Adding Mass Detection to Monitor Peptides in Biopharmaceutical Development & QC

©2015 Waters Corporation 1

Adding Mass Detection to Routine

Peptide Level Biotherapeutic Analysis


Presentation Outline

The unmet need

The ACQUITY® QDa® Detector – a brief overview

Demonstrating broad utility for peptide monitoring

A clear path to increased productivity


What we’ve been hearing…

“We’re doing a lot of routine MS monitoring in support of pilot

operations, manufacturing and QC, and foresee doing more in the

future.”

“Relying on our core MS resources and high end equipment for

this testing is expensive and creates a bottleneck.”

“We’d like to empower traditional LC analytical support labs to

generate this data, thus reducing the burden on core MS resources

and improving our overall workflow and productivity.”

The bottom line: Biopharmaceutical laboratories want to better utilize analytical resources and improve productivity.


The ACQUITY® QDa® Mass Detector A pioneering product with mass appeal

Innovative design focused on

ease-of-use for chromatographers

Empowering analytical chemists

everywhere with orthogonal mass

detection – added information with

every sample

Compact, robust and affordable:

Built for constant use with a wide

variety of chromatographic

conditions

Seamlessly integrates with HPLC &

UPLC® systems that use Empower ®

Chromatography Data Software

www.waters.com/qda

http://www.waters.com/qda


Easily Add to Existing LC Systems

Existing stack

Easy to deploy

Fully integrated with Empower® CDS Software

Minimal training required

Qualification documents / procedures ready-to-go

110/220V operation

Minimal maintenance

ACQUITY QDa

Existing stack +1upgrade


Familiar Graphical User Interface for Ease-Of-Use and Fast Adoption

Empower GUI identical to

that of a PDA for setup,

data viewing and reporting

Very little training needed

Quick addendum to current lab

SOPs


Automated Start Up ProvidesRobust, Reproducible Performance

Automated resolution and calibration occurs with each

start-up, ensuring mass information is accurate and precise

ESI interface optimized for UPLC performance to ensure

chromatographic resolution, sensitivity and throughput is

preserved

The Graphic ACQUITY QDamonitor display enables easy viewing and adjustment of system parameters


Disposable Sample Aperture and Capillary for Easy Maintenance

Sample Aperture:

As simple as replacing a detector lamp

Capillary:

No cutting or assembly required


ACQUITY QDa Detector in summary

A pioneering product that is empowering analytical chemists

and chromatographers everywhere to take advantage of the

power of mass detection

Brings greater insight into every peak, for enhanced and

streamlined monitoring workflows for improved productivity

Easy to deploy, simple to use and maintain, compact, robust

and affordable!

www.waters.com/qdabiopharm

http://www.waters.com/qdabiopharm


ACQUITY QDa for Peptides

The following data demonstrates that this easy-to-sue

mass detector can:

Detect and monitor peptides over a wide molecular weight range

Quantify peptide variants with enhanced specificity

Monitor components below optical detector sensitivity

Selectively detect and monitor coeluting components

Work with both TFA and Formic Acid based separations


Extracted spectrum

ACQUITY QDa

ACQUITY QDa Detects Peptides Over a Wide Mass Range

3 µg injection

Peak Peptideaverage

mass [M+] M+2] [M+3] [M+4] [M+5]

1 Angiotensin frag. 1-7 899.0 900.0 450.5 300.6 225.7 180.8

2 Bradykinin 1060.2 1061.2 531.1 354.4 266.0 213.0

3 Angiotensin II 1046.2 1047.1 524.1 349.7 262.5 210.2

4 Angiotensin I 1296.5 1297.4 649.2 433.1 325.1 260.3

5 Renin substrate 1759.0 1760.0 880.5 587.3 440.7 352.8

6 Enolase T35 1873.2 1874.2 937.6 625.4 469.3 375.6

7 Melittin 2847.5 2848.4 1424.7 950.1 712.8 570.5

Charge State

1 2

3

4

5

67

[M+4]

[M+3]

712.7

949.9

[M+5]

570.4


ACQUITY QDa Mass Chromatograms of Trastuzumab Digest

ACQUITY UPLC BEH Column 0.1% TFA, 4-µg injection

ACQUITY UPLC BEH Column 0.1% FA, 4-µg injection

Digest 5BEH 130Å 100mmGradient: 97-65% A

Digest 1BEH 300Å 150mmGradient: 97-65% A


Trastuzumab – Heavy Chain Peptideswith TFA vs. FA as Acid Modifier

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

Trifluoroaceticacid

Formic acid

= not observed

Legend:

90 % Coverage


Trastuzumab – Light Chain Peptideswith TFA vs. FA as Acid Modifier

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

T2* 748.9 749.9 375.5 250.6 188.2 150.8 125.8 108.0 94.6 84.2 75.9

T19-20* 868.9 869.9 435.5 290.6 218.2 174.8 145.8 125.1 109.6 97.5 87.9

T15 1502.6 1503.6 752.3 501.9 376.7 301.5 251.4 215.7 188.8 168.0 151.3

T5 1773.1 1774.1 887.5 592.0 444.3 355.6 296.5 254.3 222.6 198.0 178.3

T11* 1798.1 1799.1 900.0 600.4 450.5 360.6 300.7 257.9 225.8 200.8 180.8

T18* 1876.1 1877.1 939.1 626.4 470.0 376.2 313.7 269.0 235.5 209.5 188.6

T1 1879.0 1880.0 940.5 627.3 470.8 376.8 314.2 269.4 235.9 209.8 188.9

T10 1946.2 1947.2 974.1 649.7 487.6 390.2 325.4 279.0 244.3 217.2 195.6

T3 1991.2 1992.2 996.6 664.7 498.8 399.2 332.9 285.5 249.9 222.2 200.1

T14 2136.2 2137.2 1069.1 713.1 535.0 428.2 357.0 306.2 268.0 238.4 214.6

T17-18* 2141.4 2142.4 1071.7 714.8 536.4 429.3 357.9 306.9 268.7 238.9 215.1

T3-4 2287.6 2288.6 1144.8 763.5 572.9 458.5 382.3 327.8 286.9 255.2 229.8

T7* 4189.5 4190.5 2095.8 1397.5 1048.4 838.9 699.3 599.5 524.7 466.5 420.0

Charge State

Trifluoroaceticacid

Formic acid

= not observed

Legend:

Key Takeaways: Compatible with TFA and FA based separations Multiple charge states detected /peptide

92% Coverage


HC Peptide T15* Mass Spectrum

DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK

Average mass = 6716.5 Da

[M+6H]+6

[M+7H]+7

[M+8H]+8

[M+9H]+9

[M+10H]+10

[M+11H]+11

m/z

In

ten

sit

y

Key Takeaway~7,000 Da peptide detected with multiple charge states.


Mass Measurement Accuracy

0.00

20.00

40.00

60.00

80.00

100.00

0.00 0.05 0.10 0.15 0.20

Cu

mu

lati

ve

Erro

r (

%)

Absolute Mass Error

Cumulative Error Distribution Plot

-0.30

-0.20

-0.10

0.00

0.10

0.20

0.30

0 1,000 2,000 3,000 4,000 5,000 6,000 7,000

Mass D

iffe

ren

ce

Theoretical Average Molecular Weight (Da)

Mass Error Vs. Molecular Weight

Instru

ment S

pec.

Key TakeawayMass measurement accuracy of all reported peptides within instrument specification (+/- 0.2 Da).


Specificity:Accurate Peak Area Determinations

T10

T26

T26: Area 9.1105 (59%)T10: Area 6.3105 (41%)

T26: Area 1.6108 (62%)T10: Area 9.9107 (38%)

TUV

XIC

T10

T26

Key TakeawayMS detection reduces variability when integrating closely eluting components.


Specificity: Resolving Coelutions

XIC

SIR

Peak 2

Peak 2

Peak 1

Peak 1&2

Peak 1 [M+4H]+4

536.3 m/z

Peak 2 [M+2H]+2

426.5 m/z

TUV

Peak 2426.5 m/z

Key Takeaway MS reveals coelutions and enables you to independently monitor both species.


ACQUITY QDa Greatly Extends Linear Dynamic Range (ACQUITY UPLC BEH w/ 0.1% TFA)

Mass LoadDilution ng pmol

1:1 1025.00 6961.19

1:2 512.50 3480.59

1:4 256.25 1740.30

1:8 128.13 870.15

1:16 64.06 435.07

1:32 32.03 217.54

1:64 16.02 108.77

1:128 8.01 54.38

1:256 4.00 27.19

1:512 2.00 13.60

1:1,024 1.00 6.80

y = 74103x - 6636.1

R² = 0.9995

0.00

0.04

0.08

0.12

0.00 5.00 10.00 15.00

Area

x 1

0000000

Mass Load (ng)

BEH SIR Area Vs. Mass Load

SIRTUV


1:1 1025.00 6961.19

1:2 512.50 3480.59

1:4 256.25 1740.30

1:8 128.13 870.15

1:16 64.06 435.07

1:32 32.03 217.54

1:64 16.02 108.77

1:128 8.01 54.38

1:256 4.00 27.19

1:512 2.00 13.60

1:1,024 1.00 6.80


1:1 1025.00 6961.19

1:2 512.50 3480.59

1:4 256.25 1740.30

1:8 128.13 870.15

1:16 64.06 435.07

1:32 32.03 217.54

1:64 16.02 108.77

1:128 8.01 54.38

1:256 4.00 27.19

1:512 2.00 13.60

1:1,024 1.00 6.80

1:2,048 0.50 3.40

1:4,096 0.25 1.70

1:8,192 0.13 0.85

1:16,384 0.06 0.42

TIC

Lin

ear

Lin

ear

Lin

ear


Extend the Linear Dynamic Range Farther Using ACQUITY UPLC CSH w/ 0.1% FA

SIR


1:64 16 109

1:128 8 54.4

1:256 4 27.2

1:512 2 13.6

1:1024 1 6.80

1:2,048 0.50 3.40

1:4,096 0.25 1.70

1:8,192 0.12 0.849

1:16,384 0.063 0.425

1:32,768 0.031 0.212

1:65,536 0.016 0.106

1:131,072 0.0078 0.0531

1:262,144 0.0039 0.0266

1:524,288 0.0020 0.0133

Lin

ear

R² = 0.9993

0

4

8

12

16

20

0.00 2.00 4.00 6.00 8.00 10.00

Area M

illions

Mass Load (ng)

CSH 0.1% FA SIR Area Vs. Mass Load

2.0 pg (13fmol)

7.8 pg (53fmol)

16 pg (106fmol)

31 pg (212fmol)

SIR

1:32,768

1:65,536

1:131,0721:524,288

S/N 171

S/N 88

S/N 28

S/N 15

dilution


Quantification

T21-Oxidized T21

5.5%

94.5%

SIR

TUV

TIC

Key TakeawayMS provides the specificity and sensitivity for relative quantification of peptides.


CDR Peptide Monitoring

TIC

XIC

1

23

4

5

6

7

Key TakeawayMS enables targeted monitoring of peptides for product ID testing.

Light Chain1: ASQDVNTAVAWYQQKPGK2: LLIYSASFLYSGVPSR3: SGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTK

Heavy Chain4: DTYIHWVR5: IYPTNGYTR/(6)YADSVKG7: WGGDGFYAMDYWGQGTLVTVSSASTK

Harris, R.J., Kabakoff, B., Macchi, F.D., Shen, F.J. Kwong, M., Andya, J.D., Shire, S.J., Bjork, N., Totpal, K., Chen, A.B., Identification of multiple sources of charge heterogeneity in a recombinant antibody. J. Chromatogr. B 752 (2001) 233-245.


Deamidation Monitoring (CDR peptide)

TIC

XIC

572.8 m/z

PeptideAverage

MassXICm/z

T3-4 2,287.5 572.8

T18* 1,876.1 626.3

T2* 1,167.3 584.6

T5 8,29.9 415.9

T3-4* 2,288.5 573.1

T5

T3-4

T2*

T18*

T3-4: ASQDVNTAVAWYQQKPGKAPK

Deamidated

XICT3-4

573.1 m/zT3-4*(D)

Key TakeawayWhen chromatographically resolved, deamidated species can be monitored.

Harris, R.J., Kabakoff, B., Macchi, F.D., Shen, F.J. Kwong, M., Andya, J.D., Shire, S.J., Bjork, N., Totpal, K., Chen, A.B., Identification of multiple sources of charge heterogeneity in a recombinant antibody. J. Chromatogr. B 752 (2001) 233-245.

T3-4*(iso)573.1 m/z


Detect and monitor peptides

over a wide molecular weight

range

Quantify peptide variants with

enhanced specificity

Monitor components below

optical detector sensitivity

Selectively detect and monitor

coeluting components

Compatible with both TFA and

Formic Acid based separations

ACQUITY QDa – Fit for Purpose

When incorporated into Empower-based chromatographic workflows, the ACQUITY QDa Detector provides enhanced specificity, selectivity,

and quantification for greater productivity in biotherapeutic development, production and QC.


http://www.waters.com/waters/nav.htm?cid=134837932


Addendum: User Case Study

Customer X – a large biopharmaceutical developer and

manufacturer – wanted to further explore the potential of the

QDa to meet their analytical needs.

We were supplied with a range of samples unknown to us at the

time and were requested to analyze these samples so they

could compare to their own internal results.

The following slides show ACQUITY QDa data we generated

from these samples and how the data compared to customer

generated results shared with us afterwards.


ACQUITY QDa Detector

LysC digest of mAb* (120 min run)

ACQUITY UPLC TUV DetectorMethod Summary** ACQUITY UPLC H-Class

with TUV and ACQUITY QDa detectors run with Empower 3, SR2

ACQUITY UPLC CSH C18 130Å 1.7-µm column, 2.1x100 mm

Acid Modifier: FA

Notes:* Later revealed to be Humira®

** See speaker notes for gradient info and LC-MS settings

Comments:Good agreement between UV trace and TIC trace


Zoom-in comparison:0-30 min

LysC digest of mAb (0-30 minutes)

TUV

Mass


Zoom-in comparison:30-60 min


TUV

Mass


Zoom-in comparison: 60-90 min


TUV

Mass


Zoom-in comparison: 90-120 min


TUV

Mass


LysC digest: SYNAPT vs. ACQUITY QDa(0-30 minutes)

ACQUITY QDa

SYNAPT (data offset by 2.4 min for comparison)

SYNAPT vs. ACQUITY QDaZoom-in comparison:0-30 min



ACQUITY QDa

SYNAPT




ACQUITY QDa

SYNAPT




ACQUITY QDa

SYNAPT K7 Peptide (6,714 kDa)

+6

+7+8

+9

+10

SYANPT vs. ACQUITY QDaZoom in comparison:90-120min

Raw spectrum


Consistent Repeatable Performance

[M+6H]+6

[M+7H]+7

[M+8H]+8

[M+9H]+9

[M+10H]+10

[M+11H]+11

m/z

In

ten

sit

ySame peptide analyzed from two different samples on different days using different mobile phases…

Same Result!

LysC Digest HC Peptide: K7 FA modifier

Trypsin Digest HC Peptide: T15* TFA modifier


QDa = 96.8% (X = 97.5%)

QDa = 3.2%(X =2.5%)

H21

H21-Ox

SIRH21: M+2 [418.4]H21-Ox: M+2 [426.4]

Relative Quantification Native vs. Oxidized Peptide

Chromatographic Resolution

m/z Resolution

+ +

Scenario 1:

Large Δ in m/z Single charge state Well resolved

Comments: Ideal scenario Integration on same

data channel


H6

Relative QuantificationDeamidated vs. Non-Deamidated (1)

H6D

H6D

SIR m/z

M+3 [362.4]M+2 [543.1]

Non-deamidated

SIR m/z

M+3 [362.7]M+2 [543.6]

DeamidatedCombined:QDa = 43.2% (X = 43.6%)

31.9%

QDa = 56.8% (X = 56.4%)

11.3%


m/z Resolution

+ -

Scenario 2:

Small Δ in m/z Multiple charge states Well resolved

Comments: Run w/two SIR channels Record area from

individual SIRs Calculate % area Cross channel

processing enabled through Empower


Relative QuantificationDeamidated vs. Non-Deamidated (2)


m/z Resolution

+ -

Scenario 2(B):

Small Δ in m/z Single charge state Well resolved

Comments: Run single SIR channel,

but with time dependant windows

Record area from individual SIRs

Calculate % area

H37

0-65 minNon-deamidated SIR: [849.2]

65-140 minDeamidated SIR: [849.6]

QDa = 89.1%(X = 89.2%)

QDa = 10.9%(X = 10.8%)H37D


IdeS Digest: Humira (adalimumab)

TimeFlow

(mL/min)% A % B %C % D

Initial 0.200 0 0 95 5

3.00 0.200 0 0 77 23

5.00 0.200 0 0 77 23

35.00 0.200 0 0 67 33

38.00 0.200 0 0 20 80

40.00 0.200 0 0 20 80

43.00 0.200 0 0 95 5

46.00 0.200 0 0 95 5

Mobile phase:

A: H2O, 0.1 % TFA

B: Acetonitrile, 0.1 % TFA

C: H2O, 0.1 % FA

D: Acetonitrile, 0.1 % FA

TUV

Mass

FC

Fab

FC

Fab

Notes:See speaker notes for LC-MS settings


IdeS Digest: Humira (2)

Focused gradient on FC fragment

1

2

3

4 5

1

2

3

4

5


Trypsin digest: Glycopeptides

PeptideAverage

massM+3

H25 1189.2 397.4

G0F 2633.7 878.9

G1F 2795.8 932.9

G2F 2957.8 986.9

G0 2487.7 830.2

G1 2649.7 884.2

Man5 2405.6 802.9

G2F +NeuAc 3248.9 1084.0

G0F 51.1% (X 50.2*)

* Adjusted percent

G1F 42.8% (X 43.7*)

G2F 6.1% (X 6.2*)

G0G1

G1Man5 Man5

H25

Zoom-in

ACQUITY QDa: SIR full scan

G2F+NeuAC


Detect and monitor peptides

over a wide molecular weight

range

Quantify peptide variants with

enhanced specificity

Monitor components below

optical detector sensitivity

Selectively detect and monitor

coeluting components

Compatible with both TFA and

Formic Acid based separations

ACQUITY QDa – Fit for Purpose

When incorporated into Empower-based chromatographic workflows, the ACQUITY QDa Detector provides enhanced specificity, selectivity,

and quantification for greater productivity in biotherapeutic development, production and QC.




More info on the ACQUITY QDA Detector for Biopharm: www.waters.com/qdabiopharm

Details on the ACQUITY QDa Detector: www.waters.com/qda



Science

Adding Mass Detection to Monitor Peptides in Biopharmaceutical Development & QC