Automated Acoustic Dispensing for the Serial Dilution of Peptide Agonists in Potency Determination Assays

Summary

Peptide adsorption to plasticware during traditional tip-based serial dilutions can significantly impact potency determination and confound the understanding of structure-activity relationships used for lead identification and lead optimization phases of drug discovery. Here methods for automated acoustic non-contact serial dilution of peptide samples are described.

Abstract

As with small molecule drug discovery, screening for peptide agonists requires the serial dilution of peptides to produce concentration-response curves. Screening peptides affords an additional layer of complexity as conventional tip-based sample handling methods expose peptides to a large surface area of plasticware, providing an increased opportunity for peptide loss via adsorption. Preventing excessive exposure to plasticware reduces peptide loss via adherence to plastics and thus minimizes inaccuracies in potency prediction, and we have previously described the benefits of non-contact acoustic dispensing for in vitro high-throughput screening of peptide agonists¹. Here we discuss a fully integrated automation solution for non-contact acoustic preparation of peptide serial dilutions in microtiter plates utilizing the example of screening for peptide agonists at the mouse glucagon-like peptide-1 receptor (GLP-1R). Our methods allow for high-throughput cell-based assays to screen for agonists and are easily scalable to support increased sample throughput, or to allow for increased numbers of assay plate copies (e.g., for a panel of more target cell lines).

Introduction

The GLP-1R is an established drug target in the treatment of type 2 diabetes². The native peptide agonist for this receptor, GLP-1, has an in vivo half-life of 2-3 min³. The binding of GLP-1 to its G protein coupled target receptor results in the downstream production of the second messenger cAMP through native G protein coupling to the activation of adenylyl cyclase. Measurement of the accumulated cAMP provides a robust assay to monitor receptor activation and to screen for active GLP-1 analogues with preferred physicochemical properties. Such an assay requires the serial dilution of test samples to construct concentration-response curves, and this is particularly complicated when handing peptide samples. Potential errors from tip-based serial dilution preparation have been described previously^1,4,5. Peptides will adsorb to plasticware, resulting in unreliable potency estimations. Peptide loss can be minimized through the inclusion of bovine serum albumin (BSA) in buffers and the use of siliconized plasticware, yet protein binding remains unpredictable. In particular, the variation in binding of GLP-1 to experimental containers has been described⁶. There is a further complication in that stabilization agents used in laboratory plasticware can leach from tips and microtiter plates into aqueous assay buffers and interfere with protein function^{7, 8}. Therefore, methods to reduce exposure to plasticware are necessary to increase the accuracy of measurements.

Acoustic liquid dispensers focus a high-frequency acoustic signal onto the surface of a fluid sample, resulting in the ejection of precise nanoliter droplets into an adjacent assay plate⁹. The use of acoustic ejection is standard in the pharmaceutical industry for the preparation and screening of large synthetic compound libraries, and the technology has been well validated for small molecules¹⁰. To our knowledge, we are the first group to describe acoustic dispensing for the preparation of recombinant and synthetic peptides and we have previously reported the improved accuracy compared to conventional tip-based methods¹.

This article describes the integration of the preparation of peptide serial and direct dilutions by non-contact acoustic transfer onto a fully automated plate handling robotics system. A number of methods encompassing acoustic transfer of samples have been described previously¹¹. We utilize a two-step method to prepare intermediate stock concentrations and to serially dilute peptide analogues for the generation of the full dose-response curve. The prepared peptides are incubated with cells expressing the target mouse GLP-1R, and we use a commercially available homogenous time-resolved fluorescence (HTRF) assay to measure cAMP accumulation within these cells as a readout of peptide agonist activity. The assay is robust and amenable to a high-throughput 384-well format and routinely applied to both assay development and drug screening projects¹².

Protocol

1. Peptide Serial Dilution

1. Prepare assay buffer: Hanks buffered salt solution (HBSS) supplemented with 25 mM HEPES, 0.1% BSA and 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), pH 7.4.
2. Use a bulk reagent dispenser to systematically add 5 µl of assay buffer to each well of five 384-well low volume assay plates.
   1. Use internal software to create a dispensing program for 5 µl volume addition to every well of a 384-well plate as per manufacturer's instructions.
   2. Immerse dispensing cassette tubing in assay buffer and prime fluid.
   3. Place 384-well low volume assay plate on plate carrier.
   4. Press start.
3. Dilute all peptide samples, regardless of storage vehicle, into assay buffer to produce a 100x peptide stock.
   NOTE: Peptides requiring screening may be provided in Phosphate-buffered saline (PBS) or Dimethyl sulfoxide (DMSO) as is deemed appropriate (e.g., DMSO will have a deleterious effect on peptides with secondary modifications such as PEGylation).
4. Dispense 25 µl 100x peptide stocks into columns 1-5 of an acoustically qualified 384-well polypropylene microplate. This plate is designated 'source plate A'. Ensure that source plates, but not destination plates, are flat bottomed and conform to specific acoustic tolerances as defined by the manufacturer of acoustic instruments.
5. Dispense 25 µl 100x reference control into wells A23 and A24 of source plate A.
6. Dispense 10 µl assay buffer into columns 11-15 and 30 µl assay buffer into columns 21-22 of source plate A.
7. Dispense 10 µl assay buffer into columns 6-10 and columns 16-20 of a second acoustically qualified 384-well polypropylene microplate. This plate is designated 'source plate B'.
8. Centrifuge source plates A and B at 300 x g for 1 min. Include an appropriate balance plate.
9. Use acoustic fluid dispensers integrated into an automated robotic system to prepare three sequential 1:100 intermediate dilutions (in assay buffer) of the 100x peptide stocks from column 1 in source A.
   NOTE: Programming requires plate reformatting and dose response software (as provided by the manufacturer of the acoustic fluid dispenser) to allow three acoustic transfers between source A and source B (see Figure 1 for plate layouts). Step 1.9 details specifically the use of fluid dispensers used in this laboratory under automated robotic control (see Materials Table):
   1. Load source plates and assay plates into plate hotel section of robotics.
   2. Open automated robotic software and load plate reformatting and dose response program expansion protocols. Click 'Run'.
      NOTE: Automation instructs an acoustic fluid dispenser to transfer 250 nl from columns 1-5 of source plate A into columns 6-10 of source plate B, and a second acoustic fluid dispenser capable of handling larger fluid volumes to backfill with 15 µl assay buffer to mix. Automation then transfers source plate B to integrated centrifuge and centrifuges at 300 x g for 1 min (an appropriate balance plate is included for all centrifugation steps). Source plate B is returned to the acoustic fluid dispenser for the transfer of 250 nl from columns 6-10 of source plate B into columns 11-15 of source plate A and backfill with 15 µl assay buffer to mix. Automation transfers source plate A to integrated centrifuge and centrifuges at 300 x g for 1 min. Source plate A is then returned to the acoustic fluid dispenser for the transfer of 250 nl from columns 11-15 of source plate A into columns 16-20 of source plate B and backfill with 15 µl assay buffer to mix. Automation then transfers source plate B to integrated centrifuge and centrifuges at 300 x g for 1 min.
      NOTE: Finally the dose response protocol directs acoustic dispensing to transfer the required volume from each of the 4 serially diluted source plate wells (in both source plate A and source plate B) to construct a full 11-point curve in duplicate in assay plates (pre-filled with 5 µl assay buffer in step 1.2 above). Automation transfers assay plate to integrated centrifuge and centrifuges at 300 x g for 1 min.

2. Cell Preparation

1. Thaw cryopreserved Chinese hamster ovary (CHO) cells expressing the target mouse GLP-1 receptor rapidly in a 37 ºC water bath and resuspend in 20 ml assay buffer.
2. Centrifuge cell suspension for 5 min at 200 x g at room temperature (RT). Include an appropriate balance.
3. Discard supernatant and resuspend cell pellet in 10 ml assay buffer.
4. Dilute cell stock 1:1 in Trypan blue and determine viable cell density using an automated cell counter. Resuspend cells in assay buffer at 1.6 x 10⁶ cells per ml (equivalent to 8,000 cells per well of assay plates).
5. Use a bulk reagent dispenser to add 5 µl of cell suspension to each well of the assay plates (containing serially diluted peptides in 5 µl assay buffer) and incubate at RT for 30 min.

3. HTRF cAMP Detection Assay

1. Bring HTRF cAMP assay kit to RT for 30 min prior to use.
2. Prepare each HTRF reagent (cryptate and d2) separately at 1:20 dilution in lysis buffer (proprietary formulation, provided by manufacturer of cAMP detection assay).
   NOTE: CAUTION: Lysis buffer contains potassium fluoride (KF) which is toxic and a teratogen¹³. For disposal, assay plates containing KF should be sealed and incinerated, and any liquid waste must be diluted to <1 mmol/L with water prior to disposal down the sink.
3. Use a bulk reagent dispenser to add 5 µl cryptate reagent to all wells of the assay plates.
4. Use a bulk reagent dispenser to add 5 µl d2 reagent to columns 1-22 of the assay plates.
5. Immediately manually pipette 5 µl lysis buffer into wells A23-D24 of the assay plates to give non-specific binding (NSB) control wells, and 5 µl of d2 reagent into wells E23-P24.
6. Centrifuge the assay plates for 1 min at 200 x g at RT to mix the wells.
7. Cover the assay plates to minimize evaporation and photo-bleaching and incubate at RT for 1 hr.
8. Measure fluorescence resonance energy transfer (FRET) signal using excitation at 320 nm and emission at 620 nm and 665 nm using a plate reader.

4. Data Analysis

1. Use mean NSB to subtract background from all wells.
2. Calculate %Delta F from the 665 nm/620 nm ratio as follows:
   Ratio = (A_665nm/A_620nm) x 10⁴
   Delta F = ((Sample Ratio - Ratio_NSB)/Ratio_NSB)) x 100
   where Ratio_NSB = wells with no d2 reagent.
3. Calculate % activation between unstimulated cells and cells stimulated with maximum GLP-1 ligand as follows:
   % activation = (%Delta Fsample - %DeltaFunstimulated cells)/(%DeltaFGLP-1 stimulated cells - %DeltaF unstimulated cells) x 100
4. Analyze concentration-response curves via 4-parameter logistical analysis, and graph as % activation as defined by reference control¹.

Results

We routinely use a two-step method to dilute peptides via acoustic transfer. For the first step, an acoustic dispenser aligned with automation is used to create four stock peptide intermediate dilutions across two source plates (Figure 1a, b). For the second step, we use an acoustic dispenser to further dilute stock dilutions from source plates A and B to create an 11-point concentration range for each test peptide (Figure 1c). Each pepti...

Discussion

This protocol describes the successful application of automated acoustic dispensing to serially dilute peptide samples over a concentration range of 3 x 10⁶ requiring less than 1 µl of sample. The major advantage of this method is to increase data quality through minimizing peptide adsorption to plasticware via reduced exposure of samples to experimental containers and plasticware (such as pipette tips) that are normally required for reagent transfer and mixing. While acoustic dispensing does not complete...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Hanks’ Balanced Salt solution	Sigma-Aldrich	H8264
HEPES	Sigma-Aldrich	H3375
Bovine Serum Albumin	Sigma-Aldrich	A9418
3-Isobutyl-1-methylxanthine	Sigma-Aldrich	I7018	Prepared as a 0.5 M stock in DMSO
GLP-1 (7-36) amide	Bachem	H-6795	Prepared as a 1 mg/ml stock in PBS, referred to as '100x reference control'
Test peptides	Produced in-house at MedImmune		Supplied at various concentrations in DMSO or PBS as appropriate
100x peptide stock	Produced in-house at MedImmune		Test peptide diluted into assay buffer to 100x final required concentration
Trypan Blue Solution, 0.4%	Thermo Fisher Scientific	15250-061
Cedex XS Cell Analyzer	Innovatis
Corning 384 well plates, low volume	Sigma-Aldrich	4514
Echo Qualified 384-Well Polypropylene Microplate	Labcyte Inc.	P-05525
Echo Qualified Reservoir	Labcyte Inc.	ER-0055
Echo 550 Liquid Handler	Labcyte Inc.		Droplet transfer volumes in increments of 2.5 nl
Echo 525 Liquid Handler	Labcyte Inc.		Droplet transfer volumes in increments of 25 nl
ACell Benchtop Automation	HighRes Biosolutions	MC522
Cellario Lab Automation Scheduling software for Life Science Robotics	HighRes Biosolutions
MultidropCombi Reagent Dispenser	ThermFisher Scientific	5840300	Referred to as 'bulk reagent dispenser'
HTRF cAMP Dynamic 2 kit	Cisbio Bioassays	62AM4PEJ
EnVision Multilabel Reader	PerkinElmer