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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Bioengineering

Microbial Control and Monitoring Strategies for Cleanroom Environments and Cellular Therapies

Published: March 17th, 2023

DOI:

10.3791/65209

1Sterility Testing Service, Department of Laboratory Medicine, Clinical Center, National Institutes of Health

The protocol summarizes the best practices to minimize microbial bioburden in a cleanroom environment and includes strategies such as environmental monitoring, process monitoring, and product sterility testing. It is relevant for manufacturing and testing facilities that are required to meet current good tissue practice standards and current good manufacturing practice standards.

A well-validated and holistic program that incorporates robust gowning, cleaning, environmental monitoring, and personnel monitoring measures is critical for minimizing the microbial bioburden in cellular therapy manufacturing suites and the corresponding testing laboratories to ensure that the facilities are operating in a state of control. Ensuring product safety via quality control measures, such as sterility testing, is a regulatory requirement for both minimally manipulated (section 361) and more than minimally manipulated (section 351) human cells, tissues, and cellular and tissue-based products (HCT/Ps). In this video, we provide a stepwise guide for how to develop and incorporate the best aseptic practices for operating in a cleanroom environment, including gowning, cleaning, staging of materials, environmental monitoring, process monitoring, and product sterility testing using direct inoculation, provided by the United States Pharmacopeia (USP<71>) and the National Institutes of Health (NIH) Alternative Sterility Testing Method. This protocol is intended as a reference guide for establishments expected to meet current good tissue practices (cGTP) and current good manufacturing practices (cGMP).

Implementing a strong microbial monitoring program through environmental monitoring (EM), process monitoring, and product sterility testing is a regulatory requirement for current good tissue practices (cGTP) and current good manufacturing practices (cGMP) in cellular therapy laboratories1. Additionally, the United States Food and Drug Administration (FDA) expects that the laboratory performing the quality control (QC) testing of the product should also employ facilities and controls comparable to those used for aseptic filling operations2.

This protocol has four main sections: 1) Aseptic pra....

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1. Aseptic practices

  1. Personnel gowning for a cleanroom space
    NOTE: This procedure is based on initial gowning in an unclassified space, followed by entry into an International Organization for Standardization (ISO) 8 area and then an ISO7 area. This procedure is relevant for laboratories attempting to convert existing space into a cleanroom function. Ideally, all initial gowning would occur in an ISO8 classified space (not in an unclassified space).
    1. Secure loose hair. Wash .......

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The expected results are described in Table 1. The EM data should be reviewed and followed up with an appropriate investigation and response to action, alert, or ISO limit excursions. If an excursion occurs for non-viable particles, one should proceed as per ISO 14644-Annex A, sec A.5.57. If the excursion can be attributed to an immediately identifiable abnormal occurrence, the original sampling results should be documented, a note should be added to disregard the original results.......

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There are several critical areas in this protocol, including the maintenance of aseptic technique and unidirectional airflow within cleanrooms and the BSCs. Best practices include moving slowly and deliberately to minimize turbulence. Aseptic manipulations should be performed from the side of the product, not from above. Closed system processing and the use of terminally sterilized raw materials are recommended. Speaking in critical areas and leaning against walls or equipment should be avoided. Similarly, unnecessary to.......

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This work was supported by the Intramural Research Program of the National Institutes of Health Clinical Center. The content is solely the responsibility of the authors and does not represent the official views of the National Institutes of Health.

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Name Company Catalog Number Comments
20-25°C Incubator Lab preference
30-35°C Incubator Lab preference
Alcohol-based hand sanitizer Lab preference
BacT/ALERT Dual-T instrument BioMerieux Industry
Beard cover Lab preference
Biosafety cabinet (BSC) Lab preference
Cleanroom shoes Lab preference
Fluidthioglycollate medium (FTM) Hardy Diagnostics  U84 USP
Handheld cleaning mop Contec 2665LF
Hypodermic needle Lab preference
iFA+ BacT/ALERT bottle Biomerieux 412990
iFN+ BacT/ALERT bottle Biomerieux 412991
Isokinetic head Lab preference
Laser particle counter TSI Incorporated 9500-01
LpH III Steris 1S16CX
Mirror Lab preference
Non-sterile bouffant Lab preference
Non-sterile gloves Lab preference
Non-sterile shoe covers Lab preference
Non-sterile sleeve covers Lab preference
Parafilm Lab preference
Peridox RTU Contec CR85335IR
Plastic bag Lab preference
Sabouraud Dextrose Agar with Lecithinase and Tween (SABLT) Hardy Diagnostics  P595 USP, irradiated
Sabouraurd Dextrose Agar (SAB) Hardy Diagnostics  W565 USP, irradiated
Safety glasses Lab preference
Scrubs (top and bottom) Lab preference
Spor-Klenx RTU Steris 6525M2
Sterile 70% isopropyl alcohol (IPA) Decon CiDehol 8316
Sterile alcohol wipe Lab preference
Sterile boot covers Kimberly Clark Cat# varies based on size
Sterile coveralls Kimberly Clark Cat# varies based on size
Sterile face mask Lab preference
Sterile gloves Lab preference
Sterile hood Kimberly Clark Cat# varies based on size
Sterile low-lint wipes Texwipe TX3210
Sterile mop cleaning pads Contec MEQT0002SZ
Sterile sleeve covers Kimberly Clark 36077
Sterile spreading rod Fisher Scientific 14665231
Sterile syringe Lab preference
Tacky mats Lab preference
Tryptic Soy Agar (TSA) Hardy Diagnostics  W570R USP, irradiated
Tryptic Soy Agar with Lecithinase and Tween (TSALT) Hardy Diagnostics  P520R USP, irradiated
Tryptic Soy Broth (TSB) Hardy Diagnostics  U46 USP
Tubing Lab preference
Vesphene III Steris 1S15CX
Viable air sampler Hardy Diagnostics  BAS22K
Vortex Lab preference

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  2. United States Food and Drug Administration. Guidance for Industry. Sterile Drug Products Produced by Aseptic Processing - Current Good Manufacturing Practice. United States Food and Drug Administration. , (2004).
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  12. Anders, H. J., et al. Multisite qualification of an automated incubator and colony counter for environmental and bioburden applications in pharmaceutical microbiology. PDA Journal of Pharmaceutical Science and Technology. , (2022).
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  19. United States Pharmacopeia - National Formulary. USP<825> Radiopharmaceuticals - Preparation, Compounding, Dispensing, and Repackaging in USP43-NF38 2S. United States Pharmacopeia - National Formulary. , (2020).

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