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Lyophillizer Validations, Aseptic Process simulations design-Lyo process and Rationale for Hold duration of Partially Stoppered Media Filled Vials-In compliance with Revised EU Annex-1 2022 (part I)

Author: Varadharaj Vijayakumar - Aseptic Technology, Isolators & Lyophilization

After two draft documents from 2017 and 2020, more than 6,000 comments and much speculation about the final publication date, it is finally here: Yes, the long-awaited final version of the new 58 pages of EU Annex 1 "Manufacture of Sterile Medicinal Products" was published on August 25, 2022. The deadline for entry into force was August 25, 2023.

The document has increased in size by another six pages from the draft version, but its basic structure into eleven areas has remained the same. Among these, sections 8.121 to 8.126 on freeze-drying and product transfer were covered in this article and the deadline for entry into force is two years and will thus become mandatory on August 25, 2024.


The scope of Annex 1 applies to all sterile medicinal products manufactured in the European Union and the UK, as well as those manufactured elsewhere and exported into the European Union.

As we all know, the pharmaceutical industry is facing the new challenges of integrating the new EU GMP Annex 1 requirements into its current and new facilities.

The purpose of this white paper is to introduce Annex 1 and to describe the significant changes and the scientific/regulatory reasons behind the design of aseptic process simulation for lyophilized drug products with special considerations in key validation aspects of lyophillizer.


The “Media Fill,” or “Broth Fill,” technique, is one in which a liquid microbiological nutrient growth medium is prepared and filled in a simulation of a normal manufacturing operation. The nutrient medium processed and handled in a manner which simulates the “normal” manufacturing process as closely as possible with the same exposure to contamination (from operators, environment, equipment, and surfaces) as may occur during routine manufacture. The sealed containers of medium thus produced are then incubated under prescribed conditions and examined for evidence of microbial growth, and thus can obtain indication of the level of contaminated units produced.


Purpose of Aseptic Process simulation

The goal of a media fill is to demonstrate that the manufacturer can follow the routine aseptic production process using sterile media without contamination (evaluate and justify the aseptic capabilities of the process, the people and the system). Hence in this article, we ensure lyo media fill should primarily validate the filling, transportation and loading and unloading aseptic operations with certain duration of filled vials in the lyophillizers.

During aseptic process simulation for lyophilized product, filled vials are aseptically filled in the normal way (similar to liquid filling), but the closures (which are of a single slotted or double slotted) are not fully inserted in the filled vials and transported aseptically through conveyor/ALUS/robotics to the lyophilizer shelves under Grade A atmosphere.


Loading of media filled vials in the lyophilizer

Shelf to be pre-cooled to 20-25°C. The media filled vials are loaded in the sterilized lyophilizer (using suitable in-house designs such as automatic loading/Manual loading using fences). At the completion of the loading process, it is important to ensure all filled, partially stoppered vials are loaded into the lyophilization chamber.


Product lyophilization process

Lyophilization or freeze drying is a process in which water is removed from a product after it is frozen and placed under a vacuum, allowing the ice to change directly from solid to vapor without passing through a liquid phase.


Entire Lyophillizer needs to be simulated by loading?

Instead of filling and loading all the shelves with media filled vials, covering top, middle and bottom shelves can be preferred.  These shelves shall be loaded with filled partially stoppered vials to mimic the routine process. The shelf numbers 1, 3, 6, and 10 shall be selected to cover the loading activity (top to bottom loading) if the design of lyophilizer is with 10 shelves and this process simulation will cover all the shelves are loaded into the freeze dryer to cover the whole freeze-drying chamber and process.


What will happen if it is simulated for longer hours of holding similar to product lyophilization?

Exact simulation of media fill as that of product lyophilization will have possible reduction of microbiological levels after aseptic manipulation which will not solve the purpose of aseptic process simulation.

Carrying out a lyophilization cycle and freezing the media will be same simulation as that of product lyophilization, however this freezing of media will reduce microbial levels of some contaminants.


In aseptic process simulation of lyo process the scope is not to check the lethality of freezing and its effect on microorganisms that might be present. Hence freezing of media and the formation of ice crystals is unfavorable to microorganisms hence this should be avoided.


Unfrozen media and complete vacuum

If media is not frozen, then the lyophilizer may get contaminated due to unfrozen media which is left under vacuum similar to the product lyophilization vacuum level. If complete vacuum is drawn then it may cause the media solution to get out from the containers and contaminate the lyophilizer as well fluid loss from the containers and finally this may have serious effect on the viability of the microorganism and the ability of the media to support microbial growth will be impacted this will in turn make aseptic process simulation invalid.

Hence a complete vacuum as specified for the lyophilization process should not be drawn during the media fill.


Hold time duration in the lyophillzer

In general, the course and processing time should be long enough to challenge or stress the process, interventions, the supporting environment, and the operators. All efforts should be made to perform all routine interventions. The filled partially stoppered vials will remain inside the lyophilizer chamber for minimum 12 hours (Author’s experience for hold time duration).

The duration of the media filling stated above represents lyophilization cycle for one shift.


The integrity of the lyophilizer chamber is tested as a part of preparation process (ie) during post sterilization cycle. Therefore, no additional benefit is drawn by holding the media filled vials for longer duration (such as product lyophillization cycle), as the lyophilizer’s integrity is maintained throughout this cycle. Hence, it is not necessary to carry out lyophilization cycle as per the actual drug product.


Secondly, as compared to lyophilization hold duration for filled vials in lyophillzer the chances of contamination for partial stoppered vials is more during the process stages such as empty vial transportation, media fillings and transportations, interventions, samplings, half stoppering, loading of vials into lyophillizer, etc.


So how long the APS process shall be simulated?

Typically 600 mbar to 700mbar, few manufacturers used maximum of 900 mbar of partial vacuum and held for about 12 hours and few manufacturers held it for two to three hours and then vacuum in chamber gets broken by sterile filtered compressed air (used to back fill the chamber, instead of the nitrogen) and full stoppering of vials shall be performed using the stoppering mechanism at ambient pressure and open the door.


Finally unload the vials aseptically and transfer to sealing machine. All these activities shall be performed in class 100 (Grade A).

Possible questions from regulatory agencies for Lyo media fills


Ø  Is the aseptic handling of lyophilized products validated by media fills?

Ø  In the aseptic process, is lyophilization simulation performed during media fill?

Ø  Is the maximum amount of time the vials are held prior to lyophilization simulated during media fills. If vials are not sealed in lyophilization chamber, is the maximum hold time prior to stoppering simulated in media fills?

Ø  During validation, what level of vacuum is pulled on the lyophilization chamber?

Ø  How long do media fill vials remain in the lyophilization chamber under vacuum? How does this compare to commercial lots?

Ø  Does the process simulation result in freezing of the media? Note that this process simulation should not include freezing of the media.

Ø  Is environmental monitoring performed during loading of the lyophilizer both during production and as well as during validation?

Ø  Does the firm have data on growth promotion of the media? Are growth promotion tests done on vials after incubation is completed?

Ø  Is environmental monitoring performed during unloading of the chamber during production as well as during media fill validation?

Ø  What is used to break the vacuum during media fills (nitrogen, air, other gas)?

In my view, with practical experiences in conducting and designing several aseptic process simulation programs- would recommend few key rationales for bracketing concepts:

Vial and Stopper Size

The neck size of the vial is considered as a deciding parameter for the media fill and other parameters like body diameter are considered as non-deciding parameters for the media fill. The vials having neck diameters of 13 mm, 20 mm and 32 mm are generally used in industry. The size of the vial shall be considered while deciding the worst case for the media fill.


The size of the rubber stopper is deciding parameter of the media fill and hence is considered as a critical parameter. The rubber stoppers of size 13 mm, 20 mm and 32 mm are generally used in industry shall be considered while deciding the worst cases for the media fill.


Type of Vial and Stoppers

The tubular or molded vials are in use. For the media fill the tubular vials are considered as worst case since the tubular vials are having lesser weight and have larger movement or tendency to toppl.


The stoppers of the Bromo butyl and chlorobutyl are generally used and the type of the stopper is not a deciding parameter for the media fill and stopper of any type can be used for the media fill.


Source of Vial

The source of vials is not a deciding parameter for the media fill and hence any source of vials can be used.

Finishing and color of Vials and Stoppers

The finish of vial viz: Blow back or non-blow back and treated or non-treated does not play a deciding role in media fill and hence the vial of any finish can be used. The clear glass vials shall be used for the media fill to facilitate the visual inspection of the vials.


Design of stopper

The rubber stoppers coated and noncoated are used generally but not as deciding parameter for the media fill. But design of stoppers are important and plays a deciding factors e.g. 2-leg, 3-leg igloo or flat-teflon, serum etc


Filling Speed 

Slowest line speed with the highest opening diameter, i.e long time the unit is open and a wide area, where contamination could fall into the unit. Also, usage of slow line speed is generally appropriate for evaluating manufacturing processes with prolonged exposure of the sterile drug product and containers/closures in the aseptic area.


Conduct each simulation study run with a single line speed by simulating the worst-case condition. E.g.: using the slowest fill speed for the biggest container size and fastest speed for the smallest container size.


Fill Volume

The volume of medium must be sufficient to provide contact with all container closure seal surface on inversion and visibility for the detection of microbial growth. The quantity of sterile SCDM medium shall be considered for filling in the vial with standard different sizes are as follows:


Vial Size

Fill Volume

5 mL 13 mm

3 ml to 4 ml

10 mL 20 mm

6 mL to 8 ml

20 ml 20 mm

12 mL

30 ml 20 mm

15ml to 20 mL

Pulling and Releasing of Partial Vacuum

Pull the condenser of lyo to -40 °C and start the vacuum pumps. The loaded vials are exposed to the partial vacuum cycle.


First Pull of Vacuum 

Pull the vacuum to 500-700[1] mbar, after achieving the vacuum of 600 mbar, and maintain it for 15 minutes to one hour after that release the vacuum gradually using filtered (0.22-micron sterile filter) compressed air up to atmospheric pressure. The half-stoppered vials will be held at atmospheric pressure for approx. 15 minutes to one hour.


Second Pull of Vacuum 

Pull the vacuum to 500-700 mbar after about one hour of first pull of vacuum and after achieving the vacuum of 600 mbar and maintain it for 15 minutes to one hour after this release the vacuum gradually using filtered (0.22-micron sterile filter) compressed air up to atmospheric pressure.

The half-stoppered vials will be held at atmospheric pressure for approx.15 minutes to one hour, after this period perform the stoppering of vials into the chamber atmospheric pressure.


Note: In case of anaerobic media fill, break the vacuum of lyophilizer using filtered nitrogen gas, instead of filtered compressed air.


How many Vials shall be loaded?


1.     Minimum incubation quantity of 10000 vials. Accordingly, batch size shall be designed during media fill.

2.     Minimum number of vials to be filled shall be decided based on equipment constraint, where number of vials shall be filled considering the maximum capacity of the equipment.


Note: The media fill duration shall be sufficient to cover actual duration of the filling of the drug product. The leftover media after completion of the filling shall be measured and discarded.


Visual Inspection of filled vials 

All filled and sealed units shall be collected in sequence with respect to filling order. All the units should be inspected 100% prior to incubation. Defected vial which integrity is breached, are counted and deducted fromthe acceptable units filled.  Do not reject units for low or high fill volume any cosmetic defect and other rejection, which integrity is not breached. The filled vials must be inverted or otherwise physically manipulated to ensure that all surfaces including the internal surfaces of the stopper are thoroughly wetted by the medium. 


Handling of online rejects (Liquid and Lyo APS) generated in the aseptic processing area during media fill run 

Units removed during an APS should not be more than units removed in routine production run. The quantity and rationale for manually or automatically removed units from the filling line must be documented.


Good Practices to be considered for rejects 

Ø  The rejects of filled units generated at all the stages of processing during media fill runs and those rejects in unstoppered state (missing or displaced stoppers) shall be considered as non-integral rejects as they are not intact (lacks integrity). These non-integral rejects shall be rejected online during media fills and shall not be considered for incubation.


Ø  The half stoppered or partially stoppered filled vials are considered as unstoppered reject units. Hence rejects of these categories during media fill runs shall be considered as non-integral rejects as they are not intact (lacks integrity). These non-integral rejects shall be rejected online during media fills and shall not be considered for incubation.


Ø  The fully stoppered filled reject units generated at all the stages of processing during media fill runs to be recorded and can be incubated. This is for investigation purpose only. (in view of container closureconfiguration of aseptic products manufactured for commercialization, these rejects are considered asnon – integral units and hence are rejected and destroyed routinely).


Ø To enhance the assessment of contamination risks and obtain valuable information that may havepotential contamination from the simulated activities/interventions during aseptic productsmanufacturing, the media filled fully stoppered rejects shall be preserved in the aseptic area during media fill run. These reject units shall be subjected to sealing at the end of the media fill run and shall be incubated separately with identification details after subjecting to inspection.


Ø  The fully stoppered vial with partial sealing (improper sealed unit without leak) generated during media fill runs shall be considered as non-integral rejects (in view of container closure configuration of aseptic products manufactured for commercialization, these rejects are considered as non – integral units and hence are rejected and destroyed routinely). These rejects shall be incubated separately withidentification details after subjecting to inspection.


Ø  Assess the contamination risks and obtain valuable information, the above stated rejects shall be incubated separately for information purpose only. Any positive results of these rejects shall not beconsidered for interpretation of media fill results as these units will not provide meaningful measure ofthe aseptic process and do not represent either acceptable production practices, or acceptable container closure integrity. However, failures if any (positives) in the above rejects shall be investigated separately through deviation to determine the origin of contamination and the scope of the problem if any.

[1]The minimum vacuum level observed in lyophillizer is 500 mbarA, where the no fallen rubber stopper and spillage of solution of half stoppered vials was observed.With 500 mbarA in half stoppered vials will not inhibit the microbial growth was observed. The approach of simulated load / unload with shortened hold time has been followed as per PDA Technical Report No. 22.


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