DEPLOYING MS IN LATE DEVELOPMENT AND QC: DEFINING FIT FOR PURPOSE Scott J Berger, Jing Fang, Brooke Koshel, Robert Birdsall, Min Du, Ying Qing Yu
Waters Corporation, Milford, MA, United States
• The proposal of “Multi-Attribute Method (MAM)” based LCMS peptide
mapping methods for semi-targeted monitoring of biotherapeutic protein
attributes has been greeted with both excitement regarding reducing the
dependence on a cadre of low information content assays, and concerns
over their appropriateness for deployment into regulated development
and QC/lot release roles.
• The selectivity of High-Resolution-MS (HRMS) methodologies must be
weighed against the challenges of deploying and operating these
complex systems in regulated environments, and more established and
routine nominal mass detection approaches require more rigorous
evaluation for establishing the extent to which they can be applied to
MAM based analysis.
• In this study, we have generated a common set of Trastuzumab forced
degradation samples, subjected to various levels of oxidative and high
pH stress.
• Characterization results from the reference samples were parsed to
select peptides for targeted monitoring of product attributes using both
HRMS and nominal mass detection strategies.
• Results from these studies have been compiled to enable data-based
discussions of fit-for-purpose MS for implementing peptide map based
attribute monitoring in regulated environments.
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INTRODUCTION
METHODS
Sample Preparation:
Trastuzumab samples were treated with alkaline
and oxidation stress, followed by denaturation,
alkylation and tryptic digestion.
LC/MS:
LC System: ACQUTIY UPLC H-Class Bio System
Column: ACQUITY UPLC CSH300 C18, 1.7 µm, 2.1 x 100 mm
Column temperature: 65 oC
Mobile phase: A. 0.1% FA in water,
B. 0.1% FA in acetonitrile
Gradient: 3-33 %B over 120 min
TUV Detection: 215 nm
HRMS System: Vion IMS QTof MS
Data Acquisition: MSE Mode: ESI positive mode
Capillary Voltage: 3.0 kV Cone Voltage: 30 V
Source Temperature:100 oC
Desolvation Temperature: 250 oC
Mass Range (m/z): 100-2000
Lock mass used: LeuEnk ([M+2H]2+, 556.2763)
MSE settings:
Scan rate for alternating low/high Energy: 0.5 sec
Low energy: 6 V
High energy ramp: 20-45 V
Nominal Mass System: ACQUITY QDa Mass Detector
Sample Rate: 2points/sec Mode: ESI positive mode
Capillary Voltage: 1.5 kV Cone Voltage: 15 V
Probe Temperature:500 oC Desolvation Temperature: 250 oC
Mass Range (m/z): 350 – 1250
Informatics:
UNIFI Scientific Information System v1.8 Service Release 2
• Peptide mapping (MSE) workflow
• Accurate mass screening workflow
• UNIFI scientific library
Empower 3 Chromatography Data Software
Figure 1: Two compliant-ready LC/UV/MS solutions for flexible deploying MS in Biopharmaceutical late development and QC laboratories.
• Using UNIFI/HRMS platform, product attribute characterization and monitoring data can be acquired using a single common mapping data acquisition methodology (UPLC/UV/MSE), but different informatics processing workflows optimized for each analysis. This enables a common platform for both analyses, and efficient transfer of analytical platforms and methods between groups responsible for their execution.
• Such transferability is facilitated by:
A compliant-ready UNIFI platform
Robust Multi-Channel Quantification (UV and MS)
Ability to rapidly update the multi-attribute screening method as new product knowledge is acquired.
RESULTS AND DISCUSSION
Monitoring Product Attributes using the UNIFI Accurate Mass Screening Workflow
Figure 3: The UNIFI MSE peptide mapping workflow was used to define trastuzumab reference sample attributes, confirm protein identity/sequence, and determine product modification variants. High MS sensitivity, large dynamic range, robust accurate mass measurement (A), and high MSMS fragmentation efficiency of Vion IMS QTof results in high sequence coverage, confident assignment of peptides and their post-translational and chemical modifications (B).
Figure 4: The UNIFI Scientific Library is a repository where information from multiple characterization runs can be aggregated and managed to produce target lists for subsequent screening based analyses.
A B
A B Figure 8: Setting limits and system suitability criteria (A) enables color coded highlights for samples or batches that exceed data quality criteria (B), or breach expected limits for component ranges (C).
Peptide Mapping Workflow
Characterization
Peptide Assignment
Sequence Coverage
Modification Profiling
Reference Batch
Sample Prep : Reduction, Alkylation, Trypsin Digest
Accurate Mass Screening Workflow
Attribute Monitoring
Peptide Monitoring
Limit Checking
New Peak Detection
Reference and Unk. Batch
UNIFI Library
Acquisition
Processing
Review Reporting
UNIFI
UPLC Peptide Map with UV and MSE Detection
Single HRMS Platform Solution for Peptide Map Characterization and Monitoring
UPLC/High Resolution MS:
Biopharmaceutical Platform Solution with UNIFI
UPLC/Mass Detection:
ACQUITY UPLC, TUV, QDa Mass Detection with Empower CDS
Characterizing Product Attributes using the UNIFI Peptide Mapping Workflow
Unmodified
Iso-D form
D form
1%
UV&MS Response % Mod
XICChromatogram
MSMS Fragmentation Annotation
Figure 2: Transitioning from characterization to attribute monitoring workflows within the UNIFI Platform Solution.
Sequence
Neutral Mass
Formula
RTChargeIntensity
Detection Results
Send to UNIFI Scientific Library
Import
RT ChargeFragmentUV
Wavelength
Neutral Mass
Figure 5: The UNIFI accurate mass screening workflow enabled targeted multiattribute monitoring, providing rapid qualitative assessment with an option to flag signature fragment ions (oxonium ions for N-Glycopeptides) for increased confidence of assignments (A) of each sample, and for robust quantification and comparison across this larger multi-batch data set (B).
A
B
D f
orm
Iso
-D f
orm
0.12%
HC: T10 Deamidation (NTAYLQMN84SLR)
0.58%
1.11%
1.58%
ControlpH 9
H2O2
0.13%
0.40%
0.72%
1.03%
Figure 6: Monitoring variation in asparagine deamidaton. A) Reviewing processed and integrated results in UNIFI can be obtained for optical and MS data channels, including eXtracted Ion Chromatograms (XIC) for each targeted component. B) Summary plots demonstrate relative % abundance of UV and MS response for Light Chain CDR domain peptide (ASQDVN30TAVAWYQQKPGK). C) Robust quantification was also obtained for peptides Less susceptible to alkaline stress, such as Heavy Chain peptide (NTAYLQMN84SLR).
A
B
C TUV
BPI
XIC(Iso-D Form)
XIC(Unmodified)
XIC(D Form)
LC: CDR T3 Deamidation (ASQDVN30TAVAWYQQKPGK)
25%
ControlpH 9H2O2
% MS Response % UV Response
Iso
-D
Un
mo
dif
ied
ControlpH 9H2O2
40%
61%
24%
40%
59%
74%59%
39%
74%60%
41%
LC: CDR T3 Deamidation (ASQDVN30TAVAWYQQKPGK)
Monitoring Product Attributes using the Empower/QDa Platform
% Oxidation (MS Response)
HC: T21 Oxidation (DTLM255ISR)
2.5%
Control pH 9 H2O2
2.5%
21%
42%
53%
Warning and Error Level Settings
Error min Warning min Warning max Error max
Chromatographic width
UV response
A
C
B
Figure 9: UNIFI allows creating customizable report templates for automated report generation based on attributes of interest. In an attribute monitoring workflow, most likely each attribute would have its own report chapter with a common set of report objects.
HC: T41 Oxidation
(WQQGNVFSCSVM431HEALHNHYTQK)
Control H2O
0.3%
2.4%
5.9%
8.1%
0.4%
3.1%
7.6%
10.6%
% Oxidation (MS Response)
Fragment
Average
Mass [CH+1H]+1 [CH+2H]+2 [CH+3H]+3 [CH+4H]+4 [CH+5H]+5 [CH+6H]+6 [CH+7H]+7 [CH+8H]+8 [CH+9H]+9 [CH+10H]+10
T39 574.3 575.3 288.2 192.4 144.6 115.9 96.7 83.0 72.8 64.8 58.4
T7 681.3 682.3 341.7 228.1 171.3 137.3 114.6 98.3 86.2 76.7 69.1
T5 830.0 831.0 416.0 277.7 208.5 167.0 139.3 119.6 104.7 93.2 84.0
T21 835.0 836.0 418.5 279.3 209.7 168.0 140.2 120.3 105.4 93.8 84.5
T30 838.0 839.0 420.0 280.3 210.5 168.6 140.7 120.7 105.8 94.1 84.8
T9 969.1 970.1 485.5 324.0 243.3 194.8 162.5 139.4 122.1 108.7 97.9
T6 1084.2 1085.2 543.1 362.4 272.1 217.8 181.7 155.9 136.5 121.5 109.4
T3 1089.2 1090.2 545.6 364.1 273.3 218.8 182.5 156.6 137.2 122.0 109.9
T36* 1161.4 1162.4 581.7 388.1 291.3 233.3 194.6 166.9 146.2 130.0 117.1
T2* 1167.4 1168.4 584.7 390.1 292.8 234.5 195.6 167.8 146.9 130.7 117.7
T8-9 1182.3 1183.3 592.2 395.1 296.6 237.5 198.1 169.9 148.8 132.4 119.2
T13 1186.4 1187.4 594.2 396.5 297.6 238.3 198.7 170.5 149.3 132.8 119.6
T10 1310.5 1311.5 656.3 437.8 328.6 263.1 219.4 188.2 164.8 146.6 132.1
T4-5 1311.5 1312.5 656.8 438.2 328.9 263.3 219.6 188.4 164.9 146.7 132.2
T14* 1321.5 1322.5 661.8 441.5 331.4 265.3 221.3 189.8 166.2 147.8 133.2
T11* 1334.4 1335.4 668.2 445.8 334.6 267.9 223.4 191.6 167.8 149.3 134.4
T23 1677.8 1678.8 839.9 560.3 420.5 336.6 280.6 240.7 210.7 187.4 168.8
T33-34 1724.9 1725.9 863.5 576.0 432.2 346.0 288.5 247.4 216.6 192.7 173.5
T26 1808.1 1809.1 905.1 603.7 453.0 362.6 302.4 259.3 227.0 201.9 181.8
T38 1874.1 1875.1 938.0 625.7 469.5 375.8 313.3 268.7 235.3 209.2 188.4
T1 1882.1 1883.1 942.1 628.4 471.5 377.4 314.7 269.9 236.3 210.1 189.2
T22* 2139.4 2140.4 1070.7 714.1 535.8 428.9 357.6 306.6 268.4 238.7 214.9
T26-27 2228.6 2229.6 1115.3 743.9 558.1 446.7 372.4 319.4 279.6 248.6 223.9
T2-3* 2238.6 2239.6 1120.3 747.2 560.6 448.7 374.1 320.8 280.8 249.7 224.9
T37 2544.7 2545.7 1273.3 849.2 637.2 509.9 425.1 364.5 319.1 283.7 255.5
T12 2785.0 2786.0 1393.5 929.3 697.3 558.0 465.2 398.9 349.1 310.4 279.5
T41* 2802.1 2803.1 1402.1 935.0 701.5 561.4 468.0 401.3 351.3 312.3 281.2
T19-20* 3335.9 3336.9 1669.0 1113.0 835.0 668.2 557.0 477.6 418.0 371.7 334.6
T15* 6716.5 6717.5 3359.2 2239.8 1680.1 1344.3 1120.4 960.5 840.6 747.3 672.6
T15-16* 7058.9 7059.9 3530.4 2354.0 1765.7 1412.8 1177.5 1009.4 883.4 785.3 706.9
T15-17* 7187.0 7188.0 3594.5 2396.7 1797.8 1438.4 1198.8 1027.7 899.4 799.6 719.7
Table 1: Peptide map charge states table. Multiple charge states observed for heavy chain tryptic peptides of trastuzumab using TFA and FA based methods, affords significant flexibility in method development of monitoring assays using the ACQUITY QDa.
Figure 10: Peptide mass accuracy. The ACQUITY QDa is capable of Providing mass information for peptides over a broad molecular weight range in assays routinely employed during the analysis of biotherapeutics.
Figure 7: Monitoring variation in Methionine oxidation levels across control and stressed samples. A) Significant increase level of oxidized heavy chain peptide (DTLM255ISR).
B)The oxidized heavy chain peptide T41 (WQQGNVFSCSVM431HEALHNHYTQK) is monitored and quantified as diastereomers.
TIC
XIC
1
23
4
5
6
7
Light Chain 1: ASQDVNTAVAWYQQKPGK 2: LLIYSASFLYSGVPSR 3: SGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTK
Heavy Chain 4: DTYIHWVR 5: IYPTNGYTR/(6)YADSVKG 7: WGGDGFYAMDYWGQGTLVTVSSASTK
CDR Peptide Monitoring using ACQUITY QDa
Figure 11: Complementary determining region (CDR) peptide profiles unique to trastuzumab are effectively extracted from the MS full scan chromatography for the rapid determination of product identity.
Inte
nsity
0.0
20000
40000
60000
80000
100000
120000
Retention Time (min)
3.00 3.50 4.00 4.50 5.00 5.50 6.00
Man5
G0 G1
G0F
G1F
G2F
Component
Modification (%)
HRMS QDa + SIRs
G0 4.7 6.0
G0F 41.2 44.9
G2F 7.6 6.2
G1 2.2 2.7
G1F 42.2 38.7
Man5 2.2 1.4
QDa SIRsControl sample
Glycopeptide Monitoring using ACQUITY QDa
Figure 12: Glycopeptide profiles are obtained using the MS SIR scan for enhanced sensitivity to monitor co-eluted peptides with improved quantification robustness. Quantification using QDa with SIR scan provide compatible glycopeptide profiles vs UNIFI/HRMS results.
Monitoring LC: CDR T3 Deamidation
(ASQDVN30TAVAWYQQKPGK)
Inte
nsity
0
1.0x106
2.0x106
3.0x106
4.0x106
Retention Time (min)33.00 34.00 35.00 36.00 37.00 38.00
Unmodified
Iso-DD
QDa XICControl sample
Iso
-D
Un
mo
dif
ied
91.9 92.2 92.3
74.1 75.0 75.1
60.1 59.1 61.1
39.8 39.2 40.4
0
25
50
75
100
Contr
ol
Contr
ol
Contr
ol
Deam
_1
Deam
_1
Deam
_1
Deam
_2
Deam
_2
Deam
_2
Deam
_3
Deam
_3
Deam
_3
75%
60%
40%
7.7 7.2 7.0
25.4 24.5 24.3
39.7 40.738.6
60.0 60.7 59.4
0
20
40
60
80
Contr
ol
Contr
ol
Contr
ol
Deam
_1
Deam
_1
Deam
_1
Deam
_2
Deam
_2
Deam
_2
Deam
_3
Deam
_3
Deam
_3
74%
59%
39%
92%
91% HRMS
QDa
25%
40%
60%
25%
40%
61%
7%
8%
% Deamidation
(MS Response)
Figure 13: Quantification using QDa with MS full scan provide compatible deamidation profiles (Green) of the CDR domain peptide T3, compared to the UNIFI/HRMS results (Pink).
SUMMARY
Figure 14: Reports can be automatically generated when linked to the acquisition through the method set, automating the monitoring process in a regulated environment.
• The AQUITY QDa mass detector can be easily added to existing Empower/ACQUITY UPLC/UV systems, with minimal maintenance and training requirements for analysts.
• The QDa based system is capable of detecting peptides over a large MW range with nominal mass, and expanding the dynamic range for attribute monitoring over optical only assays.
Compliant-ready Empower platform
Accessible technology, small footprint
Easier deployment
System suitability and limit check capabilities
New/differential peak detection
Import into Accurate Mass Screening Method
UV
TIC
XIC
OxoniumIons
MS Ions
%Peptide M
od (
MS)
Control pH 9H2O2
Glycopeptide Profiles Monitoring Deamidated Peptide Monitoring
Sample List / Experimental
Supporting LC/UV/MS Data
TUV
XIC
Summary ResultsAttribute Focused Results
XIC
BPI
XIC
Setting Limits and System Suitability
Attribute Centric Reporting
Oxidized Peptide Monitoring
High Sequence Coverage Robust Accurate Mass Measurement
2 ppm95%
3 ppm99%
0.5% 0.1%3.2%
20.0%
36.9%
29.9%
7.9%
1.3% 0.7% 0.3%0%
5%
10%
15%
20%
25%
30%
35%
40%
-4 -3 -2 -1 0 1 2 3 4 5
% o
f P
ep
tid
es
Mass Errors (PPM)
1 ppm67%
5ppm
-5ppm
Overlay 10 Peptide Mass Error (10 injections)Large dynamic range