# Presentation: 589336

## Aseptic Filler Validation

- John W. Larkin
- Aseptic Processing Class

## Title 21 CFR 113

- 21 CFR 113.40(g)(2)(ii) – “Before the start of packaging operations, both the container and closure sterilizing system shall be brought to a condition of commercial sterility.”

**Notes:**
When dealing with aseptic equipment the expectation is that the filling operation is conducted at all times in an aseptic fashion.  This means that the packaging system must be brought to a sterile condition and maintained in this sterile condition for the duration of its operation.  If at any time the sterility of the packaging system is lost the system will need to be shut down and re-sterilized.

## Need for Validation

- The regulation (21 CFR 113) has some very specific elements for the sterilizer and very general ones for the packaging equipment
- Reason for the general nature of the regulation for packaging equipment is the numerous filler designs
- Regulations for packaging equipment focus on validation of equipment

**Notes:**
The majority of the GMP requirements within the regulation are specific to the food sterilization equipment, such as a temperature recorder-controller device at the end of the heater, a temperature recorder at the end of the hold tube, a positive pressure gradient in any product-to-product heat exchanger, a temperature reference device at the end of the hold tube, a positive pressure flow device, and a means to segregate under-processed product.  The GMP regulations for the packaging equipment are very general.  The packaging system must have a recording device(s) that records the delivery of the process, a method must be installed to control the timing of the system, and the system must be operated in such a way as to properly establish and maintain sterility.  The reason for the general nature of the regulation for the packaging equipment is because the packaging equipment can take many different designs.  The regulation thus deals more with the necessity of documenting that the packaging equipment has operated in the proper manner, i.e., the need for validation and monitoring of the packaging system.

## Intent of System Validation

- Develop documentation to demonstrate that the system, according to its design specifications, is able to produce product that is commercially sterile
- Filed as supplemental information using a sub-SID

**Notes:**
Over the years the processing industry has agreed to have the FDA review the validation documentation of an aseptic system before it is placed into production.  This review/discussion with the FDA is a review of the documentation that the processor has developed in conjunction with their process authority to demonstrate that the operating conditions of the system are such that they are able to establish an aseptic environment within the filler, sterilize all incoming packaging material and then aseptically fill the product and seal it.  The complete documentation is filled with the FDA as supplemental information with the aseptic filing form as part of the supplemental SID for the filler.  This documentation covers a number of areas, which I will highlight in the rest of the presentation.  Probably the best way to look at this supplemental information is to view it as the documentation of a validation plan.

## Validation Steps

- Develop a Hazard analysis/HACCP plan
- pre-purchase review of equipment
- define the sterile zone
- identify the critical factors/limits
- Conduct challenge study
- establish the necessary indicator organism log reductions
- measure indicator organism thermal/chemical resistance
- use appropriate test procedures
- Document the results

**Notes:**
Validation of an aseptic system is not much different than what you would do to validate any other type of system.  There needs to be an analysis of the system where all of the critical elements of the process are identified.  Often this is done in conjunction with the equipment manufacturer.  As a matter of fact, it is very difficult to do this without the involvement of the equipment manufacturer.  Once all of the system operation elements have been established and there is a working knowledge of the critical operating parameters, there needs to be an actual challenge of the system using biological tests.  Then finally all of this information is written up into a document that can be used to demonstrate that the system will always operate in a safe manner.

## 1. Develop a Hazard Analysis/HACCP Plan

**Notes:**
Step number 1

## Pre-Purchase Review of Equipment

**Notes:**
The important first steps. – Pre Review and negotiations with the equipment supplier.

## Pre-Purchase Review of Equipment

- Assemble the HACCP team (i.e., process authority team)
- Describe the food and its distribution
- Identify equipment manufacturer
- Identify intended use and consumers of the food
- Develop flow diagram, indicating each piece of equipment

**Notes:**
The formation of the HACCP team has to be the most important step that a processor does.  This HACCP team needs to be selected such that it contains all of the technical experts that are going to be needed.  This team should always include the process authority – which may be an outside consultant.  Before a company goes forward with an installation, the HACCP team will review the needs of the company, the projected safety considerations, future training issues, and the an overall ability of the aseptic equipment to work in a safe manner according to the needs of the company.  One of the major elements of the final documentation will be construction during the initiation phase of the installation, and that is a complete description of the equipment, including diagrams depicting all of the different operating conditions and product flows.  These diagrams need to be used in conjunction with a narrative describing the complete operation of the system.  The focus here is to be able to describe to an outsider how the system operates in such detail for them to understand each working state and what is critical for each operational step.

## Pre-Purchase Review of Equipment (cont’d)

- Verify flow diagram
- Define the sterile zone
- Develop HACCP plan
- identify and list steps in the process where the hazards of potential significance occur
- list all identified hazards associated with each step
- list preventive measures to control hazards

**Notes:**
With every HACCP analysis comes a HACCP plan.  This should be a natural outcome of the HACCP teams evaluation of the system.  The HACCP plan is not usually supplied with the sub-SID supplemental documentation, but elements of it most like will find their way into the document.  Probably the most important step during the initial evaluation of the system is a clear understanding of where the aseptic zone (or sterile) zone is.  The reason for this is that all of the other steps of the validation of the system will be clearly dependent on the definition of the aseptic zone.

## Define theSterile Zone

**Notes:**
Let’s take a closer look at the aseptic zone.

## Integrity of Sterile Zone

- The sterile zone must be well-defined
- The sterile zone does not have to be composed of physical barriers
- Laminar flow air can be used as a barrier
- Need to account for possible line stoppages and product flow through the sterile zone
- Aseptic clearing of line jams may not require resterilization – ASK yourself has the aseptic zone been compromised?

**Notes:**
Aseptic zone
Defining the aseptic zone is very important, because once the sterility of the aseptic zone has been established, it must be maintained.  I would like to emphasize the “must be maintained.”  After the presterilization of the packaging equipment the aseptic zone must remain sterile; anything entering the zone must be sterile.  The FDA considers the integrity of this zone to be very important.   The FDA has agreed that the sterile zone does not have to be defined using physical barriers on every side.  FDA understands that laminar air can be used as a barrier for one side of the aseptic zone.  This laminar flow barrier needs to be designed so that any and all potential elements that might interrupt the integrity of this laminar flow have been addressed.  For example, line stoppages, how does a line stoppage effect the laminar barrier.  

## Integrity of Sterile Zone (cont’d)

- The sterile zone must be presterilized to a condition of commercial sterility (i.e., nothing can grow)
- Once the sterility of the sterile zone has been established, it must be maintained
- Everything entering the zone must be sterile
- Integrity of the sterile zone is critical
- Clean rooms do NOT meet the definition of commercial sterility
- Steilizing media must be an approved under 21 CFR 178.

**Notes:**
When a sterile zone is created it must be maintained.  There have been numerous times where because of a chain, or other item, exiting and then reentering the sterile zone there has been an issue of has the chain been resterilized to the same condition as it was when it entered the zone.

In other areas of the manufacturing world clean rooms are used to create an aseptic environment.  These clean rooms almost always have people working within them.  It is the opinion of FDA that when people are working within an environment the area can not be properly sterilized to a condition of commercial sterility. Therefore, a clean room under the definitions accepted by the clean room industry (even a class 100 room) can not be part of the aseptic zone if people are working within the clean room.  It would be advisable for an equipment manufacturer to try and make the aseptic zone as small as possible within the design of the aseptic system.

Not only does FDA regulate the GMPs associated with the operation of an LACF system, but all food additives are required to be preapproved for food use.  This includes sterilizing chemicals for the package material.  The concern is that indirect additives by way of the package material being sterilized will get into the food and create a potential public health hazard.  At this time (5-4-2004) the only chemical that has been approved for sterilization of food contact surfaces is hydrogen peroxide.  This means, at present, the chemical complex known as Oxoniatm (or Oxonia Active) cannot be used in the US for sterilization of packaging material.  It is, however, approved as a sanitizer for extended shelf-life products.  Another issue that a food processor will want to take into consideration is the validation of the integrity of any air filters used on the system.  Integrity testing of sterilizing filters must be a part of the GMPs of the system.  This includes both before and after use.  After use validation is very critical, for this is the only way to be able to document (create an accurate record) the critical factor of proper operation of the filter.  There are no other known ways of recording the proper operation of the sterilizing filters.  Dual filter systems for air sterilization lines do allow for an improved assurance that the air was properly sterilized.  Dual filter usage is becoming more common on most aseptic equipment (Jornitz, M.W. and Meltzer, T.H. 1998. Sterile double filtration. Pharmaceutical Technology Vol 22, No. 10, pg 92-100 ). 

## Sample Aseptic Filler

**Sterile**
**Zone**

** **

**Peroxide**

** ****Sterile Air**

** ****Product**

** **

**Sealer**

**Air**

**Notes:**
Here is an example of a preformed cup filling machine where the aseptic zone is clearly identified, and where one of the boundaries of the zone is maintained by a laminar air flow.  Notice that the aseptic zone is defined half way down the outside of the cup and above the carrier.

## Does the transport enter and then exit the sterile zone?

**Notes:**
I have presented a very simple design of a preformed package system, where there is a carrier used to transport the preformed package through the aseptic zone.  Here is a system where there is something entering and exiting the filling area.  The question is: Does the carrier enter the aseptic zone or not?  This is not an easy question to answer.  The answer to the question will be different for each design of the equipment.  The issue that must be addressed is:  “Does the aseptic zone ever become contaminated with product that has not been sterilized?”  For this design the aseptic zone has been defined as half way down the container and the partitioned filling and sealing area.  For this system there is no physical barrier between the transport system and the rest of the sterile zone.  Some systems have been designed where the containers are formed out of a web and the remaining web between each formed container acts as a barrier.  For this design the packages have been preformed and there is a skid transporting the packages through the filling area.

The barrier between the aseptic zone and the non-aseptic zone is not a physical one from the standpoint of it being a plate or some other material from the filler or the package.  Here the barrier is the sterile air flow pattern.  The air flow past the packages has been designed (and validated) to ensure that it passes the packages in a laminar flow pattern and there is no opportunity for the contamination on the outside of the bottom of the package to enter back into the aseptic zone and contaminate any product.  A system like this requires careful validation work by the manufacturer since there isn’t a physical barrier.  Also, line stoppages and product flow through the filling area have to be taken into account when the system is validated.  As you might have determined by now, all of the outside of these packages are not sterilized.  This is because the bottom of the packages do not enter the aseptic zone.  If the bottoms of the packages had entered the aseptic zone, they would have needed to have been sterilized.

## Identify the Critical Factors/Limits

**Notes:**
Next, identify the all the critical factors and their limits.

## What is a Critical Factor?

- "Any property, characteristic, condition, aspect, or other parameter, variation of which may affect the scheduled process and the attainment of commercial sterility."  (21 CFR 113.3)

**Notes:**
21 CFR 113 defines a critical factor as anything that will effect the delivery of the scheduled process to create a commercially sterile product. 

## Information Needed About Critical Factors

- "The type, range, and combination of variations encountered in commercial production shall be adequately provided for in establishing the scheduled process."  (21 CFR 113.83)

**Notes:**
21 CFR 113 indicates that for a critical factor the type, range and any possible variation about its value must be taken into account when establishing the process.

## Traditional Thermal Process Bioburden (Canning)

- Raw Food

- Preformed Container

- Lid

- Commercially
- Sterile Food

**Notes:**
For those familiar with retort operations, they know that there are limited number of critical factors to the delivery of a process.

## Aseptic Process Bioburden

- Processing Equipment

- Raw Food

- Aseptic Filler

- Web/Container

- Lid Stock

- Commercially
- Sterile Food*

- *Number of critical factors for an aseptic system might be an order of magnitude higher than for traditional canning

**Notes:**
For aseptic system the number of critical factors increases, sometimes significantly, due to the added complexity of the process.

## Conduct Challenge Study

**Notes:**
2.  After the HACCP team has analyzed the system and documented its proper operation, the critical factors, the critical limits, and any other critical elements to the process, the next step is to biologically challenge the system.  This assumes that the equipment has been shipped and installed correctly.  This can usually be demonstrated by way of a installation qualification (IQ).

## Establish the Necessary Indicator Organism Log Reductions

**Notes:**
When conducting the challenge study – the process authority is going to want to be able to measure if the system has been brought to a point of commercial sterility.  This will require an understanding as to the number of log reductions that the indicator organism will need to be reduced from the artificially high test concentration to some measurable level that can be correlated to commercial sterility.  This target may be different for different steps of the filler validation, dependent on the results of the hazard analysis.

## Log Reduction

- Must be able to correlate test organism with organism of concern (*C. bot., spoilage*)
- Kinetics of test organism must be similar to organism of concern
- Must be able to demonstrate commercial sterility

**Notes:**
The target log reduction needs to be set for what is necessary to deliver a commercially sterile environment, but it also should take into consideration the most resistant pathogen of concern. 

The process authority needs to understand that when dealing with new technologies the target organism and the target log reduction is most likely going to be different than what the targets are for a more traditional processing technology, such as for thermal processing.  The is especially true since dependent on the technologies used to destroy the microorganisms the mechanism that destroys the organisms may change and the procedures used to validate say a thermal type system is going to be different than the procedures to validate a high pressure system.  One of the first questions to ask is what indicator organism can I use to demonstrate the ability to commercially sterilize the system, and that will also allow me to know how it relates to the ability to destroy the most resistant pathogen.  (i.e., C. bot).

## Test Organisms for Aseptic Systems*

- Medium
- Superheated steam
- Dry heat
- H2O2 + heat
- H2O2 + U.V.
- Heat of formation
- Gamma Radiation
- Wet heat

- Organism
- B. stearothermophilus
- B. stearothermophilus
- B. subtilis A
- B. subtilis A
- B. stearothermophilus
- B. pumilus
- B. stearothermophilus

- *Bernard et al., 1987, Principles of Aseptic Processing and Packaging, FPI

**Notes:**
This is a list of indicator organisms that have been traditionally used for different types of destruction mechanisms.

## Log Reduction

**Notes:**
The destruction efficiency of a process is often referred to the number of logs of microorganisms that are destroyed.  This would be the number of logs in difference between the starting and ending micro counts.

## Log Reduction

- In the original 1978 Tetra Pak filing for web fed system, there was an understanding that the package material was relatively clean (1 spore/1 liter package; which resulted in the target of a 6 log reduction of *Clostridium botulinum*)
- There was a carryover of this to blank fed systems, and the sterilization of the sterile zone

**Notes:**
For the very first aseptic filling equipment validation experiments it was realized that the spore load of the packaging and aseptic zone where going to be low.  From actual experiments it was found that the microbial load on the packaging material was on the order of 1 organism per 1 liter package.  From this it was agreed that a 6 log reduction in C. bot would result in an acceptable assurance of safety.  

## Log Reduction (cont’d)

- Indicator organism most often used is *B. subtilis *var. *globigii *for H2O2 treatments
- Over the years the industry has tended to target a log reduction of *B subtilis* var. *globigii *of 4 or more, for both the aseptic chamber and the packaging material
- Reuter, 1993, *Aseptic Processing of Foods*, pg 159. For LACF aseptic products a *B. subtilis *LCR of 5-6 is recommended

**Notes:**
From this expected 6 log reduction in C. bot it was shown that a 4 log reduction in B. subtilis A (or ver. Globigii) for H2O2+heat sterilization would be comparable.  Thus, the traditional expected minimum log reduction for both the aseptic zone and the packaging material when using H2O2 and heat is 4 logs of B. subtilis.  This also assumes that the B. subtilis being used is of sufficient H2O2 resistance.  There are very few studies undertaken so as to investigate what the target should be.  However, in 1993 there was a recommendation that was published that indicated that a 5-6 log reduction of B. subtilis should be the target.

## Concept of PNSU

- PNSU – probability of a non-sterile unit
- Establishes a target end point for the lethal treatment (i.e., 1 out of 10-6 non-sterile units)
- Pflug* recommended end points of:
- public health end point of  10-9 for *C. bot*
- mesophilic spoilage end point of 10-6
- thermophilic spoilage end point of 10-3 for low temperature storage or 10-6 for high temperature storage

- *Pflug, 1987, *Endpoint of a preservation process*, J. of Food Protection, Vol 50, 347-351

**Notes:**
A log reduction (or process treatment) mentality assumes that there is some know worst case and from that known worst case there is a needed log reduction in order to have some assurance that the process will always deliver a commercially sterile product.  This process treatment mentality has been used within the food industry for a long time with great success.  This however, is not the only type of logic that is used when determining the target treatment for a system.  Within the pharmaceutical industry the mentality is not on log reductions, but on establishing some level of safety or probability of a non-sterile unit.  This has developed into the expectation that a 1 out of every 1,000,000 (10^-6) units produced may be non-sterile.   The outgrowth of this is that the process must be designed so that all of the produced units have a sterilization expectation of 1 in 1,000,000.  In order to work this way the processor needs to have some level of knowledge as to the incoming raw product contamination, which may change from vendor to vendor, and from raw ingredients to raw ingredients.  In 1987 Dr. Pflug used this concept for the sterilization of food products and recommended a 1 in 1,000,0000 sterility assurance level for typical mesophilicic spoilage spores and 1in 1,000,000,000 for pathogenic C. bot.

## Concept of PNSU (cont’d)

- For PNSU to work, the initial load of the product usually becomes a critical factor
- The concept of PNSU is used within the medical drug side of FDA for aseptic processing – with a target of 10-6

**Notes:**
Using a PSNU type of process establishment means then that the initial concentration of the raw material may become a critical factor and would need to be monitored and controlled.  Most food companies would most likely not want to work this way and this may be why the LCR method of process establishment has worked so well.  I would like to point out that the PSNU concept is very close to the Food Safety Objective (FSO) concept.

## Measure Indicator Organism Thermal/Chemical Resistance

**Notes:**
Now, lets talk about the actual biological test.

## A. Sterilization of sterile zone

**Notes:**
First - the sterilization of the aseptic zone.

## Sterilization of Sterile Zone

- Organism used for inoculation study must be calibrated
- Inoculation method
- strips and discs
- not direct application to machine surface
- recovery method needs to account for all remaining surviving organisms
- Test worst case – at critical limits

**Notes:**
As indicated when we talked about the indicator organism it must be of the same resistance as the test organisms that were used when establishing the initial 4 log reduction policy.  The process authorities that work in this area all have developed transfer/growing procedures for the indicator organism to ensure its resistance.  Each spore crop is tested, and these results become part of the sub-SID, before the inoculation tests.

Each process authority has develop procedures for inoculation and recovery of the test organisms.  It is important here to make sure that correct laboratory procedures are used to inoculate and recover the test organisms.  Care needs to be used to make sure that appropriate quantitative procedures are used.  If possible it is not advisable to place organisms directly onto the filler equipment.  The reason being recovery, usually done using swabs, is almost impossible.  Swabbing is not an acceptable analytical technique.  Swabbing is good for qualitative tests but not quantitative ones.  If direct application is necessary then recovery tests must be conducted.

The Idea of validation of the aseptic zone is to try and find the area within the aseptic zone that is the most difficult to sterilize, and to do this with the machine running at or below the critical limits of the critical factors.

## Sterilization of Sterile Zone (cont’d)

- Environmental conditions - worst case
- Building construction
- Operation conditions
- Equipment preconditioning
- Computer program validation
- Normal startup - bring to normal conditions between tests
- Separate conditions for resterilization
- Filter presterilization
- Document filter sterilization

**Notes:**
Thought needs to be given to the effect of any possible environmental factors that could influence the tests.  This would include operational conditions in and around the filler and any possible building construction activities that would cause a difference.

All of the equipment components should be validated separately – for example the computerized controller.  It is expected that during the test the equipment is running correctly and this will require a separate validation of the process controller.  The system should be brought to a condition of normal operation and tested with the worst case possible.  This may include the need to evaluation the effect the cleaning cycle has on the initial condition of the filler.  The process authority may want to precondition the filler to a dirty state before cleaning and sterilization to test the ability of the sterilization cycle to make sure that the worst case has been tested.

Any possible restart, short start, etc. that the system may be operated in should be evaluated to make sure that the validation tests represent the appropriate challenge of the system.

Sterilization of the aseptic zone must also include sterilization of any aseptic filtration system used.

## Sterilization of Sterile Zone (cont’d)

- “disks” placed throughout system
- next to large metal fixtures
- look for heat sinks
- shielded areas from vapor/spray
- Multiple levels of test organism concentration (i.e., 104, 105, 106)
- Need to find lower bound of LCR (i.e., clean interval)
- Sufficient test repetition to understand system

**Notes:**
When placing tags within the aseptic zone, care needs to be given to make sure that sufficient tags are used to make sure that any possible heat sink or shielded area is tested.

When conducting this test there is only going to be one spot where the minimum sterilization treatment is delivered.  The point of this test to try and find this minimum sterilization zone.  The problem is that with only one spot being the minimum treatment zone it becomes very difficult to statistically evaluate if the test conducted has reliably determine where that spot is and if the equipment is sterile.  With both a desire to measure the actual processing effect and to get a better handle on the reliability of the system multiple levels of test organisms are used when conducting these tests.

Since theory and actual practice don’t always coincide it is the opinion of the FDA that to appropriately demonstrate the ability to sterilize the aseptic zone the minimum acceptable LCR for the test must be conducted where there are no survivors i.e., the minimum acceptable LCR needs to be a clean interval.

## Sterilization of Sterile Zone

**Peroxide**

** ****Sterile Air**

** ****Product**

**Sealer**

**Air**

**Tags**

**Notes:**
An example of where test tags might be placed.

## Sterilization of Sterile Zone (cont’d)

- There is only one spot within the sterile zone that receives the minimum lethal treatment
- Usually complete incubation of each tag with numeration of positive and negative tags
- cannot use inoculated pack type procedures to enumerate

**Notes:**
With there only being one spot where the minimum treatment to the aseptic zone is delivered the other test locations can not be used as part of a measurement of the lethal treatment of the sterilization of the aseptic zone.  You can not use the most probable number type test analysis (inoculated pack) for testing of the aseptic zone.

Testing of the aseptic zone is dependent on a strong understanding of the system, its critical factors, and demonstration of a clean minimum LCR test run.  There should also be some consideration as to repeatability over time an condition of the equipment. 

## B. Sterilization of package material

**Notes:**
The next validation test is validation of the sterilization of the packaging material.

## Sterilization of Package

- Inoculation method
- find location on package of minimum sterilizing capacity
- application of inoculum must be appropriate for sterilization system (spray vs. spot inoculation)
- may need to challenge outside of package
- for radiation sterilization, traceable dosimetry can be used
- recovery method needs to account for all remaining surviving organisms
- recovery of  inoculum around fitments may be a problem

**Notes:**
Just like there are variations is treatment within the aseptic zone the preformed, or form-fill-seal packaging material will also have a minimum treatment zone.  Web material should have a uniform treatment across the web.  For cup material - care needs to be used to try and find where the minimum treatment zone is within the packaging material. 

Both point and spray inoculation procedures have been used on packaging material.  It is expected that the process authority has the necessary understanding at to which procedure should be used for the system being tested.

Depending on the definition of the aseptic zone the outside of the package may needed to be sterilized.  If the complete package enters the aseptic zone – all of the package material must be brought to a condition of commercial sterility (inside and out).

When testing radiation systems there is sufficient evidence to indicated that dosimetry test procedures can be used in place of biological testing.

Just like for the aseptic zone the process authority should try and not use swabbing as an analytical tool.  If it is required, there needs to be some recovery test conducted to demonstrate the ability of the method to quantify the number of remaining organisms.  Sometimes the best way to measure the remaining organisms is to use a MPN type method where the actual material is cut out and placed into tubes to measure the remaining organisms.  This way you will be able to show that the treatment is at least able to deliver a process equal or greater that the initial load when no growth is detected after incubation.

## Organism used in inoculated pack study must be calibrated
For enumeration - usually use media fill with direct incubation
Use growth media that supports growth of viable and injured spores.
Run with worst case critical limits.
Often run 100-200 packages through system

- For enumeration - usually use media fill with direct incubation
- Use growth media that supports growth of viable and injured spores.
- Run with worst case critical limits.
- Often run 100-200 packages through system

**Sterilization of Package ****(cont’d)**

**Notes:**
For the inside of a cup - inoculation media fills, or MPN type testing is the best.  Test runs of 100-200 packages can be used to measure the sterilization of the packaging material.

As in the case of the aseptic zone the test organism must be calibrated to insure that the LCR will reflect the necessary lethal treatment.

Sometimes to get the system to run at or below the critical limits, the system may have to be adjusted so as to ignore the alarm conditions.  Also, a processor may fine that some tests need to be run with different test conditions in order to be able to find if the critical factors interact with each other and that the worst operating case is not different from the set of critical limits identify by looking at each critical factor individually.

## Statistical Evaluation of the Biological Validation of a Continuous Aseptic Process

**Notes:**
For the validation of the packaging there are some statistical procedures that can be used to determine if the test results are reasonable.

## Impact of Statistical Assumptions for Inoculated Pack Studies

- Each sample used to determine the lethal treatment delivered must have the exact same thermal treatment as all the other samples.

**Notes:**
Traditional test procedures are based around the inoculated pack procedure used within the canning industry.  However, case needs to be given to makes sure that the assumptions of the inoculated pack test are maintained in the test procedure.  The most important assumption is that each and every package has received the exact same treatment as all the others.  There is no worst case treated package.

## Underlying Assumptions

- "The first is that the organisms are distributed randomly throughout the sample.  This means that the organism level is equally likely to be the same in each sample."
- "The second assumption is that each sample when incubated is certain to exhibit growth whenever the sample contains one or more organisms."

- W.G. Cochran (1950) - "There are two principle assumptions."

**Notes:**
This condition of equal treatments come from the fundamental statistical bases for the most probable number (MPN) procedure.  MPN procedures are based on two assumptions.  The first being that all organisms are evenly distributed within the sample.  The second assumption is that when a sample is incubated and demonstrates growth there was one or more organisms within the sample to start with.  The MPN procedure has been used for years to measure microbial loads in samples.  Usually MPN methods use 3, 5, or 10 tubes as the replications to determine the most probable surviving number.  For inoculated pack often the replications are 100-200 samples.  By using these high replications the test procedure is able to determine a high possible LCR than with the traditional MPN 3,5, or 10 tube replications.

## How do You Know When an Inculcated Pack Procedure can be used for the Inoculation Study?

**Notes:**
The process authority needs to make sure that they ask themselves the questions – how do I know that the results of the inoculation pack test are appropriate and that all the assumptions to the test procedure have been fulfilled?  The idea here is – how do you know that the results measured are from equal treatments and not from some possible sporadic contamination that results from improper design or operation of the system.  The best way to do this is to test the likelihood of the MPN result that was obtained when starting with different initial counts.

## Log Count Reduction (LCR) as a Property of the Challenge Level*

*** ****Moruzzi et al., Food Control, 2000, page 57**

**Log Count Reduction (LCR) as a Property of the Challenge Level*******

**Notes:**
Moruzzi, et al., published an excellent article on MLE of an inoculated pack test.  Depicted here is the assumed test condition.  Where for 4 different initial loads the log reduction of the system is identical and would allow the process authority to back calculate the effectiveness of the process.  You can see that there is a artificially high challenge level that is used to extrapolate back to the low loads of reality.  The question is how can I tell if the data form the test will allow me to extrapolate back to reality.

## LCR at different challenge  levels is not constant;

**extrapolation to "reality" is impossible**

**LCR at different challenge  ****levels is constant;**
**LCR is a property of the ****process, and extrapolation to "reality" can be tried**

**"GOOD DATA"**

**"BAD DATA"**

*** ****Moruzzi et al., Food Control, 2000, page 57**

**Notes:**
Here we see two sets of test results – one being a good set of data and the other being a bad set of data.  We are not going to go into the procedure used to determine if a test produces bad or good data, but the process authority needs to make sure for any inoculated pack test there is a measurement of the likelihood of the result.

## Sterilization of Package (cont’d)

- Applicability of MPN method (i.e., inoculated pack)
- MPN assumes equal treatment for each test sample
- may not be applicable to multi-lane machines
- each lane needs to be shown to have equal treatments
- test each lane, or find worst lane
- need to demonstrate acceptance of data analysis assumptions before tests

**Notes:**
Since MPN, or inoculated pack, assumes equal treatments a multi-lane process will need to be demonstrated as each lane being equal before the lanes can be ignored as a possible worst case.  If the lanes are not equal then the lane that delivers the worst treatment must be tested to show that it is able to deliver the minimum LCR.

## Document the Results

**Notes:**
Finally all of the description of the equipment, identification of the critical factors and their limits, and the test producers and results must be put together as part of the sub-SID – in addition to the records and documents that are created by the operation of the system.

## Records

- Proper training of operator must be done
- There needs to be control of the software used in all of the automated control systems
- There needs to be version control on all operation documentation
- Thorough record review needs to be done (cannot depend on a perceived reliability of the equipment, i.e., computers)

**Notes:**
The sub-SID should include information about operator training, software validation if required, what type of records are created by the system and how are they reviewed.  Example charts should be included.

## Records (cont’d)

- Product sterilization is submitted on form 2541c
- Filler sterilization procedure is filed as a supplement to the process filing
- One supplement filing for a filler may be used for many different products as long as the processing and filling operation is unchanged

**Notes:**
When a processor is ready to produce product the process is filed on form 2541c and the sub-SID additional documentation.  The sub-SID may be used on more than one product filing as long as the filling operation does not change.  That is as long as the validation of the filing system still represents the critical limits of the system.  For example, if the filling equipment is to be slowed down for one product verses another and all of the critical factors for the operation remain the same for both products, the same sub-SID can be used.

When looking at establishing a new aseptic filler, it is best for the processor and process authority to notify FDA of how there equipment operates a number of different times verses only when ready to go into production.  This way the processor can get feedback as they develop their system and not be so surprised when concerns are raised right before they planned to produce a large quantity of product.  An informed FDA is better than an uninformed one.