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ANNEX 1: MAKING SENSE OF

EM MICROBIAL TRENDING AND

DRIVING EFFECTIVE CAPA PLANS

Dr Ulrich Herber

Director of Technology and Market Development

Charles River - Microbial Solutions

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ANNEX 1: FOCUS ON ENVIRONMENTAL PROGRAM

Environmental Monitoring is a focus across Annex 1 revision document sections…

3.2 Investigations should be performed into non-conformities, such as sterility test failures or environmental monitoring

excursions…

5.12 ... visualization studies should be considered when establishing the facility's environmental monitoring program.

5.28 Clean room qualification (including classification) should be clearly differentiated from operational process

environmental monitoring.

6.3 …performed in the clean area then precautions such as additional disinfection and additional environmental

monitoring should be considered

8.45 These items, and others representing potential routes of contamination, should be included in the environmental

monitoring program.

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APPROPRIATE & CURRENT TECHNOLOGIES

2 Principle

The use of appropriate current technologies should be implemented to ensure

protection and control of the product from potential extraneous sources of particulate and microbial

contamination such as personnel, materials and the surrounding environment.

Improve protection and control using current technologies

A contamination control strategy should be implemented across the facility in order to assess the effectiveness

of all the control and monitoring measures employed. Elements to be considered …

m) Monitoring systems - including an assessment of the feasibility of the introduction of

scientifically sound, modern methods.

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WHAT THE REGULATORS ARE TELLING US…Regulators are encouraging the industry to adopt current technology!

(1) Dr. P Hargreaves, MHRA Unit Manager, Technical & Operations Coordination Inspection, PDA/EMEA Joint Conference, February 2008, Budapest

“It is a legal requirement to keep up to date, however 20th century technology for counting and ID of microorganisms has not been introduced by the majority of the industry” (1)

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ANNEX 1: PROCESS MONITORINGFocus on EM, trending and ID

9. Viable and non-viable environment & process monitoring

9.4 In order to establish a robust environmental monitoring program, i.e. locations,

frequency of monitoring and incubation conditions (e.g. time, temperature(s) and aerobic

and or anaerobic), appropriate risk assessments should be conducted based on detailed

knowledge of the process inputs, the facility, equipment, specific processes, operations

involved and knowledge of the typical microbial flora found, consideration of other

aspects such as air visualization studies should also be included.

These risk assessments should be re-evaluated at defined intervals in order to confirm the

effectiveness of the site’s environmental monitoring program, and they should be

considered in the overall context of the trend analysis and the contamination control

strategy for the site.

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9. Environmental Monitoring

9.32 Monitoring procedures should define the approach to trending.

Trends can include but are not limited to:

a) Increasing numbers of action or alert limit breaches.

b) Consecutive breaches or alert limits.

c) Regular but isolated breaches of limits that may have a common cause, for example

single excursions that always follow planned preventative maintenance.

d) Changes in flora type and numbers.

9.33 If microorganisms are detected in a grade A or B zone, they should be identified to

species level and the impact of such microorganisms on product quality (for each batch

implicated) and state of control should be evaluated. Consideration may also be given to the

identification of grade C and D contaminants and the requirements should be defined in the

contamination control strategy.

ANNEX 1: ENVIRONMENTAL MONITORING, CONT.

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ANNEX 1: QC CONT.Focus on EM and TRENDING supporting RMM

10 Quality Control (QC)

10.10 Environmental monitoring data generated in grade A and B areas should be reviewed

as part of product batch release. A written plan should be available that describes the actions

to be taken when data from environmental monitoring are found out of trend or out of

specification.

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ENVIRONMENTAL CONTROL IS MANDATED BY REGULATORSReal world citation

Proper microbial identification:

“Your revised procedure is inadequate for the following reason… You did not provide an adequate program for identifying organisms in the environment. You currently selectively identify certain microbes (e.g., gram-positive spore-formers, filamentous fungi) and only when they reach microbial action limits.” –FDA Warning Letter issued to Aplicare, Inc., 12/15/16

“You did not appear to routinely identify (i.e., to species level) bacterial and fungal isolates recovered during environmental monitoring of your aseptic processing room.” –FDA Warning Letter to USV Limited, 3/10/17

“The firm's practices for microbial identification do not provide information related to the microbiological populations found in manufacturing areas where sterile drug products are manufactured. For example, microorganisms are randomly selected and sent for identification on a (readacted) basis and when an investigation occurred… the majority of … isolates were not identified.” –FDA Form 483 issued to CAPS, 2/19/13

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DATA INTEGRITY CONSIDERATIONS

Are you attributing the right root cause for an OOS investigation /

product contamination event?

ALCOA

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REGULATORY EXPECTATIONSFDA Guidance

FDA Guidance for industry 2004 XI Sterility testing

1. Identification (speciation) of the organism in the sterility test

Sterility test isolates should be identified to the species level. Microbiological monitoring data should

be reviewed to determine if the organism is also found in laboratory and production environments,

personnel, or product bioburden. Advanced identification methods (e.g., nucleic acid based) are

valuable for investigational purposes. When comparing results from

environmental monitoring and sterility positives, both identifications

should be performed using the same methodology.

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Environmental control is mandated by regulators

Real world citation

Appropriate data analysis: “You did not utilize environmental monitoring data to identify environmental control issues and identify appropriate follow-up actions…you did not examine the data for trends or take appropriate follow-up action.”

Your evaluation of environmental microbial data should not be narrowly limited to specific lots or events. Trend analysis, identifying sources of contamination, and risk assessment are essential to maintain adequate microbiological control.”

–FDA Warning Letter issued to Mylan Laboratories Limited, 8/6/15

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PDA TECHNICAL REPORT 80

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Case Studies

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FUNGAL TRENDINGTop 5 Most Frequently Identified Fungi 2019

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TOP 10 BACTERIA LISTS ACROSS THE GLOBE

North America

Micrococcus luteusStaphylococcus epidermidisStaphylococcus hominisBacillus cereus groupStaphylococcus capitisCorynebacterium tuberculostearicumBacillus amyloliquefaciens / siamensis / velezensisBacillus halotolerans / mojavensis / subtilis / tequilensisKocuria rhizophilaRalstonia pickettii

Europe

Micrococcus luteusStaphylococcus epidermidisBacillus cereus groupMoraxella osloensisGeobacillus stearothermophilusBacillus halotolerans / mojavensis / subtilis / tequilensisBacillus amyloliquefaciens / siamensis / velezensisKocuria rhizophilaStaphylococcus hominisBradyrhizobium embrapense / mercantei / viridifuturi

APAC

Micrococcus luteusStaphylococcus epidermidisBacillus cereus groupBacillus halotolerans / mojavensis / subtilis / tequilensisStaphylococcus capitisGeobacillus stearothermophilusStaphylococcus hominisStaphylococcus cohniiBordetella bronchiseptica / parapertussis / pertussisMoraxella osloensis

Nov 2019 – Sep 2020, N = 114.038 Spore former Gram negative

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FREQUENTLY OCCURRING ORGANISMSRegional variability

Plant A

Staphylococcus

Micrococcus

Bacillus

Misc

Plant B

Pseudomonas

Serratia

Bacillus

Staphylococcus

Microbacterium

Misc

Plant C

Bacillus

Micrococcus

Pseudomonas

Ralstonia

Methylobacterium

Staphylococcus

Misc

Plant D

Staphylococcus

Bacillus

Micrococcus

Misc

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CASE STUDY – MANAGING RISK

Background:

• Large manufacturer of personal care products

• Routine EM program in place

– Limits were set at NMT 100 cfu

– Counts began to increase to around 60 cfu

– Action was not taken immediately since counts were not in excess of limit

– A sister site with more experience was consulted, and their

recommendation was to ID

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CASE STUDY - SITUATION

• By the time IDs were begun, counts

had reached the limit of NMT 100

• Once Action Limit was reached,

facility’s production had to be halted

• Estimated cost of this closure was

$10M

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CASE STUDY - INVESTIGATION

• Investigation work commenced

and additional Environmental

Monitoring plates were taken

around areas all over the facility,

including near HVAC

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CASE STUDY - INVESTIGATION

• The ID work revealed presence of Bacillus thuringiensis

• Bacillus thuringiensis was not part of the baseline study results

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BACILLUS THURINGIENSIS

Gram-positive, soil-dwelling

microorganism

During sporulation, produces

a crystalline protein toxin

which is insecticidal

The most successful

commercial insecticide

Also referred to as “Bt”

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CASE STUDY - CONCLUSION

• Clear that the Bt was coming in

through the HVAC system and that

it was being overloaded

• Further strain work was performed

to determine whether the strain

found might be one that’s

characterized as a commercial

pesticide

• Correlated back to crop spraying in

the area

• Developed CAPA

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How to implement

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86% Gram+

and 11%

Gram-

35% Gram+

and 65%

Gram-

EXAMPLE 1: RATIO BETWEEN GRAM+ AND GRAM-

A very basic indicator for changes is a change in the ratio between

Gram+ and Gram- isolates over time.

Where does this higher number of Gram- come from?

Quick overview reports can prompt a deeper dive into EM sample

reports and metadata (sample source, sample method, location,

operator).

25EXAMPLE 2: ORGANISMS OF CONCERN

• Example clearly shows the trend of Bacillus

cereus over time period.

• Proactively address before it becomes an issue;

find root cause using metadata (source, location,

etc.) and monitor for future reoccurrences.

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26EXAMPLE 3: FREQUENTLY OCCURRING ORGANISMS

Example shows two significantly different

Frequently Occurring Organism lists.

This might have an impact on cleaning and

disinfection programs, or which microorganisms

need to be considered for product testing

validations.

Summer

FOO

Winter

FOO

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Increasing accuracy and consistency supports better investigations

MODERN ID METHODS SUPPORT ANNEX 1 REQUIREMENTS

Performing identifications and trending with

modern methods allows for higher reportability,

accuracy, and consistency in routine EM work as

well as root cause investigations.

Better trending can be achieved when:

• able to avoid “no result” or “inconclusive result”

outcomes.

• ID results are accurate and able to be trusted.

• ID results are consistent.

The goal of microbiological monitoring is to reproducibly detect microorganisms for purposes of monitoring the state of environmental

control. Consistent methods will yield a database that allows for sound data comparisons and interpretations. FDA Guidance for Industry. Sterile Drug Products Produced by Aseptic Processing – Current Good Manufacturing Practice. (2004)

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CONTACT MEDr Ulrich Herber

Director of Technology and Market Development

Charles River - Microbial Solutions

Website: www.criver.com/microbial

Email:ulrich.herber@crl.com

Phone:+49.172.3146948

www.linkedin.com/in/ulrich-herber