Written by: Vanessa Yoon Calvelo, PhD Published: June 6, 2023 Updated: July 7, 2024 Contents Introduction Early Discovery and Development of Therapeutic Antibodies Target Identification and Validation Antibody Discovery and Expression Lead Characterization and Selection Lead Engineering and Optimization Candidate Selection and Downstream Characterization Introduction [...]
Written by: Vanessa Yoon Calvelo, PhD Published: April 24, 2023 Contents Introduction Functions of Therapeutic Antibodies Functional Assays for Therapeutic Antibodies Functional Characterization in the Therapeutic Antibody Discovery Process Introduction Monoclonal antibodies (mAbs) and related biological products often present as ideal therapeutics largely due to: Their [...]
Written by: Vanessa Yoon Calvelo, PhD Published: April 14, 2023 Contents Introduction Immunochemical Properties of Therapeutic Antibodies Rapid Immunochemical Characterization of Antibodies Introduction Therapeutic antibodies, predominantly monoclonal antibodies (mAbs), are a rapidly expanding class of drugs with over 100 mAb-based biologics now approved for the treatment [...]
The great debate on the use of in vivo versus in vitro sources and strategies for antibody discovery and generation continues to thrive among antibody research groups. On one side of the debate is the argument for non-animal-derived antibodies due to the technical advancements of current in vitro technologies, and the moral obligation to reduce animal usage. On the other side of the debate is the counterargument for animal-derived antibodies due to their better performance in affinity, specificity, and reduced immunogenicity risk.
Webinar Highlights You will learn: Introduction to the structure and function of the tuberous sclerosis protein complex (pTSC) A novel strategy for isolating and purifying protein complexes from native sources using recombinantly produced Fabs How Next Generation Protein Sequencing (NGPS) works Applications of NGPS to aid design and engineering of recombinant antibody [...]
Characterization of proteins and protein complexes is a major keystone of structural biology. As our understanding of cellular processes continues to evolve from simple pathways to complicated networks, our need for advanced analytical methods is quite apparent. Mass spectrometry (MS)-based structural approaches can be used to study protein conformational changes and dynamics, protein motion/flexibility, ligand-protein binding, and protein-protein interfaces.
Written by: Vanessa Yoon Calvelo, PhD Published: July 11, 2022 Contents What is Gene Therapy? What are Adeno-Associated Viruses? Engineering of AAVs for Gene Therapy Engineering AAVs for Improved Transduction Engineering AAVs for Improved Immunogenicity De Novo Protein Sequencing Applications in AAV Characterization and Development What is Gene [...]
As nearly all individuals have pre-existing immunity to influenza viruses, influenza-specific memory B cells will likely be recalled upon COBRA HA vaccination. By comparing the antibody response towards specific wild-type influenza strains and COBRA antigens, we can begin to understand the potential for COBRA-based vaccines to be used in the clinic.
Written by: Vanessa Yoon Calvelo, PhD Published: June 13, 2022 Contents What is CAR-T Cell Therapy? CAR Structure and Function CAR-T Cell Development Engineering Strategies for CAR-T Cells De Novo Protein Sequencing Applications in CAR-T Cell Development What is CAR-T Cell Therapy? The infusion of T cells [...]
Written by: Vanessa Yoon Calvelo, PhD Updated: January 19, 2023 (Published: June 2, 2022) Contents What are post-translational modifications (PTMs)? Impact of PTMs Types of PTMs PTMs increase microheterogeneity of antibodies PTM Analysis by LC-MS The Importance of Post-Translational Modifications (PTMs) Post-translational modifications (PTMs) are processing [...]
Written by: Yuning Wang, PhD Updated: January 26, 2023 (Published: June 3, 2022) Contents Introduction The Four Levels of Protein Structure How are Protein Structures Studied? Introduction Structural information provides a great deal of understanding of how a protein works, which can allow us to [...]
The most straightforward solution would be to determine sequences of the dominating antibody forms in a polyclonal mixture to enable recombinant antibody generation and ensure reproducibility. This was recently made possible by the development of polyclonal antibody sequencing technology, which will be reviewed in this article.
The origin of hydrogen-deuterium exchange (HDX) dates back to the 1950s, when protein scientist Linderstrøm-Lang created a method involving protein deuteration to distinguish amide hydrogens participating in secondary structures. Today, scientists frequently rely on HDX data to investigate protein structure, conformational dynamics, and protein-ligand interaction.
Known, high-performing and well used antibodies against useful targets on CAR-T cells can be examined for mechanism of action using proteomics and mass spectrometry. Knowledge of the antibody sequences via Next Generation Protein Sequencing (NGPS) can be useful for humanizing or otherwise engineering constructs. Rapid Epitope mapping by HDX can be useful for both epitope and paratope engineering strategies.
The acronym “CDR” stands for complementarity determining region, a variable sequence of amino acids that folds into loops capable of binding to an antigenic amino acid sequence, also known as an epitope
To develop robust mAb biologics, it is vital to fully characterize the protein, including its primary sequence, mutations, and important post-translational modifications
Research Challenges in Veterinary Medicine Since 2006, the One Health Initiative (OHI)’s goal has been to demonstrate the inextricable link between humans, animals, and the environment.
To develop robust mAb biologics, it is vital to fully characterize the protein, including its primary sequence, mutations, and important post-translational modifications
Written by: Yuning Wang, PhD Updated: January 18, 2023 (Published: January 21, 2022) Contents Discovery of Camelid Antibodies What are Camelid Antibodies? Structure of Camelid Antibodies and Nanobodies Advantages of Camelid Antibodies and Nanobodies Camelid Antibodies and Nanobodies for Therapeutic and Research Applications How are Camelid Antibodies [...]
Non-invasive, sensitive, accurate, and easy-to-manufacture IVDs will be critical to detect and monitor diseases where misfolded proteins play a significant role such as neurological illnesses like Alzheimer’s, cancers like multiple myeloma, among others.
There have been cases where researchers have trusted a kit, and they see a positive reaction only to, unfortunately, realize that the kit was actually detecting another protein because the kit’s antibody lot changed. Such was the case for the University of Toronto’s Diamandis team who spent half a million dollars and nearly two years due to an unreliable antibody. A certificate proving that the protein sequence of an antibody remains unchanged would have easily avoided the aforementioned mishaps.
Polyclonal antibodies are popular research reagents for their high sensitivity and robust cross-platform performance. But few companies consider them viable for therapeutic applications as they are almost impossible to characterize. Additionally, they suffer from a lack of reproducibility and limited supply. Monoclonal antibodies (mAbs) can be reliably characterized and produced for therapeutic applications, but are more costly to discover and develop. Rapid Novor’s REpAb technology can overcome these limitations by capturing the sequences of the most abundant IgG in a pAb and enabling indefinite antibody production. Here we report the first successful conversion of a goat polyclonal antibody into a cocktail of recombinant mAbs using only the pAb protein sample.
Recombinant antibodies are artificially synthesized antibodies. Recombinant antibodies are generated from expression systems (e.g., E.coli, yeast, mammalian cell lines) via transfection with two separate plasmids encoding the amino acid sequences for the light and heavy chains, respectively. In order to recombinantly produce mAbs, the amino acid sequence of the light and heavy chains must be known. There are many ways to obtain the sequence of an antibody.
Over the past several years Rapid Novor has been developing the world's best antibody protein sequencing platform, with over 2700 monoclonal antibodies and proteins sequenced. In 2020, they unveiled their most advanced technology to date- REpAb® polyclonal antibody sequencing. The platform combines the world's best protein sequencing technology and NGS to comprehensively mine the antigen specific antibody repertoire present in rabbit and human patient samples. By leveraging the platform, teams can build robust antibody assays and therapeutic leads derived from patient's blood.
Since 2006, the One Health Initiative (OHI)’s goal has been to demonstrate the inextricable link between humans, animals, and the environment. Certainly, the current global pandemic is a great testament to the ties between climate change, humans, and animals that OHI has been working to highlight. The rise of other zoonotic diseases (e.g., Hendra, and Nipah viruses) not only directly affect humans through disease transmission but may also result in deep impacts to the food supply
In this study, we conducted a large-scale statistical analysis of protein sequencing data from samples digested with multiple proteases to understand the impact of using different combinations of proteases to improve the depth of sequence coverage in the application of de novo protein sequencing.
Peptide mapping is a widely used analytical technique to verify the primary structure (amino acid sequence) and characterize the chemical modifications of a protein. It analyzes peptides generated from the digestion of an isolated protein, or a protein mixture
Anti-drug antibody (ADA) assays are critical to assess the clinical efficacy and safety of a biological drug and rely on control reagents that mimic the ADA response to the biological drug being tested. These positive controls typically consist of animal-derived pooled polyclonal antibodies or human monoclonal antibody reference panels against the target protein drug.
Amino acids are small organic molecules that make up peptides and proteins. All living organisms share the same set of amino acids. Amino acids come together in different orders (sequences) to form proteins. As such, each type of protein has a different three-dimensional structure and biological activity.
Our team, along with four other industry panelists, discuss ways to safeguard their research through recombinant antibodies, cell culturing procedures, antibody protein sequences, and reference identifiers.
Protein sequencing is a method that typically utilizes mass spectrometry (MS) to determine the amino acid code of a protein1. Prior to the development of mass spectrometry, Edman degradation, a method involving the stepwise degradation of peptides to derive the order of amino acids, was the mainstream approach. Nowadays, mass spectrometry is favored due to its ease of use and high throughput capabilities, though Edman degradation is still employed for specific applications in which the technique is well suited.
Amino acids (aa)—the building blocks of proteins—are simple molecules characterized by a variable R group flanked either side by an amino group and a carboxyl group. With around 20 different commonly found amino acids, each one can bond with another to produce chains that can be classified as peptides (typically below 50 aa) and proteins (sequences above 50 aa)—molecules ubiquitous to every known organism.
Amino acid sequencing is commonly performed using Edman degradation or mass spectrometry (MS). While mass spectrometry is favoured for its high throughput capabilities and ease of use, both techniques possess their own features and limitations. This article summarizes some of the key pain points inherent in the two methodologies when determining the amino acid sequence.
Antibodies are used in a variety of ways in academia and industry, from tools to therapeutics. Because antibodies are produced using live processes, which are naturally error-prone, validation is required from time to time. Furthermore, to develop biological therapeutics, the protein sequence must be confirmed as part of the regulatory process.
DNA sequencing is the process of determining the precise order of four nucleotides bases—adenine (A), guanine (G), cytosine (C), and thymine (T)—that make up the DNA molecule. From Sanger sequencing to next-generation sequencing (NGS), DNA sequencing’s accessibility and ease of use make it one of the most widely used technologies in life sciences.
As proteins are assembled, they fold into different structural orders: from primary to quaternary. The exact sequence of the primary structure (the amino acid sequence) will dictate how a protein will fold and function. The importance of the primary structure has been noted in several studies, where changes in the original amino acid sequence have resulted in affinity problems, binding disruption, reduced half-life, and higher aggregation odds.
Of interest to human and veterinary drug development scientists, biologics and biosimilars development scientists, scientists performing pre-clinical assay development, immunotherapy researchers, oncolytic therapy development scientists, gene therapy development scientists, gene therapy, and oncolytic therapy researchers, CAR-T, and CAR-NK development scientists
Proteins are composed of peptide chains, which in turn are made up of a string or linear sequence of amino acids. Every amino acid has a basic structure containing an amino (-NH2) group and a carboxylic (-COOH) group (Figure 1B). To form a peptide, amino acids link to each other via a peptide bond, which involves the reaction between the carboxylic group of one amino acid and the amine group of another amino acid.
Protein mass spectrometry refers to the use of mass spectrometry in studying and characterizing proteins, including their quantification, profiling, interaction mapping, and identification of their post-translational modifications. Protein mass spectrometry may also be referred to as mass spectrometry-based proteomics. Mass spectrometry-based proteomics comprises three approaches: top-down, middle-down, and bottom-up proteomics.
The protein sequence is key to understanding the function of a protein target and is critical to therapeutic and diagnostic development. This is particularly important for antibodies whose code diversity and glycosylation impact both function, and stability.
This webinar offers insight into how DNA and protein sequencing compare to each other.
Antibody sequences are critical for antibody engineering and protein characterization in therapeutic development. For antibody reagent users, knowing the sequences allows them to perform sequence analysis/alignment to identify binding and cross-reactivity so they can conduct rational experiment design.
Because they share the same mass, isoleucine and leucine are known as isobaric amino acids. Conventional mass spectrometry-based proteomics cannot be easily used to distinguish between isoleucine and leucine.
Amino acid sequencing is the process of identifying the arrangement of amino acids in proteins and peptides. Numerous distinct amino acids have been discovered in nature but all proteins in the human body are comprised of just twenty different types.
Bispecific therapeutics are monoclonal antibodies that carry a specific antigen-binding capability on each arm. Bispecifics are thus capable of having two specificities that can either double the binding affinity of the antibody toward the same antigen (increased avidity), or can now bind to two targets. Bispecifics are most often described as two types: trispecifics and bispecific T-cell engaged antibodies (BiTE).
Written by: María Gerpe, PhD Updated: January 27, 2023 (Published: June 25, 2021) Contents Introduction Types of Antibody Structures Functions of Antibodies Introduction Antibodies or immunoglobulins (Ig) are Y-shaped glycoproteins produced by the adaptive immune system in response to antigens - substances or molecules the immune [...]
Antibodies are integral to life sciences research and therapeutic and diagnostics discovery and development. However, they are inherently prone to variability.
Monoclonal antibodies (mAbs) are widely used in research, diagnosis, and pharmaceutical purposes. Lately, the relatively lower quality of research-purpose mAbs is a point of concern within the research community.
Written by María Gerpe, PhD June 18, 2021 Introduction Research publications represent an additional source of validation proof for commercially available antibodies. As such, academic and industry scientists often also rely on publication references to decide which commercial antibody to purchase. Several independent efforts exist to compile such information. For instance, [...]
Over the past 5 years Rapid Novor has perfected monoclonal antibody sequencing, and is now sequencing mAbs from polyclonal mixtures using REpAb®. After successfully launching their proteogenomics based sequencing technology to deconvolute the immune response, the team has further evolved the technology and has derived the most abundant mAb sequences directly from rabbit blood using only proteomics. The talk will surround the development, progress and use cases for REpAb®.
Other than a paucity in widespread use, such efforts still do not address the main issue: the uniqueness of antibodies. Unlike currency, antibodies are non-fungible. To manufacture a replica of a specific antibody, we must first know its sequence. Sometimes, scientists may obtain the sequence through nucleotide sequencing means.
Over the past several years Rapid Novor has been developing the world’s best antibody protein sequencing platform, sequencing over 2700 monoclonal antibodies and proteins. In 2020, they unveiled their most advanced technology to date - REpAb® polyclonal antibody sequencing. The platform combines the world’s best protein sequencing technology and NGS to comprehensively mine the antigen-specific antibody repertoire present in rabbit and human patient samples. By leveraging the platform, teams can build robust antibody assays and therapeutic leads derived from patients’ blood.
The DNA sequences of antibodies are highly diverse due to the V-(D)-J recombination and hypersomatic mutations. As such, relying on homology-based searches to sequence novel antibodies can introduce bias to sequences obtained from proteomics approaches.
Mouse monoclonal antibodies (mAbs) are highly attractive for manipulation for therapeutic applications as their manufacturing is relatively easy and well-established compared to mAbs derived from larger animal models. However, they also pose several challenges which limit their use as therapeutic agents.
In-vitro diagnostics (IVDs) are one of the most commonly used tools to diagnose conditions and guide treatment decisions and are often considered the “silent champion” of healthcare. They work by detecting the absence or presence of particular markers or by measuring the concentration of analytes or specific substances.
When it comes to polyclonal antibodies, how they are discovered can be just as important as how they are reproduced. In our talk originally presented at Antibody Engineering & Therapeutics Digital 2021, we highlighted the latest technology that’s capable of capturing the most abundant and high-affinity monoclonal antibodies directly from a poly mixture.
At PepTalk 2021, we discussed the importance of antibody standardization and explained why it’s crucial for the longevity of your research. You can listen to the full on-demand video here.
To develop robust mAb biologics, it is vital to fully characterize the protein, including its primary sequence, mutations, and important post-translational modifications
If you could have guaranteed stability, certainty, and reproducibility for your research, would you be interested? Imagine this, if you’re 2 years into your project and your freezer died along with all of your important cell lines, what would you do? This is just one of the situations covered in this webinar, along with many other solutions researchers have begun to implement to safeguard their efforts. Whether you’re looking to proceed with stability and certainty or you’re looking for an immediate solution for your current reproducibility challenges, protein sequencing may be the answer.
Our team has perfected the art of monoclonal antibody sequencing and is now ready to demonstrate our ability to sequence mAbs from polyclonal mixtures. In this talk, Anthony will walk through our new polyclonal sequencing platform that uses both proteomics and genomics to sequence the most abundant antibodies found in polyclonal sera.
In this on-demand webinar, our scientific sales executive Jennifer, will briefly cover the fundamentals of protein sequencing, how researchers have benefited from implementing protein sequencing into their pipelines, and discuss how Rapid Novor is able to routinely and robustly achieve 100% accuracy and 100% coverage for both monoclonal and oligoclonal antibodies.
Nowadays, DNA sequencing is so popular that it is easy to forget that the first sequenced biological material was protein – insulin, by Sanger. Sanger, and another researcher, Edman, separately pioneered protein sequencing.
In this on-demand webinar, we discuss why it is important to characterize antibodies based on their physical properties not just by what they bind, and how you can easily do the former via mass spectrometry-based protein sequencing.
One of the most important pieces of information researchers need to know during early stage antibody drug research and development is the sequence information of the antibody protein. With the advancement of mass spectrometry instrumentation and technologies, it is helpful, and sometimes critical, to conduct sequence analysis using mass spectrometry experiments.
Recombinant Monoclonal Antibodies (rAbs) are highly reproducible, customizable and pure alternatives to the traditional antibodies produced by hybridomas. Get the antibody protein sequence, either by DNA sequencing or the de novo protein sequencing technology, you can rest assured that you can have the exact antibody made recombinantly anytime in the future.