USP <1116> Microbiological Control and

Monitoring of Aseptic Processing Environments

Gilberto Dalmaso
Senior Advisor and Pharma Customer Advisory Team Manager
gdalmaso@pmeasuring.com

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 Introduction

• <1116> provides information and recommendations for

environments where the risk of microbial contamination is
controlled through aseptic processing.
• It is one of the most comprehensive informational chapters from
the USP.
• Proposes measurement of microbial contamination based on
Contamination Recovery Rates (CRR) rather than the
conventional enumeration of colony forming units (CFU).
• Aseptic guidance microbial limits using CFUs, are replaced in
<1116> with CRR values
• CRRs are expressed as a maximum allowed percentage of
contaminated samples over a certain period of time.
• The chapter provides recommendations not yet adopted and not
enforceable by the U.S. FDA or any other government agency.

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 Main Changes in Current Version

• Title: The previous title of <1116> was “Microbial Control and Monitoring Environments
Used for the Manufacture of Healthcare Products” while the revised title is
“Microbiological Control and Monitoring of Aseptic Processing Environments.”
• Scope: It has been narrowed to be applicable to the following
products manufactured in aseptic processing environments:
• Pharmaceutical sterile products
• Bulk sterile drug substances
• Sterile intermediates
• Excipients
• Some medical devices
• Type of environments covered in <1116> are:
• Conventional cleanroom with unidirectional airflow
• Blow/fill/seal technology
• RABS (Restricted Access Barrier Systems) For other changes see “USP <1116> and its
Implications for Measuring Microbial Recovery Rates”.
• Isolators Denoya and Dalmaso, PDA Letter, Summer 2015.

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 Microbiological Control Reality Check

• Particulate and microbial counts vary with sampling locations,

manufacturing activities and human interventions conducted during
sampling
• Real Time Active Monitoring of Total Particulate Count: does not provide
direct information on the microbiological content of the environment
• Monitoring of Airborne Microorganisms: CFU-based, with results
representing a narrow period of time, and delayed by incubation

Real Time Monitoring Limited & Delayed Monitoring

Particulates Microbial

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 How to Improve Microbial Monitoring?

Analyzing data New instrumentation

in new ways providing real time or near
real time data

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 USP <1116> General Comments
• In Grade A/ISO 5 environments, zero
contamination is impractical due to
operator intervention in
environmental monitoring (EM).
• There is no way to prove Grade A/ISO
5 environments are 100% sterile.
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“In any environment where human
operators are present, microbial
contamination at some level is
inevitable. Thus, an expectation of zero
contamination at all locations during
every aseptic processing operation is
technically not possible and thus is
unrealistic. There are no means to
demonstrate that an aseptic processing
environment and the product-contact
surfaces within that environment are
sterile.”
“Environmental monitoring is usually
performed by personnel and thus
requires operator intervention.” -
USP <1116>
 USP <1116> General Comments
• It is possible for microorganisms
to not grow on plates if they are
stressed.
• Alerts and action limits should be
determined independently.
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“As a result, environmental monitoring can both
increase the risk of contamination and also give
false-positive results. Thus, intensive monitoring
is unwarranted, particularly in the ISO 5
environments that are used in the most critical
zones of aseptic processing.”
“At present, nearly all of the sampling methods
rely on the growth and recovery of
microorganisms, many of which can be in a
damaged state caused by environmental stress
and therefore may be difficult to recover.”
“The numerical values for air, surface, and
personnel monitoring included in this chapter
are not intended to represent limits or
specifications but are strictly informational.”
- USP <1116>
 USP <1116> General Comments for Air Sampling
• Monitoring technology cannot
be standardized due to
specification differences
(sensitivity, precision, detection
limits, etc.).
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“There are no standard methods for air
sampling, and available literature indicates that
air-sampling methods are highly variable.”
“It should not be assumed that similar sample
volumes taken by different methods will produce
similar rates of recovery.”
“Many factors can affect microbial recovery and
survival, and different air sampler suppliers may
have designed their systems to meet different
requirements.”
“Limited data are available regarding the
accuracy, precision, sensitivity, and limits of
detection of monitoring methods used in the
aseptic processing of healthcare products.”
- USP <1116>
 USP <1116> General Comments for Surface Monitoring
• Surface monitoring cannot be
standardized due to the variance
in methods (different types of
swabs, contact plates, etc.).
• Lower recoveries have been
demonstrated with surface
monitoring methods.
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“Surface sampling methods are also not
standardized.”
“Different media are employed, and in the case
of swabs, different results have been reported
for wet and dry swab methods and contact
plates.”
“In general, surface monitoring has been found
to recover <50%, even when used with relatively
high inoculum levels on standardized
coupons…In actual production environments
where organisms are stressed to varying
degrees, recovery rates may be lower.”
- USP <1116>
 Advance Aseptic Technologies
• Methods that remove operator
intervention are supported by
USP <1116>, because they offer a
greater ability to control
microbiological contamination.
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“Advanced aseptic technologies can be defined as
those that do not rely on the direct intervention
of human operators during processing.”
“At present, technologies such as isolators,
blow/fill/seal, and closed RABS (designs that are
never opened during setup or operation) may be
considered advanced aseptic technologies,
provided that direct intervention by gowned
personnel is disallowed during processing.”
“Because these systems substantially reduce
contamination risk, their microbiological control
levels are higher than those of conventional clean
rooms that have comparable particulate air
classification level, for example, ISO 5.”
- USP <1116>
 Cleanroom Classification for Aseptic Processing
Environments
ISO Class Particles ≥ 0.5 um/m3
• Main points from USP <1116>: ISO 5 3520
• All particulate shedding from equipment is taken as ISO 6 35200
nonviable. ISO 7 352000
• Total particle counts cannot be segmented into those ISO 8 3520000
produced by mechanical operations and those produced by
operators. Table 1 in USP <1116>
• Cleanroom EMPs should have both a total particle and microbiological component.
• As seen in Table 1, aseptic cleanroom classifications typically used are ISO 5-8 (defined in ISO 14644-1).
• Isolators and closed RABS lower microbial contamination risk because they allow personnel to make
manipulations outside the environment using specialized garments.

• Further classification considerations from USP <1116>:

• Air changes (typical minimums are 20 for ISO 8, 50 for ISO 7 and 100 for ISO 5)
• Air velocity
• Airflow (should be analyzed using smoke studies)
• Risk assessment (reduces contamination risk)

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 Importance of a Microbiological Evaluation Program for
Controlled Environments
• Main points from USP <1116>:
• Total particle counts ≠ microbiological activity
 Not even when monitoring continuously in a controlled environment
• Counts vary with sampling location and its associated activities
 Applies to both particulate counts and microbial counts
 Total particle counts are more useful in isolators/closed RABS due to lack of human
activity
• Cleaning and sanitization practices (including the personnel who perform them)
should be assessed as part of the Environmental Monitoring Program
• It isn’t possible to identify and qualify every microbe present in a controlled
environment due to limitations in sampling equipment.

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 Personnel in the Aseptic Environment
• Main points from USP <1116>:
• Due to the criticality of actions performed in aseptic environments, all
relevant personnel (including those responsible for EM) should be
rigorously trained and supervised.
• Personnel are the only significant sources of microbial contamination.
 Those permitted in controlled environments should be healthy, have good
hygiene, and be detail-oriented when gowning (NO exposed skin).
 Poor sampling techniques and personnel near critical zones has the potential
to create microbiological contamination.
 Personnel training should include aseptic technique principles,
manufacturing and handling, and contamination sources. Supervisors and
testers should have academic training in medical or environmental
microbiology.
 All cleanroom personnel are responsible for minimizing microbial
contamination.
 Periodic media-fill, process simulation studies and thorough ongoing
supervision ensure the process is controlled and proper techniques are being
used by trained personnel.

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 Critical Factors in the Design and Implementation of a
Microbiological Environmental Monitoring Program
• Growth media selection
• Good catch-all media (ex. soybean–casein digest medium (SCDM), good for bacteria,
yeast, and molds)
• Specialized media for molds and yeasts (ex. Sabouraud’s)
• Media can come with additional ingredients to mitigate
affects of common sanitizing agents/antibiotics.
• Anaerobes are not monitored (these organisms
do not survive in ambient air).
 Microbes that grow with low amounts of oxygen
 (micro-aerophilic) could be monitored if those
conditions exist in the process environment.
• From USP <71>, all media used should be evaluated on
their ability to support growth.
• Media usage (incubation, processing, manipulation) should
follow proper techniques (<1117> and <71>).

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 Establishment of Sampling Plan and Sites

• Main points from USP <1116>:

• Critical zones (where product is exposed to
environment) are always ISO 5.
 For aseptic operations, the entire critical zone
should be treated as a sterile field.
• Sterile components (filling needles, equipment
that is in the product-delivery pathway) should
not be touched by operators/EM personnel.
 Surface monitoring should be performed when
production is not occurring.
• EM plans should be flexible.
 Should be adjustable based on risk analysis
• Sampling too much is just as bad as not
sampling enough.

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 Microbiological Control Parameters in Clean Rooms,
Isolators, and RABS
• Main points from USP <1116>:
• The difference between alert and action limits are small, especially for ISO 5.
• Normal variability (± 50%) is common in growth/recovery assays, with an high as 10x
for some sampling devices.
 Ex. An alert level of 1 CFU and action level of 3 CFU are not analytically significant.
• Contamination frequency can be analyzed over CFU counts due to limited accuracy
and precision in microbial growth/recovery assays.
• 1 CFU ≠ # microorganisms present
 Colony forming units (CFUs) are used to estimate the number of viable organisms that may be
present.

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 Contamination Trends and the Case for Contamination
Recovery Rates (CRR) in <1116>

Analyzing Data in New Ways

• How about trending all contamination events (≥ 1 CFU) ?

• Could this trending help to improve data analysis and help to maintain a
continuous state of control ?
• <1116> proposes a new perspective on environmental control relying on
incident rates rather than CFU-based Action and Alert levels.
• Rather that isolated events, analysis of data in relation to time would
detect changes in the contamination recovery rate that may be indicative of
changes in the state of control within the environment.

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 Microbial Limits During Operation

Air Sample Settle Plates Contact Plates Glove Print

EU 2009 Grade cfu/m³ (Ø 90 mm), (Ø 55 mm), 5 fingers
cfu/4 hours cfu/plate cfu/glove
WHO 2010
A <1 <1 <1 <1
B 10 5 5 5
C 100 50 25 -
D 200 100 50 -

Microbiological Microbiological
Clean Area
Active Air Settling Plate
Classification ISO Designation
Action Levels Action Level
(0.5 µm particles/ft3)
(CFU/m3) (Ø 90 mm; cfu/4hr
100 ISO 5 1 1
Reference: “Microbial Control and
FDA 2004
1000 ISO 6 7 3 Monitoring in Aseptic Processing
Cleanrooms”
10000 ISO 7 10 5 Gilberto Dalmaso and Claudio
Denoya. Controlled Environments,
100000 ISO 8 100 50 Jan 12, 2015

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 Suggested Initial Contamination Recovery Rates in Aseptic
Environments (a) (b)

Active Air Settle Plate (9 Contact Plate or

Room Glove or
Sample cm) 4h Exposure Swab
Classification Garment (%)
(%) (%) (%)

Isolator/Closed
RABS (ISO 5 or <0.1 <0.1 <0.1 <0.1
better)
ISO 5 <1 <1 <1 <1
ISO 6 <3 <3 <3 <3
ISO 7 <5 <5 <5 <5
ISO 8 <10 <10 <10 <10

(a) All operators are aseptically gowned in these environments (with the exception of
background environments for isolators). These recommendations do not apply to
production areas for non-sterile products or other classified environments in which
fully aseptic gowns are not donned.
(b) From USP 36/37 <1116>, Table 3

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 Incidents and Contamination Recovery Rates

• Contamination recovery rates are more useful to analyze than trending CFU
counts.
• Variability in sampling methods make CFU counts hard to predict.
• Incident rates are the number of environmental samples found to contain
microbial contamination (≥ 1 CFU) out of the total samples taken.
• Ex. “An incident rate of 1% would mean that only 1% of the samples taken have
any contamination, regardless of colony number. In other words, 99% of the
samples taken are completely free of contamination.” <USP 1116>

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 Case Study 1: USP <1116> Contamination Recovery Rates
Grade B Routine Active Air (12 month, 2011)
Number of Cumulative
Count (CFU)
Samples percentage
0 1240 80.78
1 191 93.22
2 52 96.61
3 20 97.92
4 8 98.44
5 7 98.89
6 2 99.02
7 3 99.22
8 2 99.35
9 1 99.41

Limit EU GMP 10 0 99.41

Exceed EU GM Limit > 10 9 100.00

CRR: < 5% Annual Total 1535 Adapted from Tim Sandle’s
webinar, 2012

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 USP <1116> Contamination Recovery Rates

Trend Analysis - Grade B AFS routine active air (12 months)

35

30
Count (cfu per cubic metre)

25

20

15

10

0
1 105 209 313 417 521 625 729 833 937 1041 1145 1249 1353 1457
No. samples / time
Month* 1 2 3 4 5 6 7 8 9 10 11 12

(*) On average 128 sample/month

Adapted from Tim Sandle’s
webinar, 2012

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 Excursions and Data Interpretation

• Any ISO 5 excursion of >1 CFU, whether from

airborne, surface, or personnel sources, should
prompt a careful and thorough investigation.
• Investigate if the incident was isolated or can be
correlated with other recoveries that might indicate
an unusual pattern.
• Microbiologists should use practical scientific
judgment in their approach to excursions.
• Environmental monitoring can only assure that a
production system is in a consistent, validated state
of control.

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 USP Contamination Rates

• Corrective actions may include:

• Sanitization program including types
of disinfectants, application
methods, and frequencies
• Personnel practices by supervisory
staff
• Microbiological sampling methods
and techniques
• Training on gowning practices

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 Summary Conclusions on CRR

1. Current guidance on microbial limits for aseptic processing environments is based on CFU.
2. <1116> proposes a new way to look at microbiological data by adopting CRR as percentage
value of maximum allowed contaminated samples (those with ≥1 CFU).
3. The case study illustrated that depending on how the data is evaluated, an environment that
was compliant under CFU-based limits, could become noncompliant under the new CRR (or
vice-versa in some cases)
4. At low counts, Alert or Action Levels are not statistically different. Instead, emphasis should
be on incidents, even those having just 1 CFU.
5. Percentage incident rates force us to look historically at least 100 samples back, instead of
focusing on just a single current incident, or only on samples showing contamination above
Action Levels.
6. It also helps to focus on all samples that have any contamination regardless of colony
number. There could be a trend indicative of loss of control.
7. Even if CRR are adopted as a way to analyze microbial contamination, <1116> emphasizes
that for an ISO 5 cleanroom, any excursion of ≥1 CFU, and particularly those >15 CFU, should
be investigated.
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 Sampling Airborne Microorganisms

• Slit-to-Agar Air Sampler (STA) • Settling Plates

• Sieve Impactor • Contact Plates
• Centrifugal Sampler • Swabs
• Sterilizable Microbiological Atrium • Touch method
• Surface Air System Sampler
• Gelatin Filter Sampler

• Main points from USP <1116>:

• The sampling time chosen must be a strong representation of the current
environment.
 Sampling times can exceed 15 minutes.
• High sampling volumes can disrupt airflow.
• Turbulence and its presence in critical areas from high sampling volumes should
be considered.

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 Additional Conclusions Regarding Real Time Monitoring

• Pharmaceutical Industry is moving toward continuous manufacturing and

control to ensure full understanding of the aseptic manufacturing process
and product quality.
• Environmental monitoring trend analysis provides an enhanced way to
detect process changes before they impact the product.
• Used in conjunction with rapid microbiological test methods, such as those
based on laser-induced fluorescence for air monitoring, valuable process
information can be obtained in real time or in a short period of time.

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 Summary

• Key points from revised USP chapter

• Compared aspects with EU GMP
• Focused on areas of disagreement with EU GMP e.g. incident rates vs counts
• Is a harmonized approach possible?

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 Any questions?

Thank you

Gilberto Dalmaso

Email: gdalmaso@pmeasuring.com

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