USP <1116> and Contamination

Recovery Rates
Scott Sutton

ABSTRACT regulatory thinking regarding microbiological moni-

United States Pharmacopeia (USP) <1116> "Microbio- toring of aseptic areas. This shift leads away from
logical Control and Monitoring of Aseptic Processing arbitrary numerical levels in these extremely clean
Environments" approaches analysis of environmen- environments to a more qualitative trending meth-
tal monitoring (EM) data in the aseptic core from a odology. In addition to the important information
perspective of "contamination recovery rates" while in this chapter on new ways to set alert and action
noting a need to improve EM data analysis. levels for environmental monitoring (EM) programs,
There are two difficulties in using EM data from this chapter also stresses the separate and important
the aseptic core: task of controlling these environments. The following
• Most of the plate counts should be "zero" outlines this chapter:
• The actual numerical plate counts seen are • Introduction
far below the limit of quantification (LOQ) • Clean Room Classification for Aseptic Pro-
for the plate count method. cessing Environments
• Importance of a Microbiological Evaluation
USP <1116> suggests using "percent contamination Program for Controlled Environments
recovery rate" as the measure, but other options are • Physical Evaluation of Contamination Con-
available: trol Effectiveness
• The use of quality control (QC) control charts • Training or Personnel
has been suggested for these data • Critical Factors in the Design and Implemen-
• The use of most probable number (MPN) tation of a Microbiological Environmental
analysis has been suggested for analysis of Monitoring Program
these data-this may be a more appropriate • Selection of Growth Media
method given the Poisson distribution of the • Selection of Culture Conditions
data and its very low numbers. • Establishment of Sampling Plan and Sites
• Selection of Sample Sites Within Clean
Limitations of this approach should also be con- Rooms and Aseptic Processing Areas
sidered; some tracking of magnitude of excursions as • Microbiological Control Parameters in Clean
well as trending of microorganism identity throughout Rooms, Isolators, and RABS
the facility is needed. • Significant Excursions
• Further Considerations About Data
INTRODUCTION Interpretation
The United States Pharmacopeia (USP) <1116> "Micro- • Sampling Airborne Microorganisms
biological Control and Monitoring of Aseptic Process- • Surface Sampling
ing Environments" (1) marks a significant shift in • Culture Media and Diluents

ABOUT THE AUTHOR

For more Author
Scott Sutton, Ph.D., is the founder and principal consultant of Microbiology Network, Inc (http://bit.ly/
in1orma\ion,
n62yFG). He is a recognized consultant and trainer with emphasis in GMP, investigations, environmental
go to
monitoring, and contamination control as well as microbiology laboratory audits and operations. He may
gxpandjvt.com/bios
be reached by e-mail at [email protected] .

gxpandjvt.com ]OURNAL OF VALIDATION TECHNOLOGY (AUTUMN 2012) 79

 • Identification of Microbial Isolates "The data presented herein are not necessarily appli-
• Conclusion cable to other systems. For automated equipment,
• Appendix/Glossary. the optimum range may well vary with the instru-
ment ... Furthermore, even if automation is not used
USP <1116> MICROBIOLOGICAL CON- appropriate numbers of colonies that should be on a
TROL AND MONITORING OF ASEPTIC countable plate can very widely, depending on many
PROCESSING ENVIRONMENTS other variables. With soil fungi for example ... (3)"
The concern about reliable alert and action levels for
the aseptic core hinge on two considerations: USP has recently harmonized a microbial enumera-
• The limit of quantification for the plate count tion test (4). This test recommends, "Select the plates
method corresponding to a given dilution and showing the
• The prevalence of "zero" in the data set. highest number of colonies less than 250 for [total
, aerobic microbial count] TAMC and 50 for [total com-
This paper will first look at the issues in the limit bined yeast and mold] TYMC. In determination of
of quantification. the resistance of biological indicators, USP recom-
mends a range of "20 to 300 colonies, but not less
LIMIT OF DETECTION VERSUS LIMIT OF than 6 (5)." However, the most complete description
QUANTIFICATION of the countable range is found in the informational
The general ranges in common acceptance for count- chapter <1227> (6).
able numbers of colonies on a plate are 30-300 or
25-250 colony forming units (cfu). Breed and Dot- "The accepted range for countable colonies on a
terrer noted that, "the kind of bacteria in the mate- standard agar plate is between 25 and 250 for most
rial under examination will have an influence on bacteria and Candida albicans. This range was estab-
the size of the colonies, and consequently, on the lished in the food industry for counting coliform bac-
number that can develop on a plate." They also teria in milk. The range is acceptable for compendia!
noted that food supply can be an issue, that colonies organisms, except for fungi. It is not optimal for
close to each other on the plate may merge, and that counting all environmental monitoring isolates. The
neighbor colonies may inhibit growth or conversely recommended range for Aspergillus niger is between
stimulate growth. 8 to 80 cfu per plate. The use of membrane filtra-
tion to recover challenge organisms, or the use of
"Because of these and other difficulties certain environmental isolates as challenge organisms in the
plates in any series made from a given sample are antimicrobial effectiveness testing, requires validation
more satisfactory for use in computing a total than of the countable range (6)."
are others. The matter of selecting plates to be used
in computing a count becomes therefore a matter ASTM provides countable ranges of 20-80 cfujmem-
requiring considerable judgment (2)." brane, 20-200 for spread plates, and 30-300 for pour
plates (7). The US Food and Drug Administration's
This study determined that plates with more Bacterial Analytical Manual (BAM) recommends 25-250
than 400 cfu were unsatisfactory, as were those of cfujplate as a countable range (8).
less than 30 cfu, with best results in the range of The general consensus of these documents is that,
50-200 cfujplate (2). From this study originated at best, the limit of quantification (LOQ) for the plate
the 30-300 rule for the "countable" range of colo- count method is no less than 20 cfujplate. This leads
nies on a plate. to direct problems with the currently accepted regula-
Tomasiewicz et al reevaluated the question of tory levels that are set near the limit of detection (1
countable range on a plate, again taking data from cfujplate) rather than the LOQ (9).
colony counts of raw milk. This study used data from
three different experiments (each dilution plated in CONTAMINATION RECOVERY RATES
triplicate) to determine a mean-squared-error of the VERSUS NUMERICAL LEVELS
estimate for all plates. Their recommendation, at the Contamination recovery rates in USP <1116> are
end of the study, was for a countable range of 25-250 defined as the percentage of plates that show any
cfujplate in triplicate. This study is also notable for microbial recovery irrespective of number of cfu.
excellent cautionary advice. This term is defined in the glossary of USP <1116>.
80 ]OURNAL OF VALIDATION TECHNOLOGY (AUTUMN 2012) ivtnetwork.com
 SCOTT SUTTON

Table: Suggested initi~l ~ontamination recovery rates in_ aseptic ~nvironments.

Active Air Settle Plate (9 em) Contact Plate Glove or
Room Classification
Sample (%) 4 hr exposure (%) or Swab (%) Garment (%)
- -
Isolator/Closed RABS
<0.1 <0.1 <0.1 <0.1
{ISO 5 or better)
- .j.
- - !----- ------
IS05 <1 <1 <1 <1
---~----
IS06 <3 <3 <3 <3
- ~ - ----
I
ISO? <5 <5 <5 <5
ISO 8 <10 <10 <10 <10
This table is reproduced from Table 3 of USP <1116> (1)

"The contamination recovery rate is the rate at per plate. EM alert and action levels in the 1-10 cfu
which environmental samples are found to contain range are therefore of questionable accuracy.
any level of contamination. For example, and incident There is a real need for better quality tools, and this
rate of 1% would mean that only 1% of the samples need has led to the shift to contamination recovery
taken have any contamination regardless of colony rates rather than arbitrary cfu numbers as proposed
number." levels. This chapter is now official (1), and these con-
tamination recovery rates appear in tables of suggested
The alert and action levels are then defined relative levels for different classes.
to these percentages. The user is encouraged to collect The table on suggested initial contamination recov-
data and set these averages for the specific facility ery rates in aseptic environments (reproduced above)
and sample site (see Table for suggested contamina- suggests initial rates (percent contamination-non-
tion rates). zero-samples) in different areas. The obvious method
This is in sharp contrast to currently accepted levels of implementation for these rates is on a rolling aver-
of contamination listed in the 2004 FDA guidance age, but it is left to the operator to determine the
(10) as well as other guidance documents where, in appropriate interval for this average.
a grade B (class 1000, International Organization for In addition to the contamination recovery percent-
Standardization [ISO] cleanroom six), great signifi- age, the role of "significant excursions" (i.e., excur-
cance is placed on a result of 6 cfu versus 7 cfu (pass/ sions of approximately 15 cfu on a plate) is discussed.
fail) in active air monitoring or 2 cfu versus 3 cfu in The chapter provides a good discussion of how to
settling plates. All of these numbers are well within evaluate these events for significance, a general input
the noise level of the plate count method. Note, the on methodology, and a glossary of terms.
FDA Aseptic Processing Guide is used only for illustra- While there may be some difficulty with using this
tive purposes without any intention of singling this particular measure (contamination recovery rates) as
document out for special mention. All current regula- a trending tool (see discussion below on EM data as
tory guidance in aseptic processing from Europe and normally distributed or in a Poisson distribution),
the United States, as well as trade industry technical the great contribution of this chapter has to be the
reports, repeat these, or very similar, action levels. recognition that current EM criteria in the aseptic core
This break with accepted dogma may be the single is completely arbitrary and contrary to good science.
greatest contribution of this chapter revision. Making critical decisions on the state of control of a
facility based on numbers well into the noise range
WHY THE MAJOR CHANGE IN FOCUS? of the assay is unwise. A different method of analysis
The problem with looking at numerical limits for for these data should be developed, and USP <1116>
microbiological tests is that the levels have to be rea- describes one such method; it is the first regulato-
sonable in terms of the capability of the method. USP ry document to address this question from a valid
<1227> (1) relies heavily on the established scientific estimation of the plate count method's capabilities.
literature in its discussion of this range of countable In addition, this analysis fits in well with the FDA
colonies on a plate (2, 3) to note that colonies have recommendation that, "Increased incidence of con-
a lower LOQ of approximately 25 colonies per plate. tamination over a given period is an equal or more
This is opposed to the limit of detection of one colony significant trend to be tracked (10)."

gxpandjvt.com ]OURNAL OF VALIDATION TECHNOLOGY (AUTUMN 2012) 81

PEER REVIEWED: MICROBIOLOGY

There are other recommendations in the literature on • It yields numerical estimates more accurate
how to address EM data from aseptic core areas where (and more sensitive) than averaging when
the predominant result will be "zero cfu." In specifically the contamination rate is <0.2%.
considering environmental monitoring data, one can • It allows trending of "numerical" data.
look at two publications in the recent decade.
They tested this method using data generated from
OTHER METHODS OF TRENDING two main areas. EM data from an aseptic manufacturing
"NON-ZEROES" suite (grade B) that included active air monitoring data
and surface (floor) samples; EM data from a laminar flow
Caputo and Huffman hood in a test lab that was collected passive monitoring
Caputo and Huffman propose two methods to trend (settle plates) collected. All data sets met the chi-squared
EM data from highly aseptic areas. Like USP <1116> test for goodness-of-fit with a Poisson distribution.
they note that most data are "zero" from these areas, The method is used," ... to analyze the trend of the
and this makes any type of data analysis difficult. environmental monitoring data. The raw ... are grouped
They also stress that, in many cases, the magnitude of choosing the minimum total sampling number n, and at
an individual excursion is less informative than the least including one positive sample. This calculation will
frequency with which contamination occurs. result a maximum MPN when a positive count is observed,
Both of the methods proposed use well-known quality thus increasing the sensitivity of the monitoring (12)."
control (QC) graphing techniques, the individual value/ The MPN equation used here is:
moving range (1-MR) control chart, and the exponen-
tially weighted moving average (EWMA) control chart. MPN = 2.303*1og<,o> Total.i~.Group
To test their proposed methods, Caputo and Huffman Zeroes.m.Group
generated a normally distributed data set of values
around 10-day (n=100) and eight-day intervals (n=85) Or, the MPN estimate is equal to 2.303 times the
intervals of "non-zero" readings. As both of the graphing log 10 value of the ratio of the total number of readings
methods are appropriate for normally distributed data, in the group divided by the number of readings in
both methods worked admirably with this data set. the group of no recovery. Now, while this method is
This study is noteworthy as it is the first formal described in the text as a method to trend by dates,
treatment of the use of contamination rate (e.g., the this can also be used to trend by operators, locations,
frequency of "non-zero" readings) to trend EM data. and other factors.
In this, it is a great step forward beyond the use of
arbitrary numbers located deep in the noise range of Other Trending Requirements in Aseptic
the plate count method (11). Core
The difficulty with this method is that, while it It is important to remember that this method is restricted
is admirably suited for used with data that follow a to trending and alert/action levels for "quantitative" EM
"normal" distribution, it may not be appropriate for only. While it will admirably suit FDA expectations for
data that follow a Poisson distribution (such as EM trending of" data generated by location, shift, room, opera-
data) (12). A more appropriate model might be that tor, or other parameters (10)," it will not meet expectations
recommended by Sun et al (13). for trending programs of microorganisms by identity or by
characteristic (e.g., tending spore forming microorganisms
Sun et al as a check on the sanitization program). In addition, as
Sun's group described the use of most probable num- pointed out in USP <1116>, it is also important to track and
ber (MPN) technology for trending bacterial counts trend significant excursions as part of the EM program.
in EM data. Their discussion begins with an excel-
lent introduction to the MPN method with specific CONCLUSIONS
emphasis on the Halverson and Ziegler equation (14); The common regulatory method of setting microbio-
this forms the basis for their data analysis. From this logical alert and action levels for the environmental
base, they develop a compelling argument for the use monitoring program is not scientifically defensible as
of this MPN method: the arbitrary levels are set well within the noise level
• It is appropriate for data following a Poisson of the plate count assay. The recent revision of USP
distribution. <1116> "Microbiological Control and Monitoring of
• It is computationally straightforward. Aseptic Processing Environments" addresses this with
82 ]OURNAL OF VALIDATION TECHNOLOGY [AUTUMN 2012] ivtnetwork.com
 SCOTT SUTTON

the strong recommendation that these questionable 7. ASTM, "05465-93 (1998)," Standard Practice for Deter-
levels be replaced by trending "zero" and "non-zero" mining Microbial Colony Counts from Waters Analyzed by
in these cases. The trending tool recommended is the Plating Methods, 1998.
use of "recovery frequency" measures. 8 . L.J. Maturin and J.T. Peeler. "Aerobic Plate Count," in
Two other methods of analysis are discussed in this Bacteriological Analytical Manual (FDA, Rockville, MD,
review that might be useful in trending data from the Nov. 10, 2012) available at: http://www.fda.gov/Food/
aseptic core and in qualifying the alert and action ScienceResearch/LaboratoryMethods/BacteriologicalA-
levels. One method uses QC charts (11) and the other nalyticalManualBAM/ucm063346.htm.
a variant of the MPN method (13) . The MPN method 9. S. Sutton, "The Environmental Monitoring Program In
was validated using actual EM data, and it would be a GMP Environment," Journal of GXP Compliance 14 (3),
this paper's recommendation for a useful method 22-30, 2010.
of qualifying alert and action levels and trending 10. FDA, Guidance for Industry: Sterile Drug Products Produced
microbiological EM data in the aseptic core. by Aseptic Processing - Current Good Manufacturing Prac-
tice (Rockville, MD, Sept. 2004) .
11. R.A. Caputo and A. Huffman, "Environmental Moni-
REFERENCES toring: Data Trending Using a Frequency Model," PDA
1. USP, "USP <1116> Microbiological Control and Moni- Journal of Pharmaceutical Science Technology 58 (5), 254-
toring of Aseptic Processing Environments," USP 35 260, 2004.
1, United States Pharmacopeia! Convention, 697-707, 12. J. Wilson, "Setting Alert/Action Limits for Environmen-
2012. tal Monitoring Programs," PDAJournal of Pharmaceu-
2. R. Breed and W.O. Dotterrer, "The Number of Colonies tical Science Technology 51 (4), 161-162, 1997.
Allowable On Satisfactory Agar Plates," Journal of Bacte- 13. X. Sun et al, "The Expanded Application of Most Prob-
riolology l, 321-331, 1916. able Number to the Quantitative Evaluation of Ex-
3. D.M. Tomasiewicz et al, "The Most Suitable Number tremely Low Microbial Count," PDA Journal of Pharma-
of Colonies On Plates for Counting," Journal of Food ceutical Science Technology 60 (2), 124-134, 2006 .
Protection 43 (4), 282-286, 1980. 14. H .O. Halvorson and N.R. Ziegler, "Application of Statis-
4. USP, "USP <61> Microbial Examination of Nonster- tics to Problems In Bacteriology: II A Consideration of
ile Products: Microbial Enumeration Tests," USP 35 the Accuracy of Dilution Data Using a Single Dilution,"
1, United States Pharmacopeia! Convention, 56-60, Journal of Bacteriolology 26 (4), 331-339, 1933.
2012b.
5. USP, "USP <55> Biological Indicators- Resistance Per- GENERAL REFERENCES
formance Tests," USP 35 1, United States Pharmacopeia! PDA, "PDA Comments On USP In-Process Revision
Convention, 54-56, 2012c. <1116> Microbiological Evaluation of Clean Rooms
6. USP, "USP <1227> Validation of Microbial Recovery and Other Controlled Environments," PDA Journal of
from Pharmacopeia! Articles," USP 35 1, United States Pharmaceutical Science Technology 49 (6), 264-266, 1995.
Pharmacopeia! Convention, 883-885, 2012d. JVT

gxpandjvt.com /OURNAL OF VALIDATION TECHNOLOGY (AUTUMN 2012] 83