LCMS Based Quantitation of Intact Proteins for ... · of Intact Proteins for Bioanalytical Applications Alex Zhu, Ph.D. Agilent Technologies Wilmington, DE ASMS，2017-06 June 14,

LC/MS Based Quantitation of Intact Proteins for Bioanalytical Applications

Alex Zhu, Ph.D.

Agilent Technologies

Wilmington, DE

ASMS，2017-06

June 14, 2017

For Research Use Only. Not For Use in Diagnostic

Procedures.

1

Outline

• Introduction on intact protein quantitation

• Agilent Solutions for intact protein quantitation (standard flow

UHPLC/6545XT)

• Quantitation of intact mAb in neat solution

• Quantitation of intact mAb in rat plasma

• Quantitation of antibody drug conjugate in rat serum

June 14, 2017


Procedures.

2

Current Techniques for Protein Quantitation in Biological Matrices

• Ligand-binding assays (LBAs)

- High sensitivity; high throughput

- Require suitable capture and detection reagents; less specific, can be

affected by the presence of anti-drug antibodies in the samples

• MS based

- High specificity; wide dynamic range; faster method development;

quantify multiple proteins simultaneously; quantify PTMs, degradation

products, metabolites simultaneously

June 14, 2017


Procedures.

3

MS Based Protein Quantitation Strategies

• MS based

• Peptide approach

- Digestion is time consuming resulting in limited throughput

- Digestion efficiency and reproducibility requires extensive evaluation

- Selection of surrogate peptide is not simple

- Optimization of MRM method requires a lot of work

- Loss of essential information of the intact protein can lead to ambiguous

quantification

• Intact approach

- Higher throughput

- Specificity allows differentiation between closely related proteoforms

- Offering unique possibility to quantify therapeutic or clinically relevant

metabolites as well as postdose or pose-translational modified proteins

- More accurate quantitation

June 14, 2017


Procedures.

4

Challenges for Intact mAb Quantitation Using LC/MS

June 14, 2017


Procedures.

5

Challenges Causes Possible Solutions

Sensitivity “Diluted” peak intensity due to

formation of multiple charge

states

More sensitive instruments;

ways to reduce number of

charge states

Specificity Interference from

endogenous IgGs

Sample preparation

techniques; middle-down

approaches; Higher res instr.

(>600k) with fast enough

acquisition rate for quant

Chromatography Limited separation efficiency

and bad peak shape due to

heterogeneity

Better column; Alternative

separation mechanisms

(HILIC, etc.)

Data Analysis Single charge state?

Summation of several charge

states? Deconvoluted

Spectrum?

Need evaluation

Example: J&J (Jun, 2016)

J&J

Sample

Intact mAb in Plasma (Sigma

SiluLiteMab and SiluMab, SiluMab is

heavy labeled and used as IS)

Affinity

Purification Magnetic Beads

Flow rate Standard flow, 0.4 ml/min

Mass Spec TripleTOF API 5600

Quant

analysis Using height of deconvoluted spectra

LLOQ 60 ng on-column (1 µg/ml with 60 µl

inj)

Spectrum at

LLOQ level

Yes (large mass difference >100ppm

at all levels)

Linear range 1 order (due to limit of binding

capacity on beads)

June 14, 2017


Procedures.

6

Example: Novartis (Jan 2017)

Novartis’ Article

Sample Intact InfliximAb (Remicade) in rat

serum

Affinity

Purification

LB-MSIA （Ligand Binding-Mass

Spectrometric Immunoassay)

Flow rate 0.2 ml/min

Mass Spec QE

Quant

analysis Using sum of EICs

LLOQ 6 ng on-column

Spectrum

at LLOQ

level

Yes

Linear

range

2 orders (0.1-10 µg/ml with 60 µl

injection)

June 14, 2017


Procedures.

7

6545XT Features for Large Biomolecule Analysis

• Excellent protein spectral clarity from ultra-low TOF vacuum (10E-8)

• One-click optimization for large molecules with SWARM autotune

• Capable of analyzing very large molecules, with a variable mass range

up to 30k m/z

• Ease of maintenance with vent-free capillary removal

• Protein performance verification at install, and includes quick-start

protein method

June 14, 2017


Procedures.

8

Intact Protein Raw Spectral Quality

50+ 49+ 48+

Intact NIST mAb Analysis (0.5 µg injection)

Glycosylated Intact

Excellent raw spectral quality to

detect and identify minor isoforms

such as loss of amino acids, minor

glycoforms, and other PTMs.

June 14, 2017


Procedures.

9

Intact Protein Deconvoluted Mass Accuracy

6/14/2017


Procedures.

10

MaxEnt Deconvolution

- 0.64 ppm

1.44 ppm

- 0.06 ppm

0.78 ppm

- 3.72 ppm

G0F + G0F

G0F + G1F

G1F + G1F

G1F + G2F

G2F + G2F

3.54 ppm

(G0F+G0F)

- GlcNAc

(G0F+G1F)

- GlcNAc

(G1F+G1F)

- GlcNAc

(G1F+G2F)

- GlcNAc (G2F+G2F)

- GlcNAc

Intact NIST mAb Analysis (0.5 µg injection)

As seen in the raw spectra, the

increased spectral clarity leads

to improved deconvolved results,

both in accuracy and detection of

minor isoforms.

Maximum Entropy deconvolution

preserves low intensity signals

for investigation of heterogeneity.

How to Obtain Good Intact Protein (mAb) Spectra?

• Issues/complaints:

- Bad peak to hump

- Broad spectral peaks

- Loss of resolution of distinct

glyco-forms

- Bad mass accuracy

- Cleaning optics, changing

parts don’t help

- …

What’s Wrong??

June 14, 2017


Procedures.

11

Key: Cleanliness of the LC!!! Check Your LC Carefully Before Blaming the MS

• Contaminants in the LC may not ionize, so won’t be seen

directly in the MS spectra, but could form complexes

(adducts) with the proteins, causing the issues!

- Contaminated bottles

- Buffer (water & organic)

- Tubing

- Column

- Valves

- Fittings/capillaries

June 14, 2017


Procedures.

12

One Example: Contaminants from Newly Opened HPLC Grade Bottled Water

Using Milli Q Water

Using HPLC Grade Bottled Water

June 14, 2017


Procedures.

13

Quantitation of Intact Herceptin In Neat Solution ASMS Poster: WP-637

June 14, 2017


Procedures.

14

Chromatography Optimization

Issue: Broad chromatographic peak, tailing, making it difficult for accurate quant at low levels.

From J&J Article

June 14, 2017


Procedures.

15

From Agilent app note (5991-6296EN)

Typical TIC for intact mAb, showing

board peak (>8s FWHM) and tailing.

Chromatography Optimization: Column Temperature (Herceptin)

RT

80 °C

FWHM = 11s with significant tailing

FWHM = 5.5s with some tailing

June 14, 2017


Procedures.

16

Chromatography Optimization (Herceptin)

Optimized Chromatography

FWHM = 5.5s with some tailing

FWHM = 2.1s with no tailing

June 14, 2017


Procedures.

17

Sensitivity and Linear Range of Glycosylated Intact mAb

6/14/2017


Procedures.

18

Trastuzumab

EIC Sum

Calibration Concentration (ng on-column)

0.0316 0.1 0.316 1 3.16 10 31.6 50

Accuracy (%, n=6) 102.5 94.1 95.1 95.6 101.7 110.4 105.4 95.4

Cal. Conc. %RSD (n=6) 7.40 3.83 5.48 4.37 6.56 2.20 0.68 2.68

Zoom in

Linear Range: > 3.2 orders

(31.6 pg50 ng or 200 amol350 fmol)

Standard Flow (0.4 ml/min)

Data from Trastuzumab Dilution Series

6/14/2017


Procedures.

19

2x10

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

1.2

1.3

1.4

1.5

1.6

1.7

1.8

148053.51

148218.01

148379.79

Counts vs. Deconvoluted Mass (amu)147600 147700 147800 147900 148000 148100 148200 148300 148400 148500 148600 148700 148800 148900

3x10

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

1.2

1.3

1.4

1.5

1.6

1.7

1.8

1.9

2

2.1

2.2

2.3

2.4

148380.27

148221.24148062.96

148545.73

147911.38 148790.16148476.09

Counts vs. Deconvoluted Mass (amu)147600 147700 147800 147900 148000 148100 148200 148300 148400 148500 148600 148700 148800 1489003x10

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

5.5

6

6.5

7

7.5

8

8.5

148221.02

148061.71 148380.15

148545.18

148486.09148280.42147917.18148150.08 148675.17

148855.77


4x10

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

1.2

1.3

1.4

1.5

1.6

1.7

1.8

1.9

2

2.1

2.2

2.3

2.4

2.5 148220.09

148061.10 148382.80

148544.58

148320.56148469.44148149.73

147911.58148680.35

147992.93 148837.89147713.22


0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

5.5

6

6.5

7

7.5

8 148220.86

148381.80148059.52

148542.68

148316.78147912.51

148156.22148479.32

148703.37147852.26148830.20147705.77


5x10

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

2.2

2.4

2.6

2.8

148220.75

148382.11148059.51

148543.19

147913.28148317.32148156.92

148480.57148703.56147853.32 148004.83

147706.53 148832.85


5x10

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

5.5

6

6.5148220.75

148381.86148059.73

148543.56

147913.20148318.84

148157.23 148480.89148705.04

147852.78 148003.31147707.42 148837.62


0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0.5

0.55

0.6

0.65

0.7

0.75

0.8

0.85

0.9

0.95

1148220.64

148382.11148059.57

148543.22

147912.85148318.49

148159.05 148480.61148703.77147852.08 148002.42

147708.19 148835.66


0.0316 ng

0.1 ng

0.316 ng

1.0 ng

3.16 ng

10 ng

31.6 ng

50 ng

2x10

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

1.2

1.3

1.4

1.5

1.6

1.7

1.8

148053.51

148218.01

148379.79


3x10

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

1.2

1.3

1.4

1.5

1.6

1.7

1.8

1.9

2

2.1

2.2

2.3

2.4

148380.27

148221.24148062.96

148545.73

147911.38 148790.16148476.09


0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

5.5

6

6.5

7

7.5

8

8.5

148221.02

148061.71 148380.15

148545.18

148486.09148280.42147917.18148150.08 148675.17

148855.77


4x10

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

1.2

1.3

1.4

1.5

1.6

1.7

1.8

1.9

2

2.1

2.2

2.3

2.4

2.5 148220.09

148061.10 148382.80

148544.58

148320.56148469.44148149.73

147911.58148680.35

147992.93 148837.89147713.22


0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

5.5

6

6.5

7

7.5

8 148220.86

148381.80148059.52

148542.68

148316.78147912.51

148156.22148479.32

148703.37147852.26148830.20147705.77


5x10

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

2.2

2.4

2.6

2.8

148220.75

148382.11148059.51

148543.19

147913.28148317.32148156.92

148480.57148703.56147853.32 148004.83

147706.53 148832.85


5x10

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

5.5

6

6.5148220.75

148381.86148059.73

148543.56

147913.20148318.84

148157.23 148480.89148705.04

147852.78 148003.31147707.42 148837.62


0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0.5

0.55

0.6

0.65

0.7

0.75

0.8

0.85

0.9

0.95

1148220.64

148382.11148059.57

148543.22

147912.85148318.49

148159.05 148480.61148703.77147852.08 148002.42

147708.19 148835.66


0.0316 ng

0.1 ng

0.316 ng

1.0 ng

3.16 ng

10 ng

31.6 ng

50 ng

Relative Abundances of Different Glyco-forms Across the Linear Dynamic Range (0.0316 - 50 ng)

June 14, 2017


Procedures.

20

0.0

5.0

10.0

15.0

20.0

25.0

30.0

35.0

40.0

45.0

Rel

ativ

e ab

un

dan

ces

of

maj

or

glyc

o-f

orm

s

50ng 31.6ng 10ng 3.16ng 1ng 0.316ng 0.1ng 0.0316ng

G0/G0F

G0F/G0F

G0F/G1F

G0F/G2F

G1F/G2F

G2F/G2F

Relative abundances of 6 major glyco-forms for

Herceptin at different levels (0.0316 – 50 ng)

Mass Accuracy Reproducibility (Accuracy and Precision) Across the Linear Dynamic Range (0.0316 - 50 ng)

June 14, 2017


Procedures.

21

-60

-40

-20

0

20

40

60

Mas

s Er

ror,

pp

m

G0F/G1F G0F/G0F G0F/G2F G1F/G2F G0/G0F G2F/G2F

0.0316 ng 0.1 ng 0.316 ng 1 ng 3.16 ng 10 ng 31.6 ng 50 ng

Excellent mass accuracy and reproducibility across the linear range

• For most abundant glyco-form G0F+G1F (148220.9758):

- Average mass error: -1.78 ppm

- Standard Deviation: 1.58 ppm

- Mass error range (-5.05 to 2.09 ppm)

Mass Accuracy Reproducibility (Accuracy and Precision) 100 Replicate Runs of 10 ng Injections

6/14/2017


Procedures.

22

10 ng on-column

Response Reproducibility 100 Replicate Runs of 10 ng Injections

6/14/2017


Procedures.

23

Norm

aliz

ed Inte

nsity

# of Runs

For 100 replicate runs, sum of EICs

of most abundant 12 spectral peaks:

- Standard deviation = 1.5% #40 Run

2.0s FWHM

Quantitation of Intact Herceptin in Rat Plasma

June 14, 2017


Procedures.

24

Acknowledgement: Kevin Bateman and Lisa Varicek (Merck) for

data acquisition and discussions

Experimental Flow

June 14, 2017


Procedures.

25

Rat plasma

(for serial

dilutions)

Trastuzumab

spiked Rat

plasma

SA-

W SA-

W

1290 Infinity II

6545XT AdvanceBio

LC/Q-TOF

MassHunter Quant

AssayMAP Bravo

Amount on Column

June 14, 2017


Procedures.

26

Starting

Concentration in

Plasma (ug/mL)

Initial Quantity in

30uL of plasma used

(ng)

AssayMAP Elution

Concentration

(ug/mL)

Amount on column

with 2uL injection

(ng)

0.05 1.5 0.06 0.12

0.1 3 0.12 0.24

0.5 15 0.6 1.2

1 30 1.2 2.4

5 150 6 12

10 300 12 24

25 750 30 60

100 3000 120 240

Calibration Curve EICs (Three at Each Level)

June 14, 2017


Procedures.

27

Calibration Curve

June 14, 2017


Procedures.

28

y = 33916x + 4748.5R² = 0.9999

0.0E+00

5.0E+05

1.0E+06

1.5E+06

2.0E+06

2.5E+06

0 10 20 30 40 50 60 70

Zoom of bottom 4 points

0.0E+00

2.0E+04

4.0E+04

6.0E+04

8.0E+04

1.0E+05

1.2E+05

0 0.5 1 1.5 2 2.5 3

Agilent

Sample Intact Herceptin in rat plasma

Affinity

Purification AssayMAP


Mass Spec 6545XT AdvanceBio Q-TOF

Quant

analysis Using sum of EICs

LLOQ 0.12 ng on-column

Linear

range 2.7 orders

Quantitation of Intact ADC (T-DM1) in Rat Serum ASMS Poster MP132

June 14, 2017


Procedures.

29

Calibration Curve for Deglycosylated T-DM1

June 14, 2017


Procedures.

30

Agilent

Sample Intact deglycosylated T-DM1 in rat

serum

Affinity

Purification AssayMAP


Mass Spec 6545XT AdvanceBio Q-TOF

Quant

analysis

Using peak areas from

deconvoluted spectra

LLOQ 2 ng on-column for

deglycosylated T-DM1

Linear

range

2 orders (2-200 ng on-column,

assuming 100% recovery)

Example Spectra and DAR Calculation Deglycosylated T-DM1

June 14, 2017


Procedures.

31

2ng On-column 6.4 ng On-column

DAR Distribution at Different Levels

June 14, 2017


Procedures.

32

Summary

June 14, 2017


Procedures.

33

Neat

Herceptin

Herceptin in Rat

Plasma

T-DM1 (ADC) in Rat

Serum

Sample Prep NA AssayMAP for affinity

purification

AssayMAP for affinity

purification and deglycosylation

Flow rate Standard flow, 0.5 ml/min Standard flow, 0.5 ml/min Standard flow, 0.5 ml/min

Mass Spec 6545XT 6545XT 6545XT

LLOQ 31.6 pg on-column

(Herceptin)

120 pg on-column

(Herceptin)

2 ng on-column

(Deglycosylated T-DM1)

Spectral

fidelity

Accurate glyco profile down to

LLOQ at 31 pg on-column

Accurate glyfo profile down to

LLOQ at 120 pg on-column

Accurate DAR calculation down

to LLOQ at 2 ng on-column

Linear range 3.2 orders (0.031 – 50 ng on-

column, Herceptin)

2.7 orders (120 pg – 60 ng on-

column, Herceptin)

2 orders (2 - 200 ng on-column,

deglycosylated T-DM1)

Summary

• 6545XT AdvanceBio QTOF coupled to 1290 infinity II

UHPLC provides you:

• Significant improvement on spectral quality

• Best sensitivity for intact mAb quantitation achieved using standard flow

• Best linear range

• Excellent reproducibility on both mass accuracy and response at

different levels including the LLOQ level

• Accurate measurement of glyco-form relative abundances across the

linear range including the LLOQ level

June 14, 2017


Procedures.

34

Documents

LCMS Based Quantitation of Intact Proteins for ... · of Intact Proteins for Bioanalytical Applications Alex Zhu, Ph.D. Agilent Technologies Wilmington, DE ASMS，2017-06 June 14,