Real-time monitoring of non-viable airborne particles correlates with airborne colonies and represents an acceptable surrogate for daily assessment of cell-processing cleanroom performance

Abstract

Background aims: Airborne particulate monitoring is mandated as a component of good manufacturing practice. We present a procedure developed to monitor and interpret airborne particulates in an International Organization for Standardization (ISO) class 7 cleanroom used for the cell processing of Section 351 and Section 361 products.

Methods: We collected paired viable and non-viable airborne particle data over a period of 1 year in locations chosen to provide a range of air quality. We used receiver operator characteristic (ROC) analysis to determine empirically the relationship between non-viable and viable airborne particle counts.

Results: Viable and non-viable particles were well-correlated (r(2) = 0.78), with outlier observations at the low end of the scale (non-viable particles without detectable airborne colonies). ROC analysis predicted viable counts ≥ 0.5/feet(3) (a limit set by the United States Pharmacopeia) at an action limit of ≥ 32 000 particles (≥ 0.5 µ)/feet(3), with 95.6% sensitivity and 50% specificity. This limit was exceeded 2.6 times during 18 months of retrospective daily cleanroom data (an expected false alarm rate of 1.3 times/year). After implementing this action limit, we were alerted in real time to an air-handling failure undetected by our hospital facilities management.

Conclusions: A rational action limit for non-viable particles was determined based on the correlation with airborne colonies. Reaching or exceeding the action limit of 32 000 non-viable particles/feet(3) triggers suspension of cleanroom cell-processing activities, deep cleaning, investigation of air handling, and a deviation management process. Our full procedure for particle monitoring is available as an online supplement.

Figure 1

Laboratory schematic showing testing sites. The sampling locations are denoted by capital letters. B, biologic safety cabinet; C and V, cleanroom (two locations); T, testing laboratory; A, anteroom; H, hallway.

Figure 2

Non-viable and viable airborne particle monitoring. We performed simultaneous non-viable (≥5 μ ; 227B laser particle counter; MetOne, Grants Pass, OR, USA) and viable (Biotest HYCON RCS, high-flow viable particle counter, Biotest agar strips TCI- γ ; Bioteet AG, Landsteinerster, Germany) particle measurements over a period of 1 year. A total of 20 paired measurements was made in five different laboratory locations that varied with respect to extent of air treatment (hallway, testing laboratory, anteroom between testing laboratory and cleanroom, ISO class 7 cleanroom, and class II biologic safety cabinet within the cleanroom). Non-viable particle counts (A), expressed in counts ≥5 μ /feet ³ of air, ranged from 0 (class II biosafety cabinet) to 10 240 000. Viable counts (B) measured in colonies/feet ³ of air after 3 days of incubation, ranged from 0 (biosafety cabinet, cleanroom) to 2.5.

Figure 3

Correlation of viable and non-viable particle counts. Linear regression analysis was performed on paired viable and non-viable count data. A log scale with broken axes shows zero (undetectable) counts. The line of best fit and the 95% confidence intervals are shown. Data points are shown in upper case letters corresponding to the locations shown in In the biologic safety cabinet (B), all points overlap at 0,0.

Figure 4

ROC curve relating non-viable and viable airborne particulates sampled concurrently at the same locations. A viable particle count less than 0.5/feet ³ was chosen as the maximum acceptable level for an ISO class 7 cleanroom. Non-viable particle counts were binned in 1/2 log ₁₀ increments (with two added points where data were most dense). A non-viable particle count of <32 000 particles/feet ³ was chosen as the action limit based on sensitivity (95.6%) and specificity (dashed line, 50%) considerations.

Figure 5

Retrospective raw count data for pass-through nonviable counts, n = 1586 (200 determinations, eight replicates). The 99th percentile is 33 778 counts/feet ³ . The alert limit from ROC is 32 000 counts/feet ³ . At the alert limit, 50% would be false alarms, but 95.6% of all true contaminations (defined as 0.5 colonies/feet ³ on day 3) would be detected.

Figure 6

Daily particle readings in the cleanroom (Figure 1C) and class II biosafety cabinet ( Figure 1B). Daily particle counts were performed during working days. The bars show the standard errors associated with replicate determinations. The upper dashed line indicates the action limit for the cleanroom and the lower dashed line indicates the alert limit (10 000/feet ³ , lower dashed line). On 2 September 2011 the particle counts in the cleanroom exceeded the action limit (upper dashed line) and appropriate action was taken according to the protocol. Particles were not detected in the biosafety cabinet at the time of the malfunction.