h d dWhy do LC‐MS and LBA results differ?results differ?

A literature evaluation

18 November 2015

Nico van de Merbel

An overviewAn overview

In most published BA methods for large molecules, no comparison of platforms (LBA vs LBA, LBA vs LCMS or LCMS vs LCMS) is undertaken

If there is a comparison, in just a few cases there is a difference of <20%, this is mainly forthis is mainly for • smaller proteins• spiked samples • incurred samples after short exposure

Differences usually are not convincingly explainedDifferences usually are not convincingly explained

Bults et al, Expert Rev. Proteomics, 12 (2015) 355-374

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Proteins vs small moleculesProteins vs small molecules

Small molecules are well-defined, single species

Proteins are often heterogeneous and may occur in several different (iso)forms

Small molecules are often loosely bound to plasma proteins and the binding is easily broken during analysis

Proteins may form very stable complexes with other plasma proteins and the binding can be difficult to disrupt during analysis

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Protein formsProtein forms

Modifications:

CHCH CH3

S

CH3

SCH3

S

C=ONH2

native deamidation proteolysis

C=OOH

C=ONH2

native deamidation proteolysis

CH3 CH3

S=OCH3

S

oxidation 3D changeC=ONH2

C=ONH2

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g

Protein formsProtein forms

Binding:

CH CHCH3

SCH3

S

unbound target bound

C=ONH2

C=ONH2

unbound target-bound

CH3 CH3CH3CH3

SCH3

S S

aggregated ADA-boundC=ONH2

C=ONH2

C=ONH2

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gg g

Protein quantificationProtein quantification

Both LBA and LC-MS/MS respond to only a (small) part of the protein

Depending on the analytical mechanism, modifications and/or binding events may or may not be picked up

LBA LC-MS/MS

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LBA LC MS/MS

Some literature examplesSome literature examples

• LBA vs LBA

• LC-MS vs LC-MSLC MS vs LC MS

• LBA vs LC-MS

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LBA vs LBALBA vs LBA

Influence of capturing antibody: monoclonal vs polyclonal

Example: ocrelizumab

Concentrations in patient plasma ± 2–fold lower with mAB than with pAB

pAB

mAB

pAB

mAB

Fischer et al, mAbs, 4 (2012) 623-631

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Reproduced with permission, copyright Taylor & Francis, 2012

LBA vs LBALBA vs LBA

Pharmacological target CD20 and monoclonal capturing antibody bind to the exact same epitope of ocrelizumab

• no (or reduced) analyte recognition by ELISA when target-bound

Polyclonal capturing antibody binds to multiple epitopes

• also (partial) analyte recognition by ELISA when target-boundalso (partial) analyte recognition by ELISA when target bound

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LBA vs LBALBA vs LBA

No such effect for another CD20-directed therapeutic antibody

The nature of the capturing reagent and the exactreagent and the exact epitope to which it binds determine the concentrationdetermine the concentration result

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Reproduced with permission, copyright Taylor & Francis, 2012

LBA vs LBALBA vs LBA

Influence of detection antibody: different specificities of commercial ELISAs

Example: PTH

Concentrations depend on detection antibodyspecificity

150

200

pons

e fo

rm

a (%

)specificity

50

100

lativ

e re

spH

in p

lasm

01-34 1-12 1-6

Detection antibody raised against amino acids

Rel

PTH

Sukovaty et al, J. Pharm. Biomed. Anal. 42 (2006) 261-271

Detection antibody raised against amino acids

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LBA vs LBALBA vs LBA

The truncated forms of PTH, [3-84] and [7-84], are present in plasma at similar concentrations as full length PTH [1-84]

• They cross-react with the detection antibodies directed to amino acids 1-34 and 1-12, which leads to overestimation of PTH concentrations

• They don’t cross-react with the detection antibody directed to amino acids 1-6

The nature of the capturing reagent and the exact epitope to which it binds determine the concentration result

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LC-MS vs LC-MSLC MS vs LC MS

Influence of signature peptide: stable vs unstable

Example: trastuzumab

Concentrations in patient plasma lower with unstable signature peptide

400

300

350

400

/mL)

150

200

250 Series1

Series2

Series3

Series4

FTISADTSKIYPTNGYTRIYPTDGYTRIYPTisoDGYTRnt

ratio

n (µ

g/

0

50

100Series4IYPTisoDGYTR

Con

cen

Bults et al, submitted for publication

00 200 400 600 800

Time (h)

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LC-MS vs LC-MSLC MS vs LC MS

Stable part of the protein is not metabolized (total trastuzumab),

Unstable part is in vivo deamidated and decreases in concentration (non-deamidated trastuzumab)

IYPTDGYTRIYPTDGYTR

IYPTisoDGYTRIYPTsuccGYTRIYPTNGYTR

IYPTisoDGYTR

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LC-MS vs LC-MSLC MS vs LC MS

Influence of extraction approach

Example PEGylated protein:Digestion without further treatment gives up to 35% higher concentrations than immunocapture (PEG-directed) plus digestion

Xu et al, Anal. Chem. 82 (2010) 6877-6886

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Reproduced with permission, copyright ACS, 2010

LC-MS vs LC-MSLC MS vs LC MS

In vivo dePEGylation to a product that has the same signature peptide:

HAibDGTFTSDYSKYLDSRRAQDFVQWLMNTKRNNIAC-PEG

HAibDGTFTSDYSKYLDSRRAQDFVQWLMNTKRNNIACHAibDGTFTSDYSKYLDSRRAQDFVQWLMNTKRNNIAC

Direct digestion: concentration of PEGylated plus dePEGylated formsDirect digestion: concentration of PEGylated plus dePEGylated formsImmunocapture: concentration of PEGylated form only

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LBA vs LC-MSLBA vs LC MS

Influence of anti-drug antibodies

Example: PEGylated protein:

Lower concentrations (up to 8-fold) with ELISA than with LC-MS/MS

Reproduced with permission, copyright Springer, 2012

Wang et al Anal Bioanal Chem 402 (2012) 1229-1239

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Wang et al, Anal. Bioanal. Chem., 402 (2012) 1229 1239.

LBA vs LC-MSLBA vs LC MS

LC-MS/MS:

• Extraction into isopropanol, digestion

ELISA:

C t i ith h l i l t t d t ti ith ti PEG• Capturing with pharmacological target, detection with anti-PEG antibody

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LBA vs LC-MSLBA vs LC MS

Presence of ADAs specific to target binding site was demonstrated

These interfere with the ELISA capturing step and decrease the detectable concentration, but do not interfere with extraction and digestion in the LC-MS/MS assay

Note: discrepancy between ELISA and LC-MS/MS only at later time-points

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LBA vs LC-MSLBA vs LC MS

Influence of circulating target

Example: monoclonal antibody:

Lower concentrations (up to 3-fold) with ELISA than with LC-MS/MS

Reproduced with permission, copyright ACS, 2010

Zhang et al, Anal. Chem., 86 (2014) 8776-8784.

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LBA vs LC-MSLBA vs LC MS

LC-MS/MS:

• Immunocapture with anti-Fc antibody, digestion

YELISA:

• Anti-idiotypic antibodies for capturing and detection

Y11/23/201521

Y

LBA vs LC-MSLBA vs LC MS

Presence of target does not interfere with the LC-MS capturing step and digestion

YPresence of target interferes with the ELISA capturing and/or detectionstep and decreases the detectable concentrationstep and decreases the detectable concentration

Y11/23/201522

Y

LBA vs LC-MSLBA vs LC MS

Influence of structural modification

Example: trastuzumab:

Lower concentrations with ELISA than with LC-MS/MS

LC-MS/MS: FTISADTSK

LC-MS/MS: IYPTNGYTR

ELISA

Bults et al, submitted for publication

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LBA vs LC-MSLBA vs LC MS

Epitope / signature peptide is deamidated

This leads to a decrease in concentration for LC-MS/MS and a two-fold faster decrease for ELISA due to lost recognition

LC-MS/MS (YPTNGYTR):Non deamidated: full response

ELISA:Non deamidated: full responseNon-deamidated: full response

Singly deamidated: half responseDoubly deamidated: no response

Non-deamidated: full responseSingly deamidated: no responseDoubly deamidated: no response

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Conclusion

• Proteins are large complex molecules which often occur in multiple

Conclusion

• Proteins are large, complex molecules which often occur in multiple structural forms and may be bound to other proteins in a sample

• Quantification by LC MS and LBA is based on only a smart part of a• Quantification by LC-MS and LBA is based on only a smart part of a protein structure and disregards a major part of the analyte molecule

• The design of an analytical method determines whether or not a• The design of an analytical method determines whether or not a structural modification or binding event is picked up

• “The” protein concentration is not a meaningful result unless it is• The protein concentration is not a meaningful result, unless it is defined which molecular property the method responds to.

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AcknowledgmentAcknowledgment

Kees Bronsema Daniel Wilffert Samenwerkingsverband

Peter Bults Rainer Bischoff Noord-Nederland

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