Immunocapture-LC/MS Detection of Anti-Drug AntibodiesDave Roos, Elsy Philip, Linzhi ChenBoehringer Ingelheim Pharmaceuticals, Ridgefield, CT

CASSS21-Sep-

2017

Outline

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• Introduction

• Immunocapture-LC/MS methodology

• Case studies:

• Detection of pre-existing ADA (PEA) in blank human plasma

• Detection of ADA in support of a cynomolgus monkey toxicity study

• Conclusion

Introduction

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• ADA• Produced by immune systems against drugs• Can lead to immune-mediated adverse reactions• Neutralizing ADA reduce or eliminate drug efficacy• Can interfere with PK assays

• ADA detection• Immunoassay• Drug interference/tolerance can be an issue• Presenting here:

– Complementary methodology for ADA detection– An immunocapture-LC/MS assay to overcome drug interference

Human Immunoglobulins

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Isotype Subclass Description

IgG

IgG1 (65%)IgG2 (25%)IgG3 (5%)IgG4 (5%)

Provides the majority of antibody-based immunity against invading pathogens. The only antibody capable of crossing the placenta to give passive immunity to the fetus.

IgAIgA1 (serum)IgA2 (secretion)

Found mostly in mucosal areas, such as the gut, respiratory tract and urogenital tract, and prevents colonization by pathogens.

IgE - Binds to allergens and triggers histamine release from mast cells and basophils, and is involved in allergy.

IgM -

Expressed on the surface of B cells (monomer) and in a secreted form (pentamer) with very high avidity. Eliminates pathogens in the early stages of B cell-mediated immunity before there is sufficient IgG.

IgD - Functions mainly as an antigen receptor on B cells that have not been exposed to antigens (mostly membrane-bound)

IgG, IgE, IgD(MW 150, 190, 175K)

IgA(MW 390K)

IgM(MW 950K)

Immunocapture-LC/MS ADA Assay Workflow

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Immunocapture 1 Immunocapture 2

Human Universal Peptide for LC/MS Detection

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• Unique for each Ig isotype/subclass

• From constant region

• No allotype variable AAs

• No glycosylation AAs

• High sensitivity with minimal matrix interferences

Universal Peptide List

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Human Ig Unique Peptide MRM PairsMonitoring Peptide

IgG1 GPSVFPLAPSSK 593.83 → 699.40IgG2 GLPAPIEK 412.75 → 654.38IgG3 WYVDGVEVHNAK 708.85 → 698.48IgG4 GLPSSIEK 415.73 → 660.36IgE AEWEQK 395.69 → 590.29IgM GQPLSPEK 428.23 → 670.38

IgA1,2 YLTWASR 448.73 → 620.32Confirmation Peptide

IgG1,3 ALPAPIEK 419.76 → 654.38IgG1,3,4 VVSVLTVLHQDWLNGK 603.34 → 1110.57

IgE LEVTR 309.18 → 375.24IgM VSVFVPPR 450.77 → 615.36

IgA1,2 VAAEDWK 409.71 → 648.30IgA1 TFTC[CAM]TAAYPESK 688.31 → 765.38

LC/MS System

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HPLC

• BEH C18, 1.7 µm, 1 x 50 mm• 60°C• 60 µl/min

MS/MS

• AB Sciex Triple Quad 6500• Scheduled MRM• 7.5 min run

Case Studies Using Immunocapture-LC/MS

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Detection of pre-existing ADA (PEA) in blank human plasma

Using drug as a capture reagent

Detection of monkey ADA in the presence of high-level drug in support of a 13-wk toxicity study

Using mouse mAb as capture reagent to overcome drug interference

Immunocapture-LC/MS Detection of Human PEA

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• Drug: BI proprietary Protein XYZ (contains no human Ig Fc)• 20 blank human plasma lots already assayed by ECL

• 9 PEA- by ECL• 11 PEA+ by ECL

• 9 PEA- human blank plasma lots for cut point determination

• Immunocapture-LC/MS• Drug as immunocapture reagent

Representative Chromatograms for IgG1- Drug as Capture Reagent

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PEA- human Plasma

PEA+ Lot 3

PEA+ Lot 11

Peptide GPSVFPLAPSSK

LC/MS Response of PEA- Human Plasma for Cut-point Determination - Drug as ADA Capture Reagent

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Plasma Lot #

IgG1 IgG2 IgG3 IgG4 IgE IgM IgA1+A2

Peak Area Ratios

1 0.0277 0.0099 - 0.0107 - 0.1130 0.00412 0.0107 0.0008 - 0.0019 - - 0.00593 0.0263 - - 0.0012 0.0150 0.0084 0.00784 0.0093 0.0023 - 0.0053 - 0.0072 0.00575 0.0249 0.0244 0.0210 0.0099 0.0110 0.0474 0.01366 0.0060 0.0044 0.0110 0.0035 - 0.0326 -7 0.0129 - - 0.0037 0.0160 0.0159 0.00628 0.0637 0.0205 0.0570 0.0353 0.0140 0.0066 0.01749 0.0127 0.0008 - 0.0111 - 0.0035 0.0075

mean 0.0216 0.0070 0.0099 0.0092 0.0061 0.0261 0.0086SD 0.0177 0.0093 0.0192 0.0105 0.0074 0.0361 0.0043

Cut point (95%)

0.0507 0.0224 0.0416 0.0265 0.0183 0.0854 0.0160

LC/MS Response of PEA+ Human Blank Plasma- Drug as ADA Capture Reagent

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Lot #IgG1 IgG2 IgG3 IgG4 IgE IgM IgA1+A2

Peak Area Ratios1 0.0874 0.0033 0.0476 0.0096 - 0.0499 0.04412 0.0838 0.0452 0.0762 0.0070 - 0.0421 0.04223 0.4200 0.0061 0.0263 0.0088 - 0.0111 0.08474 0.1970 0.0047 - 0.0020 0.0113 0.0068 0.04975 0.0239 0.0044 0.0119 0.0020 - 0.0007 0.04806 0.0827 0.0141 0.0205 0.0192 0.0054 0.0316 0.07717 0.0188 0.0041 - - - 0.0127 0.02398 0.0150 0.0109 0.0315 0.0017 0.0119 0.0027 0.07129 0.0788 0.0009 - 0.0009 0.0150 0.0099 0.0727

10 0.0641 0.0076 0.0192 - 0.0155 0.0314 0.042411 0.4330 0.0041 0.0303 0.0031 - 0.1010 0.0652

Cut point 0.0507 0.0224 0.0416 0.0265 0.0183 0.0854 0.0160

Summary of LC/MS Analysis of PEA

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• Detected PEA in all of the 11 PEA+ blank human plasma; mostly IgG1, IgA1+A2

• Lots 3 and 11 had the highest PEA levels

Case Studies Using Immunocapture-LC/MS

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Detection of pre-existing ADA (PEA) in blank human plasma

Using drug as a capture reagent

Detection of monkey ADA in the presence of high-level drug in support of a 13-wk toxicity study

Using mouse mAb as capture reagent to overcome drug interference

Immunocapture-LC/MS Detection of Monkey ADA

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• A 13-wk tox study was conducted with BI experimental drug Protein XYZ

• ADA were expected in many of the plasma samples

• High drug levels (up to 1.53 mg/mL) prevented ADA detection by traditional ECL bridging assay

• Immunocapture-LC/MS assay and a new ECL assay with sample pretreatment using protein A were developed in parallel to overcome drug interference issue

Immunocapture-LC/MS Detection of Monkey ADA

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• Spiked extra drug to ensure that all ADA were converted to drug-ADA complex

• A mouse mAb for Protein XYZ as the capture reagent

• Drug interference no longer an issue

• Control group and pre-drug treatment samples used for LC/MS assay cut-point determination

• Peptide VVSVLTVTHQDWLNGK (monkey IgG 1-4) for LC/MS detection

• LC/MS results were compared with those from the new ECL assay

Representative LC/MS Chromatograms

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Blank monkey plasma Neat 100 ng/mL monkey IgG1

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Monkey # 1302526

Week 3Week 1

Monkey # 1302534Week 3Week 1

Sample LC/MS Chromatograms

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Comparison Between Control and Drug Treated Groups

- Control group Drug treated groups Week-3 or later time points Week 1

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Dosemg/kg/

wk

Male FemaleSample LC/MS ECL Sample LC/MS ECL

Subject Week Peak Area Result Result Subject WeekPeak Area Result Result

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13025141 1140 - -

13025131 1530 - -

3 1820 - + 3 2940 - +6 3750 + + 6 2400 - -

13025161 1220 - -

13025151 1400 - -

3 4660 + + 3 3430 + +6 4650 + + 6 5320 + +

13025181 745 - +

13025171 1720 - -

3 6900 + + 3 6580 + -6 11600 + + 6 18800 + +

50

13025201 1020 - -

13025191 1030 - -

3 4860 + + 3 5850 + +6 7020 + + 6 4720 + +

13025221 1540 - -

13025211 945 - -

3 1800 - - 3 1090 - -6 3020 - - 6 1840 - -

13025241 1340 - -

13025231 2440 - +

3 1840 - - 3 4690 + +6 2190 - - 6 5540 + +

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Dosemg/kg/wk

Males FemaleSample LC/MS ECL Sample LC/MS ECL

Subject Week Peak Area Result Result Subject Week Peak Area Result Result

100

13025261 879 - +

13025251 1190 - -

3 10300 + + 3 1990 - -6 23100 + + 6 7670 + +

13025281 565 - -

13025271 1150 - -

3 3980 + - 3 2170 - -6 7210 + + 6 1380 - -

13025301 980 - -

13025291 1510 - -

3 3020 - - 3 3520 + -6 7560 + + 6 3620 + -

1302532

1 2440 - -

1302531

1 1080 - -3 1640 - - 3 1490 - -6 1700 - - 6 1580 - -9 2520 - - 9 1250 - -

12 2020 - - 12 1190 - -15 2900 - - 15 917 - -18 1830 - - 18 1080 - -

1302534

1 1640 - -

1302533

1 1580 - -3 8350 + - 3 20800 + -6 8040 + + 6 24300 + +9 5180 + + 9 8930 + +

12 5460 + + 12 6070 + +15 5090 + + 15 4460 + +18 5490 + + 18 2240 - +

Summary of LC/MS Detection of Monkey ADA

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• LC/MS successfully detected ADA in the presence of very high drug levels in monkey plasma

• LC/MS results were in excellent agreement with those from an ECL immunoassay

• Both assays were used in support of a 13-wk tox study

Conclusion

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• LC/MS assays were developed for analyzing human and cynomolgus monkey ADAs and were applied for detection of PEA in blank human plasma and emerging ADA in monkey plasma

• Using mAb as capture reagent eliminated drug interference issue in the presence of very high drug levels in the monkey study samples

• LC/MS assay was in excellent agreement with ECL drug bridging assay

• LC/MS represents a complementary assay platform for ADA analysis and isotyping

Acknowledgements

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Jeff DugganSteve NorrisChristine GrimaldiCheikh KaneRajeev VesapoguShirin PagelsTom ChanEd GumpVictor ChenCaitlin QuatranoHongbin Yu

Immunocapture-LC/MS Detection of Anti-Drug AntibodiesOutlineIntroductionHuman ImmunoglobulinsImmunocapture-LC/MS ADA Assay Workflow�Human Universal Peptide for LC/MS Detection Universal Peptide ListLC/MS SystemCase Studies Using Immunocapture-LC/MSImmunocapture-LC/MS Detection of Human PEARepresentative Chromatograms for IgG1�- Drug as Capture ReagentLC/MS Response of PEA- Human Plasma for Cut-point Determination - Drug as ADA Capture ReagentLC/MS Response of PEA+ Human Blank Plasma�- Drug as ADA Capture ReagentSummary of LC/MS Analysis of PEACase Studies Using Immunocapture-LC/MSImmunocapture-LC/MS Detection of Monkey ADAImmunocapture-LC/MS Detection of Monkey ADARepresentative LC/MS ChromatogramsSample LC/MS ChromatogramsSlide Number 20Slide Number 21Slide Number 22Summary of LC/MS Detection of Monkey ADAConclusionAcknowledgements