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The manufacture of sterile pharmaceutical product requires specific attention regarding environment, premises and practice of the personnel. Regulations in place to be used by the Pharmaceutical Industry give requirements on the environmental monitoring program. FDA with the publication of Warning Letters and 483 form shows that environmental monitoring procedure and practice of the personnel belong to top 10 of the gaps found during inspections.
This article, for the Environmental Monitoring program (EM) in aseptic processing as well in Conventional Clean Rooms (CCR) or using Isolator/RABS technologies: describes the main expectations of the regulations in place, presents the hot topics in microbiology and gives solutions for implementation of best practices. From the gaps highlighted by FDA during inspections, the following Microbiology topics will be described: samples location justification, missing samples and data interpretation, trending system and CAPA system.
1. 1 Environmental Monitoring in CCR – Key Expectations
Regulations[(1)(2)(3)(4)(5)(6)(7)(8)] require the following key points to be performed during the EM for filling operations in CCR:
– Filling in aseptic processing in Conventional Clean Room (CCR) should be classified Class ISO 5 (Grade A).
– Surrounding room should be classified Class ISO 5 (Grade B at rest).
– Surrounding room should be maintained Class ISO 7 (Grade B in operation).
– Environmental Monitoring Program (EM) should be in operation.
– EM locations should be based on a formal risk analysis study and from the results.
– For CCR control of the personnel is required Gloves/Garment depend on the room class.
– For CCR Active Air Sample (AAS), Passive Air Sample (PAS) & Surfaces Monitoring (SM) should be performed.
– Surfaces should be monitored after completion of the working operation
– Gloves of the personnel should be monitored at each exit and after critical interventions
– Garment of the personnel (Grades A/B) should be monitored for each shift
– Limits for each sample type (Refer to EU/GMPs/Annex 1)
– Trending system using CRR (Refer to U.S.P.<1116>).
1.2. Environmental Monitoring using Isolator/closed RABS technologies – Key Expectations
Regulations [(1)(2)(3)(4)(5)(6)(7)(8)] require the following key points to be performed during the EM for filling operations using isolator or closed RABS technologies:
– Isolator should be classified Class ISO 5 (Grade A).
– Surrounding room should be classified Class ISO 8 (C/D).
– Environmental Monitoring (EM) should be in operation.
– EM locations should be based on a formal risk analysis study and from the results obtained both during Qualification Program & in Routine.
– For isolator control of the personnel is not required.
– For isolator AAS, PAS & Surfaces should be performed.
– Surfaces should be monitored after completion of the filling operation or campaign.
– Critical gloves & half-suits should be monitored at the end of each filling operation.
– Limits for each sample type (Refer to EU/GMPs/Annex 1).
– Trending system using CRR (Refer to U.S.P.<1116>).
2. Hot Topics in Microbiology & Best Practices
2. 1 Samples Location Justification
Samples are generally described in the procedures for Environmental Monitoring program. But the exact location for each is not always clearly defined and/or justified.
FDA Warning Letter 2011: « Your Environmental monitoring program does not give assurance that environmental contaminants are reliably detected:
– Your practice of collecting samples from the gloves of operators from left & right hands on alternative days is unacceptable.
– Your SOP fails to include instructions for the location and duration of samples collected in the critical aseptic processing areas. «
What are the expectations of the authorities?
FDA – Guidance for Industry – Aseptic Processing – 2004:
– « A vigilant and responsive personnel monitoring program should be established. Monitoring should be accomplished by obtaining surface samples of each operator’s gloves on a daily basis, or in association with each lot. This sampling should be accompanied by an appropriate sampling frequency for other strategically selected locations of the gown. The quality control unit should establish a more comprehensive monitoring program for operators involved in operations which are especially labor intensive (i.e., those requiring repeated or complex aseptic manipulations). «
– « We recommend that this assessment include microbiological surface sampling of several locations on a gown (e.g., glove fingers, facemask, forearm, chest). Sampling sites should be justified. Following an initial assessment of gowning, periodic requalification will provide for the monitoring of various gowning locations over a suitable period to ensure consistent acceptability of aseptic gowning techniques. »
– « The monitoring program should cover all production shifts and include air, floors, walls, and equipment surfaces, including the critical surfaces that come in contact with the product, container, and closures »
– « Written procedures should include a list of locations to be sampled.«
– « Sample timing, frequency, and location should be carefully selected based upon their relationship to the operation performed.«
– « Samples should be taken throughout the classified areas of the aseptic processing facility (e.g., aseptic corridors, gowning rooms) using scientifically sound sampling procedures. «
– « Air and surface samples should be taken at the locations where significant activity or product exposure occurs during production. «
– « Critical surfaces that come in contact with the sterile product should remain sterile throughout an operation. »
– « When identifying critical sites to be sampled, consideration should be given to the points of contamination risk in a process, including factors such as difficulty of setup, length of processing time, and impact of interventions.«
– « All environmental monitoring locations should be described in SOPs with sufficient detail to allow for reproducible sampling of a given location surveyed.«
– « Written SOPs should also address elements such as frequency of sampling, when the samples are taken (i.e., during or at the conclusion of operations), duration of sampling, sample size (e.g., surface area, air volume), specific sampling equipment and techniques, alert and action levels, and appropriate response to deviations from alert or action levels. »
– « Surfaces – Environmental monitoring involves sampling various surfaces for microbiological quality. For example, product contact surfaces, floors, walls, and equipment should be tested on a regular basis. »
– « Active Air Sampling – We recommend that such devices be used during each production shift to evaluate aseptic processing areas at carefully chosen locations. »
– « Passive Air Sampling – Their value in critical areas will be enhanced by ensuring that plates are positioned in locations posing the greatest risk of product contamination. »
ISO 13408 – Part 1:
« Gloved fingerprints of personnel present in the direct support zone and/or critical processing zone shall be monitored daily.
At defined intervals samples from the gowns shall also be taken (e.g. both forearms, chest, hood.
NOTE: The frequency of sampling of gowns and gloves is based on the nature of the activities performed. «
ISO 14698 – Part 1:
« The following are examples of elements to be considered: Choice of the sampling location, taking account of the location and function of the risk zone. »
« A documented risk assessment for the selection of each process should be made some examples of risk factors to consider in selecting sites for surveillance are as follow:
1. Sites or processes in which microbial contamination would most likely have an adverse effect on product quality
2. Sites that would most likely demonstrate the heaviest microbial proliferation during actual production
3. Whether site selection should involve a statistical design or should be made on the basis of grid profiling
4. Whether routine monitoring sites should be rotated
5. Sites that represent the most inaccessible or difficult areas to clean and disinfect
6. Modes of microbe dispensal in the environment
7. Sampling at a given site that may disturb the environment sufficiently to cause erroneous data to be collected or to contaminate product. »
– « Monitoring locations should be determined based upon a assessment of risk. »
– « Locations considered should include those in proximity of the exposed product, containers, closures, and product contact surfaces.«
– « The user should conduct a prospective risk analysis and develop a rationale for the sampling locations and frequencies for each controlled environment. »
« Clean rooms and clean air devices should be routinely monitored in operation and the monitoring locations based on a formal risk analysis study and the results obtained during the classification of rooms and/or clean air devices. »
How to justify samples location?
Apply a risk based approach for the analysis methodology combining:
– Process knowledge and understanding including support with smoke test studies
– Evaluation of process criticality
– Critical control points (CCP) definition and location
– Sampling plan program description
The risk analysis evaluation approach should follow the following steps:
a/ Identify the potential sources/causes of contamination:
– Ishikawa fishbone diagram (Figure 3),
– Smoke test studies,
– Data from the initial EM Qualification.
b/ Risk assessment: Levels of Severity and Occurrence evaluation tables and quotation for each intervention at risk.
c/ Mapping of potential sources/causes of contamination with risk evaluation for each process operation: Critical Control Points (CCPs) approach & Total environmental risk evaluation.
Figures 4, 5 & 6 describe an example of definition of the samples locations from an aseptic filling process including closure of vials with rubber stoppers.
Figure 4 describes an aseptic filling process in CCR with the following flows:– Product to be filled– Primary packaging components (sterile & pyrogen free vials after direct transfer from the oven)– Stoppering components after transfer by the personnel from sterile bags.
Figure 5 describes the potential samples locations where a sample point is defined normally not more than 1 foot dimension (FDA requirement) away from the work site, within the airflow, and during filling/closing operations versus their respective areas in the perimeter of intervention of the personnel (orange triangles).
All critical control points (CCP) are defined as full red spots.
Figure 6 describes the exact samples location for all samples types (Active air Sampling using an impaction system, Passive air Sampling using sedimentation settle plates 90mm, Surface Monitoring using contact plates 25cm2).
2.2. Missing Samples
FDA – Warning Letter 2011: « Your firm has not thoroughly
investigate any unexplained discrepancies, for example for missing environmental samples. »
FDA – Warning Letter 2012: « Approximately 846 EM samples were not collected in the grades A & C areas from March 2010 to Feb 2012.
This substantial number of missed samples suggests a pattern that raises concerns regarding your environmental program. Collecting scheduled EM samples is critical aspect of any environmental control program at an aseptic manufacturing facility. «
What is the expectation of the authorities?
FDA: Missing samples is a deviation.
What to do?
If immediately concerned:
– This incident must be managed as a deviation involving the performance of an investigation.
– Evaluate the impact of the batch.
– In grades C & D the impact should be low. But in grades A & B surround A the impact is high up to batch destruction.
– Review of the other results and trend at the same location on a period of time.
– Review of the results of the other location the day of the missing sample.
– If the origin is a damage of the plate, a solution could be incubation of this plate with an information of the risk to have contamination because of this damage. If important growth with result different from the trend, evaluate after investigation the possibility for invalidation.
– A CAPA must be defined and scheduled including an action for traceability of all samples and the possibility to know the status of each sample at each critical step such as sampling, transfer, incubation, reading (Refer to Figure 7).
It’s essential to keep and manage the all traceability of the sampling program from the initial step (preparation of samples devices), the incubation operations, to the final step (results and data interpretation)
Figure 7 gives an example of the management of the samples including the use of a tablet PC and a scanning bar code system.
2.3. Data Interpretation – Trending System & CAPA
Interpretation of the EM results should be described in a procedure but Tends are not always easy to evaluate.
Health Canada – 2012: « The approach to EM trending was quite broad and was not necessarily sensitive enough to identify localized trends, nor would it necessarily identify trends for specific objectionable organisms. »
What are the expectations of the authorities?
ISO 14698 – Part 1:
« To assist in interpretation, results shall be reviewed over extended periods to determine trends. Based on the review of these investigations and specific testing results, decisions shall be made on the significance of unusual results, and the acceptability of the operations or products processed under those conditions. »
ISO 14698 – Part 2:
« Management of microbiological results from risk zones should take the following factors into account: …;
– trend analysis; … »
« Data coming from a single sample are often not significant; furthermore, microbiological monitoring techniques may have serious shortcomings that result in a high degree of variability. Therefore, graphic presentation of the results collected over a period of time may be useful in distinguishing sampling variation from trends, or in indicating that a significant change has occurred, even though the results fall within the specified limits. «
ISO 13408 – Part 1:
« Environmental data (both counts and the type of microbial isolates) shall be analyzed for trends on a routine basis. »
« A trend report giving an overview of all environmental observations and trends shall be issued in fixed intervals. »
« Trend reports should include data generated by location, shift, room, operator or other parameters. When indicated by individual excursions and/or trend data, an investigation shall be initiated. »
« Examples of trends leading to an investigation include:
a) A trend towards higher numbers of microorganisms at a sampling site;
b) Repeated occurrence of microorganisms not often encountered. »
FDA/G for I/Aseptic Proc./2004:
« Adverse trends:
Because false negatives can occur, consecutive growth results are only one type of adverse trend. Increased incidence of contamination over a given period is an equal or more significant trend to be tracked. In the absence of any adverse trend, a single result above an action level should trigger an evaluation and a determination about whether remedial measures may be appropriate. In all room classes, remedial measures should be taken in response to unfavorable trends. The quality control unit should provide routine oversight of near-term (e.g., daily, weekly, monthly, quarterly) and long-term trends in environmental and personnel monitoring data. «
« Trend reports should include data generated by location, shift, room, operator, or other parameters«
« The quality control unit should be responsible for producing specialized data reports (e.g., a search on a particular isolate over a year period) with the goal of investigating results beyond established levels and identifying any appropriate follow-up actions. Significant changes in microbial flora should be considered in the review of the ongoing environmental monitoring data. »
«Routine review and analysis of environmental monitoring data for trends at an appropriate frequency is essential to aid in the interpretation of process stability and assess overall environmental control performance. »
« Recovery rate may be used for trending and control of the overall microbial load in the classified environment, equipment surfaces, material, and garment (Refer to U.S.P.<1116>). »
« The recommended recovery rate for ISO 5 environments can be achieved; … »
« It is recommended that companies develop their own recovery rate criteria for ISO 6, 7 and 8 environment depending on the activities and processes conducted in these areas. «
« The analysis of contamination trends in an aseptic environment has long been a component of the environmental control program. »
« Because of the inherent variability of microbial sampling methods, contamination recovery rates are a more useful measure of trending results than is focusing on the number of colonies recovered from a given sample. «
Table describing contamination recovery rates for aseptic processes.
« Trend Analysis: Data from a routine microbial environmental monitoring program that can be related to time, shift, facility, etc.
This information is periodically evaluated to establish the status or pattern of that program to ascertain whether it is under adequate control. A trend analysis is used to facilitate decision-making for requalification of a controlled environment or for maintenance and sanitization schedules. «
How to build a trend analysis system considering the severity and the occurrence of the microbial contamination in the objective to evaluate its risk fo the process, the product, the patient?
Written procedures should be established, detailing data review frequency and actions to be taken. The quality control unit should provide routine oversight of near-term (e.g., daily, weekly, monthly, quarterly) and long-term trends in environmental and personnel monitoring data.
Because of the limited accuracy and precision of microbial growth and recovery assays, analysts can consider the frequency with which contamination is detected rather than absolute numbers of cfu detected in any single sample. Implementation of the Contamination Recovery Rate (CRR) based on the following
Number of results with positive growth
CRR = ____________________________________
Total number of samples
The objective of each user should be to use CRRs to track ongoing performance and to refine the microbiological control program to foster improvement.
Action should be required when the CRR trends above the recommendations (U.S.P.<1116>) for a significant time.
Corrective actions may include but are not limited to the following:
– Review the sanitization program, including cleaning procedure, application methods, and frequencies
– Reinforcement of personnel training
– Increased surveillance of personnel practices
– Review of microbiological sampling methods, techniques, frequency, number of samples.
– When higher-than-typical recovery levels for gloves contamination are observed, additional action should be defined including the evaluation of gloves integrity (isolator technology).
As a conclusion regulation does not always detail all requirements for Environmental Monitoring Program. Nevertheless whatever the case each individual practice in place must be justified supported when possible by a risk based approach.
When “hot topics” in Microbiology emerge from return of inspections from authorities or/and during self-inspection, it should be a good opportunity to evaluate the local performance for a challenge to access to “best practices”.
Benoit RAMOND – SANOFI
(1) European Community – EudraLex – The Rules Governing Medicinal Products in the European Union, Volume 4 – EU Guidelines to Good Manufacturing Practice Medicinal Products for Human and Veterinary Use – Annex 1 – Manufacture of Sterile Medicinal Products.
(2) FDA – Guidance for industry – Sterile Drug Products Produced by Aseptic Processing – Current Good Manufacturing Practices – September 2004.
(3) U.S.P.<1116> – Microbiological Control and Monitoring of Aseptic Processing Environments.
(4) ISO 14644-1 – Cleanrooms and associated controlled environments – Part 1: Classification of air cleanliness
(5) ISO 14698-1 – Cleanrooms and associated controlled environments Biocontamination control – Part 1: General principles and methods.
(6) ISO 14698-2 – Cleanrooms and associated controlled environments Biocontamination control – Part 2: Evaluation and interpretation of biocontamination data.
(7) ISO 13408-1 – Aseptic processing of health care products – Part 1: General requirements.
(8) PDA – TR N°13 (Revised) – Fundamentals of an Environmental Monitoring Program – 2014.