Lyostat 4 freeze drying microscope

Contents

• History of Biopharma Technology

• How to determine the critical temperatures

• Lyostat LED Fourth Generation Freeze-Drying

Microscope

• Lyotherm – Frozen-State Analysis

• Consulting and R&D Services Overview

• Case Studies

• Conclusions

• Questions

History of Biopharma Technology

• Biopharma Technology Ltd (BTL) was founded in

1997

• Part of the Biopharma Group

• To date, BTL has worked with over 1800 products,

from small molecules to proteins, organisms, whole

cells, collagen products, blood and archaeological

samples

• BTL has expertise in the formulation of products,

accounting for the lyophilization (freeze drying) steps

involved in the development and/or production

process. Acting at early stage minimizes issues

otherwise encountered later in scale-up and

downstream process

Why are Critical Temperatures

Important in Freeze Drying?

• Freeze drying above the

product critical

temperature can lead to:

– Loss of physical structure

– Incomplete drying (high

moisture content)

– Decreased solubility

– Reduced activity and/or

stability

• Freeze drying too far

below the product critical

temperature can lead to:

– Poor efficiency

– High running costs

– Longer cycles than

necessary

Critical Temperatures for

Freeze Drying

• 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 from forming after the

solvent has been removed

• Glass Transition (Tg’)

- This is the temperature at which the frozen glass first

exhibits a change in viscosity from a brittle solid into a

soft solid.

Effect on Formulation Components

on Critical Temperature

• 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 ≈ -30°C

– Lactose + NaCl (1:0.3) Tc ≈ -45°C

Tc vs Tg’

• Many cycles use Tg’ as the critical temperature

for freeze drying cycle development

• However Tg’ can be significantly lower than Tc

• Lower primary drying temperatures mean slower

processing and more expensive cycles

• Although Tg’ is significant, many formulations

can be dried safely above this point

• Knowledge of Tc is strongly recommended for

formulation and cycle development

• BTL has used Lyostat to analyze over 1800 different formulations, from small drug molecules to large complex biomolecules

Lyostat LED Fourth Generation

Freeze-Drying Microscope

• Lyostat was developed by BTL to provide a rational scientific basis for freeze drying cycle development. Combined with BTL’s extensive experience in the freeze-drying industry, Lyostat provides a powerful tool for freeze-drying R&D Scientists

Freeze Drying Microscopy

• Real-time observation of the behavior of your

formulation during freeze drying

• By observing the sample structure the exact

point of collapse or eutectic melt can be

observed

Benefits of Using Lyostat

• Help to improve old and develop new formulations

• Provide essential data to develop cycles on a rational scientific basis

• 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

Lyostat System Components

System Components

• Lyostat 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 with LED Lamp

– 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

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

capture.

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

for closer inspection.

• Vacuum Pump

• Vacuum Gauge

• Optional Motorized Vacuum Control System

– Software-controlled motorized valve controls chamber pressure

– Enables close investigation of the effects of pressure on sample

collapse

– Chamber pressure displayed in Torr, microns, mBar and Pa

– Pressure plotted with temperature against time and can be

saved for later analysis.

System Components

Lyostat Software

LinkSys

Workstation

Software &

Control

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 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

Block

Sample

Holder

Stage Lid

Temperature

Controlled Block

Light Source

(from below)Aperture

Quartz cover slip (16 mm

dia.)

Glass cover slip (13 mm dia.)

2µl of sample

Objective Lens (usually 10x)

Metal Spacer (70µm thick)

Sample Holder

Assay Procedure

Sample Loading

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 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

Freezing

Assay Procedure

Drying – Amorphous Product

When drying, the sublimation

front will move from one side

of the sample to the other. In

front of it will be the frozen

material and behind will be

the dried material

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

until product collapse is observed

Sublimation

Front

Movement of

sublimation Front

Dried

Amorphous

Material

Frozen

Amorphous

Material

Assay Procedure

Drying – Amorphous Product

Raising the temperature above the

Tc of the sample will cause it to

undergo collapse

A precise Tc can be found by going

back and viewing the images

recorded during the analytical run

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

(Teu) is obtained instead

Dried

Amorphous

Material

Collapsed

Material

Frozen

Amorphous

Material

Assay Procedure

Drying - Crystalline Product

Frozen

Crystalline

Material

Dried

Crystalline

Material

Sublimation

Front

Eutectic melt of material at

interface

Change in gross morphology of

frozen phase to give more liquid

appearance

Increasing

Temperature

Create MovieView Pictures

Data Overview

This file contains all the

information from the analytical

run

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

Results Data

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

between points of interest on the image

(e.g. ice crystal size)

Date, time, vacuum, temperature and ramp

information are displayed under the image

Results Data

Image Gallery

The gallery view enables

fast sorting of images,

showing how the drying

progresses across a longer

timeframe

Gallery Navigation

Zoom

Image Number

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

are available

Results Data

Crust Formation

The black line around the sample

edge is an area of higher solute

concentration that impedes vapor

flow and inhibits drying

Image of a solution showing evidence of crust formation

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

Eutectic Temperature of a

Crystalline Material

Lyostat FDM

Screenshots of Lyostat analysis, showing frozen product (1), dried with

good structure (2) and collapse (3)

1

2

3

Case Study 1- Using FDM to design

a more efficient freeze drying cycle

Customer Issue

BTL was asked to reduce the freeze drying time for a product that

currently had a cycle duration of 61 hours. The formulation was

analysed using BTL’s freeze drying microscope Lyostat. The FDM

analysis identified a Tc range of -18.6°C to -17.4°C. Differential

thermal analysis and impedance analysis was carried out using

Lyotherm2. This identified a softening event in the impedance at -

25°C. A freeze drying cycle was carried out that was designed to

maintain the product temperature below -30°C, allowing a 5.0°C

safety margin below the lowest critical temperature identified. This new

cycle, designed by BTL, was 45 hours long. This was 16 hours shorter

than the original cycle, resulting in a significant time and energy saving

for the manufacturer.

Case Study 2 – Using FDM to

improve cycles

Customer IssueA customer approached BTL with a product that was being freeze-dried using a

generic cycle (which was applied to a number of other products). It was failing

to meet the required moisture threshold (no more than 2%) and each batch was

therefore rejected. FDM analysis was proposed to ascertain the lyophilisation

behaviour of the product and to see if any improvements could be made on the

existing cycle.

FDM analysis using the BTL Lyostat was carried out on the sample supplied.

Once the critical temperatures had been established a critical review of the

existing cycle was carried out to see what changes could be made to prevent

future batch failure.

Following the FDM analysis and the critical review of the existing cycle, BTL

recommended that the temperature used in primary drying was kept constant

about 5.0°C below Tc. This was implemented by the customer and it

immediately led to a reduced moisture content and thus no more batch failures.

Case Study 3 – Formulation

Development

Customer Issue• The customer wanted to develop a freeze drying cycle for a product.

However freeze drying microscopy had identified a low collapse

temperature of –42°C.

• Low collapse temperatures require cycles that are slow and lengthy and

energy expensive.

• BTL were able to reformulate to replace some of the sucrose, which has a

low collapse temperature, with dextran. Dextran can affect biological

products but the small amount used ensured there were no detrimental

effects to the active ingredient.

• The final formulation had an overall collapse temperature of -23°C, a

significant improvement over the original.

Lyostat provides you with essential information on the critical

temperatures of your formulation, enabling you to get the best results

from your product

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

Other Products and

Services from BTL

• Lyotherm frozen-state analyzer

– Provides detailed information about behavior of the

product in the frozen state, including Tg’,

crystallizations, melts, molecular mobility

• Training courses in freeze drying technology

– Regular scheduled courses held worldwide

– Customizable courses held at any preferred location

• Product and process development

– Experience with over 1800 different products

– Standalone facility for cytotoxic / potent products

intelligentfreezedrying.com