adc, qc 3.5 ug/ml, direct 5 step, no spe, 1€¦ · std. dev. %cv mean % accuracy 0.65 0.65 0.05...

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INTRODUCTION

Monoclonal antibodies (mAbs), and antibody-drug conjugates

(ADCs) represent a growing class of therapeutics due to their

target specificity, lower toxicity and higher potency. As such,

the desire for LC/MS bioanalytical quantification in support of

drug development is also increasing. However, it is not

without its challenges. There is no single standardized

workflow and the various workflow options can be complex

and laborious, making it difficult for the novice bioanalytical

scientist to achieve success.

This work aims to provide a practical, broadly applicable,

strategy to simplify and streamline LC/MS protein

bioanalysis workflows, using a kit-based approach,

universal protocol and lot-traceable, pre-measured

reagents, to accurately and reproducibly quantify several

protein therapeutics in plasma.

DEVELOPMENT OF A GENERIC KIT BASED APPROACH FOR QUANTIFYING PROTEIN THERAPEUTICS IN BIOLOGICAL MATRICES BY LC-MS/MS Mary E. Lame, Hua Yang, Paula Orens, Erin E. Chambers, and Sherri Naughton

Waters Corporation

METHODS

Sample Preparation

Infliximab, adalimumab, bevacizumab, trastuzumab, and

trastuzumab emtansine (T-DM1) were spiked into plasma.

Plasma samples (35 μL), with or without generic affinity

purification , were prepared for LC-MS analysis using the

ProteinWorks eXpress Digest Kits and Protocols. After

digestion, peptides were cleaned-up using the ProteinWorks

μElution SPE Clean-up Kit and Protocol.

LC-MS Conditions

LC-MS/MS quantification of signature peptides was performed

using a Waters Xevo TQ-S triple quadrupole MS (ESI+).

Chromatographic separation was achieved using an ACQUITY

UPLC system with an ACQUITY UPLC Peptide BEH C18, 300A,

1.7 μm, 2.1 mm x 150 mm column and 0.1% formic acid in

water and acetonitrile mobile phases. MS conditions are

summarized in Table 1.

Table 1. mAb, ADC, and Internal Standard MRM

conditions.

Protein Peptide MRM Transition

Cone Voltage

(V)

Collision Energy

(eV)

Infliximab SINSATHYAESVK 469.60>603.80 40 10

Infliximab DILLTQSPAILSVSPGER 633.10>731.80 31 21

Bevacizumab FTFSLDTSK 523.30>797.48 16 14

Adalimumab APYTFGQGTK 535.30>901.44 40 24

Trastuzumab FTISADTSK 485.20>721.40 28 20

Generic IgG DSTYSLSSTLTLSK 751.88>836.47 31 24

murine mAb SVSELPIMHQDWLNGK (ISTD) 618.64>834.41 16 12

murine mAb MNSLQTDDTAK (ISTD) 612.30>978.56 20 20

RESULTS I. Quantification of multiple mAbs through

direct digestion

Protein Peptide

Std. curve range

(ug/mL) Weighting Linear fit (r2)

Mean % accuracy of

all points

Infliximab SINSATHYAESVK 0.25-250 1/X 0.996 101.74

Bevacizumab FTFSLDTSK 0.50-500 1/X 0.999 100.00

Adalimumab APYTFGQGTK 2.50-500 1/X2

0.997 99.99

Trastuzumab FTISADTSK 2.50-500 1/X2

0.997 100.01

Table 2. Standard curve statistics for infliximab,

adalimumab, trastuzumab, and bevacizumab in human

plasma, digested and extracted using a protein

quantification direct digestion kit.

spe QC 3.5 ug/ml

Time0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50

%

0

100

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50

%

0

100

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50

%

0

100

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50

%

0

100

16Oct2015_4mAb_HumanPL_3St_direct_1031 Sm (Mn, 1x2) MRM of 15 Channels ES+ 535.3 > 901.44 (Humira APYTFGQGTK LC)

2.45e4Area

384

16Oct2015_4mAb_HumanPL_3St_direct_1030 Sm (Mn, 2x3) MRM of 15 Channels ES+ 523.3 > 797.48 (Avastin FTFSLDTSK HC)

5.10e5Area

11154

16Oct2015_4mAb_HumanPL_3St_direct_1030 Sm (Mn, 2x3) MRM of 15 Channels ES+ 469.6 > 603.8 (Remicade SINSATHYAESVK )

1.01e6Area

46370

16Oct2015_4mAb_HumanPL_3St_direct_1030 Sm (Mn, 2x3) MRM of 15 Channels ES+ 485.2 > 721.4 (Herceptin FTISADTSK HC)

2.79e4Area

600

APYTFGQGTK

SINSATHYAESVK

FTISADTSK

FTFSLDTSK

Figure 1: Low QC chromatograms (3.5 g/mL) for

bevacizumab, adalimumab, infliximab, and

trastuzumab, in plasma digested and extracted using a

protein quantification direct digestion kit.

Figure 2: Chromatogram of 10 ng/infliximab in rat

plasma, as compared to blank rat plasma that was

immunopurified (Protein A), digested and extracted

using a protein quantification digestion kit.

Remicade, 0.01 ug/ml, ProA, SPE, 1

Time1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00

%

0

100

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00

%

0

100

092315_WAA678_CD_006a Sm (Mn, 2x3) MRM of 11 Channels ES+ 469.6 > 603.8 (Remicade SINSATHYAESVK )

2.68e4Area

4.15398

092315_WAA678_CD_004a Sm (Mn, 2x3) MRM of 11 Channels ES+ 469.6 > 603.8 (Remicade SINSATHYAESVK )

2.68e4Area

10 ng/mL infliximab extracted from plasma

Blank plasma

Peptide

Std. Curve Range

(ug/mL) Weighting Linear fit (r2)

Mean % Accuracy

of all points

DILLTQSPAILSVSPGER* 0.05-250 1/X 0.998 100.00

SINSATHYAESVK* 0.01-100 1/X2 0.995 98.47

DSTYSLSSTLTLSK 0.10-500 1/X20.997 99.34

* Unique Signature Peptide

Table 4. Infliximab standard curve statistics for

signature peptides in rat plasma that was

immunopurified (Protein A), then digested and

extracted using a protein quantification digestion kit.

Table 3: mAb QC sample statistics in human plasma di-

gested and extracted using a protein quantification di-

rect digestion kit.

II. High sensitivity quantification of the mAb, Infliximab through generic affinity purification

and digestion

QC 0.035 ug/mL

Time-0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00

%

0

100

-0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00

%

0

100

-0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00%

0

100

-0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00

%

0

100

-0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00

%

0

100

-0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00

%

0

100

14Aug2015_RemicadeSPE_ProteinA_01005 MRM of 11 Channels ES+ 469.6 > 603.8 (Remicade SINSATHYAESVK )

2.19e4Area


2.76e4Area

1143


2.40e5Area

10119


2.28e6Area

95867


2.05e7Area

857961


1.32e8Area

6321264

350.0 g/mL

35.0 g/mL

3.5 g/mL

0.35 g/mL

Blank plasma

0.035 g/mL

Figure 3: QC chromatograms of infliximab

(SINSATHYAESVK) in rat plasma, that was

immunopurified (Protein A), digested and extracted

using a protein quantification digestion kit.

III. Quantification of the ADC T-DM1 and mAb trastuzumab through direct digestion

Figure 4: Chromatograms of T-DM1 and

trastuzumab (3.5 g/mL) using the peptides

IYPTNGYTR, FTISADTSK and GPSVFPLAPSSK,

Panels A-C, respectively.

ADC, QC 3.5 ug/ml, Direct 5 step, no SPE, 1

Time8.25 8.50 8.75 9.00 9.25

%

1

8.25 8.50 8.75 9.00 9.25

%

1

110415_WAA678_CD_033b Sm (Mn, 2x1) F1593.83 > 699.4 (Generic GPSVFPLAPSSK)

2.18e5Area

8.886788

110415_WAA678_CD_032b Sm (Mn, 2x1) F1593.83 > 699.4 (Generic GPSVFPLAPSSK)

2.26e5Area8.87

8408


Time5.00 5.50 6.00 6.50

%

0

5.00 5.50 6.00 6.50

%

0

110415_WAA678_CD_033b Sm (Mn, 2x1) F1542.77 > 808.4 (Herceptin IYPTNGYTR 1)

2.50e5Area5.89

7147

110415_WAA678_CD_032b Sm (Mn, 2x1) F1542.77 > 808.4 (Herceptin IYPTNGYTR 1)

2.50e5Area

5.89;7818


Time5.00 6.00 7.00

%

0

100

5.00 6.00 7.00

%

0

100

110415_WAA678_CD_033b Sm (Mn, 2x1) F1485.2 > 721.4 (Herceptin FTISADTSK HC)

5.00e4Area6.64

1181

110415_WAA678_CD_032b Sm (Mn, 2x1) F1485.2 > 721.4 (Herceptin FTISADTSK HC)

5.00e4Area

T-DM1

Trastuzuamb

A B C

IYPTNGYTR

(T-DM1/Trastuzumab)

T-DM1 T-DM1

Trastuzuamb Trastuzuamb

GPSVFPLAPSSK (Trastuzumab)

FTISADTSK

(Trastuzumab)

Table 5. Linear dynamic range, weighting, and

average accuracy for standard curves from

trastuzumab, used to quantify trastuzumab and

T-DM1 in plasma digested and extracted using a

protein quantification digestion kit.

Peptide

Std. curve range

(g/mL) Weighting Linear fit (r2)

Mean % accuracy

of all points

IYPTNGYTR 0.25-250 1/X2 0.995 100.01

FTISADTSK 0.50-500 1/X 0.999 100.01

GPSVFPLAPSSK 2.50-500 1/X20.990 100.00

*Generic IgG peptide

Table 6: Statistics for QC samples of

trastuzumab and T-DM1, in plasma digested

and extracted using a protein quantification

digestion kit.

mAb/ADCPeptide

QC conc.

(µg/ml)

Mean cal.

conc.(µg/ml)Std. dev. %CV

Mean %

accuracy

0.65 0.64 0.03 4.58 99.77

3.5 3.25 0.19 5.96 92.90

Trastuzumab IYPTNGYTR 1 6.5 6.83 0.16 2.29 105.13

35 36.41 0.42 1.16 104.03

65 63.31 2.18 3.44 97.40

350 345.64 18.66 5.40 98.73

mAb/ADCPeptide

QC conc.

(µg/ml)

Mean cal.


Mean %

accuracy

0.65 0.65 0.05 6.94 100.50

3.5 3.36 0.24 7.10 95.87

6.5 7.1 0.05 0.66 109.20

T-DM1 IYPTNGYTR 1 35 34.51 1.09 3.17 98.57

65 59.74 3.72 6.22 91.90

350 324.72 17.06 5.25 92.80

mAb/ADC

Peptide QC conc.

(µg/ml)

Mean cal.


Mean %

accuracy

0.65 0.66 0.05 7.97 100.87

3.5 3.04 0.07 2.29 86.90

Trastuzumab GPSVFPLAPSSK 6.5 6.27 0.09 1.41 96.50

35 35.5 1.62 4.55 101.43

65 71.38 3.04 4.26 109.83

350 379.79 21.64 5.70 108.50

mAb/ADCPeptide

QC conc.

(µg/ml)

Mean cal.


Mean %

accuracy

0.65 0.67 0.02 2.84 103.30

3.5 3.1 0.06 1.80 88.47

T-DM1 GPSVFPLAPSSK 6.5 6.19 0.31 4.98 95.15

35 33.55 1.44 4.29 95.87

65 63.08 4.04 6.40 97.03

350 336.36 15.35 4.56 96.10

Blank rat plasma digest, w/ IS, Direct 5 step, no SPE, 1

Time12.25 12.50 12.75 13.00 13.25 13.50 13.75 14.00 14.25 14.50 14.75 15.00 15.25 15.50 15.75 16.00

%

0

12.25 12.50 12.75 13.00 13.25 13.50 13.75 14.00 14.25 14.50 14.75 15.00 15.25 15.50 15.75 16.00

%

1

12.25 12.50 12.75 13.00 13.25 13.50 13.75 14.00 14.25 14.50 14.75 15.00 15.25 15.50 15.75 16.00

%

2

12.25 12.50 12.75 13.00 13.25 13.50 13.75 14.00 14.25 14.50 14.75 15.00 15.25 15.50 15.75 16.00

%

2

12.21

14.60

13.273538

13.392722

13.275091

13.384398

13.2729006 13.39

23826

ADC, QC 35 ug/ml, Direct 5 step, no SPE, 2

Time12.25 12.50 12.75 13.00 13.25 13.50 13.75 14.00 14.25 14.50 14.75 15.00 15.25 15.50 15.75 16.00

%

1

12.25 12.50 12.75 13.00 13.25 13.50 13.75 14.00 14.25 14.50 14.75 15.00 15.25 15.50 15.75 16.00

%

2

12.25 12.50 12.75 13.00 13.25 13.50 13.75 14.00 14.25 14.50 14.75 15.00 15.25 15.50 15.75 16.00

%

2

12.25 12.50 12.75 13.00 13.25 13.50 13.75 14.00 14.25 14.50 14.75 15.00 15.25 15.50 15.75 16.00

%

2

12.21

14.60

13.273538

13.392722

Blank Rat Plasma

35.0 µg/mL

65.0 µg/mL

350.0 µg/mL

FTISADTSKNTAYLQMNSLR

Miscleavage peptide of T-DM1

MRM: 1073.167 > 547.20

Figure 5: Chromatograms demonstrating increase of the

miscleavage peptide of T-DM1 with small drug attached

(FTISADTSKNTAYLQMNSLR), when digested and

extracted using a protein quantification digestion kit.

Protein Peptide

QC Conc

(g/mL)

Mean Cal.

Conc (g/mL) Std. Dev. %CV Mean Accuracy

Infliximab SINSATHYAESVK 0.350 0.333 0.010 3.10 95.0

3.500 3.816 0.098 2.56 109.0

35.000 36.075 0.576 1.60 103.1

350.000 359.301 19.892 5.54 102.6

Bevacizumab FTFSLDTSK 0.350 0.356 0.004 1.08 101.7

3.500 3.393 0.196 5.78 96.9

35.000 38.461 1.282 3.33 109.9

350.000 369.788 28.066 7.59 105.6

Adalimumab APYTFGQGTK 0.350 - - - -

3.500 3.978 0.570 14.34 113.7

35.000 36.567 1.023 2.80 104.5

350.000 380.963 18.143 4.76 108.8

Trastuzumab FTISADTSK 0.350 - - - -

3.500 3.663 0.067 1.82 104.7

35.000 39.182 2.389 6.10 112.0

350.000 374.080 14.01 3.75 106.9

DISCUSSION

Using commercially available protein quantification digestion kits

and generic protocols:

Simultaneous quantification of multiple mAbs in plasma was

achieved (250 ng/mL-2.5 g/mL). Analytical performance is

highlighted in Tables 2 and 3, and illustrated in Figure 1.

Incorporating an immunopurification step (Protein A), followed

by digestion, detection limits of 10 ng/mL for infliximab were

achieved (Figure 2). Accuracy and precision for the QC samples

was excellent with %CVs all <6%(data not shown). Linearity

and accuracy of the standard curves arising from each peptide

of infliximab are summarized in Table 4 and highlighted in

Figure 3.

Total antibody quantification for T-DM1: quantification limits of

0.5-1 µg/mL were achieved in plasma for trastuzumab and the

ADC (Table 5). QCs of trastuzumab and T-DM1, were within

15% accuracy, and are shown in Table 6. Representative QC

spectra for T-DM1 and trastuzumab are highlighted in Figure 4.

Detection and increasing concentration of “miscleavage”

peptides of T-DM1, with small drug attached (Figure 5). These

hydrophobic peptides contained a common drug fragment 547.2

m/z and eluted later in the chromatographic run as

diastereomeric pairs.

CONCLUSION

In this work, commercially available protein quantification

digestion kits were successfully used to quantify multiple

mAb therapeutic drugs and the ADC, trastuzumab

emtansine. The universal, kit-based approach allows

scientists to achieve high sensitivity (10 ng/mL) with a

simple step-wise protocol and standardized, pre-measured

reagents, ensuring both the sensitivity and reproducibility

required in discovery studies to make time sensitive and

critical project decisions.

adc, qc 3.5 ug/ml, direct 5 step, no spe, 1€¦ · std. dev. %cv mean % accuracy 0.65 0.65 0.05...

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