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Bioanalysis of PEGylated Proteinsby LC-MS
Jonathan St-Germain
S i S i ti t Senior Scientist,
Bioanalytical Services, Algorithme Pharma
Polyethylene Glycol (PEG)
O
H
O
OH
• Water soluble (flexible and hydrated polymeric backbone)
H OHn
• Water soluble (flexible and hydrated polymeric backbone)
• Soluble in several organic solventsBi tibl• Biocompatible
• FDA-approved for dermal, oral and intravenous applications
F d i l i h ki l i• Found in laxatives, toothpastes, skin creams, electroniccigarettes, paintball filling, etc.
But for our application of interest…
22 Bioanalysis of PEGylated Proteins by LC-MS
PEGylated ProteinsPEGylated Proteins
Pasut and Veronese (2012) J Control Release.
33 Bioanalysis of PEGylated Proteins by LC-MS
Improved Pharmacological Propertiesp g pof Protein Therapeutics
Veronese and Pasut (2005) Drug Discov Today.
44 Bioanalysis of PEGylated Proteins by LC-MS
Improved Pharmacokinetics
Human blood levels after subcutaneous injection of IFN-α2a and PEG-IFN-α2a.
Interferon-α2a PEG-Interferon-α2a.
Harris and Chess (2003) Nat Rev Drug Discov.
5 Bioanalysis of PEGylated Proteins by LC-MS
Better pharmacodynamics
TNF-related apoptosis-inducing ligand (TRAIL)
TRAIL 30K-PEG-TRAIL
Kim et al. (2011) Bioconjug Chem.
66 Bioanalysis of PEGylated Proteins by LC-MS
PEGylated Protein Therapeuticsy p
Great for patients but what aboutGreat for patients…but what about analytical biochemists?
Bioanalysis of PEGylated Proteins by LC-MS7
General Protein Quantification by LC MSGeneral Protein Quantification by LC-MS
Protein in Biological Matrix
Capture No Capture
Digestion No Digestion Digestion No Digestion
LC-MS
8 Bioanalysis of PEGylated Proteins by LC-MS
PEGylated Protein Quantification by LC MSPEGylated Protein Quantification by LC-MS
Protein in Biological Matrix
Capture No Capture
Digestion
LC-MS
9 Bioanalysis of PEGylated Proteins by LC-MS
Protein PEGylation relatedProtein PEGylation-relatedLC-MS Analytical Challenges
• PEG size polydispersity – prevents intact proteinquantification by LC-MS
1010 Bioanalysis of PEGylated Proteins by LC-MS
Protein PEGylation-related Analytical ChallengesIntact PEGylated Protein by MS
Charge sates
Si l dil ti
Charge sates
Signal dilution over
PEG size
Forstenlehner (2014) Anal Chem.
Isotope distribution
11 Bioanalysis of PEGylated Proteins by LC-MS
Protein PEGylation related Analytical ChallengesProtein PEGylation-related Analytical Challenges
• PEG size polydispersity – prevents intact proteinquantification by LC-MS
• PEG attachment site - limits choice of signature peptide
1212 Bioanalysis of PEGylated Proteins by LC-MS
PEGylated ProteinsPEGylated Proteins
Pasut and Veronese (2012) J Control Release.
1313 Bioanalysis of PEGylated Proteins by LC-MS
Impact of PEGylation on Tryptic Digestion
hGH
PEGylated hGH
Trypsin digestion impeded at PEGylated lysines resulting in miscuts,
Clark et al. (1996) J Biol Chem.
yp g p y y g ,absent peptides and altered digestion profile
1414 Bioanalysis of PEGylated Proteins by LC-MS
Protein PEGylation related Analytical ChallengesProtein PEGylation-related Analytical Challenges
• PEG size polydispersity – prevents intact proteinquantification by LC-MS
• PEG attachment site - limits choice of signature peptide
• Internal Standard unavailability – risk of variability
1515 Bioanalysis of PEGylated Proteins by LC-MS
Extraction Methods for PEGylated Proteins
Protein precipitation Immunoaffinity purification
PEGylated protein solubility in organic solvents allows protein precipitation for extraction of PEGylated proteins and reduced sample complexity
Xu et al. (2010) Anal. Chem.
1616 Bioanalysis of PEGylated Proteins by LC-MS
Case Study: LC-MS Quantification of Pegvisomant
• PEGylated human growth hormone (hGH) structural analog• PEGylated human growth hormone (hGH) structural analog• hGH Receptor antagonist• Used in treatment of acromegaly (excess pituitary hGH production)
Kopchick et al. (2013) Mol Cell Endo.
1717 Bioanalysis of PEGylated Proteins by LC-MS
LC-MS quantification of Pegvisomant:q gTryptic digestion of a Lys-PEGylated protein
Surrogate peptide should be:Surrogate peptide should be:
• Arginine-flanked
P i t ifi• Pegvisomant-specific
ArgArg ValThrLeu Phe Ser -NC-
-CN- ArgThr LeuPheSerValArg RT LFSV Pegvisomantgg g
RT LFEV hGH
1818 Bioanalysis of PEGylated Proteins by LC-MS
Di T i Di i f P iDirect Tryptic Digestion of Pegvisomant
Pegvisomant-spiked rat plasma
Dilution in NH4HCO3
Reduction (DTT) / Alkylation (IAA)
Tryptic digestionTryptic digestion
LC-MS/MS
19 Bioanalysis of PEGylated Proteins by LC-MS
LC-MS Pegvisomant Quantification in Rat PlasmaLC MS Pegvisomant Quantification in Rat Plasma
Blank LLOQ(50 ng/mL)
ULOQ(50 µg/mL)
Inte
nsity
(cps
)
Time (minutes)
20 Bioanalysis of PEGylated Proteins by LC-MS
LC-MS Pegvisomant Quantification in Rat Plasma
C lib tNominal
CCalculated
CNominal
C CV*C lib tNominal
CCalculated
CNominal
C CV*
LC MS Pegvisomant Quantification in Rat Plasma
Calibrant Conc. Conc. Conc.(ng/mL) (ng/mL) (%) (%)
P1 (LLOQ) 50 51 103 9.4P2 100 110 110 6 1
Calibrant Conc. Conc. Conc.(ng/mL) (ng/mL) (%) (%)
P1 (LLOQ) 50 51 103 9.4P2 100 110 110 6 1P2 100 110 110 6.1P3 250 247 99 2.5P4 500 470 94 6.0
P2 100 110 110 6.1P3 250 247 99 2.5P4 500 470 94 6.0P5 1000 976 98 5.7P6 2500 2589 104 4.1P7 7500 7639 102 4.1
P5 1000 976 98 5.7P6 2500 2589 104 4.1P7 7500 7639 102 4.1P8 12500 12372 99 4.7P9 20000 21056 105 2.4P10 35000 34667 99 1.7
P8 12500 12372 99 4.7P9 20000 21056 105 2.4P10 35000 34667 99 1.7P11 42500 42833 101 3.8P12 (ULOQ) 50000 49056 98 2.6*n=3
P11 42500 42833 101 3.8P12 (ULOQ) 50000 49056 98 2.6*n=3
What if I need more sensitivity and selectivity?21 Bioanalysis of PEGylated Proteins by LC-MS
Immunoprecipitation (IP)Immunoprecipitation (IP)
anti-Protein Antibody
PEGylatedProteinProtein
anti-PEG Antibody
22 Bioanalysis of PEGylated Proteins by LC-MS
Immunoprecipitation (IP) + LCMS
PEGylatedProtein
Wash, Elute and/or Digest
LC-MS
23 Bioanalysis of PEGylated Proteins by LC-MS
Automated IP Sample Processing
KingFisher® Flex: Magnetic Particle Processor
24 Bioanalysis of PEGylated Proteins by LC-MS
Hybrid Extraction vs Direct DigestionHybrid Extraction vs Direct Digestion
Rat Whole Blood Spiked with Pegvisomant at 5 µg/mL
Blood Plasma Separation
ti PEG IP ti hGH IPanti-PEG IP anti-hGH IP DilutionReduction (DTT) / Alkylation (IAA)( ) y ( )
Tryptic digestion
LC-MS/MS
25 Bioanalysis of PEGylated Proteins by LC-MS
Hybrid Extraction vs Direct DigestionHybrid Extraction vs Direct Digestion
anti-PEG IP anti-hGH IP Direct Digest
sity
(cps
)A
rea
Inte
nsA
Pegvisomant Concentration (µg/mL)
26 Bioanalysis of PEGylated Proteins by LC-MS
Hybrid Extraction vs Direct DigestionHybrid Extraction vs Direct Digestion
anti-PEG IP anti-hGH IP Direct Digest
ps)
Blank
nten
sity
(cp
In
LLOQ (50ng/mL)
Time (min)
27 Bioanalysis of PEGylated Proteins by LC-MS
Case study: PEGylated PeptideCase study: PEGylated Peptide
38 amino acids
NH2- -COOH
20-kDa PEGRegion of interest
28 Bioanalysis of PEGylated Proteins by LC-MS
PEGylated Peptide Extraction: Protein PrecipitationPEGylated Peptide Extraction: Protein Precipitation
Protein Precipitation
Keep Supernatant
E t DEvaporate Dry
Resuspend in Digestion Bufferp g
Digest
LC-MS
29 Bioanalysis of PEGylated Proteins by LC-MS
PEG l t d P tid E t ti P t i P i it tiPEGylated Peptide Extraction: Protein Precipitation
Blank LLOQ (0.50 ng/mL) ULOQ (50.0 ng/mL)
S/N=5.1
30 Bioanalysis of PEGylated Proteins by LC-MS
PEGylated Peptide Extraction:PEGylated Peptide Extraction: Protein Precipitation + Immunopurification
Protein Precipitation
Keep Supernatant
E t DEvaporate Dry
Resuspend in IP Bufferp
IP with anti-peptide antibody
On-bead digestion
LC-MSLC MS
31 Bioanalysis of PEGylated Proteins by LC-MS
PEGylated Peptide Extraction: y pProtein Precipitation + Immunopurification
S/N=15.3
32 Bioanalysis of PEGylated Proteins by LC-MS
Precision and Accuracy:Precision and Accuracy:Protein Precipitation + Immunopurification
Line # LOQ QC0.500 Line # QC1
1.500 Line # QC215.000 Line # QC3
37.50013 0.566 34 1.538 18 12.060 28 32.107
Concentration (ng/mL)CurveCode
13 0.566 34 1.538 18 12.060 28 32.10714 0.507 24 1.906 26 14.599 19 29.65622 0.568 15 1.530 17 13.811 29 39.89823 0.568 16 1.670 27 15.912 36 34.82033 0 551 25 1 249 35 16 000 37 30 268
ALG12
33 0.551 25 1.249 35 16.000 37 30.268Mean 0.552 1.579 14.476 33.350S.D. 0.026 0.239 1.634 4.175
N 5 5 5 5% C V 4 7 15 1 11 3 12 5% C.V. 4.7 15.1 11.3 12.5
% Nominal 110.4 105.2 96.5 88.9
33 Bioanalysis of PEGylated Proteins by LC-MS
Summary
PEGylated Protein Bioanalysis by LC-MS
PEG:U i A id iUse it or Avoid it
U it A id itUse it Avoid it
PEG IPProtein Precipitation
Protein IPDirect DigestionProtein Precipitation Direct Digestion
Peptide SelectionImplications for Assay Validation?
3434 Bioanalysis of PEGylated Proteins by LC-MS
AcknowledgementsAcknowledgements
Algorithme Pharma’s R&D Group
35 Bioanalysis of PEGylated Proteins by LC-MS