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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
14Aug2015_RemicadeSPE_ProteinA_01010 MRM of 11 Channels ES+ 469.6 > 603.8 (Remicade SINSATHYAESVK )
2.76e4Area
1143
14Aug2015_RemicadeSPE_ProteinA_01019 MRM of 11 Channels ES+ 469.6 > 603.8 (Remicade SINSATHYAESVK )
2.40e5Area
10119
14Aug2015_RemicadeSPE_ProteinA_01028 MRM of 11 Channels ES+ 469.6 > 603.8 (Remicade SINSATHYAESVK )
2.28e6Area
95867
14Aug2015_RemicadeSPE_ProteinA_01035 MRM of 11 Channels ES+ 469.6 > 603.8 (Remicade SINSATHYAESVK )
2.05e7Area
857961
14Aug2015_RemicadeSPE_ProteinA_01045 MRM of 11 Channels ES+ 469.6 > 603.8 (Remicade SINSATHYAESVK )
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
ADC, QC 3.5 ug/ml, Direct 5 step, no SPE, 1
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
ADC, QC 3.5 ug/ml, Direct 5 step, no SPE, 1
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.
conc.(µg/ml)Std. dev. %CV
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.
conc.(µg/ml)Std. dev. %CV
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.
conc.(µg/ml)Std. dev. %CV
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.