quantification of an oligonucleotide

Transcription

1 Challenges with a LCMS method for quantification of an oligonucleotide Lieve Dillen Regulated Bioanalysis Pictured above: IV absorption

2 utline Introduction to the compound Anticipated challenges LC-MS/MS method development Extraction from plasma 2

3 G phosphorothioate linkage T 2 2 5' P - P S - A G P - 2 P S - 2 G P - 2 P S - 2 G 2 3' 3 3'

4 G with Palmitoyl tail 5 3 4

5 Further background In licensed compound ligonucleotide therapeutic 13 mer MW 4610 AS (antisense oligo) for cancer treatment API also referred to as FLP-PS (Full Length Product- PS). PS indicates the bonding segment -P=S 5

6 Quantitative assay Program CR with hybridization Elisa assay Complementary LCMS(MS) assay(s) Quantification of UD in plasma (range 0.5 g/ml 200 g/ml) Investigation of metabolites Challenges Large acidic molecules Specificity/selectivity (a adducts, fragmentation) Adsorption protein binding Chromatography ion pairing Sample preparation 6

7 Tuning: theoretical charge state distribution ame G sequence MW average G Average m/z of charge states FLP-PS TAGGGTTAGACAA (8-) 13 mer (7-) (6-) (5-) Reference ATCTATACAAGCTGTC (9-) 16 mer (8-) (7-) (6-) (5-) (4-) 7

8 Tuning: negative mode 3.4e7 3.0e e In ntensity, cp ps 2.2e7 1.8e7 1.4e7 1.0e7 6.0e6 2.0e g/ml in 2 /Me/FIP/TEA (70/30/1/0.1) 1) G e e66 4.0e6 2.0e AS m/z, Da m/z, Da 8

9 PIS of m/z Inte ensity, cps 1.00e6 8.00e5 6.00e5 4.00e5 2.00e5 T A G e APS In ntensity y, cps e5 4.0e5 2.0e5 GPS

10 Chromatography Shimadzu LC30AD and SIL30ACMP Column Acquity BE C x 50 mm 1.7 µ Flow rate Solvent A Solvent B 300 µl/min Water/FIP/TEA (100/1/0.1; v/v/v/v) Me/TF (70/30; v/v) Time (min) Solvent A Solvent B API4000 Q1 Mass (m/z) Q3 Mass (m/z) Dwell time (msec)

11 Chromatogram 4.44 Int tensity, cps 3.2e > > > > e4 2.4e4 2.0e4 1.6e4 12e4 1.2e4 G AS Time, min 11

12 Chromatography bservations: Shift in retention times 50% decrease in response vs start end analytical l run Variations: AC vs Me as solvent B Removal of TF in solvent B Addition of 1% FIP and 0.1% TEA to B Varying concentration of FIP and TEA Different modifiers/ion pairing reagents evaluated (DIEA, A) Results: o substantial improvements 12

13 Chromatography o FIP in mobile phase (post column addition) -> p on column increases Improved sensitivity Response drift over analytical run Peak shape deterioration and pressure increase Current method: with addition of FIP and TEA in solvents volatility of FIP requires frequent refreshment of solvents Time (min) Solvent A Solvent B Shimadzu LC30AD and SIL30ACMP Column Acquity BE C x 50 mm 1.7 µ, 300A Flow rate 300 µl/min Solvent A Water/FIP/TEA (100/0.5/0.1) Solvent B Me/TF/FIP/TEA (70/30/0.5/0.1)

14 Considerations for sample treatment Adsorption - Lobind material Stability/solubility Water soluble uclease enzymes Protein binding (Gs ionically bound) 5% 3 P 4 Chaotropic reagents (ureum, guanidine, lysis buffer) Proteinase K Extraction from the matrix (plasma) LL(chloroform/phenol) SPE (ion pairing, ion exchange) Et precipitation it ti Affinity purification 14

15 Sample preparation LLE + SPE Extraction from plasma Phenol chloroform SPE-LB Protocol adapted from Ewles et al Bioanalysis, 6, 447 LLE 200 L L plasma + AS L Water/ 3 (95/5) L ice-cold phenol/chloroform/iaa Mix 20 min centrifuge transfer 600 L aqueous phase SPE (LB) L Water/FIP/TEA 100/2/0.2 - mix Condition SPE transfer sample Wash Elute in 500 L Water/AC/TEA 40/60/1 evaporate Reconstitute t in 200 L L water/me/fip/tea 70/30/1/0.11 Low recovery 15

16 % recovery in chloroform/phenol extracts sample 2 layer rganic layer AS G AS G Water + 4 * plasma * Ratio aqueous/organic 8/1 16

17 Sample preparation SPE-AEX 200 L plasma L lysis buffer + AS Condition SPE column Apply sample Wash 2 x 2 ml wash buffer elute 2 x 1 ml elution buffer Evaporate - not complete dryness Reconstitute in 300 L water/fip/tea Clarity TX SPE columns (buffers delivered with starter kit) recovery only 7 % for AS (50 % for reference G) Evaporation and reconstitution -> 30 % loss p 5.5 is critical for binding to Clarity TX 17

18 Sample preparation SPE-TX Sample description 20µl plasma + G mix + 200µl Lysisbuffer Clarity 20µl plasma + G mix + 20µl Guanidine 6M + 180µl PBS 20µl plasma + G mix + 2µl Protease K + 200µl PBS 20µl plasma + G mix + 200µl Lysisbuffer Clarity Lysis % recovery % recovery condition AS reference G 30 min RT 15 min 50 C <5 min, RT min, 50 C

19 ther strategies considered Protein removal by precipitation Add 4 and C 3 C Evaporate Ethanol precipitation of DA Protein removal chloroform/phenol extraction Ethanol precipitation of DA* ybridization purification with complementary biotinylated probe Streptavidin MSIA tips or streptavidin magnetic beads Elution of the beads * Chen and Bartlett, J Chrom A, 2013,

20 ybridization with biotinylated capture probe Capture probe biotinylated at 3 biotin streptavidine t 20

21 ybridization Protocol Plasma + Binding buffer + AS Add capture probe and heat up to > Tm Cool to allow annealing Transfer to streptavidine coated MSIA or magnetic beads Wash with ice-cold cold buffers Elute Under basic conditions (a M or ) 4 At elevated temperature Initial Results: Streptavidin biotin reaction successful o capture probe left following incubation with SA beads ybridization as evaluated by mass spec not (yet) successful 21

22 Conclusions LC-MSMS development of this AS is extremely challenging LC conditions controlled but robustness still dependent on the quality of the sample prep Sample preparation is critical step +/- 60% extraction recovery (variable) with TX protocol but no robust LCMS response protein binding removal of proteins before DA isolation Will hybridization offer the solution? Full scan RMS or MALDI MS beneficial to evaluate shifts in charge state distribution or a adducts or formation of degradation products? 22

23 Akcnowledgements Luc Sips Tony Greway Tom Verhaeghe Thank you