Characterization of a Heavily Glycosylated Fusion Protein ...€¦ · 4.00 6.00 8.00 10.00 12.00...

Keith A. Johnson, Himakshi Patel, Lisa A. Marzilli, and Jason C. Rouse

Pfizer, Inc.

BioTherapeutics Pharmaceutical Sciences

Analytical Research and Development

Mass Spectrometry and Biophysical Characterization Group

Pfizer Andover, MA

CASSS 2012, San Diego, CA

Characterization of a Heavily Glycosylated Fusion

Protein with Unique Modifications

1

Characterization of a Heavily Glycosylated Fusion

Protein with Unique Modifications: Abstract• Recombinant fusion proteins result from attaching the amino acid sequences of more than

one protein together to form a single, chimeric protein. In the pharmaceutical industry, fusion

proteins can be used to target more than one indication or, in the case of the receptor-Fc

protein described here, to increase the half-life of the circulating biotherapeutic. Receptor-Fc

proteins typically have molecular masses that are less than monoclonal antibodies (mAbs),

but the extensive structural heterogeneity of receptor-Fc proteins makes their analytical

analysis difficult. This complexity can be predicted from the amino acid sequence from the

presence of N-glycosylation consensus sites and other known sequence motifs for additional

posttranslational modifications. Some posttranslational modifications to the protein backbone

(e.g. O-glycosylation) are not predictable based on the amino acid sequence alone. The

routine high-resolution mass spectrometry methods for mAbs make a good starting point for

receptor-Fc protein characterization, but many of these methods must be optimized, further

developed, or in some cases replaced with new techniques. This presentation will

demonstrate the adaptability of some mAb mass spectrometry-based methods to this

receptor-Fc protein, as well as highlight other off-platform methods that were used for the

successful characterization of the receptor-Fc biotherapeutic. This presentation will also

illustrate how complex proteins still test the limits of state-of-the-art mass spectrometers.

Finally, an assessment of the methods and their applicability to future complex

biotherapeutics will be summarized.

2

Mass Spectrometry and Biophysical

Characterization Group

• Apply multiple state-of-art technologies, including mass spectrometry and

biophysical characterization, to characterize biologics and vaccines from

primary structure to higher order structure

• Provide support to bioprocess and pharmaceutical development groups

• Support analytical method development and method characterization to

ensure accuracy and specificity

• Provide in-depth structural characterization for agency filings from IND

through BLA and beyond, in addition to comparability assessments.

• Investigate and evaluate new instruments, methods and technologies, as

well as CRO support.

Exploratory DiscoveryPre-

DevelopmentDevelopment

TrackPhase 1 Phase 2 Registration

Research Development

(Pharmaceutical Sciences)

Commercial

MarketPhase 3

3

Mass Spectrometry Characterization Methods for

Monoclonal Antibodies (mAbs)

Intact mAbMass

Analysis

nanoESI or LC/MS

Subunit (2 or 3-Part) mAb

Mass Analysis by

LC/MS

Peptide Mapping LC/MS

N-glycanMapping LC/MS

25225 25235 2524522425 22435 2244525060 25070

Subunit analysis:

Ides enzymatic digestion and

disulfide bond reduction

and/or accurate mass

analysis of light and heavy

chains to verify correct

composition and intended

sequence.

Intact mAb analysis: Verify

correct mAb was

manufactured with 4-chain

architecture, intended

primary sequence, and

expected post-

translational

modifications

AntiAB_SEC_LMW20.000000005.00000000

10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00 65.00 70.00 75.00 80.00Time0

100

%

0

100

%

LC/MS of released 2-AB

Labeled N-glycans:

Elucidation of N-glycans and

abundance. Complementary to

other mAb methods.

Reduction, alkylation,

enzymatic digestion. Sequence

coverage at the peptide level.

Useful for understanding

post-translational modifications and

their locations.

148200 148400 148600 148800 149000 149200 149400 149600 149800 150000

mass0

100

%

148400 148800 149200 149600 mass

G0F/G0F

G0F/G1FG0F/G2F

G1F/G1F

G1F/G2F

EU

0.00

500.00

1000.00

1500.00

2000.00

EU

0.00

500.00

1000.00

1500.00

2000.00

Minutes

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G0F

G1F

G2FG0G0F-GlcNAc Man5

[

4

Structure of a Receptor-Fc Protein

Extracellular

Domain

Monoclonal Antibody

C=carbohydrate: N-glycans

Although receptor-Fc proteins may be similar in size to antibodies, they usually have

significantly more complexity (heterogeneity) that makes analysis difficult by

platform methods.

C

C

NH2

C

CC C

C

C

C

C

H2N

Receptor-Fc

Linker peptide

Fc

Domain

C=carbohydrate: N-glycans

5

Challenges and Goals of Receptor-Fc Therapeutics Characterization

Characterization Challenges• For most receptor-Fc therapeutics

and extracellular domains,

characterization and structural

results are not published.

• Molecular complexity exceeds MS

capabilities

• Starting with an unknown

modification landscape

• N-glycosylation

• O-glycosylation

• Other unique modifications

Characterization Goals• Molecular mass of intact/reduced

protein where the heterogeneity

increases the complexity

• Determine primary structure

• Localize modifications

• Elucidate post-translational

modifications

• Carbohydrates

• Expected modifications

• Unexpected modifications

• Establish a receptor-Fc

characterization platform!

Intact Protein Mass

Analysis

(very complex)

MALDI-TOF MS

Reduced Protein Mass

Analysis

(still complex)

LC/MS



6

Intact Receptor-Fc Analysis by Mass Spectrometry:

Matrix-Assisted Laser Desorption/Ionization Time-of-

Flight Mass Spectrometry (MALDI-TOF MS)

[M+H]+

131822

[M+2H]2+

•For characterization, MALDI-TOF MS provides an

average molecular mass for highly modified protein (12

N-glycans).

•The mass, peak shape and width of the peak suggest

that the protein is heavily glycosylated.

60000 70000 80000 90000 100000 110000 120000 130000 140000 150000 m/z

Intact Protein

7

N-Linked Oligosaccharide Profiling: Comparison

• mAbs

• Platform LC/MS method for mAbs.

• Size-based separation.

• Resolves and detects all N-

glycans in mAbs.

• 100% MS-compatible.

• Receptor-Fc

• Size- and charge-based method

• Resolves and detects most N-

glycans: chromatographic

resolution in charge groupings,

but all N-glycans detected by MS.

• Semi-MS compatible.

ESI MS allows the separated N-glycans to be identified with exact monosaccharide compositions

derived from accurate mass measurements afforded by quadrupole time-of-flight instruments.

2AB2AB 2AB2AB 2AB 2AB2AB 2AB

EU

0.00

500.00

1000.00

1500.00

2000.00

EU

0.00

500.00

1000.00

1500.00

2000.00

Minutes

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G0F

G1F

G2FG0G0F-GlcNAc Man5

[

Project Name: 2012 Q1 ADW\PPL3\GlycanReported by User: Himakshi Patel (hpatel)

Report Method: Compare_Landscape Date Printed:

5092 Wednesday, March 21, 2012Report Method ID: 5092

8:08:18 AM US/Eastern

0.0

4000.0

8000.0

12000.0

0.00 15.00 30.00 45.00 60.00 75.00

Time (minutes)

Fluo

resc

ence

(mV)

Excess 2-AB and

Buf fer components

Colum

n stri

p

2AB

2AB

2AB2AB

2AB

2AB

2AB

2AB

2AB

2AB

2AB

2AB

2AB 2AB

2AB

2AB

2AB

2AB

2AB

2AB

2AB2AB

2AB

2AB 2AB

2AB 2AB 2AB

2AB

core

G0-GlcNAc G0F-GlcNAc

G0 G0F

Man5

G1F

8





0.0

4000.0

8000.0

12000.0

0.00 15.00 30.00 45.00 60.00 75.00

Time (minutes)

Flu

ore

sce

nce (

mV

)

Excess 2-AB and

Buffer components

Colu

mn s

trip

2A

B2A

B2A

B2A

B

2A

B

BiF + 1 NeuAc

BiF + 2 NeuAc

BiF (asialo,

agalactosylated)

TriF + 3 NeuAc

TetraF + 4 NeuAc

BiF (asialo,

agalactosylated)

BiF + 1 NeuAc

BiF + 2 NeuAc

TriF + 3 NeuAc

TetraF + 4 NeuAc

Mannose

Galactose

Fucose

GlcNAc

GalNAc

NeuAc

N-linked Oligosaccharide Profiling by 2-AB labeling/

HILIC with MS Detection

9

N-linked Oligosaccharide Profile of Receptor-Fc





0.0

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8000.0

12000.0

0.00 15.00 30.00 45.00 60.00 75.00

Time (minutes)

Flu

ore

sce

nce (

mV

)

Excess 2-AB and

Buffer components

Colu

mn s

trip

2AB

2AB

2AB2AB

2AB

2AB

2AB

2AB

2AB

2AB

2AB

2AB

2AB 2AB

2AB

2AB

2AB

2AB

2AB

2AB

2AB2AB

2AB

2AB 2AB

2AB 2AB 2AB

2AB

core

G0-GlcNAc G0F-GlcNAc

G0 G0F

Man5

G1F

10

Intact Protein Analysis by Mass Spectrometry:

MALDI-TOF MS

Based on N-glycan Profile:

Within Range of

Expected Glycosylation

(12 N-glycans)

Can be used to determine lot-to-lot consistency in terms of potential

sialylation and site occupancy changes. For MS analysis,

reduce the complexity.

[M+H]+

131822

[M+2H]2+

60000 70000 80000 90000 100000 110000 120000 130000 140000 150000 m/z

No

gly

co

syla

tion

12 te

tra-a

nte

nn

ary

sia

lyla

ted

gly

ca

ns

Intact Protein

N-glycans

11

Peptide Mapping LC/MS: Confirmation of Results

• Intact protein, and N-

glycan analyses can be

verified through analysis

and interpretation of the

peptide map.

• We want to:

• Verify primary

structure

• Elucidate expected

and unexpected

PTMs

• Localize PTMs to

peptide or amino acid

level

• Determine site

occupancy and

microheterogeneity

Intact Protein Mass Analysis

(very complex)

MALDI-TOFMS




12

Lysyl Endoproteinase “Lys-C” Peptide Mapping

LC/MS: Receptor-Fc Protein• K7 Peptide:

C-mannosylation

consensus sequence

WSEWS partially

occupied.

• K13K14 N-glycans:

Typical Fc N-glycoform

profile was observed:

G0F, G1F major forms

and sialylated

glycoforms were trace

level.

• The heavily N-

glycosylated

glycopeptide in the

extracellular domain

was not recovered in

the peptide map. The

glycopeptide was

fractionated (size-

based), de-N-

glycosylated, and

analyzed by

nanoelectrospray

ionization mass

spectrometry. Total

sequence coverage

was 97%.

Project Name: 2012 Q1 ADW\PPL3\Peptide MapReported by User: Himakshi Patel (hpatel)


2778 Friday, April 06, 2012Report Method ID: 2778


0.00

0.03

0.06

16.80 21.00 25.20 29.40 33.60 37.80





0.00

0.20

0.40

0.00 30.00 60.00 90.00 120.00 150.00

Ab

so

rba

nce

21

4 n

m

Time (minutes)

K25

K8

Modif ied K8

1

23

4

5

67

891011 K7

K7

mod

ifie

d

K13K

14

N-g

lycan

s

K8 Peptide: Contains linker

peptide between binding

domain and Fc region.

Potential for O-glycosylation

(Ser, Thr).

C

C

NH2

C

CC C

C

C

C

C

H2N

13

Fragmentation (MS/MS) of a Modified Peptide

3400 3600 3800 4000 4200 4400mass0

100

%

T19 + 915 Da

4415.98

4336.94

P or S

79.92

HexNAc

203.04

4133.90

3795.81

HexNAc

HexA

176.07

Hex

162.01

Hex + HexA

3633.78

Hex

162.03

Hex

T19

3500.73

Pent

132.05

PentP/S

MS/MS reveals

structure of

modification

[ ]Ser

SO3

PO4

n

Xylose glucuronic acid galactose acetylgalactosamine

14

Glycosaminoglycan (GAG)

N Engl. J. Med. 354:8 Feb 23, 2006

CH2

Glucuronic acid

HexNAc

O

Xylose

Galactose

Galactose 162.053

162.053

132.042

176.032

Core Protein

+ SO3 or HPO3

203.079

79.957

632.1799

CH2

O

Xylose

Galactose

Galactose

Glucuronic Acid

HexNAc

Glucuronic Acid

n

Ser

Tetrasaccharide

core

Repeating

disaccharide

units

15

K8 Peptide Region: Immature Glycosaminoglycans

0

500

1000

1500

2000

2500

3000

Intens.

0

200

400

600

0

50

100

150

200

2000 2500 3000 3500 4000 m/z

ABC

C

B

A K8

30 32 34 36 38 40 42 44 Time [min]

0.25

0.50

0.75

1.00

1.25

5x10

Intens.

TIC

+

+SO3

+

+

+

+SO3

+

+SO3

[ ]

[ ]+SO3

[ ]+SO3

[ ]

[ ]

[ ]

+SO3[ ]

*

*

*

* Background ions

Unmodified K8 linker-

containing peptide

…EGWNPGSGEGEGSEGSGK…

Xylose glucuronic acid galactose acetylgalactosamine N-acetylneuraminic acid

…XGSGX…XGSG…

Unmodified K8 Peptide

+632.1786

+632.1790

C

C

NH2

C

CC C

C

C

C

C

H2N

16

Glycosaminoglycan (GAG) Structure: Chondroitin

Sulfate

n[ ]

Tetrasaccharide Core

632.1799 Da

Mature form

40 to >100disaccharides

Sulfated

*Sulfation: GlcA-2-SO4-GalNAc; GlcA-GalNAc-4-SO4; GlcA-GalNAc-6-SO4; GlcA-GalNAc-4,6-SO4

Modification Structure Observed Abundance

Pent Minor

Sulfate SO3 Trace

Pent-Hex-NeuAc Trace

Pent-Hex-Hex-HexA Major

Pent-Hex-Hex-HexA-

[HexNAc-HexA]1

Minor

Pent-Hex-Hex-HexA + sulfate Trace

Pent-Hex-Hex-HexA-

[HexNAc-HexA]1-HexNAc

Trace

[ ]1

+ SO3

[ ]1+ SO3

xylose

galactose

glucuronic acid

acetylgalactosamine

N-acetylneuraminic acid

key


GAG Name Linkage Geometry Unique Features

Chondroitin Sulfate -4GlcUAβ1-3GalNAcβ1- Most prevalent GAG

Dermatan Sulfate -4IdoUAβ1-3GalNAcβ1- Distinguished from chondroitin sulfate by the presence of

iduronic acid.

Keratan Sulfate -3Gal(6S)β1-4GlcNAc(6S)β1- Keratan sulfate type II may be fucosylated

Heparan -4IdoUA(2S)α1-4GlcNS(6S)α1- Highest negative charge density of any known biological

molecule

Heparan Sulfate -4GlcUAβ1-4GlcNAcα1- Highly similar in structure to heparin.

Hyaluronan -4GlcUAβ1-3GlcNAcβ1- non-sulfated

Tabulated information obtained from www.wikipedia.org

17

De-N-Glycosylated Receptor-Fc: Electrospray Ionization Mass

Spectrometry (online oSEC/ESI MS) Profile

101500 102000 102500 103000 103500 104000 104500 m/z

102094.6

*

102726.1

103359.8*

*

Theoretical Mr

102093.8 Da(7.8 ppm error) C-mannosylation

* sodiated

After treatment with PNGase F to remove 12 N-glycans.

Δ 632.3 Da

Δ 633.7 DaTheoretical

Mass:

632.1799 Da


12

1. + 2 xylose

2. + xylose and

C-mannosylation

De-N-gly

“intact”

18

De-N-Glycosylated, Disulfide Bond Reduced Receptor-Fc: Online

oSEC/ESI MS Profile

50500 51000 51500 52000 52500 53000 m/z

51058.6

51691.3

*

*

*

Theoretical Mr

51059.0 Da(-7.8 ppm error)

* sodiated

After treatment with PNGase F to remove 12 N-glycans. Further

reduce complexity by disufide bond reduction.

+SO4

Δ 632.7 Da

Δ 631.3 Da

Theoretical

Mass:

632.1799 Da


Disulfide

reduced

(polypeptide

chain)

19

Chondroitinase ABC Digestion of Receptor-Fc Containing

Mature Glycosaminoglycans

Sample Background:

•Treated with chondroitinase ABC ( )

•The enzyme incubation step also generated LMMS.

•The generation of the LMMS is not related to the enzyme,

but rather to the incubation step at 37 C.

Chondroitinase ABC-treated

material

Enzyme-related peak

(blue trace: enzyme blank)

Some LMMS is generated

during the 37 C incubation

step

0.00

Protein with large

GAGs elutes as a broad

shoulder peak

during SE-HPLC

monomer

EU

0.0

6000.0

12000.0

Minutes

5.00 10.00 15.00 20.00 25.00 30.00

[ ]n[ ] x n

+Ser

Ser


20

HILIC: 2-AB Labeled Disaccharides After Digestion



4674 Wednesday, June 13, 2012Report Method ID: 4674


0.0

4000.0

8000.0

0.00

250.00

500.00

10.00 12.50 15.00 17.50 20.00 22.50 25.00

Flu

ore

sce

nce

(m

V)

Time (minutes)

△UA→GalNAc(△Di-0S)

△UA→GalNAc-4S(△Di-4S)

Most commonly

found (90%) in

chondroitin sulfate A, B

△UA→GalNAc-6S(△Di-6S)

Most commonly

found (90%) in

chondroitin sulfate C

Chondroitin Sulfate

Disaccharide

Standard Mix

Released

Disaccharides from

Chondroitinase ABC

Treated Protein A

Purified Material

Buffer

component

*Glycoform quantification of chondroitin/dermatan sulfate using an LC-tandem MS platform.

A.M. Hitchcock, C.E.Costello and J. Zaia. Biochem. 2006, 45, 2350-2361.

*

*

[ ]

[ ] [ ]

21

Implementing Purification Steps to Remove Mature

GAGs

Project Name: 2011 Q3 ADW\ADBC\IL21R-fcReported by User: Himakshi Patel (hpatel)


1799 Monday, August 01, 2011Report Method ID: 1799

3:41:50 PM US/Eastern

SampleName 29Jul11_SEC_TS1

SampleName 29Jul11_SEC_TS1C

AU

0.00

0.09

0.18

Minutes

0.00 5.00 10.00 15.00 20.00 25.00 30.00

Material containing

mature GAGs

before additional

purification steps.

Monomer containing

immature GAGs.

Purified material.

Sample Background:

•Material containing mature

GAGs elutes as a broad peak by

size-exclusion HPLC.

•Purification steps implemented

to remove mature GAGs.

22

New Mass Spectrometry Characterization Roadmap

For Receptor-Fc Proteins

Intact protein mass analysis

MALDI MS and SEC/MS after de-N-glycosylation

De-N-glycosylated, disulfide bond reduced mass

analysis: online

SEC/MS

Peptide mapping LC/MS

sequence coverage and

PTMs

N-glycanmapping LC/MS

glycan and abundance assignment

Method optimization.

nanoESI = needs desalt

LC/MS = poor recovery

oSEC/MS = chosen

Chose Lys-C due to

good recovery of

peptides with minimal

method artifacts.

Fractionation of

glycopeptides was

required.

Complex N-glycan

profile required a size-

and charge-based

separation.

MALDI MS or reduce

complexity[M+H]+

131822.2

[M+2H]2+

0

100

200

300

400

40000 60000 80000 100000 120000 140000 m/z

101500 102000 102500 103000 103500 104000 104500 m/z

102094.6

*

102726.1

103359.8*

*

Theoretical Mr

102093.8 Da(7.8 ppm error) C-mannosylation

* sodiated

50500 51000 51500 52000 52500 53000 m/z

51058.6

51691.3

*

1

*

*

Theoretical Mr

51059.0 Da(-7.8 ppm error)

+SO4

* sodiated





0.00

0.03

0.06

16.80 21.00 25.20 29.40 33.60 37.80





0.00

0.20

0.40

0.00 30.00 60.00 90.00 120.00 150.00

Ab

so

rba

nce

21

4 n

m

Time (minutes)

K25

K8

Modif ied K8

1

23

4

5

67

891011 K7

K7

mod

ifie

d

K13K

14

N-g

lycan

s





0.0

4000.0

8000.0

12000.0

0.00 15.00 30.00 45.00 60.00 75.00

Time (minutes)

Flu

ore

sce

nce

(m

V)

Excess 2-AB and

Buffer components

Co

lum

n s

trip

2A

B2A

B2A

B2A

B

2A

B

BiF + 1 NeuAc

BiF + 2 NeuAc

BiF (agalactosylated)

TriF + 3 NeuAc

TetraF + 4 NeuAc

BiF (agalactosylated)

BiF + 1 NeuAc

BiF + 2 NeuAc

TriF + 3 NeuAc

TetraF + 4 NeuAc

Mannose

Galactose

Fucose

GlcNAc

GalNAc

NeuAc

Intact Reduced

N-Glycans Primary

Structure

23

Summary

• The predicted (and unpredicted in some cases) posttranslational

modification heterogeneity makes characterization complicated.

• C-mannosylation (1 consensus sequence per polypeptide chain)

• N-glycosylation (12 N-glycans, 6 N-glycans per polypeptide chain)

• O-glycosylation (2 immature glycosaminoglycans) – Unanticipated!

• Traditional platform methods used with mAbs can be adapted for primary

structure characterization of more complex proteins such as the receptor-Fc

protein described in this presentation.

• additional methods: de-N-glycosylation, GAG digestion and disaccharide

analysis.

• method optimization: online oSEC/MS versus nanoESI MS versus RP-LC/MS

• method development: recovery of extracellular domain glycopeptide, size- and

charge-based N-glycan profiling.

• Platform methods are adaptable case-by-case for receptor-Fc proteins, and it

is important that we have solid platform assays capable of detecting new

species that give us a head start in developing new or refining old assays.

• Identification and verification of GAGs by MS analysis and enzyme treatment

impacted how the process and the product were developed.

24

Acknowledgments

• Jason Rouse – Director MSBC

• Lisa Marzilli

• Heather DeGruttola

• Dan Haq

• Keith Johnson

• Andrew Saati

• Matt Thompson

• James Carroll

• Paul Brown

• Kathleen Cornelius

• Olga Friese

• Justin Sperry

• Jacquelynn Smith

Analytical R&D

• Marta Czupryn

• Chee-Keng Ng

• Mike Jankowski

Project Team

• Denise Pretzer

• Tina Kneeland

• Dick Wright

• Michelle Lisowski

• Chris Morrison

Characterization of a Heavily Glycosylated Fusion Protein ...€¦ · 4.00 6.00 8.00 10.00 12.00...

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