Sandwich automated buffer exchange into protein, AAV or LNP workflows with Big Tuna

insights from industryUnchained Labs

Buffer exchange is an essential part of biologics and gene therapy workflows, particularly for formulation development, characterization, purification, and sample preparation.

Due to recent gene therapy advances, buffer exchange and concentration have become critical for more molecules beyond proteins and antibodies. Researchers working with nucleic acids, lipid nanoparticles (LNPs), viral vectors and adeno-associated viruses (AAVs) need compatible tools and techniques.

The tools that researchers currently have at their disposal tend to be the same as those that were developed decades ago, which are primarily for protein and highly labor-intensive.

For example, dialysis takes time to set up and provides results reasonably slowly. Centrifugation ultrafiltration can be fast, but sample recovery suffers from clogged membranes. Tangential flow filtration (TFF) can require large sample volumes and can also be challenging to optimize to a biomolecule of interest.

All these manual methods have one thing in common: they are difficult to scale up to work with many different samples.   

Can Big Tuna be used to automate these processes with gene therapy and other biomolecule samples?

Big Tuna handles all biologic and gene therapy biomolecules, performing both buffer exchange and concentration on up to 96 samples in parallel. The system is easy to use and quick to set up. Most importantly, Big Tuna allows users to scale up the number of samples processed with ease.

Big Tuna is ideal for tackling protein concentration and buffer exchange needed for formulation development. This process typically involves finding the perfect set of conditions for the specific storage buffers being used, often requiring rapid testing of multiple buffers and samples. For manual methods, this would require significant hands-on time. Big Tuna automates this process with the added capability of working towards specific target concentrations.

For example, a pH study with monoclonal antibodies may involve using a buffer exchange for 12 different samples over 7 hours. Rather than using a manual method, Big Tuna can compeletly automate this process leaving the lab technician or researcher with 7 hours of free time for other work.

Big Tuna is perfect for working with nucleic acids too. Researchers in gene therapy tend to generate many different samples which need to be transferred into a storage buffer at a working concentration. Big Tuna can handle both buffer exchange and concentrating samples to a specific target while achieving a level of precise control that is not possible with dialysis or centrifugation.

AAVs need to be buffer exchanged differently due to their typically dilute concentration after production. Big Tuna software has sample presets that allows users to process different biomolecules easily. A Reduce Sample Volume application is also available for cases where it is necessary to concentrate a large volume of AAVs to a target value.

Anyone working with LNPs knows the importance of buffer exchange, as these biomolecules are typically manufactured in a harsh buffer. Buffer exchange must be performed quickly but also carefully so sample integrity is not compromised. Big Tuna completes buffer exchange and concentration applications quickly while ensuring high sample recovery.

Big Tuna offers excellent opportunities to simplify sample preparation. It can handle virtually any gene therapy biomolecule sample type and is able to exchange or concentrate protein, DNA, RNA, AAVs or LNPs. It is fast without sacrificing ease of use, sample recovery or quality.

What factors influence Big Tuna’s ability to deliver superior results compared to other automated buffer exchange techniques?

Big Tuna’s plate-based format, the Unfilter, is the secret to its superior buffer exchange capabilities. They come in two different layouts that let you exchange and concentrate 24 or 96 samples in parallel. Unfilter 96 can exchange as little as 100 μL for up to 96 different samples in a run. Unfilter 24 lets you exchange as much as 8 mL on up to 24 samples. Both are available in various molecular weight cutoffs and use a regenerated cellulose membrane, ensuring a low cost per sample.

Another of Big Tuna’s distinctions is its unique approach of automated pressure-based ultrafiltration (UF) and diafiltration (DF).

Big Tuna’s UF/DF begins by measuring the volume of each sample in an Unfilter before commencing a buffer exchange cycle in which uniform pressure is applied to each sample. The buffer exchange cycle is adaptive and will re-measure the volume of each individual well to negate the possibility of it drying out and losing precious sample.

This approach also avoids some of the limitations of other techniques. For example, there is a common problem with dead-end ultrafiltration where proteins may aggregate, leading them to clump up at the membrane interface and have an impact sample recovery.

Big Tuna uses a gentle orbital mixing that is programmable – set by either the user or via presets designed for a biomolecule of interest. This stops any clogging and ensures higher sample recovery and faster exchanges.

Combining individual volume tracking, uniform pressure and gentle mixing enables Big Tuna to deliver superior uniform UF/DF results.

What process parameters can be controlled and managed via Big Tuna, and how can this be accomplished?

Big Tuna gives users precise control over buffer exchange parameters, which can be programmed in advance. Programmable parameters include percent removal, percent exchange and final concentration. This precise control over buffer exchange and sample concentration parameters enables Big Tuna to scale up processing multiple samples in parallel without sacrificing quality or recovery.

Big Tuna's software is tailored to easily conduct buffer exchange and concentration. It makes use of a wizard format, guiding users to simply select the application they want to run and their molecule of interest. Sample presets are optimized to certain molecules and concentrations but can be easily modified to specific parameters for any sample. Everyone in the lab can walk up and use Big Tuna software, partly due to how straightforward it is to map samples and buffers to an Unfilter. Every buffer exchange and sample concentration experiment is validated by the software, granting additional peace of mind to automating workflows.

The software keeps users updated on progress – both overall and the progress of the filter cycle at the level of each specific sample. Unlike other manual methods, users will know exactly when the buffer exchange and concentration runs will finish, making it easier to plan further steps.

One of Big Tuna’s most useful features is its presentation of results and reports. Results are provided in a context format, reporting results of experiments well-by-well or sample-by-sample. It also records the exact conditions that the experiment was run under, helping to maintain consistency – something that would not be possible using a manual technique.

How does Big Tuna integrate with existing platforms or workflows?

Buffer exchange and concentration of the biomolecule samples used in gene therapy are never done in isolation. Whether someone works in discovery, development or manufacturing, downstream and upstream processes depend on this. Automation is often used to link samples between these interconnected workflows.

Big Tuna offers several features to integrate it into other automated workflows or systems. It offers easy sample tracking, meaning that when doing buffer exchange, it is simple to monitor where samples have come from and where they are going after exchange.

Big Tuna maintains comprehensive sample and buffer lists that can be converted and then imported directly, making it much faster to set up runs. Once runs are completed, results can be exported in either PDF or text-based format, allowing easy transfer into other software.

The Big Tuna API supercharges integration by enabling full automation of buffer exchange, concentration, and reduce sample volume workflows.

The Big Tuna API conforms to the SiLA 2 lab automation standard, making it quicker and easier to integrate Big Tuna into other software. Using this level of integration, it is possible to remotely check the status of an experiment, start and unload experiments or automate user input.

Robotic integration of Big Tuna is as straightforward as delivering Unfilters to and from the buffer exchange chamber, which can be done without losing manual user interaction.  

Big Tuna is the superior solution for high-throughput buffer exchange and concentration. With intuitive software control and a system designed for all biologic and gene therapy biomolecule samples, Big Tuna saves time and optimizes sample recovery. Finally, the new Big Tuna API makes it easy to integrate it into your automated workflows.

About Unchained Labs

Unchained Labs is all about helping biologics and gene therapy researchers break free from tools that just don’t cut it. Unleashing problem-tackling products that make a huge difference in the real science they do every day. That’s their mantra, their promise and they own it.


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