EQUIPMENT VALIDATION OF LYOPHILIZER AND ...

EQUIPMENT VALIDATION OF LYOPHILIZER AND

AUTOCLAVE IN THE MANUFACTURW OF STERILE

PHARMACEUTICALS

By

M.RAJESWARI DEVI

Dissertation submitted to the

Rajiv Gandhi University of Health Sciences,

Karnataka, Bangalore

In partial fulfillment

of the requirements for the degree of

MASTER OF PHARMACY

In

QUALITY ASSURANCE

Under the guidance of

Mr. CHANDRA MOULI R

Department of Quality Assurance

Krupanidhi College of Pharmacy

Bangalore-35

MARCH -2012

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES

BANGALORE, KARNATAKA.

DECLARATION BY THE CANDIDATE

I hereby declare that this dissertation entitled “Equipment Validation of Lyophilizer

and Autoclave in the Manufacture of Sterile Pharmaceuticals” is a bonafide and

genuine research work carried out by me under the guidance of Mr. Chandramouli R,

Assistant Professor, Department of Quality Assurance, Krupanidhi College of

Pharmacy, Bangalore.

Date: M.RAJESWARI DEVI

Place: Bangalore Candidate

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES

BANGALORE, KARNATAKA.

COPY RIGHT

Declaration by the Candidate

I hereby declare that the Rajiv Gandhi University of Health Sciences, Karnataka shall

have all the rights to preserve, use and disseminate this dissertation/thesis in print or

electronic format for academic/research purpose.

Date: M.RAJESWARI

Place: Bangalore Candidate

© Rajiv Gandhi University of Health Sciences, Karnataka

INTRODUCTION

OBJECTIVE

REVIEW OF

LITERATURE

METHODOLOGY

RESULTS

DISCUSSION

CONCLUSION

SUMMARY

BIBLIOGRAPHY

Acknowledgement

Dept. of Quality Assurance, KCP, Bangalore i

ACKNOWLEDGEMENT

None of my own work or the work cited in this dissertation would be possible

without the blessings of God and my Parents it gives me an immense pleasure to

acknowledge with gratitude the help and guidance rendered to me by a host of people to

whom I owe a substantial measure in the completion of my project work.

I take this golden opportunity to express my deepest gratitude and respect

to my research guide, Mr. Chandramouli for his invaluable guidance, constant

encouragement and marvelous support throughout my project work.

I wish to sincerely thank our Dr. N. Prem Kumar, Dean and Professor Syed

Mohammed Basheeruddin Asdaq, Principal, Krupanidhi College of Pharmacy for

providing adequate facilities for the successful completion of my work.

I owe my sincere gratitude to Suresh Nagpal, Chairman and Professor Sunil

Dhamanigi, Secretary Krupanidhi institutions for the infrastructure and all the other

essential facilities and encouragement given during my project work.

I earnestly thank my industrial guide, Mr. Biju Mathews, Strides Arco Lab

Limited to motivate me for selecting a novel project and clearing all the doubts related. I

thank him for the freedom of thought, expression and his trust generously bestowed upon

me.

It gives me immense pleasure to owe my gratitude to Mr. Arun M.P and Mr.

Uma Mahesh Babu for his support and encouragement in industry.

Acknowledgement

Dept. of Quality Assurance, KCP, Bangalore ii

My heartfelt thanks to all the teaching staff of Krupanidhi College of Pharmacy,

especially Mrs. Naira Nayeem, Mr. S. Srinivasan and Mr. Harish kumar D. R. for

their guidance and help.

I am pleased to thank our librarian, Mr. Vasanth for their availability, kindness

and cooperation.

I wish to thank the all the non-teaching staff, especially Ravi and Bhaskar for

their support through my work.

I express my deepest gratitude to all my classmates Abhinandana, Anupam,

Abhijit, Mehul, Hardik, Purvish, for their valuable and timely help and cooperation

during the project work. I also wish to thank my other batch mates Spoorthi, Anuradha

for their help.

I can hardly find any words to acknowledge the love and support of my beloved

Parents and my younger brother Adhikhya for their prayers and encouragement at each

& every front of my life to transfer my dreams in to reality. They scold me, raised me,

supported me, taught me, loved me and sacrifice a lot for me.

Last but not least, I want to thank all of my well-wishers who helped me directly

or indirectly, again who have been and continue to be a constant source of inspiration

and insight.

Date:

Place: Bangalore. M. Rajeswari Devi

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List of Abbreviations used

Dept. of Quality Assurance, KCP, Bangalore iii

LIST OF ABBREVATIONS USED

Approx. : Approximately

avg. : Average

ºC : Celsius

cGMP : Current Good Manufacturing Practice

cm : Centi Meter

DQ : Design Qualification

e.g. : For Example

eq. : Equivalent

etc. : Et cetera

EU : European Union

FDA : Food and Drug Administration

FAT : Factory acceptance test

gm : Gram

GMP : Good Manufacturing Practice

ICH : International Conference on Harmonisation

i.e. : that is

IQ : Installation Qualification

kg : Kilo Gram

LOD : Loss on Drying

LVP : Large Volume Parenteral

Max. : Maximum

mg : Milli Gram

min. : Minute

ml : Milli Leter

mm : Milli Meter

NLT : Not Less Than

NMT : Not More Than

No. : Number

OQ : Operation Qualification

PQ : Performance Qualification

List of Abbreviations used

Dept. of Quality Assurance, KCP, Bangalore iv

QA : Quality Assurance

QC : Quality Control

QRM : Quality risk management

q.s. : Quantity Sufficient

qty. : Quantity

RQ : Requalification

Sl. : Serial

SOP : Standard Operating Procedure

Spec. : Specification

std. : Standard

UK : United Kingdom

USFDA : United States Food and Drug Administration

USP : United States Pharmacopoeia

URS : User requirement specification

v/s : Versus

WHO : World Health Organization

wt. : Weight

& : And

µg : Micro Gram

# : Number

% : Percentage

Abstract

Dept. of Quality Assurance, KCP, Bangalore v

ABSTRACT

Assurance of quality, reduce rework, robustness and batch-to-batch consistency are most

important factor to maintain profitability for pharmaceutical industry and can be achieve

only by incorporation of equipment validation. In the presented work, equipment

validation of lyophilizer and autoclave is done to reduce the stability problem for

sensitive products and achieve the sterility of the final product. The presented

investigation was to carry out the validation of 3 consecutive runs of having same process

parameter. All the prerequisites have been checked and precalibration and post

calibration of data loggers and temperature sensors have been done and found complying

with the acceptance criteria. All results of validation like vacuum rate (12-15 mins),

Maximum vacuum capacity (0.0080 -0.0093 mbar), vacuum leakage test (0.0000222-

0.000029), heating rate (1.05- 1.380C) and cooling rate (1.52-2.29

0C), temperature

uniformity(0.7-1.30C), autoclave vacuum leak was (0.00-0.004) found well within the

acceptance criteria. Based on the results of the validation for 3 runs, it was concluded that

by following the process parameters consistently producing the stable product meeting its

pre-determined specifications and quality attributes.

List of contents

Dept. of Quality Assurance, KCP, Bangalore vi

LIST OF CONTENTS

Contents Page No.

1. INTRODUCTION 1-18

1.1. Definitions of Validation 1

1.2. Principle Elements of Validation 2-3

1.2.1. Documented Evidence 2

1.2.2. High Degree of Assurance 2

1.2.3. Specific Process 2

1.2.4. Consistency 2

1.2.5. Predetermined Specifications 3

1.3 Reasons for Validation 3-4

1.3 1 Compliance 3

1.3.2 Quality Assurance 3

1.3.3 Economics 3

1.3.4 Regulatory Requirements 4

1.4 Benefits of Validation 4-6

1.4.1 Quality 4

1.4.2 Understanding Equipment, System and Process 4

1.4.3 Regulatory Benefits 4

1.4.4 Cost Reduction 5

1.4.5 Time Saving 5

1.5 Equipment validation 5-14

1.5.1 User required specifications 7

1.5.2 Design qualification 7

1.5.3 Factory acceptance test 8

1.5.4 Quality risk management 8

1.5.5 Installation qualification 8

1.5.6 Operation qualification 9

1.5.7 Performance qualification 11

1.5.8 Requalification 12

List of contents

Dept. of Quality Assurance, KCP, Bangalore vii

1.6 Lyophilizer validation 14-17

1.6.1 Maximum vacuum capacity and vacuum leakage rate test 15

1.6.2 Isolator valve integrity test 16

1.6.3 Shelf heating and cooling rate 16

1.6.4 Sterilization in place test 16

1.6.5 Condenser capacity test 17

1.6.6 Placebo test 17

1.7 Equipment validation of autoclave 17-18

1.8.1 Vacuum leak test 18

1.8.2 Air removal test 18

1.8.3 Heat distribution and penetration studies 18

2 OBJECTIVE 19-21

3 REVIEW OF LITERATURE 22-36

3.4 Literature Review on Validation of equipment 22-23

3.5 Literature Review on Validation of lyophilizer 23-31

3.6 Literature Review on Validation of autoclave 21-36

4 METHODOLOGY 37-68

4.1 Prerequisites for equipment validation 37

4.1.1 Validation Approach 37

4.1.2 Documents Required 37

4.1.3 Instructions followed during validation of equipment 38

4.1.4 Requirement during equipment validation of lyophilizer 38

and autoclave

4.2 Operation qualification of lyophilizer: 39-48

4.2.1 Verification of key functionality, safety features and 39

Emergency Stop features

4.2.2 Verification of operator interface, display, automation 39

and control requirements.

4.2.3 Software operation qualification 43

List of contents

Dept. of Quality Assurance, KCP, Bangalore viii

4.2.4 Alarm categorization and verification 45

4.3 Performance qualification of lyophilizer 49-57

4.3.1 Vacuum rate, maximum vacuum capacity and vacuum 49

leakage rate test

4.3.2 Isolator Valve Integrity Tests 50

4.3.3 Shelf Cooling and Heating Rate Test 50

4.3.4 Shelf Temperature Uniformity Test 51

4.3.5 Sterilization in place test 53

4.3.6 Condenser capacity test 54

4.3.7 Cleaning In Place (CIP) Test 55

4.3.8 Placebo test 56

4.4 Operation qualification of autoclave 58-63

4.4.1 Verification of key functionality 58

4.4.2 Verification of safety feature 59

4.4.3 Verification of Alarms And Warnings 60

4.5 Performance qualification of lyophilizer 64-68

4.5.1 Vacuum leak test for sterilizer chamber 64


4.5.3 Heat distribution and penetration studies 65

5 RESULTS 69-95

5.1 Prerequisites for equipment Validation 69

5.2 Operation qualification lyophilizer and autoclave 69

5.3 performance qualification of lyophilizer 70

List of contents

Dept. of Quality Assurance, KCP, Bangalore ix

5.3.1 Vacuum rate, maximum vacuum capacity and 70

Vacuum leakage test

5.3.2 Isolated valve integrity test 70

5.3.3 Heating and cooling 71

5.3.4 Shelf temperature uniformity test 72

5.3.5 Cleaning In Place 74

5.3.6 Sterilization in place 74

5.3.7 Condenser Capacity 73

5.3.8 Placebo Test 74

5.4 Performance qualification of autoclave 75-76


5.4.2 Vacuum leak test 75

5.4.3 Heat distribution and penetration study 76

6 DISCUSSION 96-100

6.1 Prerequisites for Process Validation 96

6.1 Operation qualification of lyophilizer and autoclave 96

6.2 Performance qualification of lyophilizer and autoclave 97

7 CONCLUSION 101-102

8 SUMMARY 103-107

9 BIBLIOGRAPHY 108- 111

List of tables

Dept. of Quality Assurance, KCP, Bangalore x

LIST OF TABLES

Sl. No. Title of Table Page

No.

4.1 Equipment details of Lyophilizer and Autoclave 37

4.2

Verification list of key functionality, safety and emergency stop

features of lyophilizes

39

4.3

Verification list of operator interface, display, automation and control

requirements of lyophilizer.

39

4.4

Verification of software operation qualification

43

4.5 Alarm categorization and verification list 45

4.6 Placebo test steps 57

4.7 verification list of key functionality of autoclave 58

4.8 Verification list of safety feature 59

4.9 Verification list of alarms and warnings of autoclave. 61

4.10 Verification list of emergency stop features 63

5.1 OQ results of lyophilizer 69

5.2 OQ results of autoclave 69

List of tables

Dept. of Quality Assurance, KCP, Bangalore xi

Sl. No. Title of Table Page

No.

5.3 Results of Vacuum rate and leakage, maximum vacuum capacity test 70

5.4 Results of isolated valve integrity test. 70

5.5 Results of heating and cooling rates.

71

5.6 Results of shelf temperature uniformity test. 72

5.7 Results of clean in place. 74

5.8 Results of condenser capacity

73

5.9 . Results of sterilization in place. 74

5.10 Results of lyophilization process test. 75

5.11 Results of data logger and RTD sensors calibration. 75

5.12 Results of vacuum leak test. 75

5.13 Results of air removal tests. 76

5.14 Results of heat distribution studies 76

5.12 Results of heat penetration studies with BI challenge. 77

List of figures

Dept. of Quality Assurance, KCP, Bangalore xii

LIST OF FIGURES

S. No. List of Figures Page No.

1.1 Typical equipment qualification lifecycle 14

2.1 Best method to plan for equipment validation 22

4.1 Location of probes in the lyophilizer for uniformity test 52

4.2 Temperature Sensor location Diagram for SIP Test 54

4.3 load pattern diagram with probe location 66

S. No. List of reports Page No.

5.1 Temperature Sensors precalibration report of Autoclave 77

5.2 Data Loggers Precalibration report of Autoclave 78

5.3 Temperature Sensor precalibration report of Lyophilizer 79

5.4 Datalogger precalibration report of Lyophilizer 81

5.5

Maximum vacuum capacity, vacuum leak rate and Isolation valve

integrity report

82

5.6 Heating and Cooling rate report

83

5.7 Shelf temperature uniformity test report

85

5.8 Sterilization in Place test report

88

5.9 Vacuum leak test report

92

5.10 Air removal test report

93

5.11 Heat distribution and penetration study reports

94

List of figures

Dept. of Quality Assurance, KCP, Bangalore xiii

S.No. List of graphs

Page no.

5.1 Shelf temperature uniformity test graph

88

5.2 Sterilization in Place test graph

88

5.3 Placebo test report

91

Chapter-1 Introduction

Dept. of Quality Assurance, KCP, Bangalore 1

1. INTRODUCTION

The origins of validation in the global healthcare industry can be traced to terminal

sterilization process failures in the early 1970s. Individuals in the United States point

to the LVP sterilization problems of Abbott and Baxter, while those in the UK cite the

Davenport incident. Each incident was a result of a non-obvious fault coupled with

the inherent limitations of the end-product sterility test. As a consequence of these

events, non-sterile materials were released to the market, deaths occurred, and

regulatory investigations were launched. The outcome of this was the introduction by

the regulators of the concept of ―Validation. It is necessary, before approval of a new

drug, that an accurate and reliable assessment for its effectiveness and safety for the

intended indication and target patient population is demonstrated. Pharmaceutical

validation which includes assay validation, cleaning validation, equipment validation

as well as the overall process validation is crucial in stability analysis, animal studies

and early phases of clinical development such as bioavailability/bioequivalence

studies. 1

1.1 Definitions of Validation:

Validation is defined as follows by different agencies:

Food and Drug Administration (FDA):

―Establishing documentation evidence, which provides a high degree of assurance

that specific process, will consistently produce a product meeting its predetermined

specification and quality attributes‖.

World Health Organization (WHO):

―Action of providing that any procedure, process, equipment, material, activity, or

system actually leads to the expected results‖.

http://www.pharmainfo.net/cleaning-validation-articles



European Union (EU):

―Action of providing in accordance with the principles of good manufacturing

practice, that any procedure, process, equipment material, activity or system actually

lead to the expected results‖.

In brief validation is a key process for effective Quality Assurance.2

1.2 Principle Elements of Validation:

1.2.1 Documented Evidence:

Validation requires thorough documentation. Everything that is not documented is

considered incomplete.

1.2.2 High Degree of Assurance:

The assumption is that a large software package as used in complex computerized

systems is rarely free of errors. Frequently, there is a perception that validation means

error-free. This assumption is wrong. During the validation process, everything

realistically possible should be done to reduce errors to a high degree.

1.2.3 Specific Process:

The overall validation of software is process related, not product related. For example,

the development and testing activities performed prior to releasing the software for

manufacture are validated once for a series of products characterized by the serial

number. Some subparts of validation, such as the qualifications (installation,

operation, and performance) are product-specific and have to be done for each system.

1.2.4 Consistency:

Validation is not a one-time event. The performance of the equipment has to be

controlled during the entire life of the product.



1.2.5 Predetermined Specifications:

Validation activities start with the definition of specifications. The performance of the

equipment is then verified against these specifications. Acceptance criteria must be

defined prior to testing.2

1.3 Reasons for Validation:

To show that a process can ―consistently produce what it purports to do‖ validation is

vital requirements for any pharmaceutical industry. The four basic reasons for

validation are compliance, quality assurance, economics and regulatory requirements.

1.3.1 Compliance:

Pharmaceutical manufacturers are directed by GMP and CGMP guidelines, which

they are bound to follow. Validation is the medium with which compliance to these

guidelines is attained and presented in a systemic way. Validation requirements in

industry is supported by EC, GMP, WHO and further supported by FDA Guidelines

on General Principles of Process Validation.

1.3.2 Quality Assurance:

The second and most compelling reason for validation should be to guarantee, as far

as possible. That all processes and equipment in the pharmaceutical manufacturing

process are being used in a way that will ensure the safety, integrity, purity, quality

and strength of a product for use by the general public. Validation therefore

challenges the adequacy and reliability of a system or process to meet predetermined

criteria on a consistent basis from batch to batch.

1.3.3 Economics:

Aside from the above reasons, validation is astute business practice. It prompts

appraisal and reappraisal of every activity involved in a process and, almost



inevitably, improvements are made. As a result of these validation activities, indirect

economic benefits may arise.3

1.3.4 Regulatory Requirements:

Fourth, and certainly foremost, among the reasons for validation is that it is a

regulatory requirement for virtually every process in the global health care industry-

for pharmaceuticals, biologics, and medical devices. Regulatory agencies across the

world expect firms to validate their processes. The continuing trend toward

harmonization of requirements will eventually result in a common level of

expectation for validations worldwide. Utility for validation beyond compliance is

certainly available. The emphasis placed on compliance as a rationale has reduced the

visibility of the other advantages a firm gleans from having a sound validation

program.

1.4 Benefits of Validation:

1.4.1 Quality:

Customer – patient satisfaction

It has been built into the product

1.4.2 Understanding Equipment, System and Process:

Process improvement, technology transfer, rapid failure investigations

Improve employee awareness and increased outputs

Easier maintenance of the equipment

Fewer complaints about process related failures

1.4.3 Regulatory Benefits:

Successful inspections

Approved products

Ability to export



1.4.4 Cost Reduction:

Fewer rejects and reworks and avoidance of capital expenditures

Increased efficiency, shortening lead time resulting in lower inventories

Longer equipment life by operating the equipment as per manufacturer‘s

specifications and the establishing of cost effective preventive maintenance

schedules

Reduction in utility costs

1.4.5 Time Saving:

Possible reduced testing of raw materials bulk formulations and finished products.

Reduced testing in process and finished goods

More rapid and accurate investigations into process deviations

More rapid and reliable startup of new equipment

More rapid automation3

In the intervening years, there has been repeated affirmation of those expectations at

other firms, large and small. Regrettably, there has been little quantification of these

benefits. The predominance of compliance-based validation initiatives generally

restricts objective discussion of cost implications for any initiative. But once a

process/product is properly validated, it would seem that reduced sample size and

intervals could be easily justified, and thus provide a measurable return on the

validation effort. Aside from utility systems, this is hardly ever realized and represents

one of the major failings relative to the implementation of validation in our industry.

Flawless and predictable qualities are cornerstones of successful production of

medicaments. To streamline processes, minimize material loss and stay within legal

requirements, advanced quality assurance tools are needed in order to guarantee



customer satisfaction and to produce under optimum conditions with maximum

efficiency.

Assurance of product quality is derived from careful and systemic attention to a

number of important factors like selection of quality components and materials,

adequate product and process design, and statistical control of the process through in

process and end-product testing. Thus it is through careful design qualification and

validation of both the process and its control systems that a high degree of confidence

can be established that all succession of batches that meet specifications will be

acceptable.

1.5 Equipment validation:

Validation is a quantitative approach which is needed to prove quality, functionality

and performance of a pharmaceutical/biotechnological manufacturing process. This

proved approach will be applied to individual pieces of equipment as well as the

manufacturing process. Guidelines for equipment validation are set by the multiple

regulatory agencies like USFDA but specifications of validation are determined by

the pharmaceutical/biotechnological company.

Validation of equipment‘s is also known as qualification. The importance of the

qualification process of technical systems in the pharmaceutical industries has been

steadily increasing over the last 10 years. Bringing any new pharmaceutical to market

requires coordinated efforts in product design, formulation development and process

engineering throughout the development phase. Moving that new product to

commercial-scale manufacturing requires careful equipment validation. New methods

and tools must be implemented to reach the goals of qualifying a technical system

while minimizing effort. The main aspect is the trend for quality assurance

departments to evolve from being mere controllers of product quality to delivering



tools and methods to other departments, thus helping them to design a better

production process. The goal is to improve overall production reliability and

availability4.

Equipment validation involves the following steps:

1.5.1 User requirement specification:

The purpose of User Requirement Specification (URS) is to provide appropriate

design and performance requirements for procurement of equipment/instrument/

system including major add-on component or major modification/expansion of area so

as to meet in-house requirements as well as compliance with current Good

Manufacturing Practices. The requirement for preparation of URS shall be evaluated

at initial stage during procurement phase. The preparation of URS shall be applicable

to the items intended for use as part of pharmaceutical/nutraceutical manufacturing

and control and which impacts GMP. Wherever, an integrated line is to be procured

from same vendor, a single URS shall be considered acceptable. Wherever,

manufacturing line is to be integrated with equipment from other vendors, the same

shall be described in URS, with integration and scope requirements.

1.5.2 Design qualification:

The purpose of Design Qualification (DQ) is to qualify hardware, functional and

performance requirements for procurement of equipment/instrument/system so as

to meet in-house requirements, regulatory requirements as well as compliance with

current Good Manufacturing Practices.

During Design Qualification, specifications or technical information provided by

vendor/supplier/manufacturer shall be studied against company URS and the non-

conformance if any, shall be addressed appropriately. At a minimum, design



specifications submitted by the prospective vendor shall be reviewed and approved

by company prior to releasing purchase order. The design specification shall be

reviewed and approved by the same authorities that reviewed and approved URS.

1.5.3 Factory Acceptance Test:

Factory Acceptance Test (FAT) is required as determined during Design

Qualification, the same shall be done in accordance with a pre-approved protocol.

FAT shall provide an opportunity to company, to identify those discrepancies (if

any) at Vendor‘s site that can be resolved more effectively prior to its dispatch to

user‘s facility. FAT shall be executed at Vendor‘s site, and FAT report shall be

made available along with qualification documents of respective equipment.

1.5.4. Quality risk management:

During qualification phase, all ‗Direct impacting‘ equipment/systems shall undergo

the exercise of QRM. The QRM shall be performed in accordance with the SOP on

QRM as recommended through various regulatory requirements like ICH Q9 etc.

1.5.5 Installation qualification:

The purpose of Installation Qualification (IQ) is to demonstrate that the equipment

is installed and meets approved design specification, supplier‘s recommendations,

and drawings and that they are correctly interfaced with factory systems.

Prior to beginning IQ exercise, all requirements, as specified in URS and agreed

upon by Vendor/supplier/manufacturer shall be verified including availability

documentation and FAT report (if applicable). Installation of the

equipment/instrument/system shall be done as per recommendation of

vendor/supplier/manufacturer.



IQ protocol shall be prepared by including all design specifications as agreed

through URS, Vendors technical information and DQ. All major components of

equipment‘s (as specified by vendor) shall be verified for calibration certificates by

IQ protocol shall be executed on installed item. At a minimum, following criteria

are verified during IQ.

IQ prerequisites

Installation checklist

List of major components for verification

MoC verification

Verification of supporting utilities

Identification of SOPs (e.g. Operation and cleaning, calibration and

preventive maintenance SOPs)

Instruments to be used for calibration during OQ, their calibration status

Instruments to be calibrated during OQ, their criticality,

All the risk mitigation actions (those are supposed to be taken during IQ) shall be

executed during IQ and documented/closed accordingly in IQ report.

1.5.6 Operation qualification:

The purpose of Operational Qualification (OQ) is to demonstrate that

equipment/system/instrument is operational within its predetermined operational

range and tolerances, and meets functional requirements.

Execution of OQ necessitates testing of those parameters that regulate/control the

process or product quality. Proper operation of controllers, indicators, recorders,

alarms, and interlocks, shall be verified and documented during OQ.



OQ phase shall include calibration/testing of instruments those are identified

during. During OQ it shall also be verified that the system functions in a

predefined sequence or operating steps, all interlocking, alarms and safety

functions are functioning as specified.

A Standard Operating Procedure (SOP) shall be drafted (and/or approved) prior to

Operational Qualification and shall be verified for correctness and understanding

during OQ. OQ test criteria shall include testing equipment without any load to

verify any engineering defects (abnormal sound, wear / tear, vibrations, etc) and is

termed as ―No Load Trials.‖

OQ protocol shall be prepared by including all functional requirements of URS and

functional specification/operating manual, etc.

OQ protocol shall be executed after IQ is completed, and as recommended through

IQ report. At minimum following criteria are verified during OQ.

OQ prerequisites

Standard test instrument/devices details

Calibration details if instruments/devices on the equipment

No Load trials & ease of operation

Functionality test (operating steps, recommended speed

range, interlocking, alarms, etc)

Challenge tests at extreme operating range as recommended

through QRM

Safety checks

SOP verification (At least draft SOPs shall be made

available for verification)



OQ report shall include a statement recommending whether or not to proceed to

PQ based on the results of operational tests performed and future work (if

required).

All risk mitigation actions (those are supposed to be taken during OQ) shall be

executed during OQ and documented/closed accordingly in OQ report

1.5.7 Performance qualification:

The purpose of Performance Qualification (PQ) is to demonstrate that

equipment/system/instrument is performing as per its predetermined performance

criteria and yields an output meeting its quality and performance attributes as

specified in URS.

Performance Qualification is an amalgamation of evaluation of approved operating

procedures, personnel, systems, and materials in an integrated approach so as to

verify that pharmaceutical grade utility, environment, equipment, support system,

etc., produces the required output. It shall be performed through multiple sets of

tests to demonstrate its consistency and reproducibility.

PQ execution involves handling of dummy material or product for conducting

tests. This methodology shall be described in respective PQ protocol. PQ report

shall also document the destruction of material used during PQ execution, which

may include actives, excipients, components, etc.

Any discrepancy during PQ shall be evaluated for its impact on Quality of product.

In such a case, in addition to documenting the discrepancy in qualification report, a

deviation report shall be initiated in accordance with the Quality Management



System. Discrepancies having no impact on quality can be documented, resolved

and closed through qualification report itself.

PQ protocol shall include the following but not limited to:

PQ prerequisites

Experimental Plan & Procedure

Sampling Plan (if any)

Acceptance criteria

Observations

Conclusions /Certifications

PQ report shall include a statement recommending whether the

equipment/instrument/system is acceptable for its intended use.

1.5.8 Requalification and revalidation of Equipment/Instrument/ System:

An Equipment Requalification shall be done based in the following situations

Modification of equipment/instrument/system

Modifications to, or relocation of equipment shall follow satisfactory review and

authorization of the documented change proposal through change control

procedure. The qualified movable equipment can be moved from one place to

another in the same facility, for such movable equipment‘s no qualification is

required after the movement.

The review of changes shall include impact assessment because of

change/modification and consideration of qualification of equipment. In such cases

IQ and/or OQ and/or PQ (or process validation) shall be performed before



releasing the equipment for routine use. The scope of qualification shall be

mentioned in the applicable change control.

Changes of equipment which involve replacement of component on a "like for

like" basis would not require a re-validation. For all other replacements, an

evaluation shall be made (in change control) to determine requirement of

validation.

Periodic Re-Qualification and annual validation plan

The purpose of Requalification or program is to demonstrate that the

equipment/system/instrument is maintained consistently over a period of time and

can be utilized for its intended purpose. The objective of RQ is also to evaluate that

support systems are working as intended and eventually complements in

maintaining facility in validated state. RQ system through this process shall

identify any deficiency, and suggest appropriate remedial or corrective action.



Figure 1.1 Typical equipment qualification lifecycle

1.6 Validation of lyophilizer:

Lyophilization or freeze drying is defined as a process in which water is removed

from a product after it is frozen and placed under a vacuum, allowing the ice to

change directly from solid to vapor without passing through a liquid phase. The

process consists of three separate, unique, and interdependent processes; freezing,

primary drying (sublimation), and secondary drying (desorption).

Lyophilization, or freeze-drying, is becoming more important in developing and

manufacturing unstable, sensitive pharmaceuticals. However, lyophilization poses

special challenges to achieving and ensuring batch uniformity. First, the product itself

is sensitive to the presence of water and to process conditions. Then, the process

User Requirement Specification (Including functional requirements if applicable)

Design Qualification &

Impact assessment

Installation Qualification

Risk Management

Operational Qualification Standard Operating

Procedure

Performance Qualification Process Validation Cleaning Validation

Change Management

Routine operation

Periodic Revalidation / Requalification

FAT execution



involves manipulating subambient temperature and sub-atmospheric pressure

conditions. Success requires close control of process parameters such as temperature

and time, and equipment operating performance. It also depends on understanding

factors unique to each lyophilizer — even a vial‘s position on the freeze dryer tray

can have a major impact on product quality and batch uniformity. Controlling critical

processing parameters is imperative to ensuring that batches are uniform and the

process reproducible from batch to batch. Completing a comprehensive Installation

Qualification (IQ), Operational Qualification (OQ) and Performance Qualification

(PQ) assures that the equipment can produce material of sufficient quality. The

performance capabilities of each individual freeze dryer will influence process

reproducibility, batch uniformity, and consistency of the finished product. Qualifying

equipment performance is, thus, an integral part of assuring reproducibility,

consistency and uniformity. Complete and comprehensive Equipment Qualification

studies are necessary, including Installation and Operational Qualification, which

ensures that the equipment has been properly installed, adequate utilities are available,

and the lyophilizer is functioning properly.

As demand for parenterals and biologically-derived products expands, companies are

widely using Lyophilization to protect and stabilize their sensitive pharmaceutical and

biologic products. Freeze-dryer performance is playing an important role in achieving

the required activity, stability, quality, and shelf-life for the finished products5.

Performance qualification of lyophilizer includes

1.6.1 Maximum Vacuum Capacity and Vacuum Leakage Rate Test:

Objective

This test is to verify the final pressure that system can reach, and to verify the leak

rate of the system is as per design specification.



Scope

The test is to be performed with an empty and dry chamber to minimize evaporative

pressure gains.

1.6.2 Isolator Valve Integrity Test:

Objective

To checks the integrity of the isolation valve between the ice condenser and the

drying chamber.

Scope

This test is applicable to each Lyophilizer and to be performed successfully minimum

three consecutive times.

1.6.3 Shelf Heating and Cooling Rate Test:

Objective

This test is to ensure the proper functioning of heater and compressor with respect

to their capacity and user requirement.

Scope

This test is applicable to each Lyophilizer and to be performed successfully

minimum three consecutive times. This test is applicable to Lyophilizer with an

empty chamber.

1.6.4 SIP (Sterilization in Place) Test:

Objective

The test is to perform to evaluate the efficiency of the Sterilization cycle/SIP cycle.

Scope

This test is applicable to test Lyophilizer with an empty chamber.



1.6.5 Condenser capacity test:

Objective

The test function will verify that the ice capacity of the condenser operates as

required and to record functional drying operations and vacuum drying conditions.

Scope

This test is applicable to each Lyophilizer and to be performed once successfully.

16.6 Placebo test:

Objective

The test is to ensure the finale moisture content and physical appearance of the cake

is as per the requirements.

Scope

This test is applicable to each Lyophilizer and to be performed one successful run.

1.7 Equipment validation of autoclave:

It was found that validation of steam sterilization in autoclaves is the most-studied

validation problem faced by the pharmaceutical industry. There was a failure in

sterilizing certain LVP solutions that resulted in several patients‘ deaths which in turn

led for the call of validation of sterilization process by the U.S. FDA. So, the

autoclave Validation / Qualification have become mandatory for all machines used for

biological sterilization, in the biomedical and pharmaceutical industries. Autoclaving

is the fastest and most reliable, so there is always need in scrutinizing autoclave

validation / Qualification activities.

http://en.wikipedia.org/wiki/Sterilization_(microbiology)



1.7 Performance qualification of autoclave:

1.7.1 Vacuum leak test:

Objective

This test is performed to ensure that the sterilizer complies to leak test requirements

indicating that the integrity of the objects being sterilized is maintained during

application of vacuum cycle after the sterilization cycle.

Scope

This test is applicable for all sterilizers where vacuum cycle is applied after

sterilization

1.7.2 Air removal test (Bowie-dick test):

Objective

The air removal test is performed to ensure that the air is removed completely after

the vacuum is applied to the sterilizer

Scope

The test is applicable to all sterilization cycle where vacuum is applied prior to

sterilization.

1.7.3 Heat distribution and heat penetration studies with loaded chamber.

Objective

This test ensures that there is uniform distribution of heat in the chamber and ensure

that the sterilizer meets the temperature profile requirements, sterility assurance

requirements during the sterilization as per various load patterns.

Chapter 2 Objective


2. OBJECTIVE

The basic purpose of carrying out a validation of the manufacturing process is to

establish documented evidence that provides a high degree of assurance that the

process consistently produces a product meeting its predetermined specifications and

quality attributes. Validation is the integral part of GMP and now-a-days, it is

mandatory to incorporate validation activity in the premises for all pharmaceutical

industries. It has been found that by performing the validation activity one can omit

serious manufacturing problems. Main objective of this study to conduct validation on

lyophilizers and autoclaves in the manufacture of sterile pharmaceuticals

Figure 2.1 Best method to plan for equipment validation

It shows a pyramid, which is the best way in which to plan a qualification/validation

project. Investing more time in the first phases will save time and money in later and

critical phases. If inadequate investment is made during the start-up of a project, the

later phases of installation qualification (IQ), operational qualification (OQ), and

Preliminaries, including design qualification (D.Q)

Maintenance change control requalification

Operation qualification

Performance qualifications

Installation qualification

Chapter 2 Objective


performance qualification (PQ) will necessarily require an inordinate amount of time

and money.

As demand for parenteral and biologically-derived products expands, companies are

increasingly using Lyophilization and autoclaves to protect and stabilize their

sensitive pharmaceutical and biologic products. It was found that validation of steam

sterilization in autoclaves is the most-studied validation problem faced by the

pharmaceutical industry.

Validation of lyophilizers and autoclaves is done by:

Operational qualification - (OQ) for Autoclaves and lyophilizer:

Process control limits (time, temperature, pressure, line speed, setup conditions, etc.)

Checking alarms.

Process operating procedures.

Material handling requirement.

Operational qualification - (OQ) for Lyophilizer

Control functions such as shelf temperature control, pressure control, process

monitoring, sequence functions etc.

Checking alarm.

Material handling requirement.

Process operating procedures

Performance qualification - (PQ) of autoclave:

Following are the tests performed in performance qualification:

Vacuum leak test of sterilizing chamber.

Air removal test.

Heat distribution study and heat penetration study with loaded chamber.

Chapter 2 Objective


Performance qualification of lyophilizer:

Following are the tests performed in performance qualification:

Vacuum rate, maximum vacuum capacity and vacuum leakage rate test

Isolator valve integrity test

Shelf Cooling and Heating Rate Test

Shelf Temperature Uniformity Test

Cleaning In Place Test (CIP)

Condenser capacity test

Sterility in place test (SIP)

Placebo test

Chapter-3 Review of literature


3. REVIEW OF LITERATURE

A Literature survey on equipment validation of lyophilizer and autoclave in manufacture

of sterile pharmaceutical from various books, journals and published works gives much

information related to the objective of study. Some of the most relevant ones are

discussed below:

Literature review on validation of equipment:

The main aim in qualifying laboratory equipment is to ensure the validity of the data .It

has been reported that the current equipment validation programs and procedures used

within the pharmaceutical industry are mainly based on regulatory requirements,

voluntary standards, vendor practices, and industry practices .This in turn leads to

considerable variation in the way pharmaceutical companies approach the qualification of

laboratory equipment and how they interpret the unclear requirements. The authors

summarized that Pharmaceutical research and manufacturing association of America

(PhRMA) Workshop was an Acceptable Analytical Practices for the topic “Qualification

of Laboratory Equipment”6.

Equipment qualification protocol is one of the greatest value added services which

provide the verification of accurate system and/or equipment drawings. It has been

reported that most often contractors and vendors supply „as-builts‟ as part of a job or

project. Changes were also made even after the as-builts were delivered or

errors/omissions occurred in the original drawings unfortunately. In order to verify the

accuracy of the as-built drawing a test procedure from which could be included in the

installation qualification has been described by them7.



It is essential that the performance of the calibration is tested with a set of samples which

are typical and independent. They have calibrated on lyophilizers and they have preferred

a name called "Validation set". Researchers have developed a multivariate calibration and

have also said that, if they are satisfied with the predictions on these samples then they

will claim that they have "Validated" the calibration, which suggests that they expect it to

continue to give useful results in the future8.

According to the literature survey a significant changes were made in production system

i.e. manufacturing execution system (MES) on a production floor. It has been said that

there is a need to conduct high confidence testing the correction and performance of the

correctness and performance of the MES. It was found that a new approach was devised

to provide a highly realistic testing environment by simulating FAB wide equipments

interacting with the MES system via the equipment integration (EI) components just as it

would in actual production. This in turn led to validate the target production system with

high confidence without the need of actual equipment time9.

Equipment validation of lyophilizer:

The review deals with validation of the dynamic parameters estimation (DPE) method,

which is a non-invasive in-line monitoring technique for the freeze-drying process of

pharmaceutical products which only requires pressure measurement; simulations and

experimental data of pharmaceutical solutions in vials, are used to validate lyophilizer.

The above method used has considered as an improvement of analogous techniques, such

as the manometric temperature measurement (MTM) method, which is also based on the

pressure rise analysis concept. The approach proposed by them was able to estimate not

only the temperature profile inside the frozen product at any time during the pressure rise



test, but also the position of the moving interface of Sublimation, the external heat

transfer coefficient, and the effective diffusivity coefficient in the dried product, which

are necessary for a predictive model-based control algorithm. This in turn got good

estimations of the product temperature and of the transport parameters, as well as of the

position of the interface of sublimation, which has directly related to the state of

progression of the primary drying phase10

.

Lyophilizer refrigeration system's process capabilities and performance are critical to a

successful commercial Lyophilization operation because lyophilization dries a product

from the frozen state under temperature controlled conditions. Recent trends in

pharmaceutical product manufacturing and their impact on the evolution of refrigeration

technology in lyophilization were outlined. It has also explained about the advantages

and disadvantages of choosing a refrigeration system that uses liquid nitrogen instead of

mechanical compressors, and its affect on the overall operation of lyophilization process.

It was concluded that cryogenic nitrogen refrigeration has gained favor over mechanical

refrigeration because of its inherent reliability and responsiveness to meet stringent and

flexible cooling profiles while achieving ultra-low shelf and condenser temperatures11

.

ISO 9000 and FDA requirements mandate that any measurement be traceable to national

standards. The researchers have said that to ensure traceability, performance, and

reliability it is necessary to calibration the equipment. The review has explained the

proper method for the in situ, on-site, or off-site calibration of lyophilizer pressure gauges

and has also offered some important guidelines to ensure the accuracy of a capacitance

manometer. This calibration of lyophilizer pressure gauge resulted in assuring quality and

cost effective production12

.

http://www.fda.gov/



The success for the transfer and validation of a lyophilization process requires a thorough

characterization of the process and the equipment. The article has discussed the various

elements such as the development and characterization of the lyophilization process,

performance of the equipment, identification of the critical process parameters, etc.,

which can potentially impact the successful transfer and validation efforts13

.

Researchers have described about the commissioning and qualification of a technically

upgraded lyophilizer after the performance of defined changes. This in turn gives a

practical report of the qualification activities (IQ/OQ/PQ) performed on the upgraded

equipment. They have also proved from experience of several recent FDA-inspections,

that the lyophilizer qualification has become one of the most discussed topics and

Lyophilizers are amongst the highly complex equipment used for production. Literature

survey also says that for the qualification of lyophilizers, requires the knowledge in many

diverse fields including computer and software validation, mechanical and electrical

engineering, refrigeration engineering and steam sterilization. Article presented the

results of a hands-on approach, by their report of experiences, to the efficient and reliable

qualification of lyophilizers14

.

A Guide to Inspections of Lyophilization of Parenterals has stated that there are many

new parenteral products which are manufactured as lyophilized products. Many of the

investigators have disclosed potency, sterility and stability problems associated with the

manufacture and control of lyophilized parenteral products. There are some of the

important aspects of these operations like the formulation of the solution, filling of vials

and validation of the filling operations, sterilization and engineering aspects of the

lyophilizes, scale up and validation of the lyophilization cycle and testing of the product.



This will help in knowing some of the problems associated with the manufacture and

control of a lyophilized dosage form15

.

The evolution of product temperature and of residual ice content in the various vials of a

batch during a freeze-drying process is significantly affected by local conditions around

each vial. The researchers has developed dual-scale model that can significantly improve

the understanding for pharmaceuticals freeze-drying processes: it couples a three-

dimensional model, describing the fluid dynamics in the chamber, and a second

mathematical model, either mono- or bi-dimensional, describing the drying of the product

in each vial. Thus, it can be profitably used to gain knowledge about process dynamics,

and to improve the design of the equipment, as well as the performance of the control

system of the process16

.

According to the literature survey in lyophilization of pharmaceuticals, the product

sublimation interface temperature must be kept below the product collapse temperature to

achieve pharmaceutical elegance and assure stability. It was reviewed that currently,

meaningful equipment controls are only available for chamber pressure and shelf

temperature. The review derives and explains the use of the heat and mass transfer

equation for predicting these control parameters in a manner that meets the interface

temperature condition. It presented the derivation and solution of those equations which

can be used to find a suitable shelf temperature and chamber pressure after knowing a

product collapse temperature. This paper has shown both a derivation for the central

coupled heat and mass transfer equation, as well as methods for its solution. Other

solutions and considerable complexity can be introduced. This method is not intended to

represent a complete simulation of the lyophilization process. It was nothing more than



an analysis physical primary drying parameters. A major consideration for the use of this

analysis is that a vial-package heat transfer coefficient must be obtained as a function of

pressure. Such work can either be done experimentally or by estimate. Also, the dry

product layer resistance was treated as a single average number, when in fact it is known

to continuously increase throughout the sublimation. Still, the method is greatly superior

to having no analysis and no understanding of existing product lyophilization cycles. It

was concluded that the use of this method has substantially reduced the amount of trial

and error associated with lyophilization cycle development17

.

Article discusses the issues involved in achieving batch uniformity for lyophilized

pharmaceuticals summarizes important research and suggest strategies, at every step of

the process, for ensuring the batch uniformity of lyophilized products. Critical parameters

such as shelf temperature, chamber pressure, and time should be accurately and precisely

controlled. It has been said that even less critical factors such as product temperatures

should be monitored, since temperature data can help assess processing conditions. The

sublimation/condensation test challenges the shelves‟ ability to provide sufficient heat to

achieve acceptable sublimation rates. The test also demonstrates the condensation rate

and ice-load capacity of the condenser. Verifying equipment‟s pressure-control capability

is another importance factor in maintaining batch uniformity. Some of OQ tests that

should be tested are cooling and heating rates control at set point, temperature uniformity.

It was concluded that Success requires precise control of process conditions, extensive

equipment testing, and analysis of bulk solution, dry and reconstituted product18

.



According to the literature survey successful transfer and validation of a lyophilization

process requires thorough characterization of the process and the equipment. The article

discussed the various elements such as the development and characterization of the

lyophilization process, performance of the equipment, identification of the critical

process parameters, etc., that can potentially impact the successful transfer and validation

efforts. It was said that for most protein formulations, annealing temperature and time,

freezing rates, shelf temperature, and chamber pressure are generally considered to be the

critical process parameters since they directly influence the quality of the drug product.

These limits need to be established through the robustness studies and should serve as the

basis for validation. It was said that a process can be said to be successfully transferred

and validated when one can demonstrate that the process can be performed consistently

in three consecutive runs at production scale while meeting the pre-determined

acceptance criteria relating to process parameters and product quality attributes. This

means that the acceptance criteria must be well defined and clearly laid out in the

validation protocol before executing the validation during the design of the validation

strategy, one needs to keep in mind to include batch load range, the minimum and the

maximum. The regulatory agencies require pharmaceutical companies to demonstrate the

consistency of the drying process, in which case the design of the sampling plan and the

analytical testing must be in accordance with the current agency expectations i.e. star-

shaped sampling plan, demonstration of uniformity across the shelves in terms of content,

potency, particulate distribution, reconstitution time, residual moisture, etc. It was

concluded that awareness and understanding of the challenges associated with



commercial manufacturing environment and production freeze dryer design are central to

the development of a robust freeze drying process. 19

According to the literature survey the comparison between the bulks freeze-drying

process of glass container to membrane trays was done. The sublimation phase is an

endothermic process which is controlled predominantly by shelf temperature and

chamber pressure. Numerous other factors (such as container heat transfer coefficient,

container geometry, stopper design, freeze-dryer geometry, freezing behavior, fill depth,

formulation type and concentration, and the robustness of the filling process) also are

important and together contribute to the product temperature at the interface where the

water crystals sublimate. Tests like freezing time, primary drying cycle length; glass

integrity- lensing, product was compared between the two non- optimized process and

new process. It was found that there was a quick freezing effect in membrane trays so

that defined product characteristics can be achieved and there was lack of product

homogeneity in glass bottles when compared with membrane trays. Another difference

between large glass bottles and membrane trays is stoppering, which creates additional

stresses within glass vials. Indeed, the tensions accumulated inside the glass may be

released when force is applied to the bottom surface, resulting in glass breakage, which

can have a huge impact on product yields. Freeze-drying process transfer from glass

bottles to single-use ePTFE membrane trays is feasible and profitable in terms of freeze-

drying capacity for this specific project. It has been concluded that robust process

validation is a key element to success and a good understanding of the freeze-drying

process is advantageous in speeding up the transfer process and releasing a quality

product20

.



Successful scale-up of a lyophilization cycle from laboratory to pilot or commercial scale

requires an understanding of relative performance characteristics of the lyophilizers at

each scale. These characteristics may significantly alter the product temperature profile

upon scale-up if not properly accounted for. Several cycle scale-up scenarios are

discussed with the aid of a steady-state model of lyophilization. In this work, they

attempted to summarize and utilize the best practice of primary drying scale-up by

applying extensive lyophilizer characterization and freeze-drying process modeling. In

combination, these aspects of process design should help in building between laboratory

and any similarly characterized commercial or pilot lyophilizers. This could allow for the

rational design of scaled-up processes minimizing the number of test cycles.

Understanding the capability of lyophilizers and product behavior under different process

conditions in laboratory, pilot and commercial lyophilizers should also facilitate process

robustness design. It was said of potential product impact can be calculated ahead of time

to establish the magnitude of cycle parameter deviations examined during lyophilization

cycle robustness studies. It was concluded that if one compares the estimated product

temperature profile during the process deviation with robustness data performed at lab

scale, one can better understand any possible effect the process deviation may have on

the product quality21

.

The article summarizes and clarifies terms and issues related to vacuum integrity testing

of lyophilizer. It has been reviewed that vacuum integrity test is an integral part of the

quality assurance of lyophilized product. Among these challenges are measurement of

system tightness and the establishment of inleakage criterion that maintains a reasonable

assurance of product stability. There are many factors which one needs to be aware when



performing this qualification such as influence of time temperature, start pressure and

virtual leaks. To compare integrity of vessels one must have same temperature, pressure

and time if the volumes of vessels are dissimilar then one must specify the volume based

leak rate. It was concluded that vacuum integrity test is an important part of any factory

acceptance test (FAT), site acceptance test (SAT) and operation qualification (OQ) 22

.

Validation of autoclave:

According to the literature survey the article has provided an update of the validation of

moist heat sterilization. It has brought together the practical information one needs when

validating an autoclave from procurement through routine use. This article has described

about sterility concept, sterilization principles, development of sterilization cycle and

measurement of sterilization efficiency are measured. It was concluded that the above

described concepts are very much useful in validation of moist heat sterilization in

reliable and cost-effective manner23

.

The qualification of autoclave has been the topic of numerous articles, books, symposia

in the past decade autoclave qualifications shortcomings are among the leading source of

FDA 483 citations reported for the biotech industry. It has been assumed that the readers

are somewhat familiar with the basic theoretical development of steam sterilization and

equipment qualification. It was summarized that the authors has attempted to present

hands- on approach to efficiently and reliably qualify autoclaves24

.

Literature survey explains about the significance of Steam sterilization which remains an

issue for regulatory bodies, particularly for the processes associated with high risk in



terms of probability and severity of infections. Author says that failure to address this

requirement may place the public at risk and lead to regulatory action and in addition to

potential business liabilities there may be significant cost associated with the validation

process. It was said that if appropriate consideration is not given to employing the correct

approach unnecessary ongoing operational cost may result. Article explained the about

basic validation test to be done in IQ, OQ, PQ and set the acceptance criteria based on

European standards and also gave some tips and cautions to be followed during the

validation of steam sterilizer. It was concluded that practical experience that the

document will provide assistance in ensuring an effective, efficient validation for steam

sterilization and that end result provide the best possible validated cycle to meet the needs

of specific application25

.

According to the health science guideline the efficacy of a given sterilization process for

a specific drug product is evaluated on the basis of a series of protocols and scientific

experiments designed to demonstrate that the sterilization process and associated control

procedures can reproducibly deliver a sterile product. The guideline reviews the

autoclave process, performance specifications, autoclave loading patterns methods,

controls to monitor production cycles, requalification of production autoclaves and heat

distribution and penetration studies. Thermal effects of loading, identification and

characterisation of bioburden organisms, specifications for bioburden identification,

resistance and stability of biological indicators relative to that of bioburden

microbiological challenge studies should be checked. Microbiological monitoring of the

environment, steam quality test, bacterial endotoxins test and method sterility test

methods and release criteria should be done for autoclave validation. Data derived from



experiments and control procedures allow conclusions to be drawn about the probability

of nonsterile product units (sterility assurance level). Based on the scientific validity of

the protocols and methods, as well as on the scientific validity of the results and

conclusions, the manufacturer can conclude that the efficacy of the sterilisation process is

validated26

.

The Health Products and Food Branch Inspectorate (HPFBI) of Health Canada

recognizes that terminal moist heat sterilization, when practical, was presently considered

the method of choice to ensure sterility. Principles outlined in the document are shared

with other methods of sterilization; those processes require control and assessment of

different parameters. According the indicating devices which are used in the validation

studies or used as part of post-validation monitoring or requalification must be calibrated.

Two basic approaches are employed to develop sterilization cycles for moist heat

processes are Overkill and Probability of Survival. Prior to commencing heat distribution,

heat penetration and/or biological challenge reduction studies, it is necessary that the

equipment be checked and certified as properly installed, equipped and functioning as per

its design. The guidelines provided the information regarding heat distribution studies,

heat penetration studies, biological challenge reduction studies, and post -validation

monitoring studies. It has been concluded that a written evaluation of the entire study was

carried out utilizing the various validation protocols should be prepared and the

conclusions drawn at each stage stated. The final conclusion should clearly reflect

whether the validation protocol requirements were met27

.

This document is intended to provide guidance for the submission of information and

data in support of the efficacy of sterilization processes in drug applications for both



human and veterinary drugs. Guidelines gives the information regarding the sterilization

process. For the validation of terminal sterilization autoclave description of autoclave

Process and performance Specifications of the autoclave process which includes the

pertinent information such as cycle type (e.g., saturated steam, water immersion, and

water spray), cycle parameters and performance specifications including temperature,

pressure, time, and minimum and maximum Fo, autoclave Loading Patterns, methods

and controls used to monitor routine production cycles (e.g., thermocouples, pilot bottles,

and biological indicators) including the number and location of each as well as

Acceptance and rejection specifications are required . The guideline also contains

information regarding heat distribution and heat penetration studies, thermal Monitors the

Effects of Loading on thermal input, microbiological Efficacy of the Cycle and

microbiological Monitoring of the Environment. . It has been summarised that the

efficacy of a given sterilization process for a specific drug product is evaluated on the

basis of a series of protocols and scientific experiments designed to demonstrate that the

sterilization process and associated control procedures can reproducibly deliver a sterile

product. Data derived from experiments and control procedures allow conclusions to be

drawn about the probability of nonsterile product units (sterility assurance level). Based

on the scientific validity of the protocols and methods, as well as on the scientific validity

of the results and conclusions, the agency concludes that the efficacy of the sterilization

process is validated28

.

According to literature survey Qualification of High Pressure High Vacuum (HPHV)

Steam Sterilizer in Pharmaceuticals is done by pre and post calibration of temperature

sensors b , Vacuum Leak test (3 Trials without probe & 3 Trials with probe), Bowie –



Dick Test for Steam penetration ( 3 trials on 3 different days), Steam qualification tests (3

trials on 3 different days), Empty Chamber Heat Distribution studies (3 trials) with

temperature mapping probes at different locations of the sterilizer chamber, Loaded

Chamber heat penetration studies (3 trials) for each sterilization load of fixed loading

pattern, with temperature mapping probes inside the innermost possible layer of the load

subjected for sterilization, Bio-Challenge studies using Bacillus stearothemophilus spore

strips (containing 106 or more spores per strip) during the heat distribution & heat

penetration studies, Estimation of the FO value achieved during the sterilization hold

period at each temperature-mapping probe, Quality of the steam condensate collected

from the sampling point provided in the sterilizer chamber condensate drain line for all

loads, Vacuum break filter integrity testing and the results found complying with the

acceptance criteria29

.

The article explained the importance of air removal. It was said that to achieve effective

steam sterilization, dry saturated steam must contact the surfaces to be sterilised so that

energy can be transferred. It follows, therefore, that nothing must come between the

steam and the surface to be sterilized. Most of the equipment they seek to sterilize (filters,

tubing, vessels, filling needles, etc) contain vast quantities of air. If this air is not

removed, then it can act as an insulating barrier between steam and equipment and thus

compromise the sterilising process. Article explained the methods of air removal and

means of confirming effective air removal such as a drain-mounted air detector, the

Bowie-Dick test (or equivalent), chamber leak rate test Air detectors30

.

According to literature survey it was reviewed that from many years steam autoclaves

have been used in many different industries. Their primary task is to sterilize items so



that these same items can be used in situations where the introduction of micro-organisms

would pose a health-risk. This paper describes these tests, and how often should be

performed in order for the user to be confident that their autoclave is functioning properly

and within the requirements of the regulatory bodies. A thermometric test to determine

the temperature profile inside the chamber throughout the holding time, using a data

logging system and multiple probes placed throughout the chamber volume.

Simultaneous monitoring of the chamber pressure is also useful in checking the

efficiency of the air removal/replacement system. During the load testing, biological

indicators may also be placed in the load to determine if sterilization is in fact taking

place. If the autoclave is to be used to sterilize folded cloth, such as garments, a Bowie

Dick test should be performed to ensure complete penetration of the steam into the cloth.

It was concluded that ensuring sterilization is occurring in the autoclave is however of

prime importance thus such costs should be viewed in the light of human safety rather

than that of economics.31

Chapter-4 Methodology


4. METHODOLOGY

4.1 Prerequisites for equipment Validation:

4.1.1 Validation Approach:

The validation of lyophilizer and autoclave includes two complimentary aspects such

as examination of process parameters and examination of product characters. In order

to substantiate process reproducibility, three consecutive runs were performed.

Sr.

No.

Equipment particulars Details

1 Equipment Name Lyophilizer Autoclave

2 Equipment Capacity 5 m2

970 liters

3 Model Lyo-5.0(SIP,CIP)

4 Manufacturer Tofflon Machin fabrik

6 Category of equipment Direct Impact

Direct impact

Table 4.1 Equipment details of Lyophilizer and Autoclave

4.1.2 Documents Required:

Following documents were required for equipment validation.

Design qualification document (DQ).

User required specifications (URS).

Equipment manuals.

Related SOPs for the qualification of the equipment.

Quality risk management document

Factory acceptance test document

Calibration certificates of temperature sensors, gauges, data loggers.



Installation qualification documents

Personal training regards documents.

4.1.3 Instructions Followed During Validation Of Equipment.

Validation and samples must be collected by trained QA persons only.

Temperature probes and data loggers must be calibrated before and after post after

the validation for the accuracy and precision.

Spore count enumeration and purity test of biological indicator must be done

before use.

Probes must be ensured that they are in same location through out the validation.

The validity of the Bowie and Dick test kit to be use was checked with respect to

its expiry date.

4.1.4 Requirements during the equipment validation of Lyophilizer and

Autoclaves:

Data loggers

Temperature sensors

Biological indicator (Geobacillus sterothermophilus)

1% Nacl

1% talc

Mannitol

Water for injection

Bowie and Dick test kit



4.2 Operation qualification of lyophilizer:

4.2.1 Verification of key functionality, safety features and emergency stop

features:

Sr.

No. Function / Verification Procedure Acceptance Criteria

1

Interlocking of air inlet1 and air inlet 2 and

vacuum pump was checked.

Pump shall not switch on when

the valves are open and vice

versa.

2 Heater and valve allowing the chilled gas

supply to heat exchanger was checked.

Open only if circulation pump is

in running condition.

3

Checked when water pressure is below

2kg/cm2 and compressors stopped

working.

alarm should start

4 Checked when pressure setted is below 6

kg/cm2.

alarm should start

5 Vacuum pressure was kept to cross the set

parameters alarm should start

6 The temperature of oil was set to cross the

parameter and heater is turned off. alarm should start

7 SIP Start function was checked. Start only if all the pneumatic

door lock is activated.

Table 4.2 Verification list of key functionality, safety and emergency stop

features.

4.2.2 Verification of operator interface, display, automation and control

requirements.

Function Verification Procedure Acceptance Criteria

Switch control power

key

Switched off Control power indicator light off

Switched on Control power indicator light on

Alarm siren Alarm was activated. Siren on if there is an alarm




Button for eliminating

main alarm lamp and

siren

The button was pushed

when alarm siren is on

Siren off. The main alarm lamp

is on if there is alarming still.

And the main alarm lamp is off

if there is no alarm.

Remote/local key switch

Remote switch on Touch screen can be run and PC

can be not run

Local switch on PC can be run and touch screen

can be not run

Emergent button The button was pressed The PLC stops exporting control

signal

Circulation pump1

The air breaker of

circulation pump 1 was

closed

Choice circulation pump 1 at

computer, it can not be activated.

The air breaker of pump

1 was opened.


computer, it can be activated.

Circulation pump 2

The air breaker of


closed



The air breaker of


opened



Vacuum pump1

The air breaker of

vacuum pump 1 was

closed

Choice vacuum pump 1 at


The air breaker of

vacuum pump 1 was

opened






Vacuum pump 2

The air breaker of

vacuum pump 2 was

closed



The air breaker of

vacuum pump 2 was

opened

Choice vacuum pump 12at


Hydraulic unit

The air breaker of

shelves up and down at

control panel was closed

Choice button for shelves up and

down at control panel, shelves

can not move.

The air breaker of

shelves up and down at

control panel was

opened

Choice button for shelves up and

down at control panel, shelves

up and down.

Heater

The air breaker of heater

was closed

Regulate shelves temperature

after setting higher temperature

of shelves, heater can not be

started.

The air breaker of heater

was opened

Regulate shelves temperature

after setting higher temperature

of shelves, heater can be started.

Compresor 1 The air breaker of

compressor 1was closed

Choice compressor 1 at


Compresor 1

The air breaker of

compressor 1 was

opened



Compresor 2

The air breaker of

compressor 2 was closed



The air breaker of

compressor was opened






Chamber sight lamp

The button of chamber

lamp was clicked Chamber lamp on.

The button was

loosened

Chamber lamp off after 60

seconds.

Condenser sight lamp

The button of

condenser lamp was

clicked

Condenser lamp on.

The button was loosened Condenser lamp off after 60

seconds.

Shelves up button

The up button was

pressed

Hydraulic pump can be started

and shelves up.

The button was loosened

Shelves stop increase and

hydraulic pump stops after

delaying 3 minutes.

Shelves down button

The down button was

pressed

Hydraulic pump can be started

and shelves down.

The button was loosened

Shelves stop decline and

hydraulic pump stops after

delaying 3 minutes.

Chamber air inlet

button

The button was clicked

to open

Chamber air inlet valve is

opened


to close

Chamber air inlet valve was

closed.

Condenser air inlet

button

Click button was clicked

to open

Condenser air inlet valve is

opened

Condenser air inlet

button


to close

Condenser air inlet inlet valve is

closed

Door close button Door button was

clicked The door is closed




Door open button Click door open button The door is opened

Camber sight lamp

Click the button of

chamber lamp Chamber lamp on.

Loosen the button Chamber lamp off after 60

seconds.

Table 4.3 Verification list for operator interface, display, automation and control

requirements.

4.2.3 Software operation qualification

Table 4.4 verification of software operation qualification

Main Interface


Login

Double click icon of

LyoMaster4000.

There should be login

window.

Input user name and password and

confirm.

Enter control main

interface.

Login system with administrator

and press “user configuration”

button.

Display configuration user

dialog box. Change and add

user through this function.

Parameter

management

interface

Click Parameter management

button of main interface

Enter parameter

management interface

Lyophilization Click Lyophilization button of

main interface.

Enter lyophilization

interface

Sterilization Click SIP button of main interface Enter SIP interface

Cleaning Click CIP button of mainterface Enter CIP interface



History data:


History Data

Click History Data button on

control interface (taskbar).

Display History Data

interface

Click lyophilization and

sterilization table and trend button

separately. Input batch condition.

User can query

lyophilization and

sterilization data table and

trend.

Press print preview button on

taskbar.

Preview content that will

be printed.

History Data

Press print button on taskbar.

If install printer, there

should be print dialog

box. After confirming

print the data.

Press parameter setting button on

taskbar.

Display history server

setting interface

Press exit button on taskbar. Exit history data inquiry

interface.

Operation Data Inquiry


Operation

Data

Click Operation Data button on

control interface (events log on

taskbar).

Display events log interface.

Click events report button. Input

date condition.

User can query events

according to date.

Press print preview button on

taskbar.

Preview content that will be

printed.

Press print button on taskbar. There should be print dialog

box.



4.2.4 Alarm categorization and verification:

Sr.

No.

Alarm

Description

Alarms & Warnings Verification

Procedure

Acceptance

Criteria

1. Power abnormal

alarm

Main power switch was turned off

manually.

Alarm lamp on,

siren on and stop

the equipment.

2.

Water pressure

abnormal alarm

Water inlet or outlet was closed.

Alarm lamp on,

siren on and stop

the compressor.

3. Air pressure

abnormal alarm.

Higher setting value for air pressure

relay was adjusted manually.

Alarm lamp on,

siren on.

4.

Circulation

pressure abnormal

alarm.

Higher setting value for circulation

pressure relay was adjusted manually

(setting value is more than actual

value).

Alarm lamp on,

siren on. Switch

circulation pump

automatically.

5.

Compressor1

highpressure

relay1 abnormal

alarm.

Lower setting value for low pressure

relay was adjusted manually (setting

value is less than actual value).

Alarm lamp on,

siren on.

6.

Compressor1 oil

pressure relay 1

abnormal alarm.

Compressor 1 power was turned off.

Alarm lamp on,

siren on. Stop

compressor after

120 seconds.

7.

Compressor 1

electric heat

protection

abnormal alarm.

Power of compressor 1 electric heat

protector relay was turned off.

Alarm lamp on,

siren on. Stop

compressor 1



Sr.

No.

Alarm

Description

Alarms & Warnings

Verification Procedure Acceptance Criteria

8.

Compressor 2

high pressure

relay 1 abnormal

alarm.

Lower setting value for low

pressure relay was adjusted

manually (setting value is less

than actual value).

Alarm lamp on, siren on.

9.

Compressor 2 oil

pressure relay 1

abnormal alarm.

Compressor 2 power was turned

off.


Stop compressor after 120

seconds.

10.

Compressor 2

electric heat

protection

abnormal alarm.

Power of compressor 2 electric

heat protector relay was turned

off.


Stop compressor 2.

11.

All of vacuum

pumps off

abnormal alarm.

Close all vacuum pumps′ air

break forcibly during process. Alarm lamp on, siren on.

12. Vacuum

abnormal alarm.

The vacuum exceeds setting

value during process.


Stop heater.

13.

Chamber-

condenser

isolation valve

open abnormal

alarm.

Supply compressed air valve was

turned off, compressed air signal

was made short and isolation

valve was opened.


After 5 seconds

14.

Chamber-

condenser

isolation valve

close abnormal

alarm.


turned off; compressed air signal

was made short after opening

isolation valve and isolation

valve was closed


After 5 seconds



Sr.

No.

Alarm

Description

Alarms & Warnings


15.

Pump isolation

valve open

abnormal alarm.

Supply compressed air valve

was turned off, compressed air

signal short and isolation valve

was opened.


After 5 seconds

16.

Pump isolation

valve close

abnormal alarm.



was made short, after opening

pump isolation valve and

isolation valve was closed.

Alarm lamp on, siren on

after 5 seconds

17.

Safety isolation

valve open

abnormal alarm.


turned off, Compressed air signal

was turned off short and safety

isolation valve was opened.


after 5 seconds

18.

Safety isolation

valve close

abnormal alarm.



was turned off short after

opening isolation valve and

safety isolation valve was closed.


after 5 seconds

5

19.

Temperature

control abnormal

alarm.

Little time to reach higher

temperature was set during

automatic lyophilization.


20.

Pressure rising

test abnormal

alarm.

The difference of pressure rise

test was set minimum and the

time is ten minutes at least.




Sr.

No.

Alarm

Description

Alarms & Warnings


21. Door latch in

abnormal alarm

Door bolt inlet valve was opened

during non vacuumizing

condition


and action stop after 5

seconds

22. Door latch out

abnormal alarm.

After there is door bolt inlet

signal, door bolt outlet valve

was opened during normal

pressure.


and action stop after 5

seconds

23. Compressor 1

overloading alarm

Compressor 1 air breaker was

switched off manually.


Air breaker is off and stops

compressor 1.

24. Compressor 2

overloading alarm

Compressor 2 air breaker was



Air breaker is off and stops

compressor 2.

25. Vacuum pump 1

overloading alarm

Vacuum pump 1 air breaker was



Air breaker is off and stop

Vacuum pump 1.

26. Vacuum pump 2

overloading alarm

Vacuum pump 2 air breaker was




Vacuum pump 2.

27.

Circulation

pumps 1

overloading

alarm.

Circulation pump 1 air breaker

was switched off manually.



Circulation pump 1.

28.

Circulation pump

2 overloading

alarm.

Circulation pump 2 air breaker

was switched off manually.



Circulation pump 2.

29.

Hydraulic pump

overloading

alarm.

Hydraulic pump air breaker was




hydraulic pump.

Table 4.5 Alarm categorization and verification list



4.3 Performance qualification of lyophilizer:

4.3.1 Vacuum rate, maximum vacuum capacity and vacuum leakage rate test:

The compressor was switched on and the condenser temperature was allowed to

reach up to ≤ - 45ºC.

The vacuum pump was started and start time of cycle was recorded.

The time when vacuum reaches to 0.1mbar (10 Pascal) was recorded.

The cycle was run continuously for about 2 to 6 hours and the highest vacuum

reading was recorded.

The valves between condenser & vacuum pumps were closed.

The vacuum pumps, compressor were turned off, initial chamber vacuum

readings and time were recorded.

After 20 minutes the chamber vacuum reading was noted and the difference was

calculated.

The chamber vacuum leakage rate was calculated by using the formula -

= (Difference in chamber vacuum in mbar X vol. of chamber in m3)

Time in seconds

Acceptance criteria:

The vacuum /evacuation rate i.e. time taken to reach chamber vacuum upto

0.1mbar (10 Pascal) from atmospheric pressure should be NMT 30 min.

Maximum vacuum achieved shall be ≤ 1.0 pa (0.01 mbar) or as specified in

respective protocol. (maximum vacuum achieved shall be less than or equal to

lowest pressure defined in any lyophilization recipe for the respective lyophilizer

and shall be addressed in respective protocol)

The chamber vacuum leakage rate calculated shall be ≤ 0.00003

mbars.m3/second (0.003 pa m3/second) or as specified in respective protocol.



4.3.2 Isolator Valve Integrity Tests

The air inlet valves were made to open and the chamber vacuum was adjusted to

0.1 mbar ± 0.02 mbar.

The initial vacuum and time was recorded.

Then the isolator valve was made to close and the vacuum pump was started.

The finale vacuum was recorded after 10 minutes.

The drop in vacuum was calculated.

Acceptance criteria

The chamber vacuum pressure shall not drop below the initial vacuum pressure.

4.3.3 Shelf Cooling and Heating Rate Test (Oil in Temperature)

Cooling rate was measured from room temperature to the maximum operational

freezing temperature and heating rate was measured from lowest to the highest

operating temperatures of lyophilizer.

The time of initial shelf „oil in‟ temperature was noted and the freezing run

was started, functioned at maximum operational freezing temperature (~-500C).

After temperature reaching to a desired the temperature (~-40oC) for freezing

rate and (~+600C ) for heating rate was hold for about half and hour.

After half an hour, the drying run was started, functioned at maximum

operational temperature (~+600C ).

The cooling and heating rate (average temperature change per min) was

calculated by using formula.

Cooling rate/ heating rate (ºC/min) = Difference in temp from initial to final in 0C

Time taken to achieve the desire temperature

In minutes




Shelves inlet temperature (oil in temperature) cooling and heating rate shall be

≥1.0 °C /min. or as specified in the respective protocol.

4.3.4 Shelf Temperature Uniformity Test (During Freezing and Drying)

Temperature sensors were labelled with number.

The pre calibrated temperature sensors were connected with data logger, and

inserted into the lyophilizer chamber through suitable port ensuring that there is

no leak through the port of sensor insertion into the chamber.

The sensors were distributed on the different shelves at pre identified locations,

ensuring that the complete dryness of shelves before placing sensors on the

shelves.

The temperature sensors were kept such a way that they were in contact with the

shelf surface.

The identification number of each temperature sensors on the diagram after

placement of temperature sensors was noted.

The print interval and file recording interval for 1 min was set. Logged data

included the time, temperature and channel number labels of each temperature

sensor during each minute print interval.

The data logger time with the equipment time was checked and made

synchronise.

The temperature was monitored at the three target set points considering the

overall temperature range used for process (worst –case of all recipes) .

After placement of temperature sensors the doors of lyophilizer were closed.

The operating the lyophilizer was done in manual mode.



After all the temperature sensors placed on the shelves reached to 3.0 C of

target set temperature, the „oil in‟ temperature was maintained for about 30 min.

After completion of about 30 min, the drying run was started with 0°C

temperature set point.

After all the temperature sensors placed on the shelves reached to 3.0 C , the

„oil in‟ temperature was maintained for about another 30 min

After completion of about 30 min, drying run was continued with maximum

temperature set point of 60°C.

After all the temperature sensors placed on the shelves reached to 3.0 C, the

„oil in‟ temperature was maintained for about another 30 min.

Figure 4.1 Location of probes in the lyophilizer for uniformity test.

Thermal

balance shelf

Shelf-0

Shelf-1

Shelf-2

Shelf-3

Shelf-4

Shelf-5

Shelf-6

Shelf-7

1 2

3 4 5

6 10 7

9 8

11 12

14 13 15

16 17

19 18 20

21 22

24 23 25

26 27

29 28 30

31 36

33 35

34



: : Indicates location for Probe placement on each Shelf


The maximum variation of temperature measured across the all shelves shall be

within 3.0 C from the average temperature at a single point of time, during

respective hold period of each target set temperature, or shall comply.

4.3.5 SIP (Sterilization in place) test:

20 temperature sensors were identified and labelled with number.

Print interval and file recording interval was set 1 minute for Shelf temperature

uniformity and 5 seconds for SIP cycle.

Three Temperature sensors were placed inside the Lyophilizer chamber as

shown in probe location diagram mentioned below.

Pre evaluated biological indicator were placed near temperature sensor.

The sterilization cycle was carried out at set temperature 121.5°C at 15 minutes

After completion of sterilization cycle, the Data loggers are stopped and the door

of Lyophilizer was opened.

The Biological indicators were taken out from the chamber, and sent the

Biological indicators to Microbiological Lab for testing.

The Temperature profile from the Data logger and temperature recorder were

taken out.


The temperature measured (all Temperature sensors), throughout the sterilization

holding time, should be NLT121°C



The F0 - value at all locations of temperature sensor probes should be ≥15

minutes, Biological indicator should not show any growth during or after

completion of defined incubation. (i.e. no growth after incubation).

Figure 4.2 Temperature Sensors location Diagram for SIP Test

Probe shall be placed inside the vacuum break line filter along with a

biological indicator.

4.3.6 Condenser capacity test

The temperature of the Condenser was verified to be maintained lower than – 40

0C throughout the drying cycle and the vacuum of chamber was under 0.3mbar.



The maximum load to be tested was based on the rated maximum load of the

condenser.

The SS trays was loaded with measured amount of filtered water for injection

on eight shelves

Cycle was programmed and run with freezing at – 40 0C for duration of

minimum three hours followed by primary drying time of NLT 21 hours and

secondary drying time of NLT 21 hours.

When the cycle was completed, vacuum was break and the remaining ice in the

trays was melted. The remaining water was measured.


Amount of water sublimed from trays and deposited on condenser shall not less

than of rated capacity of the condenser.

4.3.7 Cleaning In Place (CIP) Test

1% NaCl solution (using WFI) was prepared.

The sample of (about 100 mL) of prepared 1% NaCl solution was collected and

sent to QC to check for presence of chlorides.

The chamber (inside) and shelves was spiked with 1% NaCl solution using the

spray bottle, ensuring that all parts of chamber were covered.

Allowed to dry for about two to three hours with lyophilizer door open.

After drying close the doors and perform CIP cycle.

At the end of the CIP cycle final chamber rinse water sample was collected from

the sampling point provided in clean and dry glass bottle (about 100 mL).



The sample bottle was labelled with sampling details and sent it to QC lab to

check for traces of chlorides.

Acceptance criteria

During chloride test solution should not show any change in appearance for at

least 15 minutes indicating the absence of chlorides.

4.3.8 Placebo Test :

The 30 mL vials were filled with 15 mL fill of 2.5% mannitol solution.

The vials were operated and loaded into Lyophilizer as per respective SOP.

In “Recipe Data” window, the following parameters were set.

Condenser cooling: Final Temperature = - 50 C, Ramp Duration = 05 minutes

Chamber evacuation

Alarm P1 set point = 0.5, Alarm P2 set point = 0.8, Continue evacuation = 30

Pre-cooling

Final temp

(0C)

Ramp duration

in mins

Soak duration

in mins

Ramp & Soak 1 0 30 15

Ramp & Soak 2 -35 60 180

Primary drying

Final

temp(0C)

Ramp

duration

in mins

Soak duration in

mins

Pressure

in mbar

Ramp & Soak 1 10 225 1140 0.4000

Ramp & Soak 2 20 15 240 0.2000

Ramp & Soak 3 20 0 0 0



Secondary drying

Final temp in

(0C)

(0C)

temp(0C)

Ramp in mins Soak in

mins

Pressure in

mbar

Ramp & Soak 1 30 30 150 0.0700

Ramp & Soak 2 38 15 300 0.0500

Ramp & Soak 12 38 0 0 0

Table 4.6 steps for placebo test

Plug the rubber bungs by operating the shelf moving switch.

Check the bunging quality in each shelf after unloading

After the completion of each trial, collect one vial each from four corners

and center of each shelf (55 vials), and pick two vials randomly from

eleven shelves (22 vials) to check Moisture content Cake height.

Pick two vials randomly from each shelve to check solubility

Send all samples to QC with proper identification to check for Moisture

content, Cake height and solubility.


The Moisture content measured shall not be more than 2.0 %

Cake height observed shall be uniform

Cake should dissolve within 30 seconds after addition of 15 mL water in it.



4.4 Operation qualification of autoclave

4.4.1 Verification of key functionality:

Sr.

No. Procedure Acceptance criteria

1.

Controller time accuracy

The parameter in PLC was set and

sterilization cycle was runned, the

hold time was checked with the help

of stop watch.

The controller sterilization time

as indicated in sterilizer/printout

should be within 1% of the

intended exposure period.

2.

Door precondition indicator

Both the doors of the autoclave were

closed.

When door precondition is

fulfilled the indicator should

glow

3. Alarm Indication Switch

An alarm was generated.

An audio visual alarm should

generate and the alarm indicator

should glow.

4.

Emergency Stop Push Switch

Emergency Stop Push Button was

pressed.

The equipment should stop at

current stage and all controls

and display is off.

5.

Non sterile Door open Push Button

Press the Non sterile door opens Push

Button when equipment is not in

process.

The non-sterile door should

open.

6.

Non Sterile close Push Button

Non sterile Door close

Push Button was pressed when door

is opened

The non-sterile door should

close

7.

Sterile Door close Push Button

Sterile side Door close Push Button

was pressed when sterile side door is

opened

Sterile side door should close



Sr.

No. Procedure Acceptance criteria

8.

Sterile Door close Push Button

Sterile side Door close Push Button

was pressed when sterile side door is

opened

Sterile side door should close

9.

Sterile Door open Push Button

Sterile Door open Push Button was

pressed when equipment is not in

process

Sterile side door should open

Table 4.7 verification list of key functionality of autoclave

4.4.2 Verification of safety feature

Sr.

No.

Safety Features

Description Function / Verification Procedure

Acceptance

Criteria

1

Check that both the

doors should not open at

the same time.

– First sterile door was opened and

then non sterile door open push

button was pressed to open it.

Non sterile door

should not open.

Sterile door was closed and non

sterile door was opened, then sterile

door push open button was pressed

again to open it.

Sterile door

should not open.

2

Check for the opening

of doors during manual

& auto mode of

operation

During auto or manual mode of

operation sterile and non-sterile

side doors buttons were pressed to

open.

Both doors

should not open.

3. Check for sterile door

operation

During operation the cycle was

aborted and sterile door open push

button wad pressed.

Sterile door

should not open



Sr.

No.

Safety Features

Description Function / Verification Procedure

Acceptance

Criteria

4

Process should not start

in auto or manual mode

if either door is open.

Sterile door was kept open and then

the process was tried to start, and

after that non sterile door was kept

opened and tried to start the

process again.-

Process should

not start

5

Process should not start

in manual mode if the

door precondition is not

fulfilled.

Manual mode was selected & do

not pressure non sterile door

gasket, then try to start the process

Process should

not start on both

occasions and

indicates with

alarm

Now Select manual mode & do not

pressure sterile door gasket, then

try to start the process

6 Working of safety valves

Increase the chamber pressure by

15% of the working pressure

Chamber steam

will blow off

through safety

valve

Increase the jacket pressure by

15% of the working pressure

Jacket steam will

blow off through

safety valve

7 Door Obstruction

When door is moving obstruct the

door with hand or material

Door should

move back



4.4.3 Verification of Alarms and Warnings

Sr.

No. Procedure Acceptance Criteria

1.

Leak Test Fail

During vacuum hold period the filter air

in valve was opened manually.

Vacuum should break.

Audio & Visual alarm will be generated

2.

Temperature over shooting

protection.

The over shoot temp.was set. Set point

was kept 2°C more than Sterilization

temperature & the process was run.

Audio & Visual alarm will generate The

exhaust valve should open , when

temperature goes beyond the over shoot

temperature set point

3.

Sterilization Hold period counting

stop

During Sterilization hold, after five

minutes the chamber steam inlet valve

was closed.

Alarm will be heard, message will be

displayed on the MMI and counting will

stop when the chamber temp fall down

below sterilizing Temp

The chamber steam inlet valve was

opened.

When the chamber temp. attain the

sterilization temp counting will start

further where it was stopped and alarm

will stop

4.

Sterilization Hold Period Counting

Reset

During the Sterilization Hold period,

chamber steam supply valve was closed

and waited till temperature fall down

below Sterilization reset Temp set point

Alarm should be generate and counting

will reset to zero

Open the chamber steam supply valve

When the chamber attains sterilization

temperature the counting will start

freshly and alarm will stop



Sr.


5.

Pure steam pressure low

During the process, manually close the

steam supply valve

Drop in steam pressure will be sensed by

pressure switch. Audio Visual alarm

will generate and message will be

displayed on MMI

6.

Process air pressure low

During the process, manually close the

air supply valve.

Drop in air pressure will be sensed by

pressure switch. Audio Visual alarm

will generate and message will be

displayed on MMI

7.

Compressed air pressure low

During the process, increase setting of

pressure switch mounted on

compressed air line

Audio Visual alarm will generate and

message will be displayed on MMI

8.

Vacuum Pump trip

Trip the pump manually by the

override provided on overload relay.

Alarm shall generate & message will be

displayed on MMI

9.

Door precondition.

During the process , put off compressed

air utility supply, built in pressure drop

below door precondition pressure

Alarm shall generate and message will

be displayed on MMI

10.

Purified water pressure low.

During process manually close the

Purified water valve.

Drop in purified water pressure will be

sensed by pressure switch. Audio Visual

alarm will generate and message will be

displayed on MMI.

11.

Cooling water pressure low.

During process manually close the

Purified water valve.

Drop in cooling water pressure will be

sensed by pressure switch. Audio Visual

alarm will generate and message will be

displayed on MMI



Sr.


12.

Chamber Pressure High

Allow the chamber pressure to rise

more than chamber pressure high set

point by opening the steam in valve

manually

Audio, visual alarm should be generated

and message shall be displayed on MMI

13.

Too Long Time For Pre vacuum

Set Too Long time for pre vacuum set

point less than the Actual set point



14.

Too Long Time For Post vacuum

Set Too Long time for post vacuum set




15.

Too Long Time For Heat up

Set Too Long time for Heat up set




Table 4.9 verification of alarms and warnings of autoclave

4.4.4 Verification of emergency stop features

4.4.3 Table 4.10 verification of emergency stop features

Sr.

No.

Emergency Stop Verification Procedure Acceptance Criteria

1.

Emergency Stop Push Switch

During the process on, Push the

Emergency Stop Switch

The process should stop

at the current stage in safe

mode



4.5 Performance qualification of autoclave:

4.5.1 Vacuum leak test for sterilizer chamber:

The vacuum leak test parameters were checked and ensured are as mentioned in

the authorized protocol.

The cycle for “Vacuum leak test” in PLC was selected and performed.

The process was monitored and observations were collected.

The vacuum leak test was performed before and after the validation execution to

ensure the chamber integrity.

Acceptance Criteria:

The drop in the vacuum shall not be more than 0.013 bar after 10 minutes of hold

period.

4.5.2 Air removal test :

A ready “Bowie and Dick test kit” was used to perform the test..

The Bowie and Dick test cycle in PLC was selected and the test was performed

as per the following parameters.



After completion of the cycle, the test sheet was retrieved and the compliance

against the acceptance criteria was examined.


The indicator colour shall change from light brown to dark brown / black in the

Bowie-Dick test sheet & shall be uniform throughout the sheet.

4.5.3 Heat distribution and heat penetration studies with loaded chamber.

Multi point data logger was used with T- type RTD for heat penetration studies.

Number of temperature sensors were used to check heat distribution in loaded

chamber (including one at drain point), and rest 12 probes were be used to check

heat penetration inside the load.



Load pattern was arranged as per the diagram.

Figure 4.3 load pattern diagram with probe location.

The temperature sensors were placed with identification number inside the

sterilizer chamber and inside the sterilization load where the steam penetration

could be difficult. The overall rationale for probe placement would be

combination of two or more of the following as appropriate:



Corners of sterilizer

Cartage filters, Tubing‟s / Product contact parts

Center of sterilizer and loaded units.

The temperature sensors were placed with identification number inside the

sterilizer chamber and inside the sterilization load as per the temperature sensor

location diagram.

pre enumerated biological indicators of Geobacillus stearothermophilus

(minimum10 spores per strip) were placed with properly marked details in the

middle of the sterilization load near each temperature sensor kept to check heat

penetration, and also at the locations where sensor can not be placed but the

location is defined as difficult for steam penetration .

The sterilization cycle was selected and cycle parameters are verified.

The heat penetration and distribution studies were carried out by using the Multi

point data logger with temperature logging interval of 10 seconds.

After completion of sterilization cycle the data logger was stopped and the non

sterile door of the sterilizer was opened.

The Biological indicators were taken out from the load and sent to

Microbiological Lab.

The Temperature profile from the Data logger and temperature recorder was taken

out.

The F0 value for each temperature sensor of data logger was checked. Calculation

of F0 value is based on the given formula. Check the results against the

acceptance criteria for compliance.



Formula: F0 = dt 10 (Ta- Tb)/Z

Where, Tb = 121.1°C,

Z = 10°C

Ta = Actual temperature

dt = Time interval between two successive temperature measurements.

After getting the reports of biological indicator check it for compliance against

acceptance criteria and attach with the qualification report.

Temperature sensors which are used are be calibrated after completion of activity

(post calibration) to ensure that the temperature measurement system is accurate

and precise.


The F0 - value of all the temperature sensor of data logger should be equal to

or more than sterilization hold time set. (except liquid or nutrient media load)

Chapter-5 Results

Dept. Of Quality Assurance KCP Bangalore Page 69

5. RESULTS

5.1 Prerequisites for equipment validation:

All prerequisites were checked prior to validation of lyophilizer and autoclave.

Process flowcharts and procedures were followed according to literatures followed.

5.2 Operation qualification of lyophilizer.

Sr.No Features verified Complies/ not complies

1.

Verification of key functionality,

safety features and emergency stop

features:

complies

2.

Verification of operator interface,

display, automation and control

requirements.

complies

3. Software operation qualification complies

4. Alarm categorization and verification

complies

Table 5.1 Results of operation qualification of lyophilizer

5.3 Operation qualification of autoclave.

Sr.No Features verified Complies/ not complies

1.

Key functionality

Complies

2. Safety features

complies

3. Operator interface, display,

automation and control requirements

complies

4. Alarms and warnings

Complies

5. Emergency stop features

complies

Table 5.2 OQ results of autoclave

Chapter-5 Results


4.4 5.3 performance qualification of lyophilizer

4.4 5.3.1 Vacuum rate, maximum vacuum capacity and vacuum leakage

test

Table 5.3 Results of Vacuum rate and leakage, maximum vacuum capacity test

5.3.2 Isolated valve integrity test

Table 5.4 Results of isolated valve integrity test

Test Acceptance

criteria

Observation

Run 1 Run 2 Run 3

Vacuum

rate

NMT 30 mins to

reach 0.1 mbar

15mins

0.0896 mbar

16mins1sec

0.0859 mbar

12 mins

0.1018 mbar

Maximum vacuum capacity

≤ 0.01mbar 0.0093 mbar 0.0089 mbar 0.0080 mbar

Vacuum

leakage test

≤ 0.00003

mbar.m3/sec

0.0151 x 2.38 1201 =0.000029 mbar.m3/sec

(0.00410.0178)x2.38 1200

=0.000027 mbar.m3/sec

0.0112x2.38 1200 =0.0000222 mbar.m3/sec

Test Acceptance

criteria

Observation

Run 1 Run 2 Run 3

chamber

vacuum

pressure

Chamber

vacuum

pressure shall

not drop below

the initial

vacuum

pressure

initial=0.0893 Final= 0.1127

Initial=0.1028 Final=0.1196

Initial=0.094 Final=0.102

Chapter-5 Results


5.3.3 Heating and cooling rate

Description Observations

Run 1 Run2 Run-3

Shelf

Cooling

Rate

Initial temp. (~+25

oC)

and time.

21.50C

7:43:12

21.50C

00:06:37

21.20C

07:20:08

Finale temp. (~-40oC)

and time

-40.80C

8:10:14

-40.10C

00:47:15

-40.10C

07:54:38

Difference of

Temp(T1)

62.30C

61.6

0C 61.3

0C

Time taken to achieve

target temp.(T2) 27.2 mins 40.38 mins 34.30 mins

Shelf Cooling Rate =

T1 / T2 °C per min. 2.29

0C/min 1.52

0C/min

1.790C/

min

Shelves

inlet

lowest

tempera

ture

4.3.2 Initial

temp.(~-

40oC) and

time

-40.80C,

08:10:14

-40.10C;

00:47:15

-40.10C;

07:54:38

4.3.2 Finale temp. (~-55oC)

and time

-550C,

08:49:16

-550C,

02:02:23

-55.10C;

08:43:23

4.3.2 Total time taken to

achieve ~-55oC temp.

39.2 mins 75.08 mins 48.45 mins

Shelf

Heating

Rate

Initial temp. (~-40oC)

and Time

-40.40C,

09:01:16

-40.00C,

03:53:05

-40.70C;

09:03:40

Finale temp. (~+60°C)

and Time

400C,

10:17:20

41.00C,

03:53:05

410C;

10:03:10

Difference of temp.

from ~-40oC to~

+60°C (T1)

80.40C 81

0C, 81.7

0C


temp. of ~+60°C (T2) 76.4 mins 59.56 mins 59.30 mins

Shelf Heating Rate =

T1 / T2 °C per min 1.05

0C/min

1.350C/min

1.380C/

min

Highest heating temp.

continued up to

(~+60°C) and Time

60.10C,

10:41:21

60.2°C,

04:10:21

60.30C;

10:20:20

Difference of Temp.

from ~-40oC

to~+60°C(F1)

100.50C 100.2

0C 101

0C


temp. of~+60°C (F2) 100.5 mins 78.13 mins 76.40 mins

Shelf Heating Rate=

F1/F2 °C per min. 1

0C/min 1.28

0C/min 1.32/min

Table 5.5 Results of heating and cooling rates

Chapter-5 Results


5.3.4 Shelf temperature uniformity test results at -550

C

Description

Observation

Run 1 Run 2 Run 3

Minimum temp. observed on the shelf during hold period

-55.10C -56.30C, -55.90C

Probe location of Minimum temp. & shelf number

T12 , 3rd shelf

T04; 1st shelf

T08 and T16; 2nd,4th shelf

Maximum temp. observed on the shelf during hold period

-52.60C -53.60C -53.20C

Probe location of Maximum temp. &

shelf number T35, 7th Shelf

T50; 10th

Shelf T25; 5th shelf

Acceptance criteria: Temp. on each shelf and across all the shelves during the hold period should be with in ± 3.0oC

from the Average

0.70C 1.30C 1.00C

Table 5.6 Results of Shelf temperature uniformity study results at -55C

5.3.4 Shelf temperature uniformity test results at 00

C

Description

Observation

RSD

Run 1

Run 2 Run 3


-1.00C -1.00C -0.90C

<2%

Probe location of Minimum temp. & shelf number

T10, 2nd shelf

13th

probe; 3rd shelf

T41,T44, T47,T49,T51; 5th shelf


0.60C 0.80C 0.60C

Probe location of Maximum temp. & shelf number

T23, 5th shelf

18th

probe; 4th shelf

T21,T22,T28, T48,T50; 5th, 6th,9th, 10th shelf

Acceptance criteria: Temp. on each shelf and across all the shelves during the hold period are with in ± 3.0oC from the Average

0.80C 0.70C 0.80C

Table 5.7 Results of Shelf temperature uniformity study results at 0C

Chapter-5 Results


5.3.4 Shelf temperature uniformity test results at 600

C

Description

Observation

Run1 Run 2 Run 3


57.10C 58.10C 58.10C

Probe location of Minimum Temperature & shelf number

T18, Shelf 4th

7th probe; 2nd shelf

T21; 5th shelf


59.20C 60.10C 60.30C

Probe location of Maximum Temperature

& shelf number T07, Shelf 2nd

T1,T15,T38,T40 ; 1st, 3rd, 8th shelf

T44; 9th shelf

Acceptance criteria: Temp on each shelf and across all the shelves during the hold period are with in ± 3.0oC from the Average

1.10C 0.90C 0.90C

Table 5.8 Results of shelf temperature uniformity study at +60C

5.3.5 Condenser Capacity

Description Acceptance Criteria/ Set

Value

Observation

Rated Capacity of condenser Not Applicable 100 kg

Total Amount of water loaded in Lyophilizer (A)

NLT rated capacity of condenser

112 Kg

Duration of freezing at -40℃ NLT 3 hours 70 min

Duration of Primary Drying NLT 21 hours 960 min

Duration of secondary drying NLT 21 hours 960 min

Maximum vacuum during drying:

Not less than 0.3 mbar 0.2367 mbar

Total water remaining in trays

at end of cycle (B) Not Applicable 0.300 kg

Total amount of water sublimed from trays and deposited on condenser (A-B)

Should not be less than the rated capacity of the condenser

111.7 kg

Table 5.9 Results of condenser capacity

Chapter-5 Results


5.3.6 Sterilization in Place

Description Acceptance

Criteria

Observation

Run 1 Run 2 Run 3

Cycle started at Not applicable 12:44:40 23:15:15 9:04:25

Sterilization Set temperature

NLT 121.5 0C 121.30C 121.30C 121.30C

Sterilization Hold

started at Not applicable 13:08:00 23:46:45 09:43:40

Sterilization hold end at

Not applicable 13:24:15 23:46:45 9:59:45

Total sterilization time

NLT 15 min. 16 mins 15 secs 16 mins 05 secs

16 mins 05 secs

Temperature band during sterilization Hold time

121.0 0C to 124.0 0C

Minimum temperature= 121.30C Maximum temperature= 123.80C

Minimum temperature= 121.50C Maximum temperature= 123.90C

Minimum temperature=121.30C Maximum temperature

=123.80C

Minimum F0 value calculated

NLT 15 min 23.7 mins 21.6 mins 22.4 mins

Biological Indicator recovery

No growth after defined

incubation No growth No growth No growth

Table 5.10 Results of sterilization in place

5.3.7 Cleaning In Place (CIP)

Table 5.11 Results of clean in place

Description Acceptance

Criteria

Observation

Run 1 Run 2 Run 3

Concentration of NaCl solution spiked

1 % 1 % 1 % 1 %

pH of finale drain water 5.0 to 7.0 5.87 5.82 5.95

Conductivity of finale drain water

NMT 1.3 μS 0.77 0.90 0.77

TOC of finale drain water NMT 500 ppb 32.8 28.2 180

Complies/ does not complies Shall Comply complies complies complies

Chapter-5 Results


5.3.8 Lyophilization process Test Results:

Description Acceptance Criteria Observation

Moisture content measured

NMT 2.0 % 0.61%

Cake Height Should be uniform Uniform cake height

observed

Dissolution time NMT 30 sec. 7Seconds

Complies or doesn’t complies

Shall comply complies

Table 5.12 Results of Lyophilization process test

5.4 Performance qualification reports of autoclave

4.3.2 5.4.1 Verification of Data logger and RTD Sensors calibration (Pre

calibration and post calibration details):

Sr.

No

Instrume

nt

Acceptanc

e criteria

complies/

doesn’t

complies

1 Data

Logger

+ 0.20C

compli

es

2 RTD

sensors

+0.25%

compli

es

Table 5.13 Results of data logger and RTD sensors calibration

5.4.2 Vacuum Leak test -

Sr.No

Run No.

Max Leakage observed (bar)

Acceptance criteria Complies/Doesn’t

complies

1. 1 0.004 The drop in vacuum should not be more than 0.013 bars during 10 minutes hold period

complies

2. 2 0.000 complies

3. 3 0.000 complies

Table 5.14 Results of vacuum leak test

Sr.

No

Instrum

ent

Acceptanc

e criteria

complies/

doesn’t

complies

1 Data

Logger

+ 0.20C

compli

es

2 RTD

sensors

+0.25%

compli

es

Chapter-5 Results


5.4.3 Air Removal Test

Sr.no

Run No.

Test Observations Acceptance criteria Inference

1. 1 Uniform dark black color observed

Bowie-Dick test kit should show uniform dark brown/ black color

development

Complies


complies


complies

Table 5.15 Results of air removal test

5.4.4 Heat Distribution and Penetration Studies

Table 5.16 Results of Heat Distribution and Heat Penetration Studies

Steril

izer

Run

No.

Observations

Min.

temp

Max.

temp

Min.

F0

value

Equilibr

ium

time

Max.

deviation

from average

temp

Max.

fluctuation

of temp on

a

RTDsensors

Max.

difference

between

two RTD

sensors

Result

1 121 122.

9

32.

4 30 complies

complies complies complies

2 121 123 32 30 complies complies complies complies

3 121 122.9 32.6 30 complies complies complies complies

Chapter-5 Results


Report 5.1 Temperature Sensors precalibration report of Autoclave

Chapter-5 Results

Dept of Quality Assurance KCP Bangalore Page 78

Report 5.2 Data Loggers Precalibration report of Autoclave

Chapter-5 Results


Report 5.3 Temperature Sensor precalibration report of Lyophilizer

Chapter-5 Results


Report 5.4 Datalogger precalibration report of Lyophilizer

Chapter-5 Results


Report 5.5 Maximum vacuum capacity, vacuum leak rate and Isolation valve integrity

report

Chapter-5 Results


Report 5.6 Heating and Cooling rate report

Chapter-5 Results


Report 5.7 Shelf temperature uniformity test report

Chapter-5 Results


Report 5.7 Shelf temperature uniformity test reports at 0

0C

Chapter-5 Results


Report 5.7 Shelf uniformity test reports at 600C

Chapter-5 Results


Graph 5.1 Shelf temperature uniformity test graph

Graph 5.2 Sterilization in Place test graph

Chapter-5 Results


Report 5.8 Sterilization in Place test report

Chapter-5 Results


Graph 5.3 Placebo test report

Chapter-5 Results


Report 5.9 Vacuum leak test report

Chapter-5 Results


Report 5.10 Air removal test report

Chapter-5 Results


Report 5.11 Heat distribution and penetration study reports

Chapter-5 Results


Graph 5.4 heat distribution and penetration study graph

Chapter-6 Discussion


6. DISCUSSION

All equipment’s to be used for the manufacture of sterile products must be validated to

demonstrate that the product can be manufactured in a reliable and reproducible manner

so as to reach the desired quality. Hence, the present work was undertaken with a goal to

carry out equipment validation for three consecutive times to prove that system remains

in control and process operating parameters are capable of consistently meeting the

predetermined acceptance criteria to produce a quality product.

6.1 Prerequisites for equipment validation:

Checking of approved documents, pre and post calibration of temperature sensors and

data loggers, review of prepared flow charts, instructions to be followed during the

validation availability of materials required and facility required, procedure for validation

are most important prerequisites for any effective equipment validation study. If any

single aspect is found missing during the validation study, it may lead to either delay in

validation study or deficient validation program. In this study all prerequisites were

followed and found ok prior to start the validation program.

6.2 Operation qualification of lyophilizer and qualification:

Operational qualification of the equipment ensures that the equipment is in accordance

with Design Qualification (DQ), functional requirements and meets CGMP and

regulatory expectations. This test verifies that the utility supplies are adequate and meet

the demands of the operating system. The OQ also evaluates each equipment function

and the capacity to meet the performance standards. After OQ of lyophilizer and

autoclave it has been found that equipment’s are made functional and tested for

functional requirements like alarm, safety, and interlocking features interface, display,



automation and control requirements, key functionality Software operation qualification

and found complying with acceptance criteria. Thus the Equipment meets its operational

qualification requirement.

6.3 Performance qualification of lyophilizer

The main purpose of performance qualification is to confirm through the provision of

objective evidence, that the performance requirements for the Lyophilizer meet its

intended use. It was said that for most protein formulations, annealing temperature and

time, freezing rates, shelf temperature, and chamber pressure are generally considered to

be the critical process parameters since they directly influence the quality of the drug

product. These limits need to be established through the robustness studies and should

serve as the basis for validation. Equipment is said to be validated when one can

demonstrate that the process can be performed consistently in three consecutive runs at

product while meeting the pre-determined acceptance criteria relating to process

parameters and product quality attributes.

Vacuum rate, maximum vacuum capacity and vacuum leakage rate test

The application of vacuum is the important step in lyophilization process to remove the

sublimated vapor from the chamber so finding the vacuum rate plays an important role in

validation. Implementation of calibrated leaks during primary drying offers a number of

advantages. The primary drying time of the product is shortened, and therefore a few

hours can be saved on the overall cycle time. In the absence of maximum vacuum i.e.,

pressure generated in the lyophilization chamber is sustained only by ice sublimation. A

leak in the lyophilizer probably leads to growth of microbes and product instability. So it



was found appropriate to do three consecutive runs for vacuum rate, maximum vacuum

capacity and vacuum leakage rate test in validation of Lyophilizer. Critical parameters

such as shelf temperature, chamber pressure, and time were accurately and precisely

controlled.

Isolated valve integrity test

Isolated valve connects the chamber with condenser. The integrity of the isolation valve

between the ice condenser and the drying chamber was maintained.

Shelf Cooling and Heating Rate Test

The rate at which the product is frozen can have a significant impact on the product

quality and should be controlled. Very fast freezing rate may lead to hard ice crystal so

that may be come difficult for the mass transfer. At the beginning of the primary drying,

the shelf heating rate should not be too high promote product melting at the base of the

cake. At the end of primary drying the ramping rate should not be too high so as to lead

to collapse or retrograde collapse.

Shelf Temperature Uniformity Test

It is needed to ensure that the temperature applied to the product vials kept across the

shelf during routine operation is uniformly heating as well as cooling during the

Lyophilization cycle. Many batches of the product lack the product uniformity because of

un uniform heat distribution. The regulatory agencies require pharmaceutical companies

to demonstrate the consistency of the drying process, in which case the design of the

sampling plan and the analytical testing must be in accordance with the current agency

expectations i.e. star-shaped sampling plan, demonstration of uniformity across the



shelves in terms of content, potency, particulate distribution, reconstitution time, residual

moisture, etc. are challenging.

Cleaning in Place (CIP) Test and sterilization in place (SIP)

CIP is required to evaluate the efficiency of the cleaning cycle. Improper cleaning of the

equipment leads to cross -contamination of the product. So the cleaning cycle must be

properly validated with spiking with 1% NaCl. SIP is required to check the sterility level

of the lyophilizer in accordance to its acceptance criteria level.

Placebo test

It is done to check the variation in residual moisture, cake height, dissolution time the

placebo test is being done using the 2.5% manitol. Thus the product uniformity and batch

stability can be assessed using the placebo test.

6.4 Performance qualification of autoclave

Vacuum leak test for sterilizer chamber

This test must be to assess the leakage in the autoclave chamber. Leak may lead to the

microbial growth thus in turn effects the sterility and quality o the product. Another

major problem caused due to leakage is formation of air pockets, which leads to in

adequate sterilization process. So it was found appropriate to do vacuum leak test while

validating the autoclave

Air removal test

This test is to check the uniform steam penetration throughout the chamber / load. The

main concern with steam sterilization is the complete removal of air from the chamber

and replacement with saturated steam.



Heat distribution and heat penetration studies:

This is the most critical component of the entire validation process. The key is to identify

reproducible basis the location of cool spot , the effect of the load size and configuration

on the cool spot location, and to ensure the sterility assurance level.

Chapter-7 Conclusion


7. CONCLUSION

Equipment validation of autoclave and lyophilizer in the manufacture of sterile

pharmaceuticals has been carried out as per approved validation protocol, process flow

chart and sampling plan.

All the data loggers and temperature used were calibrated and qualified before and after

the validation. Spore count enumeration and purity test of biological indicator was done

before the use. All the in-process parameters and process variables were checked and

found well within the limit. Samples were collected as per the approved sampling plan

and were submitted to QC for analysis. BIs are collected and were sent to the

microbiology lab. All the results were found well meeting the pre-defined acceptance

criteria.

No significant deviation in any process parameters like time, temperature and pressure

was observed during entire validation study. Hence all the specified process parameters

were consistent as per acceptance criteria.

Confidence in the ability of the equipment to create these necessary environmental

conditions is achieved through the successful completion of a comprehensive IQ and OQ.

Based on the review of the compiled reports and samples it was found that the obtained

values were found meeting the acceptance criteria specified in the protocol. Samples

submitted in the placebo test results for lyophilizer found the uniformity in the cake

formation and moisture content is maintained as per acceptance criteria. BI reports for

heat penetration study has assured the complete sterilization.

Based on the results of the validation data for 3 runs, it is concluded that the selected

cycle is therefore supposed to represent the target working conditions that will be applied

Chapter-7 Conclusion


for the future production. Validation of the equipment’s has lead development of the

target cycle to perform a batch-to –batch consistency in achieving the product sterility,

stability, dose Uniformity and safety. Thus the above validation provides an additional of

assurance that process is under control.

Chapter–8 Summary


8. SUMMARY

Equipment validation is a fundamental concept of cGMP. Lyophilization has become the

important process in the manufacture of sterile pharmaceuticals such as antibiotics and

proteins i.e. which are unstable in solution form. Overkill approach i.e. achieving the

sterility of final product has become a challenging part through autoclaving. So in order

to maintain the stability and sterility in pharmaceutical products the equipment validation

of lyophilizer and autoclave was chosen.

The ultimate objective of presented work was to carry validation of three consecutive

runs of having same equipment and same process parameters.

The entire validation and sampling procedure was adopted with total adherence to the

approved validation protocol and sampling plan. The Critical process parameters like

temperature, time, pressure, for all three runs were studied with respect to its effect on the

quality of the final product.

Operation qualification of lyophilizer and autoclave:

Verification of key functionality, safety features and alarms were checked and found

complying with the acceptance criteria

Operator interface, display automation, control requirements and security levels were

verified and found complying with the acceptance criteria.

Emergency stop features were verified and found complying with acceptance criteria.

Performance qualification of lyophilizer:

Vacuum rate, maximum vacuum capacity and vacuum leakage rate test:

The test was performed starting with an empty and dry chamber for eliminating any

vapours that may outgas or contribute to the evaporative pressure gains. The

Chapter–8 Summary


compressor was switched on and the condenser temperature was allowed to reach up to

≤ - 45ºC to absorb the moisture from the chamber. The vacuum was then started so that

it won’t get damaged due to moisture.

Vacuum rate: 12-16.1 minutes ( limit NMT 30 minutes )

Maximum vacuum capacity: 0.0080-0.0093 (limit ≤ 0.1 pa)

Vacuum leakage test: 0.0000222-0.000029 mbar.m3/sec ((limit ≤ 0.00003 mbar.m3/sec)

Isolated valve integrity test:

Chamber vacuum pressure: No drop in the vacuum pressure below the initial vacuum

pressure.

Shelf Cooling and Heating Rate Test (Oil In Temperature)

Cooling rate was measured from room temperature to the maximum operational

freezing temperature and heating rate was measured from lowest to the highest

operating temperatures. The maximum temperature was changed to 600C from 40

0C

in order to meet the current product temperature requirements. The results found

complying with acceptance criteria.

Shelf cooling rate: 1.520C/min-2.290C/min (limit ≥1.0 °C /min)

Shelf temperature uniformity test:

Monitoring of temperature was done using pre calibrated multi channel data loggers and

temperature sensors. The print interval and file recording interval for NMT 1 min was

set and temperature was recorded in data loggers. Monitoring of temperature was done

using pre calibrated multi channel data loggers and temperature sensors (minimum three

sensors for each operational shelf). The maximum temperature was changed to 600C

Chapter–8 Summary


from 400C in order to meet the current product temperature requirements. The results

found complying with acceptance criteria.

Shelf temperature uniformity results at -55 C : 0.70C-1.30C (limit ± 30C from the

Average)

Shelf temperature uniformity results at 0 C : 0.7-0.8 (limit ±30C from the Average)

Shelf temperature uniformity study results at +60 C : 0.9-1.1 (limit ± 30C from the

Average)

Cleaning In Place (CIP):

1% NaCl solution (using PW or WFI) was prepared. The sample of (about 100 mL)

prepared 1% NaCl solution was collected and sent to QC to check for presence of

chlorides.

Concentration of NaCl solution spiked: 1% (limit 1%)

pH of finale drain water: 5.87-5.95 (limit 5.0 to 7.0 )

Conductivity of finale drain water : 0.77-0.92 μS (limit NMT 1.3 μS)

TOC of finale drain : 28.2-180 (limit NMT 500 ppb)

Sterilization In Place:

Logged data will include the time, temperature and channel number labels of each

temperature sensor during each minute print interval. Synchronize clocks on all

equipment used in this test.

Minimum F0 value calculated: 21.6 mins-23.7 mins (limits NLT 15 min)

Biological Indicator recovery: No growth

Chapter–8 Summary


Performance qualification of autoclave:

Vacuum leak test for sterilizer chamber:

Vacuum leak test was performed before and after the validation to ensure the

integrity of the autoclave chamber. The vacuum was hold for 10 minutes to check

the leak after attaining the desired pressure.

Max Leakage observed: 0.0-0.004bar (limit NMT 0.013 bar)

Air removal test:

Air must be completely replaced with the saturated steam to produce the complete

sterility.

Uniform dark black color observed (limit: Bowie-Dick test kit should show uniform

dark brown/ black color development)

Heat distribution and penetration study:

Data loggers used are calibrated before and after the validation of the autoclave.

T-type RTD sensors are used instead of thermocouples because sensitivity is more

for RTD sensors.

Minimum F0 value was found between 32-32.60C (Limit: above the sterilization

hold time).

Results of BI were found complying with the acceptance criteria.

On assessment of the data for various physicochemical test parameters it was summarized

that the process, parameters, specifications and controls have been adequate to show the

total conformance of the product to specifications. So presented study has revealed that

Chapter–8 Summary


the set process parameters for the validation of lyophilizer and autoclave could be

reproduced during the process resulting in the product meeting the specifications.

.

Chapter–9 Bibliography


9. BIBLIOGRAPHY

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