Buffer exchange is an essential part of the biological and gene therapy workflow, especially for formulation development, characterization, purification and sample preparation.
With recent advances in gene therapy, 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 currently available to researchers are usually the same as those developed decades ago, which are primarily for proteins and are very labor intensive.
For example, dialysis takes time to set up and is relatively slow to produce results. Centrifugal ultrafiltration can be fast, but sample recovery suffers from clogged membranes. Tangential flow filtration (TFF) can require large sample volumes and can also be difficult to optimize for a biomolecule of interest.
All of these manual methods have one thing in common: they are difficult to scale to work with many different samples.
Can Big Tuna be used to automate these processes with gene therapy and other biomolecule samples?
Big Tuna processes all biological and gene therapeutic biomolecules and carries out buffer exchange and concentration on up to 96 samples at the same time. The system is easy to use and quick to set up. Most importantly, Big Tuna allows users to easily increase the number of samples processed.
Big Tuna is ideal for controlling the protein concentration and buffer exchange required for formulation development. This process typically involves finding the perfect conditions for the specific storage buffers being used, which often requires rapid testing of multiple buffers and samples. With manual methods, this would require considerable practical time. Big Tuna automates this process with the added ability to work towards specific target concentrations.
For example, a pH study with monoclonal antibodies may involve buffer exchange for 12 different samples over 7 hours. Instead of using a manual method, Big Tuna can completely automate this process, leaving the lab technician or researcher 7 hours free time to do other work.
Big Tuna is also perfect for working with nucleic acids. Gene therapy researchers tend to generate many different samples that must be transferred to a storage buffer at a working concentration. Big Tuna can handle both buffer exchange and sample concentration to a specific target, while achieving a level of precision control that dialysis or centrifugation cannot.
Because of their typically dilute concentration, AAVs have to be buffered differently after production. Big Tuna software has pattern presets that allow users to easily process different biomolecules. In cases where it is necessary to concentrate a large volume of AAVs on a target value, an application for reducing the sample volume is also available.
Anyone who works with LNPs knows the importance of buffer exchange, as these biomolecules are typically made in a hard buffer. The buffer exchange must be carried out quickly but also carefully so that the 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 process virtually all types of gene therapy biomolecule samples and is able to exchange or concentrate protein, DNA, RNA, AAVs or LNPs. It’s fast without sacrificing ease of use, sample recovery, or quality.
What factors affect Big Tuna’s ability to provide superior results when compared to other automated buffer exchange techniques?
Big Tuna’s disk-based format, the Unfilter, is the secret of its superior buffer exchange capabilities. They are available in two different layouts, with which you can exchange and concentrate 24 or 96 samples in parallel. Unfilter 96 can only exchange 100 μL for up to 96 different samples in one run. With Unfilter 24 you can exchange up to 8 ml of up to 24 samples. Both are available with different molecular weight limits and use a regenerated cellulose membrane, which ensures a low cost per sample.
Another special feature of Big Tuna is its unique approach of automated pressure-based ultrafiltration (UF) and diafiltration (DF).
Big Tunas UF / DF begins by measuring the volume of each sample in an unfilter before initiating a buffer exchange cycle in which an even pressure is applied to each sample. The buffer exchange cycle is adaptable and measures the volume of each individual well again to prevent drying out and loss of valuable samples.
This approach also avoids some of the limitations of other techniques. For example, a common problem with dead-end ultrafiltration is that proteins can aggregate, causing them to clump at the membrane interface and interfere with sample collection.
Big Tuna uses a gentle orbital mixture that is programmable – either set by the user or via presets for a biomolecule of interest. This prevents any clogging and ensures higher sample recovery and faster replacement.
The combination of custom volume tracking, even pressure and gentle mixing enables Big Tuna to deliver superior even UF / DF results.
Which process parameters can be controlled and managed via Big Tuna and how can this be achieved?
Big Tuna gives the user precise control over buffer exchange parameters that can be programmed in advance. Programmable parameters include percent removal, percent replacement, and final concentration. This precise control over buffer exchange and sample concentration parameters enables Big Tuna to be able to upscale the parallel processing of multiple samples without sacrificing quality or recovery.
Big Tuna software is tailored to make buffer exchange and concentration easy. It uses a wizard format that guides users to simply select the application they want to run and the molecule of interest. Sample presets are optimized for specific molecules and concentrations, but can easily be changed to specific parameters for each sample. Anyone in the lab can use Big Tuna software, in part because of how easy it is to map samples and buffers to an unfilter. Every buffer exchange and sample concentration experiment is validated by the software, which makes the automation of workflows even easier.
The software keeps users informed of the progress – both overall and the progress of the filter cycle at the level of each individual sample. Unlike other manual methods, users know exactly when the buffer swaps and concentration runs are over, which makes it easier to plan further steps.
One of the most useful functions of Big Tuna is the presentation of results and reports. Results are provided in a contextual format in which the results of experiments well by well or sample by sample are presented. It also records the exact conditions under which the experiment was conducted, helping to maintain consistency – something that would not be possible with a manual technique.
How can Big Tuna be integrated into existing platforms or workflows?
Buffer exchange and concentration of the biomolecule samples used in gene therapy are never done in isolation. Upstream and downstream processes depend on whether someone works in discovery, development or production. Automation is often used to link samples between these interconnected workflows.
Big Tuna offers several functions to integrate with other automated workflows or systems. It offers easy sample tracking, which means that when exchanging buffers, it is easy to monitor where the samples came from and where they go after the exchange.
Big Tuna maintains extensive sample and buffer lists that can be converted and then imported directly, making it much faster to set up runs. Once the runs are complete, the results can be exported in either PDF or text-based format, which allows easy transfer to other software.
The Big Tuna API speeds integration by enabling full automation of buffer exchange, concentration, and workflows to reduce sample volume.
The Big Tuna API complies with the laboratory automation standard SiLA 2, which makes it faster and easier to integrate Big Tuna into other software. With this level of integration it is possible to check the status of an experiment remotely, to start and unload experiments or to automate user input.
Big Tuna robotic integration is as simple as delivering unfiltered filters to and from the buffer exchange chamber, which can be done without loss of manual user interaction.
Big Tuna is the superior solution for high throughput buffer exchange and concentration. With intuitive software control and a system that was developed for all biological and gene therapy biomolecule samples, Big Tuna saves time and optimizes sample recovery. Finally, the new Big Tuna API makes it easier to integrate into your automated workflows.
About Unchained Labs
Unchained Labs is about helping biologics and gene therapy researchers break free of tools that just can’t make it. Unleashing problem solving products that make a huge difference in the real science they do every day. This is their mantra, their promise, and they own it.