Novartis Presentation AWS
Novartis Institutes for BioMedical Research / PK Sciences / Safety/ADME Bioanalysis
Comparison of LC-MS and LBA workflows and the impact of ADA on PK drug quantification Presenter: Jim Glick, Ph.D. Contributors: Adam Bentley, Yilin Feng, Gisela Peraus, Jie Zhang and Jimmy Flarakos November 12, 2017
Session description and objectives
• This presentation will provide an overview of different aspects of the LC-MS workflow applied to the analysis of protein therapeutics and their impact on drug quantification
• Review the different workflows applied to protein therapeutics
• Discuss issues related to the bioanalytical strategy for the application of LC-MS for protein therapeutics
• Cover real-world examples of LC-MS applied to protein therapeutics
AAPS Workshop on LBA and LC-MS - 11/2017 2
Public
Biography and contact information •
24 years of analytical chemistry experience with the last 17 years in mass spectrometry
•
Former Director of Barnett Institute's Core Mass Spectrometry facility at Northeastern University
•
4 years at Novartis as a Sr. Investigator/Lab Head supporting preclinical and clinical small and large molecule programs
•
Contact me at [email protected]
AAPS Workshop on LBA and LC-MS - 11/2017 3
Public
Comparison of LC-MS workflows Pellet Digestion LC-MS/MS workflow
AAPS Workshop on LBA and LC-MS - 11/2017 4
Public
Immunoaffinity LC-MS/MS workflow
LC-MS workflow options • Pellet Digestion – solvent protein precipitation followed by trypsin digestion – Generic method – LC-MS/MS quantitation with a Fc region peptide (pre-clinical studies)
– Specific method – LC-MS/MS quantitation with a CDR region peptide (clinical studies)
• Immunocapture – capture and wash followed by on-bead digestion – Generic method – Capture reagent targeted to Fc region – mouse/goat/rabbit-anti-human-IgG Fc (preclinical studies)
– Specific method – Capture regent targeted to CDR region – anti-idiotypic (clinical studies)
• Solid phase extraction (SPE) clean-up may be added after each method to reduce matrix effects or improve sensitivity AAPS Workshop on LBA and LC-MS - 11/2017 5
Public
Typical LBA workflows Homogeneous bridging ELISA TMB substrate
MSD (ECL)
AAPS Workshop on LBA and LC-MS - 11/2017 6
Public
Colored product
Gyros
Bioanalytical strategy considerations • What phase of development is the drug candidate? – Discovery vs. pre-clinical vs. clinical – – – – – –
Fit-for-purpose Throughput Sensitivity Validation Program timelines Cost
• What are you trying to measure? – – – – –
Combination studies (2 proteins vs. protein and small molecule) Generic methods vs. specific (Fc peptide vs. CDR) Capture scheme: drug vs. target capture Species matrix impact (rodent vs. cyno vs. human) Free vs. total drug
• What are you going to do with the data? – – – –
Candidate selection PK/PD assessment Toxicology Dose selection
AAPS Workshop on LBA and LC-MS - 11/2017 7
Public
What is being measured by LC-MS/MS?
• LC-MS/MS typically believed it to be a total assay method – Immunoaffinity LC-MS/MS can be tailored to be either a free or total method depending on capture reagent
• Pellet digestion must be a total drug method BUT… Could it be measuring free drug...
AAPS Workshop on LBA and LC-MS - 11/2017 8
Public
Case study 1 – NOV001, IgG mAb Comparison of LC-MS and LBA methods Pellet digestion
Immunoaffinity LC-MS/MS
Gyros
AAPS Workshop on LBA and LC-MS - 11/2017 9
Public
Case study 1 – Concentration-Time profiles 2 doses (first dose shown), 3 animals/group A
B
C
AAPS Workshop on LBA and LC-MS - 11/2017 10
Public
Case study 1 – Animal 1001 Comparisons of different methods
AAPS Workshop on LBA and LC-MS - 11/2017 11
Public
Case study 1 - Cross Correlation Plots Regression calculated across the entire range
R2 = 0.9935
R2 = 0.9976
R2 = 0.9891
AAPS Workshop on LBA and LC-MS - 11/2017 12
Public
Case study 2 – NOV002, IgG mAb
Influence of ADA on the Concentration-Time profile Immunoaffinity LC-MS/MS
AAPS Workshop on LBA and LC-MS - 11/2017 13
Public
Case study 2 – NOV002, IgG mAb
Concentration-Time profile changes due to ADA C o n c e n tra tio n o f N O V 0 0 2 ( g /m L )
10000
1000
100 D o s e 1 ( m g /k g / w e e k ) D o s e 2 ( m g /k g / w e e k )
10
D o s e 3 ( m g /k g / w e e k ) 1
0 .1 0
6
12
18 T im e (d a y s )
AAPS Workshop on LBA and LC-MS - 11/2017 14
Public
24
30
36
Case study 2 – NVS002, IgG mAb
Detection of ADA in Dose 2 group animal 1003 10000
4
C o n c e n tr a tio n o f N O V 0 0 2 ( g / m L )
1000
3
100
2
10
1
1
0 0
6
12
18 T im e (d a y s )
AAPS Workshop on LBA and LC-MS - 11/2017 15
Public
24
30
36
A n ti-N O V 0 0 2 a s s a y s ig n a l (O .D .)
D o s e 2 , A n im a l 1 0 0 3
Case study 3 – NVS002, IgG mAb
Influence of ADA on the Concentration-Time profile Pellet digestion
AAPS Workshop on LBA and LC-MS - 11/2017 16
Public
Case study 3 – NOV002, IgG mAb
Concentration-Time profile changes due to ADA
C o n c e n tra tio n o f N O V 0 0 2 ( g /m L )
10000
1000 D o s e 1 ( m g /k g /w e e k ) D o s e 2 ( m g /k g /w e e k ) 100
10
1 1
8
43
45
T im e (d a y s )
AAPS Workshop on LBA and LC-MS - 11/2017 17
Public
92
99
Case study 3 – NVS002, IgG mAb
Influence of ADA on the Concentration-Time profile 4
1000
3
100
2 D o s e 2 , A n im a l 2 0 0 2 D o s e 2 , A n im a l 2 0 0 3
10
1
1
0 1
8
43
45
T im e (d a y s p o s t-d o s e )
AAPS Workshop on LBA and LC-MS - 11/2017 18
Public
92
99
A n ti-N O V 0 0 2 a s s a y s ig n a l (O .D .)
C o n c e n tr a tio n o f N O V 0 0 2 ( g / m L )
10000
Case study 4 – NVS003, Fab construct Influence of dilution on the C-T profile of a biomarker
Immunoaffinity LC-MS/MS
• Method was setup as a multiplex assay (3 analytes) • Sample pre-dilution was required
NOV010 + BM1
NOV010-BM1 + BM1 + NOV003
NVS010-NVS003 + NOV003 + NOV010 + NOV10-BM1 + BM1
AAPS Workshop on LBA and LC-MS - 11/2017 19
Public
Case study 4 – NOV003, Fab construct C o n c e n tr a tio n o f B io m a r k e r 1 (n g /m L )
Influence of dilution on the C-T profile of a biomarker •
3 .0
2 .5
1.6x
1.3x
2 .0
Biomarker levels after treatment were above basal level
Nominal • Sample predilution resulted basal in complex level dissociation
1 .5
1 .0
•
0 .5
Over-estimation of biomarker concentration
0 .0 0
4
8
12
16
20
24
28
32
36
T im e ( h o u r s )
NVS010-NVS003 + NOV003 + NOV010 + NOV10-BM1 + BM1 20
Public
AAPS Workshop on LBA and LC-MS - 11/2017
Case study 5 – NOV004, IgG mAb
Influence of soluble receptor and sample treatment on quantitation using pellet digestion LC-MS workflow
P e a k a re a ra tio o f N O V 0 0 4
0 .1 5
N o a c id d is s o c ia tio n
0 .1 0
c v
c v
c v
c v
c v
c v
0 .0 5
0 .0 0 0
5
10
20
40
80
C o n c e n t r a t i o n o f s o l u b l e r e c e p t o r ( g /m L )
AAPS Workshop on LBA and LC-MS - 11/2017 21
Public
• NOV004 (10 µM) incubated with increasing concentration of soluble receptor
A c id d is s o c ia tio n p r e - tr e a tm e n t
• Pellet digestion LCMS workflow used • Acid dissociation increases recovery of NVS004
Case study 5 – NOV004, IgG mAb
Influence of protein precipitation solvent on complex dissociation using pellet digestion LC-MS workflow
AAPS Workshop on LBA and LC-MS - 11/2017 22
Public
Conclusions • LC-MS methods represent a complementary technique to LBA for large molecule quantitation
– Flexible options for quantitation, analyte and sensitivity are available – Sample handling needs to be considered in complex samples – Immunoaffinity LC-MS workflows need to be carefully evaluated
• LC-MS can measure free and total drug – – – –
Depends on the analyte of interest (soluble or receptor bound) Depends on the method selected (PD or IA) Depends on the reagents used (solvents and capture) Depends on the binding affinity for the target
• LC-MS and LBA have been shown to provide similar concentration results
– Discrepancies exist between the methods (both positive and negative bias of LC-MS results vs. LBA have been reported) – The intended use of the data can help drive the BA strategy AAPS Workshop on LBA and LC-MS - 11/2017 23
Public
Acknowledgements Safety/ADME colleagues Adam Bentley, Ann Draghi, Yilin Feng, Yunlin Fu, Xinliu Gao, Sagar Kawle, Shaoyong Li, Wenkui Li, Hui Lin, Irina Vinar, Bin Yang, Wei Zhou, Jie Zhang and Jimmy Flarakos
BxBA colleagues Gisela Peraus, Julie De Gagne, Carsten Krantz and Sebastian Spindeldreher
Preclinical Safety colleagues Serge Cote, Peter Hoffmann, Alexander Koch, Brain Stoll, and Nancy Yao
AAPS Workshop on LBA and LC-MS - 11/2017 24
Public
Questions
AAPS Workshop on LBA and LC-MS - 11/2017 25
Public
Comparison of LC-MS and LBA workflows and the impact of ADA on PK drug quantification Presenter: Jim Glick, Ph.D. Contributors: Adam Bentley, Yilin Feng, Gisela Peraus, Jie Zhang and Jimmy Flarakos November 12, 2017
Session description and objectives
• This presentation will provide an overview of different aspects of the LC-MS workflow applied to the analysis of protein therapeutics and their impact on drug quantification
• Review the different workflows applied to protein therapeutics
• Discuss issues related to the bioanalytical strategy for the application of LC-MS for protein therapeutics
• Cover real-world examples of LC-MS applied to protein therapeutics
AAPS Workshop on LBA and LC-MS - 11/2017 2
Public
Biography and contact information •
24 years of analytical chemistry experience with the last 17 years in mass spectrometry
•
Former Director of Barnett Institute's Core Mass Spectrometry facility at Northeastern University
•
4 years at Novartis as a Sr. Investigator/Lab Head supporting preclinical and clinical small and large molecule programs
•
Contact me at [email protected]
AAPS Workshop on LBA and LC-MS - 11/2017 3
Public
Comparison of LC-MS workflows Pellet Digestion LC-MS/MS workflow
AAPS Workshop on LBA and LC-MS - 11/2017 4
Public
Immunoaffinity LC-MS/MS workflow
LC-MS workflow options • Pellet Digestion – solvent protein precipitation followed by trypsin digestion – Generic method – LC-MS/MS quantitation with a Fc region peptide (pre-clinical studies)
– Specific method – LC-MS/MS quantitation with a CDR region peptide (clinical studies)
• Immunocapture – capture and wash followed by on-bead digestion – Generic method – Capture reagent targeted to Fc region – mouse/goat/rabbit-anti-human-IgG Fc (preclinical studies)
– Specific method – Capture regent targeted to CDR region – anti-idiotypic (clinical studies)
• Solid phase extraction (SPE) clean-up may be added after each method to reduce matrix effects or improve sensitivity AAPS Workshop on LBA and LC-MS - 11/2017 5
Public
Typical LBA workflows Homogeneous bridging ELISA TMB substrate
MSD (ECL)
AAPS Workshop on LBA and LC-MS - 11/2017 6
Public
Colored product
Gyros
Bioanalytical strategy considerations • What phase of development is the drug candidate? – Discovery vs. pre-clinical vs. clinical – – – – – –
Fit-for-purpose Throughput Sensitivity Validation Program timelines Cost
• What are you trying to measure? – – – – –
Combination studies (2 proteins vs. protein and small molecule) Generic methods vs. specific (Fc peptide vs. CDR) Capture scheme: drug vs. target capture Species matrix impact (rodent vs. cyno vs. human) Free vs. total drug
• What are you going to do with the data? – – – –
Candidate selection PK/PD assessment Toxicology Dose selection
AAPS Workshop on LBA and LC-MS - 11/2017 7
Public
What is being measured by LC-MS/MS?
• LC-MS/MS typically believed it to be a total assay method – Immunoaffinity LC-MS/MS can be tailored to be either a free or total method depending on capture reagent
• Pellet digestion must be a total drug method BUT… Could it be measuring free drug...
AAPS Workshop on LBA and LC-MS - 11/2017 8
Public
Case study 1 – NOV001, IgG mAb Comparison of LC-MS and LBA methods Pellet digestion
Immunoaffinity LC-MS/MS
Gyros
AAPS Workshop on LBA and LC-MS - 11/2017 9
Public
Case study 1 – Concentration-Time profiles 2 doses (first dose shown), 3 animals/group A
B
C
AAPS Workshop on LBA and LC-MS - 11/2017 10
Public
Case study 1 – Animal 1001 Comparisons of different methods
AAPS Workshop on LBA and LC-MS - 11/2017 11
Public
Case study 1 - Cross Correlation Plots Regression calculated across the entire range
R2 = 0.9935
R2 = 0.9976
R2 = 0.9891
AAPS Workshop on LBA and LC-MS - 11/2017 12
Public
Case study 2 – NOV002, IgG mAb
Influence of ADA on the Concentration-Time profile Immunoaffinity LC-MS/MS
AAPS Workshop on LBA and LC-MS - 11/2017 13
Public
Case study 2 – NOV002, IgG mAb
Concentration-Time profile changes due to ADA C o n c e n tra tio n o f N O V 0 0 2 ( g /m L )
10000
1000
100 D o s e 1 ( m g /k g / w e e k ) D o s e 2 ( m g /k g / w e e k )
10
D o s e 3 ( m g /k g / w e e k ) 1
0 .1 0
6
12
18 T im e (d a y s )
AAPS Workshop on LBA and LC-MS - 11/2017 14
Public
24
30
36
Case study 2 – NVS002, IgG mAb
Detection of ADA in Dose 2 group animal 1003 10000
4
C o n c e n tr a tio n o f N O V 0 0 2 ( g / m L )
1000
3
100
2
10
1
1
0 0
6
12
18 T im e (d a y s )
AAPS Workshop on LBA and LC-MS - 11/2017 15
Public
24
30
36
A n ti-N O V 0 0 2 a s s a y s ig n a l (O .D .)
D o s e 2 , A n im a l 1 0 0 3
Case study 3 – NVS002, IgG mAb
Influence of ADA on the Concentration-Time profile Pellet digestion
AAPS Workshop on LBA and LC-MS - 11/2017 16
Public
Case study 3 – NOV002, IgG mAb
Concentration-Time profile changes due to ADA
C o n c e n tra tio n o f N O V 0 0 2 ( g /m L )
10000
1000 D o s e 1 ( m g /k g /w e e k ) D o s e 2 ( m g /k g /w e e k ) 100
10
1 1
8
43
45
T im e (d a y s )
AAPS Workshop on LBA and LC-MS - 11/2017 17
Public
92
99
Case study 3 – NVS002, IgG mAb
Influence of ADA on the Concentration-Time profile 4
1000
3
100
2 D o s e 2 , A n im a l 2 0 0 2 D o s e 2 , A n im a l 2 0 0 3
10
1
1
0 1
8
43
45
T im e (d a y s p o s t-d o s e )
AAPS Workshop on LBA and LC-MS - 11/2017 18
Public
92
99
A n ti-N O V 0 0 2 a s s a y s ig n a l (O .D .)
C o n c e n tr a tio n o f N O V 0 0 2 ( g / m L )
10000
Case study 4 – NVS003, Fab construct Influence of dilution on the C-T profile of a biomarker
Immunoaffinity LC-MS/MS
• Method was setup as a multiplex assay (3 analytes) • Sample pre-dilution was required
NOV010 + BM1
NOV010-BM1 + BM1 + NOV003
NVS010-NVS003 + NOV003 + NOV010 + NOV10-BM1 + BM1
AAPS Workshop on LBA and LC-MS - 11/2017 19
Public
Case study 4 – NOV003, Fab construct C o n c e n tr a tio n o f B io m a r k e r 1 (n g /m L )
Influence of dilution on the C-T profile of a biomarker •
3 .0
2 .5
1.6x
1.3x
2 .0
Biomarker levels after treatment were above basal level
Nominal • Sample predilution resulted basal in complex level dissociation
1 .5
1 .0
•
0 .5
Over-estimation of biomarker concentration
0 .0 0
4
8
12
16
20
24
28
32
36
T im e ( h o u r s )
NVS010-NVS003 + NOV003 + NOV010 + NOV10-BM1 + BM1 20
Public
AAPS Workshop on LBA and LC-MS - 11/2017
Case study 5 – NOV004, IgG mAb
Influence of soluble receptor and sample treatment on quantitation using pellet digestion LC-MS workflow
P e a k a re a ra tio o f N O V 0 0 4
0 .1 5
N o a c id d is s o c ia tio n
0 .1 0
c v
c v
c v
c v
c v
c v
0 .0 5
0 .0 0 0
5
10
20
40
80
C o n c e n t r a t i o n o f s o l u b l e r e c e p t o r ( g /m L )
AAPS Workshop on LBA and LC-MS - 11/2017 21
Public
• NOV004 (10 µM) incubated with increasing concentration of soluble receptor
A c id d is s o c ia tio n p r e - tr e a tm e n t
• Pellet digestion LCMS workflow used • Acid dissociation increases recovery of NVS004
Case study 5 – NOV004, IgG mAb
Influence of protein precipitation solvent on complex dissociation using pellet digestion LC-MS workflow
AAPS Workshop on LBA and LC-MS - 11/2017 22
Public
Conclusions • LC-MS methods represent a complementary technique to LBA for large molecule quantitation
– Flexible options for quantitation, analyte and sensitivity are available – Sample handling needs to be considered in complex samples – Immunoaffinity LC-MS workflows need to be carefully evaluated
• LC-MS can measure free and total drug – – – –
Depends on the analyte of interest (soluble or receptor bound) Depends on the method selected (PD or IA) Depends on the reagents used (solvents and capture) Depends on the binding affinity for the target
• LC-MS and LBA have been shown to provide similar concentration results
– Discrepancies exist between the methods (both positive and negative bias of LC-MS results vs. LBA have been reported) – The intended use of the data can help drive the BA strategy AAPS Workshop on LBA and LC-MS - 11/2017 23
Public
Acknowledgements Safety/ADME colleagues Adam Bentley, Ann Draghi, Yilin Feng, Yunlin Fu, Xinliu Gao, Sagar Kawle, Shaoyong Li, Wenkui Li, Hui Lin, Irina Vinar, Bin Yang, Wei Zhou, Jie Zhang and Jimmy Flarakos
BxBA colleagues Gisela Peraus, Julie De Gagne, Carsten Krantz and Sebastian Spindeldreher
Preclinical Safety colleagues Serge Cote, Peter Hoffmann, Alexander Koch, Brain Stoll, and Nancy Yao
AAPS Workshop on LBA and LC-MS - 11/2017 24
Public
Questions
AAPS Workshop on LBA and LC-MS - 11/2017 25
Public
