Environmental Monitoring Program for Aseptic Vaccine Products

Introduction

This article is primarily about the Environmental Monitoring (EM) program that should be in place for biological products including bacterial and viral vaccines, although there is also mention of testing of raw materials and finished product as well. Biological products are derived from cells, tissues or microorganisms through cell culture growth/fermentation, extraction of substances of interest, isolation and purification leading to formulation and filling. Products include allergens, antigens, hormones, enzymes, monoclonal antibodies, immune animal sera, fermentation products and bacterial and viral vaccines.¹

Much of the article will apply to environmental monitoring during formulation and filling of aseptic biological products in general. Aseptic processing is principally what is employed to ensure assurance of sterility, because some vaccines are not filterable or cannot be sterilized by heat, gas or radiation.² Axenic products (sterile except for the organism of choice in the vaccine) must use aseptic processing as well as environmental controls/monitoring, in-situ cleaning/sterilization of tanks and other equipment, closed systems where possible and even disposable single use systems.¹

Therefore, there should be close collaboration between biosafety and cGMP systems to assure control of product contamination or cross contamination and to ensure acceptable environmental control levels. This would apply especially where there are live or spore forming organisms for vaccine preparation and especially for growth of organisms at Bio-safety levels 3 and 4 (negative pressure areas). Per World Health Organization (WHO) documents, no other infectious or live materials should be worked with in the same area during establishment of seed lots and cell banks. Consider the use of dedicated facilities for live or attenuated vaccines and different means of inactivation (chemicals, heat, etc.), while still maintaining antigenicity.¹

Environmental Control Measures

Control measures of the environment include differential pressures, air changes, unidirectional air, closed manufacturing processes wherever possible, qualification of processes such as gowning, performance of media fills, and control of environmental bio-burden. HEPA air filters should be qualified every six months and smoke studies should be performed for the highest controlled areas (Grade A) every three years. Define EM sample sites, types and frequency of sampling and alert/action levels and trends. Investigations should be performed if levels are exceeded or trends are detected (to be discussed further).^3,4

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Material and personnel flows must be well defined with traffic starting in Grade D areas, into Grade C, then B and finally Grade A with gowned personnel. Specific PPE for C and D includes masks and gloves and usually something like bulk-sterilized jumpsuits. There must be limits to the numbers of people allowed in the Aseptic Processing Area (APA) at any one time, how long they may stay in the APA and control of interventions (inherent/routine and corrective).⁵ Gowning performance must be qualified before personnel are allowed into the APA and they must show adequate aseptic technique during media fills prior to being allowed to fill aseptic batches of product.³

Bio-burden of air, people, water and gases must be controlled. Samples sites, types of sampling and sampling frequency should be defined in Standard Operating Procedures (SOPs). Levels/limits must be defined in SOPs along with steps to be taken if levels are exceeded (e.g., performance of investigations) or trends are detected.^3-7

Disinfectants may be qualifi ed by outside contract labs using coupon surfaces present in the facility or by in-situ testing. Approved disinfectants, their use-dilution and appropriate dwell (contact) time must be listed in SOPs.⁸

Failure Modes and Effects Analysis

An FMEA (Risk Assessment) for evaluating sources of microbial contamination is a valuable living document to determine what actions/changes should be taken or made to avoid ingress of organisms into an aseptic processing area. Risk criteria should be defined up front (scoring and acceptability/threshold levels). The Risk Priority Number (RPN) is calculated as follows: Severity x Probability of Occurrence x Detection = RPN

• Determine the significance of identified risks (high, medium, low risk) using your thresholds.
• Prioritize your risks based on the outcome of the assessment (i.e. high risk, high priority) for mitigation.

Mitigation decisions and activities should be determined by a cross-functional team of SMEs consisting of: Sterility Assurance, Quality Assurance, Quality Control, Compliance, Production, Engineering and Validation.^9,10

Testing of Raw Materials, Bulk and Finished Product

Starting with raw materials, sera, media and other regents, perform adventitious agent testing and assure absence of Transmissible Spongiform Encephalopathy (TSE). Bulk bioburden is a helpful, but not required test. Finished product sterility testing, adventitious agent testing showing absence of extraneous viruses and absence of Mycoplasma should be performed.

According to “EM of Clean Rooms in Vaccine Manufacturing Facilities” (WHO 2012), use a risk-based approach to determine clean room grades for different steps in vaccine production. Raw materials should be sampled in Grades A or at the most B or Grade A isolator (in a Grade D background). If final product will be filter-sterilized, use Grade C at a minimum for sampling or other processing steps.⁵

If vaccines are live pathogenic organisms, cross-contamination must be avoided by using unidirectional airflow Grade A conditions or barrier systems to protect product from the environment and people and to protect employees from pathogenic infection. Consider the use of dedicated facilities in this case or consider creating attenuated vaccines and antibiotics or preservatives in the product. Attenuated vaccines must be tested for reversion to wild type (e.g., old, discontinued oral Polio vaccine).^1,5

Environmental monitoring should indicate contamination control of formulation and filling areas, and from that, protection of a batch from contamination can be indicated. There is a lack of direct correlation between Environmental Monitoring (EM) levels and batch sterility. Environmental monitoring results are a reflection of microbial control of an area.⁷ However, batch impact must be evaluated based on EM and Personnel Monitoring (PM) results.^4,7 A risk assessment could be written to document that evaluation.

Cleanroom Standards

Cleanroom standards have been defined by the FDA (2004 Aseptic Guidance document), USP <1116> and both EU Annex 1 and WHO (Annex 4 and Annex 6, update) for non-viable particle limits (NVP) and microbial levels for Active Air, Passive Air (Settle Plates), Surface (RODAC) and Glove samples. Recently, the USP switched from numerical levels to a recommended recovery rate per month per sample type, per room and per grade, although it is considered abnormal if an excursion of 15 CFU is found.⁷

Non-Viable Particle (NVP) Monitoring

For NVP monitoring, the US (FDA Aseptic Guidance and USP) only requires monitoring of 0.5 μm particles but not 5 μm particles, but the EU (Annex 1) requires that the larger particles are monitored. However, this requirement has been removed in their recent proposed update to Annex 1.^3,5,7 It is critical that NVP values from multiple sites (determined by a grid system and Risk Assessment) not be averaged. Probe locations should be near open product (about 30 cm from critical filling area) and worst case locations for possible product contamination assessments. Use ISO 14644 as a guide to determine locations for NVP monitoring. Schematic drawings of rooms with sampling locations should be provided.¹¹

Grade A areas should be monitored for the full duration of operational activities. Continuous monitoring is performed using fixed NVP monitoring counters. Portable counters can be placed at additional locations and can be used to replace continuous monitors, when necessary. Dedicated particle counters must be used for segregated areas such as for spore-forming organisms or the higher grades of Biosafety facilities.⁶

Facilities should be qualified to meet Grade specific limits at a minimum annually and obviously to verify room classification of a new area. For Grades A and B, filter integrity testing should be performed every six months. Any failures require investigation and requalification. There should be limits developed beyond which patching of filters is not performed, but rather the filter is replaced.^3-5,11

Viable Monitoring

Viable monitoring assures microbial control of the environment and is used for aseptic products, vaccines (the subject of this article) and pharmaceuticals. Active air sampling pulls in a specified volume of air (1 m³) in a certain time period, so it is the most quantitative method. Settle plates are semi-quantitative and are exposed for a period of four hours. Most pharmaceutical plants test during the full duration of operation (per WHO requirements⁶) or twice to cover the duration of a shift (per USP <1116>⁷) RODAC (contact plates) are used for surfaces to check cleaning efficacy (ensure that media plates have the correct neutralizers for the disinfectants used) of equipment at the end of operations and for sampling of personnel in the aseptic processing area. Routine monitoring should be done on fingerprints, forearms and chest, and fingerprints should be done prior to changing gloves to go into a different filling room and prior to sanitizing with alcohol.^4-7

In the Grade A area, Active Air (volumetric sampling) should be done every four hours, settle plates may be left open for that whole period, and contact plates should be done after operations (or during static monitoring) and to sample people prior to sanitization or changing outer gloves.^4-7

The plan for Environmental Monitoring (EM) Process Qualification must be documented for a new facility or after construction in an older facility.¹² Sites should be picked by risk assessment of product exposure, highest traffic and most difficult-to-clean areas. It is best to collect 100 data points for all sites, especially for determination of alert levels (as action levels are defined in guidance documents). Alert levels should be used to determine adverse trends and can be modified based on statistical analysis after accumulation of a lot of data.⁷

Static monitoring (at rest) testing provides information about disinfectant efficacy and should be performed before production personnel enter the facility, either monthly, quarterly or semi-annually after a shutdown; define this in your SOPs. In operation (dynamic) testing provides information regarding control of the environment. Establish a database of environmental and people isolates to be used for Growth Promotion Testing and Disinfectant Efficacy Testing.⁷

Investigations

It is not necessary to investigate alert level excursions, but this must be defined in your SOPs. Alert levels are always lower than action levels. Action levels are defined in regulatory guidance documents- e.g., USP, Annex 1, PDA Technical Report 13^4,5,7 and require an investigation if exceeded by Grade, Room or Sample type. Trends can also be included in investigations by Grade or Room and also organism type. Trends are usually defined as three or more consecutive points in a specified time period. Trends usually are considered adverse if they show an increase in counts, a change to repetitive organism recoveries or multiple counts in the same room.^4,5,7

Investigations should be performed using various root cause analysis techniques including the Fishbone Method, 5 Whys, and Kepner-Tragoe Analytical Trouble-Shooting, but root cause is not likely to be found for a recovery of 1 CFU.7 Risk Assessment is a useful tool to determine product impact of an excursion or adverse trend, including a review of organism type (Gram-negative organisms could leave residual endotoxin in product and mold could leave allergens), sterilization method, location of excursion, etc.^9,10 When root cause has been determined, there should be CAPAs developed (Corrective and Preventive Actions). If there is likelihood that a batch could have been contaminated, even if no root cause was uncovered, then that batch should be rejected to err on the side of caution.

Controls that reduce the likelihood of the occurrence or increase detectability are considered as reasonable follow-up action. High risk activities (if a Risk Assessment is written) should lead to remedial (corrective actions), preventive actions and eventually should be evaluated for CAPA effectiveness.

Media Fills

Semi-annual media fills (aseptic simulations) with growth media are necessary for showing control of manufacturing processes such as duration of fill, qualified interventions, maximum number of people in the room, etc. Failures put into question all lots filled since the last successful run and must be investigated. Then three more passing runs must be performed.^3,5

As for pharmaceuticals, the best systems for filling vaccine products have avoidance of close human contact: closed systems, RABs, Isolators, etc. because humans are the biggest source of contamination in an aseptic processing area.

References

1. WHO Good Manufacturing Practices for Biological Products 2016.
2. WHO Technical Report Series, No 957, WHO Good Manufacturing Practices for Sterile Pharmaceutical Products, Annex 4 2010 and No. 961, Annex 6 2011.
3. Sterile Drug Products Produced by Aseptic Processing- Current Good Manufacturing Practice 2004.
4. PDA Technical Report No. 13 (Revised) Fundamentals of a Microbiological Environmental Monitoring Program 2014.
5. Volume 4- EU Guidelines to Good Manufacturing Practice Medicinal Products for Human and Veterinary Use- Annex 1 Manufacture of Sterile Medicinal Products (corrected version)- Effective March 1, 2009.
6. WHO Environmental Monitoring of Clean Rooms in Vaccine Manufacturing Facilities 2012.
7. United States Pharmacopoeia (current Edition 41) <1116> General Chapter-Microbiological Evaluation of Clean Rooms and Other Controlled Environments 2018.
8. United States Pharmacopoeia (current Edition 41) <1072> Disinfectants and Antiseptics, 2018.
9. ICH Q9 Quality Risk Management 2005.
10. Hutt, R. Environmental Monitoring and its Impact on Sterility. In: Moldenhauer, J., Environmental Monitoring, A Comprehensive Handbook, Vol. 8, 2017: 153-165.
11. International Organization for Standardization (ISO) 14644 Cleanrooms and Associated Controlled Environments 2015.
12. Qualification of an Environmental Monitoring Program Spring 2010, S. Sutton, J. of Validation Technology Vol. 16, #2: 78-82.