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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…