Basis Validation of Biomanufacturing Processes

Validation Of Pharmabio-Validation Of Pharmabio-
manufacture Processesmanufacture Processes
Angel L. Salaman, PhDAngel L. Salaman, PhD
angelsalaman@yahoo.comangelsalaman@yahoo.com

What is Validation?What is Validation?
Validation – An Essential Part of GMPs!Validation – An Essential Part of GMPs!
Validation is the scientific study of a systemValidation is the scientific study of a system
 To prove that the facility/system/equipment/method isTo prove that the facility/system/equipment/method is
consistently doing what it is supposed to do (i.e., that theconsistently doing what it is supposed to do (i.e., that the
process is under control).process is under control).
– We want to make decisions based on good science andWe want to make decisions based on good science and
not hunches and assumptions!not hunches and assumptions!
 To determine the process variables and acceptable limits forTo determine the process variables and acceptable limits for
these variables, and to set-up appropriate in-processthese variables, and to set-up appropriate in-process
controls.controls.
– Is it ok if the wash from a chromatography column is pHIs it ok if the wash from a chromatography column is pH
6.8 vs. 7.0 ?6.8 vs. 7.0 ?

FDA definition ofFDA definition of
validationvalidation
““Validation is a process ofValidation is a process of
demonstratingdemonstrating, through, through documenteddocumented
evidenceevidence, that a process, procedure,, that a process, procedure,
method, piece of equipment, ormethod, piece of equipment, or
facility willfacility will consistentlyconsistently produce aproduce a
product or result that meetsproduct or result that meets
predetermined specificationspredetermined specifications andand
quality attributesquality attributes.”.”

Quality AttributesQuality Attributes??
IdentityIdentity
– 21 CFR 211.84 (d) at least one test shall be conducted to verify the identity of each21 CFR 211.84 (d) at least one test shall be conducted to verify the identity of each
component of a drug product.component of a drug product.
– Chemical, biological, ImmunologicalChemical, biological, Immunological
– Raw materials, In-process intermediates, final products.Raw materials, In-process intermediates, final products.
 SafetySafety
– 21 CFR 600.3 (p) safety as the relative freedom from harmful effect to persons affected,21 CFR 600.3 (p) safety as the relative freedom from harmful effect to persons affected,
directly or indirectly, by a product when prudently administered, taking into considerationdirectly or indirectly, by a product when prudently administered, taking into consideration
the character of the product in relationship to the condition of the recipient at the time.the character of the product in relationship to the condition of the recipient at the time.
 Activity of active ingredientsActivity of active ingredients
 Activity of the excipients or additivesActivity of the excipients or additives
 Activity of process related impuritiesActivity of process related impurities
 EfficacyEfficacy
– Effectiveness of the product in achieving its medicinal purpose (therapeutic, prophylactic,Effectiveness of the product in achieving its medicinal purpose (therapeutic, prophylactic,
diagnostic). Gathered at phase II and Phase III trials.diagnostic). Gathered at phase II and Phase III trials.
 PotencyPotency
– 21 CFR 600.3 (s) specific ability or capacity of the product, as indicated by its appropriate21 CFR 600.3 (s) specific ability or capacity of the product, as indicated by its appropriate
laboratory tests or by adequately controlled clinical data obtained through the administrationlaboratory tests or by adequately controlled clinical data obtained through the administration
of the product in the manner indicated to effect the given result.of the product in the manner indicated to effect the given result.
 PurityPurity
– 21 CFR 600.3 (r) relative freedom from extraneous matters in the finished product, whether21 CFR 600.3 (r) relative freedom from extraneous matters in the finished product, whether
or not harmful to the recipient or deleterious to the product.or not harmful to the recipient or deleterious to the product.
 Cleaning ProceduresCleaning Procedures
 StabilityStability
– 21 CFR 211.137 (a) to assure that a drug product meets applicable standards of identity,21 CFR 211.137 (a) to assure that a drug product meets applicable standards of identity,
quality, and purity at the time of use; it shall bear an expiration date determined by stabilityquality, and purity at the time of use; it shall bear an expiration date determined by stability
testing. Drugs may use accelerated time studies, biologics must use real time studies.testing. Drugs may use accelerated time studies, biologics must use real time studies.
 ConsistencyConsistency
– The ability of the product and/or process to reliably possess specified quality attributes on anThe ability of the product and/or process to reliably possess specified quality attributes on an
ongoing basis. 3 consecutive batches of product meeting predetermined specifications isongoing basis. 3 consecutive batches of product meeting predetermined specifications is
accepted as proof that a process is consistent. However, in NDA data from up to twentyaccepted as proof that a process is consistent. However, in NDA data from up to twenty
batches may be submitted.batches may be submitted.

BiomanufacturingBiomanufacturing
 Biomanufacturing is a complex process involvingBiomanufacturing is a complex process involving
multiple unit operations many of which aremultiple unit operations many of which are criticalcritical
to insuring patient safety and product efficacyto insuring patient safety and product efficacy

Inoculums
Seed
Fermentation
Production
Fermentation
Harvest
Ultrafiltration
1
Chrom. 1
1
Ultrafiltration
2
Chrom.
2
Viral
Filtration
Chrom.
3
Ultrafiltration
3
Final Formulation/
Sterile Filtration
Sterile Fill
UPSTREAM
DOWN-
STREAM
VIRAL
NON-VIRAL
Block Flow Diagram of a typical Production Process

Historical Basis forHistorical Basis for
ValidationValidation
 AssumptionsAssumptions concerning virus inactivationconcerning virus inactivation
resulted in ten deaths and 200 childrenresulted in ten deaths and 200 children
becoming paralyzed, from a supposedlybecoming paralyzed, from a supposedly
“inactivated” polio vaccine.“inactivated” polio vaccine.
 AssumptionsAssumptions about sterilization causedabout sterilization caused
severe infections among burn victims givensevere infections among burn victims given
supposedly sterile solutions.supposedly sterile solutions.
 Validation eliminates assumptions and reliesValidation eliminates assumptions and relies
on experimental proof!on experimental proof!

Quality by DesignQuality by Design
 A central concept in quality is thatA central concept in quality is that quality can not bequality can not be
tested fortested for.. Quality must be designed and built into theQuality must be designed and built into the
production process.production process.
– Requires careful attention to raw materialRequires careful attention to raw material
specificationsspecifications, in process material, in process material specificationsspecifications,,
and final productand final product specificationsspecifications..

Validation andValidation and
QualityQuality
 Validating the performance of unit operations,Validating the performance of unit operations,
analytical methods, and critical process pointsanalytical methods, and critical process points
(sterilization, viral inactivation, cleaning(sterilization, viral inactivation, cleaning
procedures) is essential in insuring that theprocedures) is essential in insuring that the
process generates a quality product.process generates a quality product.

Validation in BiomanufacturingValidation in Biomanufacturing
 Validation does not replace testing, but it doesValidation does not replace testing, but it does
reduce the testing burden for raw materials,reduce the testing burden for raw materials,
in-process materials, and final productin-process materials, and final product

 Validation itself is a process that evolves withValidation itself is a process that evolves with
the product.the product.
– Validation requirements for production ofValidation requirements for production of
pre-clinical material less stringent then forpre-clinical material less stringent then for
phase III clinical material.phase III clinical material.
– Critical operations must be validatedCritical operations must be validated: For: For
example: raw materials, analyticalexample: raw materials, analytical
methods, viral clearance, sterilization,methods, viral clearance, sterilization,
cleaning.cleaning.

 A fully validated process is “locked in”A fully validated process is “locked in”
 Any change outside of the validated spaceAny change outside of the validated space
invalidates processinvalidates process
 Change must be evaluated for effect on patientChange must be evaluated for effect on patient
safety and product efficacy-safety and product efficacy-Change control !Change control !
Validated Production Process
Δ

Less
More
Difficulty in Changing and Binding Authority
LAWS
Regulation
s
Guidance
Less
More
LAWS
Regulation
s
Guidance
Scientific Content & Detail about Implementation

Regulatory requirementsRegulatory requirements
for validationfor validation
21 CFR 211 Subpart H- Holding and Distribution21 CFR 211 Subpart H- Holding and Distribution
– 211.165 – Testing and release for211.165 – Testing and release for
distributiondistribution
 ““Requires that the accuracy, sensitivity,Requires that the accuracy, sensitivity,
specificity, and reproducibility of testspecificity, and reproducibility of test
methods employed by the firm shall bemethods employed by the firm shall be
established and documented.established and documented. Such validationSuch validation
and documentation may be accomplished inand documentation may be accomplished in
accordance with 21 CFR 211.194 (a)(2)”accordance with 21 CFR 211.194 (a)(2)”

 Sec. 211.113 Control of microbiologicalSec. 211.113 Control of microbiological
contamination.contamination.
 (a) Appropriate written procedures, designed to(a) Appropriate written procedures, designed to
prevent objectionable microorganisms in drugprevent objectionable microorganisms in drug
products not required to be sterile, shall beproducts not required to be sterile, shall be
established and followed.established and followed.
 (b) Appropriate written procedures, designed to(b) Appropriate written procedures, designed to
prevent microbiological contamination of drugprevent microbiological contamination of drug
products purporting to be sterile, shall be establishedproducts purporting to be sterile, shall be established
and followed.and followed. Such procedures shall include validationSuch procedures shall include validation
of any sterilization process.of any sterilization process.
Regulatory requirementsRegulatory requirements
for validationfor validation

What does “validation of anyWhat does “validation of any
sterilization process” mean ?sterilization process” mean ?
 What parameters are critical to sterilization?What parameters are critical to sterilization?
– Temperatures, pressures, time, pore sizeTemperatures, pressures, time, pore size
(filtration), radiation dosage, chemical(filtration), radiation dosage, chemical
concentration.concentration.
 Must demonstrate that your autoclave reaches theMust demonstrate that your autoclave reaches the
temperatures, pressures, and times necessary fortemperatures, pressures, and times necessary for
sterilization.sterilization.
 Must demonstrate that items representing real worldMust demonstrate that items representing real world
samples achieve those conditions ( 20 ft of 1 ½ hose;samples achieve those conditions ( 20 ft of 1 ½ hose;
a 20 L carboy; a 500 ml bottle).a 20 L carboy; a 500 ml bottle).
 Must challenge with worse case scenario (may takeMust challenge with worse case scenario (may take
place in pilot plant if scalability demonstrated).place in pilot plant if scalability demonstrated).

Regulatory guidance onRegulatory guidance on
 Guideline on General Principals of Process ValidationGuideline on General Principals of Process Validation
http://www.fda.gov/cder/guidance/pv.htmhttp://www.fda.gov/cder/guidance/pv.htm
 Guidance for Industry: For the Submission DocumentationGuidance for Industry: For the Submission Documentation
for Sterilization Process Validation in Applications for Humanfor Sterilization Process Validation in Applications for Human
and Veterinary Drug Products. CDER CVM November 1994.and Veterinary Drug Products. CDER CVM November 1994.
www.fda.gov/CDER/GUIDANCE/cmc2.pdfwww.fda.gov/CDER/GUIDANCE/cmc2.pdf
 Working Party on Control of Medicines and InspectionsWorking Party on Control of Medicines and Inspections
 Final Version of Annex 15Final Version of Annex 15 to the EU Guide to Goodto the EU Guide to Good
Manufacturing PracticeManufacturing Practice
 Title: Qualification and validationTitle: Qualification and validation
 http://pharmacos.eudra.org/F2/eudralex/vol-4/pdfs-http://pharmacos.eudra.org/F2/eudralex/vol-4/pdfs-
en/v4an15.pdfen/v4an15.pdf
 ICH Q7a Section 12 on validationICH Q7a Section 12 on validation
 http://www.fda.gov/cder/meeting/ICH_Q7A/index.htmhttp://www.fda.gov/cder/meeting/ICH_Q7A/index.htm
 A WHO guide to good manufacturing practice (GMP)A WHO guide to good manufacturing practice (GMP)
requirements. Part 2: Validationrequirements. Part 2: Validation
 Chaloner-Larsson, G., Anderson, R., and Egan, A. 1997.Chaloner-Larsson, G., Anderson, R., and Egan, A. 1997.
World Health Organization, Geneva.World Health Organization, Geneva.

Critical Operations inCritical Operations in
BiomanufacturingBiomanufacturing
 Some operations are more critical than others.Some operations are more critical than others.
– Viral filtration, sterilization, cleaning, analyticalViral filtration, sterilization, cleaning, analytical
methods.methods.
– These operations will require greater validationThese operations will require greater validation
efforts then less critical operations (mediaefforts then less critical operations (media
blending).blending).

TestingTesting
 Usually done by the Quality ControlUsually done by the Quality Control
LaboratoryLaboratory
– CFR requires thatCFR requires that quality unitquality unit be underbe under
independent supervision and reportindependent supervision and report
directly to senior managementdirectly to senior management

QualityQuality
AssuranceAssurance
 Reviews records from quality control and productionReviews records from quality control and production
departmentsdepartments
– Verifies that all specifications and productionVerifies that all specifications and production
operations met / performedoperations met / performed
– Investigations necessary for any deviationsInvestigations necessary for any deviations
 Root causeRoot cause
 Affect on qualityAffect on quality
 Corrective action (CAPA)Corrective action (CAPA)
– Approves final release of productApproves final release of product

Designing Quality intoDesigning Quality into
the Productthe Product
 Design of production process and specificationsDesign of production process and specifications
all contribute to a quality product:all contribute to a quality product:
– Absence of contaminationAbsence of contamination
 Clean rooms, closed systems, use of BSCClean rooms, closed systems, use of BSC
for critical operations.for critical operations.
– PurityPurity
 Separation process (chromatography)Separation process (chromatography)
designed to remove potentialdesigned to remove potential
contaminantscontaminants
 Viral purification / inactivationViral purification / inactivation

PlanPlan
 Organizations must define anOrganizations must define an
approach towards validationapproach towards validation
– What is to be validatedWhat is to be validated
– How is it to be validatedHow is it to be validated
– Who is to validate itWho is to validate it
– Who is to approve the validationWho is to approve the validation
– When it must be revalidatedWhen it must be revalidated

Examples of individualExamples of individual
systems subject tosystems subject to
validation:validation:
HVAC systemsHVAC systems
AutoclavesAutoclaves
pH meterspH meters
Depyrogenation OvensDepyrogenation Ovens
LyopholyzersLyopholyzers
CentrifugesCentrifuges
Steam generatorsSteam generators
Water systemsWater systems
Compressed air systemsCompressed air systems
Vacuum systemsVacuum systems

Validation PlanValidation Plan
 Regulatory agencies (FDA, EMEA, WHO, etc) identifyRegulatory agencies (FDA, EMEA, WHO, etc) identify
minimum components of validation.minimum components of validation.
 ““Industry standards” (the c in cGMP) can increaseIndustry standards” (the c in cGMP) can increase
validation requirements.validation requirements.
 New & Novel processes / equipment require greaterNew & Novel processes / equipment require greater
scrutiny then established processes / equipment.scrutiny then established processes / equipment.
 Validation requirements increase as a product movesValidation requirements increase as a product moves
through development (phase I, phase II, phase III).through development (phase I, phase II, phase III).

Validation PlansValidation Plans
The Validation Master PlanThe Validation Master Plan
– A high level document that outlines theA high level document that outlines the
organizations philosophical approach to validationorganizations philosophical approach to validation
and revalidation. The master validation planand revalidation. The master validation plan
becomes a guideline by which individual validationbecomes a guideline by which individual validation
protocol are developed and implemented.protocol are developed and implemented.
– May contain a flow chart or other diagram of theMay contain a flow chart or other diagram of the
validation processvalidation process

A prospectiveA prospective
validation studyvalidation study
IQ
OQ
Calibration
PQ protocol
approval
PQ protocol
execution
Data Analysis
Validation
Report
Approve
Conclusions

DevelopmentalDevelopmental
studiesstudies
 Experiments designed to explore and define the limits ofExperiments designed to explore and define the limits of
the system to be validatedthe system to be validated
– Sterilization developmental studies may focus onSterilization developmental studies may focus on
“worst case ” or hard to sterilize items“worst case ” or hard to sterilize items
– Cleaning developmental studies may focus on “worstCleaning developmental studies may focus on “worst
case” or hard to clean itemscase” or hard to clean items
– Analytical methods may focus on defining the limitsAnalytical methods may focus on defining the limits
of the procedure (range, recovery, etc)of the procedure (range, recovery, etc)
– Developmental studies then used to developDevelopmental studies then used to develop
validation protocols and refine SOP’svalidation protocols and refine SOP’s

Validation ProtocolValidation Protocol
 Specific protocols (SOP’s) that provide detailedSpecific protocols (SOP’s) that provide detailed
information on what is to be validated.information on what is to be validated.
 Validation Protocols consist of:Validation Protocols consist of:
– A description of the process, equipment, or methodA description of the process, equipment, or method
to be validated.to be validated.
– A description of the validation method.A description of the validation method.
– A description of the sampling procedure including theA description of the sampling procedure including the
kind and number of samples.kind and number of samples.
– Acceptance criteria for test results.Acceptance criteria for test results.
– Schedule or criteria for revalidation.Schedule or criteria for revalidation.

Example of a protocol for the
IQ component of validating a
pH meter
As with all other SOP’s this
document will contain an
Objective, scope, and
responsibility
Section.

 Validation Protocols may consist of multipleValidation Protocols may consist of multiple
SOP’s each describing specific steps in theSOP’s each describing specific steps in the
validation processvalidation process

Critical SystemsCritical Systems
 How critical is the system being validated to finalHow critical is the system being validated to final
product quality?product quality?
– Media blending systems for cell growth vs. final fill &Media blending systems for cell growth vs. final fill &
finish operationsfinish operations
 Demonstrating that the device which fills, labels,Demonstrating that the device which fills, labels,
and caps the final product will require moreand caps the final product will require more
extensive validation then the blenders used toextensive validation then the blenders used to
prepare media for bioreactors.prepare media for bioreactors.
 Validation of complex devices may take years!Validation of complex devices may take years!

 Proceeds in stages with new facilities /Proceeds in stages with new facilities /
equipment.equipment.
 Planning for validation should start withPlanning for validation should start with
the design process.the design process.
 Leaving validation to the last minute isLeaving validation to the last minute is
asking for trouble.asking for trouble.

Stages of ValidationStages of Validation
 Starts with Design & Receipt:Starts with Design & Receipt:
– Does the equipment meet the needs (is theDoes the equipment meet the needs (is the
autoclave big enough?)autoclave big enough?)
– Do you have the manuals, spare parts, can youDo you have the manuals, spare parts, can you
plug it in?plug it in?
– Is it installed properly (drain lines, vents, etc)Is it installed properly (drain lines, vents, etc)
 Does it work?Does it work?
– Does the autoclave reach the necessary temp.Does the autoclave reach the necessary temp.
and pressure?and pressure?
– Can the autoclave sterilize your equipmentCan the autoclave sterilize your equipment
(worse case situation)?(worse case situation)?
 How does it work in the manufacturing process?How does it work in the manufacturing process?
– Can it handle production quantities?Can it handle production quantities?
– Will failure compromise product quality?Will failure compromise product quality?

IQ, OQ, PQ ?IQ, OQ, PQ ?
Installation Qualification (IQ)Installation Qualification (IQ)
A process used to document that the piece of equipmentA process used to document that the piece of equipment
was supplied and installed properly and that appropriatewas supplied and installed properly and that appropriate
utilities, i.e., electrical, steam, gas, etc. are available toutilities, i.e., electrical, steam, gas, etc. are available to
operate the equipment according to the manufacturersoperate the equipment according to the manufacturers
specifications.specifications.
Operational Qualification (OQ)Operational Qualification (OQ)
A process designed to supply the documented evidenceA process designed to supply the documented evidence
that a piece of equipment operates as it is intendedthat a piece of equipment operates as it is intended
through all anticipated operational ranges.through all anticipated operational ranges.
Performance (Process) Qualification (PQ)Performance (Process) Qualification (PQ)
Verifies that a process / piece of equipment performs as itVerifies that a process / piece of equipment performs as it
is intended to in the manufacturing process and producesis intended to in the manufacturing process and produces
product (in process or final) meeting predeterminedproduct (in process or final) meeting predetermined
specifications.specifications.

Typical information in anTypical information in an
IQ protocolIQ protocol
 Name and description of equipment, including modelName and description of equipment, including model
numbersnumbers
 Identification, including model and serial numbersIdentification, including model and serial numbers
 Location of the equipmentLocation of the equipment
 Any utility requirements, i.e. electrical voltage, steamAny utility requirements, i.e. electrical voltage, steam
or water pressure, etc.or water pressure, etc.
 Any safety features of the equipment, includingAny safety features of the equipment, including
alarms, interlocks, or relief valves.alarms, interlocks, or relief valves.
 That all documentation, including manufacturersThat all documentation, including manufacturers
contact information, spare parts inventory, operationalcontact information, spare parts inventory, operational
manual, and installation drawings are available onmanual, and installation drawings are available on
site.site.

 ObjectiveObjective
 ResponsibilityResponsibility
 Equipment required (Calibration verification &Equipment required (Calibration verification &
Traceability)Traceability)
 SOP(s) usedSOP(s) used
 Equipment IdentificationEquipment Identification
 Parameters measured (Specifications)Parameters measured (Specifications)
 DocumentationDocumentation
Typical information in anTypical information in an
IQ protocolIQ protocol

 Ideally validation takes place prior to actual production runs,Ideally validation takes place prior to actual production runs,
however in some cases validation may take place as producthowever in some cases validation may take place as product
is produced, or past production runs may be used to provideis produced, or past production runs may be used to provide
validation data.validation data.
 Prospective ValidationProspective Validation
 Concurrent ValidationConcurrent Validation
 Retrospective ValidationRetrospective Validation

RevalidationRevalidation
 Is the initial validation of a piece of equipment the end?Is the initial validation of a piece of equipment the end?
– No!No!
– Periodic revalidation may be necessary depending onPeriodic revalidation may be necessary depending on
the criticality of the equipmentthe criticality of the equipment
– Changes need to be evaluated for their impact onChanges need to be evaluated for their impact on
– Deviations from specifications may require revalidationDeviations from specifications may require revalidation
– Revalidation spelled out in Master Validation PlanRevalidation spelled out in Master Validation Plan

Change ControlChange Control
 Must assess impact of changes on FDA complianceMust assess impact of changes on FDA compliance
and validation state.and validation state.
 Change control is a formal process defined in companyChange control is a formal process defined in company
SOP on how process/equipment changes areSOP on how process/equipment changes are
evaluated.evaluated.
 Any change that takes place outside the changeAny change that takes place outside the change
control process can jeopardize product quality (patientcontrol process can jeopardize product quality (patient
safety).safety).

Autoclave ValidationAutoclave Validation
 IQ-IQ-
– Design specifications meet users needsDesign specifications meet users needs
– Proper installation, utilities, manuals,Proper installation, utilities, manuals,
spare partsspare parts

The DQ, IQ, OQ process
insures that this autoclave
will meet the needs of the
manufacturing group.

Sample Format for Installation
Qualification of an autoclave.
Courtesy of WHO. Chaloner-Larsson, G.,
Anderson, R., Egan, A. 1997. A WHO guide to good
manufacturing practice (GMP) requirements Part 2:
Validation .
World Health Organization, Geneva
. www.who.int/vaccines-documents/DocsPDF/www9666.pdf
Accessed on October 2nd, 2006.

Autoclave ValidationAutoclave Validation
 OQOQ
– Does it operate properlyDoes it operate properly
 Does it reach the specified temperatureDoes it reach the specified temperature
and pressureand pressure
 Do timers workDo timers work
 Does the operator interface panel workDoes the operator interface panel work
 Are safety interlocks functionalAre safety interlocks functional

Sample Format for Operational
Qualification of an autoclave.
Courtesy of WHO. Chaloner-Larsson, G.,
Anderson, R., Egan, A. 1997. A WHO guide to good
manufacturing practice (GMP) requirements Part 2: Validation .
World Health Organization, Geneva
. www.who.int/vaccines-documents/DocsPDF/www9666.pdf A
ccessed on October 2nd, 2006.

CalibrationCalibration
Figure 1: Operational
qualification of an
autoclave requires the
calibration of instruments
against traceable
standards. This figure
shows the calibration of a
validator (out of view)
against the IRTD probe.

Monitoring TemperatureMonitoring Temperature
Figure 2: A validator, used in the
operational qualification of an
autoclave. The validator is
attached to individual
thermocouples by wires coming
from the rear of the instrument
(arrow). Tha validator has been
previously calibrated and the data
gathered from the thermocouples
will be logged on the laptop
computer. The software on the
computer is also subject to
validation requirements.

The validator is attached to
individual thermocouples
(TC) by thin wires that pass
through the wall of the
autoclave through a
specially designed port
(arrow). This picture shows
the back side of the
autoclave. The validator is
out of view at the lower left.

The inside of the autoclave
showing the maze of wiring
connecting the individual
TC’s to the validator. The
port through which the wires
pass is visible in the middle
left of the picture. The
individual TC’s will be
placed in various areas of
the autoclave or equipment
being autoclaved to
generate a thermal map of
the interior of the autoclave.

Output from the validator. The
temperature at each
connected thermocouple is
displayed. Accumulated
lethality (F 0 ) may also be
displayed. Notice how some
TC have failed and will not
record a temperature.
Accounting for TC failure is
necessary to keep from
having to repeat a study.

Proving that sufficient lethality
(F0) is achieved within the nooks
and crannies of
biomanufacturing equipment
requires the placement of TC’s
in hard to reach areas, in this
case deep within a piece of
tubing. Special gaskets with
openings for the TC are used to
insert the TC within pieces of
equipment.

Developmental studiesDevelopmental studies
 Experiments designed to explore and define theExperiments designed to explore and define the
limits of the system to be validatedlimits of the system to be validated
– Sterilization developmental studies may focusSterilization developmental studies may focus
on “worst case ” or hard to sterilize itemson “worst case ” or hard to sterilize items
– Cleaning developmental studies may focus onCleaning developmental studies may focus on
“worst case” or hard to clean items“worst case” or hard to clean items
– Analytical methods may focus on defining theAnalytical methods may focus on defining the
limits of the procedure (range, recovery, etc)limits of the procedure (range, recovery, etc)
 Developmental studies then used to developDevelopmental studies then used to develop
validation protocols and refine SOP’svalidation protocols and refine SOP’s

Developmental StudiesDevelopmental Studies
Developmental studies are
used to identify hard to
autoclave items and to
test if item preparation has
an effect on ability to be
sterilized

Every little nook and cranny
Needs to be assessed ! We
have to prove that the inside
of the pipe reaches sufficient
temperature, for a long
enough time to insure sterility!

Does bagging make a difference?

Cleaning ValidationCleaning Validation
Validating the cleaning cycle
on a loaded dishwasher.
Notice the various pipes and
parts with narrow openings.
Identification of hard to clean
and easy to clean areas starts
with developmental studies

How do we validate aHow do we validate a
dishwasher ?dishwasher ?
 How do we identify hard to clean & easy toHow do we identify hard to clean & easy to
clean items?clean items?
 How do we test to see if they are cleaned?How do we test to see if they are cleaned?

Cleaning ValidationCleaning Validation
Sampling, documenting, and
verifying is a labor intensive
process
Why is cleaning considered a
critical process?

SamplingSampling
Swabbing is commonly
used to sample the surface
of cleaned materials. The
swab is then placed in a
sample vial and sent to the
Quality Control lab for
analysis. Proper technique
is essential in order to
evaluate the effectiveness
of cleaning techniques.
Even so, swabbing results
are typically corrected for
known deficiencies in
recovery.

CleaningCleaning
Analysis of rinse water for
residual cleaning agents or
process materials is an
essential component of
cleaning validation. Insuring
that the sample is not
contaminated requires
vigilance and properly
following the relevant SOP’s.

Analytical methods validationAnalytical methods validation
 Considered a critical step in the manufacturing processConsidered a critical step in the manufacturing process
– Requirements for validated analytical methodsRequirements for validated analytical methods
explicitly written into the CFR’sexplicitly written into the CFR’s
– 211.165 – Testing and release for distribution211.165 – Testing and release for distribution
 ““Requires that the accuracy, sensitivity, specificity, andRequires that the accuracy, sensitivity, specificity, and
reproducibility of test methods employed by the firmreproducibility of test methods employed by the firm
shall be established and documented. Such validationshall be established and documented. Such validation
and documentation may be accomplished in accordanceand documentation may be accomplished in accordance
with 21 CFR 211.194 (a)(2)”with 21 CFR 211.194 (a)(2)”

Testing For IdentityTesting For Identity
 Requires the development of validated analyticalRequires the development of validated analytical
methods that can determine identity.methods that can determine identity.
 Chemical Tests:Chemical Tests:
– Is the molecule chemically what it is supposed toIs the molecule chemically what it is supposed to
be?be?
 Biological Activity Tests:Biological Activity Tests:
– Does the molecules have theDoes the molecules have the biologic activitybiologic activity that itthat it
is supposed to have?is supposed to have?
 Immunogenic Tests:Immunogenic Tests:
– Is the molecule immunogenic (allergic)?Is the molecule immunogenic (allergic)?

IdentityIdentity
 21 CFR requires testing of raw materials:21 CFR requires testing of raw materials:
– Raw materials quarantined until identity verifiedRaw materials quarantined until identity verified
– Raw materials must meetRaw materials must meet predeterminedpredetermined
specificationsspecifications
– Vendors (and alternates) specified in BLAVendors (and alternates) specified in BLA
(NDA)(NDA)

IdentityIdentity
 21 CFR requires testing of in-process21 CFR requires testing of in-process
materials:materials:
– Product from bioreactor / fermentorProduct from bioreactor / fermentor
– Product from purification stepsProduct from purification steps
– Waste products from aboveWaste products from above
Must meet specifications, if not - stopMust meet specifications, if not - stop
thethe
process to investigate take correctiveprocess to investigate take corrective
actionaction

 Specific protocol based on developmental studiesSpecific protocol based on developmental studies
 Protocol is written, reviewed and approvedProtocol is written, reviewed and approved
 Protocol is executedProtocol is executed
 Report written and approvedReport written and approved
 System is validatedSystem is validated

Analytical Methods ValidationAnalytical Methods Validation
 Being a critical component of productionBeing a critical component of production
process analytical methods must beprocess analytical methods must be
validatedvalidated
– Raw material testingRaw material testing
– In process materialsIn process materials
– Final product specificationsFinal product specifications

What must beWhat must be
demonstrateddemonstrated
 Selectivity (specificity)Selectivity (specificity)
 AccuracyAccuracy
 PrecisionPrecision
 Linear RangeLinear Range
 Limit of detection (LOD)Limit of detection (LOD)
 Limit of quantification (LOQ orLimit of quantification (LOQ or
LLOQ)LLOQ)
 RobustnessRobustness

ValidatedValidated
methodsmethods
 USP NF (United States Pharmacopeia NationalUSP NF (United States Pharmacopeia National
Formulary) contains “validated analytical methods”Formulary) contains “validated analytical methods”
 Use of a USP method does not eliminate theUse of a USP method does not eliminate the
organizations obligation to demonstrate that theorganizations obligation to demonstrate that the
method performs adequetlymethod performs adequetly

SelectivitySelectivity
(specificity)(specificity)
 Does the analytical method detect the component it is supposedDoes the analytical method detect the component it is supposed
to detect?to detect?
– Cross reactivity in antibody based methodsCross reactivity in antibody based methods
 Demonstrate specificity by conducting analytical method onDemonstrate specificity by conducting analytical method on
materials that may mimic analyte of interestmaterials that may mimic analyte of interest
– Looking for “false positives”Looking for “false positives”

AccurAccur
acyacy
 Ability of analytical method to accurately determineAbility of analytical method to accurately determine
the presence and amount of the analyte of interestthe presence and amount of the analyte of interest
– Typically done by analyzing a traceable standardTypically done by analyzing a traceable standard

RecovRecov
eryery
 Can we recover all of the analyte from a complex matrixCan we recover all of the analyte from a complex matrix
 May reflect sample preparation problemsMay reflect sample preparation problems
 Typical recovery studies done using “spiked” samplesTypical recovery studies done using “spiked” samples
 Final results may be corrected by the % recoveryFinal results may be corrected by the % recovery
– Swab samples typically corrected to reflect recoverySwab samples typically corrected to reflect recovery

LinearLinear
RangeRange
 Must define the linear range of aMust define the linear range of a
methodmethod
– Assay may have multiple linearAssay may have multiple linear
rangesranges

PrecisioPrecisio
nn
 How much variability does the assay exhibit when analyzingHow much variability does the assay exhibit when analyzing
the same samplethe same sample
– Typically demonstrated by analyzing multiple aliquots of aTypically demonstrated by analyzing multiple aliquots of a
homogenous samplehomogenous sample
– Acceptance criteria will depend on the assay and theAcceptance criteria will depend on the assay and the
material being assayed (2-20% RSD)material being assayed (2-20% RSD)
– Typically expressed as % RSD (relative standard deviation)Typically expressed as % RSD (relative standard deviation)

Limit of DetectionLimit of Detection
(LOD)(LOD)
 Lowest level at which method can detectLowest level at which method can detect
analyteanalyte
– Results reported as less than LODResults reported as less than LOD
– Based on signal to noise specificationBased on signal to noise specification
(10:1, 20:1)(10:1, 20:1)

RobustnessRobustness
studiesstudies
 How sensitive is the method to minor variations in methodHow sensitive is the method to minor variations in method
– Pipetting variationPipetting variation
– Temperature fluctuationTemperature fluctuation
– Reagent stabilityReagent stability
– Etc.Etc.
Detailed robustness studies will be reflected in final SOP’sDetailed robustness studies will be reflected in final SOP’s

Limit ofLimit of
Quantification (LOQ)Quantification (LOQ)
 Lowest level at which method can accurately quantify analyteLowest level at which method can accurately quantify analyte
– Based on signal-to-noise ratio specification (10:1, 20:1)Based on signal-to-noise ratio specification (10:1, 20:1)
and precision specificationand precision specification
 Precision and LOQ relatedPrecision and LOQ related
– Lower LOQ will typically result in lower precisionLower LOQ will typically result in lower precision

BradfordBradford
AssayAssay
for totalfor total
proteinprotein
Well known colorimetric assay that relies on the binding ofWell known colorimetric assay that relies on the binding of
Commassie G-250 dye to the proteins in an acidic solutionCommassie G-250 dye to the proteins in an acidic solution
– Dye binding proportional to number of positive charges inDye binding proportional to number of positive charges in
proteinprotein
– Proteins >3000 dal not detectedProteins >3000 dal not detected
 Simple, quick, wide range, few interfering agentsSimple, quick, wide range, few interfering agents

DisadvantaDisadvanta
gesges
 Incompatability with surfactantsIncompatability with surfactants
 Staining of glass and quartz cuvettesStaining of glass and quartz cuvettes
– Use disposable polystyrene cuvettesUse disposable polystyrene cuvettes
– Or wash with strong detergents and methanolOr wash with strong detergents and methanol

Validating theValidating the
BradfordBradford
 Selectivity (specificity)Selectivity (specificity)
 AccuracyAccuracy
 RecoveryRecovery
 PrecisionPrecision
 Linear RangeLinear Range
 Limit of detection (LOD)Limit of detection (LOD)
 Limit of quantification (LOQ or LLOQ)Limit of quantification (LOQ or LLOQ)
 RobustnessRobustness

Some QuestionsSome Questions
 A valve used to transfer material from a holding tank to theA valve used to transfer material from a holding tank to the
purification suite jam’s closed. You have a spare valve that ispurification suite jam’s closed. You have a spare valve that is
an identical model. Can you change this valve with the sparean identical model. Can you change this valve with the spare
and continue operations? What if the valve is from a differentand continue operations? What if the valve is from a different
manufacturer?manufacturer?
 You notice that your autoclave loading plan leaves room forYou notice that your autoclave loading plan leaves room for
additional material. Realizing that increasing that amount ofadditional material. Realizing that increasing that amount of
material in the autoclave will shorten the turn around timematerial in the autoclave will shorten the turn around time
for the production line you contemplate increasing thefor the production line you contemplate increasing the
amount of material loaded into the autoclave then specifiedamount of material loaded into the autoclave then specified
by the loading plan. What should you do? What will beby the loading plan. What should you do? What will be
required to implement this change?required to implement this change?
 An SOP for calibration of a pH meter calls for a two pointAn SOP for calibration of a pH meter calls for a two point
calibration at pH 4 and pH 7. You notice that a single pointcalibration at pH 4 and pH 7. You notice that a single point
calibration at pH 7 produces the same result from pHcalibration at pH 7 produces the same result from pH
measurements of your buffer solutions and allows you tomeasurements of your buffer solutions and allows you to
take a longer break. Is it Ok to do the one point calibrationtake a longer break. Is it Ok to do the one point calibration
when the SOP calls for a two point calibration? How wouldwhen the SOP calls for a two point calibration? How would
you go about changing the SOP to allow for a one pointyou go about changing the SOP to allow for a one point
calibration?calibration?

 What documents would provide information concerning theWhat documents would provide information concerning the
make and model of a particular valve used to regulate themake and model of a particular valve used to regulate the
transfer of material from a holding tank to the purificationtransfer of material from a holding tank to the purification
suite?suite?
 Your supervisor is concerned that the fermentation vesselYour supervisor is concerned that the fermentation vessel
is not providing sufficient aeration of the culture to getis not providing sufficient aeration of the culture to get
optimal growth and suggests installing a different kind ofoptimal growth and suggests installing a different kind of
baffle in the vessel. How would you demonstrate that thisbaffle in the vessel. How would you demonstrate that this
change has no effect on product quality?change has no effect on product quality?

ReferencesReferences
 Pharmaceutical Manufacturers Association’s (Pharmaceutical Research and Manufacturers ofPharmaceutical Manufacturers Association’s (Pharmaceutical Research and Manufacturers of
America) Validation Advisory Committee “Process Validation Concepts for Drug Products”America) Validation Advisory Committee “Process Validation Concepts for Drug Products”
Pharmaceutical Technology, September 1985 p 82.Pharmaceutical Technology, September 1985 p 82.
 Bismuth, G. Cleaning Validation: A Practical Approach. CRC Press, 2000. ISBN 1574911082.Bismuth, G. Cleaning Validation: A Practical Approach. CRC Press, 2000. ISBN 1574911082.
 Pharmaceutical Process Validation, 3rd Ed. Edited by Robert Nash and Alfred Wachter, MarcelPharmaceutical Process Validation, 3rd Ed. Edited by Robert Nash and Alfred Wachter, Marcel
Decker, 2003. ISBN 082470838-5Decker, 2003. ISBN 082470838-5
 Validation of Pharmaceutical Processes: Sterile Products. 1998. 2nd Edition. Edited by Frederick J.Validation of Pharmaceutical Processes: Sterile Products. 1998. 2nd Edition. Edited by Frederick J.
Carlton and James Agalloco. Marcel Decker, 1998. ISBN 0824793846.Carlton and James Agalloco. Marcel Decker, 1998. ISBN 0824793846.
 Validation Standard Operating Procedures: A step by Step Guide for Achieving Compliance in theValidation Standard Operating Procedures: A step by Step Guide for Achieving Compliance in the
Pharmaceutical, Medical Device, and Biotech Industries, Syed Imtiaz Haider, St. Lucie Press, 2002.Pharmaceutical, Medical Device, and Biotech Industries, Syed Imtiaz Haider, St. Lucie Press, 2002.
ISBN 1574443313.ISBN 1574443313.
 Good Manufacturing Practices for Pharmaceuticals: A Plan for Total Quality Control FromGood Manufacturing Practices for Pharmaceuticals: A Plan for Total Quality Control From
Manufacturer to Consumer, Sidney J. Willig. Marcel Decker, 2000. ISBN 0824704258.Manufacturer to Consumer, Sidney J. Willig. Marcel Decker, 2000. ISBN 0824704258.
 Voss, J. Cleaning and Cleaning Validation: A Biotechnology Perspective. CRC Press, 1995. ISBNVoss, J. Cleaning and Cleaning Validation: A Biotechnology Perspective. CRC Press, 1995. ISBN
0939459507.0939459507.
 LeBlanc, D.A. 2000. Validated Cleaning Technologies for Pharmaceutical Manufacturing. CRCLeBlanc, D.A. 2000. Validated Cleaning Technologies for Pharmaceutical Manufacturing. CRC
Press. ISBN 1574911163.Press. ISBN 1574911163.
 Cloud, P. 1998. Pharmaceutical Equipment Validation: The Ultimate Qualification Guidebook. CRCCloud, P. 1998. Pharmaceutical Equipment Validation: The Ultimate Qualification Guidebook. CRC
Press. ISBN 1574910795.Press. ISBN 1574910795.
 Juran, Quality Control Handbook, 4th Edition., McGraw-Hill, 1988.Juran, Quality Control Handbook, 4th Edition., McGraw-Hill, 1988.
 DeSain C, Sutton C. (1995). Process development that supports process validation.DeSain C, Sutton C. (1995). Process development that supports process validation.
Pharmaceutical Technology 19 (Oct.): 130-136, 1995.Pharmaceutical Technology 19 (Oct.): 130-136, 1995.
 Garcia T, Wilkinson S, Scott J. The development of a blend-sampling technique to assess theGarcia T, Wilkinson S, Scott J. The development of a blend-sampling technique to assess the
uniformity of a powder mixture.uniformity of a powder mixture. Drug Development and Industrial PharmacyDrug Development and Industrial Pharmacy 27(4): 297-307,27(4): 297-307,
2001.2001.
 Chaloner-Larsson, G., Anderson, R., Egan, A. 1997. A WHO guide to good manufacturing practiceChaloner-Larsson, G., Anderson, R., Egan, A. 1997. A WHO guide to good manufacturing practice
(GMP) requirements Part 2: Validation . World Health Organization, Geneva.(GMP) requirements Part 2: Validation . World Health Organization, Geneva.
www.who.int/vaccines-documents/DocsPDF/www9666.pdf Accessed on October 2nd, 2006.www.who.int/vaccines-documents/DocsPDF/www9666.pdf Accessed on October 2nd, 2006.
 Brown, F. 1993. Review of accidents caused by incomplete inactivation of viruses.Brown, F. 1993. Review of accidents caused by incomplete inactivation of viruses. Dev. Biol.Dev. Biol.
Stand.Stand. 81:81: 103-7103-7
 Nathanson, N. and Langmuir, A.D. 1995Nathanson, N. and Langmuir, A.D. 1995.. The Cutter incident. Poliomyelitis followingThe Cutter incident. Poliomyelitis following
formaldehyde-inactivated poliovirus vaccination in the United States during the Spring of 1955. II.formaldehyde-inactivated poliovirus vaccination in the United States during the Spring of 1955. II.
Relationship of poliomyelitis to Cutter vaccine. 1963.Relationship of poliomyelitis to Cutter vaccine. 1963. Am. J. Epidemiol.Am. J. Epidemiol. 142:109-40142:109-40..

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