Immunoaffinity LC/MS/MS in peptide bioanalysis...November 8th, 2011 2nd Nordic MS Symposium Carsten...

Immunoaffinity –LC/MS/MS in peptide bioanalysisNovember 8th, 2011

2nd Nordic MS SymposiumCarsten Boye Knudsen, Head of Bioanalysis & Pharmacokinetics

Private and Confidential

AGENDA

Introduction

Immunoaffinity purification

Quantitative Applications

2

T U R N I N G P E P T I D E S I N T O D R U G S

Peptides in Clinical Development

Source: Development trends in peptide therapeutics, Peptide therapeutics foundation, 2010


Assays in Peptide Bioanalysis

• Peptides < 20 amino acids are typically analyzed by LC/MS/MS

• Due to decreased MS/MS sensitivity, larger peptides are primarily analyzed by immunoassays

• Sensitivity seems to be driving the selection of assay platform

Peptide Drug ELISA (LLOQ) LC/MS/MS (LLOQ)

Exenatide 2.3 pM 25-50 pM

Glucagon 2.9 pM ≈ 30 pM

Liraglutide 18 pM 100-200 pM


Ligand Binding Asssys and LC/MS/MS

Ligand Binding Assays

• Advantages

• High sensitivity

• Limited sample preparation

• High throughput

• Inexpensive Instrumentation

• Newer platforms more expensive

• Disadvantages

• Requires specific reagents

• Selectivity not given

• Narrow dynamic range (old platforms)

• Reagent batch variability

• Addition of IS not possible

LC/MS/MS

• Advantages

• High sensitivity, but…

• High selectivity

• Broad dynamic range

• Correction by IS possible

• Multiplexing

• Disadvantages

• Sensitivity limited for high molecular weight compounds

• Expensive instrumentation

• Sample preparation required

• Lower throughput


Immunoaffinity-LC/MS/MS

• Advantages

• High sensitivity

• Limited sample preparation

• High throughput

• Inexpensive Instrumentation

• Newer platforms more expensive

• Disadvantages

• Requires specific reagents

• Selectivity not given

• Narrow dynamic range (old platforms)

• Reagent batch variability

• Addition of IS not possible

• Advantages

• High sensitivity, but…

• High selectivity

• Broad dynamic range

• Correction by IS possible

• Multiplexing

• Disadvantages

• Sensitivity limited for high molecular weight compounds

• Expensive instrumentation

• Sample preparation required

• Lower throughput


Immunoaffinity Purification

7


Antibodies

• Epitopes• Specific binding regions on target • 6-12 AA linear or 3D sequences

• Polyclonal Antibodies (PAb)• Multiple Ab’s that bind to multiple epitopes• May include non-target epitopes (purification

dependent)

• Monoclonal Antibodies (MAb)• Single Ab that bind to a single epitope

• Epitopes should be carefully considered when selecting antibodies for an assay


0.00 0.50 1.00 1.50

Relative response

0.00 0.50 1.00 1.50

Relative response

0.00 0.50 1.00 1.50

Relative response

0.00 0.50 1.00 1.50

1-15

3-18

6-21

9-24

12-27

15-30

18-33

21-36

Relative response

Am

ino A

cid

Sequ

ence

MAb-1 MAb-2 MAb-3 PAb

H-

-NH21-15

3-18

9-24

6-2112-27

15-30

18-33

21-36

Epitope Mapping


Affinity is Critical for Sensitivity

0

2000

4000

6000

8000

10000

12000

0 3 6 9 12Conc nM

Re

sp

on

se

Mab 1

Mab 2

Mab 1+2

Antibody Titer

Mab-1 230 ng/mL

Mab-2 22 ng/mL

rateoffk

rateonk

LA

AL

k

kAffinity

kALkLA

d

a

d

a

da


blank dogpl

Time8.00 10.00 12.00 14.00 16.00 18.00 20.00

%

0

100

8.00 10.00 12.00 14.00 16.00 18.00 20.00

%

0

100

8.00 10.00 12.00 14.00 16.00 18.00 20.00

%

0

100

ZP1848_08102101_ALJ_07 1: TOF MS ES+ BPI

1.42e3

12.49

12.18

8.19 11.5810.518.88

13.24

17.22

13.95 15.5414.66 15.99

17.87

18.3019.84


1.42e3

15.75

10.98

9.89 11.20 14.5112.14

17.5416.89

18.05

18.55


1.42e3

15.82

15.17

bl pl zp1848 mix01 dyna 2431

m/z600 800 1000 1200 1400 1600 1800

%

0

100%

0

100

%

0

100

ZP1848_08102101_ALJ_07 454 (16.625) Cm (118:586) 1: TOF MS ES+ 5.64e3573 618 880853

804

782

761

709

619

908

938970

1005 1042

1082

11261172

127913401407 1481

ZP1848_08112601_ALJ_04 420 (15.753) Cm (117:587) 1: TOF MS ES+ 5.64e3

520570

608

652

795696

ZP1848_08082901_ALJ_04 371 (14.656) Cm (111:578) 1: TOF MS ES+ 5.64e3

657569 1259881783658 948 982 1193 1333 1416 188817431511

Dynabead Tosyl

Immunobead MS Background

Dynabead Epoxy

Agarose

Blank plasmaΣ-spectra 8-20min

Ion current


Wash Conditions

0.5% Triton (wash)

0.5% Tween (wash)

10 nM pl extended wash 3 0.5 % triton

Time8.00 10.00 12.00 14.00 16.00 18.00 20.00

%

0

100


1.70e3

12.52

12.19

8.06 11.4810.369.83

13.03 13.27

17.2015.92

13.98 14.65

17.88

18.26

19.8818.65

10 nM pl extended wash 3 0.5 % triton

m/z700 800 900 1000 1100 1200 1300

%

0

100

ZP1848_08102801_ALJ_06 454 (16.572) Cm (435:454) 1: TOF MS ES+ 586

920

920

736

736

687 736

920

737

793 830 908855

920

921

921

1226921 948970 1024 1106

10471115 1275

*

10 nM pl extended wash 5 0.5 % tween

Time8.00 10.00 12.00 14.00 16.00 18.00 20.00

%

0

100


1.58e3

12.32

11.96

7.89 11.2710.129.68

13.02

17.5516.88

15.7113.65 14.4017.94

19.42

10 nM pl extended wash 5 0.5 % tween

m/z700 800 900 1000 1100 1200 1300

%

0

100

ZP1848_08102801_ALJ_08 434 (16.111) Cm (429:443) 1: TOF MS ES+ 553

920

920

880803781

736

721

852

853

907

880

882

920

1275920

969

939

12741004

10411082 1124

1275

1277

*

Σ of 20 spectra @16.5 minIon current


General IA–LC/MS/MS procedure

Sample (Urine, Plasma, Tissue homogenate)

Wash cycles (2-5)

Immobilized anti-target antibody in binding buffer (ex. T-PBS)

4 C-37 C for 1-24h

Elution (Acidic/Organic/Denaturing), min-h

LC/MS/MS

Internal Standard

Protease

inhibitors


Quantitative Application - 1

14


Case 1

• Therapeutic peptide, Mw ≈ 4700 g/mol, pI ≈ 10.3

• SPE-LC/MS/MS range 1-1000 nM

• Expected therapeutic levels 10-50 pM

• A single and high affinity antibody was available

• Method needed for evaluation of transdermal delivery


Method

Immunoaffinity purification:

• Dynabead-Epoxy

• Single MAb directed against the C-term.

• 400 µL Plasma: 800 µL T-PBS (1h, RT)

• 3 wash cycles (PBS)

• Elution: 100 µL (40% MeCN, 0.25%TFA)

LC/MS/MS (UPLC-Quattro Ultima):

• Poros R/10, 10 µm, 30x2.0 mm

• 13-54% MeCN, 0.2%FA (3 min)

• Inj. 30 µL

• Analyte: 3 MRM of +7 ion,

• IS (Structural analogue): 2 MRM of +7 ion

• Range: 10-100.000 pM

ZP131 0.01 nM minipig plasma

Time0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50

%

0

100

ALJ_B030_10070101_13 2: MRM of 3 Channels ES+ TIC

4.63e42.61

Plasma + 10 pM

Plasma


Extended Column Wash

60% ACN, 0.2 FA

Time0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50

%

0

100


3.19e40.64

2.65

2.88 3.12

4.793.69

60% ACN, 0.2 FA

Time0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50

%

0

100


3.19e4

Analyte Trace IS Trace

1) 60% MeCN, 0.2% FA

2) 60% MeCN, 0.2% FA

3) 40% MeCN, 0.25% TFA

4) 40% MeCN, 0.25% TFA

Analyte


Results

• Therapeutic peptide detected in all pigs

• Immunoaffinity purification step increased method sensitivity 100-fold

• Cal. and QC samples ≤15% over 2 analytical runs

• Transdermal delivery was evaluated sucessfully

1

10

100

1000

0 1 2 3 4 5 6 7 8 9 10

Time (hr)

1

2

3

4

Transdermal delivery of

therapeutic peptide to mini-pigs


Quantitative Application -2

19


Case 2

• A clinical assay was needed for a therapeutic peptide and two metabolites having pI’s between 4.6 to 10.4 with Mw’s from 3.5-4.3 kDa.

• 2 existing preclinical SPE-LC/MS/MS methods (1 for the parent and 1 for the metabolites) had inadequate sensitivities (LLOQ= 10 nM)

• Precipitation and other pre-concentration attempts suffered from low recovery

• 2 monoclonal antibodies recognizing all compounds were available


Method

Immunoaffinity purification:

• Dynabead-Epoxy

• Dual MAb directed against the N-term. and central sequence

• 400 µL Plasma: 800 µL T-PBS (1h, RT)

• 4 wash cycles (PBS)

• Elution: 100 µL (40% MeCN, 0.25%TFA)

LC/MS/MS (UPLC-Quattro Ultima):

• Xterra C8, 2.5 µm, 50x2.0 mm

• 18-90% MeCN, 0.2%FA (6 min)

• Inj. 30 µL

• Analytes: 1 MRM from each analyte

• IS: First: SA 1 MRM, later: SIL for all compounds 1 MRM/IS

• Range: 50-10.000 pM

10nM ZP1848 mix04 in buffer (BEH 130)

Time0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00

%

0

100

KAO_ZP1848_11090501_13 MRM of 6 Channels ES+ 735.9 > 877.6 (ZP2469)

1.93e6

2.92


Time0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00

%

0

100


1.86e6

2.92


Time0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00

%

0

100


2.20e6

2.95

Parent

M1

M2

New column

Injection # 77

After Plasma Load


min2.0 4.0 6.0 8.0 10.0

%

0

100

MRM of 4 channels,ES+

720.1 > 818.4

P1848_09021001ALJ_08 Smooth(Mn,2x1)

blank pl

4.239e+0035.71

5.58

4.663.33

5.77

6.37

7.21

7.687.90

min2.0 4.0 6.0 8.0 10.0

%

0

100


720.1 > 818.4

P1848_09021001ALJ_09 Smooth(Mn,2x1)

ZP1848_mix04 plasma 0.05 nM

1.371e+0045.16

1568.53

4.37

5.73

6.76

7.56

min2.0 4.0 6.0 8.0 10.0

%

0

100


736 > 877.5

P1848_09021001ALJ_08 Smooth(Mn,2x1)

blank pl

1.026e+0036.565.69

113.71*

5.69

113.71*

4.70

3.133.02

2.050.09

7.847.05

11.81

10.919.598.06

min2.0 4.0 6.0 8.0 10.0

%

0

100


736 > 877.5

P1848_09021001ALJ_09 Smooth(Mn,2x1)


1.530e+0045.81

1387.33

5.68

6.55 7.357.93

min2.0 4.0 6.0 8.0 10.0

%

0

100


761.5 > 920.3

P1848_09021001ALJ_08 Smooth(Mn,2x1)

blank pl

2.791e+0035.63

399.20*

5.03

4.78

3.00 4.29

6.396.82

7.45

8.01

9.35

11.17

min2.0 4.0 6.0 8.0 10.0

%

0

100


761.5 > 920.3

P1848_09021001ALJ_09 Smooth(Mn,2x1)


1.278e+0045.71

1607.68

5.86

6.927.59

7.98

Parent M1 M2

Blank plasma

Spiked plasma (50 pM)


Accuracy & Precision (%)

QC level Parent M1 M2

pM Accuracy Precision Accuracy Precision Accuracy Precision

10093.4

(87.0-99.7)

9.6

(4.3-12)

108

(107-110)

6.5

(5.5-7.9)

105

(101-108)

8.8

(7.0-11)

30099.3

(96.6-104)

7.3

(3.1-10)

107

(105-109)

5.8

(3.3-8.2)

103

(98-108)

6.8

(5.1-6.5)

2000107

(103-110)

6.4

(3.3-4.4)

111

(106-115)

6.8

(4.8-8.1)

108

(105-115)

6.7

(3.7-4.1)

6000106

(103-110)

6.0

(4.8-6.1)

110

(108-111)

4.5

(2.3-5.1)

107

(106-109)

4.1

(1.6-5.4)


MAb2432-M-F1-18 (ng/ml)

10 100 1000 10000

0

1

2

3

4

y = ( (A - D)/(1 + (x/C)^B ) ) + D: A B C D R^2

1 (Coat: ZP1848, t3: Concentration vs MeanValue) 0,018 2,181 35,672 3,785 0,998

2 (Coat: ZP1848, t3+t4: Concentration vs MeanVal... 0,018 2,025 38,493 3,797 0,998

3 (Coat: ZP1848, fældet Ab: Concentration vs Mea... 0,064 1,559 182,799 3,767 0,997

4 (Coat: ZP1848, fældet+oprenset Ab: Concentrati... 0,013 2,31 33,146 3,762 1

5 (Coat: Zp1848, t5 5.opr: Concentration vs Mean... 0,079 1,658 159,043 3,707 0,998

Batch Production of Antibodies

Small Batch Purification

Small Batch Purification

Large Batch Before New Purification

Large Batch New Purification

Large Batch Original Purification

Large Scale

New Purif.


Results

• Method was successfully validated and applied in a clinical study

• Assay success rate ranged from 81-88%

• 8 different antibody coated bead batches were produced during the study

• Sensitivity was enhanced 200 times relative to the initial SPE-LC/MS/MS method

• Lessons learned

• Stable isotope labeled internal standards for each analyte are recommended

• Production, purification and test of antibodies are important to verify prior to validation


Perspectives of Immunoaffinity-LC/MS/MS

• Attributes

• Increased sensitivity relative to LC/MS/MS

• Pre-concentration

• Cleaner extracts

• Increased selectivity relative to Immunoassays

• MS/MS driven

• Can be applied to both qualitative and quantitative analysis when antibodies are available

• Information to design the right immunoassay for improved selectivity

• Prerequisites

• Close collaboration between immunoassay and LC/MS/MS scientists


Acknowledgement

• Kamilla Rolsted

• Arne Lindhardt Jensen

• Hanne Rasmussen

• Lone Eske Hansen

• Evelyn Kury

• Bjarne Due Larsen

• Lars Bo Hansen

• Karina Odefey

Immunoaffinity LC/MS/MS in peptide bioanalysis...November 8th, 2011 2nd Nordic MS Symposium Carsten...

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