new combined use of immunoassay and two-dimensional liquid … · 2018. 4. 2. · combined use of...

Combined use of Immunoassay and Two-Dimensional Liquid Chromatography Mass Spectrometry for the Detection and Identification of Metabolites from

Biotherapeutic Pharmacokinetic Samples

September 15, 2011

Robert E. Murphy, Arvind Kinhikar, Joselyn Del Rosario, Ryan Preston,

Mike Shields, and Nancy Levin

CovX, Pfizer BioTherapeutics Research and Development

1

Agenda

CovX Technology

Anti-Idiotype Capture Reagent

Immunoassays Developed and PK Results

Two-dimensional Liquid Chromatography/MS method

2DLC/MS PK Results and Intact Protein Metabolite Identification

Immunoassay and 2DLC/MS correlation

Summary

2

CovX TechnologyPharmacophore fusion onto catalytic antibody

ACTIVEMOLECULE

ANTIBODY

SPONTANEOUSCOVALENT ASSEMBLY

IN SOLUTION

HOMOGENEOUS FUSION PROTEIN

3

A closer look at the fusion reactionIntact SEC/MS characterization

T0 (minutes after addition)

Mid-reaction time point

Final product (2 peptides fused)

Deconvoluted Mass Spectra (149000 to 155000 amu)

4

CovX-bodies EvolutionPeptides to proteins to bi-functional bioconjugates

Mono-functional peptide CovX-Bodies

Substitute protein for peptides: protein CovX-Bodies

Bi-functional peptide CovX-Bodies

55

Transforming Peptides into Drugs

Antibody and peptide are both key CovX-Body design elements

Peptide and linker design dramatically impacts CovX-Body properties

6

GLP-1 Model

HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR

HGEGTFTSDLSKQMEEEAVR LFIEWLKNGGPSSGAPPPSG EGTFTSDLSKQMEEEAVRLF IEWLKNGGPSSGAPPPS

EGTFTSDVSSYLEGQAAKEFIAWLVKGR

DPP-4 enzyme

-HA

Exendin-4

GLP-1

7

Anti-Idiotype• Antibody that binds to the

variable domains of another antibody

• Develop non-blocking “private” anti-idiotype that is specific for CovX body

Utility• Generic capture or detection

reagent for all CovX Bodies • More sensitive assays; lower

backgrounds• Single format for preclinical

and clinical assays• Vendor independence

CovX Body

Peptide

Fc Domain

Fab

Anti-Idiotype MAb

VariableConstant

Anti-Idiotype Development:Specific and Universal Capture Reagent

8

PK Immunoassays Developed

Block

3) HRP-Anti-human IgG

Total

Block

1) Anti-Id

2) CovX body

TMBColor

HRP

Block BlockBlock

1) Anti-Id

2) CovX body

SA-HRP

TMBColor

3) Anti-peptide(N-term Specific)

Active

BlockBlock

9

Mouse PK Immunoassay Results

0.00

100.00

200.00

300.00

400.00

500.00

600.00

0.00 10.00 20.00 30.00 40.00 50.00 60.00

Co

nc

(u

g/m

L)

Time (hrs)

0.00

100.00

200.00

300.00

400.00

500.00

600.00

0.00 10.00 20.00 30.00 40.00 50.00 60.00

Co

nc

(u

g/m

L)

Time (hrs)

0.00

100.00

200.00

300.00

400.00

500.00

600.00

0.00 10.00 20.00 30.00 40.00 50.00 60.00

Co

nc

(u

g/m

L)

Time (hrs)

GLP-1 CovX-body Exendin-4 CovX-body

Modified Exendin-4 CovX-bodyBlue Total

Green Active

10

Anti-Idiotype Column Preparation

-38 umoles of resin + 12 nmoles of anti-Id in pH 9.0 buffer

-react for 2 hrs, then quench with 1M tris

-wash with PBS and 1M NaCl

-pack 2 x 50 mm column with 150uL of resin

1111

Two-Dimensional Liquid Chromatography with UV and MS Detection

V:P2

UV

200uL loop

Step 1:• Anti-Id Analysis

and condition RPLC

RPLC

Pump 2

QTOF

Waste

Anti-IdAuto

Pump 1

V:P1

Step 3: •RPLC/MS analysis

Pump 2

QTOF

Pump 1 UV

Step 2: • Collect fraction

33-37 minutes

Pump 2

QTOF

Pump 1 UV

V:P2

V:P1

1212

First Dimension Anti-ID Chromatography with UV Detection

CovX Body Std in mouse serum, UV at 280 nm

Anti-Id column

Protein A column

1313

First Dimension Anti-ID Chromatography with UV Detection

0

100

200

300

400

500

600

700

0.00 10.00 20.00 30.00 40.00 50.00 60.00

Co

nc

(ug/

mL)

Time (hrs)

Mouse PK Curves of Three related CovX Bodies using Anti-ID Column and UV Detection at 280 nm

GLP-1 Exendin-4 Modified Exendin-4

1414

Two-Dimensional Liquid Chromatography with MS Detection

PK at 6 hrs

PK at 1 hr

PK at 5 min

Dosing solution

GLP-1 CovX-body PK samples

1515


PK at 6 hrs

PK at 1 hr

PK at 5 min

Dosing solutionAb+GLP-1

Immunoassay Results

GLP-1 CovX-body PK samplesAb+GLP-1 -210 Da

0.00

50.00

100.00

150.00

200.00

250.00

300.00

350.00

0.00 10.00 20.00 30.00 40.00 50.00 60.00

Co

nc

(u

g/m

L)

Time (hrs)

GLP-1 Total

GLP-1 Active

HAEGTFTSDVSSYLEGQAAK EFIAWLVKGR

1616

Peptide Stability in DPP-4by RPLC/MS and MS/MS

with DPP

mass of 3087.2

(1227-210.5)

without DPP

mass of 3297.7

CVX1227 with DPP enzyme, day 14 7.00000000

m/z1000 1010 1020 1030 1040 1050 1060 1070 1080 1090 1100 1110 1120 1130 1140

%

0

100

%

0

100

14June07a23 257 (5.018) Cm (246:264) TOF MS ES+ 1.16e31030.41

1155

1030.07717

1024.40272

1024.06156

1030.721022

1031.07648

1031.41325

1031.74149 1126.99

108

14June07a16 254 (4.973) Cm (247:261) TOF MS ES+ 9151100.52

915

1100.22563

1100.57891

1101.23639

1101.55324

1105.89144

1106.5599

NSA KT AF D AL RH GE V Q VIKT EY WA LESS GFGNH2

NS KT AF D AL RGE V Q VIKT EY WA LESS GFGNH2

Molecular Weight =3297.70Exact Mass =3294Molecular Formula =C149H226N40O45

Molecular Weight =3089.48Exact Mass =3086Molecular Formula =C140H214N36O43

GLP-1 peptide -208 Da (-HA)

GLP-1 peptide

Incubated with DPP-4 overnight

HAEGTFTSDVSSYLEGQAAK EFIAWLVKGR

1717


Exendin-4 CovX-body PK samples5.00000000CVX1844_2010 300ug/mL water

mass146000 147000 148000 149000 150000 151000 152000 153000 154000 155000 156000 157000

%

0

100

%

0

100

%

0

100

%

0

100

31Aug09a15 1236 (64.205) M1 [Ev-64554,It33] (Gs,2.000,2817:4000,10.00,L33,R33); Cm (1211:1479) TOF MS ES+ 9.07e3156530.00

9070

152740.003236151620.00

2024149540.00

1874

149080.001683146940.00

1591148180.00

1321

147820.001151

150200.001619

150580.001202

156140.003024155180.00

2305153360.00

2144154720.00

1573

156620.002990


22170

156320.0011674

152710.005031

151570.003870

148970.002606

146730.002529

147870.001645

150370.001544

149880.001454

156120.002840154950.00

2568153360.00

1746

154670.001595

156600.005482


22891

156300.009998

152690.005459146720.00

2460

147090.002234

151600.002232

148860.001966

147950.001538

150150.001592

150560.001383

156200.002782155160.00

2012

154840.001698

153580.001691

156590.005353


110126

152700.0020237151590.00

3948

156400.0010477

156590.0031284

PK at 48hrs

PK at 31 hrs

PK at 24 hrs

Dosing solution

1818


Ab+2xpeptides -200 and 400 Da5.00000000CVX1844_2010 300ug/mL water

mass155000 155200 155400 155600 155800 156000 156200 156400 156600 156800 157000 157200 157400 157600 157800

%

0

100

%

0

100

%

0

100

%

0

100


9070156330.00

7414

156140.003024155180.00

2305155390.00

1765

155480.001451

155700.001380

155860.001365

156430.002837

156620.002990

157430.001239

156800.001109

156990.001106

157190.001000

157870.001152

157690.001119


22170

156320.0011674

156120.002840

155050.002378 155580.00

1822155380.00

1526

155670.001700

156030.001696

156410.005840

156600.005482

157650.001805

157430.001640

157160.001243

157860.001591


22891

156300.009998

156200.002782

156100.002405155160.00

2012155850.00

1305

156590.005353 156680.00

2506 157760.001645

157650.001633157410.00

1276156930.00

1236


110126

156400.0010477

156590.0031284

156680.00;12208 157640.005251

PK at 48hrs

PK at 31 hrs

PK at 24 hrs

Dosing solutionAb+2xpeptides

Immunoassay Results

Exendin-4 CovX-body PK samples

0.00

50.00

100.00

150.00

200.00

250.00

300.00

350.00

400.00

450.00

500.00

0.00 10.00 20.00 30.00 40.00 50.00 60.00

Co

nc

(u

g/m

L)

Time (hrs)

Total Active

-HG

HGEGTFTSDLSKQMEEEAVR LFIEWLKNGGPSSGAPPPSG EGTFTSDLSKQMEEEAVRLF IEWLKNGGPSSGAPPPS

1919


5.00000000CVX2025_2010 PK at 6hr

mass146000 147000 148000 149000 150000 151000 152000 153000 154000 155000 156000 157000

%

0

100

%

0

100

%

0

100

%

0

100


53701

155640.0022315

151480.002742

146750.002132

155540.002471

156220.005230

156420.009609


76340

155630.0017204

151480.004738

146740.003454

147110.003002 155530.00;3279

156410.0015540


90083

155630.0018838

151490.004673

146740.003190

155530.00;3120

156410.0018246


222715

156200.0015804

155630.0010935

151480.009802

156410.0038738

PK at 48 hrs

PK at 31 hrs

PK at 24 hrs

PK at 6 hrs

Modified Exendin-4 CovX-body

2020


5.00000000CVX2025_2010 PK at 6hr

mass155000 155200 155400 155600 155800 156000 156200 156400 156600 156800 157000

%

0

100

%

0

100

%

0

100

%

0

100


53701

155640.0022315

155540.002471

156220.005230

155740.005143

155830.003008

156420.009609

156500.005422


76340

155630.0017204155530.00

3279156210.00

6700

155730.004620 155910.00

3929156020.00

2729

156410.0015540

156500.007719


90083

155630.0018838155530.00

3120156210.00

7950

155720.004584

155910.004116

156410.0018246

156490.008143


222715

156200.0015804

155910.0014224

155630.0010935

156000.008251

156410.0038738

156490.0020497

PK at 48 hrs

PK at 31 hrs

PK at 24 hrs

PK at 6 hrs

Immunoassay Results

Modified Exendin-4 CovX-body

Ab+2xpeptides

Ab+2xpeptides -680 Da

0.00

100.00

200.00

300.00

400.00

500.00

600.00

0.00 10.00 20.00 30.00 40.00 50.00 60.00

Co

nc

(u

g/m

L)

Time (hrs)

Total Active

2121

Immunoassay Results using C-terminal Specific Ab

Immunoassay ResultsModified Exendin-4 CovX-body

Using C-Term Specific Ab

0.00

100.00

200.00

300.00

400.00

500.00

600.00

0.00 10.00 20.00 30.00 40.00 50.00 60.00

Co

nc

(u

g/m

L)

Time (hrs)

Total

Active

Exendin C-term

BlockBlock

1) Anti-Id

2) CovX body

SA-HRP

TMBColor

3) Anti-peptide(C-term Specific)

BlockBlock

22

Peptide Optimization Results Immunoassay and 2DLC/MS Comparison

GLP-1 CovX-Body

-Poor PK and half-life

-Degraded quickly

-Immunoassay (IA) active and 1 peptide addition (PA) by MS correlate

Modified Exendin-4 CovX-Body

-Excellent PK and half-life

-Some degradation (not active)

-Immunoassay active and 2 peptide additions by MS correlate

0

50

100

150

200

250

300

0.00

100.00

200.00

300.00

400.00

500.00

600.00

0.00 10.00 20.00 30.00 40.00 50.00 60.00

Co

nc

(u

g/m

L)

Time (hrs)

IA Total

IA Active

MS 2PA-680

MS 2PA

0.00

20.00

40.00

60.00

80.00

100.00

120.00

140.00

0.00

50.00

100.00

150.00

200.00

250.00

300.00

350.00

0.00 10.00 20.00 30.00 40.00 50.00 60.00

Co

nc

(u

g/m

L)

Time (hrs)

IA Total

IA Active

MS 1PA-210

MS 1PA

23

Summary

Anti-Idiotype reagent greatly reduces backgroundinterference in immunoassay and 2DLC/MS and is a universal reagent for CovX-Body capture

2DLC/MS is a powerful technique for the automated analysis of intact proteins from serum

2DLC/MS can be used to confirm immunoassay resultswhen using the same reagents to capture CovX-Bodies

Mass spectrometry of intact proteins is useful for the identification of in-vivo metabolites

Analysis of PK samples for intact biotherapeutics and in-vivo metabolites is a powerful research tool for improving drug development optimization

24

Questions/Discussion

Acknowledgements:

•James Kerr•Gary Ward•Rodney Lappe

CHAPTER 5: INSTRUMENTATION FOR COMPREHENSIVE MULTIDIMENSIONAL LIQUID CHROMATOGRAPHY

CHAPTER 6: METHOD DEVELOPMENT IN COMPREHENSIVE MULTIDIMENSIONAL LIQUID CHROMATOGRAPHY

CHAPTER 18 THE ANALYSIS OF SURFACTANTS BY MULTIDIMENSIONAL LIQUID CHROMATOGRAPHY

new combined use of immunoassay and two-dimensional liquid … · 2018. 4. 2. · combined use of...

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