Microbial Control and Monitoring Strategies for Cleanroom Environments and Cellular Therapies

Summary

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.

Abstract

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).

Introduction

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....

Protocol

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 hands, and change into scrubs (long-sleeve scrub tops and full-length scrub pants are preferable). Gather and change into cleanroom shoes with shoe cove....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

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