Global Aseptic Seminar

Kalamazoo
June 2014

Instructor Bio

Dona Reber, MS, SM (NCRM)

Over 30 years of academic and industrial experience in

microbiology labs and mfg support
Currently in Microbiology and Aseptic Support group,
Global Quality Operations at Pfizer, assessing aseptic
processing facilities, investigations, policy documents,
and network aseptic processing training
Directs the MicroNet, Pfizer's microbiology network,
sterile and non sterile manufacturing support

PDA member, working on TR Disinfection, member for recently completed TR

Objectionable Organisms
Provided training for new FDA Inspectors on EM
PMEDG Chair
Publications/presentations: environmental monitoring, microbial identifications and
microbiology risk assessments. PDA book: Microbial Identifications: The Keys to a
Successful Program, October 2012
NOTE: Presentation is PDAs and my own work and opinion, does not necessarily reflect Pfizer
policy.
1

Question:
Why is environmental monitoring performed?

Why is Environmental Monitoring performed?

TO:
- Demonstrate control of aseptic processing
- Identify problems in facility or process
- Identify trends more than individual events
- Gather data to support root cause analysis
and/ or follow-up on CAPAs of contamination
events

What Environmental Monitoring is not. . .

A release test
An analytical test
Highly recoverable
Always repeatable
Always linked to direct cause and effect
The total picture for control of the APA

PDA Technical Report #13

Key Revisions and Changes
Rev1 (2001)
EM Classifications (US
FDA/USP/EU/ISO)
Surveillance Support

Cleaning and Disinfection

Sampling Selection and
Frequency
Alert and Action levels
Data Management
IDs
Investigations

System Surveillance
Types of Monitoring
Validation

Rev 2 (2014)
EM Classifications (US
FDA/EU/JP/WHO/ISO)
Environmental Monitoring

Site selection and frequency

Alert and action levels, USP shift
to Excursion Rates
recommended scheme for IDs
Investigations Elements
Data Management/EM
documentation records

EM by application
Types of Monitoring
Terminal Sterilization
Aseptic Processing
Isolation Technology

Environmental Monitoring:
Key Regulations and Guidelines

Excerpt from EU Annex1

Excerpt from FDA Aseptic Guidelines

In aseptic processing, one of the most important laboratory controls is the
environmental monitoring program. This program provides meaningful information on
the quality of the aseptic processing environment (e.g., when a given batch is being
manufactured) as well as environmental trends of ancillary clean areas. Environmental
monitoring should promptly identify potential routes of contamination, allowing for
implementation of corrections before product contamination occurs (211.42 and 211.113).

Types of Environmental Monitoring

Total Airborne (Non-Viable) Particulate Monitoring
Active Viable Air Monitoring
Passive Viable Air Monitoring
Surface Monitoring
Personnel Monitoring
Water Testing
Room Temperature and Relative Humidity
Room Differential Air Pressure

FDA Aseptic Guidelines Limits

EU Annex 1 Limits

EU Action Limits

What is meant by
average value?
10

EU Annex 1 non viable limits

Reference PDA Technical Report #13:

Fundamentals of an Environmental Monitoring Program
USP <1116>

EU Annex 1
and WHO Annex 4

Japan
(Aseptic
Processing
Guidance)

JP XVI

ISO 5 /Class 100,

ISO 5/Class 100

Grade A
Grade B (at rest)

Grade A
Grade B (at rest)

Grade A
Grade B (at rest)

3520

3520

3520

3500

3520

3520

29

Not specified

Not specified

20

20

Not specified

ISO 6

ISO 6/Class 1000

ISO 6/Class 1000

NA

NA

NA

0.5 m

35,200

35,200

35,200

NA

NA

NA

5 m

290

Not specified

Not specified

NA

NA

NA

ISO 7

ISO 7/Class 10,000

ISO 7/Class 10,000

Grade B (operation)
Grade C (at rest)

Grade B (operation)
Grade C (at rest)

Grade B (operation)
Grade C (at rest)

0.5 m

352,000

352,000

352,000

350,000

352,000

352,000

5 m

2,900

Not specified

Not specified

2,900

2,900

Not specified

ISO 8

Class 100,000

ISO 8/Class 100,000

Grade C(operation)
Grade D (at rest)

Grade C (operation)
Grade D (at rest)

Grade C(operation)
Grade D (at rest)

0.5 m

3,520,000

3,520,000

3,520,000

3,500,000

3,520,000

3,520,000

5 m

29,000

Not specified

Not specified

29,000

29,000

Not specified

ISO 14644

US FDA
(Aseptic
Processing
Guidance)

ISO 5

0.5 m
5 m

Particle
Size

[1]

Class 100 and Grade A are defined as requiring unidirectional flow by all applicable guidelines
Obsolete U.S. Federal Standard 209E classification added for continuity
[3] Class titles for US FDA and USP indicate equivalent particle counts per ft3
[4] ISO 4.8 based upon reduced limit for particles 5 m
[5] Grade D operational particulate counts are dependent upon the operation and are not defined by any guideline
[2]

Why are 0.5um and 5.0 um

size particles important?

Bakers Yeast

Pin Head
Ragweed
Pollen

Rhinovirus

Human Hair
E. coli

0.03

0.05

0.01

0.3
0.1

Limit of Light
Microscopy

0.5

Red Blood Cells

3
1

30

50

10

300

500

100

Most Bacteria

1000

Limit of
Human Vision
13
13

Total Airborne Particulate Monitoring

Climet Fixed Remote Air Sampler

Discrete laser particle counting technology

Monitor 0.5 m and 5.0 m particles (EU only)
MetOne Air Fixed Sampler

Grade A frequent or continuous monitoring

Fixed sampling rates (cfm)
Varying sampling volumes. (0.5 or 1 cu meter)

Lasair III Portable Air Sampler

Fixed or portable equipment

Isokinetic heads
Additional considerations for manifolds

Manifold Air Sampler

Active Viable Air Monitoring

SAS

Impaction of organisms on
solid growth media
Slit to agar
Sieve
Centrifugal

Delay timers

AirIdeal 3

Fixed volume sampled cubic

foot or cubic meter.

R2S Slit to Agar

Fixed or portable equipment

MAS 100

RCS Plus
SMA-1

Passive Viable Air Monitoring

Settle Plates, continuous
Exposure limit per 4 hours in Annex 1
Qualify organism recovery at >maximum exposure time

What if the
process is
less than
four hours.
Do I adjust
the limits?

Volumetric SMA-1 and settle plate

16

Surface Monitoring: Plates

Replicate Organism Detection and Counting
(RODAC)
plates and swabs
Fixed area sampled ~20 cm2
Qualify recovery, including disinfectant
exposure

Day Engley (D/E)

Neutralizing Agar

Soybean Casein Digest Agar

with polysorbate and tween

Surface Monitoring - Swabs

Used for areas that are hard to sample with a RODAC plate:
lumens, small items, stopper bowl, filling needles
Usually wetted with buffer solution (e.g., Ringers solution) or
directly with broth.
Samples may be:
placed immediately in broth (presence/absence testing) for Grade A,
or
taken to lab for filtration and plating (quantitation, Grades B, C, D)

Qualify recovery, including surface disinfectant exposure

Becton Dickenson

AES Chemunex

Personnel Monitoring
Typical sites: gloves forearms and chest
Sample upon exit from APA
Self sampling only on exception, discouraged by
regulators!
Assure all finger pads and thumb
are sampled , one plate per glove

Water Testing: WFI

Assures microbiological and chemical quality of the water
TOC (on or off line meters)
Conductivity (on or off line meters)
Endotoxin
Total Heterotrophic Plate Count
DI, purified, Feed water also tested

Water Sampling
Representative locations appropriately labeled and monitored on routine basis
Frequency may vary depending on usage.
Containers used for Microbiological sampling must be sterile.
The water sampling is the same as routine use, including use of hoses.
Excerpt from Water and Wastewater, USP<1231>
IF IT IS NOT POSSIBLE TO TEST THE SAMPLE WITHIN ABOUT 2 HRS OF
COLLECTION, THE SAMPLE SHOULD BE HELD AT REFRIGERATED
TEMPERATURES (2-8C) FOR A MAXIMUM OF ABOUT 12 HRS TO
MAINTAIN THE MICROBIAL ATTRIBUTES UNTIL ANALYSIS. IN SITUATIONS
WHERE EVEN THIS IS NOT POSSIBLE (SUCH AS WHEN USING OFF-SITE
CONTRACT LABS), TESTING OF THESE REFRIGERATED SAMPLES
SHOULD BE PERFORMEDWITHIN 48 HRS AFTER SAMPLE COLLECTION.

Environmental Monitoring:
Calibration, Qualification and Training
Instrument calibration generally performed by vendor

(volume and time).

Method qualification for viables based on recovery studies of

broad range of microorganisms, including representative

environmental isolates-FDA Aseptic Guidance, ISO 14698
(AnnexA)
Analysts trained on methods including cGLP and aseptic

technique, plate counting.

Operators or quality samplers trained on aseptic sampling

technique, equipment. Quality oversight.

New Technologies for EM

Technology
BioVigilant IMD-Air
350/350A

Milliflex Rapid
Growth Direct

How it Works
Real time particle
monitoring, detects
biological fluorescence and
size.
ATP Bioluminescence

EM Application
Investigations, area
qualifications, training

Water bioburden
screening
Automated incubator / plate Viable environmental
reader/non destructive
monitoring screening

Charles River Endosafe Kinetic chromogenic, color

PTS MTS
intensity measurement,
early endotoxin detection

Automated WFI testing

Precautions When Conducting

Environmental Monitoring

Sampling of water same as manufacturing usage

More Precautions When Conducting

Environmental Monitoring

Is it okay to refrigerate plates before incubation?

EM Program:
Elements and Rationale

EM Program: Location Considerations

Critical product or sterile component exposure examples:

Filling needles
Stopper bowls
Vial Turntable

High potential for microbial/particle presence

Elevated recoveries observed during qualification or from

historical trend analysis

Difficult to clean or sanitize
High traffic (e.g. doors, RABS door handles, intercom)
Smoke studies indicate areas of eddies or turbulence

27

EM Excursions and Limits

Investigation

Limits are defined as when exceeded

28

When to identify organisms in an EM Program

All isolates found in Grade A and B are identified to the species

level where possible.

All isolates found in Grades C and D with results above the action

level should be identified to the species level.

Periodically, isolates found in Grades C and D should be identified

to at least the genus level to develop trending of common isolates.

Lets look at some examples of EM sampling of

common items. . .

Trending of EM Data
Purpose: To provide assurance that a continued state

of control is being maintained. Knowledge for

response to adverse trends before control is lost.
Observe changes in both numbers and types of

organisms.
Develop Trending Rules: By Grade and Location (room

or sampling site)
Reviewed periodically (e.g. monthly, quarterly,

annually)

Trending of EM Data, Examples

A running pattern, suggests short episodic trends that should be

investigated before becoming problematic.

Action level

Alert level

Is this data in control?

32

A trending pattern is when there is a continued increase or

decrease in the results, a problem that is not being addressed
Action level

Alert level

Is this data in control?

33

A cyclic pattern is when a series of data points show the same trend at
periodic time intervals. This could be an indicator of some systematic
error. Perhaps it is temperature fluctuation in the room, humidity issues
or seasonal variations
Action level
Alert level

Is this data in control?

34

What comment might an inspector make about this data?

Alert level does not reflect real data, difficult to identify trends

Action level
Alert level

Is this data in control?

35

Trending by Genus and species

Corynebacterium
tuberculostearicum
2.9%
Bacillus cereus,
4.3%
Staphylococcus
hominis, 5.6%

Staphylococcus
epidermidis, 8.7%

Staphylococcus
luteus, 9.6%

Staphylococcus
capitis, 2.8% Bacillus pumilus,
2.7%
Staphylococcus
warneri, 2.5%
Bacillus subtilis,
2.4%
Staphylococcus
haemolyticus, 2.2%
Ralstonia pikettii,
1.7%
Paenubacillus
glucanolyticus,
1.5%
Bacillus
megaterium, 1.5%
Bacillus
Proprionibacterium
licheniformis, 0.9%
acnes, 1.0%

Bacteria Most Often Submitted for Identification Testing During 2010, Barry A. Friedman, posted May 17, 2011

Trending by Types of Microorganisms

5-10% water/liquid source
Gram negative rod Organisms such as
Pseudomonas and Acinetobactor species

15-20% air/soil
source
Organisms such
as large Gram
positive rods,
spore formers,
Bacillus and
fungi

Data from previous

slide shows a similar
distribution:
Common APA Bacteria
People: 35.3%
Air soil: 13.3%
Liquid water: 1.7%
70+% People
Organisms, such as,
Staphylococcus,
Micrococcus and
Propionibacterium
spp.

Sources of Contamination: PEER

38

Investigation: Examples, What to Look for

Some Inspection Observations

Questions Asked/Comments During

FDA Inspector Training, 2013-2014
How do I know if locations/samples are adequate?

How are environmental monitoring methods qualified?

What are factors to consider for investigations?
What is expected for trending?
Are some air samplers/swabs/em equipment better than others?
Help! Incubation temperatures/times are not always the same!

How to get to the issuesSo much data and so little time!

EM INVESTIGATION Study:
What Would YOU Do?

While reading plates, the analyst realizes that the agar

lot used for EM sampling was expired prior to use for EM
sampling
Invalidate the results???????

42

Discussion Points for Case Study

If the EM sample can be repeated, Resample with unexpired media
If the EM sample cannot be repeated (e.g., time sensitive activity), perform
investigation: Points to consider
If expired media lot is available, perform growth promotion testing to
determine if recovery is satisfactory, demonstrating results are valid
If expired media is not available, hold a subsequent same type of media lot
to expiry and perform growth promotion to demonstrate recovery is
satisfactory

QUESTION: Should counts be invalidated on expired media for samples in

Grade A during filling?
WHY or WHY NOT?
43

Sources of Contamination: PEER

44

Group Exercise:
Discuss Potential EM sources, think PEER
Sphingomonas species (water bug) on a water sample plate

Environment, Equipment

Staphylococcus species (people bug) on a water sample plate

Increase in spore formers on EM plates in

raw materials sampling area
Water organisms on surface RODAC plates
Increase in viable counts in manufacturing area
during construction of adjacent area
Particle counter exceeding limits when area
not in use

Same organism on multiple days, multiple

locations, first in Grade C and moving to Grade A

People

Raw Materials, Environment, Equipment

People, Equipment

x
Environment

People, Environment

Equipment

Questions?

46

Personnel Behaviors:
The Good and the Bad

Hold Up the Green Card

Hold Up the Pink Card

47

Operator leans on equipment used for processing

while working in the APA.

1.
2.

Good
Bad

48

EM sampling locations are clearly defined and

posted.

1.
2.

Good
Bad

49

Items are picked up from the floor during aseptic

processing.
1.
2.

Good
Bad

50

Gloved hands are sanitized with alcohol

frequently & prior to any intervention during
aseptic processing.
1.
2.

Good
Bad

51

Operators discuss where to go for lunch while in the

APA.
1.
2.

Good
Bad

52

Alcohol is used for disinfection instead of a

sporicidal agent when transferring materials from
the warehouse to the Grade D area
1.
2.

Good
Bad

53

Entries and exits to the APA are limited to essential

activities.

1.
2.

Good
Bad

54

EM Alert levels are set at meaningful levels.

1.
2.

Good
Bad

55

70% Isopropyl Alcohol containers are changed daily

using sterile containers and fresh sterile alcohol.

1.
2.

Good
Bad

56

Alarms for non viable monitoring are at a central

location, in another room from the APA

1.
2.

Good
Bad

57

Operator needs to reach over open product to

perform an intervention.

1.
2.

Good
Bad

58

Sterile equipment is protected and dedicated to the

Grade A area.

1.
2.

Good
Bad

59

Operator notifies co-operator before entering APA

that her gown has a rip in the sleeve

1.
2.

Good
Bad

60

Cart is blocking the air return.

1.
2.

Good
Bad

61

Sterile tools (e.g. scissors) used in Grade A are

placed on a non-sterile surface between uses.

1.
2.

Good
Bad

62

Non viable monitoring heads are covered

during cleaning of the APA

1.
2.

Good
Bad

63

Sterile tools/extenders are used to contact

critical surfaces instead of gloved hands.
1.
2.

Good
Bad

64

Operator stands with arms folded across

chest.

1.
2.

Good
Bad

65

The sterile connection hose is too short to reach the

equipment through the designated wall orifice, so the
Grade B door is propped open to allow the hose to reach
1.
2.

Good
Bad

66

Sterile garments (e.g. beard covers, goggles) are removed

during manufacturing process.

1.
2.

Good
Bad

67

EM Trend results are reviewed with management and

operators

1.
2.

Good
Bad

68

Dancing is practiced when the filling line is running

smoothly in the aseptic processing area

1.
2.

Good
Bad

69

Thank You and the PDA

70