LYOPHILIZATION. Introduction to Freeze-Drying Third of Four Lectures. Freezing, Annealing, Primary, and Secondary Drying. J. Jeff Schwegman, Ph.D.

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1 LYOPHILIZATION Introduction to Freeze-Drying Third of Four Lectures Freezing, Annealing, Primary, and Secondary Drying J. Jeff Schwegman, Ph.D. AB BioTechnologies P.O. Box 1430 Bloomington, Indiana,

2 Process Development Characterize the Thermal Properties of the Frozen System (Eutectic Melt or Glass Transition Identify Critical Process Variables Freezing Rate The need for annealing Primary drying time, temperature & pressure Secondary drying time, temperature & pressure

3 Freezing What Occurs in the Freezing Phase? Ice nucleation Ice Crystal Growth Solute Concentration Formation of Glasses Solutes Crystallize (pure crystals) Formation of Eutectics

4 The Physical State of the Solute Has a Significant Impact on Secondary Drying Behavior Simple Vaporization of Non Frozen Water Diffusion Followed by Vaporization of Non Frozen Water

5 Freezing and Vapor Pressure Decreased Freezing Point There are several factors that prevent an aqueous solution from freezing at 0 C: Freezing Point Depression (if added solutes) Supercooling Need to be aware of these during cycle development as they can impact product stability and how the product behaves in the freeze-dryer

6 Freezing and Vapor Pressure Freezing point depression results from a change in the escaping tendency or vapor pressure due to an added additional species At the triple point of water, the molecules have the same tendency to go from the solid phase, to the liquid phase, to the vapor phase. **Consider interaction of solute and solvent molecules

7 Freezing and Vapor Pressure A solute dissolved in a pure solvent at the triple point decreases the escaping tendency (vapor pressure). In order to remain in an equilibrium state, the temperature of the triple point must decrease.

8 Types of Nucleation There are 2 types of nucleation: Homogenous Nucleation Nucleation by aggregation of pure material (one component): Never occurs in practical freeze-drying Heterogeneous Nucleation Aggregate formed on Foreign solid (dirt, vial wall, etc). Foreign particle acts as template (lowers G of surface energy)

9 Types of Nucleation Ice nucleation occurs randomly during freezing so that different amounts of supercooling build up in each vial leading to differences in freezing rates. Ice nucleation during freezing is the only step in the freeze-drying process that cannot be controlled or is it??

10 Types of Nucleation Praxair (Liquified Gas Company) has developed a way to control the nucleation of every vial in a freeze-dryer that is better than other methods Ice Fog Vibration Sonic Waves

11 Uncontrolled Nucleation Nucleation Temperatures for 3 wt% Mannitol Solutions Problems (1) Very cold nucleation (2) Widely varying product temperature paths

12 Controlled Nucleation with ControLyo Technology Nucleation Temperatures for 3 wt% Mannitol Solutions Improvements (1) Warmer nucleation (2) Much more uniform product temperature paths

13 Altered Pore Sizes Example 3 wt% mannitol Controlled Uncontrolled BET Surface Area [m2/g] Residual Moisture [%]

14 Issues Associated with Freezing Freezing affects thickness of channel walls and size of pores, this can eventually affect rate of reconstitution of dry powder Freezing can denature proteins during formation of the ice-water interface Rate of freezing matters

15 The rate of freezing influence ice crystal size Slow Freeze Large ice crystals Solution Fast Freeze Small ice crystals The smaller the ice crystal, the smaller pores for water vapor to escape, and the slower rate of drying!

16 Issues Associated with Freezing Protein solution 10x freeze-thaw Freeze-Thaw Native State

17 Issues Associated with Freezing In the interstitial space, be aware of: Significant increase in solute concentration Increases in ionic strength Significant shifts in ph

18 Issues Associated with Freezing When designing a formulation destined for being freeze-dried always think about what you are adding to the formulation and how the product may be affected initially in the solution state, super concentrated in the interstitial space, and in the dried product. Amount added and the form the solids take can be critical to the success or failure of a formulation

19 Annealing Metastable glasses can form during freezing. These can be a factor of the freezing rate and/or the excipients in the formulation. It is extremely important not to have a metastable glass in your product because it will eventually crystallize

20 Annealing What is Annealing? Annealing is the process of warming a sample above it s glass transition temperature (but below the eutectic and/or ice melting temperature) and allowing the glass to relax and crystallize (mannitol and glycine are good examples)

21 Macroscopic View of Annealed vs. Unannealed Sucrose/Glycine Formulations

22 Annealing Studies Studies can be conducted to determine both annealing time and temperature required to crystallize metastable glasses. DSC Freeze-Dry Microscopy

23 Annealing Studies

24 Annealing Annealing is the act of causing an amorphous phase to crystallize at temperatures above Tg Annealing normalizes ice-crystal size through Ostwald ripening Causes bulking agents such and mannitol and glycine (which can freeze as amorphous phases) to crystallize Allows primary drying to proceed at a much higher and more efficient temperature

25 Primary Drying Driving Force The driving force of primary drying (sublimation) is the pressure differential between the vapor pressure of ice at the sublimation front and the partial pressure of water vapor in the lyophilization chamber. It is false to assume that temperature is the driving force

26 The Driving Force = P Every 5 degree C increase in ice temperature results in roughly a 75% increase in the vapor pressure of ice! Describes perfectly why lyophilization cycles need to be optimized wasting time and resources if drying too far below critical temperatures

27 Primary Drying Sublimation Rate of Ice The sublimation rate is a force (ΔP) divided by a resistance. Two forms of resistance (described below in detail) include: Resistance to heat transfer (many factors contribute) Resistance to mass transfer (many factors contribute)

28 Schematic of Heat and Mass Transfer in the Freeze Dryer Temperature difference between chamber and condenser and pressure differential between solution in vials and vacuum pump drives ice out of vial and onto the condenser Conversion of solid (ice) to vapor in chamber called sublimation Heat Transfer Mass Transfer ΔP Dry Cake Sublimation Front Frozen Solution Condenser Vacuum Pump Pressure gradient between sublimation front and chamber Thermal Fluid Shelf Thermal fluid circulates within the shelves to control temperature in chamber

29 Primary Drying Do I need to determine if my process is heat transfer or mass transfer limited to develop of good lyophilization cycle? No, just need to take steps to minimize resistances from heat and mass transfer (more rule of thumb principles apply) (Most products are heat transfer limited)

30 Primary Drying Heat Transfer Transfer of heat from the heat source (heated shelf) to the sublimation front is in most cases the rate limiting step in freeze-drying Resistance to heat transfer from Thermal fluid through shelf Through shelf to bottom of vial From bottom of vial through glass to bottom of ice in vial From bottom of ice in vial to sublimation front

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32 Primary Drying Heat transfer By far, the largest resistance to heat transfer (>90%) is the gap between the shelf and the bottom of the vial due to the extremely poor thermal conductivity of gas in a vacuum

33 Primary Drying Mass Transfer Mass transfer during primary drying refers to the transfer of water vapor from the sublimation front through the pores created in the dried layer, out of the vial, and to the condenser. Sub lim ation Rate Pr essure Difference Re sis tan ce

34 Primary Drying Mass Transfer As with Heat Transfer, the total resistance to mass transfer is a sum of individual resistances Dried Product (R p ): (P i -P v )/Sublimation Rate Vial/Stopper (R v ): (P v -P c )/Sublimation Rate Chamber (R c ): (P c -P cd )/Sublimation Rate

35 Primary Drying Limiting resistance to mass transfer is water vapor transfer through the dried layer of the cake. This is important to understand for cycle development

36 Primary Drying Obvious take home message is that fill height affects the vapor flow through the cake lower fill volume = faster drying Not so obvious is the fact that sample concentration can affect the sublimation rate by increasing resistance to mass flow (water vapor flow through dried layer)

37 Primary Drying Concentration and cake depth both influence the rate of sublimation

38 Primary Drying Vapor Pressure of Ice vs. Temperature Ice Temp, C P i of Ice (µm) 25% of P i (µm)

39 Primary Drying Ice Temperature vs. Ln 25% Vapor Pressure y = e (0.0999x ) ln Ice Pi Temperature, C

40 Primary Drying After the freezing step (and annealing if used), set chamber pressure to 25% of the vapor pressure of ice at the predetermined product temperature (determined from thermal analysis studies) Raise shelf temperature until required product temperature is achieved (allowing 30 minutes of equilibration time between changes)

41 Lyophilization SHELF IN Thermocouple 1 Thermocouple 2 Thermocouple 3 Thermocouple 4 Thermocouple 5 Condenser Pirani, mtorr Temperature, C Time, minutes

42 Secondary Drying Product temperature is raised to drive off adsorbed water (crystalline systems) or nonfrozen water in the glassy phase (amorphous systems) Rate of product temperature increase varies greatly depending on the phase of the solids Generally speaking, residual moisture levels of less than 1% Time and temperature determined using sample thief, Karl Fischer and potency/purity/activity

43 Secondary Drying It is critical before starting secondary drying to understand what solid phases comprise the cake after primary drying from thermal analysis studies Crystalline Amorphous (Tg instead of Tg ) Partially Amorphous

44 Secondary Drying Temperature, C Shelf, C Thermocouple 1 Thermocouple 2 Thermocouple 3 Thermocouple 4 Condenser, C Pirani, mtorr mtorr Time, minutes

45 Secondary Drying How long do I hold the formulation at secondary drying temperature? Pull samples on a regular basis using a sample thief and measure water content and potency/purity/activity

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47 Secondary Drying For most products, a moisture level of <1% is targeted. Certain formulations (biological) may require higher moisture levels to maintain stability

48 Thank you. The next lecture in this series is April 25 th at 6:30 am & 10:00 am (NY time) Experts in Formulation, Lyophilization Cycle Design/Optimization, Thermal Characterization, and Education and Training in the Development of Injectable Drug Products and Diagnostics J. Jeff Schwegman, Ph.D. For more information about AB Biotechnologies and their services, go to 48