Establishment and implementation of a determination methodof nusinersen by peptide adsorption-controlled-LC-MS/MS
in rat samples obtained by a microsampling technique〇 Rika Dosho1, Shin-ichiro Nitta1, Kazuaki Takahashi2, Ryuta Hosogai1, Ryoya Goda3, Sun Yuchen4, Kosuke Saito4, Yoshiro Saito4 and Kodo Miura1
1: LSI Medience Corporation, 2: LSIM Safety Institute Corporation, 3: Daiichi Sankyo Company, Limited, 4: National Institute of Health Sciences

Introduction

Microsampling contributes to 3Rs (Replacement, Reduction and Refinement) of animals and it also enables to evaluate the relationship directly between thesafety data and drug exposure in the same animal. Previously, much sampling blood volume was required for the determination of drugs in toxicokinetics
analysis, but recent progress in sensitivity of analytical instruments contributed to the spread of sample reducing techniques. We focused on oligonucleotide
therapeutics because relatively less number of microsampling applications are reported in the field. In order to determine nusinersen1) concentration in rat
plasma obtained by microsampling, a determination method was developed using peptide adsorption-controlled-liquid chromatography (PAC-LC)2) combinedwith a mass spectrometer. Then, we measured the nusinersen concentration in rat plasma actually obtained by microsampling.

Validation Study1) : antisense oligonucleotides for spinal muscular atrophy treatment

Result:About PAC-LC
PAC-LC is a LC system in which the connection of the liquid line is changed
as shown in the illustration below, and enables the complete retention ofanalytes on the column while preventing adsorption during handling. Inthe system, after the sample is loaded into the line for mobile phase B, theanalytes can be mixed with mobile phase A to recover their ability toretain in the column, and continue the retention until the organic solvent
content of the eluent reaches a critical point. Due to this sample injection
mechanism, the injectable sample volume of PAC-LC is theoreticallyunlimited.
Conventional-LC

PAC-LC

Mixing Autodevice sampler

Pump B

Organicsolvent

Pump A

ColumnAutoMixing
sampler device

to MS
or waste

Column oven

Aqueoussolvent

ColumnPump B

Organicsolvent

Pump Ato MS
or waste

Validation tests of a highly-sensitivity determination method using
PAC-LC-MS/MS were conducted. Good linearity, coefficient ofvariation (CV) and relative error (RE) were observed over theconcentration range of 0.1 to 50 ng/mL in the diluted plasma.
Nusinersen in the diluted plasma was stable under various storageconditions. The results of selectivity of 6 lots plasma and dilution
integrity up to 1000-fold were also good (data not shown). About 70%
area of LLOQ was detected in the blank matrix measured
immediately after injection of the ULOQ. The carry-over peak could
be washed away by once additional injection of blank sample. Thereis probably room for improvements in the current method, in order tomitigate sample carryover.
Equipment and Software:
Triple TOF®5600, Analyst®TF (ver. 1.6), PeakViewTM (ver. 1.2), and MultiQuantTM (ver. 2.1.1) (SCIEX)

Column oven

Linearity

Aqueoussolvent

Modified citation from Goda R., Pharmacia, Vol 56, No.6 (2020)

Sample preparation method
About dilution in microsampling :For easy handling (operating or storage) or to obtain enough sample
volume to re-analysis, the dilution is useful for microsampling. To reduce
the amount of animal plasma used, we diluted plasma with L-consera®,
which is based on human serum, as an alternative matrix.

Column temp.
Mobile phase (pump A)
Mobile phase (pump B)
Run time
RemarkMS

XIC
Remark

0.317
0.318
0.294
0.345
0.322

4.03
3.95
3.96
4.51
4.49

46.2
41.9
43.9
42.4
44.1

Day 2
Determined
concentration*(ng/mL)

0.327
0.277
0.280
0.295
0.281

3.78
4.13
4.10
4.09
4.04

42.8
40.0
41.8
40.2
42.5

0.283
0.248
0.262
0.278
0.2420.291
0.030
-3.0
10.3

3.35
3.54
3.38
3.34
3.49
3.88
0.39
-3.0
10.1

36.4
35.7
36.4
34.7
37.9
40.5
3.5
1.3
8.6

Day 3
Determined
concentration*(ng/mL)RE

Stability in the diluted plasma
Nominalconcentration*(ng/mL)RE

Storage condition

Low QC(0.300 ng/mL)
and High QC(40.0 ng/mL)[each n=3](%)

Room temp.
24 h

6.0 and 9.8

4°C
24 h

3.0 and 13.8

Freeze and thaw
3 cycles

8.0 and 13.0

-80°C
161 days12.3 and 13.0

Processed sample
10°C
72 h2.7 and 6.0Sample

Rat plasma
volume(μL)

L-consera®
volume(μL)

RECV(%)(%)

High volume
n=518162-4.0

7.3

Low volume
n=5872-11.5

8.5

Microsampling
Sampling Method and Sampling Preparation:Blood (50 μL) was collected without anesthesia from the subclavian vein using the FN syringe containing EDTA-2K
solution and 27G needles. The collected blood was transferred to a polypropylene tube, and centrifuged (4°C,10000×g, 3 minutes) to obtain plasma (>10 μL each). L-consera® was added to the obtained plasma volume so as tobe diluted 10-fold at the test facility. The dilution operation was confirmed before this study started, and the dilution
reproducibility of the two volumes both gave reasonable results. (The results are shown in validation study.)

Quantitative Analysis of Nusinersen

Result:Concentrations of nusinersen in plasma tend to decrease until 48 hours after administration. The incurred sample
reanalysis (ISR) results showed the assay variability※ of -12.7% to 7.2% (data not shown), confirming themeasurement reproducibility of the actual sample.
100000※:Assay variability (%) =[(Concentration obtained by ISR - Concentration in the original analysis)/Mean of the two] × 100.
(MHLW, "Guideline on Bioanalytical Method Validation in Pharmaceutical Development", July 11 (2013))

AnimalNo.

Triple TOF®5600 and Analyst®TF (ver. 1.6)
(SCIEX, Framingham, MA)

600°C
AnalytePrecursor (m/z) DP
CE
Nusinersen 889.7-130-95
I.S.
881.7-130-90
94.940±0.015 Da
Nusinersen-OMe, bearing the identical basesequence as nusinersen but with 2'-O-methyl (2OMe) modifications in place of 2-O-methoxyethyl(2’-MOE) modifications, was used as an internal
standard (I.S.).

-2.1
7.5-8.4
0.5-4.2
1.5
4.8

Day 1
Determined
concentration*(ng/mL)

In Vivo Study | Administration: Crl:CD(SD), intravenous, 10 mg/kg, single dosing, n=3

123
MeanSD

Scan type
Product ion scan
Polarity
Negative
Ion spray voltage Floating -4500 V
TEM
Monitoring ions

0.0979
0.215
0.458
2.01
4.79
20.3
52.4

High QC
40.0

Nominal plasma concentration: 200 ng/mL

LC-MS/MS Condition
CAPCELL PAK INERT C18 MGⅢ, 3μm, 2×50mm(OSAKA SODA CO., LTD., Osaka, Japan)
50°C
Water/TEA/HFIP/acetylacetone(1000:2:30:0.05, v/v/v/v)
Methanol/water/TEA/HFIP/acetylacetone(500:500:2:30:0.05, v/v/v/v/v)
12.5 min
The mobile phase was used with N2 gas bubbling.
Patent pending: WO2021/172380

0.100
0.200
0.500
2.00
5.00
20.0
50.0

Medium QC
4.00

Dilution integrity at test facility

Rat plasma (Anticoagulant: EDTA-2K) / L-Consera® (NISSUI PHARMACEUTICAL CO.,LTD., Tokyo,Japan) (1:9,v/v). L-consera® was added to plasma at test facility.
**: Phenomenex, Torrance, CAColumn(%)

Low QC
0.300

Mean* (ng/mL)
SD (ng/mL)
RE (%)CV (%)

Typical chromatograms*:

Shimadzu XR system (Shimadzu, Kyoto, Japan)

Back calculated
concentration*(ng/mL)

Nominalconcentration*(ng/mL)*: Concentration in the diluted plasma

50 μL of rat sample (the diluted plasma*)↓ Add lysis buffer (Clarity®OTX** kit reagent)
Mixture↓ Load to Clarity®OTX 25 mg plate↓ Wash with 50 mM NaH2PO4 (pH5.5)↓ Wash with 500 mM NaH2PO4 (pH5.5)/water/ACN (10/40/50, v/v/v)↓ Elute with 400mM NH4HCO3/ACN (50/50, v/v)
Eluent↓ Add Tris-EDTA buffer and mix Due to the advantages of PAC-LC,↓ Inject (150 μL) to LC-MS/MS
the injection volume could be↓ Dry under N2 streamincreased.
Residue↓ Reconstitute with 50 μL of TE buffer/methanol (70:30, v/v)
Time reduction of about 3 hours↓ Filterof the sample preparation methodFiltrate↓ Inject (7 μL) to LC-MS/MS

PAC-LC

Nominalconcentration*(ng/mL)

Between-run

0.25 h23200 #1
23400 #1
21700 #1
22800
900

0.5 h
9240 #2
9570 #29800 #29540
280

Plasma concentration (ng/mL)
1h2h4h4480 #2 2450 #3870 #35630 #2 2890 #3 1160 #35130 #2 2990 #3 1240 #35080
2780
1090
580
290
190

8h
286 #3330 #3352 #3323
3424 h
38.2
35.1
32.0
35.1
3.1

48 h
19.6
16.0
15.9
17.2
2.1

Mean of concentrations (ng/mL)2): Goda R., Sudo K., Biomed. Chromatogr., 21, 1005-1015 (2007)

10000
1000
100
10108

16
24
3240
Sampling point (h)48Some samples were further diluted by L-consera® prior to the sample preparation (#1: 1000-fold dilution, #2: 100-fold dilution, #3: 10-fold dilution)

Conclusions

We developed highly-sensitivity determination method of nusinersen using PAC-LC-MS/MS.
 The PAC-LC-MS/MS method has the advantages of reducing time for sample preparation andincreasing injection volume. In addition, improvements are considered to be required toreduce carryover.
 We could properly determine the plasma concentration of nusinersen in the samples
obtained by microsampling.
