In the beginning the universe was created, this made a lot of people very angry and was widely regarded as a bad move (Adams, 1979).
Similar misinformed ideas include that subvisible and visible particle analysis and characterization are purely the preserve of the quality controller and the formulator – that the overhead and material required to accurately determine the content of potentially immunologically activating contaminants is too demanding for early-stage development. Instead, developers determining optimum formulation conditions for their protein candidates will perform ancillary measurements in the hope that they will be predictive of drug product particulate formation. Often relying on conformationally driven thermal unfolding (Tm), determination of the propensity for colloidal interaction using measurements at unrepresentative concentrations (B22), or simply looking for small things with dynamic light scattering (DLS), size exclusion chromatography (SEC), or mass photometry (MP) with the expectation that there will be a direct causation effect.
The literature is clear that this isn’t the case. However, it has been the best way forward for some time, as sparing 1 mL of precious material for light obscuration (LO) or flow imaging (FI) simply hasn’t been practical.
Advancements in Particle Analysis Enabling Ultra-Low Volume Assays
The advent of novel technologies based on compendial membrane microscopies enables all these experiments to be carried out with volumes as low as 5 µL, making it possible to perform reliable aggregate and particulate measurements at the earliest stage rather than try to predict them poorly. This approach realizes the potential of biophysical characterization at the very beginning of drug candidate selection and late-stage research when the product is scarce.
One of those technologies is Backgrounded Membrane Imaging (BMI), which is an automated form of USP788 method 2, the recommended method for liposomal drug preparations and those containing translucent particles that are typically formed as proteins aggregate in solution.
Automation and Robotics Requires Novel Approaches to Analysis
In addition to volume constraints, one key barrier to a technology is the overhead required to measure the typically large number of drug candidates, formulations, processing conditions, and drug substance formulations. Automation helps significantly optimize and simplify the workflow and provides the opportunity for improvements in the precision and accuracy of liquid handling.
Aura® from Halo Labs, which is BMI-enabled, has been incorporated into a variety of automation workflows and has an application program interface (API) that can be used to develop a driver to plug into your favorite robotics platform.
The BMI Workflow
BMI provides the ability to count and size particles from our high-resolution microscopy images and to use specific fluorescence labels to determine their properties.
The workflow can be broken down into a number of simple steps:
- Backgrounding
- Sample Handling
- Measurement
- Data handling
1. Backgrounding
Backgrounding involves loading an unused plate into Aura and measuring it. In the automation workflow this is most easily broken down into:
- Open tray
- Take plate from stack (we recommend 0.8 µm pore plates for protein work)
- Place plate in Aura and close tray
- Configure protocol (select wells, name samples, provide volume information)
- Background wells
2. Sample handling
- Open the tray and remove the plate
- Move plate to sample loading manifold
- USP 788 pre-wet step – load 40 µL of water for injection (WFI) on the selected wells – don’t forget to include a control! (See USP 788 pages for details regarding the reasons you may want to do the prewet and wash steps.)
- Open the vacuum valve/turn the vacuum on for 15 seconds
- Apply 5 µL (or your selected volume) to all selected wells, either individually or by transferring from a 96-well plate
- Open the vacuum valve/turn the vacuum on for 15 seconds
- USP 788 wash steps
- Apply 40 µL of WFI on all selected wells
- Open the vacuum valve/turn the vacuum on for 15 seconds
- Repeat 3 times
- Move plate to drying manifold
- Open the vacuum valve/turn the vacuum on for 60 seconds
Sample handling is the process by which the samples are loaded onto the plate and the plates are prepared for measuring. All samples are different and this is our recommended protocol to start with. Fewer or additional steps may be required as per your assay development/optimization. Your FAS can help with that, they do it every day!
At this point, your samples are ready to be measured.
3. Measurement
This will be familiar, as it’s identical to the backgrounding:
- Open tray
- Move your plate from the manifold, place in Aura, and close tray
- Measure the samples
4. Data handling
Data are automatically analyzed with the dedicated Aura software, Particle Vue. The software flags any wells that need reviewing so that you can check the images before confirming the data for the report. All data are saved as images and data files in a repository folder. Aura provides indirect integration with automation software systems, so you can set your laboratory information management systems (LIMS) or other software to watch the folder and automatically import it.
Is My Robot Compatible?
Yes!
We have users with all major platforms, including Tecan, Hamilton, Biosero… with our SDK it’s simple and straightforward to write your own driver or have your automation vendor do it for you.
For those of you with Tecan systems, the best gripper is:
PN 10614007, RoMa Fingers, Centric for Gripping Plates, set of 2
Related
References
Adams, D. (1979). The hitchhiker’s guide to the galaxy. New York: Harmon Books.
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