Direct Bioanalysis of ADCs using Affinity Capture-LC-HRAMS Affinity Capture-LC-HRAMS Techniques for Characterization and Quantification—a Progress UpdateUpdate

Rand Jenkins et al

EBF 8th Open SymposiumBa celona Spain 18 No 2015Barcelona Spain, 18 Nov 2015

Overview

1

2

Biotherapeutics diversity

h h d d2

3

Why new BA approaches are needed

LC MS protein bioanalysis workflows3

4

LC-MS protein bioanalysis workflows

ADC characterization4

5

ADC characterization

Intact ADC quantification5

6

Intact ADC quantification

Open questions & final thoughtsp q g

2

Biotherapeutics diversityp y

peptidespeptidesmAbs

bispecifics (bsAbs, DART®s, BiTE®s)ADCsADCs

prodrug/carrier conjugates (albumin, PEG)f i t ifusion proteins

messenger RNA therapeutics™oligonucleotides (ssDNA, siRNA, mRNA)

CARsCARs

3

Alternative formats for bispecifics

Ch i t h S i Qi ti Zh i P l J C t Alt ti l l f t d th ti

4

Christoph Spiess, Qianting Zhai, Paul J. Carter, Alternative molecular formats and therapeutic applications for bispecific antibodies, Molecular Immunology 67 (2015) 95–106

Antibody-Drug Conjugatesy g j g

K S l Bi l i (2013) 5(2) 201 226

5

Kaur S et al. Bioanalysis, (2013) 5(2), 201–226

GlycoConnect ADC: a new glycan conjugation ideay g y j g

Remon van Geel et al, Chemoenzymatic Conjugation of Toxic Payloads to the Globally Conserved N-Glycan of Native

6

y j g y y ymAbs Provides Homogeneous and Highly Efficacious Antibody−Drug Conjugates, Bioconjugate Chem. 2015, in press

Why new BA approaches are needed

+ Biotherapeutics innovation is producing increasingly complex

y pp

+ Biotherapeutics innovation is producing increasingly complex molecular constructs

+ Their inherent heterogeneity and potential to undergo stability- and/or catabolism-related changes are driving the need for greater characterization

+ Conventional Ligand Binding Assays (LBA) are limited in their + Conventional Ligand Binding Assays (LBA) are limited in their ability to provide data reflecting molecular structure (e.g. Drug Antibody Ratio (DAR) distribution for ADCs)g y ( ) )

+ MS-based methods, including affinity capture-liquid h t h hi h l ti t t t chromatography-high resolution accurate mass spectrometry

(AC-LC-HRAMS), are emerging as versatile tools

7

LC MS protein bioanalysis workflowsLC-MS protein bioanalysis workflows

8

Protein LC-MS/MS Bioanalysis General Strategy/ y gy

Protein/Peptide in Biomatrix Sample

MW < ~10 kDa MW > ~10 kDa

Indirect Measurement(Proteolytic Peptides)

Direct Measurement (Intact Analyte) (Proteolytic Peptides)(Intact Analyte)

Extraction/Enrichment Extraction/Enrichment/(Chemical (e.g. PPT, SPE) 

or Affinity Capture) 

/(Chemical (e.g. PPT)or Affinity Capture)

ProteolyticDigestion

LC‐MS/MSClean up (SPE or 

LC‐MS/MS

DigestionPeptide AC)

9

Protein LC-HRAMS Bioanalysis Alternative Strategyy gy

Protein/Peptide in Biomatrix Sample

MW < ??? kDa MW > ~10 kDa

Indirect Measurement(Proteolytic Peptides)

Direct Measurement (Intact Analyte) (Proteolytic Peptides)(Intact Analyte)

Enrichment Extraction/EnrichmentAffinity Capture

/(Chemical (e.g. PPT)or Affinity Capture)

ProteolyticDigestion

LC‐HRAMS Clean up (SPE or 

LC‐HRAMS

DigestionPeptide AC)

10

ADC characterizationADC characterization

11

ADC in vivo changesg

K S l Bi l i (2013) 5(2) 201 226

12

Kaur S et al. Bioanalysis, (2013) 5(2), 201–226

LC-MS ADC characterization and beyondy

Assay Technology

Drug or drug-linker metabolitesWhich species exist and are important?

LC-MS/MS

Catabolites (ADC or mAb) Affinity capture LC HRAMSCatabolites (ADC or mAb)Which species exist and are important?

Affinity capture LC-HRAMS

DAR Characterization Affinity capture LC-HRAMSDAR CharacterizationAssess ADC stability (in vitro / in vivo)

Affinity capture LC HRAMSor HIC

Critical reagent DAR specificity Affinity capture LC-HRAMSHICAssess LBA reagents or HIC

13

LC-MS ADC characterization and beyondy

Assay Technology

Drug or drug-linker metabolitesWhich species exist and are important?

LC-MS/MS

Catabolites (ADC or mAb) Affinity capture LC HRAMSCatabolites (ADC or mAb)Which species exist and are important?

Affinity capture LC-HRAMS

DAR Characterization Affinity capture LC-HRAMSDAR CharacterizationAssess ADC stability (in vitro / in vivo)

Affinity capture LC HRAMSor HIC

Critical reagent DAR specificity Affinity capture LC-HRAMSHICAssess LBA reagents or HIC

Intact ADC quantificationUseful to measure individual DARs?

Affinity capture LC-HRAMSUseful to measure individual DARs?

14

AC-LC-HRAMS to assess intact ADC changesg

X K Li L Saad O et al Anal Biochem 412 56 66 (2011)

15

Xu K, Liu L, Saad O, et al, Anal Biochem, 412, 56–66 (2011).

in vivo ADC characterization1 day post-dose

Fig.6. Drug release observed in a multiple-dose toxicokinetic study of anti-MUC16 TDC in vivo. Anti-MUC16 TDC (DAR 2) was administered to cynomolgusmonkeys intravenously once every 3 weeks for a total of four doses. Three dose groups at 6, 10, and 20 mg/kg were evaluated. Deconvoluted mass spectra of the TDC species of a representative

7 days post-dose

animal receiving the first dose of 6 mg/kg anti-MUC16 TDC show the drug release from DAR 2 to form DAR 1 and DAR 0 with time: (A) 1 day postdose; (B) 7 days postdose; (C) 21 days postdose.

21 days post-dose21 days post-dose

Xu K et al, Anal. Biochem, 412, 56–66 (2011)

16

Engineered ADC (DAR4) in vitro plasma stabilityg ( ) p y

Controlt=0 hr

DAR 4t 0 hr

37° C DAR 337 Ct=72 hr

DAR 3

37° Ct=7d

DAR 2

17

Non-cleavable ADC with site-specific catabolismp

Magdalena Dorywalska et al, Site-Dependent Degradation of a Non-Cleavable Auristatin-Based Linker-Payload in Rodent Plasma and Its Effect on ADC Efficacy PLOS ONE | DOI:10 1371/journal pone 0132282 July 10 2015

18

Rodent Plasma and Its Effect on ADC Efficacy, PLOS ONE | DOI:10.1371/journal.pone.0132282 July 10, 2015

Non-cleavable ADC with site-specific catabolismp

Magdalena Dorywalska et al, Site-Dependent Degradation of a Non-Cleavable Auristatin-Based Linker-Payload in Rodent Plasma and Its Effect on ADC Efficacy PLOS ONE | DOI:10 1371/journal pone 0132282 July 10 2015

Fig 2. Mass spectrometric analysis of non-cleavable conjugates

19

Rodent Plasma and Its Effect on ADC Efficacy, PLOS ONE | DOI:10.1371/journal.pone.0132282 July 10, 2015

Intact ADC quantificationIntact ADC quantification

20

LC-HRAMS quantitation data processing

Potential options using full spectrum (FS) data acquisition

q p g

Potential options using full spectrum (FS) data acquisition

+ XICs and traditional peak integration+ XICs and traditional peak integration- select m/z charge state(s), individual or summed- use narrow data extraction window (e.g. 5 mDa)- integrate XIC peak area

+ Deconvoluted composite spectrum+ Deconvoluted composite spectrum- average/sum FS across entire chromatographic peak- peak area = “zero-charge” state responsep g p

+ Deconvoluted individual FSt “ h ” t t XIC- generate “zero-charge” state XIC

- integrate XIC peak area

21

Raw HR mass spectrump

3066 42

90

1003066.42

2948.52Engineered DAR4 ADC

(mcMMAF conjugate)

70

80

nce 3194.13

2839.33

50

60

Abu

ndan

2737.99

30

40

Rel

ativ

e A

3332.962643.61

2555 51

10

20

30R 2555.51

2500 2600 2700 2800 2900 3000 3100 3200 3300 3400 3500 3600m/z

0

10

22

m/z

Intact analysis 1003066.42

ADC80

100an

ce10.53

80

ance 2839.33

TIC

40

60

ve A

bund

40

60

ve A

bund

a

3332.962643.61

6 8 10 12 140

20

Rel

ati

9.92

2500 2800 3000 3200 3400 36000

20

Rel

ativ

1002937.40

6 8 10 12 14Time (min)

2500 2800 3000 3200 3400 3600m/z

SILu™Lite (IS)60

80

bund

ance

2719.89

3192.73 Sample: 50 µg/mL ADC standard

20

40

Rel

ativ

e A

b

2532.25

p µgMethod:

• 30 µL mouse EDTA plasma• Add 2 µg/mL mAb IS

2500 2700 2900 3100 3300 3500m/z

0

R µg• AC with anti-hFc agarose beads• No deglycosylation• Elute and analyzey

Summed XICs approach (with mAb IS)pp ( )

Calibration Curve (XICs: m/z 2839, 2892, 2948, 3006, 3066)

120

y = 1.1969x - 0.805r=0.997

80

100

a R

atio

60

te/IS

Are

a

20

40

Ana

lyt

00 20 40 60 80 100

ADC Conc. (ug/mL)

24

A&P - Summed XICs approach (with mAb IS)pp ( )

Cal Conc Nominal ConcQCs Analyte/IS

Cal. Conc. (ug/mL)

Nominal Conc. (ug/mL) Accuracy CV%

QC 1-1 3.00 1.83 1.6 115QC 1 2 2 79 1 66 1 6 104QC 1-2 2.79 1.66 1.6 104QC 1-3 2.49 1.41 1.6 88.1QC 1-4 2.51 1.42 1.6 89.0QC 1 5 2 56 1 47 1 6 91 9

11.7

QC 1-5 2.56 1.47 1.6 91.9QC 2-1 17.3 13.8 12.5 110QC 2-2 15.2 12.0 12.5 96.3QC 2 3 14 9 11 8 12 5 94 1 8 35QC 2-3 14.9 11.8 12.5 94.1QC 2-4 17.8 14.2 12.5 114QC 2-5 16.0 12.7 12.5 101QC 3 1

8.35

QC 3-1 108 89.3 100 89.3QC 3-2 106 88.2 100 88.2QC 3-3 108 89.9 100 89.9QC 3 4

3.01QC 3-4 102 84.3 100 84.3QC 3-5 102 84.6 100 84.6

25

Deconvoluted mass spectrump

Engineered DAR4 ADC(mcMMAF conjugate)

glycoforms

26

Deconvoluted MS approach (no IS)pp ( )

Calibration curve of DAR 4 mass 153430, 2nd glycoform

2.50E+08

y = 2197502x - 123876r=0.9972.00E+08

gnal

1.00E+08

1.50E+08

volv

edSi

g

5.00E+07Dec

onv

0.00E+000 20 40 60 80 100

ADC Conc. (μg/mL)(μg )

27

A&P - Deconvoluted MS approach (no IS)pp ( )

Cal Conc Nominal ConcQCs Intensity

Cal. Conc. (ug/mL)

Nominal Conc. (ug/mL) Accuracy CV%

QC 1-1 3040465 1.44 1.60 90.0QC 1 2 3171365 1 50 1 60 93 7QC 1-2 3171365 1.50 1.60 93.7QC 1-3 3384557 1.60 1.60 99.8QC 1-4 2532637 1.21 1.60 75.6QC 1 5 3566412 1 68 1 60 105 0

12.1

QC 1-5 3566412 1.68 1.60 105.0QC 2-1 28494807 13.0 12.5 104.2QC 2-2 26480701 12.1 12.5 96.9QC 2 3 25990923 11 9 12 5 95 1 3 99QC 2-3 25990923 11.9 12.5 95.1QC 2-4 28256266 12.9 12.5 103.3QC 2-5 27660784 12.6 12.5 101.1QC 3 1 201954923 92 0 100 92 0

3.99

QC 3-1 201954923 92.0 100 92.0QC 3-2 202279068 92.1 100 92.1QC 3-3 200604530 91.3 100 91.3QC 3 4 210591547 95 9 100 95 9

2.03QC 3-4 210591547 95.9 100 95.9QC 3-5 206835150 94.2 100 94.2

28

MSIA D.A.R.T.’S – Efficient Analyte Capture

MSIA D.A.R.T.’S: Proprietary Affinity TechnologyDescribed is an affinity micro-column comprising a high surface area material,

hi h h hi h fl ti d l d d l t i d ithiwhich has high flow properties and a low dead volume, contained within a housing and having affinity reagents bound to the surface of the high surface area material that are either activated or activatable. The affinity reagents bound to the surface of the affinity micro-column further comprise affinity receptors for the integration into high throughput analysis of biomolecules.

MSIA D.A.R.T.’SStreptavidin (96 units)Streptavidin (96 units)

• Improved assaying performance• Decreased matrix carryover or loss• Decreased non-specific binding• Flexibility in assay sample volume

29

y y p

Flexible Solution: Top-Down LB-MSIA (Intact)Evolution of the Ligand Binding Assay

DataDataStartStart InjectInjectStartStart

Evolution of the Ligand Binding AssayCaptureCapture

80

100148080.48

lativ

e In

tens

ity

40

60

80

148244 48

Rel

0

20

148244.48

148202.77 148407.38147859.85 148537.11

147600 148000 148400 148800

Therapeutic mAb Capture

Eluted analyteIntact Detection

Intact mAb data content

HRAM MS detection

Streptavidin –CaptureSelect

Mass

Pre-Analytical Analytical - Detection

Hybrid MS Workflow Solution

30

ADC Bioanalysis

ADC masses Utilized for XIC PlotsIntact DetectionADC masses Utilized for XIC Plots

9

100 2740.542807.27 2860.19

2759 22+53

+53+54+55

+55+54

+54 DAR0

80859095 2759.22 2843.912775.16 2898.56

2876.492791.232707.03 2823.272771.902724.79 2743.32 2827.582722 44

+53

+54 +53+54

+55

+55

+55 +55+54 DAR1

DAR260657075

danc

e

2827.582722.442881.962691.60

2865.352787.592709.17 2904.572811.282676.16 2839 25 2892 782848 76

+53+53 +53+54

+55DAR2

DAR3

40455055

Rel

ativ

e Ab

und 2839.25 2892.782848.762794.94

2693.442737.87

2803.38

2727 77

2830.76

2814 41

DAR4

20253035 2727.77

2778.162762.202746.212712.08

2814.41

2679.13

DAR5

DAR6

2680 2700 2720 2740 2760 2780 2800 2820 2840 2860 2880 290005

1015

DAR6

31

2680 2700 2720 2740 2760 2780 2800 2820 2840 2860 2880 2900m/z

ADC Standard Curve in Rat Plasma

25

S)

Affinity capture: anti-hFc – MSIA D.A.R.T.’S™

y = 0.0635x - 0.0201R² = 0 9978

20

as (A

DC

/I

R² = 0.9978

15

peak

are

a

10

Rat

io o

f

5 Concentration range: 2.5-320 µg/mL

00 50 100 150 200 250 300 350

Concentration of ADC (µg/mL)Concentration of ADC (µg/mL)

Curve: XIC-based Quantification32

ADC Bioanalysis

Deconvoluted SpectrumIntact DetectionDeconvoluted Spectrum

DAR3~19%

~17%

DAR2

DAR3DAR4 DAR5

60

150671.68151536.65 152399.82

149808.02~10%

~14%17%

~16%

~10%DAR1 DAR6

DAR040 153263.71148943.91148080.48un

danc

e ~8%

~4%

20 154126.74154992.64

DAR7DAR8

Rel

ativ

e ab

u

~3%

0

148000 149000 150000 151000 152000 153000 154000 155000

Mass

33

ADC Standard Curve in Rat Plasma

Affinity capture: anti-hFc – MSIA D.A.R.T.’S™ 16

y = 0.0453x - 0.1606R² = 0.9997

12

14

(ADC/IS)

R 0.9997

8

10

peak areas 

6

Ratio

 of p

2

4

Concentration range: 2.5-320 µg/mL

00 50 100 150 200 250 300 350

Concentration of ADC (µg/mL)Concentration of ADC (µg/mL)

Curve: Deconvoluted-based Quantification34

Assay Characteristics

S l A

Deconvoluted-based QuantificationSample 

Concentration (µg/mL)

Average concentration (µg/mL) (n=5)

% CV (n=5)

% Accuracy (n=5)

5.0 4.5 7.2 ‐10.713.5 14.2 5.9 5.2

Day 1

53.5 53.4 5.0 ‐0.1213.5 225.6 4.9 5.4

Day 25.0 5.3 23.2 5.613.5 13.2 15.3 ‐2.653.5 52.4 8.3 ‐2

y

53.5 52.4 8.3 2213.5 237.6 3.8 11.3

5 0 5 4 9 4 8 5Day 3

5.0 5.4 9.4 8.513.5 12.5 13.1 ‐7.553.5 59.5 13.7 11.2213 5 238 8 0 11 5

35

213.5 238 8.0 11.5

Individual ADC DAR Species Quantification (DAR3)

y = 0.0143x - 0.0009R² = 0.9921

0.40

0.50

ak a

reas

/ I

S) Experimental Measurement

0.10

0.20

0.30

Rat

io o

f pea

(DA

R3

/ pQCs: 4 levels (n=5 replicates)Deconvolution approach

0.000 5 10 15 20 25 30 35

Concentration of DAR3 ADC (µg/mL)

QC % CV % Accuracy(µg/mL) % CV % Accuracy

0.43 15.5 91.7

1.02 13.5 110

6.45 3.35 86.8

27.8 4.80 93.4

Note: DAR3 theoretical concentrations basedon 19% of total ADC concentration

36

Open questionsOpen questions

37

AC-LC-HRAMS intact analysis open questions

+ Technical:

y p q

+ Technical:

• Sensitivity good enough?

• Mass resolution required to ensure specificity?

• How to best process raw FS data?p

• Reference standards heterogeneity/species purity?

What about internal standards?• What about internal standards?

• How critical is affinity capture specificity/efficiency?

• How important is chromatography?

• What to do with so much data?What to do with so much data?

38

AC-LC-HRAMS intact analysis open questions

+ Biological:

y p q

+ Biological:• Multiple detected species, which are important to measure?

Pharmacologically active (total)?

Potentially toxic or off-target effects?y g

Potentially immunogenic?

PTMs or glycosylation changes possible are they relevant?• PTMs or glycosylation changes possible, are they relevant?

• Peptide modifications (e.g. deamidation, oxidation)?

• Structural liabilities (e.g. terminal clipping, labile linkers)?

39

Final thoughts

+ Keeping up with biotherapeutics innovation is a challenge for

g

+ Keeping up with biotherapeutics innovation is a challenge for both analytics (CMC) and bioanalysis

+ Effective discovery and development of complex biologics requires both LBA and LC-MS assay technologies

+ Inherent heterogeneity of biologics and potential dynamic changes in vivo require in-depth characterizationchanges in vivo require in depth characterization

+ Regulators are aware and likely to increase their expectations

+ AC-LC-HRAMS techniques have significantly advanced, providing both characterization and sensitive intactproviding both characterization and sensitive intactquantification to aid biotherapeutics development

40

It is difficult to say whatis impossible, for theIt is difficult to say whatis impossible, for the““ p ,dream of yesterday is thehope of today and the

p ,dream of yesterday is thehope of today and thep yreality of tomorrow.

—Dr. Robert H. Goddard

p yreality of tomorrow.

—Dr. Robert H. Goddard””(1882‐1945)(1882‐1945)

16 March 1926Auburn Massachusetts

16 March 1926Auburn MassachusettsAuburn, Massachusetts

USAAuburn, Massachusetts

USA

16 March 1926Auburn, Massachusetts

USA

NASA New Horizons19 January 2006

USA

43

Pluto from 280,000 miles

13 J l 2015

44

13 July 2015

midnight EDT on 15 July 2015(3.6 billion miles away)

Farewell to Pluto from New Horizonslooking back from 1.25 million miles

Replica of J.J. Thomson'st t (1912)mass spectrometer (1912)

ThermoFisher ScientificOrbitrap analyzers (2012)

20Ne, 22Ne

Acknowledgementsg

GenentechPPD® Laboratories Genentech• Keyang Xu• Surinder Kaur

PPD Laboratories • Dongliang Zhan• Diego Cortes

Willi M l ttSeattle Genetics

• Shawna Hengel

• William Mylott• Pat Bennett

• Steve Alley• Kwasi Antwi• Jonathan Josephs Jonathan Josephs • Urban A. Kiernan• Keeley Murphy

Eric E Niederkofler• Eric E. Niederkofler• Jessica Wang

…and many others

48

Thank You

49

midnight EDT on 15 July 2015

Looking back…