Advance Your Bioprocess Workflows with Simple Western Capillary Immunoassays

Advanced Analytical Solutions from Start to Finish

The Simple Western™ family is made up of automated, capillary-based immunoassay platforms that combine the power of CE-SDS or cIEF with the sensitivity of immunodetection, enabling size- and charge-based screening of complex sample types.

• Separate and analyze proteins by either immunoassay or total protein content, from 2 kDa to 440 kDa or from a pI of 3 to 10.
• Analyze crude or purified samples for proteins of interest or biocontaminants.
• Quantitate expression levels, isoform distribution, and fragmentation in a gel- free, blot-free format.
• Choose from five instruments of differing throughput and separation mode options.

Bioprocess Contaminant Detection: Identification and Quantification of Process-Related Impurities

The identification and quantitation of bioprocess-related impurities during biotherapeutic development is critical to demonstrating the quality, efficacy and safety of the therapeutic agent. These impurities are typically residuals originating from the production cell line, the reagents and components used throughout cell culture/maintenance or during downstream bioprocess. Residual proteins that go undetected can be recognized as foreign antigens in vivo, triggering a potentially fatal immune response. Additionally, traces of process-specific reagents or components may alter the product stability and reduce therapeutic efficacy. Therefore, the accurate detection of impurities is imperative but complicated, given the range of potential impurity sources or types, their presence in trace amounts and the complex matrix of the sample in which they exist.

In this application note, we focus on the accurate detection of four candidate contaminants that may be present during various stages of the therapeutic protein and vaccine development bioprocess: host cell protein (HCP), Protein A, green fluorescence protein (GFP) and bovine serum albumin (BSA) impurities. Current methods for assessing the concentration of such residuals, like enzyme-linked immunosorbent assays (ELISAs), flow cytometry and traditional Western blotting are labor-intensive and prone to error. These traditional methods can also be misleading due to a lack of specificity and flexibility required to obtain a comprehensive contaminant profile and may exclude relevant critical information like Size-based results, oligomerization state and low limit of detection. We’ll show you how highly sensitive and analytical Simple Western immunoassays deliver where others come up short.

A Sensitive and Reproducible Multi-Attribute Tool for AAV Capsid Content Characterization that Scales with AAV Manufacturing Workflows

Adeno-associated viruses (AAVs) are frequently used as viral vectors in gene therapies to address human diseases, with over 200 studies around the world conducting active clinical Phase 1-3 trials. As gene delivery systems, AAVs include a gene of interest encoded in plasmid DNA that can be up to 5kb in length. AAVs can exist as a heterogeneous population, giving a final sample that is, for example, 30% full (including the desired plasmid DNA) and 70% empty or partially empty (devoid of the desired plasmid DNA). These empty or partially empty AAV particles can impact viral vector potency and immunogenicity and thus are unwanted byproducts of the AAV manufacturing bioprocess. Traditional analytical tools such as transmission electron microscopy (TEM), analytical ultracentrifugation (AUC), and ion-exchange chromatography (IEX) can be used to characterize capsid content but are complex, labor-intensive, and pose challenges in data reproducibility, throughput, and scalability. Therefore, there exists a need for better methods and systems for the efficient and sensitive quantification of Empty/Full status of AAV samples to be used for gene delivery.

In this Application Note, we developed a novel method for quantifying the DNA content of AAV particles with Simple Western, a next-generation biomolecular analytical tool that seamlessly combines capillary electrophoresis and immunodetection with conventional Western blot antibodies. Here, the Simple Western method automatically separates AAV samples by Size or Charge followed by specific and sensitive detection using anti-DNA and anti-VP1/2/3 antibodies directly in the capillary for quantitative and reproducible measurement of these central AAV components. For each antibody, we identify the range in which the signal intensity is linearly related to the amount of sample loaded, resulting in a rapid and sensitive assay for accurately quantifying the ratio of % full AAVs to total AAVs in a sample (also known as the Content Ratio).

Concentrating on AAV Impurities with Ultrasensitive Total Protein Detection on Simple Western

Impurities in protein products can be dangerous and impact efficacy. For example, protein impurities in final drug products could lead to undesirable immune responses in patients, so detecting total protein is critical for revealing impurities in preparative protein production. Traditional methods for total protein detection rely on SDS-PAGE with dyes like Coomassie Blue, SYPRO Ruby, or silver stain. However, SDS-PAGE requires large sample volumes, a lot of hands-on time, and it is poorly reproducible. Also, the use of staining dyes often comes with a lot of waste and can require specialized imaging equipment to which not every researcher has access.

Conversely, Simple Western assays on instruments like Jess offer fully automated protein separation and quantification with small sample volumes, and sensitive chemiluminescent-based immunodetection and total protein detection. While immunoassays on Simple Western allow target-specific detection, the Total Protein Detection Module allows for all proteins to be labeled and detected, which is ideal for monitoring impurities. Here, we show that total protein detection with Simple Western can be even more sensitive by using 5 times more concentrated biotin labeling reagent, resulting in protein detection that surpasses the sensitivity of protein stains like SYPRO Ruby. While SYPRO Ruby requires at least 1 ng of protein for reliable detection, Simple Western can reliably detect as little as 150 pg. This SYPRO Ruby-beating sensitivity improvement makes Simple Western well suited for the analysis of precious samples such as AAV samples used in Cell & Gene Therapy workflows.

Identity and purity are among the critical quality attributes (CQAs) that must be monitored during AAV manufacturing. While ELISA can provide identity information, it cannot provide purity information in the same assay. In this regard, ELISA is a single-attribute method. By contrast, Simple Western is a multi-attribute method because it can deliver both identity and purity in the same assay. For example, identity can be achieved with antibodies specific to VP1/VP2/VP3 capsid proteins, while purity may be achieved with total protein detection, which now rivals the sensitivity of the best gel staining techniques. In this Application Note, we show how to achieve ultrasensitive total protein detection using the 5X biotin labeling reagent with a focus on AAV analysis.

Shining New Light on Pharmacokinetic Assays with Simple Western

An important aspect of therapeutic drug characterization is pharmacokinetic (PK) studies, which are used to understand the fate of an administered drug. Typically, PK studies are conducted to look at drug absorption, bioavailability, distribution, metabolism, and excretion. These studies historically have utilized ELISA-based methods to quantify the remaining levels of drug in a patient post-administration. However, given that biological drugs are quite complicated, and are known to change in response to stress, a one-dimensional test like ELISA may only provide limited information.

Instead, Simple Western is a capillary-based immunoassay that separates proteins based on Charge (i.e. isoelectric point) or Size (i.e. molecular weight) followed seamlessly by immunodetection directly in the capillary (think of an ELISA, but with protein separation by Charge or Size). Because Simple Western assays rely on the specificity of antibodies recognizing separated proteins, followed by highly sensitive (low picogram) chemiluminescence detection, Simple Western is capable of protein analysis in highly complex samples like human serum. Further, Simple Western can process up to 96 samples in one overnight run, enabling relative quantitation of protein expression across samples or absolute quantitation of protein expression by incorporating standard curves alongside sample panels. This combination of highly specific measurements in complex samples and sample throughput make Simple Western an ideal tool for PK studies.

In this Application Note, we use Simple Western Charge and Size assays to evaluate the PK properties of adalimumab and two adalimumab biosimilars over the course of nine weeks in human serum. Adalimumab reduces inflammatory responses by inhibiting the binding of TNFα to its receptor. It was the bestselling biopharmaceutical until its patent expired in 2016, reaching $16 billion in global sales annually. Several biosimilars have since been approved in the USA and Europe. Simple Western Size can detect changes like antibody aggregates and antibody fragmentation. Simple Western Charge is a powerful tool for charge heterogeneity characterization including protein post translational modifications screening.

From Start to Finish, Bio-Techne Solutions for Bioprocessing

As the pace of biotherapeutic approvals increases, so does the pressure on makers of new biologics to complete their development bioprocess faster and more efficiently. The success of the drug development bioprocess is directly proportional to the utilization of accurate, sensitive and fast methods for selection, identification, quantitation, purity and other analyses of the drug components and the final drug product. The common adage, “time is money” has never been more applicable than to today’s biopharmaceutical industry and its focus on improving bioprocessing technologies to reduce costs and increase efficiencies. By offering solutions that address the key stages of bioprocessing—from cell line development to process optimization and formulation to final product characterization—Bio-Techne and its family of brands streamlines measurement and quantification of the complex interactions between a drug product and its manufacturing process. Let us show you how our technologies and systems like Simple Western increase productivity and reveal new insights to accelerate the time to market for new biologics.

Learning Objectives:
1. Gain more insight into the current state of the bioprocessing industry
2. Learn how researchers can overcome even the most difficult bioprocessing challenges with Bio-Techne’s products and services