Incorporating Validation Concepts into the Biotechnology

Curriculum (or minding your P’s and Q’s)

Thomas Burkett, Ph.D.

The Community College of Baltimore County tburkett@ccbcmd.edu

Aims

• Introduce the role of validation in biomanufacturing

• Explain basic validation concepts and terminology

• Provide an example of how validation is incorporated into the biotech. curriculum at CCBC

• Provide examples of validation exercises • Provide examples of validation resources

Validation in Biomanufacturing

• Biomanufacturing is a complex process involving multiple unit operations many of which are critical to insuring patient safety

and product efficacy

InnoculumSeed

Fermentation

Production Fermentati

onHarvest

Ultrafiltration1

Chrom. 11

Ultrafiltration2

Chrom. 2

Viral Filtration

Chrom.3

Ultrafiltration3

Final Formulation/

Sterile Filtration

Sterile Fill

UPSTREAM

DOWN-STREAM

VIRAL

NON-VIRAL

Block Flow Diagram of a typical Production Process

Mammalian Antibody Production – Cell Culture

Vial Thaw / Inoculum Expansion

20,000-Liter

5000-Liter

500-Liter

50-Liter

HEAT COOL

MediaPasteurizer

Media Prep

Mammalian Process Flow- Upstream Diagram

rProtein A Dia: 1 meterCV: 236 LBed: 30cm

Virus Inactivation

Disc-StackCentrifuge

Depth Filter75m2

Transfer to Purification

Suite

Mammalian Antibody Production - Downstream Processing

BulkFiltration

(BDS)3m2

IntermediateStorage

UF/DF Step80m2

IntermediateStorage

IntermediateStorage

ViralFiltration

20m2

Cation ExchangeDia: 1.6mCV: 600LBed: 30cm

Anion ExchangeDia: 1.6mCV: 600LBed:30cm

Hydrophobic InteractionDia: 1.6mCV: 600LBed: 30cm

I.B.ICryoPreservation

System

Validation in Biomanufacturing

• A central concept in quality is that quality can not be tested for. Quality must be designed and built into the production process.

• Requires careful attention to raw material specifications, in process material specifications, and final product specifications.

Validation in Biomanufacturing

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

Validation in Biomanufacturing

• Validation does not replace testing, but it does reduce the testing burden for raw materials, in-process materials, and final product

Validation in Biomanufacturing

• Validation itself is a process that evolves with the product.

• Validation requirements for production of pre-clinical material much less stringent then for phase III clinical material.

• Critical operations: raw materials, analytical methods, viral clearance, sterilization, cleaning.

Validation in Biomanufacturing

• A fully validated process is “locked in” • Any change outside of the validated space

invalidates process

• Change must be evaluated for effect on patient safety and product efficacy

Validated Production Process

Δ

Option A - 1:5 Split

Option B - 1:10 Split

Stream Number >>> 201 202 203 204 205 206 207 208 209 210 211A 211B 211C 420

Process Step >>>

Media (Bolus) Liters

Air SLPM

CO2 SLPM

N2 SLPM

O2 SLPM

Bottom Aeration

SLPM

Top Aeration

SLPMInoculum

LitersNutrients

LitersBase Liters Waste

Flask & Roller bottle Media

Component

Basal Media (L) 75.0

Air SLPM 110.0 10.0 100.0

CO2 SLPM 10.0 10.0

N2 SLPM 110.0 10.0 100.0

O2 SLPM 10.0 10.0

Nutrients (L) 5.0

1N NaOH (L) 5.0

Media with Cells (L) 15.0 75.0 75.0 25.0

Mab Grams

Harvest Liters

BSC-xxxBiosafety Cabinet

Air6 x 1 L Spinner

R-12000100L Seed

Bioreactor Package

To 400 liter

BioReactor

D-C-1201

1 ml Ampules

15 x 1 L Spinner

Glycol Supply

Glycol Return

Vent

CIPS

CO2

N2

O2

Gas

Man

ifold

PD

3x500 ml Flask125 ml Flask

203

204

202

205

211A

208

207

206

M

209 210

211B To 2000 liter

BioReactor

D-C-1202

Released Mobil Dew arLN2 Freezer

INC-xxxIncubator

M

PU-xxxMedia Pump

FermentationInoculation/Seed Lab

From Media Prep

D-C-1101

From Bags

CIPR

SC

SCC

201

516A,B

211C

Plant Steam

Te

mp

era

ture

C

on

tro

l M

od

ule

Process Flow Diagram

Regulatory requirement for validation

21 CFR 211 Subpart F –Production and Process Controls• 211.100 –Written procedures; deviations• (a) Requires written procedures for production and process control designed to assure that products possess the

quality attributes that they purport or are represented to possess.• (b) Requires that any deviations from written production and process control procedures be recorded and justified. • 211.101 – Change in of components• 211.103 – Calculation of yield• 211.105 – Equipment identification• 211.110 – Sampling and testing of in-process materials and drug products• “Requires that control procedures be established to monitor the output and validate the performance of those

manufacturing processes that may be responsible for causing variability of in process material and drug product.”• 211.111 – Time limit on production• 211.113 – Control of microbiological contamination• “Requires that sterilization processes be validated”• 211.115 – Reprocessing21 CFR 211 Subpart H- Holding and Distribution• 211.165 – Testing and release for distribution• “Requires that the accuracy, sensitivity, specificity, and reproducibility of test methods employed by the firm shall

be established and documented. Such validation and documentation may be accomplished in accordance with 21 CFR 211.194 (a)(2)”

21 CFR 211 Subpart I- Laboratory Controls21 CFR 211 Subpart J – Record and Reports21 CFR 820 Quality Systems Regulations

Regulatory requirement for validation

• Sec. 211.113 Control of microbiological contamination.

• (a) Appropriate written procedures, designed to prevent objectionable microorganisms in drug products not required to be sterile, shall be established and followed.

• (b) Appropriate written procedures, designed to prevent microbiological contamination of drug products purporting to be sterile, shall be established and followed. Such procedures shall include validation of any sterilization process.

What does “validation of any sterilization process” mean ?

• What parameters are critical to sterilization?

– Temperatures, pressures, time, pore size (filtration), radiation dosage, chemical concentration.

• Must demonstrate that your autoclave reaches the temperatures, pressures, and times necessary for sterilization.

• Must demonstrate that items representing real world samples achieve those conditions ( 20 ft of 1 ½ hose; a 20 L carboy; a 500 ml bottle).

• Must challenge with worse case scenario (may take place in pilot plant if scalability demonstrated).

•

FDA definition of validation

“Validation is a process of demonstrating, through documented evidence, that a process, procedure, method, piece of equipment, or facility will consistently produce a product or result that meets predetermined specifications and quality attributes.”

Regulatory guidance on validation

• Guideline on General Principals of Process Validation http://www.fda.gov/cder/guidance/pv.htm

• Guidance for Industry: For the Submission Documentation for Sterilization Process Validation in Applications for Human and Veterinary Drug Products. CDER CVM November 1994. www.fda.gov/CDER/GUIDANCE/cmc2.pdf

• Working Party on Control of Medicines and Inspections• Final Version of Annex 15 to the EU Guide to Good Manufacturing Practice• Title: Qualification and validation• http://pharmacos.eudra.org/F2/eudralex/vol-4/pdfs-en/v4an15.pdf• ICH Q7a Section 12 on validation • http://www.fda.gov/cder/meeting/ICH_Q7A/index.htm• A WHO guide to good manufacturing practice (GMP) requirements. Part 2:

Validation• Chaloner-Larsson, G., Anderson, R., and Egan, A. 1997. World Health

Organization, Geneva.

Critical Operations in Biomanufacturing

• Some operations are more critical than others. – Viral filtration, sterilization, cleaning, analytical

methods.

– These operations will require greater validation efforts then less critical operations (media blending).

Validation in the biotech. curriculum at CCBC

• Validation introduced as part of “quality systems” section in intro. Course

• First lecture is on “concepts of quality” and “quality attributes

• Second lecture introduces validation as part of the production process

• Lab exercises varies. Past examples include validation protocol for an autoclave; validation of bioreactor sterilization.

Learning Objectives • Upon completion of this module students should: • Be familiar with the various government and third party literature pertaining

to validation.• Understand how component, process, and methods validation fits into the

overall quality system.• Be aware of pertinent regulations that apply to validation strategies.• Understand concept of criticality and be able to identify points in the

production process that are critical to product quality.• Be able to distinguish between installation qualification, operation

qualification, and performance qualification (IQ, OQ, PQ).• Given the function of a piece of equipment used in biomanufacturing,

discuss valtidation issues related to that specific piece of equipment. • Be aware of the vendor, installation, and maintenance documentation

required for initiating the validation process.• Follow a validation SOP• Be able to design a validation protocol for an individual piece of equipment.

Concepts of Quality

Biomanufacturing

Quality Attributes • Identity

– 21 CFR 211.84 (d) at least one test shall be conducted to verify the identity of each component of a drug product.

– Chemical, biological, Immunological– Raw materials, In-process intermediates, final products.

• Safety– 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 consideration the character of the product in relationship to the condition of the recipient at the time.

• Activity of active ingredients• Activity of the excipients or additives• Activity of process related impurities

• Efficacy– Effectiveness of the product in achieving its medicinal purpose (therapeutic, prophylactic, diagnostic).

Gathered at phase II and Phase III trials.• Potency

– 21 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 administration of the product in the manner indicated to effect the given result.

• Purity– 21 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.• Cleaning Procedures

• Stability– 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 stability testing. Drugs may use accelerated time studies, biologics must use real time studies.

• Consistency– The 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 is accepted as proof that a process is consistent. However, in NDA data from up to twenty batches may be submitted.

Designing Quality into the product• A central concept is that quality can not be tested for!

– Testing programs are based on testing a statistically significant number of samples

• However to be absolutely sure that all of your product meets specifications you would have to test everything.

– Testing by itself will not insure quality and is inefficient

– Testing is required under the GMP’s• Raw materials• In-process samples• Final Product

– Quality (identity, safety, efficacy, potency, purity, stability, consistency) must be designed into the production process

– Begins with predetermined specifications• Raw material specifications• In-process material specifications• Final Product Specifications

Predetermined specifications

• Identity: – Size, amino acid sequence, presence of post

translational modifications, 3-D structure.

Predetermined specifications

Identity:

21 CFR 211.84 (d) at least one test shall be conducted to verify the identity of each component of a drug product. Tests consist of Chemical, biological, and Immunological methods.

Requires testing of Raw materials, In-process intermediates, final products.

Testing For Identity• Requires the development of validated

analytical methods that can determine identity.

• Chemical Tests: – Is the molecule chemically what it is supposed

to be?

• Biological Activity Tests:– Does the molecules have the biologic activity

that it is supposed to have?

• Immunogenic Tests:– Is the molecule immunogenic (allergic)?

Identity

• 21 CFR requires testing of raw materials:

– Raw materials quarantined until identity verified

– Raw materials must meet predetermined specifications

– Vendors (and alternates) specified in BLA (NDA)

Identity

• 21 CFR requires testing of in-process materials:– Product from bioreactor / fermentor– Product from purification steps– Waste products from above

Must meet specifications, if not - stop the

process to investigate take corrective action

Testing

• Usually done by the Quality Control Laboratory

– CFR requires that quality unit be under independent supervision and report directly to senior management

Quality Assurance

• Reviews records from quality control and production departments– Verifies that all specifications and production

operations met / performed– Investigations necessary for any deviations

• Root cause• Affect on quality• Corrective action (CAPA)

– Approves final release of product

Designing Quality into the Product

• Design of production process and specifications all contribute to a quality product: – Absence of contamination

• Clean rooms, closed systems, use of BSC for critical operations.

– Purity• Separation process (chromatography) designed to

remove potential contaminants• Viral purification / inactivation

Insuring the Production of a Quality Product - II

Validation & its role in quality

What is Validation

Validation – An Essential Part of GMPs!

Validation is the scientific study of a system • To prove that the facility/system/equipment/method is consistently

doing what it is supposed to do (i.e., that the process is under control).

– We want to make decisions based on good science and not hunches and assumptions!

• To determine the process variables and acceptable limits for these variables, and to set-up appropriate in-process controls.

– Is it ok if the wash from a chromatography column is pH 6.8 vs. 7.0 ?

Validation • The FDA’s definition of validation:

“Validation is a process of demonstrating, through documented evidence, that a process, procedure, method, piece of equipment, or facility will consistently produce a product or result that meets predetermined specifications and quality attributes.”

Quality Attributes Remember these?

• Identity

– 21 CFR 211.84 (d) at least one test shall be conducted to verify the identity of each component of a drug product.

– Chemical, biological, Immunological– Raw materials, In-process intermediates, final products.

• Safety– 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 consideration the character of the product in relationship to the condition of the recipient at the time.

• Activity of active ingredients• Activity of the excipients or additives• Activity of process related impurities

• Efficacy– Effectiveness of the product in achieving its medicinal purpose (therapeutic, prophylactic, diagnostic).

Gathered at phase II and Phase III trials.• Potency

– 21 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 administration of the product in the manner indicated to effect the given result.

• Purity– 21 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.• Cleaning Procedures

• Stability– 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 stability testing. Drugs may use accelerated time studies, biologics must use real time studies.

• Consistency– The 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 is accepted as proof that a process is consistent. However, in NDA data from up to twenty batches may be submitted.

Historical Basis for Validation

• Assumptions concerning virus inactivation resulted in ten deaths and 200 children becoming paralyzed, from a supposedly “inactivated” polio vaccine.

• Assumptions about sterilization caused severe infections among burn victims given supposedly sterile solutions.

• Validation eliminates assumptions and relies on experimental proof!

Validation Plan

• Organizations must define an approach towards validation

– What is to be validated – How is it to be validated– Who is to validate it – Who is to approve the validation– When it must be revalidated

Validation Plan

• Regulatory agencies (FDA, EMEA, WHO, etc) identify minimum components of validation.

• “Industry standards” (the c in cGMP) can increase validation requirements.

• New & Novel processes / equipment require greater scrutiny then established processes / equipment.

• Validation requirements increase as a product moves through development (phase I, phase II, phase III).

Validation Plans

The Validation Master Plan

– A high level document that outlines the organizations philosophical approach to validation and revalidation. The master validation plan becomes a guideline by which individual validation protocol are developed and implemented.

– May contain a flow chart or other diagram of the validation process

Validation Protocol

• Specific protocols (SOP’s) that provide detailed information on what is to be validated.

• Validation Protocols consist of: – A description of the process, equipment, or method to

be validated.– A description of the validation method.– A description of the sampling procedure including the

kind and number of samples.– Acceptance criteria for test results. – Schedule or criteria for revalidation.

Example of a protocol for the IQ component of validating apH meter

As with all other SOP’s this document will contain an Objective, scope, and responsibilitySection.

Validation Protocol

• Validation Protocols may consist of multiple SOP’s each describing specific steps in the validation process

Validation Examples of individual systems subject to validation:

HVAC systemsAutoclavespH meters

Depyrogenation OvensLyopholyzersCentrifuges

Steam generatorsWater systems

Compressed air systemsVacuum systems

Critical Systems• How critical is the system being validated

to final product quality?

– Media blending systems for cell growth vs. final fill & finish operations

• Demonstrating that the device which fills, labels, and caps the final product will require more extensive validation then the blenders used to prepare media for bioreactors.

• Validation of complex devices can take years!

Validation

• Proceeds in stages with new facilities / equipment.

• Planning for validation should start with the design process.

• Leaving validation to the last minute is asking for trouble.

Stages of Validation

• Starts with Design & Receipt:– Does the equipment meet the needs (is the autoclave big

enough?)– Do you have the manuals, spare parts, can you plug it in? – Is it installed properly (drain lines, vents, etc)

• Does it work? – Does the autoclave reach the necessary temp. and pressure? – Can the autoclave sterilize your equipment (worse case

situation)?

• How does it work in the manufacturing process?– Can it handle production quantities? – Will failure compromise product quality?

IQ, OQ, PQ ? Installation Qualification (IQ)

A process used to document that the piece of equipment was supplied and installed properly and that appropriate utilities, i.e., electrical, steam, gas, etc. are available to operate the equipment according to the manufacturers specifications.

Operational Qualification (OQ)A process designed to supply the documented evidence that a piece of equipment operates as it is intended through all anticipated operational ranges.

Performance (Process) Qualification (PQ)Verifies that a process / piece of equipment performs as it is intended to in the manufacturing process and produces product (in process or final) meeting predetermined specifications.

Example of a protocol for the IQ component of validating apH meter

As with all other SOP’s this document will contain an Objective, scope, and responsibilitySection.

Typical information in an IQ protocol

• Name and description of equipment, including model numbers

• Identification, including model and serial numbers• Location of the equipment• Any utility requirements, i.e. electrical voltage, steam or

water pressure, etc.• Any safety features of the equipment, including alarms,

interlocks, or relief valves.• That all documentation, including manufacturers contact

information, spare parts inventory, operational manual, and installation drawings are available on site.

OQ ProtocolExample of a protocol for the OQ component of validating apH meter

As with all other SOP’s this document will contain an Objective, scope, and responsibilitySection.

OQ ProtocolExample of a protocol for the OQ component of validating anautoclave

As with all other SOP’s this document will contain an Objective, scope, and responsibilitySection.

Typical OQ Protocol Components

• Objective• Responsibility• Equipment required (Calibration

verification & Traceability)• SOP(s) used• Equipment Identification• Parameters measured (Specifications)• Documentation

Validation

• Ideally validation takes place prior to actual production runs, however in some cases validation may take place as product is produced, or past production runs may be used to provide validation data.

• Prospective Validation

• Concurrent Validation

• Retrospective Validation

A prospective validation study

IQ

OQ

Calibration

PQ protocol approval

PQ protocol execution

Data Analysis

Validation Report

Approve Conclusions

A concurent / retrospective validation study

Are systems

qualified?

NoCalibrations

Correct ?

Data Analysis

Qualify systemCalibrate system

No

Yes

Yes

Approval

57

The V-ModelUser

RequirementsSpecification

(URS)

FunctionalSpecification

Detail Design

Implement/Build

InstallationQualification

OperationalQualification

PerformanceQualification

Related to

Related to

Related to

Project PlanAgreed by team members

Details phases, activities, and milestones

Gantt Chart most commonly used

ID Task Name Start Finish DurationJan 2003

1/12 1/19

1 15d1/31/031/13/03Design

5 5d3/28/033/24/03Obtain Funding

6 15d4/18/033/31/03Construct

7 10d5/2/034/21/03Commission

10d6/5/035/23/03Validate

2 10d2/14/032/3/03Prepare Quality Plan

3 5d2/21/032/17/03Prepare URS

4 20d3/21/032/24/03Prepare Project JustificationDocument

Feb 2003 Mar 2003 Apr 2003 May 2003

1/26 2/2 2/9 2/16 2/23 3/2 3/9 3/16 3/23 3/30 4/6 4/13 4/20 4/27 5/4 5/11

9

8

1d6/6/036/6/03Turnover - Project Complete

Planning for Validation

59

Putting it

all together

GoodEngineering

Practice

TheCompliance

Pyramid

Revalidation

• Is the initial validation of a piece of equipment the end? – No! – Periodic revalidation may be necessary depending on

the criticality of the equipment– Changes need to be evaluated for their impact on

validation– Deviations from specifications may require

revalidation– Revalidation spelled out in Master Validation Plan

Change Control

• Must assess impact of changes on FDA compliance and validation state.

• Change control is a formal process defined in company SOP on how process/equipment changes are evaluated.

• Any change that takes place outside the change control process can jeopardize product quality (patient safety).

An example of a facility / process validation

• Remicade® (infliximab) is a chimeric mAb* directed against TNF-α.

• Approved in 1998 (US) and 1999 (EU) to treat Crohn’s disease, and RA.

• Produced by Centocor, Inc. in Malvern, PA

* Contains mouse variable domains and human constant domains

(IgG1)

Antibodies

• Proteins– 2 heavy Chains

– 2 Light Chains

– Disulfide Bonds

• Variable region– Recognizes

antigen

• Constant region– Effecter function– Classes &

subclasses

Ig G class

Production of Remicade®

• BLA approved in August 1998 (FDA), 1999 (EMEA).

• First site for bulk manufacture was Leiden, The Netherlands.

• Process was transferred to Malvern, PA in April 2002*.

• Process changes, including larger bioreactors, external spin filters, and a change in media components were introduced to meet increased demand.

– Not only did a new facility have to be validated, but also the changes to the manufacturing process had to be validated.

– Necessary to demonstrate that product produced under these new conditions had same quality attributes as product produced in Leiden.

• An unanticipated consequence of increased product yield was a change in chromatography conditions due to product breakthrough under old conditions.

– Minor changes can have unanticipated consequences on product quality!

• A new facility for production of remicade is being constructed in the Republic of Ireland and should be on line in 2007

Changes in Production Process in Malvern, PA

Example of a 1000 L Bioreactor with an external spin filter used in the production of Remicade® in Malvern, PA

Remicade Production

These tanks are used for the holding of material from the bioreactors prior to product capture and initial chromatography.

What performance aspects of these tanks do you think need to be validated?

How does cleaning of these tanks between use affect validation?

Some Questions• A valve used to transfer material from a holding tank to the purification

suite jam’s closed. You have a spare valve that is an identical model. Can you change this valve with the spare and continue operations? What if the valve is from a different manufacturer?

• You notice that your autoclave loading plan leaves room for additional material. Realizing that increasing that amount of material in the autoclave will shorten the turn around time for the production line you contemplate increasing the amount of material loaded into the autoclave then specified by the loading plan. What should you do? What will be required to implement this change?

• An SOP for calibration of a pH meter calls for a two point calibration at pH 4 and pH 7. You notice that a single point calibration at pH 7 produces the same result from pH measurements of your buffer solutions and allows you to take a longer break. Is it Ok to do the one point calibration when the SOP calls for a two point calibration? How would you go about changing the SOP to allow for a one point calibration?

• What documents would provide information concerning the make and model of a particular valve used to regulate the transfer of material from a holding tank to the purification suite?

• Your supervisor is concerned that the fermentation vessel is not providing sufficient aeration of the culture to get optimal growth and suggests installing a different kind of baffle in the vessel. How would you demonstrate that this change has no effect on product quality?

References

• Pharmaceutical Manufacturers Association’s (Pharmaceutical Research and Manufacturers of America) Validation Advisory Committee “Process Validation Concepts for Drug Products” Pharmaceutical Technology, September 1985 p 82.

• Bismuth, G. Cleaning Validation: A Practical Approach. CRC Press, 2000. ISBN 1574911082.• Pharmaceutical Process Validation, 3rd Ed. Edited by Robert Nash and Alfred Wachter, Marcel Decker, 2003. ISBN 082470838-5• Validation of Pharmaceutical Processes: Sterile Products. 1998. 2nd Edition. Edited by Frederick J. Carlton and James Agalloco. Marcel

Decker, 1998. ISBN 0824793846.• Validation 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. ISBN 1574443313.• Good Manufacturing Practices for Pharmaceuticals: A Plan for Total Quality Control From Manufacturer to Consumer, Sidney J. Willig.

Marcel Decker, 2000. ISBN 0824704258. • Voss, J. Cleaning and Cleaning Validation: A Biotechnology Perspective. CRC Press, 1995. ISBN 0939459507. • LeBlanc, D.A. 2000. Validated Cleaning Technologies for Pharmaceutical Manufacturing. CRC Press. ISBN 1574911163. • Cloud, P. 1998. Pharmaceutical Equipment Validation: The Ultimate Qualification Guidebook. CRC Press. ISBN 1574910795. • Juran, Quality Control Handbook, 4th Edition., McGraw-Hill, 1988.• DeSain C, Sutton C. (1995). Process development that supports process validation. Pharmaceutical Technology 19 (Oct.): 130-136,

1995.• Garcia T, Wilkinson S, Scott J. The development of a blend-sampling technique to assess the uniformity of a powder mixture.

Drug Development and Industrial Pharmacy 27(4): 297-307, 2001. • 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.

• Brown, F. 1993. Review of accidents caused by incomplete inactivation of viruses. Dev. Biol. Stand. 81: 103-7• Nathanson, N. and Langmuir, A.D. 1995. The Cutter incident. Poliomyelitis following formaldehyde-inactivated poliovirus vaccination in

the United States during the Spring of 1955. II. Relationship of poliomyelitis to Cutter vaccine. 1963. Am. J. Epidemiol. 142:109-40.

Laboratory Activities

• Students develop and carry out a simplified validation plan / protocol– Autoclave validation– Bioreactor sterilization

– Bioreactor cleaning– Spectroscopy– Chromatography– Plasmid construct

Prepare Fermentation Vessel as described in SOP P002

Retrieve B. subtillus culture from -80o Freezer. Inoculate liquid culture & grow overnight at

37o.

Dilute overnight culture into fresh media. Monitor OD as described in SOP P008

Day 1

Day 2 Prepare media as described in SOP P006

When OD is between 0.5-0.75 pool cultures and mix.

Add media to fermentation vessel as described in SOP

P003

Add 50 mls of culture material to fermentation vessel. Mix for 10 minutes. Collect sample as per

SOPP005

Autoclave fermentation vessel as per SOP P004

Determine the number of viable B. subtillus cells as

per SOP Q001

Allow fermentation vessel to cool to room temperature. Collect sample

as per SOP P005

Determine the number of viable B. subtillus cells as

per SOP Q001

Day 3 Determine the number of viable cells present pre and post autoclave as per SOP Q001. Record

results and forward to Validation.

Items in orange are completed by Production. Items in turquoise are completed by Quality Control