OligoWorks™ Sample Pretreatment and SPE Extraction

Oligonucleotide therapeutics (ONTs) are a key focus area for many drug developers today given their powerful ability to address disease biology at the level of gene transcription and translation, and for their high target specificity and low toxicity. As the pipeline for this therapeutic class of drugs continues to expand, so does the need for sensitive, accurate and robust bioanalytical assays to support this drug discovery and development pipeline. LC-MS detection and quantification (Figure 1) is a widely accepted technology for bioanalytical studies, for the many benefits it affords (i.e., broad drug applicability, sensitivity, selectivity, and broad linear dynamic range). However, achieving reproducible performance with LC-MS based bioanalytical assays can be challenging. In general, the greatest source of variability for these assays arises from the sample preparation needed to extract the drug and its metabolites from biofluids, and this is especially true for oligonucleotide extractions. 

Waters OligoWorks™ SPE kits (and components) are designed to accelerate your oligonucleotide bioanalysis projects and shorten the gap between the delivery of samples to your lab and obtaining the high confidence results you need to understand dosing, metabolism, and tissue adsorption of a drug candidate. 

OligoWorks SPE kits address key challenges encountered during oligonucleotide bioanalysis by:

• Simplifying previously complex and laborious sample preparation workflows
• Improving day-to-day and user-to-user robustness and repeatability
• Addressing poor and inconsistent recoveries across a diverse range of oligonucleotide therapeutics
• Enabling greater MS sensitivity and accuracy for oligonucleotide bioanalysis

Figure 1: Graphical illustration of oligonucleotide bioanalytical quantification sample preparation, extraction, and LC-MS workflow.

OligoWorks SPE Kits include sample pretreatment and SPE protocols (Figure 2) that have been optimized for performance using starting plasma/whole serum volumes between 12 and 325 µL. The recommended reagent volumes to be added are proportional to the starting plasma/whole serum sample volume. The following protocol is based on a starting plasma sample volume of 100µL. If starting with a different sample volume, please refer to OligoWorks Pretreatment Volume Guidance Chart (Table 1) and Recommended Volumes for OligoWorks SPE Protocol Across Device Formats (Table 2) for the correct volume of reagents to add.

Optimum digestion times have been found to be 40-60 minutes. Consider using a 60-minute digestion time when working with highly proteinaceous samples and oligonucleotides known to exhibit pronounced protein binding.

Figure 2: Graphical representation of the OligoWorks Kit Protocol (P/N 186010614), optimized for 100 µL starting plasma/sera sample.

Table 1: OligoWorks Pretreatment Volume Guidance Chart

Table 2: Recommended Volumes for OligoWorks SPE Protocol Across Device Formats

Assay notes

OligoWorks Kits are simple, standardized, flexible and automation friendly sample preparation kits designed to enable accurate and robust LC-MS based bioanalytical quantitation across a diversity of oligonucleotides. The kits use an effective enzyme-based digestion sample pretreatment step with RapiZyme proteinase K digestion module to effectively disrupt oligonucleotide-biomatrix protein binding followed by selective purification using the OligoWorks SPE device, which contains a mixed-mode anion exchange SPE sorbent, designed, and QC verified for oligonucleotide performance. Each kit contains pre-measured, lot traceable, detergent free reagents and a universal protocol to streamline the

oligonucleotide sample preparation workflow and facilitate implementation by users at all experience levels.

Protocol 1: OligoWorks pretreatment – Rapizyme Proteinase K Digestion method

Figure 3: Andrew+ OneLab deck set up for the OligoWorks pretreatment – Rapizyme Proteinase K Digestion method. Samples and reagents locations on Andrew+ deck: [1-2] Tips, [3] Deepwell Domino, [4-5] 8-channel Reservoir Domino, [6] Heater Shaker+ w/ Deepwell Plate Adaptor

Protocol 2: OligoWorks WAX SPE Microplate method

Figure 4: Andrew+ OneLab deck set up for the OligoWorks WAX SPE method. Samples and reagents locations on Andrew+ deck: [1-2] Tips, [3-4] Deepwell Domino, [5] Extraction+, [6] Heater Shaker+ w/ Deepwell Plate Adaptor

Ordering information

Andrew+ System Components: Dominos, Devices, Electronic Pipettes & Tips

• Andrew+ Pipetting Robot
• OneLab software
• 2× Tip Insertion System Domino | p/n 186009612
• Deepwell Domino | p/n 186009597
• 2× 8-Channel Pipette Reservoir Domino | p/n 186009613
• Heater Shaker+ w/ Deepwell Plate Adaptor | p/n 186010426
• Extraction+ Base Kit with Gripper | p/n 176005201
• Andrew Alliance Bluetooth Electronic Pipette, 8-ch 120 μL | p/n 186009605
• Andrew Alliance Bluetooth Electronic Pipette, 8-ch 300 μL | p/n 186009607
• 2× Pipette Adaptor Multi-Channel | p/n 186009591

Additional suggested items

• 2 additional Tip Insertion System Domino | p/n 186009612; ℹ️ It will reduce the manual refill of tip boxes during the execution of the full workflow protocol (pretreatment and SPE).

Automation kit options

• Andrew+ OligoWorks Digestion Method Kit - MicroElution Plates | p/n 176005357
• Andrew+ OligoWorks SPE Method Kit - MicroElution Plates | p/n 176005358
• Andrew+ OligoWorks Digestion & SPE Kit - MicroElution Plates | p/n 176005359

Recommended consumables

• Waters QuanRecovery 96-Well Plate, 700mL | p/n 186009184
• Waters Polypropylene Cap Mat for 96-well Plate | p/n 186002483
• Waters QuanRecovery Vials with MaxPeak pre-slit PTFE silicone cap and septum | p/n 176009186
• Eppendorf 1 mL 96-deep well protein LoBind® plate | p/n 0030504216
• INTEGRA 10 mL multichannel reservoir | p/n 4332
• DNase/RNase-Free Distilled Water, Invitrogen | Fisher Scientific Catalog number 10977015

References

1. OligoWorks Care & Use Document (720008066EN)
2. An Automated, Standardized, Kit-Based Sample Preparation Workflow for Bioanalytical Quantification of Therapeutic Oligonucleotides (720008068EN)
3. Development of a Standardized, Kit-Based Approach for Selective and Reproducible Sample Preparation and Extraction for Therapeutic Oligonucleotides from Biological Matrices (720008067EN)