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