Thermal Characterization as Part of an Empirical Process for Developing Optimized Formulations and Lyophilization Cycles

Jeff Schwegman, CEOLabyrinth BioPharma, LLCjjschwegman@lab-bp.com

Lyophilization

What is Lyophilization?

Lyophilization is the process we use to remove water from a formulation at low temperatures (prevents thermal degradation) through a process of sublimation.

Lyophilization

Why do we Lyophilize (Freeze-Dry)?

Products are not stable (< 10% degradation) in the solution state at controlled room temperature for at least 2 years*

What is Freeze-Drying?

Vapor

Liquid

Solid

Temperature, °C

Triple point(0oC, 4.5 mm Hg)

Pre

ssu

re (

atm

)

0 100

1

Choice of Dryer Parameters

“The choice of values for the operating parameters of the cycle must be based on sound physical characterization of the formulation and understanding of how these parameters impact the final product”

- John Carpenter/Byeong Chang

John Carpenter/Byeong Chang. 1996. Biotechnology and Biopharmaceutical Manufacturing, Processing and Preservation. Buffalo Grove, IL: Interpharm Press.

Why Lyophilization Works

Sublimation: 1: (chemistry) a change directly from the solid to the gaseous state without becoming liquid

Ice sublimes in an attempt to achieve vapor equilibrium. When chamber pressure = ice vapor pressure, sublimation stops

The Driving Force = ∆P

Every 5 degree C increase inice temperature results inroughly a 75% increase inthe vapor pressure of ice!

Describes perfectly whylyophilization cycles need tobe optimized…wasting timeand resources if drying too farbelow critical temperatures

Lyophilization

Steps of Lyophilization

1. Product Freezing (I & II)2. Thermal Treatment (Annealing)3. System Evacuation (Vacuum)4. Primary Drying (Removal of Ice)5. Secondary Drying (Removal of Unfrozen

Water)6. Backfill with Inert Gas

Stages of Freeze-Drying

Freezing

PrimaryDrying

SecondaryDrying

Freezing

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

Freezing of Aqueous Systems

Solution

Solute crystallizes?

Ice Nucleation

Ice crystal growth, freezeconcentration

Solute Crystallizes,eutectic formation

MetastableGlass

Stable Glass

LyotropicLiquid Crystal

Temperature

Definitions

A eutectic mixture is an intimate mixture of 2 (or more) crystalline species that are in such close contact that they melt like a single, pure substance.

A glass is an amorphous species (randomly oriented molecules) which forms a solid, non flowing mass below its Tg’

Thermal Transition Terms Important in Freeze-Drying

•Eutectic temperature (Te)• Refers to crystalline systems and measured by thermal

or thermoelectric analyses

•Glass transition temperature (Tg’ and Tg)• Refers to amorphous systems and also measured by

thermal or thermoelectric analyses

•Collapse temperature (Tc)•Essentially the same as Tg’, but measure by freeze-dry

microscopy.

Eutectics and Glasses

Why are These Important?The eutectic or glass transition temperatures determine maximum temperature that the product can withstand during primary drying without loss of structure

Unfrozen Water

The amorphous solute, when in equilibrium with ice contains large amounts (10-50%) of unfrozen water which is dissolved in the solute phase

The crystalline solute contains no unfrozen water except surface adsorbed water on crystal surface or occluded water in crystals (<1%)

Eutectics and Glasses

These two different species dry very differently both in primary and secondary drying.

Cycle development conditions must take this into consideration

Eutectics and Glasses

In primary drying, must stay below Tg’ or Te or complete structural loss or the product will occur (Tg’typically much lower than Te)

Water embedded in glass while adsorbed to the surface of crystals

Eutectics and GlassesInterstitial space must be rigid enough to support itself after water (acting as the scaffolding) sublimes away

Eutectics and Glasses

In secondary drying:•Water must diffuse to surface of glass

before vaporization (very slow)•Water easily vaporized off of the

crystal surface (very fast)

Collapse

Complete Collapse of the Product

Melt Back

Cake Shrinkage – Partial and Complete

DSCIn DSC, the sample and a reference are subjected to a carefully controlled temperature program each with their own furnace.

When a transition occurs that results in a change in sample temperature, heat is added or taken away from the sample so that both cells remain at the same temperature.

This heat is equal to the energy of the transition.

DSC

DSC

DSC

DSC

•Measures thermal transitions in materials

•Useful for characterization of both frozen samples and freeze-dried solids

DSC

When using DSC for conducting thermal analysis studies for lyophilization cycle development, discard the cooling curve and only focus on the heating curve.

The sample has the potential for “supercooling” and gives misleading data.

DSC

DSC Thermal Transitions

DSC

DSC

Freeze-Dry Microcopy

Direct examination of freezing and

freeze-drying via a special microscope

and thermal stage

Compliments and supports the

information gained from the DSC

Equipment

Thermal Stage

Linkam FDCS 196 Freeze-Dry Microscopy Stage

Thermal Stage

Linkam FDCS 196 Freeze-Dry Microscopy Stage

PLM Theory

In addition to determining critical

temperatures, it is possible to tell if

your sample is crystalline or partially

crystalline due to the birefringence of

anisotropic crystals within the frozen

matrix

PLM Theory

Crystals are classified as being either isotropic or anisotropic.

Amorphous species have no ordered structure.

Equipment

Necessary equipment includes:

• Polarized Light Microscope

• Liquid nitrogen cooled thermal stage

• Vacuum pump

• Complete systems available at McCrone Microscopes in Chicago, IL

PL Microscopy

Necessary equipment includes:

• Optical Microscope (Olympus BX51)

• Bertrand Lens

• Long working distance objectives

• Polarizer and analyzer

• Condenser extension lens

What have we learned?

Thermal analysis studies (DSC/Freeze-Dry microscopy have given us several key pieces of information:• Tells us if the system is amorphous, crystalline,

or partially crystalline

• Tells us our critical temperatures (Tg’, Te)

• Tells us if we need to anneal the system and approximately what those conditions are

What have we learned?

Thermal analysis studies allow us to take an empirical approach to lyophilization cycle development as opposed to using trial-and-error which has been the dominant means of development in the past. These techniques also allow us to better troubleshoot problem cycles and formulations

What have we learned?

Questions?

www.lab-bp.comjjschwegman@lab-bp.com