Critical temperatures like the collapse temperature (Tc) and eutectic temperature (Teu) are important for freeze drying. Exceeding these temperatures can damage the product structure and properties. Analytical instruments like the Lyostat2 freeze drying microscope and Lyotherm2 DTA/impedance analyzer help determine these temperatures. A case study showed how determining the Tc of -45.7°C helped develop a cycle, but annealing increased Tc to -31.4°C, allowing faster drying at a higher temperature and shorter cycle time, saving costs. Intelligent analysis of critical temperatures thus allows optimizing freeze drying cycles.

1. Freeze Drying Critical Temperatures

2. BTL – Intelligent Freeze Drying Solutions Our advantage is a wealth of experience and knowledge of product formulation and process development, particularly in the field of pharmaceuticals and biotechnology

3. Why are These Temperatures Critical? Freeze-dry above the product critical temperature Loss of physical structure Incomplete drying (high moisture content) Decreased solubility Reduced activity and/or stability Freeze-drying below the product critical temperature Poor efficiency High Costs Longer cycles than necessary

4. Collapse Temperature (Tc) This is the temperature at which the material softens to the point of not being able to support its own structure Eutectic Temperature (Teu) This is the temperature at which the solute material melts, preventing any structure forming after the solvent has been removed All formulations can be described as having either a collapse temperature or a eutectic temperature Critical Temperatures for Freeze-drying

5. Formulation Components Impact Critical Temperatures Higher molecular weight components such as polymers tend to have higher critical temperatures Lower molecular weight components such as salts and small sugars tend to have lower critical temperatures Additionally, crystalline / amorphous mix can have a major impact on critical temperature: Lactose + NaCl (1:1) Tc = approx. -30°C Lactose + NaCl (1:0.3) Tc = approx. -45°C

6. Lyostat2Freeze-drying Microscope Lyotherm2 DTA & Impedance Analyser Critical Temperature Determination Our unique analytical instruments determine critical temperatures They bring scientific understanding and a rational approach to freeze-drying cycle development Offering an intelligent approach to cycle development and optimiasation

7. Lyostat2 – Freeze-Drying Microscope Enables real-time observation of the behaviour of your formulation during freeze-drying Enables temperature control between -196°C and +125°C to an accuracy of 0.1°C By observing the sample structure during drying as the temperature is raised, the exact point of collapse or eutectic melt can be observed under the microscope Critical Temperature Determination

8. Lyotherm2 – DTA and Impedance Analyser Provides an integrated Differential Thermal Analyser (DTA) and Electrical Impedance analyser (Zsinφ) capability in one instrument Can measure critical events in the frozen material that are undetectable by standard thermal analysis techniques Enables characterisation of the required freezing parameters that are essential to a successful freeze-drying cycle Critical Temperature Determination

9. Critical temperature Application Cycle Development From analysis of the product we now know: The maximum product temperature we can freeze-dry at before the product is damaged, allowing us to set the primary drying temperature with confidence – from Lyostat2 analysis What events occur in the frozen state that affect the freezing stage of the cycle, allowing us to add in any thermal treatment steps such as annealing – from Lyotherm2 analysis

10. Case Study Product Cycle Development A customer approached BTL with a product that was being freeze-dried using a cycle borrowed from another product They were discarding a high percentage of each batch due to defects occurring during freeze-drying

11. Case Study Product Cycle Development

12. Case Study Sample dries well at -50.0°C, but collapse starts as the temperature is increased to -45.7°C. This can be identified by defects appearing in the dried material As the temperature increases to -39.6°C the structure continues to weaken and collapse becomes more evident Lyostat2 Freeze-Drying Microscopy Analysis

13. Case Study The sample is repeated but this time with an annealing step – frozen and cooled to -50.0°C, warmed to -15.0°C and re-cooled to -50.0°C before drying. The sample dries with good structure until the temperature reaches -31.4°C and defects appear At -30.8°C the sample is too weak to keep any structure as the water is removed Lyostat2 Freeze-Drying Microscopy Analysis

14. Case Study 1 2 3 See full labels 1 – 4 on next slide 4 Lyotherm2 DTA and Impedance Analysis

15. Lyotherm2 DTA and Impedance Analysis Case Study Exotherm in DTA and increase in Impedance indicating a stabilisation / rearrangement of the frozen structure Increase in downward gradient of Impedance curve indicating a softening of the frozen material Onset of a sharp endotherm consistent with the melting of the ice Minimum Impedance indicating complete mobility within the solute structure

16. Case Study Interpretation of Analysis Results From the results of these analyses, we could make the following deductions: The inclusion of an annealing step resulted in an increase in the collapse temperature of the formulation from -45.7°C to -31.4°C, as well as increasing ice crystal size and networking Therefore, the maximum allowable product temperature during sublimation (to avoid collapse) was raised by 14.3°C by the use of annealing, thereby allowing drying to be carried out at higher temperatures, for a more efficient cycle. The higher the product temperature during drying, the faster the drying rate.

17. Case Study 1 2 3 +20°C A -15°C -40°C Tc = -45.7°C -50°C Shelf Temperature Product Temperature Chamber Pressure 1 – Freezing 2 – Primary Drying 3 – Secondary Drying A – Product at risk of collapse Existing Customer Cycle – 70 hours

18. Modified Cycle Created By BTL – 42 hours Case Study 1 2 3 4 +20°C -15°C Tc = -31.4°C -35°C -50°C Shelf Temperature Product Temperature Chamber Pressure 1 – Freezing 2 – Annealing 3 – Primary Drying 4 – Secondary Drying

19. Case Study 3 The Sublimation Cooling Effect The lowering of product temperature caused by the sublimation of ice +20°C -15°C Tc = -31.4°C -35°C -50°C Shelf Temperature Product Temperature Chamber Pressure 1 – Freezing 2 – Annealing 3 – Primary Drying 4 – Secondary Drying Zoom in on Previous Graph

20. The Next steps Case Study From the previous run we now know: The extent of sublimation cooling, allowing us to increase the shelf temperature / chamber pressure as high as possible whilst sublimation cooling keeps the product temperature below Tc When sublimation was complete in temperature-probed samples (when product temperature = shelf temperature) The physical appearance of the cakes produced by the cycle Residual moisture was measured in the final product, in order to establish whether the extent of secondary drying was sufficient

21. Intelligent Freeze Drying Delivers A lyo-cycle with increased efficiency, reduced costs and no product rejects Another happy customer!

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