Freeze-drying (lyophilization) is a means of extending a perishable product’s shelf life by stabilizing it. It is commonly utilized in the pharmaceutical industry for drugs, antibodies, and other biological materials.
The freeze-drying process has a number of steps that need careful optimization to keep a product’s function and quality after drying. Optimization conditions may differ with each product and these may also alter when scaling up to product manufacturing.
There are a number of assumptions based on prior experience or knowledge that are frequently made, but multiple misconceptions remain that persist in this process, as with many methods that have become well-developed over time.
Dr. Andrew Bright, Ph.D., Senior Scientist at Biopharma Group, UK, recently presented a two-part series of webinars outlining ten assumptions made when developing the freeze-drying process for pharmaceutical products and how these can be counteracted. This article outlines both webinars.
1. ‘Monitoring product temperature using conventional probes will represent product temperature in all vials’
Conventional probes that are placed in a vial during freeze-drying may influence the product temperature so that the measurement recorded may not represent all of the other vials in the same batch. There are a number of factors that could influence these differing results.
Putting a probe in different places within a vial may alter the measurements from within the vial. A probe that is placed at the bottom and center of a vial will optimize the length of time data can be gathered, which indicates this position should be utilized for all experiments.
The presence of the probe itself can reduce cake resistance by heightening the size of pores in the ice, growing the sublimation rate, and generating a bigger cooling effect. This could lead to the recording of cooler temperatures than other vials in the same batch.
The presence of the probe can also create a nucleation site where ice crystals will form, altering the crystal structure and so differing from the surrounding vials.
Non-invasive instruments like LyoFLux, Tunable Diode Laser Absorption Spectroscopy (TDLAS) from SP can be utilized to alleviate these issues associated with conventional probes. They monitor vapor flow and utilize this data to quantify the batch average product temperature.
Manometric Temperature Measurement (MTM), which forms part of the SMART software, is another option. The MTM calculates the mean dry front temperature from a fast pressure rise test and numerous known product batch parameters.
2. ‘Less complex formulations are easier and cheaper to freeze-dry’
Utilizing fewer formulation ingredients in a pharmaceutical product is thought to decrease the time and cost of freeze-drying. Some stabilizers and excipients like cyclodextrins can be more expensive than the active ingredient itself, so omitting these can reduce manufacturing costs substantially.
Simpler formulations can also heighten the drying rate in the lyophilization process because of the lower impedance to sublimation from the lower density of the dry layer and potentially result in faster reconstitution.
A simple formulation is not always best, as the active substance may require further ingredients, like cryo-/lyo- protectants or buffers to stay stable throughout the freeze-drying process.
These protectants and other ingredients can also act as thermal stabilizers, heightening the critical temperature of the product in the frozen and dried state to enable increased temperature to be used while freeze-drying, which will reduce the time needed.
Some small molecules may have different polymorphs or crystallization issues, which are difficult to control by freeze-drying alone, so may require excipients to influence the polymorph form or to inhibit the crystallization process completely.
In some instances, the formation of a cohesive cake may not happen without excipients, like when the product is at a low concentration, so bulking agents may be needed.
Figure 1. Observation of collapse using FDM. Image Credit: SP Scientific Products
3. ‘An iterative development process is not necessary for cycle development’
An iterative process involves data collected from one cycle to refine the subsequent one. This stepwise method helps to identify issues early and prioritize what to focus on in subsequent cycles, which will decrease the risk of product failure.
For instance, if drying is not efficient, but the product passes all testing, it might be worth increasing the product temperature in primary drying next time. Although part of a Quality by Design technique, this iterative concept does not utilize the Design of Experiment (DoE) idea, which would have completed numerous runs before gathering data on drying efficiency.
Using Process Analytical Tools (PAT) and SMART™ technology can grow the data and information about the product at every cycle. This can help grow the understanding of the process and allow robustness to be built into the developmental stages for optimum scaling up of the process.
A conventional lyophilization cycle lasted 120 hours with primary drying of 90 hours in a case study of a product with mixed amorphous and crystalline components and surfactant.
This frequently provided variable appearance, product quality, and moisture content. The cycle was analyzed with the utilization of SMART software to optimize shelf temperature and pressure. The primary drying time was reduced in the following cycle to 37 hours (67% shorter) and the total cycle time to 60 hours (59% shorter) (Figure 2).
Figure 2. Case Study: SMART software with low Tc. Image Credit: SP Scientific Products
4. ‘Everyone uses +20 °C and low pressure for secondary drying so these must be the best conditions’
Secondary drying is usually carried out at +20 °C with a lower pressure than in primary drying, yet studies have shown that the rate of secondary drying is influenced more by temperature than pressure.
Some proteins are unable to withstand temperatures of +20 °C as they will aggregate at these conditions and so may require a lower temperature to keep their stability. Low pressure may also not be needed, as higher pressures during secondary drying can create better heat transfer and more gas collisions in a product.
A more scientific method is required to establish the optimal temperature and pressure to be applied during secondary drying, which determines the relationship between moisture levels after primary drying (and during secondary drying) and the dry state Tg. This can be calculated mathematically or performed experimentally.
5. ‘The shelf temperature is a measure of the surface of the shelf during the freeze-drying process’
The actual measurement is based on the temperature of the inlet fluid. The shelf temperature of a freeze dryer is not a direct measurement of the temperature at the surface of the shelf.
There is usually a big difference between several degrees when comparing this to the outlet temperature, as seen in Figure 3. During the drying cycle, the temperature of the shelf itself will differ over the surface, although each shelf in the dryer should behave similarly to each other.
Figure 3. Differences in temperature between shelf inlet and outlet. Image Credit: SP Scientific Products
6. ‘There is not much difference between utilizing the collapse temperature (Tc) or the glass transition temperature (Tg’) of a product to measure the critical formulation temperature’
Although the Tc and Tg’ of a product can happen at the same temperature, they are not measuring the same parameters – for some formulations, the Tc values have been reported to be many times higher than the corresponding Tg’ values.
Tc is based on the temperature at which the viscosity of the product decreases to a point at which it is no longer able to support itself and loses structure (Figure 1), whereas Tg’ is the temperature at which an amorphous frozen system changes from a brittle to a flexible state.
It is a standard practice because of this to establish the Tc value at the onset of collapse using Freeze-Drying Microscopy (FDM, LyoStat). Tg’ is typically based on the influence of the phase transition rather than the transition itself, depending on how the Tg’ is established and who is evaluating it, this causes considerable variability in results.
The most common technique used to establish Tg’ is Differential Scanning Calorimetry (DSC), which relies on identifying the alteration in heat capacity accompanying the glass transition.
Other techniques look at alterations in the mechanical properties (Thermal Mechanical Analysis and Dynamic Mechanical Analysis) or softening events (Atomic Force Microscopy).
Biopharma Group has developed Lyotherm, their own instrument, which is able to measure Tg’ by combining Differential Thermal Analysis (DTA) and impedance analysis to find thermal and electrical changes within a sample. This allows a more complete picture of the physical and thermal characteristics.
It is required that the product temperature is kept at least below its Tc when successfully freeze-drying most products. In some instances, where the product might be highly unstable below its Tg’, increasing the molecular mobility can potentially heighten the rate of degradation, eventually leading to loss of cake structure.
7. ‘The lower the temperature of the condenser surface, the better’
It is thought that colder condenser temperatures equal quicker freeze-drying by removing the water from the product more quickly. It has been shown that it is not the condenser temperature alone but the difference in vapor pressure between condenser and product, which increases the speed of the drying process.
As the condenser temperature is decreased, the vapor pressure also decreases so that the difference in temperature is not enough to drive the process. It is also worth remembering that unnecessarily cold condensers will heighten the complexity and cost of equipment, in addition to the running costs.
8. ‘Shelf and product temperatures optimized for small scale freeze-drying can be used when scaling up the product manufacturing process’
It seems obvious that the shelf temperatures optimized in small-scale studies should be scalable for larger product manufacturing, but there are a number of influences that should be considered.
The performance of the freeze dryer itself could influence the scalability of the conditions. For instance, due to the capabilities of the condenser or the differential temperatures between inlet and outlet, the performance may differ more widely between freeze dryers as some may struggle with lower temperatures.
Different volumes and load sizes may also alter the optimal parameters required during the drying process. The product temperature is also not necessarily a truly scalable parameter.
Temperature is the net difference between heat in and heat out and these parameters can be influenced by impurities in a sample, leading to more nucleation points and ultimately resulting in alterations in drying times and sublimation rates. If you are scaling up from R&D where there could be more impurities in a sample than in the subsequent stages of development, this could be an issue.
Figure 4a. Pirani v Capacitance Manometer. Image Credit: SP Scientific Products
Figure 4b. Pirani v Capacitance Manometer. Image Credit: SP Scientific Products
9. ‘Repeatability is the same as robustness’
Showing repeatable results numerous times does not necessarily mean the process is robust enough to cope with unintended internal changes, e.g., temperature excursions or external factors, or reproducible enough to cope with any alterations in any single input parameter.
A robust freeze-drying process can create a quality product even when there are small deviations in critical parameters. Establishing the optimal design space parameters into cycle development will benefit the robustness of the setup and maintain batch consistency.
10. ‘Any measurement of pressure can be used when scaling up product development’
Pressure depends on the type and position of the pressure gauge. A capacitance manometer (e.g., MKS) supplies a direct measurement of the pressure inside the freeze dryer. It is tolerant to small alterations and it is unaffected by different gases.
This contrasts with the thermocouple gauge (e.g., Pirani), which will deviate from capacitance and is influenced by water vapor (Figure 4a and 4b).
The endpoint of the primary drying stage can be established by identifying when the capacitance and thermocouple gauge measurements meet, i.e., in the absence of any remaining water vapor that causes deviation in the thermocouple gauge measurement.
A balance of various factors is required for Successful freeze-drying. These can be complicated to manage and time-consuming to optimize. More experience has been gained over the years, and advanced technology has allowed more detailed analysis to be gathered.
This has resulted in the questioning of numerous assumptions, some of which have been mentioned in this article and many have also been discussed in a dedicated booklet ‘Misconceptions in Freeze-Drying’ written by Biopharma Group.
Produced from materials originally authored by Dr. Andrew Bright Ph. D from Biopharma Group, UK.
About SP Scientific Products
SP is a synergistic collection of well-known, well-established, and highly regarded scientific equipment brands — SP VirTis, SP FTS, SP Hotpack, SP Hull, SP Genevac, SP PennTech, and most recently SP i-Dositecno — joined to create one of the largest and most experienced companies in freeze-drying/lyophilization, complete aseptic fill-finish production lines, centrifugal evaporation and concentration, temperature control/thermal management, glassware washers and controlled environments.
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