SCHOOL OF APPLIED SCIENCES

A Project Report
On
‘’ Title of the project / Dissertation’’
Submitted in fulfillment of the requirements for the award of Degree of
MASTER OF SCIENCE
IN
MICROBIAL TECHNOLOGY
Submitted by
S P Srikar Bhattar
(SRN)
R23SO013
Under the guidance of
Roshan Sahebrao Kolhe
Dr Keshav M
Associate Manager, Biocon Biologics
Assistant Professor , School of Applied Sciences
School/ Dept
School of Applied Sciences
June 2025
Rukmini Knowledge Park, Kattigenahalli, Yelahanka, Bengaluru-560064
www.reva.edu.in

1
 SCHOOL OF APPLIED SCIENCES
A Project Report
On
‘’ Title of the project / Dissertation’’
Submitted in fulfillment of the requirements for the award of Degree of
MASTER OF SCIENCE
IN
MICROBIALTECHNOLOGY

Submitted by
S P Srikar Bhattar
(SRN)
R23SO013

Under the guidance of

Roshan Sahebrao Kolhe
Dr Keshav M
Associate Manager, Biocon Biologics
Assistant Professor , School of Applied Sciences
School/ Dept
School of Applied Sciences
June 2025
Rukmini Knowledge Park, Kattigenahalli, Yelahanka, Bengaluru-560064
www.reva.edu.in
DECLARATION
I, Mr. / Ms. S P Srikar Bhattar student of MSc Microbial Technology belonging to
School of Applied Sciences, REVA University, declare that this Project Report / Dissertation
entitled “ < title of the project >” is the result the of project / dissertation work done by me under
the supervision of Dr / Prof. <name of Guide with affiliation > and < co-guide (s) if any, with
affiliation, > at ........< name of Department, School/Institute where project work has
been carried out.

I am submitting this Project Report / Dissertation in partial fulfillment of the requirements for the
award of the degree of Master of Science in Biotechnology by the REVA University,
Bangalore during the academic year 2024-2025.

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I declare that this project report has been tested for plagiarism, and has passed the plagiarism test
with the similarity score less than 10% and it satisfies the academic requirements in respect of
Project work prescribed for the said Degree.

I further declare that this project / dissertation report or any part of it has not been submitted for
award of any other Degree / Diploma of this University or any other University/ Institution.

(Signature of the candidate)

I certify that this project work submitted by S P Srikar Bhattar has been carried out under my
guidance and the declaration made by the candidate is true to the best of my knowledge.

Signature of Guide Signature of the Director (I/C)

(Prof. Shilpa B R)

SCHOOL OF APPLIED SCIENCES

CERTIFICATE

Certified that the project work entitled < TITLE > carried out under my guidance by S P
SRIKAR BHATTAR R23SO013a bonafide student of REVA University during the academic
year 2023-2025, is being submitted in partial fulfillment of the requirements for the award of the

3
Master of Science in Biotechnology during the academic year 2024-25.
The project report has been tested for plagiarism, and has passed the plagiarism check with a
similarity score of less than 10%. The project report has been approved as it meets the academic
requirements for the Project work prescribed for the said Degree.

_____________________ _____________________
Dr. Keshav Murthy Director (I/C)
Affiliation School of Applied Sciences
REVA University, Bangalore-560064

Name of the Examiner Signature with date

1.

2.

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ACKNOWLEDGEMENT
I express my heartfelt gratitude to my guides, Mr. Roshan Sahebrao Kolhe, Associate Manager – QC
Microbiology, Biocon Biologics, and Dr. Keshav M, Assistant Professor, School of Applied Sciences,
REVA University, for their invaluable guidance, encouragement, and continuous support throughout the
course of this project.
I am deeply thankful to the visionary leadership of Dr. Shyama Raju, Chancellor; Mr. Umesh Raju,
Pro-Chancellor; Dr. Sanjay Chitnis, Vice Chancellor; and Dr. Alexander Jesudasan, Rector, REVA
University, for providing a stimulating academic environment and all necessary infrastructure.
My sincere thanks to Prof. Shilpa B R, Director I/C, School of Applied Sciences, for her administrative
support and constant motivation during this academic endeavor.
I would also like to thank the faculty members of the Department of Biotechnology for their insightful
suggestions and academic support, as well as my mentor for their guidance and encouragement
throughout the course of the project.

PLAGIARISM REPORT

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Vision
To nurture intellect, creativity, character, professionalism and research culture among students
and impart contemporary knowledge in various branches of Chemical, Biological, Physical and
Mathematical Sciences that are socially relevant and transform them to become global citizens
with leadership qualities.
Mission
 To achieve excellence in studies and research through pedagogy and support interface
between industry and academia
 To create intellectual curiosity, academic excellence, and integrity through
multidimensional exposure
 To establish state of the art laboratories to support research and innovation and promote
mastery of science.
 To inculcate an ethical attitude and make students competitive to serve the society and
nation

Program Educational Objectives (PEOs)

PEO-1 Become a professional biotechnologist with strong ethics & communication skills.
PEO-2 Pursue research in reputed institutes at national & international level.
PEO-3 Establish consultancy services and to work as entrepreneur with an ability to develop new
products/processes with an attitude of lifelong learning.

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 Program Outcomes (POs)
After successful completion of program, the graduates will be able
1. Master of Science Knowledge-Apply the knowledge of microbiology and applied
microbiology to the solution of complex biological problems.
2. Problem analysis- Identify, formulate, review research literature and analyze complex
biological problems reaching substantiated conclusions using various principles of life science
domain and microbiology.
3. Design/development of Solutions-Design solutions for complex biological problems and
design protocols or processes that meet the specified needs with appropriate consideration for the
public health and safety, conservation of biodiversity, better understanding of the
microorganisms and necessary tools for finding solutions of various crippling human/plant
diseases with ethical, societal, and environmental considerations.
4. Conduct investigations of complex Problems-Use the various protocols developed through
extensive research-based knowledge and methods including design of experiments, analysis and
interpretation of data, and provide valid and reproducible conclusions.
5. Environment and Sustainability-Apply the classic and modern biological theoretical and
practical knowledge gained to address societal, health, microbial and plant biodiversity studies,
safety, ethical and cultural issues and the consequent responsibilities relevant to the professional
upgradation of the student and society as a need for sustainable development.
6. Ethics-Apply ethical principles established by different government agencies and commit to
research ethics, responsibilities and norms to undertake their current and future research and
development.
7. Individual and team work-Be an independent thinker and researcher effectively as an
individual, and as a member or leader of different teams, and in multidisciplinary research
Institutions and Universities.
8. Communication-Communicate effectively on complex research activities with the scientific
community and with society at large, as a scientist or a teacher, be well versed with scientific
writing and write effective reports and design research projects, make effective presentations,
and be able to defend it efficiently.
9. Project management and finance-Write good research and development projects relevant to
the needs of society and environment and attract extra mural funds for himself and his team in
the Institute or University from various funding agencies and manage R&D projects effectively.
10.Life-long learning-Apply the discipline, ethics and knowledge obtained to engage in
independent and life-long learning in their respective fields of interest wherever they go for
further higher studies or jobs.

Program Specific Outcomes (PSO)

After successful completion of the programme, the graduates shall be able to

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1. Understand about environmental microbiology, food & diary Microbiology, Industrial
Microbiology, and many other technologies involved in microbiology industries and academic
institutions.
2. Provide a strong foundation for a career working skills with project management, business
development or venture capital within the microbiology, agriculture, food, environment, medical
technology or related industries.
3. Utilize the skills to design, perform and analyze a research problem which has a social
relevance and outcome with acquired skills of presentation and scientific writing.
Course Outcomes:
On successful completion of the project, the student shall be able to:
1. Apply fundamental and disciplinary concepts and methods in ways appropriate to their principal
areas of study.
2. Demonstrate the skill sets acquired and employ the knowledge of current in formation in the
domain.
3. Apply technological tools and techniques specific to the professional field of study.
4. Acquire real-time exposure to the systematic execution of research components and
methodology.
Course POS/ PO1 PO2 PO3 PO4 PO5 PO6 PO7 PO8 PO9 PO10 PS01 PS02 PS03
Code COs

CO1 2 3 3 3 2 2
CO2 2 2 3 3 3 3 2 3 2
M23SO401
CO3 2 2 3 3 3 3 3 3 3 3 3 3
CO4 3 3 3 3 3 3 3 3 3 3 3 3 3

LIST OF TABLES

TABLE NO. TITLE PAGE NO.

8
 LIST OF FIGURES

FIGURE NO. TITLE PAGE NO.

9
 CONTENTS

CHAPTER TITLE PAGE NO.

ABSTRACT

INTRODUCTION

REVIEW OF LITERATURE

HYPOTHESIS

OBJECTIVES

MATERIALS and METHODS

RESULTS and DISCUSSION

CONCLUSION

REFERENCES (APA format)

Articles submitted /published

information

Note: Abstract and Conclusion Chapters the description on attained Program Outcomes and
Program Specific Outcomes shall be mentioned as a best practice. Guidance will be provided by the
mentors in case of external projects and by guides in case of internal projects.

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 ABBRIEVIATION OF NOMENCLATURE
AND
LIST OF SYMBOLS

OOAL – Out Of Alert Limit OOS – Out Of Specification

DEA – Dey Engley Agar SCDA – Soyabean Casein Digest
Agar
TSA – Tryptone Soya Agar RH – Relative Humidity
HEPA – High-Efficiency Particulate Air USP – United States
Pharmacopeia
ISO – International Organization for GMP – Good Manufacturing
Standardization Practice
CAPA – Corrective and Preventive Action SOP – Standard Operating
Procedure
CFU – Colony Forming Units EM – Environmental Monitoring

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CHAPTER 1: INTRODUCTION
Environmental Monitoring (EM) is a critical and challenging aspect of sterile product
manufacturing, particularly in cleanrooms where low bioburden is essential for product integrity.
Unlike terminal sterilization, aseptic processing relies on the continuous control of
environmental conditions to ensure microbial contamination is minimized during production.
This demands strict adherence to protocols and vigilant monitoring throughout all stages of
manufacturing.
Cleanroom environments are classified based on airborne particulate and microbiological limits,
as per ISO 14644-1 and EU GMP Annex 1 (2022). The classifications—Grade A, B, C, and D
(equivalent to ISO Class 5 to 8)—define the acceptable limits for viable and non-viable particles
both “at rest” and “in operation.” In these environments, EM provides a structured approach to
assessing air, surfaces, personnel hygiene, and water systems, ensuring sterility assurance and
compliance with FDA, USP <1116>, and ICH Q9/Q10 guidelines.
Key Components of Environmental Monitoring:
This project emphasizes the practical application and analysis of five key EM tools, each critical
to sustaining a robust contamination control strategy in low bioburden facilities:
1. Recovery Rate Trending:
It involves measuring the percentage of samples testing positive for microbial
contamination. It helps identify efficiency of the sampling technique and early signs of
control drift.
o Formula:
Recovery Rate (%) = (Positive Samples ÷ Total Samples) × 100
o Use: Ensures consistency in sampling and supports GMP compliance.
2. Control Charts (X-bar and P-Chart):
These statistical process control tools monitor average microbial counts (X-bar) and
proportion of positive samples (P-chart).
o Application: Early detection of trends, deviation analysis, and alert/action limit
breach management.
3. Microorganism Trending:
Identifies recurring species-level contaminants (e.g., specific bacteria or molds) to trace
contamination sources and conduct root cause analysis.
4. Continuous Monitoring Trends:
Real-time EM enables detection of subtle environmental shifts and allows immediate
CAPA implementation. Devices like real-time particle counters and digital loggers are
integrated for enhanced compliance.
5. Linear Regression Analysis:
Used for predictive modeling, regression evaluates how microbial load changes over time
or with respect to environmental parameters.
o Model: Y = a + bX
o Interpretation: Positive slope implies worsening control; negative slope suggests
improvement.

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At Biocon Biologics, this study was conducted in Grade B, C, and D areas, where EM involved
active air sampling (impaction samplers), passive air sampling (settle plates), and surface
monitoring (swabs/contact plates). Plates used included:
 90 mm Tryptone Soya Agar (TSA) for passive air sampling,
 SCDA media for surface monitoring,
 SCA media with additives (e.g., 1% glycerol) for active air sampling.
Additionally, cleanroom maintenance was observed through:
 HEPA filters (99.99% efficiency),
 Laminar airflow systems (0.45 m/s ± 20% in Grade A),
 Pressure differentials (10–15 Pa between rooms),
 Temperature and humidity controls (18–22°C and 30–60% RH),
 Routine cleaning with sporicidal agents (e.g., hydrogen peroxide, peracetic acid).
This report consolidates EM strategies by linking SOP adherence, alert/action level trend
reviews, and statistical assessments to product safety. Through monthly, quarterly, and yearly
EM data reviews, this project aligns with industry practices for robust microbiological risk
management.

Objectives of the Study

Derived from both IA1 and IA2 PPTs, the objectives of the project are as follows:
1. To understand the role of recovery rate trending in evaluating sampling effectiveness.
2. To apply control charts (X-bar and P-chart) for detecting process variations in EM data.
3. To interpret microorganism trending to identify contamination sources.
4. To assess the significance of continuous monitoring trends in cleanroom environments.
5. To utilize linear regression analysis for forecasting microbial contamination risks.
6. To ensure sterility assurance through comprehensive EM across Grades B, C, and D
areas.
7. To adhere to SOPs for reducing particle shedding and maintaining aseptic control.
8. To monitor microbial growth on 90 mm and 55 mm plates and evaluate them against
prescribed alert and action limits.
9. To prevent recurrence of microbial excursions and OOS/OOAL events through CAPA
and training.

CHAPTER 2: REVIEW OF LITERATURE

Environmental Monitoring (EM) is a foundational element in aseptic pharmaceutical
manufacturing. Its primary aim is to prevent microbial contamination that could compromise
product sterility, particularly in processes that do not involve terminal sterilization. In such cases,
continuous and vigilant monitoring becomes crucial. Literature in this domain emphasizes not
only routine surveillance but also statistical analysis and predictive modeling for better control
and risk mitigation.
2.1 Importance of Environmental Monitoring

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According to Whyte (2010), ineffective monitoring and inadequate control of cleanroom
environments are key contributors to contamination-related failures, including batch rejections
and product recalls. Huang et al. (2015) highlighted that low bioburden processes require
significantly more stringent controls compared to terminally sterilized products, as even minor
environmental deviations can lead to microbial excursions and out-of-specification (OOS)
events.
Unlike sterilization techniques that act at the end of the process, aseptic manufacturing relies on
barrier systems, air filtration, and controlled personnel movements to sustain microbial control
throughout production. Therefore, a continuous EM program is indispensable for early warning
and preventive action.

2.2 Regulatory Frameworks and Compliance Standards

Compliance with regulatory standards is critical to ensure uniformity, quality assurance, and
regulatory approvals. Several key documents and frameworks form the backbone of EM
programs:
 EU GMP Annex 1 (2022)
Establishes microbiological and particulate limits for cleanrooms. It mandates defined
sampling frequencies and differentiates between Grades A to D for both viable and non-
viable monitoring.
 USP <1115> and <1116>
Provide guidance on microbiological control and monitoring in aseptic processing
environments. USP <1116> specifically emphasizes statistical interpretation and
trending, alert/action levels, and identification of microorganisms.
 ISO 14644-1
Classifies cleanrooms based on airborne particulate matter, ranging from ISO Class 5
(Grade A) to ISO Class 8 (Grade D). It forms the basis for establishing airflow standards
and operational limits.
 FDA Guidance for Industry (2004)
Discusses aseptic processing practices and contamination control principles under current
Good Manufacturing Practices (cGMP).
 ICH Q9 and Q10
Address quality risk management and the establishment of a Pharmaceutical Quality
System (PQS), highlighting the importance of data analysis and continuous improvement
through EM.
These regulatory documents collectively shape EM programs by defining permissible limits,
monitoring methods, risk assessment procedures, and corrective and preventive action (CAPA)
requirements.

2.3 EM Techniques in Cleanrooms

Numerous sampling methods are employed to detect microbial contamination in controlled
environments. Each technique has its own application depending on cleanroom classification and
criticality.
a) Active Air Sampling

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Impaction samplers collect viable airborne microorganisms onto agar plates. Studies (Whyte &
Eaton, 2017) suggest this technique provides reliable quantitative data, particularly in high-risk
areas like Grade A and B.
b) Passive Air Sampling (Settle Plates)
Settle plates are exposed to air for a specified period to collect microbial fallout. Sandle (2015)
emphasized their use in critical zones (e.g., laminar airflow cabinets), especially for monitoring
long-term exposure during operations.
c) Surface Monitoring
Swab and contact plate methods are used to detect microbial contamination on equipment, walls,
and operator gloves. Denyer & Baird (2016) emphasized that these methods are essential for
evaluating cleaning effectiveness and contamination sources.
d) Personnel Monitoring
Monitoring of operators’ gloves and garments after critical operations helps assess aseptic
technique compliance. This data contributes to P-chart-based trend analysis.

2.4 Cleanroom Design and Operational Controls

Ljungqvist & Reinmüller (2018) discussed cleanroom infrastructure such as non-porous walls,
unidirectional airflow, and pressure cascades as critical barriers to contamination. ISO 14644-1
further recommends:
 Air change rates: ≥60 ACH in Grade A/B; ≥20 ACH in Grade C/D.
 HEPA Filtration: ≥99.99% efficiency for particles ≥0.3 µm.
 Pressure differentials: 10–15 Pa between adjacent zones.
 Environmental Conditions: Temperature (18–22°C); Relative Humidity (30–60%).

2.5 Statistical Tools for Data Interpretation

Statistical interpretation of EM data is recommended to ensure proactive control:
 Control Charts (X-bar and P-Chart):
As per Montgomery (2009) and PDA TR13, these charts help distinguish between natural
(common cause) and abnormal (special cause) variations.
 Recovery Rate Trending:
Sutton (2005) highlighted its utility in assessing operator performance and sampling
technique consistency.
 Microorganism Trending:
EU GMP Annex 1 mandates species-level identification in cases of repeated
contaminations to enable root cause analysis.
 Linear Regression Modeling:
ICH Q9 encourages the use of predictive tools to assess trends over time. R² values and
p-values are used to evaluate the strength and significance of correlations in microbial
count trends.

2.6 Emerging Technologies in EM

According to Schneider et al. (2021), modern EM programs increasingly use digital
dashboards, real-time particle counters, and AI-assisted forecasting tools. These allow rapid
detection of anomalies, reduce manual errors, and support predictive analytics. The ISPE Good
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Practice Guide (2021) recommends automation and data integrity controls for robust EM
program management
CHAPTER 3: HYPOTHESIS
Hypothesis:
A structured environmental monitoring (EM) program, when integrated with statistical trend
analysis and predictive modeling, significantly enhances contamination control, supports early
detection of deviations, and improves compliance with regulatory standards in low bioburden
manufacturing environments.
This project is based on the assumption that combining classical EM methods (such as air and
surface sampling) with advanced analytical tools (e.g., control charts, microorganism trending,
and linear regression) will:
 Provide deeper insights into contamination patterns,
 Enable early corrective and preventive actions (CAPA),
 Reduce the incidence of microbial excursions,
 Strengthen the sterility assurance level of pharmaceutical cleanrooms.

CHAPTER 4: OBJECTIVES
The specific objectives of this project, aligned with both theoretical principles and industry
practices, are as follows:
1. To understand the role of recovery rate trending in evaluating sampling effectiveness
and consistency.
2. To apply control charts (X-bar and P-chart) for detecting and interpreting process
variations in EM data.
3. To interpret microorganism trending for identifying contamination sources and
establishing control strategies.
4. To assess the significance of continuous monitoring trends in maintaining real-time
GMP compliance.
5. To utilize linear regression analysis for forecasting microbial contamination risks and
supporting CAPA planning.
6. To ensure sterility assurance through rigorous environmental monitoring of Grade B, C,
and D cleanroom areas.
7. To adhere to validated protocols and SOPs to minimize contamination risks and maintain
low bioburden.
8. To analyze microbial loads on agar plates and interpret results against established alert
and action limits.
9. To prevent recurrence of microbial excursions and OOS/OOAL events by implementing
root cause analysis and CAPA.

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 CHAPTER 5: MATERIALS AND METHODS
5.1 Overview
Environmental Monitoring (EM) was conducted at Biocon Biologics Ltd., in classified
cleanroom areas designated for low bioburden sterile product manufacturing. Monitoring
covered Grade B, C, and D zones and employed multiple sampling techniques to ensure
compliance with EU GMP Annex 1 (2022), USP <1116>, and ISO 14644-1 standards.
Collected data were used for statistical trend analysis and risk-based CAPA.

5.2 Sampling Techniques and Detailed Procedures

5.2.1 Passive Air Sampling (Settle Plates)
1. Plate Preparation
o Use pre-sterilized 90 mm TSA plates containing neutralizers (e.g., lecithin, Tween
80).
o Label underside with date, time, location, and operator initials.
o Equilibrate in a low-traffic staging area for 15 minutes.
2. Placement in Cleanroom
o Don full cleanroom attire (coverall, hood, mask, gloves).
o Transport plates in a sterile container to monitoring sites.
o Open cover no more than 1 cm; place plates 1 m above floor and 1 m from walls
in Grade A/B zones, especially near filling lines or critical airflow areas.
3. Exposure
o Expose plates for 4 hours during normal operations.
o Record precise start/end times.
4. Recovery and Incubation
o Reseal plates, transport to lab in a sterile tray.
o Incubate inverted at 30–35 °C for 48–72 h (bacteria) and 20–25 °C for 5–7 days
(fungi).
5. Enumeration and Reporting
o Count colonies, distinguishing bacterial vs. fungal morphology.
o Log results (cfu/plate) in EM records; flag any breaches of alert/action limits
(e.g., ≥1 cfu for Grade A).
o Dispose as biohazard waste.

5.2.2 Active Air Sampling

1. Equipment Setup
o Calibrate impaction or slit-to-agar sampler to 100 L/min.
o Perform pre-sampling flow verification.
2. Media Loading and Labeling
o Place a sterile 90 mm SCA plate (with 1% glycerol) in sampler.
o Label date, time, volume (1,000 L), location, and operator.
3. Sampling Procedure

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 o Position sampler at operator breathing height (~1.2 m) or per SOP mapping.
o Sample 1,000 L of air (≈10 minutes).
o Record sampled volume and environmental parameters (temperature, RH,
pressure).
4. Post-Sampling Handling
o Remove and seal plate; transport immediately to lab.
5. Incubation and Analysis
o Incubate as for settle plates.
o Calculate cfu/m³:
cfu/m³=Total coloniesVolume sampled (m³) \text{cfu/m³} = \frac{\text{Total
colonies}}{\text{Volume sampled
(m³)}}cfu/m³=Volume sampled (m³)Total colonies
o Log results; compare to limits (e.g., ≤10 cfu/m³ for Grade A).

5.2.3 Surface Monitoring

a) Contact Plate Method
1. Use 55 mm SCDA or DEA contact plates; label underside with site, date, time, operator.
2. Remove cover; press agar onto surface (∼25 cm²) for 10 seconds with even pressure.
3. Replace cover, seal, and transport to lab.
b) Swab Method
1. Pre-moisten sterile swab with neutralizing solution.
2. Swab defined 25 cm² area in a ‘Z’ pattern, rotating to use all sides.
3. Place swab tip into 10 mL neutralizer broth; break off tip, cap tube.
Incubation
 Contact plates: incubate inverted at 30–35 °C (48–72 h) then 20–25 °C (5–7 days).
 Swabs: vortex tubes 30 s, plate 0.1 mL onto TSA in duplicate, incubate as above.
Enumeration
 Contact: report cfu/plate.
 Swabs: calculate cfu/cm²:
cfu/cm2=Mean colonies×Dilution factorArea swabbed (cm2) \text{cfu/cm}^2 = \frac{\
text{Mean colonies} \times \text{Dilution factor}}{\text{Area swabbed
(cm}^2)}cfu/cm2=Area swabbed (cm2)Mean colonies×Dilution factor
 Log data; flag excursions (e.g., >5 cfu/plate for Grade A surfaces).

5.3 Cleanroom Classification and Standards

5.3.1 Particle Limits
Grad ≥0.5 µm Particles/m³ (At Rest) ≥0.5 µm Particles/m³ (In Operation)
e
A ≤3,520 ≤3,520
B ≤3,520 ≤352,000
C ≤352,000 ≤3,520,000

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 D ≤3,520,000 Not specified
5.3.2 Viable Microbial Action Limits
TVC Limits
Test Type Grade Grade B Grade C Grade D
A
Settle Plates (cfu) ≥1 5 50 100
Active Air (cfu/m³) 10 100 200 200
Surface (cfu/plate) 5 25 50 100
Yeast & Mold Limits are tracked similarly using the same sampling methods and mold-
optimized incubation.

5.4 Cleanroom Operational Controls

 HEPA Filtration: ≥99.99% efficiency for ≥0.3 µm particles; validated with DOP/PAO.
 Laminar Airflow: 0.45 m/s ±20% at work height.
 Air Changes: ≥60 ACH (Grade A/B), ≥20 ACH (Grade C/D).
 Pressure Differentials: 10–15 Pa between zones; monitored via gauges.
 Environmental Conditions: 18–22 °C, 30–60% RH.
 Disinfection: Rotating sporicides (hydrogen peroxide, peracetic acid) with efficacy
validation.

5.5 Media and Materials

Sample Type Media & Additives Plate Size
Active Air SCA + 1% glycerol 90 mm
Settle Plates TSA + neutralizers 90 mm
Surface Contact SCDA or DEA (neutralizer) 55 mm
Personnel TSA or SCDA 55 mm
Swabs Sterile swabs + neutralizer broth N/A

5.6 Data Analysis and Trending

 Recovery Rate: (Positive samples/Total samples)×100%(\text{Positive samples}/\
text{Total samples}) \times 100\%(Positive samples/Total samples)×100%.
 Control Charts: X-bar and P-charts with ±2σ (alert) and ±3σ (action) limits.
 Microorganism ID: Gram stain, biochemical tests, and VITEK or molecular methods.
 Regression: Y=a+bXY = a + bXY=a+bX; interpret R² > 0.8 and p-value < 0.05 for trend
strength.

5.7 CAPA and Review Schedule

Frequenc Activity
y
Monthly Review trend charts; minor CAPA

19
 Quarterly Regression updates; SOP reviews; alert/action level validation
Yearly Comprehensive performance audit; contamination pattern analysis; training

Month Grade B Grade C Grade D

Jan 4.8 11.9 21.0
Feb 5.8 14.5 21.5
Mar 8.3 14.0 22.7
CHAPTER 6: RESULTS AND DISCUSSION – Sample data
6.1 Active Air Sampling Trends
The following table summarizes the average CFU/m³ recovered from active air sampling in
Grades B, C, and D over a three-month period:
Mont Grade B (CFU/m³) Grade C Grade D (CFU/m³)
h (CFU/m³)
Jan 4.8 11.9 21.0
Feb 5.8 14.5 21.5
Mar 8.3 14.0 22.7
Discussion:
 Grade B shows a gradual increase from 4.8 to 8.3 CFU/m³, remaining well below the
Action Limit (e.g., 100 CFU/m³) but indicating a rising trend that warrants review of
gown-change and cleaning frequency.
 Grade C peaked in February (14.5 CFU/m³) before slightly decreasing in March,
suggesting that interim corrective actions (e.g., filter integrity checks) may have
mitigated further excursions.
 Grade D remained relatively stable, around 21 CFU/m³, reflecting consistent
environmental conditions in less critical zones.
6.2 Recovery Rate Analysis
Recovery Rate (%) = (Number of positive samples ÷ Total samples) × 100
Sample Jan (%) Feb (%) Mar (%)
Type
Active Air 100 100 100
Settle Plates 90 85 95
Surface 40 35 45
Glove Tips 20 18 25
 Settle Plates: The March uptick to 95% positive plates aligns with seasonal humidity,
underscoring the need for enhanced environmental controls.
 Surface & Gloves: Low recovery rates (<50%) indicate effective disinfection and aseptic
technique; the slight March rise suggests refresher training may be beneficial.
6.3 Control Charts & Excursion Management

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  X-bar Chart (Active Air)
Mean CFU/m³ across all months: 13.1 CFU/m³
UCL (Mean + 3σ): 35 CFU/m³ | LCL: 0 CFU/m³
No Grade B or C samples approached the UCL; two Grade D samples in March (23 and
25 CFU/m³) triggered a brief investigation, which identified a transient HEPA bypass
during maintenance. Filters were recertified immediately.
 P-Chart (Surface Positives)
CL: 40% | Action Limit (3σ): ~60%
Surface positives never exceeded 50%, confirming continued process control.
6.4 Microorganism Trending
Species Jan Fe Mar
b
Bacillus spp. 11 15 7
Staphylococcus 9 7 4
epidermidis
Penicillium spp. 6 6 15
Other (e.g., Cladosporium) 4 2 4
 Observations:
o A spike in Bacillus in February prompted a review of gown sterilization
protocols.
o A notable increase in Penicillium in March suggests reviewing HVAC intake
filters or humidity controls.
6.5 Predictive Regression Model
Linear regression of mean active-air CFU over months (Jan = 1, Mar = 3):
CFU=12.8+0.5×(Month),R2=0.19\text{CFU} = 12.8 + 0.5 \times (\text{Month}),\quad R^2 =
0.19CFU=12.8+0.5×(Month),R2=0.19
 Interpretation:
The positive slope (0.5 CFU/m³ per month) indicates a slight upward trend. Although R²
is modest, it supports proactive scheduling of preventive maintenance prior to quarterly
reviews.

CHAPTER 7: PROGRESS ACHIEVED AND FURTHER

PLAN OF WORK
Progress to Date:
 Established routine EM sampling across Grades B, C, and D using standardized settle-
plate, active-air, and surface methods.
 Implemented control-chart dashboards and regression analytics for trend visualization.
 Conducted initial CAPA for identified excursions (gowning and filter maintenance).
Further Plan:
1. Real-Time Monitoring: Integrate digital particle counters in Grade C/D zones to reduce
sampling latency.

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 2. Predictive Alerts: Incorporate regression and control-chart thresholds into a live
dashboard with automated email alerts.
3. Process Optimization: Evaluate HVAC intake filtration and environmental controls to
mitigate fungal trends.
4. Training Refreshers: Conduct targeted aseptic technique workshops focused on
gowning and surface disinfection.
5. Yearly Review: Revalidate alert/action limits annually based on 12-month EM data and
update SOPs accordingly.

CHAPTER 8: CONCLUSION
This study demonstrates how a robust EM program—combining classical sampling (settle plates,
active air, surface monitoring) with statistical tools (control charts, recovery rates, regression)—
can effectively track and control microbial contamination in low bioburden cleanroom
environments. Mock data illustrate clear trends, guide CAPA, and support continuous
improvement. Future integration of real-time monitoring and predictive analytics will further
strengthen contamination prevention and ensure ongoing GMP compliance.

REFERENCES
1. Parenteral Drug Association. (2013). PDA Technical Report No. 13: Fundamentals of an
Environmental Monitoring Program. PDA Publishing.
2. United States Pharmacopeia. (2022). USP <1116> Microbiological Control and
Monitoring of Aseptic Processing Environments.
3. European Medicines Agency. (2022). EU GMP Annex 1: Manufacture of Sterile
Medicinal Products.
4. International Organization for Standardization. (2015). ISO 14644-1: Cleanrooms and
Associated Controlled Environments – Classification of Air Cleanliness.
5. Whyte, W., & Eaton, T. (2017). “Cleanroom Monitoring Methods,” Pharmaceutical
Technology Europe, 29(4), 56–63.
6. Sandle, T. (2015). Microbial Monitoring in Aseptic Processing Environments. PDA
Journal of Pharmaceutical Science and Technology.
7. Montgomery, D.C. (2009). Introduction to Statistical Quality Control, 6th ed. Wiley.
8. International Council for Harmonisation. (2005). ICH Q9: Quality Risk Management.
9. International Council for Harmonisation. (2008). ICH Q10: Pharmaceutical Quality
System.

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