Products like biosimilars or parenteral are heat sensitive. Thus, require utmost care while handling and processing. Also, any heat sensitive healthcare product may require drying for final bulk packaging. Normal drying processes involve the application of high heat, which is not suitable for such products. Therefore, the low heat applied drying process also known as freeze-drying or Lyophilization used for the removal of solvents from products.
Lyophilization is a simple dehydration technique in which the product mixture firstly frozen and then applied with a vacuum for the sublimation of solvents. Ice under vacuum directly changes phase to vapor without attaining liquid phase. This condition helps the product to stabilize and minimizes the impact of oxidation, which may degrade the product quality.
Lyophilization process involves three main sub steps:
- Freezing – Ice formation
- Primary Drying – Sublimation
- Secondary Drying – Desorption
One of the major advantages of using Lyophilization is it consumes less amount of heat for drying the product. Also, the final powder we harvest has enhanced stability and product integrity. The major downfall of this process however is increased process cycle time. Equipment used to conduct the lyophilization process called Lyophilizer.
Before moving on to the steps involved in freeze-drying or lyophilization, let us understand the pre-treatment operations that might be performed to outcome maximum product yield.
We perform this activity to ensure the product undergoing lyophilization has maximum yield and integrity. Following are the pre-treatment examples.
Preserving Appearance of the Product
Sometimes, physical properties of the product are extremely important, such as cosmetics once the formulated product enters the commercial market. For lower product concentrations, lyophilization may cause even a slight settling of the product in vials or bottles. For such products, we add third party agents known as bulking agents, to preserve the appearance of the product through-out.
Stabilization of the Reactive Products
Particularly, bio-similar products are prone to witness irreversible changes in their molecular structure or degradation during lyophilization. This happens because lyophilization processes carried out at very much lower temperatures. To rescue the product from such serious consequences, stabilizers known as lyo-protectants are introduced, which ensure stabilization during the entire lyophilization process. One of the crucial factor contributing to the degradation is Moisture. Hence, along with such stabilizers, bracketing (window for low and high level) the moisture content becomes equally important.
Reduced Application of High Vapor Pressure Solvents
High vapor pressure solvents have very low freezing points. During the freeze-drying step, these solvents will come-off at first rapidly. Also, for an excessive quantity of the solvent, a product may melt at high pressures in the chamber. To remove such excessive solvents, pre-treatment involves the application of equipment like rota-vapor or centrifuge as applicable or required. When the low temperature of the product becomes a priority, we can use solvent traps such as liquid nitrogen traps in a vacuum line to remove excessive amounts of solvents.
Lyophilizer typically has following components.
- Trays (required for large volume processing) or Vials
- Heat transfer fluid recirculation pump and its stand-by
- Vacuum Pump
- Hydraulic Cylinders
- Plate Type Heat Exchangers
- Steam supply for sterilization
Shelf life co-relates directly with the amount of moisture present in the powdered product. Excessive water may deteriorate the product later. Therefore, considering a suitable drying technique preserves its own importance. The Lyophilization process commonly used for manufacturing pharmaceutical drug products with sterile requirements or heat-sensitive products such as biotechnology-related products. If not handled effectively, Lyophilization cycles would lead to days and weeks to complete.
Effective control of the cycle requires a QbD (Quality by Design) approach. QbD means a systematic approach to process development with already defined objectives that focus on the study of product, process, and process controls. Obliviously, this requires a scientific and risk-based approach. Important aspects of QbD include Critical Quality Attributes (CQAs), Critical Process Parameters (CPPs), and Critical Material Attributes (CMAs).
As per ICH Q8, Pharmaceutical Development Guidance, fundamental elements of QbD include Design Space and Process Analytical Technology, which were defined in 2004 by the FDA. These aspects then in agreement with ICH (International Conference for Harmonization) have updated to the QbD approach later in 2009-10. Each of the above has its own significance in the design of Lyophilizer described further.
Design Space is a multi-dimensional blend of input variables and process parameters showed to provide quality assurance.Design Space as outlined in ICH Q8, Pharmaceutical Development Guidance
Process Analytical Technology is a risk-based framework which includes a set of scientific tools to support innovation and regulatory implementation strategy to allow the innovation.Guidance for Industry PAT, FDA
Critical Quality Attributes (CQAs)
These attributes include physical, chemical, or biological characteristics required to ensure the desired quality of the product. Deriving and defining these attributes has a basis for proper scientific experiments considering suitable risk assessments as per ICH Q9. Therefore, this approach yields effective and robust control strategies.
Examples for Lyophilization include:
- Appearance of the material
- Allied substances
- Moisture Content
Critical Process Parameters (CPPs)
These parameters define the way the process behaves as it progresses further which directly affects the quality of the product.
- Temperature of Freezing the product
- Primary Drying temperature and pressure (vacuum)
- Secondary Drying temperature and pressure (vacuum)
Critical Material Attributes (CMAs)
Attributes, as mentioned, define the criticality of properties of materials required to produce high-quality products.
- Heat transfer capability of the shelves
- Product and Eutectic temperature
- Product Morphology
- Conductivity of the water vapors
- In case of vials, heat transfer coefficient of the glass and
- In case of trays, heat transfer coefficient of the SS316L
Lyophilization Process Steps
Let us understand each phase of lyophilization mentioned earlier in this article. Prior to that, refer to the phase diagram for the lyophilization process.
1st Black Spot – Freezing Point, 2nd – Triple Point, and 3rd one – Critical Point
Two water kinds present inside the material being lyophilized.
- Free Moisture (Unbound Moisture)
- Bound Moisture
Free moisture freezes at lower temperatures, while bound moisture doesn’t easily.
Lyophilization: Diagram of Lyophilizer
The best suitable heat transfer fluid considered to be Silicone Oil, which has a less freezing point than the product. This heat transferring fluid circulates inside the shelves with the help of a circulation pump installed outside the controlled area. Shelves contain high-grade material with enhanced thermal conductivity for better heat transfer. Vials typically have glass as their MOC whereas trays have SS316L. Apart from that, instrumentation and process control may include;
- Compound Pressure and Temperature Transmitter
- PLC and HMI or dedicated SCADA System
- Chart recorders
With the help of a controlling system, manual intervention remains mainly during the initial stage of the cycle. The operator loads the pre-saved recipe parameters with the unique batch ID. Also, inserts the temperature probes in the vials or trays as applicable.
Recipes may include parameters such as,
- Shelf Temperature
- Product Temperature
- Chamber Pressure
- Secondary Drying – Shelf Temperature
- Secondary Drying times etc.
Obliviously, validated recipes get practiced for this. Sometimes these recipe parameters may change from time to time based on the dynamics of the process. In that case, the process considered non-validated.
Before initiating the Lyophilization process, Cleaning in Place, Pressure Leak Test, and Sterilization in Place cycles run. This ensures the readiness of the system for further product processing.
Step 1 of Lyophilization: Freezing
We consider this stage as most critical in terms of productivity. The product is frozen below the eutectic point. A Eutectic Point means the point in the phase diagram where all three phases co-exist and the temperature and composition of the liquid phase can not alter without disappearing one of the phases. Any product will experience the heat of fusion at lowered temperatures.
Liquid becomes solid in two different ways.
- Liquid to solid crystals @ non-steady rate (Crystallization)
- Liquid to solid (non-crystal) @ steady rate (Vitrification OR Glass Transition)
Equally important, the system is required in place to ensure uniform temperature distribution across the complete product setup. This may involve a sufficient and proper distribution of temperature probes across the shelves.
Temperature control completely depends on the behavior of the water. Challenge lies in maintaining the super-cooled state of water. A slow rate of freezing creates large-sized ice crystals. This makes up an effective lyophilization process. The slow rate may lead to void spaces allowing water to escape during drying. The rapid rate of freezing creates small-sized ice crystals, which makes the lyophilization process cumbersome.
Once the temperature of the solution reduced continuously under atmospheric pressure and below freezing temperature, the solution enters a super-cooled state.
Note that proper thermal analysis will help in mapping the way the freeze-drying would proceed. Therefore, this must include time vs. temperature curve and proper heat studies.
Step 2 of Lyophilization: Primary Drying
This is the second stage of the lyophilization process where sublimation starts. At first, the vacuum pump removes non-condensable vapors from the lyophilizer. The ice formed during the previous stage gets removed in this stage directly sublimating to vapor under vacuum. This easy removal considered being unbound removal, either of the solvent or of the water.
A key driving force for sublimation during primary drying is the pressure differential between product and condenser. As the temperature of the solvent decreases, the pressure also decreases. Therefore, the temperature of the condenser must be lower than the temperature of the product which will drive the vapors from the chamber towards the condenser.
In sublimation, this ice (solid phase) directly transforms to vapors without attaining the liquid phase. Vapors then collected and condensed to liquid in the pre-cooled condenser in the service area. When performed continuously, this process results in a dried product.
During drying, both heat and mass transfer goes in parallel. The design of the chamber should promote the flow of the vapors towards the condenser. Also, the position of vials plays an important role in design space consideration. When placed centrally, vials do not get influenced by the radiations imposed by chamber walls. Whereas, when placed off-center, radiation imposed by chamber walls influence the vials.
The rate of sublimation requires proper optimization for condenser sizing and heat load. A higher rate of sublimation may cause less efficient condensation. Ultimately, the ice formed inside the condenser may disturb the pressure inside the condenser, melting the product in the chamber. Manufacturers suitably measure vacuum either in mbar or in mtorr (Torricelli).
Step 3 of Lyophilization: Secondary Drying
This stage considered as bound moisture or solvent removal stage, where residual water gets converted to vapor and removed from the vial. This water has lower vapor pressure than that of unbound property. Product in secondary drying often appear dry but it is not.
During this stage, temperature elevates, and pressure reduces without affecting the quality of the product under lyophilization. This effect in the breakdown of molecular bonding between the product and excess bound moisture. This process is called desorption. Finally, the pressure slightly elevates up-till atmospheric pressure and thus the process completes upon following conditions.
- Product Temperature = Shelf Temperature
- Meaning: Heat transfer has reached its equilibrium
- Significant decrease in vapor molecules
- Return of temperature to its default value for condenser
The system then brought back to atmospheric pressure. The trays are unloaded and stored as soon as possible to avoid moisture absorbance.
Lyophilizers for parenteral, biotech, and other aseptic products kept under Laminar Air Flow Units (Class A) to maintain the aseptic conditions. Surrounding area supposed to include a controlled atmosphere of Class B.
Heat Transfer Modes During Lyophilization Process
Considering lyophilization process for vials, three different modes of heat transfer occur.
- Conduction (Solid to Solid, Solid to Liquid)
- Convection (Liquid to Liquid)
- Radiation (Thermal Energy Transmission by Electromagnetic waves)
Vials are solid glass containers containing material inside them. Therefore, vials and their material come in direct thermal contact with the shelves on which they are placed. Heat transfer starts from shelves, then to vials, and then to subsequent material under drying.
This occurs because of bulk fluid motions. Circulating pressure enhance the rate of convective heat transfer, which speed-up the drying process.
Microwaves or thermal radiation occurs inside the lyophilization chamber because of the heat emitted by the open space of the shelves. The vacuum also plays an important role in carrying electromagnetic waves.
Equipment Breakdown and Maintenance
A risk assessment would help in identifying the potential troubles and associated corrective actions. Probably, Process FMEA (Failure Mode Effect Analysis) would serve the purpose in case of potential malfunction. Suitable in-process checks also help in identifying the leakages inside the chamber, such as oil leakage and associated impact on the product.
To sum up, the following aspects must be considered for troubleshooting breakdown or maintenance related activities,
- Risk Assessment (pFMEA)
- Multiple In-Process checks
- Periodic Preventive Maintenance and Logs
- Equipment Discrepancy Records
- Change Control Log
- Power failure behavior
- Regular defrosting of the condenser to ensure stuck-up ice removal
Validation Approach for Lyophilization
As mentioned earlier, the eutectic point has its own importance while validating the lyophilization cycle. Manufacturers may find specific products as difficult to derive the eutectic point that leads to cycle failures. Also, the rate of freezing and temperature ramp-up plays an important role while developing lyophilization cycles on commercial levels. For the multi-product facilities, a single recipe may not provide a solution for successful lyophilization process cycles for different products. Hence, every product may have its own cycle parameters to consider while developing a recipe.
Manufacturers must consider these concerns for validation. Also, as per FDA recommendation, manufacturers are advised to conduct frequent discussions on Interim validation reports for troubleshooting of the concerned parameters and hassle-free investigations.
Sterilization of Lyophilizer
To demonstrate the validated state, equipment design must promote the QbD aspect. This may include inaccessible locations such as nitrogen lines or process airlines. Hence, steam may require a simple design of the equipment with fewer bends and zero dead legs.
In spite, Ethylene Oxide gas may be considered for sterilization but with some limitations. Humidification becomes mandatory to ensure uniform and sterile moisture distribution, which puts more challenges. Being a gas, ethylene oxide may stagnate in nitrogen and process piping. This imposes risk in ensuring complete removal of the gas.
Hence, clean steam is practiced for sterilization with the simpler piping design of the equipment. The setup is required to conduct steam sterilization follows the same as that of an autoclave. Apart from regular temperature probes, dedicated ones require installation in a chamber and low drain points to control hot and cold spots. Finally, the sterilization cycle is developed and the effectiveness of the steam sterilization is evaluated.
Once the sterilization cycle completes, performing proper air flushing ensures the system gets dry. This whole thing may achieve through the application of the automated sequence for steam sterilization. The frequency for sterilization preferred to post each batch unless otherwise specified. Like process validation, the steam sterilization cycles also require documented validation status.
Regularly testing the filter integrity is necessary for steam supply filters if applicable. Once the sterilization ends, positive pressure maintained inside the system to avoid entrance of the contaminated air and system going in vacuum.
Why is lyophilization important?
Drying without heating application, simple handling with aseptic requirements and improved product stability are key highlights of using lyophilization. Apart from that, products which are heat sensitive and can sustain only in low temperatures, lyophilization comes out as a reliable and proven technique to get things done. Also, this process has some disadvantages. This process is expensive, tricky and increased cycle times.
Does lyophilization kill bacteria?
Mostly No. It only de-activates them because of lowered temperatures. Once the temperature rise, they re-activate. Exceptionally, very few of them may get killed based on their family and origin. Low temperatures affect their cell wall structure or even may get burst.
Is lyophilization the same as freeze drying?
Yes. They are same terms used interchangeably as per relevance to the specific applications. Lyophilization derived from the word lyophilic, meaning a colloid of the particles having strong affinity for the liquid in which it gets dispersed.
Why is lyophilization of parenteral drugs required?
In simple words, to increase the shelf life of the pharmaceutical product. Also, lyophilization is well known to the fact of keeping the product valued and in quality.
What is the principle of lyophilization?
Concept of Sublimation. Sublimation is the change of phase from solid to gas without attaining liquid state. This sublimation is the effect of the applied vacuum to the system. Ice formation, followed by reduced pressure sequentially, triggers the sublimation.
How long does it take to Lyophilize?
Depends on type of equipment, process, product and application. It takes anywhere between 24 to 72 hrs. of cycle time to complete the lyophilization process. Again, automation and complexity may decrease or increase the cycle time, respectively.