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Multicenter Study on Incubation Conditions for Environmental

Monitoring and Aseptic Process Simulation
Roland Guinet, Nicole Berthoumieu, Philippe Dutot, et al.

PDA J Pharm Sci and Tech 2017, 71 43-49

Access the most recent version at doi:10.5731/pdajpst.2016.006791
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RESEARCH

Multicenter Study on Incubation Conditions for

Environmental Monitoring and Aseptic Process Simulation
ROLAND GUINET*, NICOLE BERTHOUMIEU1, PHILIPPE DUTOT2, JULIEN TRIQUET3, MEDHI RATAJCZAK3,
MICHEL THIBAUDON4, PHILIPPE BECHAUD5, CHRISTOPHE ARLIAUD5, EDITH MICLET5,
FLORINE GIORDANO5, MARJORIE LARCON5, and CATHERINE ARTHAUD5
1
Leo-Pharma, Vernouillet, France; 2NovoNordisk Productions SAS, Chartres, France; 3Aspen, Notre Dame de
Bondeville, France; 4Indicia, Saint Genis l’Argentière, France; and 5Biomerieux, Craponne, France ©PDA, Inc. 2017

ABSTRACT: Environmental monitoring and aseptic process simulations represent an integral part of the microbio-
logical quality control system of sterile pharmaceutical products manufacturing operations. However, guidance
documents and manufacturers practices differ regarding recommendations for incubation time and incubation
temperature, and, consequently, the environmental monitoring and aseptic process simulation incubation strategy should
be supported by validation data. To avoid any bias coming from in vitro studies or from single-site manufacturing in situ
studies, we performed a collaborative study at four manufacturing sites with four samples at each location. The
environmental monitoring study was performed with tryptic soy agar settle plates and contact plates, and the aseptic process
simulation study was performed with tryptic soy broth and thioglycolate broth. The highest recovery rate was obtained with
settle plates (97.7%) followed by contact plates (65.4%) and was less than 20% for liquid media (tryptic soy broth 19%
and thioglycolate broth 17%). Gram-positive cocci and non-spore-forming Gram-positive rods were largely predominant
with more than 95% of growth and recovered best at 32.5 °C. The highest recovery of molds was obtained at 22.5 °C alone
or as the first incubation temperature. Strict anaerobes were not recovered. At the end of the five days of incubation no
significant statistical difference was obtained between the four conditions. Based on these data a single incubation
temperature at 32.5 °C could be recommended for these four manufacturing sites for both environmental monitoring and
aseptic process simulation, and a second plate could be used, periodically incubated at 22.5 °C. Similar studies should be
considered for all manufacturing facilities in order to determine the optimal incubation temperature regime for both viable
environmental monitoring and aseptic process simulation.

KEYWORDS: Environmental monitoring, Aseptic process simulation, Primary isolation of microorganisms, Incu-
bation conditions, Incubation temperature, Microbiology, Cleanroom.

LAY ABSTRACT: Microbiological environmental monitoring and aseptic process simulation confirm that pharma-
ceutical cleanrooms are in an appropriate hygienic condition for manufacturing of sterile drug products. Guidance
documents from different health authorities or expert groups differ regarding recommendation of the applied
incubation time and incubation temperature, leading to variable manufacturers practices. Some recent publications
have demonstrated that laboratory studies are not relevant to determine the best incubation regime and that in situ
manufacturing site studies should be used. To solve any possible bias coming from laboratory studies or single-site
in situ studies, we conducted a multicenter study at four manufacturing sites with a significant amount of real
environmental monitoring samples collected directly from the environment in pharmaceutical production during
manufacturing operations with four solid and liquid nutrient media. These samples were then incubated under four
different conditions suggested in the guidance documents. We believe that the results of our multicenter study
confirming recent other single-site in situ studies could be the basis of the strategy to determine the best incubation
regime for both viable environmental monitoring and aseptic process simulation in any manufacturing facility.

* Corresponding Author: RGmp Compliance and A3P, 73 chemin du Crêt de Montcher, 69210 Lentilly, France.
Telephone: ⫹33986282593; e-mail: [email protected]
doi: 10.5731/pdajpst.2016.006791

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Introduction – even if laboratory strains of molds can grow at

different temperatures, including 30 –35 °C, at least
The evaluation of the microbiological quality of some environmental strains of molds in primary
sterile pharmaceutical products is mainly based on isolation prefer 20 –25 °C.
sterility tests, environmental monitoring (EM), and
aseptic process simulations (APS), but the incuba- The A3P Annex 1 Common Interest Group would like
tion program of culture media used for EM and APS to include in its Annex 1 modification proposals (15)
can differ depending on recommendations or man- recommendations for the incubation program of EM
ufacturers, although nothing is indicated in the cur- and APS, and this strategy was approved during the
rent EU Annex 1 (1). The sterility tests performed roundtable of the 2014 international meeting (16). In
following the harmonized monograph 2.6.1 of Eu- order to base these recommendations on experimental
ropean Pharmacopoiea (2) must be incubated at data, an in situ multicenter study was proposed for the
20 –25 °C for tryptic soy broth (TSB) and at 30 –35 °C incubation regimes both for EM with solid culture
for thioglycolate broth (THIO), at least 14 days media (EM study) and for APS with a liquid medium
each. For APS, the Pharmaceutical Inspection Co- (settle tube study ⫽ ST study). In addition, the pres-
operation Scheme (PIC/S) recommendation (3) is to ence of strict anaerobic bacteria in pharmaceutical
incubate first for 7 days at 20 –25 °C and then for 7 manufacturing environments was assessed with a liq-
days at 30 –35 °C although other guidance docu- uid thioglycolate medium.
ments recommend only incubation at a single tem-
Material and Methods
perature between 20 °C and 35 °C (4, 5). For EM,
recommendations are made to incubate the sample
1. EM Study
at a single temperature between 20 °C and 35 °C (6)
or at two temperatures, 20 –25 °C and 30 –35 °C (4,
Media: Media used provided by bioMérieux with con-
7, 8) although only a few manufacturers take two
form growth promotion tests were 90 mm TSA settle
samples at the same location, one incubated at 20 –
plates (SPs) bioMérieux and 55 mm TSA contact
25 °C and the other at 30 –35 °C (9). However, the
plates (CPs) containing four neutralizing agents: lec-
incubation of the same sample at two different tem-
ithin, Tween 80, sodium thiosulphate, and L-histi-
peratures may result in a thermal shock, reducing
dine).
the total recovery rate (10, 11). In PDA Technical
Report No. 13 (12) incubation conditions for micro- Incubation Conditions:
biological EM samples are not addressed.
– temperature a: 22.5 °C (20 to 25°C) for 5 days.
Recent studies performed by Symonds et al. (13) and
Gordon et al. (14) on incubation regimes of EM as – temperature b: 32.5 °C (30 to 35 °C) for 5 days.
well as Pharmaceutical and Healthcare Sciences Soci-
ety (PHSS) monograph 20 (11) and the 2012 A3P – temperatures c: 22.5 °C for 3 days then 32.5 °C for
survey (9) showed that: 2 days for a total of 5 days.

– the highest quantitative results were obtained – temperatures d: 32.5 °C for 2 days then 22.5 °C for
when using two samples at the same location, 3 days for a total of 5 days.
one incubated at 20 –25 °C and the other at
30 –35 °C; A first reading L1 was made after 2 or 3 days at
32.5 °C and 22.5 °C, including plates incubated only
– there is no significant difference between tryptic soy at one temperature.
agar (TSA) and Sabouraud dextrose agar (SDA) for
the recovery of molds even if SDA was found to be Samples:
slightly superior (14);
– 10 sequences of 3 samples were made in quadruple
– only one sample incubated at two successive tem- replicates in order to incubate each sample at 4
peratures, either 20 –25 °C and then 30 –35 °C or the incubation regimes, thus 12 samples were taken for
reverse, was found to be less effective; SPs or CPs in each sequence.

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– SPs were exposed during exactly 4 h in Grade C, D, Incubation Conditions:

or controlled non-classified (CNC) rooms and sup-
portive zones mid-shift during normal operating a ⫽ 14 days minimum at 22.5 °C (20 –25 °C).
conditions.
b ⫽ 14 days minimum at 32.5 °C (30 –35 °C).
– CPs were applied with a bioMérieux device during 10 s
with a 500 g pressure in Grade C, D, or CNC rooms c ⫽ 7 days minimum at 22.5 °C then a first reading
and supportive zones on walls, surfaces, or floors. followed by 7 days minimum at 32.5 °C.

Recommendations were made to arrange the sam- d ⫽ 7 days minimum at 32.5 °C then a first reading
ples in a pattern designed to minimize any bias that followed by 7 days minimum at 22.5 °C.
their location would have on the capture of envi-
ronmental microorganisms as shown by Symonds A first reading L1 was made after 7 days of incubation,
et al. (13). For this, each sampling sequence including regimes at only one temperature.
grouped 3 samples in quadruplicate with a total of
12 plates arranged in 4 columns of 3 plates, and the Microbial Characterization: Microbial Characteriza-
opening and closing of plates was performed in tion of Molds, yeasts, bacteria (rods or cocci, Gram-
positive or Gram-negative), Bacillus, and strict anaer-
order to avoid that any part of the operator could be
obes was performed.
over an open SP.

Results
Data: Any bacterial colony-forming units (CFUs) de-
tected at the first reading L1 or at the end of the
1. EM Study
incubation period, second reading, L2, were catego-
rized as Bacillus spp., Gram-negative (B–) or non-
The CFUs recovered for each incubation regime and
spore-forming Gram-positive rods (B⫹) rods, Gram-
each manufacturing site are given for each group of
negative (C–) or Gram-positive (C⫹) cocci, molds, or
microorganisms for SPs and for CPs in Tables I and II,
yeasts. All data were reported as total numbers of
respectively. The overall numbers of plates with growth
CFUs recovered for each category with each medium
was 97.7% for SPs, with two sites at 100%, and only
SP or CP per incubation condition. 65.4% for CPs. Gram-positive cocci (C⫹) were highly
predominant, representing more than 80% for SPs and
Good Manufacturing Practice Requirements: This 87% for CPs, followed by non-spore-forming Gram-
study was performed in addition to the routine regu- positive rods (B⫹), with more than 15.5% for SPs and
latory EM and thus the results could not have been 8% for CPs. Thus, human commensals (total C⫹ and
followed by investigations and corrective actions in B⫹) were found in more than 95% for SPs and 97% for
case of nonconformities detected. CPs. Excluding Gram-negative rods (B–) found mainly
at one site (139 CFUs in a total of 180 for the four sites),
2. Settle Tube Study the other groups of microorganisms were found only in a
few occasions, with less than 0.5% for each group, or
Media and Samples: Eleven millimeter diameter never found.
tubes of liquid media provided by bioMérieux with
conform growth promotion tests were 4 ⫻ 30 TSB The four manufacturing sites found a high majority of
and 4 ⫻ 30 THIO. The surface exposed with these human commensals. However, important differences
settle tubes is around 70 times less than with 90 mm were observed between sites in the overall CFU num-
SPs. bers. For C⫹ the results were 477 to 1618 with SPs and
273 to 1244 with CPs, and for B⫹, 91 to 406 with SPs
Grade C, D, or CNC rooms and supportive areas and 3 to 92 with CPs. The sites with the highest CFU
known to have some bacteria and fungi were sampled numbers were not the same based on media SP or CP or
for exactly 4 h with 8 tubes at each location, 4 TSB on groups of bacteria C⫹ or B⫹.
and 4 THIO, with the same recommendations as for
solid media to avoid any possible bias coming from Statistical Analyses for Settle Plates: A highly sig-
the sampling. nificant difference was observed (Table III) between the

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TABLE I
Settle Plate (SP) Detailed Results in CFU Numbers per Site and Microorganism Categories

1
a, b, c and d ⫽ incubation regimes as inducated in material and methods.
2
Number ⫽ total CFUs numbers for 120 settle plates per site.
3
S ⫽ overall total of CFUs numbers for 480 settle plates for the 4 sites.

two incubation conditions, with the most important conditions. The difference between L1 and L2 showed
recovery rate for 32.5 °C during 2 days compared to a nonsignificant increase for L2 only for C⫹ and only
22.5 °C during 3 days (first reading L1), and this was for two of the four sites.
coming from B⫹ and C⫹. For the overall results for the
four sites after 5 days incubation (last reading L2), the Molds and Bacillus: A statistical analysis was diffi-
Kruskal-Wallis test did not demonstrate a significant cult due to the low numbers of CFUs. However, Ta-
difference between the four incubation conditions, but bles I and II show that of the 26 molds found (17
some differences were observed depending on sites and SPs ⫹ 9 CPs), 23 were detected at 22.5 °C alone or at
microorganism groups. The difference between readings the beginning. For 40 Bacillus detected (32 SPs ⫹ 8
L1 and L2 was not statistically significant and was ob- CPs), no significant difference was observed between
served only for C⫹ for two of the four sites and for the four conditions a, b, c, or d, with a total SPs ⫹ CPs
22.5 °C alone or followed by 32.5 °C. of 7, 12, 8, and 13, respectively.

Statistical Analyses for Contact Plates: For the first 2. Settle Tube Study
reading L1 (after 3 days at 22.5 °C or 2 days at 32.5 °C)
a statistical difference was observed for B⫹ only for The overall growth was very low, with less than one of
one site and for C⫹ for all sites. For the last reading the five tubes with growth (TSB 19%, THIO 17%)
L2, the Kruskal-Wallis test did not show a significant even after 4 h exposure in Grade C, D, or CNC during
difference, at a 5% ␣ risk, between the four incubation normal operations, and statistical analyses were not

TABLE II
Contact Plate (CP) Detailed Results in CFU Numbers per Site and Microorganism Categories

1
a, b, c and d ⫽ incubation regimes as inducated in material and methods.
2
Number ⫽ total CFUs numbers for 120 settle plates per site.
3
S ⫽ overall total of CFUs numbers for 480 settle plates for the 4 sites.

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TABLE III TABLE IV

Settle Plates First Reading L1. Statistical Gram-Positive Rods (Bⴙ) without Bacillus and
Differences between 22.5 °C for 3 Days and Gram-Positive Cocci (Cⴙ) Growth Depending on
32.5 °C for 2 Days Incubation Conditions and Liquid Medium

formed with in situ samples did not give rise to the

same conclusion for incubation conditions at two tem-
peratures because there was no difference in incuba-
tion conditions at a single temperature for Gordon
et al. (14) but was found less appropriate for Symonds
et al. (13). Our study could not observe a statistically
possible due to these low numbers of tubes with significant difference between the four incubation con-
growth. The difference after 7 days and 14 days of ditions used at the end of the 5 days of incubation for
incubation was very low with less than 2%. Gram- the total numbers of CFUs, according to Gordon et al.
positive cocci were highly predominant, representing (14).
more than 70% with THIO and more than 84% with
TSB. Yeasts, molds, Gram-negative cocci, and strict As for these two previous studies (13, 14), for bacteria
anaerobes were never found and only one tube showed of human origin (non-spore-forming Gram-positive
Bacillus. One site has recovered 75% of Gram-posi- rods and Gram-positive cocci) the incubation condi-
tive rods. Human commensals (B⫹ and C⫹) were tions at 32.5 °C allow for better growths after the first
observed in 95.9% and the growth was better at reading after 2 or 3 days for solid media (SPs and CPs)
32.5 °C alone or as the first incubation temperature for and also for liquid media (TSB and THIO) after 7 or
TSB as well as for THIO (Table IV), with 63.8% 14 days of incubation (Table IV). This confirms that in
together, around two times better than at 22.5 °C alone liquid media as well as in solid media human com-
or as the first incubation temperature. The growth of mensals are better recovered after incubation at
these human commensals was almost equivalent be- 32.5 °C, as suggested by Symonds et al. (13). These
tween TSB (50.3%) and THIO (49.7%). human commensals were highly predominant and this
was the case for the four manufacturing sites accord-
Discussion ing to previous studies, and this was regarding the
important operator activities. Consequently, except for
There is no consensus regarding the incubation con- one site with Gram-negative rods in a washing room,
ditions used for EM of cleanrooms (6 – 8, 10, 11) and the other categories of microorganisms were not
for the APS (3–5). This multicenter study was per- found, such as yeasts and strict anaerobes, or only in
formed in order to try to propose a strategy for the very few occasions, such as Gram-negative cocci,
determination of the best incubation condition in a molds, or Bacillus.
given environment or for specific processes while also
minimizing the possible bias coming from a single-site Our multicenter study confirmed also that the primary
in situ study. The two recent studies (13, 14) per- isolations from manufacturing environments of molds

Vol. 71, No. 1, January–February 2017 47

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are never obtained after incubation at 32.5 °C alone Conclusion

but only after 22.5 °C alone or at the beginning, and
this deficiency could not be rectified by subsequent Recommendation for the Choice of an Incubation
incubation at 20 –25 °C, as shown previously (14). As Regime for EM and APS
already observed by Horn et al. (10) and Gordon et al.
(14), a first incubation period for only 2 or 3 days at The EM of cleanrooms and the APS are looking for the
32.5 °C is able to inhibit further growth for a great same microbial contaminants and thus it seems justified
majority of mold strains in primary isolation. This to use the same incubation condition for the culture
inhibited growth is also observed in our settle tube media engaged for their recovery. As for the four man-
study for human commensals because we observed ufacturing sites in our study, the cleanrooms with impor-
more than two times the growth at 32.5 °C (57 tubes) tant human activities should have a highly predominant
than at 22.5 °C, followed by 32.5 °C (27 tubes), and human commensals– based flora, which should be better
thus the PIC/S recommendation (3) to incubate the recovered after incubations of TSA or TSB at 30 –35 °C.
TSB-filled units first 7 days at 20 –25 °C followed by During the initial qualification of these cleanrooms, and
7 days at 30 –35 °C is not appropriate for these envi- periodically or after important changes or at risk events,
ronments and processes. the other categories of microorganisms such as yeasts,
molds, or Bacillus should be confirmed as either not
Different media (13, 18, 19) and temperatures (13, 14, recovered or recovered only in few occasions with a
17) were evaluated for the recovery of yeast and molds second medium incubated at 20 –25 °C. Thus, in contrast
and, because the results are still not conclusive, sup- with Symonds et al. (13), incubation conditions at two
plementary in situ studies are needed to determine the different temperatures, 20 –25 °C followed by 30 –35 °C
optimal conditions for the primary isolation of fungi in with TSA settle and CPs for EM, could be possible in
pharmaceutical environments. If a temperature be- such environments. However, for consistency with TSB-
tween 20 –25 °C and 30 –35 °C could be recom- filled units for APS, a single incubation condition at
mended, the impact on the incubation temperatures of 32.5 °C for both EM and APS could be recommended.
the sterility tests should be discussed.
Acknowledgments
For Bacillus our study showed only a trend toward
better growth at 32.5 °C alone or at the beginning, We thank A3P Services and the board of directors of
although for Symonds et al. (13), 20 –25 °C was better the A3P Association for supporting our multi-center
at least in the first part of their study. study.

It should be noted that the total numbers of growth are Conflict of Interest Declaration
linked with the diameter of the exposure or contact
because for 90 mm SPs almost 100% was obtained The authors declare that they have no competing in-
instead of only 66% for 55 mm CPs, and less than 20% terests.
was obtained with 11 mm diameter liquid media tubes,
the exposed surface of which being around 60 times References
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