indirect uv detection and applications - applica-analytik.ch · ce - qk tests: monoclonal antibody...
TRANSCRIPT
University of Applied Sciences
Western Switzerland, Wallis, Sion Institute of
Life Technologies
«Stand und Anwendungen der Kapillarelektrophorese
in Forschung & Industrie»
Franka Kálmán
Electrophorèse capillaire (CE): histoire
2
Jorgensen & Lukacs:
1981:
einfaches CE-Gerät mit
hochsensitivem Fl-Detektor
(HPLC) & Quartzkapillaren
Lit:
Jorgensen et al, Anal. Chem.
53, 1298 (1981)
20 – Hjerten (rotierende Kapillaren (3 mm))
23 – Jorgenson & Lukacs (Injektion, Detektion an Kapillaren)
28 – Gassmann (laser induced fluorescence, attomol range)
[1] “Recent advances in capillary
electrophoretic migration techniques for
pharmaceutical analysis”, S.El Deeb et
al. ELECTROPHORESIS, Reviews
2014, Volume 35, Issue 1, pages 170–
189, January 2014
© Solvias AG 3
Capillary Electrophoresis Instrumentation
3
various detectors
UV-VIS, DAD, MS, LIF, Fl,
CCD, indirect UV detection
(no chromophors)
4
Agilent 7100 CE Capillary Electrophoresis
e.g. DNA sequencing: Capillary Arrays
8, 16, 32, 64 & 96 capillary units e.g. Polymicro
Human Genome Project HUGO:
~13% conventional techniques,
remaining CE...
5
6
Beispiel: MC-CE: Integration in Lab-on-a-Chip: Untersuchung des Gehirns
Konzentrationsbestimmung von sogenannten Neurotransmittern
sehr geringe Konzentration
Matrix sehr komplex
nötig ”real time” Messungen (=> schnell)
viele Schritte (Probenvorbereitung und Analyse):
1. Mikrodialyse
+ on-column Derivatization
+ Trennung
+ Detektion
Neurotransmitter in Gehirnsäften: z.B. Glutamate
7
1-Mercaptoethanol-2-alkylisoindol
Derivatization of glutamate
derivatisation time: ~ 1min (on-column derivatisation)
OPA
+ H2O
Lit.: Roth et al
Beispiel: MC-CE _ Lab-on-a-Chip: Untersuchung des Gehirns
8
1.) Mikrodialyse:
semipermeable
Membrane
3 μl / min flow
Beispiel: MC-CE _ Lab-on-a-Chip: Untersuchung des Gehirns
9
2.) on-column Derivatisation
3.) Trennung
9 cm Trennkanal ~ 105 N (glutamate)
Beispiel: MC-CE _ Lab-on-a-Chip: Untersuchung des Gehirns
example: glutamate: LOD ~ 0.2 μmol
ex.: 330 nm (Ar laser: 351 nm), em.: 460 nm
Example: MC-CE _ Lab-on-a-Chip: probing brain Chemistry
10
11
Example: MC-CE _ Lab-on-a-Chip: Leben auf dem Mars
ExoMars mission: (Exobiology on Mars): Von Europa geleitete
Robotermission zum Mars, im Augenblick in Entwicklung
European Space Agency (ESA) & NASA
Ziel: Suche nach Leben auf dem Mars:
Lab-on-a-Chip = “Instrument” zum Messen
Amine, Aminosäuren,
Enantiomere von Aminosäuren:
LOD < ppt
(1000 x lower than “Viking gas chromatography-MS”
1976 on Mars)
Electrophorèse: principes
Dissociation, effective charge, ionization, size
12
or by additives e.g. SDS, Tensides,
CDs, Micelles ……..
e.g. sieving gels
=> various separation modi for CE:
CZE, MEKC, cGE, cIEF, chiral CE, CEC, affinity CE, cIT…….
Capillary Electrophoresis Capillary Electrophoresis
Sugars, sialic acids,
complex N-glycans
Anions / Cations
CGE-SDS (MW)
(red. / non red.)
CGE:
Oligo-nucleotides,
DNA, Polymere,
Sugars (anternary)
siRNA
CZE:
Small organic compounds,
MAB, EPO…
(assay, identity)
Chiral
separation
cIEF (identity,
charge distribution)
DNA sequencing
13
MECK: neutral compounds,
small organic compounds
14
Fab
Fc
Light chain
Heavy chain
variable region
constant region
KKK KKK
150 000 Dalton ≈ 1000 amino acids degradation products…
Testing Monograph drug substance: Example MAb
very complex manufacture process: what is needed, what does one
do to ensure constant quality of the product?
KK KK K K
Matrix: cells and medium with 100 - 1000s compounds
180 g / mol
CE - QK Tests: Monoclonal Antibody
=> generische, quantitative, einfach durchzuführende, moderne
Testmethoden, multi-funktionelle Instrumente"
CE - 1 Instrument - viele Methoden
- CGE-SDS, nicht-reduzierend: Reinheitstest, Bruchstücke, Aggregate
(ersetzt SDS-PAGE)
- CGE-SDS, reduzierend: Verhältnis leichte Kette / schwere Kette
Verhältnis nicht glykosylierte / glykosylierte schwere Kette
(ersetzt SDS-PAGE)
- CZE: Identität (ersetzt tryptischen Verdau)
- CZE: Zusammensetzung und Gehalt der Monosaccharide (ersetzt HPAEC-PAD)
- CZE, CGE: (de)sialo N-Glykan Zusammensetzung (ersetzt HPAEC-PAD, RP, NP,
HILIC-HPLC)
- cIEF, CZE: pI, Ladungsheterogenität (ersetzt IEC)
- CZE: Gegenionen, Verunreinigungen (UV direkt, indirekt), (ersetzt IEC) 15
CZE – generischer QK Identitätstest
Monoklonaler Antikörper
KKK
16
Bestimmung relatives Verhältniss der
geladenen Isoformen eines MAKs mit CZE / CGE
QK: Identität, Reinheit
Separation conditions: ε-aminocaproic acid, pH = 4.5 + 0.1% HPMC, UV detection at 280 nm, neutral
coated capillaries, normal polarity
Sample preparation: no sample preparation needed
Isoforms differ in the
number of C-terminal
lysines
KK
17
CGE-SDS: Today’s main applications of CE:
18
CGE-SDS in Biotech: e.g. MABs purity
UV detection (Coomasie Blue staining sensitivity)
CGE-SDS Brief Introduction
Proteins are denatured by heat in the presence of SDS
SDS binds to proteins in a constant ratio of 1:1.4 giving a constant size:charge ratio
Separation performed in a sieving matrix and is based on size
CGE commonly uses a replaceable sieving matrix e.g. linear polyacrylamide
1. http://bitesizebio.s3.amazonaws.com/content/uploads/2008/05/sds-protein-denaturation.gif 2. Dale R. Baker, Capillary Electrophoresis, John Wiley & Sons, Inc 1995.
Detector
Detector
q / r = constant
= separation only based on size
(sieving matrix)
20
Beckman Coulter, PA 800 plus Pharmaceutical Analysis System Also Agilent (Bioanalyser)and Caliper……..
Impurity (degradation) Analyse MAK- reduziert –
Produkt Identität, HC/LC, degHC/HC
4 3
2
1
kD
200
97.4
66.3
55.4
36.5
31
21.5
116.3
Minutes
13 14 15 16 17 18 19 20 21 22
AU
0.000
0.002
0.004
0.006
0.008
5
4 3 2 5
6
6
1
Separation conditions CGE: SDS-MW Gel Buffer (Beckman), UV at 220 nm, 480V/cm, Polymicro
capillary (fused silica)
Sample preparation: exchange of formulation buffer to NaCO3 buffer, reduction with 2-mercaptoethanol
and addition of SDS Sample Buffer; sample heated at 90 °C for 3 min
© Beckman Coulter
gHC
LC
degHC
21
DETECTION FOR CE-SDS:
LIF VERSUS UV
Minutes
15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
AU
-0.01
0.00
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
0.09
0.10
AU
-0.01
0.00
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
0.09
0.10
PDA - 220nm
BSA a
LIF - Channel 1
BSA a100x
LIF detection with FQ labeling BSA concentration 2 µg/ml
UV detection at 220 nm BSA concentration 600 µg/ml
LIF detection Sensitivity ~10 ng/ml => ~300* increase in sensitivity with LIF over UV;
Silver stain sensitivity
Silver stain Sensitivity ~20-200ng/ml
Coomassie stain Sensitivity ~5 µg/ml
22
LIF detection Sensitivity LOQ ~ 10 ng/ml
FQ: 5-FUROYLQUINOLINE-3-
CARBOXALDEHYDE
Fluorogenic reagent: react with primary amines to give a highly fluorescent product
very low background as only becomes fluorescent on reaction with primary amines
High sensitivity (attomole)
Excitation: Use Argon ion, 488 nm laser
Emission: 590 nm bandpass filter
CGE-SDS IN PHARMA
•The suitability of CGE-LIF for mAb characterisation has already been shown/validated by Hunt and Nashabeh 1999 using the dye 5-TAMRA.SE (Hunt G, Nashabeh W. Anal. Chem. 1999 71, 2390-2397).
•In 2007 Michels improved this with introduction of the dye FQ (Michels DA, Brady LJ, Guo A, Balland A. Anal. Chem. 2007 79(15), 5963-5971).
CE-SDS-UV CE-SDS-LIF
Not seen with
CE-SDS-UV
ESTIMATION OF QUANTITY OF UNKNOWN
PROTEINS IN COMPLEX MATRICES
1. What affect does protein structure have? 2. Does reduction improve the fit?
R² = 0,9983
R² = 0,9998
R² = 0,9995
0
1
2
3
4
5
6
7
8
9
0 2 4
Pe
ak
are
a
x 1
000
00
Concentration [ug/ml]
B-Lactoglobulin
Egg Albumin_1
BSA
R² = 0,9983
R² = 0,9998
R² = 0,9995
0
1
2
3
4
5
6
7
8
9
0 0,0000001 0,0000002
Pe
ak
are
a
x 1
00
00
0
Concentration [M]
B-Lactoglobulin
Egg Albumin
BSA
R² = 0,998
R² = 0,9997
R² = 0,9992
0
1
2
3
4
5
6
7
8
9
0,00E+00 2,00E-06 4,00E-06
Pe
ak
are
a x 1
00
00
0
Molar primary amine concentration
B-Lactoglobulin
Egg Albumin1
BSA
BSA: 59 lysines, MW 66432 Da Egg albumin: 20 lysines, MW 42750 Da β-lactoglobulin: 15 lysines, MW 18281 Da
Aim Quantify unknown proteins from molar lysine curve
Open Questions
a. b. c.
OPTIMIZATION OF FQ LABELING
PROCEDURE
Protein
Reduce
Alkylate
Label
Quench
Analyse CGE
Add 20ul 200mM DTT Incubate 70oC ,10 mins
20ul 10mM FQ and 15ul 20mM KCN Incubate 50oC,10 mins
Add 30ul 150mM NEM in 2% SDS Incubate 70oC, 5 mins
Add 0.6ml 1% SDS
In 250ul 100mM sodium phosphate buffer pH 6.7
Based on procedures by Michels DA, Brady LJ, Guo A, Balland A. Anal. Chem. 2007 79(15), 5963-5971 and Michels DA, Parker M, Salas-Solano, O. Electrophoresis 2012 33, 815-826
i.e. 1. Reduction 2. Labeling of dilute samples
For wide applicability in
REAL biological samples
some things need
optimizing....
OPTIMIZATION OF
REDUCTION
-2
0
2
4
6
8
10
12
14
0 5 10 15
Peak
Heig
ht
(RF
U)
DTT (mM)
Minutes
12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
RFU
0
5
10
15
20
25
30
35
40
45
50
55
60 LIF - Channel 1
BSA DTT 20ul 6/5/2013 1:21:11 PM
LIF - Channel 1
BSA DTT 15ul6/5/2013 2:11:08 PM
LIF - Channel 1
BSA DTT 10ul6/5/2013 3:01:08 PM
LIF - Channel 1
BSA DTT 5ul6/5/2013 4:21:37 PM
LIF - Channel 1
BSA DTT 2ul6/5/2013 5:11:35 PM
LIF - Channel 1
BSA No DTT 6/5/2013 8:31:28 PM
DTT can cause a reduction in fluorescence signal. Optimal DTT concentration 3 mM, protein reduced without intensity loss
Plot of peak height vs. DTT concentration
0 mM DTT
2.8 mM DTT
5.7 mM DTT
8.5 mM DTT
1.1mM DTT
11.3 mM DTT
1 Michels DA, Brady LJ, Guo A, Balland A. Anal. Chem. 2007 79(15), 5963-5971
Initial condition. Based on1
BSA labeled at 160ug/ml then diluted to 1.6ug/ml in 0.1% SDS
LOD calculated 0.7ug/ml ~ 100* loss in sensitivity compared to unreduced
~ 100* gain in sensitivity compared to reduction with 11 mM DTT
OPTIMIZATION OF LABELING
BUFFER
• suitable for both labeling and CGE. • lower concentration gives improved signal in CE due to stacking • pH 8-9 optimal for FQ reaction but risk of disulfide scrambling
Minutes
12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
RFU
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
2.2
2.4
2.6
2.8
3.0 LIF - Channel 1
BSA B10 7/10/2013 5:14:59 PM
LIF - Channel 1
BSA B100 7/10/2013 6:54:56 PM
LIF - Channel 1
BSA P10 7/10/2013 4:25:01 PM
LIF - Channel 1
BSA P100 7/10/2013 6:04:57 PM
10 mM Sod. Borate pH 8.5
100 mM Sod. Borate pH 8.5
10 mM Sod. Phos pH 6.7
100 mM Sod. Phos pH 6.71
~36* improvement in sensitivity use for reduced samples
~18* improvement in sensitivity
~11* improvement in sensitivity use for unreduced samples
1Michels DA, Parker M, Salas-Solano, O. Electrophoresis 2012 33, 815-826
Initial condition1 BSA labeled and analysed at 1.6ug/ml without reduction
29
CZE - INDIRECT UV
DETECTION
anions, organic acids, cations, aliphatic amines
with no UV absorption
Na, K, Cl, Po4, SO4………
T o g e t h e r b e t h e b e s t 9/18/2014 30 30
DETECTION: INDIRECT-UV
reverse of detector signal
/ subtraction of reference channel
Auto zero
T o g e t h e r b e t h e b e s t 9/18/2014 31 31 9/18/2014 31
ANIONS
Ion Analysis using CE 31
Minutes
4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 11.5
AU
-0.005
0.000
0.005
0.010
0.015
0.020
AU
-0.005
0.000
0.005
0.010
0.015
0.020
UV - 254nm
std 5
Name
UV - 254nm
std 4
UV - 254nm
std 3
UV - 254nm
std 2
UV - 254nm
std 1
T o g e t h e r b e t h e b e s t 9/18/2014 32 9/18/2014 32
Minutes 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5
AU
-0.004
-0.002
0.000
0.002
0.004
BGE effect of pH
pH 5.6
pH 8.2
1 Formic 3.74 4 Butyric 4.87 7 Heptanoic 4.89 10 Capric 4.9
2 Acetic 4.76 5 Valeric 4.81 8 Caprylic 4.89 11 -
3 Propionic 4.87 6 Caproic 4.80 9 Pelargonic 4.96 12 Lauric 4.9
T o g e t h e r b e t h e b e s t 9/18/2014 33 33 9/18/2014 33
EXAMPLE CATION TESTMIX
Minutes
2.0 3.0 4.0
AU
0.010
NH
4
2.4
38
Na
2.8
75
K
3.0
87
L
i 3.1
88
Mg
3.3
75
Ca
3.5
38
Minutes
2.0 3.0 4.0
AU
0.010
NH
4
2.4
38
Na
2.8
75
K
3.0
87
L
i 3.1
88
Mg
3.3
75
Ca
3.5
38
Indirect UV: 200 nm
4-Aminopyridine buffer pH 4.2
with Crown-ether
T o g e t h e r b e t h e b e s t 9/18/2014 34 34
COMMERCIAL BUFFERS
Fluka: Anion HPCE-kit
Agilent: Organic Acid, Inorganic Acid, Cation Solution, Forensic
Anion, Plating Bath
Waters: IonSelect OFM, High Mobility Anion Electrolyte, Low Mobility
Cation Electrolyte
Beckman Coulter: Anion Analysis kit (PDC buffer pH 5.6), Cation Analysis kit
(4-Aminopyridine with Crown ether, pH 4.2)
MicroSolv Tech / Analis: CElixir OA 2.5, 5.4 and 8.2. CElixir plus
titrationP
o4, C
l,
citra
te,
aceta
te, T
FA
,
CO
3-, S
CN
Na, K
, Tris
ACN,
PG
Gly
cin
e / H
is
HCO3
Tween
80
CE Kit 1
anions
CE Kit 2
cations
GC-
head-
AAA
(generic
1 NaCl 0.145M / PB 50mM / PG 10% X PO4, Cl Na PG 0 0 0 75 150 32 40 40 48 xx x x
2 NaCl 1.4M / PB 20mM / PG 45% X PO4, Cl Na PG 0 0 0 0 0 32 0 0 48 xx x x
0 0 0
3 Sodium Phosphate 10 mM X PO4 Na 0 0 0 0 0 16 0 0 0 x x
4 Potassium Phosphate 50 mM X PO4 K 0 0 0 37.5 0 16 0 0 0 x x
5 Sodium Phosphate 4mM / histidine 10 mM X PO4, Cl Na Histidine 0 0 0 8 0 16 16 16 16 xx x x
6 Sodium Phosphate 50mM X PO4 Na0 0 0 0 0 16 0 0 0 x x
0 0 0
7 Citric acid 0.1M X citrate 0 0 0 37.5 37.5 8 0 0 0 x
8 Citrate 10mM / NaCl 145 mM X Citrate, Cl Na 0 0 0 0 0 16 0 0 0 xx x
9 Sodium citrate 300 mM X citrate Na0 0 0 0 0 16 0 0 0 x x
10 Citric acid 10mM / glycine 100mM / NaCl
100mM
X Citrate, Cl Na Glycine0 0 0 0 0 16 16 37.5 16 xx x x0 0 0
11 Acetic Acid 1M X X acetate 0 0 0 37.5 37.5 8 0 0 0 x
12 Acetic acid 20 mM X acetate 0 0 0 0 0 16 0 0 0 x
13 Sodium Acetate 0.5M X acetate Na 0 0 0 0 0 8 0 0 0 x x
14 Sodium Acetate 14.5 mM X acetate Na 0 0 0 0 0 8 0 0 0 x x0 0 0
15 Tris base 1M X X Tris 0 0 0 37.5 24 24 0 0 0
16 NaSCN 0.7M / tris HCl 0.05M X SCN Na, Tris 0 0 0 37.5 37.5 24 0 0 0 x xx0 0 0
17 WFI / TFA 0.1% X TFA 0 0 0 24 24 16 0 0 0 x
18 TFA 0.1% in ACN pure X TFA ACN 0 0 0 37.5 37.5 8 0 0 0 x x
19 ACN 30% / TFA 0.1% X TFA ACN 0 0 0 0 0 8 40 40 48 x x
20 ACN 60% / TFA 0.1% / WFI X TFA ACN 0 0 0 0 0 8 0 0 48 x x
21 NaHCO3 0.67M X HCO3 Na 20 30 24 10 10 8 0 0 0 x x
22 10 % Tween 80 in water X tween 30 40 48 0 0 0 0 0 0 x
Sum
(hours) 50 70 72 342 358 320 112 133.5 224
An
aly
sis
( 8
sa
mp
les
)
probable generic separation system
Me
tho
d V
alid
ati
on
An
aly
sis
( 8
sa
mp
les
)
Me
tho
d D
ev
elo
pm
en
t
Me
tho
d V
alid
ati
on
Assay GC-headspace
or AAA
# Name
pro
du
ct 1
pro
du
ct 2
pro
du
ct 3
pro
du
ct 4
anion
TitrationAssay and
degardation
kation
neutral /
AAA
Me
tho
d D
ev
elo
pm
en
t
Me
tho
d V
alid
ati
on
An
aly
sis
Me
tho
d D
ev
elo
pm
en
t
Development of a validation program for all buffers / solutions prepared @
PHARMACEUTICAL COMPANY XYZ with respect to stability, composition,
identity
35
Bestimmung von TFA in einer Protein DS gelöst in PBS:
(150 mM NaCl, 2.83 mM NaH2PO4, 7.14 mM Na2HPO4, pH = 7.2 (1 M NaOH))
• LOQ: ≈ 25 ppm
• Matrix Ionen nicht abgetrennt
• nicht validierbar
• teure Ionenaustausch Säule
• “Spezial”-Instrument
• Adsorption der Proteinmatrix an der
stationären Phase (Reproduzierbarkeit)
• LOQ: 5 ppm
• Matrix Ionen abgetrennt
• validiert
• billige Puffer und Kapillare
• Multiuse Instrument
IEC Cl-
PO43-
CE
Cl- PO4
3-
Analyse von TFA mittels CE / indirekte UV Detektion
36 oder CE Kit (CElixierOATM pH 5.4)
37
SO42
-
Cl-
tartarate
malate
lactate
phosphate citrate gluconate
University: CZE of ions in white wine in a bare quartz
capillary , CTAB (Cethyl-trimethyl-ammonium bromide),
buffer, Pyridine-2,6-dicarboxylic pH 5.6
« Dissoudre : 668 mg acide Pyridine-2,6-
dicarboxylique (acide dipicolinique) + 364
mg de bromure d’hexadécyl-triméthyl-
ammonium (CTAB : Cethyl Trimethyl
Ammonim Bromide) + 20 ml d’acétonitrile
dans 160 ml d’eau. Amener le pH à 5.64
avec NaOH-1M puis NaOH-0.1M.
Compléter à 200 ml avec de l’eau, Filtrer
sur 0.45 mm
38
Zuckeranalyse in komplexen Matrices
T o g e t h e r b e t h e b e s t 9/18/2014 39 39 9/18/2014 39
pH 12.6
Direct Monosaccharide detection @ pH 12.6
(on-column reaction in the DAD window )
Kenner, J. et al. 1954 J. Chem. Soc. Ziderman, I. et al. 1975 Carbohydr. Res.
UV active Enolate formation
Production of Polyhydroxyalkanoates (PHAs) in Pseudomonas
putida (Gram negative bacteria) from sustainable resources
Bacterial polyesters are
accumulated as intracellular
granules in the cytoplasm
purification
During cell lysis endotoxin
(ET) contamination of the
hydrophobic product PHA
40
Fruit
pomace
Feed /
Ferm
enta
tion
*Fruit pomace and waste frying oil as sustainable resources for the bioproduction of medium-chain-length polyhydroxyalkanoates, Stéphanie
Folloniera et al, International Journal of Biological Macromolecules, 2014, in press
BT Testmethodenentwicklung mit CE, indirekte UV Detektion, sugar –
kit (Analis) used in polyhydroxyalkanoate (PHA) production from
sustainable resources)
41
Aminex HPX-87H Column (BioRad),
eluent 5 mM H2SO4, flow rate 0.45 ml/min
Glucose produced by enzymatic pre-
treatment of apricot pomace is
consumed by the cells during
fermentation.
HPLC
CE: apricot pomace CE
BT Testmethodenentwicklung mit CE, indirekte UV Detektion, (organic
acid-kit (Analis) used in polyhydroxyalkanoate (PHA) production from
sustainable resources)
42 CE-samples were prepared by filtering through a 0.45um filter, followed by 10 fold
dilution in water to give concentrations of approximately 100 ug/ml.
*Fruit pomace and waste frying oil as
sustainable resources for the
bioproduction of medium-chain-length
polyhydroxyalkanoates, Stéphanie
Folloniera et al, International Journal of
Biological Macromolecules, 2014, in
press
Background buffer: 36 mM Na2HPO4/90 mM NaOH pH=12,6
CE: 17 kV, 20°C, 270 nm, Capillary: Ldet/Ltot=52.5 cm/60 cm, I.D.: 50 mm,
0,5 mg/ml sugar
standards
EOF
EOF
2.
1.
3.
2. 3.
1. 2.
3.
EOF
1. Galactose
2. Glucose
3. Rhamnose
Hydrolyzed ET (LPS) analysis with CZE @-DAD
1 mg /ml E. coli O55:B5
1 mg /ml Pseudomonas
a.
LPS
Monosaccharide
KDO??
~ 0,01 mg/ml KDO => 500,000 EU/ml
LOD: 0,005 mg/mL (approx. 250 000 EU ET/ ml)
=> quantification method «all» ETs for process
development
2M TFA
16 h 100°C
* Specification sheet L8643 Sigma Aldrich pruduct
43
3-Deoxy-D-manno-oct-2-ulosonic acid
Chiral CE
44
Sulfated Cyclodextrins (1 to 10% @)
Properties
a (6) , b (7), and g (8) glucose
units
negatively charged
high mobility
readily soluble
good selectivity generating
high resolution
proven, tested quality each
batch
a-HSCD: 11 sulfates C36H49O63S11: FW:1841
b-HSCD: 12 sulfates C42H58O63S12: FW:2082
g-HSCD: 13 sulfates C48H67O63S13: FW:2323
© 2006
neutral
cations
anions
Migration of differently charged molecules:
sulfated ((highly)-charged) cyclodextrins
migration of molecules according to the charge of the whole complex (anion)
with the neutral but also positive / negative molecule in "backpack"
"Universal system" for all water soluble substances
46
© Solvias AG
Using 2 tested generic chiral analysis systems (screening systems):
CE-system 1: 5% (w/v) g-HSCD CE-system 2: 5% (w/v) b-HSCD
in 25 mM phosphate buffer pH 2.5
• 200nm, -12kV, 15°C, 50 mm ID, 31/39.5cm on a HP3D-CE
• c = 1 mg / ml in run buffer
• capillary conditioning: NaOH 1M, H2O, cap. cond. sol., HCl 1M, run buffer
"Universal system" for all model substances
47
© Solvias AG
0
50
100
150
200
250
0 5 10 15 20 25 30 35 min
mAU
Chiral separation of 16 water soluble model
substances using two CE-systems
0
50
100
150
200
250
0 10 20 30 40 50 60 70 min
mAUCE-system 1:
5% (w/v) g-HSCD
CE-system 2:
5% (w/v) b-HSCD PTS* PTS*
250
200
150
100
50
0
Abs.
COOH*
R1
COOH
E1
O
O
OH
*
R2
O
O
O
E2
NH*
R6
N
E6
OH
O
O
*
R9
O
O
O
E9
N
O
O
O*
OH*
R16
OH
E16
OH
O
N
O
*
R19
OH
O
N
O
E19
OH
O
*
R23
OH
O
E23
OH*
R16
OH
E16
OH
O
N
O
*
R19
OH
O
N
O
E19
0 10 20 30 40 50 60 70 min 0 5 10 15 20 25 30 min
COOH*
OH
*
© Solvias AG
Reproducibility CE-system 2: overlay of 25 runs
NH
*
R5
N
E5
CE-system 2 (sample in 10% (v/v) DMSO): 5% (w/v) b-HSCD
enantiomer e2 enantiomer e1
PTS
0 1 2 3 4 5 6 7 8 9 min
Abs.
600
500
400
300
200
100
0
• Thanks to Mirian Goyder, Blanka Buscela and Antoine Fornage at HES-SO Valais/Wallis
• Thanks to Oscar Salas Solano for his helpful advice on FQ
• Thanks to François de l’Escaille (Analis R & D), for providing the ion & sugar kits
Thank you for your attention !!!
QUESTIONS?
50