indirect uv detection and applications - applica-analytik.ch · ce - qk tests: monoclonal antibody...

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

Jorgensen & Lukacs:

einfaches CE-Gerät mit

hochsensitivem Fl-Detektor

(HPLC) & Quartzkapillaren

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

Capillary Electrophoresis Instrumentation

various detectors

UV-VIS, DAD, MS, LIF, Fl,

CCD, indirect UV detection

(no chromophors)

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...

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

1-Mercaptoethanol-2-alkylisoindol

Derivatization of glutamate

derivatisation time: ~ 1min (on-column derivatisation)

Lit.: Roth et al

Beispiel: MC-CE _ Lab-on-a-Chip: Untersuchung des Gehirns

1.) Mikrodialyse:

semipermeable

Membrane

3 μl / min flow

2.) on-column Derivatisation

3.) Trennung

9 cm Trennkanal ~ 105 N (glutamate)

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

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

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.)

Oligo-nucleotides,

DNA, Polymere,

Sugars (anternary)

Small organic compounds,

MAB, EPO…

(assay, identity)

Chiral

separation

cIEF (identity,

charge distribution)

DNA sequencing

MECK: neutral compounds,

small organic compounds

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

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

CGE-SDS: Today’s main applications of CE:

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

q / r = constant

= separation only based on size

(sieving matrix)

Beckman Coulter, PA 800 plus Pharmaceutical Analysis System Also Agilent (Bioanalyser)and Caliper……..

Impurity (degradation) Analyse MAK- reduziert –

Produkt Identität, HC/LC, degHC/HC

Minutes

13 14 15 16 17 18 19 20 21 22

4 3 2 5

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

DETECTION FOR CE-SDS:

LIF VERSUS UV

Minutes

15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

PDA - 220nm

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

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

Concentration [ug/ml]

B-Lactoglobulin

Egg Albumin_1

R² = 0,9983

R² = 0,9998

R² = 0,9995

0 0,0000001 0,0000002

Concentration [M]

B-Lactoglobulin

Egg Albumin

R² = 0,998

R² = 0,9997

R² = 0,9992

0,00E+00 2,00E-06 4,00E-06

Molar primary amine concentration

B-Lactoglobulin

Egg Albumin1

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

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

0 5 10 15

DTT (mM)

Minutes

12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

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

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

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

-0.005

UV - 254nm

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

-0.004

-0.002

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

EXAMPLE CATION TESTMIX

Minutes

2.0 3.0 4.0

Minutes

2.0 3.0 4.0

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

, Tris

CE Kit 1

anions

CE Kit 2

cations

(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

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

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

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

(hours) 50 70 72 342 358 320 112 133.5 224

probable generic separation system

Assay GC-headspace

or AAA

# Name

TitrationAssay and

degardation

kation

neutral /

Development of a validation program for all buffers / solutions prepared @

PHARMACEUTICAL COMPANY XYZ with respect to stability, composition,

identity

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-

Cl- PO4

Analyse von TFA mittels CE / indirekte UV Detektion

36 oder CE Kit (CElixierOATM pH 5.4)

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

Zuckeranalyse in komplexen Matrices

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

pomace

Feed /

*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)

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.

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

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

1. Galactose

2. Glucose

3. Rhamnose

Hydrolyzed ET (LPS) analysis with CZE @-DAD

1 mg /ml E. coli O55:B5

1 mg /ml Pseudomonas

Monosaccharide

~ 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

3-Deoxy-D-manno-oct-2-ulosonic acid

Chiral CE

Sulfated Cyclodextrins (1 to 10% @)

Properties

a (6) , b (7), and g (8) glucose

negatively charged

high mobility

readily soluble

good selectivity generating

high resolution

proven, tested quality each

a-HSCD: 11 sulfates C36H49O63S11: FW:1841

b-HSCD: 12 sulfates C42H58O63S12: FW:2082

g-HSCD: 13 sulfates C48H67O63S13: FW:2323

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

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

0 5 10 15 20 25 30 35 min

Chiral separation of 16 water soluble model

substances using two CE-systems

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*

0 10 20 30 40 50 60 70 min 0 5 10 15 20 25 30 min

Reproducibility CE-system 2: overlay of 25 runs

CE-system 2 (sample in 10% (v/v) DMSO): 5% (w/v) b-HSCD

enantiomer e2 enantiomer e1

0 1 2 3 4 5 6 7 8 9 min

• 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?

indirect uv detection and applications - applica-analytik.ch · ce - qk tests: monoclonal antibody...

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