Modernizing the Platform Characterization Peptide Map for
Accurate Assessment of Deamidation and
Isomerization by LC-MS/MS
Lisa A. Marzilli, Elaine Stephens, Michelle English,
Shibu Philip* and Jason C Rouse
Analytical R&D, Pharmaceutical Sciences, Andover, MA, *Pearl River, NY
Analytical Technology-CASSS March 16, 2017
Antibody Considerations as a Drug Candidate
Typical parameters in selecting lead antibody:
2
affinity
selectivity
activity manufacturability
stability
poly-reactivity
aggregation
propensity
viscosity
Antibodies have a propensity to degrade:
manufacturing, storage and in vivo administration
Determine Degradation
Hotspots
Sequence Liabilities or
CQAs?
Engineer to Eliminate Sequence Liability?
Early Product Development Challenge
3
Oxidation: Met & Trp
Deamidation: Asn and Gln
Isomerization: Asp
Glycation: Lys
Truncations: Asp-Pro
Molecular modeling
Peptide mapping
Does hotspot impact
antigen binding?
Focus on
complementarity
determining regions
(CDRs)
Constant region
hotspots may be
potential liabilities
Deamidation/Isomerization Scheme
4
Asn deamidation reactions proceed more
quickly at elevated pH and temperature
N+1 residue (NG, NS, NT, and NN)
Geiger T. and Clarke S., J of Biol. Chem., (1987), 262 (2), 785.
Asn, N
Asp, D
IsoAsp, isoD
Asp isomerization reaction rate most
impacted by lower pH and N+1 residue
N+1 residue (DG, DS, DT, and DD)
Li et al, J. Pharm. Sci., (2013), 102 (3), 947.
The Ideal Peptide Map
• Robust and easy to perform
• Complete sequence coverage
• High quality separations (in a MS-friendly solvent)
• High quality UV chromatograms
• Minimal method-induced artifacts (short digestion times, low pH)
• Accurate targeted quantitation (Extracted Ion Chromatograms)
5
Developed in-house, automated software to
highlight potential “hotspots”
6
Trastuzumab L Chain CDR Hotspots
1 DIQMTQSPSSLSASVGDRVTITCRASQDVN 30
31 TAVAWYQQKPGKAPKLLIYSASFLYSGVPS 60
61 RFSGSRSGTDFTLTISSLQPEDFATYYCQQ 90
91 HYTTPPTFGQGTKVEIKR
Trastuzumab H Chain CDR Hotspots
1 EVQLVESGGGLVQPGGSLRLSCAASGFNIK 30
31 DTYIHWVRQAPGKGLEWVARIYPTNGYTRY 60
61 ADSVKGRFTISADTSKNTAYLQMNSLRAED 90
91 TAVYYCSRWGGDGFYAMDYWGQGTLVTVSS 120
121
Automatically
underlines
CDR1, CDR2
and CDR3
Highlights
potential
hotspots in
blue
Case Study #1 Using Trastuzumab (Herceptin®) as
a Benchmark for Peptide Map Method Development
7
Trastuzumab L Chain CDR Hotspots Rel. Abundance
1 DIQMTQSPSSLSASVGDRVTITCRASQDVN 30 High level (>5%)
31 TAVAWYQQKPGKAPKLLIYSASFLYSGVPS 60 Low level (1-5%)
61 RFSGSRSGTDFTLTISSLQPEDFATYYCQQ 90 Potential (<1%)
91 HYTTPPTFGQGTKVEIKR
Trastuzumab H Chain CDR Hotspots Rel. Abundance
1 EVQLVESGGGLVQPGGSLRLSCAASGFNIK 30 High level (>5%)
31 DTYIHWVRQAPGKGLEWVARIYPTNGYTRY 60 Low level (1-5%)
61 ADSVKGRFTISADTSKNTAYLQMNSLRAED 90 Potential (<1%)
91 TAVYYCSRWGGDGFYAMDYWGQGTLVTVSS 120
Case Study #1 Using Trastuzumab (Herceptin®) as
a Benchmark for Peptide Map Method Development
8
Trastuzumab L Chain CDR Hotspots Rel. Abundance
1 DIQMTQSPSSLSASVGDRVTITCRASQDVN 30 High level (>5%)
31 TAVAWYQQKPGKAPKLLIYSASFLYSGVPS 60 Low level (1-5%)
61 RFSGSRSGTDFTLTISSLQPEDFATYYCQQ 90 Potential (<1%)
91 HYTTPPTFGQGTKVEIKR
Trastuzumab H Chain CDR Hotspots Rel. Abundance
1 EVQLVESGGGLVQPGGSLRLSCAASGFNIK 30 High level (>5%)
31 DTYIHWVRQAPGKGLEWVARIYPTNGYTRY 60 Low level (1-5%)
61 ADSVKGRFTISADTSKNTAYLQMNSLRAED 90 Potential (<1%)
91 TAVYYCSRWGGDGFYAMDYWGQGTLVTVSS 120
3
Digestion Time (h)
% I
so
me
riza
tio
n / S
ucc
inim
ide
Digestion Time (h)
D102G (H-CDR3)
% I
so
me
riza
tio
n / S
ucc
inim
ide
0
2
4
6
8
10
12
0 10 20
pH 7.5
pH 8.2
Platform
0
2
4
6
8
10
12
0 1 2
pH 7.5
pH 8.2
2
Digestion Time (h)
% D
ea
mid
ati
on
/ S
uc
cin
imid
e
Digestion Time (h)
N30T (L-CDR1)
% D
ea
mid
ati
on
/ S
uc
cin
imid
e
0
2
4
6
8
10
12
14
0 10 20
pH 7.5
pH 8.2
Platform
0
2
4
6
8
10
12
0 1 2
pH 7.5
pH 8.2
N55G (H-CDR2)
0
10
20
30
40
50
0 10 20
pH7.7
pH8.2
Platform
0
1
2
3
4
5
6
7
8
9
0 1 2
pH 7.5
pH 8.2
Digestion Time (h)
Asn Deamidation and Asp Isomerization vs. Trypsin
Digestion Time (Time Course) for Trastuzumab (Herceptin®)
Digestion Time Can Influence Level of Deamidation
9
%D
eam
idati
on
/ S
uccin
imid
e
%D
eam
idati
on
/ S
uccin
imid
e
Deamidation for Innovator Trastuzumab
10 10 10
Digestion Time (h)
% D
ea
mid
ati
on
/ S
ucc
inim
ide
Digestion Time (h)
Pfizer: N55G (H-CDR2) %
Dea
mid
ati
on
/ S
ucc
inim
ide
5
•Time-course digestion facilitates the
accurate measurement of deamidation at
T=0
•Digest for 30, 60 and 120 min
•Report accurate deamidation levels
Literature
% Deamidation Level at
N55G
(H-CDR2)
Genentech Harris
2001 ~1 (CEX-HPLC)
Roche Sydow
2014 4 (pH 6.0 map)
Pfizer Results
Innovator
Trastuzumab
44 to 2
New Trypsin Peptide Map
Shorter Digestion Times and Time-Course
11
•Time-course digestion facilitates the accurate
measurement of deamidation at T=0
•Digest for 30, 60 and 120 min
• Efficient reduction, alkylation and digestion with
Lys-C/trypsin (Promega) (pH 8.2)
• Waters XSelect XP CSH C18 column(formic acid)
• Reproducible UV chromatographic profiles
• Analyzed on a Thermo Orbitrap Fusion™ Tribrid™
Mass Spectrometer
• Unprecedented depth of analysis
• High quality MS/MS data (CID, ETD)
• Use Fusion for PTM discovery
Peptide Mapping
Goal: minimize
method-related
artifacts
Low-artifact Trypsin Digest Methods
12
12
Denaturation /
Reduction Alkylation Tryptic Digestion
Buffer exchange
Using Bio-Spin 6
7M GdHCl /
100mM Tris pH 8.2
DTT
30 min at 37 ˚C
IAA
30 min, dark,
RT
Buffer exchange
Bio-Spin 6 column
(Bio-Rad)
130 µL load volume
Digest at
pH 8.2
7M GdHCl /
20 mM MES (pH 6.0)
TCEP
60 min at 37 ˚C
Lys-C/trypsin (Promega)
18 hours at 37˚C in 0.5 mM TCEP
in 20 mM MES (pH 6.0)
1:10 enzyme: protein (w:w).
Digestion quenched using 1% TFA
Buffer exchange
Bio-Spin 6 column
(Bio-Rad)
130 µL load volume
Digest at
pH 6.0
Lys-C/trypsin (Promega)
30, 60, and 120 min
37˚C in 100 mM Tris (pH 8.2),
1:10; enzyme: protein (w:w).
Digestion quenched using 1% TFA
0
1
2
3
4
5
6
0 1 2
pH 7.5
pH 8.2
Time (h)
% d
ea
mid
atio
n
Elaine Stephens
Asn Deamidation and Asp Isomerization in
Trastuzumab (Herceptin®) Using Various Methods
13
Site Modification Method % Modification
N30T (L-CDR1) Deamidation
CEX-HPLC (UV)1 ~15
Trypsin pH 6.0 (overnight) map 2 11
PFE-Trypsin pH 7.5 (overnight) map 12
PFE-Lys-C/Trypsin pH 8.2 (T=0) map 10
N55G (H-CDR2) Deamidation
CEX-HPLC (UV)1 ~1
Trypsin pH 6.0 (overnight) map 2 4
PFE-Trypsin pH 7.5 (overnight) map 44
PFE-Lys-C/Trypsin pH 8.2 (T=0) map 2
D102G (H-CDR3) Isomerization
CEX-HPLC (UV)1 ~9
Trypsin pH 6.0 (overnight) map 2 7
PFE-Trypsin pH 7.5 (overnight) map Not detected
PFE-Lys-C/Trypsin pH 8.2 (T=0) map 8
1. R.J. Harris, B. Kabakoff, F.D. et al, J of Chromat. B, Biomedical sciences and applications, 752, 233-245 (2001). 2. J.F. Sydow, F. Lipsmeier, V. et al, PLoS One, 9, e100736 (2014).
Efficient Separation and ETD Spectra
Confirm ID of H12 (D102G) and H12 (iso-D102G)
14
400 600 800 1000 1200 1400 1600 1800 2000
m/z
697.3
929.3
1393.9
1155.4660.5
892.7 958.31328.4477.3
1466.7753.7 1234.7564.4390.3 1786.01506.9 1915.9
697.2
929.4
1394.01045.4
892.6660.6 1155.51466.7477.3 753.8
1785.01506.91916.9
z4
z5z6
[M+4H]4+
[M+3H]3+
[M+2H]2+
z17 z18z15
c12
C232+
z15
Z182+ C25
2+
H12 (D102G)
Peak 1
H12 (iso-D102G)Peak 2
C3+57 [Z23-57]2+
H12 peptide (WGGD102GFYAMDYWGQGTLVTVSSASTK)
* *
C242+C20
2+
Z172+
318.3
318.2
c4433.3
c3
490.3
490.3
c5
564.3
of in H-
Peak 1
H12 (D102G)
Peak 2
H12 (Iso-D102G)
Non-stressed Stressed
(14 days
@ pH8)
Rela
tive A
bun
dance
Time (min) Time (min)
Peak 1
H12 (D102G)
Peak 2
H12 (Iso-D102G)
• Waters XSelect XP CSH
C18 column with formic acid
can resolve isomerized peak
Extracted Ion
Chromatogram (EIC) for H12
Isomerization of Asp-102 led to lower potency (Harris et al, 2001)
Diagnostic ion (C3 + 57) Confirms Presence of
isoAsp in Trastuzumab
15
C N
H
C
O
C H
H
N
CH2
COOH
O
c4 c3
C N
H
C
O
C H
H
N CH2
COOH
O
c4 c3 c3 + 57 = 375
Iso-D102G
Peak 2
D102G
Peak 1
350 400 450 m/z
100
100
Rela
tive A
bundance
z5
477.3
z4
390.3 c4
433.3
477.3
c3+57 375.2
Rela
tive A
bundance
c3
318.3
c3
318.3
z4
390.3 c4
433.3
a5
a5
z3
z3
J.J. Cournoyer, J.L. Pittman, V.B. Ivleva, E. Fallows, L. Waskell, C.E. Costello, P.B. O'Connor, Prot. Sci., 14,
452-463 (2005).
of in H-
Peak 1
H12 (D102G)
Peak 2
H12 (Iso-D102G)
Non-stressed Stressed
(14 days
@ pH8)
Rela
tive A
bun
dance
Time (min) Time (min)
Peak 1
H12 (D102G)
Peak 2
H12 (Iso-D102G)
• Waters XSelect XP CSH
C18 column can resolve
isomerized peak
Extracted Ion
Chromatogram (EIC) for H12
Case Study #1
Peptide Map of Trastuzumab (Herceptin®)
16
Trastuzumab (Herceptin®) is useful as a benchmark for
peptide map method development
Artifactual deamidation is a real possibility
Some motifs are sensitive to digestion time and pH (ex.
N55G in H-CDR2)
Resolution of Asp isomerized peptides (D102G vs isoD102G)
is challenging
Peptides have the same mass
Can only quantitate Asp/isoAsp if separated
17
L Chain CDR Hotspots
1 XXXXXXXXXXXXXXXXXXXXXXXRSSQSLA 30
31 NSYGNTFLSXXXXXXXXXXXXXXXGISNRF 60
61 SXXXXXXXXXXXXXXXXXXXXXXXXXXXXX 90
91 XXXLQGTHQPYTXXXXXXXX
H Chain CDR Hotspots
1 XXXXXXXXXXXXXXXXXXXXXXXXXGYRFT 30
31 NYWIHXXXXXXXXXXXXXXGINPGNNYATY 60
61 RRKFQGXXXXXXXXXXXXXXXXXXXXXXXX 90
91 XXXXXXXXEGYGNYGAWFAYXXXXXXXXXX 120
121
Case Study #2: Deamidation in an IgG4 mAb
Case Study #2: Deamidation in an IgG4 mAb
18
Three distinct acidic isoforms (~15%)
Acidic fractions collected and characterized for structure/function
Acidic
CEX-HPLC Profile
Bulk
B Ma
in
Should
er
A3 A2 A1 AA
AU
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
0.16
0.18
0.20
Minutes
120.00 121.00 122.00 123.00 124.00 125.00 126.00 127.00 128.00 129.00 130.00 131.00 132.00 133.00 134.00 135.00
Case Study #2: UV Chromatograms of Trypsin
Peptide Maps
H12 (N103Y)
A1 H12 (D/isoD103Y
A2 H12 (D/isoD103Y)
CEX-HPLC Fraction A1 and A2 show an increase in H12 deamidation
Antigen binding impacted A1 charge variant
CEX-HPLC Profile
Red = CEX Main Black = CEX A1 Blue = CEX A2 Green = CEX A3
19
20
Case Study #2: New Peptide Map Column for
Enhanced Chromatographic Separation
0
10
20
30
40
50
60
70
80
90
100
Rela
tive
Ab
un
da
nce
7.0% Deamidation
H12
LAM02OCT2012000002.d: EIC 967.4557 +All MS
LAM02OCT2012000002.d: EIC 967.7837 +All MS0.0
0.2
0.4
0.6
0.8
1.0
5x10
Intens.
0.0
0.5
1.0
1.5
5x10
120 121 122 123 124 125 126 127 128 Time [min]
H12
Deamidation ?
Previous Trypsin Map
Waters BEH C18, TFA
New Trypsin Map
Waters XSelect XP CSH C18, FA
H12 (N103Y) H12 (N103Y)
IgG4 mAb: H chain deamidation in CDR3
Localized deamidation site to: EGYGN103YGAWFAYWGQGTLVTVSSASTK
NY site not expected to deamidate (NG, NS, NT, NN)
Causes loss of target binding
104 106 108 110 112 114 116 118 120
Time (min)
7.0% Deamidation
Case Study #2: Deamidation of an IgG4 mAb
21
New HPLC column allows for better separation and accurate quantitation
of deamidation
Detected unexpected N103Y deamidation hot spot (beyond NG, NS, NT,
and NN)
In process of updating software reflect new findings
Site Modification Modification
(total Asp and
iso-Asp, %)
CEX Fraction
N33S (L-CDR1) Deamidation 3.2 A3
N55N (H-CDR2) Deamidation 0.1 - - -
N103Y (H-CDR3) Deamidation 7.0 A1 and A2
Case Study #3
Forced Deamidation Study of IgG1 mAb
22
Stress
Study
• Incubation at 25 ºC and 37 ºC in 100 mM sodium phosphate, pH 8.0
• T=0, 2 and 4 weeks
Peptide Map
Method
• New Lys-C/trypsin map, pH 8.2, 30, 60 and 120 min
• Waters XSelect XP CSH C18 column, FA
MS Analysis
• LC-MS/MS and EICs
• Collision induced dissociation (CID)
• Targeted electron transfer dissociation (ETD)
Case Study #3: Forced Deamidation Study of IgG1
mAb
23
IgG1 L Chain CDR Hotspots Rel. Abundance
1 DIVMTQTPLSLSVTPGQPASISCRSSQSLV 30 High level (>5%)
31 HSNGNTFLYWYLQKPGQSPQLLIYRVSNRF 60 Low level (1-5%)
61 SGVPDRFSGSGSGTDFTLKISRVEAEDVGV 90 Potential (<1%)
91 YYCFQATHVPWTFGGGTKVEIKR
IgG1 H Chain CDR Hotspots Rel. Abundance
1 QVQLVQSGAEVKKPGASVKVSCKASGYTFT 30 High level (>5%)
31 GYYIHWVRQAPGQGLEWMGWIYPGNFNTKY 60 Low level (1-5%)
61 NERFKGRVTMTTDTSTSTAYMELRSLRSDD 90 Potential (<1%)
91 TAVYYCAREDGSPYYAMDYWGQGTSVTVSS 120
Deamidation of Light Chain (L2 peptide with CDR1) 25SSQSLVHSN33GNTFLYWYLQKPGQSPQ50LLIYR55
24 99 100 101 102 103 104 105 106 107 108 109
37˚C
4 weeks
L2 (D33G)
3.3%
L2 (isoD33G)
9.5%
25˚C
4 weeks
T=0
L2(N33G)
120 min EICs shown
L2 (D33G)
13.0%
L2 (isoD33G)
30.0%
L2 (D33G)
0.7%
L2 (isoD33G)
2.1%
L2 (E50L)
1.5%
Deamidation of Heavy Chain (H5 with CDR2) 39QAPGQ43GLEWMGWIYPGN55FNTK59
25
114 115 116 117 118 119 120 121 122
Time (min)
0
H5(E43G) H5(D55F or isoD55F)
1.0%
37˚C
4 weeks
25˚C
4 weeks
T=0
H5(N55F)
120 min EICs shown
H5(D55F or isoD55F)
4.0%
H5(D55F or isoD55F)
12.9%
26
CID Spectra Showing Deamidation at Asn-55 39QAPGQGLEWMGWIYPGN55FNTK59
39QAPGQGLEWMGWIYPGNFNTK59
b14
b14
b13
b13
b12
b12
b11
b11
y9
y9
y8
y8
y7
y7
b132+
b132+
b122+
b122+
y82+
y82+
y72+
y72+
y4+
y4
39QAPGQGLEWMGWIYPGDFNTK59
b14
y7
300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000
m/z
0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
50
60
70
80
90
100
Rela
tive A
bundance
777.4454
940.5127
728.0817
671.4913
1341.7339 389.3110 1053.6013 470.8820 1155.6390
1454.8182 967.5503
1618.9397 620.4687 809.5502 1296.7688 357.1390 1582.9045 1767.1686 1887.0905
778.4114
941.5096
728.0490
671.4405
1054.6180 1341.7526 471.3714 1155.6852
389.7402 1455.8529
967.5021 1617.8817 621.0251 1297.8234 809.6602
244.1232 1492.6409 1745.0511 1845.8401
CID did not provide conclusive evidence for deamidation site
ETD Fragmentation Confirms Deamidation Site
at Asn-55 (N55F)
27
400 600 800 1000 1200 1400 1600 1800 2000
m/z
0
1
2
3
4
5
6
7
8
9
10
11
12
0
1
2
3
4
5
6
7
8
9
10
11
12
Re
lative
Ab
un
da
nce
940.4541z=1
1472.7207z=1
493.2530z=1
669.3685z=1346.1862
z=1 1727.7738z=1
607.2953z=1
1053.5317z=1
1903.9133z=?1359.6283
z=1 1591.7782z=?
393.6677z=?
903.3638z=? 1244.7306
z=?
1937.8198z=?
742.7191z=?
346.1848z=1
941.4358z=1
493.2527z=1
1471.7155z=1
669.3676z=?
984.4893z=1
1904.8741z=1
1618.7538z=1
1413.6531z=1
1728.7710z=1
1126.5248z=2
1297.6061z=?
427.2351z=?
843.7211z=?
1952.6699z=?
NL: 1.24E6
03Aug16_EFNA4_HR_ETD#36869-36989 RT: 116.56-116.94 AV: 15 F: FTMS + p ESI Full ms2 [email protected] [250.0000-2000.0000]
NL: 1.94E5
03Aug16_EFNA4_HR_ETD#37814-37974 RT: 120.12-120.67 AV: 20 F: FTMS + p ESI Full ms2 [email protected] [250.0000-2000.0000]
39QAPGQGLEWMGWIYPGDFNTK59
39QAPGQGLEWMGWIYPGNFNTK59
z3
z3
z4
z4
608.2818
z5
z5
c13
c13
c17
c17
z14
z14 z4
z4 z5
z5 z3
z3
Asn Deamidation and Asp Isomerization Results for
IgG1 mAb using New Low Artifact Trypsin Map
28
Site Modification Modification (total Asp and iso-Asp, %)
Unstressed Stressed 4
weeks at 25 C
Stressed 4
weeks at 37 C
N33G (L-CDR1) Deamidation 2.8 12.8 43.0
N55F (H-CDR2) Deamidation
1.0 4.0 12.9
D100G (H-CDR3) Isomerization 0.7 1.0 1.7
Important to analyze stressed material for hotspots (2.8 to 43.0%)
Detected unexpected N55F deamidation hot spot
D100G not a hotspot despite motif and location in H-CDR3
Summary
• New trypsin peptide map-shorter digestion times, efficient enzyme, time-course elements and updated C18 column with formic acid
– Detects method-induced artifacts
– Improved resolution of deamidated and isomerized peptides
– Accurate quantitation of deamidation/isomerization via time course
– Fragmentation (CID and ETD) determine sites of modification
• ETD also distinguishes Asp vs iso-Asp
• Deamidation and isomerization hotspots are sequence and location dependent and less predictable in proteins
– Detected unexpected deamidation hot spots (beyond NG, NS, NT, and NN)
• N103Y in heavy chain CDR3
• N55F in heavy chain CDR2
• Low and constant level of Gln deamidation (Q50L, Q40G)
29
Summary of Hotspots
30
Case Study 1
Trastuzumab
Originator
Case Study 2
IgG4 mAb
Ref Material
Case Study 3
IgG1 mAb-stressed
Ref Material -
Stressed
Rel. Abundance
N30T (L-CDR1)
N33S (L-CDR1) N33G (L-CDR1)
N33G (L-CDR1) High level (>5%)
N55G (H-CDR2)
N55N (H-CDR2) N55F (H-CDR2)
N55F (H-CDR2) Low level (1-5%)
D102G (H-CDR3)
N103Y (H-CDR3) D100G (H-CDR3)
D100G (H-CDR3) Potential (<1%)
Forced degradation studies help identify potential sequence liabilities
Acknowledgements
31
Tao He
Eric Sousa
Yan Zhang
Laura Lin
Bekah Ward
Neil Steinmeyer
Tom Porter
Meg Ruesch
Jason Rouse-co-author
Elaine Stephens-co-author
Michelle English-co-author
Shibu Philip-co-author
Robert Kutlik (software)
Kristin Boggio
Heather DeGruttola
Jennifer Ide
Natalia Kozlova
Mellisa Ly
Andrew Saati
Roger Theberge
Matt Thompson
Ying Zhang