p - Waters

Transcription

p - Waters

Bioanalysis of Biotherapeutics
Summary
Peter Milland
Acknowledgment to:
©2012 Waters Corporation
Joanne Mather, Waters Corp.
1
Overview
 Status of Biotherapeutics Drugs
– Past, present, and future
 Challenges in Biotherapeutics Bioanalysis
– Key
K
challenges
h ll
iin Biotherapeutics
Bi th
ti Bioanalysis
Bi
l i
 Waters Solution
– Benefits
B
fit offered
ff
d by
b LC/MS/MS over ELISA
– SPE / LC/ MS
– Accurate Mass
2
Waters and Peptide
p
Bioanalysis
y
Simple Peptides
Desmopressin, Somatostatin,
Angiotensin Bivalirudin
Angiotensin,
Mol Wt ~ 2000
Amyloid B
Biomarker
Mol Wt ~ 4500
Company Confidential
Complicated peptides
Teriparatide (Icon), Exenatide,
Enfuvirtide Mol Wt ~ 4900
Enfuvirtide,
Insulins
Insulin glargine, detemir, aspart,
glulisine
Mol Wt ~ 6000
3
Why?
y

Why quantitative analysis for peptides?
1. Drug discovery/development activities need to be performed
o
PK/PD, metabolic fate, bioequivalence, drug monitoring
2. Peptides as biomarkers
o
Examples: Angiotensin II* used to monitor cardiovascular health
and
d natriuretic
t i
ti peptides*
tid * are biomarkers
bi
k
for
f cardiovascular
di
l
disease
3 Key peptides
3.
id can b
be used
d to quantitate
i
protein
i d
drugs and
d bi
biomarkers
k
in complex matrices, after digestion of the sample
4
Why
y LCLC-MS/MS?
 Why an LC-MS/MS based assay?
– ELISA assays not practical for discovery, no
antibodies available yet
– Challenges with ELISA assays
o time consuming, expensive to develop
o require separate assay for each peptide
o limited linear dynamic range
o Possible cross reactivity
 Benefits of LC-MS/MS for peptides
–
–
–
–
–
–
LCMSMS provides single assay for multiple peptides
Broad linear dynamic range
Accurate, precise
Universal
Faster, cheaper method development
Meeting matrix effects and ISR acceptance criteria
5
Overview
– Past,
P t present,
t and
d future
f t
– Key
y challenges
g in Biotherapeutics
p
Bioanalysis
y
 Waters Solution
– Benefits offered by LC/MS/MS over ELISA
– SPE / LC/ MS
– Accurate Mass
 Competition
 Collateral
6
Challenges
g
Challenges faced;
 SPE
 Sensitivity
y & Clean up
p
 Understanding your peptide
 Where to start?
 LC
 Parameters that worked for small molecules do not work for peptides
 Slower flow rates
 Carryover & Sensitivity
 MS
 Charge states
 Products m/z
/ larger
g than p
precursors,, many
y more MRMs
 Tuning: Specificity & Selectivity. Peptides can form many low abundance
product ions.
 Sensitivity
 Method Development
 Compliance & validation – have to meet industry guidelines!
7
Improving Chromatography:
Amyloid Beta Peptide 11-40, MW
4330
48% increase in area counts at lower flow rate, carryover also reduced
%
0
MRM of 1 Channel ES+
1.32e4
Area
385
100
400 µL/min
0.20
0.40
0.60
0.80
1.00
1.20
1.40
1.60
1.80
2.00
2.20
2.40
2.60
2.80
3.00
MRM of 1 Channel ES+
100
1.32e4
Area
%
568
200 µL/min
Improved Solubility/Diffusivity for Larger Peptides
0
0.20
0.40
0.60
0.80
1.00
1.20
1.40
1.60
1.80
2.00
2.20
2.40
2.60
2.80
Time
3.00
Data courtesy of Erin Chambers
8
Precursor Identification
Enfuvirtide
 MW 4492
 Calculate possibilities
ENFUVIRTIDE
100
– 3+ at m/z 1498
– 4+ at m/z 1124
1498.64
1498.01
 Perform MS scan
 3+ most intense
%
1498.89
– Requires at least 1500 amu
on first quad
1498
100
1499.34
Scan
ES+
9.91e7
%
0
0 400 600 800 10001200140016001800
m/z
Scan ES+
1498.32
1494 1495 1496 1497 1498 1499 1500 1501 1502 1503
m/z
Why could this peptide be
a challenge to regulated
bioanalysis?
9
Higher
g
Fragment
g
m/z
/
 Bivalirudin
– Major singly charged fragments at m/z 650 and m/z 1530
– Precursor appears at lower m/z even though MW is higher
– Higher m/z fragments require adequate mass range on 2nd quadrupole
650
Singly charged fragments
1091
1530
Result:
2 MRM Transitions identified
1091 >650
1091->650
1091-> 1530
Doubly charged precursor
10
Overview
– Past,
P t present,
t and
d future
f t
– Key
y challenges
g in Biotherapeutics
p
Bioanalysis
y
 Waters Solution
– Benefits offered by LC/MS/MS over ELISA
– SPE / LC/ MS
– Accurate Mass
 Competition
 Collateral
11
Bioanalytical Method Development
for Peptide
p
Therapeutics:
p
The Tools
ACQUITY II Class
Cl
UPLC®
X
Xevo™
™ TQ-S
TQ S MS
Method
Development Kits
12
Components of Waters Biotherapeutics
Bioanalysis
y
13
Sample
p Preparation
p
Requirements
q
 Provides maximum analyte recovery
 Minimizes matrix effects
 Provides significant increase in sample concentration to meet
detection limits
 Reproducible
 Straightforward method development
 Selectively separates peptides from matrix components
 Fast
14
Performance of Current Sample
Preparation
p
Techniques
q
% Analyte Recovery
Methods Used:
100
90
80
70
60
50
40
30
20
10
0
Reversed –phase only SPE
Basic starting protocol provided
by
y manufacturer
*
Reversed-phase
SPE
PPT
Bivalirudin (acidic)
LLE
Desmopressin (basic)
Protein Precipitation (PPT)
3:1 acetonitrile:plasma
Liquid Liquid Extraction (LLE)
5:1 ethyl acetate:plasma (most
common in literature)
Moderate to poor peptide recovery
*
< 1% recovery for LLE
15
Oasis® PST SPE Protocol for Peptides
p
Oasis® WCX
µElution
Oasis® MAX
µElution
Protocol
Dilute plasma with
4% H3PO4
Condition MeOH/Equilibrate H2O
Load Diluted Plasma
Wash 1:
5% NH4OH
Wash 2:
20% ACN
Elution:
1% TFA in 75/25 ACN/H2O
Dilute:
H2O
16
The Peptides Used
Peptide
MW
pI
# of Residues
HPLC Index*
O t
Octreotide
tid
1019
93
9.3
8
40 8
40.8
Angiotensin II
1046
7.35
8
38.3
Desmopressin
1069
8.6
9
16.8
Vasopressin
1084
9.1
9
7.6
Goserelin
1270
7.3
10
31.7
Angiotensin I
1296
7.51
10
56.2
Somatostatin
1638
10.4
14
52.6
Neurotensin
1673
8.93
13
44.4
Bi li di
Bivalirudin
2180
3 87
3.87
20
46 2
46.2
BNP
3464
12
32
15.9
Teriparatide
4118
9.1
34
90.4
Enfuvirtide
4492
4.06
36
155.9
*higher number = more hydrophobic
17
Final SPE Results after BNP, Enfuvirtide
and Somatostatin Methods Optimized
p
% SPE Rec
covery
120
100
80
60
Screening Protocol
Modified Protocol
40
20
0
Great results for diverse peptides:
Screening protocol results in method for 75% of peptides!
Minor, compound specific, modifications for 3 peptides
result in excellent recovery for all peptides
18
Final SPE Summary
y
pI
MW
% SPE
Recovery
Octreotide
93
9.3
1019
88
<10%
Angiotensin II
7.35
1046
82
8%
Desmopressin
8.6
1069
104
<11%
Vasopressin
91
9.1
1084
100
-3%
3%
Goserelin
7.3
1270
100
-2%
Angiotensin I
7.51
1296
109
*
S
Somatostatin
t t ti
10 4
10.4
1638
94
*
Neurotensin
8.93
1673
114
6%
Bivalirudin
3.87
2180
100
10%
BNP
12
3464
84
*
Teriparatide
9.1
4118
97
9%
Enfuvirtide
4.06
4492
102
*
Peptide
% Matrix
Effects
Maximum recovery = enhanced sensitivity
Minimum matrix effects = selectivity and sensitivity
*= data being generated
19
Summary
y of Sample
p Preparation
p
 An efficient SPE screening strategy, based on 1 protocol and 2
Oasis® mixed-mode sorbents, simplifies method development
f extraction
for
t
ti
off peptides
tid from
f
human
h
plasma
l
 Mixed-mode SPE provides selective clean-up of peptides from
human plasma
– Matrix effects are <11% for those peptides tested
 Oasis® µElution format SPE provides significant benefits for
peptide extraction
– No evaporation step
– Up to 15X concentration, without evaporation
– 96 samples processed in <30 minutes, < 20 seconds per sample
20
Bioanalysis
y
I-Class
21
Key Considerations for LC Screening
Protocol
 Mass spectrometry compatible solvents and modifiers
 Acceptable peak shapes even for large (~5000 MW) peptides
 Short run times
 Resolution from endogenous compounds
 Maximize sensitivity
 Ensure analyte solubility at all concentrations
– Injection solvent and needle washes contain 5-10% organic and
0.025-1% acid
22
UPLC® Technology for Peptides:
Chromatographic
g p
Screening
g Protocol
 ACQUITY UPLC® BEH300 C18 2.1 X 50 mm, 1.7 µm Peptide Separation
Technology (PST) Column
– Columns
C l
are QC tested
t t d with
ith peptide
tid standards
t d d
– 300Å PST column gave overall best performance (peak shape) for
diverse peptides
– 2.1 X 50 mm provides adequate throughput
 Generic gradient
o
Mobile phase A = 0.1% formic acid
o
Mobile phase B = 0.1% formic acid in acetonitrile
o
Flow rate = 0.4 mL/min
o
15% B to 75% B over 2 minutes
• Start at 5% B for polar peptides
o
Total cycle time 3.5
3 5 minutes
23
Single Screening Method:
Diverse Peptides
p
Analyte
MW
1. Vasopressin
1084
2. Angiotensin
g
II
1046
3. Desmopressin
1069
4. Bivalirudin
2180
5. Enfuvirtide
4492
Broad molecular weight range
2 very similar peptides; differ only by amino group
24
Single Screening Method:
Diverse Peptides
p
Resolution between similar peptides
1 2 3 4
5
MS Data
P i t
Points
Across
Peak
Analyte
MW
Peak
P
k
Width
(seconds)
1. Vasopressin
p
1084
1.8
15
2. Angiotensin II
1046
2.2
15
3. Desmopressin
1069
2.2
18
4. Bivalirudin
2180
2.4
18
5. Enfuvirtide
4492
2.1
16
Peak widths 2-3
2 3 seconds wide at base
Adequate MS data points
Short run times (3.5 min cycle time)
25
PST Method Development
p
Kits
 UPLC® PST Therapeutic Peptide Method Development Kit: Part#
176001835
 ACQUITY UPLC® BEH300 C18 1.7
1 7 µm column
l
 Oasis® µElution PST Method Development Plate
 96-well 1 mL Collection Plate and Cap Mat
 Detailed
D t il d LC/SPE S
Screening
i
Protocol
P t
l
HPLC PST Therapeutic
h
i Peptide
id Method
h d Development
l
Kit:
i Part#
176001836
 XBridge BEH300 C18 3.5 µm column
 Oasis® µElution
El tion PST Method De
Development
elopment Plate
 96-well 1 mL Collection Plate and Cap Mat
 Detailed LC/SPE Screening Protocol
26
Bioanalysis
y
Xevo TQTQ-S
UNIFI
27
Why
y Mass Spectrometry
p
y
 Specificity
— Complex biological matrices
 Sensitivity
S
iti it
— Multiply charged species, requires high sensitivity
— Accurate pK profiles
 Rapid Method Development
 Dynamic range
— 3-4 orders magnitude
 Reproducibility
p odu b y
— Multi-analyte assays
 Accuracy
 One analytical technique for many diverse peptides
 Common, comfortable technology
 Follows familiar regulatory guidelines
28
Compound Optimisation
Optimisation::
Built--in Knowledge
Built
g of Peptides
p
Typical
yp
optimization
p
for small
molecules concentrates on
the most intense transition
1
2
3
4
For peptides
F
tid
thi
this is
i
a common
mistake!
Company Confidential
29
Compound Optimisation
Optimisation::
Built--in Knowledge
Built
g of Peptides
p
1
2
3
Intelligent
Compound
O ti i ti
Optimization
1
4
4
3
Automatically
chooses the
optimum
peptide
id
transition
30
Compound Optimisation Bivalirudin
31
Compound Optimisation Example Bivalirudin
Note the
masses!
32
Method development tools – Promoted
Parameters
33
Method development tools – Promoted
Parameters (
(Templates)
p
)
Capillary Screen
Column Temperature
p
Screen
Mobile Phase Screen
Create templates that can be ‘re-purposed’ and re-used over
and over for automated method development
34
Summing MRMs across Charge
States
Import MRMs directly from Scientific Library
35
Reviewing
g Data
36
ToF Vs Quad
Q
Tandem quadrupole LC/MS/MS is traditionally the technology of choice for
quantification of analytes
- Specificity
- Linearity
- Sensitivity
Accurate mass MS instrumentation usually reserved for qualitative data
analysis
The latent full-scan data from a ToF can be of significant use in
understanding the reasons behind issues such as poor exposure or rapid
elimination.
The application of accurate mass MS for quantification in DMPK has
attracted considerable interest in recent years, allowing simultaneous
quantitative and qualitative analysis.
The b
Th
benefits
fit off accurate
t mass iinstrumentation
t
t ti
over tandem
t d
quadrupole
d
l
include; faster method development, non targeted analysis and the ability
to screen for unknowns.
37
LC/MS/MS Nominal and Accurate
Mass
Bioanalysis. 2012 Mar;4(5):605-15.
doi: 10.4155/bio.12.15.
38
Synapt G2 HDMS
20mDa Extract Mass Window 616.3821
Blank
Bl k
10ng/mL
Standard
39
Synapt G2 HDMS
Extract Mass Window 616.3821
40
Xevo TQ
TQ--S
Extracted Standard and Plasma Blank
1ng/mL
Standard
10 x
Plasma
Blank
Comparison of the quantification of a therapeutic protein using nominal and accurate mass MS/MS
Robert S Plumb, Gordon Fujimoto, Joanne Mather, Warren B Potts, Paul D Rainville, Nicholas J Ellor, Christopher Evans, Jonathan R Kehler, Matthew E Szapa
Bioanalysis, Mar 2012, Vol. 4, No. 5, Pages 605-615.
41
High Sensitivity for Biotherapeutics
Bioanalysis
Desmopressin
p
in human p
plasma with Xevo TQTQ
Q-S
Compound optimization results
obtained from UNIFI
Blank plasma
1 pg/mL
5 pg/mL
1 – 20000 pg/mL
42

p - Waters

Transcription

Similar documents

nemic lambda malaysia

low carbon cities framework and assessment system

070 Marcos - Latin American Journal of Aquatic Mammals

IBM DB2 Analytics Accelerator for z/OS (IDAA)

TM-U675/U675P

r in gkasaneksekut if re - EKAS

curriculum vitae prof. vincenzo pavone

Your Partner in The Filtration and Separation Industry.

EN TECHNICAL CATALOGUE SPECO WASTEMASTER TSF V01

Muat turun

FIS Outdoor February 2014 Prices

- Magic Theatre

norme di condotta

Ferrante d`Aragona e la ricerca di un`egemonia politica napoletana

Trading Partners