protein and peptide therapeutics: addressing … and peptide therapeutics: addressing bioanalytical...
TRANSCRIPT
©2012 Waters Corporation 1
Protein and Peptide Therapeutics: Addressing
Bioanalytical Demands of Complicated Peptides
Tips and Tricks for Troubleshooting and
Optimization During Method Development
Mary E. Lame
Senior Applications Chemist
Waters Corporation
Milford, MA
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©2012 Waters Corporation 2
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©2012 Waters Corporation 3
Mary Lame, Sr. Applications Chemist
B.S. in Chemistry from the Western Connecticut State
University (Danbury, CT)
M.S. in Chemistry from Central Connecticut State University
(New Britain, CT).
She began her bioanalytical career in 1997 with a global
Pharmaceutical company where she supported R&D, DMPK and
bioanalytical groups until 2011.
Mary joined Waters in 2011 as a Senior Applications Chemist in
Waters Applied Technology Group. Mary’s primary role is to
support discovery bioanalysis, and method development large
and small molecules. She is responsible for sample preparation,
mass spectrometry, and LC method development, and also
provides in-house customer training on these topics. Her most
recent focus has been on Peptide bioanalysis.
©2012 Waters Corporation 4
Outline
Introduction
— Basic screening methods
— Typical challenges faced
Understanding your peptide
o pI, HPLC index, hydrophobic/hydrophilic
Understanding the challenges: symptoms,
examples, and solutions for common problems
Conclusions
©2012 Waters Corporation 5
Introduction
Why bioanalysis for peptides?
1. Drug discovery/development activities need to be performed
o PK/PD, metabolic fate, bioequivalence, drug monitoring
2. Peptides as biomarkers
3. Signature peptides can be used to quantitate protein drugs and
biomarkers in complex matrices, after digestion of the sample
4. >40 peptide drugs already in clinical market, >400 peptide
drugs in advanced pre-clinical stages1
5. By 2014, 5 of the top 5 selling drugs will be proteins2
1Bioscience Technology, January 2009 2Evaluate Pharma
©2012 Waters Corporation 6
Why LC-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
©2012 Waters Corporation 7
Chromatographic Screening Protocol for Peptides
ACQUITY UPLC® BEH300 C18 2.1 X 50 mm, 1.7 µm
Peptide Separation Technology (PST) Column
—Columns are QC tested with peptide standards
—300Å PST column gave overall best performance (peak
shape) for diverse peptides
—2.1 X 50 mm provides adequate throughput
Generic gradients
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 minutes
Note: formic acid used in mobile phase to avoid MS suppression associated with TFA
©2012 Waters Corporation 8
Single Screening Method: Diverse Peptides
Time 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
%
0
MRM of 5 Channels ES+ 1 3 2 4
5
Peak widths 2-3 seconds wide at base Adequate MS data points Short run times (3.5 min cycle time)
Analyte MW
Peak
Width
(seconds)
MS Data
Points
Across
Peak
1. Vasopressin 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
©2012 Waters Corporation 9
SPE Screening Protocol for Peptides: Screening Complementary Sorbents
Oasis® WCX µElution
Oasis® MAX µElution
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
Protocol
©2012 Waters Corporation 10
SPE Format : Oasis® µElution Plates
Oasis® µElution plate technology
Up to 15X concentration without evaporation
— Concentration often necessary to reach LOD’s with peptides
Minimizes analyte loss
— Minimizes sticking to walls of collection plates
— Eliminates problems re-solubilizing after dry-down
— Beneficial for thermally unstable peptides
Speed: 96-well plate in <30 min,
<20 seconds/sample
©2012 Waters Corporation 11
Final SPE Results after BNP, Enfuvirtide and Somatostatin Methods Optimized
Minor, compound specific, modifications for 3 peptides result in excellent recovery for all peptides
% Matrix Effects were <10% where measured
% S
PE R
ecovery
0
20
40
60
80
100
120
Screening Protocol
Oasis® MAX
Modified Protocol
Screening Protocol
Oasis® WCX
©2012 Waters Corporation 12
When the Basic Methods Don’t Work: Typical Challenges Faced
Peptide Loss
Poor Sensitivity
Solubility
Specificity/Selectivity
Chromatography
Protein Binding
Non-specific Binding
Low Extraction Recovery
Inadequate Retention
Chemical Instability
©2012 Waters Corporation 13
Outline
Introduction
— Basic screening methods
— Typical challenges faced
Understanding your peptide
o pI, HPLC index, hydrophobic/hydrophilic
Understanding the challenges: symptoms,
examples, and solutions for common problems
Conclusions
©2012 Waters Corporation 14
Understanding Your Peptide
1. Obtain the peptide sequence
2. Calculate pI and HPLC index
3. Assess nature by evaluating the residues
4. Look for any potential stability problems
©2012 Waters Corporation 15
Chemical Properties of Therapeutic Peptides
Peptide MW pI # of Residues HPLC Index*
Octreotide 1019 9.3 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
Bivalirudin 2180 3.87 20 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
©2012 Waters Corporation 16
Understanding Your Peptide: Step 1
Teriparatide (1-34 fragment of human parathyroid hormone)
example: Obtain sequence
1.
©2012 Waters Corporation 17
Understanding Your Peptide: Step 2
Teriparatide (1-34 fragment of human parathyroid hormone)
example: Calculate pI and HPLC index
2.
Teriparatide is basic and sticky
©2012 Waters Corporation 18
Understanding Your Peptide: Step 3
Teriparatide (1-34 fragment of human parathyroid hormone)
example: Assess nature by evaluating the residues
3. Teriparatide sequence: SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNF
Property Amino Acid
Hydrophobic A, F, I, L, M, P, V, W, Y
Moderate C, G
Hydrophilic D, E, H, K, N, Q, R, S, T, pyro-glutamic acid
Positive Charge K, R, H, N-terminus
Negative Charge D, E, Y, C-terminus
Degradation likely M, W
Prone to de-amidation,
dehydration, cyclization to pGlu N,Q, C-terminal amides, N-terminal Q
Prone to oxidation under mild
conditions C, M
** Teriparatide is 38% hydrophobic residues, 50% hydrophilic residues and 41% charged residues ** This information is very valuable for solubility purposes
©2012 Waters Corporation 19
Understanding Your Peptide: Step 4
Teriparatide (1-34 fragment of human parathyroid hormone)
example: Look for any potential stability problems
4. Teriparatide sequence: SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNF
Property Amino Acid
Hydrophobic A, F, I, L, M, P, V, W, Y
Moderate C, G
Hydrophilic D, E, H, K, N, Q, R, S, T, pyro-glutamic acid
Positive Charge K, R, H, N-terminus
Negative Charge D, E, Y, C-terminus
Degradation likely M, W
Prone to de-amidation,
dehydration, cyclization to pGlu N,Q, C-terminal amides, N-terminal Q
Prone to oxidation under mild
conditions C, M
Modification Residue MW change
Oxidation C, M +16 or 32
Cyclization N-term E -17
Deamidation N, Q +1
©2012 Waters Corporation 20
How Can I Use This Information?
pI
— Helps to determine solubility
— Helps identify best SPE and chromatographic conditions/solvents
HPLC Index
— Indicates if polar or more hydrophobic
o Can guide troubleshooting
• Hydrophobic: may lead to non-specific binding, protein binding, carryover, low SPE recovery
• Polar: may see breakthrough during SPE, likely soluble in aqueous solutions, probably don’t need to worry about protein binding
Size/MW
— Larger peptides tend to be more “sticky” and experience problems associated with hydrophobic peptides
Classification of residues as charged, hydrophilic, or hydrophobic
— Provides starting point for solubility
o Follow guidelines in upcoming solubility section
Identification of possible modification/instability sites
— May need to eliminate pH extremes or other conditions
— Can search for expected mass shift during MS experiment
©2012 Waters Corporation 21
Outline
Introduction
— Basic screening methods
— Typical challenges faced
Understanding your peptide
o pI, HPLC index, hydrophobic/hydrophilic
Understanding the challenges: symptoms,
examples, and solutions for common problems
— Solubility
— Specificity
— Protein Binding
— Non-specific Binding
— Peptide Loss
Conclusions
— Poor Sensitivity
— Chromatography and Carryover
— Low SPE Recovery
©2012 Waters Corporation 22
Specific Challenges in Bioanalysis of Peptides: Solubility
When is Solubility Important?
– In initial solubilization of powder
– During sample preparation
o Pre-treatment, wash steps, elution
– In mobile phase, at flow rate you are running
– In injection solvent
Symptoms of inadequate/incomplete solubility
– Poor linearity
– Irreproducible chromatography
– Low sensitivity
– Poor LC peak shape
– Peptide loss, especially at low concentrations
– LC carryover
– Increase in column back pressure
©2012 Waters Corporation 23
General Guidelines for Solubilizing Powder
1. If a peptide is < 5 residues, it will likely dissolve in aqueous
solutions unless the sequence is entirely comprised of
hydrophobic residues.
2. Peptides containing >25% charged residues and < 25%
hydrophobic residues generally dissolve in aqueous
solutions.
3. If the peptide is basic, acidic solutions (formic acid or TFA)
with a low % organic (5%) often work well. The converse is true
for acidic peptides, try solubilizing in basic solutions (1-
5% NH4OH for example) with a low % organic.
©2012 Waters Corporation 24
General Guidelines for Solubilizing Powder
4. Peptides containing >50% hydrophobic residues may be only slightly soluble or insoluble in aqueous solutions. Hydrophobic peptides are best solubilized in DMSO, DMF, strong acid solutions (TFA, formic, acetic), or isopropanol. For cysteine-
containing peptides, use DMF instead of DMSO.
5. Guanidine HCl or Urea may be necessary for those peptides that tend to aggregate and can later be removed during sample
preparation.
6. Peptides which contain >75% of S, T, E, D, K, R, H, N, Q or Y may form intramolecular hydrogen bonds and form gels in aqueous solutions. These peptides should be treated in the same manner as hydrophobic peptides (#4).
7. Addition of a carrier protein to minimize any predicted or unexpected peptide loss from NSB.
©2012 Waters Corporation 25
Teriparatide Solubilization Solvent Assessment
1% TFA, 50% ACN
DMSO
1% FA, 50% ACN
Time 0.40 0.50 0.60 0.70 0.80 0.90 1.00 1.10 1.20 1.30 1.40
%
0
100
0.40 0.50 0.60 0.70 0.80 0.90 1.00 1.10 1.20 1.30 1.40
%
0
100
0.40 0.50 0.60 0.70 0.80 0.90 1.00 1.10 1.20 1.30 1.40
%
0
100
) MRM of 4 Channels ES+ 824.32 > 983.78 (Teriparatide 5+)
6.41e5 Area
0.88 47318
MRM of 4 Channels ES+ 824.32 > 983.78 (Teriparatide 5+)
6.44e5 Area
0.86 47286
MRM of 4 Channels ES+ 824.32 > 983.78 (Teriparatide 5+)
3.66e5 Area
0.87 27262
©2012 Waters Corporation 26
Protein Precipitation and Solubility of Large Peptides
Too much organic causes peptide loss, pH impacts recovery
0
20
40
60
80
100
120
1:1 ACN 2:1 ACN 1:1, 1% FA in ACN
1:1, 1% TFA in ACN
1:1, 1% AA in ACN
1:1, 5% NH4OH
ACN
2:1, 5% NH4OH
ACN
% Teriparatide Recovery
©2012 Waters Corporation 27
Improving Solubility in Mobile Phase B, Reducing Carryover
07-Jun-2012
Time 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
%
0
100
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
%
0
100
MRM of 2 Channels ES+ 824.351 > 983.774 (Teriparatide)
3.49e5 Area
1.05 9940
MRM of 2 Channels ES+ 824.351 > 983.774 (Teriparatide)
3.49e5 Area
1.00 14237
Mobile phase B= 0.1% FA in ACN
Mobile phase B= 5% TFE in ACN
Almost 50% higher area counts!
Would this information also be valuable for SPE optimization?
©2012 Waters Corporation 28
Specific Challenges in Bioanalysis of Peptides
Specificity
Possible Causes
— Many peptides, high abundance proteins in sample
— Interference by similar endogenous compounds
— Biomarker Analysis
o Difficult to get blank matrix for use in method development/validation
• Use of stripped or surrogate matrix
• Standard curves prepared with stable labeled version of analyte of interest
Possible solutions
— Use higher m/z MRM transitions
— Avoid immonium ions
— Improve sample preparation
— Adjust pretreatment prior to extraction
©2012 Waters Corporation 29
m/z 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500
%
0
100
m/z 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500
%
0
100 2: MS2 ES+
334.8 134193152
145.0 118689384
146.9 70124136
259.1 43548620
357.1 115938712
371.1 55654400
443.0 35474636
981.4 32655092
911.2 31676710 681.2
24706662 570.2;13623731
1063.1 21816462
2: MS2 ES+
Time1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00
%
0
100
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00
%
0
100
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00
%
0
100
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00
%
0
100
1: MRM of 4 Channels ES+ TIC4.48
2.86 3.95 8.214.59
5.50 6.30 7.12
1: MRM of 4 Channels ES+ TIC5.76
4.46
3.922.884.56 8.00 8.51
HLB_postspike_TFA_081911_001 2: MS2 ES+ TIC
3.19e11
2.88
2.18
5.513.223.49 3.97
4.905.62
8.027.276.45 9.068.29
9.19
HLB_postspike_TRIS_081911_001 2: MS2 ES+ TIC
3.22e112.88
2.13
3.173.36 6.345.51
3.945.114.48 5.75
8.188.057.306.77 8.66 9.24
Time1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00
%
0
100
Time1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00
%
0
100
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00
%
0
100
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00
%
0
100
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00
%
0
100
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00
%
0
100
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00
%
0
100
1: MRM of 4 Channels ES+ TIC
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00
%
0
100
1: MRM of 4 Channels ES+ TIC4.48
2.86 3.95 8.214.59
5.50 6.30 7.12
1: MRM of 4 Channels ES+ TIC
4.48
2.86 3.95 8.214.59
5.50 6.30 7.12
1: MRM of 4 Channels ES+ TIC5.76
4.46
3.922.884.56 8.00 8.51
HLB_postspike_TFA_081911_001 2: MS2 ES+ TIC
3.19e11
5.76
4.46
3.922.884.56 8.00 8.51
HLB_postspike_TFA_081911_001 2: MS2 ES+ TIC
3.19e11
2.88
2.18
5.513.223.49 3.97
4.905.62
8.027.276.45 9.068.29
9.19
HLB_postspike_TRIS_081911_001 2: MS2 ES+ TIC
3.22e11
2.88
2.18
5.513.223.49 3.97
4.905.62
8.027.276.45 9.068.29
9.19
HLB_postspike_TRIS_081911_001 2: MS2 ES+ TIC
3.22e112.88
2.13
3.173.36 6.345.51
3.945.114.48 5.75
8.188.057.306.77 8.66 9.24
Specificity in Human Plasma: Impact of Pretreatment Prior to SPE
Plasma diluted with 1% TFA
Plasma diluted with 10mM TRIS Base
Human Serum Albumin
©2012 Waters Corporation 30
Lack of Specificity in an MRM assay for Trastuzumab
1 nM Trastuzumab in Solvent A
human serum digest
1 nM Trastuzumab in serum digest
~1500 area counts
~500-700 area counts = 2-3X lower!
Addressing the problems………
©2012 Waters Corporation 31
Mixed-mode Cation Exchange SPE Clean-up for Protein Digests
Increase in Signal Reduction in Background Removal of Digest Reagents Reduction of Plasma Phospholipids Improved Specificity Digest Concentration
Before SPE
After SPE
©2012 Waters Corporation 32
MS Specificity: Avoiding Immonium Ion Fragments
02-Mar-2012
Time 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 3.20 3.40
%
0
100
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 3.20 3.40
%
0
100
1: MRM of 4 Channels ES+ 867 > 136 (Lantus)
9.40e5 0.99
0.56
1.68
1.58
1.20 1.15
1.30 1.49 1.40
1: MRM of 4 Channels ES+ 867 > 984 (Lantus)
8.97e5 0.99
867 -> 136 (tyrosine immonium ion)
867 -> 984
Lack of Specificity
©2012 Waters Corporation 33
Time 0.50 1.00 1.50 2.00 2.50 3.00 3.50
%
0
0.50 1.00 1.50 2.00 2.50 3.00 3.50
%
0
MRM of 2 Channels ES- 1031.5 > 1026.8
3.20e4 2.48
3.36
MRM of 2 Channels ES- 825 > 821.3
3.26e4 2.48
1.67
1.22 1.63
1.96 2.49
MS: Specificity: Use of Higher m/z Precursors and Fragments
Human Plasma Extract
4th charge state 1031.5 -> 1026.8
5th charge state 825 -> 821.3
©2012 Waters Corporation 34
Specific Challenges in Bioanalysis of Peptides
Protein Binding
Symptoms
– Decreases solid phase extraction recovery
o Peptide passes through on load step with associated protein
– Results in underestimation of peptide concentration
Possible Causes
– More pronounced in stickier/more hydrophobic peptides
Possible solutions
– Can be disrupted by: acidification, basification, denaturation with
urea or guanidine HCl, organic precipitation, ZnSo4 precipitation
©2012 Waters Corporation 35
Specific Challenges in Bioanalysis of Peptides
Non-specific Binding (NSB)
Symptoms
– Peptide loss
– Loss of low end of standard curve
– Non-linearity
– Irreproducible chromatography
– Loss of chromatographic peak
– Poor LC or SPE recovery
Possible Solution
– Requires carrier protein or surfactant
©2012 Waters Corporation 36
Insulin Analogs: Testing for and Eliminating Non-Specific Binding
Time 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
%
0
100
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
%
0
100
1: MRM of 4 Channels ES+ 1011.2 > 1179 (Lantus)
Area
1.01 469
1: MRM of 4 Channels ES+ 1011.2 > 1179 (Lantus)
Area
30% MeOH, 10% acetic acid, 0.05% rat plasma
30% MeOH, 10% acetic acid
Insulin Glargine solution: <30 min. on benchtop
©2012 Waters Corporation 37
Carrier Protein Options
Teriparatide + 0.05%Rat plasma
Teriparatide + 40ug/ml Bovine Serum Albumin
Time 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 3.20 3.40
%
0
100
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 3.20 3.40
%
0
100
MRM of 4 Channels ES+ 824.25 > 983.795 (Teriparatide 5+)
5.57e5
1.06
MRM of 4 Channels ES+ 824.25 > 983.795 (Teriparatide 5+)
5.57e5 1.08
©2012 Waters Corporation 38
Specific Challenges in Bioanalysis of Peptides
Peptide loss
— Potential losses during evaporation
— Hydrophobic peptides stick to vials/collection plates
o Concentration dependent
— Addition of organic or acid helps maintain solubility for storage
— Non-specific binding
— Maintain solubility across assay concentration range
Poor Sensitivity
— Multiple charge states, lower MS response
— Peptide specific: presence of Arg or Lys improves MS sensitivity
— In general, more fragments formed than for small molecules
o Results in lower intensity for each fragment
o Larger peptides, even more fragments- even lower intensity/fragment
— Sample concentration typically required to meet detection limits
— Poorly optimized chromatographic
©2012 Waters Corporation 39
Specific Challenges in Bioanalysis of Peptides: Chromatography
Problem: Poor peak shape
— Possible Solutions
o Increase temperature
o Decrease flow rate
o Increase stationary phase pore size
o Change ligand or column chemistry
o Add carrier protein to reduce NSB
Problem: Poor Sensitivity
— Possible solutions
o Flow rate
o Temperature
o Additives to improve solubility
©2012 Waters Corporation 40
Improving Chromatography for Amyloid Beta Peptide 1-40, MW 4330: Temperature
Time 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25
%
0
100
0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25
%
0
100
MRM of 1 Channel ES+
1.32e4 Area
1.17 365
MRM of 1 Channel ES+
1.32e4 Area
1.19 240 40 C
60 C
52% increase in area counts at higher temperature, carryover also reduced
1083-> 1054 4+ precursor -> 4+ b ion fragment
365 area counts
240 area counts
©2012 Waters Corporation 41
Improving Chromatography for Teriparatide: Column Chemistry
Time 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 3.20 3.40
%
0
100
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 3.20 3.40
%
0
100
0 MRM of 4 Channels ES+ 824.32 > 983.78 (Teriparatide 5+) 1.23
1.59 1.67
MRM of 4 Channels ES+ 824.32 > 983.78 (Teriparatide 5+) 0.78
ACQUITY BEH C18 300A
ACQUITY CSH C18
©2012 Waters Corporation 42
Improving Chromatography: Ligand and Pore Size
ACQUITY BEH 1.7 µm C18 300Å
ACQUITY BEH 1.7 µm C18 130Å
ACQUITY BEH 1.7 µm C4 300Å
Enfuvirtide: MW 4492
©2012 Waters Corporation 43
Chromatography: Use of Carrier Protein to Reduce NSB
Teriparatide
14:41:2418-May-2012
Time0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 1.10 1.20 1.30 1.40 1.50 1.60 1.70 1.80 1.90 2.00 2.10 2.20 2.30 2.40 2.50 2.60 2.70 2.80 2.90 3.00 3.10 3.20 3.30 3.40 3.50 3.60 3.70 3.80 3.90 4.00 4.10 4.20
%
0
100
0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 1.10 1.20 1.30 1.40 1.50 1.60 1.70 1.80 1.90 2.00 2.10 2.20 2.30 2.40 2.50 2.60 2.70 2.80 2.90 3.00 3.10 3.20 3.30 3.40 3.50 3.60 3.70 3.80 3.90 4.00 4.10 4.20
%
0
100
extracted soln 1ng rep 1 MRM of 2 Channels ES+ TIC
2.56e5
0.95
unextracted soln rep 1 MRM of 2 Channels ES+ TIC
2.54e5
0.94
Pure solution in 30% ACN, 1% TFA
Pure solution in 30% ACN, 1% TFA +0.05% rat plasma
©2012 Waters Corporation 44
Poor Peak Shape or No Peak: Column Conditioning
Time 0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25
%
0
100
0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25
%
0
100
0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25
%
0
100
MRM of 10 Channels ES+ 866.8 > 984 (Lantus)
2.85e6
MRM of 10 Channels ES+ 866.8 > 984 (Lantus)
2.85e6
MRM of 10 Channels ES+ 866.8 > 984 (Lantus)
2.85e6
New column:1st injection after solvent blanks
2nd injection after solvent blanks
After 9 injections of precipitated plasma
Insulin Glargine
©2012 Waters Corporation 45
Chromatographic Carryover
Problem: Carryover
Possible Causes of carryover
— Improper injection solvent
— Stuck on column
— Stuck to LC tubing, needle etc.
Possible Solutions
— Stuck on column: increase temperature, decrease flow rate,
change mobile phase modifier, chromatographic pore size, CSH
column, shallow gradient
— Injection solvent: adjust organic content, adjust modifier content,
add carrier protein
— Stuck to tubing, needle: modify injection solvent, modify wash
solvents (increase % or acid/base, add TFE)
©2012 Waters Corporation 46
Improving Chromatography for Amyloid Beta Peptide 1-40, MW 4330: Flow Rate
Time 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
%
0
100
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
%
0
100 MRM of 1 Channel ES+
1.32e4 Area
385
MRM of 1 Channel ES+
1.32e4 Area
568
400 µL/min
Improved Solubility/Diffusivity for Larger Peptides
200 µL/min
48% increase in area counts at lower flow rate, carryover also reduced
©2012 Waters Corporation 47
Time 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 3.20 3.40
%
0
100
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 3.20 3.40
%
0
100
MRM of 4 Channels ES+ 824.25 > 983.795 (Teriparatide 5+)
2.98e5 Area
1.08 22737
MRM of 4 Channels ES+ 824.25 > 983.795 (Teriparatide 5+)
1.88e5 Area
0.98 13238
Teriparatide Chromatography: Improving Diffusion/Solubility
15-50% B in 2 min 72% Increase in area counts
15-75% B in 2 min
Column temp 60C Flow rate 0.4ml/min
©2012 Waters Corporation 48
Low SPE Recovery
Problem: Low SPE Recovery
Possible Causes of Low Recovery
– Extraction Device Capacity
– Poor Retention
– Chemical Stability
o Apparent low recovery
– Non-specific binding
– Protein binding
– Not strong enough elution solvent or enough volume
©2012 Waters Corporation 49
Low SPE Recovery
Possible Solutions for Low Recovery
– Extraction Device Capacity
o Load less sample, increase sorbent bed mass
o Change pre-treatment to eliminate interferences
– Poor Retention
o Use appropriate mixed-mode sorbent and ensure charge through proper pretreatment
o Eliminate protein binding
– Chemical Stability
o Run MS scan and look for expected mass shifts
– Non-specific binding
o Eliminate any 100% aqueous steps
o Add TWEEN or carrier protein to solvent standards or matrices with low protein content (CSF, urine, etc)
– Protein binding
o Change pretreatment
• Protein precipitation, denaturation with guanidine HCl or urea, stronger acid or base
©2012 Waters Corporation 50
Troubleshooting Recovery
Time0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00
%
1
1ngmL_5050_N4OH_020810_001 MRM of 3 Channels ES- TIC
4.23e4
5.69
5.42
5.40
5.39
5.38
4.704.60
5.44
5.44
5.45
5.67
5.71
5.88
5.89
5.92
5.936.21
6.956.77 7.527.077.75
SPE Recovery = 94%
SPE Recovery = 92%
SPE Recovery = 64%
3 peptides of the same class, extracted from human CSF Elution solvent contains 65% ACN and 5% NH4OH
©2012 Waters Corporation 51
Oasis® MCX μElution Plate
Condition, Equilibrate
Load: 150 μL diluted, pretreated sample
Wash 1 and 2
Elute: 2 X 25 µL 75/10/15 ACN/conc. NH4OH/water (by volume)
Dilute: 25 µL water
Inject: 10 µL
Sample Extraction: Final Method
Amyloid β
Peptide
% SPE
Recovery
1-38 94%
1-40 92%
1-42 90%
Fully solubilizes aβ 1-42
©2012 Waters Corporation 52
Troubleshooting Recovery
Recovery is 94% in human CSF but 40% in human plasma
What is the problem?
— Most likely to be protein binding
What can we do?
Modify pretreatment
— Dilute with stronger acid
— Dilute with base
— Denature with guanidine HCl (typically 5M, 9:1
guanidine:plasma)
©2012 Waters Corporation 53
Amyloid Peptides: Troubleshooting Non-specific Binding in SPE Recovery
Solvent standards used for initial SPE method development — SPE recovery = 60%
Amyloid Beta Peptides — >4000 MW
— > 50% hydrophobic residues
Mass Balance Results — Nothing in load fraction or wash fractions
Possible Causes? — Elution Solvent too weak or not enough volume
— Non-specific binding
0
10
20
30
40
50
60
70
80
90
100
Solvent Std Solvent Std + 0.05% Rat Plasma
% SPE Recovery
©2012 Waters Corporation 54
Outline
Introduction
— Basic screening methods
— Typical challenges faced
Understanding your peptide
o pI, HPLC index, hydrophobic/hydrophilic
Understanding the challenges: symptoms,
examples, and solutions for common problems
Conclusions
©2012 Waters Corporation 55
Conclusions
Using predefined guidelines to learn about your peptide can help
predict potential problems and can guide you towards solutions.
Many problems stem from 5 major causes: protein binding,
non-specific binding, solubility, specificity and stability.
If you are aware of what the challenges might be, there is often
a well-defined solution and/or decision-making flow chart to
resolve them.
©2012 Waters Corporation 56
Acknowledgements
Waters Corporation
Erin Chambers
Debadeep Battacharya
Catalin Doneanu
Kenneth Fountain
Gordon Fujimoto
Joanne Mather
Paul Rainville
Martha Stapels
Hua Yang
ICON PLC
Jon Bardsley
Eileen Collins
Sally Hannam
Liz Thomas
©2012 Waters Corporation 57
Thank You!
Questions?
Landing Page…
http://www.waters.com/May1
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Separation and Sample Preparation
–Full Webinar Recording of Today’s Session
–PDF Slide Deck
–Compilation of Literature, White Papers, Brochures,
Application Notes
General Questions – [email protected]