Sterilization Cycles in a GMP Pharmaceutical Environment

Autoclave steam sterilization process relies on monitoring three parameters: time, temperature, and pressure.

We use heat to perform sterilization and our carrier is moisture in an exact value. In order to achieve an effective sterilization process, we should gain control on each one of the 3 parameters combined, in order to produce saturated steam. A controlled process will produce the exact percentage of saturation that will carry the heat unto the microbes. It is also important to maintain this control over time and over the entire sterilization volume, as we learn from the ideal Gas equation:

The cycle requirements for every load type can, however, vary significantly. This article introduces five different sterilization cycles commonly used in a cGMP pharmaceutical setting. Three sterilization cycles: Gravity, Pre-vacuum and Air Steam Mixture Cycle, and two test cycles: Bowie and Dick and Leak Test.

Fundamentals of sterilization

Gravity cycle is the simplest cycle; ideal for sterilizing liquids, media, glassware & plastic, culture plates and unwrapped instruments.

Gravity cycle (real graph)

The cycle begins with a short heating phase as steam is introduced into the chamber (Up to around 1000 seconds in the above graph). As steam fills the chamber, the air is forced out through a drain vent. By pushing the air out, the steam directly contacts the load and begins to sterilize it. Sterilization occurs when a pressure of 208 kPa and a temperature of 121℃ are reached. Sterilization phase (Seconds 3000-4000) is approximately 1000 seconds long. Afterwards, the system and load are cooled down at a very slow pace that can take up to 10 hours. The slow cooldown prevents liquids from boiling over and spilling. There is an option to add a fast cooling system that decreases cycle time by up to 80%.

Used to Sterilize:

  • Liquids
  • Media
  • Glassware & plastic- without hollows
  • Culture plates
  • Unwrapped instruments
  • Incomplete Steam Penetration
  • Wet loads at the end of cycle (Not ideal for soft packaging)
  • Longer duration with high load volume, not recommended for hollow or porous loads
  • Sealed liquid parenteral in polymer packaging

  • Ideal for sterilizing liquids
  • Very simple process
  • Low cost autoclave

When dealing with hollow loads, solids, wrapped packages or porous loads, effective sterilization means removing as much air as possible in the first stage, prior to sterilization. For those loads the Pre-Vacuum cycle was designed.

Pre-Vacuum cycle graph

The Pre-vacuum cycle actively removes air from the chamber prior to sterilization phase, using a vacuum pump. It is used for sterilizing hollow loads, porous loads and wrapped packages. The cycle begins with 4 pulses of vacuum, gradually removing air from the chamber and inserting steam into it. Eventually more than 99% of the air is removed and we are left with 0.016% of initial air (after 400 seconds in the above graph). After that we are gradually heating the chamber, creating a homogenous temperature all over the volume. Sterilization then occurs (Seconds 1000 to 1200).

After sterilization, the chamber and load are cooled down. When the pressure drops, all residual moisture boils and is transformed into a gaseous state. When pressure is released from the chamber, the moisture exits as well, leaving our product sterilized and dry.

Used to Sterilize:

  • Porous loads
  • Hollow loads
  • Solid loads
  • Wrapped packages

  • Air removal of more than 99%
  • Uniform heat distribution and temperature homogeneity

  • Cannot sterilize liquids

Air steam mixture cycle (ASM) is used to sterilize liquids that are sealed in a package. This include contact lenses, glass bottles, ampoules and infusion bags. It is designed to sterilize large quantities; for example, 25,000 ampoules, 20,000 infusion bags or 30,000 syringes. Packaging integrity is maintained and there is no deformation of elastic packages.

Air Steam mixture (ASM) cycle graph

In ASM cycle the sterilization media is a mixture of steam and air – i.e. air removal is not an issue as the loads are sealed (Ampoule for example). Throughout the cycle overpressure is maintained; the pressure external to the load is higher than internal pressure. Temperature is gradually increased to achieve homogenic temperature increment (Seconds 0-2000 in the above graph). This state of overpressure is maintained throughout the sterilization process. After sterilization cycle ends, temperature is slowly reduced by water that are introduced into the jacket. We start the cooling using air, while maintaining constant high pressure and thus avoiding boiling (low pressure means lower boiling temperature). Once the temperature is below 80℃, the air is released and the load is ready for usage. The end result is a sterile product that is dry and ready for packaging.


  • Aqueous solutions in sealed containers
  • Glass bottles
  • Bags
  • Vials / Ampoules
  • LVS- Large volume syringes
  • PFS- Pre-filled syringes
  • Contact lenses

  • Uniform heat penetration (heating/hold stage)
  • Packaging integrity is maintained
  • No deformation of elastic packages
  • Avoidance of direct steam exposure

The Bowie & Dick test (B&D) indicates proper air removal from the chamber of a pre-vacuum autoclave. It ensures that all sterilization parameters and processes are in order. The B&D test is a chemical indicator inserted in a test pack. The test pack is placed into an empty chamber, and a pre-vacuum cycle is then activated. If the test fails, it indicates that the autoclave has leak problems.

Bowie and Dick test indicators
As we can see in the picture above:

  • Unexposed: Initial color stays the same
  • Pass: Homogenous color change
  • Fail: Inhomogeneous color change

Leak test or Vacuum test is designed to examine air-tight integrity and leaks in an autoclave chamber and pipes.

The chamber is brought to vacuum conditions of 7 kPa (after approximately 200 seconds in the above graph), and all valves and motors are closed for 5 minutes, enabling pressure stabilization. A typical Vacuum Leak Test Cycle will consist of three alternating vacuum and pressure pulses, followed by a 15-minute dwell period at deep vacuum. The permitted pressure change over the following 10 minutes is 1.3 kPa or less.

The cycles described in this article are the basics of sterilization processes in a pharmaceutical autoclave. Different loads require different sterilization cycles and in order to support this we offer the following options as well:

  1. Bio-Hazard cycle for waste sterilization
  2. Hot Water Shower - Ideal process for large scale terminal sterilization of mid/large size sealed liquid finished products (I.V. Bags act.). Clean steam is not required as it is a water-based process
  3. Isothermal Cycle - A process which keeps the load temperature between 80 to 105°C.

These are only some of the processes available with Tuttnauer's Autoclaves and there is a fit for every possible load.

About Tuttnauer - the Leading Pharmaceutical Autoclave Manufacturer
Tuttnauer is a leading manufacturer of infection control solutions for Pharmaceutical, Life Science & Healthcare industries, with over 90 years of sterilization experience and 350,000 worldwide installations. Tuttnauer designs pharmaceutical autoclaves in compliance with GMP regulations, to meet the technical challenges of pharma and biotech. Tuttnauer provides the highest quality products and full documentation. This article addresses the typical cycles used in pharmaceutical autoclaves.


Yuval Shilderman has over 20 years of experience in Product Management and is the head of the Tuttnauer Research & Development department. Yuval is the inventor of an applied patent for an Ozone Sterilizer.  He has developed several sterilization processes as well as a miniature steam generator.  Yuval holds a BSc. in Electro optics Eng. from Hadassah College.

Elran Melul has 13 years of diversified technical, Sales and Marketing experience - Designing, selling and marketing of Capital equipment. He previously worked for Johnson and Johnson, Cook Medical and HP Indigo. Elran holds a B.Sc. in Mechanical Engineering, and a MBA (HMBA). Elran is the global Pharma line sales manager in Tuttnauer.



Sterilization Methods Summary

We have reached the end of the Sterilization Methods Series where we explored the different ways to kill or remove deadly bacteria, viruses, and harmful pathogens.

How do you select the appropriate sterilization method when there are so many choices available? Your decision will depend on many different parameters, including:

  • The purpose of sterilization. Sterilization in the lab is different than in hospitals, which is also different than pharmaceutical sterilization.  Each industry and application have their own unique requirements.
  • The material that needs to be sterilized. Is the material heat sensitive? Is it sensitive to moisture, gas or radiation?
  • The nature of the microorganisms that need to be destroyed or removed.
  • Additional considerations are: time, safety and budget.

The sterilization methods series can help you make an informed decision. It includes 12 blog posts and covers 9 different sterilization methods that can be divided into two major categories: heat and non-heat sterilization methods.

Sterilization methods that use heat can be further sub-categorized into:

 Non-heat sterilization methods are further divided into four categories of low temperature methods that use gas:

The last category is physical sterilization methods:

This table summarizes each method:

Sterilization Method Advantages Disadvantages Common Uses

Cost effective Short cycle times

High temperature
High moisture levels
Dental, Medical, Sterile Processing, Laboratory, Pharmaceutical, small clinics and more

Fast Cost Effective

Applied on the instrument directly
Applicable on metallic devices only
Small metal or glass objects in microbiology labs
Incineration Load volume decreases by 90% Total destruction of load
High construction and installation costs
Waste processing
Dry Heat Relatively low cost
Ideal for moisture sensitive Items
Long cycle duration Small clinics, metals, ceramics
Formaldehyde After sterilization, most loads are available for immediate use Long cycle duration Endoscopes

High efficiency Large sterilizing volume

MutagenicCarcinogenic Excessively Long cycle

PVC, Rubber, Endoscopes
Filtration Used for sterilizing gases and liquids Does not differentiate between viable and non viable particles Heat sensitive injections, solutions and air (venting systems)
Radiation High penetration power Expensive
Can produce undesirable changes in irradiated products

Industrial sterilization of heat sensitive products Biomedical devices

Ozone Uses medical grade oxygen Long cycle time Medical
Plasma Does not leave chemical residues Cannot sterilize: liquids, powders, and strong absorbers Endoscopes
Soft contact lenses


This infographic provides a visual overview of each method


The sterilization methods series has come to an end. We’d like to hear from you and learn which is your preferred sterilization methods and why? Please post your answers in the comments section below.


How to Choose the Right Autoclave for your Dental Clinic?

How to Choose the Right Autoclave for your Dental Clinic?

As a dentist, dental hygienist, or support staff at a dental clinic, you know that having a reliable, effective autoclave is essential to the smooth operation of the clinic. Also called a steam sterilizer, the autoclave is the machine that harnesses the power of high temperature and high-pressure steam in order to sterilize germs on dental equipment, such as forceps, intended for reuse, thus ensuring its safety for the next patient.

If you’re looking to buy a new autoclave for your dental practice, what questions should you consider as part of this important purchase? In this blog post, we will take a look at five general questions to start the discussion, and then we’ll look at three possible responses to why you are buying a new autoclave, found in the flowchart below. The questions and answers we provide here will help you make an informed decision.

Six General Questions to Start the Discussion​

  1. Is the autoclave manufacturer ​a well-known provider with many years of success and experience in the field?
    The manufacturer you are looking to buy your autoclave from should have a good name and reputation, and FDA-approved autoclaves. If you don’t have a machine that is safe it could be extremely dangerous for you, your staff and your patients.
  2. Will the distributor you deal with give you all the options for buying an autoclave or just push the one he wants to sell on you?
    Doing your own research on what is out there is always a good idea. This will ensure you know about the products and can talk to your distributor representative about what you prefer.
  3. Does the manufacturer offer the type of autoclave and features you need?
    The manufacturer you are looking to buy your autoclave from should have a good name and reputation, and FDA-approved autoclaves. If you don’t have a machine that is safe it could be extremely dangerous for you, your staff and your patients.
  4. What is the volume of items that needs to be sterilized?
    Has the volume of items sterilized increased? The volume of items that needs to be sterilized will determine the size of your autoclave. An experienced distributor can recommend the ideal autoclave according to chamber size..
  5. Which autoclave delivers the best reliability for your investment?
    Things to look for: What is the warranty? How does service work? Is the sales rep available if you have any questions about using the machine?
  6. Is the autoclave you are considering purchasing easy to operate?
    You have a busy practice. Can you set the autoclave and come back to find the instruments sterile and completely dry, ready to use or do you have to constantly monitor the unit?

Now let’s take a look at a flow chart of the most commonly asked questions related to why you need a new sterilizer.

As you can see from our flow chart, there are many questions and angles to consider. Replacing a broken autoclave, buying a new autoclave, or adding another autoclave requires research, forethought, and planning. We hope that this post helps you begin the autoclave purchase process, and we wish you continued successful sterilization for a smoothly operating dental clinic.

Your questions and comments are welcome below in the Comments section.