particles must be carried out throughout the whole process. In this field, a viable particle is a particle that contains one or more living microorganisms. A nonviable particle is a particle that does not contain a living microorganism.
The environmental monitoring should include a series of physical controls (concentration of particles in the air, flow of air, integrity of high efficiency particulate air (HEPA) filters, differential pressure, temperature, and relative humidity) and microbiological tests [
Regular monitoring of the environment, process, and finished product with MSCs must occur according to a written procedure and in line with the published written standards and guidelines [
For a descriptive overview of the regulatory authorities and documents the following classification is presented below. However, this is a difficult task by the range of different regulatory documents and standards [
The Food and Drug Administration (FDA) publishes guidance documents (not mandatory) to provide general requirements for investigators from the US Code of Federal Regulations (CFR). CFR is a compilation of all published federal laws in USA. All food and drug related laws are contained in its Title 21. Within this, part 211 is as follows: “Current Good Manufacturing Practice for finished Pharmaceuticals” [
The body of European Union legislation in the pharmaceutical sector is compiled in the publication “The Rules Governing Medicinal Products in the European Union” published by the European Commission [
The European Medicines Agency (EMA) is the responsible public body for the scientific evaluation of medicines. Important documents of this regulation are Volume 4, annex 1: “Manufacture of Sterile Medicinal Products” [
The WHO was the first international organization who established detailed guidelines for GMP. GMP guidelines for biological products were approved in 1992 by both the WHO Expert Committee on Biological Standardization and the WHO Expert Committee on Specifications for Pharmaceutical Preparations [
The Pharmaceutical Inspection Convention and Pharmaceutical Inspection Cooperation Scheme are two international bodies, made up of 46 representatives participating authorities from different countries with competencies in the field of GMP. The PIC/S aim to harmonize inspection procedures by developing common standards of GMP. They also aim to facilitate cooperation and contacts between the competent authorities, regional and international organizations, thereby increasing mutual trust. As GMP guide of interest for this article was that issued by PIC/S is the “Guide to GMP for Medicinal Products” PE 009 and revisions [
Originally, the PIC/S GMP guide (“PIC Basic Standards” of 1972) derived from the WHO-GMP guide. However, it was further adapted and expanded to satisfy the requirements of states taking part in PIC/S. In 1989, the EU adopted its own GMP guide. Since then the EU and PIC/S GMP guidelines have been developed in parallel but differ on small points such as expressions or references to Pharmacopeias.
ISO is an independent, nongovernmental membership organization developer of voluntary international standards. Its main aim is to promote the development of worldwide harmonization of standards. ISO publishes numerous standards of relevance to pharmaceutical manufacturing, but not all of these standards are associated with GMP conditions. The most important GMP guide related to the topic at hand is the standard ISO 14644: “Cleanrooms and Associated Controlled Environments” and its series [
Harmonization of regulatory requirements was pioneered by the European Union (formerly European Community) in the 1980s moved towards the development of a single market for pharmaceuticals. At the same time, bilateral meetings between Europe, Japan, and the USA took place. Finally, at the WHO Conference of Drug Regulatory Authorities, in Paris (1989), clear statements began to materialize.
It publishes quality and GMP documentation. Launched in 1990, ICH is a unique undertaking that brings together the drug regulatory authorities and the pharmaceutical industry of Europe, Japan, and the USA. Among others, an important document regarding environment monitoring is “Good Manufacturing Practice Guide for Active Pharmaceutical Ingredients Q7” [
The main international Pharmacopeias regarding this field are European Pharmacopeia (EP), Japanese Pharmacopeia (JP), and the United States Pharmacopeia (USP). Pharmacopeias issue some aspects with direct relevance mainly to sterility testing and other laboratory test methods. An example is the
Some countries possess national regulatory agencies that publish additional documents of guidance such as Australia, Canada, Japan, and Singapore. These agencies include Parenteral Drug Association (PDA), American Society for Testing and Materials (ASTM), Pharmaceutical Microbiology Interest Group (Pharmig), Pharmaceutical and Healthcare Sciences and Society (PHSS), IPSE (International Society for Pharmaceutical Engineering) [
A MSCs production laboratory for clinical use must meet the minimum requirements for the product sterility manufacture. These facilities are called cleanrooms or clean areas. Environmental parameters such as size and number of airborne particulates, temperature, humidity, air pressure, airflow patterns (speed and direction), air motion, vibration, noise, viable (living) organisms, radiation, and lighting must be strictly controlled [
According to the degree of purity of air three different international standards have been proposed and only particle contamination is used for classification purposes.
This standard was first published in 1963 in the USA entitled “Cleanroom and Work Station Controlled Environments” and posteriorly revised five times until 1992. Finally, it was canceled in 2001. The Federal Standard categorized cleanrooms in six general classes, depending on the particle count (particles per cubic foot) and size in
Federal Standard 209E. Class limits are given for each class name.
Class name | Class limits | ||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|
≥0.1 | ≥0.2 | ≥0.3 | ≥0.5 | ≥5 | |||||||
SI | English | m3 | ft3 | m3 | ft3 | m3 | ft3 | m3 | ft3 | m3 | ft3 |
M1 | 350 | 9.91 | 75.7 | 2.14 | 30.9 | 0.875 | 10.0 | 0.283 | |||
M1.5 | 1 | 1,240 | 35 | 265 | 7.50 | 106 | 3.00 | 35.3 | 1.00 | ||
M2 | 3,500 | 99.1 | 757 | 21.4 | 309 | 8.75 | 100 | 2.83 | |||
M2.5 | 10 | 12,400 | 350 | 2,650 | 75.0 | 1,060 | 30.0 | 353 | 10.0 | ||
M3 | 35,000 | 991 | 7,570 | 214 | 3,090 | 87.5 | 1,000 | 28.3 | |||
M3.5 | 100 | 26,500 | 750 | 10,600 | 300 | 3,530 | 100 | ||||
M4 | 75,700 | 2,140 | 30,900 | 875 | 10,000 | 283 | |||||
M4.5 | 1,000 | 35,300 | 1,000 | 247 | 7.00 | ||||||
M5 | 100,000 | 2,830 | 618 | 17.5 | |||||||
M5.5 | 10,000 | 353,000 | 10,000 | 2,470 | 70.0 | ||||||
M6 | 1,000,000 | 28,300 | 6,180 | 175 | |||||||
M6.5 | 100,000 | 3,350,000 | 100,000 | 24,700 | 700 | ||||||
M7 | 10,000,000 | 283,000 | 61,800 | 1,750 |
Cleanrooms are classified according to the air cleanliness. In the international domain, the ISO Technical Committee 209 decided to draft an international standard on these cleanrooms, whose mission was to establish the criteria that should govern the cleanrooms without making specific reference to a particular through the ISO 14644 series [
ISO-14664, cleanrooms, and associated controlled environments (particles/m3).
ISO classification number ( | Class limits | |||||
---|---|---|---|---|---|---|
≥0.1 | ≥0.2 | ≥0.3 | ≥0.5 | ≥1.0 | ≥5.0 | |
1 | 10 | 2 | ||||
2 | 100 | 24 | 10 | 4 | ||
3 | 1,000 | 237 | 102 | 35 | 8 | |
4 | 10,000 | 2,370 | 1,020 | 352 | 83 | |
5 | 100,000 | 23,700 | 10,200 | 3,520 | 832 | 29 |
6 | 1,000,000 | 237,000 | 102,000 | 352,000 | 8,320 | 293 |
7 | 3,520,000 | 83,200 | 2,930 | |||
8 | 35,200,000 | 832,000 | 29,300 | |||
9 | 8,320,000 | 293,000 |
Each manufacturing operation requires an appropriate level of environmental cleaning to minimize the risk of microbial contamination or particles in the product or materials being handled. EU-GMP, annex 1: “Manufacture of Sterile Medicinal Products of GMP” [
Two conditions are defined depending on the manufacturing activity: “
Airborne particulate classification for these grades, according to the PIC/S GMP and EU-GMP.
Grade | Maximum number of particles permitted/m3 | |||
---|---|---|---|---|
At rest | In operation | |||
≥0.5 | ≥5.0 | ≥0.5 | ≥5.0 | |
A | 3,520 | 20 | 3,520 | 20 |
B | 3,520 | 29 | 352,000 | 2,900 |
C | 352,000 | 2,900 | 3,520,000 | 29,000 |
D | 3,520,000 | 29,000 | Not defined | Not defined |
To achieve the degree of air A, B, C, and D, the number of air changes should be related to the size of the room and the equipment and personnel present in it; the air system must have appropriate filters such as HEPA grades A, B, and C. The HEPA filter is not mentioned for grade D.
Airborne particles can be shaped and composed of different materials. They can also act as “carriers” for bacteria and other microorganisms. Hence, to distinguish between viable particles and inert particles (nonviable), analysis methods in a cleanroom can be classified as microbiological and physical tests. Microbiological tests consist of viable particles counting in both air and surfaces. Physical tests consist of air nonviable particles counting, pressure, and temperature analysis. Monitoring of both physical and microbiological contamination remains essential in aseptic operations to provide ongoing information on the maintenance of a stable and suitable environment for the aseptic preparation of products for administration to patients. It is vital that test methodologies exist as part of the environmental monitoring programme. Each test method selected for routine monitoring should be validated [
Scheme of environmental control requirements for the manufacture of SCMPs in cell therapy laboratories for the monitorization of viable and nonviable particles.
ISO 14644 specifies basic requirements for cleanroom operations. This standard considers all classes of cleanrooms used to produce all types of products and does not address specific requirements for the pharmaceutical industry. A total of thirteen tests are described in this standard. However, only specific tests for cleanrooms intended for the production of SCMPs are commented on in the following sections. Some of them are mandatory but others are voluntary. The key controlling factors in the quality level of any cleanroom are the owner’s requirements and what measurements are necessary to achieve that level of performance.
The frequency of environmental testing should have a direct relationship to the operations performed and be sufficient to allow for meaningful statistical calculations. FDA-cGMP, EU-GMP, USP, or ISO do not provide specific references for that issue but rather general recommendations as shown in Table
Recommended frequency of environmental monitoring testing.
Clean area type | Frequency sampling | |||||
---|---|---|---|---|---|---|
FDA-cGMP | EU-GMP | USP | ||||
FS209E | ISO | EU-GMP | At rest | In operation | ||
M3.5 (100) | 5 | A | Should cover all production shifts | Frequent to detect system deterioration | For the duration of critical operations | Each operating shift |
M4.5 (1,000) | 6 | — | Should cover all production shifts | — | — | Each operating shift |
M5.5 (10,000) | 7 | B | Should cover all production shifts | Frequent to detect system deterioration | For the duration of critical operations | Each operating shift |
M6.5 (100,000) | 8 | C | Should cover all production shifts | Frequent to detect system deterioration | In line with quality risk management | Twice a week |
D | — | In line with quality risk management | In line with quality risk management | — |
The minimum number of sampling point locations (NL, rounded up to a whole number) is defined by ISO 14644-1, annex B, through the following equation:
Diagram of cleanrooms and sampling points of environmental monitoring for stem cell units. Sampling N should be carried out whenever an activity is performed (in operation). The sampling rate for the points A, P, S, and W must be previously validated according to the requirements of the operations.
Measurement and determination of different physical operation aspects of the cleanroom are essential to ensure that a suitable environment is maintained for the preparation of aseptically prepared products.
For the measurement of particle concentration in grade A and B areas a continuous system should be used, with the establishment of the required frequency and alert limits. The volume of the air sample should not be less than 1 m3 in both areas and also in grade C areas. Table
The locations of the monitoring systems of particles (according to risk analysis and classification results) should be next point to the product on display and working height, point of greater transfer of personnel and/or material, point on the remote environment of the area of influence of flow, and points with less effectively treated air flow (measured by the smoke test). Risk analysis is the quantitative or qualitative estimation of the likelihood associated with the previously identified hazards. A documented risk analysis to try to identify, evaluate, measure, and prevent possible failures that can initiate and trigger undesired events should be conducted by the manufacturer for ascertaining the appropriate GMP. Each cleanroom is different and therefore each of them should analyze all aspects related to the required environment. The risk analysis should consider all foreseeable hazards that may cause the input of pollutants. The location chosen for monitoring should be checked to ensure that the positions reflect the worst case. For room monitoring, the counts should be performed in locations where there is most operator activity. For the filling environment the counts should be performed adjacent to the filling zone and where components are exposed in such way as to detect operator activity within these areas.
Monitoring systems airborne particle counters may consist of independent particles, a network of sampling points for sequential access by a collector connected to a single particle counter, or a combination of both. The selected system must be appropriate to the particle size considered. It should be noted that sampling cannot compromise the laminar airflow in the critical zone and that the counting device is oriented in the direction of air flow input. It is standard practice to utilize modern technology and use an optical particle counter where the air sample is drawn into the instrument and passed through a light scattering device.
The terminology of ISO 14644-7 “Cleanrooms and Associate Controlled Environments” is “
Temperature and pressure devices are used to monitor the process. Automatic systems should be previously validated. The air pressure values will depend on the laboratory design, but a differential pressure from the most critical room to the outside of at least 30 Pa and 10–15 Pa between rooms is recommended [
In grade A cleanrooms should be provided with laminar air flow with air speed of 0.36–0.54 m/s with regular validation [
Verification laminar flow protection systems and the suitability of the containment conditions are performed to control the airflow velocity to be measured according to ISO 14644-3, annex B4 [
Both tests should be performed in all cleanrooms at maximal period of twelve months as a reference in the operational and the at rest state. These tests could be performed by the installation of anemometers (direct air velocity measurement), manometers (indirect air velocity measurement), and pitot tube (single-point probe).
Other optional tests such as installed filter leakage, airflow visualization, recovery, and containment leakage are defined in the ISO 14644-3 and suggest a retesting interval of 24 months.
Any air admitted should be passed through a HEPA filter [
The first
The second method offered in the standard is the particle counting method. This method also requires that the filter be evenly challenged with a known recorded concentration of aerosol, an aerosol diluter, and a discrete particle counter (volumetric testing). This procedure is implemented by using dissolution chambers and other devices that minimize the exposure of the delicate optical part of the device [
This gives some idea as to how quickly contamination may be removed from the cleanroom provided that there is acceptable mixing of air in the room. An assessment of air flows (from clean to dirty areas) is a specification for the manufacture of sterile products, to evaluate ISO class 5 (Grade A zone) and the surrounding ISO class 7 (Grade B) room and uniformly from unidirectional air flow units. This is undertaken by visualizing actual or video-taped the air flow with the use of smoke in accordance with ISO 14644-3, annex B7 [
Also known as the clean-up time, recovery is the time elapsed in a cleanroom to return to the static condition (in terms of particulates), according to its classification, after an incident. In accordance with ISO 14644-3, annex B13 [
It is designed to ensure that no airborne contamination can occur via leaks from higher pressure work areas to others adjacent to it. Airborne contamination can come into a cleanroom from less clean adjacent areas and pass through doors and hatches, as well as through holes and cracks in the walls, ceilings, and other parts of the cleanroom. In this way the absence of cross-contamination can be verified by the airflow direction smoke tests and room air pressures measurement in accordance to ISO 14644-3, annex B4 [
A correct air quantity is necessary to displace particles, pressurize required spaces, and control temperature and humidity. This parameter is calculated as air changes per hour. According to ISO specifications it should be >120 air changes/h, >40 air changes/h, and >20 air changes/h for 100, 10,000, and 100,000 class cleanroom or clean area class, respectively. Airflow can also be used to determine the number of air changes that occur in a space over a period of one hour. This is accomplished by determining the supply (cm3/h) and dividing it by the total volume of a space (length × width × height) to come up with the number of air exchanges per hour.
Cleanrooms should have other requirements as temperature and humidity. These measurements will also assure the correct performance of the heating, ventilation, and air-conditioning (HVAC) system. However, some process steps require appropriate temperature. Moreover, the personnel commodity wearing special clothing should be taken into consideration. Relative humidity also affects occupant comfort, productivity, and operating costs. In general acceptance criteria are 22 ± 3°C (72 ± 5°F) temperature and 30–50% relative humidity.
On the other hand, the illuminance should be in accordance with the task to be performed. A range of 400 to 750 lux is recommended [
Finally, other physical tests for parameters as noise, vibration, or radiation have little or no applicability in cleanrooms for the processing of SCMPs.
A major consideration in the operation of cleanroom technology for aseptic dispensing is the monitoring of viable contamination within clean environments [
The alert and action limits, expressed in cfu, should be established on the basis of levels of detection of microbial contamination. Action levels for nonviable particles are defined in the various regulatory and compendial documents for each room or area classification. Action levels are those that, when exceeded, indicate the appropriate corrective measure to return to the appropriate environmental safety. USA and European regulations, as well as, in the USP, chapter 1116, “Microbiological Control and Monitoring Environments Used for the Manufacture of Healthcare Products,” established the acceptable number of viable particles per m3 that can be found in determined cleanroom or clean area. WHO adopted the European standards. However, each company should set its own microbiological levels based on the aseptic requirements of it production. ISO does not refer to microbiological levels.
The methods used for microbiological monitoring include active air sampling (air sampler), passive air sampling (settle plates), surface sampling (contact plates and swabs), and personnel sampling (finger plates/plates of gowns). In order to carry out these operations the licensed manufacturer must be certified as a GMP manufacturer accredited by a recognized certification body in accordance with ISO 17025 or equivalent. However, it is not possible in Europe, where the GMP manufacturing is authorized by the national competent authority and recognized across the border on the basis of an international treaty. Currently, the US GMP authorization by FDA is not recognized in Europe and vice versa. The use of outside laboratories to carry out microbiological analysis can be accepted for particular reasons, as many companies are outsourcing technical testing activities and reducing in-house capabilities in an effort to control costs, but this should be stated in the quality control records. Manufacturers should use a risk-based approach to determine whether a preapproval audit is required before approving a contract laboratory. Various Agency guidance documents indicate how quality management principles relate to contract these operations. The ICH guidance for industry Q7 [
The sampling plan for viable particles should define the number of points sampled in each of the areas of a cleanroom and determine how often to perform the sampling. According to the FDA-cGMP monitoring locations that present the highest potential contamination risk to the product and trending performance should be selected by assessing the critical activities taking place, the flow of personnel in the processing area, and the position of filters to determine the most potential high risk contamination locations. This approach is also stated in EU-GMP and ICH Q9 recommendations [
As discussed, the ISO 14644-1 guideline [
Regarding the sampling frequency, it depends on the classification: the lower the maximum permitted particulate the higher the frequency of monitoring. GMP guidelines do not go into details. Table
The selection of the growth media should assure the growth of microbes existing in the controlled environment. Thus, according to ISO 14698-1 [
Strains of the test microorganisms suitable for use in the growth promotion test and the validation test.
Microorganism | Strains | |
---|---|---|
Aerobic | | ATCC 6538, CIP 4.83, NCTC 10788, NCIMB 9518 |
| ATCC 6633, CIP 52.62, NCIMB 8054 | |
| ATCC 9027, NCIMB 8626, CIP 82.118 | |
Anaerobic | | ATCC 19404, CIP 79.3, NCTC 532 or ATCC 11437 |
Fungi | | ATCC 10231, IP 48.72, NCPF 3179 |
| ATCC 16404, IP 1431.83, IMI 149007 |
Both USP and EP describe several adequate culture media for the sampling and quantification of microorganisms. As per USP Soybean Casein Digest Agar (SCDA) is the standard medium for sampling or quantitation of microorganisms in controlled environments. Yeasts and moulds may also be specifically sought out. Sabouraud Dextrose Agar is used especially for yeasts and moulds. As per EP fluid thioglycollate medium is primarily intended for the culture of anaerobic bacteria; however, it will also detect aerobic bacteria and SCDA for the culture of both fungi and aerobic bacteria.
For “settle plate” methods, Trypticase Soy Agar (TSA) is the most recommended medium for bacteria. It contains a mixture of peptones that promote the growth of most microorganisms. Agar Sabouraud Dextrose Chloramphenicol (SDC) is the recommended medium for fungi and yeast. Its high concentration of glucose optimizes the growth of fungi and its pH and chloramphenicol content improve the selectivity.
In order to choose the most efficient parameters for the test methodology, microbiologically, the best media and incubation conditions should be previously assayed, and parameters that yield the highest microbial recovery with the shortest incubation period are chosen for routine testing. Whether surfaces of testing were treated with detergents or disinfectant products, a neutralizing agent must be included in the recovery media. In this line, an antibiotic inactivating product must be incorporated in the recovery media if the testing surfaces have been treated with antibiotics.
Total aerobic microbial count (TAMC) is determined by incubation in those media. The incubation conditions should be previously selected and validated. Culture conditions differ between microorganisms, 48 h at 32.5 ± 2.5°C for bacteria versus 72 h at 22.5 ± 2.5°C for fungi and moulds. Posteriorly, USP considered the possibility of longer incubation times. Equally, in case of absence of confirmatory evidence, one single plate may be incubated at both a low and a higher temperature. EP for its part recommends incubating the plates not more than 3 days in the case of bacteria at 30–35°C and not more than 5 days in the case of fungi at 20–25°C [
USP lists other permitted alternative media, liquid or solid. Furthermore, other alternative media to those listed can be used whether they are validated for the purpose intended.
Critical areas’ monitoring should be carried out under “worst case” conditions for contamination with process equipment running and personnel performing normal operations (“
Recommended limits for viable airborne particles in the environment according to FDA-cGMP, EU-GMP, and USP.
Clean area type | Maximal number of cfu in the environment | ||||||
---|---|---|---|---|---|---|---|
FDA-cGMP | EU-GMP | USP | |||||
FS209E | ISO | EU-GMP | Air sample (cfu/m3) | Settle plates | Air sample (cfu/m3) | Settle plates (diam. 90 mm; cfu/4 h) | Air sample (cfu/m3) |
M3.5 (100) | 5 | A | 1 | 1 | <1 | <1 | <3 |
M4.5 (1,000) | 6 | 7 | 3 | — | — | ||
M5.5 (10,000) | 7 | B | 10 | 5 | 10 | 5 | <20 |
M6.5 (100,000) | 8 | C | 100 | 50 | 100 | 50 | <100 |
D | — | — | 200 | 100 | — |
Passive air or sedimentation sampling is based on the fact that, in absence of any kind of influence, airborne microorganisms which typically are attached to large particles will deposit onto open culture plates (settle plates) [
The cleanroom should be “
EU-GMP [
Another contact method for surfaces where contact plates could not be utilized is to undertake a swabbing with sterile swabs. When swabbing is used in sampling, the area covered should be greater than or equal to 24 cm2 but no larger than 30 cm2 as stated by USP. After swabbing, the swab should be placed into a suitable culture medium or a diluent, vortexed for about 30 s, and then tested by pour-plate or membrane filtration method [
Finally, flexible films are reported by the PDA [
Recommended limits for viable airborne particles on surfaces according to EU-GMP and USP.
Clean area type | Maximal number of cfu on surfaces | ||||
---|---|---|---|---|---|
EU GMP | USP | ||||
FS209E | ISO | EU GMP | Contact plates (diam. 55 mm; cfu/plate) | Contact plates (area 24–30 cm2; cfu/plate) | |
Surfaces | Floor | ||||
M3.5 (100) | 5 | A | <1 | 3 | 3 |
M4.5 (1,000) | 6 | — | — | — | |
M5.5 (10,000) | 7 | B | 5 | 5 | 10 |
M6.5 (100,000) | 8 | C | 25 | — | — |
D | 50 | — | — |
Only personnel who are qualified and appropriately gowned should be permitted access to the aseptic manufacturing area. Personnel can significantly affect the quality of the environment in which the product is processed; for this reason only the minimum number of personnel required should be present in cleanroom. Methods for personnel microbiological testing should include gloves and protection clothes at the end of each working session prior to the operator carrying out any cleaning or tidying operations. For this the desired area of the protection clothing is placed lightly onto the surface of the agar medium of the settle plate. On the other hand, the glove finger count checking is done randomly among individuals by finger dab plates in each of the five fingers of both hands. Finger dabs can be performed using either standard 90 mm diameter settle plates or 55 mm diameter contact plates. After incubation, results are reported as number of cfu per glove according to EU-GMP or cfu/plate according to FDA-cGMP. Recommended action limits for personnel sampling monitoring of cleanrooms and clean areas are depicted in Table
Recommended limits for viable airborne particles on personnel according to FDA-cGMP, EU-GMP, and USP.
Clean area type | Maximal number of cfu | ||||
---|---|---|---|---|---|
EU-cGMP | USP | ||||
FS209E | ISO | EU GMP | Glove print (5 fingers) (cfu/glove) | cfu per contact plate | |
Gloves | Personnel clothing and garb | ||||
M3.5 (100) | 5 | A | <1 | 3 | 5 |
M5.5 (10,000) | 7 | B | 5 | 10 | 20 |
FDA-cGMP has clearly recommended the establishment of a listing of common microorganisms found in the aseptic manufacturing environment [
It is so important to have knowledge of the “normal” background flora of a cleanroom facility. Any unusual organisms or deviation from “normal” flora may require corrective actions.
ISO 14644-1 includes the elaboration of a test report after testing in this way. The results from testing each cleanroom or clean zone shall be recorded and submitted as a comprehensive report, along with a statement of compliance or noncompliance with the specified designation of airborne particulate cleanliness classification. This standard provides for the inclusion, among other information, physical description of facilities, designation criteria for the cleanroom or clean zone, test methods, and test results.
The field of MSCs manufacture includes the task of interpreting and harmonizing international guidelines to ensure their acceptable quality for translational clinical use in regenerative medicine. One of the great challenges for the future is to set a single regulatory framework for the SCMPs manufacture, through harmonization of all the requirements for their production whatever their use or intended final purpose: gene therapy, cell therapy, or tissue engineering or whenever their production: USA, Europe, Japan, and so forth.
The authors declare no conflict of interests.