Detecting Disulfide Bond Shuffling in Biologics Using Trypsin Platinum

Biologic therapeutics such as monoclonal antibodies and biosimilars are complex proteins that are susceptible to post-translational modifications (PTMs). These chemical modifications can affect the performance and activity of the biologic, potentially resulting in decreased potency and increased immunogenicity. Such modifications include glycosylation, deamidation, oxidation and disulfide bond shuffling. These PTMs can be signs of protein degradation, manufacturing issues or improper storage. Several of these modifications are well characterized, and methods exist for detecting them during biologic manufacture. However, disulfide shuffling is not particularly well characterized for biologics, and no methods exist to easily detect and quantify disulfide bond shuffling in biologics.

Disulfide bond shuffling occurs when the S-S linkage is not between a Cys and its normal partner
Disulfide bonds are important for protein conformation and function

Normally the cysteines in a protein will pair with a predictable or “normal” partner residue either within a polypeptide chain or between two polypeptide chains when they form disulfide bonds. These normal disulfide bonds are important for final protein conformation and stability. Indeed, disulfide bonds are considered an important quality indicator for biologics.

In a recently published study, Coghlan and colleagues designed a semi-automated method for characterizing disulfide bond shuffling on two IgG1 biologics: rituximab (originator drug Rituxan® and biosimilar Acellbia®) and bevacizumab (originator Avastin® and biosimilar Avegra®).

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World Art Day: Experiences of Fine Art That Disrupt and Drive Innovation

This metal sculpture adorns the parking garage that serves Promega Kornberg and Feynman buildings on the Madison, WI, USA campus.
This metal sculpture adorns the parking garage that serves Promega Kornberg and Feynman buildings on the Madison, WI, USA campus.

World Art Day embraces art as a means for nurturing creativity and innovation, gaining greater understanding of cultural experiences that are different from our own, and showcasing the contributions of art and artists to sustainable development. It is no accident that World Art Day is celebrated on the birthday of Leonardo da Vinci, the celebrated Italian polymath who used art to express emotions, articulate technological concepts well before their time, and understand the workings of human anatomy.

Takaski Mitachi, a moderator of the 2019 global conference panel on Innovation through Art: Leveraging Disruption for a Sustainable Ecosystem, said that “Art can add value so that we could drive innovation beyond logic and data.” In a sense, the fine arts are a way of expressing our observations about the world around us, similar to how an original scientific hypothesis attempts to explain phenomena we observe and want to test. While scientific investigation builds on data and logic to test a hypothesis, art gives us a different way of knowing our world that extends beyond just data and logic. When we explore the arts, we broaden our understanding and interpretation of the world and bring these new perspectives to our scientific and technological explorations. Art is a disruptor of staid ways of thinking about and approaching problems, and like many other disruptors in our world, it can drive innovation.

In an article in the MIT Technology Review, author Sarah Lewis discusses the relationship between the arts and scientific breakthroughs. She notes one study that found a disproportionately high number of Nobel Prize-winning scientists also pursue art, writing or music as serious avocations. When asked why achievement in art and science seem to go together, she replied: “What the arts allow us to do is develop the muscle required for discernment and also strengthen our sense of agency to determine for ourselves how we’re going to tackle a given problem…Ultimately it’s up to the person creating the work to determine what that path is, and that kind of agency is what’s required for innovation.”

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Using Structural Computation Models to Predict Productive PROTAC Ternary Complexes

With use and time things wear out. Tires get worn on a car, and you have the old tires removed, recycled, and replaced with new ones. Sometimes a part or piece of something isn’t made properly. For instance, if you are assembling a piece of furniture and you find a screw with no threads, you throw it out and get a screw that was made properly. The same thing holds true for cells. Components wear out (like tires) or get improperly made (a screw with no threads), or they simply have a limited lifetime so that they are available in the cell only when needed. These used and worn components need to be removed from the cell. One system that allows cells to recycle components and remove old or improperly functioning proteins is the Ubiquitin-Proteasome System (UPS).  The UPS system relies on a series of small peptide tags, ubiquitin, to mark a protein for degradation. Researchers are now harnessing the UPS to target aberrant proteins in diseased cells through PROteolysis TArgeting Chimeras or PROTACs. PROTACs hold promise as highly efficacious therapeutics that can be directed to eliminate only a single protein. To take full advantage of the power of PROTACs, researchers need to understand the molecular underpinnings that are responsible for successful protein degradation. Here we review a paper that seeks to develop a computer model for predicting whether PROTAC ternary complex formation leads to ubiquitination and successful degradation of a target protein.

Diagram of ubiquitination of a protein. ThePROTAC ternary complex is formed the E2/E3 complex, PROTAC and target protein are bound simultaneously
Proteins are targeted for degradation by the proteasome. A small chain of ubiquitin peptides (Ub) is added to available lysine residues of the target protein through the actions of three enzymes: E1, ubiquitin-activating enzyme; E2, ubiquitin-conjugating enzyme; and E3 ubiquitin ligase. After the addition of the Ub chain, the proteasome is recruited and the protein degraded.

Addressing the Intractable Target

Research to understand diseases including cancers, neurodegeneration, and auto-immune conditions has revealed that in many disease states, affected cells produce growth factors or enzymes that are constitutively active (“always on”). These proteins are targets for small molecule inhibitors that bind specific sites preventing the constitutive activity or signaling. More recently, biologics, or protein-based therapeutics, including monoclonal antibodies (mAb), have been developed that can bind and block inappropriate signaling pathways, especially those that allow cancer cells to escape immune system surveillance.

Unfortunately, up to 85% of targets have proven intractable to small molecule inhibitors, or they are not suitable for a biologics approach. Oftentimes, the target protein doesn’t have a great place to bind a small molecule, so even though inhibitors might exist they cannot bind well enough to be effective. Or, as in the case of many cancers, the diseased cell manages to overcome the effect of the inhibitor by overexpressing the target. Still other aberrant proteins associated with diseases haven’t gained function to cause a disease; they have instead, lost function, so designing an inhibitor of the protein is not a workable strategy.  Enter the PROTAC.

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Run to Remember: A Mouse-Model Study Investigating the Mechanism of Exercise-Induced Neuroprotection

Research in animal models shows physical exercise can induce changes in the brain. In humans, studies also revealed changes in brain physiology and function resulting from physical exercise, including increased hippocampal and cognitive performance (1). Several studies in mice and rats also demonstrated that exercise can improve learning and memory and decrease neuroinflammation in models of Alzheimer’s disease and other neurodegenerative pathologies (2); these benefits are tied to increased plasticity and decreased inflammation in the hippocampus in mice (2). If regular time pounding the pavement does improve brain function, what is the underlying molecular biology of exercise-induced neuroprotection? Can we identify the cellular pathways and components involved? Can we detect important components in blood plasma? And, is the benefit of these components transferrable between organisms? De Miguel and colleagues set out to answer these questions and describe their results in a recent study published in Nature.

A recent study investigates the underlying molecular mechanisms of exercise-induced neuroprotection in a mouse model.
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How to Commit to Global Responsibility with Local Accountability

This summer, Dr. Anette Leue, Director of Digital Marketing and PR Promega GmbH, represented Promega Corporation in Sustainability Day activities sponsored by Smart Lab Connects. Dr. Leue presented Promega Corporation’s corporate responsibility activities and joined a panel discussion about global responsibility with representatives from Eppendorf, Max Planck Sustainability Network, and NIUB Sustainability Consultants.

Dr. Anette Leue, pictured, talked about global responsibility as part of Sustainability Day.

As the Sustainability Day activities progressed, what became apparent is that calls for sustainable business growth are coming from all directions. Customers of life sciences companies are asking, “what are you doing to be a responsible company”? And, employees also are asking the same question of their employers. This interest sustainability and global responsibility by customers, employees and local communities is bringing into sharp focus the activities of companies to be good corporate citizens. Sustainability and global responsibility programs are no longer nice extras for life science companies, but rather are requirements for doing business.

“Sustainability is not a “nice to have”, but something that should be intrinsically implemented in the companies.”

Dr. Anette Leue
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The Stories in the Bones: DNA Forensic Analysis 20 Years after 9/11

September 11, 2001 is the day that will live in infamy for my generation. On that beautiful late summer day, I was at my desk working on the Fall issue of Neural Notes magazine when a colleague learned of the first plane hitting the World Trade Center. As the morning wore on, we learned quickly that it wasn’t just one plane, and it wasn’t just the World Trade Center.

Two beams of light recognized the site of the World Trade Center attack. Today DNA forensic analysis applies new technologies to bring closure to families of victims.

Information was sparse. The world wide web was incredibly slow, and social media wasn’t much of a thing—nothing more than a few listservs for the life sciences. Someone managed to find a TV with a rabbit-eared, foil-covered antenna, and we gathered in the cafeteria of Promega headquarters—our shock growing as more footage became available. At Promega, conversation immediately turned to how we could bring our DNA forensic analysis expertise to help and support the authorities with the identification of victims and cataloguing of reference samples.

Just as the internet and social media have evolved into faster and more powerful means of communication—no longer do we rely on TVs with antennas for breaking news—the technology that is used to identify victims of a tragedy from partial remains like bone fragments and teeth has also evolved to be faster and more powerful.

Teeth and Bones: Then and Now

“Bones tell me the story of a person’s life—how old they were, what their gender was, their ancestral background.”  Kathy Reichs

Many stories, both fact and fiction, start with a discovery of bones from a burial site or other scene. Bones can be recovered from harsh environments, having been exposed to extreme heat, time, acidic soils, swamps, chemicals, animal activities, water, or fires and explosions. These exposures degrade the sample and make recovering DNA from the cells deep within the bone matrix difficult.

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Science and Journalism – Opposites or Not So Much?

This blog was written in collaboration with our partners at Promega GmbH.

Scientists are comfortable speaking to people who know their field. Speaking to scientists outside of their field of expertise can become a little more challenging, and many find the greatest challenge of all is speaking to people who do not have a science background and are hearing about a scientific concept for the first time, such as journalists in the popular media. What can scientists and journalists do to make the most of the interface of science and journalism?

Digital image depicting the intersection of science and journalism.

The importance of the interface between science and journalism is increasingly visible with scientific topics appearing on the national news more frequently due to COVID-19, climate change, and diseases like cancer. So, where can journalists go to learn best practices for interviewing scientists and writing about scientific topics? Promega GmbH offers a platform in which scientists and journalists come together and learn from each other in a constructive exchange. In this workshop setting, scientists speak about a certain topic, and journalists from all kinds of backgrounds can ask questions. When the journalist authors an article about what they learned in that workshop, both sides benefit. The scientists’ work becomes visible, and society learns more about scientific research and discovery that can help all of us to better understand the world and contribute to a brighter future.

Here we describe several common themes that have emerged from these science journalism workshops that may help you the next time you find yourself trying to explain your research to someone unfamiliar with your field.

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HPK1 Identified as Emerging Immuno-oncology Drug Target

Antibody-based immune checkpoint inhibitors remain a major focus of immuno-oncology drug research and development efforts because of their recent success in providing long-term anti-tumor responses. However, the range of response of different tumor types to these drugs is hugely varied. Small molecule kinase inhibitors that block signaling pathways involved in regulation of tumor immunity at multiple points in the “cancer immunity cycle” may provide alternate, effective therapeutics. One kinase that may be a target for such small molecule inhibitors is Hematopoietic Progenitor Kinase 1 or HPK1; the potential of this kinase as a therapeutic target was reviewed by Sawasdikosol and Burakoff (1). HPK1, also known as MAP4K1, is a member of the MAP kinase protein kinase family that negatively regulates signal transduction in T-cells, B-cells and dendritic cells of the immune system.

Artist rendering of what target engagement might look like for kinases like HPK1.
NanoBRET™ Target Engagement Assay (artist rendering)
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The Work of Emmett Chappelle: Lighting Up the Search for Extraterrestrial Life

What do the workings of red blood cells, ensuring breathable air for astronauts, and scraping soil off NASA’s Viking spacecraft have in common? The sharp thinking of biochemist Emmett Chappelle.

Image of Emmett Chappelle working with other scientists.
Emmett Chappelle conducting research.

February is Black History Month in the US—a time to reflect on the contributions of African Americans in all fields and celebrate their accomplishments while recognizing the adversity they had to overcome in American society.

2021 also marks 30 years since the first firefly luciferase reporter vectors and detection reagents became available as products. There’s no better person to highlight this month than Emmett Chappelle, whose work with the luciferase reaction is still used for many applications today.

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Screen Media in the Time of COVID-19: Should You Be Reading this Blog?

Screen Media. Cell phones. Social media accounts. If you are a parent, you have probably discussed rules of engagement with your children about these things. All of our modern social media platforms are designed to keep us engaged with them by showing us the latest post, the next video or the people now online. Work emails give us notifications when something arrives in our Inbox. Business software platforms like Microsoft Teams send us notifications whenever someone comments in a conversation we have ever been part of. There are many siren signals pulling us toward our screens.

Enter COVID-19, the flu-like illness caused by the SARS-CoV-2 virus that has already claimed the lives of 210,000 people in the United States, and leaving countless others permanently affected by other long-term health consequences. Spread by aerosol, COVID-19 is most dangerous in places where lots of people congregate in a small area, particularly if they are talking to each other. Consequently, office buildings are empty as many of us work or go to school remotely.

Before COVID-19, if I had a day full of meetings at work, I was running from conference room to conference room, two miles, uphill, in the snow between buildings. Now, a day full of meetings means sitting in front of a computer monitor, trying to figure out how I will get any kind of break between calls. The average number of steps recorded by my pedometer has decreased markedly since March when our remote work started.

Technology has been an incredible blessing during this pandemic—allowing us to continue to work and stay connected with friends and family. Technology is the only way that some people can connect with loved ones in long-term care facilities. It allows students to continue learning through remote classrooms and chats.

But what has been the effect of the increased time spent on screens during this pandemic?

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