Automated Disulfide Mapping - Waters Corporation...Automatic Disulfide Bond Mapping and Reporting...

Automatic Disulfide Bond Mapping and Reporting

for Biotherapeutics by High Resolution LCMS

Oral presentation at ASMS 2011 – Denver - Wed p.m. WOF

©2011 Waters Corporation 1

St John Skilton, Hongwei Xie, Scott Berger and Weibin Chen

Why are Disulfide Bonds Important?y p

Organisations need to show that they have understood their process and/ or product mapping the disulfide bonds is process and/ or product - mapping the disulfide bonds is essential to demonstrate this knowledge to the regulator

Arrangement of bonds may affect the efficacy of a biotherapeutic product

May relate to stability of a biotherapeutic protein

P R O C E S SS T R U C T U R E

Primary Assembly

P R O C E S SS T R U C T U R E

Secondary

Tertiary

Folding

Packing


Quaternary

ac g

Interaction

Relevance to the Field of Protein Analytics – Regulatory Authoritiesy g y

“Our current ability to predict the potency of biologics would be

enhanced if we had improved ability to measure and quantify

the correct (major) three-dimensional structure, aberrant

three-dimensional structures (misfolding), and the distribution

of different three-dimensional structures”.

• [TESTIMONY BEFORE THE SUBCOMMITTEE ON TECHNOLOGY AND INNOVATION COMMITTEE ON SCIENCE AND TECHNOLOGY U.S.

]HOUSE OF REPRESENTATIVES. SEPTEMBER 24, 2009]


Steven Kozlowski, M.D.Director, Office of Biotechnology Products, Office of Pharmaceutical Science. CDER

Previous and Recent Work - ASMS 2010

Disulfide bonds by ‘differential’ MS (Yi and Hoang, Merck; ASMS 2010): reduced and non-reduced in multiple runs— ‘Software tools are highly in demand to predict theoretical

fragment ion masses to facilitate the disulfide containing peptide identification and characterization’

Manual interpretation precludes ‘routine’ application


Obtaining Disulfide Bond Informationg

‘Prerequisites’:— Use of chemical/ enzymatic knowledge – e.g. LysC Use of chemical/ enzymatic knowledge e.g. LysC

cleavage at Lysine useful to create large fragments of IgG molecules

— ‘Effective’ resolution: 20,000 FWHM on Xevo™ G2 Tof, f f l h lQTof or Synapt™ G2 facilitates this analysis

— Wide Mass Range: large and small (e.g. 15kDa peptide vs 400 Da) peptides are analysed identically on a G2 QTof/ SynaptQTof/ Synapt

Analysis tools:— Simultaneous MS/MS capability to confirm assignmentsSimultaneous MS/MS capability to confirm assignments

o UPLC/MSE is the leading tool on the market

— Logical workflow to effectively perform the automated assignment of species


o BiopharmaLynx to process the data automatically

Challenges in Computing Disulfide Bonded Peptides – Simple Casesp p

Type 1a: Intra-Peptide (‘Simplest’ Case): only one species to consider

d t ti t i i lXXCXXCXX

Disulfide Bond

and computation trivial

Type 1b: Inter-Peptide: Computationally more demanding, b t li it d b f

Disulfide Bonded Peptides

but a very limited number of possibilities

XXCXX XXCXXXX

[Segment removed by


Enzymatic cleavages, e.g. Trypsin, LysC, …]

Challenges in Computing Disulfide Bonded Peptides – Complex Casesp p

Type 2: One or more peptide has two Cys

— All options for S-S bonds have to be computed but possibilities are limited XXCXXCXX XXCXXXX

— Peptides have to be considered together and separately

Cases of different bond re arrangement

XXCXX

— Cases of different bond re-arrangement may become ‘Type 1’

XXCXXCXX XXCXX


XXCXX

Challenges in Computing Disulfide Bonded Peptides – Highly Complex CasesPeptides – Highly Complex Cases

Type 3: One or more peptide has XXCXXXXCXXCXXCXX

three or more Cys residues

These present a ‘Combinatorial’ Challenge as connection possibilities

XXCXX

XXCXXCXXCXX XXCXX

XXCXXCXXCXX

g pproliferate:

— Multiple possible arrangements for bondsbonds

o Internal to peptide

o External to other peptides

Multiple missing bonds possible

XXCXCXX

XXCXXCXXCXX

XXCXXCXXCXX

— Multiple missing bonds possible

— Potentially more than a hundred possibilities from just 4 bonds (E.g. G pta et al Anal Chem 2010 82

XXCXXCXXCXX

XXCXX


Gupta et al, Anal. Chem. 2010, 82, 8313–8319) (etc.)

Classes of Compound that Exhibit Multiply-bonded Peptidesp y p

BSA, monoclonal Antibodies all have types 1 and 2

— BSA illustrates the variety of bondsS ust ates t e a ety o bo ds

— IgGs illustrate the applicability to large, therapeutic proteins

XXCXXCXX XXCXX

XXCXX XXCXXXXCXXCXX

XXCXXCXX XXCXX

XXCXX

IgGs also have Type 3 intramolecular bonds between Heavy and Light Chains

BSA has a type 3 variant where one bond is ‘nested’ within another BSA has a type 3 variant where one bond is nested within another peptide:

T28-T37 CASIQK


ECCHGDLLECADDTryptic Peptide 28 is linked to Tryptic Peptide 37

Expected Bonds in IgG1 after LysC digestion

Trastuzumab (IgG1) S-S bonds are predominantly type 1

g

14 Expected Bonds in ‘Canonical’ scheme

Symmetry of molecule means that some LysC peptides are identical irrespective of chain so that there are 8 unique peptidesidentical irrespective of chain so that there are 8 unique peptides

Light Chain (1)1:K01 1:K04 1:K07 1:K13 1:K14

Disulfide Bond

Light Chain (1)

Heavy Chain (2)

2:K052:K01 2:K212:K16 2:K312:K272:K132:K082:K07 2:K14

1:K01 1:K04 1:K07 1:K13 1:K14

Peptides from LysC digestion (K)

4:K084:K07 4:K214:K16 4:K314:K274:K13

Heavy Chain (4)

4:K054:K01 4:K14


3:K01 3:K04 3:K07 3:K13 3:K14 Light Chain (3)

Heavy Chain (4)

BSA – Schematic Representation of Theoretical S-S Tryptic Peptidesyp p

TCVADESHAGCEKT07

T08-T10-T12 NECFLSHKSLHTLFGDELCK ETYGDMADCCEK

T14-T21-T22 LKPDPNTLCDEFK YNGVFQECCQAEDK GACLLPK

CASIQK ECCHGDLLECADDT28-T37

T39-T41-T42 YICDNQDTISSK SHCIAEVEK ECCDKPLLEK

T44-T50-T51 DVCK EYEATLEECCAK DDPHACYSTVFDK

T54-T61-T62 QNCDQFEK MPCTEDYLSLILNRVGTRCCTKPESER


RPCFSALTPDETYVPKLCVLHEK CCTESLVNRT63-T66-T67

T69-T76-T77 LFTFHADICTLPDTEK CCAADDK EACFAVEGPK

Complexity of Raw Data: Timep y

Typical LC/MS peptide map data with alkylated, non-reduced protein digestreduced protein digest— Method needs to cope with many disulfide peptides in BSA of

different types

W d i ll d i ll id — We need to automatically process and assign all peptide types … All in a single run


Complexity of Raw Data: Spectral Interpretation

Raw Spectral Data is very complexM l i t t ti i hibiti l ti i

p

— Manual interpretation is prohibitively time-consuming

Each Peak would have to be deconvoluted Separately in a manual analysis


Complexity of Raw Data: Sequence Confirmation by MS/MS

Each Peptide Generated with Sulfide Bonds creates multiple termini this creates a high computational searching load

y

termini - this creates a high computational searching load for MS/MS data [manually prohibitive]

NECFLSHKSLHTLFGDELCK ETYGDMADCCEKXX XX

Each Peptide Created leads to multiple new peptide termini to

T08-T10-T12 NECFLSHKSLHTLFGDELCK ETYGDMADCCEKXX XX

compute and match from Complex, Overlapping MS/MS spectra


A d P i d A i b Bi h L ™Automated Processing and Annotation by BiopharmaLynx™


Automated Workflow to Assign Peptides in a Peptide Map – for ALL typesp p yp

Same rules need to be applied whether Peptides are S-S bonded or not

In-silico Digestion of Peptides are S-S bonded or not

Workflow must be transparent and not introduce bias— No database is used: calculations

Sequence

— No database is used: calculations performed on the fly to create a list of potential matches

— Matches are made to the ‘list’ using

Assign Peptides by Accurate Mass

accurate mass and then multiple other criteria

Validate Assignments Allow Alternatives within Criteria (mass using MS/MS

(error, modifications, RT window,

fragment ions)


Tabulate and Display

C4/ C18 BEH Peptide columns - UPLC MSE

– Xevo or Synapt Qtof – BiopharmaLynx

Basics of BiopharmaLynx™ Displayp y p y

Entire Data Set Tabulated Here

Each Processed Peak labelled Each Processed Peak labelled with Peptide Number‘Control’ Sample Display

for Chromatogram (processed)

Hoverbox for

‘Analyte’ Sample Display

‘=‘ Symbol represents


Peptide Information S-S bond

BSA Results - Automated assignment of Expected Peptides from Tryptic Digest p p yp g

With BiopharmaLynx™ All 9 peptides with expected S-S bonds are assigned automaticallyare assigned automatically

‘hoverboxes’ show sequences at processed peak apex

T08 T10 T12

T14-T21-T22T39-T41-T42

T07

T08-T10-T12

T63-T66-T67

T28-T37 T44-T50-T51 T54-T61-T62

©2011 Waters Corporation 18T69-T76-T77

Results of Automated Search for low intensity S-S peptide in BSAy p p

T69-T76-T77 is at lowest intensity of expected S-S peptides

— 0.47% of highest intensity peptide (T14-T21-T22)

o NB: Ionisation efficiencies vary for different S-S peptides

Unique Match (2ppm from theoretical)— Unique Match (2ppm from theoretical)

T69-T76-T77


‘Non-canonical’ proposals – example from BSA

Some additional proposals are plausible and supported

T62

are plausible and supported by fragment ion information— Can now be investigated

further

T67

— Manual investigation may not have identified this


Automated Mapping of an IgG1

(Alkylated to protect exposed Cysteine residues and inhibit alternative S-S bond formation)alternative S S bond formation)


IgG1 - Seven of Eight Expected Peptides assigned automaticallyg y

Only one S-S peptide not assigned – not detected in the raw data [hydrophilic peptide elutes in void volume]data [hydrophilic peptide elutes in void volume]

2:K14

4:K142:K212:K16

1:K07 1:K13

2:K082:K07

1:K01 1:K04

2:K052:K01


2:K312:K27

Automated Validation of Assignments –Expected S-S peptide in IgG1p p p g

BiopharmaLynx provides additional evidence with Fragment Ion data even with large peptides (8kDa)data even with large peptides (8kDa)

2:K082:K07


Automated Mapping of Scrambled S-S Bonds

(Non-alkylated IgG1 – alternative S-S bonds may form)(Non alkylated IgG1 alternative S S bonds may form)


Method editor allows a choice of ‘Strict’ or ‘Relaxed’ Search Criteria

Strict Criteria generally usable for IgGs where bonds should be well characterizedwell-characterizedRelaxed Criteria for unknown conditions where criteria allow multiple possibilities for further investigationTi k b t ll f bl d di lfidTick-box to allow for scrambled disulfides— IgG1 Sample not alkylated prior to digestion to allow disulfide bond

scrambling


Scrambled Disulfide Bond Sample –IgG1g

7 New Species identified with high confidence (more than 5 fragment ions in MS/MS spectrum)fragment ions in MS/MS spectrum)

1 Canonical S-S bonded peptide no longer detected (2:K01-2:K05)

1:K01 1:K04 1:K07 1:K13 1:K14 Light Chain (1)

Heavy Chain (2)

2:K052:K01 2:K14 2:K212:K16 2:K312:K272:K132:K082:K07


4:K14

Heavy Chain (4)

Scrambled Bonds in Non-alkylated IgG1 – New Species in processed datap p

dd l bl d b d

2:K162:K13

Additional Scrambled S-S bond Assignments Made Automatically in non-alkylated Sample

2:K132:K08

2 K011 K13

1:K07 2:K16

1:K07 2:K07

2:K07 2:K16

2:K011:K13

2:K161:K13

1:K07 2:K16


Appearance of New Assignment in Non-alkylated Sample within Heavy Chainy p y

E d B d

2:K212:K16

1 K07 1:K13

Expected Bond Expected Bond

1:K07 1:K13

2:K162:K13

Additional Scrambled S-S bond in non-alkylated sample


Automated Validation of Assignments –Scrambled S-S peptide in IgG1p p g

BiopharmaLynx provides additional evidence also for scrambled disulfides with fragment ion information obtained disulfides with fragment ion information obtained simultaneously


Conclusions

A bioinformatics approach for confirmation of known disulfide bonds, and search for scrambled disulfides has been developed.

The approach automatically assigned disulfides of several The approach automatically assigned disulfides of several structural classes within BSA and IgG digests.

Such analyses can be executed from a single analytical run.Such analyses can be executed from a single analytical run.

We believe this tool will allow biopharmaceutical organisations and researchers to more quickly satisfy increasing regulatory requirements for higher order structural analysis of new biotherapeutics and biosimilars.


Acknowledgmentsg

Richard DennyWeibin Chen

Scott BergerHongwei Xie

y

g

Barry Dyson

Keith Richardson


Keith Richardson

Automated Disulfide Mapping - Waters Corporation...Automatic Disulfide Bond Mapping and Reporting...

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