A Review on Freeze-drying: A Stability Enhancement Technique


Silambarasan I1*, Rajalakshmi AN2

Department of Pharmaceutics, Mother Theresa Post Graduate and Research Institute of Health Sciences,

Indira Nagar, Gorimedu, Puducherry - 605006, India.

*Corresponding Author E-mail: simbupharma12@gmail.com



Freeze-drying is effective technique used in pharmaceutical industry to increase the efficacy, shelf-life of formulations and makes easier to transport and storage. Freeze-drying applies to the product that is thermolabile and unstable in solution form, because of utilization of low temperature and pressure. Nowadays freeze-drying gained so much attention in the development of parenteral formulation especially vaccines. The selection of excipients plays a key role in development of freeze-dried product and used to improve the shelf life and makes the product cost effective. The structure of cake not only focuses on appearance of product and also provide information about issues on drying stage that impact quality of product. Tray style freeze dryer helps in development of long-term storage vaccines and labile drugs. It performs freeze drying in containers or in vials on large quantities. This makes tray style freeze dryer become most common for developing lyophilized product. The need for improve the shelf-life of protein-based drug and antibiotics formulations makes tremendous growth of freeze-dried product market. This review focuses on the lyophilization process, design of freeze dryer, role of excipients in lyophilization process, decision of selecting the excipients, importance of cake structure helps to develop stable lyophilized drug product for long-term usage and applications of lyophilization technology.


KEYWORDS: Freezing, Primary drying, Secondary drying, Excipients, Freezing, Vacuum, Cake structure.




Lyophilization is a popularly used technique in Pharmaceuticals to improve the stability of drug and make them use for long-time1. The term lyophilization means a “product that loves the dry state”. In lyophilization or freeze-drying, water is frozen first and then eliminated from the product by primary drying (Sublimation) and then through secondary drying (Desorption). It is applied to those products that are heat-sensitive and aqueous-sensitive, but in the dry state it looks stable. Other than freeze-drying, several methods for manufacturing sterile dry powder drug products include spray-drying, powder-filling and sterile crystallization2. The process can also be used to stabilize living materials like microbial cultures, animal specimen preservation and concentrate and recover reaction synthesized products3.



Arsened Arsonval and his assistant Frederic Bordas invented the freeze-drying process in 1906 at the College de France's laboratory of biophysics in Paris. Downey Harris and Shackle introduced the lyophilization method for maintaining live rabies virus in 1911, which contributed to the development of the first antirabies vaccine. In World War II, the freeze-drying technique helped to transport serum. The freeze-drying method was soon introduced to bone and penicillin, and lyophilization was considered as an important technique for biological preservation.



The basic principle in the lyophilization process is “sublimation”. Water sublimates below triple point, whereas temperature of 0.009⁰C and pressure of 4579 μm (Figure. 1)4. Because of its weak intermolecular force, increased vapor pressure converts solid directly into a vapor state. In this process, sample is frozen first. So, the frozen sample undergoes sublimation and leaves solid only. The driving force for water removal is the “concentration gradient of water vapor between the drying front and condenser”5. The whole process is carried out at low pressure and low temperature, so it is appropriate for heat sensitive drugs. Freezing, primary drying and secondary drying are the three important stages involved in lyophilization6.



Figure. 1: Phase diagram of water



The advantages of Freeze-drying of product are low chances of oxidation, minimal chemical degradation, less chance for contamination, improved sterility of product, quick reconstitution time, increased shelf life of product, loading quantity can be accurate and content uniformity, low moisture content.



The disadvantages of Freeze-drying of product are removal of volatile compound under vacuum, time consumption is high, most expensive process, reconstitution requires sterile diluent.



The ideal characteristics of freeze-dried product are adequate strength, uniform colour, sufficiently dry, chemically stable, intact cake, sufficiently porous.


Application of lyophilization technology5:

The important application of lyophilization technology and list of drugs marketed as lyophilized form is shown in Table 1.


Food industry:

Freeze-drying is a way of storing food and making it very light.

Example: Astronaut food.


Pharmaceutical and biotechnology:

In Biotechnology and Pharmaceutical field, freeze-drying technique used for extension of shelf life of vaccines, development of lyophilized nasal inserts, development of lyophilized solid protein product, development of micro and nanoparticles drying.


Technological industry:

To increase the stability of chemically synthesized products.


Table 1. List of drugs marketed in lyophilized form

S. No

Drug Name

Company Name



Hetero Labs



Orchid Pharmaceuticals



Neon Antibiotics



Lupin Pharmaceuticals Pvt Ltd



Natco Pharma Ltd



Glaxo Smithkline


Zoledronic Acid

Natco Pharma Ltd



Aristo Pharmaceuticals



Natco Pharma Ltd



Natco Pharma Ltd



Natco Pharma Ltd


Cefazolin Sodium




Natco Pharma Ltd



Chandra Bhagat Pharma Pvt Ltd



Anacalima Lifesciences Ltd



The entire drying process is classified into four different stages are Pre-treatment, Freezing, Primary drying and Secondary drying.



Pre-treatment is the first step involved in drying process. Concentrating the product, lowering the high vapor pressure of solvent, formulation revision and increasing surface area are some of pre-treatment techniques. The decision of pre-treatment is directed by freeze-drying theoretical knowledge6.



The first stage of the lyophilization process is freezing which converts liquid to solid, where water is transferred to ice by using low temperature8. The nature of solvents and excipients are essential to consider the complete freezing of the product1. For complete solidification, the product must be frozen completely9. The quality of the final product, as well as the primary and secondary drying rate will be based on microstructure formed during freezing. Slow cooling brings huge ice crystals and produced free pores in the matrix. Fast cooling brings little ice crystals, helpful in protecting structure to be examined microscopically leads to the difficulty in freeze dry8. Most freeze-dried products are mainly made up of water (solvent) and ingredients suspended in the water (solute)9. The majority of lyophilized samples are eutectics means collection of compounds that freeze below surrounding water temperature. The temperature at which the entire eutectic mixture is frozen is called eutectic temperature6.


Pharmaceutical products stabilized by formation of large ice crystals8. The concept of eutectic temperature and glass transition temperature as shown in Figure.2. The correct cooling rate of the freeze-dried product is 1⁰C /min. Fill volume of product also affect freezing rate. More time is required to freeze the product of larger fill volume1.


Freezing point is determined by using various techniques like theoretical thermodynamic value, Cryo-microscope, Differential scanning calorimetry, Measurement of temperature and resistance during freezing6.


Figure. 2: Concept of Eutectic Temperature and Glass transition temperature



Annealing is a process in which a material is kept above its final freezing point for a set period of time in order to crystallize the crystalline compound10. Solute converts into a crystalline form then it is called eutectic temperature. On the other hand, if amorphous form is formed is called glass transition temperature11. Primary during time is reduced by placing the product above glass transition temperature12,13. Annealing time is based on property and ratio of bulking agent used14. X-ray diffraction and Differential scanning calorimetry are used to study annealing15.


Primary drying:

Once the freezing process has been completed, the pressure within the freeze dryer reduced by vacuum pump. Chamber pressure reduced below the ice vapour pressure at sublimation interface to allow sublimation of ice. Based on product temperature and nature of the container, chamber pressure ranges from 30 and 300m torr. No mass transfer happens when chamber pressure is greater than vapor pressure. The shelves can be heated to a temperature to provide energy for continuing sublimation. Product temperature is most significant process parameter during freeze-drying16. Low product temperature and resulting low vapor pressure takes a long primary drying time. A 1⁰C increase in product temperature minimizes 13% primary drying time6. If critical formulation temperature is crossed above, dried pore structure near to sublimation front leads to hole development and pore fusion in the cake structure. Further cause decrease in inner surface area and increased moisture content that affect reconstitution time and API stability17. Critical formulation temperature is determined by freeze dry microscopy18. Collapse temperature is the temperature above which product collapses, so kept the temperature below 2-3⁰C below collapse temperature to avoid the product collapse.


Secondary drying:

Residual moisture upto 8% still persists in the product after primary drying, so continuous drying is required. The process of desorption of bound water is known as “Isothermal desorption”6. Depending on product, secondary drying time varies between 10⁰C to 50⁰C14. Secondary drying takes around half of the time of primary drying. During secondary drying, chamber pressure is decreased to the possible lowest level and shelf temperature is increased, to favour desorption of water6. For amorphous materials, ramp rate should be between 0.10 to 0.15⁰C/min. During secondary drying, more energy required to remove bound water. Secondary drying aims to reduce the level of water content upto 1-2%. The moisture content of the product is determined by Near IR spectroscopy,


Thermo Gravimetric Analysis and Karl Fischer Titration14.



The Three major types of freeze dryer are Manifold freeze-dryer, Rotary freeze dryer and Tray style freeze dryer.


Manifold freeze dryer:

Manifold freeze dryer have small circular tube helps to attach the dried product container with the condenser19. It is used to dry liquid substance in less time6. Moisture content of the product is reduced upto 5% by using this dryer20.


Rotary freeze dryer:

Rotary freeze dryer comprises of large rotating cylindrical reservoir used for uniform drying of pellets, cubes and other pourable substances21.


Tray style freeze dryer:

Tray style freeze dryer comprises of shelves attached rectangular reservoir in which product placed in trays and vials6. It has a stoppering mechanism to seal the vial to avoid atmosphere exposure. It dries large variety of materials for long-term storage like vaccines21.



The container should have good thermal conductivity, reduced moisture penetration in walls and seal6. Size, shape, quality of container affects thermal               conductivity 22. Usually Type I tubular vial is used 23.




The chamber has shelves to process the product. A stoppering unit made of stainless steel is also installed in the chamber. Door is locked by hydraulic motor. The functions of chamber are impart protection to the product and impart necessary temperature and pressure to perform process.



Shelves serve as heat exchanger. Silicone oil system attached to shelves by flexible or fixed hoses. Shelves with a surface area of up to 4m2 can be manufactured1.


Process condenser:

Process condenser is otherwise called cold trap. During drying process, it acts as solvent trapper. Based on the position of condenser, it is categorized as external or internal. The trapping performance does not affect by position.


Refrigeration system:

The energy for cooling is provided liquid nitrogen or compressor. Mostly multiple compressors are used because they function to cool the condenser and shelves.


Vacuum system:

The vacuum can be employed during the drying process to remove the solvent in sufficient time. Vacuum is often used between 50 to 100μbar. A two-stage rotary vacuum pump is used to produce such a low vacuum. Multiple pumps can be used in large chambers.


Shelf fluid system:

The whole freeze-drying technique requires energy. The requirement of energy is achieved by circulating fluid through shelves through pump at low pressure. The fluid normally used is silicone oil6.



Temperature and pressure must be measured during the entire process. Resistance thermometers or Thermocouples (normally type T) measures shelf temperature, product temperature and condenser temperature. Capacitance manometers (frequently used), Thermoelectric or Pirani gauge measures chamber pressure1.



Pharmaceutical excipients are essential components in lyophilized formulation. They are added to improve bulkiness, increase stability, improve drug safety in freeze-dried formulation24. The decision tree for selection of excipients as shown in Figure. 3.


Figure. 3: Decision tree for selection of Excipients


Bulking agent:

Bulking agents create bulkiness to the product and form an adequate cake structure. Mainly it is used for low-dose drugs. Bulking agent is utilized, when total solid content is below 2% in the formulation. Proper cake consists of proper pores, which enables escape of vapor during drying. Elegant cake structure with excellent mechanical properties is created by crystalline bulking agents.

Example: Mannitol, Sucrose, Lactose and Sorbitol25.


Buffering agent:

In Pharmaceutical formulations, buffers are needed to stabilize pH. Low concentration buffers that cause minimum pH change during freezing are generally preferred. Buffering agents should have high collapse temperature (Tc), high glass transition temperature (Tg), non-volatile to avoid pH shift.

Example: Sodium citrate, Sodium phosphate, Sodium hydroxide, Tris-acetate26.


Collapse temperature modifiers:

During primary drying of an amorphous substance, the temperature of primary drying is held below collapse temperature. Some amorphous excipients have low collapse temperature, which leads to extended time of primary drying. To increase the collapse temperature of the product, collapse temperature modifiers are used.

Example: Dextran, Gelatin, Hydroxyethyl starch1.


Tonicity modifiers:

To prevent tissue injury, isotonicity of parenteral formulation should be maintained with plasma. The addition of tonicity modifiers cause decrease in the collapse temperature leading to an increase in primary drying time. So, tonicity modifier is added to reconstitution diluent.

Example: Dextrose, Glycerol and Sodium chloride1.



The most common solvent used in lyophilization is water. Organic solvents often added to improve product solubility, increase primary drying rate leads to increase in product stability, decrease reconstitution time27. The addition of non-aqueous solvents will delay the degradation rate of active constituents in water.

Example: Tert-butanol/water combination28,29.



Collapsed Cake:

The collapsed cake happens when the temperature higher than the collapse temperature (Tc), usually few degrees above glass transition temperature during primary drying30. It may cause decrease in surface area results in increased moisture content and reconstitution time31.



Meltback is typically collapse described as the existence of ice at final point of primary drying and starting of secondary drying. It may be due to inadequate vial contact and reduced heat transmission to the vial.


Product ejection:

The powder is ejected from the vial during primary drying process is called product ejection. It is commonly occurred in formulation consist of solvents like ethanol, t-butanol and low dissolved solids. It is indicated by the appearance of solid in the shoulder and neck of vial32.


Slanted cake:

The slanted cake is vial loading issue. It is due to placing the vial at an improper angle on the shelf. The drying behavior of slanted cake may vary, resulting in high residual moisture.


Cake shrinkage:

Cake shrinkage means reducing cake volume due to taking away of cake from the wall of vial. During drying, stress built in the cake. This stress is released by cake contracting or cracking33.




Lyophilization technique gained special attention in the manufacture of injectable formulation and new biologic drugs. It occupies around 50 % of the formulations in the pharmaceutical market. Freeze-drying significantly reduces the chemical and physical degradation and increases the long-term stability of product. The knowledge of different stages of the freeze-drying process and the correct selection of excipients dramatically reduces the cost and formulation time.



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Received on 06.07.2021            Modified on 11.10.2021

Accepted on 22.11.2021           © RJPT All right reserved

Research J. Pharm. and Tech 2022; 15(10):4841-4846.

DOI: 10.52711/0974-360X.2022.00813