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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 II)

Author: Varadharaj Vijayakumar - Aseptic Technology, Isolators & Lyophilization

Following our previous article part I, 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.

Validation and Revalidation strategies for lyophilizer

From my experience, I Would recommend dividing the qualification plans with certain frequency. Minimum is performance qualification and performance re qualification.

Routine Qualification

Frequency of routine qualification: Once in a six month

Some of the tests can still be relaxed to once/twice a year based on risk assessment.

Performance re-qualification criteria

  •  Changes in design or major maintenance

  •  Relocation of the equipment

Note: For the performance re-qualification of system after changes in design or major maintenance required test should be performed based on impact of change on equipment performance.

From my experience, Below tests shall be considered for initial qualification


Routine Qualification: Six Months once


Routine Qualification: Six Months once

Routine Qualification: Two years once


Hold time of SIP -In lyophillizer

The holding time between the sterilization cycle and use should be appropriately challenged during aseptic process simulation (APS).

Holding time after sterilization cycle till lyo door opening to load the media filled vials

Ø  Needs to be challenged during aseptic process simulation (APS).

Ø  How long? Based in process need and risk assessment.

Ø  24-36 Hours? (some companies simulated it as minimum hold duration)

Ø  Less than or equal to 36 hours is considered in GMP runs.


Rationale for Leak Test In lyophillizer

Assurance of the vacuum integrity of freeze - dryers used for the manufacture of sterile pharmaceutical products is essential for GMP operations.

However, there is currently no generally accepted scientific rationale for establishing acceptance criteria for such testing.

As it stands, current acceptance criteria are generally based on equipment capability – perhaps proposed by the manufacturer, or from data collected during qualification of a new freeze - dryer. The targeted specification is often one cited by The Parenteral Society.

“A frequently specified leak rate for new, clean, dry, and empty freezer dryers would be 2 x 10 - 2 mbar - liter/sec or 1 x 10 - 2 mbar - liter/sec (15 μm (mTorr) liters per sec). The leak rate should not change significantly during the life of the freeze dryer ...”.


Comparison between EU Annex-1 2008 and Annex-1 2022


What Annex-1- 2008 says about Lyophilization

Aseptic preparation: 34 Prior to the completion of stoppering, transfer of partially closed containers, as used in freeze drying should be done either in a grade A environment with grade B background or in sealed transfer trays in a grade B environment.

Finishing of sterile products: 116 Partially stoppered freezedrying vials should be maintained under Grade A conditions at all times until the stopper is fully inserted.

Finishing of sterile products: 123 Containers sealed under vacuum should be tested for maintenance of that vacuum after an appropriate, pre-determined period.

Annex-1 2008


Annex-1 2022


Key Points from Annex-1-2022 about Lyophilization

8.122 : The sterilization of the lyophilizer and associated equipment (e.g. trays, vial support rings) should be validated and the holding time between the sterilisation cycle and use appropriately challenged during APS. The lyophilizer should be sterilised regularly, based on system design. Re-sterilisation should be performed following maintenance or cleaning. Sterilised lyophilizers and associated equipment should be protected from contamination after sterilisation.

8.123: Lyophilizers and associated product transfer and loading/unloading areas should be designed to minimize operator intervention as far as possible. The frequency of lyophilizer sterilisation should be determined based on the design and risks related to system contamination during use. Lyophilizers that are manually loaded or unloaded with no barrier technology separation should be sterilised before each load.  For lyophilizers loaded and unloaded by automated systems or protected by closed barrier systems, the frequency of sterilisation should be justified and documented as part of the CCS.

Explaining 8.123 : Sterilization in Lyophillizer and its Hold time simulations

A generally recognized acceptable method of sterilizing the lyophilizer is through the use of moist steam under pressure. For the sterilisation process, saturated steam is introduced into the freeze-drying system.

In the sterilisation phase, the chamber, and the condenser along with all connected piping (including venting filters) are sterilised. The pressure is controlled by means of the steam pressure regulators according to the chamber pressure measurement. Due to high pressure requirements during steam sterilization, manufacturers must design, test, and approve both chamber, condenser as rated pressure vessel. Additionally, there are numerous requirements for other factors such as pipes lengths, bends, filters, that engineers must consider as steam must penetrate all areas of the freeze dryer in order to serve as an effective sterilizing agent.

The sterilisation temperature is measured at the following drainage points: Chamber outlet, Condenser Outlet, CIP Pump Outlet and Filter outlet.

At the end of the sterilisation process, the freeze-drying system is dried with the aid of the liquid-ring pump. The drying shelf assembly and the chamber wall are re-cooled after drying.

The sterilisation of the lyophilizer and filling line associated equipment should be validated, and sterilization frequency should be justified.

The lyophilizer chamber is to be Cleaned and sterilized between batches because of the direct means of contamination. (Expectation of cleaning and sterilization is also applicable when there is maintenance in lyophillizer).

The holding time between the sterilisation cycle and its use should be appropriately challenged during aseptic process simulation (APS).

8.124 The integrity of the lyophilizer should be maintained following sterilisation and during lyophilization. The filter used to maintain lyophilizer integrity should be sterilised before each use of the system and its integrity testing results should be part of the batch certification/release. The frequency of vacuum/leak integrity testing of the chamber should be documented and the maximum permitted leakage of air into the lyophilizer should be specified and checked at the start of every cycle.

8.125 Lyophilization trays should be checked regularly to ensure that they are not misshapen or damaged.


8.126 Points to consider for the design of loading (and unloading, where the lyophilised material is still unsealed and exposed), include but are not limited to:


i. The loading pattern within the lyophilizer should be specified and documented.

ii. The transfer of partially closed containers to a lyophilizer should be undertaken under grade A conditions at all times and handled in a manner designed to minimize direct operator intervention. Technologies such as conveyor systems or portable transfer systems (e.g. clean air transfer carts, portable unidirectional airflow workstations) should be used to ensure that the cleanliness of the system used to transfer the partially closed containers is maintained. Alternatively, where supported by validation, trays closed in grade A and not reopened whilst in the grade B area may be used to protect partially stoppered vials (e.g. appropriately closed boxes).

iii. Airflow patterns should not be adversely affected by transport devices and venting of the loading zone.

iv. Unsealed containers (such as partially stoppered vials) should be maintained under grade A conditions and should normally be separated from operators by physical barrier technology or any other appropriate measures.

v. Where seating of the stoppers is not completed prior to opening the lyophilizer chamber, product removed from the lyophilizer should remain under grade A conditions during subsequent handling.

vi. Utensils used during loading and unloading of the lyophilizer (e.g. trays, bags, placing devices, tweezers) should be sterile.


Lyophilization is a critical process step and all activities that can affect the sterility of the product or material need to be regarded as extensions of the aseptic processing of the sterilised product. The lyophilization equipment and its processes should be designed to ensure that product or material sterility is maintained during lyophilization by preventing microbial and particle contamination between the filling of products for lyophilization, and completion of lyophilization process. All control measures in place should be determined by the site’s CCS.

From Lyo APS Design point of view,the duration of the media filling stated above represents overnight lyophilization cycle. We ensure the integrity of the lyophilizer chamber by testing the lyophilizer post SIP cycle, therefore no additional benefit is drawn by holding the filled vials for longer duration since the lyophilizer’s integrity is maintained throughout cycle.


Hence, it is not necessary to carry out lyophilization cycle as per the actual drug product lyophilization cycle. As compared to lyophilization hold period, probability of contamination of vials is more during the process stages such as filling, stoppering etc and also As per regulations from few regulatory authorities it’s clear that hold time does not need to be the actual duration of lyophilization cycle.


There should be written justification for the hold duration of media fill vials in the aseptic simulation process of freeze drying process.


Hence, A balance risk and science-based approach is needed to simulate the process as closely as possible, and rationale for holding the vials in the lyophillizer need to be presented which is the expectation from the regulators.


Finally in case of failure in the media it necessary to diagnose and prove the source of contamination accurately so that robust corrective or preventive actions get implemented.



1.     Freezing should not be simulated: 24 of 26 manufacturers using lyophilization who responded to the PDA's 1996 survey of aseptic manufacture claimed not to freeze their media fill vials (PDA, 1996).

2.     Lyophilization Validation: A Regulatory Perspective by Ellen Huang CBER/OCBQ/DMPQ CASSS CMC Strategy Forum July 19, 2016” slide 22 states that “Hold time does not need to be the actual duration of lyophilization cycle”

3.     FDA, Guidance for Industry: Sterile Drug Products Produced by Aseptic Processing–Current Good Manufacturing Practice, 2004

4.     Medicines and Healthcare products Regulatory Agency: Rules and Guidance for Pharmaceutical Manufacturing and Distributors, current, Annex-1: Manufacture of Sterile Medicinal Products.

5.     The Rules Governing Medicinal Products in the European Union Volume 4 EU Guidelines for Good Manufacturing Practice for Medicinal Products for Human and Veterinary Use Brussels, 22.8.2022.

6.     Pharmaceutical Inspection Convention and Pharmaceutical Inspection Co-operation Scheme: Recommendation on the Validation of Aseptic Processing, 01Jan 2011.

7.     Health Products and Food Branch Inspectorate, Good Manufacturing Practices (GMP) Guidelines – 2009, Edition GUI – 0001, November 8, 2009

8.     The Parenteral Society, Technical Monograph No.7: Leak Testing of Freeze Dryers (Wilshire, England: The Parenteral Society ),1995.”


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