lyophilization of proteinslyophilization of proteins ...presentation – 5th islfd annual meeting...

Lyophilization of ProteinsLyophilization of Proteins ––Lyophilization of Proteins Lyophilization of Proteins ––FTIRFTIR--Guided Formulation DevelopmentGuided Formulation Development

Dr HeikoDr Heiko A SchiffterA SchiffterDr. Heiko Dr. Heiko A. SchiffterA. [email protected]@gmail.comhttp://http://www.ibme.ox.ac.ukwww.ibme.ox.ac.uk (until 30(until 30thth April 2012)April 2012)

Dr. Heiko A. Schiffter – IBME OxfordPresentation – 5th ISLFD Annual Meeting – 30th March 2012

pp (( p )p )

Table Table ofof contentscontentsTable Table ofof contentscontents

Part 1: FTIR Microscopy and Chemical Imaging• Idea and Objective• FTIR protein spectrum and analysisp p y• Multivariate data analysis• In situ freeze-drying formulation development• In situ study of phase separationIn situ study of phase separation

Part 2: Multiphoton MicroscopyMPM d FD Mi S t• MPM and FD Microscopy Setup

• 3D Imaging of FD stage samples using MPM• 3D Imaging of Phase Separation using MPM• In-situ Study of Phase Separation using MPM

Summary and ConclusionsSummary and Conclusions


Part IPart I ::Part IPart I ::Part IPart I ::

FTIR FTIR MicroscopyMicroscopy andand Chemical ImagingChemical ImagingPart IPart I ::

FTIR FTIR MicroscopyMicroscopy andand Chemical ImagingChemical Imaging

• Idea and Objective

• FTIR protein spectrum and analysisp p y

• Multivariate data analysis

• In situ freeze-drying formulation development• In situ freeze-drying formulation development

• In situ study of phase separation


Th B k d IdTh B k d IdTh B k d IdTh B k d Id

Part 1: FTIR spectroscopyPart 1: FTIR spectroscopy

The Background IdeaThe Background IdeaThe Background IdeaThe Background Idea

• Spray-drying is a complex drying process in regards to the drying kinetics and product g y g pmorphology

• Single Droplet Drying of proteins and protein formulations via acoustic levitation for theformulations via acoustic levitation for the determination of drying kinetics and product characteristics in spray-drying

• What is acoustic levitation?


Th B k d IdTh B k d IdTh B k d IdTh B k d Id


The Background IdeaThe Background IdeaThe Background IdeaThe Background Idea• Single Droplet Drying using acoustic levitationSingle Droplet Drying using acoustic levitation

• for drying kinetics analysis• A better understanding for the product at hand g p

micrometerscrew

transducer

piezo-crystallevitation chamber

CCD-camera

micrometersample

back lightillumination

bellows macrolens

reflector

micrometerscrew Controlled

evaporationmixer

CEM

air flow

liquid flow

to PC withimaging software

Today used by GEA NiroGEA Niro


F l i f Bi h i lF l i f Bi h i lF l i f Bi h i lF l i f Bi h i l


Formulation of BiopharmaceuticalsFormulation of BiopharmaceuticalsFormulation of BiopharmaceuticalsFormulation of Biopharmaceuticals

McNally, E.J. and Lockwood, C.E., The importance of a thorough


y, , , p gpreformulation study (illustrated by Leigh Rodano, BI Pharmaceuticals, Inc.)


FreezeFreeze--drying basicsdrying basicsFreezeFreeze--drying basicsdrying basics

• Freeze-drying is a complex multistage process

• Risk of product instability during each process step!(Freeze-concentration, phase separation, interfacial adsorption, dehydration)(Freeze concentration, phase separation, interfacial adsorption, dehydration)

• Objective:To develop a tool for the in-situ study of product stability and excipient behaviour at the small scale during freeze-drying


FPAFPA FTIR fFTIR f d id i ii


FPAFPA--FTIR freezeFTIR freeze--drying drying mmicroscopy icroscopy ssystemystem

ObjectiveSample & Spacer CaF2 window

• FTIR microscope with focal plane array (FPA, 64 x 64) detector

CasingCaF2CoverSlips

Vacuum

• Integrated miniature freeze-drying system

Thermal Stage

Slips

CaF2 window


FTIRFTIR iiFTIRFTIR ii


FTIR FTIR proteinprotein spectrumspectrumFTIR FTIR proteinprotein spectrumspectrumQualitative and quantitative information about the protein secondary structure isQualitative and quantitative information about the protein secondary structure is obtained from the Amide bands of the infrared spectrum

α helix

0.12

α-helix

0.08

0.10

e

0 04

0.06A

bsor

banc

e

+ further vibrationsβ-sheet

0.02

0.04A

1900 1800 1700 1600 1500 1400 1300 1200

0.00

Wavenumber [cm-1]


Wavenumber [cm ]

FTIRFTIR ii l il iFTIRFTIR ii l il i


FTIR FTIR proteinprotein spectrumspectrum analysisanalysisFTIR FTIR proteinprotein spectrumspectrum analysisanalysisAnalysis methods:Analysis methods:

• Subtraction spectrum

A f l• Area of overlap

• 2nd derivative spectrumNo quantitative secondary structure data

• Peak fitting (Gaussian curve fitting)

Time intensive and highly subjective( g)

0.025

0.030 Amide I band observed Amide I band generated Peak 1Peak 2

Structure Wavenumber [cm-1]

0.010

0.015

0.020

sorb

ance

[AU

] Peak 3 Peak 4 α-helix 1648 – 1657β-sheet 1623 – 1641

1674 – 1695

1720 1700 1680 1660 1640 1620 1600 1580

0.000

0.005

Abs 1674 1695

unordered 1642 – 1657turn 1662 – 1686


1720 1700 1680 1660 1640 1620 1600 1580

Wavenumber [cm-1]


FTIRFTIR-- spectra evaluationsspectra evaluationsusing multivariate data analysisusing multivariate data analysis

FTIRFTIR-- spectra evaluationsspectra evaluationsusing multivariate data analysisusing multivariate data analysisg yg yg yg y

• Objective and fast approach towards spectra evaluation by multivariate data analysis using a partial least square (PLS) algorithmy g p q ( ) g

Relates a known concentration (here amount of secondary structure) to absorbencies to generate a calibration curve

Quantitative decomposition technique

Decomposes spectral data and structural content into two sets of vectors (variance spectra – eigenvectors) and scores (critical shapes – loading factors)

As spectral information and secondary structure are connected, the two sets of scores can be related to each other by regression and a calibration model canscores can be related to each other by regression and a calibration model can be constructed.

Spectral decomposition and regression are performed in one stepSpectral decomposition and regression are performed in one step



Multivariate Multivariate datadata analysisanalysis -- pure pure componentcomponent spectraspectraMultivariate Multivariate datadata analysisanalysis -- pure pure componentcomponent spectraspectraαα--helix (1660helix (1660 –– 1650 cm1650 cm--11))αα helix (1660 helix (1660 1650 cm1650 cm ))

Intramolecular Intramolecular ββ--sheet (1695 sheet (1695 –– 1683 cm1683 cm--11 and 1644 and 1644 –– 1620 cm1620 cm--11))

Intermolecular Intermolecular ββ--sheet (1620 sheet (1620 –– 1595 cm1595 cm--11))

0.4

0.6 α−helix intramol. β-sheet intermol. β-sheet0.025

0.030 Carboxypeptidase A Concanavalin A HSA Chymotrypsin

0.0

0.2

orba

nce

0.015

0.020

sorb

ance

Insulin

0 4

-0.2

0.0

Abso

0.005

0.010Abs

1720 1700 1680 1660 1640 1620 1600 1580-0.6

-0.4

Wavenumber

1720 1700 1680 1660 1640 1620 1600 1580

0.000

Wavenumber


WavenumberWavenumber


Protein α-helix (actual)α-helix

(calculated)intra β-sheet

(actual)intra β-sheet (calculated)

inter β-sheet (actual)

inter β-sheet (calculated)

alkaline phosphatase 36.21% 37.68% 34.49% 35.15% 4.26% 4.66%

bovine serum albumin 69.64% 71.19% 17.39% 13.95% 1.78% 2.28%

carbonic anhydrase 14.05% 15.99% 46.29% 47.59% 7.15% 6.44%

carboxypeptidase A 35.04% 37.45% 28.18% 27.74% 6.56% 6.03%

catalase 35.86% 32.32% 38.91% 36.55% 6.64% 6.69%

concanavalin A 0.00% 0.00% 60.45% 63.89% 9.45% 9.70%concanavalin A 0.00% 0.00% 60.45% 63.89% 9.45% 9.70%

α-chymotrypsin 10.00% 13.61% 49.00% 52.47% n/a 5.59%

β-galactosidase 42.78% 39.51% 36.05% 35.47% 3.63% 4.72%

glucagon 59.12% 57.15% 15.88% 17.05% 0.42% 0.23%

haemoglobin 74.68% 79.59% 10.63% 10.64% 2.79% 2.09%

human serum albumin 74 48% 77 62% 13 81% 10 76% 0 57% 1 00%human serum albumin 74.48% 77.62% 13.81% 10.76% 0.57% 1.00%

insulin 56.14% 51.23% 23.92% 22.46% 5.00% 4.79%

lactate dehydrogenase 56.81% 61.39% 25.32% 23.46% 2.92% 1.94%

lysozyme 51.92% 49.57% 21.84% 21.84% 4.17% 4.03%

myoglobin 80.71% 78.04% 9.68% 12.56% 2.25% 2.28%

ib l A 24 78% 23 39% 40 26% 42 09% 2 92% 3 65%


ribonuclease A 24.78% 23.39% 40.26% 42.09% 2.92% 3.65%


Multivariate Multivariate datadata analysisanalysis -- FTIR FTIR calibrationcalibration curvescurvesMultivariate Multivariate datadata analysisanalysis -- FTIR FTIR calibrationcalibration curvescurves8090

α-helix 70 Intramolecular β-sheet

50607080

d [%

]

α e

40

50

60

d [%

]

β

10203040

calc

ulat

ed

10

20

30

calc

ulat

ed

0 10 20 30 40 50 60 70 80

0

actual [%]

r = 0.992

10 20 30 40 50 60 700

10 r = 0.992

actual [%]

8

10 intermolecular β-sheet

2

4

6

calc

ulat

ed

0 2 4 6 8 10

0

2

r = 0.979


actual

II ii ff d id i ff l il i dd ll


InIn--situsitu freezefreeze--drying drying fformulation ormulation ddevelopmentevelopment40°C 2nd Drying20°C

30mins-40°C

60mins100mTorr

2nd Drying20°C 60mins

100mTorrFreezing 1°C/min 1°C/min 1°C/min1st Drying

–27°C 60mins

Chymotrypsin (unformulated) 20 mg/ml; freezing rate: 0 250 25°°C/minC/min andChymotrypsin (unformulated) 20 mg/ml; freezing rate: 0.250.25 C/minC/min and 11°°C/minC/min; Sample volume: 2.5 μl

αα--helixhelix IntramolIntramol IntermolIntermol αα--helixhelix ββ--sheetsheet ββ--sheetsheet

UnprocessedUnprocessed 13.6%13.6% 52.5%52.5% 5.6%5.6%0.020

unprocessed cooled at 0°C frozen at 1°C/min frozen at 0.25°C/min

CooledCooled –– 0.2%0.2% + 0.3%+ 0.3% +/+/–– 0%0%

FrozenFrozen atat(1(1°°C/ i )C/ i ) –– 3.2%3.2% –– 2.9%2.9% + 4.2%+ 4.2%0.010

0.015

freeze-thawed

banc

e [A

U]

(1(1°°C/min)C/min) %% %% %%

Frozen at Frozen at (0.25(0.25°°C/min)C/min) –– 4.8%4.8% –– 3.0%3.0% + 5.9%+ 5.9%0.005

Abso

rb

After freezeAfter freeze--thawing at thawing at 0.250.25°°C/minC/min

–– 1.9%1.9% –– 1.1%1.1% + 1.9%+ 1.9%1700 1680 1660 1640 1620 1600

0.000

Wavelenth [cm-1 ]


Wavelenth [cm ]




30mins-40°C

60mins100mTorr



–27°C 60mins

Chymotrypsin (unformulated) 20 mg/ml; freezing rate: 0.250.25°°C/minC/min;

y yp ( ) g ; g ;sample volume: 2.5 μl

0 015

0.020 unprocessed frozen at -40°C freeze-dried rehydrated

αα--helixhelix IntramolIntramolββ--sheetsheet

IntermoIntermoll ββ--sheetsheet

0.010

0.015

rban

ce [A

U]

UnprocessedUnprocessed 13.6%13.6% 52.5%52.5% 5.6%5.6%

Frozen atFrozen at

0 000

0.005Abs

or Frozen at Frozen at 0.250.25°°C/minC/min –– 4.8%4.8% –– 3.0%3.0% + 5.9%+ 5.9%

FreezeFreeze--drieddried –– 6.6%6.6% –– 3.1%3.1% + 8.2%+ 8.2%

1700 1675 1650 1625 1600

0.000

Wavelength [cm-1 ]RehydratedRehydrated –– 3.2%3.2% –– 2.1%2.1% + 4.1%+ 4.1%


g [ ]




30mins-40°C

60mins100mTorr



–27°C 60mins

Chymotrypsin (formulated) 20 mg/ml; concentration excipients:y yp ( ) g ; p30 mg/ml; freezing rate: 0.250.25°°C/minC/min and 11°°C/minC/min;sample volume: 2.5 μl

IntramolIntramol IntermoIntermo αα--helixhelix IntramolIntramolββ--sheetsheet ll ββ--sheetsheet

UnprocessedUnprocessed 13 6%13 6% 52 5%52 5% 5 6%5 6%

0.020 unprocessed cooled at 0°C frozen at 1°C/min frozen at 0.25°C/minf th UnprocessedUnprocessed 13.6%13.6% 52.5%52.5% 5.6%5.6%

CooledCooled –– 0.2%0.2% + 0.2%+ 0.2% +/+/–– 0%0%

FF tt0.010

0.015

banc

e [A

U] freeze-thaw

FrozenFrozen at at 11°°C/minC/min –– 1.9%1.9% + 0.3%+ 0.3% + 0.4%+ 0.4%

Frozen at Frozen at 0 250 25°°C/ iC/ i –– 2.9%2.9% –– 1.61.6%% + 3.1%+ 3.1%

0.005

Abs

orb

0.250.25°°C/minC/min 2.9%2.9% 1.61.6%% 3.1% 3.1%

After freezeAfter freeze--thawing at thawing at

°°C/C/–– 0.5%0.5% –– 0.2%0.2% + 0.7%+ 0.7%1700 1680 1660 1640 1620 1600

0.000

Wavelength [cm-1 ]


0.250.25°°C/minC/minWavelength [cm ]




30mins-40°C

60mins100mTorr



–27°C 60mins

Chymotrypsin (formulated) 20 mg/ml; concentration excipients:

Chymotrypsin (formulated) 20 mg/ml; concentration excipients:30 mg/ml; freezing rate: 0.250.25°°C/minC/min; sample volume: 2.5 μl

αα--helixhelix IntramolIntramolββ--sheetsheet

IntermoIntermoll ββ--sheetsheet

0.020 unprocessed frozen at 0.25°/min freeze-dried rehydrated

UnprocessedUnprocessed 13.6%13.6% 52.5%52.5% 5.6%5.6%

Frozen atFrozen at0.010

0.015

rban

ce [A

U]

Frozen at Frozen at 0.250.25°°C/minC/min –– 2.9%2.9% –– 1.61.6%% + 3.1%+ 3.1%

FreezeFreeze--drieddried –– 3.5%3.5% –– 2.1%2.1% + 4.5%+ 4.5%0.005

Abso

RehydratedRehydrated –– 1.1%1.1% –– 0.4%0.4% + 1.0%+ 1.0%1700 1675 1650 1625 16000.000

Wavelength [cm-1 ]


g [ ]




30mins-40°C

60mins100mTorr



–27°C 60mins

20mg/ml rh albumin in 145 mM NaCl solution at pH 7 120mg/ml rh albumin in 145 mM NaCl solution at pH 7 120mg/ml rh albumin in 145 mM NaCl solution at pH 7 1

Solution at 20°C 100% 20mg/ml rh albumin in 145 mM NaCl solution20mg/ml rh albumin in 145 mM NaCl solution with 15µg/L T80 & 30mM Octanoate

20mg/ml rh albumin in 145 mM NaCl solution at pH 7.120mg/ml rh albumin in 145 mM NaCl solution at pH 7.1,with 30mM Octanoate, 15µg/L Polysorbate 8020mg/ml rh albumin in 145 mM NaCl solution at pH 7.1,with 5% w/w Sucrose

Solution at 20 C 100% Frozen at -40°C 89.36% Dried at 20°C 91.82%

Solution at 20°C 100% Frozen at -40°C 90.70% Dried at 20°C 92.37%

Solution at 20°C 100% Frozen at -40°C 95.46% Dried at 20°C 95.10% 10

20mg/ml rh albumin in 145 mM NaCl solution, with 15µg/L T80 & 30mM Octanoate 20mg/ml rh albumin in 145 mM NaCl solution, with 5% w/wSucrose

th

6

8

ge C

ompa

red

wit

20 D

egre

e C

(%)

2

4

Stuc

ture

Cha

ngS

olut

ion

at 2

Frozen (at -40 Degree C) Dried (at 20 Degree C)0

Physical States



C i f f l ti b f & ft FDC i f f l ti b f & ft FDComparison of formulations before & after FDComparison of formulations before & after FD

7.0

Loss of alpha-helix structure (%) Shift in fluorescence emision (nm)Increase in agregate conten (%)7.0

Loss of alpha-helix structure (%) Shift of fluorescence emission (nm)I i t t t (%)

1 5

2.0

6.5

7.0 Increase in agregate conten (%)

1 5

2.0

6.5

7.0 Increase in aggregate content (%)

0 0

0.5

1.0

1.5

0 0

0.5

1.0

1.5

20 (f) rhA 50 (f) rhA 50 (f) rhA+Suc 50 (f) rhA+Treh-1.0

-0.5

0.0

20 rhA 50 rhA 20 rhA+Suc 50 rhA+Treh-1.0

-0.5

0.0

Comparison of defatted rh albumin at 20 mg/ml, Comparison of formulated rhA at 20 mg/ml 50 mg/mlp g ,50 mg/ml, 50 mg/ml with 5% (w/v) sucrose, and50 mg/ml with 5% (w/v) trehalose, (alpha-helix, fluorescence emission and aggregate, before and after freeze-drying)

Comparison of formulated rhA at 20 mg/ml, 50 mg/ml, 50 mg/ml with 5% (w/v) sucrose, and 50 mg/ml with 5% (w/v) trehalose, (alpha-helix, fluorescence emission and aggregate, before and after freeze-drying)


TT dd TT ’ D i i’ D i i


TTCC and and TgTg’ Determination’ Determination– 28.7°C Example:

– 25.9°C

Example:50 mg/ml rh albumin formulatedwith 50 mg/ml trehalose

– 23.8°C

– 21.2°C

Tg‘-25.24°C



II itit t d f ht d f h ti d i l hili titi d i l hili ti

2nd Drying1st Drying

InIn--situ situ study of phase study of phase sseparation during lyophilizationeparation during lyophilization

20°C30mins

-40°C60mins


100mTorrFreezing 1°C/min 1°C/min 1°C/min

1st Drying -27°C 60mins

100mTorr

5% (w/w) PEG 4000 and 5% (w/w) Dextran 500 kDa5% (w/w) PEG 4000 and 5% (w/w) Dextran 500 kDain 145 mM NaCl solution at pH 7.1

Visible light image of freeze-dried mixture of PEG

4000 and Dextran 500

FPA-FTIR image ofDextran 500 distribution

within the freeze-dried mixture

FPA-FTIR image ofPEG 4000 distribution

within freeze-dried mixture






20°C30mins

-40°C60mins




100mTorr

20 mg/ml rh albumin in 145 mM NaCl solution at pH 7.1,5% (w/w) PEG 4000 and 5% (w/w) Dextran 500 kDa

e of

re

of

an50

0

of

4000

m

ixtu

re

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light

imag

ee-

drie

d m

ixtu

r00

and

Dex

tra

A-F

TIR

imag

e ut

ion

of P

EG4

reez

e-dr

ied

m

PEG rich area

Visi

bfr

eeze

PEG

400

0 e

FPA

dist

ribu

with

in fr

Dextran rich area

R im

age

of

f Dex

tran

500

drie

d m

ixtu

re

R im

age

of

utio

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with

in

ed m

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Mix area

FPA

-FTI

Rdi

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rieDr. Heiko A. Schiffter – IBME OxfordPresentation – 5th ISLFD Annual Meeting – 30th March 2012

d w





20°C30mins

-40°C60mins




100mTorr

20 mg/ml rh albumin in 145 mM NaCl solution at pH 7.1,

PEG rich area

5% (w/w) PEG 4000 and 5% (w/w) Dextran 500 kDa

PEG rich area

Dextran rich area

Mix areaMix area

A id 1 i f h lb iAmide 1 region of rh albumin at different sample position



II itit t d f ht d f h ti d i l hili titi d i l hili tiInIn--situ situ study of phase study of phase sseparation during lyophilizationeparation during lyophilization

20°C10mins

-40°C60mins




10mTorr

Dextran ProteinDextran ProteinDextran ProteinDextran Protein

PEG VisiblePEGProtein 2nd Structural Change DistributionPEG

Protein 2nd Structural Change DistributionPEGProtein 2nd Structural Change Distribution


20°C-20°C-40°C20°C

Part IIPart II ::Part IIPart II ::Part IIPart II ::

MPM MPM MicroscopyMicroscopyPart IIPart II ::

MPM MPM MicroscopyMicroscopy

• MPM and FD Microscopy Setup

• 3D Imaging of FD stage samples using MPM3D Imaging of FD stage samples using MPM

• 3D Imaging of Phase Separation using MPM

I it St d f Ph S ti i MPM• In-situ Study of Phase Separation using MPM


M l i hM l i h Mi &Mi &

Part 2: Part 2: MultiphotonMultiphoton MicroscopyMicroscopy

MultiphotonMultiphoton Microscopy &Microscopy &FreezeFreeze--Drying Microscopy System SetDrying Microscopy System Set--upup

Laser Adjust SystemMPM

Laser Control System

y

CyrostageControl SystemControl System

Cyrostage

MPM ControlSystem

LN2 Tank


3D I i f l i3D I i f l i


3D Imaging of samples in a 3D Imaging of samples in a cryostagecryostageusing MPMusing MPM

33µm33µm

3D MPM images of freeze-dried mixture of10% w/v FITC-PEG5000 and 10% w/v Cascade Blue dextran (10 kDa)

(size 207 μm × 207 μm resolution 0 2 μm × 0 2 μm × 0 5μm)

3D MPM images of freeze-dried mixture of10% w/v FITC-PEG5000 and 10% w/v Cascade Blue dextran (10 kDa)

(size 207 μm × 207 μm resolution 0 2 μm × 0 2 μm × 0 5μm)


(size 207 μm × 207 μm, resolution 0.2 μm × 0.2 μm × 0.5μm).(size 207 μm × 207 μm, resolution 0.2 μm × 0.2 μm × 0.5μm).

3D I i f Ph S i3D I i f Ph S i


3D Imaging of Phase Separation3D Imaging of Phase Separationduring Freezeduring Freeze--DryingDrying

Freeze-Dried Sample10% w/v FITC-PEG5000 + 10% w/v TRITC-dextran155,000(Size 621μm×621μm,

Freeze-Dried Sample10% w/v FITC-PEG5000 + 10% w/v TRITC-dextran155,000(Size 621μm×621μm,

Frozen and annealed for 240mins at -40°C 10% w/v FITC-PEG5000 + 10% w/v Cascade Blue dextran 10,000 + 20mg/ml Texas Red BSA

Frozen and annealed for 240mins at -40°C 10% w/v FITC-PEG5000 + 10% w/v Cascade Blue dextran 10,000 + 20mg/ml Texas Red BSA

Frozen and annealed for 240mins at -40°C 10% w/v FITC-PEG5000 + 10% w/v Cascade Blue dextran 10,000 + 20mg/ml Texas Red BSA(Size 621μm×621μm,

Frozen and annealed for 240mins at -40°C 10% w/v FITC-PEG5000 + 10% w/v Cascade Blue dextran 10,000 + 20mg/ml Texas Red BSA(Size 621μm×621μm,

resolution 0.6 μm×0.6μm×1.5μm)resolution 0.6 μm×0.6μm×1.5μm) (Size 148μm×148μm,resolution 0.145μm×0.145μm)(Size 148μm×148μm,resolution 0.145μm×0.145μm)

resolution 0.6 μm×0.6μm×1.5μm)resolution 0.6 μm×0.6μm×1.5μm)


S f SS f S


InIn--situsitu Study of Phase Separation using MPMStudy of Phase Separation using MPM

Freeze-Drying Cycle with Annealing at -9°C for 240 mins10% w/v FITC-PEG5000 + 10% w/v Cascade Blue dextran 10,000 + 20mg/ml Texas Red BSA



Visible Light Pictures 1000μm×1000μm (Resolution 1μm×1μm)Visible Light Pictures 1000μm×1000μm (Resolution 1μm×1μm)

S f SS f S


InIn--situsitu Study of Phase Separation using MPMStudy of Phase Separation using MPM




3D MPM Images 621μm×621μm (Resolution 0.6 μm×0.6μm×1.5μm)3D MPM Images 621μm×621μm (Resolution 0.6 μm×0.6μm×1.5μm) Zoom-In MPM Slice Images 148μm×148μm (Resolution 0.145μm×0.145μm)Zoom-In MPM Slice Images 148μm×148μm (Resolution 0.145μm×0.145μm)

SSSummary and conclusionsSummary and conclusions• FTIR spectroscopy can be used for a range of protein applications including the• FTIR spectroscopy can be used for a range of protein applications including the

formulation development for freeze-drying

• Multivariate data analysis by PLS algorithm provides a rapid and objective way ofMultivariate data analysis by PLS algorithm provides a rapid and objective way of quantitative evaluation of protein FTIR spectra

• The combination of FTIR and freeze-drying microscopy enables the in-situ study of protein stability during lyophilization in the small scale

• MPM enables to look into the pore formation and structure during freeze-drying

• Both, FTIR microscopy and MPM are expensive specialist techniques and l ti l li t d d t l i t h irelatively complicated data analysis techniques are necessary

• Only ultra small scale analysis possible and the question in regards to scalability is so far unansweredis so far unanswered

Final question: Will this ever be used wide spread?


• Final question: Will this ever be used wide spread?

AcknowledgementsAcknowledgementsAcknowledgementsAcknowledgements

• Dr. Renchen Liu

• Mr Vivasvat Kaul

Institute of Biomedical Engineering,University of Oxford

• Prof. Geoffrey Lee

• Prof. Zhanfeng Cui

• Dr. Sebastian Vonhoff

• Mr Felix Wolf Division of Pharmaceutics University of Erlangen• Phil Morton

Division of Pharmaceutics, University of Erlangen

Thank you very much for your attentionThank you very much for your attentionThank you very much for your attentionThank you very much for your attention


y y yy y yy y yy y y

lyophilization of proteinslyophilization of proteins ...presentation – 5th islfd annual meeting...

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