APA 2019 -- Duchenne muscular dystrophy (DMD) is a rare X-linked recessive neuromuscular disease characterized by progressive severe muscle wasting and weakness. DMD is ultimately fatal, with patients typically dying from respiratory or cardiac complications in their mid- to late-20s. Exon skipping by phosphorodiamidate morpholino oligomer (PMO) is considered a promising, disease-modifying approach to treat the underlying cause of DMD. PMO was conjugated to a proprietary peptide to enhance tissue uptake, providing a PPMO. This poster describes the development and validation of a sensitive, selective and high-throughput liquid chromatography-tandem high resolution-accurate mass (LC/HR-AM) method for the quantitation of the PPMO in human muscle tissue using an analogue as the internal standard (ISTD). A key modification to the PMO is the addition of a proprietary peptide that provides specificity to binding and due to the metabolism of the peptide several entities of the PMO will be present in the muscle tissue, in order to quantitate the total amount of the PPMO in the muscle. The extraction undergoes a peptide digestion step to form an end product of PMO-A prior to the analysis.

1. Presented at APA 2019
Quantitative Analysis of Oligonucleotides in Human Muscle Tissue Using Liquid Chromatography
Coupled with High Resolution/Accurate Mass (HR/AM) Mass Spectrometry
Nidhi Jaiswal1, Jianbo Zhang2, Sarah Kriger1, Jeremy Elison1, Brandon Wilcock1, Yuanfeng Wu1, Scott Reuschel1 and Troy Voelker1; 1Covance Laboratories Inc., Salt Lake City, UT; 2Sarepta Therapeutics, Inc., Cambridge, MA
Introduction
Duchenne muscular dystrophy (DMD) is a rare X-linked recessive neuromuscular
disease characterized by progressive severe muscle wasting and weakness.
DMD is ultimately fatal, with patients typically dying from respiratory or cardiac
complications in their mid- to late-20s. Exon skipping by phosphorodiamidate
morpholino oligomer (PMO) is considered a promising, disease-modifying
approach to treat the underlying cause of DMD. PMO was conjugated to a
proprietary peptide to enhance tissue uptake, providing a PPMO. This poster
describes the development and validation of a sensitive, selective and high-
throughput liquid chromatography-tandem high resolution-accurate mass (LC/HR-
AM) method for the quantitation of the PPMO in human muscle tissue using an
analogue as the internal standard (ISTD). A key modification to the PMO is the
addition of a proprietary peptide that provides specificity to binding and due to the
metabolism of the peptide several entities of the PMO will be present in the
muscle tissue, in order to quantitate the total amount of the PPMO in the muscle.
The extraction undergoes a peptide digestion step to form an end product of
PMO-A prior to the analysis.
Methodology
LC-HRAM
Mass Spec: Q Exactive
Source and Ionization: HESI (positive ion mode)
Column: C18, 2.1 x 50 mm
Flow Rate: 0.400 mL/min
Mobile Phase: A: formic acid in water; B: formic acid in acetonitrile
LC Program: Gradient
Source Temperature: 350°C
MS Monitoring Parameters: 600-1200 m/z
SPE
Aliquot: 30 μL human muscle tissue
SPE Plate: Oasis HLB Plate
Reconstitution: 175 µL of formic acid in water
The muscle samples were digested with proteinase enzyme following
purification using solid phase extraction (SPE) and 30 µL sample volume.
Extracts were reconstituted using formic acid in water. The extracts were
analyzed by liquid chromatography coupled with high-resolution/accurate-mass
mass spectrometry (LC-HR/AM MS) i.e., Q Exactive (Thermo Fisher Scientific,
San Jose, CA).
Comparison of Full MS-SIM and tSIM
Although desired compounds and unwanted interferences can have the same nominal masses
(which may interfere with single unit resolution), their exact masses often differ by a fraction of a
mass unit, which can be resolved by HR/AM. Therefore, by coupling Full MS-SIM with HR/AM
detection, selectivity may be achieved for very complex matrices based on the charge state of the
parent mass without fragmentation. PMO-A was evaluated by full scan analysis (Full MS-SIM)
utilizing a scan range of 600-1200 m/z, which enabled the use of multiple charge states (11 and 9)
in order to maximize sensitivity. The full scan analysis also enabled future data mining for any
missed trypsin cleavage for PMO-A interest within the existing data file.
PMO-A was also evaluated by targeted selected ion monitoring (tSIM) in order to determine the
optimum scan mode for the validation. In tSIM, the mass spectrometer is independently trapping a
narrow mass range for each selected mass in the inclusion list. Only compounds with the selected
masses are detected; therefore, the overall background noise is reduced. Therefore, Full MS-SIM
was chosen as the scan mode for the validation.
Results and Discussion
Method Development
▶ SPE and LLE were explored for the extraction. However, SPE was chosen due
to cleaner extracts and better recovery.
▶ Oligo RP-2.0x50mm, C18 2.1x50mm, Max RP-30x2.0 mm were considered;
after final chromatographic development, PMO-A were baseline resolved using
a C18 2.1x50 mm column.
▶ In order to obtain improved sensitivity, multiple charge states were summed for
PMO-A.
▶ The method was validated under current regulatory guidelines.
Advantages of the Q Exactive (HRAM Method)
▶ No Tuning
− Compound tuning is not required for the Q Exactive mass spectrometer;
data was acquired in full scan mode and compared with tSIM, prm.
▶ Increased Sensitivity
− The HR/AM method provided improved signal to noise.
− Full MS-SIM HR/AM enables summing of multiple charge states to improve
sensitivity.
▶ Enhanced selectivity achieved by use of exact mass rather than fragment ions.
PMO-A
Curve
Number LLOQQC LQC MQC HQC
1 44.6 148 2280 4680
58.9 155 2240 &&4820
54.6 146 2310 4450
53.8 155 2300 4450
53.6 156 2250 4550
56.9 162 2210 4610
Intrarun Mean 53.7 154 2270 4590
Intrarun SD 4.92 5.82 38.3 143
Intrarun %CV 9.2 3.8 1.7 3.1
Intrarun %Bias 7.4 2.7 13.5 14.8
n 6 6 6 6
2 55.0 157 2110 4390
56.8 154 2110 4310
47.6 149 2140 4410
51.3 156 2010 4570
53.8 152 1960 4600
52.2 149 2060 4780
Intrarun Mean 52.8 153 2070 4510
Intrarun SD 3.21 3.43 68.9 173
Intrarun %CV 6.1 2.2 3.3 3.8
Intrarun %Bias 5.6 2.0 3.5 12.8
n 6 6 6 6
3 61.8 166 &&1550
60.4 148 2100 4580
60.9 155 &&1590 4820
60.0 147 1860 4420
&63.3 136 2060 4460
57.2 140 2120 4000
Intrarun Mean 60.6 149 1880 4460
Intrarun SD 2.04 10.8 258 299
Intrarun %CV 3.4 7.2 13.7 6.7
Intrarun %Bias 21.2 -0.7 -6.0 11.5
n 6 6 6 5
PMO-A %CV %Bias
LLOQ QC 11.50 16.60
LQC 8.20 -0.70
MQC 10.80 0.50
HQC 5.00 11.80
DQC 13.80 -0.50
PMO-A
MTX
LLOQ QC
F/T 4 -20C
LQC/5C
B/T WI
LQC
F/T 4 -20C
HQC/5C
B/T WI
HQC DQC
48.6 146 150 4300 4500 21900
46.8 162 163 4780 3720 22300
53.2 145 139 4520 4230 18800
53.6 140 146 4330 4270 20100
53.7 147 159 4290 4460 14900
59.0 162 141 4120 4540 21200
Intrarun
Mean
52.5 150 150 4390 4290 19900
Intrarun
SD
4.31 9.35 9.67 230 305 2750
Intrarun
%CV
8.2 6.2 6.4 5.2 7.1 13.8
Intrarun
%Bias
5.0 0.0 0.0 9.8 7.3 -0.5
n 6 6 6 6 6 6
Conclusion
A lower limit of quantitation of 50.0 ng/mL
was achieved with a linear 1/x2 regression
model over 2.0 orders of magnitude.
Quantification of PMO-A therapeutics
using LC-HR/AM MS was validated with
GLP acceptance criteria.
Table 1. Intra/Inter Assay Quality Control Precision (n=6)
Table 2. Inter-Assay Quality Control Precision Table 3. Quality Control Precision for MTX Eff LLOQ and
Freeze-Thaw and Bench-Top Stability (n=6)
Figure 3. Blank chromatograms of PMO-A and PMO-A -IS
from extracted human muscle tissue.
Figure 2. ULOQ chromatograms of PMO-A and PMO-A -IS
from extracted human muscle tissue.
Figure 4. QC0 chromatograms of PMO-A and PMO-A -IS from
extracted human muscle tissue.
Figure 5. Calibration curve of PMO-A from extracted human muscle tissue.
Figure 1. LLOQ chromatograms of PMO-A and PMO-A -IS
from extracted human muscle tissue.
&&: > meeting acceptance criteria
The poster is presented at Applied
Pharmaceutical Analysis Conference,
Sept 9-11, 2019, Boston , MA 02116