Witness The Revolution: Introducing Leo, the Next Leap in Simple Western Technology
Hi, my name is Kelly Gardner and I’m the Sr. Director and Western Business Unit leader at Bio-Techne. Over the last two years, the Simple Western Team has been hard at work, and we are so excited to provide an update on our progress towards simplifying complex protein analysis workflows. More specifically, we’re pleased to announce the next leap forward in Simple Western technology and how we are transforming Westerns into quantitative bioanalytical methods. First, a look at how we’ve gotten where we are today and how our teams at Bio-Techne have been working for the better part of two decades to reimagine the western blot.
Of course, everyone here knows that while genomics encodes information that dictates our biology, proteins are the central players that dictate biological function and they play a vital role in almost every biological process and disease. Western blotting or immunodetection, which allows researchers to detect specific proteins in a sample through a process that involves size-based separation of the proteins followed by immunodetection of specific protein targets – is one of the most fundamental methods used in the study of proteins. It is one of the most extensively used techniques in laboratories and if you go into pubmed and type in western blotting, there are over 400,000 research articles that use the approach. At Bio-Techne, our teams have been working passionately for nearly 20 years to innovate, simplify and improve on western-based protein detection with a goal of catalyzing advances in science and medicine. You can see snapshots of our teams over the years. Through this dedication to purposeful innovation, we’ve emerged as the market leader and sole provider of fully automated hands-free western analysis platforms. Looking at the evolution of the western blot,
it was first invented 45 years ago and the traditional manual approach utilizing slab gels and transferring to a PVDF membrane for immunostaining is still used today. Semi-automated western blotting tools started emerging around the 2000s and provided modest workflow improvements by automating portions of the workflow such as transfer or staining. Then in 2011, ProteinSimple, a Bio-Techne brand, launched the first fully automated capillary-based western blot– called Simon. 10 years ago, we launched a new and improved instrument called Wes which combined
the individual capillaries into pre-assembled cartridges and offered significant improvements in sensitivity, robustness and ease of use. In 2018 we launched Jess, which added multiplexing capabilities and in 2021 we launched Abby, a budget-friendly system which includes RePlex, a stripping & reprobing alternative to maximize data obtainable from each sample and run. The result of these efforts over the last few decades is that we’ve been able to transform western blotting results from the poorly reproducible, qualitative and slow data generated by traditional western blotting on the left to highly reproducible, sensitive, multiplexed data on the right. Simple Western technology uses capillaries to do an SDS-based size separation of proteins in a sample, immobilize them to the capillary wall and then do immunodetection with primary and secondary antibodies. There are a variety of assay types to support chemiluminescence and fluorescence readouts and different multiplexing strategies. At its core, capillaries are what enable this
advance in capability and the foundational innovation was being able to enable capillary electrophoresis and pair it with UV-activated sample immobilization to replace the variable transfer step of traditional western blotting and allow for in-capillary immunodetection. The Simple Western advantages can be summed up as offering faster time to result – as low as 3 hours to fully analyzed results - Quantitative results including both absolute and relative protein quantitation. Throughput and flexibility with 24 samples in 3 hours on our Jess, Abby and Wes platforms or 96 samples in an overnight run on our Peggy Sue and Sally Sue systems as well as multiplexing capabilities for more data per sample Reproducibility including low inter- and intra-assay CVs - And low sample volumes – starting from as little as 0.3 ug or 3 uL per well to get to pg-level sensitivity. As a result, Simple Western has grown to become a proven and trusted technology adopted globally with over 2500 citations, more than 5000 antibodies validated for Simple Western, approximately 75 pending and issued patents worldwide that protect the unique technology, and over 4000 instruments installed in over 50 countries worldwide. Whereas traditional Western Blot is typically used in basic research and early-stage development, customers are using Simple Western as a solution that can move all the way through development. While the improvements to data quality and speed have enabled customers to get better data, faster, researchers at all stages of development are being asked to do more and go faster, creating a need for even greater capabilities around quantitation, data quality and speed.
Further, Simple Western is being used to power regulatory submissions - for clinical trial endpoints, surrogate biomarkers as well as QC lot release assays. As customers develop and validate rigorous bioanalytical methods using Simple Western platforms, their needs around reproducibility, quantitation and throughput have also increased. Simple Western technology has opened so many doors to discovery, but there is so much more potential. So, what's next? Our vision of the future of Simple Western is to enable enhanced quantitation and throughput so researchers can use western-based readouts for applications they would never consider using traditional western blots for including both upstream and downstream development and commercial-stage assays. By enhancing reproducibility, quantitation and throughput, Simple Western can open up entirely new applications for customers and bring western blotting into the world of quantitative immunoassays. - Imagine ELISA like protein quantitation with the specificity and protein size resolution of western blotting. - By
bringing the benefits of immunoassays and western blotting together, Simple Western will enable researchers to do things they couldn’t do before, such as: quantification of protein isoforms and size variants that can now be separated by size to resolve the different protein forms before immunodetection. Enhanced detection specificity in complex matrices by leveraging size separation to resolve non-specific binding and quantify only the target protein at the correct molecular weight, and speeding up method development compared to custom ELISA development timescales since you now no longer have to waste time and money searching for a suitable antibody pair. What people love about western blots is that it’s easy to create your own assay but it’s only qualitative. What people like about ELISA is that is shown to be highly quantitative, but it can be challenging to develop especially in complex matrices. Our vision is to bring you the best of both worlds. So, as we continue our drive to innovate to meet the needs of scientists, we are excited today to announce the next leap forward for Simple Western which will provide the throughput and experimental size needed for enhanced quantitation. We look
forward to partnering with scientists around the world who can leverage this new capability to transform the future of medicine by accelerating the next generation of therapies to market. With that, I would like to introduce you to Chris Heger, our director of applications science, who will share some of the exciting applications that Simple Western is enabling in accelerating therapies to market and then we’ll reveal the new instrument. Thanks, Kelly. I’m happy to be here with you to talk about how Simple Western is enabling new applications for western-based assays. Researchers are using Simple Western in a wide variety of applications where traditional western struggles to deliver with enough speed and quantitation. These include High Throughput Targeted Protein Degradation,
Relative Potency assays for the lot release of product in QC, in both Cell and Gene therapies and for measuring biomarkers in clinical trials. Researchers are also using Simple Western for other diverse applications including Vaccine Development, Bioprocess impurity testing, pharmacokinetic/pharmacodynamics, and for measuring target expression in different tissues. I’d like to briefly dive a little deeper into a few of these, with specific examples of how Simple Western is making amazing research possible. Targeted protein degradation harnesses the cell’s own machinery to degrade proteins that previously couldn’t be targeted, called ‘undruggable’ proteins. Compounds called degraders recruit an E3 ligase to target specific proteins for degradation. Western blotting is commonly used to measure degradation, but suffers from the common
Western blot challenges we are all familiar with. Simple Western, on the otherhand provides amazing dose-response curves or degrader curves. Here’s the setup for the data I’ll show you – a company called Aurelia Biosciences used a CDK9 PROTAC, which is a type of degrader, from Bio-Techne to degrade CDK9, which is a cell-cycle protein kinase critical in regulating RNA Pol II. - They dosed
7 PROTAC concentrations and included a DMSO control. Shown in black in the Simple Western lane view is the CDK9 degradation, and actin was multiplexed as a loading control, shown in red, which was unaffected by the degrader. These data can be plotted, and a DC50 value of 11nM can easily be obtained. Simple Western offers throughput and quantitation to measure degradation, and is an ideal tool for this. Gene therapy, which harnesses the power of viruses, has exploded over the last few years. Today, there are hundreds of clinical trials
leveraging viruses to treat patients, but the challenge is….only a handful of these therapies have been FDA-approved. Researchers need better and faster tools to accelerate their pipelines to bring more therapies to market sooner. Simple Western
is a sensitive multi-attribute bioanalytical tool that conserves precious samples and can measure up to 6 quality attributes including viral titer, viral identity, capsid protein ratios, and Empty/Full ratio. Any of these attributes can be measured from only 3uL of any process step sample. The end product of gene therapy is often the expression of a protein that imparts new function to the cells and restoring the disease. Measuring the relative protein expression between different samples, or even lots of AAV, is a powerful use of Simple Western. In the first example,
we used Simple Western to look at the relative potency between two different AAVs. We compared transgene expression, shown in green, to the total protein of the cell as a loading control, shown in blue. From those data, we were able to perform parallel line analysis to calculate the potency in the middle panel. This measurement was highly reproducible over different days, resulting in potency CVs under 5%. Duchenne’s muscular dystrophy is a devastating diseased
resulting from the loss of the dystrophin protein. There are a few therapies now trying to target dystrophin. At ASGCT this year, Dr Hiren Patel of Regenyxbio presented on their Bioanalytical testing of dystrophin levels. Bioanalytical testing involves using a validated bioanalytical method for the quantitative evaluation of analyte(s) in the given biological matrix. This is critical for the successful conduct of nonclinical, biopharmaceutics, and clinical pharmacology studies. Dr Patel showed that they had successfully demonstration of parallelism indicated that the RGX-202 µDys calibration curve is appropriate for the relative quantitation of endogenous dystrophin. Cell therapy is an exciting approach, where
scientists are harnessing the power of the cell to fight cancer. Simple Western can profile signaling and differentiation states in diverse cell therapy applications, is sensitive, and is ideal for small samples. Immune cell therapy today is mostly achieved by using lentivirus to transform T cells into amazing tumor killers. A key to using lentivirus is understanding the titer of the lentivirus you are using. The capsid protein p24 is used to measure LVV titer and LVV titer is
commonly measured by ELISA. Our Simple Western p24 assay has low ng/mL p24 sensitivity but due to the size separation, also shows several p24 precursors that can be measured independently from the p24 protein and providing more accurate quantitation and characterization of the lentiviral sample. It’s important to characterize CAR-T cells for mechanism of action – to understand how they work. That can include studying CAR expression and trafficking and characterizing the signaling networks downstream of the CAR. The data in the middle panel shows activation of two pathways by stimulating the CD19 CAR. GADS and SLP76 are in the same pathway, directly beneath the CAR intracellular domains. These targets showed maximal phosphorylation at 4hrs,
whereas ERK activation was much faster and occurs through a second pathway. A quick last example for regenerative medicine – a type of cell therapy that uses stem cells. Stem cells, called iPSCs can be differentiated into new cell types like cardiomyocytes and skin cells and Simple Western can be used to track the differentiation, by measuring stemness markers, like Nanog and OCT3/4. Here Nanog expression, shown in red, was nearly undetectable in 2 of the 3 differentiated lines, and at low levels in the 3rd after allowing the cells to complete their differentiation.
OCT3/4 expression, shown in orange below, was more persistent in each differentiated lines, consistent with what’s been observed by others. To show how Simple Western offers throughput and sensitivity for rapid validation of target expression in iPSC knockouts during discovery – we targeted proteins in the HLA-1, HLA-2, and KAR ligand families labeled in blue arrows in the diagram and in table below in attempt to generate more universal iPSCs. Because 3 of these proteins are surface-exposed proteins, we compared knockout/knockdown by both flow cytometry and Simple Western shown on the top right. B2M was nearly undetectable in 10 clones and knocked down in 11. Fewer knockdowns were achieved for either CD155 or MICA. Targeting intracellular proteins by flow cytometry essentially a non-starter, so we couldn’t see C2TA or BAT3 by flow. However,
by Simple Western, several knockdowns and a few knockouts were observed. Overall, with Simple Western, we were able to rapidly screen 23 iPSC clones for the expression of 5 targets in about 15hrs across 5 runs. Another crucial application supported by Simple Western is vaccine development. Vaccines are biological drugs that prevent communicable diseases in both humans and animals and are developed like other therapeutics. Vaccine development starts with candidates screening – as shown in the first example by Merck, where they screened 219 antigens across 90 pulmonary TB patients by Simple Western in their effort to develop a tuberculosis vaccine. After identifying your lead candidate, the vaccine needs to be optimized for improved activity. - A great example from researchers
at the National Institute of Animal Health in Japan is shown in the middle, where they sought to improve an avian recombinant vaccine by the addition of untranslated regions. They measured both Hemagglutinin and Nucleocapsid expression by Simple Western, and showed maximal HA expression when the NP-UTR was introduced. - With a well-optimized vaccine, vaccine manufacturers work to develop robust processes to produce it. Here’s a great example, again from Merck,
where they used Simple Western for Ebola vaccine production optimization. In this work, summarized on the right, they compared the harvest approaches for 4 lots of the vaccine and compared levels of GP1, soluble GP1 and GP2/GP2 Delta. Clearly, the RVH samples in the last 4 lanes are lacking the higher GP1 species, demonstrating how harvest conditions can impact vaccine recovery. ___ A biomarker is a measurable substance in an organism whose presence is indicative of some phenomenon such as disease or infection. - Biomarkers can be measured by several techniques including ELISA and Western blot, but as Kelly talked about, Simple Western is an ideal combination of those two older technologies and offers specificity, reproducibility and quantitation for tissue biomarker detection in support of clinical trials. Several companies are using Simple Western to take their
lead compound through to the clinic. Regulus is currently in a Phase 1b clinical trial for ADPKD, a devastating kidney disease. They’ve just completed their 3rd cohort in the study for a total of 42 subjects across the 3 cohorts, across 8 time points. On Simple Western, 3 targets were
analyzed across all the samples. Shown below is a snapshot of some of those data from the Cohort 2 data, showing significant increases in both targets PC1 and PC2 at the 2mg/kg dose, shown in red. These targets serve as biomarkers, and informed Regulus on dosing for the 3rd cohort. Despite all the great data I just showed you, the experimental size you can run on a Jess/Wes/Abby is limited for quantitative assay designs that include standard curves, QC samples, blanks, duplicates and reference standards to 24 samples per run. - And if I’ve got one instrument,
it would take me 12 hrs to get 96 samples processed. - You could get 96 samples on 4 Jess in parallel, but this requires more bench space, and requires run to run comparisons, but that’s about to change. I’d like to now introduce Dr. Madhuri Ganta, Director of Product Management for the Simple Western product family, to tell you about the next leap in Simple Western technology. - Madhuri. Thanks Chris. The future of high throughput quantitative westerns is here - check it out! I am so excited to introduce Leo, the newest member of the Simple Western family. Four times
the throughput, one quarter the time, same great quantitative western results – the next leap in Simple Western Technology. Leo offers a simple way to maximize throughput for quantitative western results and will be available in 2025. Leo provides all the benefits of existing Simple Western platforms like Jess and Abby, fully automating protein separation and immunodetection. Leo works with small sample volumes to provide reproducible protein quantitation with high dynamic range and fast time to results. Leo goes even further by providing additional capabilities. Some of the key features of Leo include the ability to process up to 100 capillaries in a single run, with a ~3 hour turnaround time, the flexibility to run 25-100 capillaries per run and increased multiplexing capability, and Leo does all of this with efficient reagent usage. Leo will support chemiluminescence at launch and will be upgradeable to support fluorescence later.
Leo is built on proven and trusted Simple Western technology. Leo has 4 capillary cartridge holders, holds 4 peel and go pre-filled reagent plates and a new sample plate with room to accommodate 96 samples and 4 molecular weight ladders. Supporting Leo is a full set of reagents and consumables in new kit configurations and Compass for Simple Western software version 7.0. The capillary cartridges move back and forth between pre-filled reagent plates and sample plates to load samples and reagents into the capillaries, and perform protein separation, immobilization, and detection.
The Leo workflow will be familiar to those who currently use Simple Western. New to Simple Western – don’t worry – setting up a run on Leo is simple. Prepare and load your sample plate with samples, primary and secondary antibodies, and detection reagents. Insert capillary cartridges, pre-filled peel and go reagent plates that contain the separation and stacking matrices and required buffers, and your sample plate. Start run and walk away! Leo allows you to maximize throughput per run and process more samples in a day. Leo can process up to 100 capillaries in a single 3 hour run. This is 4 times the number of
capillaries per run with the same turn around time as Jess. Processing 96 samples on a Jess system requires 4 runs and 12 hours, whereas with Leo you can process 96 samples in just 3 hours. Processing 96 samples on a Peggy Sue or a Sally Sue takes about 14-15 hours, contrast that with just 3 hours, or 1/3rd the time on a Leo. Leo provides 4X the experimental size of Jess
for enhanced quantitation. Shown below are results from 4 Jess runs required to process 96 samples, vs results from a single Leo run. The Leo run lane view shows sample data from a single Leo run, with 96 samples of HeLa cell lysate for analysis of 4 targets and 4 lanes of molecular weight ladder.
ELISA is a powerful and commonly used analytical method for the quantitation of proteins. ELISAs are commonly run in 96 well plates, with standard curves to enable quantitation. Standard curves are usually generated using concentration and optical density. With 96 sample throughput, Leo can now match the throughput of ELISA and offer an alternative option for a quantitative immunoassay where ELISAs are not suitable. Standard curves, for use in quantitation can be generated using concentration and peak areas. - ELISA provides a cumulative signal from all proteins detected in a well, Simple Western separates proteins by size providing specificity and size resolution that can be crucial when working with complex lysates or tissue homogenates. The size resolution
allows Simple Western assays to distinguish between intact and degraded proteins, isoforms, post translational modifications and more. - ELISA requires well characterized antibody pairs. Simple Western requires only one antibody as proteins are immobilized to capillary surface, therefore capture antibodies are not needed. Simple Western assay development is therefore
much faster than custom ELISA development as screening of antibody pairs is not necessary. Leo offers flexibility and scalability by allowing the use of 25-100 capillaries per run. Specifying the number of cartridges to use per run is simple – simply check the number of cartridges that will be used in Compass for Simple Western software. The modular approach allows you to use only what you need without any wastage. For e.g. running one capillary cartridge requires only one reagent plate, 2 cartridges require 2, 3 require 3 and 4 cartridges requires 4 reagent plates. A single
sample plate can be used for up to 96 samples. Leo combines RePlex and resampling offering powerful multiplex options that allow you to get the most data from your precious samples. RePlex enables serial immunoprobing – 2 immunoassays or an immunoassay and total protein per cartridge.
By resampling, the same sample row up to 4 times, with the 4 cartridges available, Leo allows you to get data from up to 8 target proteins, or 4 target proteins and total protein. - Total protein detection happens in the same capillary on the same sample and offers more reliable protein quantification since it normalizes out experimental variation in protein loading. Each resampling is performed with a different cartridge eliminating concerns with antibody cross-reactivity. By directing all 4 cartridges to the same row of primary and secondary antibodies, you can save up to 50% on your primary and secondary antibodies. You can define your Simple Western assay in a single day on Leo. You can screen up to 48 antibodies against positive and negative controls to find antibodies that meets your needs.
To find the appropriate concentration of antibody to use in your Simple Western assay, we recommend running a 3*3 matrix, 3 sample concentrations, 3 antibody concentrations, no primary ab control and no lysate control. This requires 11 capillaries, which means you can optimize two antibodies in a single Jess run. With Leo you can optimize up to 8 antibodies in a single run! Leo enables you to get to your next milestone faster with faster method validation to support regulatory submissions. For e.g. an accuracy and precision study following FDA guidelines
for bioanalytical method validation would require 6 days and 12 runs to complete on a Jess. These same experiments can be completed in half the time with half the number of runs on Leo. Leo meets your needs through the full drug development process, offering one platform that can travel with you from discovery to development and beyond. Leo provides you the flexibility and multiplexing capability you may need in early stages of discovery and pre-clinical research, allowing you to quickly iterate on multiple assays, or conduct in-depth mechanism of action studies, with a small number of samples. When you are ready to move to validation, use Leo for the throughput, ease of use and software that enables 21 CFR Part 11 compliance. Avoid having to transfer assays between platforms from discovery to analytical validation which can be time consuming and costly.
Summarizing the advantages of Leo – maximize throughput and process more samples in a day, run larger experiments for analytical grade protein quantitation, design high specificity quantitative immunoassays in crude lysates, quantify protein isoforms and size variants. In Leo, you have a single platform that meets your needs from discovery, through analytical development and commercialization, offers you the confidence to meet regulatory guidelines for bioanalytical method validation and allows you to get to your next milestone faster! We hope you are as excited as we are about Leo, and we can’t wait to see all the incredible applications and research you create with this next generation simple western instrument. Thank you for watching!
2024-08-11 06:04
