Lyostat3Third GenerationFreeze-Drying Microscope

Lyostat3

• Lyostat3 was developed by Biopharma Technology Ltd to provide a rational scientific basis for freeze-drying cycle development. The Lyostat3 combined with BTL’s extensive experience in the freeze-drying industry provides a powerful tool for freeze-drying R&D Scientists

• Biopharma Technology Ltd has used Lyostat3 to analyse over 700 different formulations, from small drug molecules to large complex biomolecules for biopharmaceutical companies worldwide

Freeze-drying microscopy

• Enables real-time observation of the behaviour of your formulation during freeze-drying

• Obtains vital information on the critical temperatures of your product relevant to freeze-temperatures of your product relevant to freeze-drying

• Enables you to develop a safe and efficient freeze-drying cycle specific your product

Benefits of using Lyostat3

• It can help improve old and develop new formulations

• It can provide essential data to develop cycles on a rational scientific basis

• It can save valuable development time and money• Enables rapid determination of collapse, melting and

eutectic event temperatures• Archives data and image capture for future

reference• Only 2µl of sample material is required to determine

the key parameters

Lyostat3 System Components

System Components

• Lyostat3 Freeze-Drying Cryostage– Temperature range -196°C to +125°C– Up to 130°C per min heating/cooling rates– Silver heating block for high thermal conductivity

• Innovative New Imaging Station– Purpose Built– Small benchtop footprint – Higher resolution than standard compound microscopes – Tilting lens and camera for easy loading of samples

System Components

• Temperature Control System– Temperature is controlled by carefully balancing heat

input from a heating element and the precise control of liquid nitrogen through the silver block

– Temperature is measured to 0.01°C by a Pt100 – Temperature is measured to 0.01°C by a Pt100 resistor mounted close to the sample and controlled to 0.1°C even down to pressures as low as 10-3mBar

System Components

• High Performance Digital Firewire Camera• Dedicated Digital Image Capture Software

– Displays real-time temperature and pressure information and gives the user full control heating rate, limit and hold time of up to 100 ramps within the temperature profile.

– Image capture is integrated with temperature programmer data – Image capture is integrated with temperature programmer data capture.

– Images can be loaded into a gallery of either 2,8 or 32 images, for closer inspection.

System Components• Vacuum Pump

• Vacuum Gauge

• Optional Motorised Vacuum Control System– Software-controlled motorized valve controls chamber pressure– Enables close investigation of the effects of pressure on sample

collapsecollapse– Chamber pressure displayed in Torr, microns, mBar and Pa– Pressure plotted with temperature against time and can be

saved for later analysis.

Assay Procedure

• Unscrew lid and side door• Place sample holder on

block and close door• Place drop of silicone oil

on sample block• Place quartz cover slip in

Sample Block

• Place quartz cover slip in the sample holder

• Place spacer ring on quartz cover slip

• Pipette 2µl of sample onto quartz cover slip

• Place a smaller cover slip on top of the spacer ring

Sample Holder

Stage Lid

Quartz cover slip (16 mm dia.)

Glass cover slip (13 mm dia.)

2µl of sample

Objective Lens (usually 10x)Metal Spacer (70µm thick)

Assay ProcedureSample Loading

Drop of silicone oil for low

Temperature Controlled Block

Light Source (from below)

Aperture

dia.)

Sample Holder

Drop of silicone oil for low temperature lubrication

Assay Procedure

• Once sample is loaded onto stage it can be viewed in real-time on the computer screen

• Program your freezing temperature and initiate

Freezing

temperature and initiate the pump with one click

• When your freezing temperature is attained it can be held to ensure complete freezing

• Turn on the vacuum pump to begin drying the sample

Assay ProcedureDrying – Amorphous Product

Frozen Amorphous

Material

When drying, the sublimation

front will move from one side

of the sample to the other. In

front of it will be the frozen

Dried Amorphous

Material

Movement of sublimation Front

front of it will be the frozen

material and behind will be

the dried material

Sublimation Front

The collapse temperature (Tc) is obtained by gradually increasing the temperature until product collapse is observed

Assay ProcedureDrying – Amorphous Product

Frozen Amorphous

Material

Raising the temperature above the

Tc of the sample will cause it to

undergo collapse

A precise T can be found by going

Dried Amorphous

Material

A precise Tc can be found by going

back and viewing the images

recorded during the analytical runCollapsed Material

For crystalline products the same method is applied but a eutectic temperature

(Teu) is obtained instead

Assay ProcedureDrying - Crystalline Product

Sublimation Front

Eutectic melt of material at interface

Frozen Crystalline Material

Dried Crystalline Material

Change in gross morphology of frozen phase to give more liquid

appearance

Increasing Temperature

Crust Formation

Image of a solution showing evidence of crust formation

The black line around the sample

edge is an area of higher solute

concentration that impedes vapour

flow and inhibits drying

When the crust ruptures the sample will

dry from the site of the break. In this

case the temperature had been raised

above the sample Tc and so shows

evidence of collapse

Micro-collapse

Image of a 2% Mannitol + 1% Glucose solution drying under vacuum at -41.8°C, around the Tc of Glucose

Frozen material

Movement of sublimation front

Dried material

Mannitol retains

structure

Glucose collapses

within mannitol structure

Create MovieView Pictures

Data Overview

This file contains all the

information from the analytical

run

Results Data

From here you can view the

pictures recorded in a gallery

or put them together to create

a movie (.avi format)

The data chart shows temperature and pressure information throughout the

procedure

Data OverviewEach point on the purple image line

represents a single image viewed by

selecting it with the cursor

The single image view enables the user to

draw lines and a size bar to measure

Results Data

draw lines and a size bar to measure

between points of interest on the image

(e.g. ice crystal size)

Date, time, vacuum, temperature and ramp

information are displayed under the image

Image Gallery

The gallery view enables

fast sorting of images,

showing how the drying

Gallery Navigation

ZoomImage Number

Results Data

showing how the drying

progresses across a longer

timeframe

For larger images the gallery can be viewed showing less images – 32, 8 and 2

are available

Determination of critical temperatures

‘Trial and Error’ Cycle resulting in loss of product or costly inefficient cycle

New cycle based on actual

parameters

Problem Solved

Lyostat3 provides you with essential information on the critical temperatures of your formulation, enabling you to get the best results

from your product

Biopharma House, Winnall Valley Road, Winchester SO23 0LD, UK

Tel: +44 (0)1962 841092 Web: www.btl-solutions.net