Mass Spectrometry Application: Antibody Quantitation for Preclinical PK studies

Isoform_Antibodies_LinkedInTherapeutic monoclonal antibodies (mAbs) represent the majority of therapeutics biologics now on the market, with more than 20 mAbs approved as drugs (1–3). During preclinical development of therapeutic antibodies, multiple variants of each antibody are assessed for pharmacokinetic (PK) characteristics across model systems such as rodents, beagles and  primates. Ligand-binding assays (LBA) are the standard technology used to perform the PK studies for mAb candidates (4). Ligand-binding assays (LBAs) are methods used  to detect and measure a macromolecular interaction between a ligand and a binding molecule. In LBAs, a therapeutic monoclonal antibody is considered to be the ligand, or analyte of interest, while the binding molecule is usually a target protein.

LBAs have certain well-documented limitations (5). Specific assay reagents are often not available early in a program. Interferences from endogenous proteins, antidrug antibodies, and soluble target ligands are potential complicating factors.

Liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS)-based methods represent a viable and complementary addition to LBA for mAb quantification in biological matrixes. LC–MS/MS provides specificity, sensitivity, and multiplexing capability.

A recent reference (6) illustrates an automated method to perform LC–MS/MS-based quantitation, with IgG1 conserved peptides, a heavy isotope labeled mAb internal standard,and anti-human Fc enrichment. The method was applied to the pharmacokinetic study of a mAb dosed in cynomolgus monkey, and the results were compared with the immunoassay data. The interesting finding of the difference between ELISA and LC–MRM-MS data indicated that those two methods can provide complementary information regarding the drug’s PK profile.

Literature Cited

  1. Mao, T. et al. (2013) Top-Down Structural Analysis of an Intact Monoclonal Antibody by Electron Capture Dissociation-Fourier Transform Ion Cyclotron Resonance-Mass Spectrometry. Anal.Chem. 85, 4239–46.
  2. Weiner, L. M. et al. (2010) Monoclonal antibodies: versatile platforms for cancer immunotherapy. Nat. Rev. Immunol. 10, 317–27.
  3. Nelson, A. et al. (2010) Development trends for human monoclonal antibody therapeutics. Nat. Rev. Drug Discovery. 9, 767–74.
  4. DeSilva, B. et al. (2003) Recommendations for the Bioanalytical Method Validation of Ligand-Binding Assays to Support Pharmacokinetic Assessments of MacromoleculesPharm. Res. 20, 1885–00.
  5. Ezan, E.et al. (2009) Critical comparison of MS and immunoassays for the bioanalysis of therapeutic antibodiesBioanalysis 1, 1375–88.
  6. Zhang, Q. et al. (2014) Generic Automated Method for Liquid Chromatography–Multiple Reaction Monitoring Mass Spectrometry Based Monoclonal Antibody Quantitation for Preclinical Pharmacokinetic Studies. Anal.Chem. 86, 8776–84.

Tips for Successful Dual-Reporter Assays

Dual-Reporter-AssayRecently, one of my fellow bloggers described some of the advantages of using dual-reporter assays (including our Dual-Luciferase®, Dual-Glo® Luciferase and our new NanoDLR™ assay debuting soon). These assays are relatively easy to understand in principle. Use a primary and secondary reporter vector transiently transfected into your favorite mammalian cell line. The primary reporter is commonly used as a marker for a gene, promoter, or response element of interest. The secondary reporter drives a steady level of expression of a different marker. We can use that second marker to normalize the changes in expression of the primary under the assumption that the secondary marker is unaffected by what is being experimentally manipulated.

While there are many advantages to dual-reporter assays, they require careful planning to avoid common pitfalls. Here’s what you can do to avoid repeating some of the common mistakes we see with new users: Continue reading “Tips for Successful Dual-Reporter Assays”

Reflections on Summer 2014 Courses

Group Photo from the 2014 Core Techniques in Protein and Genetic Engineering Course Held at the BTC Institute July 14-18. Photo credit: BTCI
Group Photo from the 2014 Core Techniques in Protein and Genetic Engineering Course Held at the BTC Institute July 14-18. Photo credit: BTCI
One of the things that I encourage all of the students I interact with in BTC Institute courses to do in order to boost retention and make meaning out of the activities that we do in class is to reflect on their experiences. Reflection is one way to connect new knowledge to past experience and get it to really stick in the brain, among other things.

Taking my own advice to heart, I use this space to ponder some interesting aspects of these experiences from my own perspective. This summer, I worked with 65 students and over 25 instructors to deliver four weeks of intensive instruction in molecular biology applied to a wide range of research areas. Continue reading “Reflections on Summer 2014 Courses”

Cell free application: Sumoylation characterization

Small Ubiquitin-like Modifier (or SUMO) proteins are a family of small proteins that are covalently attached to and detached from other proteins in cells to modify their function. SUMOylation is a post-translational modification involved in various cellular processes, such as nuclear-cytosolic transport,
transcriptional regulation, apoptosis, protein stability and response to stress.

In contrast to ubiquitin, SUMO is not used to tag proteins for degradation. Mature SUMO is produced when the last four amino acids of the C-terminus have been cleaved off to allow formation of an isopeptide bond between the C-terminal glycine residue of SUMO and an acceptor lysine on the target protein.

Cell free expression can be used to characterize sumoylation of proteins. Target proteins are expressed in a rabbit reticulocyte cell free system (supplemented with necessary addition components,). Proteins that have been modified can be analyzed by a shift in migration on polyacrylamide gels, when compared to control reactions.

The following references illustrate the use of cell free expression for this application.

Brandl, A. et al. (2012) Dynamically regulated sumoylation of HDAC2 controls p53 deacetylation and restricts apoptosis following genotoxic stress. J. Mol. Cell. Biol. (online only)

Janer, A. et al. (2010). SUMOylation attenuates the aggregation propensity and cellular toxicity of the polyglutamine expanded ataxin-7. Human. Mol. Gen. 19, 181—95.

Rytinki, M. et al. (2009) SUMOylation attenuates the function of PGC-1alpha. J. Biol. Chem. 284, 26184-93.

Klein, U. et al. (2009) RanBP2 and SENP3 function in a mitotic SUMO2/3 conjugation-deconjugation cycle on Borealin. Mol. Cell. Biol. 20, 410–18.

Seo, W. and Ziltener, H. (2009) CD43 processing and nuclear translocation of CD43 cytoplasmic tail are required for cell homeostasis. Blood, 114, 3567–77.

A New Method that Marks Proteins for Destruction

The ability to manipulate genes and proteins and observe the effects of specific changes is a foundational aspect of molecular biology. From the first site-directed mutagenesis systems to the development of knockout mice and RNA interference, technologies for making targeted changes to specific proteins to eliminate their expression or alter their function have made tremendous contributions to scientific discovery.

A recent paper highlights a novel application of HaloTag technology to enable the targeted destruction of specific HaloTag fusion proteins in vivo. The paper, published online in the July issue of Nature Chemical Biology, details a promising new method with application for validation of potential drug targets by specific in vivo inhibition, and for studying the function of specific genes in organogenesis or disease development. Continue reading “A New Method that Marks Proteins for Destruction”

The Benefits of Hindsight

One of the nice things about working in a biotechnology company is the opportunity to learn about new products as they are developed and to get exposed to scientific disciplines outside of my original area of expertise. Since I came to Promega I have had the opportunity to learn about a large number of products for widely differing research applications including cell biology, forensics, nucleic acid purification, and drug screening.

Over the years I have seen quite a few products that made me think “I wish I had that back when I was in the lab”. Even although there are a lot of “sexier” products around, the number 1 item that would have made my life in the lab better is still the 10-minute plasmid prep kit. In the labs I worked in we tried to save money and do our own minipreps, and it was tedious indeed. It turns out I am not alone in this sentiment, home-made plasmid minipreps came in at #9 on this list of “techniques we are most glad we don’t have to do any more” on BiteSize bio.

Home made minipreps. I may not have been the greenest-fingered scientist that ever lived but the failure rate of my home-made minipreps was pretty big, especially when I did many of them at once. Thank goodness for miniprep kits.

I can add a hearty Amen to that. Continue reading “The Benefits of Hindsight”

Use Words That You Understand

Getting What You Want from Your Science Writing Part VI

A friend of mine told me about an incident that happened during a speech crafting workshop for professionals. One of the members was given the task of selecting a word to introduce and define for the group. The other members of the group were supposed to incorporate that word into their conversation during the workshop. Continue reading “Use Words That You Understand”

Describing Life and Death in the Cell

4621CALife is complicated. So is death. And when the cells in your multiwell plate die after compound treatment, it’s not enough to know that they died. You need to know how they died: apoptosis or necrosis? Or, have you really just reduced viability, rather than induced death? Is the cytotoxicity you see dose-dependent? If you look earlier during drug treatment of your cells, do you see markers of apoptosis? If you wait longer, do you observe necrosis? If you reduce the dosage of your test compound, is it still cytotoxic? Continue reading “Describing Life and Death in the Cell”

Efficient Cloning and Expression of High Protein Yields Using KRX Cells

Escherichia coli remains the first choice of many researchers for producing recombinant protein for functional studies due to its ease of use, well established protocols, rapid cell growth and low cost of culturing. Researchers often need to clone using an E. coli host with good transformation characteristics first, then transfer the desired clone to the expression host. We have developed a new E. coli host KRX, that provides protein yields comparable to those of BL21(DE3) but with much higher transformation efficiencies. Continue reading “Efficient Cloning and Expression of High Protein Yields Using KRX Cells”

Representations of Science

As a storyteller, I notice that there are two stories to most life science research. The first is the story of process, and the second is that of results. Coming from a liberal arts background, the nuance of the second – how chemistry, concentration, base pairs and phosphorylated molecules interact to deliver a measurable result – often escapes me.

I can appreciate process: an idea, followed by a near-endless series of small tests to evaluate the viability of that idea. I can appreciate the sifting of signal from noise, and the dangerous seduction of a tangent, a curious detail, and the empty promise of a lead. Conceptually, these things make art and science close cousins. I can walk comfortably on a conceptual ground, but the hard grammar delivers a path beyond my training and knowledge.

To my eye, this research looks like nothing more than the transfer of clear liquids from tube-to-tube. Sometimes the application of heat is involved, and refrigeration is essential. Visually, the two most exciting things you’ll encounter are a slight color change in a microcentrifuge tube or a bar graph. At the cellular level, we may find a grand narrative of transformation , death, and recovery; at the molecular level we can witness attraction, separation, reconciliation. Yet capturing these events requires either a miracle or a mountain of costly resources. When we want to visually communicate the story of pure DNA , we are left with the story of pipetting liquids.

In developing video protocols for our products, I’ve attempted to keep that point in mind. There are things I cannot practically show, and what I can show needs to be carefully presented in order to remain both accurate and interesting. I’ve also tried to cut details that work better in text without introducing confusion for end users.

Here’s one of the results:

[wpvideo pAyMVHa2]

I invite you to keep an eye out for future video protocols. In the meantime, you should leave a comment and let me know what procedures and products you think would benefit most from this kind of treatment.