Validation in Biomanufacturing by nikeborome

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									                   Validation in
What is Validation?
Validation is a key ingredient in producing a biopharmaceutical that meets the quality
attributes stipulated by The FDA. What validation does, is force the manufacturer to
examine assumptions about equipment, materials, procedures, and even entire processes
used in the production of the biopharmaceutical. By examining these assumptions and
providing proof that the equipment, test method, or production process do what they are
supposed to do and perform the way they are supposed to perform we eliminate areas of
uncertainty in the production process and increase the likelihood of making a product that
meets our predetermined design specifications. For example, In order to produce a
product that is free from any viral contamination you design a heat inactivation step into
your production process. The design specifications require that you achieve and hold a
temperature of 37o for 60 minutes. Now you have two areas to validate; first of all you
must validate that in fact holding your process material at 370 for 60 minutes will in fact
inactivate any and all contaminating viruses, and secondly, you must demonstrate that the
container you are doing your virus inactivation in does in fact allow your materials to
achieve a temperature of 370.

The FDA has a somewhat more succinct way of describing Validation. In their words:

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.

The above definition determines what needs to be validated, i.e. any processes,
procedures, methods, equipment, or facilities that are used to produce the product. It also
describes how they must be validated, i.e. through documented evidence. In other words
we have to prove that a process, method, procedure, piece of equipment, or software
package does what it says it will do. Often times, because of the nature of what we are
producing we must go through several validation runs in order to prove that our process
consistently produces a product that meets our predetermined specifications and quality
attributes. Documenting these validation runs is an essential aspect of demonstrating that
our process, method, procedure, or equipment produces a quality product. A lack of
documentation, or the failure to investigate an out of specification result (OOSR) during a
validation test will most surely win you a citation when the FDA comes around to
investigate. For example the following citation was written by the FDA concerning a visit
to a soft contact lens maker:
1. Failure to validate with a high degree of assurance a process that cannot be fully verified by
subsequent inspection and test, and to document and approve the activities and results of the
validation, as required by 21 CFR 820.75(a). For example:

       Your firm failed to adequately validate the terminal steam autoclave sterilization process.
                      o There was no validation protocol or procedure for the original sterilization
                      o There was no evidence that the sterilization process validation had any
                           established acceptance criteria prior to validation efforts.
                      o The validation study documentation discussed the results of bio-indicators
                           placed in the autoclave during the validation study, but the documentation
                           did not establish if there were positive or negative controls utilized in the
                      o There was no documentation that the sterilization validation results were
                           acceptable and approved.

As you can see the FDA takes validations seriously, especially steam sterilization, or
autoclaving of product which is specifically mentioned in the CFR’s. Notice that the
above citation specifically cites the lack of documented evidence, and the lack of
predetermined specifications, two critical aspects of validation. Additionally, every good
scientist knows of the need for controls in their experiment. However the company cited
failed to use controls (or failed to write down that they used controls). Conducting a
validation exercises is an experiment and with out the proper controls it’s impossible to
evaluate any experiment.

Why Do We Validate?
Historical Basis for Validation Requirements

As is the case with many government regulations, tragic accidents that could have been
prevented are the basis for process and equipment validation requirements in the
pharmaceutical and biopharmaceutical industry. Two of these bear mention, because they
illustrate how false assumptions that have not been proven through rigorous scientific
study can have a fatal impact.

The Cutter Incident: Unless you are of a particular age, you probably consider polio to be
a disease largely confined to developing countries and having little impact in more
developed countries, such as the United States. However, prior to the development of
effective vaccination strategies, the mention of polio struck fear into the hearts of
Americans. Visions of young adults confined to “iron lungs”, or permanently requiring
metal braces to walk were splashed across the pages of mid 20th century news magazines.
The development of an effective vaccine was a significant accomplishment in the fight
against infectious disease, by the end of the first year after introduction in 1955, over 10
million children in five countries had been inoculated. A number of companies were
recruited to produce the batches of vaccine necessary for this massive vaccination effort,
including Cutter Laboratories.
In April of 1955, during this mass vaccination drive, public health officials in California
noticed an increase in reported cases of polio. Subsequent investigation showed that over
200,000 people had been vaccinated with a preparation from Cutter Laboratories that
contained live polio virus. By the time it was over, the “Cutter Incident” resulted in 200
children developing permanent paralysis, and 10 deaths.

Assumptions about inactivation rates: Vaccines are often prepared from either attenuated
(weakened), or killed infectious agents. In the case of the polio vaccine, live virus was
prepared and then inactivated by treatment with formaldehyde. The inactivation of live
virus with formaldehyde was assumed to proceed with first order kinetics, meaning that
inactivation proceeded at a rate that was proportional to the concentration of
formaldehyde; this means that the rate will slow down as the chemical reaction proceeds,
but the slowing will take place in a predictable manner. What was found after the
discovery of live virus was that inactivation proceeded by second order kinetics, or the
rate varied with the square of the concentration. The practical effect was that the reaction
slowed much faster then if the reaction was to proceed by first order kinetics. So if we
assume first order kinetics we might devise a viral inactivation protocol that would
involve treating our batch of virus for six hours with a given concentration of
formaldehyde, and would result in complete inactivation of all viruses. However, if
inactivation proceeded by second order kinetics then six hours might not be long enough
to inactivate all the virus and our supposedly inactive vaccine preparations would contain
live virus that could infect people.

In the “Cutter incident” we see what can happen when a particular process, in this case
viral inactivation, is assumed to proceed in one way but actually proceeds in a different
manner. Small scale experiments, such as might be done in a test tube or on the
laboratory batch may not actually reproduce, or be informative as to the actual
manufacturing process. If Cutter Laboratories had actually tried to validated their
inactivation process on a manufacturing scale, they would have seen that the conditions
sufficient to inactivate virus on a small scale were not sufficient when scaled up. The
consequence is the requirement that actual production scale manufacturing processes be
validated to insure product quality.

Terminal Sterilization: A common process used to prepare solutions that are to be
injected, or used to treat wounds, and that are not affected by the high temperatures used
in autoclaves is terminal sterilization. Terminal Sterilization is not used in the
production of biopharmaceuticals since protein based therapeutics are degraded by the
high temperatures and pressures used in autoclaves. Instead, biopharmaceuticals are
prepared via aseptic processing, where filtration, clean rooms, and biological safety
cabinets are used to insure sterility of the end product. An incident that occurred in 1971
with terminally sterilized solutions that were incompletely sterilized, as with the Cutter
incident, pointed out the need to use actual conditions to demonstrate critical aspects of
pharmaceutical production. In addition, this incident illustrates how quality control
testing alone is ineffective in guaranteeing a safe product. Today, the concept that quality
needs to be designed into the production process and that testing by itself is not adequate
is a central concept in the field of quality management, and pharmaceutical production.

In 1971, supposedly sterile solutions used in burn wards were incompletely sterilized and
contaminated with Pseudomonas spp. The administration of these solutions to burn
patients resulted in a number of infections and several deaths. A subsequent investigation
of the manufacturer showed that autoclave loading and the sterilization cycles used
needed to be carefully matched to the material being sterilized to insure complete
sterilization. In this incident air caught between the metal crimp used to seal the container
and the rubber bung, served to insulate residual Pseudomonas bacteria, and allowed them
to survive the autoclave cycles used.

The lessons from the “Cutter Incident” and pseudomonas contamination led to the
concept of validation; that we must demonstrate, 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. In the
case of pharmaceutical or biopharmaceutical production we must demonstrate through
documented evidence that our process, procedure, method, equipment, and facilities
consistently produce products that meet predetermined specifications for identity, safety,
efficacy, potency, purity, stability, and consistency.

                                          Attributes of Quality
              Identity
                   o    21 CFR 211.84 (d) at least one test shall be conducted to verify the identity of
                        each component of a drug product.
                   o    Chemical, biological, Immunological
                   o    Raw materials, In-process intermediates, final products.
              Safety
                   o    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
                   o    Effectiveness of the product in achieving its medicinal purpose (therapeutic,
                        prophylactic, diagnostic). Gathered at phase II and Phase III trials.
              Potency
                   o    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
                   o    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
                   o   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
                   o   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.

How Do We Validate?
The Production of Sterile Water – an example in validation: Let’s say we are in the
business of making sterile water and selling it for use in research laboratories. Now we
could take tap water, boil it, and then bottle it and sell it that way. However, we decide
that we want to produce a quality product that our customers will buy, so we poll our
customers to find out what specifications they would require. After talking to several
potential customers we find out that they want water that has:

                          No visible solids
                          No detectable salts or ions
                          No detectable organic compounds
                          No detectable microorganisms

These will be the specifications that our final product will have to meet before we can sell
it. One of the things that we will have to do is define what “no detectable” means. This
will depend upon our customer’s requirements as well as the technology available to
detect organic compounds, salts, ions, or microorganisms.

A key concept in quality control and quality assurance is that testing, by and of itself is
insufficient to insure quality. Instead we must design our manufacturing process to insure
that our final product meets its predetermined specifications. Not only must we design the
process to meet these specifications we also have to prove through documented evidence
that our designed process produces a product that meets those specifications. This is the
essential feature of validation, conducting experiments that provide the documented

Process Design: The next step is to design a process that will produce water that meets
these specifications. The first step is to draw a Block Flow Diagram that diagrams the
major steps in our production process (Figure 1). An example of a block flow diagram
applied to biomanufacturing is provided in Figure 2. Once we have our major steps
defined we will construct a process flow diagram that will identify specific pieces of
equipment, process flow and quantities of process material. An example of a process flow
diagram for biomanufacturing is provided in Figure 3. Once the process flow diagram is
completed we can work on the most detailed engineering drawing, the Piping &
Instrumentation Diagram (PID). The PID will provide information on the routing of all
process piping, branch lines, vents, valves and other connections which connect the
process equipment together and carry the flow of process material. As we will see later
on, all of these documents will be referred to as we build and validate our production

Now consider what our manufacturing process would look like if we were making water
that would be part of a medicine. To start with, we would have to make sure that we
followed the Good Manufacturing Practices outlined in the Code of Federal Regulations
Volume 21 parts 210 and 211 (21 CFR 210 & 211). As we discussed earlier, the GMP’s
were established in 1978 and constitute “the minimum current good manufacturing
practices for manufacturing processing, packing, or holding of drug products or devices.”
The GMP’s are regulations written by the regulatory agencies (in this case FDA) in
response to laws passed by Congress and signed by the President. The GMP’s are
purposely vague so that they will not need to be changed with every new development. In
order to provide specific guidelines to companies, the FDA (and other regulatory
agencies) publish guidance documents (See Appendix III for some of the official
guidance documents that apply to validation). Unlike the regulations published in the
Federal Register and The Code of Federal Regulations, these guidance documents do not
have the force of law, but they do represent the agencies “current thinking about a
particular topic”. Even guidance documents can be vague, offering guidance on how to
remain or become compliant with the GMP’s, but little in the way of specific approaches.

The following are just a few of the GMP’s that apply to our water production system:

      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.
      21 CFR 211.113
              Requires that sterilization processes be validated
      21 CFR 211.165
              Requires that the accuracy, sensitivity, specificity, and reproducibility of
              test methods employed by the firm shall be established and documented.

Note that the GMP’s are constantly changing, which explains the use of the abbreviation
“cGMP” or “CGMP” the C standing for current in current good manufacturing practices.
The change reflects not only changing regulations, but also the best practices of the
industry. For instance if an technology that can measure organic contaminants down to
the ppt (part per trillion, or 1 femtogram /ml) is readily available and has been used by
others in the industry, your company would be hard pressed to justify using technology
that can only detect down to the ppb (part per billion, or 1 picogram/ml) level-particularly
if contaminants at the ppt level could affect product quality (safety, potency, identity,
stability, etc.).

A good example of the changing nature of the CGMP’s is found in some amendments
that were originally proposed in 1996. These amendments addressed the field of
validation and sought to introduce some definitions, terminology and requirements for
validation and periodic revalidation of the production process and procedures and
equipment associated with that process. These amendments can be found in the Federal
Register Vol 61, page 20103.

Making the Process of water production CGMP compliant: So what changes would
we have to make to our production system to insure that it complied with the GMP’s ?

      Put in place written procedures.
      Record and investigate any deviations from the written procedures.
      Validate our sterilization process.
      Document and establish specifications for our test methods.

Written Procedures: The first step we will have to do is to develop written procedures,
in the form of SOP’s that describe how to perform the steps of our process from start to
finish, and including the methods used for our quality control procedures. Included in
these written documents will be several that pertain to validation, or the process we will
go through to establish “through documented evidence a high degree of assurance that a
specific process will consistently produce a product that meets its predetermined
specifications and quality characteristics.” These documents will include a) Master
Validation Plan, and b) Validation Protocol(s). The Master Validation Plan is, in most
cases, a document that outlines the company’s philosophical approach to validation and
revalidation. In essence the master validation plan becomes a guideline by which
individual validation protocol are developed and implemented in house.

Individual validation protocols are in many ways like individual SOP’s, in that different
SOP’s will apply to different procedures. In this case different validation protocols will
apply to different processes, pieces of equipment, analytical test methods, and even
software. The types of information that we could expect to find in a validation protocol
                     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.
A sample validation protocol: Let’s look at one step of our production process and the
steps that we would have to take to validate that particular operation. One of our
specifications requires that our product be sterile, or that there are no detectable
microorganisms present. In addition, according to 21 CFR 211.113, there is an explicit
requirement for the validation of sterilization steps. In our case we have elected to
terminally sterilize our product using heat and pressure in an autoclave.

Validating our autoclave and sterilization protocol will allow us to examine some other
terminology that is commonly found in the validation field; Installation Qualification,
Operational Qualification, and Process Qualification often referred to as IQ, OQ, and
PQ. Recently, a third term Design Qualification, or DQ, has been used to refer to the
process of outlining the design specifications of the equipment based on what it is
intended to accomplish.

Installation Qualification, or IQ is a processes used to document that the piece of
equipment was installed properly and that appropriate utilities, i.e., electrical, steam, gas,
etc. are available to operate the equipment according to the manufacturers specifications.
Systems and equipment that are critical to the production of a quality product need to
undergo IQ. Examples of such equipment include:

                                  HVAC systems
                                  Autoclaves
                                  pH meters
                                  Depyrogenation Ovens
                                  Lyopholyzers
                                  Centrifuges
                                  Steam generators
                                  Water systems
                                  Compressed air systems
                                  Vacume systems

In the case of our hypothetical autoclave we would need to reference additional
documents such as operational manuals, piping and instrumentation diagrams, utilities,
and any software programs used in programmable logic controllers (PLC) used to operate
the autoclave. If we are building an entirely new plant we can use the documents that
were generated in the commissioning of the plant, which will simplify the entire IQ
procedure. However, if we are adding a new piece of equipment to an existing process
such as our water production, then we will have to conduct a more elaborate IQ protocol
to insure that our equipment is installed properly. Typical information found in the IQ for
a piece of equipment includes:

      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
      That all documentation, including manufacturers contact information, spare parts
       inventory, operational manual, and installation drawings are available on site.

In addition to documenting our installation, we also have to calibrate our test equipment.
In the case of an autoclave we will want to be able to measure the temperature and the
pressure within the autoclave during its operation, so will need thermocouples for the
measurement of temperature, and pressure gages for the measurement of pressure. In
order to insure that our test equipment will produce accurate results it’s necessary that the
test equipment be calibrated using standards traceable to the National Institute of
Standards and Technology (NIST), a government organization that produces extremely
accurate calibration standards for use in science and industry. An example of a general
format for IQ of equipment is provided in appendix IV, a specific IQ protocol utilized for
an autoclave IQ is provided in Appendix V.

Operational Qualification, or OQ is a process designed to supply the documented
evidence that a piece of equipment operates as it is intended to. For example, our
autoclave will be expected to reach a certain temperature, and produce a specified
pressure within the vessel for a certain amount of time. The manufacturer of our
autoclave provided us with the design specifications. However, it is up to use to insure
that the autoclave actually reaches those design specifications and operates as it is
supposed to.

In order to test our autoclave we are going to have to use test equipment such as
temperature probes, flow meters, vacuum gauges, pressure gauges, and so on. The IQ
protocol will specify the actual test equipment, placement of that equipment, and the
methods for calibrating that equipment. Appendix VII outlines the components of a
typical autoclave OQ protocol. In this protocol, temperature gauges, pressure gauges,
switches and alarms are all checked and documented that the autoclave is performing as it
is supposed to.

Performance Qualification, ( Process Qualification ), or PQ is a demonstration that the
autoclave actually performs as it is intended to in the manufacturing process. In our
case of producing sterile water we would autoclave actual production samples and then
test them to insure that sterilization had occurred. This might involve challenge testing,
where, in our case, a known amount of microbial contamination is introduced into the
water and we prove that our autoclave process actually kills those bacteria. Challenge
testing generally involves worst case scenarios, or “to the edge of failure” to prove that
even under these extreme conditions the process produces an acceptable product. In
addition it is necessary that we test our autoclave under all possible loading scenarios, for
instance when we only have a few bottles of water to autoclave and when we have the
autoclave jammed packed. The results of this qualification may dictate that a specific
loading plan be utilized for the autoclave. Failure to follow this loading plan will
compromise the validated state of that autoclave and we will be unable to prove that our
autoclaving actually sterilizes our bottled water.
It’s important to realize that the validated state is one that is “locked in”. It is not possible
to make changes unless those changes have been previously validated. For instance a
minor piece of equipment may break or be unavailable and under pressure to adhere to a
production schedule we may substitute another piece of equipment or a part that will do
the job. The problem is that if the process has not been validated with that substitute
equipment you will have compromised the validated state of that process and can no
longer guarantee that your product meets those predetermined specifications.

Remember also that we have to demonstrate that our equipment, process, and methods
consistently produce a product meeting our predetermined specifications. To prove
consistency means that we must perform multiple validation runs and show that our
product meets those specifications every time. Generally speaking if you can show that
your process produces an acceptable product three times in a row this is taken as
evidence that your process is in control and consistent. However, if you do five validation
runs and the first, third, and fifth produce an acceptable product, but the second and
fourth do not meet specifications then you will be hard pressed to prove that your process
is under control and consistent.

Approaches to Validation
With our relatively simple water production system it would be conceivable to build the
system, go through the IQ and OQ for the components and then do a PQ on the entire
process before we start marketing or distributing our product. Such validation studies that
take place prior to product distribution are referred to as prospective validation.
Biopharmaceutical production processes are much more complicated then our simple
water system, and the production process often changes as these products progress
through the clinical trials necessary for marketing approval. For example, in early phase
1 trials where the drug product is being assessed for safety in humans, relatively small
amounts of materials are required and at that stage the manufacturing process may not be
completely worked out. Due to the uncertainty, risk, complexity, and economics of
biopharamaceutical development regulatory agencies allow other validation strategies
that are based on historical data from past production runs, as well as studies that take
place at the same time as production. These validation strategies are referred to as
retrospective validation for those studies relying on historical data, and concurrent
validation for those studies that rely on validation during the production process. In the
majority of cases a mixture of prospective, concurrent, and retrospective validation
strategies are used.

Table 1 illustrates the progressive nature of validation as biopharmaceutical products
progress through the development phases. In early stages of development relatively small
amounts of products may be required, through critical systems that may affect product
quality such as equipment cleaning and the analytical methods used to test the product
need to be validated, other areas will require less validation and data generated from
production runs may be used for subsequent validation studies.
Figure 4 illustrates the process of a prospective validation study. In this case the IQ, OQ,
and PQ are performed prior to product distribution. Figure 5 illustrates the
implementation of a retrospective validation study. Notice how the data generated during
previous production runs are analyzed, including all calibrations. In this case an emphasis
is placed on proving that all subsystems are individually qualified, calibrations are in
order, and that control parameters (in this case analytical test methods) are proven.

Development                                Areas to be validated
   Phase          Facilities     Equipment         Man.        Analytical          Raw
                                                 Process       Methods           Materials
   Phase I        Complete        Critical        Safety,       Develop          ID & test
                                   Issues       Cleaning                       specifications
   Phase II       Complete         Major         Expand         Expand         Vendor Audit
  Phase III       Complete           All        Extensive      Complete          Vendor

Table 1. Validation Progression during drug development. Major areas of the
production process such as plant facilities, production equipment, manufacturing process,
analytical methods, and raw materials are subject to progressively stricter validation
criteria as the product moves through the production process. Some validation studies
may be completed by an examination of historical production data including quality
control testing. Retrospective validation typically requires data from larger numbers of
production runs then prospective studies. Critical areas such as sterilization must be
prospectively validated.

Revalidation & Change Control
Document Changes

Engineering Changes

Planned Changes and Unplanned Changes
Problems & Student Exercises
  1. 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?

  2. 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?

  3. 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

  4. 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?

  5. 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?


                                      VIII. Appendices
               1. Definitions
               2. Regulatory Documents applicable to validation
               3. Regulations that apply to the …………….
               4. General IQ Format
               5. Example of Autoclave IQ protocol
               6. Example of pH meter IQ protocol
               7. Example of an Autoclave OQ protocol
               8. Example of a pH meter OQ protocol
               9. Example of a Change Control Form
               10. Warning Letter
               11. Example of engineering diagram

         The Community College of Baltimore County
               Biomanufacturing Laboratory

          Validation Plan-Fermentation Vessel Sterilization

 Subject: Sterilization of BioFlow 110 3 Liter             Prepared By:_______________________
 Fermentation Vessel with media.                                             Name / Signature / Date

 Document #: VP001                                         Reviewed By:______________________
 Revison:0.0                                                                 Name / Signature / Date
 Effective Date: January 1, 2006
                                                           Approved By:______________________
                                                                             Name / Signature / Date

   1. Scope: This document covers validation of the fermentation vessel sterilization process as
described in SOP P004. Validation of the bioreactor sterilization procedure is conducted in order to
provide documented evidence that the process described in SOP P004 will effectively sterilize
growth media contained in the New Brunswick Scientific BioFlow 110 3 liter fermentation vessel
and kill any microorganisms inadvertently introduced into the fermentation vessel during the
preparation of media and the vessel.
2. Definitions:

2.1    NBS – New Brunswick Scientific
2.2    CFU -Colony Forming Units
2.3    CFU/ml – Clolony Forming Units per milliliter
2.3    rGFP – Recombinant Green Fluorescent Protein
2.4    D.I. H2O – Deionized water
2.5    LB – Luria Bertani media
2.6    ml(s) – Milliliter(s) (10-3 Liters)
2.7    O.D.600 – Optical Density at 600 nanometers
2.8    μl – microliter (10-6 Liters )

3. References:

3.1    Guide to Operations: BioFlow 110 Modular Benchtop Fermentor Manual # M1273 0054
       Revision E
3.2    Guide to Operations: AMSCO Eagle Series Autoclave Model 3011 Revision 4.1
3.3    SOP P001 - Cleaning of NBS Bioflow 110 BioFlow 110 Modular Benchtop Fermentor 3
       Liter vessel between batches of same product.
3.4    SOP P002 –Preparation of NEB BioFlow 110 Modular Benchtop Fermentor 3 Liter
       vessel for growth of Saccharomyces cerevisiae.
3.5    SOP P003 Addition of Media to NEB BioFlow 110 Modular Benchtop Fermentor 3 Liter
3.6    SOP P004 – Autoclaving of NEB BioFlow 110 Modular Benchtop Fermentor 3 Liter
3.7    SOP P005 – Collection of In-process samples from NEB BioFlow 110 Modular Benchtop
       Fermentor 3 Liter vessel.
3.8    SOP Q001 – Determination of the number of viable bacterial cells through serial dilution
       and growth on Luria-Bertani media.
3.9    SOP Q002 - Gram Staining.
3.10   SOP P006 – Preparation of Luria - Bertani liquid media.
3.11   SOP P007 – Preparation of Luria – Bertani solid media.
3.12   SOP P008 – Determination of the Optical Density of a bacterial culture

4. Reagents:

4.1    Luria-Bertani liquid media
4.2    Luria Bertani solid media
4.3    Bioreactor cleaning solution
4.4    Silicone Grease

5. Responsibility:

5.1    It is the responsibility of all personnel validating the performance of the equipment
       covered by this validation plan to read and understand the validation plan.
5.2    It is the responsibility of the Quality Control Laboratory to perform the viable cell counts
       on pre and post autoclaved material described in this validation plan.

5.3    It is the responsibility of the validation department to supervise the described validation
       exercises in accordance with the validation schedule, in response to replacement or
       modification to autoclaves used for the purpose of sterilizing fermentation vessels, or, in
       response to modification or replacement of SOP P002, P003, or P004.

5.4    It is the responsibility of the Production Department to perform the described validation
       exercise and to deliver samples to the Quality Control Department for analysis.

5.5    The Quality Assurance Department will review all validation data generated in response
       to this validation plan and will have final sign-off on all validation certification.

6. Hazard Communication:

6.1    Fermentation Vessels, if not properly vented, may build up sufficient pressure during the
       autoclave process to explode. All personnel should wear appropriate safety clothing when
       removing fermentation vessels from the autoclave.
6.2    Autoclaved liquids may boil over and leak from the fermentation vessel. All personnel
       should wear appropriate safety clothing when removing fermentation vessels from the

6.3    Baccillus subtilus

7. Attachments:

7.1    Validation Data Sheet
7.2    Validation Process Flow Chart

8. Procedures:

Growth of Bacillus subtillus

8.1    Assemble materials necessary for the inoculation of liquid cultures (innoculating loop,
       Bunsen burner, culture tube with liquid media, shaking water bath at 37oC.)

8.2    Obtain a frozen vial of Baccilus subtilus from the -80 freezer.

8.3    Record the lot number of the Baccilus subtilus cells.

8.4    Thaw the frozen vial on ice for 10 – 20 minutes.
8.5    Innoculate 2 culture tubes, each containing 10 mls of LB media, with 100 μl of thawed B.
       subtilus cells for each culture tube.

8.6    Place the culture tube in the shaking water bath set at 370C.

8.7    Grow the cells at 370C overnight (minimum of 12 hours).

8.8    Using the overnight liquid culture, inoculate a 500 ml baffled cultured flask containing
       100 mls of sterile LB media with 10 mls of the overnight culture material.

8.9    Place the inoculated 500 ml baffled culture flask in a 370C shaking water bath.

8.10   Determine the OD600 of the culture as described in SOP P008.

8.11   Grow the culture until an O.D.600 of between 0.5 – 0.75 is reached.

8.12   Pool the two 50 ml cultures into 1 baffled shaking flask.

       Preparation of Bioreactor

8.12   Prepare the NEB BioFlow 110 3 Liter vessel as described in SOP P002.

8.13   Prepare 2 Liters of LB liquid media as described in SOP P006.

8.14   Add the LB Liquid media to the fermentation vessel.

8.15   Innoculate the fermentation vessel with 50 mls of culture obtained from step 8.12

8.16   Close the fermentation vessel and agitate for 10 minutes at 500 rpm.

8.17   Collect a 15 ml sample from the autoclave as described in SOP P005.

Viable Cell Count in pre-autoclaved bioreactor

8.18   Prepare a serial dilution series using LB media as the diluant, from 1:10 to 1:108 of the
       sample taken in step 8.17.

8.19   Place 100 μl from the 1:103 through 1:108 dilutions on a pietrie plate containing LB solid

8.20   Repeat step 8.18 two times on two different pietri plates containing LB solid media.

8.21   Add 100 μl of the LB media diluant to each of two plates. Label these plates pre
       autoclave control 1 and control 2.
8.22   Add 10-20 sterile glass beads to each plate and to two uninnoculated plates containing
       LB solid media.

8.23   Recover the plates and swirl the glass beads to spread the culture material over the
       surface of the plate.

8.24   Remove the glass beads from the plates by briefly uncovering the plates and taping the
       bottom of the plate against the top edge of a clean 500 ml beaker.

8.25   Incubate the plates at 370C overnight.

8.26   Determine the number of CFU in the starting culture by counting the number of colonies
       visible on each pietri plate. Average the results of the three plates for each dilution.
       Record the results on Validation Data Sheet.

8.27   The number of CFUs’/ ml is the number of bacterial colonies multiplied by the dilution
       factor multiplied by 10.

Determination of viable cells in post-autoclaved bioreactor.

8.28   Remove fermentation vessel from autoclave and allow to cool to room temperature.

8.29   Collect a 15 ml sample from the autoclaved fermentation vessel as described in SOP

8.30   Prepare a serial dilution series using LB media as the diluant, from 1:10 to 1:108 of the
       sample taken in step 8.29.

8.31   Repeat steps 8.18 through 8.27.

9. Acceptance Criteria

9.1    All control plates should show no bacterial growth after incubation at 370 for 16

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