Protein and Peptide Therapeutics: Addressing … and Peptide Therapeutics: Addressing Bioanalytical Demands of ... and solutions for common problems ... tend to be more “sticky”

©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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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.


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


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


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


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


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


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


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


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


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


Outline

Introduction







Conclusions


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


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


Understanding Your Peptide: Step 1

Teriparatide (1-34 fragment of human parathyroid hormone)

example: Obtain sequence

1.




example: Calculate pI and HPLC index

2.

Teriparatide is basic and sticky




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




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


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


Outline

Introduction







— Solubility

— Specificity

— Protein Binding

— Non-specific Binding

— Peptide Loss

Conclusions

— Poor Sensitivity

— Chromatography and Carryover

— Low SPE Recovery


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


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.


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.


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


3.66e5 Area

0.87 27262


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


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?


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


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


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


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


4.46

3.922.884.56 8.00 8.51


3.19e11

5.76

4.46

3.922.884.56 8.00 8.51


3.19e11

2.88

2.18

5.513.223.49 3.97

4.905.62

8.027.276.45 9.068.29

9.19


3.22e11

2.88

2.18

5.513.223.49 3.97

4.905.62

8.027.276.45 9.068.29

9.19


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


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………


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


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


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



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



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


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


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


5.57e5

1.06


5.57e5 1.08



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


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


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


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


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


Improving Chromatography: Ligand and Pore Size

ACQUITY BEH 1.7 µm C18 300Å



Enfuvirtide: MW 4492


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


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


2.85e6


2.85e6

New column:1st injection after solvent blanks

2nd injection after solvent blanks

After 9 injections of precipitated plasma

Insulin Glargine


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)


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


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


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


2.98e5 Area

1.08 22737


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


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


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


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


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


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)


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


Outline

Introduction







Conclusions


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.


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


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]

Documents

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