Bioanalysis of free antisense oligonucleotide payload from antibody–oligonucleotide conjugate by hybridization LC-MS/MS

Figures & data

Figure 1. Scheme of ASO1, ASO1-linker and Fab–ASO1.

Table 1. Analyte and IS information.

Name	MW (kDa)	Sequence length	Chemistry	Usage
ASO1	5.4	16	Mixed PS and PO backbone	Analyte
ASO-IS	7.9	20	Uniform MOE with PS backbone	IS

IS: Internal standard; MOE: 2′-O-(2-Methoxyethyl)-oligonucleotide; PO: Phosphate linkage; PS: Phosphorothioate linkage.

Table 2. MS parameters of ASO analyte and internal standard.

Name	Charge state	Q1 mass (m/z)	Q3 mass (m/z)	DP (V)	CE (eV)	CXP (V)	EP (V)	Purpose
ASO1	-9	594.3	95.0	-45	-110	-12	-10	Qualifier
	-8	668.7	95.0	-60	-120	-12	-10	Quantifier
	-7	764.5	95.0	-60	-130	-12	-10	Qualifier
ASO-IS	-9	879.5	95.0	-60	-150	-12	-10	IS

ASO: Antisense oligonucleotide; IS: Internal standard.

Figure 2. Effect of proteinase K digestion and hybridization extraction on Fab–ASO1 and ASO1-linker. (A) Peak area of ASO1 in Fab–ASO1 and ASO1-linker with no treatment, hybridization only and proteinase K digestion followed by hybridization; (B) Peak area of ASO1-linker in Fab–ASO1 and ASO1-linker with no treatment, hybridization only and proteinase K digestion followed by hybridization.

Figure 3. Recovery of ASO1 in mouse plasma and brain homogenate at different MRDs.

Figure 4. LC-MS/MS chromatograms of ASO1 and the IS, ASO-IS from (A) double blank, (B) blank spiked with ASO-IS only, (C) LLOQ and (D) ULOQ in mouse liver and gastrocnemius combine homogenate.

IS: Internal standard; LLOQ: Lower limit of quantitation; ULOQ: Upper limit of quantitation.

Table 3. Precision and accuracy of ASO1 in mouse liver and gastrocnemius combine homogenate.

No.	LLOQ QC 0.50 ng/ml	Low QC 1.50 ng/ml	Mid QC 40.0 ng/ml	High QC 375 ng/ml
1	0.428	1.60	41.5	365
2	0.485	1.18^†	40.0	342
3	0.485	1.31	38.5	362
4	0.434	1.35	42.5	398
5	0.494	1.68	36.8	371
6	0.455	1.60	32.9^†	354
Mean	0.464	1.45	38.7	365
SD	0.028	0.20	3.50	19.0
CV%	6.2	13.8	9.0	5.2
Accuracy%	92.7	96.9	96.8	97.4

† Individual QC samples beyond ±15% of the nominal value, included in the statistics.

QC: Quality control; SD: Standard deviation.