Biotherapeutics Quantification in Discovery and Early ... · • Comparing TQ-XS and G2-XS • Optimization produced same transition using similar collision energy (CE) • ≥ 4

©2017 Waters Corporation Company Confidential 1

Biotherapeutics Quantification in Discovery and Early Development:Comparison of LC-MS Techniques for both Digested and Intact Quantification

John C Gebler, PhDDirector, Biopharma


Outline

§ Introduction§ Surrogate Peptide Quantification

§ Sample preparation§ Platform comparison between QQQ and QTof

§ Intact Level Protein Quantification§ BSA solution – optimizing instrumentation§ In Plasma – handling the real world

§ Summary and future directions


Strategies for mAb Bioanalysis

Intact mass analysis (LBA, LC/MS)(m/z 150 kDa)

Surrogate Peptides(m/z 1000’s Da)

reduction

digestion

Middle down (Subunit analysis)(m/z 20-50 kDa)

reduction

IdeS

reduction


Trastuzumab Quantification viaSurrogate Peptide Approach, Sample Preparation

Trastuzumab(Infliximab as ISTD)

Rat Plasma (50 µL)(ProteinWorks eXpress Digest Kit)Protein Digestion

(ProteinWorks µElution SPE Clean-Up)

Peptide Purification

(Protein A)Affinity Purification

and

TQ-XS G2-XS

90 µL final volume

Same LC (H-Class ACQUITY)


Blank Rat Plasma - Rep 2

Time4.60 4.80 5.00 5.20 5.40 5.60 5.80 6.00 6.20 6.40 6.60 6.80 7.00 7.20 7.40

%

0

100

4.60 4.80 5.00 5.20 5.40 5.60 5.80 6.00 6.20 6.40 6.60 6.80 7.00 7.20 7.40

%

0

100

4.60 4.80 5.00 5.20 5.40 5.60 5.80 6.00 6.20 6.40 6.60 6.80 7.00 7.20 7.40

%

0

100

4.60 4.80 5.00 5.20 5.40 5.60 5.80 6.00 6.20 6.40 6.60 6.80 7.00 7.20 7.40

%

0

100

Blank Rat Plasma - Rep 2

Time4.60 4.80 5.00 5.20 5.40 5.60 5.80 6.00 6.20 6.40 6.60 6.80 7.00 7.20 7.40

%

0

100

4.60 4.80 5.00 5.20 5.40 5.60 5.80 6.00 6.20 6.40 6.60 6.80 7.00 7.20 7.40

%

0

100

4.60 4.80 5.00 5.20 5.40 5.60 5.80 6.00 6.20 6.40 6.60 6.80 7.00 7.20 7.40

%

0

100

4.60 4.80 5.00 5.20 5.40 5.60 5.80 6.00 6.20 6.40 6.60 6.80 7.00 7.20 7.40

%

0

100

Representative Trastuzumab Chromatograms

FTISADTSKTQ-XS(MRM)(485.2 > 721.4, CE = 15)

0.010 µg/mL

0.050 µg/mL

0.025 µg/mL

Blank

G2-XS(Tof-MRM)(485.2 > 721.373, CE=16)

0.010 µg/mL

0.050 µg/mL

0.025 µg/mL

Blank

LOD

LOQ

LOQ


FTISADTSK DTYIHWVR

Xevo TQXS QQQ Xevo G2XS QTof Xevo TQXS QQQ Xevo G2XS QTof

Transition (CE) 485.2 > 721.4 (CE=15)

485.2 > 721.373(CE=16)

545.3 > 597.3(CE=24)

545.3 > 597.326(CE=23)

LOD (µg/mL) 0.005 0.010 0.025 0.050

LLOQ (µg/mL) 0.010 0.025 0.025 0.050

Curve (µg/mL) 0.010 – 250 0.025 - 500 0.025 – 500 0.050 - 500

Log10 Range 4.4 4.3 4.3 4

Linear Fit (R2) 0.988 0.991 0.992 0.995

% Accuracy Range 85.0 – 111.6 89.2 - 114.4 89.0 – 108.3 95.5 - 105.6

Weighting 1/X2 1/X2 1/X2 1/X2

Trastuzumab Peptide Level Quantification

• Comparing TQ-XS and G2-XS• Optimization produced same transition using similar collision energy (CE)• ≥ 4 linear dynamic range on both platforms

• In general, TQ-XS is 2X more sensitive than G2-XS QTof • LOQs 0.01-0.025 µg/mL (TQ-XS) vs. 0.025-0.050 µg/mL (G2-XS)


Outline

§ Introduction§ Surrogate Peptide Quantification

§ Sample preparation§ Platform comparison between QQQ and QTof

§ Intact Level Protein Quantification§ 0.1 mg/mL BSA solution – optimizing instrumentation§ In Plasma – handling the real world

§ Summary and future directions


LC-MS/MS and ELISA results in plasma obtained from a breast cancer patient on long-term treatment with trastuzumab. LC-MS/MS concentrations obtained for peptides FTISADTSK, IYPTNGYTR, IYPTDGYTR, and IYPTisoDGYTR.

“LC-MS/MS-Based Monitoring of In Vivo Protein Biotransformation: Quantitative Determination of Trastuzumab and Its Deamidation Products in Human Plasma”P. Bults, R. Bischoff, H. Bakker, J. A. Gietema, N. C. van de Merbel, Anal. Chem., 2016, 88 (3), 1871.

Total trastuzumab (T-mAb)

T-mAb that has not been deamidated at Asn55

Immunoractive T-mAb (ELISA)

“What is the true concentration?”


¡ W. Jian, et al, Bioanalysis 2016, 8(16), 1679-1691 – Sigma mAb in plasma– LOQ = 1 µg/mL at 60 µL injection volume, linear range 1-30 µg/mL

¡ C. Lanshoeft, et al, Anal. Chem. 2017, 89(4), 2628-2635– hIgG1 in plasma– LOQ = 0.1 µg/mL at 60 µL injection volume, linear range 0.1-10 µg/mL

Challenges of Intact Level Protein Quantification by LC/MS

¡ Signal dilution with multiple charge state¡ Charge states shift with varying concentrations¡ Relatively low sensitivity¡ Relatively narrow calibration range¡ Relatively broad LC peak¡ Sensitive to LC settings¡ Sensitive to sample preparation

Recent Literature:


Trastuzumab 100 µg/mL in BSA:MS full scan, 1 µL inj

T-mAb

BSABaseline separation of BSA and T-mAb & minimal mass overlap

BSA

T-mAb

TIC Chromatogram

MAbPac RPCustomer sample,


Spectra at 100 ug/mLDeconvoluted

SpectrumZoomed Charge States

Left peak(G0F/G0F)

Middlepeak(G0F/G1F)

Right peak(G1F/G1F)

Quantification Options

Single m/z Sum of six m/z Wide 1000 Da window

Quantify based on deconvoluted spectrum


Linearity Based on XICs of the Sum of 6 Masses 1 µL injection Volume, LOQ = 0.1 µg/mL, Linear range = 0.1 – 100 µg/mL

0.1 µg/mL

0.5 µg/mL

1.0 µg/mL

Blank

S/N= 9.8

S/N= 16

S/N= 20


Trastuzumab in BSA Second Dilution Series: Dilution range 0.01 – 100 µg/mL, 10 µL injection volume

BSAblank 0.01 0.02 0.04 0.1

0.2 0.4 0.8 1.6 3.1

6.3 12.5 25 50 100

LOQ = 0.01 µg/mL, linear range = 0.01 – 0.8 µg/mL, R2 = 0.97

(in µg/mL) LC/MS system No. 2


Reproducibility at LOQ of 0.012 µg/mL

0.012 µg/mLInj 1

0.012 µg/mLInj 2

0.012 µg/mLInj 3

XIC SpectrumDeconvoluted

spectrum

10 µL injection Volume


Trastuzumab in Mouse Plasma

Sample preparation: can be simpler than surrogate peptide approach

Trastuzumab

Mouse Plasma (50 µL)

Goat Anti-Human IgG FCMagnetic bead

Final volume (50 µL)

TIC Spectrum


Systematic LC Method development


35% 40% 45% 50%

20%

25%

30%

Chromatographic Method Development Results

Sample: T-mAb in plasma 50 µg/mL, 0.2 mL/min

Final ACN concentration

0.4 mL/min


Quantification Results in Mouse PlasmaXIC of m/z 2907 with 2 Da window

BK0.1 0.25

0.5 1.0

2.5

5

10 25

Sample range: 0.1-50 µg/mLInjection volume = 1 µL

Linear range: 0.25 - 25 µg/mLLLOQ: 0.25 µg/mL

%accuracy range: 85-118%R2 = 0.98

S/N=9

Plasma blank

0.10 µg/mL

0.25 µg/mL

0.50 µg/mL

1.0 µg/mL

in µg/mL

XIC Spectrum

5


Quantification from Deconvoluted Spectrum

Same quantification Results

LLOQ = 0.25 µg/mLLinear range = 0.25 - 25 µg/mL

0.5 µg/mL

1.0 µg/mL

2.5 µg/mL

5.0 µg/mL

10 µg/mL

25 µg/mL

Consistent relative percent of the glycoforms

0.25 µg/mL


Sample Quantification method LLOQ S/N at LOD Linear range

In BSA1 µL injection

Single m/z 2 Da window

0.1 µg/mL 5.5 0.1 - 100 µg/mL

Sum of six m/z 0.1 µg/mL 9.8 0.1 - 100 µg/mL

1000 Da window 0.1 µg/mL 8.8 0.1 - 100 µg/mL

In BSA 10 µL injection

2 Da or 1000 Da window 0.01 µg/mL 16 0.01-0.78 µg/mL

Mouse Plasma 1 µL injection

Single m/z or from deconvoluted

spectrum0.25 µg/mL 9.5 0.25-25 µg/mL

• Flexible quantification method based on peak selection or from deconvoluted spectrum.

Intact Protein Quantification Data Summary

LC separation and sample preparation are critically important for sensitive intact protein quantification


FTISADTSK DTYIHWVR Intact (Plasma)

Xevo TQ-XS QQQ

Xevo G2-XS QTof

Xevo TQ-XS QQQ

Xevo G2-XS QTof

VionQTof

LOD (µg/mL) 0.005 0.010 0.025 0.050 0.25

LLOQ (µg/mL) 0.010 0.025 0.025 0.050 0.25

Inj Vol (µL) 8 8 8 8 1

Curve (µg/mL) 0.010 – 250 0.025 - 500 0.025 – 500 0.050 - 500 0.25-25

Log10 Range 4.4 4.3 4.3 4 2

Linear Fit (R2) 0.988 0.991 0.992 0.995 0.985

% Accuracy Range 85 – 112 89 - 114 89 – 108 95 - 105 85-118

Weighting 1/X2 1/X2 1/X2 1/X2 1/X2

Comparison of Peptide and Intact Level Quantification

• Peptide level quantification is ~5-25x more sensitive than intact trastuzumab.• MRM based peptide quantification has wider linear dynamic range than MS scan based quantification (both for peptide and intact).


¡ HRMS workflows are compatible with traditional bioanalysis¡ Surrogate peptide: LOQ = 0.01-0.025 µg/mL and >4 orders linear dynamic

range¡ Intact Level: LOQ = 0.25 µg/mL and 2 orders linear dynamic range¡ Sample preparation is especially important for intact level analysis to

minimize matrix interferences¡ Both peptide and intact level quantification are viable options for

measuring protein levels in plasma

¡ Work in process– Quantification post deglycosylation– Quantification via subunit analysis– Evaluating sample preparation approaches

Summary


Acknowledgements

Yun Wang AlelyunasPrincipal Scientist

¡ Henry Shion¡ Mary Lame¡ Caitlin Dunning¡ Gregory Roman¡ Jing Fang¡ Catalin Doneanu¡ Kerri Smith¡ Nilini Ranbaduge¡ Nikunj Tanna

Merck§ Lisa A Vasicek§ Kevin Bateman

§ Kelly Doering§ Logan Umberger§ YingQing Yu§ Weibin Chen§ Mark Wrona§ Erin Chambers

Documents

Biotherapeutics Quantification in Discovery and Early ... · • Comparing TQ-XS and G2-XS • Optimization produced same transition using similar collision energy (CE) • ≥ 4