VALIDATION AND QUALIFICATION

Lizette Caballero, B.S., M.T. (ASCP)

Laboratory Manager
Cellular Therapy Laboratory
Florida Hospital Cancer Institute
Orlando, FL 1
 Learning Objectives
• Validation of Equipment
– Provide an overview of equipment validation
– Outline the components of a validation plan
– Provide a specific example of an equipment
validation

• Validation of Process
– Provide overview for process validation
– Outline the components of a process validation
– Provide a specific example of a process validation

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 When do we do it?
• Applies to new or significantly changed
equipment or processes that affect
donor or patient safety, or the safety,
purity or potency of products.
• Examples include:
– Equipment used during product processing
– Transport procedures, including transport
carrier
– New or revised processing procedures

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Equipment Validation or Qualification
• Phase I
– Equipment Installation Qualification (IQ)
• Identify the equipment
• Installation/Maintenance/Calibration
requirements verified with Biomed
• SOP written to include maintenance /calibration
requirements
• Equipment added to Preventive maintenance
list
• Supply vendor qualification check completed
• Supplies added to order list
• Individual(s) responsible for completing this
phase

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Equipment Validation or Qualification
Validation Checklist
Instrument: _____________________ SN: _________________

Directions: Initial each item as it is completed.

Initials/Date Checklist Item

Equipment Installation Qualification (IQ)
Identify Equipment
Installation / Maintenance / Calibration requirements verified
with Biomed
SOP Written
SOP includes maintenance / calibration requirements
Equipment added to PM list
Supply vendor qualification check completed
Supplies added to order list
IQ validation plan completed
Operation Qualification (OQ)
Operational variables / critical control points identified
SOP(s) written
SOP specifies expected outcomes
Training and competency records completed
OQ validation plan completed
Performance Qualification (PQ)
Product performance (specifications / outcome) identified
Monitoring process in place
SOP written
SOP includes tolerance limits and action for non-conformities
PQ validation plan completed

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Equipment Validation or Qualification
• Phase II
– Operation Qualification (OQ)
• Operational Variable/Critical control points
identified
• Does the instrument function as described by
manufacturer?
• SOP written
• SOP specifies expected outcomes
• Training and competency records completed
• Individual(s) responsible for completing this
phase

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Equipment Validation or Qualification
Validation Checklist
Instrument: _____________________ SN: _________________

Directions: Initial each item as it is completed.

Initials/Date Checklist Item

Equipment Installation Qualification (IQ)
Identify Equipment
Installation / Maintenance / Calibration requirements verified
with Biomed
SOP Written
SOP includes maintenance / calibration requirements
Equipment added to PM list
Supply vendor qualification check completed
Supplies added to order list
IQ validation plan completed
Operation Qualification (OQ)
Operational variables / critical control points identified
SOP(s) written
SOP specifies expected outcomes
Training and competency records completed
OQ validation plan completed
Performance Qualification (PQ)
Product performance (specifications / outcome) identified
Monitoring process in place
SOP written
SOP includes tolerance limits and action for non-conformities
PQ validation plan completed

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Equipment Validation or Qualification
• Phase III
– Performance Qualification
• Does the equipment function correctly and
consistently for the intended application (Mock
products)
• Monitoring process in place
• SOP written

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Equipment Validation or Qualification
Validation Checklist
Instrument: _____________________ SN: _________________

Directions: Initial each item as it is completed.

Initials/Date Checklist Item

Equipment Installation Qualification (IQ)
Identify Equipment
Installation / Maintenance / Calibration requirements verified
with Biomed
SOP Written
SOP includes maintenance / calibration requirements
Equipment added to PM list
Supply vendor qualification check completed
Supplies added to order list
IQ validation plan completed
Operation Qualification (OQ)
Operational variables / critical control points identified
SOP(s) written
SOP specifies expected outcomes
Training and competency records completed
OQ validation plan completed
Performance Qualification (PQ)
Product performance (specifications / outcome) identified
Monitoring process in place
SOP written
SOP includes tolerance limits and action for non-conformities
PQ validation plan completed

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Equipment Validation or Qualification
• Example of Equipment Validation-
Steps to follow:
– Identify equipment used to be implemented
or improved.

– Select equipment and determine which

elements require validation or qualification.

– Write an SOP according to procedure

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 Writing The Validation Plan
• Validation Title

• Assign Validation Number

• Type of Validation (IQ, OQ or PQ)

• Purpose of the Validation

• System Description- Define the scope or

beginning and ending steps of the validation.
Include identification of equipment.

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Example of Control Rate Freezer Validation

Validation Plan

VALIDATION TITLE: MRV Controller Installation Validation

VALIDATION NUMBER: V193.433.1

Validation of: Equipment Process Product (Check all that apply)

Type: (IQ) Installation Qualification (OQ) Operation Qualification (PQ) Performance Qualification

I. PURPOSE OF VALIDATION
To prospectively ensure that the MRV controller performs as expected after installation and prior to
freezing of patient products.

II. SYSTEM DESCRIPTION

Manufacturer: Planner
Model MRV RV
Serial Number: 23226
Controller for Control Rate
Type of Equipment System
The Control Rate Freezer system (Chamber and
MRV controller) is used to freeze Hematopoetic
Progenitor Cells (HPC) at a slow rate, usually 1o
C/min. The freezing controller is programmed to
Description of Features and Capabilities: control the procedure and the freezing chamber
provides the environment for the controlled
freezing. The chart record provides readout of the
temperature and rate of freezing or cooling for both
chamber and sample
Date of purchase/receipt: Purchased: 11/19/2008 Receipt: 1/30/2009
Cryo Associates
Supplier Contact Information: 301-279-2864 (phone)
Contact Person: Billy
In the Cellular Therapy Laboratory, next to
Number and Location of user manuals:
instrument.

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Equipment Validation or Qualification
• Responsibility assignment

• List of SOPs, personnel, equipment and

supplies required:
– Include SOPs for operation, maintenance,
quality control and supplies if required
– Review current SOPs and list SOPs
requiring revision
– List all equipment used in validation
– List supplies- including labels and training
forms

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 Equipment Validation or Qualification
III. RESPONSIBILITY ASSIGNMENT- Document name in table (Signatures follow plan and results).
Validation Plan written by: Lizette Caballero, MT
Validation Plan reviewed by: Susan Ingersoll, Ph.D.
Validation Plan approved by (medical director): Vijay Reddy, M.D., Ph.D.
Installation Performed by: Robert Conine, Cryo Associates Representative
Validation performed by: Anginett Batista, Lizette Caballero
Validation Results evaluated by: Lizette Caballero, MT
Validation Results reviewed by: Lizette Caballero, MT
Validation Results approved by (medical director): Vijay Reddy, M.D., Ph.D.
Validation Results approved by (QA Manager): Marie Fuentes- Rivera, RN

IV. VALIDATION PLAN

A. List SOPs, personnel, equipment, and supplies required.
A.1. Installation Qualification
SOP
Validation of Equipment, Process or Product SOP# 193.493
Personnel
Robert Conine- Cryo Associates
Lizette Caballero- CTL Supervisor
Equipment
MRV Controller SN: 23226
Kryo 10-16 Chamber
Liquid Nitrogen Tank
A.2. Operation Qualification
SOP’s
Draft Control Rate Freezer Operation (Kryo 10-16) SOP 193.433
Personnel
Anginett Batista
Lizette Caballero
Equipment
MRV Controller SN:23226
Kryo 10-16 Chamber
Liquid Nitrogen Tank

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Equipment Validation or Qualification

A.3. Performance Qualification

SOP’s
Draft Control Rate Freezer Operation (Kryo 10-16) SOP 193.433
HPC Cryopreservation With or Without Volume Depletion SOP 193.432
Viability Test Procedure SOP 193.406
Personnel
Anginett Batista
Lizette Caballero
Equipment
MRV Controller SN:23226
Kryo 10-16 Chamber
Kryo 10-16 Probe
Liquid Nitrogen Tank
Cell-Dyn 1700
Beckman Centrifuge
Supplies and Reagents
Cryovials
Plasma-Lyte A
DMSO
EDTA Tubes
Peripheral Blood from donor
Pipet
12X75 Tubes
Tips
Trypan Blue
Hemacytometer

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Equipment Validation or Qualification

• Establish the number of test samples

required for validation
– This may be determined by the manufacturer or
regulations, but in any case must be adequate to
assure a high degree of confidence in the
validation results (for most cases no less than 3
samples)

• Establish testing conditions

– Step by step directions to perform validation
– Consult with department director/designee or
regulatory specialist

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Equipment Validation or Qualification

A. Establish the number of test samples required.

B.1. Installation Qualification
Not applicable
B.2. Operation Qualification
One test run will be performed with the freezer empty to ensure that the freezer runs properly.
B.3. Performance Qualification

Three test runs will be performed using conditions similar to a stem cell product prior to using the
freezer to cryopreserve any patient products and 20 patient products will be prospectively analyzed to
ensure that the freezer is working properly after installation.

C. Establish testing conditions (Step-by-step directions for validation.)

C.1. Installation Qualification
1. Identify Equipment
2. Check SN and add to Bio Med Surveillance program
3. Check SOP for changes if applicable
4. Get completed IQ paperwork from Cryo Associate Representative

C.2. Operation Qualification

1. Program new controller using program described on Draft SOP 193.433 Control Rate Freezer
Operation.
2. One test run will be performed with the freezer empty to ensure that the freezer runs properly.
The printed graph will be reviewed for accuracy and reproducibility of cryopreservation
program.
3. Perform personnel training

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Equipment Validation or Qualification
C.3. Performance Qualification

Three test runs will be performed prior to using the freezer to cryopreserve any patient products and 20
patient products will be prospectively analyzed to ensure that the freezer is working properly after
installation.
Part I. Perform three test runs following this procedure:
A. Collect peripheral blood using 5 EDTA tubes from a donor.
B. Prepare a buffy coat with the EDTA blood sample by centrifuging for 5 minutes at 1000
RPM’s.
C. Save red cells and plasma.
D. Label two cryovials with: Donor’s name, Date of testing, test sample number.
E. Make a hole in one of the cryovial’s lid to allow chamber probe placement.
F. Using a 12 X 75 plastic tube mix:
1. buffy coat cells from step B.
2. 0.4ml plasma from step C.
3. 0.4mlPlasma-Lyte A
4. 0.2ml DMSO
G. Run WBC (using SOP# 193.455 “Cell-Dyn Analyzer Operation” and viability using SOP# 193.406
“Viability Test procedure”)
H. Transfer mix from step F to 2 cryovials from step D (1.0ml each).
I. Insert Chamber probe trough hole in lid of cryiovial containing cells and freezing media.
J. Place both cryovials inside chamber
K. Start cryopreservation procedure following SOP 193.433 “Control Rate Freezer Operation”.
L. Move sample without probe to Liquid Nitrogen Tank. Discard 2nd cryovial (the one with the hole
on the lid).
M. Save print out of cryopreservation graph.
N. Repeat steps 1-10 using two more donors.
O. Keep cryovial for at least 24 hours before testing.
P. After 24 hrs thaw cryovial using waterbath and perform:
1. Viability test
2. WBC counts

Part II. Collect the following data from 20 consecutive products:

A. WBC counts pre and post.
B. Viability post thaw.
C. ANC and PLT engraftment.

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Equipment Validation or Qualification
• List of data/records to be collected
Examples include:
– New or revised SOPs, labels and forms
– Training and competency records
– Checklists
– Quality Control and outcome
measurements

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Equipment Validation or Qualification

C. Determine data/records to be collected.

D.1. Installation Qualification
1. When and where received.
2. Condition upon receipt.
3. Installation paperwork by Cryo Associates representative to include: start up, self checks,
calibration, etc.
4. Sticker provided by Bio Med with assigned ESN number.
D.2. Operation Qualification
1. After mock run the print out of graph will be reviewed and saved.
D.3. Performance Qualification
1. The following will be checked for acceptable performance for Part I and II :
a. Viability post thaw
b. WBC counts pre and post thawed
c. ANC and PLT Engraftment for products from 20 patients.
2. Prepare table with Pre and Post values.
3. Prepare graphs to show results.

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Equipment Validation or Qualification
• Establish Acceptance criteria for each
data/record collected
– Must be defined and measurable

• List of references

• Validation Plan Signatures

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Equipment Validation or Qualification
C. Establish acceptance criteria.
E.1. Installation Qualification
1. Instrument turn on and off.
2. Control Panel, Displays indicators working properly.
3. Alarm sound is working properly when activated.
4. Controller pass calibration testing performed by Cryo Associates Rep.
5. Printer works properly

E.2. Operation Qualification

1. Temperatures are accurate.
2. Instrument able to follow programmed temperatures.
E.3. Performance Qualification
Expected Results:
Part I:
1. WBC recovery within 20% of pre cryopreservation value.
2. Post thawed viability result >50%.

Part II.
1. WBC recovery within 20% of pre cryopreservation value.
2. Post thawed viability result >50%.
3. ANC engraftment < 12 days and PLT engraftment < 15.

Validation Plan written by: Date:

Validation Plan reviewed by: Date:

Validation Plan approved: YES NO (if not approved, attach recommendations.)

Validation Plan approved by: Date:

Quality Approval: Date:

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 Validation Results

DATE(S) OF VALIDATION:
VI. VALIDATION RESULTS
A. List SOPs, personnel, equipment, supplies, or procedure steps that deviated from or
were added to original validation plan. State reason for change and document approval
of change.

B. Record the number of test samples used.

C. Record data/records collected. (Attach separate sheet or data-collection forms if

necessary.)

VII. CONCLUSION
A. Validation data evaluation and determination of acceptance.
Data must meet pre-determined acceptance criteria.

B. Comments/Actions.

VIII. VALIDATION RESULTS SIGNATURES

Validation performed by: Date:
Validation results evaluated by: Date:
Validation reviewed by: Date:
Validation Results approved: YES NO (if not approved, attach revised plan.)
Medical Director Approval: Date:

Quality Approval: Date:

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FDA Guidance for Industry Process Validation: General
Principles and Practices

• Process validation is defined as the collection and

evaluation of data, from the process design stage
throughout production, which establishes scientific
evidence that a process is capable of consistently
delivering quality products

• Process validation activities:

– Process Design: The commercial process is defined during
this stage based on knowledge gained through development
and scale-up activities
– Process Qualification: During this stage, the process design
is confirmed as being capable of reproducible commercial
manufacturing
– Continued Process Verification: Ongoing assurance is
gained during routine production that the process remains in
a state of control

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 Example of Process Validation

• Validation of change in sampling for bacterial and

fungal cultures
Current process is to draw 0.2ml of final product (post
processing) and 2.8 ml of concurrent plasma. Then
Bacterial and Fungal culture bottles are inoculated
with 1.5ml of mixture in each bottle.

We want to change this process to be able to test a

representative specimen of the cryopreserved
product by collecting 3.5 ml of freezing media
(Plasma-Lyte A, concurrent plasma and DMSO) and
mix with final product prior to inoculation of culture
bottles.

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 Process Validation

• Validation Title: Validation of Sample to be Used for Microbial

and Fungal Cultures

• Purpose of Validation: To determine if the addition of DMSO to

the Microbial and Fungal Culture inhibits the growth of
contaminants

• System Description: HPC, aphereis products are tested for

microbial and fungal contamination. This process is used to
determine if the HPC, apheresis product is contaminated during
the cryopreservation process or apheresis process
The cultures will grow under the proper conditions in the
presence of 20% or 10% DMSO

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 Process Validation

• Validation Plan: List of SOPs, personnel, equipment

and supplies to be used during validation

• Establish the number of test samples required:

– Three independent test runs are required to assure that the
addition of DMSO to the Microbial and Fungal Cultures do
not inhibit the growth. If the results of the three independent
tests are identical then the results will be considered
acceptable; if not then 2 more independent experiments will
be performed

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 Process Validation
• Establish Testing Conditions:
– Preparation of the Inoculums (Staphylococcus epidermidis
and Candida albicans)
• Dilute each culture to 0.5 O.D. Dilute the bacteria with sterile
saline and the fungus with sterile water. This 0.5 dilution
represents approximately 108 CFU/ml
• Dilute 1:100 to give 106 CFU/ml
• Dilute to 3 X 103 CFU/ml by adding 3ul of the 108 dilution to 1
ml of diluent

– Label 4 BACT Myco/F-Lytic and PEDS PLUS/F bottles with

date, Experiment # and: 20%, 10%, No DMSO, Negative
Control

– Collect 4 tubes of blood in ACD

– Spin 3 tubes at 3000RPM for 10 minutes

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 Process Validation
• Draw up 3ml of the remaining blood in 3cc syringe
and inoculate the negative control bottles with 1.5ml
of blood

• Take off 3ml of the remaining blood and place 1.5ml

each into two sterile tubes
– Spike one tube with 75 CFU of Staphylococcus epidermidis
(25ul of the 3 X 103 CFU/ml dilution of each organism)
– Spike the other tube with Candida albicans

• This result in a bottle being inoculated with 10 CFU of

the appropriate organism

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 Process Validation
• Make up freezing media using supernatant
plasma, plasma Lyte A and DMSO

• Prepare test samples for Bacteria and Fungal

organism
– 20% DMSO Bacteria Test sample
– 10% DMSO Bacteria test Sample
– No DMSO Bacteria Test Sample

• Inoculate bottles with each sample (bacterial

and fungal)

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 Process Validation
• Data to be Collected: reports of growth from
both bacterial and fungal cultures

• Expected Results:
– The negative control will no grow any microbial or
fungal culture
– The No DMSO control will grow both bacteria and
fungus
– The 10% and 20% DMSO test samples will grow
the bacteria and fungus the blood was inoculated
with.

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 Process Validation

Days to Grow th in Culture

2.5

1.5
S. epidermidis
Days

C. albicans
1

0.5

0
0% 10% 20%
DMSO Concentration

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 Process Validation

Conditions Acceptance Criteria Experiment 1 Experiment 2 Experiment 3

10% DMSO Sample Bacterial Growth PASS PASS FAIL

20% DMSO Sample Bacterial Growth PASS PASS PASS

No DMSO Sample Bacterial Growth PASS PASS FAIL

Negative Control No Growth of Bacteria PASS PASS PASS

10% DMSO Sample Fungus Growth PASS PASS PASS

20% DMSO Sample Fungus Growth PASS PASS PASS

No DMSO Sample Fungus Growth PASS PASS PASS

Negative Control No Growth of Fungus PASS PASS PASS

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 Process Validation

• Acceptance Criteria: If the 20% DMSO is deemed

acceptable then 20% DMSO will be added to the
micro cultures mix with the product during future HPC
processing

• Validation Data Evaluation and Determination of

Acceptance: We have shown that the addition of 20%
DMSO to the sample that is used for the inoculation
of the microbiology culture bottles does not inhibit
growth. Therefore is acceptable to add freeze media,
containing DMSO, to the culture inocolum

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 Summary
• Perform validation according to
Validation Plan
• Data/records evaluation and
determination of acceptance must meet
pre-determined test criteria
• Ensure new or revised SOPs, forms or
labels are in place for use
• Ensure completion of training and
competency records
• Ensure ongoing monitoring is in place

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 Contact Information
Lizette Caballero
MT(ASCP)
2501 North Orange Ave Ste 786
Orlando, FL 32817
407-303-2442
Lizette.Caballero@flhosp.org

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